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heegyu

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elsevier-oa-cc-by

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[ "ENGI", "MATE", "PHYS" ]

[ "CAN", "Kissing bond", "NDE", "NDT", "Non-collinear", "Nonlinear", "Ultrasonic" ]

[ "2210", "2500", "3104" ]

[ "Kissing bonds, two surfaces in intimate contact but not bonded together, can be difficult to detect with the non-destructive testing (NDT) techniques that are standard in industry today [1,2].", "For this reason, some structures are over-engineered to allow for the safe failure of an adhesive joint; ‘chicken rivets’ in aeronautical structures are an example of this.", "Kissing bonds are hard to detect with conventional ultrasound techniques because the kissing interface has a transmission coefficient very similar to the properly bonded case.", "This is particularly true when the interface is under compressive load.", "If enough acoustic stress can be applied to the interface the kissing bond will open during the tensile part of the wave.", "This opening and closing of the interface causes contact acoustic nonlinearity (CAN), clipping parts of the waveforms and transferring energy into other harmonics [3,4].", "The research presented here aims to investigate this CAN behaviour in order to create a method for reliable, spatially sensitive, detection of kissing bonds.", "There are many possible ways to detect the acoustic nonlinearity of a kissing bond.", "Measuring the change in transmission/reflection of the fundamental frequency is the simplest but it is insensitive due to the small changes involved [4,5].", "Detecting the harmonics produced is more sensitive [1] but the harmonics often have other potential sources such as the amplifiers, transducers, couplant or the bulk materials themselves [6,7].", "To overcome these problems a more advanced technique is required such as non-collinear mixing, pioneered by Jones and Kobett, and Rollins [8–10] in the 1960s.", "In non-collinear mixing two beams follow different paths that overlap in an area of interest.", "In this overlap region nonlinearities can cause the two waves to interact with each other producing a new one.", "The scattered beam travels in a different direction from the input beams separating its signal from the system harmonics present in the input beams that might otherwise obscure it.", "This creates a method which is spatially selective and when combined with filtration techniques makes it highly sensitive.", "One of the conditions that must be met for bulk nonlinear mixing to occur is that the geometry of the input beams' interference pattern is such that the spacing of the antinodes is the same as the wavelength corresponding to the sum or difference of the input frequencies.", "The two key parameters that control the geometry of the interference pattern are the angle at which the two beams overlap (referred to as interaction angle) and the ratio of their frequencies.", "The optimal conditions were defined as ‘resonant conditions’ in Ref. [8].", "Within the volume of interaction there are two main sources of nonlinearity; the classical nonlinearity of the solids [11], corresponding to the intrinsic bulk nonlinearity, which allows for the mixing of the two input beams as described by Refs. [8,9], and the CAN.", "CAN generates a signal from the kissing bond in the non-collinear case by the combined acoustic forces of the two input waves opening, closing, or unloading the interface enough to allow them to slip when it would be in a different state if only a single wave were applied.", "This modulation generates harmonics in a similar way to the single beam case.", "These perturbations effectively create an array of acoustic sources on the interface which together produce plane waves.", "Another difference between bulk and CAN mixing is that the latter produces scattered beams in both directions from the interface [12,13].", "This can be thought of as being caused by the reflection from the interface when the two overlapping waves open it when it would be closed in the single beam case.", "This effect was not exploited in the following research due to difficulties in positioning an array between the input transducers but the results from transmission testing should be informative of likely reflective behaviour which would be useful for developing a one-sided NDT inspection tool.", "Non-collinear mixing has been used to investigate the state of many different materials including; physical ageing of thermoplastics [14], epoxy curing [14], fatigue in aluminium [15], and oxidative aging of asphalt [16].", "Research into the behaviour of kissing bonds with non-collinear ultrasonic mixing is limited.", "Demčenko et al. conducted testing on PVC plates [17], and there has been modelling conducted by Blanloeuil et al. [13], and Zhang et al. [18].", "The modelling by Zhang et al. focuses on an infinite interface with nonlinear stiffness terms in one case, and a thin region of hyperelastic solid in another case.", "These differ from the work presented here as their interfaces never open but the results are similar in many ways.", "In Demčenko's work the interaction between shear and longitudinal beams overlapping at a kissing bond at fixed angles is investigated.", "If the interface is defined as the x-z plane then the input beams were tested with interaction planes of x-z and y-z.", "When operating in the y-z plane the beams approached the interface from opposite sides.", "The study showed that the interface led to a reduction in nonlinear wave signal in both interaction planes.", "In the work presented here the input beams are in the y-z plane but both approach the interface from the same side.", "Current methods consider the response for single values of interaction angle, ϕ, and frequency ratio, a, usually selected to satisfy the resonance criteria.", "The scattered wave amplitude however may be evaluated for a range of these parameter values, producing a surface within the a-ϕ parameter space.", "There is more information about the material contained within the full parameter space than can be recovered from a single experimental operating point.", "For classical nonlinearity, this parameter space has a characteristic shape, governed by the resonant phasing-matching condition.", "It has previously been observed by Blanloeuil et al. [13] in a numerical study that production of a sum-frequency wave from shear-shear mixing is also predicted by a contact-acoustic nonlinearity.", "The hypothesis examined in this work is that CAN will produce a response within the wave mixing parameter space that is characteristically different from that produced by classical nonlinear terms and that, consequently, analysis of the full parameter space allows the underlying nonlinear mechanics to be identified in addition to the magnitude of nonlinearity.", "Further, by evaluating elastic nonlinearity using the shape of this surface, the measurements become much less sensitive to incident wave amplitude.", "This offers the potential for more experimentally robust nonlinear measurements.", "Herein the shape of the parameter-space response shall be referred to as the ‘fingerprint’ of the nonlinear interaction.", "This study first identifies, through numerical modelling, the expected fingerprints for the shear-shear to longitudinal interaction for the cases of classical and contact-acoustic nonlinearity.", "An experimental program is then undertaken to acquire fingerprints for wave interactions within both bulk material and at an interface.", "Good agreement is found between theoretical and experimental fingerprints.", "The fingerprints of the classical and contact-acoustic interactions are found to be characteristically different in shape, supporting the hypothesis that the fingerprint is a useful tool for the detection of kissing bonds and, more generally, the characterisation of nonlinearity.", "To investigate the parameter space efficiently a computer-controlled, motorised rig was developed.", "The angle of each transducer is independently set, their lateral separation can also be controlled, allowing a constant interaction depth to be maintained with varying interaction angle.", "This is shown schematically in Fig. 1 (a).", "The sample was mounted below the transducers, with an ultrasonic phased array below it in contact with its bottom surface.", "An array was used because as the frequency ratio is changed so is the scattering angle of the produced beam.", "The scattering angle for bulk mixing can be predicted by using the relevant equation from Table 1 of [8].", "The 40 mm length of the array was enough to capture the signal of interest in nearly all cases within the desired parameter space.", "The assembly was placed in a water tank, submerging the input transducers, sample, and array to minimise the coupling variation.", "The temperature of the water was controlled with 0.1 °C precision to maintain a constant speed of sound in water, ensuring reliable refraction angles into the sample.", "The input pulses were generated using Agilent 33250A arbitrary waveform generators and amplified with Amplifier Research 75A250A/100A400 amplifiers.", "The input transducers were Olympus V551-SM's which have an active diameter of 10 mm, a peak frequency of 4.7 MHz, and a −6 dB bandwidth of 3.4 MHz.", "In the testing one transducer was always used at 5 MHz, and the other was varied between 3 MHz and 7.5 MHz.", "This results in the frequency ratio being coupled with the average input frequency which could have an additional impact on the measured fingerprints.", "In future work it might be better to avoid this coupling by changing both the input frequencies in order to keep a constant output.", "To detect the nonlinear signal an Imasonic 10 MHz linear array with a −6 dB bandwidth of 9 MHz was used.", "This array had 128 elements at a pitch of 0.3 mm, and was used in conjunction with a MicroPulseFMC array controller.", "These wide bandwidth transducers allowed frequency ratios between 0.6 and 1.5 to be tested with enough sensitivity to detect scattering even at the extremes.", "The samples discussed in this report were both 2024 T351 aluminium blocks with outer dimensions of 120 × 80 × 60 mm.", "A solid block was used as a reference and another block cut into two halves and compressively loaded together with bolts to simulate a kissing bond.", "The reference block allows measurements of just the bulk nonlinear behaviour.", "The interface testing was conducted with the contacting surfaces finely ground using P1000 grit wet and dry paper (18 μm average particle size).", "Different results would be expected with different surface finishes due to changes in the fraction of the surfaces in contact and the range of angles at which they meet, although this is not tested in this work.", "The torque on the bolts of the two-part block was varied, using a torque wrench, altering the loading on the interface, see Fig. 1 (b).", "The use of bolts along the sides allows unobstructed ultrasonic access to a large section of the block, this gives greater flexibility in the measurements that can be made when compared to the conventional universal testing machine method of loading.", "The main negative of the technique was the random error in loading magnitude due to the unreliable frictional behaviour of the nuts/bolts and the systematic error due to the difficulty in directly measuring the applied load.", "Another limitation was the loading range due to the 5 to 50 Nm torque range.", "Lower torques than this were very inaccurate due to frictional effects, and larger torques would be hard to apply manually.", "The interface sample was sealed with silicone to prevent water ingress when immersed.", "FE modelling, in Abaqus, was conducted to verify that the dimensions of the blocks and bolts should give an even interface loading along the centre.", "This modelling predicted that a torque of 5 Nm should produce a compressive load of 2 MPa in the region of inspection, however due to the experimental samples not being perfectly flat there is likely to be some error in this.", "It should be noted that the approximation of a kissing bond by the compressive loading of the two plates is not intended to produce an interface that is undetectable to conventional linear methods.", "The focus here is on measuring the CAN mixing behaviour in a simplified scenario so that the knowledge can then be applied to the detection of more realistic invisible kissing bonds in later research.", "There are many modes of non-collinear mixing possible [8], investigated in this work is the interaction of two shear waves producing a longitudinal wave at the sum of the two incident frequencies.", "This mode was used mainly due to the simplicity of producing exclusively shear waves over a wide range of angles, and because it allows for the generation of mixing from both the bulk nonlinearities and CAN which enables the bulk signal to be used as a reference for the CAN signal amplitude.", "If the aim of the experiment were to avoid the production of bulk scattering and only produce CAN scattering a different interaction mode, such as the mixing of two longitudinal waves, would be preferred.", "20-cycle Hann-windowed pulses were used for both input transducers.", "These long pulses create a narrow frequency bandwidth which makes the experiment more sensitive to frequency ratio and improves the filtering of the output signal because the energy is within a smaller frequency window.", "The Hann window is used to reduce frequency sidebands.", "For each combination of interaction angle and frequency ratio three measurements were taken; signal with both transducers emitting, signal with just the left transducer, and signal with just the right.", "The signals received from the left and right were subtracted from the case where both were emitting simultaneously (Fig. 2 shows examples of the time data at various points of acquisition and processing).", "In plots a, b, and c of Fig. 2 the side lobes of the input pulses dominate but after subtraction, shown in plot d, the scattered pulse becomes visible.", "Note the different colour scales.", "Filtering at the sum frequency, Fig. 2 (e), removes nearly all of the remaining unwanted signal allowing the pulse of interest and its echoes to be clearly seen.", "Pulse inversion is a commonly used technique in nonlinear ultrasonics [19–21] as it can be used to remove either the even or odd harmonics from the signal.", "However, it is less useful in sum-frequency non-collinear mixing since the signal of interest is at a similar frequency to the second harmonic of the input beams when the frequency ratio is close to one.", "In non-collinear mixing the second harmonic component of the input beams' side lobes is commonly the largest source of unwanted signal that remains after processing in the way detailed in the previous paragraph.", "Conventional pulse inversion is not able to remove these side lobes while enhancing the sum-frequency scattered wave.", "There is a more advanced form of pulse inversion where all combinations of inversions of the input pulse are applied, requiring a total of four firings [22].", "This method was not used in the experimentation presented here but it looks very promising for future work.", "A window of the data in time and space was selected based upon the predicted time of arrival and angle of scattering, as stated in Ref. [8].", "This window removed most of the unwanted signal from the sidelobes of the input beams that normally arrived later than the signal of interest.", "Focusing on reception was then performed to enhance the measurement of the wave scattered by the interface.", "To do so a delay is applied to each element's response, depending on the position of the element within the array and its location with respect to the interaction volume.", "The remaining signal was then summed element-wise to complete the focusing operation.", "Finally, the Hilbert transform was used to acquire the envelope of the signal and the peak value of this was recorded.", "This value is used as the metric of scattering and referred to in later figures as ‘peak scattering amplitude’.", "By recording this scattering value for the range of input parameters a ‘fingerprint’ can be made.", "These steps are shown as a flowchart in Fig. 3.", "A program of numerical modelling was undertaken in order to determine the independent contribution of both the classical and contact acoustic nonlinearity on the wave mixing parameter space.", "The modelling is also useful to inform which areas of the parameter space are likely to be of interest so that the experiment can be designed to include these ranges.", "Knowledge of the experimental geometry, apparatus, and processing techniques is used in the production of models that more accurately relate to the experimental measurements.", "Many factors such as transducer bandwidth, mode conversion at the water-aluminium interface, and interaction volume have significant impacts on the resulting fingerprints so are included in the following results.", "It can be seen in Fig. 4 that the strongest mixing response is predicted at 118° and a frequency ratio of 1.06.", "This is approximately the same angle as the resonance angle given by the equation stated in Ref. [9], 120°.", "There are also two secondary lobes of nonlinear scattering that can be seen at smaller interaction angles, peaking at around 100° and 85°.", "The reduction in amplitude at frequency ratios far from 1 is due mainly to the bandwidth of the transducers, and the cut off at angles smaller than 60° is caused by very little production of shear waves at the water/aluminium interface below the first critical angle.", "These results predict that there are multiple features in the fingerprint within the 60°–140° that might interfere with the CAN signals of interest presented in the following section.", "The nonlinearity of the kissing interface is very different from the classical bulk nonlinearity, as such it is not obvious based upon previously established theory that the interface would cause two incident shear waves to interact to produce a scattered longitudinal wave.", "The modelling conducted in this section shows that a kissing interface can cause non-collinear mixing, as others have done previously, and it explores the parametric sensitivity of the mixing.", "The behaviour of a contacting interface requires a model that can accurately capture how the interface can be in one of three states, strongly closed (transferring transverse and normal stresses), slipping (transferring only normal stress), and open.", "This was achieved using a 2D plane strain FE model.", "The model is similar to the one reported in Ref. [13], with differences in terms of geometry and incident frequencies.", "The main characteristics of the FE model are detailed below for completeness.", "This model does not include the higher order elastic terms so classical bulk mixing should not occur.", "The model consisted of 720000 elements, 723002 nodes (each node has two degrees of freedom) and took about 11 h to solve for each parametric point on an average desktop PC.", "Since 130 different points in the parameter space were investigated over 1000 h of computation time was required to generate the fingerprint.", "The code does not currently make use of parallel or GPU computing so it might be possible to reduce the time requirements by these methods in the future.", "Since the model used for this work is presented in other publications further details will not be shown or discussed here.", "The following is about the resulting fingerprint produced when the time signals from an array of points below the crack are processed in the same way as defined in the experimental methods section.", "The modelling demonstrated the possibility of bulk mixing happening at smaller interaction angles than its resonance condition.", "This could potentially obscure interface mixing measurements so testing of solid material must be done first to understand its influence on later interface fingerprints.", "Fig. 6 shows the nonlinear response of aluminium 2024 T351, with the interaction volume's centre at 18 mm below the surface of the 60 mm thick block.", "In this most simple case the fingerprint has only one peak, at the angle predicted by the classical equations [8,10].", "Fingerprints have been taken at various input power levels and depths (10 mm–30 mm) into the material, despite these changes the fingerprint remains largely unchanged in shape.", "The intensity of the pattern is proportional to the product of the input beams' amplitudes, as expected.", "Fig. 7 shows the fingerprint of the solid aluminium at a depth of 30 mm (the centre of the sample).", "The fingerprint is quite similar to that taken at 18 mm, again showing only one peak in response approximately at the resonant condition.", "There are some slight differences between measurements at 18 mm and 30 mm however.", "The decrease in intensity at angles greater than 125° at 30 mm deep is due to a geometric limitation that reduces the fraction of the beams able to propagate into the sample.", "This is caused by the larger input beam separations required for deeper interactions.", "Another notable difference between the two fingerprints is their overall amplitude; at 18 mm deep the scattering response is nearly twice that at 30 mm.", "This is mainly due to beam divergence as they propagate through the sample, reducing beam amplitude but increasing volume of interaction.", "The scattering amplitude is proportional to the interaction volume and the square of the input amplitude.", "The combination of these two factors results in scattering amplitude being proportional to the inverse of beam radius at the interaction point.", "The classical modelling predicted that there would be a primary mixing peak at 118° ranging from 110° to 130°, this matches the experimental data very well, Fig. 6.", "It also predicted the existence of smaller peaks in mixing at angles of 100° and 85°, Fig. 4, with the 100° peak having a quarter of the magnitude of the main mixing region.", "It does not look like these secondary peaks are present in the experimental fingerprint.", "There is some signal visible between 95° and 106° experimentally but it is much smaller than predicted and is likely due to poor filtration of input beam side lobes at frequency ratios close to 1.", "Otherwise the model and experiments agree well showing a main mixing region between 110° and 130° and similar behaviour in terms of frequency ratio.", "Now that a benchmark for solid aluminium mixing has been obtained the interface sample can be studied for comparison.", "Fig. 8 shows a fingerprint of the compression loaded interface sample, with the volume of interaction centred on the interface.", "The reduction in signal seen at 125° and greater is due to the geometric limitation mentioned previously in Section 4.1.", "A peak in mixing behaviour is observed at around 75° and a frequency ratio of 0.9 in this case.", "There is a much smaller peak at around 100°, and a very slight peak at frequency ratios around 0.85 at 120°.", "Figs. 6–8 were normalised to the maximum scattering amplitude of the three which occurred in the interface case.", "The maximum scattering amplitude from the interface was an order of magnitude larger than that from the solid sample at the same depth.", "The most useful trends in the fingerprints appear to occur in the interaction angle dimension therefore further testing was conducted at a single frequency ratio, 0.9.", "This was selected as it was near the peak response points of both solid and interface samples and far enough away from 1.0 that it had reduced noise from the frequency filtering.", "Values for the peak scattering amplitude are presented in two ways in the following sections.", "In part a of the figures the values have been normalised by the peak value obtained in the kissing interface fingerprint, Fig. 8.", "In part b the data is normalised by the peak scattering of each parametric sweep.", "The former is to allow for absolute amplitude trends to be compared and the latter for comparison of fingerprint shapes.", "Fig. 9 (a) shows the scattering response of the interface region at a frequency ratio of 0.9 with bolts torqued between 10 Nm and 40 Nm.", "This range was used because very little signal was observable with the torque below 10 Nm, and 40 Nm was as much as could be applied to the sample with the torque wrench.", "Since it is very difficult to know accurately the interface pressure with this experimental method bolt torque will be referred to as the controlled variable.", "The two are predicted to be directly proportional, ignoring microscopic contact changes.", "The sample was preloaded to 40 Nm before the two full loading cycles, ‘a’ and ‘b’, were tested.", "For the cycles the bolts were torqued to 10 Nm initially then increased in steps of 10 Nm up to 40 Nm.", "As the loading was increased the amount of mixing increased.", "When 10 Nm was applied the main CAN related peak is seen at around 76°, this shifted approximately 2° towards smaller interaction angles as the load increased.", "This plot also shows that there was an overall trend of increased scattering with each loading cycle.", "This can be explained by the fact that the interface was never fully unloaded during these cycles, each bolt was unloaded from 40 Nm and re-tightened to 10 Nm in turn, keeping the faces in constant contact.", "This method was intended to stop the faces moving relative to each other between each cycle, keeping the same parts of the interface in contact.", "Due to this it is expected that the surface asperities will gradually deform to match each other with each cycle, increasing the contact between the two faces and thus the transmission.", "In the paper by Blanloeuil et al. [13] the FE modelling predicted that the maximal mixing response occurs when the interface load is 0.25 that of the peak combined acoustic loading.", "The experimental acoustic loading was estimated by using a laser vibrometer.", "A measurement was taken with one of the input beams at normal incidence on a 30 mm thick aluminium sample.", "The surface deflection due to the longitudinal wave that propagated through the sample was converted into an acoustic stress.", "Using mode conversion calculations an estimate was made of the combined acoustic stress of two shear waves in the aluminium that would be created when an interaction angle of about 80° is used.", "The resulting value was 0.1 MPa, but due to the many approximations involved this is probably only accurate to an order of magnitude.", "Using this value of acoustic stress gives an expected peak response at 0.025 MPa interface loading which corresponds to bolts torqued to 0.05 Nm.", "This is much smaller than the experimentally tested range of 10–40 Nm in which the mixing was observed increasing with load.", "The laser vibrometer measurement and torque to interface pressure estimations were quite rough but a disagreement of at least three orders of magnitude suggest that there is likely another source of difference between the model and experimental measurements.", "One possible difference is the smoothness of the interface with the modelling being perfectly flat and having completely evenly distributed loading.", "To demonstrate the repeatability of the method a plot of measurements taken at 40 Nm torque is shown in Fig. 10.", "It contains a parametric sweep taken after the plates were first loaded to 40 Nm (the pretest measurement), another taken after the load was released and then reapplied on each bolt in turn (a), and two after a second load cycle (b).", "The sample was removed from the immersion tank and replaced between the two ‘b’ tests.", "It can be seen in Fig. 10 (a) that the pretest measurement had amplitudes 25% smaller than the cycles that followed, and that there was about a 10% variation in amplitudes of cycles a and b.", "The variation in amplitude with cycle number is expected as surface asperities are altered by each successive cycle, although most of the deformation occurs during the first [4].", "Initially the surfaces only contact where they are locally raised.", "Due to the small area in contact this area is under high load and unable to be overcome by the acoustic stress.", "The remaining troughs are not in contact so transmit no signal.", "The combination of these factors leads to small CAN signals when the plates are first brought together but cause the signal to increase as the surfaces conform to each other.", "This process is expected to be more dominant in a roughly ground interface case than for a polished interface because the asperities of the polished interface should be much smaller and form a better match initially.", "The normalised data in Fig. 10 (b) displays very good agreement between the measurements, only the pretest response significantly differed from the others, having a smaller 120° to main peak ratio.", "Some difference would be expected due to the changing interface condition discussed above.", "This data gives an indication of the repeatability of the measurement, showing that peak normalisation results in consistent parametric trends when the sample is unaltered.", "Measurements taken consecutively without the removal of the sample were conducted, these showed even smaller variation than seen above, leading to the conclusion that positioning of the sample is the primary cause of the slight variation observed in ‘40Nm b’ trends in Fig. 10 (b).", "The impact of positioning is explored in the following section.", "There is almost certainly some variation in the average surface height of the blocks between points a few millimeters apart due to the limitations of the production method used, therefore it is expected that some macroscopic regions of the interface will be under greater average load than others despite efforts to design a geometry that minimises loading variability.", "Due to this it might be expected that testing a different region of the interface could yield a fingerprint that resembles another taken at a different torque setting.", "To investigate this the interface sample had scattering measurements taken at various points along the central axis of the sample, specifically at 0, 2, 4, and 5 mm from the centre.", "In Fig. 11 (a) the unadjusted arbitrary amplitudes can be seen.", "The bolt torque was 40 Nm for this testing.", "The reduction in signal observed at above 120° for increasing displacements is related to the input beam clipping issue mentioned previously.", "The largest difference between measurement points in Fig. 11 (a) was the drop in amplitude when displacing from the central position.", "Moving only 2 mm caused a 25% drop in signal.", "The diameter of the interaction area on the interface at the -3 dB limits is estimated to be 21 mm by beam divergence calculations (-3 dB was selected rather than -6 dB due to the scattering being a product of the square of the input amplitude).", "In the 2 mm translation roughly 12% of the initial surface area moved outside of the new overlap area.", "It is possible that a highly CAN active area of the interface was moved outside the interaction region and that the new area was not very active, but the disproportionately large change of 25% means this is unlikely.", "Another possible explanation is proposed at the end of this section.", "Fig. 11 (b) also contains some interesting trends.", "The width of the main peak is much larger at 4 mm displacement and it has a rounded peak.", "The peak response interaction angle varies by about 4° between the tests and the smaller peak at 100° does not exist other than at 0 mm.", "Some of these trends are similar to those observed as load was varied in Fig. 9, such as peak shifting, but others are quite different, e.g. the large peak width changes.", "This implies that the shape of the response must be related to more than just average interface loading within the interaction area, indicating that there are other factor(s) causing the variation despite the surfaces being uniformly rough.", "The combination of the rapidly changing amplitudes and shapes of the parametric trend therefore probably have a more complex cause than has been discussed above.", "One explanation is that the overlapping input shear waves constructively produce lines of positive and negative tensile stress on the interface with regions of destructive interference in between.", "It is these lines where the waves cancel each other that create transmission at the interface when otherwise it might be open due to the tensile forces of the individual beams, thus these lines are the sources of the non-collinearly mixed signal.", "The lines have a spacing of approximately 1 mm (dependent on interaction angle and frequency ratio) and are at fixed places on the interface when the frequency ratio is one.", "At other ratios these sampling lines sweep across the interface during the pulse, sweeping faster the further ratio is away from one.", "For the case of a 0.9 ratio, as used in this study, the sampling lines will shift back to the starting pattern over the course of 10 cycles of the reference input beam (at 5 MHz in this research).", "Therefore, the experiment's sensitivity is biased towards the lines of the interface that are sampled when the input pulses are near their maxima due to the peak scattering amplitude being used as the measurement metric.", "Movement of the sample or change in the interaction angle causes the position of the sampling lines to be altered resulting in the complex parametric-space response that was observed.", "The non-collinear interaction of two shear waves in a dry, aluminium, compression loaded interface has been studied over a wide range of interaction angles and frequency ratios in the sum-frequency, shear-shear mixing regime at around 5 MHz, forming ‘fingerprints’ of the interface.", "This sample is intended to simulate an acoustically simple case of a kissing bond to allow the fundamentals of a non-collinear approach to detecting more realistic kissing bonds to be developed.", "The kissing interface sample displayed nonlinear scattering fingerprints very different from reference solid sample, producing signal at interaction angles between 60° and 120°.", "At all points in the loading range investigated a characteristic shape was produced, peaking at around 75°.", "This fingerprint was similar to that predicted by Blanloeuil's FE modelling [13] except for the secondary peaks in the 100°–120° region in some cases.", "These peaks were most prominent at higher compressive loads and their cause is unclear.", "Frequency ratio was not studied in detail in this work as the initial fingerprints had few apparent features in this dimension.", "It has been shown that mixing behaviour away from the peak conditions may contain useful information about the interface; e.g. the trends at around the 100° region relating to the interface loading.", "The interaction angle of peak mixing may be another indicator of interface loading.", "In this study the peak amplitude correlated well with the interface loading but this trend is not expected to continue at higher contact pressures/lower acoustic pressures, as the interface becomes too highly loaded to be separated by the acoustic waves.", "There is potential benefit to measuring multiple fingerprint features related to the same interface/material parameter as it would improve the robustness of the method.", "When different regions of the interface were probed the parameter space changed in ways that did not match with the changes observed due to varied loading.", "Therefore, further testing of different samples and parts of their interfaces is required to understand the general parametric behaviour of kissing interfaces.", "It is hypothesised that the position sensitivity is partly due to the non-collinear method only sampling the regions of the interface where the component of stress normal to the interface of the input beams cancel, forming an array of sampling lines.", "Further testing of a smoother interface in terms of position sensitivity would be of interest in relation to this phenomenon as it would be expected to have properties that vary less spatially.", "It would also be of interest to test kissing interfaces at higher mechanical compressive loading to investigate the point at which the loading becomes too great for the acoustic waves to separate the surfaces.", "The secondary peaks in the interface fingerprints were not predicted by the FE modelling or observed in the solid sample.", "The precise nature of their source is not known but could be linked to the sampling lines theory mentioned in the previous paragraph.", "It might be that particular interaction angles sampled the interface at more active regions creating the peaks in response, in a similar way to how the peaks changed when the sample was moved.", "At low loads the interface had a smoother parametric response.", "Combining this fact with the sampling theory suggests that the interface has a more uniform contact profile at low loads.", "Another possible explanation relating to the load based behaviour is that at low loads the interfaces meet more unevenly and the increased deviation in contact angle from the macroscopic surface normal causes a smoothing of the response due to a wider distribution of interaction angles experienced at the microscopic level.", "This concept alone does not explain the existence of secondary peaks at higher loads however so perhaps it is a combination of effects.", "These behaviours indicate that the system is highly complex, probably requiring more advanced models and further experimentation to fully understand the impact of kissing bond parameters on their fingerprints.", "In the future use of focusing on input beams would allow interaction regions with far fewer overlapping wavefronts to be made.", "This would probe the interface in greater detail and might confirm if the position sensitivity trends observed with larger interaction areas were a result of interface properties varying on a wavelength scale.", "If using unfocused beams sweeping the interaction nodes along the interface, perhaps by altering the phase of the beams, and summing the responses together might be a route to measuring a more averaged scattering value for the interaction area.", "Alternatively frequency ratio ratios further from one with longer pulses could achieve a similar level of sampling coverage.", "This could be useful if a faster measurement is required than scanning a focus across the whole area and would also ensure that no parts of the interface are unsampled.", "In this work there was only one interface at a known depth, in this case a non-collinear c-scan could have been conducted by moving the input transducers and array along the sample.", "If the defect is at an unknown depth the technique could be easily extended to 3D by sweeping the depth of the interaction volume.", "The fingerprint at each location might then be analysed to identify the properties of the sample within the interaction volume, allowing 3D positional detection of kissing bonds." ]

[]

[ "AGRI", "EART", "ENVI" ]

[ "Dark incubation", "Esterase substrate", "Flow cytometry", "Inactivation", "Tetraselmis suecica", "Ultraviolet irradiation" ]

[ "1104", "1910", "2310" ]

[ "Ships use water as ballast to ensure stability and trim during the voyage, and ambient water is pumped into ballast tanks in the hull of the ships.", "It is traditionally discharged without any treatment and represents a global vector for aquatic invasion.", "A multitude of organisms like virus, bacteria, algae and zooplankton are carried around the world in ship's ballast tanks (David et al., 2007; Drake et al., 2007; Hallegraeff and Bolch, 1991).", "Some organisms survive in ballast tanks and are released into new environments.", "If nonindigenous species adapt and establish in a new environment, they might have an impact on the native species and cause ecological change in the ocean (Gollasch et al., 2015; Ruiz et al., 1997).", "It is of importance to minimize and prevent dispersal of species by ballast water discharge to hinder potential harm to ecosystems, the economy, or human health (Ruiz et al., 2000).", "In 2004 the International Maritime Organization (IMO) established standards for ballast water treatment through the International Convention for the Control and Management of Ship's Ballast Water and Sediments (International Maritime Organization, 2004).", "Regulation D-2 of the Convention sets the standard regarding category and concentration of organisms at discharge.", "The Convention will enter into force 12 months after being ratified by 30 States representing 35% of the merchant shipping tonnage.", "In August 2015 44 States, representing 32.86% of the world tonnage, have ratified the Convention.", "The upcoming IMO regulations have led to development of various ballast water treatment systems (BWTS) that facilitate disinfection of ballast water (David and Gollasch, 2015; Delacroix et al., 2013; Lloyd's Register Marine's, 2015a, 2015b; Stehouwer et al., 2015; Werschkun et al., 2012, 2014).", "All BWTS have to be approved by national authorities according to IMO regulations and/or the regulations of other national bodies (e.g. U.S. Coast Guard (USCG)).", "When selecting and installing a BWTS, the shipping companies have to consider different technical and operational aspects (Lloyd's Register Marine's, 2015a, 2015b).", "The BWTS use a range of different treatment technologies, from processing the water with solid–liquid separation to chemical- (active substances) and/or physical disinfection (e.g. UV).", "The main operational cost for UV based BWTS is related to power consumption (Werschkun et al., 2014).", "Ship owners can reduce such costs by lowering the UV intensity, providing that the ship's discharged ballast water still complies to Regulation D-2 (International Maritime Organization, 2008a).", "It is therefore of interest to determine the lowest lethal UV dose and to estimate the time required for inactivation when stored in ballast tanks after irradiation.", "UV irradiation is performed either by low pressure (LP) or medium pressure (MP) UV lamps (Oguma et al., 2002; Werschkun et al., 2012; Zimmer and Slawson, 2002).", "LP lamps emit UV-C radiation, primarily at 254 nm, which is most efficiently absorbed by nucleic acids and causes DNA damages (Sinha and Häder, 2002).", "UV induced DNA damages can be reversed by DNA repair mechanisms, referred to as photoreactivation and dark repair (Sancar and Sancar, 1988; Sinha and Häder, 2002).", "MP UV lamps emit radiation spanning the UV-A, -B and -C bands causing additional damage to proteins and enzymes.", "For instance, UV-B radiation can affect key components in photosynthesis (Fiscus and Booker, 1995; Holzinger and Lütz, 2006; Kottuparambil et al., 2012), causing energy deprivation in phytoplankton cells.", "Thus, it has been argued that MP UV lamps can cause a higher degree of inactivation compared to LP UV lamps (Kalisvaart, 2001; Oguma et al., 2002).", "UV irradiation can leave cells in different conditions (live, dead or damaged), whereof the viability of damaged cells at discharge is uncertain (Olsen et al., 2015).", "Damaged cells can be unculturable, though they can be metabolic active and may pose a health risk (Oliver, 2010).", "Further, cellular DNA repair mechanisms can restore the genetic information (Sancar and Sancar, 1988; Sinha and Häder, 2002; Zimmer and Slawson, 2002) causing the cell to grow and replicate after discharge (Liebich et al., 2012; Martínez et al., 2012, 2013).", "Additionally, the terminology describing the organisms at discharge can be confusing or unclear.", "The IMO Convention refers to “viable” organisms (International Maritime Organization, 2004), and the Guidelines for approval of ballast water management systems (G8) define “viable organisms” as “organisms and any life stages thereof that are living” (International Maritime Organization, 2008a).", "USCG also uses the term “living” (United States Coast Guard, 2012).", "Determining the condition of UV irradiated cells is a complex task.", "On the other hand, cheap, fast and reliable methods to analyze ballast water are necessary for approval of BWTS technologies and for compliance testing of ballast water discharge (International Maritime Organization, 2013).", "Testing for compliance can be performed in two steps; an indicative and a detailed analyses.", "An indicative analysis is a relatively simple and quick measurement that gives a rough estimate of the number of viable organisms in the ballast water at discharge.", "Examples of indicative analysis methods are e.g. BallastCAM and various fluorescence or ATP detections (Drake et al., 2014; First and Drake, 2013, 2014; Gollasch and David, 2012, 2015; van Slooten et al., 2015).", "If an indicative analysis shows compliance to Regulation D-2, there is no need for a detailed analysis.", "Should the indicative analyses be non-compliant, however, a detailed analysis must be undertaken to give robust and direct measurements determining the concentration of viable organism in ballast water discharge according to Regulation D-2.", "Quantification of live bacteria traditionally relies on cultivation methods, which is time-consuming and may give false negatives as several species are uncultivable although viable (Roszak and Colwell, 1987; Staley and Konopka, 1985).", "Flow cytometry (FCM) has been suggested as a promising method for detailed analysis (International Maritime Organization, 2013; Peperzak and Gollasch, 2013).", "FCM facilitates rapid detection, enumeration and characterization of organisms in combination with fluorescent dyes, and enables to study populations and communities indirectly (Peperzak and Brussaard, 2011; Shapiro, 2000).", "Previously a FCM protocol was developed to distinguish between live and dead Tetraselmis suecica cells (Olsen et al., 2015).", "For UV irradiated samples the FCM protocol could not distinguish between live and damaged cells, as the latter contain both dying and repairable cells.", "The current study uses the FCM protocol to elaborate on different UV doses and the effect of dark incubation on inactivation of the algae T. suecica, to simulate a ballast water treatment and subsequent transport.", "Our specific objectives were to:", "Determine the minimum UV dose that permanently inactivates the algae.", "Quantify effects of different UV doses on T. suecica.", "Estimate the time of dark incubation required to permanently inactivate the algae treated with UV doses lower than minimum permanently inactivation dose.", "Provide recommendations for ballast water management.", "The phytoplankter specie T. suecica (Strain K-0297, Scandinavian Culture Collection of Alga and Protozoa, University of Copenhagen, Denmark) was selected as a test organism.", "It was cultured in 24 PPT artificial sea water (Marine SeaSalt, Tetra, Melle, Germany) added 0.12% Substral (The Scotts Company (Nordic) A/S, Naverland, Glostrup, Denmark), at 15 °C, 100 rpm, 14:10 light:dark cycle and 90 lx light intensity (Flora-Glo, T8, 20 W).", "The culture was diluted in growth medium to a density of 104 live cells ml− 1 prior to irradiation, monitored by FCM.", "Irradiation was performed using a collimated beam MP UV lamp (800 W) (BestUV, Hazerswoude, The Netherlands) (Olsen et al., 2015).", "For each experiment three samples of 15 ml diluted T. suecica culture were irradiated with the same UV dose in a petri dish (inner diameter 6 cm, culture depth 7 mm) while mixed with a 1 × 0.4 cm magnetic stir bar at 200 rpm in room temperature (RT).", "The intensity (mW/cm2) of the UV lamp was fixed and the exposure times used were 155, 233, 311, 622 and 1244 s for UV doses 100, 150, 200, 400 and 800 mJ/cm2, respectively.", "The irradiated samples were transferred to sterile 50 ml polypropylene tubes (Fisher Scientific), so was the control samples, including 2 × 15 ml non-irradiated cells and 10 ml dead cells.", "The dead cells were killed by fixation with formaldehyde at 5% final concentration (36.5–38% formaldehyde, Sigma-Aldrich).", "All tubes were wrapped in aluminum foil and incubated in the dark with loosened lids at 15 °C.", "First, a pre-study over 5 days was performed to observe the inactivation effect of different UV doses and dark incubation, and to test whether this effect was interpretable with FCM.", "This was followed by two complete experiments, denoted as exp-I and exp-II, and an overview of the set-up for these experiments is given in Fig. 1.", "For FCM analysis, the samples were stained with 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) and analyzed with an Attune Acoustic Focusing Cytometer (Olsen et al., 2015).", "The samples in the pre-study were analyzed at days 1, 3 and 5 after treatment.", "In exp-I samples were analyzed at days 1, 3, 6, 9, 13 and 22, and in exp-II samples were analyzed at days 1, 3, 6, 10, 15 and 22 after treatment (Fig. 1).", "The samples in exp-I and -II were analyzed at different intervals due to logistics.", "A previously defined gate (i.e. a collection of single cell FCM-signals) in the FCM dot plots was used for analysis.", "The gate was defined based on plate count results from non-irradiated cells, and regression analysis was used for validation of the gate (Olsen et al., 2015).", "To determine the number of culturable cells, plate count analysis was performed 1 day after UV treatment (Olsen et al., 2015).", "Also at day 1, a most probable number (MPN) analysis was performed.", "The samples were diluted in growth medium (24 PPT artificial sea water added 0.12% Substral) in 10-fold series up to 10− 4 dilution to a total volume of 1 ml pr.", "well in 48 well plates (Greiner Bio-One, Austria) and incubated at 15 °C, 14:10 light:dark cycle and 90 lx light intensity.", "Positive growth was determined by a change in color into green as detected by the eye, and scored against the MPN table for a three-replicate design from FDA's Bacterial Analytical Manual (U. S. Food and Drug Administration (FDA), 2010), which gives rough results in intervals.", "When the numbers of live/damaged cell signals in FCM dot plots were approximately 10% of the total number of cells, a regrowth check was performed for verification.", "For exp-I this procedure was carried out at day 22 for samples treated with 100–200 mJ/cm2 and at day 2 for samples treated with 400 and 800 mJ/cm2, and for exp-II at day 20 for samples treated with 100–200 mJ/cm2 and at day 3 for samples treated with 400 and 800 mJ/cm2.", "1 ml of the sample was added to 9 ml growth medium in 50 ml Erlenmeyer flasks.", "No visible change to green color indicated that there were no reproductive cells in the sample.", "The flasks, trays and plates were incubated at 15 °C in the dark for 3 weeks.", "FCM analysis in the pre-study showed that inactivation increased with higher UV doses and during the dark incubation period (data not shown).", "Based on these results, two complete experiments were carried out (exp-I and -II), and these results are presented below.", "UV irradiation with doses ≥ 400 mJ/cm2 inactivated the algae permanently as demonstrated by all analysis methods.", "FCM analyses (Fig. 2b, d) of samples irradiated with 400 and 800 mJ/cm2 displayed < 4% and ≤ 0.1% live/damaged cell signals after 1 day, respectively.", "The numbers of live and damaged cells remained at this level or were further reduced during the incubation period.", "Plate count and MPN did not show any growth at UV doses ≥ 400 mJ/cm2 and neither did regrowth check performed at days 2 and 3 for exp-I and exp-II, respectively.", "As observed in the pre-study, FCM analysis of UV irradiated samples showed a relationship between inactivation and UV doses.", "This was examined at day 1, when an immediate effect of UV treatment was observed, and again the number of live/damaged cells decreased with higher UV doses.", "Table 1 shows a comparison of the results from FCM, MPN and plate count from exp-I and -II.", "The control samples were all in the same range, but MPN results showed > 11,000 cells ml− 1, indicating that the samples should have been diluted further.", "The results for the UV irradiated samples, varied when using different analysis methods.", "In exp-I there were good agreements between live/damaged cell numbers obtained by FCM and MPN analysis, but the plate count analysis resulted in considerably lower numbers at day 1.", "For exp-II, comparable results were obtained using plate count and MPN analysis whereas the results using FCM gave considerable higher live/damaged cell numbers.", "For samples treated with UV doses 100, 150 and 200 mJ/cm2 inactivation of cells was dependent on time of dark incubation as demonstrated by FCM (Fig. 2a, c).", "Generally, the numbers of live/damaged cells decreased during incubation, and were fewer in UV irradiated samples than in the stained controls.", "In exp-I the percentage of live/damaged cells in the UV irradiated samples (Fig. 2a) decreased throughout the incubation period and amounted to ≤ 3% at day 22.", "The samples treated with 100 mJ/cm2 behaved similar to the stained controls during incubation, but at day 22 the percentage of live/damaged cells was lower than the stained controls also for samples treated with this UV dose.", "In exp-II (Fig. 2c) inactivation increased during incubation, and < 3% live/damaged cells were observed at days 22, 10 and 3 in the samples treated with 100, 150 and 200 mJ/cm2, respectively.", "Regrowth checks performed at days 22 and 20 for exp-I and exp-II, respectively, were negative for all UV irradiated samples and positive for the control samples (data not shown).", "Considerable variations were observed between the results from the two experiments when looking at a detailed level.", "Firstly, the percentage of live/damaged cells in the stained controls varied at day 1 (Fig. 2a, c), being 92% and 54% in exp-I and exp-II, respectively.", "Secondly, for the samples UV irradiated with ≤ 200 mJ/cm2, the inactivation rate varied between the experiments (Fig. 2a, c) and the number of FCM live/damaged cells fluctuated (Table 1).", "Thirdly, some replicates showed large standard deviations; most evident in exp-I (Fig. 2a).", "Fourthly, at the first days after UV irradiation, it was observed more cells in the treated samples than in the controls (Table 1).", "The aim of this study was to evaluate inactivation by different UV doses and dark incubation on the algae T. suecica, to give recommendations for ballast water management regarding treatment and transport.", "UV doses 400 and 800 mJ/cm2 rendered T. suecica cells unculturable and without esterase activity 1 day after irradiation whereas doses ≤ 200 mJ/cm2 did not necessarily inactivate the cells.", "This indicates that the minimum UV dose that permanently inactivates this algal specie is somewhere between 200 and 400 mJ/cm2 which is similar to the dose Ou et al. (2012) found to be lethal after UV-C radiating the cyanobacteria Microcystis aeruginosa (Ou et al., 2012).", "The samples irradiated with UV doses 100, 150 and 200 mJ/cm2 contained culturable and esterase active T. suecica cells 1 day after irradiation, but the inactivation increased with higher UV doses.", "Our results are in line with previous studies of freshwater green algae Chlorella ellipsoidea, Chlorella vulgaris, and Scenedesmus quadricanda, and the cyanobacteria M. aeruginosa which showed limited sensitivity to UV-C irradiation with doses ≤ 200 mJ/cm2 (Ou et al., 2012; Tao et al., 2010).", "Comparing the different analysis methods at day 1 for the samples UV irradiated with doses ≤ 200 mJ/cm2 revealed that the numbers of FCM gated cells were higher than the numbers of cfu detected by plate count.", "Such discrepancy between plate count and FCM results has also been observed in other studies of UV irradiated bacteria and alga (Kramer and Muranyi, 2014; Olsen et al., 2015; Schenk et al., 2011).", "UV induced DNA damage can block transcription and replication, inhibiting growth and reproduction (Oguma et al., 2002; Sinha and Häder, 2002).", "DNA damaged cells are not detected as live by growth assays, though they can express activity (Davey, 2011; Hammes et al., 2011; Villarino et al., 2003); explaining the contradicting results from FCM and plate count analysis.", "Plate count and MPN analysis are based on reproductive capacity and one could expect these growth assays to give comparable results.", "However, the results obtained by the MPN were similar to the plate count results in exp-II and to FCM in exp-I.", "This illustrates the challenge of getting reproducible results when analyzing UV irradiated organisms with methods that analyze different cellular characteristics.", "Further, growth assays may introduce errors as a majority of the microbes are uncultivable (Roszak and Colwell, 1987; Staley and Konopka, 1985).", "In addition, the MPN positive growth was determined by the eye, though fluorometry determination would have been a more objective analysis method and could potentially have given a slightly different outcome.", "However, the MPN method was validated by another researcher and by microscopy.", "The FCM results showed that inactivation increased with the time of dark incubation.", "Phytoplankton are photosynthetic organisms requiring light as an energy source (McMinn and Martin, 2013) and dark incubation will naturally affect their viability over time (Gollasch and David, 2010), especially if the cells continue with unchanged activity causing them to run out of energy (Jochem, 1999).", "Additionally, incubation in darkness will limit photoreactivation; the repair mechanism which removes UV induced DNA lesions and reverses damages by using the energy of light (Sinha and Häder, 2002), leaving the cells unrepaired and dying.", "Other studies have reported limited or no photoreactivation in UV irradiated Escherichia coli during incubation at darkness (Oguma et al., 2001; Yin et al., 2015; Zimmer and Slawson, 2002).", "In agreement with other studies the control samples contained reproducible cells even after 3 weeks of dark incubation (Gollasch and David, 2010; Olsen et al., 2015).", "T. suecica's ability to survive in darkness over time implies it does not overrate effects of UV treatments and makes it an appropriate indicator organism for ballast water monitoring.", "Some of the variation on results between exp-I and -II, such as variation in the stained controls day 1, difference in inactivation rate when irradiated with doses ≤ 200 mJ/cm2, large standard deviations and more cells in treated samples than in untreated ones, indicates that the algal culture used in exp-I contained a greater portion of fresh and healthy cells than the culture used in exp-II.", "Natural sea water shows great variability and contains a diverse community of algae species in different cellular phases varying with time and space and may have different tolerance and response to the same UV doses applied (Rastogi et al., 2010; Sinha et al., 1998; Xiong et al., 1997).", "Although not intentionally, our study indeed demonstrated that the UV treatment/dark incubation required for inactivation is dependent on the status of the ballast water inhabiting organisms.", "Therefore, our results imply that organisms in ballast water treated with UV doses 100–200 mJ/cm2 are inactivated when left in dark ballast tanks over a period of time.", "Our results also demonstrate the challenge of giving recommendations regarding duration of dark incubation needed for inactivation when using lower UV doses.", "It is, however, important to keep in mind that controllable experiments in a laboratory differ from a flow-through chamber in a commercial BWTS and that a BWTS comprise two or more treatment stages, enhancing the inactivation efficiency.", "Other factors can, however, also influence the inactivation efficiency in a BWTS, like biotic and abiotic particles in the sea water protecting the microbes during UV irradiation (Hess-Erga et al., 2008; Tang et al., 2011) and a BWTS has to be optimal under the prevailing circumstances whatever factors exist that may prevent inactivation.", "UV irradiation as a treatment technology has been criticized due to the uncertainty regarding inactivation at discharge but is more environmental friendly than chemical disinfection and creates no harmful by-products (Jung et al., 2012).", "In addition, UV irradiation represents little risk for the operators and less training is required to run the systems (International Maritime Organization, 2008b).", "This study was performed in a laboratory with cultures of T. suecica and with the MP UV lamp as the sole treatment source.", "For future studies it would be of interest to do experiments with T. suecica in real BWTS with MP UV technology.", "Such studies would improve our ability to give recommendations regarding UV doses and duration of dark incubation of T. suecica.", "To evaluate whether our FCM protocol is applicable to other microbes, laboratory studies should be performed with natural sea water irradiated with different UV doses.", "If analysis of natural sea water is feasible with our FCM protocol, the experiment needs to be repeated in a BWTS to evaluate inactivation in a system where it is intended to be used.", "The microbial community in ballast water is very diverse and organisms will vary with season, location and environmental conditions.", "The level of metabolic activity can vary between various algae species, and the response to environmental changes and inactivation treatments can differ (Jochem, 1999, 2000; Olsen et al., 2015).", "Although there are indications that the majority of phytoplankton species can be detected by the esterase substrates fluorescein diacetate (FDA) and 5-chloromethylfluoorescein diacetate (CMFDA) (Peperzak and Brussaard, 2011), we are therefore aware that fluorescing signal from esterase substrates can vary over a large range of intensities (Dorsey et al., 1989).", "Our recommendation at this point is thus to treat the ballast water with UV doses close to 400 mJ/cm2 in order to permanently inactivate the organisms.", "The variable results for UV doses 100–200 mJ/cm2 demonstrate the challenge of giving unambiguous recommendations on duration of dark incubation needed for inactivation." ]

[ "Response of T. suecica to different UV doses and dark incubation was determined.", "Flow cytometry and standard culturing methods were used to determine inactivation.", "Inactivation increased with increasing doses and with time of dark incubation after treatment.", "UV doses ≥ 400 mJ/cm2 permanently inactivated the algae.", "It is a challenge to give advices on dark incubation times for UV doses ≤ 200 mJ/cm2." ]

[ "MULT" ]

[ "Bioengineering", "Biotechnology", "Chemical engineering", "Komagataella phaffii", "Pichia pastoris", "Transaminase", "Whole cell biocatalyst" ]

[ "1000" ]

[ "The unicellular methylotrophic yeast Komagataella phaffii (K. phaffii), formerly known as Pichia pastoris (Kurtzman, 2009), is a well-established platform for heterologous protein production (Byrne, 2015; Macauley-Patrick et al., 2005).", "The initial genome sequence of K. phaffii (De Schutter et al., 2009; Küberl et al., 2011) has been further refined and annotated (Sturmberger et al., 2016), supporting the development of genetic tools for its manipulation (Ahmad et al., 2014; Vogl et al., 2016).", "K. phaffii can grow to very high cell densities, exceeding 150 g L−1 of dry cell weight, using inexpensive and simple media and carbon sources.", "Abad et al. (2010) demonstrated the effectiveness of K. phaffii as a whole cell biocatalyst by showing that cells engineered to express Trigonopsis variabilis D-amino acid oxidase could be used effectively for D-amino acid oxidation.", "Wei et al. (2018) engineered K. phaffii strain GS115 to overexpress a native transketolase and in whole cell biocatalysis the resultant strain achieved high yields of the chiral sugar L-erythrulose from prochiral substrates.", "Bea et al. (2010) showed that K. phaffii cells engineered to express an ω-transaminase from Vibrio fluvialis strain JS17 achieved kinetic resolution of racemic α-methylbenzylamine mixture to an enantiomeric excess of 99% (R)-α-methylbenzylamine.", "The ω-transaminase designated CV 2025 (Kaulmann et al., 2007) from Chromobacterium violaceum (C. violaceum) has shown promising results in the production of chiral amino-alcohols, such as 2-amino-1,3,4-butanetriol (ABT), using bacterial whole cells (Ingram et al., 2007; Rios-Solis et al., 2015).", "ABT is a key synthon in the production of many small molecule drugs (Ager et al., 1996; Sehl et al., 2015), including protease inhibitors (Kwon and Ko, 2002) and antiretrovirals (Raghavan et al., 2010).", "Bioconversion using ω-transaminases, either purified (Heintz et al., 2017) or within whole cells (Shin and Kim, 1999; Slabu et al., 2017; Gomm and O'Reilly, 2018) has also been used for production of chiral amines, another class of synthon utilised extensively in synthesis of bioactive natural products, agrochemicals and active pharmaceutical ingredients (Kelly et al., 2018).", "In this work, we engineered the K. phaffii strain BG-10, available commercially from ATUM, to overexpress the bacterial transaminase CV2025 in the new strain, designated BG-TAM.", "We established the growth performance of the BG-TAM strain and tested its whole-cell biocatalytic production of both the chiral amino alcohol ABT, and the chiral amine 1-methyl-3-phenylpropylamine (MPPA), when fed low-cost substrates.", "Komagataella phaffii strain BG-10 and plasmid pJ902-15 were purchased from ATUM.", "K. phaffii biomass concentration was measured as optical density at 600 nm (OD600) using a spectrophotometer.", "The C. violaceum CV2025 ω-transaminase gene (NCBI sequence reference WP_011135573.1) was obtained from GenScript.", "Zeocin was purchased from Thermo Fischer Scientific.", "The C. violaceum CV2025 gene was codon-optimised for expression in K. phaffii using the OptimumGene™ algorithm by GenScript.", "The optimised coding sequence is available as a public data set (Braun-Galleani et al., 2018).", "The 1.4 kb CV2025 gene was flanked by BsaI sites enabling subcloning into a lone BsaI site present in pJ902-15 to generate the new, 4.9 kb plasmid, pJ-CV2025.", "BG-10 cells were transformed using 20 μg of SacI-linearised pJ-CV2025 by electroporation (1500 V, 200 Ω, 25 μF) and subsequently plated onto YPD agar plates supplemented with 1 M sorbitol and 200–1000 μg mL−1 of zeocin to select for stable integrants.", "Colony PCR was used to confirm the presence of integrated pJ-CV2025 in the AOX1 locus in zeocin-resistant colonies using the primer, CCAAAGACGAAAGGTTGAATG, which was designed to anneal within the AOX1 promoter and the primer, GATAATTCGACAACAGCAGG, designed to anneal within the codon-optimised C. violaceum CV2025 gene at a position predicted to be 300 base pairs downstream of the AOX1 promoter only in transformants.", "Transformant colonies, which were both zeocin resistant and positive by colony PCR, were designated ‘BG-TAM’ and a master cell bank of clones was generated and cryopreserved at -80 °C.", "K. phaffii BG-10 and BG-TAM strains were grown for 18 h in 250 mL baffled flasks at 30 °C with 250 rpm to an OD600 ∼ 10 in 50 mL of BMGY medium (1% w/v yeast extract, 2% w/v peptone, 100 mM pH 6.0 potassium phosphate buffer, 0.4 μg mL−1 biotin, 1% v/v glycerol and 1.34% v/v yeast nitrogen base), a base medium for preparation of minimal and synthetic defined yeast media.", "Cells were harvested by centrifugation at 5,000 rpm and 4 °C for 5 min, washed in phosphate buffer and resuspended in 50 mL BMMY medium (same constituents as BMGY but substituting 1% v/v glycerol for 2% v/v methanol) to an OD600 = 5.", "2% v/v methanol was added every 24 h to maintain induction of the AOX1 promoter.", "Samples were harvested after 48 h of induction and used for bioconversion measurements.", "Cultivation in bioreactor was performed using a Multifors 1L device (INFORS HT).", "The Multifors 1L device has been reported previously to achieve OD600 in the range of 800–1000 and DCW of 150–200 g L−1 during K. phaffii cultivation by Templar et al. (2016) and Wei et al. (2018) respectively.", "For a starter culture, 150 mL BMGY in a 1 L baffled flask was inoculated with a 1.5 mL cryostock, and incubated at 30 °C and 250 rpm, until an OD600 = 60 was reached.", "This was used as inoculum for 600 mL of basal salt medium (BSM: 1 L with RO water with 26.7 mL 85% w/v H2PO4, 0.93 g CaSO4, 18.2 g K2SO4, 14.9 g MgSO4·7H2O, 4.13 g KOH, 40 g glycerol and 12 mL of ‘Pichia Trace Metal 1’ (PTM1) solution.", "PTM1 had the following constituents: 6.0 g L−1 CuSO4·5H2O, 0.08 g L−1 Nal, 3.0 g L−1 MnSO4·H2O, 0.2 g L−1 Na2MoO4·2H2O, 0.02 g L−1 H3BO3, 0.5 g L−1 CoCl2, 20.0 g L−1 ZnCl2, 65.0 g L−1 FeSO4·7H2O, 0.2 g L−1 biotin and 5.0 mL L−1 of 96% v/v H2SO4.", "Cultivation parameters and methanol induction conditions followed Invitrogen guidelines for Pichia fermentation (Invitrogen, 2002) with cultivation conditions of 30 °C, pH 5.0 and dissolved oxygen kept above 20% by increasing agitation speed or pure oxygen sparging.", "Briefly, cultivation was performed in batch mode for ∼18 h, until glycerol was completely consumed, and subsequently switched to a glycerol fed-batch regime (50% w/v glycerol feed containing 12 mL L−1 PTM1 trace salts, 18.15 mL h−1 L−1 initial fermentation volume) for 6 h, followed by a methanol fed-batch phase using 100% methanol supplemented with 12 mL L−1 PTM1 trace salts at the following feed rates: 3.6 mL h−1 L−1 for 1 h (M1), 7.3 mL h−1 L−1 for 2 h (M2), and final feed rate of 10.9 mL h−1 L−1, which was maintained for 48 h. Samples for growth rate monitoring were harvested at t = 0 and t = end of each feeding phase.", "The measurement of dry cell weight was carried out by putting the cell pellet of 1 mL of culture in an oven (100 °C) and measuring weight in an analytical balance every 24 h until constant (∼96 h).", "Samples for bioconversions were harvested after the initial batch stage and every 24 h after methanol induction, the supernatant removed and the cell pellet stored at -20 °C.", "All bioconversion reactions were carried out in 900 μL final volume using whole cells at 30 °C and 500 rpm in a thermomixer (Eppendorf ThermoMixer® C).", "Pelleted shake flask and bioreactor samples were thawed and re-suspended to 600 μL in a pH 7.5 solution of 0.2 mM pyridoxal phosphate (PLP) in 200 mM, 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES) buffer to allow the PLP cofactor to associate with transaminase within cells.", "These cell suspensions were then incubated in 1.5 mL glass vials for 20 m, followed by the addition of 300 μL of substrate solution, consisting of 200 mM HEPES buffer pH 7.5, 0.2 mM pyridoxal phosphate and substrate levels needed to provide the final concentrations indicated in the Results section.", "Aliquots of 100 μL were taken from the bioconversion reactions at different time points, centrifuged at 12,000 rpm for 10 m at 4 °C, before the supernatant was removed, retained and stored at -20 °C for further analysis.", "An integrated Dionex Ultimate 3000 HPLC system fitted with an ACE 5 C18 reverse phase column (150 mm × 4.6 mm, 5 μm particle size) controlled by Chromeleon 7 software (Thermo Fisher Scientific) was used to quantify MBA, ACP, PB, MPPA and ABT.", "Samples were quenched and diluted with 0.1% v/v TFA prior to analysis.", "To analyse MBA and ACP, two mobile phases were used: 100% acetonitrile (A) and 0.1% (v/v) trifluoroacetic acid (B).", "A gradient was run from 15% A/85% B to 72% A/28% B over 9 min, followed by a re-equilibration step for 2 m (oven temperature 30 °C, flow rate 1 mL/min).", "UV detection was carried out at 260 nm, with detection times of 3.6 m for MBA, and 7.8 m for ACP.", "For MPPA and PB analysis, two mobile phases were used: 0.1% (v/v) trifluoroacetic acid (C) and 0.095% trifluoroacetic acid/80% acetonitrile in MilliQ water (D).", "A gradient of 40% C/60% D to 100% D was run over 15 min, followed by 2.5 m wash with 100% D, and a subsequent re-equilibration step for 2.5 m (oven temperature 30 °C, flow rate 1 mL/min).", "UV detection was carried out at 254 nm, with retention times of 2.7 m for MPPA, and 7.7 m for PB.", "For ABT measurement, 6-aminoquinolyl-n-hydroxysuccinimidyl carbamate (AQC) was used as derivatization agent.", "AQC, provided by Dr Fabiana Subrizi from UCL Department of Chemistry, was solubilized in dry acetonitrile (10 mg mL−1) and stored in dry conditions for no longer than 2 weeks.", "Typically, a volume of 75 μL of AQC solution was added to 150 μL of bioconversion supernatant previously diluted in 0.02 M borate buffer pH 8.8, reaching a final volume of 225 uL.", "This mixture was centrifuged prior to analysis.", "To analyse ABT, two mobile phases were used: 140 mM sodium acetate, pH 5.05 (E) and 100% acetonitrile (F).", "A gradient of 85% E/15% F to 100% F over 10 m (flow rate 0.5 mL min−1), followed by a 2.5 m wash with 40% E/60% F and a 2.5 m wash with 100% E (flow rate 1 mL min−1).", "A re-equilibration step was run for 5 min.", "UV detection was carried out at 254 nm, with a retention time of 6.1 min.", "For quantification of ERY, an Aminex HPX-87H reverse phase column (300 × 7.8 mm, Bio-Rad, USA) was used.", "The system was run at 60 °C with 0.6 mL min−1 isocratic flow of 0.1% trifluoroacetic acid as mobile phase.", "UV Detection was carried out at 210 nm with a retention time of 11.5 min.", "The molar extinction coefficient (ε) at wavelength of 260 nm was determined to be 4878.05 M−1 cm−1 for MBA and 4405.29 M−1 cm−1 for ACP.", "At 254 nm ε was determined to be 171.32 M−1 cm−1 for PB, 168.86 M−1 cm−1 for MPPA and 2890.17 M−1 cm−1 for derivatised ABT.", "We tested whether transaminase overexpression impacted the growth characteristics of K. phaffii strain BG-TAM, given that endogenous transaminases form part of the native metabolic networks of the organism.", "BG-TAM and BG-10 strains were each cultivated in the presence of up to 5% v/v methanol.", "The BG-TAM growth profile (Fig. 1a) did not deviate markedly from that of the parental strain BG-10 (Fig. 1b).", "The highest concentration of 5% v/v methanol suppressed growth in both strains, whereas 2% v/v provided the best results for cell density and growth rate.", "This methanol concentration was used subsequently for induction in the shake flask experiments of this study.", "BG-10 and BG-TAM cells were cultivated in shake flasks in the presence of 2% v/v methanol for 48 h. Harvested cells were diluted or concentrated to a normalised concentration of OD600 = 39, for purposes of comparison, and incubated with substrates to assess whole cell biocatalytic activity for production of ABT and MPPA by the pathways illustrated in Fig. 2a and b, respectively.", "When cells of the BG-TAM were incubated with 10 mM MBA and 30 mM ERY, production of ACP was observed, whereas under the same conditions cells of the parental BG-10 strain resulted in no ACP production (Fig. 2c).", "We interpreted this observation as further evidence that the plasmid pJ-CV2025 had integrated at the AOX1 locus as intended and that expression of the codon-optimised CV2025 gene was responsible for the observed ACP accumulation.", "Shake flask-cultivated BG-TAM cells were prepared as above for bioconversion of MBA and ERY to ABT and also for bioconversion of MBA and PB to MPPA.", "Both bioconversions showed activity, with production of around 4mM product for both ABT (Fig. 2d) and MPPA (Fig. 2e).", "BG-TAM and parental BG-10 strain cells were cultivated in a 1L bioreactor comprising firstly a glycerol batch and fed-batch feeding to achieve high biomass, followed by fed-batch methanol feeding to induce the AOX1 promoter that controls expression of the transgene.", "Under this cultivation regime, the unmodified parental BG-10 strain and the engineered BG-TAM strain achieved cell densities of OD600 ∼ 900–1000, after 48 h of methanol induction.", "The growth profile illustrated in Fig. 3 shows cell biomass accumulation data from three cultivations performed in parallel using the Multifors 1L bioreactor system.", "Growth of the engineered BG-TAM strain was comparable to that of the unmodified strain, with matching levels of final biomass achieved at harvest.", "Given that the strains also showed similar growth performance during shake flask cultivation (Fig. 1), under methanol induction, we concluded that overexpression of the CV20205 transaminase gene in BG-TAM did not considerably perturb viability of the BG-TAM strain relative to the parental strain.", "Bioreactor-cultivated BG-TAM cells were harvested after 66 h of methanol induction (92.5 h post inoculation) when an OD600 = 1024 was achieved (final time point, Fig. 3).", "We then mapped the limits of substrate concentration that high cell density suspensions of BG-TAM cells could tolerate whilst maintaining bioconversion.", "We measured bioconversion achieved by cells incubated with 10–30 mM substrate, the concentrations we had used for shake flask samples, and then increased substrate concentration to 100 mM and finally to 0.5 M (Fig. 4).", "A concentration of 0.5 M MBA and ERY abolished production of both ACP (Fig. 4a) and ABT (Fig. 4b), establishing an upper limit on the performance of the whole cells for this reaction.", "By contrast, the whole cell biocatalyst retained a level of activity in 0.5 M substrates PB and MBA, producing 2.82 mM MPPA.", "We concluded from these observations that the upper limit for substrate tolerance for the ABT reaction is not due solely to general stress of the enzyme or living cells but is in part substrate-specific.", "At a concentration of 100 mM, all substrates were well-tolerated and resulted in the highest observed bioconversion levels of 11.48 mM for ABT, and 29.6 mM for MPPA (Fig. 4).", "The data in Fig. 4 suggest future investigations would be warranted to map biocatalysis performance for substrate concentrations between 0.1 M and 0.5 M.", "Schrewe et al. (2013) proposed a battery of performance measures best suited to whole cell biocatalysts, which could be useful for researchers or industrialists across a range of different process configurations and needs.", "In Table 1 we collated the performance metrics for BG-10 strain whole cells used in the three best-performing conditions, with respect to product concentration, observed in Fig. 4.", "Table 1 shows that BG-10 whole cells achieved a best yield on substrate (Yp/s) of 0.41 mol mol−1 (molproduct molsubstrate−1), which is comparable to the Yp/s of 0.52 mol mol−1 reported by Wei et al. (2018) for whole cell biocatalysis using a K. phaffii strain engineered to overexpress transketolase, an enzyme type that typically exhibits high activity in bioconversions.", "As ω-transaminases have been shown to be responsive to mutational approaches to increase their performance (Deszcz et al., 2015), the performance data reported here with an unmodified ω-transaminase certainly encourages further investigation of whole cell applications.", "Stephanie Braun-Galleani: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.", "Maria-José Henríquez: Contributed reagents, materials, analysis tools or data.", "Darren N. Nesbeth: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.", "This work was supported by the UK BBSRC grant BB/M004880/1 within the ERA-NET IPCRES consortium, and CONICYT-Becas Chile (Folio no. 72120390).", "The authors declare no conflict of interest.", "No additional information is available for this paper." ]

[]

[ "MULT" ]

[ "Fluid intelligence", "Independent component analysis", "Individual differences", "Neuroanatomy", "Tractography" ]

[ "1000" ]

[ "The data (Supplementary Table 1) includes cognitive and anatomical variables collected prior to a large, multi-modal cognitive training study [25].", "They include:", "Demographic measures (i.e., age, sex, and education).", "4 cognitive factors estimated via structural equation modeling [15].", "Scores from the battery of 12 cognitive tests used to estimate these factors.", "35 cortical thickness estimates and volume estimates for these same regions.", "11 sub-cortical volumetric estimates.", "Total brain and total intracranial volume estimates.", "7 estimates of ventricular size.", "5 estimates of corpus callosum.", "12 estimates of fractional anisotropy and in matter tracts.", "12 estimates of radial diffusivity in white matter tracts.", "The 190 participants consisted of 85 females, and 105 males.", "The age range in our sample was 18–44 years, with a median of 22 years, and a mean of 24.3 years.", "The mean educational level of the participants was “some college” (i.e., median score 3, mean score 3.6) as reported on a scale from 1 to 5, where 1 denoted “less than a high school diploma”, 2 denoted “high school diploma or equivalent”, 3 denoted “some college”, 4 denoted “college degree”, and 5 denoted “post-graduate education.”", "Maximal oxygen consumption (VO2max) was measured using a computerized indirect calorimetry system (ParvoMedics True Max 2400) and a modified Balke protocol [1] with averages for oxygen uptake (VO2) and respiratory exchange ratio (RER) assessed every 20 s. Participants ran on a motor-driven treadmill at a constant speed, with 2.0% increases in grade every two minutes until volitional exhaustion.", "The raw value was adjusted for body size, age, and gender to produce a VO2max percentile score.", "Participants received a battery of 12 cognitive tests designed to estimate underlying latent variables corresponding to cognitive constructs (see Table 1).", "The four latent variables of interest were fluid intelligence (gf), working memory (wm), executive function (ef), and episodic memory (em).", "Each of these latent variables was measured with three cognitive tests as follows.", "Fluid intelligence (gf) was measured by the BOMAT, number series, and letter sets tests [3,4,7].", "Working memory (wm) was measured by the reading, rotation, and symmetry span tests [8,23].", "Executive function (ef) was measured by the Garavan, Keep Track, and Stroop tests [14,22,26].", "Episodic memory (em) was measured by immediate free recall, words, pictures and paired associates tests [23,24,9].", "Using a structural equation modeling approach [15], across the larger sample of 518 participants, we extracted estimates of the four cognitive construct latent variables (i.e., gf, wm, ef, em).", "Because Garavan and Stroop produce error scores, while all others are measures of accuracy, we inverted these two values (i.e., multiplied by −1) in order to ensure all cognitive variables had the same sign.", "High resolution T1-weighted brain images were acquired using a 3D MPRAGE (Magnetization Prepared Rapid Gradient Echo Imaging) protocol with 192 contiguous axial slices, collected in ascending fashion parallel to the anterior and posterior commissures, echo time (TE)=2.32 ms, repetition time (TR)=1900 ms, field of view (FOV)=230 mm, acquisition matrix 256 mm×256 mm, slice thickness=0.90 mm, and flip angle=9°.", "All images were collected on a Siemens Magnetom Trio 3T whole-body MRI scanner.", "Automated brain tissue segmentation and reconstruction of the T1-weighted structural MRI images were performed using the standard recon-all processing pipeline in FreeSurfer, version 5.2.0 (Released May, 2013; http://surfer-nmr.mgh.harvard.edu/).", "This produced estimates of 1) cortical thickness, 2) cortical volumes, 3) sub-cortical volumes, 4) ventricles, and 5) corpus callosum [5,6,10–13].", "Segmentations and tractography were manually checked for errors.", "Estimates in the left and right hemispheres were summed to produce bilateral estimates, and all values were converted to z-scores to control for differences in scale.", "A complete list of estimated structures appears in Table 1.", "FreeSurfer produced automated segmentation that closely approximates hand tracing, but like all segmentation procedures may introduce systematic bias.", "The diffusion tensor imaging estimates for fractional anisotropy (FA) and radial diffusivity (RD) data was analyzed using tract-based spatial statistics in FSL [19–21].", "This pipeline involves fitting a tensor model to the raw diffusion data using fMRIDB׳s diffusion toolbox, and non-brain tissues were removed using FSL׳s brain extraction tool.", "All subjects׳ FA data were then aligned into a common space using the nonlinear registration tool FNIRT [18,2].", "Next, the mean FA image was created and thinned to create a mean FA skeleton that represents the centers of all tracts common to the group.", "Each subject׳s aligned FA data was then projected onto this skeleton to create an estimate of the subject-level value associated with each tract." ]

[]

[ "BIOC" ]

[ "Neonatal hypoxic/ischemic encephalopathy", "Oligodendrocyte precursor cells", "PARP inhibitors", "Remyelination" ]

[ "1307", "1309" ]

[ "Oligodendrocytes, the cells wrapping the axons of the central nervous system (CNS) with the myelin sheath, derive from the oligodendrocyte precursor cells (OPCs) generated during development from multipotent neuroectodermal derivatives in the cortex and spinal cord (Bergles and Richardson, 2015).", "These cells proliferate and migrate to populate the entire adult CNS, where they account for approximately 5–8% of the entire cell population (Dawson et al., 2003).", "New OPCs can be also generated in the adult CNS from neural stem cells (NSCs; Agathou et al., 2013) and by mitosis, as OPCs are the major proliferating population of the CNS under appropriate stimuli (Fernandez-Castaneda and Gaultier, 2016).", "These cells are responsible for myelin formation during development (Bergles and Richardson, 2015), and for myelin turnover and repair during adulthood (Young et al., 2013).", "The process of myelin formation is highly orchestrated in time and space, involves different cell types, and a key step of this process is the OPC maturation toward myelinating oligodendrocyte (Zuchero and Barres, 2013).", "Internal and external cues targeting genetic, epigenetic and cytoplasmic mechanisms provide the appropriate microenvironment that regulates the molecular machinery triggering OPCs out of the cell cycle to terminal differentiation (Zuchero and Barres, 2013; Liu et al., 2016; Fernández et al., 2016).", "It has been suggested that remyelination after lesions in the adult CNS recapitulates developmental myelination (Franklin and Hinks, 1999).", "However, substantial differences could well be expected between a normal turnover process and a repair process (Fancy et al., 2011) triggered by pathological events like inflammation and hypoxic/ischemic damage.", "These events actually mobilize a large number of inhibitory factors leading to a differentiation block of OPC (Gaesser and Fyffe-Maricich, 2016).", "Thus, there is an urgent need to more clearly elucidate the molecular bases of the relationship between OPCs proliferation and differentiation, carefully dissecting developmental myelination vs. myelin turnover in adulthood, and physiological vs. pathological conditions.", "In the present study, we investigated the role of poly(ADP-ribose) polymerases (PARPs) in OPCs survival, proliferation and differentiation.", "PARPs are members of nuclear enzyme family that catalyse the formation of (ADP-ribose)n chains from NAD+ on acceptor proteins after DNA double strands breaks (Amé et al., 2004).", "Because of the PARPs well described role in DNA repair and apoptosis induction (Heeres and Hergenrother, 2007), PARPs inhibition is currently considered a therapeutic option for cancer, including glioblastoma (Curtin and Szabo, 2013).", "Moreover, PARPs inhibition was proposed as a neuroprotective strategy for neonatal asphyxia and hypoxia/ischemia encephalopathy (Neira-Peña et al., 2015), a CNS injury that occurs during a critical period of developmental myelination (Dimou and Götz, 2014).", "In order to study the effect of PARP inhibition in all stages of differentiation, from NSCs to mature oligodendrocytes, we used OPCs-enriched cultures obtained from the fetal forebrain- and the adult sub-ventricular zone of the mouse to study the different role of PARPs in adult and fetal OPCs differentiation and maturation.", "We used PARP inhibitors having a different inhibitory concentration as pharmacological toll, including the clinically approved Olaparib (LYNPARZA™).", "All animal protocols described herein were carried out according to the European Community Council Directives (86/609/EEC), and comply with the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals.", "Fetal and adult NSCs were isolated from E.13–14 fetal mouse forebrain or 2.5 month old mice sub-ventricular zone (SVZ), following the Ahlenius and Kokaia protocol (Ahlenius and Kokaia, 2010).", "Oligodendrocyte differentiation was performed following the Chen protocol (Chen et al., 2007) with some modifications.", "Tissues were enzymatically dissociated using trypsin (SIGMA), hyaluronidase (SIGMA) and DNase (SIGMA), than mechanically dissociated by piepetting.", "The solution was filtered, centrifuged and the resulting pellet was washed twice in HBSS.", "After 7 min centrifugation at 400 × g, the cellular pellet was resuspended in serum-free medium (DMEM/F12 GlutaMAX 1 ×; 8 mmol/L HEPES; 100 U/100 μg Penicillin/Streptomycin; 0.1 × B27; 1 × N-2; 20 ng/ml bFGF; 20 ng/ml EGF) and, after cell count, cells were plated in suspension at a density of 10 cells/μl in a final volume of 3 ml in low-attachment 6-well plates (NUNC).", "Half medium was changed every three days, centrifuging the cell suspension at 300 × g for 5 min and gently resuspending the cellular pellet in fresh medium.", "Neurospheres were allowed to proliferate until they attained a diameter of about 100 μm.", "To obtain oligospheres, primary neurospheres were centrifuged at 300 × g for 5 min.", "The pellet was mechanically dissociated by pipetting and cells were counted and plated again at a density of 10 cells/μl in a final volume of 3 ml of OPCs medium (DMEM/F12 GlutaMAX 1 ×; 8 mmol/L HEPES; 100 U/100 μg Penicillin/Streptomycin; 0.1 × B27; 1 × N-2; 20 ng/ml bFGF; 20 ng/ml PDGF) in low-attachment 6-well plates (NUNC).", "Oligospheres were centrifuged and the pellet was mechanically dissociated to obtain a single cell suspension.", "After cell count, cells were plated at a density of 3000 cells/cm2 on poly-d,l-ornithine (50 μg/ml)/laminin (5 μg/ml) coating, in OPC medium.", "In order to induce oligodendrocyte differentiation and maturation, after 3 DIVs OPC medium was replaced with the oligodendrocyte differentiation medium (DMEM/F12 GlutaMAX 1 ×; 8 mmol/L HEPES; 100 U/100 μg Penicillin/Streptomycin; 0.1 × B27; 1 × N-2; 50 nM T3; 10 ng/ml CNTF; 1 × N-acetyl-l-cysteine-NAC-).", "The following PARPi (PARP inhibitors) were used in this study: TIQ-A (IC50 140–450 nM), PJ34 (IC50 20 nM) and Olaparib (IC50 5 nM; Ferraris, 2010; Hans et al., 2011).", "OPCs were treated 24 h before the T3-mediated differentiation induction (− 1 DIV), and cell viability was analysed at DIV 12 of differentiation (Fig. 1A).", "In order to study effects of PARPi on fetal and adult cultures, different treatment were performed: i) in order to study PARP1–2 expression and PARP activity, cells were analysed 24 h after treatment (DIV 0; Fig. 1A); ii) a dose-response curve of PARPi were performed, treating cultures with a series of 7 concentrations (0–10 μM).", "PARPi treatments were maintained after the oligodendrocyte differentiation medium change, until DIV 12 (Fig. 2A); iii) fetal cultures were also treated with PJ34 10 μM and analysed for cell number and cell death at DIV 0, 6 and 12 (Fig. 3A); iv) both fetal and adult cultures were treated with lower dose of PARPi (1 μM and 10 μM) and analysed for mature oligodendrocytes at DIV 12 (Fig. 4A).", "Indirect immunofluorescence was used to identify OPCs (NG2-positive cells), mature (CNPase-positive cells) and myelinating (MBP-positive cells) OLs, neurons (β-III-tubulin-positive cells) and astrocytes (GFAP-positive cells).", "The following primary antisera were used: mouse anti-β-III-tubulin (R&D system, Trento, Italy) 1:3000; rabbit anti-GFAP (Glial Fibrillary Acidic Protein, Dako) 1:1000; rabbit anti-NG2 (chondroitin sulphate proteoglycan, neural/glial antigen 2, Millipore, Merck S.p.a., Milan, Italy) 1:350; mouse anti-CNPase (2′, 3′-cyclic nucleotide 3′-phosphodiesterase, Millipore) 1:500; rabbit anti-MBP (Myelin Basic Protein, Dako) 1:500.", "Donkey Alexa 488-conjugated anti mouse and donkey Alexa 568-conjugated anti-rabbit (Invitrogen) were used as secondary antisera.", "After immunofluorescence staining, cells were incubated with the nuclear dye Hoechst 33,258 (1 μg/ml in PBS, 0.3% Triton-X 100) for 20 min at RT.", "Cells were finally washed in PBS and mounted in glycerol and PBS (3:1, v/v) containing 0.1% paraphenylendiamine.", "For HCS analysis cells were grown in 96 flat-bottom well HCS plates (NUNC).", "Analysis of condensed nuclei, cell number and lineage/differentiation markers were performed with Cell Insight™ CX5 High Content Screening (HCS; Thermo Scientific), using the Compartmental analysis BioApplication.", "Based on nuclear staining, the software is able to recognise nuclei and calculate the percentage of high intensity/small sized condensed nuclei.", "Moreover, based on the nuclei identification, the software is able to detect the presence of the marker-specific stain in the cell body, calculating the percentage of the immunoreactive cells.", "Lineage/differentiation markers analysis was performed only on cells showing intact nuclei, excluding condensed nuclei from the percentage calculation.", "Total RNA isolation was performed by using the RNeasy Mini kit (Qiagen, Milan, Italy) following manufacturer's instructions.", "Total RNA was eluted in RNase Free Water and concentration estimated through absorbance values at 260, 280 and 320 (Nanodrop 2000 spectrophotometer, Thermo Scientific).", "First strand cDNAs were obtained using the iScript™ cDNA Synthesis Kit (BioRad), incubating samples at 42 °C for 30 min.", "An RNA sample with no reverse transcriptase enzyme in the reaction mix was processed as a no-reverse transcription control sample.", "Semi-quantitative real-time PCR was performed using the CFX96 real-time PCR system (BioRad, CA, USA).", "The reactions were performed in a final volume of 20 μl consisting of 1 × SYBR Green qPCR master mix (BioRad) and 0.4 μM forward and reverse primers.", "In order to avoid possible contamination of genomic DNA in isolated RNA, the no-reverse transcriptase sample was processed in parallel with the others and tested by real-time PCR for every pair of primers used.", "All primers used were designed using Primer Blast software (NCBI, MD, USA) and synthesised by IDT (Coralville, IA, USA).", "The following primer sequences were used: PARP-1 (FW: 5′-GCCACACATCTCAGGGAGAC-3′; REV: 5′-CCCAAACCTTTGACACTGTGC-3′), PARP-2 (FW: 5′-GAAGGACGCAGACAGGACAA-3′; REV: 5′-ACATGAGCCTTTCCCAGCTT-3′).", "GAPDH (FW: 5′-GGCAAGTTCAATGGCACAGTCAAG-3′; REV: 5′-ACATACTCAGCACCAGCATCACC-3′) was used as housekeeping gene to normalize the amount of reverse-transcribed RNA used for PCR.", "Thermal profile of PCR reactions consisted first of a denaturation step (95 °C, 2 min) and 40 cycles of amplification (95 °C for 15 s and 60 °C for 60 s).", "At the end of the amplification cycles the melting curve of amplified products was performed according to the following temperature/time scheme: heating from 55 °C to 95 °C with a temperature increase of 0.5 °C/s.", "Primers efficiency values for all primers were 95–102%.", "The 2(− ΔΔCT) method was used for the calculation of gene expression.", "Fetal and adult OPCs at DIV 0, after 24 h of PARPi treatment, were lysed and proteins were quantified using the Lowry method (BioRad) following the manufacturer's instruction.", "PARP enzymatic activity was measured using HT Colorimetric PARP Apoptosis Assay Kit (Trevingen), following manufacturer's instructions.", "Data are reported as mean ± SD.", "Prism software (GraphPad) was used for statistical analyses and graph generation.", "Student's t-test or one-way ANOVA and Dunnett's multiple comparison post-hoc were used to analyse data.", "Results were considered significant when the probability of their occurrence as a result of chance alone was < 5% (P < 0.05).", "The cell systems used in this study are presented in Fig. 1.", "After cell expansion as neurospheres and oligospheres, cells were seeded for 3 DIVs in OPC medium, and then differentiated for 12 days in the presence of T3 (Fig. 1A).", "Lineage progression from NG2-positive OPC (DIV0) through CNPase-mature OL as far as myelinating MBP-positive OL is illustrated in Fig. 1C–E (representative images).", "The culture composition changes during the 12 DIV differentiation period, starting from DIV0, where the majority of cells are NG2 positive (NG2-positive cells 80%; CNPase-positive cells 5%), passing thorough DIV 6, where CNPase cells are growing in number (NG2-positive cells 60%; CNPase-positive cells 30%), reaching the end of the differentiation with CNPase/MBP positive cells representing the majority of cells.", "Culture composition at 12 DIV is presented in Fig. 1B. Adult cultures after 12 DIVs of differentiation show a less mature cell composition, with a high percentage of NG2-positive cells (P = 0.016) and less CNPase (P < 0.0001) and MBP (P = 0.008) positive cells.", "Fetal cultures show less astrocytes (GFAP-positive cells; P = 0.0023) and around 6% of neurons (β-III-tubulin positive cells) that are not detected in adult cultures (P = 0.002).", "In order to characterize the effect of PARP inhibition in fetal and adult OPCs on oligodendrocyte differentiation and maturation, cells were exposed to PARPi 24 h before differentiation induction (Fig. 1A).", "We first investigated the gene expression of the two major and more extensively studied PARPs, PARP-1 and 2 (Fig. 1F–G), and PARP activity (Fig. 1H), before the induction of differentiation, 24 h after PARPi treatment (DIV 0).", "Both genes are expressed in fetal (ΔCq = 3.28 ± 0.08) and adult (ΔCq = 5.79 ± 0.4) OPCs; gene expression is significantly higher in fetal compared to adult OPCs (PARP-1, P < 0.0001; PARP-2, P = 0.0002; Fig. 1F), and PJ34 treatment did not influence the mRNA expression levels (Fig. 1G).", "PARP activity analysis was based on the capability of PARP to parrylate histons in an ELISA-based assay.", "PARP activity was significantly higher in fetal OPCs compared to adult (P < 0.0001), and PJ34 treatment was able to decrease the activity in both cultures (fetal, P = 0.0005; adult P = 0.0284), thus confirming that the enzyme is active in both cell systems (Fig. 1H).", "In order to establish the cytostatic/toxic profile of PARPs inhibition on OPCs, we first constructed a dose-response curve on the cell culture which displayed the higher activity and expression of PARPs, i.e. fetal OPCs, using two standard PARPi, i.e. PJ34 and TIQ-A.", "The cytostatic effect is evaluated by the cell number (Fig. 2B), the cytotoxic effect by the percentage of condensed nuclei (Fig. 2C), as established by HCS in three independent experiments.", "The cytostatic effect was observed at 1 μM and 10 μM using TIQ-A (P < 0.0001); at 10 μM using PJ34 (P < 0.0001).", "Both molecules induced an increase in the percentage of condensed nuclei at the highest concentration (10 μM) according to the respective potency (TIQ-A, P < 0.0001; PJ34, P = 0.0362).", "We then investigated the effect of PJ34 and TIQ-A on adult OPCs using the toxic concentration for fetal OPCs (10 μM) finding that PJ34, but not TIQ-A, reduced the cell number in adult cultures (Fig. 2D; P = 0.0004) without affecting the percentage of condensed nuclei (Fig. 2E).", "We also tested the Olaparib, a clinically approved PARPi, showing the highest PARP inhibitory activity.", "Olaparib treatment caused a reduction in cell number in both fetal and adult cultures at low concentrations (Fig. 2F; 10− 1 nM; P < 0.0001) but is cytotoxic only for fetal OPCs (Fig. 2G; 101 nM; P < 0.0001).", "In order to better elucidate the timing of the PARP inhibition effect, we performed a time course analysis of cell number and cell death, by using PJ34 at a 10 μM toxic dose, (Fig. 3A).", "We found that after 24 h from the treatment there were no effects on cell number (Fig. 3B) and cell death (Fig. 3C).", "In vehicle treated cells, cell number increases over time, while in PARPi-treated cells remain at the same level for the whole differentiation period (Two Ways ANOVA, time F(2, 90) = 20.37, P < 0.0001; treatment F(1, 90) = 63.13, P < 0.0001; interaction F(2, 90) = 15.28, P < 0.0001), leading to a strong difference between the two groups at DIV 6 (P < 0.0001) and DIV 12 (P < 0.0001; Fig. 3B).", "In the opposite way, the percentage of condensed nuclei remain constant in the vehicle-treated, while increasing over time in the PARPi-treated group (Two Ways ANOVA, time F(2, 86) = 12.49, P < 0.0001; treatment F(1, 86) = 46.65, P < 0.0001; interaction F(2, 86) = 15.60, P < 0.0001), starting from DIV6 (P = 0.0003) and reaching > 30% at DIV 12 (P < 0.0001; Fig. 3C).", "We finally investigated the effect of PARP inhibition on the differentiation of remaining OPCs using the two highest concentrations, being either toxic or non-toxic.", "Fetal OPCs were treated with TIQ-A, PJ34 and Olaparib at 1 μM (non-toxic concentration of TIQ-A and PJ34, toxic concentration of Olaparib) and 10 μM (toxic concentration of all PARPi).", "Adult OPCs were treated with all 10 μM and 1 μM (both non-toxic concentration of all PARPi).", "Representative HCS images of CNPase/MBP-positive cells in the different treatments of both fetal and adult OPCs are presented in Fig. 4B; results of the quantitative HCS analysis in Fig. 4C (fetal) and Fig. 4D (adult).", "The number of cells included in this analysis ranged from 1199 to 7152/well.", "Three wells were included in each experiment.", "PARP inhibition impairs OPCs maturation in fetal, but not in adult, cultures.", "In cell preparations derived from fetal brain, TIQ-A, the less potent PARPi, does not affect OPC maturation, as indicated by the percentage of MBP-IR cells.", "On the contrary, PJ34 treatment, already at non-toxic concentrations (1 μM) resulted in a reduction of mature OPCs (P = 0.0013), as when used in a toxic dose (10 μM) (P < 0.0001).", "Olaparib treatment at both concentrations resulted in a strong reduction of the percentage of mature and myelinating OPCs (P < 0.0001).", "Notably, all PARPi had no effect on adult OPC differentiation (1 μM treatment, data not shown).", "Several in vivo and in vitro studies indicate that OPCs have substantially different properties according to their chronological age.", "For example, human fetal OPCs from different gestational stages exhibit differences in the myelination profile (Cui et al., 2012), such as human OPCs derived from fetal brain during the period of maximum oligoneogenesis or from adult subcortical white matter (Windrem et al., 2004).", "However, the mechanisms behind the different age-related OPC biological properties are not known.", "The aim of this study was to investigate if PARP is involved in determining the age-related OPCs properties.", "Thus, we compared OPCs derived from NSCs obtained from fetal and adult brains.", "This cell system was preferred to primary and purified OPCs to better mimic developmental biology of OPC, including lineage specification, proliferation/survival and differentiation.", "PARP is a family of enzymes comprising 17 homologues involved in multiple cell functions (Jubin et al., 2016a).", "Apart from the role in DNA repair, PARP-1 and 2 participate in cell homeostasis maintenance (Bai, 2015), intracellular transports (Abd Elmageed et al., 2012), cell cycle (Madison et al., 2011) inflammation and immunity (Bai and Virág, 2012) and gene expression regulation (Bock et al., 2015) through > 100 substrates (Hottiger, 2015).", "Moreover, PARPs are critically involved in inflammation and in the tissue damage caused by ischemia/reperfusion conditions (Li et al., 2015).", "We first showed that PARP mRNA expression and PARP activity are much higher in fetal- than in adult-derived OPCs.", "Due to the role of PARPs in DNA repair and apoptosis induction (Heeres and Hergenrother, 2007), the reduction in PARP mRNA expression level and activity observed in adult compared to fetal-derived OPC might reflect the age-related decrease of DNA repair capability (Bürkle et al., 2015).", "In fact, PARP activity also varies according to the age in different brain regions (Strosznajder et al., 2000), and PARylation is developmentally regulated (Jubin et al., 2016b).", "We then showed that PARP inhibition produces substantially different effects in OPCs derived from fetal and adult brain.", "In particular: (i) the culture treatment with PARP inhibitors is cytotoxic for OPCs derived from fetal, but not from adult, brain; (ii) PARP inhibition reduces cell number in proportion to the inhibitory potency of the compounds; (iii) the PARP inhibition effect in fetal OPCs is a slow process (iv) PARP inhibition impairs OPC maturation into myelinating OL in fetal, but not in adult OPCs, according to the inhibitory potency of the compounds.", "A primary role of PARP in the differentiation process of different cell types was recently recognized, possibly though chromatin plasticity and epigenetic regulation (Li et al., 2015).", "In embryonic stem cells, PARP interacts with the Wnt pathway, Sonic Hedgehog and Pax6 signalling (Hemberger et al., 2003; Yoo et al., 2011).", "PARP-1 has also a role in neuroectoderm differentiation (Yoo et al., 2011) and favours the transit of NSCs toward a glial fate (Plane et al., 2012).", "Here we confirmed and further extend this latter observation, showing that PARP inhibition reduced proliferation in fetal-derived neurospheres and oligospheres (see supplemental material), and reduces OPCs differentiation into mature OLs.", "Notably, key transcription factors involved in OPC maturation, i.e. retinoic acid receptors, thyroid hormone receptors and their hetero-dimerization, such as PDGF signalling, a critical pathway for OPC biology, are targets of PARP regulation (Pavri et al., 2005; Allen, 2008).", "Overall, these results suggest that a different PARP signalling in fetal and adult OPCs might be part in the biological properties of OPCs at different chronological ages.", "These results have also therapeutic implications.", "In fact, PARP inhibition has even been proposed as a pharmacological strategy in a number of acquired inflammation/demyelinating disorders in which OPCs play a key pathogenic role, occurring both in perinatal/neonatal (e.g. neonatal hypoxia/ischemia encephalopathy) and adult age (e.g. spinal cord injury and multiple sclerosis; Komjáti et al., 2005; Moroni, 2008; Cavone and Chiarugi, 2012).", "Contradictory results have been obtained in mice models of inflammatory/demyelinating diseases (Selvaraj et al., 2009; Veto et al., 2010; Casaccia, 2011; Kamboj et al., 2013).", "PARP-2 deletion in conventional transgenic mice results in a protection from experimental allergic encephalomyelitis (EAE), the most widely used animal model for multiple sclerosis (Kamboj et al., 2013).", "Conversely, knock-out of PARP-1 gene leads to an exacerbated EAE and an increase in the mortality rate (Selvaraj et al., 2009).", "Pharmacological PARP inhibition in EAE mice has a protective effect, preventing OL death and attenuating inflammation (Veto et al., 2010), although this effect is attributed to the reduction of CNS inflammation and immunomodulation (Scott et al., 2004).", "Our data, showing that PARP inhibition is cytotoxic in OPCs derived from the fetal brain, suggest that the OPCs chronological age should be taken into account in considering PARP inhibition as a neuroprotective strategy.", "In particular, PARP inhibition in ischemic damage should distinguish perinatal/neonatal from adult conditions, considering that the time-window for the treatment of neonatal hypoxia/ischemia encephalopathy is the key period for OPCs proliferation, maturation and developmental myelination." ]

[ "Oligodendrocyte precursor cell (OPC) were derived from fetal and adult neural stem cells.", "PARP activity is much higher in fetal than adult—derived OPC.", "PARP inhibition is toxic for fetal, but not adult OPC, also impairing OPC differentiation." ]

[ "AGRI", "ENVI" ]

[ "Ginseng", "Overbrowsing", "Panax quinquefolius", "Plant demography", "Refugium" ]

[ "1105", "2303", "2309" ]

[ "“Since then I have lived to see state after state extirpate its wolves.", "I have watched the face of many a newly wolfless mountain, and seen the south-facing slopes wrinkle with a maze of new deer trails.", "I have seen every edible bush and seedling browsed, first to anemic desuetude, and then to death.”", "–Aldo Leopold, from ‘Thinking Like a Mountain’, Sand County Almanac, 1949", "Since publication of Leopold's iconic Sand County Almanac, numerous studies have demonstrated the cascading effects of predator removal and predator re-introduction in a diverse array of biological communities (Pace et al., 1999).", "While many of the classic cases of trophic cascades were described in aquatic systems (Paine, 1966), profound impacts of top predator abundance have been demonstrated repeatedly in terrestrial communities as well (Schmitz et al., 2000; Terborgh et al., 2001; Rooney and Waller, 2003; Beschta and Ripple, 2009).", "In systems where herbivore abundance strongly affects vegetation, the loss of keystone predators can be particularly impactful (Côté et al., 2004).", "Overabundant herbivores can then shape the vegetation over time by profoundly altering recruitment patterns of overstory trees and reducing both abundance and diversity of the understory (Waller and Alverson, 1997; Russell et al., 2001; Côté et al., 2004; Habeck and Schultz, 2015).", "In the eastern deciduous forest of the U. S., two top predators have been functionally extinct for more than a century.", "Eastern cougars (Puma concolor) were officially declared extinct in 2011, but this reflected a condition that had existed for approximately 8 decades (U. S. Fish and Wildlife Service, 2011), and they were uncommon to rare for several decades prior to that.", "The other top predator in eastern forests, the gray wolf (Canis lupus), had a similar history of being functionally extinct in most of its former eastern range by 1900 (Carson et al., 2014).", "Hunting by humans also kept white-tailed deer populations (Odocoileus virgianus) low, along with their predators.", "Over the past several decades, management by state wildlife agencies on behalf of human hunters successfully resurrected large deer herds, ultimately resulting in densities that have far exceeded those found in pre-settlement forests in most regions (Waller and Alverson, 1997; Russell et al., 2001; Côté et al., 2004; Habeck and Schultz, 2015).", "The subsequent widespread overbrowsing of understory tree seedlings, shrubs, and herbaceous understory plants is well-documented (Rooney and Waller, 2003; Côté et al., 2004).", "Indeed, it has been argued that the absence of keystone predators along with mismanagement of their populations resulted in white-tailed deer being recognized as keystone species in its own right (Waller and Alverson, 1997) due to their broad-ranging impacts on the entire ecosystem.", "Frequently, ecologists have been concerned that common understory plant species may be gradually going extinct due to overbrowsing (Carson et al., 2014).", "Particular attention has been paid to browsing of seedlings and saplings of commercially valuable tree species that appear to be favored foods of deer (Côté et al., 2004; Bradshaw and Waller, 2016).", "However, since overstory trees persist for decades to centuries, future overstory species composition, not outright extinction, has been the primary immediate concern with trees.", "Somewhat less attention has been paid to effects of overbrowsing on forest understory herbaceous plants, probably because many have little direct economic value.", "In addition, due to the longevity of most understory herbs and the existence of perennating structures underground, the long-term effects of herbivory are difficult to observe over short time spans.", "Yet it would be valuable to identify populations in this stage of ‘anemic desuetude’ as a precursor to extinction in order to enable conservation action prior to critical endangerment.", "Among understory species that have been studied with respect to deer herbivory, Trillium, a showy and palatable understory wildflower species, has provided an excellent model species (Anderson, 1994; Augustine and Frelich, 1998; Rooney and Gross, 2003; Jenkins et al., 2007; Knight et al., 2009; Kalisz et al., 2014).", "Anderson (1994) showed that mean height of Trillium plants was a strong indicator of deer browse intensity.", "Beauvais et al. (2017) even showed that reduced plant sizes in deer-browsed sites were reflected in the record of herbarium specimens.", "Rooney and Gross (2003) demonstrated population declines of 3.5% per year, even with relatively low browse rates.", "Jenkins et al. (2007) demonstrated the effect of herbivory in truncating the age-structure of natural populations in Great Smokey Mountain National Park, TN, U.S.A. Using matrix models for 12 Pennsylvania populations, Knight et al. (2009) demonstrated that a browse rate of only 15% was sufficient to threaten Trillium's long-term persistence.", "Several approaches have been employed to evaluate deer browse effects, each with its own advantages and disadvantages.", "Some studies have compared dynamics of different populations that naturally experience contrasting browse rates (e.g., Knight et al., 2009); this has the advantage of examining realistic rates in the current environment, but may not include populations at ‘natural’ pre-settlement rates, or browse rates that would be ‘desirable’ from a conservation standpoint.", "In addition, the reasons for the differences in browse rates (e.g., proximity to edge habitat) may be correlated with other factors (e.g., light) that also influence plant population dynamics.", "Others have modeled deer browse effects by removing or including browsed plants from the data set in the calculation of vital rates (e.g., Rooney and Gross, 2003; McGraw and Furedi, 2005); this allows for easy manipulation of browse rates and allows a nice dissection of affected model parameters, though it assumes browse effects on an individual are confined to a particular year.", "Others have taken an experimental approach, fencing areas to remove deer and observing the response over time (e.g., Augustine and Frelich, 1998; Heckel et al., 2010; Kalisz et al., 2014; Sabo et al., 2017; Wilbur et al., 2017).", "Exclusion experiments permit a rigorous, spatially controlled test of browse effects and result in an entire community response; therefore they are well-suited to examining rates of recovery following release from high browsing rates.", "However, unless such experiments are very long-term, allowing re-colonization by a full complement of understory plants, this approach primarily captures the initial recovery period, rather than a continuous condition of low browse rates.", "Finally, a few studies have used naturally-occurring refugia within populations exposed to browsing (Rooney, 1997; Carson et al., 2005); if the refugia have been in existence for a long time, then plant demography in the refugium presumably reflects performance, including the whole-community effects such as increased competition, that may counter some positive effects of no browsing.", "However, in extreme refugia, browse rates may be zero, which could be unrealistically low relative to a desirable, or generally achievable, rate.", "In long-term demographic studies of the widespread medicinal plant, American ginseng, early work focused on human harvest, but deer browsing occurs at much higher rates than harvest (Furedi, 2004; McGraw and Furedi, 2005).", "Unlike Trillium, ginseng is not a ‘preferred’ food plant for deer, an idea supported by detailed analyses of interactions between deer and plants observed with wildlife cameras (Hruska et al., 2014), and the fact that deer will often only partially browse the leaves on a given plant that is within easy reach.", "However, deer will consume the aboveground stalk, leaves, flowers and fruits occasionally, especially as other understory plants become scarce.", "Though browsing rates varied among populations and years, most populations censused did experience significant loss of reproductive adult plants by the end of each growing season (Furedi, 2004).", "These losses catalyzed reduced population growth rates both through lost reproduction and reduced plant size (Furedi, 2004; Farrington et al., 2008).", "Using the data removal approach described above, McGraw and Furedi (2005) showed that virtually all ginseng populations have low population viability at current rates of deer browse, but would be viable with significant reductions in browse.", "Experimental studies confirmed that in addition to consuming leaf material, deer were ginseng seed predators, and not dispersers (Furedi and McGraw, 2004).", "Defoliation, especially early in the season, was shown to decrease plant size and reproductive output in ginseng (Furedi, 2004, Farrington et al., 2008).", "A decrease in stature of plants in herbarium specimens was also shown over the past two centuries, consistent with hypotheses about effects of both human harvest and deer browse (McGraw, 2001).", "Virtually all ginseng populations studied have experienced significant browse; therefore it is not known what characteristics a ginseng population would have in a forest with a rich, dense understory that has not been strongly impacted by deer.", "However, in one population we censused for 20 seasons, we were fortunate to discover a subset of plants growing on top of an isolated, flat-topped, 2.5–4 m high ‘boulder’ that has not experienced deer browsing in its history.", "This ‘control’ group allowed us to examine effects of chronic overbrowsing in order to characterize the nature of ‘anemic desuetude’ in the rest of the population in a demographic sense.", "Therefore, in this study, we (1) examined growth, survival, and reproduction in the two groups of plants, (2) projected relative population growth rates, (3) performed a life table response analysis, dissecting the cause of differential population growth rates, and (4) tested whether size structure differences on and off the rock could yield a static signature of browse intensity across many populations.", "Based on prior studies of browse effects, we expected that the browse-exposed population off the perched rock would experience reduced performance in all three measures, relative to the on rock (protected) subgroup, and that this would in turn reduce population growth rate in equal measure.", "We further hypothesized that demographically Aldo Leopold's ‘anemic desuetude’ would be evident in population-level performance over time, and this would be in turn manifested at a given point in time by large differences in the size structures resulting from those demographic contrasts.", "We expected that the life table response analysis would show deer effects on population growth by mechanisms related to both growth and reproduction.", "Coopers Rock State Forest is a 5158 ha forest in northern West Virginia, U. S. A.", "The primary feature of the forest is the 400 m deep canyon cut by the Cheat River.", "The upper margins of the canyon and other stream-dissected areas of the forest are rimmed by weather-resistant Pottsville sandstone.", "As streams or rivers cut away less-resistant layers beneath, large blocks of Pottsville sandstone break away to form intact blocks of rock isolated by large cracks that eventually erode to isolate the forest community on top of them.", "Such blocks are found wherever this erosion pattern exposes the edge of the resistant sandstones, and they range in size from a few square meters to >1000 m2 and 1–40 m high.", "On one such sandstone block (ca. 12 m × 26 m), the continuous 2.5–4 m high vertical wall on all sides was sufficient to prevent white-tailed deer from browsing the vegetation, resulting in sharply contrasting forest floor vegetation (Fig. 1).", "Except near the rock edges, soil depth was similar to the surrounding off-rock areas, and supported tree growth.", "While the overstory was not different from the surrounding vegetation, the herbaceous vegetative cover was continuous on the rock, and the understory flora was far taller and more diverse than the surrounding off-rock community.", "Vegetative cover in the off-rock community varied between 0 and ∼10%.", "During our early surveys of Population 27 in the off-rock surroundings, we discovered three clusters of ginseng plants on top of the rock.", "They were formally censused (procedure described below) along with the surrounding population since 1998.", "Beginning in 1998, two formal censuses were performed annually through summer, 2017.", "Since ginseng is harvested, plants were marked cryptically with subterranean aluminum nails engraved with unique identifying numbers.", "In spring (between May 25 and June 15) each year, all marked plants were visited and scored for presence, number of leaves, height of stalk, length of longest leaflet on each leaf, width of longest leaflet on each leaf, reproductive status (forming buds or not), as well as evidence of harvest, browse, insect damage and fungal disease.", "In late summer (July 25 – August 15), plants were revisited, berries and seeds were counted, and damage status was re-assessed.", "Leaf area for each plant was estimated by multiple regression using maximum leaflet lengths and widths, then summing across all leaves on the plant (Souther and McGraw, 2011a).", "Two independent estimates of annual deer browsing rates were made.", "First, monitoring every 3 wk by Furedi (2004) showed that vegetative stalks persisted for 3–4 weeks after browsing.", "Therefore, in the two censuses occurring in all other years, we estimated that we effectively sampled browsing for 6 weeks of the ca. 15 wk growing season.", "Hence, the number of observed browsed stalks was multiplied by 2.5 as one estimate of browse rate.", "This first estimate may have underestimated browse rates because some browsed stalks may have been missed in each census (they are harder to visually detect than plants with leaves).", "A second estimate of browse rate was done by adding the total number of ‘missing’ plants in the population that were known to be alive because they emerged in a later year.", "This rate probably overestimated browse because it assumed that missing plants were entirely due to deer, however other factors could have caused them to be missing in any given year (e.g., rodent, rabbit, ground hog browsing), though these other causes were likely to be infrequent.", "Both measures were used to estimate an average browse rate for ginseng in the focus population, but also to provide a comparison with 30 populations censused in the same way across 7 states (Indiana, Kentucky, Maryland, New York, Pennsylvania, Virginia, and West Virginia; McGraw et al., 2017).", "All of these populations were censused annually from 2004 to 2016.", "To estimate growth rate, we used a non-destructive approach by examining the relative growth rate of leaf area (RGR-LA) for any plant that persisted for at least 5 years in the population (McGraw and Garbutt, 1990).", "The slope of ln (leaf area) vs. year was used as the estimate of RGR-LA.", "RGR-LA for plants on and off the rock was compared with a t-test.", "Survival was assessed annually for all plants on and off the rock.", "Since survival is known to be size-dependent and to vary among years, both leaf area and year were included in the logistic model, as well as location on or off the rock with ‘survived/died’ as the dependent variable.", "Total reproductive output via seeds was broken into two components; (a) produced seeds or did not produce seeds, and (b) for seed-producing plants, the number of seeds produced.", "The former was scored at the fall census based upon the presence of forming berries and was a nominal dependent variable.", "The latter was scored by counting bulges in each forming berry and summing for each plant.", "Berries contain 1, 2, and infrequently, 3 seeds so accurate counts were readily obtained.", "Natural log of seed number was analyzed with a general linear model having plant size and year as independent variables, along with location on or off the rock.", "All one-leaf plants were excluded from both of these analyses as ginseng is not able to produce seeds at that life history stage.", "The census interval was from spring census (t) to the following spring census (t+1).", "Since new seedlings germinated in May and were counted at the spring census, this number accounted for any seedling losses prior to the census.", "Deaths were counted as plants that were alive at census t, but were missing at the second census and were not seen for at least two growing seasons later.", "Because occasional early browsing or other damage to the meristem may prevent a plant from being observed aboveground at the spring census in a given year, we waited two growing seasons to pronounce the individual as ‘dead’.", "Hence the observed population change concluded in 2015 (using the 2016 and 2017 censuses to confirm death).", "In the course of censusing, we added new (previously undiscovered, non-seedling) plants to the census as they were found.", "These new plants were not used in calculating net population change until the following year.", "The relative changes on and off the rock were illustrated by starting with a theoretical cohort of 100 in both locations, and multiplying by the observed Nt+1/Nt at each location for every year between 2005 and 2015.", "We chose to use the traditional discrete-class matrix structure (Caswell, 2001) rather than an integral projection model for this analysis because unlike some plants that have more continuous size variation, ginseng does exhibit discrete ‘behaviors’ in the early life history stages (seeds, seedling 1-leaf, and juvenile 2-leaf).", "In addition, sample sizes were limited in the adult classes, in part due to effects of herbivory (Furedi, 2004).", "The 1-leaf plants, distinctive because none produced flowers or berries, were split into new seedlings and >1 y old seedlings.", "New seedlings had lower survival and growth than older 1-leaf plants as the former are in the early phases of establishing a tap root, and are more vulnerable to mid-season drought and other stresses.", "Juvenile plants having two leaves are distinct from 1-leaf plants because most of them do produce flowers, and therefore can act as males in the population.", "However, production of the large seeds and fleshy fruit is rare to infrequent in this intermediate stage.", "The juvenile 2-leaf plants were split into equal size small (<50 cm2 leaf area) and large (>50 cm2) classes.", "Adults (3-leaf and 4-leaf plants) were partitioned into three size classes based on log-linear divisions based on leaf area (small adults, <100 cm2; medium adults, 100 cm2 < leaf area<250 cm2; large adults, >250 cm2); this partitioning accounts for the significantly greater seed production in larger adults than in smaller adults.", "Using leaf area to distinguish classes of 2-leaf and larger plants allowed for growth, stasis, or shrinkage between years; the latter was particularly important for the resulting model in determining accurate population growth rates and explaining population growth rate differences (Salguero-Gomez and Casper, 2010).", "Due to innate dormancy that is released only after two winters, all individuals pass through a seed stage in the soil.", "Although ginseng seeds have been shown to persist in the soil at some sites for up to 4 years, in Population 27, two sequential on-site seed cage studies showed persistence for only 2 years at this site.", "Therefore two seed stage classes were included in the matrix model; 9 month old and 21 month old seeds (age when censused in late spring).", "The seed cage study allowed us to calculate initial 9 month survival, survival within the seed bank from 9 to 21 months, and germination from the 9 month old and 21 month old seed pools.", "Size-specific fertilities (mean number of 9 month old seeds per adult of each size class the previous year) were determined as the product of seed number, 9 month survival observed in the seed cage study and a loss rate specific to on rock vs. off rock environments.", "This loss rate was included because seed cages protected dispersed seeds to an unknown degree.", "To calculate the loss rate we determined the ratio of observed new seedlings to the expected new seedling number based upon seed production and 9 month survival.", "The transition probability from 9 to 21 month old seeds was determined directly as the mean of the two seed cage studies (Souther and McGraw, 2011b).", "Germination rates (transition from seed to 1-leaf new seedlings) were also determined from the seed cage study.", "The results of the age-, stage- and size-based class partitioning of the life history was a 9 × 9 transition probability matrix model.", "Transition matrices were determined for each pair of years from 2005 to 2014 (ten total matrices) for the off rock subpopulation.", "The sample size ranged from 355 to 428, resulting in a well-parameterized model.", "In years prior to 2005, a small amount of harvest occurred and therefore in order to focus on deer browse effects in isolation, we did not include those years in this study.", "Transition matrices from 2015 onward could not be estimated because of the unknown mortality status of missing plants.", "The deer-isolated ‘on rock’ subpopulation numbered between 7 and 30 plants in the 20 census years.", "This number of observations was insufficient to construct a separate population projection matrix for each year.", "To construct a matrix for comparison to the off rock subpopulation, we pooled the transition observations across pairs of years to construct a single matrix.", "While the observations were therefore not entirely independent as some individuals were repeatedly sampled, these observations were made across ten widely varying growing seasons and provided a good composite measure of demographic response to the on rock environment.", "To examine how a static population measure could indicate a state of long-term overbrowsing, we examined the stable size distribution differences between the on rock composite matrix and off rock mean matrix of Population 27.", "Next, in order to test the fidelity of our proposed indicators beyond Population 27, we examined the association between several possible size structure indicators (proportion of 1-leaf seedlings, proportion of 2-leaf juveniles, proportion of adults, and ratio of non-juveniles to juveniles) and our two browse estimates in 30 natural populations over a wide area by correlation analysis.", "All asymptotic population statistics were determined using MATLAB (v. R2015b, The Mathworks, Inc.).", "The overall browse rate for Population 27 (the focus population of this study) was slightly above the 30-population median by either measure; ranking 12th (out of 30) by one measure and 14th (out of 30) by the other (Fig. 2).", "Population 27 is therefore near the middle of the set of natural populations we studied across seven eastern U.S. states.", "The focus population's annual browse rate estimate was 20–25% by the two measures used in this study.", "No plants on the rock were browsed in any of the 20 census years (1998–2017).", "Mean relative growth rate of plants that persisted at least 5 years on the rock was higher than the same measure for the surrounding population (Fig. 3A).", "Plants on the rock had an RGR significantly greater than 0 (one-sample t-test, t = 4.1514, p = 0.0013), indicating an increase in mean plant leaf area.", "Indeed, after a decade, mean leaf area of plants on the rock nearly tripled.", "However, mean RGR-LA for plants in the browse-exposed population was negative, showing a declining leaf area (one sample t-test; t = −1.6749, p = 0.0473).", "This corresponded to a 10% decline in mean leaf area over a decade in the browse-exposed portion of the population.", "Survival of individual plants to the following year was size-dependent, as expected (Leaf Area Effect, Likelihood Ratio Chi-square = 94.5375, p < 0.0001).", "There was no statistical support for a survival difference between locations (Location Effect L-R Chi-square = 1.2868, p = 0.2566).", "However, the effect of size on survival depended on location (Location x Leaf Area Effect, L-R Chi-square = 4.3360, p = 0.0373).", "Inspection of the logistic regressions of survival on size at the two locations showed that survival was lower at small sizes on the rock, but this pattern was reversed above a size threshold of ca. 37 cm2 (Fig. 3B).", "No evidence was found to support a main effect of location on components of reproduction (on vs. off the rock; p > 0.10).", "The chance that a plant would produce seeds increased only as a function of size (L-R Chi-square = 180.5527, p < 0.0001).", "In addition, the effect of size on whether a plant was reproductive did not depend on the location (p > 0.10).", "With separate logistic regressions, on rock and off rock plants showed similar patterns of sharply increasing reproductive probabilities with plant size (Fig. 3C).", "For plants that successfully produced berries, mean number of seeds was a function only of plant leaf area (FLeaf area = 84.8808, p < 0.0001) and not location (FLocation = 0.0168, p = 0.8970), but there was a trend suggesting a dependency of the size effect on location (FLeaf Area x Location = 3.3220, p = 0.0688).", "Predicted seed numbers tended to be greater for small reproductive plants on the rock than for those off the rock, but this pattern was reversed for large plants (Fig. 3D).", "The bulk of the population (91% as of 2017) was located off the rock, and this part of the population declined steadily in numbers from 2005 to 2015 at a rate equivalent to 4.5%/y (Fig. 4).", "By contrast, the small subpopulation on the rock grew haltingly with episodic recruitment, with a net increase of 50% after one decade.", "This continued for the following two summers, by which time the number of plants had nearly tripled in number due to a second large pulse of new seedlings (data not shown).", "Based on the mean population projection matrix for the off rock subpopulation, that group of plants was predicted to shrink in numbers at a rate of 4.51% per year when at the stable stage distribution (Fig. 5A).", "This was almost precisely the same as the observed annual rate of decline from 2005 to 2015.", "Meanwhile, the on rock subpopulation was predicted to increase at a rate of 3.73% per year (Fig. 5A).", "This rate would translate to a 44% rate of increase over a decade, again not very different from the observed rate from 2005 to 2015, though clearly the on rock plants were not in a stable stage distribution.", "Remarkably, the LTRE clearly showed that all six of the top positive contributors to the difference in λ between on rock and off rock subpopulations were due to contrasts in matrix parameters describing growth to larger classes (Fig. 5B).", "Growth into the largest and second largest adult classes were most important, followed by growth into the small adult class.", "Size-specific fertility parameter differences on and off the rock were not important contributors to Δλ, all having absolute values < 0.002 (data not shown).", "The stable stage distribution of the composite matrix for the on rock subpopulation and the mean matrix for the off rock subpopulations in Population 27 showed distinct differences in almost all stages (Fig. 6).", "The on rock subpopulation had more seeds in the soil, more new seedlings and more medium and large adults, as a proportion of the total in the stable stage distribution, while the off the rock subpopulation was enhanced in the older seedling and juvenile categories.", "By placing Population 27 in the broader context of 30 natural populations, we were able to examine which of the size structure indices showed a correlation to the browse indices.", "Indeed, almost all of the size structure indices showed some correlation to browse.", "For example, the larger the number of browsed stems observed with eaten stalks (browse estimate 1), the fewer 1-leaf plants were found in the population (r = −0.6222,p = 0.0002).", "The strongest association was the positive correlation shown between the second browse estimate and the proportion of the population in the 2-leaf plant classes (Fig. 7).", "Across the 30 populations, the proportion of juveniles ranged widely, from 17 to 58% of the whole population.", "At the upper end, these proportions corresponded with browse estimates of more than 40%.", "Compared with the other 29 populations, Population 27 was near the middle of the scatterplot (Fig. 7), again suggesting it was not atypical.", "Interpreting individual- and population-level contrasts between on rock and off rock subpopulations as a browse effect makes the assumption that absence or presence of deer was the only difference between the two environments.", "Sensu stricto, this is clearly not the case.", "One obvious difference is soil depth on the rock, which could in turn affect water and nutrient availability.", "In August 2017, we measured soil depth in the vicinity of ginseng plants on the rock and found an O horizon varying between 5 and 35 cm deep, with almost no other soil horizons above the perched rock surface.", "From prior experience digging ginseng roots, we find most rooting does occur within this depth range, however fine roots accessing deeper water supplies are found in larger plants at sites with deeper soils.", "The prediction of greater drought stress on the rock was reinforced by notations on our annual August census sheets of wilting or early senescence in 5 of 20 census years.", "Relatively undeveloped soil would likely reduce ginseng growth on the rock relative to an unbrowsed condition off the rock, thus underestimating the negative ‘effect of browse’ relative to what we observed; our ‘browse effect’ interpretation is likely conservative.", "Another abiotic contrast of the on rock and off rock microsites could be overstory canopy coverage.", "In other work, ginseng responded positively to smaller canopy openings (Wagner and McGraw, 2013; Chandler and McGraw, 2015, 2017).", "However, the rock surface was small, and large trees adjacent to it were overhanging the rock subpopulation.", "In addition, multiple trees up to 15 cm in diameter were growing on the rock surface itself, such that the tree canopy difference was minimal.", "Intense shading from herbaceous competitors, likely an indirect deer browse effect, would have more than compensated for any reduction in tree canopy experienced by ginseng plants on the rock (see Fig. 1), again making the abiotic environment more favorable for the off rock subpopulation with respect to light.", "We therefore conclude that browse difference between on rock and off rock subpopulations would have been even greater if the lower quality microenvironment on the rock had not tempered the contrast.", "Comisky et al. (2005) demonstrated that low rocks in the Alleghany National Forest that were exposed to browse closely resembled the off rock vegetation, while high browse-protected rocks acted as refugia, with higher species diversity and much higher reproductive output from herbaceous plants found there.", "This affirms our interpretation that the overwhelming observed difference between the high rock and the surrounding vegetation was due to deer browse.", "Over 30 wide-ranging populations, two browse estimates revealed a wide range of browse rates (1.5%–67% for one measure; 12%–44% for the other) for ginseng populations without obvious deer refugia (McGraw et al., 2017).", "No precise measure is available because populations would have to be visited at least 6 times during the season to assess actual browse rates and such a high human visitation rate could itself alter browse probability.", "Therefore, some uncertainty in natural browse rates is inevitable.", "The two estimates were similar, and near the median, for Population 27 (20–25% each season); we conclude that this population is representative of deer impacts in the middle range of those experienced by natural populations.", "The presence of a ‘control’ group of plants in unbrowsed vegetation atop a rock therefore presented a unique opportunity to examine ‘average’ effects of browsing on this species.", "There was only one such ‘control’ group within one of the 30 populations, however the fact that it was typical of other populations with respect to deer browse suggests that our findings are more broadly relevant.", "Contradicting our original hypothesis, browse effects on survival, growth, and reproductive were not spread evenly across size-dependent measures on individual plants.", "Instead, the primary effect of exposure to browse was to reduce the size-dependent growth rate (RGR-LA) of individuals in the browse-exposed region of the population to a mean that was negative; plants off the rock were actually shrinking in mean leaf area.", "This pattern of shrinkage following browse has been observed for other understory species, such as Trillium (Knight et al., 2009).", "We interpret this slow decline in mean size of plants as a primary signal of Aldo Leopold's ‘anemic desuetude’ (Leopold, 1949) at the plant level for this forest floor herb.", "Because smaller plant size results in higher mortality and lower reproductive output, browse has consequences for these components of plant performance as well at the population level, but there were only subtle differences between off rock and on rock plants in terms of their size-dependent survival and reproduction.", "Physiologically, the effect of browsing on growth is explained by the fact that it curtails energy storage in the tap root for re-growth the following growing season.", "Ginseng, like most understory perennials, exhibits deterministic growth within a growing season, and the top does not re-grow once browsed.", "Therefore, a shortened growing season effectively reduces energy storage in the form of non-structural carbohydrates in the root.", "Smaller ‘adult’ plant sizes have been identified in other deer-browsed understory species as well, including reduced height and leaf area in Trillium (Anderson, 1994, Beauvais et al., 2017), and reduced leaf size in Maianthemum (Rooney, 1997).", "Off the rock, deer may also have had nonconsumptive, indirect effects as well.", "In Arisaema triphyllum, for example, Heckel et al. (2010) found that deer activity increases soil compaction and decreases leaf litter depth, contributing to declines in A. triphyllum size and seed rain, and resulting in shifts toward male-dominated populations, even though this species was rarely consumed by deer.", "Sabo et al. (2017) further suggested that the direct effect of deer browse were compounded by deer-mediated environmental alterations, including soil compaction.", "The contrasting performance of individual plants was reflected in demographic projections that forecasted long-term increases in the rock subpopulation, and by contrast, declines in the off rock subpopulation.", "Indeed, although the browse-protected subpopulation showed episodic recruitment and stochastic fluctuations typical of low N populations, the net change over a decade nevertheless closely reflected the long-term positive trajectory predicted by the composite projection matrix model for those plants.", "The decline seen off the rock, based on larger numbers, was steadier over time, and again reflected the projections closely.", "The modeling projections assumed non-connectivity between on-rock and off-rock subpopulations, which strictly speaking is not the case.", "However, the model projections closely mirrored observed changes, so if, for example, seed production from the boulder subpopulation was altering population change off the rock, its effect was undetectable.", "The LTRE showed that greater rates of growth to larger size classes (reflecting higher RGR) was the primary reason for higher growth rate in the browse-protected group of plants on the rock within Population 27; reinforcing the primacy of growth effects at the population level.", "The contrasting demographic parameters resulted in altered stable stage distributions, which, in turn, reflected the state of ‘anemic desuetude’ in the population.", "The high proportion of ‘juvenile’ 2-leaf plants in ginseng may be the best indicator that deer have been chronically overbrowsing a population.", "By contrast, a large proportion of new seedlings (reflecting a larger seed pool) and a large proportion of large fecund adults is likely an indicator that a population is not being overbrowsed.", "A juvenile-heavy stage-distribution was also found to be a consequence of harvest by humans (Mooney and McGraw, 2009).", "However, when humans harvest plants, unlike deer, the roots are destroyed – the skewed structure in that case is a consequence of removing larger individuals from the population, not due to stunting.", "This suggests a way to distinguish anemic desuetude produced by deer and the skewed distribution produced by human harvesters: In the case of deer, a large proportion of 2-leaf ‘juvenile’ plants are likely to be old ‘stunted’ individuals, while in human harvested populations, juveniles will largely be young, growing, and in the process of transitioning from seedling to adult.", "In ginseng, ages can be determined by counting annual bud scars on the rhizome (Lewis and Zenger, 1982).", "Indeed, in the complete published data set for 30 populations of ginseng (McGraw et al., 2017), although only a small proportion of older plants were aged using the bud scar approach, 36% of the observations of plants more than 30 years old were 2-leaf ‘juvenile’ plants, and one of these was more than 50 years old.", "This pattern is not readily explained by human harvest effects but is consistent with deer browse effects.", "Refugia such as large rock or boulder tops that are out of reach of deer could be important sources of recolonization of surrounding communities if browse rates were reduced through improved habitat management in the future (Carson et al., 2005).", "Within Population 27, the single rock top subpopulation of ginseng has become more significant over the 20 year census period.", "At the beginning of our censusing efforts (1998), the boulder top subpopulation comprised 3% of the plants and 2% of the seeds produced in the larger population.", "By 2017, the boulder subpopulation accounted for 9% of the plants and 37% of the seeds produced by the overall population.", "Given the subpopulation trajectories we showed, this trend is likely to continue.", "Indeed, certain other rock top species we noted (e.g., Sanguinaria canadensis, Asarum canadense, Actaea racemosa, Urtica dioica, Polygonatum odoratum and Thalyctrum sp.) were rare or absent altogether in the off rock community, suggesting that more palatable species were farther along on this trajectory.", "Ultimately, of course, density-dependence within ginseng will likely limit the boulder top subpopulation.", "Assuming no density-dependence to this point, backward projection suggests this group of plants originated ca. 9 decades ago.", "In practical terms, most forest communities are unlikely to contain many rock refugia to act as biodiversity preservation hotspots.", "Certain other microsites (e.g., slash left by logging operations or very steep slopes; Grisez, 1960, Rooney, 1997) may be somewhat protected from overbrowsing by deer, which could provide partial refugia.", "Land managers could also create refugia by fencing areas and allowing them to recover, however this strategy requires significant initial investment and maintenance to be effective in the long term.", "Moreover, recovery could be quite slow due to the ‘ghost of herbivory past’ (Carson et al., 2005); a general problem with the exclusion approach to conservation.", "The obvious more permanent remedy is to manage deer populations with the health of the ecosystem and biodiversity preservation as the primary goal (deCalesta and Stout, 1997; Rooney, 2001; Côté et al., 2004; Latham et al., 2005).", "When human hunting is inadequate for achieving this goal, reintroduction of top predators should be seriously considered (McShea, 2012).", "The success of such reintroduction efforts in certain regions of the world provides examples that could readily be followed, even in areas close to human-dominated habitats (Creel et al., 2005; Beschta and Ripple 2009, 2010).", "Recovery of vegetation from overbrowsing would likely be fastest for species such as ginseng that are secondary food sources for deer and therefore have persisted through decades of overbrowsing, albeit in a state of ‘anemic desuetude’.", "Recovery will also be more rapid for communities that do have natural refugia as a source of recolonization.", "Beyond their immediate importance for plant conservation, refugia from overbrowsing may be important for whole-community preservation.", "For example, pollinator guilds associated with rich understory vegetation (Motten, 1986) could be preserved within refugia.", "Ginseng seeds represent a food source for thrushes and other birds (Elza et al., 2016).", "Indeed, the origin of the on rock subpopulation was very likely seed dispersal by wood thrushes as they have been observed annually at that site.", "The degree to which isolated refugia can have cascading conservation effects has not been thoroughly investigated.", "As overbrowsing by deer remains unaddressed in much of the range of white-tailed deer in the eastern U. S., the role of refugia in preservation of biodiversity at all levels will likely continue to increase.", "The funding source had no role in study design, nor in collection, analysis and interpretation of data, nor in writing of this report or the decision to submit for publication." ]

[]

[ "BUSI", "DECI", "MATH" ]

[ "Currencies", "Investment strategy", "Optimization algorithms", "Profitable portfolios" ]

[ "1408", "1803", "2606", "2613" ]

[ "The ultimate objective of any investor, trader or manager is to speculate, to generate profits in a consistent basis.", "Simsek [27] assumed that any financial innovation on portfolio risks is likely to lead to speculation rather than risk sharing due to the motives of the participants in market.", "An approach that can be implemented in order to maximize profits and simultaneously to minimize the risk of loss, is to define specific rules for buying and selling securities; rules that will be able to predict accurately the future movements of the market.", "These rules formulate the so-called trading strategy or system.", "The most common trading strategies are based on fundamental or technical analysis; this work is focused on technical trading strategies that rely on the assumption that historical data can create patterns that repeat themselves in the future.", "According to a top technical analyst (Ping [[25], p.2]) the technical analysis is defined as follows: ``The technical approach to investment is essentially a reflection of the idea that prices move in trends that are determined by the changing attitudes of investors toward a variety of economic, monetary, political, and psychological forces.", "The art of technical analysis, for it is an art, is to identify a trend reversal at a relatively early stage and ride on that trend until the weight of the evidence shows or proves that the trend has reversed.''", "The prediction of market's future movements became an important research topic for academicians into a theoretical basis and a challenging task for investors in practical level.", "One of the earliest empirical studies in this field is the one by Donchian [7] who presented the movement of the market as a channel approach focusing on the breakouts of these channels.", "Apart from this approach, there is a plethora of technical strategy types.", "Among others, trading systems that include filters were introduced (Fama and Blume [8]; Sweeney [29]), strategies that focus on the moving averages were presented (Cootner [4]; Dale and Workman [6]) and strategies based on the relative strength were studied (Jensen and Benington [13]).", "One of the most significant studies on this field was carried out by Brock et al. [3], who strongly supported the efficiency of technical strategies.", "They tested two simple technical strategies (moving average and range breakout) in Dow Jones Index in their study and using the model-based bootstrap approach they conducted statistical tests on the trading returns.", "A few years later, Bessembinder and Chan [2] confirmed the research outcomes presented by Brock et al. [3] and provided further support to the technical rules, indicating that they can predict the movement of the market and particularly those of the US Equity Index.", "In another research, Taylor [30] indicated that technical trading approach and specifically the channel style when applied to currencies can lead to profits, having also remarkable forecasting ability when the market follows almost random walk.", "Menkhoff and Taylor [21] tried to explain the continuously rising use of technical analysis and its apparent profitability.", "Among their arguments, they sustained that technical analysis could fit to the foreign exchange market due to not-fully-rational behaviour of the market and it might provide information on foreign exchange movements that cannot be explained through fundamental analysis.", "Gehrig and Menkhoff [11] also underlined the importance of technical analysis into the world of investment; especially they mentioned that it is by far the most important tool when dealing with FX and is rated second in the field of fund management.", "Later, Osler [23] showed that technical trading strategies can represent rational long run balance given the structure of the currency markets and traders’ motivations.", "Relying also on the relationship between technical analysis and the FOReign EXchange (FOREX) market, in a recent study by Smith et al. [28] it was found that during high-sentiment periods, the use of technical analysis provided an edge to the hedge funds that helped them to succeed higher performance with superior market-timing ability and at the same time achieve lower risk to their investments.", "Another study that focused on FOREX market was by Novotný et al. [22].", "They investigated a strategy based on price jump and indicated how price jumps carry a tradable signal for all currencies.", "Back to real life, the experiment performed by Richard Dennis is considered as a unique example representative of the obsession related to trading and the feverish effort to generate profits.", "Covel [5] described in his book entitled ``The Complete Turtle Trader'' that in mid-1983, the famous commodity trader Richard Dennis conducted an experiment aiming to prove that he could teach people how to become great traders.", "In order to prove his belief, he published an advertisement in Barron's, the Wall Street Journal and The New York Times seeking to recruit and train people (Michael W. Covel, was one of them).", "According to the experiment Richard Dennis provided the trainee traders with real accounts in order to trade.", "These trainee traders were called as ``Turtles'' and Dennis taught them a trend-followed system based on a channel approach.", "The ``Turtles'' succeeded to earn an aggregate sum of over $100 million dollars in the next four years and became the most famous experiment in trading history.", "Another actual example is the one by Professor Josef Lakonishok [18], who supported that value strategies can beat the market.", "Based on his study, Lakonishok decided to apply his theoretical research in real-world trading practice and turn it to an almost $70 billion dollars practice.", "His strategy is focused on identifying valued shares before the market recognize them.", "To succeed that, he proposed a system that uses valuation ratios such as price-to-book or price-to-sales and searches for companies with ratios relatively lower than their peers.", "Then, his system tries to identify possible entry points based on the price momentum of the last six-month period.", "As Pardo [24] indicated in his book ``The Evaluation and Optimization of Trading Strategies'' the first step into trading strategy design process is the formulation of the trading strategy while another extremely crucial step is the optimization of that strategy.", "Optimizing the trading rules is extremely important, since actual traders are likely to choose the best-performing rules in advance.", "The work by Jensen and Benington [13] is considered as the forerunner study in this direction, they followed an optimization and out-of-sample validation procedure for improving the performance of relative strength index based strategies.", "Later, Marshall et al. [20] tried to answer if commodity futures can be traded profitably with quantitative timing strategies and to find the most suitable trading rules for each commodity in order to provide statistically significant profits.", "Fisher [9] in his book ``The logical trader'' introduced the ACD Rules and Pivot Point System, that provided specific entry levels for buying and selling based on the opening range of virtually any security.", "Tian et al. [31] attempted to optimize the rules of ACD system in an intraday basis in order to ameliorate its performance in Chinese future market.", "Foltice and Langer [10] focused on the creation of a momentum strategy, which could be found appropriate for an individual investor.", "They developed and calibrated a simple strategy, which succeeded to outperform its benchmark and it required a small initial capital.", "The main objective of this study is to develop an empirical technical trading strategy, which could be applicable to the financial markets and lead to the construction of profitable portfolios.", "This strategy follows a channel breakout approach based on the study by Donchian [7].", "The portfolios that are formed in this work are based on the currency market.", "Barroso and Santa-Clara [1] proved that the exposure to currency can lead to portfolios with significant higher Sharpe ratio.", "The strategy developed in the current study is combined with the Modified Renko Bars (MRBs); a trading tool which responses more accurately than the normal candle sticks to the nature and characteristics of the FOREX market.", "Aiming to create an edge to the investor and to develop a profitable portfolio, an optimization problem is formulated and solutions are carried out.", "The optimization stage focuses on the calibration of the system for eight FOREX pairs (GBP/USD, USD/JPY, NZD/USD, AUD/USD, EUR/USD, USD/CAD, GBP/JPY and EUR/JPY) using three global search derivative-free optimization algorithms; a Swarm Optimization one called Pity Beetle Algorithm (PBA) along with the DIvide a hyperRECTangle (DIRECT) and Multilevel Coordinate Search (MCS) algorithms.", "Then, optimized strategy is tested to the specific pairs and based on the returns obtained two kinds of portfolios were constructed; an equally weighted portfolio and a portfolio based on Kelly Criterion.", "Finally, the performance of the portfolios is assessed based on common and widespread evaluation measures (arithmetic mean, geometric return and sharpe ratio) and then they are compared with well-known benchmarks (S&P500, Barclay CTA Index).", "Thus, the proper question that can be stated is ``how can a profitable currency portfolio be made based on a specific trading system?''", "This is the question that is answered in this study.", "The second parameter is based on the same assumption, i.e. that the market is mostly flat and therefore the probability of a strategy to have positive outcome is becoming smaller.", "Contrary to Toshchakov [32] who supports in his book entitled ``Beat the odds in FOREX trading'' that the market has two directions, we believe that the market has two conditions, the trending condition and the flat condition.", "During the trending condition, the market indeed moves up or down.", "When it comes however to the flat condition, the market presents a neutral direction, which is characterized by plenty of fluctuations.", "Therefore, the objective is to create a mechanism that diminishes the trades when the market does not move.", "In this direction, a strategy needs to be developed that would be activated only by the movement of the market, independently of the time.", "This approach targets to increase the amount of the winning trades meaning the win/loss ratio.", "Already, there are trading tools that are focused mainly on the movement of the price, the renko bars is one of these tools.", "Trading techniques that are focused on the price action suffer from the disadvantage of market's noise.", "The common candlesticks contain a lot of information that most of it, is contradictory.", "For example, one candlestick might provide a long signal and the next one a short one.", "This kind of noise that comes from the interaction between traders and institutions trying to establish their opinion for the market.", "In order for the traders to stay focused on the main trend, many tools have been created to support this approach; i.e. to smooth the movement of the market and to provide only the essential information, Heikin-Ashi and Renko charts are the most common ones.", "Both aim to remove or reduce the noise through proper manipulation of the simple candlestick.", "The main difference between Heikin-Ashi and Renko approaches is that the first one formulates bars relying on the time just like the common candlesticks.", "On the other hand, Renko bars are based on the price movement only, without taking into account the time.", "This is the main reason that this study will focus on Renko charts, since time represents a major contributor on noise creation.", "According to the Renko bars trading tool the common candlestick chart is transformed into a chart where the bars are formed based on the range that the security covers, independent of the time.", "Renko charts use price ``bricks'' that represent a fixed price move.", "As it is seen in Fig. 1, the new chart is formed up or down in 45° lines with one brick per vertical column.", "For example, in a one-hour chart, each renko bar can represent different numbers of candlesticks depending on the size of the renko bar (or brick).", "If the brick value is set to 10 points, a move of 10 points or more is required to draw another brick.", "Price movements less than 10 points will be ignored and the renko chart will remain unchanged.", "But what ``a move of 10 points'' does it mean?", "Renko charts can be either closing prices or high-low range based ones.", "In the case of a close price based renko bar, a brick is formed only when the candlestick close price covers the 10 points (for the above mentioned example), in contrary to a high-low range based renko bar where a brick is formed when the price approaches the 10 points ignoring where the candlestick will close.", "The most common mode is the closed price based renko bars since they fluctuate less than the high-low range based ones.", "Based on empirical tests on both renko charts it was observed that they have different advantages and disadvantages.", "Specifically, in a same, for instance, uptrend movement, less close-renko bars would be formed than high-low range renko bars because as it is mentioned before they require from the price to close above to a specific price level and just to reach this level.", "So in this situation, for a long trade the renko bars based on high-low range are superior creating larger margin for profit.", "But when the direction of the market changes, the high-low range renko bars become too sensitive giving wrong signals to the real shift of the direction and making the trader to close earlier the trade.", "In the other hand, the close-renko bars, moving slower, give more accurate changing of direction points.", "So, it become clear to us that any kind of renko bar by itself creates difficulties on the low RRR approach.", "To overcome this difficulty an alternative algorithm of a modified renko approach was developed herein.", "The key point of the proposed new concept is that the bricks of the same direction are formed based on the high-low range logic and the bricks when the direction changes are formed based on the closing price logic.", "The proposed new renko style (labelled as Modified Renko Bars - MRBs) creates more accurate representation of the market's true movement.", "Following the development of the MRB charts that represent a reliable trading tool, a trading strategy will be formulated aiming to achieve stable performance.", "The proposed strategy is a breakout style system applied to the modified renko chart based on the Channel Breakout systems taught by Donchian [7].", "The system consists of two different but related breakout systems: (i) System 1 - A long-term system based on a X-MRB breakout which is shown in Fig. 2 and (ii) System 2 - A short-term system based on a Y-MRB breakout, where X > Y, which can be seen in Fig. 3.", "System 2 is complementary to System 1; more specifically, System 2 aims to catch the continuation of a price movement that triggers the exit of System 2.", "System 1 Entry - Trader enters a position when the price exceeds by one MRB the previous high or the low value of the preceding X MRBs.", "If the price exceeds the previous X-MRB high, then trader buys one unit to initiate a long position in the corresponding commodity.", "If the price drops by one MRB below the previous X-MRB low, trader sells one unit to initiate a short position.", "System 2 Entry - The system 2 is activated only when the trade from System 1 has closed.", "Trader enters position when the price is exceeded by one MRB the previous high or the low of the preceding Y MRBs.", "If the price exceeds the previous Y-MRB high and at the same time the price is over the previous X-MRB high, then trader buys one unit to initiate a long position in the corresponding commodity.", "If the price drops one MRB below the previous low of the last Y MRBs and is below the previous X-MRB low, trader sells one unit to initiate a short position.", "System 1 Exit - The exit signal is the Y-MRB low for long positions and the Y-MRB high for short positions.", "The unit in the position will be exited if the price goes against the position for a Y-MRB breakout.", "System 2 Exit - which is identical with that of system 1.", "Where the exit signal is the Y-MRB low for long positions and the Y-MRB high for short positions.", "A long position would be closed if the price drops on MRB below the previous Y-MRB low.", "Respectively, the trader would be exited from a short position if the price exceeds the previous Y-MRB high by one MRB.", "The two systems are constructed in such a way that their entry signals do not affect each other.", "Although their philosophy is complementary, their execution is completely independent.", "An advantage of the proposed trading system is the range of its applicability, where input data from any kind of financial asset can be used (i.e. stocks, indexes, interest rates, commodities and currencies).", "In order to achieve the highest possible accuracy for the simulation, the results of the back-testing process and the actual trading, traders should apply the system mainly to the most liquid markets such as futures markets (commodities, indexes, currencies, etc.); otherwise it will be too difficult under real market conditions to enter and to exit positions without taking large losses.", "Futures markets have several features that make them a more attractive market for active trading strategies than stock markets.", "Specifically, transaction costs are lower and it is easier to short-sell.", "All the data that are employed in this study represents spot FX rates (the most liquid financial asset) of different currency pairs.", "Finally, worth mentioning that in order for the strategy developed in this study to be available under real market conditions, it will be applied based on spot FX market.", "The average commission per trade of this instrument is about 0.2 pips (based on the commission section of Interactive Brokers for FOREX).", "For each FX pair, the developed strategy generates about 100 trades per year.", "Hence, the transaction costs for the total trades of a year for a FX pair would be approximately 1.5% of the initial capital.", "This issue would not make any significant different in the obtained results, for this reason it was decided to make the calculation with no transaction costs in order to emphasize mainly to the more precise development of the proposed strategy.", "Concerning the fourth design variable, although, the observation that the smaller the size of the brick is, the higher the total return will be, is obvious; it is not able to check in advance if the drawdown (in pips) will be lower than the total initial capital.", "If this is not the case, then the optimized parameters resulted from the optimization of Eq. (1) will not be valuable.", "In order to deal with this issue, the fourth parameter was expressed as the percentage of the capital that each brick corresponds to.", "For this reason, the total return, that is the objective function of the optimization process to be maximized and the maximum drawdown that will be treated as a constraint are calculated at percentages.", "The assessment of the trading strategy over a set of data is carried out in two phases: the strategy optimization and portfolio construction ones, which are described in detail bellow.", "The constructed portfolios are assessed over a set of data composed by one-hour time frames corresponding to the period of 2006 to 2016 for each currency pair employed for the needs of the current study.", "The full data is composed by 65,000 candlesticks, and they are divided into years’ periods, thus each year period contains 6000 candlesticks.", "In order to achieve increased accuracy concerning the actual representation of the candlestick chart into MRB charts one-hour data are employed.", "Aiming to minimize the effect of the gaps and the closing prices one-hour data are used.", "Although, the MRB chart depends on the range, if for the same security data from a small time frame and from a higher one are used, the results will not be the same.", "This is because part of the modified renko algorithmic formulation (the change direction part) depends on the closing price of the candlesticks.", "Thus, the spread between the closing price of the MRB and the actual closing price is smaller when short time frames are used and consequently the representation of movement of the market from the modified renko chart is more normalized.", "Through a parametric study the optimization phase was decided to rely on the first 36% part of the data (24,000 candlesticks = 4 years) as a whole, aiming to achieve the highest total return and at the same time to keep the maximum drawdown lower than 100% for each currency pair so that the result to be realistic for any starting time.", "The main objective of this parametric study was to create the largest testing margin (i.e. reduce the part of the data required for the optimization phase).", "According to the parametric study, the optimization phase initially was based on the first 60% part of the data and gradually this percentage was reduced.", "It was observed that the minimum percentage of the data that is required in order for the optimization phase to converge to the optimal values of four variables is equal to 36%.", "The optimization phase is performed using the three optimization algorithms that are described in the following section.", "Relying on the first 36% part of the data (i.e. 24,000 elements), for each currency pair, optimized values for the four variables are obtained implementing each of the three algorithms.", "Out of the three algorithms the variables corresponding the highest total return are selected in order to be used in the testing phase.", "On the next phase, the calibrated strategy was tested and its performance was calculated over the remaining part of the data (i.e. elements 24,001 to 65,000 corresponding to 7 years) on a yearly basis.", "As it will be described subsequently, the derivative of the objective function F used in Eq. (1) is not analytically available, thus this type of problems is generally referred as Derivative-Free Optimization (DFO).", "We further refer to any algorithm applied to type of problems as a Derivative-Free Algorithm, which are classified as direct and model-based ones.", "Direct algorithms determine search directions by computing values of the function F directly, whereas model-based algorithms construct and utilize a surrogate model of the objective F to guide the search process.", "Algorithms are further classified as local or global, with the latter having the ability to refine the search domain arbitrarily.", "Finally, algorithms are classified as stochastic or deterministic, depending upon whether they require random search steps or not.", "In the framework of the present study three DFO global search algorithms are considered.", "The first one is called Pity Beetle Algorithm (PBA) which is metaheuristic algorithm belonging to the type of Particle Swarm Optimization algorithms that is also a global stochastic search algorithm, along with the DIvide a hyperRECTangle (DIRECT) and Multilevel Coordinate Search (MCS) algorithms that belong to the category of global deterministic search algorithms.", "A short description of all three of them is provided below.", "Swarm optimization characterize a stochastic, population-based group of algorithms inspired by the social behaviour of birds flocking, fish schooling etc., PBA proposed by the authors (Kallioras et al. [15]) belongs to this class of algorithms and its efficiency was found superior to other well established metaheuristic algorithms according to the CEC 2014 benchmark test, this is why was chosen as a representative of metaheuristics for the purposes of this study.", "It was inspired by the aggregation behaviour, searching for nest and food, of the beetle named Pityogenes chalcographus, also known as six-toothed spruce bark beetle.", "This beetle has the ability to locate and harvest on the bark of weakened trees into a forest, while when its population exceeds a specific threshold it can infest healthy and robust trees as well.", "PBA consists of three basic steps: initialization, host selection pattern and update location of broods, while a population consists of males and females; some males act as pioneer beetles that search for the most suitable (weakened) host.", "The random selection of the initial population is a common practice of the application of the metaheuristics.", "In this implementation the initial population is generated by means of Random Sampling Technique (RST) (Kallioras et al. [15]).", "In this step of the algorithm, the first beetle brood (gallery/colony) is generated randomly into the search space (first generation).", "In general, three to six broods are created (composed of Npop pioneer beetles each).", "Once all new broods are created, a host selection pattern is decided for each new brood.", "All newly-emerged beetles will fly inside the search space looking for a better solution (host tree, preferably a weakened one) in order to create their own brood.", "Based on the behaviour of the beetle described previously, five types of host selection pattern are implemented into the proposed algorithm: (i) neighbouring search flight, (ii) mid-scale search flight, (iii) large-scale search flight, (iv) global-scale search flight and (v) memory consideration search flight where the best positions found so far are used.", "The analytical description of their algorithmic implementation can be found in (Kallioras et al. [15]).", "According to the host selection pattern chosen, a search area is created around the birth position of the beetles.", "For each pattern the definition of this area is implemented using a properly selected pattern factor (fpat) and represents a parameter of PBA.", "According to every pattern, Npop new pioneer beetles are randomly positioned into this search area by means of RST.", "In the last step the location of the broods (location of mating males and females) are updated and the past ones are dropped, except those stored in memory.", "In particular, all previous broods are extinct and the new locations become birth-places for the new generations.", "Flight patterns are selected for the newborns.", "This procedure is repeated until the termination criterion of PBA is satisfied.", "The DIvide a hyperRECTangle optimization algorithm (Jones et al. [14]) was presented as an extension of Lipschitzian optimization [26] to derivative-free optimization problems.", "The DIRECT sampling algorithm consists of three basic steps: initialization, identify and divide potentially optimal hyper-rectangles.", "Initially a transformation of the problem's search domain into a normalized unit hyper-cube space is performed.", "Reference to the original search space is required only when function evaluation calls are performed.", "Multilevel coordinate search algorithm (Huyer and Neumaier [12]) was inspired by the DIRECT algorithm and based on multilevel coordinate search partitions the search space into hyper-rectangles with one evaluated base point.", "Contrary to DIRECT, MCS algorithm allows base points anywhere in the hyper-rectangles.", "Global-local search based on balanced multilevel approach is performed, where a level s is assigned to every hyper-rectangle defined as an increasing function of the number of times the hyper-rectangle has been split.", "Those with level s equal to smax are considered too small to be further split.", "The numerical investigation is composed by four parts, in the first one the trading strategy is calibrated based on three search algorithms, all belonging to the derivative-free optimization type of algorithms.", "In the second part the optimized strategies resulted from the first part are tested.", "In the third part invest portfolios are built while in the last one the constructed portfolios are compared with Benchmarks.", "The main objective for this part of the study is to identify the most suitable values of the four parameters, in order to calibrate the proposed trading strategy model for each pair of currencies.", "The calibration of the trading strategy model is performed over the first 24,000 elements for each currency pair, aiming to find the values of the parameters that produce the highest total return.", "The design bounds of the four parameters were set as follow:", "Size ∈ [5, 25]", "X ∈ [50, 200]", "Y ∈ [1, 49]", "D ∈ [1, 25]", "The performance of the search algorithms is influenced by the values of their control parameters; however, their selection is not a straightforward procedure.", "In the current study the values proposed by the developers of the three algorithms are used.", "More specifically for the case of PBA search algorithm, although it is not possible to define specific values for its algorithmic parameters that will be the proper ones for all test examples considered, the values that were used in the current study were found to be the proper ones as a balance between robustness and computational efficiency out of multiple numerical tests performed by the authors (Kallioras et al. [15]).", "The results obtained using the three optimization algorithms for each currency pair are depicted in Table 1.", "As it can be observed, the total returns of the currency pairs in the optimization level are significantly high.", "This fact provides a first evidence of the value the proposed trading strategy.", "The parameters corresponding to the highest total return for each pair will be the ones that will be used for the testing period of the 7 next years.", "Indicatively, for the GBP/USD pair among the three optimization algorithms the highest total return was obtained by PBA.", "The best total return that was achieved is equal to 280.84% with maximum drawdown equal to 35.83% and the corresponding parameters were: Size = 12 (pips), X = 138, Y = 13 and D = 2.", "Accordingly, for the USD/JPY pair among the three optimization algorithms the highest total return was achieved by DIRECT.", "The optimum total return achieved for this pair is equal to 226.59% with maximum drawdown equal to 30.00% and the corresponding parameters were: Size = 9 (pips), X = 178, Y = 17 and D = 2.", "Summarizing the results, although MCS achieved the best total return for four out of eight currency pairs, DIRECT for three out of eight ones and PBA for one out of eight ones, as shown in Table 1 all three algorithms achieved rather similar results concerning the value of the total return, establishing that the research is statistically significant.", "In previous section, it was stated that a strategy in order to be mostly profitable and valuable in a long-term basis should be adapted to the market conditions.", "In this direction, the MRB were developed that aim to make the proposed trading strategy more sustainable.", "To check if the proposed trading tool succeeded its purpose, at the optimization process we follow the exact same procedure to the same channel breakout strategy but without this time the application of the MRB.", "If the optimum performance of each pair for the 4 years (optimization period data) is better when the channel strategy is applied to the MRB than it does not, then the importance of the developed trading tool is confirmed.", "As it can be observed in Table 2, the MRB-based channel strategy provides steadily higher performance for the most of the FX pairs than the strategy without the use of the MRB.", "Specifically, the use of MRBs provides an edge in all the cases apart from USD/CAD.", "Worth mentioning also that the improvement of the total return in the cases of GBP/JPY, AUD/USD and GBP/USD is outstanding high.", "Additionally, it can be observed through Table 3 that with reference to the total return of the FX pairs, the average improvement achieved when applying the trading tool of MRBs was about 30%.", "The values of the design parameters that were identified in the first part of the numerical investigation are subsequently used in order to check the quality of the optimization process and the profitability of the developed strategy in the future (i.e. during the next seven years).", "Table 4 represents the testing results for each currency pair based on the calibration of the trading strategy model achieved through the optimization stage.", "In particular, Table 4 provides the total return and the maximum drawdown for each of the eight pairs.", "The tests were conducted at a yearly basis for the period 2010 to 2016 and each raw denotes the values of the total return and the maximum drawdown for the particular pair for a specific year.", "Table 5 shows the evaluation measures that are used to assess the total quality of the testing process for each FX pair; specifically, Total Sum, Arithmetic average, Geometric return, Standard deviation and Sharpe ratio are employed.", "For example, for the USD/JPY pair the total return observed for the 7-year period is equal to 74.86% with an arithmetic average of 10.69%, but the relatively high standard deviation of 36.05 provides geometric return of 4.23% and sharpe ratio of 0.23.", "For the USD/CAD also a negative total return is observed for the 7-year period that is equal to −29.10% with an arithmetic average of −4.16%, the relatively high standard deviation of 27.85 results into a geometric return of −7.17% and a negative sharpe ratio of −0.24.", "Moreover, for the GBP/JPY an extremely high total return was attained for the 7-year period that is equal to 217.59% with an arithmetic average of 31.08%.", "The high standard deviation of 36.35 provides a geometric return of 26.24% and a very positive sharpe ratio of 0.79.", "Table 5 depicts the values of total sum (%), arithmetic average, geometric return, standard deviation and sharpe ratio for each currency pair.", "As it can be observed from this comparative study some of the pairs performed very well and some had rather poor performance.", "As a first attempt, in order to eliminate poor performances that were related to the nature of each pair and to minimize the risk, it was decided to construct a portfolio formed by the eight currency pairs contributing with equal percentages.", "It was mentioned previously this is a simple average portfolio, where all currency pairs have exactly the same percentage in the allocation chosen that is equal to 1/8 (i.e. participation of every pair by 12.50%) and will be referenced as ``Equally Weighted'' portfolio and denoted below as EWP1.", "In order to further improve the constructed portfolio's statistics, it was decided to rely mainly on the pairs that performed better.", "In order to succeed that, the Kelly Criterion was used in order to evaluate the quality of the annual performance of each currency pair, the formulated portfolio is labelled as ``Kelly Criterion'' one and will denoted below as KCP1.", "Table 6 shows the percentages that each currency pair constitutes in Kelly Criterion portfolio for every particular year.", "All other proportions emerge from the data of the precious year.", "Specifically, the percentage of a pair in the given year is equal to its Kelly number obtained from the previous year divided by the sum of the Kelly numbers of all currency pairs for the previous year.", "In case that during the previous year a currency pair resulted into a negative Kelly number, then its percentage for the next year becomes equal to zero.", "Furthermore, the weight coefficients of each pair of the first year are equal since there are no data available for year 2009.", "The annual returns for the two constructed portfolios that are composed by the eight currency pairs are presented in Tables 7 and 8 that contain also the evaluation measure used in order to assess the performance of the two constructed portfolios.", "In the calculations presented herein an average free-risk rate equal to 2.5% was used.", "Table 8 provides the values of total sum (%), arithmetic average, geometric return, standard deviation and sharpe ratio both for EWP1 and KCP1 portfolios.", "As it can be observed in Table 8, EWP1 portfolio has a 7-year total return of 58.19% with an arithmetic average of 8.31% and a rather low standard deviation of 12.19, which results into a geometric return of 7.69% and a positive sharpe ratio of 0.47.", "As it can be seen in Table 8, KCP1 portfolio presents a significantly better performance compared to EWP1 portfolio.", "It has a 7-year total return of 98.45% with an arithmetic average of 14.06%, the standard deviation is equal to 16.6, which provides a geometric return of 10.54% and a higher sharpe ratio value equal to 0.71.", "Aiming to improve the performance of the two portfolios, the results of the above-described comparative optimization study were exploited.", "In particular, the value of the average total return was calculated based on the total return of the three optimization algorithms for each currency pair.", "It was observed that the pairs having average total return lower than the threshold value of 200% had negative performance in the testing part.", "This performance can be somehow justified, since, if a currency pair cannot generate high total returns in ideal conditions (i.e. over the period that was calibrated), then during the testing part is not expected to perform well.", "The currency pairs that had an average total return greater than 200% were GBP/USD, USD/JPY, EUR/USD, EUR/JPY and GBP/JPY.", "Based on this assumption, the proposed strategy is not suitable for NZD/USD, AUD/USD and USD/CAD pairs.", "Thus, it was decided to construct two new portfolios, a second Equally Weighted one (EWP2) and one based on Kelly Criterion (KCP2), using only the five pairs that responded better during the optimization stage.", "The structure of these portfolios is the same with the original ones, i.e. with the Equally Weighted (EWP1) and Kelly Criterion (KCP1) portfolios, varying only on the number of currency pairs, the new ones contain only five out of the eight currency pairs (i.e. GBP/USD, USD/JPY, EUR/USD, EUR/JPY and GBP/JPY).", "The difference from the original EWP1 portfolio is the value of the weight coefficients; since it is composed by five currency pairs only, the allocation chosen is equal to 1/5 (i.e. participation of 20%).", "Table 9 shows the annual proportions of each currency pair for the second Kelly Criterion portfolio.", "Similar to KCP1 the weight coefficients for each currency pair of the first year are equal since no data are available from year 2009 and the other percentages emerge from the data of the precious year.", "The only difference with KCP1 is that the new one contains only the pairs that succeeded an average total return above the threshold value of than 200% during the calibration procedure.", "The annual returns for both new portfolios that have been constructed i.e. EWP2 and KCP2 is presented in Table 10, for six out of the seven years both portfolios were improved compared to the original ones.", "Table 11 contains the evaluation measures that are used in order to assess the performance of the two new portfolios.", "An average free-risk rate equal to 2.5% is also used for the calculations.", "As it can be seen in Table 11, the new equally weighted portfolio outperformed the original one.", "Specifically, it has a total return of 141.60% with an arithmetic average of 20.23%, and a standard deviation of 21.77 that provides a geometric return of 18.47% and a positive sharpe ratio of 0.83.", "The new KC portfolio presents impressive results having a total return almost twice that of the original Kelly based portfolio.", "As it can be observed from the results shown in Table 11, it has a 7-year total return of 178.34% with an arithmetic average of 25.48%, and a standard deviation of 20.42 that provides a geometric return of 23.96% and significantly high positive sharpe ratio of 1.14.", "Another noteworthy issue is that this portfolio presents only one negative year (2015 return −4.47%).", "This means that KCP2 portfolio is characterized from a high stability in its performance.", "A measure for an investor to choose a specific portfolio over another is its performance compared to its benchmarks.", "If the portfolio has the same or poorer returns than its benchmarks then it is more rational for the investor to choose the benchmarks itself.", "The benchmarks contain larger variety of securities than a portfolio.", "Hence, a benchmark resembles a well-diversified portfolio that reduces the unsystematic risk from the specific asset, maintaining only the systematic or market risk that cannot be eliminated.", "Therefore, it can be stated that when a specific portfolio cannot generate returns higher than its benchmarks, the later ones is a better alternative solution mainly due to their lower risk.", "As Markowitz [19] supported in his book ``Modern Portfolio Theory'' that a rational investor will not invest in a portfolio, if a second portfolio exists with a more favourable risk-expected return profile.", "However, currency cannot be considered as part of a ``default market'', making the formation of currency benchmarks a challenging issue.", "Currencies were always traded in pairs; therefore, if a manager or trader is long in EUR/USD i.e. buys Euros and sell U.S. dollars.", "Investing in currencies represent an active investment decision.", "Every trade leads to be a relative value trade.", "As a result, there is no real natural market portfolio to measure and capture foreign exchange beta.", "In order to further emphasize on the significance of the results obtained, it was decided to compare the performance of the four portfolios constructed in this study with that of benchmarks that are considered as fundamentals for the investors; i.e. the Standard & Poor's 500, Barclay CTA Index, Barclay BTOP FX Index, Barclay Currency Traders Index and Barclay Systematic Traders Index benchmarks.", "The Standard & Poor's 500, often abbreviated as the S&P 500, is an American stock market index based on the market capitalizations of the 500 large companies having common stock listed on the NYSE or NASDAQ.", "Although, the specific benchmark is not directly related to the constructed portfolios since it is constituted by stocks, it was decided to use it, because is the most popular benchmark in the world of finance and it succeeds constantly high returns making the comparison with the constructed portfolios more challenging.", "The Barclay CTA Index is a leading industry benchmark of representative performance of more than 500 commodity trading advisors, while an advisor must have four years of prior performance history.", "The Barclay BTOP FX Index seeks to replicate the overall composition of the currency sector of the managed futures industry with regard to trading style and overall market exposure.", "The BTOP FX Index employs a top-down approach in selecting its constituents.", "The largest investable currency trading programs, as measured by assets under management, are selected for inclusion in the BTOP FX Index.", "The Barclay Currency Traders Index is an equal weighted composite of managed programs that trade currency futures and/or cash forwards in the interbank market.", "It contains more than 50 currency programs.", "The Barclay Systematic Traders Index is an equal weighted composite of managed programs whose approach is at least 95% systematic.", "It contains more than 300 systematic programs.", "Table 12 shows the yearly returns of the five benchmarks for the period 2010–2016, where it can be noticed that during this period (2010–2016) only S&P500 and Barclay Currency Traders Index have positive returns for every year, showing that both have a stable performance with respect to the time.", "Table 13 presents the evaluation measures that were used to assess the performance of the five benchmarks.", "The comparison was focused on the same metrics used for the constructed portfolios in order to make results directly comparable.", "As it can be observed through Table 13, S&P500 seems to have the best performance out of all benchmarks that were selected.", "It outperforms the three out of the four Barclay's indexes with reference to the total sum, arithmetic average, geometric return and sharpe ratio.", "The standard deviation is the only disadvantages for S&P500.", "It has an almost double standard deviation value compared to Barclay CTA Index, Barclay BTOP FX Index and Barclay Systematic Traders Index, worth mentioning that Barclay Currency Traders Index attains an extremely low standard deviation value equal to 1.33%.", "Among the four Barclay's indexes, Currency Traders Index is the only one that provides a positive sharpe ratio.", "Furthermore, it presents better performance than the other three indexes in all the other measures.", "Through the assessment process, the constructed portfolios were compared to S&P500 and Barclay Currency Traders Index only that proved to be the most competitive benchmarks.", "In particular, comparing Tables 7 and 10 with Table 12 for a year-per-year basis, it can be seen that EWP1 portfolio outperforms S&P500 for three out of the seven years and Barclay Currency Traders Index for four out of the seven years, while EWP2 portfolio outplays both benchmarks for five out of the seven years.", "Concerning the KCP1 portfolio, it succeeds higher returns than both benchmarks, outperforming both benchmarks for four out of the seven years and the KCP2 portfolio also outperforms S&P500 for four out of the seven years while for the case of Barclay Currency Traders Index for six out of the seven years KCP2 has better returns.", "However, comparing Tables 8 and 11 with Table 13, it can be seen that the evaluation measures provides a more detailed overview concerning the quality of the portfolios constructed in this study.", "The four constructed portfolio outperformed the Barclay Currency Traders Index with enormous difference with respect to the total sum, arithmetic average, geometric return and sharpe ratio.", "Specifically, the geometric return and sharpe ratio of KCP2 portfolio exceed the Barclay Currency Trader Index's by 924% and 418%, respectively.", "Only EWP1 portfolio failed to beat S&P500 in terms of the total sum.", "Although, the true advantage of the S&P500 is its sharpe ratio, KCP2 portfolio achieved a positive sharpe ratio of 1.14 outperforming by almost 10% the corresponding value of the S&P500 benchmark.", "Moreover, the KCP2 portfolio seems to achieve better performance with respect to all measures compared to both benchmarks.", "The only drawdown of the constructed portfolios is that they depict relatively higher standard deviation values from all selected benchmarks.", "For further elaborating on the significance of this study, the performance of the proposed MRB channel strategy system when applied to the best (derived from the optimization process) currency pairs (GBP/USD, USD/JPY, EUR/USD, GBP/JPY and EUR/JPY) is compared to the buy-and-hold (BaH) strategy.", "According to the BaH strategy a pair is bought at the beginning of the year and is held until the end of the year.", "As it can be seen in Table 14, the proposed trading strategy outperforms the BaH one in the four of the five pairs.", "Only for the case of the USD/JPY pair MRB fails to beat the BaH returns achieving, however, significant profits.", "Worth mentioning that the MRB channel strategy generates better results even for the absolute value of the returns of the buy-and-hold (assuming that a sell-and-hold approach is followed).", "The major achievement of this study was the construction of a portfolio having steadily profitable performance.", "In order to achieve this target, a friendlier and more adaptable to market conditions trading tool was developed first.", "In particular, the Modified Renko Bars (MRBs) were proposed in this study that comply much better with the market's movement and represent more accurately its true directions than the simple or the common renko bars.", "Thus, based on MRB charts a Channel breakout strategy was implemented.", "Subsequently, it was proved that in order to formulate a profitable strategy an optimization phase is necessary to be performed first.", "The optimization phase, which was carried out over a 4-year period, helped us to calibrate the parameters of the trading strategy for eight currency pairs.", "For the requirements of the optimization phase, three derivative-free algorithms were employed aiming to identify the parameters that achieve the highest total return for each currency pair; the parameters that develop the highest result among the three algorithms were chosen to be used in the testing and portfolios construction phases.", "Afterwards, the optimized parameters obtained for each pair were tested over a 7-year period.", "The results obtained, especially, for five out of the eight currency pairs were found to be impressive.", "Specifically, when a threshold value of 200% average total return for the three optimization algorithms was set for each currency pair, it was observed that five out of the eight currency pairs resulted into average total return greater than the 200% threshold value.", "This observation indicates that this kind of strategy will not fit smoothly to the three pairs that failed to achieve 200% average total return.", "Subsequently, two couples of portfolios were constructed using equally weighted proportions and based on the Kelly criterion.", "The first group was constructed using the eight currency pairs (the two portfolios of the group were denoted as EWP1 and KCP1) and the second one using the five best pairs (distinguished according to the 200% threshold value principle, and the two portfolios of the group were denoted as EWP2 and KCP2).", "Relying on the five best currency pairs, the sharpe ratio of both portfolios was improved when compared to the original ones (i.e. EWP1 and KCP1), while the total returns of the simple average portfolio (EWP2) was increased by 143% compared to EWP1 and that of the KCP2 by 66% with reference to KCP1.", "Afterwards, the portfolios constructed were compared with well-known benchmarks.", "In particular, comparing the 7-year performance of the improved Kelly Criterion portfolio (KCP2) with the corresponding one of the S&P500, Barclay CTA Index, Barclay BTOP FX Index, Barclay Currency Traders Index and Barclay Systematic Traders Index benchmarks it was observed that KCP2 outperformed the S&P500’s total return by 92.6% and that of the Barclay Currency Traders Index by 900%, the rest of the benchmarks had rather poor to very poor performances.", "In general, it can be stated that the four portfolios constructed in this study performed extremely well alongside all benchmarks selected, succeeding remarkably better results in most of the cases.", "The only disadvantage of the constructed portfolios is the higher standard deviation that is observed; however it can be justified by the larger and better diversification in terms of securities that is available by the benchmarks that leads to lower risk and mildest fluctuation.", "The development of the proposed trading system (the optimization process included) and the construction of the two portfolios derived, followed a completely systematic approach that is applicable to any financial instrument.", "A major advantage of the proposed investment philosophy is that it can be operated entirely by a computer generating the trading signals automatically.", "There is no need for investor's intervention in the trading procedure since it is strictly defined, while it composed by no ``black box'' parts.", "Worth mentioning also that, even in the case of excluding some currency pairs from the constructed portfolios due to their poor performance, the procedure followed, was also strictly mathematical and easy to be operated automatically.", "It consists from the comparison of the performance (i.e. total return achieved from the optimization process) part between relative financial instruments to their average.", "The instruments that achieve performance above average will participate on the constructed portfolios and the ones below will be excluded.", "Future work will be focused mainly on the reduction of the standard deviation of the constructed portfolios and further improvement of their performance.", "In order to achieve these objectives, machine learning techniques will be applied, trying to evaluate the quality of every produced signal from the proposed strategy and recognize those that will be more likely to end at a losing trade." ]

[ "Development of a new trading tool: the Modified Renko Bar (MRB).", "We prove that the application of MRB in eight currency pairs increases the performance of channel breakout trading strategy by an average 30%.", "Calibration of the parameters of the MRB channel strategy based on three optimization algorithms and analysis of the results.", "Construction of two types of currency portfolio: an equally weighted one and a portfolio based on Kelly Criterion.", "We succeed to beat fundamental benchmarks such as Standard & Poor's 500 and Barclay CTA Index." ]

[ "ENER" ]

[ "Case study", "Electricity balancing", "Forecast error time series", "Market analysis", "Variable renewable electricity" ]

[ "2101" ]

[ "The increasing share of renewable electricity (RES-E) deployment required to reach the sustainability targets stated by the European Union will have increasing impacts on the functioning of electricity markets across all Member States.", "The envisaged transformation is likely to require numerous regulatory adjustments and the implementation of new electricity market design elements to efficiently integrate these RES-E shares into markets and grids.", "In the view of upcoming challenges posed by the ambitious climate policy the European Commission (EC) recognized the need for action and established the concept of the Energy Union.", "In November 2016 the Commission published a communication concerning a policy package related to the newly developed concept of the Energy Union entitled “Clean Energy For All Europeans”.1", "This so-called “Winter package” incorporates among many others proposed reforms of existing directives as well as guidelines tackling issues across the board, e.g. proposals for the revision of wholesale and retail electricity market design, energy efficiency and renewable energy policies and governance issues.", "In particular, therein the EC acknowledges that “… short-term electricity markets which allow trading RES-E across borders are key for the successful integration of RES-E into the market”.2", "The short-term electricity markets, i.e. day-ahead, intra-day and balancing markets, shall provide non-discriminatory access to all technically capable technologies, should allow for cross-border trading as close as possible to real time.", "A successful integration of RES-E does also call for common rules and standards for the operation of power grids in Europe.", "The development of such rules has been initiated by the Agency for the Cooperation of Energy Regulators (ACER) that established guidelines to which these common rules and standards need to comply with.", "Based on these guidelines the European Network of Transmission System Operators for Electricity (ENTSO-E) drafted eight network codes with the aim to enable the implementation of the EU internal electricity market.", "These network codes have the legal status of directives.", "The network codes on Capacity Allocation and Congestion Management (CACM) and Electricity Balancing (EB)3 concern the design and operation of short-term electricity markets across EU Member States and thus provide a frame under which potential developments of these markets can be studied.", "However, at the time this text has been written it was still not clear how the network codes and proposed directives of the Winter package will exactly be translated into national legislation, rather than whether consensus and acceptance will be reached among Member States and stakeholders about the content of the proposals.", "Therefore, the future framework conditions related to electricity market design and accompanying regulations are subject to considerable uncertainty.", "In this context the current paper aims to contribute to developing an understanding of relevant future drivers of short-term electricity markets across Europe through the application of a large-scale European power-system model.", "In particular, the aim of this paper is to assess the future monetary relevance of balancing markets across the EU.", "In the European context the term electricity balancing market refers in its general form to all activities of transmission system operators (TSO's) related to their obligation to ensure an efficient and reliable real-time settlement of active power imbalances in their networks.", "In many countries, the concrete implementation of these activities historically evolved around the specific characteristics of system imbalances and the type of available generation facilities in their national power systems.", "Therefore, a great variety in the design and organization of balancing mechanisms and markets across Europe exists today.", "In the following, it is assumed that all kinds of balancing mechanisms in Europe converge towards energy-only real-time markets, which are operated in the time-frame of 5 min.", "We further do not differentiate between various qualities of imbalances with regard to time frame.", "We assume that all imbalances that remain after gate-closure of intra-day markets are settled in balancing markets.", "In this paper, the importance of this market as compared to day-ahead markets is assessed through the model-based analysis of potential revenue streams from both market segments under different framework conditions.", "In particular, the following research questions should be answered:", "How big is the overall market volume of balancing markets and how relevant are revenues from balancing markets for different generation technologies across EU countries in future years?", "How do currently ongoing market design trends impact these results?", "What is the benefit of market integration (i.e. cross-border participation and imbalance netting) of balancing markets within the EU internal market?", "The results of this analysis contribute to the ongoing debate about interactions of RES policy and electricity market design and the efficiency gains of market integration.", "Furthermore the results provide next to the impact assessment accompanying the EU winter package an independent estimate on the future value of flexibility and its importance for revenues of different supply technologies.", "There is plenty of literature studying electricity balancing markets across Europe from a national perspective, see e.g. Refs. [1–6].", "This is completely justified, since todays markets are merely national with a low level of international cooperation.", "Some studies focus on the impact of regional cooperation, e.g. Refs. [7,8].", "However, concerning the impact of an European-wide market coupling of balancing markets there is currently only a limited amount of literature.", "The most prominent studies have been launched by the European Commission with the aim to assess potential benefits of different design options for cooperation mechanisms between national balancing markets.", "In Ref. [9] the authors explore the advantages and disadvantages of different market design options and underpin their argumentation with a quantitative analysis relying on historical market data from 2011.", "In Ref. [10] this analysis has been fundamentally updated and the time scope has been put to the period up to 2030.", "Both impact assessments have been prepared with the intention to support decision making of European policy makers and therefore are written in a very concise form that lacks transparency concerning the concrete assumptions that were taken.", "In the scientific literature there are a number of articles concerning the 2030 time frame.", "For example [11], presents a methodology to assess the impact of different market arrangements on overall costs and benefits of electricity balancing markets.", "In this paper, the authors simply scale up historical demand for balancing energy in order to project it into the future [12].", "offers a method to assess the amount of reduced costs that might result from imbalance netting on basis of a small scale example.", "Both papers are concerned with the question of how different market design elements and internationalization of markets could impact the overall costs of electricity balancing.", "One main contribution of this work is the development of high-frequency balancing demand time series that match the characteristics of a certain future generation mix.", "Subsequently, these series are feed into a large-scale electricity market model in order to get numerical estimates on how balancing markets in major European countries might evolve in the 2030 time frame.", "We analyze the general relevance of these markets by showing how large physical and monetary volumes of these markets might become by 2030 as compared to overall electricity consumption and day-ahead volumes, respectively.", "Furthermore, we analyze the specific relevance of these markets for different types of generation technologies.", "We show the results as share of revenues from balancing markets as compared to day-ahead market revenues.", "A main contribution of this work along these objectives is that the relation between increasing shares of variable renewable electricity (vRES) in the European generation mix and the resulting demand for balancing energy has been explicitly worked out by the application of statistical models.", "Although the suitability of such models for this purpose has already been shown in the literature, cf [13].", "or [14], to the best knowledge of the authors no comprehensive and consistent quantification of the size and form of high-frequency forecast error time series for different generation technologies and all major European countries have been carried out so far for the time frame until 2030.", "We apply the electricity market model HiREPS (High Resolution Power System) developed at the Energy Economics Group of the Technische Universität Wien [15] to bring together supply, the transmission grid and demand in a way that electricity market prices for different market segments and scenarios can be derived.", "HiREPS is a large-scale mixed integer optimization model4 covering the electricity and heat sector in all EU Member States and in addition Norway, Switzerland, the Western Balkan countries, North Africa and Turkey.", "The model covers both the electricity and the heat sector.", "Thus, on the supply-side the model contains detailed techno-economic data on electricity-only, heat-only and combined-heat-and-power plants for all countries within Europe.", "Technologies are grouped by efficiency and fuel type.", "Important technical restrictions of the supply-side, e.g. minimum load, minimum on-/off-time and ramping rates, as well as startup costs are implemented in the form of classical unit-commitment modeling approaches presented in the literature, e.g. Ref. [16].", "Hydro units are aggregated by country and are grouped into run-of-river, hydro storage and pumped hydro storage plants.", "Hydro units have been implemented with exogenous natural inflow time series and corresponding hydro reservoirs by making use of a linearized representation of storages.", "A schematic overview on the model structure is given in Fig. 1.", "The model needs a comprehensive set of framework parameters that can be grouped into assumptions concerning the supply side, demand side and transmission representation of the model.", "The installed generation capacity of conventional power plants in 2030 is based on forecasts of the European transmission system operators [17].", "In addition, the model allows for additional investments in efficient gas turbines.", "With regard to Europeans long-term environmental targets investments in other fossil generation technologies have not been allowed.", "Also additional investments in storages are not allowed in the model.", "This assumption might be justified to some extent with regard to large-scale hydro power units.", "With regard to batteries and other small-scale storages it appears to be very conservative.", "To a certain extent small-scale batteries in households and electric cars have been already considered via a change of the hourly load curve.", "However, although we did not assume that these batteries participate in balancing markets we argue that additional flexible supply would simply downsize the monetary volume of short-term markets.", "Thus, the results of this analysis can be interpreted as conservative, upper limit with regard to the relevance of this market segment.", "The amount of RES generation is also exogenous to the model and are based on the results of the EU-funded project Towards2030.5", "In the scenario used for this paper, it has been assumed that the EU reach its overall RES target of 30% on gross final energy consumption in 2030 in a cost-efficient way.", "The installed capacities for renewable electricity have been derived from this scenario and are fixed as well.", "The hourly availability of renewable generating capacity as well as hourly load in 2030 has been taken from the model input parameters applied in the above mentioned project Towards2030.", "In the model a simplified representation of the European transmission grid is used to accurately calculate physical power flows across Europe.", "It has been assumed that the concept of flow-based market coupling as already implemented in some countries has been applied to the whole modeled region by 2030.", "All these assumptions are documented in more detail in Ref. [18].", "The additional input assumptions that are of specific importance for the research question posed in this paper concern demand in intra-day and electricity balancing markets.", "First, historical quarter-hourly day-ahead forecast errors time series of load, wind and PV on country-level and for the year 2016 have been collected from the ENTSO-E transparency platform.6", "Each of these three time series have been further interpolated7 to a 5-min time resolution and normalized to their peak value.", "These series were used to fit Autoregressive Integrated Moving Average (ARIMAX) models, whereas normalized total generation of wind and PV as well as normalized load has been used as explanatory variables.", "These models have been fitted with the normalized renewable generation and load assumed for the year 2030 and rescaled with actual capacity and peak load in order to derive forecast error time series that are adjusted for the new generation mix.", "Unplanned outages of conventional generation units have been simulated by Monte-Carlo analysis based on statistical outage data.", "Each of these time series were added to the corresponding day-ahead generation and load profiles.", "The so derived time series have been assumed to be the total amount of unplanned deviations from day-ahead schedules that are required to be balanced in intra-day and balancing markets.", "In order to differentiate which share of each profile can be balanced in intra-day markets the authors used forecast quality curves showing the decline of normalized root mean square error (RMSE) of generation and load forecasts for wind, PV and load subject to the forecast horizon ranging between 24-h to 1-h ahead of delivery [19,20].", "The total forecast error curve have been downscaled until the RMSE value at 1-h ahead of delivery have been reached.", "This share of the curve has been subtracted from the total curve in order to extract the share to be balanced in intra-day markets.", "A more detailed description of the applied methodology is given in Appendix A.", "Fig. 2 shows a detailed description of the model runs undertaken and corresponding assumptions in order to simulate balancing markets.", "In order to derive the desired results the HiREPS model has been first used to simulate day-ahead market prices over 1 year based on day-ahead input profiles.", "In a second run prices for intra-day markets have been simulated, whereas the day-ahead profiles have been adjusted by the corresponding share of forecast errors to be balanced in intra-day markets as described above.", "Third, the model have been again applied to simulate electricity balancing prices by again adding the remaining change of unplanned deviations to the time series of wind, PV and load.", "With regard to unplanned outages of conventional units it has been assumed that only the first hour of an unplanned outages needs to be balanced in the balancing market, leaving the remaining hours for the intra-day model runs carried out beforehand.", "In the model run for the balancing market much stricter restrictions on the flexibility of generation units have been assumed than in the model runs beforehand, because the time resolution have been increased to 5 min.", "The model results enable the authors to assess the future monetary relevance of balancing markets across selected European countries under different assumptions of framework conditions.", "In that way, it is possible to assess the benefit of market integration (i.e. cross-border participation and imbalance netting) of balancing markets within the EU internal market and to quantify for different technologies the share of revenues from balancing markets on their total revenues.", "The policy framework concerning the EU internal electricity market comprising all market segments is under constant revision.", "It is therefore at the time of writing not possible to take any harmonized market design and set of regulations as given in the 2030 time frame.", "The eight drafted network codes and guidelines of ENTSO-E as well as the statements of the European Commission can serve as an orientation towards the envisaged target model in Europe.", "However, at the time of writing it is not clear how the network codes and proposed directives of the existing policy proposals and guidelines will exactly be translated into national legislation and whether consensus and acceptance will be reached among Member States and stakeholders about the concrete content of the proposals at all.", "Therefore, the future framework conditions related to electricity market design and accompanying regulations are subject to considerable uncertainty.", "In this paper we deal with these uncertainties through the analysis of four scenarios.", "Table gives an overview on the simulated scenarios.", "They are grouped into two categories, one concerning short-term electricity markets ahead of the balancing market and the other the balancing market itself.", "Under short-term electricity markets ahead of balancing we understand efficient day-ahead markets and intra-day auctions which are held one hour-ahead of physical delivery.", "In the modeling we assume that in the more international (Mi) perspective that interconnections are extended until 2030 in accordance with the TYNDP of ENTSO-E and that all countries run Energy-Only markets.", "In the more nationalistic (Mn) perspective we assume that 30% of all planned international transmission line projects of the TYNDP are delayed and therefore are not available in 2030.", "Furthermore, we assume that in all countries in addition to Energy-Only markets some form of capacity mechanism is implemented that accounts for missing money of generators from those markets.", "Consequently, no scarcity prices occur in the respective scenarios.", "The resulting day-ahead prices are therefore lower (cf. Fig. 3).", "This means that the price-increasing effect of missing scarcity prices outweights the price-decreasing effect of transmission expansion.", "The second category of scenarios concern the design of electricity balancing markets.", "In line with efforts to open electricity markets for international competition, also TSOs were searching for possibilities to increase the efficiency of balancing markets.", "International cooperation has proven to be a fruitful option to decrease costs.", "In order to reflect the uncertainty towards the level of market coupling in 2030, two extreme assumptions have been taken.", "First, it is assumed that balancing markets are solely operated on a national level (N) and no kind of cooperation takes place.", "On the contrary, in a second assumption all TSOs across Europe cooperate on the basis of a fully integrated European balancing market (I), i.e. both imbalance netting and cross-border activation of balancing reserves.", "Both perspectives together form the four scenarios shown in Table 1.", "In section 3 it has already been outlined how electricity prices in day-ahead, intra-day and balancing markets have been simulated through the application of the electricity market model HiREPS.", "In order to model different market segments we collected historical time series for day-ahead forecasts, hour-ahead forecasts and actual data for renewable electricity generation and electricity consumption respectively.", "For each of these three sets of time series we run the model and interpreted the modeled marginal system cost of one additional unit of demand as a proxy for market prices.", "That is, the price for balancing electricity has been interpreted as the price that resulted from the model run with the time series of actual generation and load.", "In order to derive revenues and costs from that market segment we need to price deviations between forecasts and actual values of generation and load.", "We therefore shortly outline some definitions.", "The sum of unintended deviations of all generators plus deviations from planned load between hour-ahead forecasts and real-time has been considered to be the net balancing position of the overall control zone.", "If this balancing position is positive, we call the control zone oversupplied, if it is negative we call it undersupplied.", "There are different methods how to design and implement electricity balancing markets [21].", "In this paper we assume a single-price balancing market design in which deviations of market actors that are in the same direction as the deviation of the overall control zone are penalized with the market price and deviations in the opposite direction are paid to actors.", "That is, if the control zone is oversupplied all generators which have a higher output than planned have to pay the market price and all generators which have a lower output than in their forecast are paid that price.", "The same holds for deviations of load, but in inverse logic.", "We do not differentiate between unintended and intended deviations in form of counter-balancing, because with regard to revenues and costs it does not matter for what reason a deviation has happened.", "Renewable electricity generators can benefit from that, because it case an unintended deviation turns out to be in opposite direction to the overall system imbalance they earn an additional profit.", "The starting point of our discussion is the resulting generation mix and the average day-ahead market prices shown in Fig. 3.", "For each country two columns are shown.", "The left column represents the Mi-scenarios and the right column the Mn-scenarios from Table 1.", "Since only day-ahead market results are shown, the balancing market design do not impact these results.", "It can be seen that the generation mixes in the countries of interest are not significantly impacted by the assumed scenario assumptions.", "However, day-ahead electricity prices significantly differ in both scenarios, mostly due to missing scarcity prices in the Mn-scenarios.", "The share of renewable generation on gross electricity demand differ between countries and is the highest in Austria, Slovenia and Spain.", "The export of some countries in Fig. 3 flows into other European countries not shown in this Figure.", "They are not shown in this graph because their electricity balancing markets have not been assessed.", "The results of the day-ahead market clearing do reflect two possible worlds of day-ahead market development in Europe in 2030 and provide the basis for our assessment of the relevance of electricity balancing markets.", "All of the technologies in the generation mix as well as the consumption are sources of forecast errors.", "The characteristic of historical forecast errors in Europe have been projected to 2030 via the application of ARIMAX models.", "The results of this tasks are shown in Fig. 4.", "In order to assess the relative importance of each of these sources, the resulting energy content for each of the sources that together form the overall demand of balancing electricity, as well as the netted sum of forecast errors and unplanned outages are shown.", "In order to enable a cross-country comparison all numbers are displayed as share of gross electricity consumption.", "Due to netting effects the overall sum of all sources is smaller than the sum of energy contents over all sources.", "Fig. 4 therefore also shows this difference that expresses the size of this netting effect.", "The composition of sources shows that wind onshore accounts for the main source of balancing demand in many countries and ranges between −2.3 and 0.9%.", "It also becomes apparent that the amount of negative forecast errors, i.e. actual generation has been higher than predicted, exceed the amount of positive forecast errors in many countries.", "The second most important source of balancing demand are consumption forecast errors, which are in the range of 0.5% of consumption for positive errors and around −0.1 for negative errors.", "The skewness of the consumption forecast error distribution has been empirically verified by Ref. [22].", "The remaining sources of balancing demand, e.g. unplanned generator outages, forecast errors of photovoltaics and wind offshore are in relation to overall consumption rather insignificant.", "The size of netting effects stemming from counterbalances of forecast errors and outages are in the range of −/+ 0.3% of electricity consumption.", "The future monetary volume of electricity balancing markets varies according to the four selected scenarios.", "Fig. 5 shows the relative importance of balancing markets as share of market volume as compared to day-ahead market volume.", "Due to the fact that prices tend to be more volatile in electricity balancing markets than in day-ahead markets the share of monetary volumes is slightly higher than the share of physical volumes shown in Fig. 4.", "The range of outcomes significantly varies across countries.8", "The market shares for positive imbalances are in the range of 0.2–2.8% and the ones for negative balancing energy from −3.4 to 0.2%.", "In general, it can be seen that all the scenarios that limit international cooperation, i.e. scenarios 2, 3 and 4 increase the share of the balancing market, because in these cases prices are more volatile.", "However, the impact considerably varies across countries.", "Another interesting result is that the several scenarios impact both control directions of the balancing markets differently.", "Whereas the share of the positive balancing market segment is merely driven by the day-ahead market design, the international market integration of balancing markets seems to be a crucial element determining the volume of this segment.", "One major reason of this finding is that the demand for negative balancing energy was in general higher than the one for positive imbalances.", "As a result, also the impacts of imbalance nettings due to market coupling becomes larger.", "This is also reflected in the different market volumes of both control directions.", "The previous results confirm that the expected market share of electricity balancing markets might lay in the range of a few percent of day-ahead market volume.", "However, the importance of revenues from this market segment significantly varies across generation technologies.9", "In Fig. 6 the costs and benefits stemming from electricity balancing related to their revenues in day-ahead electricity markets are shown for renewable generation technologies.", "In general, costs arise from the forecast errors or unplanned outages, which are in the same direction as the imbalance of the overall control zone, whereas benefits arise next to the remuneration for balancing activities from payments related to forecast errors that act contrary to the imbalance of the overall control zone.", "Whether actual payments are positive or negative additionally depends on the sign of the corresponding prices.", "In the modeling it has been assumed that vRES generators can down-ramp their generation in case they would incur losses otherwise.", "The major result from Fig. 6 is that vRES generators significantly profit from international market coupling of electricity balancing markets.", "Because of imbalance nettings and international competition the costs for forecast errors sharply decrease and benefits of deviations that support grid stability are rewarded higher.", "This is in particular important for wind onshore and wind offshore.", "The case of photovoltaics show that because of its low forecast errors neither costs nor benefits from balancing markets might be significant for this technology.", "Biomass generators are due to their capability to adjust their power output a provider of balancing energy.", "The only source of costs are unplanned outages.", "In contrary to vRES these generators profit from national electricity balancing markets and loose in case of international market coupling.", "In a similar manner, Fig. 7 shows the relative costs and benefits of electricity balancing for relevant conventional generation technologies.", "The results for coal, gas and waste generators lead to the same conclusion as outlined above for biomass generators.", "Positive revenues arise from national balancing markets, whereas these revenues diminish in case that markets are coupled.", "In the case of a more international electricity market across the EU (Mi-I) only the costs of unplanned outages have to be borne by these technologies.", "The results show that storages are probably play a major role in electricity balancing markets.", "Unlike other technologies, storages profit from both positive and negative imbalances.", "They pay a price for negative balancing energy, however, a price that is in general lower than the one that storages could capture in day-ahead markets.", "In turn, electricity can be sold for higher prices than in day-ahead markets.", "As a result, storages realize savings in both ways and thus might become a very important market player of the future.", "The above presented results indicate that the physical volumes of control zone imbalances are still in a range of a few percent of consumption despite increasing shares of renewable electricity.", "The amount of imbalances is strongly related to the amount of variable renewable electricity that is integrated in a certain country.", "From the results can be derived that wind onshore accounts for the main share of demand for balancing energy.", "The netting effect from different sources of system imbalances has proven to be an important factor to be considered.", "However, as compared to overall physical volume of gross electricity consumption the resulting imbalances can be considered to be rather low in the mid-term.", "It should be critically noted that all results are based on historical forecast data series.", "The current work do not consider any structural changes in the forecast quality nor do they contain expected improvements of forecasting accuracy.", "These results can be therefore classified as upper boundary on the mid-term relevance of this market segment.", "The relevance of electricity balancing markets in monetary terms has been evaluated to be minor as compared to day-ahead market volumes.", "The results also confirm that the concrete size of these markets are considerably impacted different framework conditions.", "However, the impact significantly differ by country and technology.", "It can be argued that the price formation as it has been modeled is biased because the underlying assumptions on the characteristics and type of considered technologies do not adequately capture and probably will never capture the expected diversity of new market actors in the future.", "Furthermore, in systems with a large share of conventional generators with high start-up costs as well as minimum-load requirements auctions for reserve capacity might help to reduce costs of balancing.", "It should be also noted that the assumed market design in this paper is only one of several options to design balancing markets.", "The modeling results have to be interpreted in the view of these aspects.", "An appropriate interpretation of these results might consider them to be an upper boundary of the relevance of electricity balancing markets in the mid-term.", "Furthermore, also the findings on potential market revenues of different technologies from electricity balancing markets have to be interpreted against this background.", "It should be further noted that in this work revenues rather than profits have been assessed.", "The actual attractiveness of electricity balancing markets for different market actors in terms of potential additional profits under different market designs is therefore an interesting field for further research." ]

[ "Development of amount and composition of electricity imbalances by 2030.", "Assessment of the future relevance of electricity balancing markets in Europe.", "Market revenues from balancing markets for different technologies." ]

[ "BIOC", "CHEM" ]

[ "Cellular bioenergetics", "Diabetes", "Mesangial cells", "Mitochondrial dysfunction", "Renal cells", "Tubular cells" ]

[ "1308", "1605" ]

[ "Diabetic nephropathy (DN) is a kidney disease which affects approximately one-third of patients with diabetes and develops over a long period of clinical silence [1].", "Despite good metabolic control, patients with both Type 1 and Type 2 diabetes are at risk of DN [2–4].", "With the epidemic rise in the incidence of diabetes currently affecting more 350 million people worldwide, more than 100 million people are at risk of DN and approximately 30% of these are likely to progress to end stage renal failure despite therapy [5,6].", "Therefore, there is an urgent need to understand the underlying mechanisms that result in damage to the kidney in order to design novel preventative and therapeutic strategies.", "DN is associated with structural changes in the kidney manifested as thickening of the glomerular basement membrane and expansion of the extracellular matrix (ECM) with deposition of collagen type IV and VI, fibronectin and laminin [7], resulting in glomerulosclerosis and tubular interstitial fibrosis, pathological hallmarks of DN.", "Mesangial cells are specialised smooth muscle cells located around the glomerular capillaries within the renal corpuscle of the kidney and have several functions, including the deposition of ECM.", "In DN, mesangial cells contribute to the disease by responding to increased glomerular filtration pressure by the expansion of the matrix into the capillary lumens and the secretion of pro-inflammatory cytokines [8], leading to glomerulosclerosis and glomerular inflammation [9].", "Although, for a long time the major pathogenic mechanisms in DN were thought to stem from glomerulosclerosis through mesangial expansion, it is now widely accepted that tubular epithelial cells present in the lining of the nephrons are also involved and contribute to interstitial fibrosis, peritubular capillary loss and destruction of functioning nephrons due to tubular atrophy and severe tubulointerstitial lesions [10–13].", "Hyperglycemia is known to be a contributing factor in the development of DN through activation of a series of overlapping biochemical pathways [14].", "Hyperglycemia-induced oxidative stress caused by the over-production of mitochondrial reactive oxygen species (ROS) has been implicated in this process [15,16].", "Indirect evidence for mitochondrial involvement in DN from animal and in-vitro studies has been accumulating [17–20] and more recently, studies have directly demonstrated mitochondrial dysfunction in human clinical samples.", "For example Sharma et al., demonstrated that patients with DN had reduced urinary mitochondrial metabolites [21].", "We recently reported that mitochondrial DNA and mitochondrial RNAs are altered in blood cells of DN patients, and in renal cells cultured in high glucose [22].", "Despite the putative involvement of mitochondrial dysfunction in DN, the exact mechanisms involved remain unknown [17].", "Kidneys are organs requiring large amounts of energy in form of ATP due to the re-absorption processes and although their mass accounts for less than 1% of total body mass, they use almost 10% of the body's oxygen which is utilized in cellular respiration [23].", "Renal mRNA profiling shows one of the highest levels of mitochondrial transcripts, after colon, muscle and heart [24], suggesting an abundance of renal mitochondria.", "In support of this, we recently showed that mouse kidneys have one of the highest amount of MtDNA, second only to the heart, when compared to liver, lung, brain, islet and blood [25].", "Eukaryotic cells, especially those with high energy demands like kidney cells, rely mostly on ATP generated through oxidative phosphorylation (OXPHOS), which is 15 times more productive than the amount produced by aerobic glycolysis [26].", "OXPHOS takes place in the electron transport chain (ETC) located within the mitochondrial matrix and can be studied by precise measurement of oxygen consumption rate and proton leak, a process described as extracellular flux analysis, which can be used to define the bioenergetic properties of cells [27–29].", "The use of specific inhibitors of various part of the ETC, which is made of 5 complexes of proteins, allows the definition of cellular bioenergetic profiles [30] and can provide information on cellular metabolism.", "There have been very few studies of mitochondrial bioenergetics related to DN.", "We recently reported for the first time that peripheral blood mononuclear cells (PBMCs) from patients with DN have a severely diminished bioenergetic profile and reduced reserve capacity when compared to diabetes controls, the latter had a long duration of diabetes and no history of kidney disease [22].", "This deficit could contribute to energy depletion, and indeed we found that mesangial cells grown in diabetic conditions show altered mitochondrial respiration [22].", "Therefore, in the current study, we wished to test the hypothesis that diabetes can cause damage to mitochondrial respiration in kidney cells and to examine respiration in the two key cell types known to be implicated in DN, namely tubular and mesangial cells.", "Primary human mesangial cells (HMCs) were cultured and characterised as previously described [31,32] and grown in Dulbecco's Modified Eagle Medium (DMEM, Sigma Aldrich) supplemented with 10% FBS, ITS and antibiotics.", "Human cortex proximal tubular immortalized cells (HK-2, ATCC, LGC Standards) were cultured according to the manufacturer's conditions.", "Cells were seeded at an equal density (1×105/well) in DMEM containing either 5 mM (normal, NG) or 25 mM (high, HG) glucose for 4 and 8 days.", "20 mM mannitol in 5 mM glucose DMEM was used as an osmotic control in all experiments.", "The metabolic profiles of cultured HMCs and HK-2 cells were assessed using the XFe96 Seahorse analyser and XF cell mito stress test kit and XF-FluxPaks containing 96-well plates and cartridges (Agilent Technologies).", "Oxygen consumption rate (OCR), a measurement of mitochondrial respiration, and extracellular acidification rate (ECAR), were determined in the presence of specific mitochondrial activators and inhibitors.", "Oligomycin (ATP synthase blocker) was used to measure ATP turnover and to determine proton leak.", "Mitochondrial un-coupler FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone) was used to measure maximum respiratory function (maximal OCR).", "Reserve capacity was calculated as a maximal OCR minus the basal respiration.", "Rotenone (inhibitor of complex I) and antimycin A (a blocker of complex III), were injected to completely shut the mitochondrial respiration down, to confirm that any observed changes in respiration were mitochondrial [33,34].", "In order to determine optimal cell number and drug concentration, optimisation experiments with a different cell seeding number and titration of electron transport chain activators and inhibitors were carried out, to accurately assess OCR and ECAR as described by Dranka et al. [33].", "One day prior to the extracellular flux measurement, HMCs and HK-2 cells were seeded at the concentration of 30,000 and 25,000 cells per well respectively in 96-well assay plates in complete growth medium containing 5 mm, 25 mM glucose and osmotic control.", "1hr prior to the experiment the growth medium was removed, cells were washed 3 times and then incubated in low buffered assay medium (Agilent Technologies) supplemented with 5 mM glucose and 1 mM sodium pyruvate, pH 7.4.", "The microplates were then assayed in the XFe96 analyzer.", "For a measurement of steady basal respiration, 4–5 measurements were taken before injecting ATP synthase inhibitor, oligomycin at 1 μM (final concentration).", "Mitochondrial membrane uncoupler FCCP was then injected at 0.75 μM.", "Finally a mixture of rotenone and antimycin A (1 μM) was injected.", "Mitochondrial basal respiration, proton leak, spare capacity and maximal respiration were measured after correcting for non-mitochondrial respiration.", "After finishing the experiments 20 μl of cell lysis buffer [33] were added per well and protein content measured by a BCA assay (Fisher Scientific).", "OCR and ECAR rates were normalized to the protein content and presented as pmolesO2/min/μg protein and mpH/min/μg protein respectively.", "Analysis was performed using GraphPad (GraphPad Software, Inc).", "The distribution of the data was tested using the Kolmogorov-Smirnov test and histograms and parametric tests were used on raw data.", "Groups were compared using Student's t-test (2 groups) or one way ANOVA with post-hoc Tukey's multiple comparison test (>2 groups), statistical significance was considered at P<0.05.", "Data are presented as mean±SEM.", "We first characterised the bioenergetic profiles of mesangial (HMCs) and tubular (HK-2 cells) in normoglycemic conditions (5 mM glucose) using the seahorse XFe96analyser.", "Oxygen consumption rate (OCR) including basal, ATP-linked, maximal respiration and reserve capacity, and basal extracellular acidification rate (ECAR) which correlates to amount of protons released from the cell with potential contribution from glycolysis and the Krebs cycle were determined in real time (Fig. 1A-H) using specific activators and inhibitors of cellular respiration [30].", "HK-2 cells had significantly lower (P<0.001) levels of basal respiration (13 pmolesO2/min/μg of protein) when compared to HMCs (20 pmolesO2/min/μg of protein, Fig. 1A, 1H).", "Following the injection of oligomycin, an ATP synthase inhibitor, basal OCR is expected to drop and the level of drop can be used to deduce ATP-production (ATP-linked respiration).", "ATP linked respiration was significantly lower (P<0.001) in HK-2 cells (11 pmolesO2/min/μg protein) compared to HMCs (16 pmolesO2/min/μg of protein, Fig. 1B, 1H).", "Injection of mitochondrial uncoupler, FCCP caused an increase in OCR and allowed measurement of the maximal mitochondrial respiration and reserved capacity.", "Maximal OCR was significantly lower (P<0.01) in HK-2 cells (20 pmolesO2/min/μg of protein) compared to HMCs (33 pmolesO2/min/μg of protein, Fig. 1E, 1H).", "Reserve capacity was also significantly lower (P<0.01) in HK-2 cells (6.5 pmolesO2/min/μg of protein) compared to HMCs (12 pmolesO2/min/μg of protein, Fig. 1F).", "Proton leak, measured as the difference between ATP-linked and basal OCR, was significantly lower (P<0.001) in HK-2 cells (2.2 pmolesO2/min/μg of protein) compared to HMCs (4.5 pmolesO2/min/μg of protein, Fig. 1C).", "Non-mitochondrial respiration, measured as a drop in OCR following the administration of mitochondrial respiratory chain blockers, rotenone and antimycin A, did not vary significantly between the two cell types, and was measured as 4.6 and 5.6 pmolesO2/min/μg of protein for HK-2 and HMCs respectively (P>0.05, Fig. 1D).", "Basal glycolytic rates were also measured at similar levels between both experimental cell types (P>0.05, Fig. 1G).", "We next evaluated the coupling efficiency defined as a proportion of mitochondrial oxygen consumption used for the ATP synthesis (Fig. 1I).", "Coupling efficiency represents the proportion of the substrate oxidation energy used for the ATP production, while the rest is lost as a heat.", "Of all the oxygen consumed, HK-2 cells use a significantly higher rate for ATP-linked respiration relative to HMCs (P<0.05, 0.8 versus 0.75 respectively, Fig. 1I).", "The proportional distribution of oxygen consumption and utilization among the different parameters defined in the bioenergetic profile illustrate the higher reserve capacity but lower ATP-linked respiration in HMCs compared to HK-2 cells (Fig. 1J).", "The effect of hyperglycemia on cellular respiration in HK-2 cells was examined.", "Growth in high glucose caused a significant down-regulation of all respiratory parameters.", "Basal, ATP-linked and maximal respiration as well as reserve capacity were reduced within four days (P<0.01) and remained reduced after prolonged exposure over 8 days (Fig. 2A, B, E, F).", "No significant changes were detected in proton leak (P>0.05, Fig. 2C), but non-mitochondrial respiration was significantly reduced following growth in HG (P<0.05, Fig. 2D).", "Basal ECAR was significantly up-regulated after 8 days growth in HG (P<0.05, Fig. 2G).", "HK-2 cells had significantly down regulated metabolic rate (plotted as a ration of OCR and ECAR) when cultured in HG for 4 and 8 days and compared to NG controls (P<0.001, P<0.01 respectively Fig. 2I).", "Mitochondrial respiration was unaltered in osmolarity controls and was unaffected by the duration of culturing time (Supplementary Fig. 1 and Fig. 2) showing that the changes in respiration of tubular cells were a direct consequence of growth in hyperglycemia.", "Unlike tubular cells, growth of mesangial cells in HG for 4 days did not affect their bioenergetic profiles, there were no significant changes in basal, ATP-linked, maximal respiration, and basal glycolytic rate remained unchanged (P>0,05, Fig. 2A-E).", "Growth of mesangial cells in HG for 8 days still did not affect basal and ATP-linked respiration and basal glycolytic rate (P>0.05), however maximal OCR and reserve capacity were reduced (P<0.05, Fig. 2A-E and G).", "No statistically significant difference was detected in the non-mitochondrial respiration (P>0.05, Fig. 2F) and in basal glycolytic rate (P>0.05, Fig. 2H).", "The metabolic rates of mesangial cells were plotted as the ratio of OCR and ECAR under basal conditions and were unaltered in hyperglycemic conditions (P>0.05, Fig. 2I).", "Mitochondrial respiration was unaltered in osmolarity controls and was unaffected by duration of culturing time of mesangial cells (Supplementary Fig. 1 and Fig. 2).", "Since HMCs seemed more resistant to hyperglycemia than HK-2 cells, we decided to determine the effect of longer exposure to hyperglycemia, therefore HMCs were cultured for 12 days in normal and HG (Fig. 3).", "12-d exposure to HG decreased basal oxygen consumption rate in HMCs by more than 50% when compared to control (P<0.0001, Fig. 3A) and the respiration used for the ATP production reduced by 2.5 fold when compared to cells cultured in NG (P<0.0001, Fig. 3A).", "There was no difference in proton leak and in non-mitochondrial respiration (P>0.05, data not shown).", "As described above, maximal respiratory capacity of cells, was significantly lower in the cells cultured for 8 days in HG when compared to the controls, and this reduction became highly significant (P<0.0001) after 12 days of culture in HG, with 3 fold reduction of maximal OCR (P<0.0001, Fig. 3A) and 4 fold reduction in the reserve capacity when compared to cells cultured in NG (P<0.001).", "When glycolysis was assessed in these cells, there was also a significant reduction in glycolysis at basal level (P<0.05) and also after the addition of oligomycin (which stimulates glycolysis due to inhibition of ATP synthesis, P<0.01, Fig. 3B).", "Furthermore, we found that the ability of cells to respond to acute stress was also blunted after 12 days growth in hyperglycemic conditions (Fig. 4).", "HMCs grown in normoglycemic conditions for 12 days were able to respond to acute stress by altering their metabolism, whereas the hyperglycemic cells had lost this ability (Fig. 4).", "After acute glucose load, both normoglycemic and hyperglycemia HMCs switched their metabolism to glycolysis (Fig. 4), however hyperglycemic HMCs which were previously exposed to chronic hyperglycemia had lost their flexibility to respond to nutrient load, as they could not increase their glycolytic rate but at the same time mitochondrial respiration remained the same which suggested they cannot balance their energy production.", "In summary, we showed that 4 days exposure to hyperglycemia had no effect on any of the measured parameters involved in mitochondrial respiration or glycolysis in HMCs, 8 days exposure significantly decreased maximal respiration and reserve capacity but did not affect any other mitochondrial respiration parameters, whereas 12 days exposure significantly diminished both mitochondrial respiration and glycolytic capacity, suggesting an energy deficit in these cells.", "With the global rise in the incidence of diabetes, diabetic nephropathy, a major risk factor for morbidity and mortality, is likely to reach epidemic levels in the next decade.", "To combat this, there is a strong clinical need to understand the early molecular mechanisms that lead to kidney damage in diabetes.", "The kidney as an organ has a high requirement for energy in the form of ATP derived from cellular respiration to drive cellular processes [23,24].", "Pathological changes in renal mesangial and tubular cells are well known to play significant roles in the development and progression of DN and lead to glomerulosclerosis and tubulointerstitial fibrosis [10,12], both structural hallmarks of DN, but the role of energy deficit in these processes has been largely unaddressed in the past.", "Mitochondrial dysfunction in the diabetic kidney disease is increasingly being seen as a key mechanism in DN [21,22] leading us to speculate that damage to the cellular respiratory apparatus in the diabetic kidney could lead to energy deficit and cellular damage.", "In order to shed light on the bioenergetic properties of renal cells, in the current paper we sought to define the bioenergetic profiles of renal mesangial and tubular cells and to assess whether diabetes can damage cellular respiration in these cells.", "Cells need the ability to produce sufficient energy to carry out their normal cellular function, and have differing bioenergetic requirements depending on their function.", "In addition, cells also have to be able to respond to stressors by appropriately altering their metabolism.", "Our data show that both HMCs and HK-2 cells have distinct metabolic profiles and both cell types utilize more than two thirds of the consumed oxygen into ATP production.", "HMCs’ mitochondrial respiratory parameters are much higher when compared to HK-2 cells in normoglycemia, but tubular cells’ coupling efficiency is significantly higher, meaning that of all the oxygen consumed, HK-2 cells use a slightly higher proportion for ATP-linked respiration relative to HMCs and lose less energy through proton leak.", "The bioenergetic profile of both mesangial and tubular cells was negatively affected by exposure to hyperglycemia but tubular cells were more susceptible, with cellular respiration parameters being significantly altered within 4 days of exposure to high glucose, and showing severe damage to cellular respiration by 8 days.", "In contrast, mesangial cells showed little change in high glucose even after 8 days.", "After prolonged high glucose exposure of 12 days, mesangial cells showed signs of decreased mitochondrial respiratory function, reduced their reserve capacity.", "Hyperglycemia had no effect on the proton leak in HMCs and HK-2, but tubular cells had decreased non-mitochondrial respiration when exposed to high glucose.", "Therefore our data show that cellular respiration is reduced in renal cells after exposure to hyperglycemia, and that mesangial cells are more resistant to this damage than tubular cells but do become damaged and more sensitive to acute stress after prolonged exposure.", "Whilst mitochondrial and glycolytic metabolism have been proposed to be altered in metabolic disease [35], few studies have examined the effect of hyperglycemia on these parameters.", "Hyperglycemia has been previously shown to have varying effects on bioenergetics and cellular respiration in cultured cells, including decreased, unaltered and increased cellular respiration.", "For example, a decline in mitochondrial basal and maximal respiration was seen in rat retinal endothelial cells after 3 and 6 days of culture [36], and down-regulation of mitochondrial function (including reduced ATP-linked respiration and reserve capacity) was seen in primary cultured mouse mesangial cells exposed to hyperglycemia for 14 days [38], in human mesangial cells after 8 days [22].", "A faulty bioenergetic profile was observed in cultured neurons derived from streptozotocin induced diabetic rats with decreased maximal respiration and decreased spare capacity [39].", "No changes in respiration were detected in bovine aortic endothelial cells after 24 h exposure to hyperglycemia and in human platelets cultured in high glucose for 18 h [37], interestingly these studies had shorter duration of exposure to hyperglycemia than those that observed decreases.", "In contrast, Stieger at al., (2012) demonstrated an increase in the basal and maximal respiration of immortalized mouse podocytes cultured in high glucose for 10 passages. [40].", "Measurement of cellular respiration directly in live human blood cells from patients with diabetes has also been attempted by us and others.", "Increased mitochondrial basal, and maximal respiration rates were found in peripheral blood mononuclear cells (PBMCs) from patients with type 2 diabetes (T2D), when compared to healthy controls [41].", "We previously found a slight but non-significant increase in cellular respiration in PBMCs from diabetes patients without complications (both T1D and T2D) compared to healthy controls, however we found that PBMCs of patients with DN had reduced reserve capacity and increased sensitivity to stress, showing loss of metabolic flexibility [22].", "Mitochondrial respiration was also reported to be reduced in the skeletal muscle of T2D patients [42,43] and not changed in myocytes from non-diabetic, obese subjects with family history of T2D [44].", "Using isolated mitochondria from diabetic models, Ruggiero at al., (2011) reported increased levels of mitochondrial superoxide dismutase (MnSOD) and increased mitochondrial ATP-linked respiration in 12 week old diabetic animals, but this increase was not observed at later stages, [45].", "This suggests an adaptive response in mitochondrial respiration in order to cope with nutrient overload, and an increase in maximal and basal respiration which, when within threshold limit for mitochondria without diminishing spare respiratory capacity may be still tolerable.", "Longer exposure hyperglycemia might be detrimental as we have previously demonstrated that hyperglycemia can also damage MtDNA which could affect mitochondrial function over time [22].", "If there is an adaptive phase when mitochondria increase their respiration in response to nutrient overload, then this could contribute to increased ROS production and further exacerbate cellular stress.", "Anderson at al., (2009) reported increased mitochondrial H2O2 production in human and rodent muscles fed with high fat diets, which correlated with a shift in cellular redox balance, without any changes in mitochondrial respiration.", "The oxidation state of the cells was balanced following treatment with mitochondria-specific antioxidants [46].", "The data from the current study support the view that cells have different tolerance levels for chronic hyperglycemia which may lead to defective mitochondria that cannot properly respond to changes in glucose levels, and this could lead to possible disruption in glucose metabolism.", "HK-2 cells increased their basal ECAR in response to high glucose, which may be a compensatory response to the condition when cells cannot rely on their OXPHOS energy, but try to keep balance in the cellular ATP production [36,47], or it could be caused by negative regulation of the OXPHOS through glycolytic intermediates [48].", "However, when the ratio of both OCR and ECAR were compared, tubular cells seem to be metabolically less active in the presence of hyperglycemia when compared to the mesangial cells after 1 week of culture.", "Energy production in the kidneys, which are mitochondrial rich organs, is largely through respiration, and Warburg demonstrated that the total energy production in quiescent kidney cells was similar to that produced in proliferating cancer cells, which indicates how much energy the kidney requires for proper functioning [49].", "In support of this, we found that after the heart, kidney has the second highest mitochondrial DNA content when compared to other mouse organs, including liver, blood, brain, islets and lungs [25] suggesting that kidneys are rich in mitochondrial content.", "The data shown in the current study clearly show that high glucose had a toxic effect on renal cell mitochondrial metabolism, but the mechanisms by which this was caused remains unclear.", "Reserve capacity, described as a mitochondrial bioenergetic reserve, was significantly reduced in hyperglycemia in both mesangial and tubular cells.", "Reserve capacity depends on many factors, like substrate supply, energy demand and integrity of the electron transport chain.", "Dysfunction in mitochondrial metabolism could be caused by the direct effect of the glucose through modification of mitochondria-localised proteins, as described by others [50–53].", "But it could also be caused by the disruption in the synthesis of mitochondrial complexes assembly subunits, caused by mutations in MtDNA.", "We have previously detected a clear difference in metabolic function between the patients with diabetes without and with DN and we have shown that growth of renal cells in high glucose can rapidly change the amount of MtDNA and cause MtDNA mutations, leading to the proposal that the inability to cope with energy demand due to MtDNA damage could be a key factor in the progression of DN [22].", "If systemic mitochondrial dysfunction is associated with DN, then we would predict systemic changes in the body which may be the basis of renal microvascular complications in diabetes.", "In such a scenario, different cells in the body would show different levels of sensitivity to hyperglycemia induced damage, the extent of which would be related to but not exclusive to mitochondrial content and bioenergetic need.", "In conclusion, we have shown that hyperglycemia alters the metabolic profile of both mesangial and tubular cells by reducing their mitochondrial respiratory function.", "Mesangial cells have a substantial bioenergetic reserve compared to tubular cells.", "These results show that distinct cells within one organ can have different bioenergetic profiles and sensitivities to hyperglycemia.", "A thorough understanding of the bioenergetic properties of different cell types to oxidative stress and nutrient overload, present in diabetes and its complications is needed and could lead to the development of novel therapies to protect mitochondrial respiration in organs with high energy requirements." ]

[ "The kidney as an organ has high bioenergetic needs.", "Cultured renal mesangial and tubular cells have different bioenergetic profiles.", "Hyperglycemia had a toxic effect on renal cell mitochondrial metabolism.", "Mesangial cells were more resistant to diabetes induced damage to cellular respiration than tubular cells.", "Energy deficit in renal cells may be a contributing factor to renal damage in diabetic nephropathy." ]

[ "COMP" ]

[ "Bio-economic model", "Fisheries management", "Fleet dynamics", "Impact assessment", "Management strategy evaluation" ]

[ "1706", "1712" ]

[ "It describes fleet’s short term dynamics or tactical behaviour.", "Each season it models how much effort is exerted and how it is distributed along metiers.6", "In the first model, effort and its distribution along metiers are given as input data.", "Thus the effort exerted by the fleet is independent of the stock status and the management advice.", "A second model is based on the Fcube method [10] and it describes mixed fisheries 7 dynamics.", "The effort share along metiers is given as input data and in each step the total effort is estimated.", "First, the effort corresponding to the total allowable catch (TAC) share of each stock caught by the fleet is calculated.", "The final effort is chosen among previously estimated efforts using one of the options available (the minimum, the maximum or the mean of the efforts, the most similar to the previous year effort and the effort that produces a catch level equal to the quota share of one particular stock).", "An alternative model to simulate mixed fisheries dynamics calculates the total effort and the effort allocation among metiers that maximizes the profit.", "The maximization is constrained by the capacity of the fleet and by the catch quota of the stocks.", "Finally, there is a model that describes sequential fisheries 8 dynamics.", "In this model historical effort level guides the present performance of the fleet.", "Seasonally, each metier has only one target stock and its expected effort follows the historical trend, but is restricted by the total TAC share of the fleet.", "In the case where TAC share is exhausted for a particular stock, the remaining effort is reallocated among the rest of the metiers which target other stocks.", "In general, the effort exerted by the fleets is driven by the TACs of the selected stocks, hence the catch of some stocks may not coincide with the advised TAC.", "It describes the relationship between catch and effort.", "At present the only model available is the Cobb-Douglas production model [22] which is widely used in economy to describe production in industry.", "FLBEIA tries to integrate the models used by biologists and economists.", "However, the exponential survival model and the Cobb-Douglas model cannot be coupled in a natural way because the former describes catch production in a continuous way and the last discretely and instantaneously.", "To overcome this discrepancy in the models implemented, it has been assumed that the catch takes place in the middle of the season.", "It describes how price changes as a function of other factors, for example landings.", "Price varies at fleet and stock level.", "There are two models available to describe its dynamic.", "The constant model and a model where the price depends on the ratio between current landings and landings in a baseline time period [23].", "It describes the long term dynamics of the fleet or strategic behaviour; the investment or disinvestment of fishermen in new vessels or technological improvements.", "In FLBEIA the capital dynamics can be modelled through changes in fleet’s capacity or changes in fleet’s catchability (technological improvements).", "Fleet’s capacity can be modelled using a constant model or the model described by Salz et al. [24].", "However, at present, models that dynamically change catchability are not available in FLBEIA.", "Fishery systems are complex systems formed by fish populations, fleets that exploit them and the stakeholders involved in the management process.", "Fisheries need to be managed in order to ensure sustainable and efficient exploitation of marine resources [1].", "Management measures are usually based on biological criteria despite the fact that the fleets (i.e. the socio-economic dimension) are the ones directly managed.", "In recent years, driven by the ecosystem based fisheries management approach [2], the need to incorporate the economic and the social dimensions into the management process has been recognized.", "Approaches that integrate these three disciplines are therefore needed, but such approaches are currently scarce and usually case specific.", "Furthermore, scientists tend to focus on biology or economy whereas sociology is still quite undeveloped [3].", "Biological evaluation of management measures is usually conducted under a Management Strategy Evaluation (MSE) approach [4].", "It consists in simulating the fish stocks and the fleets that exploit them together with the management process.", "When a management strategy is tested, the advice is not based on the real system but on the perceived system obtained in the management process.", "Thus, not only the strategy itself is evaluated, but the entire management process which includes data collection and estimation processes.", "MSE implementations have been generally biologically oriented, single stock and single fleet.", "There are several examples of integrated bio-economic models developed under the MSE approach (see for example [5–7]).", "However, they are all case specific models built to address case specific problems.", "FLBEIA (Bio-Economic Impact Assessment using FLR) is a bio-economic simulation software, aimed at facilitating the evaluation of management strategies under the MSE approach.", "The software allows the bio-economic evaluation of a wide range of management strategies in a great variety of case studies.", "The model has been developed in a composable1 manner [8].", "One of the strengths of FLBEIA is that it is built using FLR libraries.", "FLR is a collaborative project oriented to develop quantitative fisheries management tools.", "Moreover, stock assessment methods [9], fleet dynamics models [10,11] or harvest control rules2 (HRCs) [12] have been also developed and they could be directly integrated into FLBEIA.", "FLBEIA has been used in several case studies where its utility to analyse diverse problems has been demonstrated [13–16].", "Furthermore, in [16] the model was contrasted against flexibility, applicability and utility criteria in a generic framework.", "Afterwards, the recommendations derived from this study were taken into account to improve the model development.", "FLBEIA is a R [17] package which main function is a simulation algorithm called FLBEIA (from here referred as the algorithm).", "It also provides several functions to facilitate the generation of the input data, to summarize and plot the results and to build up the algorithm.", "The main input and output (I/O) data is stored in the objects defined in FLCore and FLFleet FLR packages.", "Furthermore, FLCore provides the methods to perform basic mathematical operations with the objects defined within these packages.", "Other packages built in the FLR framework can also interact with FLBEIA.", "However, its use is case specific and not mandatory.", "The algorithm is divided into two blocks, the Operating Model (OM) and the Management Procedure (MP) (Fig. 1).", "The OM is the part of the model that simulates the true dynamics of the fishery system (the real population).", "Biological populations and fleets are its essential elements and they interact through fishing effort and catch (Fig. 1).", "The MP describes the management process and it is divided into three modules, the observation model (the link between the OM and the MP), the assessment procedure and the management advice.", "The observation model together with the assessment model generate the perceived population based on which the management advice is calculated.", "The advice is given in terms of catch and it can also be combined with technical management measures such as gear restrictions, temporal closures or capacity limitations.", "The algorithm has been developed by composition which eases checking and debugging, and allows for increased complexity adding extra modules [8].", "The fishery system has been decomposed into several processes (dark grey rectangles in Fig. 2) and several functions have been implemented to model each of them.", "The assembly of processes within FLBEIA follows a top-down approach (Fig. 2).", "The algorithm is divided into three main levels:", "The FLR functions used within the algorithm are called by the third level functions.", "However, in most of the cases third level functions only used functions in FLCore and FLFleets packages.", "FLXSA, FLSAM and other stock assessment model packages, can be used within the assessment module to generate stock status estimates.", "In turn, FLash and FLAssess packages, are used in the advice module to do some calculations in some specific Harvest Control Rules (HCR).", "The source code of these packages is available at http://github.com/flr.", "The compiled packages compatible with FLBEIA and FLBEIA itself can be downloaded from http://flbeia.azti.es/download/.", "The installation instructions of FLBEIA can be found at its website or in Github repository.", "The flow chart in Fig. 3 shows how the algorithm moves from one module to another along the simulation.", "The projection of stocks, fleets and covariates is done independently one by one.", "In the first step of the simulation the fish stocks are projected one season ahead.", "Afterwards, fleets are projected and four processes are modelled: effort allocation, catch production, price formation and capital dynamics.", "The last component projected within the OM are the covariates.", "The management procedure takes place once a year for each stock independently.", "The season when it takes place is selected by the user and can vary from stock to stock.", "First the observed data is simulated.", "The data is divided into two types: stock data and abundance indices.4", "Abundance indices are simulated by stock and each stock can have several indices.", "The observed data is then used to feed the stock assessment models which provide estimates of the real stocks simulated in the OM.", "Afterwards, a specific HCR is applied to each of the estimated stocks and the management advices are obtained.", "Finally, this information is transmitted to the fleets in the OM and the algorithm moves one season and/or year forward.", "The stochasticity in the model is introduced using Monte Carlo simulation [18].", "Uncertainty can be introduced in all the variables used to describe the system.", "The algorithm is replicated the number of times defined by the user, and the sequence in the flow chart is independently repeated each time.", "Each input variable can be conditioned using a single value or a vector, in this last case, each model replicate is conditioned taken a single value from this vector each time.", "In the following sections we describe the functions available to describe the processes.", "Their mathematical description can be found in the manual.", "The stocks can be age structured or aggregated in biomass.", "Three models are available to describe stock dynamics.", "In the first model the stock abundance in the projection is given as input data and is maintained unchanged in the simulation.", "Implicitly it assumes that population growth is independent of fleets’ catch.", "This model can be useful for example when nothing is known about the dynamics of a certain stock, but its incorporation into the model is justified due to the economic relevance of the stock for a particular fleet.", "A second model projects age structured populations one season ahead using a stock-recruitment model for incoming recruitment5 and an exponential survival model for the existing age classes [19].", "All the individuals move from one age class to the next on 1st of January regardless of the season in which they were born.", "Finally, populations aggregated in biomass are projected using a Pella–Tomlinson growth model [20].", "In the three models, the catch is assumed to take place in the middle of the season in accordance with the fleet dynamics models implemented (see Section 2.2.2).", "Fleets OM is divided into four processes: effort allocation, catch production, price formation and capital dynamics.", "The models used to describe these processes can differ from fleet to fleet.", "The role of the covariates OM is to have room to incorporate into the model variables that are not included in biological and fleet OMs but are relevant to the fishery system.", "The variables can be of any kind (biological, economic, environmental, social, …).", "But appropriate models should be implemented to simulate their dynamics and also their interaction with other model components.", "At present the only model available is the constant model.", "Its usefulness is to allow the inclusion of data into the model which has no room in the stocks and fleets data containers, for example some economic variables required in the capital model [24].", "The observation model simulates data for each stock independently.", "All the data is generated annually despite OM’s seasonal dimension and can be observed with error.", "Two types of observation errors can be introduced, errors associated with age determination and multiplicative errors associated to any other reason.", "Observed data can contain data related to fleets’ production (landing and discard data), to stock biology (natural mortality, fecundity and individual weight), to abundance indices and to stock status (number of individuals and harvest rate).", "In FLBEIA the indices are linearly related to biomass being the catchability the slope of the model.9", "Assessment models are used to obtain estimates of abundance and/or exploitation rate of the stocks and they are applied independently stock by stock.", "Any assessment model coded in R can be used within FLBEIA if the input and output data have the right shape.", "The assessment models available in FLR can be integrated in FLBEIA and some of them have been already used, for example FLXSA in [13].", "To initialize the model two types of data are needed, historical data (starting conditions) and model parameters in the projection.", "The historical data can comprise just one year or an historical period.", "It depends mainly on the management procedure.", "If the assessment model or the HCR depend on the historical period, then the historical data should include at least this time period, otherwise, just one year to start the simulation is enough.", "Theoretically FLBEIA supports unlimited number of stocks, fleets, metiers, covariates and replicates.", "In practice, memory allocation in the operating system sets the limit.", "In summary, at fishery level, the use of new reference points overcomes the loss in profits derived from the implementation of the LO.", "Furthermore, the biomass levels were similar or higher when they were used.", "However, in the long term, the less selective fleet, the gillnetters, were penalized when they were used.", "A “How to use video” (https://youtu.be/J-mSsvdeh6Q) is provided as supplementary material together with the data and the R script used (see Appendix A).", "Firstly, the video explains what FLBEIA is and how to install it.", "Secondly, it shows how to compile the input data in excel format to convert it afterwards in the FLR objects necessary to feed the algorithm.", "Then, it indicates how to generate the control objects, how to run the model and how to plot the I/O data with the functions available in FLBEIA.", "Finally, it shows how to change several input and control parameters to run different scenarios.", "FLBEIA provides a generic and extendible software to conduct integrated bio-economic evaluation of a wide range of management strategies under MSE approach.", "For example, the price models available, cannot handle substitution effects among species, which could be important when modelling mixed fisheries.", "FLBEIA allows the development and integration of a price model that could handle this issue.", "The step forward of FLBEIA is that the MP is modelled explicitly.", "This feature allows, for example, to value the information included in the MP, the value of a new a data set, the value of an abundance index obtained through a research survey, the value of using a complex versus a simpler model…", "FLBEIA avoids having to develop bio-economic models from scratch whenever you want to use an alternative model to describe a process.", "If the function required to describe it is not available, the function needed can be implemented and the existing functions can be used in the rest of the processes.", "The software has already been extensively used by the developers together with other scientist in a variety of case studies (see for example the work done for Cod stock in North West Atlantic [26], Bay of Biscay Anchovy [27], Deepfishman and Myfish European projects [13,14] or the evaluation of multiannual management plans in European South Western Waters [28]) and by external scientist in the Evaluation of multiannual management plans in European North Western Waters [28].", "Bio-economic complex models are increasingly used to evaluate the impact of new policies before they are put in place.", "The strengths of FLBEIA are that it follows MSE approach, is composable and uses FLR packages.", "The management procedure in FLBEIA does not only include the HCR as occurs in many simulation models, but it also includes the observation and assessment models.", "The composability of the software allows different functions to be selected to model each of the processes that build up the fishery system and code new functions to satisfy specific user requirements.", "The functions implemented correspond with already published models.", "FLBEIA is the first Bio-Economic MSE algorithm available in the framework of FLR.", "FLR packages have contributors across a number of institutes and universities and hence FLBEIA can automatically benefit from new developments in FLR." ]

[]

[ "MEDI", "NEUR" ]

[ "Desmetramadol", "metabolism", "poor metabolizer", "tramadol", "ultrarapid metabolizer" ]

[ "2703", "2728", "2808" ]

[ "The study design consisted of 2 consecutive randomized, double-blind, 3-period cross-over, placebo- and active comparator-controlled, single-center trials A and B performed between August 2014 and October 2014 and between October 2017 and December 2017 and conducted in a clinical research unit in Salt Lake City, Utah (ClinicalTrials.gov Identifiers: NCT02205554 and NCT03312777).", "The study was approved by an independent ethics committee and was conducted in accordance with the Declaration of Helsinki and other applicable guidelines, laws, and regulations.", "Written informed consent was obtained from all participants.", "After screening, participants in both trials A and B were randomized to 1 of 6 possible treatment sequences of placebo, 50 mg tramadol (Ultram; Janssen Ortho, LLC, Gurabo, Puerto Rico) and 20 mg desmetramadol (Syntrix Pharmaceuticals; Fig 2).", "Nine doses of each study drug were given every 6 hours in each of the 3 treatment segments, with segments separated by 1 week in trial A and 2 weeks in trial B. Participants stayed at the clinical research unit during the entirety of each treatment segment and were discharged during the time between segments and at the end of the third segment.", "For 1 hour before and 1 hour after doses 8 and 9, the participant's diet (oral intake) was limited to clear liquids only.", "Participants in trial B also received 3 consecutive 20 mg daily doses of paroxetine beginning 1 day before each treatment segment.", "Paroxetine levels were quantified by sampling blood immediately before the ninth dose of study drug in each treatment segment (Quest Diagnostics, West Valley City, Utah).", "Blood was collected to test the CYP2D6 genotype after the ninth dose of study drug of the first segment in trial A and at screening in trial B.", "The end of the study in both trials consisted of a telephone follow-up 1 week after the end of the third segment.", "Randomization to the 6 treatment sequences was in a ratio of 1:1:1:1:1:1 using a computer-generated random list of permuted blocks of 6.", "Blinding of study drug was by overencapsulation.", "Measurements were made to quantify steady-state plasma M1 and tramadol enantiomers by sampling blood immediately before and after the ninth dose of study drug.", "Cold-induced pain was measured in the cold pressor test after the ninth dose of study drug.", "Pupil diameter and abuse liability measures were assessed after the seventh dose of study drug in trial A. Adverse events (AEs) and vital signs were collected throughout each trial.", "The primary end point consisted of the steady-state minimum (Cssmin) and maximum (Cssmax) plasma concentrations of (+)-tramadol, (-)-tramadol, (+)-M1, and (-)-M1.", "Secondary end points consisted of cold-induced pain, safety, abuse liability, pupil diameter, and CYP2D6 genotype.", "The primary end point consisted of cold-induced pain perception or tolerance.", "Secondary end points consisted of Cssmin and Cssmax, and safety.", "The formal study hypothesis was that bioequivalent and equianalgesic doses of tramadol and desmetramadol in trial A will produce significantly greater plasma (+)-M1 and superior analgesia for desmetramadol compared with tramadol in trial B, where participants are metabolically deficient.", "Eligible participants were aged 18 to 55 years, of general good health, had a tolerance to cold-induced pain of ≥20 seconds and ≤120 seconds, and in trial B had a CYP2D6 genotype consistent with an intermediate metabolizer phenotype or normal metabolizer phenotype.", "Each participant's CYP2D6 genotype was determined using a multiplex PCR and allele-specific primer extension assay (xTAG Mutation Detection, Luminex Molecular Diagnostics, Austin, Texas) that identifies 17 variants (*1, *2, *3, *4, *5, *6, *7, *8, *9, *10, *11, *12, *14, *15, *17, *41, and gene duplication) and 2 gene rearrangements (Genelex, Inc., Seattle, Washington).", "The assay covers >93–97% of poor metabolizer phenotypes and has an analytical specificity and sensitivity for detection of these mutations of >99%.", "Trial A conserved statistical power to detect tramadol and desmetramadol analgesia by enrolling only males because females exhibit large variation and temporal instability to repeated cold-induced pain.41", "Further criteria for key inclusion and exclusion criteria are presented in Table 1.", "Each participant's CYP2D6 genotype was reported using the star (*) allele nomenclature and used to predict metabolizer phenotype.9", "Each star (*) allele or haplotype is defined by the presence of a specific combination of single-nucleotide polymorphisms and/or other sequence alterations within the CYP2D6 gene locus.", "The *1 allele is defined as wild type (see www.pharmvar.org for other alleles).", "Each CYP2D6 genotype is reported as a diplotype, which includes 1 maternal and 1 paternal allele (eg, *1/*4).", "Participants with >2 copies of the CYP2D6 gene are denoted by an “xN” after the allele designation; for example, a *2 × 2 haplotype is a duplication of the *2 allele.", "Each CYP2D6 allele designation was translated into an activity score, that is, 0 for nonfunctional (eg, *3, *4, or *5), .5 for reduced function (eg, *9, *10, or *17), or 1.0 for fully functional (eg, *1, *2, or *27).25", "The sum of the activity scores for each allele in the diplotype determines the participant's overall CYP2D6 activity score; for example, a *1/*1 genotype has an activity score of 2.0, a *3/*9 genotype has an activity score of .5, and a *3/*5 genotype has an activity score of .0 (www.pharmgkb.org/vip/PA166170264).", "Participants with an activity score of 0 were designated poor metabolizers (individuals carrying no functional alleles), those with a score of .5 or 1.0 were designated intermediate metabolizers, those with a score of 1.5 or 2.0 were designated normal metabolizers (individuals carrying 2 alleles with full function or 1 full function and 1 reduced function allele), and those participants with a score of >2.0 were designated as ultrarapid metabolizers (individuals carrying >2 copies of functional alleles).", "The Cssmin and Cssmax of (+)-tramadol, (-)-tramadol, (+)-M1, and (-)-M1 were measured by collecting blood immediately before the ninth study drug dose and at 1.0, 1.5, 2.0, 2.5, and 4.0 hours afterward.", "An additional sample was taken at 8.0 hours in trial B to measure the half-life (t1/2).", "The tramadol and M1 enantiomers were quantified using a chiral liquid chromatography mass spectroscopy method using a Lux Cellulose-2 (Phenomenex, California) chromatographic column and positive atmospheric pressure chemical ionization mode while operating the instrument in the multiple reaction monitoring mode.", "The assay was validated in accordance with FDA guidance.", "The lower limit of quantification for each enantiomer was 5.0 ng/mL.", "The calibration range was 5–1,000 ng/mL for each enantiomer.", "Assay accuracy for (+)-M1 was 99.3–103% and the precision (relative standard deviation [SD]) was .8–3.6%.", "Assays were conducted in the bioanalytical laboratories of IITRI Life Sciences Group (Chicago, Illinois).", "Pupillary contraction measured by pupillometry is a pharmacodynamic marker of target engagement by opioids including tramadol.24", "Pupil diameter was measured with a NeurOptic VIP 200 pupillometer (Laguna Hills, California) before the first dose and after the seventh dose of each treatment segment.", "Pupillometry was not performed in trial B because paroxetine causes pupil dilation that confounds the contractionary effect of opioids.49", "Opioids induce positive responses in subjective measures of abuse in healthy participants who are not abusing drugs.6–8,54,78,90–92", "Abuse liability assessments were performed in trial A after the seventh dose that consisted of a 100-mm visual analog scale (VAS) for each of drug liking–disliking, take drug again, and strength of drug effect measures.", "The cold pressor test is an established test model for evaluating opioid‐induced analgesia including tramadol.37,40,43", "Pain intensity (0–10 VAS) at 30 seconds and at first perception, and time (seconds) to hand withdrawal and first pain were determined at 1, 2, and 3 hours after the ninth dose of each study drug and averaged.", "Assessments of the safety and tolerability of desmetramadol and tramadol included 1) AEs and serious AEs, 2) vital signs, 3) laboratory analyses, and 4) study drug discontinuation.", "AEs were allocated to a study drug if they occurred after its first dose and before either the first dose of the next study drug or the end of the study.", "The AE relationship to blinded study drug was assessed by the investigator as either not related, unlikely related, possibly related, probably related, or definitely related.", "An AE was drug-related if it was designated as possibly, probably, or definitely related.", "The severity of AEs were graded on an FDA-specified scale for healthy adult and adolescent volunteers.23", "Vital signs included systolic and diastolic arterial blood pressures, pulse, and respiratory rate.", "Vital signs were obtained at screening baseline and before and after each study drug administration in trial A.", "In trial B, baseline vital signs were obtained once for each segment before paroxetine administration and then once after each paroxetine and study drug administration.", "Vital signs were obtained in trials A and B at the end of each treatment segment and before discharge.", "The reported peak mean pain perception to cold before and after a single 50 mg dose of tramadol was used to power the first-in-man trial A (mean [SD] pain intensity before and after tramadol of 6.3 [2.0] and 5.0 [2.3] cm on a VAS, respectively).31", "To provide ≥80% power in trial A to detect a -1.3-cm change in pain perception between desmetramadol and placebo, a sample size of 39 participants was planned.", "To provide ≥97% power in trial B to detect a -.5-cm change in pain perception between desmetramadol and tramadol at 30 seconds, a sample size of 60 participants was planned, as informed by trial A data.", "Formal statistical analysis plans were developed before unblinding trials A and B. All descriptive and inferential statistical analyses were performed using SAS version 9.3 (SAS Institute, Cary, North Carolina) or R version 3.0 (The R Foundation, Vienna, Austria).", "Continuous end points were analyzed using mixed-effects linear models.", "The appropriate covariance structure was selected using graphical tools and information criteria.", "In trial B, the overall analgesic analysis used a backward selection approach to determine the significant effects with treatment, segment, sequence, and gender as fixed effects and participant as a random effect nested within sequence.", "Segment was added as a fixed effect to find any significant first-order crossover effects.", "Otherwise, analyses used mixed effects linear models with treatment, segment, and sequence as fixed effects and participant as a random effect nested within sequence.", "Segment was again added as a fixed effect to find any significant first-order crossover effects.", "If significant treatment effects were present, least-squares means were compared between desmetramadol and placebo, or tramadol and placebo, using Dunnett's procedure, and between desmetramadol and tramadol using a paired t-test.", "In addition to overall analyses, separate analyses for measures of analgesia were performed for males and females.", "The Cssmin was specified as the smallest and the Cssmax as the largest value for each analyte in a segment.", "Bioequivalence was computed using the log-transformed Cssmin and Cssmax values for each enantiomer and claimed when the 90% confidence interval (CI) of their ratio was .8 to 1.25.22,59", "The t1/2 in trial B was computed using the elimination rate constant (ke) and analyte concentrations C4 and C8 at hour 4 (t4) and hour 8 (t8), respectively, where ke = (ln C4 – ln C8)/(t8 – t4) and t1/2 = .693/ke.", "Missing data were confirmed to be missing completely at random and excluded without imputation.", "The hypothesized superior analgesia of desmetramadol to tramadol in trial B was tested using a 1-sided test at the 5% significance level.", "All other statistical comparisons were made using 2-sided tests at the 5% significance level and all CIs for bioequivalence were calculated with a 2-sided 90% confidence level.", "The intention-to-treat (ITT) population included all participants who were randomized to treatment, and was stipulated to be the primary dataset for analysis and statistical conclusions of significance.", "The per-protocol population was composed of a subset of participants from the ITT population who completed the study with no major protocol deviations.", "A major protocol deviation was one that could adversely affect the rights, safety, or well-being of the participants and/or the quality and integrity of data.", "Protocol deviations were assigned as being major or minor before unblinding.", "The efficacy population in trial B constituted participants who received all drug doses and had cold pressor efficacy data from all 3 segments.", "A sensitivity analysis was performed by conducting the analyses for the ITT, per-protocol, and efficacy populations as defined.", "All results presented are for the ITT population; unless otherwise specified, results from the per-protocol and efficacy population analyses supported those for the ITT population.", "The safety population included all patients who received study drug.", "All safety analyses were performed on the safety population.", "Of the 300 participants screened, 103 participants were randomized in consecutive trials A and B at 1 clinical research unit in the United States (Fig 3).", "All participants who were randomized (the ITT population) received treatment with study drug and 96 participants (93%) completed the study.", "A total of 7 of the 103 participants discontinued from the study after treatment was initiated.", "In the trial A cohort, 4 participants discontinued; 1 discontinued owing to AEs (grade 1 nausea, vomiting, and dizziness after 4 doses of desmetramadol in segment 1; no blood samples were collected and the participant did not advance to the remaining segments with placebo and tramadol) and 3 were lost to follow-up.", "In the trial B cohort, 1 participant was withdrawn because of a major protocol violation involving a urine test positive for a substance of abuse (cocaine) and 2 participants were lost to follow-up.", "Participants received 1,111 doses (96%) of 1,161 possible study drug doses in trial A, and received 1,575 doses (97%) of 1,620 possible study drug doses in trial B. Participants with missing study drug doses were evenly distributed across placebo (A, B = 2, 2), desmetramadol (A, B = 1, 1), and tramadol (A, B = 3, 2).", "All participants in trial B received all planned paroxetine doses while on study, or 525 of 540 (97%) possible doses.", "Most participants were Caucasian (92%) and baseline demographic characteristics such as age and body mass index were similar in both trial A and B cohorts (Table 2).", "Normal and intermediate CYP2D6 metabolizers constituted 96% and 100% of the trial A and B cohorts, respectively (for individual CYP2D6 genotypes, see Supplementary Table 1).", "In the absence of paroxetine (trial A), 20 mg desmetramadol dosed every 6 hours replicated the mean steady-state plasma profile of (+)-M1 produced by 50 mg tramadol dosed at the same frequency (Fig 4A).", "The desmetramadol and tramadol mean Cssmin and Cssmax for (+)-M1 were statistically bioequivalent (mean [SD] = 28 [7] ng/mL vs 26 [6] ng/mL and 37 [10] ng/mL vs 36 [10] ng/mL; 90% CIs, .85–1.08 and .88–1.13, respectively; Table 3).", "The Cssmin and Cssmax for (-)-M1 were 30% lower for desmetramadol compared with tramadol and just outside statistical bioequivalence (mean [SD] = 30 [6] ng/mL vs 21 [5] ng/mL and 42 [9] ng/mL vs 30 [9] ng/mL; 90% CIs, .69–.76 and .67–.76, respectively; Fig 4B and Table 3).", "Desmetramadol produced no circulating tramadol enantiomers, as expected (Fig 4C and 4D).", "Paroxetine given in 3 daily 20-mg doses in trial B produced a similar level of circulating paroxetine in tramadol and desmetramadol dosed segments (mean [SD] = 11 [8] ng/mL vs 12 [9] ng/mL, respectively; Table 3).", "Compared with trial A, paroxetine in trial B depressed tramadol (+)-M1 Cssmin (-61%) and Cssmax (-62%), but increased desmetramadol (+)-M1 Cssmin (46%) and Cssmax (41%; Fig 4E vs Fig 4A and Table 3).", "The paroxetine-induced changes in trial B caused desmetramadol Cssmin and Cssmax for (+)-M1 to each significantly exceed by 3.5-fold the corresponding tramadol Cssmin and Cssmax (mean [SD] = 38 [9] ng/mL vs 11 [6] a ng/mL and 51 [11] ng/mL vs 14 [8] ng/mL, respectively; P < .001; Table 3).", "The (+)-M1 t1/2 after tramadol dosing was double the t1/2 after desmetramadol dosing (mean [SD] = 18 [39] hours vs 8 [6] hours; P = .065).", "Paroxetine resulted in comparatively smaller changes for (-)-M1 (Fig 4F vs Fig 4B).", "Compared with trial A, the (-)-M1 Cssmin and Cssmax were decreased 17% for tramadol and increased 14–16% for desmetramadol in the presence of paroxetine (Table 3).", "The paroxetine-induced changes in trial B had the net effect of making desmetramadol and tramadol Cssmin and Cssmax for (-)-M1 statistically bioequivalent (mean [SD] = 25 [7] ng/mL vs 25 [8] ng/mL and 35 [10] vs 35 [10] ng/mL ; 90% CIs, .95–1.09 and .93–1.07; Table 3).", "Like the positive enantiomer, the t1/2 of (-)-M1 in trial B was greater for tramadol compared with desmetramadol (mean [SD] = 12 [8] hours vs 7 [5] hours; P < .001).", "As in trial A, desmetramadol produced no circulating tramadol enantiomers in trial B (Fig 4G and 4H).", "However, compared with trial A, the mean steady-state (+)-tramadol and (-)-tramadol plasma concentration profiles after tramadol dosing were increased 2-fold in the presence of paroxetine, as were the Cssmin and Cssmax (Fig 4G vs Fig 4C and Fig 4H vs Fig 4D; Table 3).", "The Cssmin, Cssmax and t1/2 had no statistically significant sequence or segment effects in either trial A or trial B.", "There was no carryover from segment to segment in either trial A or trial B, as evidenced by no detectable M1 or tramadol enantiomers in placebo-treated segments, and no detectable tramadol enantiomers in desmetramadol treated segments.", "Compared with mean tramadol (+)-M1 in the poor metabolizer and ultrarapid metabolizer of trial A, the mean desmetramadol (+)-M1 was increased 650% (41 ng/mL vs 6.3 ng/mL) and decreased 40% (22 ng/mL vs 36 ng/mL), respectively (Supplementary Fig 1).", "The mean predose pupil diameter in trial A was similar in the placebo, tramadol, and desmetramadol dosed segments (mean [SD] =6.1 [.9], 6.1 [.9], and 6.0 [1.1] mm, respectively; Table 4).", "After dosing, tramadol and desmetramadol each caused a significant decrease in pupil diameter compared with placebo (mean paired reduction [SD] = -.7 (.6) and -1.1 (.8) mm; each P < .0001).", "Tramadol and desmetramadol dosing did not cause mean responses in the drug liking–disliking and take drug again VASs to differentiate from placebo (Table 4).", "There was a significant treatment effect (P < .0001) in the strength of drug effect VAS and mean responses after tramadol and desmetramadol dosing were significantly elevated compared with placebo (mean [SD] = 32 [29] mm vs 12 [8] mm and 29 [28] mm vs 12 [8] mm; P < .001 and P = .0004, respectively).", "There were also significant segment (P = .004) and sequence (P = .034) effects in the strength of drug effect VAS (Supplementary Table 2).", "In the male population of trial A (n = 43), there was a similar and statistically significant decrease in average cold-induced pain perception at 30 seconds in participants treated with tramadol and desmetramadol compared with placebo (mean and standard error [SE] = -.46 [.19] and -.60 [.15]; P = .022 and P = .0005, respectively; Fig 5).", "There was no significant difference between tramadol and desmetramadol in trial A (P = .47).", "In the male population of trial B (n = 42) treated identically as trial A, except for the inclusion of paroxetine, tramadol failed to statistically differentiate from placebo (P = .90).", "In contrast, desmetramadol provided pain relief that was statistically superior to both placebo (P = .036) and tramadol (P = .003).", "The average change in paired pain scores between desmetramadol and placebo, and between desmetramadol and tramadol were similar (mean [SE] = -.75 [.28] and -.62 [.20], respectively; Fig 5).", "There was no significant treatment effect in the intensity of pain at first perception by participants in either trial A or trial B (Supplementary Table 3).", "The average duration of tolerance to pain in trial A was similar for desmetramadol (63 seconds) and tramadol (62 seconds), and each was significantly greater than placebo (mean paired increase relative to placebo [SE] = 12.6 [3.8] and 12.4 [4.4] seconds; P = .006 and P = .0076, respectively; Supplementary Table 3).", "In the presence of paroxetine, male participants in trial B tolerated pain 44% longer after desmetramadol than after tramadol (mean paired increase relative to placebo [SE] = 9.9 [1.9] seconds vs 6.9 [1.6] seconds; P < .001 and P = .001, respectively).", "In both trials, the average time to the first perception of pain was similar for tramadol and desmetramadol, and each was significantly greater than placebo.", "There was no significant treatment effect in the trial B female population (see Discussion and Supplementary Table 3).", "There was no significant sequence or segment effect in any pain measure in either trial A or trial B. Results from the sensitivity analyses were consistent with the primary analgesic analyses.", "One participant in trial A discontinued owing to AEs after administration of desmetramadol in the first segment.", "No participants discontinued from trial B owing to AEs.", "After dosing with tramadol and desmetramadol in trial A, participants reported a similar qualitative and quantitative profile of drug-related AEs (Table 5).", "AEs were reported in 49% and 44% of participants after tramadol and desmetramadol, respectively, compared with 24% of participants after placebo.", "The 5 most common drug-related AEs after desmetramadol and tramadol in trial A were nausea, dizziness, headache, somnolence, and pruritus.", "The severity of drug-related AEs in trial A were all grade 1 except for a single participant who reported grade 2 headache and dizziness after tramadol.", "Drug-related AEs were reported in 27%, 50%, and 67% of participants in trial B after placebo, tramadol and desmetramadol, respectively (Table 5).", "Compared with the desmetramadol AE profile in trial B, the tramadol AE profile in the same participants featured less nausea (-50%), somnolence (-60%), headache (-40%), vomiting (-78%), presyncope (-83%), and pruritus (-75%).", "The tramadol AE profile resembled placebo except for an increased incidence of dizziness (8-fold placebo) and muscle spasticity (absent from placebo).", "The incidence of muscle spasticity after tramadol was the same as after desmetramadol.", "Female participants in trial B made up 30% of the safety population and 43% of the specified AEs.", "Drug-related AE severity in trial B was all grade 1 except for 5 participants who had grade 2 drug-related AEs after placebo (somnolence), tramadol (asthenia), and desmetramadol (nausea/vomiting, feeling hot, hypotension).", "Less common drug-related AEs reported by participants in trial A and trial B are provided in Supplementary Table 4.", "No deaths or serious AEs were reported in either trial A or trial B.", "Respiration was assessed 1,744 times in trial A and 2,510 times in trial B.", "In trial A, respiratory rate was assessed before and after each of the 9 study drug doses in each of the 3 treatment segments (Fig 6).", "Desmetramadol and tramadol had no discernable predose versus postdose effect on respiratory rate compared with placebo, or compared with each other.", "Compared with baseline screening assessments in trial A, there was no effect of placebo, tramadol, or desmetramadol on the systolic or diastolic blood pressure, pulse, or respiration at the end of each treatment segment (Supplementary Table 5).", "Paired comparisons of respiration were made between tramadol and placebo, desmetramadol and placebo, and desmetramadol and tramadol with respect to the average postdose respiration.", "Average respiration after tramadol and desmetramadol were minimally reduced compared with placebo, and this decrease was statistically significant in the presence of paroxetine, but not in its absence (trial B mean paired difference [SD] = -.34 [.99] and -.30 [.90] breaths per minute; P = .004 and P = .012, respectively; Table 6).", "In addition to a significant treatment effect (P = .004), there was a significant segment effect (P < .001).", "There was no significant difference in respiration between desmetramadol and tramadol.", "Desmetramadol provided superior analgesia to tramadol in metabolically deficient participants, the same group in which tramadol efficacy was lost.", "Desmetramadol provided the same qualitative and quantitative safety profile as tramadol in metabolically unselected participants and the same as described in the FDA-approved tramadol label.35", "Desmetramadol thus obviates the metabolic liabilities of tramadol while preserving its safety profile, because it does not rely on the activity of CYP enzymes for its activity.", "This property of desmetramadol is significant because tramadol is widely used globally with 41 million prescriptions dispensed in 2017 in the United States alone,18,28 and an estimated one-third or more of patients treated with tramadol fail to metabolize it to its active metabolite with optimal kinetics.9,35,46,50,56,65", "The metabolic liabilities of tramadol could explain the misalignment between U.S. prescriber perceptions in the United Sates of modest efficacy and its approved indication for treating moderate to moderately severe pain.35,87", "Tramadol is racemic, and the negative and positive enantiomers are metabolized in vivo to (-)-M1 and (+)-M1, respectively.29,58", "The in vitro µ-opioid receptor binding affinity (Ki) is dominated by (+)-M1 (.0034 μmol/L), compared with substantially weaker affinities for (-)-tramadol (25 μmol/L), (+)-tramadol (1.3 μmol/L), and (-)-M1 (.24 μmol/L).", "The in vitro inhibition (Ki) of serotonin uptake is dominated by (+)-tramadol (.5 μmol/L), whereas the inhibition of norepinephrine uptake is mediated by the similarly potent (-)-tramadol (.5-1.6 μmol/L) and (-)-M1 (.9-1.4 μmol/L).16,58", "The analgesia of tramadol is thought to arise from a combination of µ-opioid receptor binding and inhibition of norepinephrine uptake in the descending pain inhibitory system.58", "Serotonin is a transmitter in descending inhibitory and excitatory projections and causes antinociceptive and pronociceptive effects, respectively, leading some investigators to question its role in mediating tramadol analgesia.1,58,73", "The role played by the enantiomers of tramadol and M1 in human analgesia has been the subject of investigation.", "Controlled trials in metabolically deficient patients demonstrated that M1 is necessary for analgesia in both experimental and surgical pain.43,55,70,71,84", "Other trials used opioid and α2-adrenoceptor antagonists to demonstrate that human tramadol analgesia is mediated by both opioid receptor agonism and monoaminergic modulation.14", "The specific contribution of the tramadol enantiomers to human tramadol analgesia has not been investigated.", "To our knowledge, this is the first human study to ask whether the tramadol parent enantiomers can be discarded and tramadol analgesia replicated by (-)-M1 and (+)-M1 alone; that is, whether M1 is not only necessary, but sufficient, to replicate tramadol analgesia in both metabolically unselected and deficient participants.", "A distinguishing feature of this study from single-dose trial designs was that participants were on the study drug for >2 days (54 hours, 9 doses).", "This duration allowed systemic concentrations, including central nervous system concentrations, to equilibrate before key assessments were made.", "It also allowed for the collection of safety information that more faithfully reflects actual clinical use.", "In trial A, 50 mg tramadol and 20 mg desmetramadol dosed every 6 hours gave systemic (+)-M1 levels that were bioequivalent and (-)-M1 levels there were nearly bioequivalent.", "In the absence of circulating tramadol enantiomers, desmetramadol produced similar responses as tramadol with respect to analgesia, pupil constriction, abuse measures, AE profile, and vital signs.", "If tramadol enantiomers contributed to analgesia, participants should have experienced greater analgesia after tramadol, but they did not.", "Serotonergic agents have been reported to cause mydriasis,49 and both pronociceptive and antinociceptive effects.1,58,73", "Compared with desmetramadol, tramadol exhibited relative pupil dilation (-.4 mm; post hoc P = .0017) and muted analgesia, possibly suggestive of the serotonergic effects of (+)-tramadol.", "The most straightforward interpretation of these findings is that circulating M1 enantiomers as provided by desmetramadol are not only necessary, but are also sufficient to replicate the therapeutic pharmacology of tramadol.", "In this interpretation, tramadol provides superfluous enantiomers with (+)-tramadol contributing unwanted metabolic liabilities related to the under or over production of the (+)-M1 opioid, and unwanted serotonergic activity that may negatively influence analgesia and potentially contribute to the risk of seizure and serotonin syndrome (discussed elsewhere in this article).", "The doses of tramadol and desmetramadol in trial A were advanced into trial B, where participants were made metabolically deficient by coadministration of paroxetine, a strong inhibitor of CYP2D6 and CYP2B6.33,79", "Studies in human liver microsomes indicate that tramadol is metabolized to M1 by CYP2D6 and to N-desmethyltramadol by CYP2B6 and CYP3A4; M1 is metabolized to O,N-didesmethyltramadol (M5) by CYP2B6 and CYP3A4 (Fig 7).72,88", "Although racemic M5 does bind to the μ-opioid receptor with substantial affinity in vitro (Ki = .10 μmol/L), it is highly polar and neither crosses the blood–brain barrier nor contributes to analgesia or centrally mediated AEs in vivo.29", "Metabolism of tramadol to M1 by CYP2D6 favors the positive enantiomer.55", "Consistent with these transformations, the presence of paroxetine in trial B depressed tramadol plasma (+)-M1 by approximately 60% and increased desmetramadol plasma (+)-M1 by approximately 40%.", "The effect of paroxetine on (-)-M1 levels was less pronounced, with tramadol plasma (-)-M1 decreased and desmetramadol plasma (-)-M1 increased by approximately the same amount.", "The net paroxetine effect in trial B caused tramadol and desmetramadol (-)-M1 to assume bioequivalent levels, and for tramadol (+)-M1 to be depressed to less than one-third of the desmetramadol (+)-M1 level.", "Consistent with a paroxetine block on tramadol metabolism by CYP2D6 and CYP2B6, (-)-tramadol and (+)-tramadol levels in trial B increased to 200% of their levels in trial A in the absence of paroxetine.", "Despite the elevated levels of tramadol enantiomers and bioequivalent (-)-M1, the depression of (+)-M1 in trial B was sufficient to cause the analgesic activity of tramadol to collapse to that of placebo.", "This finding is consistent with prior studies that demonstrated that M1 is necessary for tramadol analgesia in both experimental and surgical pain.43,55,70,71,84", "The finding underscores the actual role tramadol enantiomers play in mediating analgesia, because even elevated levels could not compensate for the loss of (+)-M1.", "In contrast, desmetramadol had no corresponding metabolic liability; in metabolically deficient participants of trial B, it produced therapeutic levels of both M1 enantiomers and analgesia as effective as in the metabolically unselected participants of trial A. Desmetramadol also normalized the abnormal levels of tramadol M1 seen in genetic poor metabolizers and ultrarapid metabolizers.", "As seen in trial A, desmetramadol returned M1 to therapeutic levels in a poor metabolizer and reduced M1 exposure in an ultrarapid metabolizer.", "Mechanistically, because desmetramadol does not depend on CYP2D6 for its plasma level, it obviates the metabolic liabilities of tramadol, regardless of whether the metabolic defect is due to inhibition of CYP2D6 (eg, by paroxetine in trial B) or CYP2D6 genetics.", "The lack of statistically significant analgesia in the trial B female population dosed with either tramadol or desmetramadol was expected a priori, because normally menstruating women exhibit a variable and increasing cold-induced pain tolerance and threshold over repeated stimulation.41", "Females were enrolled in trial B to collect data for the secondary safety and pharmacokinetic end points in both sexes.", "To ensure sufficient males would be enrolled to test the formal hypothesis and primary pain end point, trial B was intentionally overpowered to 97%.", "Desmetramadol had the same safety profile in trial B as in the approved tramadol label.35", "Consistent with selective reduction of the (+)-M1 opioid, participants in trial B dosed with tramadol exhibited a safety profile that resembled placebo except for dizziness and muscle spasticity.", "The latter AEs likely resulted from persistent monoaminergic activity.", "Desmetramadol had the same incidence of muscle spasticity in trial B as tramadol.", "Muscle spasticity was more common in trial B than trial A, possibly owing to the additive effect of paroxetine.", "The major route of excretion for tramadol and its metabolites is through the kidneys, with >90% of a tramadol dose appearing in the urine.29,45", "Inhibition of CYP2B6 by paroxetine in this study increased steady-state desmetramadol levels—approximately 40% for (+)-M1 and approximately 15% for (-)-M1—consistent with a role for CYP2B6 in desmetramadol elimination by its transformation to M5 (Fig 7).", "A cross-over study in 12 participants administered tramadol with either placebo, ticlopidine (CYP2B6 and CYP2D6 inhibitor), or ticlopidine with itraconazole (CYP3A4 inhibitor) demonstrated the following.31", "Ticlopidine alone decreased the formation rate of M1 consistent with inhibition of CYP2D6.", "The addition of itraconazole had no effect on tramadol pharmacokinetics or the rate of M1 formation rate compared with ticlopidine alone, suggesting that CYP3A4 is of limited importance in the metabolism and elimination of tramadol or desmetramadol in vivo.", "Another crossover study pretreated 12 participants for 5 days with placebo or rifampicin, an inducer of CYP2B6 and CYP3A4, before the administration of 100 mg oral tramadol.61", "Induction decreased the tramadol and M1 AUC by nearly the same amount (59% and 54%) and increased the M1 formation rate by only 12%, consistent with less available CYP2D6 substrate proportionally forming less M1 as the major cause of decreased plasma M1 and to a lesser extent enhancement of the M1 to M5 reaction.", "M1 is glucuronidated in vitro most actively by the UDP-glucuronosyltransferases 2B7 and 1A8, with 2B7 having a slight preference for (-)-M1 over (+)-M1 and 1A8 exhibiting strict stereoselectivity for (+)-M1.44", "Previous human studies have reported that an oral dose of tramadol is excreted in the urine in the following forms and approximate quantitative ranges: unchanged tramadol (12–32%), unchanged M1 (>10%), M1 glucuronide (2–5%, 24%, 30%, 31%, and 48%), M1 sulphate (2–5%), unchanged N-desmethyltramadol (>10%), unchanged M5 (5–10%), M5 sulphate (5–10%), and M5 glucuronide (2–5%, 10%, 15%, and 16%).31,45,51,52,68,88", "These data collectively suggest that the glucuronidation of desmetramadol, and conversion of desmetramadol to M5 by CYP2B6 are metabolic transformations involved in the in vivo elimination of desmetramadol in the urine, together with unchanged desmetramadol.", "Seizures and serotonin syndrome after normal doses of tramadol alone are exceedingly rare.26,27,36,53", "The risk for seizure and serotonin syndrome increases with the concomitant use of serotonergic drugs, although on an absolute basis the risk remains rare and it is common clinical practice to coprescribe tramadol and serotonergic antidepressants in pain disorders.53", "Coprescribing antidepressants that are also CYP2D6 inhibitors (eg, bupropion, duloxetine, fluoxetine, or paroxetine) was among several factors associated with enhanced risk of tramadol-induced serotonin syndrome.53", "As shown in this study, CYP2D6 inhibition decreased tramadol clearance and exposed a participant to the combined serotonergic effect of the antidepressant and markedly elevated levels of the serotonergic (+)-tramadol enantiomer, which may reach supratherapeutic levels.", "Desmetramadol may have a lower risk of serotonin syndrome when combined with antidepressants because the serotonergic (+)-tramadol enantiomer is absent, and because plasma levels of its active enantiomers undergo clinically insignificant changes in response to CYP2D6 and CYP2B6 inhibition.", "A major cause of schedule II opioid lethality is respiratory depression mediated by agonism of μ-opioid receptors.63", "Participants with respiratory depression (oxygen saturation of <94%) after tramadol overdose had ingested a median dose of 2,500 mg (range = 500–4,000 mg), or 25 times the maximum approved therapeutic dose, compared with participants with no respiratory depression who had ingested a median dose of 1,000 mg (range = 450–6,000 mg).60", "These properties explain why lethal overdoses owing to tramadol alone are rare.11,12,62,64,69", "Achiral analyses of blood from fatal intoxications with tramadol and M1 found mean blood M1 levels of 1,900 and 1,300 ng/mL, or 38-fold and 26-fold the mean M1 level in this study, respectively.11,42", "Even at therapeutic doses, schedule II opioids and the biased opioid receptor ligand TRV130 caused clinically significant respiratory depression, whereas tramadol does not.3,29,34,39,48,66,67,75,76,83", "Tramadol is reported to cause a minimal and clinically insignificant decrease in respiration in healthy participants and patients at therapeutic doses.3,48,83", "In 1 study, the decrease in respiration was associated with an increase in plasma epinephrine.48", "It is unknown whether this respiratory depression by tramadol is mediated by its opioid or monoaminergic mechanism, because adrenergic agents also cause respiratory depression and naloxone failed to fully reverse it.4,5,47,77,85", "The effect of serotonin on ventilatory control is more uncertain and depends on the types of respiratory neuron and 5-hydroxytryptophan receptor.2", "In this study, mean respiration after tramadol and desmetramadol were minimally decreased compared with placebo, and this decrease was statistically significant in trial B, but not in trial A. To the extent this represents a bone fide phenomenon in this study, it is most likely attributable to the monoaminergic activities of tramadol and desmetramadol rather than their opioid activities, because the paroxetine-induced depression of the (+)-M1 opioid in trial B had no effect on its magnitude.", "Consistent with its mixed mechanism pharmacology, tramadol is less prone to abuse and diversion than schedule II opioid analgesics and was made a schedule IV controlled substance in 2014 in the United States.10,17,21,32", "The abuse potential of tramadol is attributed to the (+)-M1 opioid, which exhibits rate-limited and delayed transport into the central nervous system.32,74", "It has been suggested that experienced drug abusers are the most sensitive clinical population for assessing abuse liability.7", "However, studies have consistently shown that opioids elicit similar signals of abuse-related subjective effects in non–drug abusers and drug abusers.6,8,19,78,89–92", "Morphine, oxycodone, and hydrocodone all elicit robust and statistically significant responses in non–drug abusers in one or all of the measures for drug liking–disliking, take drug again, and strength of drug effect (Table 7).", "In trial A of this study, 50 mg tramadol and 20 mg desmetramadol exhibited similar and statistically significant responses for strength of drug effect, but were indistinguishable from placebo for drug liking–disliking and take drug again.", "Strength of drug effect seems to be the more sensitive of the 3 measures in other studies of opioids as well.", "Absent signals for drug liking–disliking and take drug again are unlikely to be due to insufficient statistical power, because the present study (trial A) distinguishes itself with a sample size that is 2–5 times larger than the size of a typical human abuse liability study.", "The lack of responses for these abuse measures is likely the result of dose.", "In recreational drug users (n = 22) who were not opioid experienced, 50 mg tramadol caused no significant subjective effects, but 100 mg caused significant responses in all 3 abuse measures.89", "In nondependent recreational opioid users (n = 8) trained to discriminate hydromorphone and methylphenidate, tramadol exhibited a positive dose-dependent trend in drug liking–disliking and strength of drug effect between 100 mg and 400 mg.19", "No dose attained statistical significance, likely because of the small sample size.", "Lower doses of tramadol (50 mg and 100 mg) were identified as placebo, whereas 200 mg and 400 mg doses were identified as hydromorphone.", "The 400 mg dose also increased scores on a stimulant scale, consistent with the monoaminergic activity of tramadol.", "For prescribers seeking to decrease the morphine milligram equivalents in their patients who require effective analgesia, tramadol is a viable option to the schedule II opioids.", "Tramadol provides analgesia for moderate to moderately severe pain but, compared with the schedule II opioids, has a lower abuse potential and a substantially wider margin of safety with respect to respiratory depression and lethality in overdose.", "Critical shortcomings of tramadol relate to its metabolic liabilities.", "The findings from this study indicate that desmetramadol offers the safety and analgesia of tramadol, but without its metabolic liabilities and related drug–drug interactions.", "Desmetramadol could, therefore, offer expanded safety and usefulness for clinicians who prescribe tramadol as an alternative to schedule II opioids." ]

[ "Desmetramadol delivers the active metabolite of tramadol.", "P450 enzyme inhibition made participants metabolically deficient.", "Tramadol lost its analgesic efficacy in metabolically deficient participants.", "Desmetramadol preserved its efficacy in metabolically deficient participants.", "Desmetramadol and tramadol had the same safety profile." ]

[ "MEDI", "NEUR" ]

[ "DAT SPECT", "Longitudinal", "Outcome prediction", "Parkinson's disease", "Radiomics", "Textural features" ]

[ "2728", "2741", "2805", "2808" ]

[ "Parkinson's disease (PD) is a progressive, degenerative movement disorder, characterized by neuronal loss in the substantia nigra with the loss of dopaminergic terminals in the basal ganglia (Brooks et al., 1990; Garnett et al., 1987; Stoessl et al., 2011).", "Given the absence of proven disease modifying therapies for PD, there is a critical need to establish biomarkers of disease progression (Marek et al., 2008); e.g. an aim of the Parkinson's Progressive Marker Initiative (PPMI) (Parkinson Progression Marker Initiative, 2011).", "Furthermore, there is significant interest in prognostication of disease outcome, to properly adapt and power clinical trial studies, as applied to appropriate patients.", "Stratification of PD based on expected prognosis would allow better designs of disease modifying trials, with greater power to ascertain efficacy.", "Imaging of the dopaminergic system with 123I-ioflupane-dopamine transporter (DAT) SPECT is now widely used (Catafau and Tolosa, 2004; Grachev et al., 2012; Kupsch et al., 2012).", "DAT SPECT images are commonly assessed visually.", "However, there are increasing efforts to quantify uptake in regions-of-interest (ROIs) in order to provide more objective measures of disease severity (Badiavas et al., 2011; Koch et al., 2005).", "Quantitative imaging biomarkers also have the potential for earlier detection of disease.", "A significant way in which DAT SPECT imaging has been helpful is to identify a subgroup of PD patients who are symptomatic without evidence of dopamine deficit (SWEDDs).", "These patients have a significantly better prognosis.", "What remains of critical importance, is to discover further subsets in the PD population of different outcomes, to enable significantly improved targeted clinical trials for the assessment of novel therapies for PD.", "Advanced radiographic metrics that quantify heterogeneity in shape and uptake have been explored, primarily in the field of oncology, in order to improve diagnosis as well as prediction of treatment response and survival outcome in different cancers (Eary et al., 2008; El Naqa et al., 2009; Hatt et al., 2015; Rahmim et al., 2016b; Soufi et al., 2017; Tixier et al., 2014; Tixier et al., 2011; van Velden et al., 2011; Vriens et al., 2012).", "Overall, the field of radiomics aims to extract a large number of quantitative features from radiological images, aiming to uncover correlates of disease characteristics that are ordinarily not visually observed or quantitatively measured (Aerts et al., 2014; Asselin et al., 2012; Chicklore et al., 2013; Kumar et al., 2012; Lambin et al., 2012).", "In a previous work, we assessed whether radiomics analysis improved correlations with clinical assessments (Rahmim et al., 2016a).", "The present work investigates whether radiomics analysis significantly adds to the ability of non-imaging and conventional measures to predict outcome.", "This is plausible since pathophysiologic studies have demonstrated very heterogeneous patterns of dopamine loss in the basal ganglia (Kish et al., 1988).", "As such, we hypothesize that radiomics analysis has the potential to significantly improve prediction of outcome in Parkinson's disease patients.", "Longitudinal data were extracted from the PPMI database (www.ppmi-info.org/data) (Parkinson Progression Marker Initiative, 2011).", "The movement disorder society – unified Parkinson's disease rating scale (MDS-UPDRS) – part III (motor) in year 4 was used as outcome (referred to as UPDRS-III from here on).", "Predictors included demographics (age, sex), as well as baseline (year 0) and year 1 DAT SPECT images and clinical measures.", "Clinical measures included: disease duration (DD) taken with respect to (a) time of diagnosis (DD-diag.)", "as well as (b) time of appearance of symptoms (DD-sympt.).", "This also included (c) the motor measure, UPDRS-III, and (d) the non-motor cognitive measure, Montreal Cognitive Assessment (MoCA), for both baseline (year 0) and year 1.", "For consistency, we only included patients with SPECT data acquired on similar kinds of scanner (Siemens, 2-headed ECAM or Symbia systems), and subjects who underwent high-resolution 3 T MRI.", "These selection criteria resulted in 64 PD subjects (38 male, 26 female; age at year 0: 61.9 ± 7.3, range [46,78]), with widely distributed year 4 outcome UPDRS-III: 27.3 ± 14.7; range [3,77].", "Imaging was performed 4 ± 0.5 h following injection of DAT SPECT (123I-Ioflupane; 111–185 MBq).", "Thyroid update was blocked via pre-treatment of subjects with saturated iodine solution (10 drops in water) or perchlorate (1000 mg) prior to injection.", "Data acquisition consisted of 128 × 128 raw SPECT projection data acquired every 3 degrees, 120 projections, 20% symmetric photopeak windows centered on 159 keV and 122 keV, and a total scan duration of ~ 30–45 min.", "A HERMES system (Hermes Medical Solutions, Stockholm, Sweden) was used to perform iterative OSEM reconstruction on the input raw SPECT projection data, for all studies to ensure consistency.", "Subsequently, PMOD (PMOD Technologies, Zurich, Switzerland) was used for attenuation correction.", "Ellipses where drawn on the images and Chang 0 attenuation correction was applied invoking a site-specific mu as empirically derived from phantom data (as acquired in site initiation for the trial).", "Following this, standard 3D Gaussian post-smoothing (6.0 mm FWHM) was applied.", "Image processing consisted of segmentation of MRI images and registration of SPECT images onto MRI images, followed by quantitative feature extraction from SPECT images, as follows:", "Segmentation: We focused on six ROIs, namely the caudate, putamen and ventral striatum (VS) (both right and left) for analysis.", "The occipital cortex was also segmented, which is commonly used as a reference region for normalization of counts (Badiavas et al., 2011; Djang et al., 2012; Koch et al., 2005).", "Segmentation consisted of: 1) affine registration of the T1-weighted MRI of the subject to the MNI305 atlas (Evans et al., 1993); 2) initial volumetric labeling of the registered MRI of the subject using the labels of the atlas; 3) correction of image inhomogeneity, i.e., variation of the image intensity due to the B1 bias field, using the results of the previous stage; 4) nonlinear volumetric alignment of the subject's affine-registered and inhomogeneity-corrected MRI to the MNI305 atlas; and 5) propagation of the atlas labels to the MRI generated in the previous step and then, mapping of the results back to the original MRI of the subject.", "The classification of each point in the space to a given label was achieved by finding the segmentation that maximized the probability of input (image) given the prior probabilities from the training set (MNI305 atlas).", "Details of this framework are presented by Fischl et al., and an implementation of the methods is available in FreeSurfer (Fischl et al., 2002; Fischl et al., 2004a; Fischl et al., 2004b).", "Registration: To register SPECT images of each subject to the MRI of the subject, we employed a rigid, information-theory-based co-registration approach (Penny et al., 2011), implemented in two steps.", "The algorithm uses the normalized mutual information as the objective function along with a Gaussian smoothing kernel with a width of 7 mm.", "It also uses two separation levels, the average distance between sampled points of 4 and 2 mm, for the coarse to fine registration of the images, as elaborated by Collignon et al. (1995).", "Since DAT SPECT images have lower spatial resolution (typically 10 mm) compared to MRI (typically 1 mm), and depict centralized hyper intense striatal uptake (with minimal uptake elsewhere), standard co-registration commonly performs poorly.", "To tackle this, in the next stage, we performed linear intensity normalization of the T1-w images so that the average intensity of white matter equals 100.", "Then, we set the intensity value of the caudate and VS to 4000 and putamen to 1000.", "Finally, we employed the rigid co-registration algorithm on the co-registered DAT SPECT images from the previous stage and the manipulated T1-w images.", "In this way, the hyper intensity regions in the SPECT image are forced to better align to the regions around the favorable structures.", "In the end, we overlaid the structures segmented in MRI on the co-registered SPECT images.", "Furthermore, the more (m) vs. less (l) affected sides of the structures were determined with reference to the SPECT uptake at the putamen.", "Feature extraction: A total of 92 imaging features were extracted, including 13 first-order intensity features, 22 morphological features, and 57 second- and higher-order textural features, describing the intensity and spatial distribution of radiotracer uptake.", "The definitions of these features are elaborated in the supplement.", "Of the 92 features, 75 of them incorporated DAT SPECT uptake information, while 17 of the morphological features were purely based on the MRI-based ROIs (see supplement).", "This latter is justified given findings of structural brain atrophy in PD patients (Brenneis et al., 2003; Zeighami et al., 2015).", "Of the 92 features, 4 features were conventional measures of SUVmax, SUVmean, SUVpeak and ROI-volume, though these were extracted for each of the 6 ROIs and on both years of imaging (year 0 and 1).", "Multivariate analysis was performed in three groups, involving: (1) use of only non-imaging features (i.e. demographics and clinical measures), (2) additional use of conventional imaging features, and (3) additional use of radiomic features.", "For each group of predictors, a single regression tree was fitted to explore interactions among variables, followed by random forest analysis (R package randomForest) to identify the top predictors.", "Random forest analysis is an ensemble non-parametric machine learning method.", "It can handle complicated interactions among large number of variables and their non-linear effects efficiently.", "In fact, it has shown favorable performance in comparison to other machine learning methods in the context of radiomics analysis (Parmar et al., 2015).", "An observation is that the prediction model cannot be visualized using a single straightforward formula because the prediction is done through a collection of trees, and each tree has its own formula, which is also the case with artificial neural networks (ANNs).", "Year 4 UPDRS-III was used as the response variable in all these analyses.", "Features with zero variance were removed before the analysis.", "For each group of predictors, 5000 bootstrap samples were randomly drawn from all patients.", "Each of these bootstrap samples included about two-third of the total number of patients, and the remaining one-third patients were called out-of-bag (OOB) for that bootstrap sample.", "A decision tree was grown from each bootstrap sample and internally validated using the corresponding OOB sample.", "This procedure was repeated for all bootstrap samples.", "The minimum node size was set to 5, and at each node, 1/3 of candidate features were randomly selected to determine how to split the node.", "To rank the importance of a predictor, all values of this predictor were permuted among all individuals in the OOB, and we put both permuted and non-permuted data down all trees to obtain their predictions.", "The average absolute difference in prediction between permuted and non-permuted OOB data was then used to measure the variable importance score and to select top predictors.", "Variables with larger difference scores were considered more important.", "Prediction accuracy of random forest analysis was assessed using leave-one-out-cross-validation (LOOCV).", "Each time, one patient was excluded from the random forest analysis.", "The developed random forest algorithm was then applied to this excluded patient to obtain a prediction of UPDRS-III score.", "We then calculated the absolute difference between the predicted and observed UPDRS-III scores for the patient.", "The process was repeated for all patients.", "The average difference Δ from all patients was used to compare predictions using different groups of predictors.", "In particular, we investigated added value of radiomics features by comparing Δ from group 2 and group 3.", "For longitudinal analysis, combined features from year 0, year 1, as well as their differences were utilized.", "To assess performance when only baseline information was included, the abovementioned analysis was repeated when data from only year 0 were used to predict outcome in year 4.", "Fig. 1 depicts 3D segmentation as performed on a typical study, as well as transaxial, coronal and sagittal slices of the DAT SPECT image overlaid with the segmentations.", "Fig. 2 shows a single decision tree (left) and its performance (right).", "Patients with baseline UPDRS-III greater than or equal to 36 had the highest median year 4 UPDRS-III score.", "For patients with baseline UPDRS-III < 36, there were interactions among different radiomic features.", "However, it is seen that using a single decision tree results in a limited number of outcomes, which is why a random forest of decision trees is utilized to provide improved performance (next).", "Fig. 3 depicts our main findings.", "Non-imaging features (specifically, demographics and clinical motor and non-motor measures) are used in all predictions.", "When only these measures are used, absolute error in prediction of outcome is 8.93 ± 0.91.", "It is seen that addition of only conventional imaging features does not improve prediction performance (9.00 ± 0.88).", "However, addition of radiomic features results in an absolute error in outcome prediction of 4.12 ± 0.43.", "Given the year 4 UPDRS-III distribution (27.3 ± 14.7), this represents a significant (p-value < 0.001) average improvement of 18% in prediction of outcome, and can be readily observed visually.", "Fig. 4 depicts the findings when data from only year 0 (baseline) are used to predict outcome.", "It is generally seen that predictions are poorer.", "What is more, in comparison to use of non-imaging features (10.77 ± 1.10), outcome prediction it not improved when adding conventional features (10.63 ± 1.07) or even by further addition of radiomic feature (9.93 ± 1.10).", "Overall, it is seen that longitudinal images (acquired at year 0 and year 1) are required to enable significantly improved prediction of motor outcome in year 4.", "Fig. 5 plots relative contribution of different predictors via a metric %IncMSE: incremental percent change in mean square error (MSE) by exclusion of a single feature.", "It is seen that UPDRS-III (motor) from year 1 and then from year 0 are the most important predictors.", "At the same time, it is seen that radiomics features were among the top 10 most important predictors.", "The critical observation, from Fig. 3, is that it is through the combination of conventional measures with radiomic features that one is able to significantly improve prediction of outcome.", "There exist two intertwined challenges in PD biomarker discovery.", "One is to identify biomarkers that track PD progression, and the other is to predict outcome (Marek et al., 2008).", "Both are very important clinically and in the discovery of disease modifying therapies.", "PD progression biomarkers are needed to assess efficacy of such therapies, while better prediction of outcome is needed in order to discover PD sub-types so as to more effectively design clinical trials in the first place.", "In the present work, we utilized motor assessment (UPDRS-III) in year 4 as the outcome measure being predicted.", "We plan to extend our work to prediction of other outcomes, including cognitive assessment (MoCA) and time to initiation of symptomatic therapy (TIST).", "In fact, it is plausible that use of a single clinical metric may not be sufficient to truly track PD progression.", "As a result, it is meaningful to use a combination of metrics, or global statistical tests for assessment of disease progression in clinical trials (Huang et al., 2009), which we plan to utilize in future efforts involving prediction of outcome in PD.", "There exist considerable difficulties and uncertainties with PD disease metrics.", "Disease duration is a particularly problematic metric, given the subtle and often nonspecific nature of early symptoms.", "The patients' abilities to detect the first symptoms is highly variable, and is impacted by a number of factors such as personality, education level and professional background, and the type of initial symptom (e.g. tremor vs. bradykinesia).", "This somewhat subjective nature of UPDRS evaluation makes the disease duration metric prone to inter-rater variability.", "UPDRS-III motor assessment itself is a highly variable measure (Horne et al., 2016; Mentzel et al., 2016), involving both patient- and rater-dependent variabilities.", "To reduce such variability, we averaged motor measures, if additionally performed up to 6 months before or after the visit.", "For instance, for year 4 outcome prediction, if a patient had a visit at 3.5 years and/or 4.5 years post-enrollment, the measures would be utilized and averaged with year 4 measures.", "In our past efforts, we investigated use of advanced texture analysis in quantitative brain PET imaging, in studies of PD (Gonzalez et al., 2013; Klyuzhin et al., 2016; Sossi et al., 2012) and Neuroinflammation (Rahmim et al., 2012).", "Furthermore, we found strong evidence that such measures retain their information even as one transitions from the higher resolution domain of PET images to the lower resolution domain of SPECT images by significant post-reconstruction blurring of PET images (e.g. up to 1 cm) (Blinder et al., 2014).", "In a subsequent DAT SPECT study (Rahmim et al., 2016a), we also demonstrated significantly improved correlation of radiomic features with clinical assessment.", "This prompted the present study, for the challenging task of predicting clinical outcome, making use of radiomic features involving DAT SPECT images.", "It is also worth noting that a potential of radiomic features is to move beyond conventional measures that require access to a good reference region for normalization, enabling more subtle detection and assessment of disease (Campbell and Shokouhi, 2017).", "The large number of features used in radiomics analysis is a concern in the context of the curse of dimensionality, and there exist increasing scrutiny (Chalkidou et al., 2015) and efforts (Kumar et al., 2012) to avoid problems of overfitting and false discovery.", "One approach is to pre-eliminate a number of features as shown in independent studies to depict poor repeatability (test-retest) or poor reproducibility to variations in image reconstruction and processing parameters (Ashrafinia et al., 2017; Doumou et al., 2015; Galavis et al., 2010; Grkovski et al., 2015; Hatt et al., 2013; Leijenaar et al., 2013; Lu et al., 2016; Lv et al., 2017; Shiri et al., 2017; Tixier et al., 2012; van Velden et al., 2016).", "An alternative approach, which we have pursued in this work, is to perform feature selection within the training dataset itself, using unsupervised methods (Bartenhagen et al., 2010; Dy, 2008; Mitra et al., 2002), to reduce the dimensionality of the data.", "This involved elimination of features that have very low dynamic range or those that are very highly correlated with other features (redundancy).", "Among predictors used in this work, we also included genomics profiling.", "In particular, we used a reduced list of 8 alpha-synuclein (SNCA) single nucleotide polymorphisms (SNPs), given previous findings on their association with increased risk of PD onset (Guella et al., 2016).", "However, no significant genomic interactions were found for progression.", "Our ongoing efforts include correlation of the SNPs with imaging phenotypes to boost statistical power, and analysis of a wider array of SNPs.", "We plan on performing extensive analysis on a larger patient dataset, including a larger training set as well as separate validation set, in order to assess reproducibility of our findings of significantly improved prognosis with inclusion of radiomic features.", "We finally note that there is increasing recognition for the critical role of standardization and reproducible research for effective progress in the hunt for and established utility of biomarkers (Collins and Tabak, 2014; Economist, 2013; Poste, 2011).", "This is equally a concern in the field of radiomics which involves complicated imaging feature definitions and analyses (Hatt et al., 2017).", "To this end, there are now significant efforts underway for standardization of imaging biomarkers (Zwanenburg et al., 2016), which are critically needed for reproducible application of radiomics features and their successful translation to clinical practice.", "This work has shown that use of longitudinal data (year 0 and year 1), combined with radiomics analysis, can result in significant improvements in prediction of outcome.", "Our results demonstrated that addition of radiomic features to conventional measures significantly improved (p < 0.001) prediction of outcome (namely that of year 4 motor performance as assessed using MDS-UPDRS-III), reducing the absolute error of prediction from 9.00 ± 0.88 to 4.12 ± 0.43 (MDS-UPDRS-III distribution: 27.3 ± 14.7).", "Radiomics analysis of DAT SPECT images holds significant potential towards development of effective biomarkers for prognostication of PD, with implications in design of clinical trials.", "None of the authors report any conflict of interest.", "The project was supported by the Michael J. Fox Foundation, including use of data available from the PPMI—a public-private partnership—funded by The Michael J. Fox Foundation for Parkinson's Research and funding partners (listed at www.ppmi-info.org/fundingpartners).", "This work was also partially supported by the Natural Sciences and Engineering Research Council of Canada, and the National Natural Science Foundation of China (grant 61628105)." ]

[ "Aim to enable image-assisted prediction of outcome in Parkinson's disease.", "Radiomics analysis of clinical dopamine transporter (DAT) SPECT images (DaTscans).", "Significant improvement in prediction of motor outcome." ]

[ "BIOC" ]

[]

[ "1300" ]

[ "T lymphocyte tolerance is essential for limiting autoimmune disease.", "Tolerance occurs “centrally” when developing thymocytes with strongly self-reactive T cell receptors (TCRs) are deleted following engagement of self-antigen-derived peptides presented by major histocompatibility complex (MHC) antigens.", "The expression of thousands of tissue-specific self-antigens (TSAs) by medullary thymic epithelial cells (mTEC) is directly promoted by AIRE, a poorly understood transcriptional regulator (Mathis and Benoist, 2009; Klein et al., 2014).", "Reflecting its importance, AIRE deficiency is defined by the APS1/APECED syndrome for which autoimmune polyendocrinopathy and chronic mucocutaneous candidiasis are pathognomonic (Nagamine et al., 1997).", "There are also several mechanisms of peripheral T cell tolerance, including requirements for co-stimulatory signals for the activation of naive T cells; the expression of molecular “brakes” (e.g., CTLA-4, PD-1) by activated T cells; and the suppression of effector T cells in trans by FOXP3-expressing T-regulatory (T-reg) cells.", "Reflecting its importance, FOXP3 deficiency is defined by early-onset, life-threatening autoimmunity (Bennett et al., 2001; Wildin et al., 2001).", "Central and peripheral tolerance mechanisms have likewise been hypothesized to shape the B cell compartment.", "Thus, self-reactive B cells developing in the bone marrow may be censored by clonal deletion, clonal anergy, or B cell receptor (BCR) editing in which secondary gene rearrangements replace the initial BCR with a new specificity (Goodnow et al., 2010; Pillai et al., 2011; Übelhart and Jumaa, 2015).", "Peripheral B cell tolerance is less well characterized, although some checkpoints have been inferred.", "For example, immature transitional B cells recently emigrated from the bone marrow contain many autoreactive and polyreactive cells, whereas there are relatively few among mature naive B cells, strongly suggesting that tolerance is imposed as transitional B cells differentiate into naive B cells (Wardemann et al., 2003).", "Interestingly, this B cell checkpoint is T cell dependent, as reflected by its impairment in patients with T-reg deficiencies (Kinnunen et al., 2013).", "Likewise, CD40L and MHC class II deficiencies that each impair T-B interactions also display more autoreactive B cells (Meffre and Wardemann, 2008).", "These considerations raise the possibility that B cell tolerance is largely governed by the state of T cell tolerance.", "Certainly, any autoreactive B cell that might progress through to the naive B cell compartment of a healthy individual should lack cognate autoreactive T cells to help it mature.", "Likewise, T cell help is required in the germinal center (GC) reaction in which B cells undergo somatic hyper-mutation (SHM) of the immunoglobulin (Ig) variable (V) region genes, thereby driving T cell-dependent selective expansion of clones with increased antigen affinity (Brink, 2014).", "The question that then arises is whether major defects in central T cell tolerance provoke wide-ranging losses of B cell tolerance at either or both of these stages.", "An approach to assessing this is to examine B cell reactivities in AIRE-deficient APS1/APECED patients whose under-expression of TSAs in the thymus is predicted to lead to increased numbers of peripheral autoreactive T cells.", "Thus, there are reports of APS1/APECED patients carrying autoantibodies against twenty-five TSAs, with prevalence ranging from 6% to 69% (Kisand and Peterson, 2015).", "Their specificities include steroidogenic enzymes, consistent with the patients’ polyendocrinopathies (Krohn et al., 1992; Uibo et al., 1994; Winqvist et al., 1993).", "In addition, most patients display autoreactivities toward type I IFNs and T helper (Th)-17-related cytokines, antibodies to which limit resistance to Candida infection (Kisand et al., 2010; Meager et al., 2006; Puel et al., 2010).", "These findings notwithstanding, there has been no large-scale analysis of the scope and nature of autoantibodies in APS1/APECED patients, thereby resolving how T cell tolerance impacts upon B cell tolerance in humans.", "By analyzing 81 APS1/APECED patients, we found that each was much more likely than a healthy relative or an unrelated control to harbor strong serum reactivities toward ∼100 human proteins.", "About 10 of those, including type I IFNs and interleukin-22 (IL22), were recognized by almost all patients, whereas others were mostly “private specificities.”", "Hence, 81 patients collectively harbored antibodies toward >3,700 human proteins.", "Focusing on antibodies to type I IFNs, IL22, and IL17, we found unexpectedly that most were reactive to conformational determinants and included highly mutated antibodies of sub-picomolar affinity.", "Because their gemline counterparts were not self-reactive, B cell autoreactivity was most probably driven by self-reactive T cells in the GC reaction.", "The autoantibodies commonly neutralized their targets in vivo, and APS1/APECED patients with signature type 1 diabetes (T1D)-associated antibodies (e.g., anti-GAD65) commonly failed to develop T1D so long as they harbored powerfully neutralizing IFNα-specific antibodies.", "Thus, autoantibodies naturally arising in subjects with defective central T cell tolerance may be disease ameliorating.", "Sera from 81 APS1/APECED patients from discrete Finnish, Norwegian, Slovenian, and Sardinian cohorts were directed against a ProtoArray displaying ∼9000 immobilized recombinant human proteins or protein fragments.", "Because some patients were sampled longitudinally, 97 sera were assayed in total.", "Control sera were from healthy first-degree relatives (n = 9) and healthy unrelated volunteers (n = 12) across the same age range.", "Data readouts for the binding of individual sera were normalized by applying robust linear modeling (Sboner et al., 2009), whereafter each signal was assigned a Z score denoting the number of standard deviations (SD) above or below the mean of the combined healthy relatives and controls.", "Most patients and the combined controls displayed Z scores of 1–2 for ∼200 proteins (Figure 1A).", "However, when the convention was employed of defining Z ≥ 3 as bona fide positives, the patients segregated from the two control cohorts, considered either jointly or separately.", "Thus, each control serum displayed reactivities of Z ≥ 3 toward an average of ≤20 proteins, with most recognizing < 10 (Figures 1A, 1B, and S1A).", "Given that there was inter-individual variation, the 21 control sera collectively displayed Z ≥ 3 reactivities toward 406 distinct proteins, i.e., ∼5% of those displayed on the array (Figure 1B).", "For only 2 proteins was Z ≥ 4, and for none was Z ≥ 5 (Figures 1A and 1B).", "Hence, as expected, the control cohorts largely lacked high-titer serum autoreactivities.", "Conversely, most patients at any one time displayed Z ≥ 3 autoreactivities toward ≥ 80 proteins (Figures 1A, 1B, and S1A).", "These data were re-analyzed with stringent procedures to minimize false-positives, including exclusion of any signals that might have arisen from cross-sample print contamination.", "With this achieved, the patients’ “private” autoantibody repertoires collectively detected 3,731 distinct targets (Figure 1B).", "Furthermore, almost all patients displayed Z ≥ 4 scores for at least 10 proteins (mean of ∼30), collectively recognizing > 1,500 proteins, and > 50% of patients displayed Z ≥ 5 scores for ≥ 10 proteins (mean of > 12), collectively recognizing 636 proteins (Figures 1A and 1B).", "Hence, high-level reactivity toward multiple self-proteins was a disease-defining property.", "This was further illustrated by the qualitative difference in Z score distribution curves for patients versus controls, which cannot simply be explained by there being 5-fold more patient sera (Figure 1C).", "Thus, whereas sampling greater numbers would likely have increased the protein species detected by control cohorts at Z ≥ 4, it would not bridge the 1,000-fold gap between two proteins detected by 21 control sera versus > 1,500 proteins detected by 97 patient sera (Figure 1B).", "In sum, 81 different patients collectively displayed strong reactivities to >40% of human proteins arrayed.", "For most proteins (blue dots 13–3731, Figure 1D), reactivities were spread across the cohort, reflecting high inter-patient variation, whereas ∼12 proteins (blue dots 1–12, Figure 1D), including several type I IFNs, were recognized by > 60% of patients, as reported (Meager et al., 2006).", "However, the “public specificities” were not enriched among the 126 reactivities shared between patients and controls at z > 3 (red dots, Figure 1D), emphasizing that their common autoantigenicity is unique to the patients.", "Patient autoreactivity frequencies were largely comparable across geographical locations, albeit somewhat less in Norway and Slovenia, and age ranges (Figures S1B and S1C).", "Indeed, most anti-IFN autoantibodies of APECED patients were reported to increase early in life and remain stable thereafter (Meager et al., 2006; Wolff et al., 2013).", "The collective targets of patient antibodies included intracellular, trans-membrane, and secreted proteins.", "Because many proteins displayed on the ProtoArray may be denatured, there may be false-negatives that underestimate patient reactivities to conformational determinants.", "Although a detailed analysis of the types of proteins targeted will be presented, it is evident that the proteins most commonly detected by patient sera included numerous cytokines, particularly type I IFNs, for which reason this study focuses on the nature of those autoreactivities.", "Human type I IFN genes include 13 IFNα genes, 1 IFNβ gene, and 1 IFNω gene.", "There is also a type II IFNγ gene and three type III IFNλ genes.", "IFNγ is largely limited to lymphocytes, whereas type I and type III IFNs are broadly expressed, with their functional uniqueness and/or redundancy unresolved (Ivashkiv and Donlin, 2014).", "As assessed by ProtoArray, patient sera showed significantly stronger reactivities than controls toward all IFNα subtypes, albeit the reactivities to some (e.g., α1/13, α5, and α14) were higher than those to others (e.g., α2, α16, and α21) (Figure 2A).", "The differential between patients versus controls was emphasized by luciferase-based immunoprecipitation (LIPS) in which many target proteins were recognized in their native conformations (Figure 2B).", "Many patients showed strong reactivities to IFNω but rarely toward IFNβ (Figure 2B) and never toward IFNκ and IFNε, two phylogenetically distant type I IFNs (data not shown).", "By contrast, patient sera harbored reactivities significantly above controls toward IL1α, IL5, IL6, IL17A, IL17F, IL20, IL22, IL28A (IFNλ2), IL28B (IFNλ3), and IL29 (IFNλ1) (Figure 2C).", "Whereas reactivities toward some targets (e.g., IL17F, IL22) were common to most patients, reactivities toward others (e.g., IL20, IL28, IL6) were not (Table S1), and with the exception of IL5, patient sera mostly did not detect either Th2 cytokines (e.g., IL4 and IL13) or IL21, a Tfh (T follicular helper) cell cytokine that drives high-affinity antibody maturation.", "There were also no reactivities toward G-CSF and GM-CSF (Table S1), which drive the development of myeloid cells associated with the patients’ inflammatory endocrinopathies.", "Cytokine reactivities were largely validated by ELISA, which confirmed that IFNγ was only rarely and weakly recognized by patient sera (Figure 2D; Table S1) and that there was no reactivity toward TNFα (data not shown).", "By contrast, ELISA revealed autoantibodies toward IL32α and IL32γ, two poorly characterized proinflammatory cytokines (Figure 2D; Table S1).", "In sum, 81 APS1/APECED patient sera collectively displayed strong reactivities to a very selective subset of human cytokines.", "To understand the nature of patient serum reactivities, nine IFNα-specific monoclonal antibodies (mAbs) were derived by limit-dilution cloning from memory B cells of four patients.", "Two were characterized in detail (26B9 and 19D11), whereas a more limited analysis of the others strongly argued that the properties of 26B9 and 19D11 were generally representative of patients’ cytokine-specific antibodies.", "First, their VH and Vκ sequences were highly mutated relative to their germline counterparts, with non-conservative replacements enriched in complementarity-determining regions (CDRs), as expected (white; Figure 3A).", "The antibodies bore no obvious resemblance to each other in V-gene segment or CDR3 usage.", "Conversely, a third anti-IFNα antibody, 50E11, shared with 19D11 the same VH (IGHV1-69) and junctional (IGHJ4) segments and a very similar light chain (IGKV3-11 versus V3-20) (Figure S2A).", "However, there were very different template-independent nucleotide insertions in the VH CDR3s of 19D11 and 50E11, and the somatic mutation patterns were different: whereas 19D11 and 26B9 showed high mutation frequencies in VH CDR2 and Vκ CDR1, 50E11 did not (Figures 3A and S2A).", "The recombinant antibodies 26B9 and 19D11 harvested from transfected CHO cells were immobilized on surface plasmon resonance (SPR) chips over which were run recombinant human IFNα2b, IFNα4, IFNα14, and IFNω, the latter being recognized by 26B9 but not by 19D11 (Figure 3B).", "These experiments revealed very slow off-rates reflecting extremely high affinities of the antibodies for their targets, ranging from KD = 3.28e−14M for 26B9 toward IFNα14 to KD = 2.09e−11M for 26B9 toward IFNα2b (Figures 3B and 3C).", "Sub-picomolar/near-femtomolar dissociation constants were likewise shown by 19D11 (Figures 3B and 3C).", "Thus, APS1/APECED patients harbor some of the strongest affinity antibodies described.", "18-mer peptides spanning IFNα2b and IFNω were used to map linear epitopes recognized by 26B9 and 19D11.", "However, no specific reactivities were detected (data not shown), consistent with the antibodies binding conformational determinants shared by several type I IFNs.", "Also, the antibodies reacted poorly or not at all to mouse IFNs (Table S2).", "To investigate the origins of the high-affinity, conformation-specific antibodies, germline counterparts for 19D11, 26B9, and 50E11, albeit with the same CDR3-VDJ sequences, were expressed and tested by LIPS against recombinant human IFNα2b, IFNα8, and IFNα14.", "There was no measurable interaction with any target (Figure 3D), although the antibodies’ quality was evident from their comparable detection by anti-human IgG (Figure S2B).", "These data argue that the strong autoreactivity toward IFNs developed de novo during affinity maturation, rather than being an intrinsic property of the germline repertoire that is enhanced by affinity maturation.", "The high affinities of 26B9 and 19D11 were not unique.", "Thus, a patient-derived IgGκ mAb (20A10) specific for IL20 (which is not a target detected by most patients; Figure 2C; Table S1) displayed a KD of 9.1e−14M, (Table S3).", "Likewise one IgGκ mAb (17E3) and one IgGλ mAb (24D3), each specific for IL17F, displayed dissociation constants of <10 pM, and one IgGκ antibody (30G1) and one IgGλ antibody (35G11) specific for IL22 displayed dissociation constants of 37 pM and 39 pM, respectively.", "As a comparison, a CHO cell-expressed form of fezakinumab, a humanized anti-IL22 mAb tested in the clinic, displayed a KD of 54 pM (Table S3).", "The only exception to this pattern was 2C2, an IgGλ mAb specific for IL32γ (for which no human antibody has been reported), which displayed nanomolar dissociation (Table S3).", "Similar to IFNα antibodies, most cytokine-specific antibodies did not detect linear peptides from relevant target proteins, strongly suggestive of complex conformational determinants (data not shown).", "The one exception was 20A10, which bound to an IL20 peptide and within which key amino acids were identified by mutagenesis (Figures S2C and S2D).", "The antibody sequences of IL17F-reactive 17E3 and 9A2 and of IL22-reactive 30G1 and 35G11 displayed myriad non-conservative mutations enriched in the CDRs.", "Again their germline counterparts did not detect the respective targets (Figures 3D, S2E, and S2F).", "Moreover, neither patient-derived antibodies nor their germline counterparts showed any general autoreactivity (judged by immunofluorescent staining of tissue sections or HEp-2 cells) or reactivity to Candida albicans, thus arguing against candida infection being the trigger for autoantibody generation (data not shown).", "The highly mutated CDRs of all studied antibodies suggested that they derived from GC reactions that partially rely on Tfh cells.", "Aberrant generation and/or activation of Tfh cells has been described in several autoimmune diseases (Ueno et al., 2015), but when four pediatric and four adult APS1/APECED were compared to controls, we found no differences in the percentages of circulating CXCR5+ Tfh cells, or their activation state, as judged by ICOS (inducible costimulator) and CCR7 levels (Figure S3).", "To test the biological activities of 19D11 and 26B9, HEK293 cells transfected with type I IFN-stimulated response elements (ISRE) fused to firefly luciferase were treated with recombinant forms of each of 12 IFNα subtypes and IFNω in the presence or absence of increasing concentrations of 19D11 or 26B9.", "Following treatment, firefly luciferase values were normalized to those of co-transfected Renilla luciferase, so as to control for variations in transfection efficiency.", "Both antibodies strongly inhibited the IFN-dependent response, with median IC50 values of 2.83 ng/ml for 26B9 and 0.9 ng/ml for 19D11 (Figure 4A; Table S4).", "By comparison, median IC50 values of 76.24 ng/ml and 10.86 ng/ml, respectively, were displayed by in-house-generated recombinant sifalimumab and rontalizumab, two anti-IFN mAbs used in clinical trials for systemic lupus erythematosus patients (Table S4).", "Predictably, the antibodies varied in their inhibition of IFN-stimulated responses.", "Thus, 26B9 neutralized IFNω, but not IFNα16, and only poorly inhibited IFNα8 (Figure 4A; Table S4).", "Likewise, in the same assay, rontalizumab failed to efficiently neutralize IFNα6, IFNα7 and IFNα10, whereas sifalimumab neutralized several IFNα subtypes only weakly.", "By contrast, 19D11 neutralized all 12 IFNα subtypes tested (Table S4).", "Patient-derived IFN-specific mAbs were also assessed for their capacity to inhibit STAT1 phosphorylation in cells treated with each of 12 IFNα subtypes, IFNω, IFNβ, or IFNγ (Figures 4B, 4C, and 4D).", "As predicted from the luciferase assay, 19D11 inhibited STAT1 phosphorylation levels (normalized to total STAT1 or tubulin) driven by all IFNα subtypes but did not affect responses to IFNω, IFNβ, or IFNγ.", "By contrast, 25C3, an additional patient-derived mAb (Table S2), was highly selective for discrete IFNα subtypes, whereas other antibodies tested, including 26B9, showed neutralization profiles between those of 19D11 and 25C3 (Figures 4B–4D).", "Only 26B9 and 31B4 neutralized IFNω, and none neutralized IFNβ or IFNγ.", "By comparison, sifalimumab, rontalizumab, and AGS-009 (another IFNα-targeting mAb in clinical development) showed variable and less uniform inhibition of STAT1 phosphorylation induced by different IFNα subtypes (Figure S4A).", "The striking biological activities of patient mAbs were not limited to those specific for IFNs in that potent functional target neutralization was shown by mAbs targeting IL17F, IL22, IL32γ, and IL20, respectively (Figure S4B).", "We next asked whether patient autoantibodies could functionally neutralize targets in vivo.", "To test this, mice were treated intraperitoneally (i.p.) with a single aliquot of antibodies 26B9, 19D11, or sifalimumab, and their ears inoculated intradermally (i.d.) on days 1, 3, 6, and 8 with recombinant human IFNα5 or IFNα14 (Figure 5A) and IFNω (data not shown).", "Relative to repeated inoculation with vehicle/PBS, the cytokines induced ear swelling, reflecting an inflammatory response that includes rapid TNFα and IFNγ induction (Figures S5A and S5B).", "This ear swelling was significantly inhibited by single injections of antibodies (Figure 5B).", "Again, neutralization varied toward the effector IFNα subtype: 26B9 and 19D11, but not sifalimumab, largely ablated the IFNα5 response, whereas all three partially yet significantly limited swelling induced by IFNα14 (Figure 5B).", "Specific, antibody-mediated neutralization in vivo was likewise seen when the same assay was applied to human IL17F or IL32γ (Figures 6A and 6B).", "For IL17 neutralization, the data are clearly consistent with the known capacity of APS1/APECED patients’ antibodies to neutralize Th17-family cytokines (Kisand et al., 2010; Puel et al., 2010), thereby predisposing to Candida infection.", "Additionally, the detection of mouse IL22 by antibody 30G1 offered an opportunity to measure its bio-activity toward endogenous IL22, a primary effector of imiquimod (IMQ)-induced dermatitis used to model psoriasis (van der Fits et al., 2009).", "IMQ-induced pathology measured by modified PASI scoring was significantly ameliorated by 30G1 relative to IgG control, particularly following an initial inflammatory response (Figures 6C and S6).", "Again, 30G1 was at least as effective as an in-house-expressed anti-IL22 antibody, fezakinumab (see above) (Figure 6C).", "Collectively these data establish the capacity of patient anti-cytokine antibodies to limit pathologies induced by their targets in vivo.", "Given the results from animal models, it was appropriate to consider the potential impact of APS1/APECED antibodies in the patients themselves.", "Because circulating IFNα levels are extremely low in human peripheral blood, even following vaccination (Sobolev et al., 2016), circulating IFN levels do not offer robust bio-markers of anti-IFNα antibodies.", "Neither does measurement of interferon-stimulated genes (ISGs) because many, e.g., CXCL10, can be upregulated by type II IFNs (Welcher et al., 2015).", "By contrast, antibody activities may be reliably reflected in discrete pathologies, as in the correlation of anti-IL22 with candidiasis.", "In this regard, many datasets, particularly in mouse models, suggest that type I IFN contributes to type 1 diabetes (T1D) (Carrero et al., 2013; Downes et al., 2010; Foulis et al., 1987; Huang et al., 1995; Li et al., 2008).", "Although APECED/APS1 patients by definition suffer from polyendocrinopathy, T1D affects only ∼10%–20% of patients and manifests primarily in adulthood (Husebye et al., 2009; Kisand and Peterson, 2015).", "This is despite the fact that radioimmunoassays have revealed that many APS1/APECED patients carry GAD65-reactive autoantibodies, a clinically applied biomarker for likely onset of T1D (Ziegler et al., 2013).", "Consistent with this, ProtoArray and LIPS data showed that many patients carried GAD65- and/or GAD67-reactive antibodies, but among them relatively few presented with T1D (red dots, Figures 7A and 7B).", "Collectively, these many observations suggest that patients at risk of T1D, as judged by anti-GAD65/GAD67, might fail to develop T1D if they harbored powerfully neutralizing anti-IFNα antibodies.", "Indeed, we reported a seemingly exceptional APS1/APECED patient, completely lacking IFNα-neutralizing antibodies and presenting with T1D (Kisand et al., 2008).", "To investigate this, the 8 patients presenting with T1D (red dots, Figure 7B; mean age ± SD, 48 ± 11 years) were compared with an available cohort of 13 patients without T1D but with strong GAD65 reactivity (relative luciferase units > 5) (blue dots, Figure 7B; mean age ± SD, 31 ± 12 years).", "Consistent with T1D developing in adult APS1/APECED patients, GAD65 reactivities mostly arose post-adolescence, and hence the patient cohorts comprised 20 adults and one 8 year old.", "As expected, all 21 patients harbored antibodies to IFNα and IFNω (see Figure 2B), but when tested for IFNα and IFNω neutralization, the antibodies showed a striking segregation with clinical status (Figures 7C, 7D, and S7A): patients without T1D collectively neutralized all IFNα subtypes, whereas those with T1D showed only low or negligible neutralization.", "Particularly strong differences were seen vis-a-vis IFNα1, IFNα2, IFNα5, IFNα8, IFNα14, and IFNα17 neutralization (Figure 7C).", "The two subgroups of the 21 patients also showed statistically significant differences in neutralizing IFNω, but the difference was weaker than for IFNα (Figure S7A).", "Interestingly, the two GAD65-reactive non-diabetics who displayed relatively low IFNα neutralization were young adults who may be en route to developing T1D.", "In a small subcohort of GAD65/67-reactive patients for whom longitudinal samples were available, the three T1D patients (red bars) again showed lower IFNα neutralization relative to the two patients without T1D.", "Moreover, one patient was able to neutralize IFNα4 in 1978 but by 2012 could no longer do so and presented with T1D (Figure S7B).", "Such striking correlations with T1D (Figure 7D) were not evident for any other naturally arising anti-cytokine antibodies, supporting the view that IFNα may contribute critically to the natural progression of T1D.", "Moreover, although the data do not prove that active anti-IFN antibodies underpin selective protection from T1D, they provide a firm foundation for exploring the potentials of APS1/APECED-derived autoantibodies to ameliorate other major diseases that are rarely if ever present in APS1/APECED patients.", "This analysis of the impact of AIRE deficiency on human B cells has revealed a signature pattern of humoral autoreactivity with general implications for our understanding of autoimmunity.", "First, the autoantibodies studied were mostly extremely high affinity and specific for native conformational epitopes.", "These properties were shared by antibodies specific for cytokines targeted by most patients (e.g., IFNα, IL17, IL22) and by antibodies specific for IL20 to which few patients displayed reactivity.", "Because such properties are very rare among antibodies raised by immunization, when B cells are primed de novo to antigen for short periods of time, it seems inappropriate to continue to model one type of mAb on the other.", "Second, essentially all 81 APS1/APECED patients studied showed strong reactivities toward a common set of 10–15 proteins, coupled with patient-specific reactivity profiles toward 80–90 additional proteins.", "This limited frequency (< 1% of proteins displayed on the array) is consistent with a recent report that B cell tolerance was not globally disrupted in 51 APS1/APECED patients sampled (Landegren et al., 2016).", "Nonetheless, the patient-to-patient variation in reactivity profiles meant that the 97 sera analyzed in our study collectively harbored antibodies toward over 3,500 proteins.", "The patient-to-patient variation argues that B cell autoimmunity resulting from AIRE deficiency is not simply an amplification of sporadic, low-level autoreactivities seen in healthy controls but has distinct origins.", "By this perspective, defects in central T cell tolerance may underpin other autoimmune and autoinflammatory pathologies attributed to high-affinity autoantibodies.", "Whereas this contrasts with the widely held view that autoimmune diseases mostly reflect peripheral tolerance defects, it aligns with data that central tolerance defects contribute to the NOD mouse model of T1D (Geng et al., 1998; Zucchelli et al., 2005).", "Moreover, wherever autoantibodies reflect central T cell tolerance defects, donor-to-donor variation is to be expected, as individuals will generate distinct TCR repertoires via quasi-random gene rearrangements, will be exposed to different physiologic and environmental triggers that promote the selective outgrowth of autoreactive T cell clones, and will differ in immune response modifier genes (e.g., HLA) that regulate the magnitude of antigen-specific responses.", "Autoantibodies to some non-tissue-restricted antigens, including multiple type I IFNα subtypes, are displayed by almost all patients, sometimes early post-partum (Wolff et al., 2013).", "Most likely, the immunogenicity of these proteins arises by mechanisms distinct from those shaping patient-specific autoantibody repertoires.", "Possibly the public autoantibodies arise from a direct impact of AIRE deficiency on B cell tolerance, for example, via the dysregulation of AIRE-expressing thymic B cells that resemble GC B cells by several criteria (Yamano et al., 2015).", "Arguing against this, however, autoantibodies to type I IFNs, Th17 cytokines, and additional self-proteins are found in thymoma patients with AIRE-sufficient B cells (Kisand et al., 2011; Meager et al., 1997; Wolff et al., 2014).", "This likewise argues against autoantibodies to type I IFNs and Th17 cytokines originating from defects in lymph node AIRE+ cells termed eTACs (Gardner et al., 2008).", "Although studies in mice have suggested tolerizing roles of eTACs, the functions of their rare human counterparts are unknown (Poliani et al., 2010).", "AIRE deficiency may, however, act indirectly on thymic B cells, for example by hyperactivity of functionally competent thymic γδ cells (Ribot et al., 2009) that may likewise be dysregulated in thymoma.", "Such cells may create an intra-thymic milieu favoring priming rather than tolerance of thymic B cells toward proteins highly expressed in the thymus (Dudakov et al., 2012; Meager et al., 2006).", "Notwithstanding this possibility, our findings suggest that high-affinity autoantibodies in APS1/APECED patients probably reflect dysregulated GC reactions, wherein autoreactive T cells, e.g., Tfh cells, that were not tolerized in the thymus promote the competitive outgrowth and affinity maturation of B cells that were initially primed to exogenous antigen(s) but whose mutated IgGs bind to self-proteins.", "Consistent with this, autoantibodies targeting thyroid-stimulating hormone receptor in Graves’ disease cross-react to Yersinia enterocolitica antigens (Brink, 2014; Hargreaves et al., 2013), and activated peripheral blood Tfh cells correlate positively with serum autoantibodies and disease activity/severity in multiple autoimmune diseases (Ueno et al., 2015).", "Although our analysis of four adult and four pediatric APS1/APECED patients revealed no alterations in Tfh cell numbers relative to age-matched healthy controls, this did not exclude Tfh cells being enriched in autoreactive specificities.", "Moreover, no patients displayed neutralizing autoantibodies to IL21, a major mediator of Tfh cells in the GC.", "This etiology of APS1/APECED B cell autoimmunity is strikingly similar to proposed origins of highly mutated anti-desmoglein-3 antibodies in autoimmune pemphigus (Di Zenzo et al., 2012) and of anti-GM-CSF antibodies pathognomonic in pulmonary alveolar proteinosis (Piccoli et al., 2015).", "In those studies, as in this, the closest germline counterparts (“unmutated common ancestors” [UCAs]) showed no reactivity toward the targets of the affinity-matured autoantibodies.", "By contrast, germline versions of antiviral antibodies showed only slightly reduced binding to target viral antigens (Corti et al., 2011, 2013).", "Moreover, it is not the case that UCAs intrinsically lack autoreactivity, as germline counterparts of some autoantibodies with few replacement mutations showed autoantigen reactivity in pemphigus patients (Cho et al., 2014).", "The underlying defect(s) in T cell tolerance that dysregulate affinity maturation in pemphigus, pulmonary alveolar proteinosis, and other organ-specific autoimmune diseases may be limited to few antigens, by contrast to broad-spectrum defects in APS1/APECED.", "That almost all APS1/APECED-derived mAbs were biologically active in vivo against a range of cytokine targets has profound implications for patients.", "Clearly, immune-effector responses may be reduced, as in the association of anti-IL22 with susceptibility to Candidiasis (Kisand et al., 2010).", "Likewise, gut barrier integrity may be compromised, leading to increased levels of anti-commensal antibodies (Hetemäki et al., 2016).", "Conversely, despite the common neutralization of IFNα and IFNω, APS1/APECED patients do not show severe viral infections, as were recently reported for a child genetically impaired in type I IFN (Ciancanelli et al., 2015).", "Possibly preserved IFNβ function mediates anti-viral protection in APS1/APECED patients.", "On the other hand, some autoantibodies may target key mediators of immunopathologies, thereby ameliorating disease.", "Thus, a unique correlation was observed between antibody-mediated neutralization of IFNα and failure to develop T1D, providing a novel strand of support for animal studies arguing that targeting type I IFNs could be effective in T1D.", "The concept that naturally arising autoantibodies may be beneficial is not widely considered, despite its underpinning the widespread use of therapeutic mAbs.", "In this regard, it is striking that despite their severe flaws in central T cell tolerance, APS1/APECED patients do not present with systemic sclerosis, Sjögren’s syndrome, MS, or SLE.", "These pathologies are considered to involve interplays of IL17/Th17 and type I IFNs—two main targets of APS1/APECED autoantibodies (Ambrosi et al., 2012).", "Likewise, Th17-driven psoriasis was diagnosed in only two of our patients, each of whom lacked autoantibodies to IL17A, IL17F, and IL22 (our unpublished data).", "Furthermore, atopy/allergy is seemingly rare among APS1/APECED patients, although whether anti-IL5 antibodies underpin this requires more study.", "For now, the data presented by this study strongly suggest that antibodies recovered from APS1/APECED patients include ones with profound therapeutic and diagnostic potential.", "More details are available in the Supplemental Experimental Procedures.", "Eighty-one APS1/APECED patients were diagnosed by mutational analysis of AIRE and by autoantibodies to type I IFNs.", "All provided informed consent, and many were analyzed previously (Kisand et al., 2011; Kluger et al., 2015; Meloni et al., 2012; Wolff et al., 2007).", "Approvals by local ethics committees are described in the Supplemental Experimental Procedures.", "Ages at serum sampling were 4–73 years; mean = 31.9.", "For protoarray there were 12 age-matched controls and 9 healthy first-degree relatives, and there were additional healthy controls for LIPS and ELISA.", "Sera of patients, healthy relatives, and controls were tested against > 9,000 human proteins displayed on the Human Protein Microarray v5.1 (ThermoFisher Scientific).", "Preprocessing methods were applied to account for technical variability.", "First, corresponding local background intensity was subtracted, whereafter values were log-transformed and subjected to robust linear normalization (Sboner et al., 2009).", "Z scores were calculated as the number of standard deviations of the signal from the mean of the corresponding controls and healthy relatives; Z ≥ 3 was considered positive.", "After scoring, stringent quality assessment was undertaken, including high correlation coefficients of duplicate spots of printed proteins (average r = 0.92), reactivity toward known autoantibody targets, and perfect correlation of signals for proteins spotted in different locations.", "Printing contaminants were identified as proteins showing high correlation coefficients with known APECED antibody targets and were further verified by cross-reference to another protoarray (5.0) used for 23 patients and 7 controls.", "Thus, 31 suspect false-positives were identified and excluded from further consideration.", "Cloning, production, and purification of human mAbs were performed as described (patent application WO2013/098419).", "In brief, memory B cells (CD22+, IgD−, IgM−, CD3−, CD8−, and CD54−) were flow-sorted (MoFlo) from patient PBMC, incubated transiently with EBV-containing B95-8 supernatant (SN) for 3.5 hr at 37°C, and then incubated in Transferrin- and CpG- supplemented IMDM at 37°C, 5% CO2, at 10 cells/well in 96-well plates coated with irradiated PBMC feeders.", "Short-term, oligoclonal B cell culture SN were analyzed for IgG and antigen-specific antibodies detected by ELISA and/or LIPS.", "Positive wells were harvested, cells single-cell-sorted into reverse transcriptase (RT) buffer (Life Technologies), and RT-PCR performed using Superscript III (Life Technologies) and random hexamers.", "IgG VH, Vλ, and Vκ regions were amplified from cDNA by two-step nested PCR reaction using Advantage 2 cDNA polymerase (Clontech) and primer mixes specific for germline families (VBASE database).", "Nested primers attached restriction sites for V-region cloning into expression vectors providing IgG1, Ig-κ, or Ig-λ constant regions.", "Recombinant antibodies were produced in HEK293T cells and antigen specificity analyzed by ELISA.", "Corresponding closest germline region sequences were identified using the VBASE2 database (Retter et al., 2005).", "CDRs were identified by IMGT definitions (Lefranc, 2003).", "Complete Ig-VH and VL regions described in US7741449 (Sifalimumab), US7087726 B2 (Rontalizumab), US8361463 (ACO-1), and US20070258982 A1 (Fezakinumab) were ordered as CHO-codon-optimized synthetic constructs (GenScript) and expressed as above.", "EC50 binding of mAbs was determined by ELISA.", "Neutralizing capacities of type I IFN-specific mAbs were studied using phospho-STAT1 quantification in immunoblot and ISRE-luciferase reporter assay.", "IL17F, IL22, IL20, and IL32 neutralization assays were performed on respective responsive cell lines.", "mAB affinities were measured with a Biacore T200 (GE Healthcare).", "Epitope mapping used overlapping 18-mer peptides.", "C57BL/6J (WT; from Charles River) mice were administered i.p. with mAbs (day 0) and inoculated i.d. on days 1, 3, 6, and 8 with cognate human cytokines, IFNα2a, IFNα2b, IFNα4, IFNα14, IL17F, and IL32γ, and their ear thicknesses measured with a micrometer.", "For IL22 mAbs cross-reactive to mouse, bioactivity was assessed in imiquimod-treated mice.", "The members of the APECED patient collaborative are Antonella Meloni, Nicolas Kluger, Eystein S. Husebye, Katarina Trebusak Podkrajsek, Tadej Battelino, Nina Bratanic, and Aleksandr Peet.", "S.M., A.M., and S.C.O. cloned monoclonal antibodies from patient samples, and K.J. and K.H. assisted.", "S.M., P.V., and A.M. characterized antibodies in vitro; M.W. and Y.H. did so in vivo.", "C.H. analyzed ProtoArray data and wrote and edited the paper.", "J.K. assayed neutralization by sera and Tfh subsets and performed LIPS.", "D.F. and H.P. analyzed ProtoArray data.", "K.M. and R.U. screened sera for T1D autoantibodies and tested germline antibody specificities.", "K. Krohn and A.R. developed the clinical database, sampled Finnish patients, and employed ELISA.", "A.S.B.W. sampled Norwegian patients, contributed to the clinical database, and assayed antibodies.", "APECED patient collaborative contributed to the clinical database and sampled respective patients.", "P.P., K. Kisand, and A.H. supervised research, reviewed data, and wrote and edited the paper." ]

[ "Each AIRE-deficient patient has a private repertoire of autoantibody reactivities", "Loss of B cell tolerance occurs during T cell-dependent somatic hypermutation", "Patient autoantibodies have unprecedented affinities for conformational epitopes", "Patient autoantibodies can display disease-ameliorating properties in vivo" ]

[ "ENGI", "PHYS" ]

[ "3D printing", "Cell homogenizer", "Customization", "Dual syringe pump", "Plasmodium falciparum cell extract" ]

[ "2204", "2205", "2209", "2210", "3105" ]

[ "Syringe pumps have a wide variety of uses across fields from engineering to biology.", "Their primary purpose is to continuously dispense precise volumes over a set amount of time.", "They save time by running unsupervised and provide more consistency than human hands.", "A dual syringe pump allows for two syringes to have coordinated actions, broadening the potential applications.", "In particular, dual syringe pumps have the power to automate a number of routine and repetitive protocols in the life sciences.", "Our lab initially conceived of building a dual syringe pump that could be used to make Plasmodium falciparum whole cell extracts used for in vitro translation.", "Previously, we generated lysates by passing purified infected red blood cells through an Isobiotech cell homogenizer using a ball bearing with 4um clearance.", "Frozen pellets of purified parasites were thawed, loaded into one 3 mL syringe, and passed through the homogenizer into a second 3 mL syringe.", "The lysate was then passed back into the first syringe.", "This back and forth cycle was repeated up to 20 times per parasite pellet [1].", "This process takes between 20 and 30 min per parasite pellet and the resistance in the device makes it physically taxing on the wrists and thumbs.", "In the lab, lysate generation was often a rate-limiting step when making in vitro translation extracts due to the manual and tedious nature of the process.", "It also entailed unacceptable amounts of user-dependent variation between lysate preparations.", "In the worst cases, syringes would break or the plunger would deform as pressure was applied unevenly.", "A customizable PDSP addresses all of these issues not only for our uses, but also for other applications of the Isobiotech Cell homogenizer such as C. elegans lysis [2] and mammalian cell culture homogenization [3].", "We reasoned that this process could be easily replaced with a programmable dual syringe pump.", "However, commercially available dual syringe pumps have a number of limitations, including cost and flexibility.", "As of this writing, commercially available dual syringe pumps cost upwards of $1500 [4] and coordinated programmable motion tends to be limited.", "Additionally, the physical configuration of commercial products makes alternative mounting options difficult.", "Previously described lab-built syringe pumps include single syringe pumps [5,6], or pumps that operate as part of a larger device, such as an auto-sampler [7], a 3D printer [8] or a pH-stat device [9].", "To our knowledge, there are no current solutions that feature a customizable graphical user interface (GUI) touchscreen, which greatly simplify use, eliminating the need for keyboard and/or mouse.", "Thus, we designed, built, and tested a PDSP that can be easily used in the lab using a touch-screen but that can also be customized for any use.", "This PDSP drives two independent pumps operating under a single microcontroller which makes it not only amenable to synchronized pumping patterns, but also makes it extensible (up to 16 pumps) for multiplexed liquid handling operations.", "Our custom PDSP is constructed on an extruded aluminum frame that can be mounted horizontally or vertically.", "For our specific application, we chose a vertical mount to allow the cell homogenizer to be immersed in ice (Fig. 1A/B).", "The PDSP utilizes two NEMA-14 stepper motors (StepperOnline) that create precise volume changes even at high-torque.", "The motors are driven by a Pi-Plate MOTORplate controller (Pi-Plates, Inc.) mated to a Raspberry Pi (v3 Model B).", "Integrated limit switches provide for simple and accurate “homing” procedures.", "To make the PDSP easy to use for routine laboratory stand-alone use, the PDSP is operated via an attached touchscreen (Landzo), without a keyboard or mouse.", "While this PDSP is specialized to lysate generation, alternative applications may have different requirements.", "We designed this device so that it could be adapted to many different environments, such as a biohazard hood, and for many different tasks, such as microfluidic experiments [5].", "To allow for maximum customization, we made the PDSP modular with 3D printed parts that can be interchanged to accommodate different volume syringes.", "By simply changing the dimensions of the printed parts the syringes can be set to any distance apart.", "The cell homogenizer holder can be interchanged with any other user-designed holder.", "Our custom Python/Tkinter interface can be easily configured to drive the stepper motors at different speeds allowing for a range of flow rates or even gradients of flow rates.", "The Pi-Plate MOTORplates can be stacked, allowing a single Pi and interface to simultaneously control up to 8 PDSPs (16 syringes) for high volume production environments.", "Table 1 contains links to the STL design files for all 3D printed parts described here:", "The Motor Base (Fig. 2A) secures the two stepper motors and holds the Aluminum Heat Sink Plate against the T-profile rails.", "The Toe Hold (Fig. 2B) positions the linear motion shafts against the T-profile rails.", "The Syringe Stabilizer (Fig. 2C) holds the syringe body in place by securing both the barrel and the barrel flange parallel to the movement direction of the plunger.", "The left and right Plunger Movers (Fig. 2D) hold the plunger flange of the syringes and move them along the linear motion shaft as the T8 threaded rods turn according to the stepper motors to change the volume.", "The Pi Base (Fig. 2E) and Cover (Fig. 2F) secure the Raspberry Pi along the T-profile rails and insulates it from short-circuiting against nearby conductive material.", "It also protects the Raspberry Pi from dust and accidental splashes from the ice bucket.", "The Screen Mount Top and Bottom (Fig. 2G) hold the touch screen at eye-level when the PDSP is constructed vertically.", "Situated in front of the motors, it keeps the touchscreen a safe distance from the generated heat.", "The Cell Homogenizer Holder (Fig. 2H) positions the cell homogenizer in alignment with luer lock syringes such that no additional tubing is necessary.", "It also allows the cell homogenizer to be in contact with ice at all times.", "The Right-Angle Brackets are used to position the syringe pump vertically on a breadboard.", "If desired, they can be 3D printed rather than purchased.", "The Aluminum Heat Sink Plate is placed between the stepper motors on the Motor Base and the T-profile rails to rapidly disperse heat generated from the stepper.", "The essential PDSP components are 3D printed.", "The remaining hardware can be acquired online from common hardware suppliers, such as McMaster-Carr.", "All electronic parts can be acquired from common electronics suppliers, such as Adafruit or Microcenter (see Section 4).", "The additional tools required for assembly include:", "Metric Hex Key Set", "Imperial Hex Key Set", "It is useful to prepare the electronic components prior to assembly.", "First, solder wires to the GND pin and the N pin on the leaf switches as outlined in Fig. 3.", "Wrap the exposed solder and pin with heat shrink to protect the connection.", "To keep the electronic wiring neat, braid the four wires of each stepper motor and twist together the two wires of each leaf switches.", "Next, attach one heat sink onto the rear of each stepper motor by applying an appropriately sized piece of thermal tape to the rear end-cap and firmly pressing the heat sink to the tape.", "To prepare the motor power supply, cut and strip the wires of the 2.1 mm female/male barrel jack extension cable to separate the power and ground wires.", "Finally, attach the Raspberry Pi onto the Pi base (Fig. 2E) using four M2.5 × 5 mm screws.", "Next, attach the PiPlates Motor Plate to the Raspberry Pi header pins by carefully applying even pressure on the plate while pushing the two components together to prevent bending any pins.", "Once attached, secure the PiPlates Motor Plate onto the Pi base using another four M2.5 × 5 mm screws.", "3D print each of the necessary parts from the provided design files, including four right-angle brackets if not using ThorLab’s precision cut components.", "We recommend printing with ABS material with a low-density fill.", "Cut the two T8 lead screws to 25 cm each and the two linear motion shafts to 21.5 cm each.", "Saw the aluminum plate to the appropriate dimensions according the Aluminum Heat Sink Plate STL file (Supplementary File 1).", "Using a drill press, drill two through holes as specified into the aluminum plate for later assembly onto the T-profile rails.", "Tap M2.5 and M3 holes into the 3D printed parts as indicated in their respective STL files.", "The complete assembly of the PDSP as described below can be seen as a time-lapse in Supplementary File 2.", "Attach the compact end-feed fastener with M5 × 5 mm screws to each through hole of the Motor Base (Fig. 2A), Toe Hold (Fig. 2B), Syringe Stabilizer (Fig. 2C), Pi Base (Fig. 2E), and Cell Homogenizer Holder (Fig. 2H).", "Attach the compact end-fasteners of two M5 × 8 mm through the previously drilled holes aluminum Heat Sink Plate (Fig. 4A).", "Attach each of the two pillow block bearings to the Extract Maker Holder (Fig. 4B) using M3 × 20 mm socket head screws.", "Insert one copper nut and one linear ball bearing into each of the Plunger Movers (Fig. 2D).", "Attach the copper nut using four M2.5 × 5 mm socket head screws per nut and secure the linear ball bearing into its designated slot with silicone if necessary (Fig. 4C).", "Thread the one T8 lead screws into the copper nut and slide one linear motion shaft through the linear ball bearing on each of the two Plunger Movers (Fig. 4C).", "Making sure each part is oriented correctly, slide the four metal bars through the Syringe Stabilizer, and secure into the Cell Homogenizer Holder.", "The arms on each of the plunger mover should be on the outside of the linear structure and each pointed towards the Syringe Stabilizer (Fig. 4B and D).", "On the other end, slide the four metal bars towards the Toe Hold making sure the linear motion shaft is secured by the Cell Homogenizer Holder on one end and by the Toe Hold on the other end (Fig. 4D).", "Slide the Toe Hold, the Syringe Holder, and the Cell Homogenizer Holder together onto the T-profile rails by correctly slotting the compact end-feeder into the rails.", "Do not tighten the M5 screws yet (Fig. 4D).", "Attach and secure the stepper motors onto the motor base using four M3 × 5 mm per motor (Fig. 4E).", "Slide the motor base with attached motors onto the T-profile rails on the side closest to the Toe Hold.", "Do not yet tighten the M5 screws.", "Attach the couplers between the T8 threaded rods and the stepper motors by carefully sliding all the moving parts along the T-profile rails towards each other.", "After making sure that the T8 threaded rods and the motors are completely aligned, secure the couplers by tightening the attached screws (Fig. 4F).", "Slide the Aluminum Heat Sink Plate onto the T-profile rails beneath the stepper motors on the Motor Base.", "If desired, ensure there is sufficient space between the Cell Homogenizer Holder and the end of the T-profile rails to place an ice bucket.", "Finally, secure all the parts to the T-profile rails by tightening all ten of the M5 screws.", "Attach the Screen Mount-Bottom (Fig. 2I) onto the Motor Base using four M2.5 × 20 mm screws.", "The correct orientation should allow the touchscreen to rest on the base without bending the ribbon cable.", "Then, sandwich the touchscreen between the Screen Mounts using four more M2.5 × 20 mm screws (Fig. 4G).", "Attach the leaf switches associated with each stepper motor to the Syringe Stabilizer on their respective sides using M2 × 12 mm screws (Fig. 4H).", "To stand the PDSP vertically, insert one M5 × 8 mm screw into the highest through hole on each of the four Right-Angle Brackets.", "Attach the compact end-feeder into each of these screws and slide them into the bottom of the T-profile rails, closest to the cell homogenizer holder.", "Do this for the four Right-Angle Brackets for a total of one each on the left and right, and two in the rear (Fig. 3I).", "Stand the PDSP up over the breadboard and secure the Right-Angle Brackets onto the base of choice using the ¼″ × ¾″ long socket screws.", "Attach the Pi Base on the rear of the PSDP such that it is flush against the top of the T-profile rails (Fig. 4I)", "Thread the wires neatly using the through holes on the Pi Base before connecting each to the Raspberry Pi according to the Electronic Wiring Schematics (Fig. 3).", "Slide on and secure the Pi Cover (Fig. 2H) to the rear of the Pi Base using M2.5 × 10 mm screws (Fig. 4I).", "Check that all the screws on the PDSP are appropriately tightened prior to use.", "To install our software and GUI, we have provided the Raspberry Pi image on our lab website (http://derisilab.ucsf.edu/).", "Download the image onto a MicroSD card.", "We suggest using the free software Etcher to create bootable SD cards [10].", "Once installed, unmount the MicroSD and insert it into the Raspberry Pi.", "Plug in the Raspberry Pi power cord to a conventional outlet to turn it on.", "To power the stepper motors, connect a conventional 7.5 V power supply to the barrel jack extension cable attached to the PiPlate Motor Plate.", "Alternatively, install the latest release of Raspbian for the Raspberry Pi, the MOTORplate drivers (via the PIP Python repository: sudo pip install pi-plates), and the source code for the PDSP (Supplementary File 3) and interface manually.", "The resolution of the Pi can be set to fit any screen by altering the/boot/config.txt file.", "For the touchscreen used here, add the following lines to the end of the config.txt file:", "hdmi_cvt 800 480 60 6 0 0 0", "We have designed a GUI optimized for safe and streamlined operation of the PDSP as an automated cell homogenizer (Fig. 5).", "The software was written with internal limitations to protect both the sample being lysed and the PDSP components.", "For the push-pull pumping motion, we strongly encourage users to consider employing our intuitive GUI to operate the PDSP.", "The complete operation instructions of the PDSP as an automated cell homogenizer is described below and can be seen as a video clip in Supplementary File 2.", "While we use the PDSP to automate P. falciparum lysis using an Isobiotech Cell Homogenizer, the PDSP is adaptable to other tasks.", "As previously described, the PDSP is programmed on and executed from a Raspberry Pi running the Raspbian operating system.", "Our software to control the PDSP is written in Python using commands from the PiPlates MOTORPlate Users Guide documentation [11].", "Our object oriented GUI is also designed in Python using Tkinter.", "To customize the PDSP for a variety of other applications, users can reference both the script that we have written as well the extensive documentation provided by PiPlate.", "The PiPlate MOTORplate is highly customizable, offering a wide variety of options for stepper movement in terms of stepper size, speed, and acceleration or deceleration.", "Furthermore, the PiPlates MOTOR library is highly compatible with the GPIO control library, allowing flexibility in the control of the stepper motors.", "While python scripts can be executed from the Raspberry Pi command line using a keyboard, users can also create a specialized GUI for their own purposes.", "The PDSP presented here is already compatible with a number of different pumping systems.", "These include, but are not limited to, continuous infusion systems, dual injection systems, and inverse linear constant flow systems.", "While this PDSP is relatively simple, we can add complexity by attaching up to eight PiPlate MOTORplates powered by one Raspberry Pi, allowing for control of up to sixteen stepper motors simultaneously.", "To characterize the PDSP, we tested the minimum and maximum flow rates provided by the stepper motors on a 3 mL syringe.", "By timing the flow of liquid, we were able to measure the consistency and the dynamic range of flow rates for the PDSP (Fig. 6A).", "We compared these values with the theoretical minimum and maximum flow rates that were calculated as described below:", "The measured flow rates were consistent with the theoretical flow rates calculated using the manufactured syringe diameters and the stepper motor step sizes.", "This gives us confidence in the PDSP as an appropriate alternative to commercial syringe pumps.", "Here we provide the dynamic range of the PDSP in terms of the theoretical minimum and maximum flow rates of various compatible syringe sizes (Table 3).", "To demonstrate the ability of the PDSP to replace manual cell lysis we compared the two methods directly.", "In brief, Plasmodium falciparum parasites were harvested as previously published [1] and split into two pools, one for manual lysis and one for lysis using the PDSP.", "We either passed the lysate back-and-forth through the Isobiotec cell homogenizer for 20 cycles by hand or the PDSP controlled by the GUI interface passed the lysate through.", "Lysates were then collected and centrifuged at 13,000×g for 10 min at 4 °C.", "The supernatant was aliquoted and flash frozen in liquid nitrogen.", "One aliquot was taken for each lysis method and used in an in vitro translation reaction as previously published with a few modifications [1].", "Each 10 μL reaction consisted of 7 μL of lysate, 10 µM amino acid mixture, 20 mM HEPES/KOH pH 8.0, 75 mM KOAc, a range of 1–5 mM Mg(OAc)2, 2 mM DTT, 0.5 mM ATP, 0.1 mM GTP, 20 mM creatine phosphate, 0.2 μg/μl creatine kinase, and 0.5 pmol of Nanoluciferase reporter RNA.", "Reactions were done in triplicate, incubated at 37 °C for 45 min, and stopped with the addition of 10 μM cycloheximide.", "The Nanoluciferase reporter RNA consists of the 130 base pairs directly 5′ of PFE_1248300 followed by the Nanoluciferase coding sequence [12] in the 3′ UTR of HRP.", "All RNA was generated off of plasmid digested with PVUII and APALl using T7 transcription (Fig. 6B).", "After transcription, the DNA template was digested with Turbo DNAse (Thermo Fisher) and the RNA was purified using RNA Clean and Concentrate-25 kit (Zymo).", "Nanoluciferase reactions were performed using Promega’s Nano-Glo Luciferase Assay System.", "In brief, 10 μL of 1:50 Nanoluciferase substrate:Nanoluciferase buffer was added to each reaction and incubated at room temperature for a minimum of 5 min before reading with a 6 s integration on a Promega GloMax-Multi microplate reader.", "Our results showed that lysates generated by hand and by the PDSP performed similarly, indicating the PDSP can be used to replace manual lysis for generating in vitro translation extracts (Fig. 6C).", "We have constructed a programmable syringe pump that can be used for biological life science applications.", "Not only is the PDSP more affordable than commercially available options, but it is also modular and programmable, allowing the user to customize the device for specific tasks or experiments.", "Here, we have demonstrated that the PDSP can be used to automate and standardize the time-consuming task of P. falciparum lysis.", "Overall, the PDSP can be used as an affordable and customizable alternative to traditional syringe pumps to automate any number of routine laboratory practices." ]

[]

[ "AGRI", "EART" ]

[ "Epifauna", "Ground fish", "Infauna", "North Sea", "Sand mining", "Sediment" ]

[ "1104", "1910" ]

[ "The demand for marine sand in the Netherlands and worldwide is strongly increasing.", "In the Netherlands, approximately 24 million m3 sand is used annually for coastal nourishments and for construction.", "An increase of annual coastline nourishments of up to 40-85 million m3 to counteract effects of future sea level rise is expected (Deltacommissie, 2008).", "For the seaward expansion of the Port of Rotterdam (Maasvlakte 2) approximately 220 million m3 of sand was extracted between 2009 and 2013 with an average extraction depth of 20 m.", "In general, only shallow sand extraction down to 2 m below the seabed and beyond the 20 m isobath is allowed in the Netherlands (IDON, 2005; V&W, 2004).", "For Maasvlakte 2 though, the Dutch government permitted sand extraction deeper than the common 2 m, primarily to reduce the surface area of direct impact.", "Fish assemblages at North Sea scale are mainly influenced by bottom water temperature, bottom water salinity, tidal stress and water depth (Callaway et al., 2002; Reiss et al., 2010).", "Furthermore, fish assemblages are linked to biotic and abiotic habitat characteristics and meso-scale bedforms (Ellis et al., 2011; Sell and Kröncke, 2013).", "Ellis et al. (2011) found that species richness of infauna, epifauna and fish was larger in the silty troughs of sandbanks off the coast of the UK than on the crests.", "Large-scale sand extraction was shown to have a negative impact on fish in the Yellow Sea (Hwang et al., 2013), a decline of more than 70% of the total number of fish and the number of species (Son and Han, 2007) and direct and indirect damages to commercial fisheries were observed (Kim and Grigalunas, 2009).", "Since the start of aggregate extraction in the Eastern Channel Region, the majority of fish species have shown marked reductions in abundance and draghead entrainment was identified as a possible cause (Drabble, 2012).", "On the other hand, aggregate extraction may also lead to new habitats and may favour macrozoobenthos and fish (Desprez, 2000).", "Ecosystem-based landscaping techniques are not commonly used to reduce the impact of sand extraction.", "In the UK, gravel-seeding techniques were tested to restore the seabed after gravel extraction (Cooper et al., 2011).", "In the Maasvlakte 2 sand extraction site, two sandbars were artificially created by selective dredging, copying naturally occurring meso-scale bedforms to increase habitat heterogeneity and thereby possibly increasing post-dredging benthic and demersal fish species richness and biomass (van Dalfsen and Aarninkhof, 2009; van Raalte et al., 2007).", "In this study, we test the hypothesis that deep and large-scale sand extraction and ecosystem-based landscaping approaches will lead to differences in fish assemblage and we are aiming to answer the following questions:", "Are there significant differences in fish species assemblage between reference area and sand extraction site, and within the extraction site?", "Are there significant temporal differences in fish assemblage, macrozoobenthos and environmental variables during the monitoring campaign?", "Which environmental variables determine the differences?", "Are ecosystem-based landscaping techniques landscaping techniques feasible and effective in influencing fish assemblages?", "The Maasvlakte 2 sand extraction site is situated in front of the Port of Rotterdam, the Netherlands, outside the 20 m depth contour (Fig. 1).", "The sand extraction site is 2 km long, 6 km wide with an average extraction depth of 20 m at an initial water depth of approximately 20 m below average sea level.", "Approximately 220 million m3 sand was extracted between 2009 and 2013, of which 170 million m3 in the first two years (Borst and Vellinga, 2012).", "Two sandbars were created in the extraction site to investigate the applicability of ecosystem-based landscaping in sand extraction projects (Figs. 1 and 2).", "One sandbar (I), parallel to the tidal current, was left behind in the seabed in spring 2010.", "This parallel sandbar has a length of 700 m, a width at the crest of 70 m and slopes of 140 m length (Fig. 2).", "The crest of the sandbar is located at a water depth of 30 m and the troughs are more than 40 m deep.", "In 2011, the second sandbar (II) was completed with an orientation oblique to the tidal current.", "The length and width are similar to the parallel sandbar but, due to time constraints, the difference in depth between crest and trough is less pronounced.", "The crest is situated at a water depth of 28 m and the northern trough is 36 m deep.", "A narrow and 32 m deep trench separates the crest from the slope of the sand extraction site.", "The volume of each sandbar is approximately 1.25 million m3 with slopes of 1:7–1:10.", "During our surveys in 2011 and 2012, two trailer suction hopper dredgers were active in the centre of the sand extraction site, extracting approximately 2 million m3 marine sand per week.", "The water depth increased from 33 m to approximately 40 m (Fig. 1) but the areas near the landscaped sandbars remained un-dredged after completion.", "A commercial fishing vessel was used, the Jan Maria, GO 29, with a length of 23 m, less than 300 horsepower and equipped with a standard commercial 4.5 m beam trawl.", "The beam trawl was equipped with four tickler chains, five flip-up ropes and diamond mesh size of 80 mm, which was applied at 4 knots fishing speed.", "The ship GPS-system logged the position of the sampling locations and water depth was determined with the ship's depth sounder.", "The maximum haul distance was one nautical mile in the reference area.", "Shorter hauls were planned within the sand extraction site; at the landscaped sandbars, hauls of approximately 700 m length were applied.", "Some of the hauls ended before the planned end-coordinates because of difficulties with fishing inside the sand extraction site due to large changes in seabed topography and sediment composition.", "In surrounding reference areas, fishing direction was generally perpendicular to the direction of naturally occurring seabed patters to ensure heterogeneous sampling of crests and troughs of sand waves.", "In the sand extraction site, fishing direction was generally parallel to the seabed structures to enable comparisons between the different locations.", "We sampled in the reference area, at the slope of the sand extraction site, two locations in the deep parts of the extraction site i.e. the south-east and north-west, in the troughs and at the crests of the sandbars (Fig. 3, Table 1).", "The fish surveys were conducted on 14 July 2010, 27–29 July 2011 and 13–15 June 2012.", "In 2010, four reference fish samples and five sand extraction site samples were collected (Table 1, Fig. 2).", "In 2011, seven samples were collected in the reference area and thirteen samples in the extraction site.", "In 2012, for samples were collected in the reference area and thirteen in the extraction site.", "Fish were sorted and length frequency distribution ‘to the cm below’ was determined.", "Abundance of fish was calculated by dividing the number of fish by the fishing surface area and expressed as number of fish per hectare (length of haul ×4.5 m × number of nets).", "Species richness of a haul (number of species haul−1) was determined and published length–weight relationships (Coull et al., 1988; Robinson et al., 2010) were used to calculate the weight of fish (WW biomass, kg ha−1).", "The average length of a fish species was calculated by dividing the sum of all multiplied size classes and their abundances with the total abundance.", "In 2012, stomach and intestine contents of on average 10 specimens of plaice (Pleuronectes platessa), dab (Limanda limanda), and shorthorn sculpin (Myoxocephalus scorpius) were taken from the south-eastern deep part and troughs to obtain a rapid indication of fish diets.", "The closest macrozoobenthos and sediment samples locations were selected to link with the fish hauls (Fig. 3).", "A boxcorer was used to sample macrobenthic infauna (>1 mm) and sediment, carried out by the Monitor Taskforce of the Royal Netherlands Institute for Sea Research (NIOZ) on 29–30 June 2010, 2–5 May 2011 and 23–25 April 2012.", "The boxcorer surface area was 0.0774 m2 with a maximum penetration depth of 30 cm (Perdon and Kaag, 2006; Craeymeersch and Escaravage, 2010).", "Infaunal ash-free dry weight biomass (g AFDW m−2) was analysed by means of loss on ignition (2 days at 80 °C followed by 2 h at 520 °C).", "A bottom sledge was used to sample larger macrobenthic fauna (0.5–10 cm), executed by the Institute for Marine Resources & Ecosystem Studies (IMARES Wageningen UR) on 7–8 July 2010, 14–15 June 2011 and 6–7 June 2012.", "The sledge was equipped with a 5 mm mesh cage.", "On average, 15 m2 was sampled during each sledge haul (∼150 m length, 10 cm width and a maximum penetration depth of ∼10 cm).", "Wet weight of larger fauna was directly measured (g WW m−2).", "Samples from the upper 5 cm were collected from untreated boxcorer samples and kept frozen until analysis.", "Sediment samples were freeze dried, homogenised and analysed with a Malvern Mastersizer 2000 particle size analyser.", "Percentile sediment grain size (D10, D50, D90) and sediment grain size distribution among the different classes; clay (<4 μm), silt (4–63 μm), mud (<63 μm), very fine sand (63 μm-125 μm), fine sand (125 μm-250 μm), medium sand (250 μm-500 μm) and coarse sand (>500 μm) were measured as percentage of total volume.", "Sediment organic matter (SOM) was analysed in 2012 by means of loss on ignition as percentage of sediment mass (freeze-dried sediment samples were placed for 2 h at 520 °C).", "Areas surrounding the extraction site are labelled as reference area (Ref), Trecent denotes sampling directly after sand extraction and for 1 and 2 year after the cessation, T1 and T2 are used.", "Fishing activity in- and outside the sand extraction site was derived from Vessel Monitoring through Satellite (VMS) data for the years of the fish survey (2010, 2011 and 2012).", "Significance of differences in fish species composition between location and time after sand extraction was tested using analysis of homogeneity by the Betadisper function of the package ‘vegan’ followed by permutation test and Tukey's HSD test posteriori multi-comparison tests of the package ‘stats’.", "We applied Dufrêne-Legendre Indicator species analysis using the indval function of the package ‘labdsv’ to determine indicator species of sub locations.", "The analysis is based on the product of relative frequency and relative average abundance of a fish species for a certain sub location (Dufrêne and Legendre, 1997).", "After checking of normality and homogeneity of univariate variables (Shapiro–Wilk, Levene's Test and diagnostic residuals plot) the parametric two-way ANOVA with interaction of variables followed by Tukey's HSD test was used.", "When the normality and homogeneity assumptions were violated, the non-parametric Kruskal–Wallis one-way multi-comparison tests of the package ‘pgirmess’ was used to determine significant differences between locations.", "We applied Non-Metric Dimensional Scaling (nMDS) using the metaMDS function in the package ‘vegan’, based on Bray–Curtis dissimilarities of fish abundance data, to visualize differences in fish assemblages in the extraction site and reference area (Oksanen, 2013).", "Environmental variables were linearly fit onto the ordination using the envfit function in the package ‘vegan’ (999 permutations).", "We used the ordisurf function to plot a smooth surface onto the ordination in the case of non-linear relationships.", "When Spearman rank correlation coefficients between a set of variables exceeded 0.9 one of the variables was dropped (Zuur et al., 2007).", "We used the mantel.correlog function in the package ‘vegan’ to check for autocorrelation between the ecological distance matrix and geographic distance matrix (Borcard and Legendre, 2012).", "For all analyses we used R: A Language and Environment for Statistical Computing, version 3.0.1 (R Core Team, 2013).", "In total, 32 fish species were identified.", "Fish assemblages in the reference area were dominated by dab (Limanda limanda), plaice (Pleuronectes platessa), scaldfish (Arnoglossus laterna), common sole (Solea solea), shorthorn sculpin (Myoxocephalus scorpius) and solenette (Buglossidium luteum).", "On average, 20.9 ± 12.2 kg WW ha−1 of fish and 13.1 ± 1.7 fish species haul−1 were caught (Table 2).", "Species assemblage and biomass at the slope of the extraction site were not significant different from the reference area (Tables 4 and 5).", "Plaice, dab, scaldfish, shorthorn sculpin and solenette were most abundant and on average 20.2 ± 6.7 kg WW ha−1 and 14.2 ± 1.9 species haul−1 were caught.", "Turbot (Psetta maxima) and brill (Scophthalmus rhombus) were Dufrêne-Legendre indicator species for the slope of the extraction site due to a higher relative frequency and average abundance compared to the other sub locations (Table 3).", "At the crests of the sandbars, plaice, dab, sole, shorthorn sculpin and hooknose (Agonus cataphractus) were most abundant and on average, 93.8 ± 47.1 kg WW ha−1 and 11.4 ± 2.1 species haul−1 were caught.", "Tub gurnard (Chelidonichthys lucerna) is a Dufrêne-Legendre indicator species of the crests of the sandbars (average 4.4 ind. ha−1).", "Species assemblage at the crest of the oblique sandbar (T1) significantly differed from the assemblage of the reference areas in 2012 (Table 4).", "Biomass at the crests showed a nearly 4.5-fold increase compared to the reference area (Table 2) and was significantly different for the comparisons between crests (T1 and T2) and reference area (Table 5).", "Plaice, dab, european flounder (Platichthys flesus), sole and hooknose dominated the troughs of the sandbars.", "On average, 164.6 ± 205.1 kg WW ha−1 and 10.2 ± 2.5 fish species haul−1 were caught (Table 2).", "The highest biomass values were found for fish sample 44 and 45 (Fig. 3) in the trough of the oblique sandbar (T1), respectively 522.1 and 484.0 kg WW ha−1 (Fig. 4).", "This is a significant 23-fold increase in biomass values compared to the reference area in 2012.", "Increased biomass of white furrow shell (Abra alba) was not detected in the accompanying infaunal sample but in a sample from the trough west of the sandbar, total infaunal biomass reached 61.9 g AFDW m−2 with 24.9 g AFDW A. alba m−2.", "In 2012, species assemblage in the south-eastern deep area significantly differed from the troughs two years after cessation of sand extraction whereas in 2011 and in troughs 1 year after cessation significant differences were absent (Tables 4 and 5).", "This difference is also clearly visible in the nMDS ordination (Fig. 6) as T2 samples from the trough ended up in the left region surrounded by reference and deep north-western samples (Trecent).", "Furthermore, fish biomass values in the troughs of the parallel sandbar (31.4 kg WW ha−1, Table 2) differed significantly from the locations sampled one year after cessation, which harboured 175.1 and 413.9 kg WW ha−1.", "The accompanying bottom sledge samples of the fish samples from the troughs of the parallel sandbar was characterised by a very high biomass of serpent star Ophiura ophiura (242.2 g WW m−2).", "The most dominant species in the south-eastern deep area of the sand extraction site were plaice, sole, European flounder, dab and shorthorn sculpin.", "European flounder and plaice are Dufrêne-Legendre indicator species (Table 3).", "In 2011, 2012, fish biomass significantly increased 20-fold and reached 413.9 and 409.5 kg WW ha−1 and 15 and 8.5 fish species were caught (Table 2, Fig. 4).", "Demersal fish biomass in south-eastern deep area remained in 2012 almost as high as in 2011 but the abundance of plaice decreased from 3520.1 in 2011 to 1771.4 ind. ha−1 in 2012.", "In 2012, the length of plaice was larger than in 2011, 23.4 cm instead of 15.5 cm, which compensated for the lower observed abundance in 2011 (Table 2, Fig. 5).", "A similar trend was found in the troughs; in 2012, the average length of plaice was 20.8 cm instead of 15.4 cm in 2011.", "The average length of plaice in the reference area in 2010, 2011 and 2012 was 17.82, 15.29 and 17.11 cm, which means that the deep areas of the extraction site attracted larger plaice specimen.", "The length of plaice in the reference area in 2011 was the smallest of the three years, which may explain the smaller length of plaice in the deep areas in 2011.", "No differences in length of the other dominant fish species were observed.", "Biomass in the north-western deep area remained relatively low but just above the reference level, 25.7 ± 15.5 kg WW ha−1.", "Species richness is significantly lower in the north-western deep area, 9 species haul−1 compared 13.1 species haul−1 in the reference area, (Tukey's HSD, p < 0.05).", "The most dominant species were plaice, dab, shorthorn sculpin, sole and hooknose.", "Fish assemblage and environmental dissimilarities revealed a significant association (Mantel: p < 0.01, r = 0.2181).", "Percentage coarse sand was dropped from the analysis because of collinearity with D50, percentage medium and fine sand and mud content were dropped because of collinearity with very fine sand.", "Mantel correlograms analysis showed that autocorrelation was below significance level.", "All nMDS ordinations had stress values below 0.07, which means an outstanding goodness of fit.", "For the 2010–2012 survey periods, the ordination showed a significant association with time after the cessation of sand extraction and water depth (Fig. 6 and Table 6).", "Median grain size D50 (μm), the fraction very fine sand and infaunal white furrow shell biomass was just above the significance level.", "In 2010, only water depth showed a significant association with the ordination.", "In 2011, the very fine sand fraction, time after the cessation of sand extraction, water depth and D50 showed significant associations with the ordination.", "In 2012, infaunal white furrow shell biomass, water depth and the fraction very fine sand showed a strong association with the ordination.", "The association of the ordination with time after the cessation of sand extraction was just above the significance level.", "No significant associations were found for total epifaunal biomass and specific species sampled with the bottom sledge, e.g. scavenging brittlestars (Ophiura sp.) and predatory flying crab (Liocarcinus sp.).", "Stomach and intestine content of plaice was dominated by undigested crushed white furrow shell remains, dab stomachs end guts were filled with remains of brittle stars (Ophiura sp.) and shorthorn sculpin stomachs were filled with whole swimming crabs (Liocarcinus sp.).", "We observed significant changes in fish assemblage in the sand extraction site, which showed a strong association with sediment composition and white furrow shell biomass.", "For the fraction of very fine sand significant differences were found between location and time after sand extraction (Kruskal–Wallis chi-squared = 27.7, df = 5, all p < 0.001).", "In 2011, a significant difference was found between reference and troughs.", "In 2012, the fraction very fines significantly differed between reference area and the south-eastern deep area.", "In general, the fraction very fines decreased at the crests of the sandbars and increased in the troughs and deep areas of the extraction site.", "D50 at the crests of the parallel sandbar increased from 165 μm in 2010 to 304 μm in 2012 and very fine sand fraction decreased from 22.5% in 2010 to 6.1% in 2012.", "We observed the opposite for the sediment in the trough of parallel sandbar, D50 decreased from 321.8 to 133.9 μm.", "Significant differences were found in white furrow shell (Abra alba) biomass, between locations and year (2011: Kruskal–Wallis chi-squared = 15.7729, df = 5, p < 0.01 and 2012: Kruskal–Wallis chi-squared = 12.25, df = 5, p < 0.05).", "In 2011, biomass of white furrow shell was significantly higher at the crests of the sandbars compared to the reference areas.", "White furrow shell is virtually absent in reference areas and the slope of the extraction site.", "After 2 years, white furrow shell biomass increased to 20.6 and 97.4 g m−2 AFDW for respectively the troughs of sandbars and the south-eastern deep area.", "Based on Vessel Monitoring through Satellite (VMS) data (Hintzen et al., 2013) seabed disturbing fishing activity in the sand extraction site was virtually absent in 2010 (Fig. 7).", "In 2011, fishing activity mainly occurred in the northern area of the northern sand extraction site.", "In 2012, fishing activity in the sand extraction site exceeded the fishing activity level of the reference area.", "Furthermore, fishing activity in 2012 almost equalled the fishing activity level before sand extraction (Steenbergen and Machiels, 2013).", "Fish biomass significantly increased 20-fold in the south-eastern deep area of the Maasvlakte 2 sand extraction site and 5-fold on the crests of the landscaped sandbars compared to the reference areas and recently extracted areas.", "This increased biomass is associated with a significant increase in biomass of infaunal white furrow shell (Abra alba).", "In the north-western deep area biomass values remained relatively low but just above the reference level, 25.7 ± 15.5 kg WW ha−1 probably due to ongoing sand extraction activities and the absence of the increase in white furrow shell biomass.", "The highest species richness, 14.2 species haul−1, was found at slope the of the extraction site with turbot (Psetta maxima) and brill (Scophthalmus rhombus) being indicator species.", "These species are at a length of 20 cm known to forage on mobile prey while severely relying on eyesight (De Groot, 1971; Western, 1971; Beyst et al., 1999; Vinagre et al., 2011).", "Instead of foraging in more turbid circumstances, the edge of the extraction site may be more suitable while still favouring from increased fish biomass in the surroundings.", "Species richness in the sand extraction site is lower compared to the reference area.", "Inside the extraction site, on average, 10.5 species per haul were found compared to 13.1 species haul−1 in reference areas.", "The lowest species richness was found in the north-western deep area, 9.0 species haul−1 probably again due to continuing sand extraction.", "Comparisons of species richness between reference area and locations in the sand extraction site are potentially biased due to differences in sampled surface area.", "Based on a study in an extraction site in France, Desprez (2000) stated that sand extraction on the long term might create new habitats such as the presence of boulders and higher heterogeneity of sediment and favour an increase in the richness of benthic fauna and fish.", "A qualitative analysis of fish assemblage was however lacking.", "In line with our results, large-scale sand extraction in the Yellow Sea and the resulting bottom disturbance was shown to have a more pronounced negative impact on fish species richness, a decline of more than 70% of the total number of fish and the number of species was observed (Son and Han, 2007).", "In the Eastern Channel Region, the majority of fish species have shown marked reductions in abundance since the start of aggregate extraction and draghead entrainment was identified as a possible cause (Drabble, 2012).", "Next to differences in biomass and species richness, significant differences in fish species assemblage between reference area and extraction site were found.", "Dab (Limanda limanda) and plaice (Pleuronectes platessa) were the most abundant species in the reference area whereas in the sand extraction site plaice was more abundant.", "This difference is again possibly due to the increase in white furrow shell biomass, which may be a more preferred prey item of plaice.", "In a smaller sand extraction site on the Belgium Continental Shelf with shallow sand extraction dab was also more abundant than plaice (Maertens, 1988) although a clear change in fish distribution was not observed possibly due to continuous sand extraction or less pronounced differences in bathymetry and sediment characteristics.", "Epibenthos in the Belgium extraction site was dominated by the predatory common starfish Asterias rubens and the scavenging serpent star Ophiura ophiura.", "The fish assemblage differed significantly between crests and troughs of the sandbars and tub gurnard (Chelidonichthys lucerna) is an indicator species of the crests which may be induced by differences in macrozoobenthic assemblage.", "The difference in fish assemblage is a first indication of the applicability of landscaping techniques to induce heterogeneity of the seabed although it remains difficult to draw a strong conclusion due the lack of replication of the experiment.", "Comparable differences in species assemblage also occur on the crests of natural sandbanks, early-life history stages of the lesser weever (Echiichthys vipera) were found to be more abundant compared to the troughs (Ellis et al., 2011).", "Several environmental variables may be responsible for the differences in fish species assemblage.", "In general, fishing activity may also play a role but, in 2012, activity in the extraction site was almost equal to the reference area (Steenbergen and Machiels, 2013).", "Therefore, differences in fish assemblage and biomass between dredged and non-dredged areas are not induced by differences in fishing intensity.", "In 2010, only water depth showed a significant association with the ordination.", "In 2011, the very fine sand fraction, time after the cessation of sand extraction, water depth and D50 showed significant associations with the ordination.", "In 2012, infaunal white furrow shell biomass, water depth and the fraction very fine sand showed a strong association with the ordination.", "In 2011, observed differences in the ordination were not yet associated with infaunal white furrow shell biomass, possibly due to the gap between the two sampling activities.", "Macrozoobenthos sampling occurred at the end of June 2010, the start of May 2011 and at the end of April 2012 while fishing occurred mid-July 2010, at the end of July 2011 and mid-June 2012.", "Sell and Kröncke (2013) concluded that fish assemblages on the Dogger Bank (North Sea) were linked to both biotic and abiotic habitat characteristics, abundance of specific fish species could be linked with individual in- and epifauna species.", "In the Western Baltic, white furrow shell comprised 24% of the diet of plaice (Arntz and Finger, 1981; Rainer, 1985) and a comparison of present-day diet and the diet of plaice at the beginning of the 20th century suggested that the preponderance of polychaetes has increased and that of bivalves decreased (Rijnsdorp and Vingerhoed, 2001).", "White furrow shell is a deposit feeding bivalve and tends to prefer fine-grained sediments with a median grain size between 50 and 250 μm and a mud content of 10–50% (Degraer et al., 2006).", "An increase of Tellinid shellfish (e.g. white furrow shell), plaice and common sole was found at deposition areas around aggregation sites in France (Desprez and Lafite, 2012).", "We found that stomach and intestines of plaice were mainly filled with undigested remains of white furrow shell.", "Stomach content analysis of the other fish species also revealed specific preference of prey items but this is not confirmed by our statistical analysis.", "Prey items of dab were dominated by brittle stars (Ophiura sp.), a similar preference was also found by other researchers (Hinz et al., 2005).", "All shorthorn sculpin stomachs contained swimming crabs (Liocarcinus sp.) and this preference was also found in other studies (Western, 1971; Link and Almeida, 2002).", "Time after the cessation of sand extraction is an important variable.", "Directly after sand extraction, fish biomass values are similar to the reference area.", "Fish biomass values increased in 2011 and 2012.", "White furrow shell biomass is showing the same pattern, median grain size in the extraction site is decreasing and the fraction of very fines in the sediment is rising.", "For shallow sand extraction in the North Sea, recovery time of benthic assemblages is estimated to be six years (van Dalfsen et al., 2000; van Dalfsen and Essink, 2001; Boyd et al., 2005).", "A study in a deep temporary sand extraction site with 6.5 million m3 sand extracted, an initial water depth of 23 m and extraction depth between 5 and 12 m revealed a sedimentation rate of 3 cm per year (Boers, 2005).", "Thatje et al. (1999) found a continuous sedimentation rate of around 50 cm per year inside a 700 m wide and 48 m deep natural seafloor crater 20 miles off the coast of Germany with an initial water depth of 34 m. Mud content inside the crater increased from 5% in 1963 up to 40% in 1995 and the benthic community was characterized as an urchin – brittle star association (Echinocardium cordatum and Amphiura Filiformis).", "Considering these sedimentation rates, backfilling of the Maasvlakte 2 sand extraction site may take decades or longer, resulting in a prolonged and more pronounced effect on macrozoobenthos and fish assemblages in the area.", "The increase in length of plaice in the troughs and south-eastern deep area (T2) may be the result of a residing cohort within the extraction site, plaice with body size 16 cm is 1.5 years old and with body size 22 cm is 2.5 years old (Bolle et al., 2004).", "The increase in length of plaice may also be related to prey-size preferences of juvenile plaice.", "In one year specimens of Abra alba reach lengths of 12–14 mm, in two years 13–16 mm and maximum length of 20–25 mm (Holtmann et al., 1996).", "All sediment parameters remained in the same range except SOM values, which was on average 3.9% in the south-eastern deep area and 6.0% in the troughs of the parallel sandbar.", "However, the statistical analysis revealed no significant association of the ordination in 2012 with SOM.", "Dissolved oxygen (DO) levels are influenced by factors such as; SOM, water depth, temperature and water circulation (Eldridge and Roelke, 2011).", "The increase in length may also be induced by avoidance of the south-eastern deep area by smaller plaice, which are more sensitive to reduced DO levels (Miller et al., 1995; Rabalais et al., 2001; Gray et al., 2002).", "DO levels may even be more reduced in the troughs resulting from the specific bathymetry and greater water depth, which may have led to the significant decrease in biomass values from the 23-fold increase in 2011 down to reference level and significant change in species assemblage in 2012.", "Fish biomass was found to be significantly correlated with oxygen concentration and a reduction in fish biomass was observed when oxygen concentration in the bottom water dropped below 3 mg l−1 (Petersen and Pihl, 1995).", "Next to the difference in SOM and possible differences in DO levels, differences in macrozoobenthic assemblage were also present.", "The accompanying bottom sledge samples of the fish samples from the troughs of the parallel sandbar were characterised by very high biomass values of serpent star Ophiura ophiura (242.2 g WW m−2).", "Studying the distribution of demersal fish for two years is not sufficient to understand the final impact of deep and large-scale sand extraction on demersal fish.", "Conclusions on sedimentary evolution are based on a relatively small sample size and short monitoring period.", "More sediment samples from the 2010–2012 surveys will be analysed in future work.", "On-going sedimentation and a rise of mud content up to 40% can be expected (Thatje et al., 1999).", "The highest encountered mud content in the second Maasvlakte extraction site was 23.3% in the south-eastern deep part in 2012.", "Fish surveys were conducted mid-July 2010, at the end of July 2011 and mid-June 2012 while the oxygen concentration reached a minimum at the end of the summer and a maximum in May (Boers, 2005).", "The occurrence of temporal hypoxia and possible detrimental effects on fish and macrozoobenthos cannot be excluded with our data.", "We recommend monitoring of demersal fish and macrozoobenthic assemblage and accompanied sediment variables for a longer period, at least for a period of six years, i.e. the estimated recovery time of shallow sand extraction.", "Six years of monitoring may be even insufficient because of larger differences resulted from the large-scale and deep sand extraction.", "An increase of annual coastline nourishments up to 40-85 million m3 per year is expected for counteracting effects of future sea level rise.", "For the seaward harbour extension of Maasvlakte 2, the Dutch government permitted deep and large-scale sand extraction to reduce the surface area of impact.", "It is questionable if the seabed of the Maasvlakte 2 extraction site will return to its original state within decades.", "We showed that deep sand extraction leads to significant differences in fish biomass between the deep extraction site and the surrounding reference.", "Our findings indicate that ecosystem-based landscaping techniques are feasible and effective in influencing fish assemblages.", "These findings have to be included in future design and permitting procedures of large-scale sand extraction projects." ]

[ "Demersal fish biomass in a deep sand extraction site increased 20-fold and fish species assemblage changed significantly.", "The most abundant fish species in the extraction site is plaice whereas dab dominated reference areas.", "Increased demersal fish biomass is closely linked to increased white furrow shell (Abra alba) biomass.", "Ecological landscaping techniques are feasible and effective in influencing fish assemblage.", "A new ecological equilibrium is not reached after 2 years since biotic and abiotic variables are still adapting." ]

[ "MULT" ]

[ "Cerium oxide nanoparticles", "LPS", "MTT", "Raw 264.7", "Sepsis", "Sprague Dawley rat" ]

[ "1000" ]

[ "This data articles contains data related to the research articles entitled “Inhibition of MAP kinase/NF-kB mediated signaling and attenuation of lipopolysaccharide induced severe sepsis by cerium oxide nanoparticles” in Biomaterials [1].", "In the present study we have confirmed how the administration of a single dose (0.5 mg/kg) of CeO2NPs intravenously into septic rats prevent the LPS induced sepsis associated physiological, blood cell count and inflammatory protein and growth factors changes.", "In addition to that we treated the macrophages cells with different concentrations of cerium oxide nanoparticle to determine the range of cytotoxic and non-cytotoxic concentrations.", "Finally we have hypothesized the potential mechanistic model of LPS induced sepsis.", "Concentration of cerium oxide nanoparticles below 1 µg/ml did not appears to significantly influence cell survival as determined by the MTT assay.", "Finally we have showed the mechanistic insight of systemic inflammatory response syndrome (SIRS) of sepsis and effect of CeO2 nanoparticles treatment.", "Male Sprague-Dawley rats weighing 300–350 g were obtained from Charles River laboratories and housed two per cage using a 12:12- h dark-light cycle at a temperature of 22±2 °C.", "Animals were provided food and water ad libitum and allowed to acclimate for at least two week prior to any experimentation.", "Animals were randomly assigned to the following groups: control group (n=6) received 1.5 ml of endotoxin free water by i.p., CeO2 nanoparticle treated group (n=6) received 1.5 ml of endotoxin free water by i.p., and CeO2 nanoparticles (0.5 mg/kg) in 200 µl of sterile distilled water via the tail vein.", "All procedures were performed as outlined in the guide for the care and use of laboratory animals as approved by the council of the American physiological society and the institutional animal use review board of Marshall University.", "Approximately 100 mg of frozen tissue was taken and pulverized in liquid nitrogen and added to 900 µl of T-PER (Pierce, Rockford, IL, USA) containing 1% protease and phosphatase inhibitors (P8340 and P5726, Sigma- Aldrich, St. Louis, MO, USA).", "Samples were homogenized and centrifuged at 13,000 rpm for 10 min at 4 °C to collect the supernatant.", "Amount of protein in the samples was estimated through 660 nm assay (Pierce, Rockford, IL, USA) and normalized with T-PER and 4× Laemlli buffer to a final equal concentration across all samples.", "Equal amount of protein was loaded in 10% PAGEr Gold Precast gel (Lonza, Rockland, ME) and transferred to nitrocellulose membranes using standard protocol.", "Membranes were block with 5% milk in TBST for 1 h at room temperature, washed thrice with TBST and probed for detection of MyD-88, total and P-p-38 MAPK, total and P- p-ERK 1/2 MAPK (44/42), iNOS, COX-2, HMGB1, Total and cleaved caspase-3, Bax and Bcl-2, GAPDH (Cell Signaling Technology, Danvers, MA).", "Membranes were incubated with primary antibody overnight at 4 °C, washed with TBST (3×5 min), and incubated with secondary anti-rabbit (Cell Signaling Technology, Danvers, MA) for 1 h at room temperature.", "Immunoreactive signal was visualized using Supersignal West Pico Chemiluminiscent substrate (Pierce, Rockford, IL, USA) and quantified using Fluorchem 9900 software (Protein Simple, Santa Clara, CA).", "Protein expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).", "TUNEL staining was performed on tissue sections (5 µm) of frozen samples, which were dewaxed and rehydrated according to the manufactures recommendation (Roche Applied Science, Indianapolis, IN).", "Additional experiments were performed in parallel using DNaseI or the omission of labeling reagent was used as positive and negative control.", "Images were captured using an EVOSfl fluorescence (Fisher Scientific, Pittsburgh, PA, USA) microscope at 200× magnification.", "TUNEL positive apoptotic nuclei were counted and expressed as the number of TUNEL-positive nuclei per 500 nuclei (TUNEL+/500 nuclei).", "RAW264.7 cells from (ATCC) were grown in 25 cm2 cell culture flasks at 30 °C with 5% CO2 in DMEM high glucose medium containing 1% Pen/Strep (10,000 U Penicillin and 10 mg Streptomycin/ml) and supplemented with 5% of fetal bovine serum.", "The cytotoxic and non-cytotoxic concentration of CeO2 nanoparticles was determined by MTT assay.", "The protective effect of CeO2 nanoparticles again LPS induced cytotoxicity was measured by MTT assay as follows.", "RAW 264.7 (1.2×105/ml) were grown in 24 well tissue culture plates until 70–80% confluence and the media was replaced with the fresh medium containing different doses of CeO2 NPs (0, 1, 5, 10, 25, 50, 100 and 1000 ng/ml) for 24 h in the absence and presence of LPS and performed MTT assay.", "To examine if the CeO2 nanoparticles exhibit the ability to sequester or neutralize LPS functionality of LPS, different doses of CeO2 NPs (0, 1, 5, 10, 25, 50, 100, or 1000 ng/ml) were incubated for 2 h at room temperature in growth medium in the presence or absence of LPS (2 µg/ml).", "After centrifugation (5000×g for 10 min) to pellet any suspended nanoparticles, the now clarified media (500 µl) was added to cultured RAW 264.7 cells for 24 h and the MTT assay performed as detailed previously.", "Reactive oxygen species (ROS) levels were determined following the addition of 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA), using the OxiSelectTM kit from Cell Bio Labs (San Diego, CA) as outlined by the manufacturer.", "The assay employs the cell-permeable fluorogenic probe (DCFH-DC).", "The DCFH-DC is diffused into cells and is deacctylated by cellular esterases to non-fluorescent DCFH, which is rapidly oxidized to highly fluorescent 2′,7′-dichlorodihydrofluorescein(DCF) by ROS.", "Briefly, RAW cells were grown in 24-well plates, after the treatment period, cells were washed with cold PBS and incubated in serum-free medium with 100 µl of 1× DCFH-DA reagent and incubated at 37 °C for 1 h. Following this incubation period, cells were washed twice with DPBS and terminated the assay by adding 100 µl of the 2× cell lysis buffer and, the fluorescence of each cell suspension was measured with a fluorometric plate reader (Spectramax, Gemini EM) at 480 nm/530 nm.", "Observation of dye uptake by cells was determined following imaging using an EVOSfl fluorescence (Fisher Scientific, Pittsburgh, PA, USA) microscope equipped with a GFP filter.", "The extent of mitochondrial membrane damage was determined by measuring mitochondrial membrane potential (Δψm), using the JC 1 dye (Cell Technology, Mountain View, CA).", "Experimentally treated cells were washed with cold PBS, removed with trypsin and centrifuged at 400×g for 10 min.", "The resulting pellets were then re-suspended in PBS and the cell density adjusted to 1×105 cells/ml.", "Aliquots (200 µl each) were then centrifuged at 400×g for 5 min, and the resulting cell pellets were assayed for mitochondrial membrane damage.", "In the absence of mitochondrial damage, the JC-1 dye accumulates in the organelle and fluoresces red.", "Conversely, an inability of the mitochondria to concentrate JC-1 dye results in the accumulation of the dye in the cytoplasm and a green fluorescence.", "Overall fluorescence was measured with a fluorometric plate reader (Spectramax, Gemini EM) at 535 nm and 600 nm for green and red fluorescence, respectively.", "Observation of dye uptake by mitochondria was determined following imaging under fluorescence (EVOSfl Model, Fisher Scientific, Pittsburgh, PA, USA).", "RAW 264.7 cells were treated with different concentration of CeO2 nanoparticles in the presence and absence of LPS (2 mg/ml) for 24 h. Nitrite production in the culture supernatants was assayed using the Griess reaction kit from Cayman Chemical Company (Ann Arbor, Michigan, USA).", "One hundred microliters was removed from the medium and incubated with an equal volume of Griess reagent for 30 min at room temperature and the absorbance was measured at 540 nm in an ELISA reader (BioTek, Instrument, Inc., Winooski, Vermont, USA) as outlined by the manufacturer.", "Nitrite concentration was calculated with reference to a standard curve obtained using NaNO2.", "Cells were cultured for 24 h in the presence of CeO2 nanoparticles with and without LPS.", "Cell culture media was recovered by centrifugation at 400×g for 10 min.", "The concentration of TNF-α, IL-6, and IL-1β in the media was measured by ELISA reagent kits (BD Bioscience, Franklin Lakes, NJ, USA) as detailed by the manufacture.", "HMGB1 concentration in the media was estimated by ELISA reagent kits (Chondrex Inc, Red mound, WA, USA)", "Cells were washed with cold PBS, collected by scraping and centrifuged at 400×g for 10 min.", "Total cell lysates, cytoplasmic, and nuclear fractions were prepared by cell lyticTM M cell lysis reagent (Sigma) and NE-PER cell lysis buffer (Thermo Scientific, Rockford, IL, USA) as outlined by the manufacturer.", "Protein content was estimated in triplicate using the Bradford reagent with bovine serum albumin as a standard.", "Western blot was performed as mentioned elsewhere.", "Fifty µg of total protein per well was then subjected to electrophoresis and transfer to nitrocellulose Hybond-C membranes (AmershamTM HybondTM) using standard conditions.", "Membranes were incubated overnight at 4 °C with the appropriate primary antibody iNOS, COX-2, IkB-α, and NF-kβ ( Cell Signaling, Dnavers, MA), washed extensively and then incubated for 1 h at room temperature with a horse radish peroxidase labeled anti-rabbit before detection by ECL (Western Blotting Detection Reagent, GE Health Care Amersham, Piscataway, NJ).", "Immunoreactive signals were quantified by densitometry using Alpha Innotech software (Santa Clara, California).", "Beta actin immunoreactivity was used for normalization between samples.", "The electromobility shift (EMSA) assay was performed using a commercially available kit (Pierce, Rockford, IL, USA) as detailed by the manufacturer Briefly, 5 µg of the nuclear protein extract was used in a binding reaction with 1× binding buffer, 2.5% glycerol, 5 mM MgCl2, 50 ng/µl of poly (dI:dC), and 0.05% Nonidet P-40.", "A double stranded 5′-biotin-NF-kB oligonucleotide probe (consensus sequence 5′-AGTTGAGGGGACTTTCCCAGGC-3′) was added to the reaction at a final concentration of 10 pM/20 µl reaction mix.", "After 30 min, 5 µl of 5× loading buffer was added to the reaction mix, and the samples were resolved on 6% polyacrylamide gels at 120 V for 55 min using 0.5% TBE before transfer to nylon membrane at 10 V for 70 min using 0.5% TBE.", "The protein-DNA probe complexes were cross-linked with a UV cross linker.", "NF-kB specific bands were detected by streptavidin-horseradish peroxidase conjugate using a chemiluminescence nucleic acid detection kit (Thermo scientific, Rockford, IL, USA).", "NF-kB reporter construct were purchase from Promega (Madison, WI, USA).", "For the report assay, cells were seeded into 24 well plates at a density of 5×105 cells per well and transiently transfected with 400 ng of luciferase reporter construct and 100 ng of internal control plasmid of the pCMV-β-galactosidase reporter plasmid from Clontech (Mountain view, CA, USA) using lipofectamin TM200 reagent according to the manufacture’s procedure (In vitrogen, Carlsbad, CA, USA).", "Twenty four hours after transfection, the cells were treated with fresh medium containing LPS and LPS +CeO2 nanoparticles for further 24 h.", "After 24 h, the luciferase activities were measured in cell lysates using luciferase and x-gal reagents following manufacture’s instruction (Promega, Madison, WI, USA and Clone tech, Mountain view, CA, USA).", "In Table 1 physiological behavior and morphological changes among different experimental groups is represented.", "In control animals, there were no changes in appearance, breathing rate, weight change, and animal behavior and, provoked reaction.", "Whereas LPS induced septic animals showed changes in animal morphology, roughened fur, fast breathing rate, slow moment and no flight reaction at all.", "In contrast, the behavior and morphological changes of animals from LPS+ CeO2 nanoparticles group was similar to that from the control animals.", "Table 2 exhibits the result of blood cells count among different experimental groups.", "Compared to control animals, the septic animal’s white blood cells count at 6 h and total and percent (%) lymphocytic count both at 6 and 24 h time points dropped significantly compared to control, whereas percent (%) granulocyte count at both time points increased significantly.", "Nanoparticle treatment increased the total and percent (%) lymphocyte count and decreased the percent (%) granulocyte count significantly.", "Table 3 demonstrates the results of inflammatory protein and growth factors among the different experimental groups.", "Compared to the data obtained from control animals, septic animals appears in increased the levels of inflammatory proteins and growth hormones such stem cell factor, myoglobin, CD-40 ligand, fibrinogen, growth hormone, hepataglobin, leptin and IP-10 both at 6 and 24 h time points.", "Nanoparticles treatment decreased LPS-induced myoglobin at 6 h and 24 h and, the stem cell factor, CD-40, fibrinogen at both time points.", "Fig. 1 reveals the cytotoxic and non-cytotoxic concentration determination in RAW cells.", "Concentration of cerium oxide nanoparticles below 1 µg/ml did not appears to significantly influence cell survival as determined by the MTT assay.", "Fig. 2 shows the mechanistic insight of systemic inflammatory response syndrome (SIRS) of sepsis and effect of CeO2 nanoparticles treatment.", "The liver plays a central role in lipopolysaccharide (LPS) induced sepsis.", "LPS from the circulation may mat illicit a response from the liver causing a release of cytokines and reactive oxygen mediators.", "Kupffer cells (macrophages) represent the main cellular mediators of the effects of LPS in the liver [3].", "LPS interacts with Toll like receptor 4 (TLR-4) which is present on cell membrane of macrophage.", "As the result of interaction, myeloid differentiation protein 88 (MyD88) initiates early activation of NF-kB.", "Upon activation TLR4 with LPS, MyD 88 activates Ikβ kinases, leading to phosphorylation and degradation of IkB and release and translocation of NF-kB to the nucleus as well as induced expression of proinflammatory gene and expression of pro-inflammatory cytokines and chemokines, which causes for liver and other organ damage [4].", "At the same time, LPS leads to secrete elevated reactive oxygen species (ROS) which trigger mitogen-activated protein kinase (MAPK) signaling pathway.", "Activated MAPK kinase plays a crucial role in the release of specific cytokines and chemokines which causes for liver and other organ damage [5].", "The authors declare that they have no competing interests." ]

[]

[ "CENG", "ENGI" ]

[ "Computational fluid dynamic (CFD)", "Fin", "Heat sink", "Thermal performance" ]

[ "1507", "2201" ]

[ "Power density in electronic and microelectronic equipment is significantly increasing due to recent advances in semiconductor technology.", "Therefore, in a highly competitive electronic equipment industry, the improvement of heat transfer rate for such devices is critically essential for long-term reliable operation.", "Cooling the electronic devices by natural convection with the help of finned heat sinks is a common practice in industry due to several advantages (e.g. easy fabrication, powerless operation and high reliability).", "Heat sinks coupled with peripherals such as jets and fans are continuously developing to meet the requirement of higher heat dissipation [1].", "Two common configuration of heat sinks are the parallel arrangement either of the rectangular cross section plate fins or with pin fins on a flat base.", "The former known as plate-fin heat sinks and the latter called pin-fin heat sinks.", "Several studies demonstrate that attempts have been made to compare the efficiency of these configurations.", "For instance, F. Forghan et al. [2] reported that the thermal performance is considerably lower in plate-fin heat sinks compared to pin-fin heat sinks for the low air velocities.", "Contrary to this, research has been developed demonstrating that under certain conditions the thermal performance of the pin-fin heat sink is superior to that of the plate-fin heat sink [3,4].", "To improve the previous understandings, Kim et al. [5] performed an experimental investigation and proposed a model based on the volume averaging method for predicting the pressure drop and the thermal resistance of both configurations.", "They concluded that when the dimensionless length of heat sinks is large and dimensionless pumping power is small, optimized pin-fin heat sinks have lower thermal resistances than optimized plate-fin heat sinks.", "However, the optimized plate-fin heat sinks possess smaller thermal resistances when dimensionless pumping power is large and the dimensionless length of heat sinks is small.", "Not limited to the common type of heat sinks, a considerable body of research has been concentrated to investigate the enhancement of heat transfer rate through applying other type of fins.", "This mainly includes hollow rectangular profile fins [6], rectangular perforation fins [7,8] square and circular perforation fins [9–11], triangular perforation fins [12], inclined plate fins [13,14] and flared fins [2].", "Beside demonstrating the effect of fin shape on the enhancement of heat transfer rate, research has been reported in the literature showing that other parameters may also have significant influence on the thermal performance of heat sinks.", "For instance, some of the above studies looked at the efficiency of different fin arrays [8,11] as well as fin size and spacing [8,13,15].", "In Ref [15], it has also been reported that the fin thickness has more influence on thermal performance of heat sinks compared to the number of fins.", "In another paper, Li et al. [16] carried out a numerical and experimental study to examine the effect of several parameters including width and height of fins, distance of impinging and jet velocity on the thermal performance of plate-fin heat sinks.", "They reported that these parameters have considerable effect on the thermal characteristics of heat sinks as well.", "To investigate the possibility of further enhance in the heat transfer efficiency of the plate-fin heat sinks, kok-cheong wong and sanjiv indran [17] studied the effect of fillet profile on the thermal performance of heat sinks.", "They reported that adding a fillet profile at the bottom of plate-fin heat sinks improves their overall thermal performance by approximately 13%.", "Above understandings reveal there is a good correlation between the design parameters of fins and thermal performance of heat sinks.", "For the remainder of this paper, plate-fin heat sinks with fillet profile are considered and compared with plate-fin heat sinks without fillet profile (as a part of numerical analysis that will be carried put in Section 4.2) since they offer simple design, easy fabrication and implementation.", "Pin-fin heat sinks, however, provide higher heat transfer coefficient that comes at the expense of higher production cost.", "Therefore, the efficiency of plate-fin heat sinks can be improved realistically by establishing an effective flow direction.", "Many researchers have considered analytical solutions for the heat transfer enhancement of plate-fin heat sinks in both parallel and impinging flow.", "The former has been studied based on the Nusselt number [18], optimisation of entropy generation [19] and a pressure drop model [20].", "In case of plate-fin heat sinks by applying impinging flow, Biber [21] developed numerically obtained correlations for pressure loss coefficient and total heat transfer in a variable-length channel that covers a wide range of practical plate-fin heat sinks.", "Later on, Saini and Webb [22] validated Biber model by experiments and made some modifications and improvements.", "In another work, Duan and Muzychka [23] developed a simple semi-empirical model for predicting the pressure drop and the heat transfer coefficient of air-cooled plate-fin heat sinks.", "However, to the best knowledge of the authors, the development of a CFD based model that can demonstrate the effect of flow direction on the plate-heat sinks with fillet profile, as a primary phase to increase the performance of conventional heat sinks in electronic devices, has not yet been reported.", "This is of high importance in engineering applications to develop effective cooling technologies as the extra heat may degrade the function of the electronic devices.", "This paper attempts to fill this gap and presents a numerical analysis to compare thermal performance of plate-fin heat sinks with fillet profile subject to parallel flow and those without fillet profile subject to impinging.", "The impact of this research work can be better understood by considering the limitations of the fan place in electronic equipment industry (e.g. the lack of available space on the side and top surfaces of the electronic box).", "With the help of this study, it is possible to improve the thermal performance of plate-fin heat sinks (as their lower production cost comes at the expense of lower heat transfer rate) based on the flow direction and fillet profile.", "In this section, the investigation develops a CFD model to analyze the convective heat transfer in plate-fin heat sinks.", "There are three main steps in CFD analysis: (a) pre-processing, (b) solver execution and (c) post-processing.", "The former step includes creating the geometry of the desired model as well as the mesh generation, whilst as expected, the results are presented in the last step.", "Boundary conditions are fed into the model in the solver execution (middle) stage.", "In this paper, two geometries i.e. plate-fin heat sinks without fillet profile and plate-fin heat sinks with fillet profile are investigated as shown in Fig. 1(a) and (b) respectively.", "For the plate-fin heat sink with fillet profile, the optimum radius of fillet profile equal to 1.5 mm is considered for the remainder of paper as detailed in Ref. [17].", "The base dimension measures 40 mm × 39.7 mm, with thickness of 5 mm.", "The channel width and thickness of fin is assumed to be constant across the length of the base and measures 3.3 mm and 1 mm respectively as shown in Fig. 1(a).", "Moreover, the fin height in Fig. 1(a) and (b) are different and measures 25 mm and 28.6 mm respectively.", "The difference is due to the compensation for the volume of solid material that removed from the base to generate fillet, as detailed in appendix A.", "For the remainder of paper, the plate-fin heat sink subject to impinging flow, in which air impinges on the heat sink along the y-axis and then flows parallel to x-axis, refers to as the conventional design.", "Proposed design refers to as a plate-fin heat sink subject to parallel flow so that the air flows into the heat sink along the x-axis, Fig. 1.", "In this paper, ANSYS FLUENT R16.0 is used to construct the computational grid as well as to discretize and to solve the governing equations as detailed in Section 2.4.", "To ensure the accuracy of the simulations, the mesh needs to be generated with care in terms of computational time.", "Moreover, to ensure the mesh independency of numerical results, grid independency test is needed to be carried out.", "Table 1 illustrates numerical results for the base temperature with different mesh configurations.", "Two cases are considered, i.e. plate-fin heat sinks with and without fillet profile.", "As it is evident, a significant increase in the number of grid elements does not lead to a notable change in the results, i.e. base temperature.", "Based on Table 1 the difference between the results, either for plate-fin heat sinks without fillet profile or plate-fin heat sinks with fillet profile, remains negligible.", "Therefore, to reduce the computation time, the mesh with lower elements will be used for the remainder of the paper.", "The exploratory experiments in present work have shown that the mesh size with 721704 elements meets the requirements of accuracy and computation time as shown in Fig. 2.", "Moreover, a good agreement is evident between the numerical results and experimental data presented in [5], as detailed in Section 4.1, that proves the choice of number of element in numerical side (CFD).", "For the pressure-velocity coupling, the SIMPLE (Semi Implicit Method for Pressure Linked Equation) algorithm is applied.", "In this research, boundary conditions are considered similar to the work published by Kim et al. [5] where a constant heat flux of 18750W/m2 along with a variable value of inlet air flow (i.e. 0.00092, 0.00218, 0.0033 and 0.00433 kg/s) has been applied.", "In their experimental work, as shown schematically in Fig. 3, the plate-fin heat sink (made of aluminium alloy 6061) is positioned in the wind tunnel (made of acrylic) and air impinges into the tunnel from a pressure tank through a mass flow meter.", "Electrical heaters warm the heat sink up and the temperature distribution at the base of the heat sink at different flow rates is measured by thermocouples.", "In order to measure the pressure difference between the inlet and the outlet of the heat sink, a pressure tap is mounted on the wind tunnel wall that is connected to a manometer.", "Interested readers are encouraged to see Ref. [5] for more details on experimental set up, measurement systems and test procedures.", "Navier-Stokes (N-S), energy and continuity equations are solved numerically to simulate the convective heat transfer process, under some simplification and presumptions as followings:", "The air flow is steady, turbulent and incompressible flow.", "All properties of coolant air depend on its mean temperature.", "The energy and momentum conservation equations are discretized in ANSYS FLUENT R16.0 by a second-order upwind interpolation scheme that is utilized to calculate cell-face pressure.", "To be fair in comparison, the boundary conditions, geometry of plate-fin heat sinks (without profile) and direction of flow (impinging flow) is considered exactly similar to the experimental study carried out in Ref [5].", "On this basis, thermal resistance and pressure drop of air at different mass flow rates are compared as illustrated in Figs. 4 and 5 respectively.", "As it is evident, good agreement is observed between the results of numerical solution (CFD) and the experimental work of Kim et al. [5].", "The maximum discrepancy is 8.8% and 12.4% for thermal resistance and pressure drop respectively.", "A plausible explanation for the difference could be, perhaps, due to the thermal losses and measurement errors.", "Therefore, it can be concluded that the developed numerical approach is accurate enough and can be effectively applied to investigate the effect of flow direction and fillet profile on the thermal performance of heat sinks, as detailed in Section 4.2.", "Having validated the numerical computations with an experimental study, this section aims to clearly identify the impact of the fillet profile and direction of flow on the efficiency of plate-fin heat sinks.", "To be robust in comparison, three sets of simulations will be detailed.", "First, the effect of fillet profile in impinging flow is investigated.", "This is next followed by a numerical study on the effect of flow direction on plate-fin heat sinks with fillet profile.", "The section concludes with a focus on the comparison of proposed and conventional designs based on numerical simulations.", "The effect of fillet profile on thermal efficiency of an air-impinged plate fin heat sink has already been investigated numerically (and validated against experimental data) in Ref [17].", "To improve previous understandings and to distinct the contribution of adding a fillet profile to the overall thermal enhancement of proposed design, plate-fin heat sinks with and without profile are compared in this section.", "Fig. 6 presents the temperature contour of the plate-fin heat sink subject to impinging flow for two different configurations, i.e. with fillet profile and without it.", "As it is expected in plate-fin heat sinks with fillet profile, due to the increase in heat transfer area, more amount of heat is removed from the hot region.", "Therefore, the base temperature for plate-fin heat sinks with fillet profile is less than those without fillet profile.", "Moreover, the heat distribution is altered by the fillet profile that proves the superior thermal performance of this configuration.", "To investigate the effect of adding a fillet profile on the efficiency of plate-fin heat sinks subject to impinging flow, the variation of heat-sink's base temperature along with thermal resistance of both configurations (i.e. with and without fillet profile) is compared at various values of the air mass flow rate in Figs. 7 and 8 respectively.", "As it is evident in figures, the base temperature and thermal resistance decrease by the mass flow rate increase in both configurations.", "However, the base temperature and thermal resistance of a heat sink with fillet profile are approximately 3.78% and 5.18% lower compared to a plate-fin heat sink without fillet profile at different mass flow rates that is in good agreement with previous works [17].", "The effect of flow direction on the thermal performance of conventional plate-fin heat sinks without fillet profile has already been studied in Refs [19–23].", "This section aims to study the effect of flow direction in plate-fin heat sinks with fillet profile and hence, provide a better understanding of its contribution to the overall thermal enhancement of proposed design (i.e. plate-fin heat sink with fillet profile subject to parallel flow) that will be detailed in next section.", "As it is evident in Fig. 9, for both configurations, the area that is directly blown by the fan remains cooler for all flow rates.", "In impinging flow, however, the hot region occupies the base of the heat sink whilst the cold region settled in the top of the fin indicating the lower thermal efficiency of this configuration.", "In parallel flow the temperature contours show more effective thermal distribution between the base and upper region of the heat sink that may lead to a notable thermal efficiency enhancement.", "To study the effect of flow direction on the thermal performance of plate-fin heat sinks, the variation of heat-sink's base temperature along with thermal resistance of both configurations (i.e. impinging flow and parallel flow) is compared at various values of the air mass flow rate in Figs. 10 and 11 respectively.", "As it is evident, the base temperature and thermal resistance of a plate-fin heat sink with fillet profile subject to parallel flow are approximately 3.85% and 5.29% lower at different mass flow rates compared to a plate-fin heat sink with fillet profile subject to impinging flow.", "Having investigated the effect of fillet profile and flow direction on thermal performance of plate-fin heat sinks, the conventional design (plate-fin heat sink without fillet profile subject to impinging flow) is compared with the proposed design (plate-fin heat sink with fillet profile subject to parallel flow) in terms of temperature difference and thermal resistance.", "Fig. 12 shows the temperature contour of the plate-fin heat sink for both proposed and conventional designs at various mass flow rates of air.", "As it is evident in conventional design, the hot region occupies the base of the heat sink whilst the cold region settled in the top of the fin indicating the lower thermal efficiency of this design.", "However, by creating a fillet, the temperature contours show an effective thermal distribution between the base and upper region of the heat sink that leads to a notable thermal efficiency enhancement as has been discussed in Section 4.2.1.", "In addition, the heat distribution is altered by the fillet profile and hence the convection is smoothened near the bottom of the plate fin.", "Moreover, since in the proposed approach the fan is located closer to the base of heat sink, therefore, more amount of heat is removed from the hot region.", "Hence, the base region remains cooler for the proposed design.", "As it has been detailed in Section 4.2.2, for both configurations, the area that is directly blown by the fan (i.e. top region in conventional design and side region in proposed design) remains cooler for all flow rates.", "However, the coldest area (dark blue region) is significantly lower for proposed design indicating the superior thermal efficiency of this model over the conventional design.", "To get insight into the efficiency of proposed design, the variation of heat-sink's base temperature of conventional model is compared with the proposed design at various values of the air mass flow rate in Fig. 13.", "As it is evident, the base temperature of heat sink is approximately 7.5% lower for the proposed approach at different mass flow rates that shows a notable improvement in design.", "This could be the effect of adding a fillet profile along with flow direction as has already been discussed in Sections 4.2.1 and 4.2.2.", "In Fig. 14, variation of thermal resistance for the conventional design is compared with the proposed approach at various values of the air mass flow rate.", "A notable difference (approximately 18%) is evident between thermal resistance of the proposed design and conventional design that may prove the efficiency of proposed approach.", "A plausible explanation for such improvement in proposed approach is that, in addition to the effect of fillet profile and flow direction that have been discussed in previous sections, the average heat transfer coefficient goes up due to the temperature gradient increase between the base of heat sink and the airflow.", "Therefore, the thermal resistance is lower as it is inversely proportional to the surface area and coefficient of heat transfer, Eq. (13).", "Thermal performance of plate-fin heat sinks with fillet profile subject to parallel flow (proposed design) and those without fillet profile subject to impinging (so called conventional design in the paper) has been compared.", "To achieve this, a CFD model for plate-fin heat sinks without fillet profile subject to impinging has been developed and validated with an experimental study from the literature.", "The obtained results demonstrated that the maximum difference between experimental data and numerical results were 12.4% and 8.8% for pressure drop and the thermal resistance respectively under same conditions.", "This proves the accuracy of the numerical analysis that has been developed in this study.", "In particular, three sets of simulations have been discussed, i.e. the effect of fillet profile on plate-fin heat sinks subject to impinging flow, the effect of flow direction on plate-fin heat sinks with fillet profile and the comparison of the proposed design and conventional design.", "The study has shown that adding a fillet profile and changing the flow direction (from impinging to the parallel flow) have a notable effect on thermal performance of plate-fin heat sinks.", "Although, this was beyond the scope of present paper to demonstrate which one has superior effect, the primary results have shown that both parameters have approximately same effect on base temperature and thermal resistance of heat sinks.", "Furthermore, the numerical results of comparison between proposed design and conventional design shown that the base temperature of heat sink in proposed design decreases by 7.5% compared to the conventional model.", "Moreover, the thermal resistance for proposed design is 18% lower in comparison with the conventional design.", "Therefore, a notable improvement in the thermal performance of the heat sink was demonstrated that might help to develop more advanced cooling technologies for electronic equipment industry." ]

[]

[ "AGRI", "BIOC" ]

[ "Bloodfeeding", "Hyaluronidase", "Pathogen transmission", "Saliva", "Tabanids" ]

[ "1109", "1303", "1312" ]

[ "Tabanids (Diptera: Tabanidae) are a cosmopolitan group of haematophagous insects comprising more than 4.400 species (Roskov et al., 2015).", "These pests are considered a serious nuisance, and are characterized by their persistent biting behavior and painful bites that may induce allergic reactions in sensitive individuals (Bircher, 2005).", "In comparison with other blood-feeding insects, however, tabanids have remained relatively neglected, even though they are efficient biological and mechanical vectors of various human and animal disease agents including viruses, bacteria and parasites (reviewed in Baldacchino et al., 2014).", "Nevertheless, over the past few decades the epidemiological relevancy of tabanids as mechanical vectors has been focused on due to the increasing incidence of some veterinary important diseases, above all equine infectious anemia (Herholz et al., 2008), bovine besnoitiosis (Alvarez-Garcia et al., 2013), and livestock trypanosomoses (Desquesnes, 2004; Desquesnes et al., 2013).", "Of the biologically transmitted pathogens, the filarial nematode Loa loa has recently re-emerged as a disease agent of public health importance, because of its negative impact on the control of onchocerciasis and lymphatic filariasis in areas of co-endemicity (Zoure et al., 2011).", "The saliva of bloodsucking insects modulates host hemostasis and immunity in order to enable successful blood-meal acquisition (Fontaine et al., 2011).", "Salivary components found in tabanids correspond entirely with their feeding strategy of engorging a large bloodmeal within a few minutes; their saliva exhibits strong anticoagulant and vasodilatory activities (Kazimirova et al., 2002; Rajska et al., 2003), together with a broad repertoire of platelet aggregation inhibitors (Reddy et al., 2000; Xu et al., 2008; Zhang et al., 2014) and immunoregulatory peptides (Yan et al., 2008; Zhao et al., 2009).", "An extreme diversity of more than 30 anti-thrombosis components has been described in the saliva of Tabanus yao (Ma et al., 2009).", "Hyaluronidase activity has been detected in the saliva of Tabanus yao and Chrysops viduatus, and transcripts coding for hyaluronidase have been found in sialotranscriptomes of the horseflies T. yao and Tabanus bromius (Ribeiro et al., 2015; Volfova et al., 2008; Xu et al., 2008).", "Hyaluronidases (hyases) are widely distributed enzymes in nature, being found in mammalian somatic tissues, multiple animal venoms and microbial toxins (El-Safory and Fazary, 2010).", "Hyase activity has also been demonstrated in various bloodsucking parasites, e.g. ticks (Neitz and Vermeulen, 1987), leeches (Hovingh and Linker, 1999), and some invasive nematodes (Hotez et al., 1994).", "Hyases preferentially degrade hyaluronic acid (HA), but also exhibit a diverse ability to digest chondroitin (Ch) and chondroitin sulphates (CS) (Honda et al., 2012; Stern and Jedrzejas, 2006).", "Their predominant substrate HA is a multifunctional high molecular weigh glycosaminoglycan ubiquitous in a vast majority of mammalian tissues (Fraser et al., 1997).", "Being a prominent component of both the pericellular coat and the extracellular matrix (ECM) of the skin, HA contributes to the skin integrity and resistance against penetration or invasion due to its unique physiochemical and biological characteristics.", "Moreover, HA also participates in numerous physiological and pathological processes including inflammation and wound healing, and the biological functions of HA polymers are dependent on the molecular weight of the molecule (for review see Anderegg et al., 2014; Stern and Maibach, 2008).", "Among insects, hyases from Hymenoptera venoms are the best characterized.", "Similarly to other arthropod venom hyases, they contribute to local tissue damage via HA degradation and serve as a spreading factor for other venomous components (reviewed by Bordon et al., 2015).", "Furthermore, these enzymes are also responsible for the cross-reactivity between Hymenoptera venoms, although their relevance differs among species (Jin et al., 2010; King et al., 1996).", "The very first hyase activity in the saliva of a haematophagous insect was documented by Charlab et al. (1999) in the sand fly Lutzomyia longipalpis, with its hyase cDNA showing high similarity to honey bee and vertebrate hyases.", "Subsequently, hyase activity has been identified in the saliva of all other sand fly species studied, and according to their physicochemical characterization and sequence analyses they have been classified in the endo-β- N acetyl-hexosdiaminidase (E.C.3.2.1.35) family (Cerna et al., 2002; Rohousova et al., 2012; Vlkova et al., 2014).", "Additionally, hyase has been confirmed as a common constituent in the saliva of other Diptera with pool-feeding modes, namely in black flies (Ribeiro et al., 2000; Volfova et al., 2008) and biting midges (Radrova et al., 2015; Russell et al., 2009).", "Degrading ECM, salivary hyases may (i) help the diffusion of other bioactive salivary substances, and (ii) promote pathogen invasion due to both increased skin permeability and the immunoregulative activities of generated HA fragments (Ribeiro et al., 2000).", "Moreover, hyases have also been suggested to contribute to pathogen transmission (Charlab et al., 1999).", "The enhancing effect of co-inoculated hyase to Leishmania major infection in murine skin has been documented by Volfova et al. (2008).", "However, the precise role of salivary hyases in pathogen transmission still needs to be elucidated.", "In this study, we demonstrated the presence of extremely high levels of hyase activity in the saliva of ten species from four tabanid genera Chrysops, Haematopota, Hybomitra and Tabanus.", "We described the basic characteristics of the enzymes, including their molecular weight under reducing and non-reducing conditions, and quantified and compared their activities to the hyases of sand flies, which are the best known among bloodsucking insects.", "Female tabanids were collected with a Malaise trap or by manual capture on human and cow bait at four trapping sites in Bohemia, the Czech Republic (Table 1).", "Females were transported to the laboratory and immediately processed as described below.", "The species used in the study are summarized in Table 2.", "The species identification was done according to the key by Chvala et al. (1980).", "Female tabanids were immobilized on ice.", "Salivary glands (SG) were dissected under a stereomicroscope in 20 mM Tris buffered saline (TBS: 150 mM NaCl, pH 7.6).", "Dissected SGs were transferred to Eppendorf tubes with TBS and stored at −80 °C in batches of 5 pairs of SGs in 20 μl (small and medium size species of Chrysops spp, Haematopota spp) or 2 pairs of SGs in 50 μl (large size species of Hybomitra spp, Tabanus spp).", "SGs of 5-day-old females of two sand fly species with known hyase activity, Phlebotomus arabicus and Phlebotomus papatasi originating from laboratory colonies at Charles University in Prague, were used as controls.", "Salivary gland extracts (SGEs) were obtained by the disruption of tissue by three freeze-thaw cycles in liquid nitrogen, mechanical homogenization and centrifugation at 12,000 g for 5 min.", "Protein concentrations in the resulting SGEs were measured by Quibit Fluorometr (Invitrogen) following the manufacturer's instructions.", "The detection of enzyme activity and determination of substrate specificity were done using the dot method on 10% polyacrylamide gels with copolymerized substrate.", "Gels were prepared using 0.1 M acetate buffer, containing 0.1 M NaCl, 0.05% Tween-20 (AA buffer) and 0.002% substrate.", "Based on our previous study (Volfova et al., 2008), pH 5.0 was chosen as optimal for the salivary hyase of tabanids.", "Hyaluronic acid (HA) potassium salt from human umbilical cord (SIGMA, #H1504) or chondroitin sulfate A (CS-A) sodium salt from bovine trachea (Sigma, #C9819) were incorporated as substrates.", "SGE samples adjusted to the same protein concentration were dotted on the gel (1.0 μg of total salivary protein in a 1 μl dot) in three independent trials.", "TBS served as a negative control, and bovine testicular hyaluronidase (SIGMA, #H3884, 1 μg in 1 μl) and Phlebotomus papatasi SGE (0.4 μg in 1 μl) were used as positive controls.", "Incubation was carried out for 24 h at 37 °C in a moist chamber.", "The gels were then washed in water, soaked in 50% formamide for 30 min and stained in Stains-all (Sigma) solution (100 μg/ml in 50% formamide) for 24 h in the dark.", "After a rinse in distilled water the gels were scanned and photographed.", "Hyase activity was visible as a pink spot on a dark blue background.", "The hyase activity in SGEs of eight tabanid species (Table 2) was quantified using a sensitive method by Frost and Stern (1997) modified by Rohousova et al. (2012).", "Biotinylated HA (bHA), prepared as described in Cerna et al. (2002), was immobilized onto Covalink NH microtiter plates (NUNC).", "Fifty μl of bHA in 1.62 mM N-hydroxysuccinimide was mixed in wells with 50 μl of 0.64 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide at a final concentration of 1 μg of bHA per well, and incubated overnight at 4 °C.", "Following the incubation, the plates were washed three times in PBS, pH 7.2, containing 2 M NaCl and 50 mM MgSO4.", "The plates with immobilized bHA were coated for 45 min with 1% BSA in PBS, then washed and equilibrated with the appropriate assay buffer (100 μl/well).", "SGE samples were incubated for 45 min at 37 °C in quadruplicates at a final concentration of 0.005 μg of total salivary protein per well.", "To obtain a standard curve ranging from 0.003 to 0.2 turbidity reducing units (TRU), bovine testicular hyase (SIGMA; 851 units/mg solid) was serially diluted in 0.1 M acetate buffer, pH 4.5, 0.1 M NaCl, 0.1% Triton X-100.", "Wells without bHA or without SGE were used as negative controls.", "The reaction was terminated by 6 M guanidine (200 μl/well).", "Plates were washed in PBS containing 2 M NaCl, 50 mM MgSO4, 0.05% Tween-20, pH 7.2.", "Then, avidin-peroxidase (SIGMA) was added at a final concentration of 0.2 μg/well and incubated for 30 min at room temperature.", "The color reaction was developed with o-phenylenediamine substrate in 0.1 M citrate-phosphate buffer, pH 5.5, and absorbance was measured at 492 nm (Tecan-Infinite M 200 Fluorometer; Schoeller Instruments).", "Raw data were evaluated by Measurement Parameters Editor Magelan 6 (Tecan).", "The obtained results were expressed as relative TRU (rTRU).", "Three independent experiments were performed with different set of SGE samples in each trial.", "The pH dependence of enzyme activities in SGE of Chrysops relictus, Ha.", "pluvialis and Hy.", "muehlfeldi hyases was tested within the pH range of 3.5–8.5.", "Four different buffers were used: 0.1 M citrate, pH 3.5; 0.1 M acetate, pH 4.0, 4.5, 5.0 and 5.5; 0.1 M bis-Tris, pH 6.0, 6,5 and 7.0; 0.1 M Tris, pH 7.5, 8.0 and 8.5; all buffers contained 0.1 M NaCl and 0.1% Triton X-100.", "Controls were performed for each pH separately.", "Two pH values, pH 4.0 and 5.0 (both 0.1 M acetate buffer) were chosen for comparisons of enzyme activities among various tabanid species.", "The hyase activities of the three most common tabanid species studied, Chrysops viduatus, Ha.", "pluvialis and Hy.", "muehlfeldi, were compared with the enzymes from the saliva of two sand fly species P. papatasi and Phlebotomus arabicus, as the salivary hyases of sand flies are the best characterized of the haematophagous insects (Cerna et al., 2002; Rohousova et al., 2012).", "Since the enzyme activity was much stronger in tabanid SGE, different protein loads were used to obtain appropriate levels of absorbance in wells: 0.03 μg in sand flies and 0.001 μg in tabanids.", "Three independent experiments were carried out on microtitration plates at pH 5.0 as described above.", "The molecular weights (MW) of hyases were determined by SDS PAGE electrophoresis carried out on 10% polyacrylamide gels (0.75 mm thick) with 0.002% HA incorporated as described previously (Volfova et al., 2008).", "Considering that the hyase activities varied among tabanid species (see results from microplate assays), different protein contents were loaded per lane in order to obtain bands of approximately equal intensity (Table 2).", "Samples run under reducing conditions were pre-incubated with sample buffer containing 2% 2-mercaptoethanol for 40 min at 45 °C.", "Following electrophoresis, gels were rinsed 2 × 20 min in 0.1 M Tris, pH 7.8, with 1% Triton X-100, 20 min in AA buffer to wash out SDS, and then incubated in AA buffer for 2 h at room temperature.", "After rinsing in distilled water, the gels were stained in Stains-all as described above.", "Hyase activity was visualized as a pink band on a dark background.", "Invitrogen BenchMark™ Protein Ladder (Invitrogen # 10747-012) stained in Coomassie Brilliant Blue R-250 was used as a standard for the MW determination.", "Additional experiments on HA substrate gels were performed to analyze the sensitivity of Tabanus bovinus salivary hyase to the denaturing effect of sodium dodecyl sulfate (SDS).", "The enzyme activity was studied in the presence and the absence of SDS on two different 10% polyacrylamide gels (0.75 mm slab) with 0.002% HA incorporated.", "Gel A was mixed using AA assay buffer, pH 5.0, as described above in the dot method.", "In gel B, the assay buffer was replaced by 1.5 M TRIS-HCl, 0.4% SDS, pH 8.8, which is routinely used for electrophoretic gels (with a final concentration of 0.1% SDS in the gel).", "T. bovinus SGEs diluted either in TBS without SDS (TAB1) or in a sample buffer containing 1% SDS (TAB2), were dotted on the gel at a concentration of 1.5 μg of the total salivary protein per 1 μl dot.", "Bovine testicular hyase (1 μg in 1 μl) with and without SDS served as a positive control.", "Incubation was carried out for 24 h at 37 °C in a moist chamber.", "Afterwards, gel A was washed and stained as described in the dot method.", "Gel B was processed as described above for the gels after the zymographic procedure.", "Then the gels were scanned and photographed.", "The protein concentrations in SGEs of the ten tabanid species studied are summarized in Table 2.", "The protein content mostly reflected the body and salivary gland size of the species and differed more than fifteen-fold between the most and the least concentrated SGE samples (Tabanus autumnalis versus C. relictus).", "The protein concentration of SGE in controls was 0.34 μg, and 0.28 μg per gland in Phlebotomus arabicus, and P. papatasi, respectively.", "The dot method on substrate gels was performed to examine the hyase activity in SGE and to study the substrate specificity of the enzyme.", "Pronounced activity was observed in the saliva of all species, and HA was found to be the preferential substrate for tabanid salivary hyases (Fig. 1A), whereas CS-A showed solely moderate hydrolysis (Fig. 1B).", "The highest degradation of HA was detected in Tabanus maculicornis and both Hybomitra species, while the activities of the other large-size tabanids T. autumnalis and T. bovinus were lower.", "Among small species, the hyase of C. relictus acted most intensively.", "The results obtained from the CS-A substrate gel were consistent with those from the HA gel, although digestion of the substrate was much slower.", "The enzymes from Hy.", "muehlfeldi, Hy.", "ciureai and T. maculicornis SGEs were the most active, followed by Chrysops caecutiens, while the lowest activity was observed in SGE samples of T. autumnalis and T. bovinus (Fig 1B).", "The pH dependence of hyase activity in SGEs of C. relictus, Ha.", "pluvialis and Hy.", "muehlfeldi was characterized using an assay on microtitration plates with incorporated bHA.", "In Ha.", "pluvialis and Hy.", "muehlfeldi considerable activities were observed within a pH range of 4.0–5.5, with pH optimums at 4.0 and 5.0, respectively.", "Nevertheless, in both of the species the activity was well detectable over a broad pH range from 3.5 to 7.5 (Fig. 2A, B).", "In C. relictus substantial activity occurred at pH 4.0 with a sharp peak at pH 5.0; at higher pH it went down rapidly, being reduced to 10% at pH 5.5 and almost undetectable under alkaline conditions (Fig. 2C).", "A sensitive assay on microtitration plates was also used to quantify the hyase activity in SGSs of eight tabanid species (T. bovinus and Ha.", "italica SGEs were not included due to the lack of material for repeated experiments).", "Since two different values of pH optima were detected in the previous tests (see above), the quantitative assays were performed at pH 4.0 and at pH 5.0.", "Average activities related to the total protein content in SGEs are summarized in Table 2, and the relative activities at both pH 4.0 and pH 5.0 are compared in Fig. 2D.", "The activity levels detected corresponded mostly to the results on HA substrate gels.", "The highest activity was seen in Hy.", "muehlfeldi, followed by other large and medium size species Hy.", "ciureai, T. maculicornis and T. autumnalis (Fig. 2D).", "In C. relictus SGE, much higher activity was found at pH 5.0 than at pH 4.0, which corresponded well with the sharp peak at the pH optima in this species (Fig. 2C).", "By contrast, insignificant differences between the activity at pH 4.0 and pH 5.0 were found in T. autumnalis.", "The lowest hydrolysis of HA was detected in C. caecutiens SGE at both pH values tested.", "Hyaluronidase from Ha.", "pluvialis SGE was the only one among the tested enzymes with an optimum at pH 4.0.", "As the hyase activity in all tabanid species appeared to be high, an additional experiment (repeated three times) was carried out on microtitration plates at pH 5.0 to compare hyase activity of three tabanid and two sand fly species.", "Significant differences between tabanids and sand flies were found: the hyase activity (expressed in rTRU per 1 μg of total salivary protein) reached 1.15 ± 0.15 in P. arabicus and 1.64 ± 0.14 in P. papatasi, whereas activities in tabanid flies were an order of magnitude higher, 11.04 ± 2.7 in C. viduatus, 15.19 ± 2.1 in Ha.", "pluvialis and 52.90 ± 4.6 in Hy.", "muehlfeldi.", "These data confirmed the extremely high hyase activity in the saliva of tabanids (Fig. 3).", "Ten tabanid species were analyzed by SDS PAGE zymography on HA substrate gels to estimate the molecular weight of their salivary hyases.", "In all but one species, marked activities were detected under both non-reducing and reducing conditions.", "In non-reduced samples enzyme activities were visualized as monomers providing a single diffuse band.", "The estimated MW of the enzymes ranged between 35 and 52 kDa (Fig. 4A).", "In Haematopota spp., the bands had a MW around 40 kDa, in Hy.", "muehlfeldi and Hy.", "ciureai 35 and 37 kDa, respectively.", "In Tabanus species, the activity bands were detected at around 38 kDa in T. autumnalis, and 48 kDa in T. maculicornis.", "The highest variability was found in Chrysops species, with MWs of 52, 48 and 40 kDa in C. viduatus, C. caecutiens and C. relictus, respectively.", "Under reducing conditions the enzymatic activity gave sharper bands that allowed more accurate estimations of MW, which ranged from 43 to 52 kDa.", "In most species the activity bands had higher MW than in non-reducing conditions, suggesting that the hyases of tabanids are monomers that run slower in the reducing environment due to conformation changes after the breakage of disulfide bonds by 2-mercaptoethanol (Fig. 4B).", "This difference was most pronounced in Hy.", "muehlfeldi with MWs around 35 and 46 kDa under non-reducing and reducing conditions, respectively.", "On the other hand, the hyaluronidase activity of C. viduatus gave a 52 kDa band in both conditions tested.", "In T. bovinus, no activity was detected when the protein content loaded per lane was similar to the other tabanids tested.", "Weak bands appeared after the protein concentration was increased tenfold, and their MW occurred in the same range as those in T. autumnalis, 38 kDa under non-reducing and 48 kDa under reducing conditions (Fig. 4A, B).", "For the protein loads applied see Table 2.", "The pronounced hydrolysis of HA found in T. bovinus SGE on substrate gels contrasted with the scarcely detectable activity found by zymographic analysis.", "To clarify this unique discrepancy, hyase of T. bovinus was additionally tested for sensitivity to SDS by the dot method on HA substrate gels in the presence and the absence of SDS.", "This experiment revealed an irreversible sensitivity of the enzyme to the denaturing effect of SDS.", "On substrate gels without any incorporated SDS, the hyase activity remained visible only in samples diluted in TBS.", "The samples prepared using a sample buffer with 1% SDS did not show any degradation of HA (Fig. 5A).", "Furthermore, on gels copolymerized with 0.1% SDS the activity was completely inhibited in both of samples, the one with added SDS and the second with TBS alone (Fig. 5B).", "In the control, the activity of bovine testicular hyase was not affected by the presence of SDS in either the substrate gels or in the sample buffer.", "We demonstrated pronounced hyase activity in ten tabanid species of the genera Chrysops, Haematopota, Hybomitra and Tabanus.", "Hyase has been previously shown to be a ubiquitous component in the saliva of bloodsucking insects with pool-feeding mode (Ribeiro et al., 2000; Volfova et al., 2008).", "However, compared to other pool-feeders, the ability of tabanid hyases to hydrolyze HA is unusually high.", "Here we showed that the enzyme activity of Hybomytra muehlfeldi is 32-fold higher than hyase of Phlebotomus papatasi, which had previously been found to be one of the most active among sand flies (Cerna et al., 2002).", "The very high levels of the enzyme activity in tabanids might be related to their aggressive biting behavior.", "Tabanids are able to ingest up to 200 mg of blood within a few minutes (Chvala et al., 1980), and the presence of strong hyase activity in their saliva supports the hypothesis about the essential role of this enzyme in the formation of the feeding hematoma in blood-sucking insects with pool-feeding mode (Ribeiro et al., 2000).", "Depolymerization of HA by tabanid salivary hyases might facilitate more the efficient spread of other bioactive salivary substances essential for a prompt blood-meal acquisition.", "We found significant differences in hyase activity among the tabanid species studied; the substrate degradation was several-fold lower in the small species than in medium- and large-sized ones.", "This finding might be explained by dissimilarities in the feeding behavior of various tabanid species.", "Small species like Chrysops are more frequently able to complete their blood-meal during one continuous feeding, while larger Hybomitra and Tabanus are usually interrupted by host defensive reactions and require several partial attempts before reaching engorgement (Desquesnes and Dia, 2003; Magnarelli and Anderson, 1980; Muzari et al., 2010; for review see Baldacchino et al., 2014).", "Hypothetically, small species may not need such potent salivary hyase for blood-meal acquisition.", "Nevertheless, the hyase activity quantified here did not entirely correlate with total salivary protein content within the studied genera, and might also be affected by adaptations to a different spectrum of hosts.", "Among Chrysops spp., the smallest species, C. relictus, displayed the highest enzyme activity.", "Similarly, medium-sized Tabanus maculicornis showed more effective degradation of HA than the large species T. autumnalis and T. bovinus.", "According to Chvala et al. (1980), C. relictus has the broadest host spectrum of the studied Chrysops species.", "Analogously, T. maculicornis feed on a wide range of large mammals, in contrast to T. autumnalis and T. bovinus.", "Therefore, the range of hosts might be another aspect of feeding strategy that affects the pharmacological potency of tabanid saliva.", "A similar observation has been published by Kazimirova et al. (2002) in a study of the anticoagulant activities in the saliva of 19 tabanid species belonging to six genera: most tabanid species with the strongest anticoagulant activities were those having a broad host spectrum.", "The DNA sequence encoding for the tabanid hyase obtained from a Tabanus yao Macquart salivary gland cDNA library shared 60% sequence similarity with hyase from wasp venoms, and has been classified to the glycoside hydrolase family 56 (Ma et al., 2011).", "In addition to the HA hydrolysis, these enzymes may also display a diverse capability to catabolize chondroitin and chondroitin sulfates, glycosaminoglycans that are structurally related to HA (El-Safory and Fazary, 2010).", "Our study demonstrated the ability of all tabanid species tested to utilize CS as an alternative substrate.", "The highest activity on CS substrate gels was observed in both Hybomitra spp. and T. maculicornis, the species with the strongest hydrolysis of HA, whereas species with lower activity on HA gels also showed weaker digestion of the alternative substrate.", "Previously, salivary extracts of various blood feeding insects has been shown to hydrolyze CS, although the intensity of digestion is usually lower compared to HA.", "Interestingly, the hydrolysis of CS significantly differs among insect taxons; pronounced degradation of CS has been detected in Culex quinquefasciatus (Volfova et al., 2008) while moderate hydrolysis has been documented in all sand flies studied (Cerna et al., 2002).", "A high variability of CS degradation has been observed within the genus Culicoides: C. newsteadi and Culicoides nubeculosus displayed strong activity, whereas the hydrolysis in C. imicola, C. pulicaris and C. punctatus was only moderate, and no activity towards CS was observed in Culicoides obsoletus (Radrova et al., 2016).", "The hydrolysis of alternative substrates by hyases might be related to the supposed ability of HA and CS to form complexes in nature and to the adaptation of these enzymes to degrade these two polymers simultaneously, at approximately a speed that reflects their relative abundance in tissues (Stern et al., 2007).", "In mammalian skin, HA is expressed in both the epidermis and dermis, in contrast to CS which is associated mainly with the upper part of the dermis (Tammi and Tammi, 1998; Coolen et al., 2010).", "Moreover, the pH optima for the hydrolytic activity of hyases toward different substrates may slightly differ (Honda et al., 2012).", "Thus, the relatively broad pH range of hyase activity detected in the saliva of some tabanid species might facilitate the efficient ECM degradation required for the successful feeding.", "A pH of 5.0 was found as the optimum for hyase activity in seven of eight tested tabanid species, with the hyase of Ha.", "pluvialis the only one tested with an optimum at pH 4.0.", "In other bloodsucking insects, salivary hyase activities have also been reported to have optima at acidic pH, which is probably an adaptation to the slightly acidic environment of the skin.", "In sand flies, Cerna et al. (2002) demonstrated HA hydrolysis over a broad pH range between 4.0 and 8.0 with an optimum at pH 5.0.", "The hyase of Simulium vittatum black fly showed substantial activity from pH 5.0–7.0 and a peak at pH 6.0 (Ribeiro et al., 2000).", "SDS PAGE zymography revealed the monomeric character of the enzymes in all tested tabanid species.", "Under non-reducing conditions, the activities were visible as single bands with estimated MW ranging between 35 and 52 kDa.", "Similar MWs have also been demonstrated for two tabanids studied previously, C. viduatus and T. yao (Volfova et al., 2008; Xu et al., 2008).", "In contrast to other the tabanid species studied, however, the salivary hyase of T. bovinus showed irreversible sensitivity to the denaturing effect of SDS.", "Such an inhibition of hyase activity by SDS has previously been reported in Culex quinquefasciatus mosquito SGE (Volfova et al., 2008) and several Culicoides species (Radrova et al., 2015).", "Likewise, irreversible denaturation in the presence of SDS was also observed by Hotez et al. (1994) in hyase from invasive stages of the gastrointestinal nematode Anisakis simplex, whereas hyase from Ancylostoma caninum remained active after exposure to SDS.", "Salivary hyases of some bloodsucking Diptera are known as medically and veterinary relevant allergens.", "In the saliva of biting midges Culicoides nubeculosus and C. obsoletus, hyases are primary allergens that induce insect bite hypersensitivity, an economically important allergic dermatitis in horses (Schaffartzik et al., 2011; van der Meide et al., 2013).", "Three allergens, hyase, antigen 5-related protein and apyrase, have been purified from the salivary glands of the horsefly Tabanus yao (An et al., 2011; Ma et al., 2011), and are the only native allergens purified and characterized from tabanids to date.", "Subsequently, An et al. (2012) confirmed hyase as one of the cross-reactive allergens between wasp venom and horsefly saliva responsible for the so-called wasp-horsefly syndrome.", "This coexistent hypersensitivity to Hymenoptera and tabanids has been demonstrated in individuals that are hypersensitive to Tabanus americanus (Freye and Litwin, 1996), Haematopota pluvialis and Chrysops spp. (Quercia et al., 2008).", "At present, the lack of well-characterized allergen extracts and commercially available tools for immunotherapy impedes the diagnosis and treatment of allergies to tabanid bites (Quercia et al., 2008).", "Thus, our demonstration of pronounced hyase activity in various tabanid species might be useful for further immunological and allergological studies.", "The hydrolysis of HA as a prominent component of the ECM and the pericellular coat might allow some pathogens to overcome that barrier and enhance their infectivity, due to reduced viscosity of the ECM, increased accessibility of cell membrane components to host cell-infectious agent interactions, and/or the supposed immunomodulatory activities of external hyases (Bookbinder et al., 2006; Cermelli et al., 2011; Huang et al., 2014).", "HA fragments generated during the degradation of the ECM display a broad spectrum of biological functions, depending upon the HA polymer size, microenvironment, localization, and availability of specific binding partners (for review see Petrey and de la Motte, 2014).", "The co-administration of recombinant human PH20 hyase was shown to significantly increase the dispersion of injected adenoviruses in vivo (Bookbinder et al., 2006), and the enhancing effect of co-inoculated bovine testicular hyaluronidase was also reported in mice infected by Leishmania major (Volfova et al., 2008).", "However, no experimental data are available for purified salivary hyases from blood-feeding insects, and it is difficult to hypothesize about an immunological role for these enzymes during the invasion of transmitted pathogens.", "Tabanids are the biological vectors of various filarial nematodes, of which Loa loa is of significant epidemiological importance (Baldacchino et al., 2014; Zoure et al., 2011).", "In contrast to biological transmission, the range of pathogens transmitted mechanically by tabanids is relatively broad, since these Diptera display all the attributes of an efficient mechanical vector: (i) high mobility and good vision, (ii) interrupted blood feeding due to host defensive reactions to painful bites, (iii) a tendency to switch hosts when dislodged, (iv) a large amount of residual blood on the mouthparts, and (v) pharmacologically active saliva (for review see Baldacchino et al., 2014; Carn, 1996).", "The very high hyase activity in tabanid saliva might be related to their high efficiency as mechanical vectors of various pathogens.", "As they are supposedly involved in the enlargement of feeding hematomas, hyases might contribute to this mechanical transmission.", "Pathogens imported on vector mouthparts, along with residual blood from the previous feeding, are co-inoculated with the saliva and may benefit from increased tissue permeability and the immunomodulatory activity of the tabanid hyase.", "Clearly, further studies on the role of vector salivary hyases in both the biological and mechanical transmission of pathogens are needed." ]

[ "Pronounced hyaluronidase activity was found in saliva of ten tabanid species studied.", "Tabanid hyaluronidases cleave chondroitin sulfate and have pH optimum at 4.0 or 5.0.", "The enzymes are monomers with estimated molecular weight between 35 and 52 kDa.", "The high activity may correspond to the high efficiency of tabanids as mechanical vectors." ]

[ "BUSI", "ENGI", "SOCI" ]

[ "Biomass equipment", "Biomass terminals", "Equipment selection", "MILP", "Terminal logistics", "Terminal operations" ]

[ "1403", "2205", "3313" ]

[ "Biomass use in the European Union (EU) is expected to significantly grow in the co-firing and heating sectors by 2030 (European Biomass Association, 2016).", "At the moment, 4% of the total biomass used for energy purposes in the EU is imported (Dafnomilis et al., 2017).", "However by 2030, this amount (both in percentage of total biomass and in absolute amounts) could substantially increase, taking into account potential supply gaps in electricity production or the closing down of coal power plants (Dafnomilis et al., 2017; Mai-Moulin and Junginger, 2016).", "The Netherlands has been relying on biomass (specifically wood pellet) imports in order to reach the renewable energy target for electricity production, and is expected to rely on them for the future as well.", "This corresponds to approximately 3.5 Mt of imports (Dafnomilis et al., 2017).", "The above throughput can substantially increase when biomass is imported to Belgium, Denmark and Germany.", "The Port of Rotterdam aims to handle up to 10 Mt of biomass by 2020, and as such assume a hub role for biomass imports to the whole of Northwestern Europe (Port of Rotterdam Authority, 2013) (du Mez, personal communication, May 2017).", "Biomass is considered a bulk material, such as coal or iron ore.", "However, unlike these products, biomass requires specific equipment and techniques used during bulk handling, transport and storage (Hancock et al., 2016).", "Use of unsuitable equipment can lead to deterioration of the product or lead to health and safety hazards, such as dust production and explosions, self-heating and ignition or respiratory issues (Dafnomilis et al., 2015).", "The equipment at a port terminal handling biomass need to match biomass's specific properties.", "This includes specifically designed equipment (e.g. grabs) that minimize product deterioration; fully covered or enclosed transportation and storage facilities; spark detectors, fire detection and suppression systems and temperature monitoring through the whole handling chain.", "This is not entirely realized at the moment; traded volumes are low, so most terminal operators choose not to invest in specialized infrastructure (Dafnomilis et al., 2017).", "This can lead to a general degradation of the product and incur significant financial losses, as well as facility and personnel hazards (Dafnomilis et al., 2017).", "Increasing the service reliability, profit margins and reducing cargo damages are therefore essential to a biomass bulk terminal and are identified as some of the most important Port Performance Indicators (PPIs) (Ha and Yang, 2017; Talley et al., 2014).", "Due to the above reasons, biomass terminal logistics are more demanding in terms of designing the terminal setup and selecting the suitable equipment to efficiently handle the product.", "The additional safety equipment and facilities increase the capital investments required, and, since only a handful of port terminals are dedicated biomass terminals, mostly in the US, Canada and the UK, terminal operators do not have a lot of information sources on which to base the required investment decisions.", "Despite the existence of dedicated biomass terminals and the expected biomass trade growth, there is currently no comprehensive method to assist terminal operators in optimizing equipment and facility selection when dealing with biomass.", "The scientific literature relating to equipment and facility deployment is minimal, and focuses on extremely particular cases or is applied on relatively small scale examples.", "The existing literature data, such as capital and operational costs of equipment and facilities are usually simplified approaches and do not reflect the actual situation within the industry.", "This is the scientific gap that this paper aims to address, by providing a model that can be used in the field of biomass terminal design, taking into account dedicated and shared equipment within the same terminal.", "The results can assist terminal operators and port authorities with strategic level planning decisions related to biomass terminal investments.", "A substantial amount of research has been performed on terminal design, both for dry bulk material and container terminals.", "Dry bulk terminals are usually characterized by the presence and size of the terminal jetty.", "Dry bulk vessels can have large draughts, because of the large cargo density and thus large tonnage, and as such, it can be more economical to realize a jetty/pier instead of a quay wall (Kox, 2017).", "Terminals located in deep waters however, such as the port of Rotterdam, can still make use of quay walls for bulk cargo handling without the need for a jetty (Port of Rotterdam Authority, 2018).", "The equipment used for each necessary function that a terminal performs, such as loading/unloading vessels, transport of material and storage is unique and more complex than the equivalent for container terminals (van Vianen, 2015).", "Most importantly however, the equipment selected and installed must take into account the numerous properties of the cargo, such as density, angle of repose, dust generation, hazardous and handling properties (Kox, 2017; Bradley, 2016).", "The selection of equipment differs per transport direction and depends on the type and quantity of the bulk material, space and environmental conditions and the intensity of operations.", "Dust generating materials like cement require enclosed transport and small terminals with low capacity requirements can make use of wheel mounted mobile installations (Kox, 2017).", "The type of storage selected is also completely dependent on the material handled, ranging from open storage, to covered storage (warehouses and sheds, to silos and domes) (Dafnomilis et al., 2015; Kox, 2017).", "Finally, the productivity of equipment used is measured in tons of material handled per hour of operation (Ligteringen, 2014; UNCTAD, 1985; PIANC, 2014).", "A comprehensive design method that still serves as an important guideline on bulk terminal design was introduced by the United Nations Conference on Trade and Development (UNCTAD, 1985, 1991) in 1985 and again in 1991, focusing on the physical characteristics, management and operation of bulk terminals.", "At the same time, the Transportation Department of the World Bank (Frankel et al., 1985) published a comprehensive report on bulk terminal development, including information on terminal logistics and mathematical models used in evaluating preliminary design options.", "Memos (2004) provided planning parameters and other bases for estimating vessel queuing times, vessel service time and estimation of storage area needed for dry bulk cargo terminals.", "Discrete-event simulation for designing and improving the operations of dry bulk terminals was used by Ottjes et al. (2007).", "Lodewijks et al. (2007) discusses the application of discrete event simulation as a tool to determine the best operational control of the terminal and the required number of equipment and their capacity.", "Cimpeanu et al. (2017) introduced a discrete event simulation model as well to analyze bulk carrier unloading and material transport, storage and discharge.", "Taneja et al. (2011) suggested that Adaptive Port Planning methods, which value flexibility of design, are better suited in times of uncertainty than the traditional methods. van Vianen (2015)", "approached the issue suggesting an expansion of existing design methods, based on stochastic variations of the operational parameters, rather than developing a new design method.", "Bruglieri et al. (2015), Babu et al. (2015) and Robenek et al. (2014), among others, have investigated yard planning problems in bulk terminals.", "The berth allocation problem has also been extensively examined by Robenek et al. (2014), Ernst et al. (2017), Umang et al. (2013) and Al-Hammadi and Diabat (2017).", "Scientific research into specific types of equipment used in dry bulk terminals has also been performed.", "Schott and Lodewijks (2007) provides an overview and analysis of the terminal facilities provided for handling, storage and processing of bulk materials.", "General information on equipment needs of dry bulk terminals has been provided by Negenborn et al. (2017).", "Research on types of equipment has been performed by Strien (2010), on equipment used in stacking, reclaiming or the combination of these 2 functions.", "Wang et al. (2011) developed a model for the optimum allocation of loading and unloading equipment at a bulk terminal.", "The unloading capacity of a bulk cargo terminal was examined by Bugaric et al. (2011) using queuing theory.", "Pratap et al. (2017, 2016) looked into crane and unloader allocation respectively in two different works.", "Wu et al. (2007) and Wu (2012) researched dedicated biomass terminals in details and provided a database of suitable equipment for biomass terminal operations.", "Studies on the selection of equipment through different software or modelling approaches have been performed by Temiz and Calis (2017) and Prasad et al. (2015) regarding equipment selection in construction sites.", "Velury and Kennedy (1992) used a similar approach to the one discussed in this work, although the depth and detail of data used were more superficial.", "Container terminal design differs from bulk terminal design in several major elements.", "Vertical quay walls directly connected to the land are used, instead of jetties in the case of bulk terminals (de Gijt, 2005, 2010).", "The storage yard in container terminals is preferably as close to the berth as possible (Quist and Wijdeven, 2014).", "The handling of containers from and to the storage yard is performed mostly via mobile equipment such as tractor trailer units, container stackers or carriers and automated guided vehicles (AGV) (PIANC, 2014).", "The storage yard is an uncovered open area, and in the design phase, the storage capacity of empty containers as well as the container freight station needs to be taken into account.", "The container yard has dedicated equipment for container handling as well (Mohseni, 2011).", "Finally, the productivity of the equipment used is measured in terms of moves performed per hour for the transportation equipment (PIANC, 2014; Agerschou et al., 2004), or average container stacking height and density for the storage yard equipment (Ligteringen, 2014; PIANC, 2014; Bose, 2011).", "Despite the differences in terminal design approach, the research into container terminal design focuses on similar approaches as the bulk terminal design field.", "Iris et al. (2015), Tao and Lee (2015) and Liu et al. (2016) investigated the berth allocation and, to a smaller extend, the quay crane and yard allocation problem in container terminals.", "Imai et al. (2014), Lau and Zhao (2008) and Liu and Ge (2017) used heuristic approaches and queuing theory to study a strategic berth scheduling problem, to integrate the scheduling of handling equipment and to model the assignment of quay cranes in a container terminal respectively.", "Martin Alcalde et al. (2015) presented a method for determining the optimal storage space utilization in a container storage yard based on a stochastic approach.", "Sun et al. (2013) developed a general simulation framework to facilitate the design and evaluation of mega-sized container terminals which require multiple berths and yards.", "Most relevant to this work, Chang et al. (2015) used a centralized data envelopment analysis to optimize resource allocation in a container terminal based on a single company’s perspective.", "Similarly, Mbiydzenyuy (2007) realized a linear programming model for a container terminal's equipment configuration, based on a case study of a small port in Sweden.", "Apart from significant research on container and bulk terminals, the field of biomass and biofuel supply chains has also been investigated by numerous researchers.", "Poudel et al. (2016a,b), Marufuzzaman and Ekşioğlu (2017), Ghaderi et al. (2016) and Quddus et al. (2018) all examined different approaches in designing and managing biomass supply networks and transportation chains. de Jong et al. (2017), Lee et al. (2017), Yue et al. (2014) and Ahmad et al. (2016)", "developed approaches for optimizing biomass to biofuel production under different conditions and scenarios.", "Stevens and Vis (2016) conceptualized port integration with biofuel supply chains on a qualitative level.", "Some of the only scientific literature that specifically deals with biomass terminals originates mostly from Scandinavian researchers such as Sikanen et al. (2016), Kons et al. (2014), Virkkunen et al. (2015) and Gautam (2016).", "However, the above works only look into land terminals located next to forested source areas and do not go into a detailed examination of the terminals beyond an assessment of scenarios with different biomass throughputs, truck capacities and transport distances.", "While most of the studies presented in Section 1.1 had a goal of providing information, improving or optimizing terminal design, the focus was asymmetrically put on the simulation field of research.", "Terminal simulation tries to measure the performance of the terminal under different scenarios, and does not necessarily consider achieving the optimum solution as its end goal.", "On the other hand, research on equipment selection usually focuses on optimizing the (un)loading operational steps only, which are deemed as the most important parts of the terminal handling chain.", "Total equipment allocation and utilization in these approaches have a second role, even though they can be equally (or more) important costs of a terminal.", "Most importantly, all the above works are lacking either in depth of data used (equipment database, parameters etc.) or in scale of application – only performed for a pre-existing site of a specific company or for a unique small scale port terminal.", "The precise equipment configuration, and the utilization of such equipment are some of the most critical decisions that dry bulk terminals must take, and affect almost every aspect of a terminal’s operation.", "The goal of an efficient equipment configuration and utilization planning is twofold: firstly, to minimize the total investment costs incurred while determining a terminal’s design; secondly, to minimize the operational costs while handling a product.", "An approach through this scope however is distinctly lacking in the present scientific literature.", "To this end, we propose an optimization model to determine the total equipment allocation and utilization in a solid bulk biomass terminal.", "The scope of the model includes the complete activities within a terminal, from the unloading of the biomass from an arriving vessel, to the loading at a vessel at the end of the handling chain.", "Our research goal is to provide a model that can be used in the field of biomass terminal design, assisting terminal operators and port authorities with biomass terminal investment decisions.", "The results can support tactical level decision planning when applied to existing bulk terminals, which may need to retrofit equipment or parts of their handling chain, but are mostly geared towards assisting in strategic level planning – investing in new terminal setups, infrastructure and equipment decisions.", "For this purpose, the equipment configuration is presented on a detailed level within the terminal bounds, and, most importantly, the utilization of this equipment is taken into account and is linked directly to the material throughput, as is the case in reality.", "The equipment allocation and utilization are optimized in order to provide a better estimation of a dedicated biomass terminal's logistics.", "In summary, our work contributes to the above in that we take a holistic approach, unbound by pre-existing conditions, and at the same time using an extensive database with data depth that is unique for the occasion.", "Consequently, to the best of the authors' knowledge, this paper provides the following novel contributions:", "A defined mathematical model for optimizing biomass terminal equipment configuration, not only for dedicated equipment, but also for equipment that is partially used and shared between different operational steps", "An approach for minimizing total (investment and operational) costs in biomass bulk terminals from a strategic planning point of view, taking into account all the discrete operations of a terminal, as well as the respective equipment needed", "Results that are based on real world data instead of assumptions or relevant experience only, thus providing increased credibility and validity of the outcomes of the proposed model", "The authors were able to collaborate with numerous industrial experts, including the biggest and most experienced solid bulk terminal operators in the port of Rotterdam, the Port of Rotterdam Authority, equipment manufacturers, power plant and energy industry stakeholders.", "Their input was used in ascertaining the accuracy of operational assumptions, as well as confirming the validity and usefulness of the results and their approximation of real-life terminal design.", "The article is organized as follows.", "The outline and context of the terminal design and setup selected for this paper is explained in Section 2.", "The mathematical model is presented in Section 3.", "The computational results are presented in Section 4, showing that our model is capable of providing significant information regarding terminal equipment configuration and utilization decisions in a reasonable amount of time.", "We conclude in Section 5 and discuss future work.", "For the purposes of this paper, the focus in terminal design is on the equipment configuration, i.e. what type of equipment to use and in what amount for each selected task.", "A proportionate amount of attention is given to equipment utilization, i.e. how much is the selected equipment used to perform its task.", "The terminal operations are assigned into 6 discrete steps: receiving and transshipment of material, first transportation, storage of material, reclaiming, second transportation and finally, loading of material onto a vessel, truck or train for further transportation outside the terminal boundaries (see Fig. 1 for an illustration of the proposed biomass bulk handling terminal setup).", "The operational steps are defined based on the specific function that is performed within their bounds, as described above.", "In order to perform said function, unique equipment associated with it needs to be present in each operational step, chosen based on their capacity, capital and operational costs, as well as potential synergy with equipment in adjacent operational steps.", "We consider a wide range of types of equipment that can be used in each discrete operational step, depending on their function.", "In total, 15 different types of equipment of 82 different sizes and capacities are in the database used for this paper at the moment, with plans to be significantly expanded (see Section 5).", "The most common equipment used in each specific bulk terminal function, i.e. operational step is included in this research.", "Grabs and grab cranes are used for the receiving step, with pneumatic unloaders as an additional option for increased capacity.", "Belt and pipe conveyors, as well as trucks are used for the two transportation steps.", "Pneumatic conveyors are present in the transportation step when a pneumatic conveyor is used as the selected receiving equipment, since they constitute a continuous closed system, expanding into both operational steps.", "Warehouses, domes, silos, bunkers and floating barges are the options under consideration for the storage of bulk material.", "Only enclosed storage options are taken into account since wood pellets require it (Hancock et al., 2016; Dafnomilis et al., 2015, 2017) (Ruijgrok, Pothoven and Lokker, personal communication, May 2017).", "Underground hoppers are used in the reclaiming step when dealing with domes or silos, since gravity reclaiming is easier performed that way.", "Respectively, front loaders are used when the storage option selected is a bunker or a warehouse.", "Finally, loaders generally consist of a feed conveyor and a chute emptying into a vessel, or, less often, a gran and grab crane that perform the same function.", "Where data was available, all additional equipment or systems related to biomass bulk terminals were included in the equipment capital and operational costs.", "A full list of the types and sizes of equipment used can be seen in Table 1 at the end of this section.", "The information presented in this section originated from an extensive literature review and industrial field investigation.", "The most detailed and relevant information came from the authors' personal visits to several of the biggest and most experienced bulk (and biomass) terminals in the Port of Rotterdam in the Netherlands.", "This provided a unique opportunity to gain a detailed account of first hand industrial conditions of biomass handling.", "Personal interviews were conducted with representatives from terminal operators that handle biomass in the Port of Rotterdam, the Port of Rotterdam Authority, and other fields closely related to biomass production and handling.", "During these interviews and visits to the facilities, equipment of the industrial stakeholders engaged in biomass handling and storage were investigated and they are presented in this section.", "A general overview of the types of equipment used in each discrete operational step can be found in Fig. 2.", "Each type of equipment in each operational step is linked to its respective capital and operational costs, relating also to its capacity and its lifetime.", "For example for the operational step ‘transportation’ there are 9 different belt conveyor capacities, ranging from 300 tph to 2500 tph capacity, and of different lengths, depending on their position and function in the terminal handling chain (see Table 1).", "Both the capacity and the length are important parameters of the model, as they directly affect the capital and operational costs of each equipment.", "In this particular case, as the capacity increases a wider belt with a more powerful drive and support structure is needed.", "Likewise, as length increases, we need additional structural elements and more powerful drives.", "Utilization of equipment is directly linked to and affects the operational expenses of a terminal.", "According to industrial experts, operational costs can actually be the biggest factor in a terminal's annual expenses, especially as the throughput and the size and capacity of the related equipment is increased; bigger and heavier equipment means larger drives, more fuel or electricity consumed while in operation and more personnel to maintain or operate them (Corbeau and Pothoven, personal communication, May 2017).", "All the additional measures needed to be taken into account for dedicated biomass handling equipment, i.e. temperature sensors, dust extraction systems, fire detection and suppression systems, covered or enclosed conveyors, incur extra costs relative to simple bulk handling equipment.", "These costs are incorporated in the capital and operational costs of the equipment used for this work, after extensive literature study and close collaboration with numerous industrial experts as stated in Section 1.2.", "The optimization approach presented in this paper is formulated as a mixed-integer linear programming (MILP) problem that minimizes terminal infrastructure and operational costs on a normalized annual basis.", "Our MILP model differs from simulation based approaches in the way that it optimizes a detailed equipment configuration solution.", "It is also, to the best of the authors' knowledge, the first terminal model to take into account shared equipment (equipment used in more than one operational step) into account, and provide information on the utilization of equipment.", "Costs related to utilization of equipment is normally calculated as a percentage of the equipment's capital costs.", "In this work, we link it to the actual throughput of the terminal in order to demonstrate its importance and effect on total costs, especially at larger terminal sizes.", "The overall goal is to minimize the total annual costs of a biomass terminal by optimizing the amount of fully utilized and shared or partially used equipment within the terminal.", "One of the most important decisions, following a functional analysis is the breakdown of terminal operations in 6 steps, as mentioned in Section 2.", "However, no matter the level of detail that can be achieved with a specific terminal design approach, certain assumptions about terminal operations had to be taken.", "Each operational step in the terminal handling chain is assumed to be independent of the others (unless specifically stated otherwise, see constraint (6) in Section 2.2), with deterministic characteristics.", "In reality, the assignment of such discrete steps within a terminal is not so straightforward.", "This is more evident when using continuous equipment such as conveyor belts, where there may be transfer stations or towers present connecting multiple equipment.", "In our model, we incorporate the transfer stations and their associated costs in the transportation steps of the terminal.", "As input for the model, equipment and terminal data need to be specified beforehand, as well as relevant assumptions.", "Based on these data, the cost function is minimized respecting certain system constraints.", "The output of the model is the optimal terminal configuration with specific installed capacities for the chosen equipment in terms of overall costs.", "The optimization performed by the model is an overall terminal logistics optimization and not a step-specific one.", "In certain cases it might seem that the results in individual operational steps are contrary to common sense or relevant experience, but that proposed solution will be the cost optimal from a terminal perspective.", "Storage constraint (4) guarantees that all the possible equipment used for storage is able to hold a percentage of the total annual throughput.", "The storage factor is defined as the ratio of storage capacity over the annual throughput between the required stockyard size and the terminal’s annual throughput (van Vianen, 2015).", "When deciding on continuous equipment, the general rule in terminal design is that there should be only one present for each discrete operational step.", "Unless looking into the far end of large scale terminals, single conveyor belts of varying capacity can handle the incoming material.", "Even when talking about redundancy of equipment in conveyor belts, it is taken into account in the form of individual components; idlers, belt fabric rolls, drive motors etc.", "Otherwise, logistics increase disproportionately and issues with available land, support structures etc. become overcomplicated.", "Constraint (5) signifies that whenever a continuous equipment is used in the 2 inter-terminal transportation steps, the amount is limited to a single type and capacity only.", "Constraint (7) guarantees that the utilization of each shared or partially used equipment can never exceed the actual amount of used equipment.", "Summing the utilization over all operational steps where shared or partial equipment may be used and denoting it to be equal or less to the units of equipment ensures that.", "Constraints (8) and (9) guarantee that the units of dedicated or shared equipment are positive integers only (including 0), and that the utilization of equipment is a real number between 0 and 1 respectively.", "Other assumptions used to calculate the values for the model constraints appear in this section.", "A considerable effort has been made for the assumptions to depict as close as possible the industrial setting reality, rather than educated guesses or literature data.", "The overall projects industrial partners were a great asset in this endeavor, proving first hand field experience and input.", "We assume a 1 km transportation distance between the arriving vessel and the storage facilities, as well as a 200 m transportation length after reclaiming and a 500 m transportation distance to the loading point, at the end of the terminal chain.", "In the context of this paper, the term equipment utilization refers to the percentage of the terminal's total operating hours that an equipment is being used.", "That means that a result of e.g. 0.5 utilization for a 7000 operating hours per year terminal, indicates that that particular piece of equipment is functioning for 3500 h throughout the year.", "The operating hours of the terminal is one of the varied parameters of the model developed in this work.", "A detailed database of the equipment types and their respective capacities used in each operational step can be found in Table 1.", "The detailed cost data for each equipment type cannot be included in this work as they are considered confidential information between the authors and industrial partners.", "Based on the input data presented in Section 3 the MILP model was solved using the inherent MATLAB MILP solver (‘intlinprog’, MATLAB R2015b, v8.6.0.267246) using a dual simplex algorithm.", "In terms of solve time, a single application of the problem is solved to optimality within 4.7 s on an Intel Core i5-4670 CPU @ 3.4 GHz processor, and 8 GB of RAM.", "The above formulation of the problem was constructed for the maximum amount of biomass equipment we could find accurate reliable data for and approximates a realistic case– 6 operational steps within a port terminal's boundaries, and 83 different types of equipment to be used for the terminal's operations.", "In order to analyze the complexity of the model, we used a randomly generated database of 10 operational steps and 1000 different types of equipment, leading to 11,000 decision variables.", "The model can solve approaches of this size to optimality within approximately 17 s, making it appropriate for larger scale problems as well.", "By applying the model to a wide range of throughputs, results for the costs per ton of throughput are obtained Fig. 3).", "Minimization of total terminal costs per ton is achieved around 5 Mt of throughput and remains relatively stable thereafter.", "As can be discerned from Fig. 3, certain differences between discrete terminal sizes do not follow the economies of scale ‘doctrine’.", "For example, Table 3 shows the results in costs per ton, and equipment selection between two terminals of 5 and 6 Mt respectively, for which an observable difference in costs (8%) per ton of throughput is present.", "The tipping points where technology and equipment selection can most evidently be observed here.", "The larger terminal while using the same equipment for transport and reclaiming (with a higher utilization), needs to use larger equipment and infrastructure for receiving, storage and loading, which drives up costs per ton.", "After that point, costs continue to decrease until the 10 Mt milestone is reached, where another major switch to larger equipment is present once more.", "The general trend however supports the industry’s experience of aggregating handling and storage facilities in order to take advantage of economies of scale (Corbeau, Bouwmeester and Pothoven, personal communication, May 2017).", "Results like this showcase the importance of economies of scale in terminal setups and suggest that the model can be an important asset in aiding stakeholders with terminal design and investment decisions.", "In the context of dedicated biomass terminals design this means that equipment selection and utilization on its own becomes less significant as the size of the terminal increases.", "Wider implications of the results suggest that a smaller number of medium to larger size terminals are probably the best solution to increasing biomass throughputs instead of multiple smaller terminals.", "For the case of Northwest Europe, as briefly discussed in Section 1, it seems that there is no considerable difference in terms of costs per ton on whether to situate biomass terminals in a central location, thus creating a central biomass hub for the whole region, or split them between the limited number of respective importing countries – as long as all respective terminals are above the 5–6 Mt throughput threshold in order to take advantage of economies of scale.", "Other important cascading factors need to be considered at the same time, such as geographical location of the terminal, further transportation connections to the hinterland, client demand and location relative to the terminals, and low port charges or environmental regulations (Tongzon, 2009).", "In any case, relevant decisions are directly related to expected throughput.", "In the case of biomass, the high uncertainty of future developments, owning to lack of long term political commitments also affects industry investments.", "Dedicated (biomass) terminals require significant investments upfront.", "If terminal operators are unable to take advantage of economies of scale over a long period of time, there is little point in proceeding with such a task.", "Easier access and encouragement of investments usually leads to reduction in logistics costs and increases in port efficiency (Chang and Tovar, 2014).", "Table 4 presents in detail the results of the optimization for the equipment selection and utilization for terminals with a throughput of 1, 5 and 10 Mt respectively.", "The equipment selection and utilization depicted here are the optimum result for each specific terminal in terms of average annual costs.", "Smaller terminals use equipment with lower capacities at a lower utilization rate, switching to heavier equipment with higher capacity that is used more as the throughput increases.", "In the storage step, combination of a bigger and a smaller type of storage offers the best results, enabled by the choice of small, floating barges to be used as extra storage facilities.", "With most types of equipment, this form of small, additional equipment types is usually unavailable.", "One exception is trucks, which are used as complimentary transport methods for small size terminals (approximately 500 kt per year throughput) as it makes no financial sense to invest in a heavy equipment like a conveyor belt yet.", "Despite their significantly higher operational costs compared to the other transportation methods, trucks also appear in larger terminals, where investing in another major transporting equipment would incur much larger investments.", "Focusing on major equipment and technology tipping points, 25 ton mobile cranes are used up to a 3 Mt terminal, 50 ton mobile cranes from 3 to 5 Mt, 25 ton gantry cranes for 6 Mt terminals, and above that size, all terminals use 50 ton gantry crane with different degrees of utilization.", "Small terminals (1 and 2 Mt throughput) use 20 kt bunkers for storage.", "100 kt silos appear at 3 Mt up to 5 Mt terminals.", "Bigger terminals, 6 Mt and above, switch to 130 kt bunkers and add extra storage infrastructures as their size increases – 15 Mt terminals need a 130 kt bunker and two 110 kt silos as storage capacity.", "From an optimization perspective, belt conveyors are always preferable to pipe conveyors for transportation of biomass.", "Due to higher investment and operational costs, pipe conveyors are usually used when specific reasons arise, such as more strict environmental regulations concerning dust emissions, proximity to populated areas etc.", "It should be noted also, that at this point the additional costs regarding covered belt conveyors are not taking into account miscellaneous equipment that should be used when dealing with biomass, such as temperature and spark monitoring throughout the belt, dust extraction or explosion prevention and suppression systems.", "Figs. 4 and 5 offer a detailed overview of the total costs for 3 individual terminal throughputs, broken down per operational step and the individual costs as a percentage of the total.", "For each operational step, the annual equivalent of the capital cost of the selected equipment along with the annual operational cost can be seen.", "In smaller terminals, the infrastructure costs dominate the total costs in every operational area of the terminal.", "This is expected, as even smaller terminal equipment have significant investment costs; since the throughput is limited the operational costs incurred are kept low.", "The importance of operational costs as the throughput (and therefore the size) of the terminals increases is obvious in Fig. 4.", "Operational costs of equipment are directly linked to throughput and directly affect the operational expenses of a terminal.", "While the amounts may seem insignificant at first for smaller size terminals, as the size and throughput of a terminal increases, so will the utilization of increasingly larger types of equipment.", "Using bigger, heavier equipment, operating longer hours or moving more material incurs much higher costs on an annual basis that the annual equivalent of each equipment's capital cost.", "Operational costs can reach up to 32% of the total terminal costs and 55% of the individual costs in certain operational steps.", "These numbers are confirmed by industry expert group the author are collaborating with on this work (Pothoven, Lokker and Theunissen, personal communication, May 2017).", "Storage costs are by far the biggest contributors to the total costs in all terminal sizes.", "This is because biomass as a bulk material requires enclosed storage, continuous temperature monitoring and safety equipment, which increases the storage infrastructure costs exponentially, especially the infrastructure costs.", "Storage costs represent already 30% of the costs in smaller terminals, increasing gradually and representing almost half the total costs at bigger size terminals Fig. 5.", "This is contrary to the expected economies of scale effect, which would suggest all costs to decrease as terminal size increases.", "The reality is that enclosed storage can only go up to a certain size before running into problems with available land use, need for support structures or material stress against the inner walls of the facility.", "A single bulk material storage facility is generally limited to a maximum size of around 130 kt (Ruijrok and Geijs, personal communication, May 2017).", "This in turn leads to multiple storage facilities of the maximum available size as terminal throughput increases, causing disproportionately high storage costs for larger terminal sizes.", "However, as seen in Fig. 3, the economies of scale in the other operational areas of a terminal are sufficient to bring about a leveling out of the costs per ton as terminals increase in size.", "Fig. 6 presents the results of optimizing for a peak capacity approach (details can be found in Section 2.2), for two specific terminals of 3.5 and 10 Mt throughput respectively.", "Smaller size terminals tend to be impacted a lot more in terms of size and utilization of equipment when designing for peak capacity.", "Bigger and heavier equipment is required to handle a specific size of vessel in a specific allotted time, leading to a significant difference in total costs.", "On the other hand, moving to bigger size terminals eliminates this difference, since the selected equipment for the non-peak approach is of sufficient capacity to handle bigger size vessel during peak approaches as well.", "The non-peak and peak cost graphs in this case are completely identical.", "Storage costs remain the same during both approaches as they are unrelated to service times of vessels, but only to total throughput of a terminal in our model.", "Additionally, the percentage breakdown of each operational step in both approaches for a terminal of 3.5 Mt of throughput can be seen in Fig. 7.", "The ‘spread’ between the individual step costs is more balanced, since the equipment in all other steps except storage either switch to bigger types, or are utilized more during the peak approach.", "The storage costs still remaining dominant, even in peak approach though.", "In Fig. 8, the effects of the annual operational hours on the decrease of costs is depicted.", "For small terminals which have already invested in small equipment with low utilization, the effect of increasing the operational hours is extremely low in terms of costs decrease (less than 2% from 6000 to 8000 operational hours).", "The terminal is able to handle all throughput even at lower available operational hours, as the minimum required equipment is sufficient.", "As terminal size increases, an increase in operational hours is significantly more impactful, leading up to 13% reduction in total annual costs in some cases.", "As the operational hours increase, the optimum terminal setup moves to bigger heavier equipment with lower utilization.", "In contrast, for lower operating hours, smaller equipment is used at a near full utilization.", "This means that while terminals will incur higher capital expenses for the heavier equipment, they will be using it much less to handle the same throughput, as their capacity also increases.", "As explained before, utilization costs have a higher impact for larger size terminals than capital costs, which leads to a total decrease of costs with an increase in operational hours.", "Table 5 provides an overview of the calibration process of the model.", "For the initial data (collected in May 2017), only values obtained from scientific literature or freely available in online or printed sources were used.", "As mentioned in Sections 1.1 and 1.2, the scientific literature directly relating to the subject is either dated (UNCTAD, 1985, 1991; Frankel et al., 1985), or contains intentionally vague data due to confidentiality or other reasons.", "However, we were able to get access to more detailed data over the course of this work (with the final data collected in October 2017).", "As a result, through this calibration process the deviation of the final results significantly decreased.", "Initially, total annual costs for a 3.5 Mt terminals amounted to 13.56 million euros, a considerable +256% difference with the final, rational value of 3.81 million euros per annum.", "Fig. 9 highlights the improvements made in calculating the costs per ton of throughput for a wide range of terminal sizes.", "When using literature data and educated assumptions, the initial absolute values are in stark contrast with the final results.", "Costs per ton handled in port terminals decrease significantly as data accuracy increased; from 3.6 €/ton for a 10 Mt terminal to 0.94 €/ton.", "Additionally, the trends of development did not resemble the economies of scale effect that was expected until late in the data collection period.", "As an additional frame of reference, the wood pellet handling prices for a small terminal (500 kt) in the Port of Rotterdam in 2014 where around 3.5–4 €/ton when intermediate storage was used.", "Our model achieves an optimal price of 2.1 €/ton, effectively decreasing costs by 39–47%.", "The final results illustrate that access to real-life detailed data relating to capital and operational costs of the equipment and infrastructure database is of paramount importance to verifying and validating such a model.", "The relevance and validity of the assumptions and results presented in this chapter were discussed with a wide range of industrial experts that the authors collaborate on for the purposes of the broader project this paper is a part of.", "They have been verified to be as close to reality as possible at this stage.", "Partners include the biggest and most experienced solid bulk terminal operators in the Port of Rotterdam, the Port of Rotterdam Authority, equipment manufacturers, power plant and energy industry stakeholders.", "Their comments and feedback were useful in figuring out the accuracy of operational assumptions, as well as confirming the validity and usefulness of the results and their approximation of real-life terminal design.", "It has been shown that the model presented and developed in this paper is fit for its intended purpose: identifying the optimal selection and utilization of equipment of a dedicated biomass terminal in terms of total annual costs.", "This manuscript is, to the best of the authors’ knowledge, the first attempt to investigate terminal equipment selection and utilization to such an extensive manner and detail.", "Computational results based on real-life input data for biomass bulk terminals indicate that the optimum size of terminals in order to achieve the minimum cost per ton of throughput is achieved at 5 Mt of throughput and beyond.", "The total optimal equipment allocation and utilization is presented alongside each specific terminal size.", "We are able to observe when a switch to a different type of technology or equipment is needed as throughput increases.", "Partially used equipment in specific steps or shared equipment between different operational steps is also taken into account with the same level of detail as dedicated equipment.", "Most relevant work in literature focused on simulation of different scenarios rather than optimization of operations; attempts at optimization of equipment and operations logistics had been limited in scope and application.", "The results also demonstrate the relevance of biomass storage needs over the total terminal logistics.", "Necessary enclosed storage can contribute to as much as 45% of the total terminal logistics, since enclosed facilities can only reach a certain size before requiring multiple units to accommodate the throughput.", "The importance of the effect of the utilization of equipment on bigger size terminals is also presented.", "Decoupled from a percentage of capital costs, operational costs have a significant role in terminal logistics, amounting to 32% of the total terminal costs in larger terminals and 55% of individual operational steps.", "The model can be used as a decision tool for practitioners and regulators in order to rationalize tactical level decision planning (when there is a chance of retrofitting or adjusting existing terminal equipment) or strategic level planning when designing a dedicated biomass port terminal.", "Its framework allows the model to potentially be used for optimization of a biomass terminal in terms of energy consumption or CO2 emissions, as long as the relevant equipment operation data are known to a similar detail.", "Further improvement of the model will include an expansion of the database with more equipment types per operational step.", "As mentioned throughout the body of the text, an effort is made to stick to data as close to real-life industrial conditions as possible, made feasible by the close cooperation of numerous industrial partners.", "This, however, means that in this 'quest' for detailed foundations, the usefulness of our model's output depends directly on the quality of the input.", "For this reason, a constant effort is made to update all relevant equipment data in order to stay relevant to current circumstances.", "The detailed cost components of support and safety systems – such as dust extraction, continuous temperature monitoring etc. – in certain types of equipment will also be implemented in the optimization routine.", "Different approaches to biomass storage will also be investigated in further work.", "Instead of a fixed percentage of the annual throughput, terminal storage facilities could be designed based on: a) The demand rate and related safety stock levels.", "This however means knowing when and how much biomass each terminal client demands and planning accordingly.", "b) The bulk carrier size and arrival intervals.", "The bulk vessel size is based on the total throughput (see Section 2.3), so the storage facilities could be designed to accommodate, e.g. one full maximum size vessel.", "This, however, means that the arrival rate must be known in detail and plan appropriately beforehand.", "Redundancy of equipment is another important cost factor that has not yet been taken into account.", "Most terminals usually plan ahead and have extra units of equipment on stand-by, in case of failure or emergency.", "Of course this is most common for smaller types of equipment, like the grabs of the grab crane, or related miscellaneous components (generators, inverters, idlers for conveyor belts etc.).", "These do not necessarily constitute terminal ‘equipment’, but can however have a substantial impact on terminal logistics.", "At a later stage, linking the interest rates and effective utilization to each specific equipment type will improve the accuracy and realism of the results as well.", "Further work on the subject will tackle the limitations of this current model, transitioning to a dynamic, multi-stage planning approach to biomass terminal investments.", "This approach is necessary as the yearly forecasted throughputs of biomass are subject to great variations, to which a static model cannot adequately adapt.", "The results will be a total optimization approach across the whole investigated time period, where investments on equipment and infrastructure will be made in anticipation of future throughputs, i.e. overdesigned or over capacity equipment may be purchased ahead of time, as future developments will make their acquirement the optimal case.", "Salvage of equipment will also be integrated as an integral decision variable in the model, adding complexity and realism to our approach.", "The input for this work will be biomass throughput scenarios, defined over a specific time horizon, over which investments in equipment and infrastructure will need to be carried out.", "This will enable us to have an overview of the investments needed in infrastructure and the expected operational costs throughout the whole time period for which we have data related to terminal throughput." ]

[ "Optimization of biomass port terminal equipment is presented.", "MILP model minimizes investment and operational costs in biomass bulk terminals.", "Real data from industry used as input.", "Results provide optimal equipment allocation and utilization, optimal terminal size and associated logistics.", "Insight on importance of storage and utilization costs when dealing with biomass." ]

[ "BIOC", "COMP" ]

[ "Energy-transfer-efficiency", "MbT-Tool", "Microbial metabolic reaction", "Microbial metabolism", "Microbial yield prediction", "Thermodynamics" ]

[ "1303", "1304", "1305", "1311", "1315", "1706" ]

[ "For the construction and development of models of living organisms, it is necessary to describe their physical constants, physiological reactions, interactions and responses to the environment.", "A robust model of any living system must include chemicals and conservation principles, and relate stoichiometric mass balance with the laws of conservation of energy.", "Most biochemical processes that involve energy transduction close to the balance can be studied using a non-equilibrium thermodynamic approach.", "Intuitively, a complete model has a number of sub-models that take into account the compartmentalized structure of living organisms; and specifically the metabolic sub-model should include descriptions of different parts and functions of their developmental stages [1–3].", "The use of microbial metabolic reactions (MMRs) that include the microbial bio-mass as a product or as a reactant is a relevant task for modelling microbial activity.", "An MMR is the written version of a biochemical process.", "The stoichiometry is a part of the chemistry that permits the writing of balanced equations because it studies the molar relation between the reactants and the products in a chemical reaction.", "There are several reasons to suggest that the presence of micro-organisms in a biochemical process complicates the stoichiometry.", "First, biochemical reactions often involve oxidation and reduction of more than one chemical species.", "Second, the micro-organisms have two different roles: they perform the reaction and also they are the final products of it.", "Third, the micro-organisms produce several chemical reactions in order to capture part of the liberated energy for cell synthesis [4,5].", "The overall procedure for representing these reactions in balanced equations is a considerable and sometimes complex task for biologists, ecologists, modellers, and engineers, among others.", "The cellular metabolism has been studied, investigated and described at many levels ranging from traditional enrichment tools, molecular and genetics tools, mathematical modelling of micro-biological multispecies systems, to non-equilibrium thermodynamics [4,6].", "In the field of non-equilibrium thermodynamics, several approaches have been reported to develop a rigorous thermodynamic description for biomass yield prediction based on the energy-transfer-efficiency (ε) between catabolism and anabolism [4,5,7–14], while other thermodynamic approaches correlate the biomass yield calculations with the Gibbs energy dissipations [6,15–21].", "In brief, the thermodynamic approaches explain how catabolic and anabolic reactions are coupled to predict the biomass yield in terms of standard Gibbs energy.", "Considering these diverse approaches in the bio-thermodynamics field, this study takes into consideration the one that is based on ε, because it includes: i) the standard Gibbs energy for cell synthesis from different carbon-sources and nitrogen-sources, ii) the energy available from substrate transformation, and iii) the ε to the overall metabolic process.", "In addition, choosing the thermodynamic approach allows the prediction of full stoichiometric relationships for biological reactions in the absence of experimental yield values [5,12].", "One powerful tool that is currently revolutionizing the study of microbial biotechnology is the mathematical and computing modelling that explicitly takes into account the microbial diversity and the exchanges of nutrients and materials within the micro-organism community and its surrounding environment [22,23].", "Models such as individual-based models (IBMs) [24–27], metabolic energy-based modelling (EBMs) [28], reaction-centric models (RCMs) [29], and even, deterministic-population models (DPMs) [30] can obtain useful information from a MMR.", "The reason is that these reactions, or the stoichiometry between chemical species described in the metabolic sub-model, can help in understanding how the microbial interactions happen and why microbial systems behave as they do.", "Environmental microbiologists could take advantage of the knowledge of metabolic reactions because usually the chemical resources for the micro-organisms are the pollutants that technicians must control [4].", "In addition, the information provided by MMRs could be of importance in other engineering fields because they provide information about the quantities of chemicals required to satisfy microbial requirements as well as the quantities of the generated end products [5].", "In this context, the principal aim of this contribution is to develop a computational tool to write MMRs for processes driven by a range of microbes based on the Thermodynamic Electron Equivalents Model (TEEM), a methodology proposed by Perry L. McCarty [4,7–9,11], which is the baseline of many current bio-technological studies [4,27,31].", "The specific objectives are: (i) to implement the tool on the open-access platform NetLogo to distribute the source-code among the scientific community, (ii) to promote its use and to encourage future extensions and adaptations; and (iii) to test the computational tool by writing of some MMRs.", "This manuscript presents the following structure: Section 2 describes the TEEM thermodynamic approach, the basic concepts about the empirical chemical composition used to represent the microorganisms and the open-access software used to implement the computational tool; Section 3 describes the design, performance and application of the computational tool and presents some MMRs for diverse metabolic functional groups of micro-organisms; and finally Section 4 presents some final remarks and possible future applications for this work.", "The Thermodynamic Electron Equivalents Model (TEEM) is designed to study the stoichiometry and kinetics of a wide variety of biological treatments of wastewaters.", "TEEM writes a stoichiometric reaction to describe the overall cellular metabolism from reduction-half-reactions for: the electron-donor (Rd), electron-acceptor (Ra) and cell synthesis (Rc).", "TEEM is based on terms of the standard Gibbs free energy involved in these reactions and in how the energy between catabolism and anabolism is coupled [4,7–11].", "For the use of TEEM, no specific and detailed knowledge of metabolism is required.", "First, we have to identify the electron donor(s) (eD) and the electron acceptor(s) (eA) and write reduction-half-reactions for each one of them.", "Second, it is necessary to establish the N-source for biomass synthesis, and third, it is essential to determine the empirical chemical formula that will represent the microbial cells.", "TEEM has two versions, the first one, TEEM1 [4], considers a realistic formulation of the anabolic reaction taking into account different N-sources such as ammonium (NH4+), nitrate (NO3−), nitrite (NO2−) and di-nitrogen (N2), and a complete explanation of ε between catabolism and anabolism.", "The second version, TEEM2 [11], complements TEEM1 because it considers oxygenase reactions involved and the aerobic heterotrophic oxidation of C1 organic compounds.", "According to TEEM, the metabolic energy is obtained from the redox reaction between an eD with an eA.", "The electrons are obtained from eD and transferred to intra-cellular intermediates.", "In TEEM1 the intermediate is the pyruvate with its half-reaction standard Gibbs free energy equal to 35.09 kJ/eeq, and in TEEM2 the intermediate is acetyl-CoA with its half-reaction standard Gibbs free energy equal to 30.9 kJ/eeq.", "The intermediate compounds bring the electrons towards the eA, which is being reduced causing the initial carrier regeneration [11].", "TEEM calculates microbial yield (Yc/c) using a relation between the standard Gibbs free energy of the catabolic and anabolic reactions and an appropriate ε value.", "The microbial catabolism is represented by the energy reaction (Re).", "To write it we have to combine Rd with Ra.", "Once Re is known, it is necessary to represent the microbial anabolism by writing the reaction for microbial biomass synthesis (Rs), and to do this we have to combine Rd with Rc.", "Rc is a hypothetical half-reaction, which considers as reactants the N-source (NH4+, NO3−, NO2− or N2), carbon dioxide (CO2) and bicarbonate (HCO3−), and as products water and the microbial biomass represented by an empirical chemical formula of cells (CnHaObNc).", "This empirical chemical formula considers the molar relationships only for four basic elements: Carbon (n), Hydrogen (a), Oxygen (b) and Nitrogen (c).", "To establish the adequacy of this formula, researchers compared theoretical thermodynamic calculations using the cell's empirical chemical formula with the thermodynamics of growth of the same micro-organism on several substrates using batch cultures growing in the exponential phase at μmax[32–35].", "If the formula only considers four elements, the fitness is close to 95%, but if we include two more elements, e.g. phosphorous and sulphur, the fitness increase its value to around 98–99% [36,37].", "To estimate the standard Gibbs free energy of Rc (∆Gpc), TEEM proposes a value of 3.33 KJ per gram cells [4,9,11], which is related to one generic microbial cell composition C5H7O2N when NH4+ serves as the N-source for cell synthesis.", "The ∆Gpc value is valid in the context in which TEEM was developed.", "This does not make a great deal of difference in calculated microbial yields, but is felt to be an acceptable theoretical choice [11].", "In other research fields, e.g. bio-geochemical processes in marine environments, and taking into account different pressure, temperature, pH and N-sources for cell synthesis the reported ∆Gpc value is 302 J per gram cells [3,37,38].", "Moreover, TEEM considers in its internal structure the possibility of using four different N-sources to write Rc [4].", "The theoretical explanation is that micro-organisms prefer to use ammonium as an inorganic nitrogen source for cell synthesis, because it is already in the (-III) oxidation state, the status of organic nitrogen within the cell.", "However, when ammonium is not available for cell synthesis, many prokaryotic cells could use oxidized forms of nitrogen as alternatives.", "Therefore, nitrate (NO3−), nitrite (NO2−) and di-nitrogen (N2) are included as nitrogen sources.", "When an oxidized form of nitrogen is used, micro-organisms must reduce it to the (-III) oxidation state of ammonium, a process that requires electrons and energy, thus reducing their availability for synthesis.", "Therefore, different N-sources will obtain different results in the microbial yield [4,11].", "To couple the energy from catabolism to anabolism, TEEM considers a relation between the electrons involved.", "The electrons that come from the eD will be divided into two portions.", "The first portion (feo) is transferred to the eA to generate energy (catabolism) and the other portion of electrons (fso) is transferred to the N-source for cell synthesis (anabolism).", "TEEM calculates the relationship between feo and fso using: (i) standard Gibbs free energy of Rd, Ra and Rc, (ii) standard Gibbs free energy of the intracellular intermediates compounds, and (iii) an appropriate ε value.", "This term is included because TEEM considers that a fraction of the thermodynamic free energy involved is lost at each energy transfer between catabolism and anabolism [13,14].", "With this information, an MMR (R = feoRa + fsoRc − Rd) is written.", "This reaction represents the full stoichiometric relationships for a biological process without the experimental yield values commonly required to obtain it [12].", "For a detailed description of this thermodynamic approach the reader can refer to McCarty (2007) and Rittmann and McCarty (2001).", "The computational tool is implemented in the widely used, free and open source program NetLogo, a multi-agent programmable modelling environment [39].", "NetLogo is licensed under GPL (GNU General Public Licence) and it was chosen mainly for the way this platform is organized: the source-code is very well documented, open and easy to read, giving the option to share this developed tool with other researchers without difficulty.", "This straightforward interaction, in the near future, will facilitate the upgrading of the computational tool by the scientific community interested in writing and using metabolic reactions for microbial processes.", "We have named the computational tool as MbT-Tool, standing for Metabolism based on Thermodynamics.", "MbT-Tool is the interactive tool developed to study and model microbial metabolism based on two versions of TEEM: TEEM1 [4] and TEEM2 [11].", "With the MbT-Tool it is possible to write MMRs conducted by microbes.", "Moreover, using the MbT-Tool it is possible to predict the microbial yield in standard thermodynamic conditions, e.g. P = 1 bar, temperature = 25 °C and pH = 7.0.", "The MbT-Tool user interface displays all variables and parameters regarding the composition of the microbial biomass and the substrates that can be selected to describe the metabolic processes, avoiding the complex and tedious calculations of the implemented thermodynamic model by the user.", "The composition of the microbial biomass, which is represented by CnHaObNc, seems to be related to the substrates where the micro-organism grows and it can be slightly different depending on the C-sources and N-sources [14].", "However, Battley (2013) asserts that the empirical composition of the same cells metabolizing any given substrate is expected to be the same.", "Therefore, the MbT-Tool considers Battley's hypothesis as valid and allows use of the same microbial biomass formula and writing MMR using different growing substrates.", "Fig. 1 shows the schematic description of how the MbT-Tool is based on TEEM with a brief explanation of TEEM concepts which are helpful in understanding the main terms involved in it, especially how energy from catabolism is coupled to anabolism and as a result of these combinations a MMR is obtained.", "Fig. 2 shows the schematic programming structure of MbT-Tool using a flow diagram.", "Fig. 3 shows a screen-print of the MbT-Tool, which includes: interface, info and code tabs.", "The interface tab is where the reader can obtain the outputs.", "It also has inputs-options that can be used to set up the MbT-Tool.", "The info tab provides an introduction to the MbT-Tool.", "It explains how it was created and how to use it.", "The code tab is where the code is stored.", "The reader can find the lists of organic and inorganic reactions, TEEM equations with theoretical half-reactions of biomass formation taking into account different N-sources, and other programmed details of the MbT-Tool in the supplementary material.", "To use the developed computational tool, it is necessary to install NetLogo (at least NetLogo 5.0) (https://ccl.northwestern.edu/netlogo/download.shtml), and download the MbT-Tool (http://mosimbio.upc.edu/en/publications/publications-by-year/years-2010-now).", "It runs on any computer that supports JAVA with an operating system (OS): Windows, Mac OS X and Linux [22,39,40].", "On opening MbT-Tool, the interface window appears (Fig. 3).", "On the first screen the user has to select: one or two electron donor(s) and one electron acceptor from the organic (in supplementary material, Table S1) or inorganic (in supplementary material, Table S2) reduction-half-reactions, and the empirical chemical formula of the cells (in supplementary material, Table S3) or can introduce the molar relationship between the four main elements (C, H, O and N), the N-source to the cell synthesis half-reaction.", "It is possible to choose between four sources, NH4+, NO3−, NO2− or N2 (please refer to the supplementary material, Table S4).", "With this setup information, the user has to select the thermodynamic approach TEEM1 or TEEM2 (in supplementary material, Table S5) to write the MMRs.", "If the user chooses TEEM2 it is necessary to define the number of oxygenase reactions per mole of substrate, introducing an integer number in the “q” parameter.", "Finally, the user has to fix the ε value for the process.", "With all this data, the MbT-Tool displays the following outputs: the Rd, Ra and Rc half-reactions, the energy reaction (Re), the synthesis reaction (Rs), feo, fso, ε, the MMR (R) and the calculated microbial yield (Fig. 3).", "This MMR is the reaction that McCarty defines as global reaction, which arises from the combination between: the catabolic reaction with the anabolic reaction and the value of ε[11].", "The user could also download these outputs in an archive with a “.", "txt” extension.", "The file name is written with the information of the Rd, Ra, Rc, N-source and the thermodynamic approach used (TEEM1 or TEEM2).", "To avoid potential errors when using the MbT-Tool, we recommend not selecting the same chemical species to the eD and to the eA; they must be different.", "If eD and eA are the same, the result of the calculations using their standard Gibbs free energy could provoke an inconsistent value on the fraction of electrons destined for cellular synthesis or electrons destined for energy.", "An inconsistent value will be obtained when these fractions of electrons are greater than one or below zero.", "If this numerical inconsistency occurs, the MbT-Tool stops and shows an alert message to the user.", "Regarding the decision of using TEEM1 or TEEM2, it all depends on: (i) the growth substrate, (ii) the microorganism(s) involved, and (iii) the metabolic pathway that will be represented using the MbT-Tool.", "For instance, if the growth substrate is a C1 compound, it is better to use TEEM2 over TEEM1.", "If one of the intermediates metabolic compounds in the pathway is pyruvate, it is better to use TEEM1 over TEEM2, If Acetyl-CoA is an intermediate compound in the pathway, it is better to use TEEM2 over TEEM1.", "Finally, if oxygenase reactions are involved, it is better to use TEEM2 over TEEM1.", "Basically, micro-organisms utilize the oxygenase reactions to create more biodegradable forms of substrates.", "Some examples are: when alkanes are hydroxylated [41], alkenes are converted into the corresponding epoxides [42], carbon monoxide is oxidized to carbon dioxide [43], ammonia is oxidized to hydroxylamine [44], and some aromatic compounds and cyclic alkanes are hydroxylated [45].", "To determine the “q” value, the user must establish if the reaction is mono-oxygenase-catalyzed or di-oxygenase-catalyzed.", "But in the most common cases, the “q” value ranges from 0 to 4.", "For a detailed description of this procedure the reader can refer to Xiao and VanBriesen (2006, 2008) [13,14].", "Some different microbial processes have been selected to demonstrate the potential and versatility of the MbT-Tool showing the output of the simulator, which is a set of MMRs with the corresponding yield prediction value.", "In the experiments carried out by Battley (2013, 2007, 1995) [32,35,36] related to the Saccharomyces cerevisiae, he has established C6.33H10.21O3.53N as the empirical chemical formula for this yeast.", "Considering this information, using the MbT-Tool it is possible to represent two metabolic pathways: the biomass synthesis from glucose, and the biomass synthesis from pyruvate to ethanol.", "Before executing the MbT-Tool, it is necessary to determine some parameters.", "We used TEEM1 because we consider that there is no oxygenase reaction involved and the c-source is not a C1 compound.", "We used NH4+ because this chemical species is the universal N-source to biomass synthesis, and for ε we used a value that allows us to obtain the reported cell yield growing on glucose of the 0.098 mol Ccells/mol Cdonor [36].", "Using the MbT-Tool with the established inputs, theoretical considerations and TEEM1 with ε value of 0.57 for the first pathway and ε value of 0.84 for the second one, the MMRs and the yields are:", "Biomass synthesis from glucose:", "C6H12O6 + 0.094 NH4+ + 2.25 HCO3− → 0.094 C6.33H10.21O3.53N + 2.16 CH3COCOO− + 1.17 CO2 + 3.59 H2O with an Yc/c = 0.099 mol Ccells/mol Cdonor.", "Biomass synthesis from pyruvate to ethanol:", "CH3COCOO− + 0.047 NH4+ + 1.325 H2O → 0.047 C6.33H10.21O3.53N + 0.734 CH3CH2OH + 0.283 CO2 + 0.953 HCO3− with an Yc/c = 0.098 mol Ccells/mol Cdonor.", "In the published research carried out by H.W. van Verseveld [46–48] related to the growth of Paracoccus denitrificans, and considering succinate as electron donor and various final electron acceptors, the reported formula for this denitrifying bacterium was established as C3H5.4O1.45N0.75.", "To write MMRs, we considered succinate as C-source, NH4+ as N-source for biomass synthesis and the main electron acceptors involved in the de-nitrification pathway.", "Taking into account this information we used the MbT-Tool with TEEM2 and different ε values to represent a sequence of four reduction reactions (NO3− → NO2− → NO → N2O → N2) using MMRs for this denitrifying bacterium.First reaction (NO3− → NO2−): (C4H4O4)2− + 0.30 NH4+ + 4.55 NO3− → 0.40 C3H5.4O1.45N0.75 + 4.55 NO2− + 1.10 CO2 + 1.70 HCO3− + 0.67 H2O.", "(ε = 0.41).", "Second reaction (NO2− → NO): (C4H4O4)2− + 0.58 NH4+ + 4.55 NO2− + 4.55 H+ → 0.77 C3H5.4O1.45N0.75 + 4.55 NO + 0.26 CO2 + 1.42 HCO3− + 2.64 H2O.", "(ε = 0.84).", "Third reaction (NO → N2O): (C4H4O4)2− + 0.58 NH4+ + 4.55 NO → 0.77 C3H5.4O1.45N0.75 + 2.28 N2O + 0.26 CO2 + 1.42 HCO3− + 0.36 H2O (ε = 0.56).", "Final reaction (N2O → N2): (C4H4O4)2− + 0.58 NH4+ + 2.28 N2O → 0.77 C3H5.4O1.45N0.75 + 2.28 N2 + 0.26 CO2 + 1.42 HCO3− + 0.36 H2O.", "(ε = 0.53).", "Using these MMRs we created an IBM called INDISIM-Paracoccus [27,31,48,49], which is designed to study the de-nitrification process carried out by the bacteria P. denitrificans in order to explore the consequence of different priorities in the individual use of electron-acceptors on the denitrification pathway [27].", "Battley (1987) [33] established C3.85H6.69O1.78N as the empirical chemical formula for Escherichia coli.", "Considering this information, using the MbT-Tool it is possible to represent its diauxic growth on glucose and lactose.", "With the obtained MMR, it is possible to begin a modelling project related to the quantitative determination of metabolic fluxes during co-utilization of two C-sources [49] or a modelling project related to the organization of metabolic reaction networks [50].", "We used TEEM2 because we consider that at least one mono-oxygenase-catalyzed reaction is involved (q = 1) [13,14].", "We used NH4+ as N-source to biomass synthesis.", "We used two eDs (glucose and lactate) and one eA (oxygen), and for ε we used a 0.37 value [11].", "Using the MbT-Tool with the established inputs, the MMRs and the yields are:C6H12O6 + CH3CHOHCOO− + 5.05 O2 + 1.018 NH4+ + 0.018 HCO3− → 1.018 C3.85H6.69O1.78N + 5.097 CO2 + 7.133 H2O.", "Bacterial yield: Yg/m = 97.139 (gramscells/moldonor); Yc/m = 3.917 (molCcells/moldonor) and Yc/c = 0.435 (molCcell/molCdonor).", "In the MbT-Tool, we selected a gram-negative bacterium with the empirical formula of C5H7O2N [5].", "This bacterium degrades nitrilotriacetic acid (NTA) in the absence of molecular oxygen.", "To write the metabolic equation using nitrate as eA, NTA is used as the eD, and TEEM1 is used as the thermodynamic approach with an ε value of 0.33 [4,8,9,11,13,14].", "Wanner et al. [51], reported a cell yield growing on NTA equal to 50.760 g cells/mol NTA for this microbial process, while the bacterial yield prediction obtained with the MbT-Tool is 51.311 g cells/mol NTA.", "All outputs are presented in Fig. 4.", "The main purpose of this contribution is to present the development of the MbT-Tool and make it available to a wide spectrum of readers, showing how this tool could be used in different research frameworks.", "This tool is a tangible way of achieving the compression of a thermodynamic application connected with microbial metabolism, therefore, it transfers a theoretical knowledge to a diverse range of applications of interest.", "The MbT-Tool is, as far as we know, the only open-access and open-source software, that allows the writing of MMRs based on thermodynamic concepts.", "To use the MbT-Tool, non-expert knowledge about microbial metabolism is necessary, and only the most basic organic and inorganic chemistry is enough.", "The MbT-Tool by itself does not simulate any microbiological, ecological or biotechnological process, but the results obtained from the MbT-Tool allow the user to start with the calculations or the simulations for the study of any of these research fields.", "In addition, the two strongest key points of this tool include: i) that the user doesn't have to pay for using the MbT-Tool, and ii) that the user is able to modify the source-code to extend the scope of the MbT-Tool into their specific research field and expertise.", "Moreover, the scope of the MbT-Tool is not limited to the number of the pre-programmed half-reactions included in this version.", "With the combination of these half-reactions, it is possible to write MMRs for diverse microbial functional groups, such as aerobic heterotrophs, nitrifiers, denitrifiers, methanogens, sulphate reducers, sulphide oxidizers and fermenters (in supplementary material, Table S6).", "Also, the power and scope of the MbT-Tool increases if we take into account the possibility of changing the molar relationship between the main four elements to define the empirical chemical formula that represents the microbial biomass.", "We consider that results obtained through the MbT-Tool could be the starting point to deal with a modelling project in the framework of microbial ecology or bio-technological processes.", "The non-expert user achieves an MMR in which one of the products is the chemical composition of the micro-organism involved in the process and some values of its bacterial yield.", "With these reactions, the user can i) start to construct a model, ii) incorporate the information into an existing model or iii) start the calculations of the mass balance for a bioreactor.", "However, this tool has a limitation: it is not possible to compare the MbT-Tool's outputs directly with experimental results.", "We consider that only after the construction of a model with the MbT-Tool's results would the non-expert user be able to interpret its results.", "A non-expert user is considered here as a person whose expertise is not in the field of non-equilibrium thermodynamics, and therefore, to develop and apply all the conceptual elements involved in this thermodynamic approach for the construction of living models could be a time-consuming task.", "In contrast, a person with experience in these MMRs could have enough criteria to use and interpret the output reactions from MbT-Tool in his academic or research context.", "In the research field on IBMs, using the INDISIM methodology as a core model, for instance [24–26,52–54], we realized that is essential to know the metabolic reactions carried out by the micro-organisms to increase or decrease their own biomass.", "These metabolic reactions could be used to design the individual metabolism model as well as to parameterize the IBM models.", "In this sense, we think that the MbT-Tool's outputs are a convenient means to advance with this type of model [27,31,55].", "Finally, we consider that the user could take advantage of the MMRs provided by the MbT-Tool in a wide range of academic or research fields, such as, for example: i) as a source to design processes that take advantage of the microbial system [4], and (ii) as the bedrock to make a connection between the microbial biomass and the substrates used by the micro-organism for pollution control [10,56].", "Additionally, the NetLogo's rather flat learning curve and comprehensive documentation [57] make this a user-friendly tool, easily accessible to chemists, biologists, ecologists, engineers and modellers, among others.", "Users without extensive programming experience can modify the code, introducing new chemical species to write new reduction-half-reactions, as well as other options for investigating alternative metabolic pathways or adapting certain processes according to specific studies.", "In conclusion, the description of the cellular metabolism by means of thermodynamic concepts is strategic for investigating microbial activity and modelling bio-technological processes.", "We are convinced that the MbT-Tool will facilitate users to think about the biochemistry of metabolism due to its simplicity of use, and its results could be an interesting starting point for a microbial modelling approach." ]

[ "Represent microbial metabolism based on thermodynamics.", "Write microbial metabolic reactions to represent environmental processes.", "An open-source computational tool to study microbial activity", "The basis of metabolic behaviour-rules embedded in an individual-based model." ]

[ "MEDI", "NEUR" ]

[ "Connectome", "Diffusion MRI", "Diffusion tensor imaging", "Lewy bodies", "Neurodegenerative disorders", "Support vector machine" ]

[ "2728", "2741", "2805", "2808" ]

[ "Parkinson's disease (PD) is the second most common neurological disorder after Alzheimer's disease, affecting 6 million individuals worldwide (Vos et al., 2016).", "The disease is characterized by motor symptoms (i.e., akinesia or bradykinesia, rigidity, and tremor) as well as non-motor symptoms such as cognitive impairment and psychiatric symptoms (Kalia and Lang, 2015).", "Motor symptoms primarily result from dopamine neurodegeneration in the substantia nigra pars compacta and striatum (putamen and caudate nucleus), which are the main targets of nigral dopaminergic axons.", "However, dopamine loss is associated with neuronal activity changes in cortico–basal ganglia–thalamocortical circuits, which are also linked to motor and non-motor symptoms (Rodriguez-Oroz et al., 2009).", "These circuits are large, functional, parallel, and segregated cortical–subcortical re-entrant circuits that are topographically and functionally divided into motor, associative, and limbic sub-circuits (Alexander et al., 1990).", "Therefore, disruption to these complex circuits may account for the many motor and non-motor symptoms observed in PD (Galvan et al., 2015).", "Evidence for brain circuit and axonal pathology has been widely reported for PD.", "The pathological hallmark of PD is the presence of Lewy bodies and Lewy neurites, which are protein inclusions whose main constituent is fibrillary-aggregated α-synuclein (Spillantini et al., 1998).", "These abnormal aggregates predominantly occur at presynaptic sites, where they cause synaptic and axonal degeneration that can potentially result in widespread macroscopic disruption in connectivity.", "The connectome is a network representation of whole-brain connectivity, which can be mapped to reveal circuit-based alterations in neurological and psychiatric conditions (Fornito et al., 2015; Fornito et al., 2012; Zalesky et al., 2016).", "Macroscale connectome studies combine diffusion-weighted magnetic resonance imaging (DW-MRI) and graph theory to generate a network representation of the brain, comprising gray matter (GM) brain regions (nodes) and axonal connections (edges) (Craddock et al., 2013).", "Recent studies have used DW-MRI data to infer interregional structural connectivity (Behrens and Sporns, 2012) when evaluating connectome dysfunction in PD (Aarabi et al., 2015; Barbagallo et al., 2017; Galantucci et al., 2017; Li et al., 2016; Nigro et al., 2016; Shah et al., 2017; Tinaz et al., 2017).", "These studies report reduced structural connectivity between the basal ganglia, thalamic, limbic, frontal, temporal, and parietal regions in PD (Aarabi et al., 2015; Barbagallo et al., 2017; Galantucci et al., 2017; Kim and Park, 2016; Li et al., 2016; Nigro et al., 2016).", "Disrupted connectivity has shown associations with clinical and non-motor symptoms of PD (Barbagallo et al., 2017; Galantucci et al., 2017; Shah et al., 2017).", "Graph theory analyses have identified reduced global clustering and efficiency, as well as increased global path length (Nigro et al., 2016).", "In addition, local network measures have been shown to be disrupted in PD, including decreased “functional segregation,” as indicated by decreased local efficiency and clustering, and decreased “functional importance,” as indicated by decreased nodal strength and betweenness centrality in the basal ganglia, thalamus, limbic, frontal, temporal, parietal, and occipital cortices (Li et al., 2016; Nigro et al., 2016).", "Together, diffusion tensor imaging-based connectome studies have identified global and local connectome disruptions in PD in vivo.", "These previous studies employed deterministic or probabilistic tractography to trace the trajectories of white matter (WM) streamlines (edges) and map structural connectomes per individual.", "A major limitation of deterministic fiber tracking based on unimodal diffusion tensor imaging is the difficulty in estimating neural fiber connections in a voxel in which there are crossing or kissing fibers (Mori and van Zijl, 2002).", "Probabilistic tractography algorithms that estimate multiple fiber directions have been proposed to overcome this limitation (Behrens et al., 2007), and one of the most commonly applied techniques is constrained spherical deconvolution (CSD), which can improve the reliability of whole-brain tractography by using a high-quality fiber orientation distribution function (fODF) (Jeurissen et al., 2011).", "However, CSD cannot yield accurate fODFs in voxels containing GM and cerebrospinal fluid (CSF) (Roine et al., 2014), and the method particularly struggles to reconstruct the GM–WM interface.", "Given that the connectome contains nodes (corresponding to GM regions) and edges between nodes (i.e., tractographic streamlines), accurate delineation of GM and WM is very important for accurate tractogram generation to be achieved.", "To circumvent the limitations of deterministic tractography and the difficulties in resolving the GM–WM interface with existing CSD methods, we employed a new, multi-shell, multi-tissue CSD (MSMT-CSD) method (Jeurissen et al., 2014) to map whole-brain connectomes.", "To estimate the ODF in multiple tissues, MSMT-CSD employs multi-shell DW-MRI data with unique b-value dependences for different macroscopic tissue types (WM, GM, or CSF) (Jeurissen et al., 2014).", "Because MSMT-CSD can produce reliable volume fraction maps of WM, GM, and CSF directly from DW-MRI data, it can substantially increase the precision of the fODF and the resulting tractograms at tissue interfaces (Jeurissen et al., 2014).", "We hypothesized that connectome disruption would be evident in PD, and that it would involve cortico–basal ganglia–thalamocortical networks (Rodriguez-Oroz et al., 2009).", "We further hypothesized that connectomes derived from the MSMT-CSD method would be more sensitive to connectivity deficits compared with conventional methods.", "To test this hypothesis, we compared connectivity strength as well as global and local graph theory metrics obtained by probabilistic MSMT-CSD with those obtained by deterministic single-shell, single tissue (SSST)-CSD tracking and probabilistic SSST-CSD tracking.", "Then, we used machine learning to evaluate which of MSMT-CSD, deterministic SSST-CSD tracking, and probabilistic SSST-CSD tracking yielded the highest accuracy for classifying cases and controls based on topological descriptors of the brain network.", "This study was approved by the institutional review board of Juntendo University Hospital, Japan, and was conducted in accordance with the criteria of the Helsinki Declaration.", "Written informed consent was obtained from all participants before evaluation.", "In total, 21 patients with PD were recruited and the diagnosis of a movement disorder was confirmed by specialists according to the United Kingdom Parkinson's Disease Society Brain Bank criteria (Hughes et al., 1992).", "Disease severity was evaluated using the motor scores of the Unified Idiopathic Parkinson's Disease Rating Scale (UPDRS)-III (Martinez-Martin et al., 1994), and the Hoehn and Yahr staging scale (Hoehn and Yahr, 1967).", "All patients were receiving levodopa in combination with a dopamine decarboxylase inhibitor (benserazide or carbidopa) at the time of scanning, were required to have responded to antiparkinsonian therapy, and were required to have remained free of atypical parkinsonism at 12 months (or longer) after diagnosis.", "For comparison, we recruited 21 age- and gender-matched healthy controls with no history of neurological disease.", "The demographic and clinical characteristics of all participants are shown in Table 1.", "Neuroimaging data were obtained on a 3.0-T system (Achieva; Philips Healthcare, Best, the Netherlands) equipped with an eight-channel head coil for sensitivity-encoding parallel imaging.", "DW-MRI was acquired at b-values of 1000 and 2000 s/mm2 along 32 uniformly distributed directions with a spin-echo echo-planar imaging scheme in an anterior–posterior phase encoding direction.", "The same diffusion directions were used for each shell.", "Each DW-MRI acquisition was complemented with a non-weighted diffusion image (b = 0 s/mm2).", "Standard- and reverse-phase encoded blipped non-diffusion-weighted images were also obtained to correct for the echo-planar imaging distortion correction (Andersson et al., 2016).", "Acquisition parameters were as follows: repetition time, 9810 ms; echo time, 100 ms; voxel size, 2.0 × 2.0 × 2.0 mm3; matrix: 128 × 128; slices, 65; number of excitations, 1; and acquisition time, 13.07 min.", "T1-weighted images (T1WI) were also acquired for structural data by three-dimensional (3D) magnetization-prepared rapid gradient-echo sequence.", "The acquisition parameters were: repetition time, 15 ms; echo time, 3.54 ms; inversion time, 1100 ms; voxel size, 0.86 × 0.86 × 0.86 mm3; and acquisition time, 5.14 min.", "Fig. 1 shows a schematic of the connectome mapping methodology.", "Preprocessing was performed using the Functional MRI of the Brain (FMRIB) Software Library, Version 5.0.9 (Greve and Fischl, 2009).", "For each subject, anatomical 3D–T1WIs were affine-aligned to the corresponding b0 maps using boundary-based registration.", "Partial volume fraction maps of WM, cortical GM, deep GM, and CSF, calculated from the T1WI data and then were processed for the multi-shell MSMT-CSD and anatomically-constrained tractography (ACT) framework.", "This involved the following steps.", "First, the Brain Extraction Tool (Smith, 2002) was used to remove non-brain tissue from the 3D-T1WIs.", "Second, the FMRIB Automated Segmentation Tool (Zhang et al., 2001) estimated the partial volume fractions of WM, cortical GM, and CSF.", "Third, the FMRIB Integrated Registration and Segmentation Tool (Patenaude et al., 2011) were used to estimate partial volume fractions of the deep GM for all voxels within the brain.", "Fourth, the GM–WM interface mask was obtained using the “5tt2gmwmi” command, which was implemented in the MRtrix (http://www.mrtrix.org/).", "Although WM masks are commonly used for seeding, over-reconstruction of streamline density for longer fiber pathways can be induced by homogeneously seeding streamlines throughout the WM (Yeh et al., 2016).", "In this study, we used the GM–WM interface as a seeding point for tractogram generation, as it is reported that this method can avoid the over-reconstruction problem (Girard et al., 2014); however, this approach can result in an underestimation of the prevalence of long-distance fibers (Zalesky and Fornito, 2009).", "DW-MRI data were checked visually in all three orthogonal views, which did not reveal severe artifacts related to gross geometric distortion, signal dropout, or bulk motion.", "The data were corrected for susceptibility-induced geometric distortions, eddy currents, and inter-volume motion using the TOPUP and EDDY toolboxes (Andersson and Sotiropoulos, 2016).", "A default reconstruction pipeline was performed in FreeSurfer (Dale et al., 1999) to obtain 84 brain nodes according to Desikan–Killiany cortical atlas segmentation (Desikan et al., 2006).", "Due to high variability in the spatial location and extent of subcortical GM segmentations produced by FreeSurfer (Dale et al., 1999), the subcortical structures were obtained from subcortical GM partial volume fraction maps using the FMRIB Integrated Registration and Segmentation Tool in the FMRIB Software Library (see the Data preprocessing section).", "Whole-brain tractograms were generated using 1) deterministic SSST-CSD tracking (b-value of 2000 s/mm2), (2) probabilistic SSST-CSD tracking (b-value of 2000 s/mm2), and (3) probabilistic MSMT-CSD probabilistic tracking from multi-shell DW-MRI data (b-values of 0, 1000, and 2000 s/mm2).", "For all three methods (deterministic SSST-CSD tracking, probabilistic SSST-CSD tracking and probabilistic MSMT-CSD probabilistic tracking), spherical deconvolution informed filtering of tractograms (Smith et al., 2013) and ACT (Smith et al., 2012) were applied to reduce bias in streamline density and to prevent biologically unrealistic connection terminations (Smith et al., 2012).", "Tractography for all three methods was performed with the MRtrix software package (Brain Research Institute, Melbourne, Australia, http://www.brain.org.au/software/).", "For conventional CSD and deterministic tractography, voxels were assigned to WM if the tissue segment WM volume fraction was > 0.95 and the fractional anisotropy (FA) was > 0.7.", "Subsequently, the DW-MRI signal was reoriented to ensure that the principal axis of diffusion was aligned.", "Finally, the anisotropic response functions for WM were estimated from single shell data (b = 2000) by averaging the DW-MRI profiles over these voxels.", "The WM fODF was obtained using the dwi2fod command with the msmt-csd option, using WM response function.", "The maximal SH order lmax = 6 for WM was used.", "We generated probabilistic tractography (CSD) and deterministic tractography using these WM fODFs.", "For MSMT-CSD (Jeurissen et al., 2014), multiple response functions were estimated as a function of b-value and tissue type.", "Specifically, voxels were assigned to WM if the tissue segment WM volume fraction based on the structural image was > 0.95 and the fractional anisotropy (FA) was > 0.7.", "Subsequently, the DW-MRI signal was reoriented to ensure alignment of the principal axis of diffusion.", "Finally, the anisotropic response functions for WM were estimated per shell by averaging the DW-MRI profiles over these voxels.", "If a tissue segmentation had a volume fraction > 95% and an FA < 0.2, voxels were assigned to GM and CSF.", "The response functions for GM and CSF were obtained by averaging DW-MRI profiles per shell.", "Finally, the WM fODF, GM fODF, and CSF fODF were obtained using the dwi2fod command with the msmt-csd option.", "A maximal spherical harmonic (SH) order of lmax = 6 for WM, lmax = 0 for GM and CSF were used.", "The WM-fODFs obtained here were used for probabilistic MSMT-CSD tracking.", "Data with a b-value of 2000 s/mm2 were used for deterministic and probabilistic SSST-CSD tracking, which is the basic requirement for ODF reconstruction of high angular resolution diffusion imaging (Pichon et al., 2005).", "In addition, data with b-values of 1000 and 2000 s/mm2 were used for probabilistic MCMT-CSD tracking.", "For deterministic SSST-CSD tracking, we employed a deterministic algorithm based on spherical deconvolution (SD) (Tournier et al., 2012), referred to as SD_STREAM in MRtrix software, with the following parameters: step size = 0.2 mm, maximum curvature = 45° per step, length = 4–200 mm, and fiber orientation distributions threshold = 0.1.", "For probabilistic SSST-CSD and MSMT-CSD tracking, second-order integration was employed over the FOD (iFOD2) algorithm (Tournier et al., 2010), using the following parameters: step size, 1.0 mm; maximum curvature, 45° per step; length, 4–200 mm; and fiber orientation distribution threshold, 0.06 for probabilistic MSMT-CSD tracking, 0.1 for deterministic and probabilistic SSST-CSD tracking.", "For all tracking methods, in total, 5 × 107 streamlines were seeded from WM fODFs.", "Seed points were determined dynamically using the Spherical deconvolution informed filtering of tractograms (SIFT) model (Smith et al., 2013).", "Furthermore, “back-tracking” was used within the ACT framework (Smith et al., 2012).", "SIFT was also applied to filter the reconstruction from 5 × 107 to 5 × 106 streamlines.", "Connectomes were constructed for each subject based on connectivity derived from the three tractography methods (i.e., deterministic SSST-CSD tracking, probabilistic SSST-CSD tracking and probabilistic MSMT-CSD probabilistic tracking).", "WM connectivity was modeled as a weighted, undirected network (connectome).", "Nodes were defined as the 84 brain regions of the Desikan–Killiany GM parcellation, and the number of streamlines interconnecting each pair of nodes was enumerated.", "Specifically, streamlines were assigned to the closest node within a 2 mm radius of each streamline endpoint in ACT (Smith et al., 2015).", "This resulted in an 84 × 84 interregional connectivity matrix, with each element populated by the number of streamlines that served as a measure of connectivity strength.", "The diagonal elements represented self-connections and were excluded from analysis.", "A connection density threshold (T) was applied to discard spurious links (Rubinov and Sporns, 2010).", "Pairs of regions with the lowest streamline counts were set to a value of zero, and the top T% of regions according to streamline count were left unaltered.", "To avoid bias from using a single threshold, global network metrics were examined across a range of thresholds (10% < T < 30% in 5% increments) (Zhang et al., 2011).", "Topological measures were analyzed for the three sets of connectivity matrices, as derived from deterministic SSST-CSD tracking, probabilistic SSST-CSD tracking and probabilistic MSMT-CSD probabilistic tracking, using the Brain Connectivity Toolbox (http://www.brain-connectivity-toolbox.net/).", "The following five global network metrics were computed: global path length, clustering coefficient, global efficiency, mean strength, and the small-worldness ratio.", "In addition, four local network metrics were computed: nodal strength, betweenness centrality, local clustering, and local efficiency.", "Detailed descriptions of the global and local network metrics are summarized in Supporting information Table S1.", "To estimate effect sizes, Cohen's d was computed for each global network metric, across the range of thresholds to discard spurious links (10% < T < 30% in 5% increments) for comparison between PD and control groups.", "Cohen's d was typically largest for a threshold of 30% (Supporting information Table S2), so this was applied as the threshold for both global and local network metrics.", "The network-based statistic (NBS) was used to identify subnetworks (clusters of nodes and edges) comprising connections with a reduced streamline count in patients with PD.", "The NBS was separately applied to the connectivity matrices derived from the three methods being compared (i.e., deterministic SSST-CSD tracking, probabilistic SSST-CSD tracking and probabilistic MSMT-CSD probabilistic tracking).", "A detailed description of NBS can be found elsewhere (Zalesky et al., 2010).", "In brief, a two-sample t-test was independently performed at each edge to test the null hypothesis of equality in mean streamline count between patients and controls.", "A set of suprathreshold edges was defined by applying a primary component-forming threshold (e.g., P = 0.005, t = 2.97, two-tailed t-test) to the test statistic computed for each edge (results across different thresholds are reported in Supporting information Table S3).", "The statistical significance of each connected component was obtained with respect to an empirical estimate of the null distribution of maximal component sizes (5000 permutations), with the component size measured as the number of edges it comprised.", "We reported any components that were significant at a P-value of 0.05 after family-wise error correction.", "Connections comprising significant components (subnetworks) were assigned to motor, associative, or limbic circuits that constitute the cortico–basal ganglia–thalamocortical network (Galvan et al., 2015; Ikemoto et al., 2015).", "Further details are provided in Supporting information Fig. S1.", "Connections comprising significant components (subnetworks) were assigned to motor, associative, or limbic circuits that constitute the cortico–basal ganglia–thalamocortical network (Galvan et al., 2015; Ikemoto et al., 2015).", "Further details are provided in Supporting information Fig. S1.", "A support vector machine (SVM) classifier was trained to predict an individual's diagnostic status (case or control), based on topological measures derived from the three tractography methods (i.e., deterministic SSST-CSD tracking, probabilistic SSST-CSD tracking and probabilistic MSMT-CSD probabilistic tracking).", "A linear kernel was used to train the SVM.", "The feature space comprised five global measures and two local measures (local clustering and local efficiency) from eight regions of interest.", "The selected regions of interest were subcortical structures spanning the cortico–basal ganglia–thalamocortical circuit (Galvan et al., 2015), including the putamen, globus pallidus, caudate, and thalamus.", "Local efficiency and clustering were used to measure the efficiency with which these regions, and by extension, the circuit of interest, could exchange neural information.", "To train the SVM and to evaluate classification accuracy, we used stratified ten-fold cross-validation.", "The patient and control groups were partitioned into validation and training subgroups.", "The validation subgroup comprised 10% of all individuals in either the patient or control group (i.e., two controls and two patients), and all other individuals were randomly assigned to the training subgroup (i.e., 19 controls and 19 patients).", "The SVM was trained to classify patients and controls in the training subgroup based on latent patterns among the topological features described above.", "The validation subgroup was then used to evaluate the classification accuracy of the SVM.", "This process was repeated for ten unique validation subgroups (i.e., ten folds), with each individual only ever assigned to one validation subgroup.", "Classification performance was then averaged over these ten folds.", "All statistical analyses were performed using IBM SPSS for Windows, Version 22.0 (IBM Corp., Armonk, NY, USA).", "Demographic and clinical variables were normally distributed, as confirmed with the Kolmogorov–Smirnov test.", "Between-group differences were analyzed by Student's t-tests for continuous variables (age, global, and local topological metrics) and by chi-squared tests for gender.", "Pearson's correlation coefficient was used to test for relationships between brain measures (e.g., connectivity strength and topological metrics) exhibiting significant between-group differences and clinical measures (e.g., disease duration and UPDRS-III score).", "The false discovery rate (FDR) was used to correct for multiple comparisons, using a significance threshold of P < 0.05.", "NBS did not identify any between-group differences when applied to connectivity matrices from deterministic SSST-CSD tracking.", "For both probabilistic SSST-CSD and MSMT-CSD tracking, the null hypothesis of equality in mean streamline count between patients with PD and controls was rejected (P < 0.05) for networks involving the basal ganglia, thalamic, limbic, frontal, temporal, and parietal areas (Fig. 4, Supporting information Table S4).", "The streamline count was reduced in the PD group relative to the control group.", "Specifically, probabilistic SSST-CSD tracking identified a comparable subnetwork of reduced connectivity comprising 44 edges connecting 43 regions (P = 0.007).", "These included 15 associative edges, 1 limbic edge, and 5 motor edges.", "By contrast, probabilistic MSMT-CSD tracking identified a significant subnetwork comprising 58 edges that connected 45 regions (P = 0.006) with a reduced streamline count in patients with PD.", "These included 17 edges classified as associative, 3 as limbic, and 7 as motor circuits.", "In addition, significant correlations were not detected between disease severity and the streamline count averaged across the subnetworks associated with significant between-group differences, although a trend involving the UPDRS-III score was evident for probabilistic MSMT-CSD tracking (Supporting information Table S5).", "No between-group differences were found in any global metrics derived with deterministic SSST-CSD tracking.", "Probabilistic SSST-CSD tracking only detected significant differences in global connectivity strength and small-worldness, which were significantly decreased in patients with PD compared with healthy controls (Table 2).", "There were significant between-group differences across the five global measures with probabilistic MSMT-CSD tracking.", "Specifically, patients with PD displayed significantly decreased global connectivity strength, efficiency, and clustering, as well as increased global path length (Table 2).", "In addition, although both patients and controls demonstrated small-world organization (σ > 1), the small-worldness ratio was significantly decreased in the PD group.", "No correlations were found between global network metrics and clinical measures (e.g., the UPDRS-III score and disease duration) in the PD group.", "Only probabilistic MSMT-CSD tracking detected significantly decreased local efficiency, predominantly localized to motor, frontal temporoparietal associative, limbic, basal ganglia, and thalamic areas (Fig. 2A, Table 3).", "The PD group also displayed reduced local efficiency, most evidently in the motor, frontal temporoparietal associative, limbic, basal ganglia, and thalamic areas (Fig. 2B, Table 4).", "Significant negative correlations were observed between UPDRS-III scores and local efficiency in the bilateral putamen, bilateral pars triangularis, right caudate nucleus, right lateral orbitofrontal gyrus, right post central gyrus, left supramarginal gyrus, left transverse temporal gyrus (Fig. 3, Table 5).", "This correlation was mainly seen in the motor area, frontal temporoparietal associative area, and basal ganglia, and no significant correlations were detected between local metrics and disease duration.", "SVM based on probabilistic MSMT-CSD tracking yielded improved classification accuracies compared to deterministic and probabilistic SSST-CSD tracking (see Table 6).", "A feature space comprising all five global measures yielded the highest classification accuracy (78.33%), precision (85.00%), recall (81.67%), and area under the curve (85.28%).", "However, a feature space comprising only two local measures (i.e., clustering and efficiency) across eight regions (i.e., bilateral putamen, globus pallidus, caudate, and thalamus) yielded lower, but reasonable accuracy (61.67%), precision (46.67%), recall (51.67%), and area under the curve (68.06%).", "SVM based on probabilistic MSMT-CSD tracking with the five global and two local measures combined yielded modest accuracy (76.67%), precision (81.67%), recall (76.67%), and area under the curve (81.39%).", "We compared DW-MRI-based connectomes derived from three different algorithms (i.e., deterministic SSST-CSD tracking, probabilistic SSST-CSD tracking, and probabilistic MSMT-CSD tracking) between patients with idiopathic PD and healthy controls.", "Across all analyses, probabilistic MSMT-CSD tracking outperformed the deterministic and probabilistic SSST-CSD tracking methods when detecting connectome abnormalities and accurately predicting PD diagnosis.", "At the global level, probabilistic MSMT-CSD tracking detected significant between-group differences across all five global measures, while probabilistic SSST-CSD tracking only detected lower global strength and small-worldness in the PD group.", "At the local level, probabilistic MSMT-CSD tracking detected significantly reduced local efficiency and clustering in patients, indicating greater functional segregation in the motor, frontal temporoparietal associative, limbic, basal ganglia, and thalamic areas.", "NBS identified subnetworks of reduced connectivity from the cortico–basal ganglia–thalamocortical network in patients with PD via both the probabilistic SSST-CSD and MSMT-CSD tracking methods.", "Our data suggest that probabilistic MSMT-CSD tracking provides improved sensitivity to the WM connectivity disruptions typically associated with PD, though it should be noted that we did not evaluate the specificity of this approach.", "The improved sensitivity of probabilistic MSMT-CSD tracking may be due to the use of more precise fODF estimates at the GM–WM interface (Jeurissen et al., 2014), which is essential for accurately characterizing both nodes (GM regions) and the edges between nodes.", "Although probabilistic SSST-CSD tracking could partially detect connectivity disruptions, deterministic SSST-CSD tracking could not detect significant between-group differences in topological measures or connectivity strength.", "This may be due to differences in the algorithms used for global tracking (e.g., edge generation).", "In the present study, we adopted a fODF-based deterministic tracking algorithm (deterministic SSST-CSD tracking).", "This tracking method provides fiber distributions that are thought to correspond more closely with known brain anatomy, compared to conventional deterministic tensor tracking algorithms (Jeurissen et al., 2011).", "However, the probabilistic fODF-based algorithm has been shown to provide more accurate tractography compared to the deterministic fODF-based algorithm (Tournier et al., 2012).", "Although deterministic tracking provides a single best fit streamline, probabilistic tracking algorithms can explore other possible directions to account for the uncertainty of fiber orientation distributions.", "Therefore, in many cases, probabilistic methods can identify peripheral branches of tracts, where these branches might be difficult to detect with deterministic tractography.", "However, an important disadvantage of probabilistic tractography is that its high sensitivity typically comes at the cost of low specificity; namely, there is an increased likelihood of identifying spurious fibers (false positives) with probabilistic methods (Knosche et al., 2015; Thomas et al., 2014).", "The estimation of spurious fibers is particularly detrimental to the characterization of the connectome's topological properties (Zalesky et al., 2016).", "For probabilistic MSMT-CSD tracking, using stratified ten-fold cross-validation, we could predict an individual's diagnostic status with modest accuracy (approximately 78%) based on graph theory.", "The most accurate prediction of diagnostic status was achieved with all global measures computed in the probabilistic MSMT-CSD tracking connectivity matrices (78.33%).", "The global measures for deterministic and probabilistic SSST-CSD tracking performed substantially less well than those in probabilistic MSMT-CSD tracking, with no parameter yielding an accuracy of > 70%.", "Although diagnostic accuracy was low when we used local graph metrics of the basal ganglia and thalamus, diagnostic accuracy (61.67%) was highest for local graph metrics derived from probabilistic MSMT-CSD tracking.", "This provides further evidence of the increased sensitivity of probabilistic MSMT-CSD tracking for detecting connectome pathology compared with deterministic and probabilistic SSST-CSD tracking.", "Although our findings indicate that PD can be predicted with modest accuracy by probabilistic MSMT-CSD tracking, further work is needed to establish whether the classifier performance is robust to alternative image acquisition sequences and alternative probabilistic tractography algorithms.", "Deep learning methods, decision trees, and other supervised learning methods appropriate for neuroimaging data might improve the achieved classification accuracies (Vieira et al., 2017).", "Indeed, a criticism of SVM is the need to extract less redundant and more informative data from the raw data during feature selection (Plis et al., 2014).", "In this study, we selected features based on subcortical regions spanning a circuit known to be affected by PD pathology.", "Deep learning methods can automatically identify the optimal representation of features within a high-dimensional dataset, without requiring prior feature selection.", "Longitudinal studies are now required to determine the accuracy with which machine learning techniques trained on probabilistic MSMT-CSD tracking data can predict patient outcome and prognosis.", "In the graph theory analysis using probabilistic MSMT-CSD tracking, we found decreased global clustering, efficiency, strength, and small-worldness, as well as increased global path length in PD.", "Reduced global clustering suggests poor network segregation (i.e., reduced specialized information processing), whereas decreased global efficiency and increased global path length indicate compromised network integration (i.e., reduced parallel information transfer).", "These global, local, and connectivity alterations are consistent with the results of previous studies (Li et al., 2016; Nigro et al., 2016) and probably reflect the presence of extensive pathological changes in the WM of PD brains.", "In particular, the α-synuclein inclusions that are deposited in the pre-synapse of patients with PD may cause synaptic collapse and impaired axonal transport, resulting in widespread axonal degeneration (Braak and Del Tredici, 2008).", "Structural WM disintegration (reduced connectivity) can impair efficient information exchange, resulting in network disorganization (poor segregation and integration).", "Previous connectome studies report unimpaired small-world organization (Li et al., 2016; Nigro et al., 2016).", "While we also found that small-world organization was maintained in PD (σ > 1), the small-world ratio measured by probabilistic MSMT-CSD tracking was decreased relative to that in controls.", "Previous studies have evaluated small-worldness by deterministic tracking based on the “Fiber Assignment by Continuous Tracking” algorithm (Li et al., 2016; Nigro et al., 2016), but this cannot accurately estimate neural fiber connections in regions with crossing and kissing fibers at the voxel level (Mori et al., 2004).", "By contrast, we applied probabilistic tracking that can deal with the crossing/kissing problem, and therefore yielded more accurate estimates of the connectome.", "Small-world organization is considered to reflect the optimal balance of functional integration and segregation, with the reduced small-world property in this study suggesting that this balance may be disrupted in PD.", "At the local level, decreased segregation was found among key components of the cortico–basal ganglia–thalamocortical network, as indicated by decreased local efficiency and clustering.", "These changes were correlated with the UPDRS-III motor score, thereby strongly supporting involvement of this network in PD pathophysiology.", "Nigro et al. reported reduced local efficiency and clustering in the globus pallidus and inferior occipital gyrus (Nigro et al., 2016), but we found similar reductions across a more extensive area, including the motor, frontal temporoparietal associative, and basal ganglia areas.", "This difference might be due to discrepant connectome generation methods (as described above); however, discrepancies in disease durations between studies may be important.", "The participants in the study by Nigro et al. (2016) had early PD (mean duration of 1 year 7 months) and had not received medications, whereas participants in our study had a mean illness duration of 5 years and all had already received levodopa treatment.", "Therefore, pathological changes may have progressed to a wider area among the patients in our study.", "Both probabilistic SSST-CSD and MSMT-CSD connectomes revealed subnetworks of reduced connectivity between key components of the cortico–basal ganglia–thalamocortical network.", "The results of NBS analysis also provide evidence supporting involvement of this network in PD pathophysiology.", "Other studies using NBS have reported reduced connectivity strength between the basal ganglia, thalamic, limbic, frontal, temporal, and parietal areas in patients with PD (Aarabi et al., 2015; Kim and Park, 2016; Li et al., 2016; Nigro et al., 2016), which is also in agreement with our results.", "This study has some limitations: First, the sample size was small and the study design was retrospective in nature.", "Larger, multicenter, prospective studies are therefore required.", "In addition, we only included patients with relatively long durations of PD, so a longitudinal study of prodromal or early PD is required to confirm whether connectome analyses based on probabilistic MSMT-CSD tracking is effective for monitoring and predicting disease progression.", "Second, we did not evaluate the non-motor symptoms of PD, such as cognitive and psychiatric symptoms.", "Because connectivity disruptions in the basal ganglia–thalamocortical circuits might contribute to non-motor symptoms in PD, future studies should investigate the relationship between non-motor symptoms and connectome dysfunction more closely.", "Finally, because the PD diagnoses were not confirmed histopathologically, misdiagnosis remains possible; however, the validity of each diagnosis was supported by continued response to therapy and continued freedom from atypical parkinsonism at 12 months after scanning.", "We mapped the connectomes of patients with PD and healthy controls to determine which of deterministic SSST-CSD tracking, probabilistic SSST-CSD tracking, and probabilistic MSMT-CSD probabilistic tracking was most sensitive for detecting associated WM connectivity disruptions.", "Probabilistic MSMT-CSD tracking detected WM disruptions most extensively among the three methods, and these disruptions were characteristic of known PD pathophysiology, including connectivity loss in cortico–basal ganglia–thalamocortical networks.", "Moreover, when compared with deterministic and probabilistic SSST-CSD tracking connectome methods, probabilistic MSMT-CSD tracking more accurately classified patients with PD from healthy comparison subjects.", "We, therefore, conclude that connectome analysis based on probabilistic MSMT-CSD tracking offers greater sensitivity and accuracy when assessing aberrant WM connectivity in cases of suspected PD.", "However, further research is needed to confirm our results in larger studies, and specific research is needed to look at the specificity of probabilistic MSMT-CSD tracking in prodromal or early stages of PD when there is diagnostic uncertainty.", "The following are the supplementary data related to this article.Fig.", "S1", "Segregated basal ganglia—thalamocortical circuit.", "The motor circuit involves projections from the supplementary motor area, arcuate premotor area, motor cortex and somatosensory cortex into the putamen, which projects into the globus pallidus which projects into the cortex through the thalamus.", "Therefore, we classified connections linking these areas as motor circuit.", "The limbic circuit involving the projections from the anterior cingulate, hippocampus, entorhinal cortex, and insula into the ventral striatum, then into the globus pallidus, followed by a loop back into the cortex through the thalamus.", "Therefore, we classified connections linking these areas as limbic circuit.", "The associative circuit proposes a pathway from the dorsolateral prefrontal cortex, lateral orbitofrontal cortex, parietal and temporal association area into the caudate, putamen, followed by a projection into the globus pallidus, followed by a loop back into the cortex through the thalamus.", "Therefore, we classified connections linking these areas as cognitive/associative circuit.", "(based on Galvan et al., 2015).", "Supplementary data to this article can be found online at https://doi.org/10.1016/j.nicl.2017.11.007." ]

[ "Connectomes mapped in Parkinson's disease (PD) using multi-shell tractography.", "Multi-shell tractography provided improved sensitivity to connectome pathology.", "Machine learning accurately predicted PD diagnosis based on connectome.", "Connectome pathology in PD was localized to basal ganglia-thalamocortical circuits." ]

[ "BIOC" ]

[ "APOBEC3B", "Ataxia-telangiectasia and Rad3-related", "Base excision repair", "Deaminase", "Genomic instability", "Uracil-DNA glycosylase" ]

[ "1303", "1304", "1307", "1312" ]

[ "Cancer development is associated with mutations and genomic instability [1,2].", "In cancers that develop under conditions of chronic inflammation, deamination-associated C>T mutations are induced massively in association with expression of deaminase APOBEC3A and B [3].", "These C>T mutations occur widely in epigenetically-methylated CpG islands; this is because deamination of 5me-cytosine yields thymine (5me-C>T transition) [4].", "While APOBEC3A is more effective in 5me-C deamination than APOBEC3B, deamination mediated C>U induction by APOBEC3A is less efficient than that by APOBEC3B [5,6].", "APOBEC3A and B targets single strand DNA (ssDNA); therefore, it plays a crucial role in restricting proliferation of retroviruses by hyperediting their complementary DNA intermediates [7].", "Deamination may also occur at exposed ssDNA sites in normal cells.", "While deamination at unpaired ssDNA loci could result in G/T-mismatches that are repairable by base excision repair (BER) (via the thymine DNA glycosylases TDG and MBD4) [8,9], deamination at exposed ssDNA sites could lead to mutational 5me-C>T transitions in addition to major C>U transitions that are repairable through UNG-mediated BER [10].", "Indeed, deamination-mediated mutagenesis, i.e., 5me-C>T transition, is elevated in APOBEC3B-expressing cells [5] and is a major pathway that results in cancer-driver mutations, such as those present in the p53 and APC genes [11].", "Deamination-mediated mutations (5me-C>T transitions) at CpG sites are induced in association with kataegis, a mutational process that results in localized hypermutations at or around genome rearrangement loci [12].", "Because genome rearrangement occurs via erroneous end-joining of double-strand breaks (DSBs), deamination-mediated hypermutation may occur during the damage response or repair processes.", "Intriguingly, deamination-mediated mutagenesis occurs in the lagging strand under replication stress in a manner that is dependent on the ataxia-telangiectasia and Rad3-related (ATR) kinase [13].", "However, the association between deamination and damage responses is still not clear.", "In addition, although deamination is usually induced at very limited genomic loci [12] (where it should also induce massive C>U transitions), it remains unclear whether such massive transitions are associated with genome integrity.", "In this study, we found that DSBs trigger the onset of APOBEC3B activation, in a manner dependent on ATR.", "Subsequently, the cells undergo massive BER via nuclear uracil-DNA glycosylase UNG2, ultimately leading to further DSB accumulation.", "HeLa and SW480 cells were obtained from ATCC and cultivated in DMEM containing 10% fetal calf serum [14].", "The cells were transfected with the APOBEC3B expression vector (pPM-APOBEC3B; abm) or the negative control vector (pPM-NC; abm) using Lipofectamine 3000 (Life Technologies).", "Treatment with olaparib (Selleckchem), KU55933 (Merck Millipore), VE-822 (Selleckchem), or NU7441 (Selleckchem) was performed where indicated.", "To knockdown nuclear UNG2, a siRNA that targets both mitochondrial UNG1 and UNG2 (Thermo Fisher; 139934) were used where indicated, comparing with a negative control siRNA (Qiagen; 1027281).", "Survival rates were determined by counting the number of viable cells 7 days after γ-ray irradiation.", "DNA damage was induced by 137Cs irradiation of cells in a Gammacell 40 Exactor (Best Theratronics).", "The induced DSBs were detected by immunostaining of γH2AX and 53BP1.", "Hydroxyurea (Sigma) was also used to induce DNA replication stress-associated DSBs.", "Statistical analysis of γH2AX foci was performed from the indicated numbers of cells.", "In Figs. 1B and 1D, statistical analyses were performed together for the experiments done in same condition.", "Antibodies against the following proteins were obtained from the indicated suppliers: γH2AX (Millipore, 05–636; and CST, 9718), β-actin (Sigma, AC-74), 53BP1 (Merck, PC712), APOBEC3B (GeneTex, GTX17214), and HA-tag (Abcam, ab49969).", "Western blotting was performed as described previously [15].", "Immunostaining was also performed as described previously [16] using a confocal laser microscope (Olympus, FV10i).", "Before immunostaining with primary and secondary antibodies, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100/PBS for 10 min.", "For confocal microscope imaging, cells were cultured on coverslips and stained as above.", "The APOBEC3B deaminase targets both cytosine and 5me-cytosine in ssDNA [17] (Fig. 1A).", "While deamination of 5me-cytosine is induced only at epigenetically-methylated DNA loci (which leads to 5me-C>T transitions), deamination of cytosine should be induced widely; this causes C>U transitions that subsequently induce UNG2-initiated BER to eliminate uracil from the DNA strands in the nucleus [10].", "To determine the deamination status of cellular APOBEC3B in response to DNA damage, DSBs that appeared as by-products of BER were analyzed in irradiated cells expressing an APOBEC3B-containing or negative control vector.", "Because large numbers of DSBs are induced during BER in association with DNA replication [18], late-onset DSB formation was evaluated 12–48 h after γ-irradiation (2 Gy) by monitoring the formation of γH2AX foci.", "As expected, after the early repair of DSBs that were directly caused by the γ-irradiation, secondary γH2AX foci were specifically observed in APOBEC3B-expressing cells 12–48 h after γ-irradiation (Fig. 1B).", "In addition, most γH2AX foci merged with 53BP1 foci (Fig. 1C), indicating that the γH2AX foci were DSBs.", "Such late-onset γH2AX foci were observed in most of the APOBEC3B-expressing cells, and the level of induction of these foci was almost identical to that caused by the γ-irradiation.", "In addition, late-onset γH2AX foci were observed continuously throughout the monitoring period, indicating that, unlike the DSBs caused by irradiation directly, they were not efficiently repaired.", "To gain further insights into the roles of deamination and BER in late-onset DSB formation, we examined the effects of siRNA-mediated knock-down of UNG2, a nuclear uracil-DNA glycosylase, on delayed γH2AX foci formation (at 24–48 h) in cells overexpressing APOBEC3B.", "As expected, the delayed formation of γH2AX foci in these cells was effectively suppressed by knock-down of UNG2 (Fig. 1D), suggesting that late-onset DSB formation is dependent on UNG2-mediated BER, thereby supporting the induction of APOBEC3B-mediated C>U transitions.", "This hypothesis is further supported by the finding that treatment of cells with olaparib, a PARP inhibitor that blocks the ligation step of BER, enhanced the accumulation of late-onset γH2AX foci (Fig. 1E).", "Taken together, these results support the hypothesis that APOBEC3B is activated by γ-irradiation and leads to C>U transitions, which subsequently result in UNG2-mediated BER and the induction of γH2AX foci as by-products.", "Unlike the early-onset γH2AX foci directly caused by γ-irradiation, the secondary γH2AX foci were continuously observed throughout the monitoring period, suggesting that the secondary damage caused by deamination and BER was persistent (Fig. 1A).", "Next, we examined the involvement of the DNA damage checkpoint response in APOBEC3B activation after γ-irradiation.", "The kinases involved in this response, namely, ataxia-telangiectasia mutated (ATM), ATR, and DNA-PK, were inhibited by treatment of APOBEC3B-expressing cells with KU55933, VE-822, and NU7441, respectively (Fig. 2A).", "The late-onset γH2AX foci formed via APOBEC3B were efficiently induced in the presence of KU55933 or NU7441; however, their formation was specifically blocked in the presence of VE-822, suggesting a requirement for ATR.", "We also examined the effects of inhibiting ATR and ATM on early DNA damage responses (Fig. 2B).", "As expected, early-onset formation of γH2AX and 53BP1 foci after γ-irradiation was reduced in the presence of KU55933 (Fig. 2B), confirming the role of ATM in immediate responses to DSBs [19].", "However, this had no effect on the frequency of late-onset DSB formation.", "Taken together, these results suggest that the deaminase APOBEC3B is activated by DSBs specifically via the ATR kinase.", "This results in C>U transitions that ultimately lead to accumulation of late-onset γH2AX-foci via UNG-mediated BER.", "Since ATR dependence is also observed during deamination-mediated mutagenesis under replication stress [20], ATR might be commonly involved in deamination.", "Deaminase-mediated mutations occur in association with kataegis (localized hypermutation at or around DNA rearrangement loci) [12], suggesting that deamination reaction is specifically induced at or around DSB sites that occur following DNA damage or during the repair process.", "To determine the damage dependence, APOBEC3B expression status was monitored after γ-irradiation, as APOBEC3B is usually not expressed in most cells and is specifically expressed in cells accumulated deamination-mediated mutations [21,22].", "As expected, the level of APOBEC3B protein was increased after γ-irradiation or treatment of hydroxyurea, an agent that causes replication stress-associated DSBs (Figs. 3A and 3B), indicating damage-dependent stabilization.", "The increase in APOBEC3B expression was observed as early as 30 min after γ-irradiation (Fig. 3C), which is much faster than the expression level changes that are usually seen following modulation of transcription and translation.", "One possible explanation is the involvement of proteasomal degradation, a process in which proteins are degraded under normal conditions.", "Such proteolysis is blocked in response to certain types of damage/stress, resulting in rapid accumulations of proteins, such as those observed for H2AX and NRF2 in response to DSBs and oxidative stress, respectively [23,24].", "To examine the hypothesis that inhibition of proteasomal degradation results in APOBEC3B accumulation, we treated SW480 cells with the proteasome inhibitor MG132 or the E1 ubiquitin ligase inhibitor PYR41.", "Remarkably, APOBEC3B was accumulated in non-irradiated cells following treatment with these agents (Fig. 3D).", "These results suggest that the deaminase APOBEC3B is continuously synthesized and is proteolyzed under normal conditions, but is immediately accumulated in response to DSBs via inhibition of the proteasomal-degradation pathway, ultimately causing deamination-mediated C>U transitions and subsequent long-lasting DSB formation.", "To determine the effect of those deaminase-mediated secondary DSBs for radiation sensitivity, we examined the killing efficiencies of cells expressing APOBEC3B and compared with the control after radiation and observed almost no difference in those two conditions (Fig. 3E).", "This suggests that the effect of such late-onset DSB formation is probably limited at least for cancer-cell killing by IR.", "The results presented here demonstrate that the deaminase APOBEC3B is activated in response to DSBs, specifically through ATR kinase.", "In addition to causing mutagenic 5me-C>T transitions, APOBEC3B-mediated deamination reactions also cause C>U transitions, which subsequently induce UNG-mediated BER to eliminate uracil from the DNA.", "As a result, cells expressing APOBEC3B accumulate late-onset γH2AX foci as by-products of BER.", "Notably, the levels of late-onset γH2AX foci observed here were comparable to those of the primary γH2AX foci caused directly by γ-irradiation (Figs. 1 and 2), suggesting massive BER induction.", "Deaminase-mediated mutagenesis is induced in association with kataegis, suggesting that deamination is very specifically induced at or around DSB sites.", "The precise mechanism by which APOBEC3B is induced is still unclear; however, the results presented here provide some insights.", "APOBEC3B is rapidly up-regulated in response to γ-irradiation.", "Such a rapid increase in expression is not possible through changes in transcription and translation rates, but can occur for factors that are continuously synthesized and degraded by the proteasome pathway.", "In fact, our experiments revealed that APOBEC3B is continuously synthesized and degraded by the proteasomal system under normal conditions.", "Notably, this type of rapid up-regulation of expression is usually seen during immediate responses to certain types of stress or damage; for example, H2AX and NRF2 are upregulated rapidly in response to DSBs and oxidative stress, respectively [23,24].", "Therefore, we suggest that APOBEC3B is regulated in a similar manner to enable immediate cellular responses to stress or damage.", "Deaminases cause C>T/U transitions often in association with hypermutation at very limited loci, as seen in inflammation-associated cancer cells and in B cells during somatic hypermutation, specifically at variable regions of immunoglobulin genes [25–27].", "Intriguingly, both are associated with DSB formation and genomic rearrangement, although the mechanism by which deamination-mediated mutagenesis is associated with DSBs is still elusive.", "UNG-mediated late-onset γH2AX foci were continuously observed in most APOBEC3B-expressing cells 12–48 h after γ-irradiation (Fig. 1), indicating that such secondary DSBs are massively induced and difficult to repair.", "This finding is unexpected because BER-mediated DSB formation occurs during replication (or sometimes in association with transcription) and is therefore expected to occur only in some cells, specifically those in which DSBs are repairable (Fig. 4A).", "However, the frequency and persistence of the DSBs observed in the current study were much higher than expected.", "It is possible that secondary DSB formation is directly associated with the mechanisms of deamination and BER.", "In fact, since deamination-mediated hypermutation is induced in very limited regions at or around genome rearrangement loci (DSB sites) [12], deamination-mediated C>U transitions should be induced in very close proximity to these sites, at which spontaneous formation of more DSBs is expected as a consequence of BER (Fig. 4B).", "Such BER-mediated formation of DSBs could induce further deamination and cause further BER-mediated DSBs, resulting in multiple rounds of DSB formation and repair (Fig. 4C).", "It is not clear whether the late-onset DSBs observed herein undergo such futile cycles; however, this type of chain reaction could certainly contribute to their persistence.", "It was recently shown that APOBEC3A/B expressing cancer cells are sensitive to ATR inhibitors owing to the resulting replication catastrophe [28].", "These findings are likely different from our own because formation of late-onset long-lasting DSBs is due primarily to γ-irradiation and is blocked by an ATR inhibitor.", "Thus, the effects of ATR-mediated deamination reactions are likely context-dependent.", "As shown in Fig. 3E, expression of APOBEC3B does not contribute to the killing effect of γ-irradiation.", "This might be due to the associated damage levels.", "In fact, when significant damage is caused by γ-irradiation, cells respond directly by inducing apoptosis via activation of damage checkpoints; therefore, in such cases, the effects of late-onset DSBs are probably limited.", "By contrast, APOBEC3B-mediated secondary DSBs arise only after repair of primary DSBs, in which damages caused by γ-irradiation must be tolerable for those cells.", "The number of secondary γH2AX foci is similar to that of primary foci; hence, the cells are expected to tolerate the damage.", "This may be why cells survive with long-lasting secondary DSBs.", "APOBEC3B-mediated long-lasting DSBs could be a risk factor for genomic rearrangements.", "In fact, deamination-mediated hypermutation is observed in association with kataegis, which is a localized hypermutation at genome rearrangement loci [12].", "This implies that the locus subjected to DSBs with massive deamination likely undergoes genomic rearrangement rather than direct end-joining repair.", "It is widely accepted that genomic rearrangements are caused by erroneous end-joining of DSBs, each of which is located originally at separated chromosomal positions.", "Such risks could increase when DSBs are not repaired effectively and/or when these DSBs occur in close proximity to other broken ends.", "Thus, formation of long-lasting DSBs likely increases the risk of genomic rearrangement.", "This study was supported by the MEXT Grants-in-Aid, Japan (20770136)." ]

[ "APOBEC3B is stabilized to induce deamination reaction in response to DSBs.", "Deamination leads to formation of late-onset DSBs through UNG2-mediated BER.", "Late-onset DSBs induced in an ATR-dependent manner are persistent." ]

[ "CENG", "COMP", "ENER", "ENGI", "ENVI", "MATE" ]

[ "City intelligence", "Construction emphases", "Ternary space", "Urban big data" ]

[ "1500", "1700", "2102", "2200", "2305", "2501" ]

[ "Amid China's rapid industrialization and urbanization, the rise in the population, manufacturing, and traffic of its cities is becoming increasingly intense and complex leading to a variety of urban diseases such as rapid population growth, traffic jams, environmental deterioration, housing shortages, employment problems, and public safety challenges.", "This is just a short list of the side effects of urbanization while there is a host of other less prominent policy problems facing Chinese policymakers.", "All of these factors have become serious constraints upon the healthy and sustainable development of China's urban ecosystems [1].", "On one hand, the urban ecosystem is composed of urban infrastructure and diverse social environment among urban residents that is becoming increasingly intricate and scaling upon a daily basis; on the other, decision makers and administrators are not fully conscious of this complexity and are consequently deficient in the efficient management of this ecosystem.", "Modern cities have been upgraded to ternary spaces from dual spaces.", "The first-dimensional space is a physical space made up solely of a physical environment with all its resources in a natural state.", "The second-dimensional space contains a human society space shaped and sustained by the culture, norms, and social interactions of urban residents.", "A third-dimensional space, unlike the previous two, is a cyber space, which is comprised of computers, internet access, and the data flowing through these systems to informationized domain [2].", "This new structural concept of urban life calls for new philosophies, theories, and practices for analyzing the structures, economic development, and governance problems of urban life in a meaningful and systematical way, to better understand the new direction of the new urban landscape in context of this technological revolution.", "The advent of “intelligent city” and “big data” provides new potential platforms for resolving various “urban diseases.”", "Intelligent cities provide an artificial nervous system based upon the blueprints of traditional urban ecosystems, while urban big data describes the physical domain of real objects (buildings, cars, roads, and so on) and social domain of urban residents in real space, all of which has a virtual form that reflects the real forms in first-dimensional space and second-dimensional space.", "These virtual forms constitute the third-dimensional space: urban big data.", "Scholar Guojie Li, member of the Chinese Academy of Engineering (CAE), believes that the role of big data is similar to the “Honeybee Model,” which is based on the value of having honey bees: The bees improve agricultural output of farms through the pollination of crop plants rather than the honey they produce [3].", "Just like the human and non-human resources of cities, urban big data has become an important strategic resource for the development of intelligent city and strategic direction.", "As a city evolves toward informatization and intelligence, numerous information bases and data centers have been emerging, which should be properly interconnected to form urban big data.", "Urban big data can be converged, analyzed, and mined with depth via the Internet of Things, cloud computing, and artificial intelligence technology to help people understand the forces guiding the development for each layer of the urban system to assist the government and society with decision-making and urban planning to achieve the goal of intelligent administration of the city.", "Meanwhile, urban big data will bring about profound changes to the operating mode of various urban sectors, and speed the transformation and upgrading of traditional industries as well as the development of emerging industries.", "Thus, urban big data is speeding up the development of city intelligence.", "Specifically, city intelligence is implemented through the combined development of the Internet, Internet of Things, telecommunication networks, radio and television networks, and wireless broadband networks; it is characterized mainly by thorough integration of information technologies and comprehensive applications of big data; it focuses on intelligent technologies, intelligent industries, intelligent services, intelligent administration, and intelligent life; lastly, this transformation is committed to building a new form of urban development that is capable of self-correction and solving critical social, economic, and ecological problems in a more automated and timely fashion [2].", "China actively encourages the development of big data and the construction of intelligent cities.", "Local governments attach great importance to the development of an information economy.", "In this context, it is necessary to correctly examine the relationship between decentralization and centralization during the development of urban big data and stick to a close combination between the various priorities: overall planning, cooperative construction of the systems, and balancing technological-push and demand-pull.", "Accordingly, the key to the development of city intelligence is enhancing the top-level design for urban big data and defining the key points of urban big data development.", "In light of China's development model for city intelligence, this study proposes the key points for the development of city intelligence are based on four perspectives: infrastructural support, urban governance, urban services, and economic development (Fig. 5).", "A city should build an Internet of Things platform for urban public facilities.", "The technological architecture of this platform is characterized by an integrated service platform and interoperable application modules, while the management system is characterized by professional operations and open services.", "The platform is intended to dynamically detect the fluctuating changes in the conditions of urban infrastructure (e.g., road facilities, water, electricity and fuel gas, and underground pipelines), be well-informed about the status and features of urban operation, accurately forecast the development trends of urban elements, and provide powerful decision-making support for urban development.", "To this end, the city should survey and determine the status quo of the citywide governmental data resources (e.g., types, scale, growth rate, ease of sharing, source, and ownership), identify the data directories and data fields that can be prioritized for sharing or opening access to, and list the data that are confidential or involve public security and personal privacy.", "The city should also build an urban governmental data architecture, and set up an urban data sharing and exchange service hub committed to the cleaning, processing, integration, mining, and sharing of urban governmental data.", "Backed by a tool platform, the service hub would focus on hierarchical data services under an appropriate mechanism to promote the integration and sharing of governmental and other data resources across the whole city.", "Finally, the city should build a unified open platform for governmental data—which would be perfect for the management specifications for governmental data resources—and develop a directory and service center allowing the public to access helpful data for everyday living such as personal credit, traffic, medicine, health, employment, social insurance, geography, culture, education, science and technology, resource, agriculture, environments, safety supervision, finance, quality assurance, statistics, meteorology, and commercial paperwork.", "Urban governance involves a variety of fields, including public security administration, urban planning and construction, market supervision, and environmental protection.", "Therefore, it is important to apply big data to urban governance.", "The big data application platform for grassroots social governance carries data in diverse fields, including civil air security, energy, electricity, environmental protection, health, traffic, municipal administration, water conservancy, safety supervision, meteorology, and earthquakes.", "Data resources should be mined, analyzed, and applied more deeply so as to identify social administration problems quickly, provide early warning, and solve the problems satisfactorily, thus enhancing the grassroots social governance capacity.", "To attain multi-planning integration and inter-departmental collaboration, a city should perfect the One-Map database of land resources, build a management platform for space-time big data and real-estate registration information, conduct big data analysis for land resources, develop a unified big data service system for land resources, and build a big data application platform for urban planning in support of inter-departmental collaboration management, so as to provide a powerful support for the whole process—from formulating and reviewing through to the implementation of urban planning.", "The city should also intensify the credit monitoring of main market players, create public and personal credit databases, and acquire big data on credit covering all credit matters (mainly enterprises, along with individuals and governments) and all categories of credit information, to make urban citizens more credit-conscious and build a favorable credit environment.", "Finally, the city should improve the layout of the urban environmental quality-monitoring network; develop a real-time merging and analysis platform for the comprehensive monitoring of big data concerning water, soil, garbage, and air pollution; and acquire information on urban environmental quality and pollution sources comprehensively and accurately in real time in order to provide decision-making support for environmental protection.", "Moreover, the city should build a big data energy management system to improve the efficiency of energy utilization, perfect the resource pricing mechanism, and innovate in energy consumption patterns.", "Urban services are comprised of a variety of fields, such as traffic, medicine, education, social welfare, culture, and tourism.", "Concerning urban traffic, a city should build a big data traffic application platform that would allow urban citizens to access various kinds of traffic information (including public transport, road condition, taxies, public transport facilities, and road maintenance) in real time.", "The platform would provide various value-added services (e.g., traffic information service and traffic guidance), aid the optimization of traffic planning, and design to boost transportation efficiency and improving the traffic experience.", "The city should set up a unified big data health application platform using collected and integrated population data, electronic health archives, and electronic medical records, and develop a big data application system for health care management and services for public health, medical services, medical insurance, and drug supply and management.", "The city should also encourage enterprises and institutions to develop innovative big data applications for health care, and enhance comprehensive health care services.", "Concerning social benefits, the city should build an integrated urban-rural big data platform for social relief, social welfare, and social security, encourage data exchange and information-sharing between related departments, and support the application of urban big data to the management of employment status and social security funds, monitoring and controlling medical services by insurance authorities, labor oversight, auditing of internal control, and the formulation of policies for human resources and social security policies—as well as the evaluation of their effects—in order to provide highly personalized and targeted services to the public.", "Finally, the city should integrate the digital cultural resources available from digital libraries, archives, museums, art galleries, mass culture sites, and technological museums, thus building a comprehensive big data service platform for cultural propagation.", "To promote tourism, the city should encourage the sharing of tourist information, integrate tourism-related data resources (e.g., public security, traffic, environmental protection, commerce, aviation, postal service, telecommunication, and meteorology), and develop a big data tourism resource pool in cooperation with the main network search engines and online tourist service providers.", "A city should encourage the application of big data to industrial development, cross-border e-commerce, logistics, and technological and knowledge services.", "To support the development of different industrial sectors, the city should take the following measures: build cloud service platforms for key sectors and promote the application of big data resources to research and development (R&D) design, data management, and systematic marketing; implement the Internet of Things cloud project “Cloud + Web + End,” introduce cloud service enterprises, and develop a cloud engineering industry that provides integrated services such as whole-process overall design, equipment manufacturing, software development, system integration, engineering installation, and network operation and maintenance; optimize the statistical standards for various industrial sectors, improve the efficiency of the acquisition and use of the big data resources of the related industrial sectors, tap the industrial value and potential of data resources, and provide a powerful support for the innovation of R&D systems, the reformation of production management modes, and the conceptualization of industrial value chain systems.", "Concerning e-commerce, the city should develop manufacturer-to-consumer (M2C) and manufacturer-to-business (M2B) services, enhance the development of cross-border e-commerce platforms, and build an integrated cross-border e-commerce service system that supports the acquisition, cleaning, integration, analysis, and presentation of data in order to provide a powerful support for cross-border e-commerce platforms as well as basic logistics and warehousing, credit rating, and comprehensive information services for such platforms.", "The city should also develop an e-marketing and price-comparison system oriented to the countries along the “Online Silk Road” to provide a convenient and fast shopping experience to global customers.", "The city should support the development of a logistics industry by building a big data logistics platform, integrate logistics data (e.g., commodities, traffic networks, freight, and goods turnover) under unified standards, and provide optimal transport routes for logistics enterprises through an all-round analysis of vehicles, routes, and commodities, thus improving logistical efficiency.", "Using logistics big data would help the city obtain up-to-date inventory information and dynamic demand information for many types of commodity in a timely manner, enabling the optimization of inventories and warehousing and dynamic allocation adjustments of logistics and warehousing resources.", "The city should promote mass entrepreneurship and innovation by encouraging people to tap into open data resources and explore new technologies and patterns of data mining, analysis, and application.", "People should also promote a close combination of big data development with scientific innovation, technological development, and government and market demand, thus establishing big data-driven innovation and facilitating open and coordinated innovation.", "Urban big data plays a core role in the development of city intelligence, the ideal cut-in point to urban development of China.", "As China's population and economic output are concentrated in cities, successful urban development would signal that the main body of China is well developed.", "Therefore, the promotion of city intelligence has bright prospects in China.", "China's macro environment of industrialization and urbanization and governmental structure are favorable to the development of urban big data.", "The use of urban big data that is successfully administered and opened will promote the development of an urban knowledge-based service industry, create new markets and business opportunities, and further promote the development of city intelligence.", "It is thus imperative that China make full use of its unique advantages to promote the development of city intelligence through urban big data.", "This work was partly supported by the Major Strategic Consulting Projects of Chinese Academy of Engineering (2012-ZD-6 and 2014-ZD-01) and the Key Consulting Project of Chinese Academy of Engineering (2015-XZ-14).", "The authors would like to thank all experts from the above projects for their contributions.", "Compliance with ethics guidelines", "Yunhe Pan, Yun Tian, Xiaolong Liu, Dedao Gu, and Gang Hua declare that they have no conflict of interest or financial conflicts to disclose.", "Urban big data is a massive amount of dynamic and static data generated from the subjects and objects including various urban facilities, organizations, and individuals, which have been being collected and collated by city governments, public institutions, enterprises, and individuals using a new generation information technologies.", "Big data can be shared, integrated, analyzed, and mined to give people a deeper understanding of the status of urban operations and help them make more informed decisions on urban administration with a more scientific approach, thereby optimizing the allocation of urban resources, reducing the operating costs of the urban system, and promoting the safe, efficient, green, harmonious, and intelligent development of the cities as a whole.", "In addition to their general features (e.g., volume, velocity, variety, veracity, and value), urban big data also has the additional features below:", "Hierarchy: For example, electronic medical records are categorized by hospital or region, while medical images are categorized in terms of individual medical devices and hospitals.", "Meanwhile, health data can be categorized in terms of individuals, hospitalized patients, communities, or health and anti-epidemic authorities.", "The hierarchy of urban big data deeply reflects the organizational hierarchy of a city's physical and social systems.", "Integrity: As an urban system evolves, the data coverage of each subsystem becomes increasingly broad.", "In recent years, for example, the inclusion of environmental protection data in China's cities has improved rapidly.", "Due to the rapid improvement of data integrity, urban big data has acquired the capacity to uncover the overall dynamics of urban development increasingly accurately.", "Correlation: Types of urban data are highly correlated with each other.", "For example, information about urban logistics is included not only in the data of logistics enterprises but also in the data of the manufacturing, commercial, and transport industries, and even in the financial industry.", "Such correlations can be used, not only for mutual corroboration, but also for cooperative reasoning and mining rules of cities operation.", "Due to these general and unique features, urban big data must be applied using a new data processing technology—targeted data extraction based on a target-driven method [4].", "The entire data process, from data acquisition to data processing to data modeling, is automated [5,6].", "This occurs as follows: The first step is acquiring and storing the original data, including pattern extraction and filtering the data obtained from the desired data source according to the target requirements and then cleaning and preprocessing the acquired data (i.e., data filling, data optimization, data merging, data normalization, data consistency check, and preliminary organization of diverse data attributes), and establishing the dataset to be processed.", "The second step is processing and analyzing the dataset (including linear analysis, nonlinear analysis, factor analysis, sequential analysis, linear regression, variable curve analysis, and bivariate statistics), and then categorizing the data and analyzing the inter-data and inter-category relationships via the support vector machine (SVM), Naïve Bayes, random forest, and logistic regression.", "The third step is identifying the inherent relationships among the categorized data and uncovering the further patterns, rules, and knowledge via an artificial neural network, genetic algorithm, and cross-media algorithm.", "Finally, the relationships among the variables are explained in an interactive and visual way to express a deeper understanding of the results.", "Urban big data describes the real-time status of various urban elements, including buildings, streets, pipelines, environments, enterprises, finance, commerce, products, markets, logistics, medicine, culture, education, traffic, public order, and population.", "As proposed in Ref. [7], urban big data can be categorized into five types: sensor data on urban infrastructure and moving objects, user data on society and humans, governmental administration data, customer and transaction record data, and arts and humanities data.", "Table 1 lists examples and user groups for the five types.", "Urban big data can be categorized in more than one way.", "In essence, data categorization is not effective without a tree structure, but urban data information is organized using a mesh structure.", "Therefore, urban big data should be categorized according to the data processing method used and the application objective.", "The big data on China's cities is typically categorized using the three methods below.", "The advent of urban big data provides not only a new approach to the in-depth study of urban operations and development [8–11], but also a new opportunity to renew the competitive advantage of cities [12].", "In the context of rapid global informatization, big data has become a vital strategic resource for every city.", "Strengthening urban competitiveness requires that every city make full use of its advantages in scale, quality, and applications, to tap into and unleash the potential value of data resources, and improve the socioeconomic benefits of big data.", "Meanwhile, big data has also become a new driving force of urban economic transformation [13].", "Specifically, big data plays an important role in the following: promoting web-based sharing, intensive integration, and collaborative utilization of production factors; facilitating innovation in the business and circulation modes for production materials, technologies, human resources, and funds; and improving enterprises' core value and strengths.", "In addition, the use of big data constantly gives birth to new business patterns and new economic growth points.", "Last, but not least, big data provides a new way to improve the administrative capacity of governments [14].", "Using big data can reveal the latent relationships beyond the reach of traditional technological methods for identifying correlations among seemingly unrelated knowledge and transform such information into new knowledge—and diagnose and evaluate urban development via qualitative and quantitative analysis.", "Accordingly, big data helps governments improve their data-driven decision-making ability and provide a new means of solving complex social problems.", "In essence, using urban big data to explore the urban mode and urbanization process is to analyze, visualize, and understand urban big data and interpret both structured and unstructured data in order to enhance the dynamic management of urban resources, knowledge creation, and in-depth analysis of the urban mode and urbanization process, the effective participation of urban residents, and reasonable urban planning and scientific analysis of urban policies.", "Fig. 1 shows the relationships among the objectives, methods, and applications of urban big data.", "Originally proposed by IBM in 2008, the “smart city” concept focuses on measurement, interconnection, and intelligence [15], aiming to apply specific information technology (IT) systems to the urban administration toolbox.", "This concept is suitable for the developed countries of Europe and North America whereas urbanization, industrialization, and agricultural modernization have been successfully accomplished.", "Smart cities of the developed countries are developing mainly in the fields of government administration and intelligent services [16,17].", "However, the mayors of China's cities must perform far more administrative functions and are responsible for more things than the mayors of cities in developed countries.", "In addition, China is at the apex of its industrialization, informatization, and urbanization while facing a range of puzzles and problems unique in both quality and quantity.", "As a result, China is following a city intelligence development path different from that of developed countries as shown in Fig. 2.", "Obviously, the development roadmap for smart cities in developed countries is not an appropriate solution for the diverse problems being encountered in China's urban development process.", "Promoting city intelligence is the process of developing intelligent cities with the aim of developing the urban ternary space (comprising urban physical facilities, human society, and urban data) with a scientific approach based on the intelligence consolidated from citizens, enterprises, and governments.", "The key is to artfully dispatch comprehensive urban resources reasonably, optimize urban economic development, urban construction and administration, improve urban development and citizens' lives constantly, and satisfy urban citizens' current and future needs more effectively [2].", "The development of city intelligence is a process from decentralization to centralization and from the surface to the depths.", "An appropriate starting point for building an intelligent city is implementing an intelligent application system based on the existing urban data.", "The subsequent tasks are enhancing the automated datafication of the physical urban infrastructure that gradually integrates and shares data with innovation in the applications of urban big data.", "The aim is to promote the deep development of macro decision-making and micro services and to promote industrial upgrading.", "Therefore, the applications of urban big data serve to advance city intelligence from a local level to a systematic and global level and produce the city intelligence suited to the users' economic, social, and ecological needs.", "As shown in Fig. 3, the development model for city intelligence and urban big data comprises five parts: infrastructural support system, application system, industrial system, index system, and operation assurance system.", "The development process occurs as follows: Massive amounts of structured and unstructured data are generated from urban sources, and consolidated into a unified urban data platform, thus generating an urban foundational and comprehensive database; by correlating, integrating, cleaning, processing, analyzing, mining, and visualizing the massive amounts of data, valuable information is obtained that can reflect the more objective course of events in cities to satisfy the needs of governmental affairs, commerce, and urban administration and improve the capacities for decision-making, knowledge discovery, and process optimization; the transformation and upgrading of other industries are promoted alongside the development of the big data industry (e.g., data acquisition, data analysis, and data exchange), and the development of urban informatization and intelligence is quickened; and indexes suitable for measuring the development effect and level of urban big data are created, and a big data operation assurance mechanism is developed to ensure the stable and reliable operation of the urban big data service architecture.", "Urban big data is derived from interchanging and integrating the data generated during the operations of numerous physical facilities and human activities in a city.", "Using appropriate processing and analysis technologies, this data can be used to extrapolate various complex relationships in the operating status of physical facilities, trends in industrial and economic developments, and the status, relationships, and rules of citizen health, education, science and technology, and culture.", "Therefore, urban big data not only lays down basic information for understanding a whole city, but also plays a core role in promoting city intelligence.", "Fig. 4 shows the infrastructural support model for urban big data.", "The sensing layer detects and acquires urban data via the Internet of Things.", "The network layer focuses on unified network construction and information convergence.", "The data layer collates the large amount of data generated by the Internet of Things and information systems, thus generating an urban public database.", "The platform layer mainly comprises various cloud computing facilities, a public information platform, and a big data analysis and processing platform.", "The key to advancing the multitude of urban interaction and sustainable development of the ternary world that comprises cyber space, physical space, and human space (CPH) uses the big data approach and technology to facilitate the informatization of the urban physical world with the following effects: breaking through information barriers; fostering an open access to the data flow, capital flow, and human resources; scientifically automating and regulating the urban physical facilities; promoting a reasonable and efficient circulation of people, material, capital, and technology; helping to solve the contradictions between urban development and environments, resources, and space; and building new-style future cities to meet the needs of citizens, enterprises, and governments.", "The use of big data is speeding the integration between ITs and diverse industrial sectors, giving birth to new forms of business and further opening up the development space of the IT industry.", "In its development process, city intelligence needs to be continuously improved by reactivating existing data resources and making full use of big data increments.", "Urban big data plays an important role in revealing the essential nature of typical urban diseases, analyzing complex systems, conducting empirical urban research, and sensing the cities collaboratively.", "Using urban big data is also an important starting point for solving various urban problems.", "In urban planning, urban big data provides powerful decision-making support via the mining of information on the natural status (including geography, meteorology, and environments) and societal information (including economy, society, culture, and population) for cities, increasing the scientific approach and foresight of urban administration.", "The applications in terms of urban planning focus on infrastructure construction, traffic administration, public facilities, and public security [18,19].", "City intelligence enables data sharing; the databases of different governmental departments can be interconnected and interoperable at an efficient level.", "Thus, inter-departmental collaboration and governmental efficiency is significantly improved while reducing the administrative costs of governance [20].", "Big data lays the foundation for the prospective “smart” life and allows people to create personalized archives and manage their daily affairs, personal health, routines, shopping, and travel intelligently.", "Furthermore, using big data strengthens the link between public services and personal lives and provides a variety of applications (e.g., information inquiry, content delivery, and mobile payment) for medicine and health, education and training, traffic, and safety.", "In this sense, big data will turn people's simple and planar lives into multi-dimensional ones [21,22] and make city intelligence serve people's livelihoods.", "Urban big data is used extensively to research and solve diverse urban problems, rapidly becoming a vital bridge and means of promoting the development of city intelligence in China.", "By kick starting out the initial stages of “digital city” and “smart city” projects, most Chinese cities have constructed an information network infrastructure, governmental information integration and sharing platforms, and intelligent application systems.", "They have also developed an information economy and accumulated a large quantity of urban data resources, thus laying a firm foundation for the further development of city intelligence.", "However, the rapid development of urban big data is also confronted by various challenges, including: The existing operating mechanisms presents certain difficulties for data integration and sharing; no standard laws or regulation systems for governing information security and sharing have been promulgated; technological innovations lag behind the development of big data; the ambiguous business mode affects the sustainable development of big data; and the shortage of qualified human resources constrains the development of big data." ]

[]

[ "CENG", "COMP", "ENER", "ENGI", "ENVI", "MATE" ]

[ "Demand-side management", "Internet of Things", "LoRa technology", "Low-power wide-area network", "Narrow-band Internet of Things" ]

[ "1500", "1700", "2102", "2200", "2305", "2501" ]

[ "Under intense environmental pressure, the global energy sector is transitioning toward clean and sustainable development.", "The concept of the smart grid has been widely accepted in the last decade as a means of integrating higher percentages of renewables [1,2].", "In 2016, China’s government announced new policies on combining the Internet with smart energy in order to demonstrate new clean energy technologies [3,4].", "The government and the energy industry have recognized that the construction of an energy-Internet backbone via smart grid is the core strategy to promote a clean energy revolution for a new era.", "A clean energy system requests a robust communication infrastructure that can accept greater variation from renewable energy inputs [5].", "From the perspective of control theory, maximizing system observabilities enhances the system controllability.", "To balance a complex energy system, it is therefore necessary to obtain abundant information from both the supply and demand side.", "The information Internet is a reliable tool that can collect information at zero marginal cost.", "Nevertheless, energy systems are still restricted by closed-information environments due to management and technical issues.", "Particularly on the demand side, for example, communication infrastructure is incomplete at the power distribution level [6], and even less communication infrastructure is available for utilization systems at lower voltage levels.", "Despite developments in the smart grid over the last decade, periphery energy networks are still out of the scope of system operators [7].", "Management is not the only problem, as technology also plays a critical role in the issue of demand-side management (DSM).", "The current power automation architecture was developed based on standards to satisfy the particular requirements of centralized generation and transmission systems [8].", "With the rapid integration of distributed energy resources, the current design cannot meet the requirements of the fast changes that are happening on the demand side.", "Meanwhile, the end users do not have the expertise required to operate and maintain such complicated systems.", "Under these circumstances, technical complexity has become a major bottleneck restricting the acceptance of DSM applications such as demand response in the real world [9,10].", "To overcome this barrier, low-power wide-area network (LPWAN) is a new solution in the context of a wireless breakthrough in the communication sector.", "Unlike WiFi and ZigBee, LPWAN enables massive wireless connections covering long distances with minimum power consumption and maintenance [11].", "Two representative technologies of LPWAN are the narrow-band Internet of Things (NB-IoT) [12] and Long Range (LoRa) technology [13].", "The NB-IoT is inherited from cellular communication, and seamlessly works on the existing global system for mobile (GSM) and long term evolution (LTE) networks in licensed frequency bands [14].", "Many telecom operators have been ambitiously working on weaving together city-scale Internet of Things (IoT) networks based on NB-IoT.", "In contrast, LoRa technology operates in the unlicensed frequency band, so that end users are free to build up LoRa gateways that are similar to house-owned WiFi routers.", "Therefore, LoRa technology is perfect for outlying regions without cellular network coverage, or for establishing private networks with specific requirements for quality and security [15].", "LPWAN provides a practical and economical way of establishing IoT networks.", "This paper presents the potential of an Internet of Energy Things (IoET) based on LPWAN as a future communication infrastructure for DSM applications.", "First, we briefly review the technologies of NB-IoT and LoRa as the representatives of LPWAN.", "Next, a new architecture is proposed for the IoET to establish wireless-to-cloud connections between end devices and the cloud computing center.", "Finally, this paper looks forward to the potential of the IoET in various DSM application scenarios.", "LPWAN represents a new trend in the evolution of IoT technologies.", "Unlike 3G/4G or WiFi, these systems do not focus on enabling high data rates per device or on minimizing latency.", "Rather, the key performance metrics defined for LPWAN are energy efficiency, scalability, and coverage.", "Many LPWAN players have come to the market, with the two most widely accepted players being the LoRa and NB-IoT technologies.", "This section briefly reviews the main features of both technologies and compares them with existing telecom and IoT technologies.", "LoRa technology, developed by Semtech, is the most widely used technology for LPWAN in the sub-GHz unlicensed band [16].", "Due to the utilization of unlicensed bands, the LoRa network is open to customers who lack authorization from radio frequency regulators.", "As a result, the LoRa network is easy to deploy over a range of more than several kilometers, and serves customers with minimum investment and maintenance costs.", "LoRa technology has made tremendous improvements to existing technology in order to achieve its target [17,18].", "The first of these is LoRa modulation based on the chirp spread spectrum (CSS) scheme, which uses broadband linear frequency-modulated pulses whose frequency increases or decreases based on the encoded information.", "The Shannon-Hartley theorem indicates that an increase in transmission channel bandwidth is a way to overcome a poor signal-to-noise ratio (SNR).", "CSS, which has been used for radar applications since the 1940s, was chosen for its inherent robustness to channel degradation mechanisms such as multipath fading, the Doppler effect, and in-band jamming interference.", "As a result, the maximum coupling loss (MCL) for the LoRa modulation reaches as high as 148 dB—20 dB greater than that of existing sub-GHz communications—in order to extend the coverage distance to kilometers and increase the capacity of the network.", "LoRa modulation features six spreading factors that result in adaptive data rates.", "This feature enables multiple differently spread signals to be transmitted at the same time on the same frequency channel.", "The other improvement is the optimization of the LoRa network protocol for energy-limited sensors because the uplink traffic usually exceeds the amount of downlink for IoT networks.", "Under this environment, the LoRa technology specification has defined three modes of different data-receiving windows for different application scenarios.", "In addition, data encryption is supported by LoRa technology to ensure channel security by means of AES-128 encrypted key pairs.", "Thus far, LoRa technology has been tested in 56 countries in demonstrations on smart meters, traffic tracking, smart appliances, and smart healthcare [19].", "In the Netherlands, the telecom operator KPN has deployed a LoRa network that covers the entire country, as has SK Telecom in Korea [20].", "In addition, a LoRa Alliance with more than 300 members is collaborating to define an open global standard for secure and carrier-grade LPWAN connectivity representing the different layers of an ecosystem, from chipsets, modules, devices, and gateways to network and application servers.", "NB-IoT is a new narrow-band IoT system built from existing LTE functionalities.", "The technology standard was announced by the 3rd generation partnership project (3GPP) in 2016, which promises to provide improved coverage for a massive number of low-throughput low-cost devices with low device power consumption in delay-tolerant applications.", "NB-IoT technology makes use of narrow-band channels to provide higher sensitivity and long range at the expense of limited data rates—typically below a few hundred bits per second (bps) [21,22].", "The demodulated spectrum is much wider than individual transmissions so that multiple uplinks can occur simultaneously.", "The base station carries the complexity to decode multiple narrow-band channels simultaneously without knowing the exact frequency of these channels.", "The advantages of NB-IoT technology include its enhanced indoor coverage, which is targeted at an MCL of 164 dB, and its ability to connect a massive number of low-throughput devices with an adapted data rate.", "As indicated by the 3GPP guideline, the design objectives of NB-IoT technology include low-cost devices, high coverage (a 20 dB improvement over the general packet radio service (GPRS)), long device battery life (more than 10 years), and massive capacity (more than 52 000 devices per channel per cell).", "Latency is relaxed, although a delay budget of 10 s is the target for exception reports.", "In addition, NB-IoT network supports three deployment operation modes to provide flexibility based on existing cellular infrastructure [23]:", "(1) Acting as a standalone and dedicated carrier.", "In standalone operation, NB-IoT network can be used as a replacement for one or more GSM carriers.", "This allows the efficient re-farming of GSM infrastructure for IoT.", "(2) Acting in-band within the reserved physical resource block (PRB) of a wideband LTE carrier.", "Here, all communication channels are shared between LTE and NB-IoT network, with the possibility of using power spectral density boosting on the NB-IoT PRB.", "(3) Acting as the guard-band of an existing LTE carrier.", "In the guard-band mode of operation, NB-IoT network utilizes new resource blocks within the guard-band of an LTE carrier.", "Compared with LoRa network, NB-IoT network is designed to work seamlessly on existing GSM and LTE networks within the licensed frequency bands, without enormous updates on the existing base stations.", "Due to its efficient utilization of existing cellular networks, many telecom manufacturers, including Huawei, Ericsson, and Nokia, support the standardization of NB-IoT network.", "Thus far, the commercialization of NB-IoT networks has been initiated, with a particular focus on applications for smart transportation, logistic management, smart grids, and smart manufacturers [24–26].", "Telecom giants such as AT&T and China Telecom have announced ambitious plans for using NB-IoT networks to implement coverage in major cities in 2017 [27].", "Before LPWAN, many IoT business applications were run on GPRS networks [28–32].", "GPRS technology is commonly referred to as “2.5G” mobile communication; the subsequent 3G and 4G technologies are targeted to high data rates per device or to minimum latency in order to support the high-quality transmittal of voice, image, and video [33–35].", "Table 1 compares the main features of GPRS and LPWAN technologies regarding the aspects of power consumption, latency, coverage, and data rate.", "End devices in LPWAN are expected to have one tenth of the energy consumption and a 20 dB improvement over GPRS networks.", "In addition, the capacity of GPRS networks is limited by communication channels, whereas both NB-IoT and LoRa networks have optimized utilization of channel composition in order to extend connection capabilities under lower data rates.", "For connections among personal devices, ZigBee [36] and WiFi [37] have dominated the current IoT market.", "These technologies possess different features and performances.", "WiFi benefits from a high data rate and low latency, but its power consumption is much higher than that of ZigBee.", "ZigBee is designed for small-scale projects that need wireless connections, and is used to create personal area networks with small, low-power consumption, such as for home automation, medical device data collection, and other low-power low-data-rate scenarios.", "Although ZigBee has been optimized for IoT networks, its prominent problem is its low coverage distance and device scalability.", "Therefore, WiFi and ZigBee are referred to as “area IoT” in that they can be used to support connections between devices within a limited area [38–40].", "Table 2 compares the communication distance, maximum connection, and data rate among WiFi, ZigBee, and LPWAN.", "LPWAN provides a much greater coverage distance and a higher connection capability for IoT networks.", "Section 2 presented the key features of LPWAN and compared LPWAN with the technologies of cellular telecom (GPRS) and area IoT (ZigBee/WiFi).", "The primary advantages of LPWAN lie in its wide communication coverage and low-power consumption; its disadvantages are its relatively low data rate and the limited computation capability of its end devices.", "A wireless-to-cloud architecture is therefore proposed for the IoET, in order to integrate cloud computing into end devices via LPWAN, as shown in Fig. 1(a).", "As shown in Fig. 1, an IoET enables communication between end devices and the cloud platform via wireless connections.", "Compared with area IoT architecture, shown in Fig. 1(b), the primary distinction is the savings on the area IoT gateway associated with the gateway network layer.", "As a result, the LPWAN-based IoET architecture becomes manageable for both network operators and end users.", "This simplified network topology conveniently extends the integration of sensing and control devices in actual energy systems.", "In addition, the functions of end devices become extendable by the computing capacity provided by the cloud center.", "The three functional layers of the wireless-to-cloud architecture, as shown in Fig. 1(a), are discussed in the following subsections.", "Actual energy-related devices are connected through the remote-sensing and control layer, which is associated with a huge amount of energy sensors, controllers, and embedded computer and wireless communication modules.", "Energy sensors monitor device statuses and send them to the cloud center, while controllers deliver the instructions provided by the cloud center.", "Embedded systems compactly incorporate embedded central processing units (CPUs), memory, periphery devices, and wireless communication modules as the carrier of energy sensors and controllers for machine-to-machine (M2M) communication conversion.", "They also control actions with quality of service (QoS) requirements.", "Therefore, in addition to sensing and control functions, real-time services are provided via embedded systems in order to enhance the sensitivity of communication QoS to wireless transmitting and cloud computing.", "This concept, which is named “fog computing,” fills in the technical gaps of cloud computing [41].", "Distributed energy devices are usually geographically dispersed, making IoT technologies such as ZigBee and WiFi difficult to use.", "Under such scenarios, LPWAN becomes the alternative.", "The LPWAN transmission layer establishes wireless channels between end devices and the cloud platform.", "As representative technologies, NB-IoT and LoRa technologies are suitable for different application scenarios.", "In outlying districts without cellular coverage, LoRa network is the practical choice, as it forms a star-shaped topology around end devices that are served by a single base station (BS).", "In the example illustrated in Fig. 2, a LoRa BS that is set up in a substation in a rural area communicates with distributed photovoltaic (PV) panels.", "In a city with cellular networks, end devices simply connect with the cloud center via NB-IoT cellular network by paying a data fee to telecom companies.", "Fig. 3 shows a scenario in which the cloud center coordinates home appliances and electric vehicles (EVs) dispersed throughout the city via the NB-IoT cellular network.", "For systems with particular security requirements, one solution is to establish a virtual private network with secured channels in an NB-IoT cellular network; another choice is to construct a private LoRa network to physically secure the connections.", "The cloud platform layer serves as the cloud platform that hosts energy applications via data exchange and communication between devices.", "More specifically, the platform corporates the functions of information conversion, integration, and interoperation, as shown in Fig. 4.", "The corresponding functions are described below.", "(1) Protocol parsing.", "An IoET must deal with communications between various end devices that have different protocols.", "It is not realistic to assume that every device understands all the protocols in the network.", "Instead, the protocols are parsed by the cloud platform, other than the field devices.", "Therefore, it is convenient to decouple actual devices and communication protocols under centralized management and maintenance.", "To achieve higher flexibility, the cloud platform also provides a programming interface to enable user-defined protocols for connections with unknown devices.", "(2) M2M communications.", "M2M communication among energy devices is the main feature of an IoET, in order to extend the interoperability of networks between devices.", "Limited by wireless bandwidth, a “subscribe/publish” pattern is suitable for M2M communications.", "Many event-driven middleware technologies have implemented this pattern, which enables almost unlimited device addressing and efficient bandwidth utilization.", "Data encryption is also supported by middleware to enhance communication security.", "(3) Big data storage and analysis.", "Access to a huge number of sensors inevitably results in massive data storage and analysis requirements.", "The cloud platform supports both real-time and historical data access for specific energy system applications.", "Real-time data is accessed by memory database in order to meet the requirements of low latency and high concurrency.", "The historical data is accessed by distributed file systems in order to meet the needs of massive data storage and big data analysis.", "(4) End user interoperations.", "At present, energy systems are restricted to closed-information environments in order to secure management operations.", "However, inflexible information exchanges will make it difficult to address diverse interoperations among various end user roles in the clean energy era.", "The cloud platform extends user interoperations by visualizing the energy devices in the cloud-based pool to permit platform-independent accessing.", "A cloud-based tool is also set up to program the working flow for user-defined operations by editing the graphics, model, and data of visualized energy devices in the platform.", "Based on LPWAN, an IoET enables extensive connections between energy devices at very low cost and without expert knowledge.", "An IoET extends the reach of the energy information system to match the requirements of DSM.", "Many DSM concepts being reported in the literature now become practical once they are supported by IoET infrastructure.", "One of the primary objectives of smart appliances is to reduce energy consumption and energy bills.", "The smart appliance market has attracted considerable attention, from manufacturing to industry; however, an obstacle remains in the form of a lack of appropriate communication channels.", "The current solution is to use ZigBee or WiFi to establish connections between appliances and the cloud platform [42].", "The drawback to this solution is that diverse communication environments usually result in unexpected failures that may challenge end users who lack patience and specific knowledge.", "Another alternative is to set up an independent gateway for appliances.", "However, this solution often results in fragmented applications for different brands and in poor user feedback.", "The NB-IoT-based IoET provides city-scale networks of devices to satisfy the requirements of smart appliances and smart home systems.", "Wireless channels established by NB-IoT networks give smart appliances access to the cloud platform without any particular configurations on the gateway.", "As long as appliances are connected, the cloud platform automatically recognizes them and provides feedback to smart home applications [12].", "Adding an appliance to the cloud platform thus becomes a simple task, and the entire process is completed without the need for expert knowledge.", "Moreover, smart home systems are prompted to carry out management based on the statuses of appliances and on historical data stored in the cloud platform.", "Microgrids and distributed energy systems aim at the high-percentage integration of clean energy and at highly reliable power supply.", "These energy systems are suitable for outlying districts such as islands, highlands, and river valleys, where large-scale power systems are too expensive to construct [43].", "The operation of a distributed energy system usually requires flexible and reliable communication systems.", "However, cable-based communications are impractical in most cases due to their complicated maintenance.", "Furthermore, the communication distances of ZigBee and WiFi are too short to be practical for an energy site [44].", "Therefore, LPWAN-based IoET becomes another option for micro-grids and distributed energy systems.", "Unlike LoRa network, an NB-IoT network must be set up within an existing cellular network.", "Therefore, LoRa technology is a more flexible tool to meet the requirements of outlying districts.", "Since LoRa network works in the unlicensed band, the site owner is allowed to set up a wireless BS to connect energy devices that are dispersed over distances of several kilometers, without authorized permission.", "A star topology is simpler than a more complex network, and is more convenient to maintain without expert skills.", "An active distribution network manages a high percentage of distributed energy resources that are integrated at the distribution and utilization systems.", "With the emergence of distributed generators and storage at the low voltage level, various smart energy devices now request the level of information-sharing that is associated with distribution system operations.", "Nevertheless, it is difficult to extend current information systems to end consumers due to complex field environments and security concerns.", "In particular, utilization networks at the lowest voltage level lack the space and communication channels required to add additional remote devices to enable interoperations with power distribution systems [45,46].", "The current solution is to use GPRS networks operated by telecom companies [47].", "The substantial data bill makes it necessary to use smart meters in order to save measurements at the gateway and send them back to the operation center when inquired for.", "To address this issue, the LPWAN-based IoET provides an economical way to reach consumers at the low voltage level.", "On the one hand, by investing in enterprise-own LoRa networks, the cloud platform receives data generated by energy consumers with no charge from telecom companies.", "On the other hand, bi-directional communication enables the aggregation of energy consumers to participate in demand response.", "Considering the reachability of LPWAN, demand-response capacities are anticipated to be comparable to peak-valley differences and renewable variations.", "Regarding the mobile energy system, EVs have been accepted as the transportation solution of the future in order to reduce fossil fuel use and carbon emissions [48].", "The large-scale integration of EVs will introduce inherent flexibility to the operations of energy systems.", "Current academic studies have demonstrated the effectiveness of EVs in balancing the control of power systems via means such as frequency regulation, peak shaving, and vehicle-to-grid (V2G) operations [49].", "An efficient communication network is a precondition to aggregate dispersed EV charging.", "However, installations of cable networks are impractical in most cases, and coverage of telecom signals is weak in some underground parking lots.", "LPWAN is a general solution for EV communications since the adapted modulation scheme is sensitive to end receivers, even with over 150 dB decay.", "Thus far, both NB-IoT and LoRa technologies have been successfully tested in actual parking lot demonstrations.", "Based on LPWAN, EVs connected to an IoET are aggregated as a storage pool to respond to energy systems and allow greater integration of variable renewables.", "Multiple forms of energy conversion, such as power-to-gas systems, power-to-heat systems, and combined heat and power systems, are integrated into energy hubs for higher energy utilization efficiency [50,51].", "Performing the role of grid nodes, energy hubs are interconnected in the energy distribution network (EDN).", "The EDN is a novel concept of integrating flexibilities in the demand side by interoperating different energies such as electricity, gas, and heat within a local area.", "With highly flexible energy interoperations, EDN can actively respond to variable renewable energy, leading to a larger renewable share in power systems.", "Energy flowing through the heat and gas networks of an EDN creates a harsher environment for sensing and communication than an electricity network.", "To tackle the issue, the ultra-low-power mode of LPWAN enables NB-IoT and LoRa modules to last for several years without battery changing.", "The reachability of IoET collects more details of multi-energy systems, allowing highly efficient interoperations.", "The NB-IoT and LoRa technologies are two representative breakthroughs of wireless LPWAN in the communication sector.", "Compared with GPRS, LPWAN is a better solution for low energy consumption and improved coverage.", "Compared with the area IoT of ZigBee and WiFi, LPWAN enables massive connections covering long distances at the cost of minimum construction work and maintenance.", "Based on LPWAN, an IoET allows extensive connections of energy devices under wireless-to-cloud architecture at very low cost and without expert knowledge.", "The IoET hence extends the reachability of the energy information system to match the requirements of demand-side applications.", "The establishment of an IoET can stimulate the DSM applications of smart appliances and smart home systems, microgrids and distributed energy systems, active distribution networks and aggregated demand response, EVs and aggregated V2G, and EDNs and multi-energy systems.", "In conclusion, the IoET paves the way to aggregate DSM flexibilities on the demand side in order to enable higher integration of renewables in energy systems.", "Yonghua Song, Jin Lin, Ming Tang, and Shufeng Dong declare that they have no conflict of interest or financial conflicts to disclose." ]

[]

[ "BIOC", "MEDI" ]

[]

[ "1311", "2716" ]

[ "Chronic kidney disease (CKD) is a major public health burden and affects nearly 10% of the global population.1", "Reduced estimated glomerular filtration rate (eGFR), a measure of kidney function used to define CKD, is associated with premature cardiovascular disease and mortality, acute kidney injury, and progression to end stage renal disease (ESRD).2", "Although individuals of African and Hispanic descent suffer the largest burden of CKD,3 the largest genome-wide association studies (GWASs) to search for kidney-function loci have been undertaken in populations of European and East Asian ancestry.4–8", "Many of these loci are characterized by common variant association signals that map to large genomic intervals, which contain many possible causal genes for eGFR, thereby limiting understanding of the downstream pathogenesis of CKD.", "To address this challenge, we have undertaken a trans-ethnic meta-analysis of nine GWASs comprising 71,638 individuals from four ancestries (African American, Hispanic, European, and East Asian), each imputed up to the phase 1 integrated (March 2012 release) multi-ethnic reference panel from the 1000 Genomes Project9, from the Continental Origins and Genetic Epidemiology Network (COGENT)-Kidney consortium.", "With these data, we aimed to (1) assess the evidence for heterogeneity in allelic effects on eGFR for lead SNPs at kidney-function loci across ethnic groups; (2) fine-map these loci by taking advantage of high-density imputation and by leveraging differences in the pattern of linkage disequilibrium (LD) between diverse populations to localize “credible sets” of variants driving eGFR association signals; (3) define potential molecular mechanisms through which eGFR association signals at these loci impact kidney function through overlap of credible variants with genomic annotation; and (4) assess possible markers for biological processes that impact kidney function and CKD in humans through targeted experimentation in model organisms.", "All human research was approved by the relevant institutional review boards and conducted according to the Declaration of Helsinki.", "All participants provided written informed consent.", "We performed trans-ethnic fine mapping of each locus in a 1 Mb genomic interval flanking the lead SNP.", "Association summary statistics for each variant in the flanking region were combined across studies with a Bayesian hybrid of fixed- and random-effects meta-analysis, as implemented in MANTRA.22", "MANTRA allows for heterogeneity in allelic effects between ancestry groups arising as a result of differences in the structure of LD between diverse populations by assigning studies to clusters according to a Bayesian partition model of relatedness between them, defined by pairwise genome-wide mean allele frequency differences (Figure S1).", "MANTRA has been demonstrated, both empirically and by simulation, to improve fine-mapping resolution, as compared to either a fixed- or random-effects meta-analysis.22–24", "Variants passing quality control in less than 50% of the total sample size across studies were excluded from the fine-mapping analysis.", "For each locus attaining genome-wide significance in our trans-ethnic meta-analysis, we obtained genomic annotations of all single-nucleotide variants in a 1 Mb interval flanking the lead SNP.", "We utilized the Ensembl Variant Effect Predictor (VEP, version 2.7), based on the Ensembl transcript set (version 69).", "By default, the VEP reports all possible annotations (transcript- and gene-specific) for each variant.", "We therefore prioritized annotations by considering the most severe consequence of all those reported.", "We then calculated the total posterior probability of driving association signals for each consequence across loci.", "We collected genomic annotations from three sources.", "First, we obtained regulatory chromatin states from the Epigenome Roadmap Project26 for 93 cell types after removing five cancer cell lines.", "For each cell type, we pooled enhancer (EnhA and EnhWk) and promoter (TssA and TssFlnk) elements into one annotation.", "Second, we obtained 145 non-redundant DNase I hypersensitivity sites (DHSs) from the ENCODE Project27 by retaining only one dataset for cell types with multiple assayed samples.", "Third, we obtained chromatin immuno-precipitation sequence (ChIP-seq) binding sites for 165 transcription factors: 161 proteins from the ENCODE Project27 and additional factors assayed in primary pancreatic islets.28", "This resulted in a total of 403 annotations for downstream enrichment analyses.", "For each annotation, we considered variants passing quality control and mapping within 1 Mb of the lead SNP attaining genome-wide significance in the trans-ethnic meta-analysis.", "Using fGWAS software, we then tested for the effect of each annotation by using the Bayes factor in favor of association.29", "We included coding exons, 3′ UTRs, 5′ UTRs, and the region within 1 kb upstream of the TSS in the model for each annotation.", "We obtained the estimated effect and 95% confidence interval (CI) from this model and considered an annotation enriched if the 95% CI did not overlap zero.", "Four y1w1 virgin females were mated with two males each of the genotypes y1w1/Y, y1w1/Y; locoEY-P283/TM3 Sb or y1w1/Y; locod06164 in rearing vials on standard cornmeal/yeast/molasses food (prepared in a central kitchen at University of Texas Southwestern Medical Center).", "The y1w1 isogenic control, in which all loci had been previously homozygosed, and to which loco mutants had been backcrossed for six generations, were obtained from Dr. Yongkyu Park (Rutgers New Jersey Medical School).30", "Separately, to obtain highly heterozygous progeny (heterogenic), virgin females from the A.R.R. lab’s wBerlin strain were mated with males of genotypes y1w1/Y, y1w1/Y; locoEY-P283/TM3 Sb or y1w1/Y; locod06164, as above.", "Adults were cleared from rearing vials on rearing day eight.", "Ten female progeny from each vial were collected within 1–3 days of eclosion and placed on food containing various concentrations of added NaCl.", "Each experimental vial contained flies from a single rearing vial.", "The number of dead flies in each vial was counted daily.", "Flies were transferred to fresh medium after day five, and again after day ten for the heterogenic flies.", "For each concentration of experimental medium, 225 g Applied Scientific Jazz-Mix Drosophila Food (Fisher, cat.", "no. AS-153) was added to 500 mL deionized water with constant stirring.", "Flasks were then placed on a hot plate at 350°C with constant stirring and heated to a slow boil (about 20–25 min).", "The heat was then turned off, 4M NaCl was added to achieve varying concentrations of added NaCl, and total volume adjusted to 900 mL with deinonized water.", "Medium was dispensed in 3–4 mL aliquots in polystyrene vials.", "All crosses and assays were performed at room temperature (∼22°C–23°C) and ambient humidity.", "We estimated the effect of the mutations on salt sensitivity by applying a Cox proportional hazards model on the fly survival data.", "The outcome was survival time, and at the end of the follow-up period, all living flies were censored.", "The data for each genetic background (heterogenic or isogenic) and NaCl concentration were analyzed separately.", "We estimated the effect on the hazard ratio of genotype (each mutation versus control as baseline).", "To account for intra-vial correlation, we used robust sandwich variance estimators in a generalized estimating equation (GEE)-like model that treats members of each vial as associated with a single cluster.", "Analyses were performed with the R “survival” package.", "129S6 mice were purchased from Taconic Biosciences and were maintained on a 12 hr light-dark cycle with free access to standard chow and water in the animal facility of the University of Virginia.", "Only male mice at 12 weeks of age were used.", "High-salt diet (HSD, 6% NaCl) in pellets was purchased from Harlan Teklad and administered in place of normal chow for two weeks.", "Experiments were carried out in accordance with local and NIH guidelines.", "To induce CKD, mice were subjected to sub-total nephrectomy (Nx) under 1.5% isoflurane anesthesia, the right kidney was removed, and the upper branch of the two main branches of the left renal artery were ligated to impede blood supply to the upper half of the kidney as previously reported.31", "Renal mRNA was extracted at the end of 2 weeks of HSD, or at 12 weeks after sub-total Nx.", "Real-time RT-PCR was performed as previously described32 with the primers listed in Table S3.", "Fluorescence detection was accomplished with Sybr Green and the iCYcler system (Bio-Rad).", "mRNA expression was normalized against mRNA expression of the Hprt housekeeping gene, and the mean at baseline was used as the reference for determination of relative expression across conditions.", "We aggregated five GWASs of individuals of European ancestry (23,553 individuals from Europe, the USA, and Australia), two GWASs of Hispanic Americans (16,325 individuals from the USA), one GWAS of individuals of East Asian ancestry (23,536 individuals from Japan), and one GWAS of African Americans (8,224 individuals from the USA).", "Study sample characteristics are presented in Table S1.", "Samples were genotyped with a variety of GWAS arrays, and quality control was undertaken within each study (Table S2).", "Sample quality control included exclusions on the basis of genome-wide call rate, extreme heterozygosity, sex discordance, cryptic relatedness, and outlying ethnicity.", "SNP quality control included exclusions on the basis of call rate across samples and extreme deviation from Hardy-Weinberg equilibrium.", "Non-autosomal SNPs were excluded from imputation and association analysis.", "Within each study, the autosomal GWAS genotype scaffold was first pre-phased10,11 with genetic maps from the International HapMap Consortium12 to model recombination rates.", "The scaffold was then imputed up to the phase 1 integrated (March 2012 release) multi-ethnic reference panel from the 1000 Genomes Project9 via IMPUTE211,13 or MaCH/Minimac11 (Table S2).", "Imputed variants were retained for downstream association analyses if they attained established GWAS quality control thresholds:14 IMPUTE2 info ≥ 0.4 or MaCH/Minimac r2 ≥ 0.3.", "Within each study, eGFR was calculated from serum creatinine (mg/dL), with adjustment for age, sex, and ethnicity by means of the four-variable MDRD (modification of diet in renal disease) equation15 to be comparable with published GWASs of kidney function.4–8", "Within each study, we tested association of eGFR with each variant passing quality control in a linear regression framework under an additive dosage model and with adjustment for study-specific covariates to account for confounding due to population structure (Table S2).", "Association summary statistics were subsequently corrected in each study for residual population structure through a first round of genomic control16 where necessary (Table S2).", "Association summary statistics were combined across studies via fixed-effects meta-analysis (inverse-variance weighting) implemented in the GWAMA software.17", "Variants passing quality control in fewer than 50% of the total sample size across studies were excluded from the meta-analysis.", "Association summary statistics from the meta-analysis were then corrected for a second round of genomic control16 (λGC = 1.028).", "Heterogeneity in allelic effects between studies at each variant was assessed by means of Cochran’s Q statistic.18", "We extracted association summary statistics for eGFR from the trans-ethnic meta-analysis for previously reported lead SNPs at established GWAS loci.", "LD, as measured by the correlation coefficient r2, was calculated on the basis of haplotypes in each ancestry group from the 1000 Genomes Project9 via LDlink.19", "To assess the evidence for distinct association signals at each locus attaining nominal significance (pCOND < 10−5, Bonferroni correction for ∼5,000 variants per locus) in our trans-ethnic meta-analysis, we performed conditional analysis in a 1 Mb genomic interval flanking the lead SNP.", "Within each study, we tested association of eGFR with each variant passing quality control in the flanking region in a linear regression framework under an additive dosage model and with adjustment for genotypes at the lead SNP, in addition to other study-specific covariates used in unconditional analysis (Table S2).", "Association summary statistics were subsequently corrected in each study for residual population structure, via the same genomic control16 correction employed for unconditional analysis (Table S2).", "These association summary statistics were combined across studies via fixed-effects meta-analysis (inverse-variance weighting) implemented in GWAMA.17", "Variants passing quality control in less than 50% of the total sample size across studies were excluded from the meta-analysis.", "Association summary statistics from the conditional meta-analysis were then corrected for a second round of genomic control,16 making use of the same adjustment as defined in the unconditional analysis (λGC = 1.028).", "We defined CKD by an eGFR < 60 mL/min/1.73 m2 (calculated with the MDRD equation defined above) and/or incidence of ESRD, if available.", "Any individual who was prospectively initiated on dialysis or received a kidney transplant (self-reported or obtained from medical records or registries) was defined as having ESRD.", "Individuals who did not develop ESRD at follow-up were considered control subjects.", "We considered the lead eGFR SNP identified at each locus attaining genome-wide significance in our trans-ethnic meta-analysis.", "Within each study, we tested association of CKD with each SNP in a logistic regression framework under an additive dosage model and with adjustment for study-specific covariates to account for confounding due to population structure (Table S2).", "Association summary statistics were combined across studies via fixed-effects meta-analysis (sample size and inverse-variance weighting) implemented in METAL20 and GWAMA.17", "We considered the lead eGFR SNP at each locus attaining genome-wide significance in our trans-ethnic meta-analysis.", "We performed a look-up of association summary statistics for eGFR in 13,158 subjects with diabetes (9,197 with type 2 diabetes [T2D] and 3,961 with type 1 diabetes [T1D]) from five studies of individuals of European ancestry from the SUMMIT Consortium.", "Within each study, the outcome variable was defined as the last measured eGFR, calculated with the MDRD equation (defined above).", "Each study was imputed up to the phase 1 integrated (March 2012 release) multi-ethnic reference panel from the 1000 Genomes Project.9", "Estimated allelic effects on eGFR were obtained from a linear mixed model and implemented in EMMAX21 with an empirical genetic relationship matrix, assuming an additive dosage of the minor allele and including sex, age at diabetes onset, and duration of diabetes as covariates.", "Association summary statistics for eGFR were combined across studies via fixed-effects meta-analysis (inverse-variance weighting) implemented in GWAMA.17", "Combined allelic effect estimates across studies were reported for T1D and T2D subjects, both separately and for all diabetic individuals combined.", "Heterogeneity in allelic effects between T1D and T2D subjects at each variant was assessed by means of Cochran’s Q statistic,18 as implemented in GWAMA.17", "For lead SNPs, we tested for a difference in the allelic effect on eGFR in the general population (from our trans-ethnic meta-analysis) and in diabetic indivuduals (combined T1D and T2D from the SUMMIT Consortium) by using a two-sample Z-test.", "We identified 20 loci attaining genome-wide-significant evidence of association with eGFR (p < 5 × 10−8) in trans-ethnic meta-analysis (Table 1, Figure S2).", "These loci have been previously reported in ethnic-specific GWASs of individuals with European and East Asian ancestry4–6,8 (Table S4).", "They include two loci discovered in a recently published meta-analysis of European ancestry GWASs: LRP2 ([MIM: 600073] rs57989581, p = 5.6 × 10−10) and NFATC1 ([MIM: 600489] rs8096658, p = 1.3 × 10−8).", "Previously reported lead SNPs at an additional 21 established kidney-function loci attained nominal evidence of association (p < 0.05) with eGFR, with consistent direction of effect (Table S4).", "As expected, lead SNPs were common across ancestry groups at all 20 loci, with each displaying modest effects on eGFR (Table S5).", "Despite substantial variability in allele frequencies between ancestry groups, we observed no evidence of trans-ethnic heterogeneity in allelic effects on eGFR for any lead SNP (Table 1, Table S5).", "Through conditional analyses (Table S6), we observed no evidence of multiple distinct signals of association for eGFR at any locus (pCOND < 10−5, Bonferroni correction for ∼5,000 variants per locus).", "Taken together, these data are consistent with a single variant driving association signals in each locus; each variant is shared across ancestry groups and has homogeneous effects on eGFR in diverse populations.", "However, we recognize that larger multi-ethnic samples will be required to detect lower frequency, population-specific distinct association signals of modest effect on kidney function.", "We assessed the impact on CKD of lead SNPs at the 20 eGFR loci in a subset of individuals (up to 3,976 cases and 55,904 controls) contributing to our trans-ethnic meta-analysis (Table S7).", "We defined CKD by eGFR < 60 mL/min/1.73 m2 and/or incidence of ESRD.", "For all 20 lead SNPs, the eGFR-decreasing allele was associated with increased risk of CKD.", "Eleven of the lead SNPs demonstrated evidence of association with CKD at nominal significance (p < 0.05), and the strongest signals were observed at UNCX (rs62435145, p = 2.2 × 10−7), ALMS1 ([MIM: 606844] rs7587577, p = 3.1 × 10−6), and PDILT-UMOD ([MIM: 191845] rs77924615, p = 4.0 × 10−6).", "We also investigated the impact of the lead SNPs on eGFR in GWASs of individuals with diabetes for whom there are different mechanisms for loss of renal function, such as diabetic nephropathy.", "We obtained association summary statistics for eGFR in 13,158 subjects of European ancestry with diabetes (9,197 with T2D and 3,961 with T1D) from the SUMMIT Consortium (Table S8).", "Consistent with previous reports,8,33 allelic effects on eGFR in diabetic individuals and our trans-ethnic meta-analysis of individuals from the general population were homogeneous (Figure S3).", "There was nominal evidence of association with eGFR (p < 0.05), with the same direction of effect, at seven loci, and the strongest signals were observed at PDILT-UMOD (p = 6.9 × 10−6), PRKAG2 ([MIM: 602743] p = 0.00013) and NFATC1 (p = 0.00045).", "We next sought to localize variants driving eGFR association signals in each of the 20 loci attaining genome-wide significance in our trans-ethnic meta-analysis.", "We utilized trans-ethnic fine mapping implemented in MANTRA,22 taking advantage of increased sample size and the expectation that patterns of LD vary between diverse populations.", "We derived credible sets of variants25 mapping within 500 kb of the lead SNP at each locus that together account for 99% of the posterior probability (πC) of driving the association signal (Table S9).", "Smaller credible sets, in terms of the number of SNPs they contain, or the genomic interval that they cover, thus correspond to more precise fine-mapping.", "The 99% credible set at the PDILT-UMOD locus included a single variant (rs77924615, πC>0.999), which maps to an intron of PDILT.", "This variant has previously been reported as driving the primary association signal for CKD at the PDILT-UMOD locus through whole-genome sequencing and long-range haplotype imputation into 194,286 Icelandic individuals with serum creatinine measurements.34", "We also observed precise localization, defined by a 99% credible set including no more than five variants (Table S10), at a five additional loci: NFATC1 (two variants, mapping to 0.4 kb), SLC34A1 ([MIM: 182309] two variants, mapping to 0.6 kb), GCKR ([MIM: 600842] three variants, mapping to 11.7 kb), DCDC5-MPPED2 ([MIM: 612321, 600911] four variants, mapping to 27.9 kb), and PIP5K1B ([MIM: 602745] five variants, mapping to 3.5 kb).", "To gain insight into the mechanisms through which association signals at the 20 GWAS loci attaining genome-wide significance in our trans-ethnic meta-analysis impact eGFR, we began by obtaining genomic annotations for all single-nucleotide variants mapping within 500 kb of lead SNPs.", "Across all 20 loci, only 5.4% of the posterior probability of driving association signals was annotated to coding variants (Table S11), the majority of which was accounted for by GCKR p.Pro446Leu (rs1260326, πC = 0.938).", "This missense variant has been shown, functionally, to result in increased de novo triglyceride and cholesterol synthesis and export and decreased plasma glucose concentrations, all of which have been associated with risk of CKD,35,36 making GCKR the likely effector transcript for eGFR at this locus.", "However, outside of the GCKR locus, variants mapping to non-coding sequence accounted for more than 99.4% of the probability of driving eGFR association, suggesting that these signals are most likely to be mediated by effects on gene regulation.", "We next investigated whether genomic annotations of regulatory chromatin state for 93 cell types,26 DHSs for 145 cell types,27 and ChIP-seq binding sites for 165 transcription factors27,28 were predictive of posterior probability of driving association signals across the 20 loci (Figure 1, Table S12).", "We observed significant effects (p < 0.00012, Bonferroni correction for 403 annotations) on posterior probability for variants in kidney DHSs, including adult renal proximal tubular epithelial cells (RPTECs; p = 3.4 × 10−8), renal cortical epithelial cells (HRCEs; p = 4.7 ×10−7), and fetal kidney cells (p = 8.8 × 10−6).", "We also observed significant effects on posterior probability for transcription-factor binding sites, most notably for HDAC8 (p = 1.1 × 10−14).", "Histone deacetylases (HDACs) are involved in kidney function and development,37 and HDAC inhibitors could be promising in the treatment of kidney disease.38", "We repeated our analyses by using fGWAS29 (Figure S4, Table S12) and observed strong correlation in the ranking of enriched annotations (r2 = 0.93).", "These results highlight that variants driving association signals with eGFR are more likely to be co-localized with annotated elements in kidney cells, thereby suggesting that gene regulation in disease-relevant tissues is a likely mechanism by which GWAS loci impact CKD.", "Lead SNPs that, by themselves, accounted for more than 80% of the posterior probability of driving association signals overlapped an enriched annotation at five loci (Table S13).", "In particular, at the SLC34A1 locus, rs35716097 (πC = 0.946) overlapped DHSs in RPTECs and HRCEs, as well as a binding site for HDAC8, while at the NFATC1 locus, rs8096658 (πC = 0.877) overlapped fetal kidney cell DHSs (Figure S5).", "At both of these loci, the lead SNPs were also expression quantitative trait loci (eQTLs) for NFATC1 and RGS14 (MIM: 602513; at the SLC34A1 locus) in multiple tissues (Table S13), highlighting these genes as likely effector transcripts through which eGFR association signals are mediated.", "NFATC1 plays a central role in inducible gene transcription during immune response and is a downstream target of the transplant immunosuppression drug cyclosporine A. RGS14 encodes a member of the regulator of G protein signaling family, which modulates downstream effects of Gα subunits and has unknown function in kidneys.", "To provide insight into the role of NFATC1 and RGS14 (at the SLC34A1 locus) in kidney physiology, we examined the function of ancestral orthologs in Drosophila melanogaster.", "The Drosophila genome encodes a single member of the NFAT family, and a previous report has demonstrated that flies with NFAT loss-of-function mutations have increased salt sensitivity, suggesting a role for this gene in ionic or osmotic regulation.39", "The closest RGS14 ortholog in Drosophila melanogaster is loco, for which reduced expression is associated with longer lifespan and stress resistance.30", "We thus conducted experiments aimed at characterizing a role for loco loss-of-function variants in salt sensitivity.", "We compared survival of two independently derived heterozygous loco mutants (y1w1; locod06164/+ and y1w1; locoEY-P283/+) with isogenic y1w1 controls after supplementing their diet with varying NaCl concentrations for 8 days (Figure 2).", "There was very little mortality of any of the genotypes on non-NaCl-supplemented food, indicating no baseline differences in viability over the time period tested.", "However, we observed significantly improved survival of the heterozygous loco mutants over controls on NaCl-supplemented food (Figure 2, Table S14), thereby indicating a role for this gene in resistance to salt stress.", "To exclude the effects of inbreeding depression on our findings, we also repeated our experiments with the same strains on a heterogenic background.", "As expected, the hybrid heterogenic strains were less salt susceptible than the isogenic strains, but the loco mutants remained salt-resistant when compared to controls of a similar genetic background (Figure 2, Table S14).", "To further investigate the role of NFATC1 and RGS14 in kidney function, we used the 129S6 mouse strain that is salt-sensitive31 and susceptible to glomerulosclerosis.40", "We compared the renal mRNA expression of Nfatc1 and Rgs14 at baseline versus (1) after a 2-week exposure to high-salt diet and (2) at 12 weeks after CKD induced by sub-total nephrectomy.", "Compared to baseline condition, Rgs14 was significantly decreased (∼75%, p = 0.01) during high-salt exposure (Figure 3).", "In the CKD model, Rgs14 expression was also reduced and approached statistical significance (p = 0.06).", "The renal mRNA expression of Nfatc1 was also significantly decreased (∼50%, p = 0.03) during high-salt exposure and trended down in CKD (p = 0.31).", "Although we cannot establish cause and effect, these data illustrate that the expression of both genes is altered during disease states.", "We have undertaken a trans-ethnic meta-analysis of GWASs of eGFR, supplemented by imputation up to the phase 1 integrated (March 2012 release) multi-ethnic reference panel from the 1000 Genomes Project.9", "With these high-density imputed data, we identified 20 loci at genome-wide significance for eGFR through trans-ethnic meta-analysis.", "Despite improved coverage of low-frequency variation offered by high-density imputation, lead SNPs were common across ancestry groups at all 20 of these kidney-function loci.", "There was also minimal evidence of trans-ethnic heterogeneity in allelic effects on eGFR at lead SNPs at kidney-function loci, thereby arguing against the “synthetic association” hypothesis.41", "It is highly unlikely that eGFR association signals at these kidney-function loci reflect unobserved lower frequency causal alleles with larger effects because (1) rare variants are unlikely to have arisen before human population migration out of Africa and thus are not anticipated to be widely shared across diverse populations9,42 and (2) LD with these variants is expected to be highly variable between ethnicities.", "Our conditional analyses did not provide evidence for multiple distinct eGFR association signals, which is consistent with a single causal variant at each of the 20 eGFR loci.", "However, we recognize that conditional analyses evaluate the evidence for residual association at the locus that cannot be ascribed to the lead SNP and do not provide a formal framework to test for the presence of multiple causal variants, for example, that are in strong LD with each other and reside on the same haplotype.", "Furthermore, larger sample sizes will be required to detect distinct association signals defined by common variants of modest effect or low-frequency variants that might be specific to particular ethnic groups.", "As with most previous GWASs of kidney function, our study was limited to a single measure of eGFR for each participant.", "We also did not adjust for diabetes or hypertension in our analyses given that these conditions are potential mediators or modifiers of the SNP-eGFR associations.", "However, despite ethnic differences in the prevalence of these conditions, we observed no evidence of heterogeneity in allelic effects on eGFR between ancestry groups.", "Exploration of context-dependent effects should be considered in future studies, for example, by using gene-environment interaction or mediation analyses.", "Given our observation that eGFR association signals are shared across ancestry groups, we next sought to take advantage of the differential patterns of LD across diverse populations to fine-map kidney-function loci.", "Credible-set variants mapped predominantly to non-coding sequence, suggesting that eGFR association signals are most likely to be mediated by effects on gene regulation, in agreement with previous reports for other complex human traits.43–45", "Through integration of genetic fine-mapping data with information from regulatory annotation resources, we have demonstrated significant enrichment of variants driving eGFR association signals with DHSs in multiple kidney cell types.", "Overlap with these enriched annotations could be used as a prior model for eGFR association signals, genome-wide, to improve power for discovery of additional kidney-function loci and further enhance trans-ethnic fine-mapping efforts.46", "Lead SNPs at kidney-function loci overlapping enriched annotations included eQTL for NFATC1 and RGS14 (at the SLC34A1 locus) in multiple tissues, pointing to likely effector transcripts through which these eGFR association signals are mediated.", "We have established that loss-of-function mutations in ancestral orthologs of both genes in Drosophila melanogaster are associated with response to salt stress.", "Although salt sensitivity has not been directly correlated with variation in eGFR in humans, it has been associated with albuminuria,47,48 elevated creatinine,48 and the subsequent development of hypertension,49 suggesting the relevance of this trait to kidney function.", "Indeed, in animal models, salt sensitivity is tightly linked with a blunted tubuloglomerular feedback (TGF) or impaired increase in GFR after salt loading.50–53", "Consistent with this, we demonstrated that renal mRNA expression of Nfatc1 and Rgs14 in a salt-sensitive mouse model was reduced after exposure to a high-salt diet and induced CKD.", "In parallel with the findings in Drosophila melanogaster, these results are consistent with the hypothesis that the capacity to reduce expression of Rgs14 and Nfatc1 determines the extent of the response to stress.", "Another possible mechanism suggested by our results in Drosophila is a role for oxidative stress, to which RGS14 ortholog mutants are resistant,30 and which has been implicated in mammalian salt sensitivity.54,55", "Establishing the functional role of these genes in salt sensitivity, TGF, GFR, oxidative stress, and CKD will require targeted in vivo studies using knockout and/or transgenic mouse models.", "In conclusion, our study demonstrates the utility of trans-ethnic fine mapping through integration of GWASs of diverse populations with genomic annotation from relevant tissues to define molecular mechanisms by which association signals exert their effect, thereby offering an exciting opportunity to elucidate the pathophysiology of complex human diseases." ]

[]

[ "COMP", "ENGI" ]

[ "Biometrics", "Convolutional neural network", "Deep learning", "Pig face recognition" ]

[ "1700", "2200" ]

[ "The need for on farm identification of individual animals has become more pressing in recent years as sustainable intensification has become commonplace, and the ability to monitor inputs to, and outputs of each animal is increasingly desired.", "The major method of livestock identification is via passive Radio Frequency IDentification (RFID) tags.", "These low-cost tags are commonly fitted to the animals’ ears through piercing – a time consuming and distressing activity for the animal.", "They also have a limited range (even long range readers state a maximum distance of 120 cm) at which they can be activated and read successfully, and multiple tags cannot be read concurrently.", "Even fitting two tags per pig (to improve the chance of a successful reading) was found to only identify the animal at close range with an accuracy of 88.6% [1].", "Common elements in the farm environment can also be detrimental to the antenna's effectiveness.", "Metal apparatus and tag readers from other equipment (e.g. shedding gates or weigh scales) can reduce the range further and interference can prevent some equipment from functioning at all.", "Human face recognition has been an active area of research for at least five decades [2].", "From geometric feature matching to holistic methods in the 1990s [3,4], the recent trend of using deep networks has advanced the state-of-the-art to near human level performance [5,6].", "It is commonly used for non-intrusive access control and monitoring/surveillance purposes, and as such represents a potentially useful research area to apply to the problem of pig identification.", "Although there has been related work to automatically identify behaviours of pigs [7] and feeding/standing of cattle [8–10], biometrics on cattle [11–14], sheep [15] and canines [16] showing promising results, to date there has been very little research into using a pig face as a biometric, although [17] show some preliminary results of applying the Eigenfaces technique to pigs and achieve a recognition performance of 77% on 10 pigs using the full manually cropped face.", "They reported better results for smaller regions (i.e. the nose, or the eyes), but this relied on further manual segmentation of the regions so is not very applicable to an on-farm system.", "They also only collected 16 sequential image frames per pig, so the generalisation of such a system to different environmental conditions when imaging the same pigs is unknown.", "This paper presents the results of three face recognition methods applied to a dataset of pig faces that have been captured on a farm under natural conditions: Fisherfaces [4], transfer learning using the pre-trained VGG-Face model [6] and our own convolutional neural network which has been trained using our own dataset captured using an off the shelf web camera at the drinker in a pen.", "This represents a machine vision application in a challenging, poorly structured environment and even though the pigs are technically under cover, ie in a shed, the subjects position and pose as well as other aspects, such as the lighting, expression, contamination from dirt, etc., are relatively uncontrolled and highly variable.", "We demonstrate the efficacy of the system on recognising unconstrained, and un-preprocessed images of pig faces and present an analysis of those features and activation areas which our system has learned in response to training.", "The rest of the paper is organised as follows: Section 2 outlines the data collection methodology, the video preprocessing approach to remove very similar frames, and the implementation details.", "Section 3 gives the background to the chosen approaches before the results being given in Section 4.", "A discussion follows which puts these results into context together with limitations and suggestions for future work.", "This section describes the data capture, data cleaning and implementation details.", "An overview of the processing pipeline can be seen in Fig. 1.", "The pigs were Large White × Landrace × Hampshire breed, approximately four months old and housed at SRUC's research farm (Midlothian, Scotland).", "The pigs were filmed using a Sogatel USB2.0 webcam, with VGA resolution (640 × 480 px) at 30 frames per second.", "The camera was connected to a Dell Precision laptop running “iSpy Connect” software to allow motion-detection capture of the pigs each time they voluntarily approached the drinker.", "The camera was positioned behind the drinking nipple as shown in Fig. 2.", "A Manfrotto universal clamp and articulated arm were used to mount the camera to the pen frame at sufficient distance to ensure it was out of reach but close enough that the bars did not obscure the pigs’ face as they drank.", "Access to the drinker was altered slightly through addition of shoulder bars which help to keep the pig face-on to the camera and other pigs from being in frame.", "No other changes were made to the drinker and the experiment was approved by the SRUC's Animal Ethics Committee.", "Data of 10 pigs were collected in 2 sessions (31/03/2017 and 03/04/2017).", "The camera was left running unattended and manually labelled afterwards in order to create the training and test data required.", "As can be seen in the images, the pigs have been spray-painted to aid manual identification, and this is not required by the automated system itself.", "It should also be noted that some care was taken to mount the camera in such a way as to ensure that direct sunlight did not fall on the pigs’ faces at the drinker as this led to saturated images.", "Other than this, natural variation in lighting levels was handled automatically by the camera.", "Examples of the pigs can be seen in Fig. 3.", "Each image is compared to subsequent images until a sufficient difference (an empirically determined threshold) is found (Fig. 4).", "The number of raw images for each pig compared with only dissimilar images can be seen in Table 1.", "The convolutional neural network was written using available libraries in Python3.5 (Keras2.0, scikit-learn0.18.2, tensorflow-gpu-1.1.0).", "All code has been run on a desktop computer with an Intel i5 processor, 16 GB RAM using Windows7 64-bit, and an NVidia Titan-X 12GB graphics card with Maxwell Architecture.", "Training our network over 500 epochs takes approximately 5 min, and results of the model from the best performing epoch on the test data is used.", "The pretrained VGG-face model was loaded using matconvnet-1.0-beta13 (a Matlab Toolbox for CNNs), and Matlab's default Linear SVM implementation was used in Matlab 2017a.", "This application was also used for the Fisherface implementation and uses N − M coefficients for PCA, to generate M − 1 LDA coefficients as per the original formulation [4], where N is the number of observations and M is the number of classes.", "This section presents the algorithms and neural network architectures used for the identification of individual animals.", "Due to the abundance of work in human face recognition [19], it is logical to apply well proven methods from that area to explore the feasibility of pig face recognition.", "In particular we use two approaches: a benchmark method known as Fisherfaces and a deep learning approach which we split into two formulations: a deep pre-trained convolutional network model that we classify using a Support Vector Machine (SVM) and our own convolutional network which uses a fully connected layer for classification.", "Fisherfaces uses a combination of Principal Component Analysis (PCA) with Fisher's Linear Discriminant (FLD).", "FLD is named after Robert Fisher who developed the technique for taxonomic classification [20].", "The key aspect to this technique is that it uses labelled data and seeks to minimise intra-class scatter and maximise inter-class scatter.", "The concept of clusters is important in FLD, and ideally the cluster for a given label (or class) is compact (small intra-class scatter) and distant from other clusters (large inter-class scatter).", "This lends itself well to face recognition as faces are labelled as being that of a certain person.", "A major assumption here is that the intra-class scatter matrix is non-singular However, when the number of variables is far greater than the number of classes – which is the case when used for face recognition (i.e. the number of pixels is far greater than the number of identities), the likelihood of the matrix being singular is extremely high.", "To overcome this, Belhumeur et al. use PCA on the image set as a means of reducing the dimensionality first before projecting into FLD space.", "It is this additional step which allows them to identify their approach as Fisherfaces.", "In their paper, Belhumeur et al. compare performance of PCA (Eigenfaces) and Fisherfaces and report far better results for Fisherfaces under varying illumination and expression (error rates half that of any other method and a third of Eigenface).", "Although this work is over 20 years old, it is still prominently used as a benchmark method due to its effectiveness, hence its inclusion in this paper.", "Neural networks have been an active area of research for many decades due to their theoretical ability to model any relationship between input and output, linear or non-linear, provided with sufficient data from which to generalise.", "As far back as 1980, Fukushima [21] proposed an architecture based on the human visual receptive fields that he named the Neocognitron.", "It described alternating layers that convolved and sub-sampled an input image.", "This architecture inspired the development of LeNet-5 [22], a 7-layer convolutional neural network (CNN) which recognised hand written digits.", "However, due to the large numbers of trainable parameters needed, it could only operate on 32 × 32 px images.", "With the growth in low cost, powerful graphics cards, it is now possible to design and train far deeper networks on desktop machines.", "For example an NVidia Corporation Titan-X card (similar to that used in this study) contains 3072 computing cores and 12 GB of onboard RAM.", "In recent years deep learning and convolutional networks have been applied in a wide range of both research areas and also in industrial settings [5,23,24] with the first reported use for face recognition by Lawrence et al. [25].", "Deep learning, and CNN approaches in particular, combine both the feature extraction and classification stages of classical pattern recognition, by propagating the training of a fully connected classifier back through the convolutional layers in order to select the best features.", "A popular publicly available model that offers close to state-of-the-art performance on the Labelled Faces in the Wild dataset [26] is called VGG-Face which is based on the VGG-Very-Deep-16 CNN described in [6] and contains 37 layers.", "The model has been trained on 2.5 million images of 2400 people.", "In doing so it will have learned discriminative features of the faces.", "We propose to use this pre-trained model to see whether we can leverage the power it shows in humans on pig faces.", "In order to do this we need to either retrain the classification layers at the end of the network with the new classes, or feed the 4096 features that are output from the final convolutional layer into a traditional classifier.", "We were unable to get a new classification layer to converge using the pig photographs (presumably because there is an insufficient number given the depth of the network), and so settled for using a Support Vector Machine (SVM) to discriminate between the 4096 features to identify individuals.", "To train a network of this depth not only takes a great deal of computing power, but also a very large amount of training data.", "In this study we have a limited amount of data (on average 150 images per pig), and so are limited in how deep we can make our own network.", "In order to boost the amount of data we perform small manipulations to increase the variance and also improve the robustness of the network (shifting the images by up to 64 pixels in the x/y plane, flipping the images horizontally and rotating up to 30°).", "The colour jpg images are 640 × 480 pixels in size and these are resized to 64 × 64 pixels (this was determined empirically to give an acceptable compromise of good performance vs speed of processing), before being fed into the first layer of the network.", "The target for each image is the identity of the pig (1–10), and these are converted to a ‘binary’ representation of 10 output nodes, where a ‘1’ in the relevant index corresponds to that identity e.g. ‘0100000000’ corresponds to Pig 2.", "In both networks, the data is split 60:40 into train and test partitions, and the results in Section 4 report performance on the test partition.", "The network we train consists of six convolutional layers, with alternating dropout and max-pooling layers in between.", "The purpose of these is to aid convergence by preventing local minima, and providing scale/location invariance.", "The classification layer consists of three fully connected layers with the final layer containing 10 outputs – corresponding to each pig.", "The architecture is shown in Fig. 5.", "Using the dissimilar dataset, the results on the test partition using the Fisherface, VGG-face pre-trained model and our own network can be seen in Table 2.", "Accuracy is given as the number of correctly identified images as a percentage of the whole dataset.", "The False Positive (FP) rate is the number of pigs that are incorrectly identified as a particular pig as a percentage of total identifications of that pig ie how many times an imposter is identified as a certain pig e.g. for pig 1, if pig 4 is identified as pig 1, then this is a false positive.", "Likewise, the False Negative (FN) rate is the number of times a particular pig is wrongly identified as another pig as a percentage of total identifications of that pig e.g. for pig 1, if pig 1 is identified as pig 4, then this would be a false negative.", "We essentially replicate the previous findings of [17] using Fisherfaces instead of Eigenfaces and achieve an accuracy of 78.4%.", "This is very similar to their reported accuracy of 77% on whole faces.", "The human face recognition literature tends to report better results for Fisherfaces over Eigenfaces and one reason we are not seeing a marked improvement may be due to their results reporting on very similar testing and training images, together with the manual cropping of the pig faces which they performed.", "However, the Fisherface results are convincingly outperformed by both deep learning approaches.", "Extremely high recognition results are seen using the CNNs with our own performing the best (just under 97%).", "The entire test set of 622 images can be identified in less than 1 s (0.002 s per image) once the network has been trained, meaning that the system can be run on a standard computer to identify animals in real time.", "The FP rates shown in Table 2 show the number of false identifications that take place.", "These are important metrics in terms of how practical the system might be in real-world use.", "The Fisherface and VGG-Face + SVM approaches show the highest FP and FN rates with over 1 in 4 pigs potentially being misclassified in the case of Fisherfaces.", "This rate is very likely too high for on farm use.", "Our proposed CNN approach does better at 5.8%, meaning that just over 1 in 17 might be misidentified.", "However, much of this error comes from the confusion between pigs 2 and 3 which have high false positive rates of 11% and 25% respectively.", "It is very interesting that the VGG-Face pre-trained model performs as well as it does given that it has only ever been trained on human faces.", "This indicates that many of the same features that the network has learned to be useful for discriminating human faces are also useful for discriminating pig faces.", "This hints toward how a trained network for faces in one species may be transferable to other species, at least in so far as using it to bootstrap a new model for a new species using a reduced training set.", "This bootstrapping approach will be the subject of future work.", "Fig. 6 shows the normalised confusion matrix for the results of our CNN.", "The pig which is misidentified the most is Pig 2 (67% accuracy), which is confused for Pig 3 in 21% of cases and Pig 6 in 12% of cases.", "Interestingly, referring to Fig. 3, these three pigs do not have pigmentation/markings on their faces (unlike the other seven pigs).", "What is uncertain is why there should be asymmetry in the confusion matrix i.e. Pigs 3 and 6 have recognition accuracies of 96% and 94% respectively, but Pig 2 only has 67%.", "The number of samples for Pig 2 is only slightly less than for Pig 3 (58 vs 66), although these two are the only two with less than 100 samples, so this could be a contributory factor.", "Indeed as discussed above, the FP rate for these two pigs is also far higher than the others; another indication that the number of samples is probably too low – something which will be addressed in further studies by increasing the size of the dataset.", "The Receiver Operating Characteristic (ROC) plot shown in Fig. 7 demonstrate the robustness of the system with extremely high areas under each curve indicating high accuracy with very low false positive rates.", "As expected, Pig 2 generates the worst performance, with four of the pigs (1, 7, 8 and 10) being correctly identified in every instance.", "In summary, the results indicate that it is possible to accurately recognise individual pigs non-invasively from a relatively unconstrained scene (i.e. the pig can present their faces in many poses).", "On average, using our own trained CNN we achieve an accuracy of 96.7% on 1553 images of 10 pigs.", "This outperforms a standard face recognition technique frequently used in automated human face recognition known as Fisherfaces and also a pre-trained human face recognition CNN, VGG-face.", "A frequently cited downside of using neural networks is that understanding exactly what they are modelling is very difficult.", "This is further compounded using deep networks.", "However, this has begun attracting a great deal of research interest, especially for CNNs to ensure that the “attention” of the network is focussed on real, discriminative features of the animal itself, rather than other parts of the image which may happen to contain discriminative information (e.g. a class label, a time stamp imprinted onto the image or even the spray-marked region of the pig).", "In [27], Selvaraju et al. present a method of producing a course localization map for a given class that the network has been trained on (Gradient-weighted Class Activated Mapping, Grad-CAM).", "Essentially this shows what regions of an input image are activating the network for a given class.", "We implement this approach and show some representative examples in Fig. 8.", "By examining Fig. 8 it is possible to see that the network learns to discriminate the pigs mainly from three regions: the snout itself and the region above it containing wrinkles, the top of the head (where the markings are most prevalent) and, to a lesser extent, the eye regions.", "Unfortunately it does not appear to give any insight into performance variations i.e. it does not tell us anything obvious about why the recognition performance on Pig 2 is poor, nor why the performance is at 100% for pigs 1, 7, 8 and 10.", "One of the main aims of this work is to assess the feasibility of on-farm livestock recognition systems.", "Although the number of animals in this study (10) is relatively small and the images have not been taken over a long enough period to see any large changes in the animal, we have shown that with only minor changes to the arrangement of the drinker, and the addition of a webcam, extremely accurate recognition performance is possible.", "A drawback with the current arrangement is that the camera is mounted some distance behind the drinker, which in practice would locate it in an adjacent pen.", "The reason for choosing this location was to maximise the opportunity of getting frontal faces of the pigs while they drank.", "From the results presented in this paper, and specifically the regions which the network uses to identify the individuals, it is reasonable to hypothesise that an entirely overhead view is likely to provide sufficiently good data without the need to put the camera in an adjacent pen.", "This could be done in conjunction with other technology such as a 3D camera which would be capable of estimating the volume and hence weight of the animal.", "While this project specifically looked at pigs there is reason to believe that such an approach may be used for other common livestock breeds e.g. cows and sheep, where precision non-invasive monitoring is required.", "While no effort mass made to ensure that the pigs’ face were not dirty in this study, none appeared to become overly muddy.", "However this need not necessarily become too much of an issue (within reason); as long as the training data shows sufficient variance of the animals with dirty faces, reliable descriptors will be extracted that do not rely on these features.", "Other factors that may affect the recognition over the duration the animal is housed are: how the animals’ face ages, how big it grows and in particular relevance to pigs, the presence of tear stains beneath the eyes which may also be used as a welfare indicator [28].", "These can have a marked effect on the appearance of the animal, and future work will look to quantify and mitigate the effects.", "In terms of an on-farm solution, one could retrain the network on a daily basis, using recently taken images.", "Assuming that any changes are slow (e.g. which seems reasonable in the case of growth/ageing) the animal will be correctly recognised and these incremental changes will become incorporated into the network's model in an unsupervised manner.", "The effects from more instantaneous changes to an animals’ appearance due to dirt are unknown but will be the focus of further work.", "It would also be beneficial to measure the effectiveness on a large number of pigs that do not have any markings and see whether the system can still learn to distinguish them based on different features.", "We might expect the VGG-face model to perform well given data of this sort as the data are arguably more similar to human faces.", "If this work shows that the proposed system is not scalable to large numbers of pigs, a potential solution would be to consider using the system at a “pen-level” rather than across the entire farm.", "This would ensure fewer pigs for each system, but still provide a feasible method of identification of individual pigs across the farm.", "Future work will investigate these effects, and test the scalability limits of the system.", "We have presented a non-invasive imaging system capable of recognising individual pigs from their faces at a minimally adjusted drinker in their pen.", "The system uses data from an unconstrained, commercial farm environment where the animals’ position and pose as well as other aspects, such as the lighting and dirt, are relatively uncontrolled and highly variable.", "Once trained it can operate in real time and accurately identify pigs with a high accuracy (96.7%).", "Although our limited dataset consists of 1553 images of 10 pigs, the excellent results presented here demonstrate the potential of our approach, and further work will look to use a more convenient overhead viewpoint and investigate the effects of more changeable aspects of the pig's appearance (e.g. ageing, dirt, tear staining).", "The study has implications for intensive livestock practices globally, allowing identification of animals without the need for RFID tags, for the purposes of welfare and growth monitoring." ]

[ "Face recognition for humans is a well-studied and proven biometric.", "Precision agriculture requires individual animals to be identified reliably.", "Current methods e.g. RFID have shortcomings (range, distressing to fit).", "We adapt approaches from human literature to on farm pig-face recognition.", "Accuracy of 96.7% is achieved on 1553 images of 10 pigs using our own CNN." ]

[ "MEDI" ]

[]

[ "2700" ]

[ "Type 1 diabetes represents 5–10% of cases with diabetes worldwide, and is presently incurable.1", "Achievement of recommended glycaemic control remains challenging across all age groups,2 in part because tight glycaemic control increases the risk of hypoglycaemia.3,4", "Over the past decade, considerable progress has been made in the development of closed-loop insulin delivery systems (the artificial pancreas), which couple continuous glucose monitoring and algorithm-directed insulin pump delivery.5", "Hybrid closed-loop systems are characterised by automated insulin delivery, apart from when the user administers insulin boosts at meal time.", "In 2017, the first hybrid closed-loop system entered clinical use on the basis of a pivotal safety non-randomised, single-arm trial of a hybrid closed-loop system in patients with type 1 diabetes.6", "Evidence before this study", "We searched PubMed for articles published up to June 12, 2018, using the terms (“artificial pancreas” OR “closed-loop”) AND (“type 1 diabetes mellitus” OR “diabetes”) AND (“outpatient” OR “home”) AND (“randomised” OR “randomised controlled trial”), for reports of randomised controlled trials published in English only.", "We identified 27 randomised trials that tested automated or semiautomated glucose control outside hospital settings.", "Outpatient use of automated insulin delivery systems is associated with an increased percentage of time during which sensor glucose is within the near normoglycaemic range, and reduced hyperglycaemia and hypoglycaemia, while modestly reducing glycated haemoglobin (HbA1c) in studies that were of long enough duration to report results for HbA1c.", "13 of the 27 trials assessed day-and-night use of closed-loop systems.", "Seven of these 13 trials tested insulin-only systems, of which one trial assessed long-term use (≥12 weeks) of 24 h per day, 7 days per week, closed-loop.", "However, this study was done in adults only, and the participant number was small (n=33).", "Added value of this study", "To our knowledge, this multinational, multicentre study is the largest randomised study of closed-loop use in outpatient settings so far.", "It is also the longest randomised outpatient study of 24 h per day, 7 days per week, closed-loop use in children as young as 6 years and older.", "We showed that compared with sensor-augmented insulin pump therapy, day-and-night hybrid closed-loop insulin delivery significantly improved the percentage of time spent within the glucose target range (3·9–10·0 mmol/L) and mean glucose concentrations, and led to a significant decrease in HbA1c while reducing hyperglycaemia and hypoglycaemia in a mixed population with suboptimally controlled type 1 diabetes.", "These improvements were seen irrespective of age.", "Implications of all the available evidence", "The use of day-and-night hybrid closed-loop insulin delivery improves glycaemic control while reducing the risk of hypoglycaemia in adults, adolescents, and children with type 1 diabetes compared with conventional pump therapy or sensor-augmented pump therapy.", "Results from our study together with those from previous studies support the adoption of closed-loop technology in clinical practice across all age groups.", "Two meta-analyses of randomised trials reported that outpatient use of closed-loop systems increases the time sensor-measured glucose is near-normoglycaemia, and reduces the risk of hyperglycaemia and hypoglycaemia.7,8", "However, most trials had a small sample size, a short intervention period, and were done predominantly in adults.", "Only two studies9,10 reported glycated haemoglobin [HbA1c] outcomes.", "This implies that effectiveness assessments from larger and appropriately designed and powered clinical trials are needed to support reimbursement and wider adoption of hybrid closed-loop systems.", "In the present multicentre randomised trial, we hypothesised that the use of a hybrid closed-loop system improves glucose control and reduces the risk of hypoglycaemia compared with sensor-augmented pump therapy in individuals with suboptimally controlled type 1 diabetes.", "Hybrid closed-loop was applied over 12 weeks in a mixed population, including adults, adolescents, and children aged 6 years and older.", "We studied people with suboptimally controlled type 1 diabetes because we anticipated that this population might accrue particular benefits subject to satisfactory compliance and regular closed-loop use.", "From May 12, 2016, to Nov 17, 2017, 114 individuals were screened.", "Nine participants did not meet inclusion criteria following screening assessment, and three withdrew before entering the run-in period.", "Another 15 participants did not successfully complete the run-in period.", "One participant was withdrawn after run-in because of non-compliance.", "86 eligible participants were randomly assigned to treatment (figure 1).", "46 participants were assigned to the closed-loop group and 40 participants to the control group.", "Of those enrolled, 44 participants were aged 22 years or older, 19 were aged 13–21 years, and 33 were aged 6–12 years.", "Baseline characteristics are summarised in table 1 (breakdown in the appendix).", "After the run-in period, no participant failed the competency assessment and no participant withdrew post randomisation.", "Primary and secondary endpoints are summarised in table 2.", "24-h sensor glucose profiles are shown in figure 2.", "The primary endpoint, the proportion of time sensor glucose was within the target range of 3·9–10·0 mmol/L, was 10·8 percentage points higher (95% CI 8·2–13·5; p<0·0001) in the closed-loop group (65%, SD 8) than in the control group (54%, SD 9).", "Improvements in time within target range were present in all three age groups (<13 years, 13–21 years, ≥22 years), in both sexes, and for both high and low baseline HbA1c (appendix), with the majority of greater improvements present in participants with high baseline HbA1c (post-hoc analysis, appendix).", "All participants in the closed-loop group had an improvement in percentage of time spent with glucose concentrations in target range compared with run-in period (appendix).", "A consistent difference of 10–15 percentage points occurred between the two groups across the whole range of time in range values, and a difference of nearly 20 percentage points among users with the highest time in range in the two groups (figure 3).", "In both groups, HbA1c concentration was reduced from screening (closed-loop 8·3% [SD 0·6], 68 mmol/mol [SD 7]; control 8·2% [SD 0·5], 66 mmol/mol [SD 5]) to post run-in assessment (closed-loop 8·0% [SD 0·6], 63 mmol/mol [SD 7]; control 7·8% [SD 0·6], 62 mmol/mol [SD 6]).", "HbA1c concentrations were significantly lower after closed-loop intervention (7·4% [SD 0·6], 57 mmol/mol [SD 7]) compared with control intervention (7·7% [SD 0·5], 60 mmol/mol [SD 6]), with a mean difference between groups favouring the closed-loop group by 0·36% (95% CI 0·19–0·53; 4·0 mmol/mol, 95% CI 2·2–5·8; p<0·0001).", "HbA1c improvements were not different among children, adolescents, and adults (appendix).", "Day-and-night closed-loop therapy significantly reduced mean glucose (p<0·0001) and time spent above target (p<0·0001) compared with the control group.", "Glucose variability, measured as the SD of sensor glucose was lower in the closed-loop group than in the control group (p<0·0001).", "The coefficient of variation of sensor glucose was not different between groups (p=0·50).", "Closed-loop therapy significantly reduced the percentage of time sensor glucose was below 3·9 mmol/L (p=0·0130).", "The percentage of time spent with sensor readings below 3·5 mmol/L and 2·8 mmol/L was low, and not different between interventions (table 2).", "The burden of hypoglycaemia, as measured by the area under the curve when sensor glucose was less than 3·5 mmol/L, was not different between the groups (p=0·08).", "Increased time when glucose was within target range, reduced mean glucose, reduced time when glucose was below target, and a reduction in HbA1c was achieved by the closed-loop group without increasing total daily insulin (p=0·09).", "Higher basal insulin delivery than the control group during closed-loop therapy (p<0·0001) was offset by lower bolus delivery (p<0·0001).", "The change in bodyweight from the screening value was not different between groups (closed-loop 2·2 [SD 2·3] vs control 1·4 [SD 2·6]; p=0·19).", "The use of the closed-loop system was not associated with any additional burden, as assessed by the participant version of PedsQL (p=0·85) or the parent version (p=0·29; table 2).", "Benefits of closed-loop therapy were prominent during the night (table 3, figure 2).", "Closed-loop therapy significantly reduced daytime and night-time mean glucose and glucose variability (for both, daytime p=0·0003, night-time p<0·0001).", "The proportion of time when glucose was within the target range, between 3·9 mmol/L and 10·0 mmol/L, was significantly greater in the closed-loop group than the control group (day and night, both p<0·0001).", "Day-and-night closed-loop therapy was used for a median of 71% (63–83) of the time over the 12-week period, and participants in the closed-loop group wore a glucose sensor for a median of 90% (83–95) of the time (appendix).", "Control group participants wore a glucose sensor over a median of 90% (81–95) of the time.", "The number of planned contacts (ie, visits, email, or phone calls) was the same in both groups.", "However, more unscheduled contacts took place in the closed-loop group than in the control group (n=69 vs n=17; appendix).", "In the closed-loop group, a greater number of threshold suspend events occurred during the day than the night (appendix).", "In a prespecified per-protocol analysis of the primary endpoint, comprising 24 participants in the closed-loop group and 39 participants in the control group (appendix), similar results to those from the intention-to-treat analysis were observed (closed-loop 68% [SD 8] vs control 54% [SD 9]; p<0·0001; appendix).", "These results did not change when less stringent per-protocol criteria were used (post-hoc analysis, appendix).", "Post randomisation, no severe hypoglycaemia occurred in either study group.", "One diabetic ketoacidosis presented in the closed-loop group due to infusion set failure, and was not related to the closed-loop therapy (table 4).", "Two participants in each study group had significant hyperglycaemia with capillary glucose greater than 16·7 mmol/L and elevated plasma ketones (>0·6 mmol/L).", "There were 13 other adverse events in the closed-loop group and three in the control group (appendix); all were unrelated to treatment.", "All participants recovered fully without clinical sequelae.", "Protocol deviations were comparable between study groups (appendix).", "In this multinational, multicentre, open-label, randomised trial, we show that 12-week use of a day-and-night hybrid closed-loop insulin delivery system, compared with sensor-augmented insulin pump therapy, was associated with an improvement in overall glucose control and a reduction in hypoglycaemia risk in suboptimally controlled type 1 diabetes in children, adolescents, and adults.", "The hybrid closed-loop system was used safely during daily living without supervision or remote monitoring.", "We report a 10·8 percentage point increase in time with glucose concentrations within the target glucose range across all age groups.", "This improvement resulted from a reduction of time spent in hyperglycaemia without change in total insulin delivery.", "We observed a lower amount of bolus insulin and a higher amount of basal insulin in the closed-loop group than in the control group.", "Lower bolus insulin requirements in the closed-loop group than in the control group could be explained by lower glucose concentrations in this group during closed-loop use, lessening the need for correction boluses.", "The insulin to carbohydrate ratio did not need to be increased, unlike in other closed-loop systems,17 simplifying clinical adoption of our closed-loop system.", "Benefits of the closed-loop were greater overnight because, even with the use of a closed-loop system, daytime control is typically confounded by meals and physical activity.", "These improvements are attributable to the use of the closed-loop system alone because no regular adjustments of insulin pump therapy driven by a health-care professional took place, unlike in another study.17", "The findings of the present study are consistent with results from our previous trials during free living in children and adolescents,9 adults with well controlled type 1 diabetes,18 and adults with less well controlled type 1 diabetes.9", "This consistency of findings underpins the robustness of our model predictive algorithm, and supports the application of our closed-loop systems across a wide range of people with type 1 diabetes.", "Use of hybrid closed-loop therapy led to a modest, but clinically significant, 0·36% reduction in HbA1c, compared with sensor-augmented pump therapy.", "This reduction was additive to that observed during the run-in phase, the latter attributable to the observer bias and initiation of continuous glucose monitoring.", "The decrease in HbA1c during closed-loop use was slightly greater than that observed in two randomised trials9,10 run for long enough to assess changes in HbA1c, both adopting sensor-augmented pump therapy as a comparator.", "Thabit and colleagues9 showed a mean reduction in HbA1c by 0·3% with day-and-night hybrid closed-loop therapy, whereas Kropff and colleagues10 reported a reduction of 0·2% for evening-and-night closed-loop application.", "These two trials were small, with approximately 30 participants per trial, and closed-loop application was restricted to adults.", "In comparison, the present study randomly assigned 86 participants and the age range was wider.", "Improvements in HbA1c in the present study were consistent across all age groups.", "The proportion of patients who experienced a hypoglycaemic event was low in the present study and comparable to other outpatient closed-loop studies.9,17", "The reduction in the proportion of time spent in hypoglycaemia below 3·9 mmol/L with closed-loop therapy was statistically significant; time below 3·5 mmol/L and 2·8 mmol/L did not reach statistical significance.", "Because no severe hypoglycaemia presented in either group, the effect of closed-loop therapy on severe hypoglycaemia remains unclear.", "Further reduction of hypoglycaemia risk might be achieved through the addition of glucagon in bihormonal closed-loop systems,19,20 particularly during exercise.21,22", "The strengths of our study are the multicentre, multinational design and the wide age range of participants, which support generalisability of study findings.", "The study was done without remote monitoring or close supervision in free-living settings, allowing for real-world assessment of performance of closed-loop systems.", "No investigator-led optimisation of insulin therapy took place, and improvements in glucose outcomes with closed-loop therapy are solely attributable to its use.", "Limitations include the number of devices comprising our hybrid closed-loop system, which increased the risk of device and connectivity problems, and resulted in more frequent non-protocol contacts to address technical issues.", "Threshold suspend and predictive low glucose suspend features4,23 were not enabled in the control group because the study objective was to compare algorithmic and non-algorithmic insulin delivery approaches.", "We excluded participants with HbA1c outside the range of 7·5–10·0% and other groups, such as those with an impaired awareness of hypoglycaemia or a history of recurrent severe hypoglycaemia, although these subgroups might benefit from use of the closed-loop system.", "In conclusion, we found that free-living use of hybrid closed-loop insulin delivery over a period of 12 weeks led to clinically meaningful improvements in glycaemic control, while reducing the risk of hypoglycaemia in suboptimally controlled type 1 diabetes in adults, adolescents, and children aged 6 years and older.", "No additional data are available for this Article.", "The study had an open-label, multicentre, multinational (the UK and the USA), randomised, parallel design.", "Insulin was delivered by contrasting day-and-night hybrid closed-loop (closed-loop group) or sensor-augmented pump therapy (control group) during free living over 12 weeks.11", "Participants were recruited from diabetes outpatient clinics at four hospitals in the UK and two centres in the USA (see below).", "Before study initialisation, approval was received from an independent research ethics committee in the UK (East of England–Cambridge East Research Ethics Committee), independent review boards in the USA (Jaeb Center for Health Research Institutional Review Board), regulatory authorities in the UK (Medicines and Healthcare products Regulatory Agency) and in the USA (Food and Drug Administration).", "Safety aspects were overseen by an independent data safety monitoring board.", "The study protocol is available online.", "Inclusion criteria included type 1 diabetes, as defined by WHO,12 for at least 1 year, insulin pump therapy for at least 3 months, and HbA1c between 7·5% and 10% (58–86 mmol/mol).", "Participants were aged 6 years or older, with an equal proportion of children and young adults aged between 6 years and 21 years, and adults aged 22 years and older.", "Key exclusion criteria included regular use of real-time continuous glucose monitoring in the preceding 3 months, history of one or more episodes of severe hypoglycaemia in the preceding 6 months, and substantially reduced hypoglycaemia awareness in participants aged 18 years and older, as defined by a Gold score of 5 or more.13", "A complete list of all inclusion and exclusion criteria is provided in the appendix.", "We identified eligible adults from diabetes clinics attending Addenbrooke's Hospital (Cambridge, UK), Manchester Royal Infirmary (Manchester, UK), International Diabetes Center at Park Nicollet (Minneapolis, MN, USA), and Barbara Davis Center for Diabetes (Aurora, CO, USA).", "Children and adolescents were recruited from paediatric diabetes centres at Addenbrooke's Hospital (Cambridge, UK), Royal Hospital for Sick Children (Edinburgh, UK), Leeds Teaching Hospital (Leeds, UK), and International Diabetes Center at Park Nicollet, (Minneapolis, MN, USA).", "Eligible children and adolescents were identified by clinical teams at each centre, and were recruited by member of the local study team.", "Study participants aged 16 years or older in the UK, 18 years or older in the USA, and parents or guardians of participants aged 15 years or younger in the UK and 17 years or younger in the USA gave written informed consent; written assent was obtained from minors.", "Eligible participants who met criteria after the run-in period (see below) were randomly assigned using central randomisation software (SAS, version 9.4) to the use of day-and-night hybrid closed-loop or sensor-augmented pump therapy.", "The randomisation was stratified within centre by low (<8·5%) or high (≥8·5%) HbA1c.", "Implicit randomisation by age applied, given that each centre recruited either children and young adults (6–21 years), or adults (≥22 years).", "PC (study statistician) generated the sequence, which was used by local research teams to enrol and assign participants to the trial groups.", "Participants in both study groups used a modified 640G insulin pump (investigational use only; Medtronic, Northridge, CA, USA), Enlite 3 glucose sensor (Medtronic), and Contour Next Link 2.4 glucometer (Ascensia Diabetes Care, Basel, Switzerland).", "Participants were not remotely monitored or supervised, and were able to do their usual activities.", "They were free to consume any meals of their choice and were allowed to participate in any indoor or outdoor physical activity.", "Participants were required to be present at regular visit intervals to receive appropriate training, and to be contactable via phone or email for scheduled study contacts to review device use.", "Data from the study insulin pump and glucometer were downloaded once per week by participants using Carelink software and stored on Carelink Clinical server (Medtronic).", "Blood samples were drawn for HbA1c measurements at the hospitals where the patients were enrolled by qualified members of the centre's study team.", "Blood samples were taken at baseline, and at the start and at the end of the respective intervention period (closed-loop intervention or control intervention).", "An age-appropriate Pedatric Quality of Life Inventory (PedsQL) questionnaire was administered to participants (participant version) and guardians of participants aged 17 years and younger (parent proxy version) before and after the intervention period.", "After training on the study pump and continuous glucose monitoring, participants underwent a run-in period of at least 4 weeks.", "During this period, participants were contacted once per week.", "Data obtained during this period could be used for adjustment of the insulin therapy.", "At the end of the run-in period, adherence to the use of study pump and continuous glucose monitoring was assessed.", "Before being randomly assigned to treatment, participants were required to show use of continuous glucose monitoring for at least 12 days, and use of the bolus calculator for at least 75% of meal boluses in the 2 weeks before randomisation.", "Participants randomly assigned to the closed-loop group attended the clinical research facility for a 2–3-h visit.", "Training was provided on initiation and discontinuation of the hybrid closed-loop system, switching between closed-loop and standard insulin pump therapy, meal bolus procedure, and the use of study devices during exercise.", "Competency on the use of the closed-loop system was assessed.", "After discharge, participants applied the closed-loop system for the following 12 weeks.", "Participants randomly assigned to the control group (sensor-augmented insulin pump therapy) received additional training on the effective use of real-time continuous glucose monitoring for optimisation of insulin therapy.", "Participants were instructed not to activate the pump's threshold suspend or predictive low glucose features.", "Participants were free to optimise their treatment independently or on advice from health-care professionals.", "Hypoglycaemia and hyperglycaemia alarms were activated according to personal preference and requirements in both study groups.", "The participants in both study groups had an identical number of planned contacts with the local study team.", "Participants were contacted within 24–48 h after the initiation of study treatment.", "During the first 2 weeks of the intervention, participants in the UK were contacted by phone or email, and those in the USA were seen in the clinic once per week.", "Thereafter, participants were contacted once per month.", "All participants were provided with a 24-h helpline to contact the study team in the event of study-related issues.", "The closed-loop system (appendix) used a model predictive control algorithm (version 0.3.46, University of Cambridge, Cambridge, UK) on a smartphone (Galaxy S4, Samsung, Seoul, South Korea).", "Every 10 min, the control algorithm calculated an insulin infusion rate, which was set on the study pump.", "The control algorithm was initialised using preprogrammed basal insulin delivery downloaded from the study pump.", "Information about the participant's bodyweight and total daily insulin dose were entered at set-up.", "The treat-to-target control algorithm aimed to achieve glucose concentrations between 5·8 mmol/L and 7·3 mmol/L, depending on the accuracy of model-based glucose predictions.", "The threshold suspend feature on the modified 640G pump was turned on during closed-loop operation and allowed insulin delivery to be suspended even when the smartphone was not within range or not operational.", "Further safety mitigations during closed-loop are detailed in the appendix.", "HbA1c was measured locally at screening, and at a central laboratory (University of Minnesota, Minneapolis, MN, USA) at the beginning and end of study interventions by use of an International Federation of Clinical Chemistry and Laboratory Medicine aligned method (Tosoh HPLC Glycohemoglobin Analyzer, Tosoh Medics, CA, USA; coefficient of variation range of 1·4–1·9%).", "The primary endpoint was the between-group difference in the proportion of time spent in the target glucose range of 3·9–10·0 mmol/L (70–180 mg/dL) based on sensor-measured glucose concentrations during the 12-week free-living phase.14", "Secondary endpoints included HbA1c concentration at 12 weeks; the mean (SD) and coefficient of variation of sensor-measured glucose concentrations over the 12-week study period; percentage of time with glucose concentrations in hypoglycaemia (<3·9 mmol/L, <3·5 mmol/L, and <2·8 mmol/L) and hyperglycaemic (>10·0 mmol/L and >16·7 mmol/L); the area under the curve below 3·5 mmol/L; insulin requirements (total, basal, and bolus); bodyweight; and participant and parent PedsQL score.", "A subset of endpoints, to restrict multiple comparisons, including the proportion of time spent in the glucose target range of 3·9–10·0 mmol/L, the percentage of time with glucose concentrations of less than 3·5 mmol/L, and the mean (SD) sensor-measured glucose concentration, was assessed during the day (0800 h to 2359 h) and night (2400 h to 0759 h).", "The utility analysis assessed the amount of sensor-measured glucose use in both study groups, and the amount of closed-loop system use in the closed-loop group.", "The safety analysis assessed the frequency of severe hypoglycaemic episodes, frequency of severe hyperglycaemia (capillary blood glucose >16·7 mmol/L) with substantial ketosis (plasma ketones >0·6 mmol/L), and nature and severity of other adverse events, including diabetic ketoacidosis.", "On the basis of previous day-and-night closed-loop studies,9,15 and an estimate of 10 percentage points (SD 14·5) improvement in time when glucose is within target range, 76 participants were required to achieve 85% power and an α level of 0·05 (two-tailed t test).", "84 participants were planned to be randomly assigned to allow for dropouts.", "Statistical analyses were done on an intention-to-treat basis.", "Mean and SD were reported for the primary and secondary outcomes, which had approximately normal distribution.", "For outcomes with skewed distribution, median and IQR are reported.", "For the primary outcome and secondary outcomes, the treatment group differences were analysed using linear models, while adjusting for HbA1c at treatment initiation, corresponding run-in values for the study outcomes (ie, adjusting time spent in target glucose range during run-in when comparing the primary endpoint), and a random site effect.", "For comparison of bodyweight at 12 weeks, age and sex were additionally adjusted in the linear model.", "Normality of the residuals was assessed; if the residuals had highly skewed distribution, then ranked normal score transformation of outcome data was applied in the regression model and point estimate with 95% CI was constructed on the basis of the rank test.16", "A per-protocol analysis was limited to participants with sensor glucose data availability for at least 50% of the time over the 12-week study period (both groups) and closed-loop use for at least 80% of the time when sensor glucose data were available (closed-loop group).", "All p values reported are two-sided, and no formal adjustments for multiple comparisons were made.", "A 5% significance level was used to declare statistical significance for the primary endpoint.", "Among the secondary endpoints, p values of less than 0·05 were used to define statistical significance for HbA1c, coefficient of variation of sensor glucose, percentage of time sensor glucose was below 3·9 mmol/L, percentage of time sensor glucose was above 10·0 mmol/L, total daily insulin, and bodyweight.", "For all other endpoints, statistical significance was defined at p values of less than 0·01.", "Separate p values for day and night glucose metrics were only calculated when the 24-h version of the same metric was statistically significant based on the above criteria.", "No formal statistical comparisons were made for safety outcomes (diabetic ketoacidosis and severe hyperglycaemia events) because of the small number of events.", "Outcomes were calculated using GStat, version 2.2.4, and statistical analyses were done using SAS, version 9.4.", "This study is registered with ClinicalTrials.gov, number NCT02523131.", "The funders of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report.", "Medtronic employees read the manuscript before submission as a courtesy.", "No changes were made in the manuscript following the review.", "The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication." ]

[]

[ "ENGI", "MEDI" ]

[ "Articular cartilage", "Post-traumatic osteoarthritis", "Quantitative susceptibility mapping", "T2* relaxation" ]

[ "2204", "2732", "2745" ]

[ "Osteoarthritis (OA) is a progressive disease that leads to restricted mobility and severe joint pain1,2.", "The onset of OA may be due to joint trauma, such as a ligamental tear or focal cartilage loss.2", "Magnetic resonance imaging (MRI) is among the best non-invasive tools available for diagnosis of OA.", "Especially, quantitative MRI methods have been proposed for diagnosis of OA.3", "However, quantitative parameters are usually time-consuming to measure in full 3-D imaging due to the requirement of multiple datasets to allow for data quantification (e.g., measurement of T1 relaxation time using gold standard inversion recovery sequence); furthermore, they usually require specific sequence design.4–7", "To overcome the limitations set by desired imaging time and resolution, we propose the use of quantitative susceptibility and T2* relaxation time mapping (QSM and T2*, respectively) that are measurable using standard multi-echo gradient echo sequences.", "QSM maps the magnetic susceptibility distribution inside the imaging target, which can be altered for example by accumulation of iron or calcifications inside the target region.8–11", "In OA, mineralization of cartilage has been reported12,13; hence QSM might be sensitive in diagnosis of OA.", "So far, mostly preliminary studies with limited data sets on QSM of articular cartilage have been reported.14–16", "Wei et al. showed that susceptibility of articular cartilage displays orientational anisotropy and that the susceptibility has depth-wise varying contrast in articular cartilage.14", "In another study, Wei et al. utilized the anisotropy of susceptibility to perform collagen fiber orientation tracking using susceptibility tensor imaging.15,17", "Nykänen et al. showed that proteoglycan loss did not affect the susceptibility in ex vivo cartilage.16", "The same study also revealed that anisotropy of QSM was seemingly different from the anisotropy of T2* relaxation, which has been linked to the structural anisotropy of the collagen network in cartilage.16,18", "Furthermore, in a recent in vivo study, Wei et al. have shown that the decrease of depth-wise variation in the susceptibility is linked to osteoarthritis in vivo.19", "In conclusion, previous studies have pointed out that the collagen fiber orientation could be the source of the QSM contrast in the cartilage.", "Besides the few studies on articular cartilage, some QSM studies have targeted the venous structures of the epiphyseal-articular cartilage complex.20–23", "T2* and related T2 relaxation time measurements have been studied more extensively in cartilage imaging and have been related to properties of the collagen network.24", "Our aim was to validate the use of QSM and T2* for prediction of cartilage biomechanical and structural properties using samples from an equine post-traumatic OA study.25", "We hypothesized (1) that cartilage properties can be predicted from depth-wise profiles of quantitative MRI-parameters using partial least squares regression (PLSR) and (2) that this is a more sophisticated way of utilizing MRI data than direct Spearman's rank correlation analysis between bulk values of quantitative MRI and reference parameters.", "Osteochondral samples were obtained from a cartilage repair study involving seven Shetland ponies (N = 7, 6 females and 1 male, Age = 8.8 ± 3.5 years).25", "For the repair study, surgical lesions were induced in the medial trochlear cartilage in both hinds and repaired with a combination of chondrons and mesenchymal stem cells in different carrier hydrogels.", "The ponies were sacrificed after 1 year.", "Wedge-shaped samples (Fig. 1) were collected from the medial trochleae post-mortem and included part of the lesions as well as surrounding cartilage.", "Stifle joints from three healthy ponies (with matching age range) were acquired from a local abattoir (van de Veen, Nijkerk, The Netherlands).", "From these stifles six control samples (one from each stifle) were taken, leading to a total of 20 samples (14 experimental, 6 control).", "The number of animals was based on the power calculations performed for the original cartilage repair study.26", "The ethical permission for animal study was given by the Ethics Committee of Utrecht University for Animal Experiments in compliance with the Institutional Guidelines on the Use of Laboratory Animals.", "The animal study was carried out in a surgical theatre at the Department of Equine Sciences, Utrecht University, The Netherlands (Permission DEC 2014.", "III.11.098).", "Prior to MRI, the samples underwent biomechanical indentation testing.", "The indentation was performed using a system that had a 250 g load cell (accuracy ± 0.25%, Model 31, Honeywell Sensotec Sensors, Columbus, OH, USA) and an actuator (displacement resolution 0.1 μm, PM500-1 A, Newport, Irvine, CA, USA).", "Several locations (proximal, central and distal with respect to the anatomical location in the joint) were investigated, because cartilage thickness and properties vary throughout the joint.", "Thus, biomechanical measurements were conducted on a pre-defined grid of 12 testing locations on each sample (Fig. 1), leading to a total number of 235 biomechanical testing locations (two samples had less than 12 locations).25", "Adjacent points could be measured after each other in quick succession since the utilized indender had a small diameter (∼0.5 mm) compared to the distance between the adjacent points (∼3–4 mm).27", "Equilibrium and dynamic moduli were determined from the biomechanical testing results.", "After the MRI measurements, the samples were fixed in formalin and then decalcified in EDTA to soften them for histological sectioning.", "Sections were cut to include two biomechanical testing locations with matching distances from the edges of the block to ensure reliable location matching for microscopical imaging.", "Sections were stained using safranin-O for digital densitometry (DD) analysis to reveal proteoglycan content.28", "The staining was done in one process, using the same batch of dye for all the slices to ensure consistency of the stain concentration.", "Unstained sections were digested with hyaluronidase to remove proteoglycans to prepare them for polarized light microscopy (PLM), which was used to reveal collagen fiber angles and anisotropy.29", "DD was performed using a light microscope equipped with a monochromatic light source and a CCD-camera.", "The settings of the microscope and the camera were the following: objective 1×, zoom 2×, binning 1 and calibration with neutral density filters of 0–3.0 optical density.", "The optical density of each sample was calculated based on the calibration set.", "To calculate the average depth-dependent profile for each measurement location, the subchondral bone was manually segmented from the images, followed by automatic extraction of profiles perpendicular to the cartilage surface and interpolation of profiles to 100 points.", "The analysis was performed with a custom Matlab algorithm (Matlab 2016b, Mathworks, Inc.).", "MRI was performed in a 9.4 T vertical bore small animal scanner using a 19-mm-diameter quadrature RF volume transceiver (Rapid Biomedical, Rimpar, Germany).", "The samples were immersed in 1HMRI-signal-free perfluoropolyether oil (Galden HS 240, Solvay Solexis, Brussels, Belgium) inside a thin latex holder.", "During MRI, the cartilage surface was oriented approximately perpendicular to the main magnetic field of the scanner with extreme care, as orientation-anisotropy has been reported for both T2* and QSM of articular cartilage.15,16,18", "Imaging was conducted using a multi-echo gradient echo sequence with six echoes.", "The first echo time of the sequence was 2.0 ms and echo spacing was 3.05 ms. Isotropic voxel size of 100 × 100 × 100 μm3 was used and the matrix size of the image was 200 × 256 × 200 voxels.", "The longest dimension, which was also the readout direction, was in line with the main magnetic field of the scanner.", "After collecting the raw MRI data, the T2* relaxation time as well as QS-maps were reconstructed.", "In QSM post-processing, the “complex fitting”–method was used to combine multi-echo data30, followed by Laplacian unwrapping to resolve residual wraps in echo combination data.31", "The unwrapped field map was then masked using cartilage segmentation.", "The susceptibility maps were calculated from masked unwrapped phase data using a total field inversion (TFI) method to limit the need of region of interest (ROI) erosion in the QSM dipole inversion.32", "The erosion of the ROI in conventional QSM methods such as projection onto dipole fields (PDF) + morphology enabled dipole inversion (MEDI)33 is depicted in supplemental material (Supplementary Fig. S1).", "The susceptibility values were referenced by setting the mean over an individual sample to zero susceptibility.", "T2* relaxation time maps were calculated using 2-parameter linearized fitting of the data.", "In the T2* fitting procedure, voxels with T2* higher than 150 ms were considered erroneous and their T2* value was set to 150 ms. Three-dimensional surface maps were gathered from MRI parameters to visualize differences between the experimental and control samples.", "Depth-wise profiles from the 3-D T2*- and QS- maps were obtained using cylindrical 3-D ROIs of 1-mm diameter and carefully matched with the biomechanical testing points based on μCT measurements and photographs of the samples.25", "Prior to the analysis, these profiles were interpolated into 100 depth-wise points.", "The profiles were then used to predict reference variables (i.e., equilibrium and dynamic moduli, proteoglycan content, collagen fiber angle and collagen anisotropy) using PLSR.", "Prior to the PLSR-analysis, depth profiles were normalized using standard normal variate.", "To avoid overfitting of the models, PLSR-analysis was conducted using 10-fold cross-validation.", "PLSR analysis was performed using a custom-made algorithm in MATLAB.", "Predictions from PLSR-analysis were compared with reference parameter values using Spearman's rank correlation analysis.", "In addition to PLSR-analysis, direct correlations were calculated between means of full and superficial (25% depth) MRI profiles and reference parameter values.", "For QSM, the range of the susceptibility values within the profile was used instead of the mean due to lack of absolute QSM referencing in the imaging.", "Statistical significance of differences in tissue properties between experimental and control groups was tested with the Mann–Whitney U test in SPSS (Version 25, SPSS Inc., IBM Company, Armonk, NY, USA).", "Statistical significance of Spearman correlations was tested using exact permutation distributions in MATLAB.", "In both tests P < 0.05 was considered as the limit for statistical significance.", "Non-parametric tests were utilized in this study due to the non-normality of the reference data (Shapiro–Wilk test P < 0.0001).", "Surface map analysis was conducted through the following steps.", "First, a tetrahedral grid was created in the cartilage mask (every 20th point of the mask was used as nodes).", "Surface triangles were then sought and their surface normals were calculated.", "After this, the 3-D QSM and T2* maps were sampled from within the volume along the surface normals.", "From these sampled profiles, the range for QSM and mean for T2* was calculated.", "Finally, the QSM range and T2* mean values were displayed on the surface triangles of the mesh to generate the parametric surface maps.", "All raw data and documentation, as well as key analysis codes used in this study, are available for download at Zenodo (https://doi.org/10.5281/zenodo.2558172).", "Differences between experimental and control specimens were noted with all reference methods (Figs. 2–4).", "In biomechanical testing, both equilibrium and dynamic moduli were decreased in the experimental specimens compared to moduli of control samples at the same location (P < 0.05, for all distal and most of the central locations) and the differences became clearer when moving towards more distal aspect of the specimens (referring to the position in the joint) (Fig. 2).", "It is also worth noting that when moving towards the lesion sites (i.e., towards the medial aspect of the femoral groove), also the moduli of the control specimens decreased (Fig. 2) (see Fig. 1 for terminology).", "Further results of the biomechanical testing have been reported in a previous study.25", "Overall, lower proteoglycan contents were observed in experimental specimens compared to controls.", "The difference between control and experimental groups was largest at the regions nearby the lesions and decreased further away from the lesions for proximal and distal locations (Figs. 3 and 4).", "Conversely, for central locations differences in PG content became clearer while moving away from the lesions.", "The collagen fiber angles in the radial zone of the experimental group were lower than in the control group and the difference between groups was highest at the locations nearby the lesions (Figs. 3 and 4), except for the proximal regions, for which no significant differences in fiber angle were observed.", "There were also differences between the collagen fiber anisotropy of the experimental and control groups, but contrary to the optical density and fiber angle, the differences became clearer when moving away from the lesion site and notable changes were only observed on the distal side of the specimens (Figs. 3 and 4).", "QSM exhibited differences between the control and experimental groups especially nearby the lesion site in the central locations and further away from the lesion site at proximal and distal locations (Figs. 5–7).", "Nearby the lesion at central locations, QSM profiles had a smaller range in the experimental samples when compared to the controls (Fig. 5).", "When moving further away from the lesion site at distal and proximal locations, the range of the profiles increased in experimental samples when compared to the control samples (Fig. 5).", "Overall, QSM seemed to have more spatial variation in the experimental samples (Fig. 7).", "The T2* profiles had qualitatively slightly lower maximum values nearby the lesion and higher values further away from the lesion site (Fig. 5).", "However, the mean value of T2* was increased nearby the lesion sites (i.e., towards the abaxial aspect of the femoral groove) in both experimental and control groups but slightly more so in the experimental specimens (Fig. 7).", "Cartilage thickness seemed to have no appreciable effect on the T2* or QSM values (Fig. 7, Supplementary Figs. S2–4).", "The absolute value of Spearman's rank correlation between MRI and reference parameters was between ρ = 0.03 and ρ = 0.46, with the highest correlations observed between bulk T2* and collagen anisotropy (ρ = −0.46) and between superficial QSM and bulk fiber angle (ρ = 0.40) (Table I).", "In general, the T2* bulk value had a stronger correlation with the reference parameters, whereas for QSM the strongest correlations were observed between the superficial QSM and the reference parameters (Table I).", "Most of the correlations in the direct correlation analysis were statistically significant, and only correlations near ρ = 0.00 were non-significant.", "PLSR-modelling results showed that the estimation of the reference parameters from MRI data was more successful using T2* or a combination of T2* and QSM (apart from the superficial collagen fiber angle and anisotropy, the correlations were between ρ = 0.49 and ρ = 0.68), and less successful using QSM results only (correlations between ρ = 0.41 and ρ = 0.55) (Table I, Supplementary Fig. S5).", "For combined T2* and QSM, mean PLM anisotropy showed the highest (ρ = 0.68) and mean superficial PLM anisotropy showed the lowest (ρ = 0.18) correlation between predicted and measured values (Table I).", "All correlations from PLSR-analysis were statistically significant.", "In this study, we examined the potential of 3-D quantitative MRI to detect post-traumatic OA (PTOA) changes in cartilage properties.", "More specifically, QSM and T2* relaxation time mapping were investigated and correlated with biomechanical parameters, proteoglycan content and collagen network properties.", "The analysis revealed weak-to-moderate direct correlations between MRI and reference parameters.", "PLSR-modelling of depth-wise MRI profiles increased the correlations between MRI and reference parameters, demonstrating that PLSR outperforms univariate analysis in quantitative MRI evaluation of articular cartilage.", "Especially biomechanical testing, but also quantitative microscopic analyses (DD and PLM) revealed differences between the experimental and control samples (Figs. 2–4).", "Interestingly, large intrasample differences in both DD and PLM were observed within both experimental and control groups (Figs. 2–4).", "This could indicate that the cartilage nearby the trochlear ridges of femur is different from the cartilage in more central locations of the knee joint (Figs. 2–4).", "Particularly interesting were the PLM results, showing that the cartilage seemed to lose its usual trilaminar appearance nearby the lesion sites in both experimental and control groups and instead had fibers directed parallel to the cartilage surface throughout the cartilage thickness (Figs. 3 and 4).", "Marked structural changes in the properties of the collagen fiber network have been demonstrated earlier at the edges of canine humeral cartilage.34", "T2* relaxation time mapping was done using linear fitting through magnitude images of each individual echo of multi-echo gradient echo sequences.", "This procedure is well-established in cartilage MRI.", "T2*-mapping revealed the usual trilaminar appearance in both experimental and control groups further away from the trochlear ridge (Fig. 6).", "However, at locations nearby the ridge or lesion site, this trilaminar appearance was less visible, especially in the experimental group (Fig. 6).", "This supports the idea that cartilage near the lesion area exhibits degenerative changes.", "It is worth noting that for T2* results, the same main relaxation contributions should apply as for the more commonly used T2 relaxation time, because cartilage does not contain high susceptibility differences or other field homogeneity issues that would differentiate T2* from T2.", "Both T2* and QSM displayed visual differences between the experimental and control samples (Figs. 5–7, Supplementary Figs. S1–3).", "The surface visualization revealed more distinct changes in QS- than in T2*-maps.", "However, these changes were difficult to quantify, which may be reflected in the poorer PLSR-modelling results.", "One notable feature in QSM was the flattening of QSM depth profiles at central measurement points in the experimental group when compared to the control samples.", "This finding is in line with a recent study, statingtural that depth-wise standard deviation of QSM is decreased in OA.19", "The direct correlations between T2* and biomechanical and structural properties of cartilage were similar to those that have previously been reported between T2 and cartilage properties.35,36", "Furthermore, the PLSR-modelling yielded higher correlations between MRI and other cartilage properties than direct univariate analysis.", "Thus, in the future, modelling approaches such as artificial neural networks and PLSR would be preferred in the analysis of quantitative MRI of cartilage.", "While the PLSR-modelling could have certainly benefitted from an even larger data set, it outperformed direct correlation (ρbulk = 0–0.46) already in this limited data set and yielded high correlations (ρPLS = 0.41–0.68) between the predicted and measured reference parameters in analyses which took full-depth profiles into account.", "In this study, QSM-post processing was based on the “complex fitting”, Laplacian unwrapping and Total Field Inversion steps.30–32", "Several studies have been dedicated to finding the most appropriate processing pathways and methods, especially for brain imaging, which has been the main target for QSM.9,37", "For cartilage, QSM has not been fully established for imaging small ex vivo cartilage specimens, a concern that was also raised in a previous study.16", "The fundamental difficulty in QSM imaging of small ex vivo cartilage samples is that regardless of the methodology, the accuracy of QSM is sub-optimal nearby the boundaries of the ROI.38,39", "Due to the thinness of cartilage, the entire ROI comprising cartilage is always near to a tissue boundary, making QSM difficult.", "Here we utilized TFI because it doesn't necessitate erosion of the already-thin cartilage mask.", "Supplementary material (Supplementary Fig. S1) provides a brief comparison of different methods and the implications with respect to small ROI.", "Encouragingly however, differences between the experimental and control groups were detected in this study even though the processing is not fully optimized for such thin tissue structures of interest.", "In vivo imaging should at least partially overcome this problem, as there will be lots of other nearby signal sources in addition to cartilage, improving the susceptibility processing.", "Another fundamental difficulty (or perhaps a property) with the current QSM processing methods is that the susceptibility values are relative to the imaged target, i.e., if susceptibility is increased by 0.05 ppm in the whole target, this change may not be detectable.", "Thus referencing (e.g., adding a capsule of oil near the target tissue) would be beneficial in the QSM studies of cartilage or of any other tissue.9,37", "The main limitation of this study is the lack of referencing in QSM reconstructions, which was partially dealt with by using the range instead of the mean in the direct analysis.", "QSM could also have benefitted from imaging in signal-producing media such as saline instead of signal-free perfluoropolyether.", "On the other hand, this would have led to lower resolution due to potential wrapping in the phase encoding direction.", "Furthermore, the measurement of the T2* relaxation time may have been suboptimal since the last echo time (17.25 ms) was considerably shorter than the longest measured T2* relaxation times (∼70 ms).", "However, this is considered to have only a small effect on the analysis of the data, since two-parameter linear fitting of T2* relaxation time is a relatively robust method.", "The robustness of the fit was further enhanced by limiting the maximum allowed value of T2* to 150 ms, which is a sufficient limit for the T2* of articular cartilage.", "The sequence parameters were optimized for QSM measurement, relieving the strain on the gradient system by reducing the number of echoes; the longest echo times were limited, thus briefly compromising the accuracy of the T2* relaxation time mapping.", "For PLSR-analysis, the dataset could have been larger, since intrasample differences were higher than expected (especially for PLM), which may affect the PLSR results due to the relatively high number of potential outliers in the data.", "While the number of animals as such may be considered low for the PLSR-analysis, the limitation was mitigated by performing multiple measurements at different locations in each sample, leading to a sufficiently high number of data points for the analysis.", "Furthermore, other qMRI parameters could have been included in the study, especially T1rho, which has been suggested as a good parameter in the evaluation of cartilage.3,40,41", "However, due to logistic reasons, MR-imaging time for the specimens was restricted and measuring additional parameters was not possible.", "In PLM, the lack of true anisotropy might have caused biased results when comparing MRI to anisotropy, since the anisotropy was calculated from the fiber angle images using image entropy.", "This study showed that evaluation of the degradative state of articular cartilage using qMRI is improved when using PLSR-modelling instead of direct correlations.", "Based on the present findings, PTOA processes change QSM-contrast in cartilage; while the exact reason remains unknown, the collagen fiber network structure appears to change as well, a finding that has been previously linked to changes in QSM contrast.15,16", "Moreover, T2* relaxation time was shown to be a more useful parameter than QSM in the evaluation of cartilage degeneration.", "Combining the results from both modalities resulted in slightly higher correlations between the measured and PLSR-predicted reference parameters, suggesting that QSM adds to qMRI evaluation of cartilage.", "Nykänen: corresponding author, QS- and T2* map calculation, data-analysis, PLM measurements, manuscript drafting and editing.", "Sarin: Data analysis, OD measurements, biomechanical measurements, sample preparation, manuscript editing.", "Leskinen: MRI ROI definitions, data-analysis, manuscript editing.", "Ketola: MRI measurements, manuscript editing.", "te Moller: animal experiments, data analysis, biomechanical measurements, manuscript editing.", "Tiitu: Histological sample preparation, manuscript editing.", "Mancini: animal experiments, design of the study, manuscript editing.", "Visser: animal experiments, design of the study, manuscript editing.", "Brommer: animal experiments, design of the study, manuscript editing.", "van Weeren: animal experiments, design of the study, manuscript editing.", "Malda: animal experiments, design of the study, manuscript editing.", "Töyräs: design of the study, manuscript editing.", "Nissi: design of the study, MRI measurements, data-analysis, manuscript drafting and editing.", "All authors: revision for intellectual content, final approval.", "Authors declare no conflicts of interest.", "Academy of Finland, Finnish Cultural Foundation, Northern Savo Regional Fund of Finnish Cultural Foundation, Dutch Arthritis Foundation.", "Funding sources had no influence on study design or interpretation." ]

[]

[ "MATE" ]

[ "Antiferromagnets", "Disorder materials", "Heat capacity", "Magnetic materials", "Spin orbital order" ]

[ "2501", "2502", "2503", "2504" ]

[ "The interplay between spin-orbital interaction and phase transitions has attracted much interest recently in strongly correlated electron systems, in particular the transition metal (TM) oxides.", "Coupling to the lattice further enriches the interplay through lattice distortions, phonons, and cooperative effects such as Jahn-Teller (JT) distortions [1].", "Cuprate superconductors and Manganites with colossal magnetoresistance belong to the TM oxides with 3d eg bands at the Fermi level.", "Perovskite Vanadates RVO3 (R = rare earth and Y) belong to the same type but with 3d t2g bands and show very different behaviour.", "The crystal structure of RVO3 is distorted from cubic to orthorhombic Pbnm symmetry by a cooperative rotation of the VO6 octahedra [2].", "A long-range magnetic ordering of the V sublattice has been observed at low temperatures for different RVO3 compounds with the magnetic structures being either being C-type with the spins parallel along the c-axis but antiparallel in the ab-plane or G-type with the spins antiparallel along all directions [3].", "In RVO3 it was found that, with decreasing Lanthanide ionic radii, the onset temperature for the orbital ordering (OO) TOO increases (137 K for LaVO3 and 179 K for LuVO3) while the spin ordering (SO) temperature TSO decreases monotonically (139 K for LaVO3 and 107 K for LuVO3).", "The crossover of TOO and TSO is between R = La and Ce.", "Recent Hartree-Fock studies [4] have shown that the C- and G-phases are energetically close, consequently, the interplay between different factors such as JT distortions, orbital quantum fluctuations, and the Dzyaloshinsky–Moriya interaction have led to very interesting properties.", "In the perovskite-type RVO3 compounds, the OO phenomenon investigated for LaVO3 and YVO3 indicated an orbitally induced-structural phase transition from orthorhombic to monoclinic when cooling through TOO.", "In LaVO3, the SO temperature TSO is 139 K which is slightly above TOO of 137 K [5], whereas it is equal to 116 K and so well below the 200 K of TOO for YVO3.", "When cooling down further, interestingly, an additional first-order phase transition appears at transition at TS = 77 K for YVO3 below which the orthorhombic phase is recovered and the magnetic structure becomes G-type [6,7].", "Concerning the high temperature phase at TS < T < TSO in YVO3, a magnetic neutron scattering study [8] has revealed some unusual features: i) the magnetic structure is non-collinear, and just more complex than previously assumed for the simple C-type; ii) The magnon band width as derived from inelastic neutron scattering along the ferromagnetic c-axis is larger than that in the antiferromagnetic ab-plane.", "This violates the standard Goodenough-Kanamoru rules according to which ferromagnetic superexchange interactions are generally substantially weaker than the antiferromagnetic interactions; iii) The spectrum is split into optical and acoustic magnons with a gap of 5 meV.", "To explain the latter feature, C. Ulrich et al. [8] proposed two different ferromagnetic exchange bonds Jc along the c-axis (i.e. dimerisation) which can be made possible by an orbital Peierls state due to the formation of an orbital singlet.", "However, Z. Fang et al. [9] argued that the splitting should be accounted for by the two different exchange interaction Jab of inequivalent VO2 layers which have different amounts of JT distortion.", "For LuVO3, an earlier powder neutron diffraction (PND) study by Zubkov et al. [3] indicated that the compound has a G-type magnetic structure at low temperature.", "Muňoz et al. [10], also using PND, studied the structural and magnetic structure in the temperature range from 2 to 300 K.", "They pointed out that LuVO3 has G-type magnetic ordering below TSO = 107 K and this magnetic structure remains stable down to 2 K.", "The material also has an OO temperature of 190 K, but without any structural phase transition at this temperature.", "The change in the crystallographic structure from orthohombic to monoclinic symmetry occurs instead between ∼82 and 94 K, which is below the SO temperature.", "Recently, we have studied this compound in detail using high quality single crystals combining a variety of experimental methods including neutron and synchrotron studies [11].", "In this work, a canted C-type magnetic structure was observed that transforms to a collinear G-type at lower temperature.", "It has also been shown that the features of orbital-Peierls state (i.e. orbital-singlet similar to spin-singlet dimers) attributed previously in YVO3 [8] are in fact a consequence of the static OO and corresponding JT distortion.", "In this contribution, we report on the magnetic, heat capacity, and neutron diffraction studies of single crystal LuVO3.", "The compound appears to be an antiferromagnet and its observed magnetic properties are consistent with the inhomogeneous nature of the compound.", "Single crystal LuVO3 was grown by means of the floating zone technique using a high temperature Xenon arc-furnace.", "At first, LuVO4 was prepared by mixing stoichiometric quantities of Lu2O3 and V2O5 (with purity of 99.9%), followed by annealing at 1100 °C for 48 h.", "The product was then reduced at 1000 °C in flowing H2 for 10 h to produce the LuVO3 powder phase.", "The LuVO3 feed and seed rods used for the single crystal growth were made by pressing the powder under hydrostatic pressure and then annealing these rods at 1500 °C under a flow of Ar.", "A similar procedure for single crystal growth is described elsewhere [12].", "Measurements of the zero-field-cooled (ZFC) [13] and field-cooled (FC) magnetisation and the magnetic isotherms were carried out in a Quantum Design SQUID magnetometer.", "Here we use zero and ZFC in italics to indicate that we neglect the small trapped field in the superconducting solenoid of the magnetometer.", "For the FC measurements, the sample was cooled from the paramagnetic region to 1.8 K in an applied field, e.g. 0.1 kOe, with the data collected (FCC), then it was warming during the measurements (FCW).", "For the ZFC measurements, the sample was cooled in zero field to 1.8 K before the magnetic field was applied.", "The data were then taken on warming.", "Heat capacity measurements of the sample were carried out in a Quantum Design Physical Property Measurement System (PPMS) with a heat capacity option using a relaxation technique.", "The magnetic structure of the compound was determined from single crystal neutron diffraction measurements on the TriCS instrument at the Paul Scherrer Institute, Switzerland using a wavelength of 1.1807 Å [13].", "In Fig. 1, we present the results of the heat capacity measurements on the LuVO3 single crystal; C vs. T (left scale) and C/T vs. T (right scale).", "Three transitions are detected and defined as the orbital ordering (OO) TOO = 179 K, the spin ordering (SO) TSO = 107 K, and To = 82.5 K manifested as a drop in the heat capacity with decreasing temperature.", "The values of these transition temperatures are in good agreement with those obtained for the polycrystalline sample [10].", "To determine the magnetic structure, the results of the neutron diffraction studies with some selected reflections as a function of temperature are presented in Fig. 2.", "Below To = 82.5 K, we observed the magnetic contribution on top of the (h k l) reflections with h zero or even, k odd and vice versa, l odd (e.g. (0 3 1) (0 1 1) reflections as seen in Fig. 2) indicating the collinear G-type magnetic structure [8].", "Between To and TSO, a magnetic contribution is seen to develop on a different set of (h k l) with h zero or even (odd), k odd (zero or even) and l zero or even characteristic of the C-type magnetic structure (see Fig. 2 for (100) and (012) reflections).", "The magnetic structure is, however, canted since the magnetic contribution due to a G-type magnetic structure is seen not to diminish completely which is also in accordance with that reported in Ref. [11].", "To explore further, the results of the FCC and FCW M(T) measured in two different magnetic fields of 0.1 kOe and 0.4 kOe along the principal axes are displayed in Fig. 3.", "There is an upturn in the magnetisation with decreasing the temperature at TSO = 107 K.", "In the SO regime, there is an additional first order transition at To of about 82.5 K, consistent with the observation of magnetic hysteresis between FCC and FCW data.", "Earlier, we reported M(T) measurements for some different RVO3 compounds [12], and showed that the form of the ZFC curves are very much dependent on the very small value of the trapped field (TF) in the superconducting magnet of the SQUID magnetometer.", "We have examined this TF carefully.", "Before each measurement, we ran a degauss sequence to minimise the TF; its absolute value was estimated to be less than 2 Oe.", "We can “generate” a TF with opposite sign by reversing the sign of the magnetic fields in the degauss sequence [12].", "In Fig. 4, it can be seen that the ZFC magnetisation measured in an applied field of 0.1 kOe observed after cooling in a positive TF (ZFC_PTF) is mirrored with that of the negative TF (ZFC_NTF) even though the TF is about two orders of magnitude smaller than the applied field used for the measurement.", "It is well known that for conventional magnetic materials, domain translation is reversible at (very) low magnetic fields [14] and so the TF of the order of a few oersteds does not have any influence on the nominal ZFC results.", "However, this is clearly not the case for LuVO3.", "It is surprising that a TF of less than 2 Oe can create the irreversible magnetisation at low temperature for this compound.", "In Fig. 5, we present the results of the reciprocal of the magnetic susceptibility as a function of temperature.", "Since there is a OO transition at a temperature TOO = 179 K accompanying a change in the crystallographic structure from a Pbnm orthorhombic space group to a monoclinic P21/b space group [11], there is a change in the slope in the χ−1(T) as well.", "We have tried to fit for the Curie–Weiss behaviour in the paramagnetic regime in two different temperature ranges, namely between 120–175 K and 185–300 K.", "The values of the effective moments μeff and the Weiss temperatures θp along different principal crystallographic axes as derived from the fitting are listed in Table 1.", "The values of μeff ranged from 2.16 to 2.61 μB/f.", "u. which is somewhat lower than the value of 2.83 μB for a free ion V3+ (spin only, S = 1).", "The Weiss temperatures θp are all negative in the range from −108.8 K to −265 K, indicating the presence of strong antiferromagnetic correlations in the compound.", "In Fig. 6, we present the magnetic isotherms measured at 1.8 K along different principal crystallographic axes.", "Despite the fact that the compound has a simple collinear G-type antiferromagnetic structure, at 1.8 K we observe open hysteresis loops along all directions with coercivities Hc being 1.7 kOe, 3 kOe, 0.2 kOe and remanent magnetisation Mr of 0.012μB, 0.0011μB, 0.0002μB along the a-, b-, and c-axes, respectively.", "This anomalous feature is indeed consistent with the inhomogeneous nature due to the defects in the spin orbital system as has been proposed recently for the RVO3 compounds [12,15–17].", "In this model, LuVO3 can be considered as an inhomogeneous antiferromagnet in which a fraction of the spins interact via weak local fields and thus they can turn easily to lie along the direction of the applied field.", "The remaining spins are strongly antiferromagnetically coupled (i.e. are hardly affected by the applied field) and are responsible for the observed SO temperature TSO and the negative Weiss temperatures.", "In addition, we would like to note that the weak local fields of the former spins also imply that the crystal field effects can lead to the reduced magnetic moment as well as the anisotropy in the magnetisation along different directions.", "In order to estimate the number of spins with weak local fields we consider the ratio between Mr/Ms where Ms is the saturation magnetisation which we assume to be 2μB of the full moment expected for V3+.", "At 1.8 K, the ratio Mr/Ms measured along the a-b-c-directions is 0.6%, 0.055% and 0.01%, respectively.", "From these, we derive the number of spins with weak local fields in respect to the applied field of 0.3% which is determined as a half of the largest value obtained along the a-axis.", "This percentage of spins with weak local field is very small and can hardly be detected using experimental techniques like neutron diffraction, but as they are embedded in a strong antiferromagnetic matrix, their effect is strong and visible on the observed magnetic properties.", "In summary, we have studied the magnetic properties of a LuVO3 single crystal using magnetometry, heat capacity and neutron diffraction measurements.", "The compound undergoes an OO transition at TOO = 179 K, followed by SO with a canted C-type magnetic structure at TSO = 109 K.", "In the SO regime, with lowering temperature there is the change in magnetic structure from C-type to G-type at To = 82.5 K.", "The open hysteresis loops observed in the collinear G-type magnetic structure are attributed to the small inhomogeneity from spins with weak local fields embedded in the majority strongly antiferromagnetic matrix." ]

[]

[ "AGRI", "BIOC" ]

[ "Active site motif", "Copper amine oxidase", "Functional classification", "Phylogenetics", "Three-dimensional structure" ]

[ "1105", "1311", "1312" ]

[ "Copper-containing amine oxidases (CAOs) are a large class of enzymes that catalyze the oxidation of primary amines to the corresponding aldehyde associated with the reduction of molecular oxygen to hydrogen peroxide (Reviewed in Klema and Wilmot, 2012).", "Because of their wide distribution in mammalian tissues, the CAO family members are involved in a range of complex processes, and changes in their expression and catalytic activity have been connected with several diseases e.g. histamine intolerance, rheumatoid arthritis, psoriasis, atopic eczema, multiple sclerosis, diabetes, respiratory diseases and cancer (Boomsma et al., 2003; Maintz and Novak, 2007; Dunkel et al., 2008; Jalkanen and Salmi, 2017).", "Mammals typically have either three (AOC1-3) or four (AOC1-4) genes encoding CAOs (Schwelberger, 2006).", "Schwelberger (2010) identified AOC1-4 genes in the genomes of cows, horses, dogs, chimpanzees and macaques.", "The complete AOC4 gene was also found in the human genome but it is disrupted by a stop codon and does not form a functional protein.", "The genomes of rat and mouse only have a partial AOC4 gene (Schwelberger, 2006).", "The AOC1 gene has five exons and it is not linked with the other AOC genes, whereas the AOC2, AOC3 and AOC4 genes are tandemly arranged on the same chromosome and each gene has four exons (Schwelberger, 2006).", "The AOC1 gene encodes diamine oxidase (DAO; hereafter called AOC1), which is expressed e.g. in placenta, kidney, gut and lung (Elmore et al., 2002).", "Decreased levels of human AOC1 (hAOC1) are correlated with histamine intolerance (Maintz and Novak, 2007).", "In accordance with the substrate preference for the aromatic diamines histamine and 1-methylhistamine (Elmore et al., 2002), hAOC1 has been suggested to have in vivo functions in cell proliferation, inflammation, allergic response and ischemia (McGrath et al., 2009).", "There is also evidence that some pharmaceuticals might interfere with the normal function of hAOC1 and, thus, cause harmful side effects (McGrath et al., 2009).", "The AOC2 gene encodes retina-specific amine oxidase (RAO; hereafter called AOC2), which was originally identified in the ganglion cell layer of the retina and has an N-terminal transmembrane segment.", "Its physiological role is uncertain (Imamura et al., 1997; Kaitaniemi et al., 2009) but human AOC2 (hAOC2) has been suggested to have a role in hereditary retinal diseases (Imamura et al., 1997, 1998).", "hAOC2 oxidizes aromatic monoamines like p-tyramine, tryptamine and 2-phenylethylamine (Kaitaniemi et al., 2009).", "The AOC3 gene encodes vascular adhesion protein-1 (VAP-1; hereafter called AOC3), which is primarily expressed on the endothelial cell surface (Salmi et al., 1993; Smith et al., 1998), but also in smooth muscle cells and adipocytes (Salmi et al., 1993; Zorzano et al., 2003).", "In addition to its amine oxidase activity, human AOC3 (hAOC3) functions as an adhesin (Smith et al., 1998; Salmi and Jalkanen, 2001).", "Upon inflammation, the expression of hAOC3 is upregulated (Salmi et al., 1993; Jaakkola et al., 2000) and it has an essential role in extravasation of leukocytes from the blood into tissues (Tohka et al., 2001; Lalor et al., 2002; Bonder et al., 2005; Merinen et al., 2005; Jalkanen et al., 2007).", "The physiological substrates of hAOC3 include methylamine and aminoacetone, it exhibits high activity towards the exogenous aromatic amine, benzylamine, and it is weakly active with 2-phenylethylamine (Smith et al., 1998; Kaitaniemi et al., 2009).", "Additionally, the Siglec-9 and Siglec-10 proteins on leukocyte surfaces have been identified as physiological ligands for the adhesive function of hAOC3 (Kivi et al., 2009; Aalto et al., 2011; Salmi and Jalkanen, 2014).", "The level of hAOC3 activity is low in healthy humans but it is elevated upon inflammation due to hAOC3 translocation to endothelial cell surface.", "Thus, a Siglec-9 derived peptide binding to hAOC3 can be used as a tracer in positron emission tomography (PET) to detect sites of inflammation in vivo (Aalto et al., 2011).", "The AOC4 gene encodes a soluble plasma amine oxidase (SAO; hereafter called AOC4) (Schwelberger, 2006).", "Bovine AOC4 (bAOC4) prefers the physiological polyamines spermidine and spermine (Di Paolo et al., 2003; Bonaiuto et al., 2010).", "It is primarily expressed in the liver from where it is secreted into the bloodstream, constituting the major part of the serum amine oxidase activity in some mammalian species (Schwelberger, 2006).", "Recently, delivery of bAOC4 to cancer cells has been reported as a method for in situ killing of cancer cells (Reviewed in Agostinelli et al., 2010).", "Cancer cells have elevated levels of endogeneous polyamines, which are catalyzed to cytotoxic reaction products that selectively reduce tumor growth upon the targeted delivery of bAOC4.", "Some mammals e.g. humans, mice and rats, do not have an AOC4 gene product.", "Therefore, the detected CAO activity in the blood of these three species is derived from AOC3, which is proteolytically released from the endothelial cell surface (Kurkijärvi et al., 1998; Abella et al., 2004; Stolen et al., 2004; Jalkanen and Salmi, 2008).", "In humans, elevated levels of plasma AOC3 activity are associated with diabetes mellitus, congestive heart failure and liver cirrhosis (Boomsma et al., 2005).", "In adipocytes, AOC3 is involved in glucose metabolism (Boomsma et al., 2005) and its activity in these cells has been shown to be down-regulated by hypoxia (Repessé et al., 2015).", "Besides having a role in various diseases and being suitable as drug-targets and therapeutics, CAOs are also effective biomarkers for several diseases including histamine intolerance, cancers and inflammatory diseases (Agostinelli et al., 2010; Mušič et al., 2013; Pannecoeck et al., 2015).", "The mammalian CAOs have a highly conserved T/S-X-X-N-Y-D motif in the active site (Mu et al., 1994; Salminen et al., 1998) where the tyrosine is post-translationally modified to 2,4,5-trihydroxyphenylalanine quinone (topaquinone; TPQ) (Janes et al., 1990).", "To date, the crystal structures of hAOC1 (McGrath et al., 2009), hAOC3 (Airenne et al., 2005; Jakobsson et al., 2005; Elovaara et al., 2011; Bligt-Lindén et al., 2013), and bAOC4 (Lunelli et al., 2005; Holt et al., 2008) are known but no X-ray structure for AOC2 has been solved.", "Based on the 68% sequence identity between hAOC2 and hAOC3, the overall structure of hAOC2 is predicted to be very similar to that of hAOC3 (Kaitaniemi et al., 2009).", "The CAOs as a family are all tightly bound homodimers with a similar three-dimensional (3D) fold consisting of the D2 and D3 domains, and the catalytic D4 domain (Fig. 1).", "The buried active site is highly conserved and has several common features: the TPQ cofactor and the conserved catalytic aspartic acid residue are both involved in the catalytic reaction, and the three conserved histidines coordinate the copper ion involved in TPQ biogenesis.", "The catalytic reaction mechanism of the CAOs is divided into two half-reactions and has been extensively studied in bacteria and yeast (Reviewed in Dawkes, 2001; Klema and Wilmot, 2012; Shepard and Dooley, 2015).", "In the reductive half-reaction, the amine substrate binds to TPQ of an oxidized enzyme forming a substrate Schiff base, and the α-carbon proton of the substrate abstracted by the catalytic base to form the product Schiff base, which is hydrolyzed to release the aldehyde product.", "In the oxidative half-reaction, the reduced TPQ is re-oxidized by molecular oxygen with the release of ammonia and hydrogen peroxide.", "CAOs are currently sub-classified by the Enzyme Commission (EC) based on their substrate preference for either primary monoamines (EC 1.4.3.21) or diamine (EC 1.4.3.22).", "Due to the lack of an appropriate classification to distinguish between AOC3 and AOC4, the majority of AOC4 proteins are currently annotated as “membrane primary amine oxidase-like” proteins in the sequence databases.", "In the earlier literature, bAOC4 and hAOC3 were often regarded as the same protein, although they are products of different genes and present clear differences e.g. in the substrate preference and enzyme kinetics (Holt et al., 2008; Bonaiuto et al., 2010).", "Similarly, hAOC2 and hAOC3 have clear differences in the substrate preference and respectively prefer aromatic and aliphatic amines (Kaitaniemi et al., 2009).", "Despite the reported differences of the EC 1.4.3.21 monoamine oxidases, there are currently no guidelines to further classify them into AOC2-4 sub-families.", "In this study, we have combined a phylogenetic study of vertebrate CAOs with analyses of the variable X1 and X2 residues from the T/S-X1-X2-N-Y-D active site motif and linked the results to known substrate preferences of hAOC1-3 and bAOC4 in order to derive general rules that aid in the classification of the CAOs to distinct sub-families of AOC1, AOC2, AOC3 and AOC4.", "Most notably, our comprehensive analysis allowed us to distinguish the poorly annotated AOC4 proteins from the highly similar AOC3s.", "By associating similarities and differences in the active sites of CAOs with the unique substrate preference of each CAO sub-family, we were able to identify residues that contribute to the substrate selectivity.", "The results from our work will benefit the design of new AOC3 sub-family specific inhibitors, which would not bind to the other CAOs, and the design of in vitro tests to detect specific CAO levels for diagnostic purposes.", "The sequences for human AOC1 (hAOC1; UniProt ID: P19801), AOC2 (hAOC2; UniProt ID: O75106), AOC3 (hAOC3; UniProt ID: Q16853) and bovine AOC4 (bAOC4; UniProt ID: Q29437) proteins (ranging from 756 to 763 residues) were obtained from UniProt Knowledgebase (Jain et al., 2009).", "To identify vertebrate CAO sequences, the hAOC1, hAOC2, hAOC3 and bAOC4 sequences were used as a query to search vertebrate genomes with protein BLAST (Altschul et al., 1990) at the National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/Blast.cgi).", "Only the full-length sequences and the sequences, which had the conserved tyrosine that is post-transcriptionally modified to TPQ, the catalytic aspartic acid and the three histidines that coordinate the copper ion, were used in the analysis.", "Gene information for the selected protein sequences was collected from NCBI gene database (https://www.ncbi.nlm.nih.gov/gene/).", "As the degree of conservation is higher at the 3D structural level than at the sequence level (Illergård et al., 2009), we built a structure-based alignment of hAOC1 (PDB: 3HI7) (McGrath et al., 2009), hAOC3 (PDB: 4BTX) (Bligt-Lindén et al., 2013), bAOC4 (PDB: 1TU5) (Lunelli et al., 2005) 3D structures in BODIL (Lehtonen et al., 2004).", "This alignment was then used as a starting point for aligning the sequences from the BLAST search to ensure the correct positioning of gaps and the conservation of structural features.", "All sequence alignments were done using the MALIGN (Johnson et al., 1996) in BODIL (Lehtonen et al., 2004) by first creating a separate multiple sequence alignments (MSAs) for the sequences retrieved by each BLAST search.", "These alignments were then combined in BODIL to generate a MSA with all studied sequences.", "The MSA was analyzed with special focus on the residue near the active site and on other functionally important residues cited in the literature to confirm that they are conserved in the final MSA.", "The Hidden Markov Model (HMM) motifs were generated with Skylign (Wheeler et al., 2014).", "Signal peptide predictions were performed in the SignalIP 4.1 server (Petersen et al., 2011) and transmembrane domain predictions were performed in the TMHMM 2.0 server (http://www.cbs.dtu.dk/services/TMHMM-2.0/) for all AOC3 and AOC4 sequences.", "Before performing the phylogenetic analysis, isoforms with the same active site motif were removed from the alignments previously generated in BODIL to avoid redundancies in the trees and to include only one representative for each sequence of each species in the study.", "The model that best fits the data was chosen and the phylogenetic reconstructions were performed in MEGA-CC 7 (Kumar et al., 2012, 2016).", "Phylogenetic tree of vertebrate CAOs were estimated using Maximum Likelihood (ML) and Neighbour-joining (NJ) (Saitou and Nei, 1987) methods.", "The NJ method is based on distance matrix calculated from the MSA, whereas the ML method uses a random tree as a starting point, which is then modified until it reaches the highest likelihood of producing the alignment that was used.", "The NL trees were generated with the Jones-Taylor-Thornton (JTT) (Jones et al., 1992) distance matrix.", "To generate the ML trees, the best fitting model for each alignment was assessed using MEGA-CC 7 (Kumar et al., 2012, 2016) and chosen based on the Bayesian Information Criteria.", "The Poisson model (Zuckerkandl and Pauling, 1965) with gamma distributions with invariant sites (i.e. P + G + I) was the best model for the vertebrate CAO alignment.", "Since the ML and NJ phylogenetic trees were congruent, we used the ML tree for further analysis (Fig. 2A; NJ in Supplementary Fig. S1).", "JTT with gamma distributions (JTT + G) was chosen as the best model for the mammalian AOC1 and AOC2 alignments while the JTT model with gamma distributions and invariant sites (JTT + G + I) fitted best for the alignment of mammalian AOC3 and AOC4 sequences.", "The areas with gaps were excluded from the phylogenetic analysis.", "Statistical support for the topology of the tree was inferred from using the bootstrap method (Felsenstein, 1985) with 500 replicates.", "In general, bootstrap values of 70–100% indicate significant support for a branch (Soltis and Soltis, 2003b).", "To generate the 3D multivariate plots, the distance matrices from mammalian MSAs were generated in MEGA-CC 7 (Kumar et al., 2012, 2016) using JTT substitution matrix.", "Thereafter, the JTT distance data was supplied to a C-program to perform a multivariate analysis (PCA, MS Johnson, unpublished).", "The program displays coordinates for each sequence and their locations such that the variance among the data is a maximum, and projections for various numbers of dimensions are possible.", "The program returns the three most informative dimensions as a pseudo-PDB coordinate file, which can be visualized in BODIL (Lehtonen et al., 2004) as multivariate 3D plots.", "We first generated a phylogenetic tree containing all vertebrate CAO sequences to aid a rough classification of the mammalian CAOs into the AOC1-4 sub-families.", "In the resulting tree, we named the proteins as AOC1 or AOC2 based on the similarity of their active site motif to hAOC1 and hAOC2, respectively.", "The proteins with an active site motif similar to either hAOC3 or bAOC4 were named as AOC3/4.", "Thereafter, individual phylogenetic trees were constructed for the AOC1 and AOC2 gene products and a joint tree of the AOC3/4 gene products that grouped on the same clade within the phylogenetic tree of vertebrate CAOs.", "With an aim to produce phylogenetic trees with improved bootstrap values, we also constructed the phylogenetic trees for the conserved D4 domain using the nucleotide sequences corresponding to the proteins listed in the Supplementary Tables S3 and S4.", "The nucleotide sequences that fulfilled the same functional criteria as we applied for protein sequences were aligned with ClustalW in MEGA-CC 7 (Kumar et al., 2012, 2016).", "The ML trees were calculated similarly as previously (the best fitting model for each alignment listed in the Supplementary Table S5).", "The original structure-based alignment was used as a starting point to generate a model for hAOC2 and the crystal structure of hAOC3 (PDB: 4BTX) (Bligt-Lindén et al., 2013) was used as a template.", "Ten models were generated using MODELLER (Sali and Blundell, 1993).", "From the generated models, the one with the lowest value of the MODELLER objective function was chosen for analysis.", "All structures were compared by superimposition using VERTAA in BODIL (Lehtonen et al., 2004) and quality of the model was evaluated using PROCHECK (Laskowski et al., 1993) and QMEAN (Benkert et al., 2009).", "Visual analysis of the model and crystal structures was performed in Pymol (The PyMOL Molecular Graphics System, Version 1.23, Schrödinger, LLC.).", "In this study, we used a large number of sequences from a variety of vertebrate species, which gives us the benefit of obtaining a more in-depth knowledge of the phylogenetic relationship of the mammalian and non-mammalian CAO sequences.", "No extensive phylogenetics analysis of the CAO family has been performed earlier, since Lai and co-workers analyzed only ten mammalian CAO sequences (Lai et al., 2012) and Schwelberger (2006, 2010) solely focused on the porcine genome.", "To collect sequences homologous to CAOs for the phylogenetic tree of vertebrate CAOs, we performed BLAST searches using hAOC1, hAOC2, hAOC3 and bAOC4 as queries against vertebrate genomes.", "The sequences without (1) the tyrosine post-transcriptionally modified to TPQ, (2) the catalytic aspartate or (3) the three histidines coordinating the copper ion (Fig. 1) were excluded to ensure that the selected sequences are catalytically active.", "Isoforms with identical active site motifs were not taken into account as highly similar sequences induce redundancy in the trees.", "The final dataset for inferring the phylogenetic relationships of the collected vertebrate CAOs consists of 369 sequences originating from 143 vertebrate species and includes 283 sequences from 90 mammalian species.", "The collected sequences share sequence identities ranging from 29.9% to 99.9% (Supplementary Tables S1 and S2).", "The AOC1, AOC2 and AOC3 proteins were found in Amphibians and Amniotes but the AOC4 proteins were exclusively found in placental mammals.", "Additionally, bony fishes had AOC1s and Cartilaginous fishes (out-group) had only AOC2s.", "We used the CAO sequences from cartilaginous fishes Rhincodon typus (whale shark) and Callorhinchus milii (elephant shark) as an out-group in the phylogenetic analysis.", "C. milii diverged from the bony vertebrates ~ 450 million years ago and has the slowest evolving genome of all known vertebrates, which makes it a good model for inferring the state of ancestral genomes (Venkatesh et al., 2014).", "Of the ancestral CAO proteins, CAO from C. milii annotated as “retina-specific amine oxidase-like” also shares the highest sequence identity with hAOC2 (43.8%) and has almost the same identity to hAOC3 (43.4%) whereas its identity to hAOC1 (40.9%) is somewhat lower.", "Similarly, R. typys CAO has the highest identities to hAOC2 and hAOC3 (42.5% and 41.8%, respectively) and somewhat lower identity to hAOC1 (39.2%).", "Since the phylogenetic trees inferred using MEGA-CC 7 (Kumar et al., 2012, 2016) with ML and NJ methods were congruent, we used the ML tree for further analysis (Fig. 2A; NJ in Supplementary Fig. S1).", "The phylogenetic tree of vertebrate CAO is divided into two major branches of the AOC1 and AOC2-4 proteins (Fig. 2A).", "The distances of the three branches, which are scaled to the relative evolutionary distance, show that the AOC2 and AOC3/AOC4 proteins are closely related but AOC1s have a longer evolutionary distance to them.", "The analysis of the gene information from the NCBI gene database (http://www.ncbi.nlm.nih.gov/gene/) revealed that the AOC2-4 genes in the studied organisms are located in the same chromosome, whereas the AOC1 gene is located in another chromosome, which is in accordance with previous studies (Schwelberger, 2006, 2010).", "In the phylogenetic tree of vertebrate CAOs, the mammalian and non-mammalian AOC1 proteins share the same clade with a strong bootstrap support of 100% (Fig. 2A).", "The phylogenetic tree suggests a similar substrate preference for AOC1 from fish, amphibians, reptiles, birds and mammals.", "Accordingly, Almeida and Beaven (1981) have reported AOC1 activity towards histamine in the brain of lower vertebrates.", "Unlike for AOC1s, the non-mammalian AOC2-4s segregate from the mammalian proteins with a bootstrap value of 86%.", "The non-mammalian AOC2 and AOC3 proteins form their own sub-branches with a bootstrap support of 85% for the node, whereas mammalian AOC2-4s are further divided into the AOC2 and AOC3/AOC4 sub-branches with high bootstrap values (Fig. 2A).", "Within the non-mammalian AOC2 clade, the crocodilian CAO-like proteins (labeled AOCX) are found in a separate clade (48% bootstrap value).", "The non-mammalian AOC3 branch is further divided with a low bootstrap support of 28% into the major AOC3 branch and the Amphibian branch including both AOC2s and AOC3s.", "The phylogenetic tree of vertebrate CAOs was also constructed using the nucleotide sequences and shows similar branching topology as the tree based on protein sequences (Supplementary Fig. S6).", "Our earlier structural and sequence analysis of CAOs indicated that variable residues X1 and X2 of the T/S-X1-X2-N-Y-D active site motif might follow the substrate preference of mammalian CAOs (Salminen et al., 1998; Airenne et al., 2005; Kaitaniemi et al., 2009).", "This led us to propose that the residues at position X1 and X2 might be useful in distinguishing the sub-families of mammalian CAOs.", "In the vertebrate CAO tree, the three clades of the mammalian CAOs (Fig. 2A) agree with their known substrate preference: (1) diamines for the AOC1 clade, (2) aromatic monoamines for the AOC2 clade, and (3) aliphatic amines for the AOC3/AOC4 clade.", "Therefore, we next focused our study at positions X1 and X2 and analyzed the collected vertebrate CAO sequences with a special focus on the mammalian CAOs to find out if these two residues are suitable for categorizing the mammalian CAO sub-families.", "When we visualized the vertebrate CAO alignment using the Hidden Markov Model (HMM) motifs (Wheeler et al., 2014) and analyzed the X1-X2-N-Y-D motif in the mammalian CAOs, the results revealed that residue X2 is always a Tyr in AOC1s, a Gly in AOC2s and a Leu in the AOC3/AOC4 proteins (Fig. 2B).", "Thus, residue X2 coincides with both the substrate preference and the phylogenetic classification of the three CAO sub-families in Mammalia (Fig. 2A).", "Consistently, all vertebrate AOC1s have a Tyr at position X2 and most of the AOC2s a Gly.", "The additional CAO-like proteins in crocodilians (labeled AOCX) have a Ser at position X2 indicating that they have a different substrate preference than the other non-mammalian AOC2s.", "In non-mammalian AOC3s, residue X2 is less conserved than the totally conserved Leu in mammalian AOC3/AOC4 proteins.", "It is always a hydrophobic residue but more frequently an Ile than a Leu and even a Val in some of the non-mammalian AOC3s.", "As mammalian AOC3s and AOC4s both have a Leu at position X2 (Fig. 2B), and they are in the same AOC3/AOC4 clade of the phylogenetic tree (Fig. 2A), their classification needed further inspection.", "Therefore, we limited the vertebrate CAO data set to mammalian sequences including 85 AOC1, 77 AOC2, 74 AOC3 and 47 AOC4 sequences from 90 different mammalian species (Supplementary Table S1) and created a multiple sequence alignment (MSA).", "The average sequence identity of AOC1s to AOC2 is 62% and to AOC3/AOC4s 60.2%, whereas the AOC2s and AOC3/AOC4 proteins share a higher average sequence identity of 73.2%.", "Furthermore, the intra-species sequence identities of AOC3/AOC4 proteins range from a high identity of 84% to an extremely high value of 98%.", "As an alternative to the phylogenetic reconstruction methods, principal components analysis (PCA) was used to provide a multivariate analysis of the pairwise sequence distance data.", "The resulted 3D multivariate plot with three clusters representing sequences similar to hAOC1, hAOC2 and hAOC3/bAOC4 (Fig. 3A) is in accordance with the phylogenetic tree of vertebrate CAOs (Fig. 2A).", "Since position X2 did not differentiate AOC3 and AOC4, we next analyzed residues at position X1.", "Comparison of hAOC3 and bAOC4 sequences revealed that residue X1 was a Leu in hAOC3 and a Met in bAOC4.", "Next we analyzed the origin of the AOC3 and AOC4 proteins using the information in the gene and sequence databases and noticed, with few exceptions (see below), that the proteins having a Leu at position X1 originated from a different gene than those with a Met.", "Furthermore, the fact that a Leu is predominantly found at position X1 in the non-mammalian AOC3s gave further support for our hypothesis that X1 might distinguish mammalian AOC3s from AOC4s.", "To test our hypothesis, we named the mammalian CAOs with a L-L-N-Y-D motif as AOC3 and those with a M-L-N-Y-D motif as AOC4 and generated a 3D multivariate plot for them (Fig. 3B).", "In the resulting plot, the AOC3 cluster separates from the AOC4 cluster suggesting that the CAOs with a Leu at position X1 should be classified as AOC3 and those with a Met as AOC4.", "Thereafter, we generated the Hidden Markov Model (HMM) motifs for the AOC1-4 subfamilies of mammalian CAOs (Wheeler et al., 2014).", "The HMM motifs (Fig. 3C) further confirmed that residue X2 of the X1-X2-N-Y-D motif reveals the substrate preference of mammalian CAOs while residue X1 classifies the mammalian AOC3 and AOC4 proteins into distinct sub-families.", "As a summary, a Tyr at position X2 is consistent with a preference for aromatic diamines (AOC1), a Gly for aromatic monoamines (AOC2) and a Leu for aliphatic amines (AOC3 and AOC4).", "A Leu at position X1 allows preference for small aliphatic monoamines (AOC3) and a Met for aliphatic polyamines (AOC4), whereas AOC1 and AOC2 that prefer aromatic substrates both have a Val at position X1.", "Our results concluded that mammalian CAOs can be classified into sub-families according to the X1 and X2 residues in the active site motif, which is (1) T-V-Y-N-Y-D in AOC1, (2) T/S-V-G-N-Y-D in AOC2s, (3) T-L-L-N-Y-D in AOC3s and (4) T-M-L-N-Y-D in AOC4s (Fig. 3C).", "To analyze the evolution of the CAO sub-families, we now generated separate trees for the AOC1, AOC2 and AOC3/4 subfamilies characterized by a Tyr, a Gly and a Leu at position X2, respectively.", "In general, the phylogenetic trees of CAO sub-families are supported by low bootstrap values (Figs. 4–6), which reflect the short evolutionary distances of CAOs in each sub-family.", "When the evolutionary time frame is short, the number of substitutions supporting each node and the dataset as a whole remain small and it is particularly sensitive to random fluctuations in observed mutations and small differences, which leads to low bootstrap values (Soltis and Soltis, 2003a).", "Indeed, CAOs were one of the few protein families accepted into a study that explicitly searched for proteins related by short evolutionary distances (Lai et al. (2012).", "Among AOC1s, the inter-species sequence identity varies between 68.3 and 99.1% (the average identity is 80.0%).", "The AOC1 tree is divided into two major branches (Fig. 4).", "One of the branches has the majority of AOC1s and the other one consists of AOC1s from Myomorpha (mouse-like rodents; Fig. 4, green).", "Thus, the rodent proteins are divided into two distinct branches, despite AOC1s from the same taxonomic order generally group together.", "The other rodent branch with AOC1s from the Hystricomorpha, Sciuromorpha and Castorimorpha suborders of rodents (Fig. 4, orange) is in the same sub-branch as primate AOC1s.", "This rodent branch also includes AOC1s from Lagomorpha, Scandentia and Dermoptera.", "In the phylogenetic tree constructed using the nucleotide sequences of the D4 domain of AOC1s (Supplementary Fig. S7), the rodent proteins are distributed among several branches and Pteropus alecto, Pteropus vampyrus and Rousettus aegyptiacus did not group together with the other Chiroptera but the topology of the tree was otherwise similar.", "Interestingly, the tricyclic antidepressant amitriptyline had an opposite effect on Rattus norvegicus (rat; Myomorpha) and Cavia porcellus (guinea pig; Hystricomorpha) AOC1s (Rajtar and Irman-Florjanc, 2007), which also belong to different branches of the AOC1 tree (Fig. 4).", "Amitriptyline was reported to act as an inhibitor of R. norvegicus AOC1, whereas it increased the activity of C. porcellus AOC1 leading to faster histamine inactivation.", "Thus, the phylogenetic relationship of the two proteins is also reflected at the molecular level in their response to ligand binding.", "The tree of the AOC2 proteins has two main branches (Fig. 5), which in turn have diverged into seven sub-branches.", "The branch length in the seven sub-branches is short and the bootstrap values supporting each branch range from 17% to 98% and the bootstrap values of the tree of AOC2 nucleotide sequences are not significantly higher for the main sub-branches (Supplementary Fig. S8).", "All the AOC2 proteins are predicted to have a N-terminal transmembrane helix.", "The prediction could not however be done for Orincus orca and Ictiodomys tridecemlinearus AOC2s, since they lack part of the N-terminal sequence.", "The inter-species sequence identity of AOC2s (67.1–100%, average 87.0%) is higher than that of AOC1s and, similar to AOC1, the sequences group in the phylogenetic tree according to the taxonomic rank of species (Fig. 4).", "Unlike AOC1s, all rodent AOC2s group together in the same sub-tree with O. cuniculus AOC2 (Lagomorpha).", "The AOC2 proteins from rodents and O. cuniculus are closely related but differ from Primates; therefore studies conducted on the physiological function of AOC2 from rodents and O. cuniculus should not be extended to Primates without careful comparison of the proteins at sequence and structural level.", "Of the rodents, R. norvegicus lacks AOC2 due to a stop codon that terminates the peptide at 127 amino acids (Zhang et al., 2003).", "The function of AOC2 is still unknown and further characterization is needed to gain insights into its contribution to the CAO activity in mammals.", "As rats only have AOC1 and AOC3, detailed and comparable studies of CAO activities in rats and mouse (AOC1-3 proteins) could give hints for the function of AOC2.", "The average sequence identity of 81.1% shows that the AOC3 and AOC4 proteins are closely related.", "The identity of all AOC3 and AOC4 proteins varies between 36.7% and 99.9% and is much higher for the catalytic D4 domain (59.5–100%).", "Consistent with the AOC1 and AOC2 trees (Figs. 4 and 5), the main branches of the AOC3/AOC4 tree (Fig. 6) have diverged over a relatively short evolutionary time frame.", "Although the main branches have low bootstrap values, the bootstrap values are on average higher within the sub-branches.", "For example, the bootstrap of the sub-branch containing Artiodactyla and Cetacea proteins is low (9%) but the bootstrap support for the node of the AOC3 proteins from Cetacea is high (99%) and, similarly, the bootstrap support for grouping the AOC3 and AOC4 proteins from Artiodactyla into different clades is 99% for the AOC3 clade and 67% for the AOC4 clade.", "In the case of AOC3/AOC4, the same taxonomic order group together much better in the tree of the nucleotide sequences (Fig. 7) than in the tree of the protein sequences (Fig. 6).", "Based on the phylogenetic trees (Figs. 6 and 7), the divergence between AOC3 and AOC4 proteins has occurred in a species-specific manner, probably due to different evolutionary pressure as no consensus among all species was found.", "In some species, a recent divergence has occurred, whereas in other species the divergence has happened earlier.", "For example, the AOC3 and AOC4 proteins from monkeys do not share the same most recent last common ancestor, whereas bovine AOC3 and AOC4 proteins do.", "Previously, it has been suggested that those species that have an AOC4 gene also have a high level of serum CAO (Schwelberger, 2006).", "Much higher levels of CAO activity ha been measured in the serum of Ovis aires (sheep), Bos taurus (cow), O. cuniculus, Capra hircus (goat), Canis lupus familiaris (dog) and Sus scrofa (pig) than in the serum of human, R. norvegicus (rat), M. musculus (mouse) and C. porcellus (Guinea pig) (Boomsma et al., 2003; Schwelberger, 2006).", "The suggested relationship is otherwise in accordance with our results but we could identify only one AOC3/4 gene in the C. lupus familiaris and S. scrofa genomes.", "The two AOC3/4 proteins from C. lupus familiaris are special since they turned out to be isoforms from the same AOC3 gene.", "They share a sequence identity of 97.8% but one of them has Leu (like AOC3) and the other one Met (like AOC4) at position X1.", "In the multivariate plot, the AOC4-like protein from C. lupus familiaris appears as an outlier clustering together with the AOC3 proteins (Clu in Fig. 3B).", "Analysis of the gene information of those species that have both AOC3 and AOC4 proteins revealed eight other species in which the AOC3/4 proteins are isoforms from the same gene (Supplementary Table S6).", "Unlike C. lupus familiaris proteins, these other proteins are not found in the same branch of the phylogenetic trees (Figs. 6 and 7).", "In the S. scrofa genome, we could identify only one protein in the AOC3/4 clade and it is also an outlier in the multivariate analysis.", "The S. scrofa protein is currently annotated as an AOC3 gene product and it consistently clusters with AOC3s (Ssc in Fig. 3B) despite it having Met at position X1 (like AOC4).", "In accordance with the multivariate plot, S. scrofa AOC3/AOC4 groups into the main clade of the AOC3 proteins in the phylogenetic tree (Fig. 7).", "In addition to C. lupus familiaris and S. scrofa proteins, there are some other similar outliers in the multivariate plot (Fig. 3B) but there is no literature on CAO serum activity levels in these organisms.", "Of these outliers, Ailuropoda melanoleuca (panda) protein (Ame in Fig. 3B) should be classified as an AOC4-like protein, since it has Met at position X1 despite it is encoded by AOC3 and has a predicted transmembrane helix.", "The additional outliers in the multivariate plot are the AOC4 proteins from Bos taurus (cattle), Bison bison bison (plains bison) and Bubalus bubalis (water buffalo), which group closer to AOC3s than AOC4s (Bta, Bbi and Bbu in Fig. 3B).", "All these species also have an AOC3 protein, which clusters together with the other AOC3 proteins from Artiodactyla (Figs. 6 and 7).", "Thus, their AOC4 gene product might still be differentiating from AOC3.", "In accordance with previous research stating that the AOC3 gene duplication is the origin of the AOC4 gene (Schwelberger, 2010), we did not find any AOC4 encoded protein with an AOC3 motif (a Leu at position X1), whereas we found the above mentioned AOC3 encoded proteins with an AOC4 motif (a Met at position X1).", "The predominant codon for a Leu is TTG, which turns to a Met coding ATG by a single nucleotide substitution.", "The need for a new protein with a unique CAO activity in mammals probably results from environmental pressure.", "For example, the AOC4 proteins from O. cuniculus and S. scrofa plasma have relatively high activity towards mescaline, which is a hallucinogenic plant-derived amine, suggesting a dietary influence on the selection of the metabolized substrates (Lyles, 1996).", "Moreover, the fact that extracellular proteins, like AOC3 and AOC4, have a constant need to adapt to new functions and niches and, therefore, evolve fast (Sojo et al., 2016), supports the inferred phylogenetic tree and the relationships between AOC3 and AOC4.", "It is interesting that there is variation in the number of AOC genes even between closely related species e.g. we found a protein-coding AOC4 gene in chimpanzees and macaques, whereas human and gorilla have a pseudo gene.", "In this study, we noticed that all species from Cetacea (Supplementary Table S1) and the majority of rodents have only the AOC3 protein and the AOC4 gene is missing or non-functional.", "In fact, Ictidomys tridecemlineatus (thirteen-lined ground squirrel) and Marmota marmota marmota are the only rodents that have both AOC3 and AOC4.", "Furthermore, our results show that it is important to analyze the genomic origin of the encoded protein.", "We suggest that a CAO with a predicted AOC4 activity (X1 = Met) but originating from AOC3 should not be named as AOC3 but rather as an AOC4-like protein.", "The proposed classification system could be used to correctly annotate new mammalian CAO sequences and revise wrongly annotated sequences.", "Next we extended our study from the phylogenetic analysis to the level of the 3D structure with a particular focus on the catalytic region near TPQ (Fig. 1).", "We used the following X-ray structures as the representative structures: hAOC1 (Protein Data Bank (PDB) Identification code (ID) 3MPH; McGrath et al., 2010), hAOC3 (PDB ID 4BTX; Bligt-Lindén et al., 2013), and bAOC4 (PDB ID 1TU5; Lunelli et al., 2005), which are homodimers consisting of the D2, D3 and D4 domains (Fig. 1).", "Since no crystal structure is available for hAOC2 we built a homology model based on the hAOC3 structure (PDB: 4BTX) (Bligt-Lindén et al., 2013).", "The high sequence identity of 68% between hAOC2 and the hAOC3 template is much higher than 30%, which is generally regarded as the sequence identity criteria for an accurate homology model (John and Sali, 2003).", "The quality of the hAOC2 model is good, since 90% of the residues in the Ramachandran plot are in the favored regions and the QMEAN quality score of 0.704 (range 0–1; where 1 is good) is within the range of scores for reference structures of the same size.", "The folds of the hAOC2 model and the X-ray structure of hAOC3 are very similar with a root mean square deviation (RMSD) of 0.67 Å for the C-alpha atoms of the superimposed structures.", "In the studied CAO structures, half of the residues near TPQ are fully conserved (white in Fig. 8).", "Most of the conserved residues are involved in TPQ biogenesis and/or in the catalytic mechanism (Airenne et al., 2005; Ernberg et al., 2010; Klema and Wilmot, 2012).", "Of these residues, the catalytic Asp (D373 in hAOC1) and a nearby Tyr (Y371 in hAOC1) form the conserved Y-X-D motif.", "The corresponding Tyr has been identified as a “gate” residue in all mammalian CAOs (Salminen et al., 1998; Lunelli et al., 2005; Kaitaniemi et al., 2009; McGrath et al., 2009).", "In the hAOC3 − 2-hydrazino-pyridine (2HP) complex, the corresponding Y384 stacks with the pyridine ring of 2HP (Jakobsson et al., 2005) and docking studies also suggested that this residue interacts with the aromatic ring of benzylamine (Kaitaniemi et al., 2009).", "Therefore, it is likely that the conserved “gate” Tyr interacts with the amine substrates of all mammalian CAOs.", "Two conserved Tyrs and an Asn (Y359, Y463 and N460 in hAOC1) orient TPQ (TPQ461 in hAOC1; (Airenne et al., 2005; Ernberg et al., 2010), whereas the conserved Ser (S456 in hAOC1) forms hydrogen bonds that stabilize the active site architecture (Fig. 8).", "The rest of the strictly conserved residues originate from the T/S-X1-X2-N-Y-D active site motif, where the first residue is a Ser in most of the AOC2 proteins, but conservatively replaced by a Thr in all the other mammalian CAOs.", "The Ser/Thr residue (T457 in hAOC1) and the Asp (D462 in hAOC1) form a hydrogen bond that stabilizes the tip of the β-hairpin formed by the active site motif.", "Both of them are also involved in an extensive network of inter-monomeric hydrogen bonds together with the fully conserved Arg (R429 in hAOC1) and His (H430 in hAOC1) from the other monomer.", "A similar hydrogen bonding network has been identified e.g. in the crystal structure of Escherichia coli amine oxidase (Parsons et al., 1995) but its functional significance is currently unknown.", "Since the unique substrate preferences of each CAO sub-family is likely determined by the active site residues, which are conserved within a sub-family but variable between the sub-families, we next investigated these residues in each CAO sub-family (Fig. 8; Table 1).", "hAOC1 has a unique preference for aromatic diamines as substrates (e.g. histamine; Elmore et al., 2002) and, accordingly, the AOC1 sub-family does not group together with the other CAO sub-families (Fig. 2).", "In addition to the residues involved in the catalytic mechanism and orienting TPQ, three other residues are strictly conserved only within the AOC1 sub-family (V458, Y459 and D186 in hAOC1) (Fig. 8A; Table 1).", "Of these residues, the totally conserved Val from the active site motif (V458 in hAOC1; X1) is also conserved in the AOC2 sub-family and, based on our results, it contributes to the preference for aromatic substrates.", "The Tyr (Y459 in hAOC1; X2) and the other aromatic residue in the active site (W376 in hAOC1; Trp/Tyr in all AOC1s) are suitably positioned to form stacking interactions with the aromatic ring of the substrate (Fig. 8A).", "In addition, D186 in hAOC1 has been predicted to be involved in histamine binding, since its position is suitable for binding the second amine group of the diamine substrates (McGrath et al., 2009).", "Our results support this prediction, as the corresponding residue is an Asp in all the studied AOC1s (Fig. 8A; Table 1).", "In the crystal structure of the hAOC1-pentamidine complex (McGrath et al., 2009), S380 forms a hydrogen bond with the amidinium group of pentamidine.", "The longer diamine substrates of hAOC1, spermidine and spermine, have nitrogen in the linker region between the primary and second amines that could interact with D186, whereas the position of S380 is perfect for binding the second amine of these longer diamines (McGrath et al., 2009).", "The role of S380 in binding the second amine is supported by the fact that the corresponding residue always has a hydroxyl group (Ser/Thr) in the studied mammalian AOC1s (Table 1).", "AOC2 prefers the aromatic amine 2-phenylethylamine and also binds the polar aromatic amines tryptamine and p-tyramine (Kaitaniemi et al., 2009).", "The small variable Gly and Val residues in the active site motif, make AOC2 optimal for aromatic amines by providing the space required for binding the aromatic ring in the substrates (G463 and V462 in hAOC2; Fig. 8B; Table 1).", "Of these, the Gly has been shown to be important for the p-tyramine activity since the replacement of the corresponding Leu in hAOC3 by a Gly (L469G) acquired enzymatic activity towards the aromatic p-tyramine, which cannot be oxidized by wild-type hAOC3 (Elovaara et al., 2011).", "In the position corresponding to the conserved Asp in AOC1s (D186 in hAOC1), AOC2s have a His (H206 in hAOC2; Fig. 8B), which could interact with polar and/or aromatic substrates.", "This His can be considered as an AOC2-specific residue as it is exclusively conserved in AOC2s (Table 1), with the exception of the bat proteins where a Gln conservatively substitutes for His.", "It is worth noticing that rats do not have a functional AOC2 and, thus, mice are preferable model organisms for AOC2 studies.", "The phylogenetic and sequence analysis of the ancestral CAOs from C. milii and R. typys suggest that they could use both diamines and monoamines as substrates.", "A Gly at position X2 classifies both C. milii and R. typys CAOs as AOC2 proteins, which is in accordance with their highest sequence identities to human AOC2 and their predicted N-terminal transmembrane helices.", "Val at position X1 makes R. typys CAO even more similar to hAOC2 than C. milii CAO, which has an Ile.", "Further comparison of C. milii and R. typys CAOs with hAOC1 (Supplementary Fig. S2) reveals that they might also have diamine oxidase activity since they have a conserved Asp at the position of D186 in hAOC1 instead of a highly conserved His (H206 in hAOC2).", "AOC3 and AOC4 proteins both prefer aliphatic amines; AOC3 prefers small aliphatic amines and AOC4 long polyamines (Kaitaniemi et al., 2009; Bonaiuto et al., 2010; Elovaara et al., 2011).", "Based on the active site comparison of hAOC3 and bAOC4 (Fig. 8C, D), both proteins have a Phe (F389 in hAOC3) in the active site channel 6 Å from TPQ towards the surface a Leu (Leu469 in hAOC3; X2), which are totally conserved in all the studied proteins (Table 1).", "The Leu has been associated in blocking/allowing access to TPQ (Airenne et al., 2005; Jakobsson et al., 2005; Kaitaniemi et al., 2009; McGrath et al., 2009), and mutational studies have proven that it is involved in the substrate preference of hAOC3 (Kaitaniemi et al., 2009; Elovaara et al., 2011).", "Unlike hAOC1 and hAOC2, both AOC3 and AOC4 prefer aliphatic amines, for which Leu and Phe provide a hydrophobic environment that can well accommodate the carbon tails of the aliphatic substrates (Fig. 8C, D).", "Residue X1 from the active site motif is the only active site residue that is not conserved between the AOC3 (Leu) and AOC4 (Met) sub-families but almost exclusively conserved in each of them.", "The Met in AOC4s is slightly more polar and much more flexible than the Leu in AOC3s.", "Similar amino acid difference was found in Arabidopsis thaliana and Euphorbia lagascae sterol carrier protein-2 in which the Met/Leu difference was respectively crucial for sterol sensitivity and insensitivity (Viitanen et al., 2006).", "However, the exact role of the X1 residue in CAOs has not been studied experimentally.", "Furthermore, it has been indicated that the K393/Y392 residue difference of hAOC3/bAOC4 contributes to the substrate preference as the positively charged Lys in hAOC3 could hinder binding of long polyamines (Bonaiuto et al., 2010).", "The high conservation of the corresponding Lys in AOC3s observed in our study lends support to this suggested role in selecting preferred substrates.", "Interestingly, the residue corresponding to D186 in AOC1 (Fig. 8A) is a highly conserved Thr in AOC3s (T212 in hAOC3; Fig. 8C) and a highly conserved Asn in most of the studied mammalian AOC4s (N211 in bAOC4; Fig. 8D).", "Mutational studies on hAOC3 (T212A) have shown that T212 slightly contributes to substrate preference but merely has a role as an additional gate residue (Elovaara et al., 2011).", "Intriguingly, both AOC3 and AOC4 from I. tridecemlineatus (rodent) have an Asn in this position, whereas all primate AOC3 and AOC4 proteins have a Thr.", "It should also be noted that only human and gorilla lack AOC4 but most of the studied Primates have it, whereas I. tridecemlineatus and M. marmota marmota are the only rodent species with AOC4.", "Therefore, based on our analysis, rats and mice are better model organisms for studying AOC3 than those Primates that have both AOC3 and AOC4.", "The substrate preference of CAOs has mainly been studied for human and rodent proteins (Lyles, 1996; Elmore et al., 2002; Zhang et al., 2003; Shen et al., 2012; Finney et al., 2014).", "Rodents have been used as model organisms in human AOC3-related diseases, drug design and in vivo imaging applications, whereas the research on AOC4 has focused on bAOC4.", "Comparison of rodent and primate CAO proteins suggests that for binding studies of small substrates and inhibitors binding covalently to TPQ, rodents and other primate species can serve as good model organisms for human CAOs.", "In drug design projects, problematic species-specific differences in ligand binding between human and mouse AOC3 have been demonstrated for a number of AOC3 inhibitors (Foot et al., 2012; Bligt-Lindén et al., 2013; Inoue et al., 2013a; Inoue et al., 2013b).", "These problems are mainly caused by residue differences in the 15 Å long active site channel of CAOs, which leads from TPQ to the surface of the protein.", "Therefore, our analysis suggests that the active site channel in CAOs from model organisms should be studied in detail when larger substrates and inhibitors are designed.", "In addition to the species-specific binding properties, the other CAO proteins expressed in the used model organism should also be taken into consideration in the CAO-related research.", "In this work, we combined the phylogenetic analysis of vertebrate CAOs with the multivariate plots of mammalian CAOs to suggest a novel way to classify mammalian CAOs based on their active site motif.", "We conclude that residue X2 from the X1-X2-N-Y-D motif separates the mammalian CAOs in the AOC1, AOC2 and AOC3/AOC4 sub-families and residue X1 can be used to discriminate between the AOC3 and AOC4 proteins.", "Moreover, we aimed to understand what makes each mammalian CAO unique and, by relating phylogenetic analyses with sequence and structural data, we gained new insights into the substrate preference of mammalian CAOs.", "Residues X1 and X2, which are conserved within each CAO sub-family and located in the vicinity of the catalytic site, can be considered as the key determinants for selective substrate binding.", "Both AOC1 and AOC2 that uniquely prefer aromatic substrates have a Val at position X1.", "In AOC1, residue X2, which is a conserved Tyr, might be the most important residue for its preference for aromatic diamines.", "Additionally, two other key positions are conserved in the AOC1 sub-family but variable in the other sub-families.", "The totally conserved Asp (D186 in hAOC1) and the conservatively replaced Ser/Thr (S380 in hAOC1) could bind the second amine group in shorter and longer diamine substrates, respectively.", "The position corresponding to the totally conserved Asp is also highly conserved in the other sub-families (a His in AOC2, a Thr in AOC3 and an Asn in AOC4) and, thus, it seems to have an effect on the substrate selectivity in the other subfamilies as well.", "In AOC2, the highly conserved His (H206 in hAOC2) and the totally conserved Gly (X2), which creates space for large aromatic substrates, form the foundation for substrate preference.", "The respective Thr and Asn residues in AOC3 (T212) and AOC4 (N211) likely contribute to their substrate selectivity together with the X1 residue (Leu in AOC3 and Met in AOC4) whereas the X2 residue (Leu) makes them distinctively prefer aliphatic rather than aromatic substrates.", "Due to the species-specific expression profile of CAOs, our results also highlight that it is important to be aware of the presence of all CAO family members expressed in the studied organism, even thought the research would focus on one of the sub-families." ]

[ "Active site motif classifies CAOs to functional sub-families.", "Variable residues from the active site motif contribute to substrate selectivity.", "One sub-family specific residue seems to be important for substrate preference.", "Expression profile of mammalian AOC1, AOC2, AOC3 and AOC4 is species-specific." ]

[ "MULT" ]

[ "Mammaplasty", "Self–esteem", "Sexual satisfaction", "Women" ]

[ "1000" ]

[ "Out of 100 investigated couples, 3% of the women were under 20 years of age, 17% between 20–30 years, 49% between 30–40 years and 31% over age of 40.", "About 57% of their spouses were employees and 43% were self-employed.", "About 19% of the couples had no children, 19% had one child, 55% had two children and 7% had more than two children.", "About 66% underwent a breast reduction surgery, 19% breast augmentation and 15% underwent mastopexy.", "About 19% predicted to achieve beauty less than 30%, 41% between 30–50%, 34% between 50–70% and 6% predicted to achieve over 70% of beauty.", "About 16% of the women had history of a plastic surgery while 84% did not.", "Meanwhile, 5% of the women had a history of plastic surgery in their immediate family members while 95% did not.", "About 53% of the women had a history of surgery in their friends and relatives while 47% mentioned no history.", "Table 1 presents mean (SD) of couples’ self-esteem and sexual satisfaction before and after mammaplasty surgery.", "Mean score of self-esteem was 18.77 (moderate) before surgery in 100 participants, and it remained steady (17.96) after the surgery (moderate).", "Table 2 shows the mean (SD) of couples’ self-esteem and sexual satisfaction before and after mammaplasty surgery.", "Women׳s sexual satisfaction was increased after mammoplasty; however, their satisfaction did not change.", "A pretest -posttest design with only one group was contacted among 100 clients referring to 5 hospitals affiliated to Kermanshah University of Medical Sciences for a mammaplasty surgery in 2013.", "100 couples were selected by purposive sampling, when referring to the hospitals affiliated to Kermanshah University of Medical Sciences.", "Participation inclusion criteria included being married with at least one year of marriage length, living with their spouse, having reading and writing literacy, being a candidate for mammaplasty surgery, and referring to hospitals in Kermanshah province.", "The couples with the history of a diagnosed mental disease, taking psychotropic medications and those with unpleasant postoperative complications were left out of study.", "The first contained participants’ demographic characteristics including women׳s and their spouses’ level of education, women׳s and their spouses’ occupational status, number of children, type of surgery, prediction of beauty percentage, history of any previous plastic surgeries among the participants and their immediate family members, relatives and friends.", "To establish the validity of demographic questionnaire, content validity was used through taking indications of 10 academic members.", "The second questionnaire was couples’ sexual satisfaction questionnaire, which is a self-assessment tool to measure the level of marital satisfaction in sexual issues and includes items on sexual satisfaction, sexual shyness, sexual trenchancy, sexual anxiety, sexual health, and fear and sexual compulsion in couples’ marital relationship.", "This questionnaire was designed in two separate forms for women and men.", "The men׳s version contains 28 items which are scored in a five point Likert scale (absolutely agree, agree, no idea, disagree, absolutely disagree) ranging between 0–4 points.", "Each subject is scored between 0–108.", "Scores 0–27 show low sexual satisfaction; 28–54 show moderate, 55–81 show good sexual satisfaction,and scores 82–108 show high sexual satisfaction.", "The women׳s version of this questionnaire contains 17 items, scored in a five- point Likert scale (absolutely agree, agree, no idea, disagree, absolutely disagree), ranging between 0–4 points.", "Participants’ total score ranges 0–68.", "Scores 0–17 show low sexual satisfaction; 18–34 show moderate; 35–51 show high and scores 52–68 show very high sexual satisfaction.", "To check reliability of the women׳s and men׳s sexual satisfaction questionnaires, Cronbach׳s alpha method was adopted.", "In the present study, reliability of this tool was calculated 0.84 through Cronbach׳s alpha in a 20- male subject sample in a pilot study and 0.83 in a 20-female sample.", "The third questionnaire was Rosenberg Body-esteem Scale containing 10 general items (five items with negative and five items with positive sentences).", "Each item is scored with a four- point Likert scale (absolutely agree, agree, disagree, absolutely disagree) ranging between 0–3 with total scores of 0–40.", "Scores 0–10 show very low self-esteem; 10–20 show moderate; 20–30 show high and score, 30–40 show very high self-esteem.", "In the present study, internal consistency of the tool was calculated 0.82 through Cronbach׳s alpha of 0.82 on 20 participants.", "After getting permission from research chancellery of Kermanshah University of Medical Sciences and attaining the consent from the participants, the questionnaires were administered among the participants in an appropriate place, and they were explained about the goals of study and the way to fill the questionnaires.", "The participants were investigated before the surgery and two months after, and finally, the obtained data were analyzed.", "All obtained data in the present study were confidential and the participants were assured about their anonymous questionnaires.", "Collected data were summarized using descriptive statistics namely absolute (n) and relative (%) frequencies for categorical variables and mean and standard deviation (SD) to study the level of women׳s self-esteem and couples’ sexual satisfaction before and after mammaplasty," ]

[]

[ "BIOC", "MEDI" ]

[ "Cardiac", "Hypoxia", "Mitochondria", "Mitofusin", "Neonatal" ]

[ "1312", "2705" ]

[ "Fetal cardiomyocytes are adapted to function in low levels of oxygen in utero, whereas adult cardiomyocytes are extremely vulnerable to hypoxia, as evidenced by high rates of mortality and morbidity caused by ischemic heart disease [1].", "Shortly after birth, cardiomyocytes lose their capability to adapt to hypoxic conditions.", "The process underlying this postnatal maturational process is not understood.", "It is thought that fetal cardiomyocytes generate ATP predominantly by glycolysis and glucose oxidation [2].", "At birth, ambient oxygen levels increase sharply as the neonate emerges into the terrestrial environment.", "Immediately after birth, cardiac output, and therefore cardiac energy requirements, increase rapidly [3].", "This increase is achieved through switching from glycolysis to β-oxidation of lipids, which generates larger amounts of ATP per unit of substrate than glycolysis [2,4], and is accompanied by an increase in mitochondrial number [5].", "Recently, our understanding of molecular mechanisms controlling postnatal cardiac bioenergetics and mitochondrial remodeling has advanced, but physiological stimuli controlling these processes remain to be defined.", "This information is of interest with respect to future strategies to protect adult cardiomyocytes in ischemic heart disease and to increase our understanding of human cardiac conditions presenting around birth.", "Mitochondrial biogenesis is upregulated following birth as a consequence of increases in Pgc1α/β expression [5], although the physiological stimuli for this are yet to be described.", "Furthermore, it is recognized that mitochondrial morphology is intimately linked with function, fusion of mitochondria being associated with increased β-oxidation of lipid [6,7].", "Fetal mitochondria are “fragmented” in appearance, whereas postnatal mitochondria appear elongated, consistent with the metabolic switch occurring in the heart flowing birth [2].", "Mitofusin proteins (MFNs) are implicated in the control of postnatal mitochondrial fusion, although, again, the relationship to physiological changes around birth is not known [8].", "We investigated the mechanisms controlling neonatal cardiac mitochondrial remodeling at birth.", "Immediately following birth, ambient levels of oxygen encountered by the neonate increase significantly [9].", "We have recently shown that the postnatal decrease in cardiac hypoxia signaling controls the lipid metabolizing gene expression program [10] and postnatal remodeling of the cardiac conduction system [11].", "Additionally, cell culture experiments have shown that overexpression of hypoxia inducible factor (HIF) leads to diminished respiratory capacity and inhibited mitochondrial biogenesis [12,13].", "We therefore hypothesized that the increase in cardiac oxygen tension acts, via HIF signaling, as a developmental “switch”, driving postnatal maturation of the heart.", "We investigated the effects of the reduction in hypoxia signaling following birth on the structure and function of cardiac mitochondria.", "Here we describe data supporting a novel hypothesis that the postnatal increase in oxygen tension encountered by the neonate acts as a physiological trigger for mitochondrial maturation in the heart.", "We found that mitochondrial morphology changes dramatically around birth, with mitochondria assuming an elongated form postnatally.", "These changes in mitochondrial morphology are dependent on a reduction in cardiac HIF signaling, and that HIF-dependent changes in expression of MFN1 and OPA1 may mediate this.", "The postnatal increase in cardiac mitochondrial biogenesis is also dependent on a decrease in HIF signaling.", "Postnatal downregulation of HIF signaling also leads to increased respiratory capacity and oxidative phosphorylation.", "This work has implications for medical conditions such as cyanotic congenital heart disease, where tissues are inadequately oxygenated following birth due to abnormal cardiac anatomy.", "This work focuses on cardiac mitochondrial changes around birth; it is not yet known how these relate to mechanisms regulating adult cardiac mitochondrial function and morphology.", "We found no evidence that the postnatal increase in transcription of Pgc1α is dependent on a decrease in HIF-signaling, in agreement with our earlier finding that upregulating of HIF1α in HL1 cells does not lead to an increase in Pgc1α protein levels [10].", "Our finding that the rise in Pgc1α mRNA and Pgc1α protein levels is attenuated in mice born into low levels of ambient oxygen agrees with the finding that Pgc1α expression is hypoxia dependent, but HIF-independent in mouse skeletal muscle [26].", "Interestingly, the changes seen in the heart at birth are the reverse of skeletal hypoxic adult skeletal muscle—i.e. hypoxia in utero inhibits expression of Pgc1α.", "This does leave the question open as to what does transcriptionally regulate Pgc1α in the heart around birth.", "Certainly increases in expression of Pgc1α are coincident with those of MFN1 and MFN2 in the heart around birth, and the phenotypes of Pgc1α/β and MFN1/2 null mice have similarities [5,8].", "However, our findings that postnatal increased Pgc1α transcription is hypoxia- but not HIF-dependent, suggest parallel regulatory pathways.", "Our finding that postnatal levels of pgc1β in the mouse heart are reduced in VHL null hearts supports the finding that HIF negatively regulates pgc1β expression in renal carcinoma cells [27].", "Immediately following birth, in addition to changes in ambient oxygen/HIF signaling, the ambient temperature of the neonate drops, and levels of several hormones and signaling pathways change, although these changes remain poorly defined.", "Many of these changes have been identified as stimuli to Pgc1α expression in the context of “stress” [28], and are thus candidates for regulatory triggers for neonatal cardiac maturation.", "We are still some way away from a complete understanding of the physiological regulation of neonatal cardiac remodeling.", "It is unclear whether the HIF system is maximally activated at 10% FiO2.", "We chose this oxygen concentration as it is the lowest level before significant mortality makes analysis impossible.", "Death rates of wide type neonatal mice at 10% FiO2 are still significantly higher than cardiac VHL null pups, however, and it is unclear whether any observed cardiac effects in these mice are cardiac-specific rather than due to the global effects of hypoxia.", "Our understanding of mitochondrial function in the heart lags behind that of mitochondria in cell-types such as neuron, possibly because of the difficulty in culturing and studying cardiomyocytes.", "We believe that study of the changes occurring in the heart around birth will afford an insight into the endogenous physiological mechanisms adapting the embryonic heart to extremely low levels of oxygen in utero.", "This information may be of use in devising novel strategies to protect vulnerable adult cardiomyocytes, for example during myocardial infarction.", "The changes in, and regulation of gene expression in the heart around birth are not fully understood.", "Our chromatin immunoprecipitation data suggest that there could be significant remodeling of cardiac chromatin immediately following birth, and that this may be HIF dependent.", "Oxygen-dependent chromatin modification has been described [29].", "The role of chromatin modification versus direct HIF1α promoter binding in the transcriptional control of Mitofusin expression is as yet unclear, and will be necessary to define completely the control of neonatal Mitofusin expression by promoter analysis.", "Analysis of genomic binding sites of HIF1α led to the conclusion that HIF1α acts as a transcriptional activator for the vast majority of putative targets [30].", "However, some examples of HIF1α acting as a repressor have been published [10,27,31].", "Our data suggest that HIF1α is acting as a transcriptional repressor in the context of neonatal cardiac Mitofusin expression, although formal proof of this is needed.", "We found no difference in HIF1α protein levels in E18.5 VHL null and control hearts, implying that the HIF system is maximally active in the fetal heart (Supplementary Fig. 6).", "This, however, remains to be investigated formally.", "Furthermore, it should be noted that deletion of the VHL gene has HIF-independent effects on gene expression [32].", "This is therefore a potential limitation of our study.", "Our data suggest that the predominant mitochondrial site of adaptation to hypoxia is electron transport complex 1.", "Complex 1 has been found to be a target for HIF signaling in cardiac cells [33,34].", "Chronic exposure of rats to low ambient levels of oxygen leads to adaptive changes in ROS generation along with decreases in activity of mitochondrial complexes I, II and IV.", "The relatively low density of mitochondria in embryonic cardiomyocytes is arguably an adaptation to low levels of oxygen, leading to lower ROS generation.", "This does raise the possibility that therapies directed towards inhibiting cardiac mitochondrial function may be beneficial in ischemic adult myocardium.", "Indeed, it could be argued that the field of ischemia protection via preconditioning and/or opening of the mitochondrial permeability transition pore (MPTP) is exactly this—inhibiting mitochondrial function in a similar manner to the embryonic cardiomyocyte.", "We found that ATP is generated in VHL-null cardiomyocytes predominantly by oxidative phosphorylation with a small contribution of glycolysis.", "Importantly, despite the inhibition of mitochondrial respiration in hypoxic cardiomyocytes, oxidative phosphorylation is not completely inhibited, and we hypothesize that hypoxic cardiomyocytes are able to utilize a number of mitochondrial substrates allowing levels of oxidative phosphorylation sufficient to maintain ATP production.", "Our work outlining a novel mechanism whereby VHL-knockdown HL1 cells generate ATP and maintain an inner mitochondrial membrane gradient on a background of constitutive hypoxia signaling provides a mechanistic framework for future studies investigating how fetal cardiomyocytes are adapted to extreme hypoxia, and potential novel therapeutic approaches to cardioprotection.", "We would predict that treatments aimed at boosting mitochondrial ATP production rather than saving oxygen might benefit ischemic cells in some circumstances.", "Finally, our data together with those of others, suggest that the embryonic myocardium is adapted to function in low levels of uterine oxygen by a number of parallel mechanisms.", "We have shown that postnatal expression of the genes encoding lipid-metabolizing genes is upregulated postnatally by an HIF-dependent mechanism [10].", "Mitochondrial number is reduced in embryonic myocardium relative to neonatal, and activity of mitochondrial complex 1 and 4 is inhibited in the embryonic heart and hypoxic cardiac tissue.", "This suggests that a combination of several therapeutic strategies may be necessary to protect ischemic adult myocardium adequately.", "In situ measurement of mitochondrial function in utero is not currently possible without exposing the cells to oxygen, leading to artifacts due to oxygen-induced maturational processes.", "We therefore studied hearts from mice born into normobaric hypoxia (Fi02 10%) and αMHC-Cre::VHL(fl/fl) mice which maintain constitutively high levels of HIF signaling in the presence of normoxia [14] in order to inhibit postnatal maturational processes dependent on postnatal reductions in HIF signaling.", "We also generated stable HL1 cell lines transfected with an shRNA construct directed against VHL mRNA, which has a similar effect of stabilizing HIF expression in normoxia (Supplementary Fig. 1).", "Perinatal changes in mitochondrial morphology have been observed in the mouse [15] and rabbit [16] from day 3 onwards and in the guinea pig [17] sheep [18], and mouse [8] around birth.", "These studies used electron microscopy, and consistently observed a shift in mitochondrial morphology from many small and ovoid to fewer long and rectangular mitochondria with an increase in proximity between mitochondria and the myofibrils with time after birth.", "In concordance with these previously published studies, we observed that mitochondria change shape from small and round before birth to larger and rectangular following birth (Fig. 1A and Supplementary Fig. 2).", "We estimated that percentage area occupied by mitochondria in the cell increases from E16.5 (10%) to P10.5 (34%), with the largest increase occurring in the first few days after birth between P0.5 and P2.5 (Fig. 1B).", "We observed that the mitochondrial cristae become more dense and uniform between E16.5 and P10.5, with an observable decrease in glycogen storage granules sequentially from E16.5 to P10.5 and lipid droplets observed on micrographs from the day of birth onwards (P0.5) (Fig. 1C and Supplementary Fig. 2).", "We noted that mitochondrial morphology changes during the perinatal period.", "Mitochondrial matrix in P2.5 mitochondria appears less dense, and sarcomeres more dense and defined on EMs compared with E18.5 mitochondria (Fig. 1D and Supplementary Fig. 2).", "We went on to examine whether perinatal changes in cardiac mitochondrial morphology are dependent on changes in ambient oxygen levels and hypoxia signaling immediately following birth.", "We found that interpreting mitochondrial morphology in electron micrographs was difficult, due to plane of sectioning issues.", "Therefore, we used primary cultures of cardiomyocytes, which enable the use of vital dyes to assess mitochondrial morphology more sensitively than electron microscopy (Fig. 2A).", "Primary culture of embryonic cardiac cells involves removal from a hypoxic environment in vivo and exposure to potentially artifactually high levels of oxygen in culture.", "We therefore assessed the effect of cardiomyocyte culture time on fusion phenotype of the heart cells.", "We digested and plated E18.5 embryonic hearts and found that the length of time in culture increased the proportion of fused mitochondria until 8 h post-plating (p = 0.004) (Fig. 2B).", "At a culture time of 18 h the association reversed.", "This emphasizes the difficulty in interpretation of mitochondrial morphology after prolonged periods of cell culture.", "Cardiomyocyte viability was not compromised by 45-min incubation with MitoTracker dye: at 24 h after MitoTracker treatment, we observed adherent cells, often displaying synchronized beating.", "Observers blinded to their origin examined primary cell cultures of cardiomyocytes, after 45-min of MitoTracker dye treatment in cells one hour after plating.", "We found an increase in the proportion of cardiac cells exhibiting mainly fused mitochondria from E18.5 to P10.5.", "We observed an increase in the numbers of fused mitochondria after birth with only 27% mitochondria being fused at E18.5 compared to 62% fused at P2.5 (p = 0.05) (Fig. 2C).", "At birth, the mammalian embryo encounters a sharp increase in ambient oxygenation [9].", "We have previously shown that cardiac levels of HIF1α protein, a key mediator of hypoxia signaling fall, but are elevated in neonatal hearts homozygous null for VHL [10].", "We therefore investigated whether neonatal changes in cardiac HIF signaling drive the changes in cardiac morphology, number and function in the neonatal heart.", "To assess the impact of hypoxia signaling on mitochondrial morphology we examined electron micrographs of hearts from postnatal day 10.5 (P10.5) αMHC-Cre::VHL(fl/fl) mice.", "Mitochondria from αMHC-Cre::VHL(fl/fl) mice appeared immature with respect to controls (Fig. 2D).", "The image in Fig. 2D is taken from a αMHC-Cre heart (constitutive cre expressing), which is on a C57BL/10 background as opposed to the (CBA/Ca × C57BL/10) strain used for the wild-type analysis outlined in Fig. 1 and Supplementary Fig. 2.", "This strain difference could explain the different mitochondrial morphology observed between these two images.", "Further examples of mitochondrial morphology are pictured in Supplementary Fig. 2.", "In primary cultures of cardiomyocytes using Mitotracker dye, we found that mitochondria from P10 αMHC-Cre::VHL(fl/fl) hearts exhibited significantly higher numbers of fragmented-appearing mitochondria compared with controls (p < 0.001) (Fig. 2E).", "Mitochondrial morphology is regulated by a family of mitochondrial fusion and fission proteins [8,19].", "We therefore investigated expression of the fusion proteins, MFN1, MFN2 and OPA1, and fission proteins, DRP1 and FIS1 at regular intervals from E16.5 to P10.5.", "Western blotting of protein extracts of ventricular tissue revealed an increase in fusion protein expression from E16.5 in a stepwise manner through to P10.5 with the largest increase taking place between P0.5 and P2.5 in MFN1 and MFN2 (p = 0.05) and between P2.5 and P10.5 in OPA1 (p = 0.05) (Figs. 3A–C, representative blots in Supplementary Fig. 3).", "There were no changes detectable in fission protein DRP1 and FIS1 levels over this time period (Supplementary Fig. 4).", "We found that in αMHC-Cre::VHL(fl/fl) hearts, expression of fusion proteins MFN1 and OPA1 was significantly reduced compared with controls (P < 0.001 and p = 0.04 respectively, 2 tailed t tests n = 4 hearts each group) (Fig. 3D).", "We analyzed cardiac mRNA expression of MFN1, MFN2 and OPA1.", "We found that levels of all three mRNAs increase significantly in the heart at birth, and this rise is significantly attenuated in hearts from αMHC-Cre::VHL(fl/fl) neonates (Figs. 3E–G).", "These data suggest that postnatal HIF-dependent mitochondrial fusion could be mediated by increases in MFN1, MFN2 and OPA1 expression.", "Interestingly, no significant change in mRNA levels of MFN1, MFN2 and OPA1 was seen in adult inducible, cardiac specific VHL deleting mercremer::VHL(fl/fl) hearts, following 5 days of tamoxifen treatment.", "We went on to examine whether HIF1α binds to the 5’ promoters of MFN1, MFN2 and OPA1.", "We analyzed published 5’ promoter sequences for these 5 genes, and found several canonical (A/G)CGTG HIF1α binding sites in each case (Fig. 4A).", "We assayed whether HIF1α directly binds these promoters using anti HIF1α serum to perform chromatin immunoprecipitation.", "We found that in each case, chromatin containing at least one HIF1α-binding site promoter fragment was enriched 20 fold over non-amplified sequences in chromatin from embryonic αMHC-Cre control hearts (Figs. 4B-D), implying that HIF1α binds to these sites in vivo.", "Chromatin Immunoprecipitation with anti-HIF1α serum in chromatin from P2.5 control hearts revealed differences in binding of HIF1α to several sites in these three promoters (Figs. 4E-F).", "These changes were not seen in their entirety in chromatin from P2.5 αMHC-Cre::VHL(fl/fl) hearts (Fig. 4H–I).", "We performed immunoprecipitation for each promoter site in e18 cardiac chromatin using an isotype control IgG.", "We found no significant specific binding of this antibody to any promoter site.", "This implies that chromatin remodeling occurs in the heart after birth, and that, to a degree, this remodeling may be affected by HIF signaling.", "We went on to investigate whether the previously observed increase in cardiac mitochondrial mass after birth is also dependent on HIF-signaling.", "We examined the mitochondrial: nuclear DNA ratio from DNA extracted from cardiac ventricular tissue at E18.5 (shortly before birth) and P2.5.", "We found a significant increase in the mitochondrial: nuclear DNA ratio between E18.5 and P2.5 in wild-type mouse hearts (Fig. 5A) (p < 0.01).", "This increase was not seen in hearts from αMHC-Cre::VHL(fl/fl) neonates or from mice born in 10% ambient oxygen, implying that postnatal cardiac mitochondrial biogenesis is dependent on downregulation of HIF signaling (Fig. 3A).", "We found that increased postnatal expression of several mitochondrial-encoded mRNAs is also inhibited in αMHC-Cre::VHL(fl/fl) hearts and hearts from P2.5 mice born and reared in 10% oxygen (hypoxia) (Fig. 5B).", "We also examined myocardial citrate synthase activity in αMHC-Cre::VHL(fl/fl) and control hearts.", "Citrate synthase activity has been shown to reflect the overall volume of mitochondria present in a sample, a proxy for mitochondrial number [20].", "We found that citrate synthase activity increases 2.5 fold between E16.5 and P2.5 (p = 0.0003, n = 6 each group) in wild type mouse hearts consistent with our finding of an increased proportion of cardiomyocytes occupied by mitochondria and increased mitochondrial: nuclear DNA ratio postnatally (Fig. 5C).", "Examination of hearts from αMHC-Cre::VHL(fl/fl) P2.5 mice revealed no postnatal increase in citrate synthase activity, implying that a drop in postnatal HIF signaling is required for increased mitochondrial biogenesis.", "The Pgc1α/β proteins have been shown to control neonatal mitochondrial biogenesis [5].", "We therefore examined whether postnatal changes in HIF signaling and/or ambient oxygen levels lead to altered expression of Pgc1α/β.", "We found a postnatal increase in Pgc1α mRNA expression following birth in control hearts, as previously reported [5]; we found no significant difference in Pgc1α mRNA expression between P2.5 αMHC-Cre::VHL(fl/fl) and control hearts (Fig. 5D).", "However, we observed a significant decrease in Pgc1α RNA expression in cardiac ventricles from mice born in 10% ambient oxygen compared with normoxic controls (p = 0.019, 2 tailed t-test, 4 hearts each group).", "Pgc1β mRNA expression was decreased in ventricles from both αMHC-Cre::VHL(fl/fl) and wild-type hypoxic mice compared with controls (Fig. 5D).", "Western blotting of protein extracts from ventricles of E16.5 and P2.5 αMHC-Cre::VHL(fl/fl) and control mouse hearts revealed that the pattern of Pgc1α/β protein expression broadly mirrors that of mRNA in these hearts (Fig. 5E).", "This implies that Pgc1α expression in the heart around birth is not dependent on HIF signaling, whereas Pgc1β expression is.", "Taken together, our data suggest that postnatal increases in mitochondrial mass and changes in mitochondrial morphology are driven by increased ambient oxygen levels and decreased HIF signaling immediately after birth.", "The exact molecular mechanisms underlying this hypoxia-dependent mitochondrial biogenesis remain unclear.", "We went on to investigate the effects of high levels of HIF signaling on mitochondrial function in the embryonic heart.", "It is well known that increases in HIF1α expression in a wide variety of cell types induce metabolic changes that lead to lower oxygen consumption [21].", "We therefore examined mitochondrial function and oxygen consumption in primary cultures from αMHC-Cre::VHL(fl/fl) and control hearts, as well as HL1 cardiomyocyte cell lines stably transfected with an shRNA construct directed against VHL.", "Application of the mitochondrial uncoupler FCCP (1 μM) to cells maximizes respiration and completely oxidizes the mitochondrial pool of NADH, manifesting as a decrease in fluorescence (taken as 0) (Fig. 5A).", "Conversely, the complex IV inhibitor NaCN (1 mM) suppresses respiration by preventing NADH oxidation and allowing the NADH pool to regenerate fully (taken as 100).", "The redox index is expressed as a percentage of basal NADH autofluorescence between 0 and 100 [22].", "Cardiomyocytes isolated from E16.5 αMHC-Cre::VHL(fl/fl) hearts exhibited an increase in mitochondrial NADH redox index when compared to controls (Figs. 6A–B) suggesting an inhibition of mitochondrial respiration.", "Interestingly, the rate of regeneration of the NADH pool in response to NaCN addition was much slower in VHL knockdown cells than the corresponding rate in control cells (Fig. 6A) suggesting a mild inhibition of NADH production in the tricarboxylic acid cycle (TCA).", "Despite the slower NADH production the total mitochondrial pool of NADH (estimated as a difference in signal between application FCCP and NaCN) was significantly higher than control cells (Fig. 6C).", "This suggests that the inhibition of mitochondrial respiration in VHL null cells (i.e. with constitutively active HIF signaling) is not due to lack of substrate for complex I.", "Mitochondrial respiration is dependent on the mitochondrial membrane potential (Δψm).", "Using live cell imaging and the fluorescent dye tetramethylrhodamine methyl ester (TMRM), we measured the Δψm, a universal indicator of mitochondrial health and the metabolic state of the cell.", "We observed a dramatic decrease in the level of Δψm in VHL-knockdown HL1 cells (to 60% actual of control, n = 14 cells per genotype) and even lower in αMHC-Cre::VHL(fl/fl) cardiomyocytes (to 25% of control, n = 12 cells; Fig. 6D).", "In healthy cells, Δψm is maintained by the activity of the mitochondrial respiratory chain.", "In the event of damage or an inhibition of mitochondrial respiration, cells may maintain Δψm using ATP hydrolysis through the ATP synthase [23].", "In order to investigate the mechanism of maintenance of Δψm in hypoxic cardiomyocytes, mitochondrial inhibitors were applied and their effects on Δψm were observed.", "Application of the F0-F1-ATP synthase inhibitor oligomycin (2 μg/ml) did not significantly change Δψm, while subsequent inhibition of complex I by rotenone (1 μM) caused a rapid loss of potential (Fig. 6E).", "Despite the low Δψm, VHL knockdown cells did not depolarize in response to oligomycin but showed a small hyperpolarization, suggesting that the mitochondria produce ATP in conditions of a low Δψm (Fig. 6F).", "Subsequent application of rotenone and FCCP led to a drop in the signal in response to the inhibitor of complex I that was greater in VHL knockdown cells when compared to control cells.", "In VHL knockdown cells, a lower response to FCCP at the end of the experiment suggests that complex II plays a smaller part in the maintenance of Δψm as previous application of oligomycin and rotenone leaves complex II as the only electron donor under these conditions (Fig. 6G).", "Measurement of cell respiration using a SeahorseXF analyzer showed lower levels of oxygen consumption in αMHC-Cre::VHL(fl/fl) cardiomyocytes (Fig. 7A).", "The inhibitory effect of oligomycin (which reflects respiration coupled to ATP production) was also significantly lower in VHL null cells in comparison to controls cells implying lower levels of oxidative phosphorylation (n = 5 assays for each genotype, experiment repeated 3 times).", "This difference was also reflected in the maximal rate of oxygen consumption (measured after stimulation with the uncoupler FCCP), which was also significantly reduced in αMHC-Cre::VHL(fl/fl) cardiomyocytes (Fig. 7A), implying that downregulation of cardiac HIF signaling is necessary for postnatal mitochondrial remodeling.", "We performed oxygen flux analysis on VHL knockdown and control HL1 cells and found a similar result (Supplementary Fig. 5).", "The reduced respiration and its insensitivity to oligomycin, lower mitochondrial membrane potential in VHL null cardiomyocytes indicate that oxidative phosphorylation could be impaired in these cells.", "We therefore compared the ATP levels in control and VHL knockdown HL1 cells with the ATP sensing probe Cox AT1.03 [24,25].", "Surprisingly, we found no significant difference in mitochondrial ATP production between control (empty vector) and VHL knockdown HL1 cells (Fig. 7B).", "We also investigated the impact on ATP production of inhibiting oxidative phosphorylation (by inhibition with oligomycin, 2 μg/ml) or glycolysis (by inhibition with iodoacetic acid (IAA), 20 μM).", "Compared to empty-vector transfected controls, the basal level of ATP in VHL knockdown cells showed no significant difference (n = 6) (Figs. 7C, D).", "Most importantly, the use of inhibitors revealed that the mechanism of ATP production differs between VHL knockdown and control cells.", "As expected, in control cells oligomycin depleted all ATP and IAA had no further effect (Fig. 7C).", "In VHL-knockdown cells, oligomycin induced a decrease in ATP levels but it also significantly induced a drop in ATP in response to the inhibitor of glycolysis IAA (Figs. 7D-E), implying that VHL-knockdown cells rely not only on oxidative phosphorylation but also heavily on glycolysis for their ATP production.", "Interestingly, pre-incubation of VHL-knockdown cells with substrates for mitochondrial respiration (5 mM glutamate, 5 mM malate, 5 mM succinate and 1 mM palmitine–carnitine) did not change the level of [ATP] but completely blocked the effect of IAA on [ATP] (Fig. 7F).", "Thus, a small proportion of the glycolysis to ATP production in VHL-knockdown cells can be inhibited by recovery of oxidative phosphorylation by addition of mitochondrial substrates.", "We suggest that provision of several substrates stimulates oxidative phosphorylation and reduces the need for glycolysis in hypoxic cardiomyocytes.", "All mouse experiments were carried out in compliance with institutional ethical and welfare standards in accordance with Home Office Animal Procedures Act (1986).", "The transgenic mouse strain, αMHC-Cre::VHL(fl/fl) on a C57BL/10 background is previously described [11].", "PCR amplification was performed on tail-derived genomic DNA to determine genotype.", "αMHC-Cre mouse hearts were used as controls in this study, unless otherwise stated.", "Tamoxifen was administered to mercremer::VHL(fl/fl) and mercremer control mice intraperitoneally dissolved in vegetable oil at a dose of 1 mg for 5 consecutive days starting at 30 days old, and were analyzed 30 days after the first tamoxifen injection.", "Hearts were isolated from E10.5–E18.5 embryos from timed-mated pregnant mice, and from newborn mice, between 0.5 and 10.5 days old.", "Paired tail samples were collected for PCR genotyping.", "Left ventricular sections were prepared for electron microscopy as described previously and imaged using a Jeol 1200EX Transmission Electron Microscope with a Gatan Orius 1000 CCD.", "Mitochondrial density was calculated by the number of mitochondria in a given area (128 μm2) at 2000 × magnification.", "The percentage area occupied by mitochondria was estimated by the density multiplied by the average length and width of the mitochondria, divided by the image area.", "An average over ten pictures was taken for each heart and three hearts were used per time point.", "Pictures were taken by serial panning and the user was blinded to the sample type.", "Hearts were isolated from E10.5–E18.5 embryos from timed-mated pregnant mice, and from new born mice, between 0.5 and 10 days old.", "Paired tail samples were collected for PCR genotyping.", "Hearts were individually placed in a labeled tube containing 300 μl of warm 1 mg/ml Collagenase II solution (Worthington Chemicals), which was then transferred to a shaking incubator (37 °C) for 10 min.", "Collagenase was then removed and the hearts washed three times in warm Claycomb medium (Sigma-Aldrich).", "MitoTracker green (Life technologies), a green-fluorescent mitochondrial stain, was prepared to a 1 μM DMSO solution as per the manufacturer's specifications, and added to tissue culture medium at a concentration of 0.2 μl/ml.", "500 μl of medium was transferred to the hearts in sterile tubes and a p1000 Pasteur pipette was used to disrupt the tissue and disaggregate the cells until there were no visible clumps of cells.", "The cells were plated onto a glass-bottomed petri dish (Mattek Corp) coated with 1% gelatin (Difco) and the volume of medium was made up to 2 ml total.", "The plate was incubated at 5% CO2 and 37 °C for 45 min after which the medium was washed and replaced with phenol-red free Dulbecco’s Modified Eagle’s Medium (DMEM).", "A Deltavision RT system based on an inverted Olympus IX70 microscope (Applied Precision Inc.), inside a heated chamber (Solent) at 37 °C, was used to capture single cell images using the Fluorescein IsothioCyanate (FITC) excitation and emitter filters and a 100× oil objective lens.", "MitoTracker Green signal is not sensitive to changes of mitochondrial conditions including membrane potential and redox status.", "Between 20 and 30 cells were imaged per heart sample.", "Images were randomized, anonymized, and then scored blindly and independently by two experts in mitochondrial morphology as either predominantly fragmented or fused.", "The scores were averaged.", "Hearts from embryos and neonatal mice were dissected in cold PBS.", "Hearts were homogenized in homogenization buffer (10% weight: volume) (PBS supplemented with protease and phosphatase inhibitors, Thermo Scientific).", "Samples were boiled and denatured in loading buffer containing 5% 2-mercaptoethanol (Invitrogen) and run on a 10% SDS PAGE gel.", "Samples were transferred to PVDF membrane and probed with antibodies for MFN1 (Abcam, UK, cat.", "no. ab57602), MFN2 (Abcam, UK, ab124773) OPA1 (Abcam, UK, ab90857) and DRP1 (Santa Cruz, USA cat.", "no. sc-271583).", "Blots were developed using ECL and hyperfilm (GE Healthcare, UK) and scanned before analysis using Image J.", "This was carried out as previously described [10], using 4 μg rabbit anti-mouse HIF1α antibody (NB100-134, Novus Biochem).", "We performed immunoprecipitation for each promoter site in e18 cardiac chromatin using an isotype control IgG (Abcam ab13347000.", "We found no significant specific binding of this antibody to any promoter site.", "Pregnant F1 (CBA/Ca x C57BL/10) females were sacrificed at 18 days post coitus.", "The embryos were removed and fostered onto a Parkes mouse, which had littered the previous day.", "Neonates were raised by a foster mother for 24 h in either normoxia or in a hypoxic chamber (Custom Glovebox, Biospherix) maintained at 10% O2.", "Prior to this, foster mothers were acclimatized to increasing hypoxia for at least three days preceding the experiment, whereas the dam carrying the experimental neonates was maintained in normoxia until she was sacrificed and embryos removed.", "This eliminated maternal and prenatal exposure to hypoxia which may have affected postnatal phenotypes.", "Hearts were snap frozen, and after PCR genotyping homogenized and subjected to colorimetric assay for citrate synthase (Biovision, California, USA) as per manufacturer’s instructions.", "Lowry protein assay was performed on the cardiac homogenate (BioRad).", "Results were normalized to protein content.", "Immediately after dissection in cold PBS, hearts were snap frozen in liquid nitrogen and stored at − 80 °C.", "RNA was extracted from hearts using Trizol reagent (Invitrogen) according to the manufacturer's instructions.", "CDNA was prepared from RNA samples using Superscript III and random primers (Invitrogen).", "Quantitative RT-PCR was carried out using an ABI Prism 7000 cycler (Applied Bio systems) with the cDNA equivalent to 30 ng of total RNA per reaction using SYBR green (Thermo Scientific), with 18 s mRNA as the normalizing control.", "All PCR primers were purchased from Qiagen.", "Primers used Complex I; NDUF8, Complex 4 mtCOX2, Complex 5 ATP5a [35].", "Embryonic cardiac myocytes were isolated from E16–18 embryos from timed-pregnant mice.", "The mother was sacrificed by cervical dislocation and embryos were taken and stored at room temperature in freshly prepared ADS buffer (116 mM NaCl, 5.4 mM KCL, 20 mM HEPES, 0.8 mM NaH2PO4, 405.7 μM MgSO4, 5.5 mM glucose, pH 7.35).", "Hearts from each embryo were excised and rinsed in ADS buffer before they were stored in labeled 1.5 ml Eppendorf tubes.", "Tails samples were also obtained for PCR analysis.", "Tissue culture dishes (6 well plates or Seahorse plates) were coated with 1% gelatin (Difco) during the isolation steps.", "The following steps were carried out under tissue culture conditions: Hearts were minced and serially digested in 1 ml digestion buffer (ABS buffer) containing 1 mg/ml Collagenase II (Worthington Chemicals) and 0.5 mg/ml Pancreatin (Sigma-Aldrich) in a shaking incubator set at 37 °C for 15 min.", "Myocytes were not removed from the first digestion therefore the supernatant from the first digest was discarded without removing the hearts.", "Fresh digestion buffer was added to each heart sample and left to incubate at 37 °C for 10 min.", "After this incubation, the digest was removed and transferred to 15 ml Falcon tubes containing 2 ml Fetal Calf serum (FCS) to stop the enzymic reaction.", "The supernatant collected from two digests were centrifuged at 1000 r.p.m for 3 min, the solution was discarded and the cell pellet was resuspended in fresh FCS.", "These incubation and centrifugation steps were repeated six to eight times until the cardiac myocytes were completely removed and the heart tissue was reduced to a single matrix conglomerate.", "The final cell pellet from each heart was resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FCS and penicillin (50 U/ml), streptomycin (50 μg/ml) (Invitrogen).", "Fibroblasts were allowed to attach for 30 min and the non-adherent (myocyte) fraction was plated into either 6 well dishes containing 22 mm glass coverslips (VWR) for mitochondrial function analysis or Seahorse plates for oxygen consumption analysis.", "After 24 h, the myocytes formed a monolayer of spontaneously beating cells; the medium was removed and replaced with DMEM containing 1% FCS and 1% P/S before further analysis.", "Hearts from Cre positive or VHL floxed embryos were used as controls to compare against mutant VHL hearts.", "HL-1 cells were transfected with shRNA construct directed against VHL mRNA using FugeneHD (Invitrogen) according to manufacturer's instructions.", "48 h after transfection, Claycomb medium was supplemented with 0.4 mg/ml G148 in order to select clones overexpressing shRNA VHL.", "Individual clones were picked and qRT-PCR and Western Blot verified expression levels of VHL.", "For oxygen consumption rate and mitochondrial function experiments, neomycin was removed several passages before analysis.", "An HL1 line stably expressing an empty vector was used as control in these studies, unless otherwise stated.", "A Seahorse Bioscience Instrument was used to measure oxygen consumption rate as per manufacturer's instructions [36].", "For a detailed protocol, please refer to Breckenridge et al. 2013 [10].", "All readings were normalized to protein content, by performing a Lowry protein content assay on wells following completion of the Seahorse assay.", "The redox state of mitochondrial NADH is a function of respiratory chain activity and substrate turnover.", "We measured the resting level of NADH autofluorescence in the cells, which was then expressed as the ‘redox index’, a ratio of the maximally oxidized and maximally reduced signals [37].", "The dynamic range of the signals was defined by obtaining the maximally oxidized signal following the response to 1 μM FCCP (which stimulates maximal respiration and fully oxidizes the mitochondrial NADH pool).", "In these conditions, mitochondrial NADH is taken as fully oxidized and defined as 0%.", "The maximally reduced signal was then defined as the response to 1 mM NaCN (which fully inhibits respiration), preventing NADH oxidation, and so promoting maximal mitochondrial NADH reduction.", "In these conditions NADH is taken as 100% reduced.", "NADH autofluorescence measurements were obtained from ShRNA-VHL cells.", "NADH autofluorescence was measured using an epifluorescence inverted microscope equipped with a 20 × fluorite objective.", "Excitation light at a wavelength of 350 nm was provided by a Xenon arc lamp, the beam passing through a monochromator (Cairn Research, Kent, UK).", "Emitted fluorescence light was reflected through a 455 nm long-pass filter to a cooled CCD camera (Retiga, QImaging, Canada) and digitized to 12 bit resolution.", "Imaging data were collected and analyzed using software from Andor (Belfast, UK).", "Mitochondrial membrane potential (Δψm) is an indicator of mitochondrial state.", "In these experiments tetramethylrhodamine methylester (TMRM) is used in the ‘redistribution mode’ to assess Δψm, and therefore a reduction in mitochondrial localized TMRM fluorescence represents mitochondrial depolarization.", "We measured Δψm, in shRNA VHL knockdown cell lines and in primary cultures.", "For measurements of ΔΨm, cells were plated on 22 mm glass coverslips and loaded for 40 min at room temperature with 25 nM TMR (Invitrogen) in a HEPES buffered saline solution (HBSS) composed of 156 mM NaCl, 3 mM KCl, 2 mM MgSO4, 1.25 mM KH2PO4, 2 mM CaCl2, 10 mM glucose and 10 mM HEPES; pH adjusted to 7.35 with NaOH.", "The dye remained present in the media at the time of recording and is used in the redistribution mode.", "Therefore a reduction in TMRM fluorescence represents Δψm depolarization.", "Confocal images were obtained using a Zeiss 710 VIS CLSM equipped with a META detection system and a 40 × oil immersion objective.", "TMRM was excited using the 560 nm laser line and fluorescence was measured above 580 nm.", "For basal ΔΨm measurements, Z-stack images were obtained by confocal microscopy and analyzed using Zeiss software [22].", "For measurements of ATP, the cells were transfected for 24 h with the ATP sensing probe Cox AT1.03 using Lipofectamine 2000 (Qiagen) according to manufacturer’s instructions [24,25].", "NADH autofluorescence was determined with excitation at 351 nm and emission at 375–470 nm [22].", "Measurements of ATP levels with Cox AT1.03 were obtained using measurement of fluorescence resonance energy transfer by exciting the cyan fluorescent protein at 405 nm (measured at 460 to 510 nm), used to excite yellow fluorescent protein, measured from 515 to 580 nm [24].", "All data presented were obtained from at least 5 coverslips and 2–3 different cell preparations.", "Statistical analysis was performed with Origin 8 (Microcal Software Inc., Northampton, MA, USA) software.", "Student’s t-test was applied.", "Means expressed ± the standard error of the mean (SEM).", "This work was part-funded by the British Heart Foundation, Project grant PG/10/51/28444 to R. B.", "T.M. is funded by the Medical Research Council (U117562103)." ]

[ "The reduction in HIF signaling encountered by the heart following birth acts as a physiological switch.", "Reduced postnatal cardiac HIF signaling affects mitochondrial number, structure and function.", "Experimental study of mitochondria is prone to artifacts due to the effect of oxygen.", "Cardiomyocytes employ multiple strategies to function in low oxygen in utero." ]

[ "MEDI" ]

[ "Critical care", "Early warning score", "Ebola virus disease", "Viral haemorrhagic fever" ]

[ "2725", "2726" ]

[ "The recent outbreak of Ebola virus disease (EVD) in West Africa was unprecedented.", "Following the first case in Guinea in December 2013, 28 616 cases were reported in Guinea, Liberia and Sierra Leone resulting in 11 310 deaths.1,2", "Fragile healthcare systems in the affected countries struggled to cope with the scale and complexity of the outbreak, and the World Health Organisation declared a Public Health Emergency of International Concern on 8 August 2014, resulting in the mobilisation of the international community to assist in the control of the EVD outbreak in West Africa.3", "Case finding and contact tracing, combined with safe and dignified burial of corpses, are key public health measures required to control EVD outbreaks, underpinned by social mobilisation.4", "Case management and isolation of cases in EVD treatment centres is the fourth key pillar of outbreak response.5,6", "Whilst there were a number of investigational therapies in development and trialled for the treatment of EVD, none were routinely available in West Africa during the recent outbreak.", "Optimising clinical outcomes therefore was entirely dependent upon the provision of high quality supportive care.", "The practical difficulties of providing parenteral fluid and electrolyte therapy in EVD treatment centres, combined with the perceived risks to healthcare workers (HCWs) of invasive clinical procedures resulted in generally poor access to enhanced levels of care across West Africa.7,8", "During the outbreak in Gulu, Uganda in 2000, higher levels of care were delivered including routine use of intravenous fluids with an overall case fatality rate of 53% (224/425, 95% CI 48%–58%).", "At the beginning of this outbreak some clinicians also endeavoured to challenge the historically poor EVD patient outcomes with improved supportive care delivery, including the first ETC in Conakry that sustained at a low CFR of 30%–40%.8,9", "Unfortunately, despite this progress some organisations have failed to learn these key clinical lessons, and still question the benefit and evidence base of intravenous fluid and electrolyte replacement in EVD.10", "As part of the international response to the EVD outbreak in West Africa, the UK built seven treatment centres in Sierra Leone,11 the first of which was commissioned on 5 November 2014 at Kerry Town, near the capital Freetown.", "This included 20 beds operated by the UK and Canadian Defence Medical Services specifically for HCWs with suspected or confirmed EVD.", "The EVD treatment unit (EVDTU) was well resourced with dedicated laboratory facilities and was capable of providing high quality medical and nursing care, fluid and electrolyte therapy, blood component transfusion, oxygen therapy and vasopressor support.", "Limited data exist describing supportive care management, laboratory abnormalities and outcomes in patients with EVD in West Africa, apart from within EVD clinical trials.", "Patient age, baseline viral load and renal dysfunction have been highlighted as key prognostic indicators in EVD.12,13", "Combined analysis of 27 exported cases of EVD to Europe and the United States of America (USA) also highlighted the prevalence of organ dysfunction in EVD and demonstrated the feasibility and importance of critical care interventions, with an associated low case fatality rate of 18.5% (95% CI 6.3%–38%).14", "We report cohort data from Sierra Leone which is the first description of the provision of enhanced EVD case management protocols in a West African setting, in a unique treatment centre dedicated to caring for infected HCWs.", "It demonstrates what can be achieved by well-resourced and committed clinical teams and pushes the boundaries of EVD supportive care levels in low-resource settings.", "Demographic, clinical and laboratory data were collected by retrospective review of clinical notes, nursing charts and laboratory records of patients with confirmed EVD admitted to the military EVDTU at Kerry Town, between 5 November 2014 and 30 June 2015.", "Clinical features, observations and laboratory test results were recorded each day on a standardised proforma.", "All clinical notes were electronically scanned on patient discharge or death, prior to destruction of the paper copies used in the EVDTU clinical area.", "All clinical samples and data were collected during routine patient care as part of the public health response and the Sierra Leone ethics and scientific review board confirmed board approval was not required.", "All information on individual patients was anonymized and recorded on standardised forms, which were kept securely.", "Patients admitted to the military EVDTU included international and national HCWs and other personnel engaged in the EVD relief effort in Sierra Leone (including doctors, nurses, community healthcare officers, ambulance drivers and hygienists).", "Local nationals not involved in healthcare were also referred and admitted on a case-by-case basis, although pregnant women and children with EVD were directed to alternative treatment centres.", "Patients were admitted directly from the community, or were transferred from other treatment centres once their status as a HCW was realised.", "Diagnosis of EVD was confirmed on admission by EBOV real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay of blood.", "Baseline data from patients subsequently aeromedically evacuated to Europe or USA is reported, but not included in longitudinal data reported or outcome dependent regression analysis.", "All patients underwent EBOV RT-PCR testing on admission, provided by the onsite Public Health England laboratory utilising an existing Trombley Ebola Zaire nucleoprotein assay or the Altona RealStar Filovirus RT-PCR Kit (Altona, Hamburg, Germany).15", "Results were reported as positive or negative, with cycle threshold (Ct) values only being obtained retrospectively.", "The onsite military laboratory provided basic haematology, clotting, clinical chemistry and blood culture capabilities.", "On admission, and when clinically indicated thereafter, blood samples were assayed in the onsite laboratory using the Piccolo Express system (Abaxis, CA, USA), Horiba ABX Micros ES60 analyser (Horiba, Montpellier, France) and Hemochron Signature Elite (Accriva Diagnostics,CA, USA).", "When laboratory facilities were not available, bedside i-STAT® (Abbott Point of Care) testing was undertaken when indicated.", "The Bact-Alert™ system was used for blood cultures, and if positive a Blood Culture Identification Detection panel was run on the BioFire FilmArray™.", "A rapid diagnostic test (RDT) was undertaken on admission to exclude malaria (BinaxNOW®).", "HIV (Alere Determine HIV 1/2 Ag/Ab) and Dengue (SD Bioline Dengue NS1 Ag Ab Combo) rapid diagnostic tests were also available.", "An EVD treatment bundle was developed to facilitate a protocolized approach to the provision of clinical care thus ensuring optimal utilisation of clinical contact time.", "Whilst acknowledging there is little evidence of efficacy of any specific intervention in the management of EVD, the elements of the EVD treatment bundle were developed by the clinical group encompassing interventions well established in the management of the critically ill and incorporating basic tenets of care provided in EVD treatment centres.", "The EVD treatment bundle evolved through regular review by clinical groups at the end of each 60-day roulement.", "In the EVD treatment bundle (Annex 1), clinical disease was defined by three stages of illness (Table 1).", "The main tenets of the EVD treatment bundle include parenteral fluid & electrolyte replacement therapy, stress-ulcer prophylaxis, empirical antibiotics and anti-helminthic medication, analgesia and standardised approaches to the management of coagulopathy & haemorrhage, encephalopathy and shock (Appendix S1).", "Venous access was routinely achieved by placement of a central venous cannula (CVC) in all patients with stage 2 or 3 disease.", "Vital signs were recorded 6 hourly with continuous monitoring of heart rate, non-invasive blood pressure and pulse oximetry undertaken when required (and facilitated remotely by closed circuit television monitoring).", "Urethral catheterisation and placement of faecal management systems were undertaken as clinically indicated.", "All staff received education in the delivery of the EVD treatment bundle prior to the opening of the EVDTU and prior to each roulement of clinical staff throughout the outbreak.16", "Maximum daily early warning scores (national early warning score – NEWS, quick Sepsis Related Organ Failure Assessment – qSOFA) were calculated retrospectively.", "The qSOFA score can be calculated using basic parameters of clinical assessment and aims to identify patients at increased risk of death due to sepsis.17", "NEWS is a scoring system (score 0–20) based upon routine clinical observations reflecting the individual's physiological response to illness.", "When a patient is admitted to a medical facility it can be used to track changes in clinical observations thus alerting the healthcare team to any deterioration and so triggering a timely escalation in clinical care.18", "Descriptive analyses are reported as frequencies and proportions for categorical variables, means or medians as appropriate for continuous variables.", "We used Fisher's exact test for comparing categorical variables, and Student's t-test and Mann–Whitney U test for comparing continuous variables.", "We assessed risk factors for mortality by univariate logistic regression and reported odds ratio of death with its 95% CI.", "No imputation for missing data was made due to small sample sizes.", "Hypothesis tests were two-tailed (p < 0.05) and we analysed data with SPSS (version 24).", "During the study period a total of 44 cases were admitted with confirmed EVD.", "Three international HCWs with confirmed EVD were initially managed in the EVDTU before being evacuated to Europe or the USA for further treatment, all of whom survived.", "The remaining cases were Sierra Leonean nationals of whom 71% (29/41) were employed in healthcare.", "The majority of patients were male (28/44) with a median age of 37 years (range 17–63).", "The mean time from onset of illness until admission was 5.3 days (SD 3.2), with 23/44 patients admitted directly to the EVDTU (Table 2).", "Excluding those who were aeromedically evacuated, the case fatality rate for those receiving care in the military EVDTU was 49%.", "At the time of admission to the EVDTU the most common clinical features were diarrhoea and vomiting (33/44), lethargy (31/44), anorexia (29/44), abdominal pain (28/44) and fever (27/44).", "Only 3/44 patients had signs or symptoms of haemorrhage at admission, with 10/44 patients reporting hiccups of which 8/10 died.", "Vital signs at admission were within normal limits in the majority of patients admitted, apart from respiratory rate (mean 24.6, SD 7.5), with mean temperature 37.5°C (SD 1.0) at admission.", "Other signs and symptoms of EVD patients admitted to the military EVDTU are shown in Table 3.", "Stage 1 disease (fever & constitutional symptoms only) was present in 9/44 patients, stage 2 disease (presence of diarrhoea and/or vomiting without organ failure) in 12/44 and stage 3 disease (presence of diarrhoea and/or vomiting with organ failure) was present in 23/44 patients with case fatality rates of those not evacuated of 14% (95% CI 1%–58%), 27% (95% CI 6%–61%), and 70% (95% CI 47%–87%) respectively (p = 0.009).", "Cycle threshold value at baseline was available for 42/44 participants with a mean of 22.7 (SD 5.2) overall, and 20.3 (SD 4.3) in fatal cases and 24.8 (SD 5.5) in survivors (p = 0.007) (Table 3).", "Clinical findings during hospitalisation are summarised in Fig. 1.", "Nearly all patients had diarrhoea (39/41), lasting a median 5 days (IQR 3–6 days) and complained of abdominal pain (38/41) lasting a median 3 days (IQR1–3 days).", "Lethargy was also very common (36/41), lasting a median 3 days (IQR 1–5), with vomiting occurring in 33/41 patients and lasting a median of 3 days (IQR 1–2).", "During hospitalisation fever (>38 °C) was recorded in 33/41 patients with the median time until resolution (<38 °C) of 9 days from the onset of symptoms.", "Haemorrhage occurred in 17/41 patients (and one HCW prior to evacuation) with encephalopathy seen in 26/41 patients.", "The lowest oxygen saturations recorded in fatal cases was lower than cases that survived (mean 86.6% versus 93.9%, respectively, p = 0.003).", "Blood was analysed using the onsite laboratory and point of care devices as clinically indicated and in accordance with the EVD treatment bundle (e.g. daily in stage 2/3 disease).", "During a total of 312 EVD patient admission days in the EVDTU, the most frequently measured electrolytes were sodium and potassium, analysed in 255/312 (81.7%) and 240/312 (76.9%) patient days respectively.", "The majority of patients had electrolyte abnormalities at admission and during hospitalisation (Supplementary Table S1).", "Hyponatraemia (<135 mmol/L) occurred in 37/40 (93%) of patients and hypokalaemia (<3.5 mmol/L) in 21/40 (53%) during hospitalisation.", "Hyperkalaemia (>5 mmol/L) occurred in 12/40 patients during admission with 3 patients recording potassium levels >6 mmol/L.", "Hypomagnesaemia (<0.7 mmol/L) and hypophosphataemia (<0.8 mmol/L) were recognised complications in 11/31 and 8/31 patients in whom magnesium and phosphate were measured, typically occurring in patients with a protracted diarrhoeal phase of illness.", "Hypoglycaemia (<4 mmol/L) occurred in 24/40 (60%) patients, with 9 patients recording blood sugars ≤2.8 mmol/L, all of whom were in the terminal stages of illness.", "Renal dysfunction frequently occurred with an elevated creatinine (>110 umol/L) in 26/41 (63%) at admission and 32/40 (80%) during hospitalisation.", "Acute kidney injury (AKI) (RIFLE criteria - creatinine > twice estimated baseline) occurred in 20/40 patients through the course of their illness, with 14/20 (70%, 95% CI 46%–88%) subsequently dying, compared to 5/20 (25%, 95% CI 9%–49%) patients who did not suffer AKI dying (p = 0.01).", "Elevated creatine kinase (>380 IU/L) developed in 33/34 (97%), with the median maximum creatine kinase (CK) observed in patients with AKI 3200 IU/L and in those without AKI, 3129 IU/L.", "Elevated alanine aminotransferase (ALT) levels (>110 IU/L) were demonstrated in all patients in whom it was measured (35/35), with aspartate aminotransferase (AST) enzyme levels raised in virtually all patients (34/35) during the course of their illness.", "Peak AST levels (median 1322 IU/L) were higher than peak ALT levels (median 352 U/L).", "Elevated bilirubin (>21 umol/L) developed in 10/34 patients with 2/34 patients having levels (>100 umol/L).", "The median C-reactive protein on admission was 30 (n = 34), with 18/35 recording a CRP >200 (limit of assay) during the course of their admission.", "Elevated activated partial thromboplastin time (31/35, APPT >37 s) was more common than elevations in prothrombin time (18/39, PT >14 s).", "Overall, 18/41 (44%) patients developed a significant derangement in one or more assays of global coagulation (Activated Partial Thromboplastin Time (APTT) > 57 s, Activated Clotting Time (ACT) > 180 s or Prothrombin Time (PT) > 23 s) during the course of their illness.", "Thrombocytopenia (<150 × 109/L) was recorded in the majority of patients (24/35), with significant thrombocytopenia (platelets < 50 × 109/L) observed in 6/35 (17%) patients.", "The majority of patients with platelets < 50 × 109/L survived (5/6) – only three of whom required platelet transfusion.", "Leucocytosis (>15 × 109/L) occurred in 16/34 patients and was associated with fatal outcome (p = 0.005).", "Malaria RDTs were performed on all patients at admission, with results available in 37 patients, of which one was positive.", "One patient was also managed empirically for falciparum malaria as a convalescent blood transfusion tested positive by RDT at the time of administration.", "Thirty-five blood cultures were taken from 27/44 patients, with one positive blood culture identified.", "Patients were on pre-existing empirical antibiotics in 19/34 negative blood cultures.", "One positive blood culture (E. coli species) was identified from an admission sample of a 48-year-old Sierra Leonean HCW, admitted 7 days after disease onset with severe EVD (stage 3 disease, NEWS 10, qSOFA 2, Ct 18.3).", "The patient presented with diarrhoea, haemorrhage and acute renal failure and died after 3 days despite supportive therapy including empirical ceftriaxone.", "At the time of admission mean NEWS score was 5.5 (SD 4.4) in fatal cases and 3 (SD 1.9) in survivors (p = 0.02), with mean qSOFA scores of 1 (SD 0.8) and 0.7 (0.6) respectively (p = 0.27).", "The highest NEWS score recorded during the course of admission was mean 7.2 (SD 3.7) in survivors vs. 12.8 (SD 3.3) in fatal cases (p < 0.001), with mean highest qSOFA scores also lower in survivors (1.2, SD 0.6) compared to fatal cases (2, SD 0.76, p < 0.001).", "Intravenous catheters were inserted in all patients, with central venous catheters placed in 37/41 patients, and 1/3 international HCWs prior to evacuation (Table 4).", "The majority CVCs were placed within 24 hours of admission, remaining in situ for a median of 5 days.", "Faecal management systems were inserted in 21/41 patients during their admission, for a median of 3 days duration (range 1–10 days).", "Urinary catheters were placed in 27/41 patients for a median duration of 2 days.", "Intravenous fluid was administered to 40/41 patients for a median duration of 5 days (range 2–17 days).", "The median maximum IV fluid volume administered to a patient in 24 hours was 3.2 L (range 2-8 L).", "Intravenous potassium replacement was administered to 31/41 patients, with intravenous magnesium and phosphate replacement administered to 18/41 and 6/41 patients respectively.", "Blood component therapy was administered to 20/41 patients, and 1 international HCW prior to aeromedical evacuation, with fresh frozen plasma the most frequently administered product.", "Convalescent whole blood transfusion was administered to 4/41 patients.", "Empirical ceftriaxone was administered to 37/41 patients for a median of 5 days duration (range 1–10).", "Oxygen supplementation was administered to 18/41 patients, for a median duration of 3 days (range 1–7), with 16/18 patients having a fatal outcome.", "Oxygen supplementation was administered to 2 survivors for 5 and 7 days, with one recording a lowest saturation of 77% on room air.", "It was provided via oxygen concentrators and as such limited to low flow rates.", "No patients received vasopressors for the management of septic shock.", "Univariate logistic regression analysis of factors associated with mortality at admission is shown in Table 5.", "The following laboratory variables were found to be associated with mortality at univariate analysis: AST greater than 1000 U/L (OR 6.0 95% CI 1.17–30.72); RIFLE criteria stage 2 (injury) (OR 4.8 95% CI 1.2–19.13); Granulocyte count greater than 7.5 × 109/L (OR 5.6 95% CI 1.16–27.08); and Ct value less than 20 (OR 19.5 95% CI 3.38–112.05).", "EVD stage greater than 2 (OR 8.0 95% CI 1.93–33.18) and NEWS greater than 5 (OR 5.19 95% CI 1.28–21.08) at admission were associated with increased mortality.", "The scale of the EVD outbreak in West Africa presented a unique opportunity to improve patient outcomes, our understanding of the natural history of disease and to evaluate novel therapeutics.", "Much has been learnt from both African cohorts and exported cases in terms of the clinical syndrome of EVD and the benefits of supportive care provision.19", "The EVDTU at Kerry Town was a small unit dedicated to providing care for HCWs in a well-resourced clinical environment, with high quality nursing care and senior clinician leadership, supported by a comprehensive deployed laboratory.", "It was committed to improving equity of access to life saving interventions and to pushing the boundaries of EVD supportive care – including central venous catheterisation, ultrasound guided fluid resuscitation, and laboratory support to guide electrolyte replacement.", "The majority of patients admitted were HCWs and represent an older and more unwell cohort, compared to other data sets reported.", "Nearly half of all patients were also transferred from other treatment centres and as such presenting later in their disease course.", "At admission the majority of the cohort had severe EVD, based on a novel EVD staging system developed by the UK DMS, underpinned by laboratory and clinical data.", "Although the total number of patients admitted with confirmed EVD was smaller than expected, the unique breadth, depth and completeness of data collected presents a unique opportunity for detailed analysis.", "It is the first description of an “EVD care bundle” based on established tenets of critical care medicine, and demonstrates the utility of both a novel EVD staging system and established early warning scores in EVD for the first time.", "The majority of the patients had renal impairment with 40% having acute kidney injury by RIFLE criteria, 17/40 with serum creatinine >350 umol/L, higher than in other published cohorts.20,21", "Electrolyte imbalance was extremely common, with hypokalaemia and hypoglycaemia occurring in the majority of patients, and is to be expected with the significant gastrointestinal involvement in EVD.", "Potassium supplementation was required by a number of patients into the recovery/convalescent phase of illness, and we believe hypokalaemia induced cardiac arrhythmia may be the cause of sudden death in recovering patients that was reported by other clinicians (Dr. M Jalloh – personal communication 2014).", "Haemorrhage and significant coagulopathy occurred in over 40% of patients, although haemorrhage was not uniformly fatal with 7/17 (41%, 95% CI 18%–67%) patients surviving to discharge.", "This rate is consistent with aggregate data of exported cases and we believe that it reflects higher staff-to-patient ratios allowing improved case observation, combined with an interventional approach of placing IV catheters.", "Encephalopathy was also frequently seen, and although it was associated with mortality, 7/26 (27%, 95% CI 12%–48%) patients with encephalopathy survived.", "The encephalopathy that we observed was an alteration in conscious level accompanied by transient cognitive impairment, consistent with diffuse cerebral dysfunction that was hypoactive in nature.", "We believe the aetiology is most likely multifactorial, incorporating metabolic and infective factors with direct viral brain involvement possibly contributing.22", "Identifying features which reliably predict poor clinical outcome at presentation and during the course of illness improve understanding of disease pathogenesis and can inform future intervention strategies.", "In our cohort, factors significantly associated with poor outcome at presentation include a low Ct value, presence of established organ dysfunction (EVD stage 3) and a high NEWS score.", "Other significant laboratory variables associated with mortality in univariate analysis at baseline include AST, ALT, creatinine, CRP and white blood count.", "Recent further analysis of clinical trial data also showed significant electrolyte abnormalities in their EVD cohort and tested a prognostic model also incorporating calcium and haemoglobin levels.23", "Early warning scores aim to identify patients at risk of severe illness or death, utilising simple physiological parameters and have long been advocated by critical care outreach and acute medical teams.18", "In keeping with other forms of critical illness, timely intervention with supportive care before organ dysfunction becomes established and has the potential to improve clinical outcomes in EVD.", "Due to the limited provision of systematic vital signs monitoring and data recording in EVD patients in West Africa, limited data existed to inform development of EVD early warning systems.", "We retrospectively analysed our data utilising the UK national early warning score system and qSOFA scores at baseline, that in comparison to the novel staging system we report they do not rely on laboratory parameters.", "Admission NEWS score was significantly higher in fatal cases, and could be utilised in the future as a prognostic indictor and planning tool.", "The qSOFA showed no utility in predicting outcome in EVD in our cohort.", "Although our data set is small it provides the first evidence of the utility of an early warning score in EVD and requires evaluation in larger cohorts, ideally prospectively.", "The scale and range of medical interventions we report highlights what is feasible in deployed low-resource settings.", "Central venous access requires skill, but facilitates fluid and concentrated electrolyte replacement, limiting invasive procedures.24", "Peripherally inserted central lines may provide an alternative, dependent on operator skill and experience and were used successfully in 40% of exported cases.14", "There were no needlestick injuries associated with central venous catheters in our staff, and one healthcare worker infection in an EVDTU nurse, the cause of which was not identified.", "Faecal management systems were also used for the first time in patients with EVD in West Africa.", "They were well tolerated by patients and used in 50% of cases, providing infection prevention and control benefits for staff and cleanliness and dignity for patients with profuse diarrhoea.", "In this cohort of patients with EVD, hypotension refractory to fluid resuscitation in keeping with the clinical syndrome of septic shock was not observed and as such vasopressors were not utilised.", "We were also fortunate to have access to comprehensive panel of blood products that were delivered to almost 50% of patients.", "The EVDTU was not a clinical trial site for novel therapeutics during the outbreak and focussed on delivery of high quality supportive care.", "However, at their own request, and through provision by referring units, three HCWs received convalescent whole blood transfusion, providing informed consent and ultimately all surviving their infection.", "Subsequent clinical trial data failed to show any clear benefit of convalescent plasma in EVD.25", "Experimental antiviral therapies had not been used for post-exposure prophylaxis (PEP) in any of this cohort, but a number of international HCWs did receive (PEP) after high risk exposures and evacuation to the UK.26", "Whilst we rightly emphasise the benefits of comprehensive supportive care for EVD patients we must also recognise the benefits of palliative care and psychological support in patients who will not survive.", "We had established protocols for end of life care and with higher staffing levels, were able to provide improved symptom management to fatal cases with 75% receiving opiate analgesia and 50% benzodiazepines, frequently through syringe drivers.", "Opiate analgesia was also utilised in over 50% of survivors, mainly as an oral formulation.", "Healthcare workers at the forefront of the fight against EVD have suffered consistently high case fatality rates.", "The reasons underlying these rates across the outbreak remain unclear, but older age, comorbidities and high viral load are consistently associated with poorer outcome and may be more common in HCWs.", "A clinical prioritization score developed by Hartley et al.,27 adjusted for age and viral load predicted a case fatality rate of 62% in our cohort (95% CI 45%–74%) at admission.", "The case fatality rate in the military EVDTU was 49% (95% CI 33%–65%) overall, but direct comparison of clinical outcomes from different treatment facilities has proved difficult.", "Rates reported from other ETCs in Sierra Leone were similar from cohorts with lower median ages and higher Ct values.", "International Medical Corps28 reported a case fatality rate of 58% (95% CI 53%–64%, median Ct 25.4), and Medicins san Frontieres,29 a case fatality rate of 51% (95% CI 47%–56%, median Ct 31 in survivors and 22 in fatal cases).", "Historical control data for 3 months preceding the JIKI trial in Guinea had similar Ct values (Adults and children ≥6 years Ct value 20.1) with a case fatality rate of 57% (272/478, 95% CI 52%–61%).30", "A study of the heterogeneity in the case fatality rates in West Africa,31 was limited by missing outcome data, but demonstrated that in the age group 35–39 years there was a case fatality rate of 67.5% (95% CI 62%–72%, n = 351).", "Analysis of supplementary data from this study also showed that in adults (≥17 years, median age 32 years) with confirmed EVD and known outcome status admitted to ETCs in Sierra Leone, there were 268/477 deaths (CFR 56%, 95% CI 52%–61%).", "Whilst the difficulty of comparing outcomes in cohorts with significantly different demographics and baseline viral loads is obvious, the importance of survivor bias due to local factors affecting patient distribution must also be considered.32", "A number of limitations of our data must be appreciated, that reflect the primary mission of the EVDTU to manage infected HCWs.", "As such paediatric or pregnant cases were not referred to the EVDTU, that may have different clinical syndromes and management approaches.", "The EVDTU was also highly resourced with its main assets being staff-to-patient ratios and laboratory support.", "The intensive medical and nursing care provided would be challenging to safely maintain in larger ETCs, but a compromise incorporating the tenets of our management approach is achievable.", "The number of HCW cases that were admitted was also lower than expected resulting in a small sample size.", "This reflects the timing of the epidemic and reduced nosocomial risk to HCWs as better treatment centres opened, and is consistent with the experience of other HCW ETCs in Guinea and Liberia.", "Small sample size has prevented us from performing multivariable analysis to looking at the risk factors of mortality at admission simultaneously.", "As a result, observed differences may be subject to possible confounding effects due to unknown or unmeasured factors.", "In resource-limited African settings, the provision of even basic supportive care to patients with EVD is difficult.", "Recommendations to limit treatment based upon perceived poor patient prognosis and risk to HCWs, perpetuate the cycle of limited care, poor outcomes and fear.33", "Whilst the provision of renal replacement therapy and mechanical ventilation have been successfully utilised in one ETC in Sierra Leone during the recent outbreak, the logistic and practical considerations of providing level 3 care, unfortunately makes it an unrealistic proposition as an established level of care for EVD in future outbreaks.", "We do recognise that when resources and appropriately trained personnel exist, this capability can be safely delivered and will improve outcomes in severe EVD for limited numbers of patients.", "We believe that the approach pioneered at our EVDTU utilising improved resources, clinical staging of disease severity and an enhanced level of protocolized care offers a blueprint for the future management of EVD in resource-limited environments.", "These processes were subsequently adopted by other non-governmental organisations in West Africa, and can form a platform for future viral haemorrhagic fever clinical care augmented by specific therapeutics when available.", "The authors declare that they have no competing financial interests.", "The content is solely the responsibility of the authors.", "No specific funding.", "TF is funded by the Wellcome Trust (104480/Z/14/Z) and the UK Ministry of Defence.", "The PHE-led EVD laboratory operation was funded through the Department for International Development." ]

[ "EVD is associated with life-threatening electrolyte imbalance and organ dysfunction.", "Clinical staging/early warning scores can be useful EVD prognostic indicators.", "Enhanced protocolized care is a blueprint for future treatment in low-resource settings." ]

[ "COMP" ]

[ "2D-3D registration", "Feature detection", "Feature matching", "Point detection", "Saliency" ]

[ "1707", "1711", "1712" ]

[ "Light Detection And Ranging (LiDAR) scanners have been used to obtain 3D data for decades, but it is only in recent years that they have seen more widespread applicability due to the high computational capacity required to cope with such large datasets.", "However, the integration of LiDAR scans with data from other modalities (e.g. images) remains a difficult problem, with many approaches relying on line features for their registration (Liu and Stamos, 2012; Wang and Neumann, 2009), which may not always be available.", "This causes significant bottlenecks in practical applications such as digital film production, where LiDAR scans and images are captured on-set to obtain data about the scene, but subsequently need to be manually registered during post-production.", "The problem is further exacerbated by the high resolution and large scale of the data, requiring scalable methods for registration that are robust to the diverse, multi-modal aspect of the data.", "To address this, here we propose a point feature detector that may be naturally and meaningfully applied between both 2D and 3D data.", "Feature detection is a typical first stage in many registration pipelines (Li et al., 2010; Liu and Stamos, 2012; Wu et al., 2008b), whereby considering only a small subset of discriminative features in each dataset the registration parameters may be obtained in a relatively straightforward manner.", "However, obtaining suitably repeatable features between both 2D and 3D data is a particularly challenging problem due to the large heterogeneity between the two modalities.", "Instead, existing point feature detection methods are typically centred around images.", "Recent advances in 3D data acquisition (e.g. Microsoft Kinect) has resulted in a significant interest in 3D feature detection (Guo et al., 2014; Tombari et al., 2013b).", "However, it is clear that the majority of 2D and 3D feature detectors are constructed in very separate ways.", "The more popular 2D feature detectors are based on the derivative of the image, and provide a principled approach to scale selection using scale-space theory (Lowe, 2004; Mikolajczyk and Schmid, 2004).", "Yet, very few may be extended to operate on 3D data, with many 3D feature detectors based on surface curvature (Tombari et al., 2013b), and since the traditional scale-space approach typically cannot be applied to 3D data without altering the geometry.", "The differences between 2D and 3D feature detectors are further exacerbated by the range of existing 3D data types (point cloud, volumetric, mesh, textured / untextured), leading to different 3D feature detectors for each case (Guo et al., 2014; Tombari et al., 2013b; Yu et al., 2013).", "As such, it is very difficult to use existing point feature detectors jointly across 2D and 3D due to the incomparable nature of their constructions, and the limited scope to which 3D detectors may be applied.", "Applications such as registration, that would typically rely on point feature detectors, instead use other techniques in the 2D-3D case (e.g. learning a bag of features across multiple viewpoints (Tombari et al., 2013a), or Mutual Information alignment (Mastin et al., 2009)).", "These approaches are not as general as their feature-based counterparts; often making restrictive assumptions about the scene, or requiring a good initial alignment.", "To address this issue, here we propose a more general approach to point feature detection, based on the Kadir-Brady (KB) saliency detector (Kadir and Brady, 2001).", "Its histogram-based approach does not exclusively depend upon data-type specific quantities such as derivatives or curvatures.", "Instead, it defines a salient point as having a high information content (as measured by the entropy of its histogram) at a particular scale.", "This histogram-based approach allows it to be formulated across different modalities in a more meaningful manner than other feature detectors due to the vast array of ways in which histograms may be constructed.", "Based upon the KB saliency detector, and inspired by the success of the 2D Harris corner detector (Aanæs et al., 2012; Harris and Stephens, 1988) we propose a novel extension to the 2D KB saliency detector.", "Whereas the original KB saliency detector constructs a histogram of pixel intensities in a circular region, we propose a derivative-based approach whereby the histogram is constructed based on the distribution of eigenvalues of the second moment matrix.", "This allows our approach to detect salient points with respect to the derivative of the image, where it may operate in a more general manner than a typical corner detector and avoid repetitive parts of the scene.", "By using the generalisable histogram-based approach of the KB saliency detector, the above approach may be naturally extended to 3D data by constructing a histogram based on the 3D second moment matrix (Sipiran and Bustos, 2010).", "Furthermore, the histogram-based approach allows for the detection of salient points based on both the geometry and texture of the scene by constructing a 2D histogram based on the texture of the 3D surface, and combining the 2D and 3D histograms.", "This allows it to operate in a meaningful manner regardless of whether or not the 3D data is textured, and is able to combine the best of both sets of features for textured data.", "The contributions of this paper are three-fold.", "Firstly, a generalisation to the KB saliency detector is formulated, demonstrating its broad applicability to operate wherever histograms may be meaningfully constructed within a metric space.", "Secondly, in light of this generalisation, we propose a 2D derivative-based KB saliency detector based on the second moment matrix.", "Thirdly, the derivative-based KB saliency detector is naturally extended to 3D, where it may operate on both textured and untextured 3D data.", "It is, to the best of our knowledge, the first 3D feature detector to operate based on both the geometry and texture of the scene simultaneously.", "The proposed detectors are evaluated in a 2D-2D and 2D-3D manner where it is shown to be more repeatable than existing detectors (Harris 2D and 3D (Harris and Stephens, 1988; Sipiran and Bustos, 2010), and SIFT 2D and 3D (Lowe, 2004; Zaharescu et al., 2012)).", "This paper is structured as follows.", "In Section 2 we describe related work in point feature detection between 2D and 3D.", "In Section 3 a description of the KB saliency detector is given, along with proposed extensions and modifications (Kadir et al., 2004; Shao and Brady, 2006; Shao et al., 2007).", "In Section 4 we propose a generalisation of KB saliency.", "The generalisation is subsequently implemented for a 2D derivative-based KB saliency detector (Section 5), and a 3D KB saliency detector (Section 6) that may operate on textured or untextured 3D data.", "In Section 7 results will be given, involving qualitative and quantitative results in both 2D and 3D; finally, conclusions and future work are presented in Section 8.", "There has been a significant amount of research in point feature detection; both in 2D (Li et al., 2015; Tuytelaars and Mikolajczyk, 2008) and in 3D (Guo et al., 2014; Tombari et al., 2013b).", "Here we aim to give a brief overview of point feature detection in each modality, describing and comparing the mechanisms involved.", "In the case of both the Harris and Hessian detectors, they may be made affine-invariant by constructing the matrices from image derivatives over an elliptical regions (Mikolajczyk and Schmid, 2004).", "Furthermore, they may be made scale-invariant by constructing the matrices over ellipses of varying size while convolving with a Gaussian kernel (Mikolajczyk and Schmid, 2004).", "It is observed that detecting keypoints based on the magnitude of the scale-normalised Laplacian of Gaussians (LoG) produces the highest percentage of correct scales.", "This has led to the popular SIFT detector (Lowe, 2004) that detects keypoints by the magnitude of the Difference of Gaussians (DoG).", "DoG is approximately equal to the scale-normalised LoG by the heat equation, hence this approach allows for LoG estimation without the need for derivatives to be computed.", "However, the DoG response is large for edge-like structures, so SIFT subsequently culls edge responses using the ratio of eigenvalues of the Hessian.", "The traditional Gaussian scale-space approach has its limitations since it blurs both noise and fine detail (e.g. edges); this has been addressed by Alcantarilla et al. (2012) who use a non-linear scale-space that respects the natural boundaries of the image.", "A secondary category of point feature detectors are those that are intensity-based.", "These detectors typically operate over a neighbouring set of pixels, but disregard the derivative of the image.", "As such, they are often more robust to noise (particularly salt-and-pepper noise) than derivative-based feature detectors.", "An early intensity-based approach is the SUSAN detector (Smith and Brady, 1997); it defines a Univalue Segment Assimilating Nucleus (USAN) as a set of neighbouring pixels that have a similar intensity value to a centre pixel.", "Corners are subsequently defined where the number of pixels in the USAN is small.", "Region detectors typically fall into the intensity-based approaches category; for example, the MSER detector (Matas et al., 2002) detects regions where pixel intensities inside the region are either higher or lower than those on its boundary.", "A subset of intensity-based approaches are the histogram-based feature detectors that detect feature points via histogram construction.", "The Kadir-Brady saliency detector (Kadir and Brady, 2001) is an example of this; it constructs a histogram of pixel intensities in a neighbourhood of a point, salient points are detected where the distribution of pixel intensities has a high entropy at a particular scale.", "It will be discussed in greater detail in the next section, where it forms the basis of the proposed 2D-3D point feature detector.", "Using the histogram-based approach, a keypoint may be detected based on the idea of self-similarity, (or lack of it) to its neighbours.", "Maver (Maver, 2010) looks for similar histograms of pixel intensities in radial and tangential regions so as to detect keypoints that exhibit different types of symmetry.", "Conversely, Lee and Chen (2009) look for a point whose histogram is significantly dissimilar from its immediate neighbours.", "Tombari and di Stefano (2014) use a similar idea, but where histogram comparison is only performed on the k-nearest neighbours and a computationally efficient implementation is proposed.", "The notion of self-similarity is very useful for multi-modal registration, since scenes may often exhibit a similar structure between modalities but lack similar finer features.", "Tombari and di Stefano (2014) show their approach to be of potential use for cross-spectral image registration, and Shechtman and Irani (2007) construct a self-similarity descriptor for cross-spectral imagery and sketch-based retrieval.", "The majority of 2D point feature detectors are focused purely within the 2D domain.", "There is evidence to suggest that histogram-based approaches are a promising avenue for multi-modal feature detection due to their general formulation.", "However, this has never been applied in a 2D-3D context, where the histogram construction process may more generally result in feature detection based on both the geometry and texture of the 3D data.", "Approaches to point feature detection in 3D vary depending upon the type of data being used.", "For volumetric 3D data many 2D feature detectors may be naturally extended, e.g. 3D SIFT (Flitton et al., 2010).", "Indeed, a performance evaluation of volumetric 3D feature detectors (Yu et al., 2013) show extensions of familiar 2D feature detectors (Harris, Hessian, MSER, etc).", "However, other representations of 3D data (point cloud or mesh) create difficulties since points are non-uniformly sampled, points may or may not be textured, and a scale-space may not be so naturally constructed.", "Point cloud representations are however the subject of this paper and as such feature detection for this representation will be reviewed here.", "Similarly to 2D feature detection, the Harris corner detector has been naturally extended to operate on 3D data (Sipiran and Bustos, 2010).", "For each point, a best fit tangent plane is first determined.", "Each neighbouring point is projected onto the plane and assigned an ‘intensity’ value for each point as its distance to the plane.", "The 2D Harris corner detector may be applied to this set of intensity values, resulting in the 3D Harris corner detector.", "Second derivative-based approaches in 3D typically manifest themselves through curvature-based approaches, while avoiding any mention of a Hessian matrix.", "For example, Chen and Bhanu (2007) propose an approach that locally estimates a quadratic surface around each vertex and uses this to obtain the principal curvatures.", "They then assign a Shape Index (SI) to each vertex based on the maximum and minimum principal curvatures.", "Points are detected based upon whether its SI is significantly bigger or smaller than the mean of a neighbourhood of SIs.", "Alternative approaches may not be derivative-based at all, taking advantage of the unordered point cloud representation of the data.", "For example, Zhong (2009) proposes Intrinsic Shape Signatures (ISS), based on the eigenvalue decomposition of the 3 × 3 covariance matrix around a point.", "They subsequently cull points whose ratio between successive eigenvalues are similar, then rank feature points in proportion to the smallest eigenvalue.", "Learning-based approaches have also been proposed, for example by Teran and Mordohai (2014), who learn across a set of geometric attributes using a random forest.", "The approach allows for specific point detection to match the criteria observed during the training phase, resulting in a more flexible approach.", "Scale-space approaches to 3D feature detection have been proposed in a number of ways.", "Castellani et al. (2008) propose to detect point features by using the Difference of Gaussians (DoG) on the set of 3D points, determining a point’s saliency by how far it moves along its normal under the DoG operator.", "However, this type of approach has been criticised since it obtains a scale-space representation by altering the geometry of the scene.", "Alternatively, a scale-space may be constructed by convolving other attributes of the 3D data.", "Such an approach is taken by Zaharescu et al. (2012): they detect keypoints in a generic way that is applicable to scalar functions of 2D manifolds, e.g. mean curvature, or the intensity (if the data is textured).", "However, it cannot detect keypoints based jointly on geometry and texture.", "Their approach is similar to SIFT, computing a scalar function at each point, using a DoG operator on the scalar function and rejecting keypoints for which the ratio of the eigenvalues of the Hessian are large.", "An approach that is very similar to SIFT is the Viewpoint Invariant Patches approach of Wu et al. (2008a), that is only applicable to textured 3D models.", "They propose to compute a local tangent plane to each 3D point, onto which a neighbouring texture patch may be orthographically projected.", "The 2D SIFT detector and descriptor may be subsequently applied on the texture patch to allow a framework for 3D-3D registration.", "Wu et al. furthermore apply their approach in a 2D-3D scenario (Wu et al., 2008b), where SIFT features are detected in both 2D and 3D data.", "They determine putative feature matches that are refined by warping the 2D SIFT features such that they approximately match the same form of the orthographic VIP SIFT features.", "A histogram-based approach to 3D point feature detection was prosed by Fiolka et al. (2012), who extend the KB saliency detector (Kadir and Brady, 2001) and construct a histogram based on the distribution of normals.", "However, their approach only detects salient features based on the geometry of the scene and does not detect those based on any available texture; as a result it does not provide a unified approach to salient point detection in 3D.", "An earlier version of this work was published in Brown et al. (2014) based on the mean curvature, however this was a purely geometry-based KB saliency detector.", "In this paper we a) propose a derivative-based 2D KB saliency detector, and b) in contrast to both Fiolka et al. (2012) and Brown et al. (2014), we consider both the geometry and texture of the scene, allowing for salient point detection based on both attributes of the data simultaneously.", "Our framework for generalisable salient point detection is evaluated between 2D and 3D on a range of synthetic and real data.", "Here an outline of the Kadir-Brady (KB) saliency detector (Kadir and Brady, 2001) and its extensions and various implementations (Kadir et al., 2004; Shao and Brady, 2006; Shao et al., 2007) are given.", "The KB saliency detector (Kadir and Brady, 2001) is originally based on the principle that the parts on an image that are highly complex are salient.", "Scale-invariance is achieved by measuring the complexity across a range of scales and only selecting points whose complexity is peaked with respect to their scale.", "To further localise its scale, it is required that the point is statistically dissimilar across its neighbouring scales, known as inter-scale saliency.", "The saliency of a point is therefore the product of two terms: its complexity and its inter-scale saliency.", "Finally, salient points are clustered into salient regions so as to be more robust to noise.", "These three stages of the KB saliency detector (complexity estimation, inter-scale saliency, and clustering) are now described in more detail:", "From these two stages, a set of keypoints - those whose entropy is peaked in scale-space - are obtained.", "They have a saliency value of H(p, σs) × W(p, σs).", "An example of histograms obtained for the first two stages is given in Fig. 1, where the advantages of determining salient points as those with a high entropy and dissimilarity across scale are demonstrated.", "Stage III: Salient regions.", "From the previous stage a great deal of salient points are returned by the detector (typically hundreds of thousands); far too many to be of use in any practical application.", "Hence, a simple clustering algorithm is proposed.", "In the original paper (Kadir and Brady, 2001) a rather complicated clustering algorithm, dependent upon two user-defined parameters, is proposed.", "However, code provided on the author’s webpage uses a greedy clustering algorithm: it iteratively takes the point with the highest saliency value and removes all other points within its scale, continuing in this fashion until no points are left.", "We have found the greedy clustering algorithm to be better in practice, as well as more general since it is parameter free.", "A deficiency in the above approach is that it is not affine-invariant: histograms are computed in a circular region around a point, rather than the full range of potential elliptical regions.", "This was addressed in Kadir et al. (2004) where a full, time-consuming search over all ellipses in the image is implemented.", "Alternatively, in Shao and Brady (2006), the authors propose to first detect affine-covariant salient regions using the original KB saliency detector, then adapt these to make them affine-invariant.", "The proposed modifications of Shao et al. (2007) result in some improvement to the performance of the KB detector, as evaluated on the dataset of Mikolajczyk et al. (2005).", "Hence, Shao et al. demonstrate the potential of the approach as a repeatable feature detector, but do not demonstrate its broad applicability.", "In the next section, we generalise the KB detector and show how it may be broadly applied across different modalities.", "The original KB saliency detector was limited in its construction and as such was only applicable to images.", "In this section we propose a much more general formulation that allows it to be applicable in a multi-modal manner.", "Subsequently, we propose a derivative-based reformulation in the 2D domain, and a 3D formulation that naturally accounts for both the geometry and texture of the scene.", "To generalise the KB saliency detector, we observe that much of its construction is based on a very general concept: points whose entropy is peaked across scale are regarded as salient.", "To illustrate how widely this concept may be applied, we shall formulate the KB saliency detector in a more general manner for points lying in a metric space.", "Based on the above formulation, the generalised KB saliency detector may be applied to a range of multi-modal data.", "In the next two subsections, we construct a derivative-based 2D KB saliency detector, as well as a 3D KB saliency detector that naturally operates on both the geometry and texture of the scene.", "In both cases, the approaches are elegantly incorporated within the generalised KB saliency framework by simply defining the metric space and constructing the mapping F.", "The original 2D KB saliency detector was constructed based on the distribution of pixel intensities in a neighbourhood of a point.", "Whilst this gives some indication of some of the more complex, salient parts of the image, it fails to detect the geometrically salient aspects.", "In particular, it rarely detects corners, for which the neighbouring complexity of pixel intensities varies little with scale.", "As a result, the original 2D KB saliency detector fails to detect repeatable features between 2D and 3D (see the results in Section 7.5); focusing more on the texture of the scene rather than the geometry.", "In light of this limitation for the original KB saliency detector and based on the preceding generalisation, in this section we propose a derivative-based KB saliency detector.", "Specifically, the histogram mapping F is modified to be a function of the derivative of the image at any given pixel.", "This allows for high-derivative points within a low-derivative neighbourhood (e.g. corners) to be deemed salient; an important outcome in low-textured scenes.", "However, it is more general than a typical corner detector, determining salient points wherever a change in image derivative with respect to scale occurs, and avoiding noisy or repetitive parts of the scene.", "For 3D KB saliency detection, we shall define the metric space and histogram construction from Section 4.", "Such a general formulation allows for a large range of potential implementations; of note is its applicability to both textureless and textured 3D data within the same framework.", "More concretely, we may use a histogram mapping F that describes both the geometry and the texture of 3D data, rendering it equally applicable regardless of whether the 3D data is textured.", "In this section, we describe the histogram construction based purely on geometry (Section 6.1), on texture (Section 6.2), and on both (Section 6.3).", "An example of histograms constructed using each approach is shown in Fig. 5.", "Initially, we describe the approach taken based purely on the geometry of the 3D data.", "To do so, we project the local surface of the 3D data to an image and apply the same techniques as performed previously (construction of the second moment matrix); a similar approach has been taken for the construction of the 3D Harris corner detector (Sipiran and Bustos, 2010).", "The image is taken to be a tangent plane to the 3D data, and the ‘intensity’ value of the image represents the distance of the 3D data to the plane.", "We take a purely derivative-based approach in this subsection; an intensity-based geometric KB detector may not be constructed since the ‘intensity’ value of every point onto its own tangent plane is always zero.", "Our framework naturally allows for the extension to detect salient points based on both the geometry and texture.", "Given that the two histograms may be constructed based on the geometry or the texture, their joint histogram may be constructed.", "The intensity texture-based KB detector may be combined with the geometry-based KB detector to produce a KrG2 histogram.", "Alternatively, the derivative texture-based KB detector may be combined with the geometry-based KB detector, to produce a rD2rG2 histogram.", "Bilinear interpolation is again performed in these histograms.", "An example of histograms constructed based on the geometry, derivative-based texture, and both, is shown in Fig. 5.", "The histograms based on both are the joint histogram of the geometry and the derivative-based texture histograms.", "They are relatively large and, in general, sparse; exhibiting a very high entropy only when caused by both the geometry and texture.", "However, this approach is able to detect salient points based on either the geometry and texture, since in either case a relatively high entropy is observed at a particular scale.", "In this section we evaluate the performance of our proposed generalised salient point detector against other approaches, with both 2D and 3D data.", "Qualitative and quantitative results are given, where the final aim is to detect highly repeatable, sparse features between 2D and 3D, that may be of use in the subsequent registration stage.", "For comparison against our approaches, there exist a large number of feature detectors in both 2D and 3D (Guo et al., 2014; Tuytelaars and Mikolajczyk, 2008), however we focus specifically on comparing against feature detectors that may be meaningfully constructed in both 2D and 3D.", "We shall first introduce the detectors in each modality before describing how they are evaluated: firstly between 2D and 2D, and secondly between 2D and 3D.", "In 2D, we consider five detectors.", "Firstly, the traditional Harris corner detector (Mikolajczyk and Schmid, 2004).", "However, it is observed that, for small numbers of features, Harris does not detect a suitable spread of features, with many corners detected in the same area (see Fig. 9).", "Therefore, we secondly evaluate the Good Features to Track algorithm (GFT) Shi and Tomasi (1994) to obtain a better, more representative set of corners.", "Thirdly, we evaluate against the state-of-the-art SIFT detector (Lowe, 2004).", "The final two detectors evaluated are the proposed derivative-based KB detector (Section 5), referred to as KBD, and the original intensity-based KB detector (Shao et al., 2007) (referred to as KBI) so as to experimentally justify the construction of the proposed KBD detector formulated in Section 5.", "In 3D, there are optional detectors available to compare against depending upon if the texture of the data is used.", "For untextured 3D data, we consider four detectors: Harris (Sipiran and Bustos, 2010), SIFT, SURE1(Fiolka et al., 2012) and the proposed derivative-based geometric KB detector (Section 6.1), referred to as KB-G.", "In 3D, Harris is not scale-invariant and performs non-maxima suppression, therefore typically detects a better spread of corners in 3D than its 2D counterpart; hence there is no need for a 3D Good Features to Track detector.", "For untextured 3D data, SIFT detects keypoints based upon the mean curvature, and will be referred to as SIFT-G.", "Both Harris and SIFT-G are implemented in Point Cloud Library.2", "Harris is extended to 3D (Filipe and Alexandre, 2014) by replacing image gradients by surface normals from which a 3D covariance matrix is constructed.", "The response value is then a function of the determinant and trace of the covariance matrix (similar to 2D).", "SIFT is extended to 3D (Hänsch et al., 2014) using either the curvature of a point or the intensity (if the 3D point cloud is textured).", "A Difference-of-Gaussians (DoG) may be applied solely on this attribute of the point cloud (curvature or intensity) that does not change the position of the points.", "Local maxima and minima may then be found by comparing to a point’s k-nearest neighbours, subsequently points with low curvature are rejected as they are deemed unstable.", "For textured 3D data, there are additional detectors that may be evaluated against.", "SIFT may detect features on textured data based on the intensity (referred to as SIFT-T).", "Alternatively, the KB approaches may be used to detect features based purely on the texture, with the intensity-based KB detector referred as KBI-T and the derivative-based KB detector for textured 3D data referred to as KBD-T.", "Only the KB approaches allow for both the texture and geometry to be combined (Section 6.3), referred to as KBI-B and KBD-B.", "From the above 2D feature detectors (Harris, GFT, SIFT, KBI, and KBD) we firstly evaluate their repeatability in a 2D-2D scenario (Section 7.4).", "Subsequently, alongside the 3D feature detectors (Harris, SIFT-G, KB-G, SURE, SIFT-T, KBI-T, KBD-T, KBI-B, and KBD-B) we evaluate their repeatability between 2D and 3D.", "For untextured 3D data, we use six 2D-3D point combinations: Harris-Harris, GFT-Harris, SIFT-SIFT-G, KBI-KB-G, KBD-SURE and KBD-KB-G.", "For textured data there are a further five 2D-3D combinations: SIFT-SIFT-T, KBI-KBI-T, KBD-KBD-T; and where both geometry and texture are considered by KB: KBI-KBI-B and KBD-KBD-B.", "Thus, where the 3D data is textured, a total of 11 2D-3D feature detector combinations will be evaluated, to compare the effects of considering the geometry, texture, or both, of the textured 3D data.", "For a fair comparison, the other approaches (SIFT, GFT, Harris, and SURE) are altered, where possible, to align with these user-defined parameters.", "For SIFT in 2D the parameters provided by Vedaldi and Fulkerson (2008) are used and by Mikolajczyk et al. (2005) for Harris; and the parameter for GFT is defined such that no two corners are within 16 pixels of each other.", "In 3D, the fixed scale of Harris is set to σ1, and for SIFT-G, SIFT-T, and SURE, 12 scales are used, with the smallest set to σ1.", "Three datasets are used: a 2D-2D dataset from Mikolajczyk et al. (2005) (shown in Fig. 6); a synthetic 2D-3D dataset (shown in Fig. 7); and a real 2D-3D dataset (shown in Fig. 8).", "The 2D dataset is taken from Mikolajczyk et al. (2005).", "It is a set of six groups of six images, with the known homography between each image in a group provided.", "Each group of images has undergone a certain transformation (blurring, scale, JPEG compression, lighting, and viewpoint (twice)), from small to large transformations.", "The first and last images in each group are shown in Fig. 6.", "For synthetic data, we use six untextured 3D models.", "The first four models in Fig. 7 are from the Stanford 3D Scanning Repository.3", "For each of these four models, 50 images were rendered using POV-Ray using a random rotation matrix (Arvo, 1992) and translation such that the model is centred in the image, using a point light source at the same location as the camera.", "The latter two models are the 3D reconstruction provided by Guillemaut and Hilton (2011) of the dinosaur and temple from Middlebury’s multi-view reconstruction dataset (Seitz et al., 2006).", "In this case, 50 images with their known projection matrix from the model are provided as part of the dataset, so there is no need for rendering using POV-Ray.", "For real data (Fig. 8), we use five textured 3D models, obtained by a colour LiDAR scanner.", "All have been obtained from Kim (2014) with the exception of room, which is from Klaudiny et al. (2014).", "The number of points and the dimensions of the 3D models is tabulated below (Table 1):", "For each model, a set of between 7 and 11 images have been taken of the scene and manually aligned.", "This has been achieved by picking pairs of image and scene points, and using the approach by Penate-Sanchez et al. (2013) to determine the pose and focal length of the camera.", "An example image of each model is shown at the bottom of Fig. 8.", "Note that for certain models this does not encapsulate much of the scene (e.g. courtyard), making 2D-3D point detection more difficult.", "It has been observed in the literature (e.g. Hauagge and Snavely, 2012; Tombari et al., 2013b) that the repeatability measure is biased towards detectors that produce a lot of features, and a measure that is invariant to the number of points detected is proposed.", "Therefore, we compute the relative repeatability: for each set of points, order them in decreasing value of their response value.", "Then, the repeatability may be determined from the top-k points, and a graph may be plotted of repeatability against the k most responsive features in each set.", "Furthermore, this is a more useful measure for the purposes of sparse 2D-3D registration, where large numbers of features will not be of use due to the computational complexity of such a registration problem.", "Qualitative results for the set of five 2D point detectors are shown in Fig. 9, for a selection of images across the three datasets used.", "It is immediately noticeable, by the size and shape of the features, that Harris is affine- and scale-invariant; SIFT, KBI and KBD are scale-invariant, and GFT is neither, being a very parameter-dependent approach.", "SIFT, and in particular Harris, evidently have a tendency to detect the same feature at multiple scales and very similar locations: this motivated the use of GFT to obtain a better spread of features (Section 7).", "KBI and KBD naturally detect a better spread of points than Harris and SIFT, while retaining a parameter-free approach to scale selection.", "As a qualitative comparison between the KB approaches; KBD detects more corners than KBI (e.g. on the cathedral) while still detecting blob-like structures (e.g. windows in the third from top image) due to the necessary change in derivative present in such features.", "In contrast, KBI does not detect as wide a range of point feature types as KBD and often detects many edges (e.g. the cathedral).", "While edges may be regarded as salient, a point on an edge is poorly localised along the edge and is not useful for registration purposes.", "Quantitative results for 2D point feature detectors are given in Fig. 10 for the 2D-2D dataset (Fig. 6).", "The top-100 features are detected in each image, and an inlier threshold of 3 pixels is used.", "It is observed that no feature detector performs the best across all transformations.", "Harris performs particularly well for scale and JPEG compression changes, but very poorly across a change in viewpoint.", "GFT generally performs very well across the range of transformations.", "Importantly, KBD outperforms KBI across a number of transformations, justifying our proposed reformulation of the 2D KB detector.", "Qualitative results for the 3D feature detectors are shown in Fig. 11 for synthetic data and Fig. 12 for real data.", "For the untextured synthetic data, Harris, SIFT-G, KB-G, and SURE may be used.", "In Fig. 11, the scale-covariant Harris detector successfully detects a number of small-scale corners but often in repetitive places (e.g. the leg of the armadillo).", "KB-G is more robust than SIFT-G, detecting a wider range of points, e.g. on the armadillo and dino.", "By contrast, SIFT-G has a tendency to detect smaller, less meaningful features, e.g on the bunny.", "SURE typically detects corner-like structures where there is a wide distribution of normals, however it often detects large features and misses smaller corners e.g. on the dragon.", "As a comparison between features detected in 3D and the qualitative 2D results (Fig. 9); 3D Harris correlates quite well with 2D GFT, however it is clear the scale-covariance of GFT is an issue on the dragon.", "SIFT and SIFT-G often do not detect geometrically meaningful entities, with some 2D SIFT features detected off the model.", "KBI and KBD have some qualitative correlation with KB-G, but KBI often detects edges and avoids corner-like structures (particularly so on the dino).", "Qualitative results for real data are given in Fig. 12, where points are detected based on geometry (Harris, SIFT-G, KB-G), texture (SIFT-T, KBI-T and KBD-T), or both (KBI-B and KBD-B).", "Similar conclusions may be drawn from the geometry-based approaches as for the synthetic results (Fig. 11): Harris is limited by its scale-covariance, KB-G is generally more robust than SIFT-G, and SURE typically detects larger features and misses the finer detail.", "For texture-based detectors, few qualitative distinctions can be made between SIFT-T and KBD-T, however KBD-T detects more textural corner-like structures than SIFT-T (the same as in 2D in Fig. 9).", "Similarly to the 2D results, KBI-T detects more edge-like structures - particularly on the pavement on the cathedral.", "Interestingly, texture-based feature detectors often detect geometrically-significant features (e.g. corners on the cathedral, and the table-leg in the room) due to a natural change in colour on the model surface, or the lighting conditions.", "Finally, it is clear that both KBI-B and KBD-B detect points based on both the geometry (corners of the cathedral) and texture (carpet and picture in room).", "Quantitative results for the synthetic dataset are presented first.", "For each model-image pair, the relative repeatability is computed using the top-k 2D points and the top-2k 3D points (since it is expected half the 3D points will be occluded by the model), for k varying between 20 and 100.", "It is computed for inlier thresholds (t) of 3 and 6 pixels and averaged across all images of the model.", "Results are given in Fig. (13), where, given the 3D data is untextured, a comparison is made between Harris-Harris, SIFT-SIFT-G, GFT-Harris, KBD-SURE, KBI-KB-G, and KBD-KB-G.", "It is observed that, in general, GFT-Harris and KBD-KB-G perform the best; between them having the highest repeatability across all six models.", "Both have repeatabilities of at least 30% for (relatively) large numbers of points; sufficiently high for subsequent 2D-3D registration.", "KBI-KB-G performs quite well, but never as well as KBD-KB-G.", "This is perhaps surprsing in comparison to the results of KBI on the 2D-2D evaluation (Fig. 10) - the derivative-based KB formulation is evidently more indicative of geometry rather than texture based on these results.", "Harris-Harris, SIFT-SIFT-G, KBD-SURE, and KBI-KB-G perform similarly poorly, rarely obtaining a repeatability of above 20%.", "Comparing between 3 pixels and 6 pixels as the inlier threshold; GFT-Harris performs slightly better than KBD-KB-G for the smaller threshold, the reverse is true of the larger threshold.", "However, the increase in inlier threshold from 3 to 6 typically results in a repeatability increase by a factor of around 2, regardless of detector or dataset.", "Fig. 13 shows that, in general, the repeatability increases with respect to the number of points detected.", "However, this is not the case with GFT-Harris which, in some circumstances, shows a decrease in repeatability for increasing numbers of points - particularly so on the armadillo, and to a lesser extent on the dino and dragon.", "Fig. 14 shows qualitative results on the armadillo for GFT-Harris and KBD-KBG for smaller quantities of points.", "For very small quantities of points (20 in 2D and 40 in 3D) GFT-Harris has a high correlation due to the relatively small number of well-defined corners on the model (toes, fingers, and ears) and hence the relative ease at which they are detected by a corner detector.", "For a higher quantity of features (60 in 2D and 120 in 3D) there are insufficient corners in the scene and so it becomes unclear why certain features should be detected by the corner detectors.", "By contrast, our saliency-based approach is more broadly defined than a corner detector allowing KBD and KBG to admit a wider range of features.", "As a result, it is relatively unlikely our approach will have a higher repeatability for small numbers of features (since salient points are not as narrowly defined as corner points) but conversely the definition of saliency extends to larger numbers of features.", "Next, quantitative results for the real dataset are presented.", "For each model-image pair, the relative repeatability is computed using the top-k 2D points and the top-2k 3D points, with the exception of the larger courtyard and reception datasets where the top-4k 3D points are used, since here it is expected the majority of the 3D points will not be projected onto the image.", "k is varied up from 20 to 200.", "Similarly to the synthetic dataset, the relative repeatability is computed for inlier thresholds of 3 and 6 pixels.", "Results are presented in Fig. 15, where a comparison is made between all 11 approaches (as described at the beginning of Section 7).", "Between the different models, the best results are obtained on reception and room, with repeatability rates of over 30% in some cases.", "However, the other three models only obtain repeatability rates of between 15% and 25%.", "Between the different point detectors, KBD-KBD-T and KBD-KBD-B generally perform the best across all models.", "GFT-Harris performs nearly as well except on the more textured models room and studio.", "KBI-KBI-T more often outperforms KBI-KBI-B, further demonstrating that KBI does not detect geometrically significant features in 2D.", "Similarly to the synthetic dataset, SIFT-SIFT-G Harris-Harris, and KBD-SURE do not perform well in general.", "As a comparison between the methods proposed here (KBD-KB-G, KBD-KB-T, and KBD-KB-B), KBD-KB-G generally does not perform as well except on the cathedral model.", "It is perhaps surprising that KBD-KB-T consistently performs well, particularly on courtyard and reception where there is little discriminating texture; however as observed in the qualitative results, geometric features are often accompanied by a change in texture.", "Furthermore, the scale selection process within the KB detector allows it to naturally avoid repetitive parts of a scene.", "KBD-B consistently performs well regardless of the scene, outperforming the other approaches on the cathedral and studio.", "In this paper we have presented a general approach to 2D-3D salient point feature detection, based on the information-theoretic Kadir-Brady saliency detector (Kadir and Brady, 2001).", "The histogram-based framework developed allows for a unified approach to feature detection in 2D, and both textured and untextured 3D data.", "Intensity-based and derivative-based approaches were proposed, where the derivative-based approaches were shown to be superior since image derivatives are more indicative of the underlying geometry of the scene.", "The results also show the proposed approach to be more repeatable than existing feature detectors that have 2D and 3D implementations (Harris and SIFT) across a range of image and LiDAR data, from both indoor and outdoor scenes.", "Furthermore, its ability to naturally operate on textured or untextured 3D data allow the approach to detect features based on both attributes simultaneously, increasing its robustness and widening its applicability.", "There is scope for improvement in our method; in particular, the qualitative results show our approach to occasionally detect edges as salient.", "While there may be some salient properties regarding the edges, a point on an edge is not well localised along the edge and may not be as useful for geometry estimation.", "This could be addressed in a similar manner to Tombari and di Stefano (2014) where histograms are compared between neighbouring points, rather than between neighbouring scales.", "Alternatively, one may consider other attributes to construct a histogram from, other than the first derivatives of the image.", "However, while the second derivatives of the image have had considerable success in feature detection via SIFT (Lowe, 2004), the blob-like features they detect are generally more indicative of texture rather than geometry.", "Future work will include the registration of points between images and 3D LiDAR data.", "In many cases, correspondences between features cannot be automatically determined, and need to be established alongside registration parameters.", "It is a computationally expensive problem (Moreno-Noguer et al., 2008), so any method that has a high repeatability for a smaller number of points will be more suited to this kind of problem.", "We furthermore plan to integrate our approach with line features (Brown et al., 2015) detected in both 2D and 3D, so as to obtain a more complete scene description and make the subsequent registration process more robust due to the complementarity of these features.", "The authors confirm that the indoor and outdoor 2D-3D datasets generated as part of this research are freely available under the terms and conditions detailed in the license agreement enclosed in the data repositories.", "Details of the data and how to obtain access are available for the Roomdataset at Klaudiny et al. (2014); and for the Cathedral, Courtyard, Reception, and Studio datasets at (Kim, 2014)." ]

[ "A novel multi-modal histogram-based salient point detector is proposed.", "The general formulation allows salient point detection in both images and 3D data.", "The derivative-based approach improves upon existing intensity based 2D approaches.", "The 3D detector naturally operates on both the geometry and texture of 3D scenes.", "Increased 2D-2D and 2D-3D repeatability demonstrated on indoor and outdoor scenes." ]

[ "ENVI" ]

[ "Crete", "Karst", "Mediterranean", "Multi-site calibration", "SWAT", "Water balance" ]

[ "2312" ]

[ "“Karst” identifies a specific geological landscape and morphology formed by the dissolving action of water on soluble carbonate rocks such as primarily limestone, but also marble, dolomite, and gypsum.", "These rocks are mechanically strong but chemically soluble with high degree of secondary porosity.", "As a consequence, the hydrological cycle provides the primary source of energy for karst formation because water is the solvent that dissolves carbonate rocks and then carries the ions away in solution (Williams, 2004).", "The process of dissolution (‘karstification’) leads to the development of caves, sinkholes, springs and sinking streams that are typical features of a karst system.", "With progressing karstification, groundwater flow in the karst aquifer develops from a flow in an interconnected fissure network to a flow concentrated in several large pipes, interconnected cavities and cave systems (EC, 2003, 2004).", "The downstream end of a karst system usually is a spring where the underground conduit reaches the surface as an output point from an extensive network of groundwater conduits (Smart and Worthington, 2004).", "In Europe, soluble carbonate rocks are widespread in Western, Southern and Eastern part covering 35% of whole Europe (Daly et al., 2002), so that the karst processes are significant components of the physical geography of Mediterranean basins.", "In particular, limestones reach great thickness in Spain, southern France, Italy, the Balkan Peninsula, Turkey and in many islands in the Mediterranean (Crete, Majorca and Sicily).", "As a consequence, karst aquifers and springs are an important source of water supply for Mediterranean countries and special strategies are required to manage the quantity and quality of their waters.", "Bakalowicz (2015) pointed out the importance to study the karst aquifer functioning and the local geological evolution in order to manage in realistic and sustainable way the water resources.", "The monitoring and management of these resources are recognized in Europe as an essential issue and the European Union prompted the creation of COST Actions 620 and 621 to develop a comprehensive methodology for risk assessment and for the sustainable management of karst systems (EC, 2003, 2004).", "Unfortunately, most countries are lacking behind in monitoring the discharge of springs or wells and the exploitation of karst aquifers in generally is inappropriate (Bakalowicz, 2015).", "In this context, large scale hydrologic models are essential tools for watershed management at regional scale.", "Regional scale models with an appropriate discretization of watershed can adequately account for the spatial heterogeneity improving water predictions (Wooldridge and Kalma, 2001).", "A variety of karst models have been developed and applied to karst watersheds (Nikolaidis et al., 2013).", "Recently, Hartmann et al. (2015) presented for the first time the simulations of groundwater recharge in Europe with a grid-hydrological model (VarKarst-R) pointing out the importance of a characterization of subsurface heterogeneity.", "Baffaut and Benson (2009) modified the SWAT (Soil and Water Assessment Tool) model to simulate faster aquifer recharge in a Missouri karst watershed (SWAT-B&B) modifying deep groundwater recharge equations, increasing the hydraulic conductivity of sinkholes simulated as ponds and losses from streams.", "After that, Yachtao (2009) further modified the SWAT model in order to improve the simulation of water quality and quantity in the Opequon Creek watershed (USA).", "The author introduced two new parameters for simulating the hydrology and nitrate transport in a sinkhole and losses from sinking streams.", "More recently, Wang and Brubaker (2014) proposed a non-linear modification of groundwater algorithm in SWAT (ISWAT) improving the recession and low-flow simulation.", "Nikolaidis et al. (2013) developed a reservoir model approach linked with SWAT to simulate karst’s behavior and the recharge of springs of Koiliaris basin in Crete adding five new parameters.", "All these studies indicate that a specific parameterization of aquifer discharge, return flow, stream losses and sinkholes is required in karst watersheds.", "In addition, Wang et al. (2014) showed that a specific database of karst SWAT soils should be used to assess the influence of soil hydrological process in a karst region.", "In this paper the SWAT model was integrated with a karst-flow model (Nikolaidis et al., 2013; Tzoraki and Nikolaidis, 2007).", "Karst-subbasins were defined in order to reproduce more accurately the water balance at regional scale.", "This integrated model, KSWAT model, was applied to the Island of Crete, one of the most intensively managed Mediterranean islands, where the major water use is irrigation (84.5% of the total consumption) and the main water source is karst aquifers.", "The growing water demand of the region makes the rational management of water resources extremely important in view of a sustainable development.", "Consequently, the specific objective of this study is the development of a large scale methodology for the quantification of a spatially explicit water balance of karst-dominated geomorphologies using the SWAT model in order to assess the sustainability of water use in the Island of Crete and potentially to other areas.", "The Island of Crete occupies the southern part of Greece and is the largest island of Greece and the fifth in the Mediterranean.", "The Island covers an area of 8336 km2 and is divided into prefectures, including from east to west: Lasithi (1810 km2), Heraklion (2626 km2), Rethymno (1487 km2) and Chania (2342 km2) (Fig. 1a).", "The maximum length of the Island is 269 km and the maximum width 60 km.", "Four main mountains run west to east: the White Mountains in the west (2453 m), Idis mountain (2456 m) in the center, Asterousian (1280 m) in south Heraklion and Dikti (2148 m) in the east (Baltas and Tzoraki, 2013).", "The Island of Crete is characterized by a dry semi-humid Mediterranean climate with dry and warm summers and humid and relatively cold winters where mean annual rainfall decreases from west to east and from north to south, but increases with altitude (MEDIWAT, 2013).", "Annual precipitations are highly variable ranging between 300 mm in coastal areas and 2000 mm in headwaters in White Mountains.", "The mean annual temperature ranges from 18.5° in the west to 20° in the south of island and decreases with altitude.", "The mountainous areas, in particular in the western part, have mountainous climate.", "As a consequence, Crete contains sub-regions with very different hydrological characteristics.", "Crete has about 2550 km2 of agriculture land, about 30% of whole Crete, with more than 1100 km2 in Heraklion, and 3800 km2 of pasture (45% of total areas of Crete).", "The main crops are olives, grapes, and the main vegetables crops are tomatoes, cucumbers, onions, potatoes, watermelons and melons.", "The demand for irrigation water is high (about 360 Mm3/y), while only 47% (1200 km2) of agricultural land is irrigated.", "In Heraklion the irrigated area is around 600 km2, followed by Lasithi and Chania with around 300 km2, while a very small area in Rethymno is irrigated (Agriculture statistics of Greece, 2005).", "The geology of Crete is composed of carbonate rocks (limestone, marble and dolomite) which allow water to penetrate, creating major karst formation (Baltas and Tzoraki, 2013).", "More than 30% of carbonate rocks cover the total area of the Island and the major carbonate rocks of Crete are located in the White mountains (Lefka Ori), Idi, Dikti and Sitia (Fig. 1b).", "The total karst area covers about 2730 km2 and the water contribution to the karst aquifer is estimated around 2000 Mm3/y which discharges out in many springs (Chartzoulakis et al., 2001).", "There are 47 gauged springs in Crete with relative large flow, which are subdivided in three main classes: freshwater springs, brackish water springs and undersea springs.", "Most of springs are karst springs and refer to the same karst hydrogeological system (Lefka Ori, Idi, Dikti and Sitia) discharging around 500 Mm3/y freshwater into rivers.", "The most important springs are Stylos, Platanos and Kourtaliotis that discharge 85 Mm3/y in the Kalami/Koiliaris basin, 67 Mm3/y in Plantanias Basin and 38 Mm3/y in Kourtaliotis basin, respectively (Fig. 1c).", "Big brackish springs, located on coastal areas, include Almiros-Heraklion that discharges around 235 Mm3/y, Almiros-Agios-Nikolaos (83 Mm3/y) and brackish springs of lower discharge include Almiros-Mallia (3.2 Mm3/y), Georgioupolis, Grammatikaki and Malavra.", "Brackish springs discharge directly about 285 Mm3/y in the sea.", "Submarine discharges can be found in the southern part of Lefka Ori, Souda bay, Bali bay, Mallia bay, Elounta, Skinia, and the eastern part of the limestones of Zakros (MEDIWAT, 2013).", "Stream and spring discharge measurements were provided by the bureau of Water Resources at the Decentralized Administration of the Region of Crete that deals with data elaboration and the assessment of their accuracy.", "The stream measurements at 25 permanent locations were conducted using a continuous graphic record of the water depth.", "For a comprehensive description of continuous measurements of water depth, the reader can refer to Rantz et al. (1982).", "These measurements were then converted to streamflow rate with an established rating curve (streamflow, m3/s vs water depth, m) calculated every year for each monitoring station following the procedure described in Buchanan and Somers (1976).", "The streamflow-water depth relationship was adopted to calculate the streamflow at daily time step, and then aggregated at monthly time step.", "The available dataset for this study included 22 gauging stations because the streamflow stations STR30 and STR29 were excluded due to their uncertain localization and drain areas.", "STR5 was also excluded because observed data were lacking during the study period 1980–2009.", "The spring’s discharges measurements were directly calculated by measuring velocity once a month on surveyed cross-sectional channel geometry at 47 permanent locations.", "The only exception was the Almiros spring for which only the calculated monthly discharge was available.", "Table 1 summarizes the streamflow and springs data utilized in this study.", "The proposed karst modeling approach links the process-based model SWAT and a karst-flow model (Nikolaidis et al., 2013).", "Fig. 2 represents the sequential steps of our approach that involved the setup of SWAT model, the identification of karst subbasins in which a modified version of SWAT model was applied, the calibration of streamflow at selected gauging stations (step-wise calibration), the regionalization of calibrated parameters for ungauged subbasins, the spring’s discharges calibration outside the SWAT model using the karst-flow model and finally the introduction of calibrated discharge of springs as a point sources in the SWAT model.", "The overall procedure included the use of hard and soft data.", "Hard data are defined as long-term, measured time series, typically at a point within a watershed, while soft data are defined as information on individual processes within a budget that may not be directly measured within the study area, generally from literature information (Arnold et al., 2015).", "In this study long time series of 22 streamflow and 47 of spring monitoring stations were used as hard data, while for instance literature information of the extension of karst recharge areas, as soft data.", "This approach enables the dialog between local experts, experimentalists and modelers, increases the reliability of the model results in ungauged areas and helps to better constrain model parameters (Seibert and McDonnell, 2002).", "The SWAT model (Arnold et al., 1998) model is a continuous-time, semi-distributed, process based river basin model, originally developed to predict the long-term impact of climate and land use management practices on water, sediment, and agricultural chemical yields in large complex basins.", "SWAT uses the water balance approach to simulate watershed hydrologic partitioning.", "In particular, the water balance is calculated considering the snow, soil, shallow aquifer and deep aquifer components.", "The watershed is first subdivided into subbasins and each subbasin into hydrologic response units (HRUs) that are a function of soil type, land use and land slope.", "The main components of the model are: weather, hydrologic mass balance, soil temperature and soil properties, plant growth, nutrients cycling, and sediments yield.", "SWAT runs on a daily time step.", "For specific aspects of SWAT, Neitsch et al. (2010) provide a complete description in the theoretical documentation of the model.", "SWAT2012 (v.622) uses two linear reservoirs to partition groundwater into two aquifer systems (Fig. 3a): a shallow aquifer which contributes baseflow to streams (GWQ1) and a deep aquifer which can also contribute baseflow to streams (GWQ2).", "The remaining portion in the deep aquifer (DA_RCHRG – GWQ2) can be considered lost from the system.", "The recharge for a specific day is calculated as a linear function of the daily seepage, the recharge of the previous day and the groundwater delay.", "Daily seepage includes seepage through the soil profile, through ponds or wetlands and loosing streams (tributaries and main channels).", "All seepage losses are added together and assumed to travel vertically to the aquifer with the same velocity.", "The baseflow from the shallow aquifer GWQ1 is calculated through the aquifer recharge (GW_RCHRG, mm H2O), the deep aquifer recharge (DA_RCHRG, mm H2O) and the baseflow recession constant (ALPHA_BF, 1/day).", "Similarly, the baseflow from the deep aquifer (GWQ2) depends on the deep aquifer recharge and on the deep-baseflow recession constant (ALPHA_BF_D, 1/day).", "In order to simulate the specific characteristics of karst aquifers (i.e. fast infiltration, movement of water in cave systems) and to calculate the contribution of the karst areas to streamflow as spring flow, the SWAT model structure (Fig. 3a) was adapted as shown in Fig. 3b.", "This new SWAT Model structure (Fig. 3b) was the result of the combination of two main studies: Baffaut and Benson (2009) and Nikolaidis et al. (2013) and represents the concept of the karst model and its hydrological pathways.", "Hereafter, this model configuration is called KSWAT and combines an adapted SWAT model and a karst-flow model.", "In particular, the adapted SWAT model considers fast infiltration through caves and sinkholes up to the deep-aquifer, while the karst-flow model represents the fast movement of water in subterranean conduits and the lower movement in narrow fractures through the interconnection of reservoirs structure.", "The adapted SWAT model consists in representing sinkholes by wetlands with small drainage area and a large hydraulic conductivity at the bottom of the wetlands and loosing streams were represented by tributary channels with high hydraulic conductivity in the stream bed.", "All the percolation in the soil profile, stream losses and seepage from the bottom of wetlands directly recharges the deep aquifer (DA_RCHRG, mm H2O).", "This is achieved by setting the deep aquifer percolation fraction (RCHRG_DP) to 1, imposing minimum groundwater delay (GW_DELAY equal to 1) and setting the groundwater coefficient of capillarity rise (“revap”) 0.1 in order to avoid that water moves from shallow into the overlying unsaturated zone while the baseflow from shallow and deep aquifers was imposed negligible (Table 2).", "The spring’s discharge was simulated with a karst-flow model developed in “Excel” environment by Nikolaidis et al. (2013).", "The input was the daily deep aquifer recharge (DA_RCHRG, mm) calculated by the SWAT model.", "The karst-flow model is composed by an upper reservoir with faster response that represents wide conduits in a karst system and a lower reservoir with a slower response that simulates narrow fractures (Kourgialas et al., 2010).", "The karst-flow model uses the deep aquifer recharge of the surrounding subbasins and involved at the same time the simulation and calibration of springs discharges.", "The procedure required the contribution/exclusion of deep aquifer recharge of nearest subbasins and the manually adjustment of five parameters of karst-flow model minimizing the Normalized Root Mean Square Errors.", "These five parameters included: Qko, the initial karst flow (m3/day), a1 the fraction of deep groundwater discharge entering the upper reservoir, a2 the fraction of flow from the upper reservoir discharge entering the lower reservoir and ku and kl were recession constants (1/d) for the upper and lower reservoirs (Table 2).", "For further details of karst-flow model equations, the reader can refer to Nikolaidis et al. (2013) who provided a complete description.", "The outputs of the karst-flow model were then introduced in SWAT as point sources with aggregated values at monthly time step.", "This modified SWAT was applied only in the karst-subbasins identified by three dominant soils that were strongly correlated to the karst areas of the Geological Map (Fig. 4b and c).", "According to the European Soil Database these are Leptosols (European Soil Portal, 2014), poorly developed and shallow soils over hard rock and comprise of very gravelly or highly calcareous material.", "However, the use of the “dominant karst soils” sometimes was limiting since in some cases the size of the subbasins (about 20 km2) and the fragmentation of soils inside the subbasin did not allow identifying it as “karst subbasin”.", "This occurred for instance in the south-central part of the Island in Kourtaliotis Basin (corresponding to Kourtaliotis river basin in Fig. 1c), where this limitation was overcome using literature information (i.e. Steiakakis et al., 2011) and the geological map.", "Thus, both karst-soils extension and the geological map were used to identify the karst-subbasins where the KSWAT model was applied.", "It is noteworthy that the extension of wetlands inside each karst-subbasin was defined using the geological map, generally covering the entire subbasins where the karst-soils are dominant, or only a percentage of subbasin area as in the case of the Kourtaliotis Basin.", "The resulted area covered by the karst-subbasins was estimated around 2600 km2.", "The SWAT model requires pedological, climatological, topographical and land use data.", "Subbasins were delineated using the ArcSWAT interface with a Digital Elevation Model of 25 m pixel size (EU-DEM; Bashfield and Keim, 2011).", "The Digital Elevation Map was obtained from a Pan-European elevation data at 1 arc-second (EU-DEM).", "Subbasins and streams were defined using a drainage area threshold of 1000 ha resulting in 352 subbasins with an average area of 19 km2 covering 6700 km2 (Fig. 4a).", "Land cover was derived from a 1 km raster map built from the combination of CAPRI (Britz, 2004), SAGE (Monfreda et al., 2008), HYDE 3 (Klein and van Drecht, 2006) and GLC (Bartholome and Belward, 2005) for the year 2005.", "Land use was obtained from the Agriculture statistics of Greece (2005).", "For each subbasin the “dominant land cover approach” was used in order to reduce the complexity and the computational level of the model.", "Six classes of dominant land cover were defined: arable land, pasture, forest, urban area, water and range grasses.", "Each subbasin with dominant arable land cover was further subdivided in three parts (15–35–50% of subbasin area) in order to distribute more accurately the crops reported by the Agricultural census.", "Soil type and characteristics were defined using a 1 km soil raster map, obtained from the Harmonized World Soil Database (HWSD) (FAO, 2008).", "From the combination of land use, dominant soils, and single slope for each subbasin the final configuration resulted in 502 HRUs (Hydrological Response Units) with an average area of 13 km2.", "The management practices for each crop included planting, fertilization, irrigation and harvesting.", "The simulated timing of plant sowing and harvesting were implemented in SWAT through daily heat unit concept.", "In this study the heat units for each crop were calculated by Bouraoui and Aloe (2007) using the PHU (Potential Heat Units) program (PHU, 2007), developed at Texas Agricultural Experiment Station.", "The PHU program calculates the total number of heat units required to bring a crop to maturity.", "For each crop the crop growing season (winter versus spring crop), the base growing temperature (°C), the optimum growing temperature (°C), the dry down fraction, the time to maturity (number of days between planting and harvesting) were considered as input in the PHU program.", "These attributes were related to the different European climatic zone.", "The amount of manure and mineral fertilization applied were retrieved from the Common Agricultural Policy Regionalized Impact (CAPRI) agro-economic model (Britz and Witzke, 2008).", "It provided at NUTS 2 administrative level (Nomenclature of Territorial Units for Statistics) the mineral and organic nitrogen and phosphorus fertilizer application estimated from sectoral statistics and animal production levels.", "A manual irrigation was applied selecting the irrigated HRUs for a total irrigated area around 25,000 ha in Chania, 9300 ha in Rethymno, 60,000 ha in Heraklion and 26,000 ha in Lasithi.", "The total abstractions for irrigation use in each Prefecture was 89 Mm3/y in Chania, 35 Mm3/y in Rethymno, 170 Mm3/y in Heraklion and 66 Mm3/y in Lasithi, respectively.", "Both irrigated areas and volumes were obtained from the Agriculture statistics of Greece (2005).", "According to the National statistics, the sources of abstraction for irrigation uses were set from deep aquifer and shallow aquifer, and from springs.", "In the model the abstraction for irrigation from springs (38 Mm3/y) was implemented as outside-watershed source in the schedule management plan and the corresponding abstractions were subtracted from the daily deep aquifer recharge (DA_RCHRG, m3/s) (outside SWAT), so that the calibration of springs discharge in the karst-flow model was performed using the net of the abstractions.", "The abstractions for drinking water, industry and olive mills (40 Mm3/y) were introduced in SWAT by subtracting water from the deep aquifer and shallow aquifer at monthly time step, while the abstractions for drinking water from the springs (24 Mm3/y) were subtracted from the daily deep aquifer recharge (DA_RCHRG) (outside SWAT).", "The climate data used in this study include 69 stations with daily data for precipitation and 21 stations for temperature from 1961 to 2009.", "Monthly statistics of solar radiation, wind speed and relative humidity were calculated using the pan European high-resolution gridded daily data set EFAS-METEO, acronym of European Flood Awareness System – METEO (Ntegeka et al., 2012) for 29 stations uniformly distributed over the modeled territory.", "The subbasins were split into elevation bands, and snow cover and snowmelt were simulated separately for each elevation band.", "In this study steep subbasins were subdivided into 4 classes of elevation.", "For the calibration and validation of the Crete SWAT model 22 stations of streamflow were used.", "Based on the simplest test technique (Klemeš, 1986), 15 of them were used for calibration and 7 for validation covering the period 1980–2009.", "The selected subbasins for calibration were headwaters and were uniformly distributed in the Island.", "The streamflow was calibrated in the 15 selected subbasins in different steps (“step-wise calibration”) according to the involved hydrological process, providing a calibrated parameter set NOP (Near Optimal Parameters set).", "The obtained NOP set was then transposed in ungauged subbasins using the “hydrological similarity approach”.", "The step-wise calibration is a procedure that allowed calibrating the different components of the streamflow according to different hydrologic processes underpinning each calibration objective.", "This procedure proposed by Pagliero et al. (2015) and Malagò et al. (2015) was performed using the SWAT-CUP (SWAT Calibration Uncertainty Procedures) programs and the SUFI-2 algorithm (Abbaspour, 2008).", "The calibration period covered 30 years from 1980 to 2009 with 10 years of warm up.", "Among several parameters in SWAT, 19 parameters (see parameters that refer to SWAT and SWAT/Adapted SWAT model in Table 2) were chosen for the streamflow calibration as the most sensitive and representative of each hydrological process involved in the calibration.", "The parameters and their initial ranges (Table 2) were selected based on preliminary model runs, literature reviews (van Griensven at al., 2006) and sensitivity test.", "It is noteworthy however that the parameters in karst-subbasins were not changed keeping the settings of the adapted SWAT model configuration as described in Table 2.", "In this work, since the streamflow data was only available at monthly resolution, the streamflow was not divided into the main components (surface runoff, lateral flow, baseflow).", "However, the calibration was still performed step wise:", "Calibration of total monthly flow in order to control the timing of runoff signal by adjusting the snow parameters by minimizing the coefficient of determination r2;", "Calibration of total monthly flow using only the parameters that regulate the surface runoff process by minimizing the coefficient of determination r2;", "Calibration of total monthly flow using only the parameters that regulate the lateral flow process by minimizing the coefficient of determination r2;", "Calibration of total monthly flow using only the parameters that regulate the baseflow process by minimizing the Nash Sutcliffe Efficiency, NSE (Nash and Sutcliffe, 1970);", "Final step, calibration of total monthly flow using all previous parameters together with a reduced uncertainty ranges by minimizing the NSE coefficient.", "The coefficient of determination r2 (Taylor, 1990) was adopted as objective function for the first three steps and the NSE coefficient (Nash and Sutcliffe, 1970) was selected for the fourth and final step as described below.", "The selection of the objective functions is the result of several tests since the differences between objective functions on parameter sets could be quite striking (Abbaspour et al., 2015).", "The calibration was performed using 1000 simulation runs at each step.", "The Final Near Optimal Parameter set (NOP) of calibrated subbasins (called “donors”) was transposed to ungauged subbasins using the hydrological similarity approach.", "The measure of hydrologic similarity between subbasins is translated into subbasin characteristics which are common for gauged and ungauged watersheds.", "These characteristics that linked the subbasin characteristics with the hydrological responses have been performed in the literature (Shamir et al., 2005; Oudin et al., 2010).", "However, for a comprehensive enlightenment of hydrological similarity approach the reader can refer to Wagener et al. (2007) and Sawicz et al. (2011).", "The calibration for the parameters of the karst-subbasins were kept unchanged using the settings of the adapted SWAT model.", "The hydrological similarity approach was performed using the PLSR (Partial Least Squares Regression) that allowed identifying similar subbasins based on the correlation between the watershed characteristics and the discharge characteristics.", "Following Malagò et al. (2015), this correlation was performed using 17 gauged independent basins using the R package “pls” (Mevic and Wehrens, 2007).", "The gauging stations selected to perform the PLSR analysis involved stations from the group selected for the calibration and some from the validation group in order to identify a group of stations with almost 8 years of continuous data from 1988 to 1995 that allowed calculating robust statistical indices.", "For the selected subbasins 20 independent variables representing the subbasin characteristics (“c” matrix) and 2 dependent flow variables (“q” matrix) were used in the PLSR analysis as the responses and regressors (q ∼ c) (Malagò et al., 2015).", "The independent variables included river length (km), minimum, maximum and average elevation (m), median slope (%), content of clay and sand in the soil, percentage of different classes of land cover (%), percentage of karst area, mean annual precipitation (mm), minimum, maximum and average temperature (°C), annual potential evapotranspiration (mm) and average number of days with precipitation.", "The dependent variables included the mean annual discharge (mm/y) and the corresponding coefficient of variation.", "The PLSR analysis defined latent variables that were used to perform the classification of subbasins.", "Due to the limited number of potential donors (15 subbasins, 4% of total number of subbasins), it was necessary to define “hydrological regions” and then the “classification” of similar subbasins was performed inside each region.", "For a review of regionalization techniques, the reader can refer to Parajka et al. (2005).", "Using the latent variables, the regionalization was performed with the Ward’s hierarchical cluster analysis (Ward, 1963) based on the Euclidean distances and the classification of subbasins in donor’s classes was pursued inside each hydrological region according to the K-Nearest-Neighbor using the R package “supclust” (Dettling and Maechler, 2012).", "The classification of each subbasin allowed the automatic assignation of the NOP of “donor subbasins” to the classified ungauged subbasins.", "In order to verify the strength of the parameters regionalization in the ungauged subbasins the model performance was evaluated using the percent bias (PBIAS; Gupta et al., 1999) between monthly observed and simulated streamflow at seven gauging stations.", "This was an intermediate validation that anticipated a final complete validation performed in all gauging stations after the introduction of springs in the model as described in the next paragraphs.", "The springs were calibrated using the daily recharge of deep aquifer (DA_RCHRG) from the adapted SWAT model, except for Spring SP54 (Almiros Heraklion) that was calibrated using DA_RCHRG at monthly time step.", "The springs were calibrated manually changing parameters of the karst-flow model in reasonable ranges: the initial karst flow (m3/day), the fractions a1, a2, and the recession constants ku and kl (1/d) for each reservoir as show in Table 2.", "This process ended when the Normalized Root Mean Square Error (NRMSE) calculated using the simulated and observed values for each spring was minimized.", "42 springs out of 47 were calibrated having hypothesized that some springs drained the same karst area.", "This assumption was strongly correlated to the neighborhood location of these springs that didn’t allow distinguishing the contribution of deep aquifer recharge from the same karst-subbasins; hence SP12 and SP13, SP44 and SP45, SP4 and SP50, SP7, SP18 and SP19 were grouped into SP12-13, SP44-45, SP18-19-7 and SP4-50.", "The springs were entered as points sources in the model and the associated calibrated monthly Qk (m3/s) were implemented as input data in SWAT.", "For each spring the karst recharge areas (inside and outside the hydrological boundary) was defined as sum of subbasins areas whose deep aquifer recharge contributes to a specific spring.", "The subbasins that contributed to spring flow through their deep aquifer recharge in the karst-flow model were not necessarily hydrologically connected.", "The linkage between these subbasins and the springs represented the movement of water through the cave systems whose spatial extent and organization was different from the surface subbasin hydrologic boundaries.", "The performances of the karst-flow model were evaluated by visual comparison of measured and simulated hydrographs of springs and using performance indicators including the Normalized Root Mean Square Error (NRMSE), the percent bias (PBIAS; Gupta et al., 1999), the coefficient of determination r2 multiplied by the slope of the regression line between simulated and observed values (br2; Krause et al., 2005) to overcome the intrinsic weakness of each indicator (Bennett et al., 2013).", "After the introduction of calibrated springs in the SWAT model as point sources, an additional manual calibration was necessary to adjust the water discharge including peaks and baseflow since the addition of the spring contribution modified the already calibrated discharge.", "In particular, the effective hydraulic conductivity in main channel (mm/hr) in Messera, Anapodiaris and in Petras Basins was adjusted (Table 2).", "Finally, the complete SWAT model was validated using the whole dataset of streamflow gauging stations in order to evaluate the model performance after the introduction of the springs.", "Similarly to calibrated streamflow and spring’s discharges, the complete SWAT model was validated comparing monthly observed and simulated streamflow in the period 1983–2009 calculating the NRMSE (%), PBIAS (%), the Nash Sutcliffe efficiency (NSE), br2 and r2.", "The performance indicators obtained comparing the monthly observed and simulated streamflow for the 22 stream gauging stations after calibration, regionalization and the introduction of the calibrated springs are summarized in Table 3.", "It is noteworthy that about 64% of the calibrated gauging streamflow stations reached satisfactory PBIAS% (values in the range ± 25%), while only 40% had NSE greater than 0.5 (see Moriasi et al., 2007 for performance rating as reference).", "Furthermore, 50% and 36% of calibrated streamflow gauging stations reached respectively r2 and br2 larger than 0.5.", "Fig. 5 shows some examples of comparison of hydrographs.", "Even though some of the statistical indicators did not score very high, the hydrographs comparison illustrates that the model was able to reproduce accurately the monthly variations over a long period of simulation even in subbasins influenced by excessive water pumping, such as STR16.", "This gauging station was fed by precipitation and discharge of spring SP16.", "After the year 1990, the spring’s discharge was diverted to supply domestic uses reducing drastically the streamflow in particular during the summer and autumn.", "It was estimated that the percentage of streamflow gauging stations that reached satisfactory PBIAS (PBIAS < ±25%, Moriasi et al., 2007) increased from 59% to 64% adding spring’s discharges in the SWAT model highlighting the significant influence of springs in the water balance of Crete.", "Fig. 6 shows the PBIAS cumulative curves of performance indicators calculated between monthly and observed streamflow before and after the introduction of springs as point sources in the SWAT model.", "In particular, streamflow discharge of gauging stations STR32 (Koiliaris), STR16 (Avli), and STR10 (Koutsoulidis) were strongly influenced by the discharge of springs SP41 (Stylos), SP14-SP25 (Migilisi and Emparos) and SP20 (Zaros), respectively.", "For STR32 the PBIAS decreased to 3% and the NSE increased to 0.68, for STR16 the PBIAS% reached 11%, and finally for STR10 the PBIAS% and NSE were also satisfactory reaching 17% and 0.83 respectively (Table 3).", "These results demonstrated that the karst-flow model correctly simulated the discharge of springs increasing the SWAT model performance.", "Fig. 7 shows some examples of comparison between daily observed and simulated spring flows (only for SP54 monthly comparison), and Table 4 summarizes the comparison between monthly and observed long-term annual discharge on the period 1983–2009 and the performance indicators for all springs.", "It is noteworthy that for more than 70% of the calibrated springs the PBIAS% is within the range of ±25%, indicating that the performance of the karst-flow model was satisfactory (Moriasi et al., 2007).", "NSE was positive for 80% of calibrated springs, while r2 and br2 had a wide range of values with around 50% of the values larger than 0.4.", "However, the karst-flow model markedly overestimated the discharge for springs SP4-50 (Agios Georgios Sitia-Sikia Sitia), SP14 (Avli Migilisi), SP16 (Mega Vrisi), SP41 (Stylos) and SP51 (Loutraki) maybe due to the quality of the observed data.", "Concerning SP41, the most studied spring in Crete (Nikolaidis et al., 2013; Moraetis et al., 2010; Kourgialas et al., 2010), was intentionally calibrated to overestimate the flow in order to take into account the contribution of ungauged springs.", "Nikolaidis et al. (2013) reported that Stylos spring has two permanent springs at elevation +17 m AMSL (with 85 Mm3/year of volume), but there was also an ungauged intermittent spring (Anavreti at elevation +24 m AMSL).", "Both contributed to Koiliaris discharge.", "The authors argued that these springs have a total average discharge around 154 Mm3/y (2007–2010), and this volume was in agreement with the total overestimated volume of Stylos in this work (150 Mm3/y, for the same period 2007–2010, 170 Mm3/y considering 1983–2009).", "Finally, it is noteworthy that the spring SP46 (Fig. 7) had two different hydrological behaviors: observed data were higher prior to year 1988, then the values decreased sharply highlighting changes in the regime of discharge.", "This may be explained by a change in the method of measurement or more probably by an excessive pumping of water as occurred for the STR16 spring.", "Fig. 8 shows some schematic examples of recharge areas of springs obtained considering the subbasins that contribute with their deep aquifer recharge to the calibration of springs.", "Table 4 summarized also the karst recharge areas (km2) for each spring or group of springs.", "The karst recharge areas of springs cover karst-subbasins and karst areas for a total of 1928 km2, about the 70% of total karst areas in Crete.", "The main karst recharge area drains more than 300 km2 into Almiros Heraklion Spring (SP54), followed by the karst recharge area of Almiros-Agios-Nikolaos Spring (SP9) that cover about 155 km2, and 130 km2 of karst recharge area of springs in Koiliaris Basin.", "To the best of our knowledge this work was the first estimation of karst recharge areas of all gauged springs in the Island of Crete, providing valuable information for water resources management.", "The only exception was the karst area drained by Almiros Spring and the knowledge of extended areas (outside the hydrological boundary of basin) of springs in Koiliaris Basin.", "Bonacci and Fistanic (2006) pointed out the difficulties to define the karst area drained by Almiros Spring due to complex geology and hydrogeology.", "There were different assumptions of its boundaries and size which varies from 300 km2 (Arfib et al., 2000) to 500 km2 (Lambrakis et al., 2000).", "However, Bonacci (1995) and Bonacci and Ljubenkov (2005) estimated the karst recharge area of Almiros of about 300 km2, similar to our finding.", "The area outside the Koiliaris Basin that contributed to the karst recharge area of Stylos Spring and other ungauged springs in Koiliaris Basin was estimated around 80 km2 (7 subbasins that contribute with 70% of their total area).", "As a consequence, more than 60% of karst recharge area of Koiliaris springs was outside the boundary of the hydrological basin.", "This result was confirmed by Nikolaidis et al. (2013) that estimated an extended karst area of springs in Koiliaris Basin of 79 km2 based on hydrologic modeling and geologic cross-sections.", "Albeit this approach may be criticized by the fact that karst recharge areas should be only delineated based on hydrogeological considerations derived, i.e. by tracer tests, it has to be taken into account that there are limits in the use of tracer studies in karst areas such as in Koiliaris basin.", "In a geological karst area which discharges a large volume of water there is a risk of high degree of dilution of tracers (Knithakis, 1995) which in turn limits the usefulness of the methods in delineating the extend area.", "In addition, breakthrough curves are highly dependent on the flow conditions, often necessitating the repetition of the tracing experiment under low flow and high flow conditions as observed by Goldscheider (2005) and Ravbar et al. (2011).", "The mean annual precipitation ranges from 550 mm (driest year) to 1400 mm (wettest year) during the period 1983–2009, with a decreasing gradient from west to east.", "The precipitation was higher at high elevation, reaching more than 2500 mm in the wet years (1996, 1997, 2001 and 2003) and around 1000 mm in the central north areas, while in the plain areas and along the south cost the average annual rainfall was below 1000 mm.", "The mean calculated actual evapotranspiration of Crete varied from 340 mm/y (driest year) to 390 mm/y (wettest year) reaching the maximum values long the north coast and, in particular in Karteros, Aposelemis, Tzermiadon, Myrtos and Anapodiaris basins (east-central part of Crete).", "Large values of surface runoff were simulated in the western part of Crete and in particular in Tavronitis Basin with maximum value in wet years, such as in 1997 and 2003 (more than 1100 mm).", "Minor contributors to the water balance include lateral flow and baseflow.", "The baseflow reached high values in Kourtaliotis, Akoumianos and Platis basins (south-central Crete).", "The annual and spatial variations of deep aquifer recharge were strongly correlated to the precipitation, as a consequence in zones with high precipitation the deep aquifer recharge reached the largest values since the main mountainous systems were associated with the most important karst systems, allowing water to penetrate directly to the deep aquifer as showed in Fig. 9.", "In particular, Fig. 9 illustrates how precipitation, evapotranspiration and recharge of the deep aquifer change during the year.", "Precipitation and deep aquifer recharge changed during the year decreasing from January (∼1120 and 463 Mm3/month respectively) to August (∼18 and 62 Mm3/month respectively) and increasing from September (∼163 and 63 Mm3/month respectively) to December (∼1250 and 416 Mm3/month respectively).", "During the wettest months, the west part of Crete reached the highest values of precipitation and also the highest value of deep aquifer recharge.", "The evapotranspiration increased from January (∼240 Mm3/month) to April (∼320 Mm3/month), in particular in subbasins long the cost, and then decreased until August (∼54 Mm3/month) and increased from September (∼71 Mm3/month) to December (∼240 Mm3/month).", "These long-term spatially and monthly variations could be very useful for planning and implementing conservations measures and programs and evaluating their performance.", "Chartzoulakis et al. (2001) and many other studies (RCG, 2002; Vardavas et al., 2004; MEDIWAT, 2013; Koutroulis et al., 2013; Baltas and Tzoraki, 2013) pointed out the importance of the evapotranspiration on the water balance of the whole Crete giving infiltration a secondary role.", "Conversely to these findings, the Crete SWAT model has allowed the estimation of the water balance of Crete resulting in significantly different estimates.", "Fig. 10 summarizes the percentage of flow contribution of the water balance main components with respect to the long-term average volumes and different hydrological conditions during the period 1983–2009.", "In addition, Fig. 11 represents the previous results following the KSWAT model approach providing a more readable and systematic information of the fluxes.", "The evapotranspiration volume ranged from around 2250 Mm3/y in the driest year to 2500 Mm3/y in the wettest year exhibiting a small variation in terms of absolute values but with a significant variation with respect to the total volume of precipitation (Fig. 10).", "In the wettest year the main component of hydrological processes was the deep aquifer recharge, while in the driest year the evapotranspiration had the main role.", "From dry to wet year the surface runoff increased from 95 Mm3/y to more than 800 Mm3/y and the total net water available in the deep aquifer increased from 550 Mm3/y to around 3500 Mm3/y in dry and wet year, respectively.", "As a consequence, during the wet conditions there was high infiltration, but also the surface runoff was larger than that during driest and normal hydrological condition.", "On the other hand, in dry years there was also significant percolation to the deep aquifer (1000 Mm3/y).", "This result was also highlighted by Hartmann et al. (2014, 2015) pointing out that karst regions might be more resilient to climate change in terms of both flooding and droughts.", "Furthermore, the authors demonstrated the existence a nonlinear relationship between precipitation and recharge rate indicating that the recharge of deep aquifer is more sensitive to a decrease than to an increase of precipitation (Hartmann et al., 2014).", "This was confirmed also in this study where the total deep aquifer recharge was estimated 28% of precipitation in driest year, while was 50% in wettest year departing of 16% and 6% from normal conditions, respectively.", "Concerning the percentage of water balance components (Fig. 11), it was estimated that from the total precipitation of 6400 Mm3/y about 40% was lost through evapotranspiration, 55% infiltrates and only 5% was surface runoff.", "The surface runoff including lateral flow (LATQ), baseflow (GWQ1) and spring’s discharge (Qkin) contribution to streamflow represents 22% of total precipitation.", "The total amount of deep aquifer recharge was around 44% of which 14% contributed as springs discharge to streamflow (Qin = 9%) or sea (Qout = 5%) (Fig. 11b).", "These percentages were valid for all years in normal hydrological conditions (for instance year 1987), but they changed in extreme hydrological conditions.", "Considering the driest year (year 1990, Fig. 11a), it was estimated that from the total precipitations of 3700 Mm3/y about 60% was lost through evapotranspiration, 37% infiltrated and only 3% was surface runoff.", "The amount of deep aquifer recharge decreased with respect to the normal hydrological condition to 28% of which 13% contributed as springs discharge to streamflow (Qin = 9%) or sea (Qout = 4%).", "The total contribution to freshwater decreased to 14% with respect to the normal condition.", "In the wettest year (year 2003, Fig. 11c), considering a total volume of precipitation around 9600 Mm3/y, evapotranspiration was estimated around 26%, 65% was infiltration and 9% was surface runoff.", "The deep aquifer recharge reached the highest percentage of 50% and the water yield increased to 28%.", "Our work has shown that the evapotranspiration ranged between 2250 Mm3/y in a driest year and of 2500 Mm3/y in wettest year, corresponding to 60% and 26% of total precipitation, respectively.", "These results suggested the importance of karst water supply during dry periods.", "The modeling approach proposed in this study allowed achieving satisfactory simulations of streamflow and springs discharges at monthly and daily time step respectively across the whole Crete Island.", "These temporal resolutions are inherently linked to the available input data (Table 1), the nature of the simulated hydrologic processes and the modeling objectives (Baffaut et al., 2015).", "Conversely to previously studies that focus mainly on annual or long-term mean annual prediction (i.e. Malard et al., 2015), here the monthly simulations of streamflow have allowed capturing the seasonal variations and gathering precious information for a better water resources management.", "Similarly, the daily simulated springs discharges provided information of each karst recharge area and the spring hydrograph, if further investigated, can also express some geometrical and hydraulic characteristics of aquifers (Fiorillo, 2014).", "Furthermore, the spatial and temporal (annual and monthly) distribution of simulated water resources (i.e. precipitation, deep aquifer recharge, evapotranspiration and runoff) and the mean annual water balances in different hydrological conditions are important benefits of the present work.", "Despite these strengths, the modeling approach suffers from some limits.", "For instance, the monthly time step of streamflow has limited the application of step-wise calibration not allowing a reliable estimation of each component of the streamflow.", "Furthermore, in data scarce areas, where only annual time step is available and spatial data has coarse resolutions, the reproducibility of the KSWAT model needs some adaptations that should be evaluated case by case.", "Likewise, the knowledge of springs discharges and their position is vital and in extremis they could be approximated using literature information.", "In this context, soft data has an important role and should be seen as injections of some insightful common sense into the automatic or semiautomatic calibration procedure (Seibert and McDonnell, 2002).", "For instance, Malard et al. (2015) used 42 alpine and peri-alpine specific springs discharges from literature as references sites in Switzerland for karst recharge assessments, and Hartmann et al. (2015) used 40 independent observations of mean annual recharge both from field and modeling studies over Europe and Mediterranean.", "Finally, albeit the identification of karst recharge areas of each spring is a valuable aspect of the KSWAT model, the associated uncertainty was not object of this study and this topic deserves further investigations that may involve climatic and topographic descriptors as pointed out in Hartmann et al. (2015).", "In this study the SWAT model was adapted and integrated with a karst-flow model to simulate the karst water resources in the Island of Crete.", "The KSWAT model allowed calibrating the daily spring discharges in 47 gauging stations, estimating their karst recharge areas and their introduction in the SWAT model increased the performance of streamflow prediction in karst-subbasins.", "These springs contributed significantly to total discharge with 300 Mm3/y in dry hydrological condition, ensuring ecological services (Malard et al., 2015), and 850 Mm3/y in wet periods.", "In addition, the seasonal variation of volume of springs suggests that these valuable sources should be conserved and preserved in particular from April to September when available volumes are the lowest and agriculture and tourism demand increases.", "The analysis of the water balance also showed that water resources are not homogeneously distributed in Crete and change significantly in different hydrological conditions.", "In particular, the western part of Crete has a surplus of water resources with respect to the eastern part where there is low water availability and high demand.", "Messara Valley (Geropotamos Basin) is an example of intensively managed basin in south-eastern Crete where an overexploitation of groundwater is occurring in a large number of wells since 1984 that continue to decrease groundwater levels at alarming proportions (Kritsotakis and Tsanis, 2009).", "In this context, the present study provides a methodology and a tool for the integrated water management of Crete and other similar areas, by providing detailed spatially distributed hydrologic balances and accurate estimation of water availability using both hard and soft data." ]

[ "SWAT model was adapted and integrated with a karst-flow model (KSWAT model).", "KSWAT has allowed calibrating 22 streamflow and 47 springs gauging stations.", "KSWAT has captured the spatial and temporal variability of water balance.", "The proposed methodology has allowed defining the karst recharge area of each spring.", "The water balance was assessed and compared in different hydrological conditions." ]

[ "ENGI" ]

[ "Buildings", "Climate change mitigation", "Construction", "Embodied carbon", "Paris Agreement", "Science Based Targets", "Whole life carbon" ]

[ "2205", "2208", "2210", "2215" ]

[ "The dangers posed by anthropogenic carbon emissions and a changing climate are well documented [1], yet in 2016 humanity emitted a further 36 GtCO2 from fossil fuels and industrial processes [2].", "In December 2015, 195 countries adopted the first legally binding global climate deal seeking to hold increases in global average temperature to “well below 2 °C above pre-industrial levels” and to “pursue efforts to limit the temperature increase to 1.5 °C” [3].", "Current ‘do nothing’ scenarios project global temperature increases of 3.2–5.4 °C by 2100 [1] and even fulfilment of all signatories’ Nationally Determined Contributions put forward as part of the Paris Agreement implies a median warming of 2.6–3.1 °C by 2100 [4].", "Limiting temperature increases to below 2 °C will likely require global emissions to peak by 2020 followed by rapid reductions [5], necessitating a significant ratcheting up of global emission abatement efforts as part of a periodic stocktake and commitment cycle.", "In addition to its headline temperature target, the Paris Agreement sets the goal of achieving “a balance between anthropogenic emissions by sources and removals by sinks of greenhouse gases in the second half of this century”, i.e. ‘net zero’ emissions.", "This is in recognition of the fact that net carbon dioxide emissions will need to fall to zero in order to stabilise global temperature.", "It is expected that wealthier developed countries will achieve this net zero goal at an earlier date in line with the principle of common but differentiated responsibilities.", "The immense scale of the challenge involved in delivering these goals is frequently understated but is clearly illustrated by a range of recent roadmaps and scenario analyses.", "For instance, Rockstrom et al. set out one roadmap with a 75% probability of limiting warming to below 2 °C, if global greenhouse gas (GHG) emissions were halved every decade [6].", "Such a radical transformation can only be achieved with the active participation of non-state actors, including corporate and privately owned companies.", "This will require companies to independently set long term reduction targets that are aligned with global mitigation goals [7,8].", "The construction sector is the largest global consumer of resources [9] and is a major contributor to climate change through the GHG emissions incurred both within its supply chains and from the operation of assets it creates [1].", "Growing demand for buildings and infrastructure is driving significant increases in material extraction and emissions [10] and further unabated growth has the potential to undermine climate targets [11–13].", "An additional challenge within this is enabling developing countries to grow, expanding infrastructure and buildings to support higher standards of living, whilst minimising the associated GHG emissions.", "Detailed analyses of the GHG emissions attributable to construction sector activities have been conducted for numerous countries, such as Sweden [14], Norway [15], China [16], Australia [17] and the UK [18].", "A common trend across countries is an increasing share of project whole life carbon emissions coming from embodied rather than operational emissions [19].", "There are a wide range of opportunities to reduce carbon emissions throughout a project's life cycle, including mitigation strategies to reduce embodied emissions in design and construction [20,21]; operation [22] and end of life management [23].", "A growing body of guidance and standards has supported some exploitation of these opportunities [24].", "Though many firms now undertake routine project carbon assessments, best practice in whole life carbon management is predominantly confined to a small number of multinational firms with significant organisational capacity and expertise.", "Even amongst these firms there is wide variation in common practices, including assessment and reporting procedures [25].", "There are many barriers to the more widespread deployment of these mitigation options [26] and additional policy support is likely to be essential in the medium to long term [27,28].", "Yet in spite of the observed barriers and limited drivers, numerous construction firms have publicly adopted carbon reduction targets.", "These targets vary widely in scope [29] and are typically determined by esoteric means, with many simply decided by individual CEOs, through comparison with competing firms, or copied verbatim from headline national mitigation commitments [30].", "Few firms have targets that are truly aligned with sectoral, national or international mitigation commitments, though demand for such alignment has been growing of late.", "The means by which such an alignment can best be achieved is a subject of ongoing debate amongst industry and academic experts.", "This paper sets out some of the possible options, their implications and shortcomings, and illustrates the resultant pathways through a case study of the UK.", "Section 2 provides context, describing the UK's national emission reduction targets and current construction industry practice.", "Section 3 discusses current approaches to target alignment and the unique challenges in aligning targets within the construction industry.", "Section 4 presents a set of illustrative sectoral trajectories and discusses their implications for industry practice.", "Section 5 concludes with a summary of the key considerations in setting an appropriate sectoral target.", "The UK construction sector faces the profound challenge of substantially reducing carbon emissions whilst meeting increasing demand for buildings and infrastructure [31].", "Over the coming years the UK faces anticipated population growth (some 14 million additional people by 2050 [32]); that will require an additional 3.2 million households by 2037 [33].", "This comes on top of an existing housing crisis with record property prices and a local authority housing waiting list exceeding 1.2 million at the time of writing [34].", "Furthermore, 8 million ‘non-decent’ homes require urgent refurbishment [35] and broader targets require the retrofit of more than one home every minute until 2050 [36].", "In the meantime an infrastructure pipeline worth around £600bn must be delivered [37], including additional investments in climate adaptation, such as flood defences, and a significant renewal and expansion of energy and communications infrastructure [31].", "Over the same period the UK Government is pursuing a legally binding target of reducing GHG emissions by 80% by 2050, set out in the 2008 Climate Change Act.", "Interim progress towards the 2050 target is aligned with a series of 5 year carbon budgets, currently set into law until 2032 (see Fig. 1).", "Existing policies are projected to be insufficient to meet the 4th and 5th Carbon Budgets and additional interventions are expected in the coming year [38].", "The UK's 2050 target is broadly expected to be elevated in light of the Paris Agreement [39], with the then Minister of State for Energy intimating that a net zero emissions objective will enter into UK law: “the question is not whether, but how we do it”.1", "Similar net zero emissions targets have already received parliamentary approval in other developed countries such as Sweden and Norway.", "In spite of this, the means by which a net zero emissions objective can be delivered and translated into specific targets has yet to be determined.", "The prospective date by which the UK should deliver net zero emissions is also heavily dependent upon interpretation of the Paris Agreement's Article 2.1a targets and the means of determining a fair allocation of the remaining global carbon budget.", "Current estimates, based on common interpretations, suggest that the target date for UK net zero carbon dioxide emissions should be within the range of 2045–2075 [39,40].", "However, the means by which net zero domestic emissions could be delivered is unknown.", "The Committee on Climate Change (CCC), who provide independent monitoring and advice to Government, currently have no scenarios under which the UK can achieve net zero domestic emissions.", "Even “a full and successful roll-out of all options” identified by the CCC, results in GHG emissions in 2050 just over 90% lower than 1990 [39].", "Achieving net zero will therefore require both deep mitigation and the widespread deployment of ‘Negative Emissions Technologies’ (NET), which extract and store carbon.", "The feasible level of NET that can be delivered in the UK is highly uncertain, with recent technical estimates of the order of 44–180 MtCO2e per year, which is around 8–32% of current total UK territorial GHG emissions [41].", "The precise ceiling of this potential deployment is likely to determine the long term sustainable level that UK emissions must be reduced to through additional mitigation measures.", "For instance if, through deployment of NET, the UK could deliver 100 MtCO2 per annum of additional carbon sinks, then it could continue to emit 100 MtCO2 per annum from hard to mitigate sources, whilst still achieving the overall net zero objective.", "Though much is unknown at the present time, it appears likely that the UK will adopt a net zero emissions target in future and all interim strategies, roadmaps and decarbonisation trajectories should account for this.", "The potential contribution of the construction industry to low carbon development has been the subject of numerous reviews and strategy documents over the past 20 years [42–48].", "Most recently the UK's principal construction strategy, Construction 2025 set a target of halving annual GHG emissions from the built environment by the middle of the next decade [49].", "It is envisaged that this can be achieved alongside significant capital cost reductions, following the Infrastructure Carbon Review's conclusion that “reducing carbon reduces cost” [47].", "Indeed, one of the principal objectives of the UK Government Construction Strategy 2016–2020 is to “enable and drive whole-life approaches to cost and carbon reduction” [50].", "In 2013, the Green Construction Board's Low Carbon Routemap for the Built Environment provided a definition and baseline for UK built environment emissions, and set out the steps required to achieve an 80% reduction by 2050 [48].", "However, a 2015 Progress Update showed the sector was already falling behind the target trajectory [51].", "In 2014 UK built environment emissions totalled some 183.5 MtCO2e [24].", "Roughly a quarter of these were attributable to embodied emissions incurred in the construction of new assets.", "The remaining three quarters were attributable to the operation of existing assets (see Fig. 2).", "Over recent years operational emissions have gradually declined owing to the retrofit of older properties, the construction of better performing new properties and decarbonisation of the electricity supply.", "Meanwhile embodied emissions have been increasing due to a growth in construction activity as the sector emerged from a deep recession in 2009, combined with the use of thicker building fabrics to meet revised Building Regulations.", "Delivering sector carbon reduction targets will require substantial reductions in both operational and embodied emissions [51].", "Progress in accounting for and reducing embodied emissions has varied widely between sub-sectors of the industry and between firms within each sub-sector.", "For instance, significant progress has been made in the water industry [52], in large part driven by leadership from a small number of firms supported by an attentive regulator.", "Significant investments have been made in additional guidance, tools and standards for carbon management, as reviewed by Giesekam and Pomponi [24] and De Wolf et al. [25].", "Sourcing accurate data reflecting the environmental impacts of products and construction processes remains a challenge [25,26,53,54]; however much recent progress has been made.", "For instance, the industry has now published over 3500 verified Environmental Product Declarations (EPDs) for construction products (with an additional 2400 unverified EPDs also available), over 40% of which were published in the last year [55].", "Reductions in operational carbon emissions have largely been driven by policy interventions, such as amendments to Part L of the Building Regulations; but the recent removal of key policies, such as Zero Carbon Homes and the Green Deal, has undermined these efforts.", "Collective industry commitments to reduce emissions have been made through initiatives such as the Infrastructure Carbon Review [47] and through organisations such as the now defunct UK Contractors Group [56].", "Most major companies are now disclosing their emissions through schemes such as the Carbon Disclosure Project and many have publicly set reduction targets.", "Three scopes are typically defined for reporting purposes.", "Scope 1 emissions are direct GHG emissions that occur from sources that are owned or controlled by the company (e.g. the direct combustion of fuel in the operation of facilities).", "Scope 2 emissions are indirect emissions from generation of energy purchased by the company (e.g. emissions incurred in the generation of electricity or heat purchased by the company).", "Scope 3 emissions are other indirect emissions incurred as a consequence of the activities of a company, but at sources not owned or controlled by the company (e.g. emissions incurred upstream in a supply chain producing base materials used in the manufacture of a company's product).", "See the GHG Protocol Corporate Accounting and Reporting Standard [79] or DEFRA's Environmental Reporting Guidelines [80] for a more detailed description of each scope.", "Annual reporting of Scope 1 and 2 emissions is now a legal requirement for many quoted companies and public bodies in the UK, with some companies choosing to voluntarily report Scope 3 emissions.", "Fig. 3 summarises the targets set by the UK's top 20 housebuilders2 and largest 50 construction firms3 by turnover, based upon a review of their annual reports and corporate social responsibility commitments conducted in July 2017.", "An equivalent figure for major infrastructure providers can be found in a recent UK Green Building Council report [30].", "A small number of firms, such as Hammerson [57], Berkeley [58] and WSP [59], have made commitments to be carbon-neutral or net-positive in future – through the use of carbon offsetting schemes – but they represent the minority of UK practice.", "It should be noted that Fig. 3 is comprised of company targets covering a variety of scopes and activities, and is not an amalgamation of the project targets being set by these companies.", "Therefore, though this figure does not reflect the full range of mitigation options being pursued, nor provide a basis for calculating a precise carbon reduction commitment from the industry, it does indicate the general scale of ambition amongst UK industry leaders.", "26 of the 664 companies reviewed had publicly set carbon reduction targets.", "2 more were in the process of preparing targets at the time of writing.", "The remaining 38 are reporting carbon in some form but have not publicly specified reduction targets.", "Most have made general commitments to carbon reduction, such as qualitative statements, undertaking exemplar projects or providing public support for regulatory ambitions.", "Even from a cursory inspection of this figure, there is a clear misalignment between the targets currently set by individual companies and the required long term emissions reduction trajectory.", "Most company targets are short term – predominantly out to 2020 – and generally exclude Scope 3 emissions.", "For many built environment firms, such as major property developers, Scope 3 emissions are significantly greater than Scope 1 and 2 emissions, and often present the greatest opportunities for mitigation through changes in design.", "The exclusion of Scope 3 emissions from reporting and associated targets may limit the range of mitigation solutions pursued and consequently reduce the rate of sectoral carbon reduction that can be achieved.", "Though a small number of firms are setting highly ambitious targets, the majority are not setting any targets.", "The targets set by housebuilders are typically less ambitious than those of the other construction firms.", "Generally most reduction plans are linked to set investment periods, with no long-term targets beyond current investment plans.", "Few targets appear to be linked to any broader sectoral or national target.", "To rectify this problem there have been calls within the industry for development of a common sector target trajectory from which commensurate targets for individual companies and projects can be derived [30].", "This begs the question, by what means should company, sectoral, national and international targets be aligned?", "A small number of companies across a range of sectors have already sought to align their carbon targets with national and international commitments [81,82].", "These firms have adopted a range of approaches from a simple replication of international targets to more nuanced schemes, such as the Science Based Targets initiative.", "The following section begins by briefly reviewing the approaches currently in use (Section 3.1).", "This is followed by a discussion of the unique challenges faced in aligning targets within the UK construction industry (Section 3.2).", "In respect to this application, the principal limitations of current approaches are further discussed in Section 3.3, before an alternative approach is proposed in Section 3.4.", "Targets can be aligned by a number of approaches, which vary in sophistication, ethical and scientific defensibility.", "At the simplest level, some companies have chosen to adopt carbon reduction targets that are copied verbatim from recommendations for global reductions based upon analysis by the IPCC of the carbon budgets that could restrict warming to 2 °C.", "Implicitly this assumes an equal proportional responsibility for emissions reduction from all sources, regardless of geography, historic responsibility or scope for mitigation.", "Other companies have copied targets from national commitments, such as the UK's 2008 Climate Change Act.", "NGOs and non-profit organisations have launched a number of initiatives encouraging companies to adopt more sophisticated approaches, such as aligning targets relative to a company's contribution to global GDP, or incorporating some consideration of historic emissions [81].", "The most prominent such initiative, which is gaining traction within UK industry, is the Science Based Targets (SBT) initiative [60].", "The SBT initiative is a partnership between CDP, UN Global Compact, WRI and WWF aimed at encouraging adoption of company carbon reduction targets consistent with keeping global temperature increases below 2 °C.", "At the time of writing in November 2017, 82 companies had approved SBT, with a further 239 committed to the development of such a target [61].", "A minority are involved in construction and property development.", "Ferrovial, Landsec [62], Gecina, TODA Corporation and Host Hotels & Resorts have already set targets, whilst a further 27 firms involved in ‘Real estate’, ‘Building products’, ‘Homebuilding’, ‘Construction materials’ and ‘Construction and engineering’ had committed to set targets at the time of writing.", "A number of these firms, such as Laing O'Rourke, ISG and Bennetts Associates, are based in the UK.", "These SBT are developed based upon a range of approved approaches, including the SDA (Sectoral Decarbonization Approach) [8], C-FACT (Corporate Finance Approach to Climate-Stabilizing Targets), CSO (Context-based carbon metric), GEVA (GHG emissions per unit of value added), and CSI (Climate Stabilization Intensity Targets) approach [63].", "Amongst these the SDA has proved the most popular so far, particularly amongst firms in the built environment.", "Though each approach is different, they predominantly follow a common sequence:", "Start from an estimate of the remaining cumulative global carbon budget (e.g. for a > 66% probability of keeping warming below 2 °C, ∼1010 GtCO2e from 2011).", "Select a future emissions scenario consistent with this budget (such as the International Energy Agency's 2 °C Scenario - IEA 2DS).", "Allocate remaining emissions between companies on a contraction or convergence basis.", "Translate this pathway into specific interim company targets for reductions in carbon intensity or absolute emissions.", "The resulting targets are submitted for validation by the initiative.", "Although longer term target setting is encouraged, the targets submitted for validation only cover up to a maximum of 15 years and progress against the targets is not currently monitored by the initiative (though annual public reports are published by all the firms involved).", "Each of these four steps involves fundamental choices with profound implications.", "For example the selection of a budget consistent with 1.5 °C or 2 °C of warming; and selection from the several allocation approaches (used to break down reductions from the sector to company level) set out in the SBT manual [63].", "The efficacy of each of these choices and approaches can be debated, but that is not the focus of this paper.", "Some within the construction industry have advocated for development of an SBT for ‘UK infrastructure’ or the ‘UK built environment’ which could in turn be translated into commensurate company targets [30].", "However, there are numerous challenges in calculating such a trajectory.", "These challenges reflect the unique characteristics of the sector's structure, product and reporting procedures, as well as its role in decarbonisation of the UK economy.", "Collectively these provide cause for concern, and any collective industry effort to establish such a target should address these challenges.", "The unique challenges in aligning targets for firms in the construction sector include:", "Poor sector representation in global pathway analyses.", "Heterogeneity of sector output and divided responsibilities.", "High proportion of Scope 3 emissions and heavy dependence upon imported materials.", "Shortage of benchmark data.", "Limited mitigation opportunities for certain critical inputs.", "Role in setting boundary conditions for decarbonisation of other sectors.", "Capacity to deliver carbon storage and negative emissions.", "This list represents the authors’ views on the main challenges facing the UK industry, based upon discussions with construction firms and early adopters of SBT.", "As more firms develop and implement SBT, it is likely that this list will evolve and grow.", "Let us consider each of these currently foreseen challenges in turn.", "Despite its significance, the construction sector is rarely an explicit sector in global emissions scenario analyses.", "These analyses tend to consider the impact of ‘buildings’ solely in operation.", "The constituent inputs to construction are usually outputs of an aggregated ‘industry’ or sequence of major producers, such as ‘steel’ and ‘cement’.", "Similarly, the transport of these materials in production and to site is usually amalgamated into an overarching ‘transport’ sector alongside domestic travel.", "Consequently in analyses which use off-the-shelf future emissions scenarios, such as the SBT SDA approach, pathways for construction firms are typically based upon categories such as the IEA's ‘Other industry’ or ‘Buildings, agriculture, fishing, non-specified other’, which includes a wide range of manufacturing and food producers in addition to construction.", "Thus, in most cases, these analyses poorly reflect the mitigation potential and associated costs within the construction sector, and portray output trajectories that are not truly indicative of what can be achieved.", "Such analyses do not provide a sound future emissions scenario upon which company targets can be based.", "The output of the construction industry is highly diverse, comprising a wide variety of buildings and infrastructure assets.", "Even defining a boundary encompassing all the firms included within the construction industry can prove challenging.", "This wide range of outputs are paralleled by an equally broad range of development approaches and ownership arrangements.", "Typically numerous actors are involved in the conception, design, delivery, ownership, use, maintenance and decommissioning of a built environment asset, and the responsibilities and influence of each party can be difficult to discern.", "For example, clients may set targets for the embodied and operational carbon of their project – which is a challenge in itself – but will then rely on the design team and contractor to be able to deliver a building that meets these targets.", "If the building is leased, the operational energy will likely be outside of the control of the original client and in the hands of the tenant, who may use the building in different ways to those anticipated.", "This model is also predicated on an informed client.", "Where the client is not setting targets, the onus is on the design team and contractor to explore opportunities for whole life carbon reduction, and then try to persuade their client of the benefits of this approach, which is a significant challenge in a predominantly cost driven industry.", "Furthermore, establishing common benchmarks across the range of actors and project types can prove difficult, with each actor and sub-sector desiring bespoke solutions that reflect their deliverables.", "For instance, carbon benchmarking can occur across a range of units, from kgCO2e/m2 GIA for a commercial property developer, to kgCO2e/km of pipeline constructed to kgCO2e/m3 of waste water treated to kgCO2e/passenger through a transport hub.", "Thus, the many challenges that come with the complexity of the sector cannot be underestimated and a means of reflecting these divided responsibilities must be considered in any collective approach to SBT development.", "Built environment assets typically have multi-decadal design lives, which they often outlive.", "The age distribution of the current UK building stock indicates that many houses and non-domestic properties constructed in previous centuries are still in use today and will likely continue to be in use into the next century.", "The same is true of major infrastructure assets which are often designed for anticipated operating periods exceeding 50 or 100 years.", "Given the longevity of these design lives, most built environment assets being designed today will still be operational in the period by which the UK must achieve net zero emissions, as indicated in Fig. 4.", "Once these assets are in place, they lock-in operational emissions that will be incurred in coming decades, barring additional retrofit measures which will incur further embodied emissions.", "Consideration also needs to be given to the adaptability and deconstructability of the buildings designed.", "In the first instance to ensure buildings can respond to changing needs, thus increasing their lifespan, and in the second instance to enable the reuse of components and materials.", "By planning effectively now, the demand for future buildings and materials can be reduced.", "This is crucial when considering that in around fifty years, at the potential end of life of these structures, humanity will need to be operating at near zero GHG emissions.", "Given the timescales involved, it is imperative that the performance of current designs is considered relative to these longer term carbon reduction targets.", "The UK construction industry is heavily dependent upon imported materials, including several million tonnes of steel per annum, over a million tonnes of cement and nearly all aluminium products.", "Consequently, in total, around 40% of the embodied emissions associated with UK construction supply chains occur outside UK borders [18].", "Furthermore, responsibility for these emissions is widely distributed along lengthy supply chains.", "As a result the Scope 3 emissions incurred in the development of a construction project will often outweigh the Scope 1 and Scope 2 emissions under the influence of an individual actor.", "Therefore the inclusion of Scope 3 emissions is an essential part of addressing the construction industry's full impacts and leveraging the greatest reduction opportunities.", "In spite of this, SBT approaches predominantly focus upon Scope 1 and Scope 2 emissions.", "Although Scope 3 emissions are included where significant (>40% of total emissions), guidance from the initiative states that “Scope 3 targets generally need not be science-based, but should be ambitious and clearly demonstrate how the company is addressing the main sources of GHG emissions within their value chain in line with current best practices” [63].", "Given the significance of Scope 3 emissions for the UK construction industry, any collective approach that failed to address these in line with a 2 °C scenario would clearly be inconsistent with national and global mitigation goals.", "The reporting boundaries for carbon emissions adopted at project, company, sector and national level are fundamentally misaligned.", "At a project level, assessors typically adopt a consumption-based assessment approach including all emissions incurred in the project supply chains regardless of the geographical point of origin.", "These project assessments will include some subset of the life cycle stages set out in BS EN 15978: Sustainability of construction works — Assessment of environmental performance of buildings — Calculation method [64], though no single common boundary is used in practice [65].", "At a company level reporting boundaries are typically dictated by regulatory requirements, which generally require only emissions incurred across a limited subset of activities.", "At a sector level the boundary in the three major UK assessments to date has varied, though all have adopted a consumption-based accounting perspective [48,66,67].", "Meanwhile, at a national level, emissions are reported on a territorial basis, omitting any emissions incurred in overseas supply chains.", "Corresponding carbon reduction targets are also set across each of these differing boundaries.", "Companies operating internationally also face the question of what boundaries should be drawn and if and how differentiated responsibilities should be taken into account depending on which country they are operating in.", "Translating between these disparate boundaries is thus a non-trivial activity and any common sectoral trajectory must clearly state the boundaries adopted.", "It remains difficult to effectively benchmark performance within the built environment, particularly for the embodied emissions associated with initial construction, which is often responsible for the largest volume of emissions that can be influenced by contractors and developers.", "Recent work by WRAP [68], the Carbon Leadership Forum [69], RICS [70] and De Wolf et al. [71], has sought to address this, but it will likely be many years before benchmark data is available for a broad range of project types.", "In the meantime it will remain difficult to set project embodied carbon reduction targets, which are a key means of delivering any company's reduction commitments [29].", "Even once this benchmark data is available, further work will be required to understand what the embodied carbon reduction potential is of different assets types.", "Though a wide range of low carbon materials can be used across numerous applications, the construction industry is fundamentally dependent upon certain carbon-intensive materials for specific tasks.", "Many of these materials, such as steel and cement, have significant emissions resulting from essential chemical processes in the production of the material, which are prohibitively expensive to capture with current technology.", "The UK Government explored a wide array of mitigation options for these materials in a set of Industrial Decarbonisation and Energy Efficiency Roadmaps published in 2015 [83].", "However, few of the options identified in this process are commercially viable, and limited near term reductions are foreseen in the recently published joint Government and industry Action Plans [84].", "In the long term significant reductions in the costs of industrial Carbon Capture and Storage (CCS) may facilitate more widespread deployment but this is only likely to cover a portion of total emissions from certain facilities that are both large in scale and geographically clustered with other emitters [72].", "Furthermore, the significant cost and competitiveness implications of attempting to deploy CCS to mitigate process emissions, particularly from cement production, mean that mitigation options in other sectors with a lower marginal cost per tonne of carbon are likely to be prioritised for support by the UK government.", "Therefore a non-trivial level of industrial emissions are likely to continue and, even in the very long term, net zero emissions are unlikely to be achieved within the construction sector and its supply chains.", "More work must be done to understand the practical limits to achieving ultra-low carbon buildings and infrastructure, and the viability of their supply chains.", "In the meantime any collective trajectory must reflect the limited options that are presently available.", "The outputs of the construction industry, in particular infrastructure assets, have a critical role in shaping future development pathways and supporting the decarbonisation of other sectors, such as transport.", "For instance, switching to lower carbon forms of transport requires the availability of different modes and additional infrastructure to ease existing congestion.", "As national infrastructure is increasingly viewed as an interdependent system-of-systems, it becomes increasingly difficult to disentangle critical infrastructure choices from decarbonisation trajectories.", "Ultimately, the long term development pathways the UK selects for its infrastructure will determine the scale and pace of emissions reduction that is achievable.", "Clearly, any desirable trajectory must not promote options that can deliver short term carbon reduction targets but lock-in high-emitting infrastructure that may ultimately prevent delivery of the long term net zero emissions goal [73].", "Though much laudable work has been done to explore different long term pathways [74], these analyses have yet to consider the emissions embodied in delivering and maintaining this new infrastructure.", "Whether these emissions are best considered separately as part of a bespoke built environment trajectory, or integrated as part of a holistic analysis of UK national emissions, remains a subject of debate.", "The achievement of net zero emissions is fundamentally dependent upon the development of additional carbon sinks at scale.", "The built environment represents one of the largest potential stores of carbon through the use of biogenic building materials, as recognised in public campaigns such as Wood for Good's ‘Build with Carbon’ [78].", "Indeed the increased use of wood in construction is now included in the CCC's UK decarbonisation scenarios [39].", "Albeit the relative costs of using biogenic materials compared with steel, concrete and masonry are highly dependent upon project circumstances, in all cases the costs of switching to lower carbon building materials are likely to be substantially cheaper than delivering other NET such as Direct Air Capture or Bioenergy with Carbon Capture and Storage (BECCS) [75].", "Thus, the delivery of a substantial built environment carbon sink represents a relatively cost effective route to achieving net zero emissions.", "The introduction of policy instruments that internalise the external costs of carbon emissions, or value the benefits of sequestered carbon, could in future encourage a structural change towards the use of wood and other biogenic building materials [76].", "Any industry wide decarbonisation trajectory should therefore recognise not just the opportunity to mitigate emissions but also the opportunity to sequester carbon.", "Furthermore, any associated action plan should seek to incentivise and allocate responsibility to those actors that can specify the use of materials that act as carbon stores.", "There are a few other reasons to be sceptical of the appropriateness of simply applying current variations of the SDA to develop a SBT for the UK built environment.", "Firstly, the SDA does not recognise the “common but differentiated responsibilities” between nations recognised in the Paris Agreement, preferring to construct pathways consistent with a global convergence, and thus inconsistent with UK national commitments.", "Secondly, this approach does not recognise the historic responsibility of companies for past emissions, as described by Bjørn et al. [82] Thirdly, the pathways calculated under the SDA are typically based upon a truncated timeline (generally to a 2050 end point) and extrapolated into linear reduction trajectories.", "This encourages an incrementalist approach to carbon reduction, which focuses on modest year on year reductions rather than promoting step changes in practice.", "Driven by a long term focus on cost and risk reduction, exacerbated by narrowing profit margins in the wake of the recession, the UK construction industry has historically adopted a cautious and highly incrementalist approach to innovation.", "This has resulted in a form of path-dependent development that has locked-in dominant technologies and processes, restricted the range of solutions explored and the capacity of construction firms to make radical changes to processes and structures [77].", "Achieving deep carbon reductions will necessitate a much more radical approach to innovation, which sectoral and company targets must reflect.", "If it is not already apparent, the significance of these unique challenges and other shortcomings is immediately evident through comparison with other industries, for example the production of chocolate bars.", "Chocolate bars have a short lifespan with high turnover, cannot be a store of carbon, and the requisite energy and material inputs could all potentially come from renewable sources.", "Changes in the production technologies and processes of chocolate bars can generally be implemented by a single or small group of actors over a short period of time.", "Furthermore, changes in the output of chocolate bars will not significantly affect the decarbonisation efforts of other industries or provide boundary conditions which limit the reductions that can ultimately be achieved.", "Therefore whilst the current SDA to SBT development may be suitable for chocolate bars, an alternative or adapted approach may be required for construction.", "One possible alternative would start from an estimate of potential carbon sinks and deployment of NET under a time frame consistent with achieving net zero emissions.", "This would provide a ceiling figure for remaining UK annual emissions under a sustainable long term net zero scenario.", "That remaining annual emissions budget would then be allocated amongst hard to mitigate sectors through a cross-sector dialogue based upon the maximum mitigation potential within each sector.", "The portion allocated to the built environment would then be translated into a long term reduction trajectory that is consistent with stabilising global temperature increases.", "Developing a detailed trajectory by such an approach would ideally require both a deeper understanding of the scope for deployment of additional carbon sinks, and a concerted effort to establish a cross-sector dialogue.", "Such a dialogue would likely need to be facilitated by a respected independent party, such as the CCC or the Carbon Trust.", "In the meantime, it is possible to calculate approximate example trajectories based upon current understanding and a series of plausible assumptions.", "By way of illustration, the impact of the various assumptions and approaches described in the preceding section can be seen in the indicative trajectories depicted in Fig. 5.", "Their bounds broadly represent the corridor within which UK built environment emissions must remain to be deemed consistent with the goals of the Paris Agreement.", "These trajectories are calculated based upon the assumptions set out in Table 1 and described in further detail in the Supporting Information.", "Table 2 sets out the corresponding carbon reduction targets against the most ambitious (UK NET ZERO 2045) and least ambitious (UK NET ZERO 2075) decarbonisation trajectories.", "Corresponding targets for the other example trajectories can be found in the Supporting Information.", "A number of simple observations can be made from Fig. 5 and Table 2.", "Firstly, all the example trajectories require a more rapid rate of carbon reduction than has been delivered historically.", "Secondly, a linear reduction trajectory from the status quo will not deliver the interim Construction 2025 target, no matter the end point of that trajectory.", "Thirdly, national decarbonisation targets will be missed if UK firms only seek to align their company targets with the IEA 2DS trajectory.", "A simple comparison between the headline carbon reduction rates presented in Table 2 and the targets currently set by the largest UK companies (summarised in Fig. 3) is also illuminating.", "18 of the 66 firms surveyed have targets which meet or exceed the headline reduction rate implied by the UK NET ZERO 2045 trajectory.", "A further 5 companies have targets which exceed the UK NET ZERO 2075 trajectory but are below the rate required by the UK NET ZERO 2045 trajectory.", "The remaining 43 companies either have targets that are less ambitious than that implied by the least ambitious example trajectory presented here, or have no target at all.", "This simple comparison does not indicate if current sector targets are collectively adequate, as it does not account for the differing market shares and accounting boundaries of the companies; however, it does provide a rough indication of how current company targets compare with plausible sectoral trajectories.", "All of these points suggest that greater ambition and additional policy support for mitigation is urgently required if sectoral targets are to be delivered.", "Furthermore, the difference in cumulative emissions between the trajectories is substantial, as summarised in Table 3.", "For instance, simply following a trajectory based upon IEA 2DS could result in over a gigatonne of additional cumulative emissions by 2050 compared with adopting the GCB ROUTEMAP trajectory.", "As the required reduction rates vary substantially between scenarios, so do the corresponding mitigation options.", "This has profound implications for the required rate of deployment of low carbon materials, technologies and practices.", "Though the trajectories are fairly similar through the early 2020 s, they diverge substantially by the mid-2030 s.", "This implies that under the more ambitious trajectories, ultra-low carbon options such as cement free concrete, industrial CCS and high rise timber structures would have to be commonplace within just two decades.", "Achieving such a fast rate of deployment in a notoriously conservative industry would likely require substantial investment in skills and training; radically increased support for innovation to demonstrate alternative materials at scale; and additional regulatory drivers for those less willing to adopt new technologies.", "Such a transition would also require a fundamental leap of faith from early adopters and a profound shift in the industry's perception of what can be delivered.", "Under the deeper decarbonisation trajectories, the “common industry view that imperceptibly slow change is typical and radical change almost unimaginable” [26] would no longer be tenable.", "Ultimately, the value in determining a sectoral carbon reduction trajectory is inextricably linked to the measures that will generate confidence that the trajectory can be delivered.", "The challenge of this shift in approach cannot be underestimated, in a large and fragmented industry, with complex international supply chains, there is an immediate need for a coordinated legislative approach, supported by investment, upskilling and technology acceleration.", "If the UK construction industry is intent on generating a credible trajectory that will meet the requisite carbon targets, it must consider the simultaneous introduction of novel measures that could support its delivery.", "Otherwise many within the industry will immediately deem the proposed trajectory implausible, or even impossible, undermining the value of its development.", "Indeed, when the Green Construction Board's Low Carbon Routemap for the Built Environment was published in 2013, the Government's then chief construction advisor Paul Morrell stated that “my personal view is that the assumptions the model makes are so heroic that I don't believe anyone will believe it will happen in the timeframe”.", "When generating a refreshed trajectory that reflects the interim changes (such as the Paris Agreement), it is imperative that the industry also generates an accompanying action plan that can provide confidence that the trajectory can be delivered.", "The full implications of the Paris Agreement for the construction industry are profound, yet poorly understood and rarely specifically interpreted into long-term targets for individual firms.", "For many countries, such as the UK, the Paris Agreement implies the transition to a net zero carbon economy shortly after mid-century.", "This will require both deeper mitigation than the currently targeted 80% national GHG emissions reduction by 2050 and substantial deployment of NET.", "Delivering such a transition requires that short term national, sectoral and company emission reduction targets are nested within longer-term pathways and strategies that are consistent with deep decarbonisation and the delivery of a net zero emissions UK.", "If strategies only focus upon the achievement of short term or interim carbon reduction goals, there is a risk that insufficient action will be taken out to mid-century and that infrastructure and technologies that are incompatible with the long term objective will be locked in.", "Given this timeline it is imperative that the construction industry urgently engage with efforts to depict a net zero emissions future for the UK, as decisions taken by the industry over the coming decades will fundamentally determine the viability of such an objective.", "This will require setting a target trajectory for the built environment that is consistent with current climate science, global and national ambitions.", "Through a review of current company carbon reduction targets, and comparison with a range of sectoral carbon reduction trajectories, this paper has highlighted that current targets set by major UK construction firms are generally insufficient to deliver national carbon reduction goals.", "The largest firms are typically setting modest short term reduction targets, and only a minority have sought to align their targets with international commitments.", "Recent initiatives have encouraged a small but growing number of construction firms to develop such targets, leading to calls for the development of sectoral targets and pathways from which commensurate company targets can be developed.", "Developing a credible common sectoral decarbonisation trajectory for the UK built environment will require the adaptation of current alignment approaches to address the range of unique challenges set out in Section 3.", "Any failure to properly attend to these details in the selection of a methodology and development of a target trajectory will have significant consequences.", "As demonstrated by the example trajectories presented in Section 4, the implications of these seemingly minor changes are profound, necessitating radically different mitigation responses and resulting in substantially different cumulative emissions totals.", "It is therefore important that the industry must engage in an open and informed debate to determine an appropriate approach that recognizes these challenges.", "This study was limited to a brief review of targets from the largest firms, and was only sufficient to provide a general indication of progress.", "A more comprehensive review of all company carbon targets, and perhaps their regular compilation in a public forum, would be a welcome extension of this work.", "The focus upon the UK industry was a further limiting factor.", "Future studies to determine target trajectories for other major construction markets would complement this work.", "A more in depth analytical study of the Science Based Targets adopted thus far by firms in other sectors would also be a welcome addition to the literature.", "As the UK industry prepares to convene roundtable discussions on the development of a common trajectory, let us conclude by recapping the pertinent features that must be included.", "Such a trajectory must deliver both the 80% emissions reduction by 2050 set out in the Climate Change Act and the longer term objective of net zero emissions shortly thereafter implied by the Paris Agreement.", "It must consider the projected level of industry output and deliver reductions in absolute emissions not just carbon intensity.", "It must include commitments on Scope 3 emissions and some consideration of future carbon storage.", "However, it must also recognise that complete mitigation of all Scope 1, 2 and 3 emissions from construction, operation and maintenance of the built environment this century is not feasible, and that, within any net zero emissions scenario, a portion of the UK's remaining national carbon budget must be allocated accordingly.", "Therefore, in developing the trajectory, the industry must engage in an active attempt to depict the long term role of a sustainable construction industry in a net zero UK.", "Critically, it must encourage the requisite step changes in practice rather than a sequence of short term incremental improvements.", "It must garner widespread support within the industry and be accompanied by an action plan which instils confidence that it can be delivered.", "Finally, beyond its initial publication, the trajectory must be maintained and reported against, ideally by a suitably well-resourced and independent body.", "If such a trajectory can be developed, it will set a powerful precedent for other nations and industries seeking to deliver upon the Paris Agreement." ]

[ "Some firms are trying to align carbon targets with (inter)national mitigation commitments.", "Construction firms face a unique set of challenges in aligning targets.", "There are a range of approaches, each with distinct limitations.", "Adopting different approaches yields different targets and corresponding mitigation options.", "Cross-sector dialogue is urgently needed to determine the best route forward." ]

[ "BIOC", "IMMU", "MEDI", "VETE" ]

[ "Subdominant T-cell epitopes", "T-cell hierarchy", "Vaccines", "Viral vectors" ]

[ "1313", "2400", "2725", "2739", "3400" ]

[ "Leading vaccine strategies aiming at inducing strong cellular immunity in humans use recombinant adenovirus (Ad) and Modified Vaccinia Ankara (MVA) [1,2].", "These vectors have been assessed since the late 90s for their capacity to induce different frequency, phenotype, function and localization of T- and B-cell responses [1,3–5].", "A consensus emerged that priming with Ad followed by boosting with MVA was optimal to maintain strong and long-lived CD8+ T-cell responses [5,6], and thus many comparisons have been performed in the context of various heterologous prime – boost regimens [7–9], with few exceptions comparing them individually [3,10,11].", "In the latter studies, T-cell responses were measured with tetramers [3], dominant epitopes [10], or peptide pools [11], but none investigated the dominance pattern of T-cell responses induced by each vaccine vector.", "However, it has been suggested that the immunogenicity of the vector is likely to have a strong influence on the transgenic antigen [12].", "The objective of this study was to identify the influence of Ad and MVA vaccine vectors on the hierarchy and breadth of the T-cell epitope responses to the vaccine immunogen, and to specifically address the following four questions: (1) Is the hierarchy of T cell responses influenced by the vector?", "(2) Does the hierarchy of T cell responses change over time after a single immunization?", "(3) Do homologous or heterologous immunizations affect the T-cell hierarchy?", "(4) Is the hierarchy influenced by the timing between prime and boost?", "Human serotype 5 Ad and MVA vectors expressing the transgene ME.TRAP have been described previously [13].", "The insert ME.TRAP encodes the Plasmodium falciparum TRAP and the immunodominant BALB/c H-2Kd epitope pb9 (CS252–260, SYIPSAEKI) from the P. berghei circumsporozoite protein.", "Animal experiments were performed in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines and EU Directive 2010/63/EU for animal experiments.", "Procedures were approved by the University of Oxford Animal Care and Ethical Review Committee.", "Groups of six 6 week-old female BALB/c mice (Harlan, UK) were injected intradermally in the ear pinna with 106 pfu for MVA or 5 × 109 vp for Ad, under short anesthesia using isoflurane.", "Ad + MVA vector mixtures (further referred as ‘Mix’) consisted of a single preparation containing both vectors at the same concentrations as each vector alone.", "All vaccines were injected in a final volume of 50 μl of phosphate-buffered saline.", "Peripheral mononuclear cells (PBMCs) were stimulated with peptides at a final concentration of 1 μg/ml.", "ELISPOT was performed as previously described [13].", "Results were expressed as the average spot forming units (SFU) per million cells.", "Peptides included the immunodominant Pb9, Ad hexon-specific H-2Kd-restricted epitope hex486–494 KYSPSNVKIA (Kia), Ad DNA-binding protein(dbp)-specific Ld-restricted epitope dbp413–421 LPKLTPFALA (Ala), MVA epitopes previously described E3, F2G [14] and SI9 [15].", "Subdominant epitopes were identified in TRAP using 20mer peptides overlapping by 10 and covering TRAP aminoacid (aa) 101–310 from strain T9/96 [16].", "This region was previously reported to elicit IFN-γ T-cell responses in BALB/c mice as opposed to peptides covering aa 1–110 and 301–564 (A. Spencer, personal communication and data not shown).", "Within this region, only epitopes 11 (IRLHSDASKNKEKALIIIRS), 12 (KEKALIIIRSLLSTNLPYGR), 17 (TDGIPDSIKDSLKESRKLSD), 20 (GQGINVAFNRFLVGCHPSDG), 22 (KCNLYADSAWENVKNVIGPF), 25 (TASCGVWDEWSPCSVTCG KG) and 30 (EPLDVPDEPEDDQPRPRGDN) induced detectable responses in at least 1 BALB/c mouse and are represented in the results.", "After a single injection with either Ad or MVA, or a mixture of both, T-cell responses were measured in blood at the peak of response, at week 1 for MVA and week 2 post-immunization for Ad [10], and after the contraction phase (week 10) (Fig. 1).", "Results are presented on a two-segment scale so that both the dominant (10,000–50,000 SFU/million cells) and subdominant (0–10,000 SFU/million cells) responses can be observed for all groups.", "One week after immunization, Ad induced responses only to the dominant epitope from the transgene, pb9, while responses induced by MVA were lower but broader, targeting peptides 22, 12 and 11 in addition (Fig. 1, top panels).", "Responses to the vector itself also differed: MVA epitope E3 was as strong as Pb9 in the MVA-immunized group, while the Ad hexon Kia was subdominant in the Ad group.", "Interestingly, the mixed modality combined the advantages of both vectors: a strong Pb9 response, detectable responses to peptides 22, 11 and 12 and low responses to the MVA vector epitopes.", "A week later (week 2), responses to the subdominant epitopes in the transgene were detected in the Ad-immunized group (peptides 22 > 20 > 12 > 17 > 25), with higher responses but similar hierarchy as compared with MVA immunization (Fig. 1, middle panels, responses to peptides ranked 22 > 20 > 12 > 17 > 25 in the MVA-immunized group).", "Of note, the response to the Ad dbp epitope Ala only appeared at week 2 and became dominant over the hexon epitope Kia, reflecting the late appearance of dbp being expressed from the vector while the hexon is present on the vaccine composition [17].", "Responses had contracted after 10 weeks from injection.", "In the MVA-vaccinated animals, viral vector epitope E3 dominated and only the transgene pb9 epitope induced a detectable response (Fig. 1, bottom panels).", "In contrast, the response remained dominated by Pb9 in the Ad-vaccinated animals, but the subdominant hierarchy was modified, with peptides 20 and 11 still inducing detectable responses, while the response to peptide 22 was lost.", "These results suggest that the hierarchy and breadth of T-cell epitope dominance in the transgene is not influenced by the vector, but rather by the timing of analysis after immunization, and the hierarchy changes overtime, which likely reflects affinity maturation and T cell proliferation [18].", "To establish whether the T-cell hierarchy is modified after homologous boosting, the same vector was administered after 10 weeks.", "Responses were measured 1 and 2 weeks post second injection and 10 weeks later (weeks 11, 12 and 20, respectively) (Fig. 2).", "A reduction in breadth of T-cell responses was observed, focusing only on the dominant epitopes (Fig. 2): pb9 in the transgene, E3 in the MVA-MVA group, and Ad hexon Kia in the Ad-Ad and Mix-Mix groups dominant over dbp at all time points.", "This reflects the neutralization of the second Ad injection, resulting in lower expression of the transgene and Ad-encoded proteins (as opposed to hexon contained in the preparation) [19] (Fig. 2).", "The Mix-Mix modality induced a broader response that included peptides 12 in addition to 11, but responses to peptides 22, 20 and 17 were not boosted at all.", "The responses detected at peak were longer lived than after a single injection, as pb9 and peptide 11-specific responses were detectable at higher levels 10 weeks post boost (week 20, Fig. 2), compared to 10 weeks post prime (Fig. 1).", "Therefore the timing difference between 1 week and 2 weeks post injection is not observed anymore after a homologous boost, reflecting the rapid expansion of memory T-cells rather than de novo induction [3].", "The T-cell hierarchy is modified by repeated homologous immunization, with a clear narrowing and focus on dominant epitopes from the transgene or the vector.", "Of note, after a third homologous injection performed at week 24, Ad was completely neutralized (no boosting effect was observed as compared with week 20), and only MVA (alone or in the Mix) was able to boost the T cell responses (Fig. 2, bottom panels), suggesting less anti-vector immunity in agreement with previous observations [13].", "To assess whether narrowing of T-cell responses could be avoided with a shorter vaccination regime, we selected a 4-weeks interval shown to be suitable for eliciting T-cell response to dominant epitopes and suitable for human vaccine deployment.", "Groups of mice were injected at week 4 with a homologous or heterologous boost (Ad-MVA, Fig. 3).", "Responses were similar in both regimens (10-weeks and 4-weeks intervals) at 1 or 2 weeks post boost (comparing Fig. 2, top panels with Fig. 3, top panels).", "The heterologous Ad-prime MVA-boost induced the strongest response to pb9 (reaching a mean of 79,715 SFU/million cells as compared with 37,848 for Mix-Mix).", "Modest responses to peptides 20, 12, 11 and 22 were detectable in this group.", "However, when a third injection was administered 4 weeks later, at week 8, the Ad-MVA-MVA regimen also induced a narrowing of the response to pb9, with responses to peptides 11 and 12 barely detectable (measured at week 10, Fig. 3, bottom panels).", "Therefore, the timing interval between prime and boost made no difference on the T-cell hierarchy and breadth.", "The heterologous prime-boost induced the strongest response to the dominant epitope, reflecting the expansion of memory response overcoming the induction of de novo response to the new vector.", "However, this regimen did not support induction of strong responses to subdominant epitopes.", "These results suggest that clinical selection of viral vectored-based vaccine regimen should be based on the type of response required for protection.", "If a strong response to a limited number of epitope targets is required, then our results support the heterologous Ad prime – MVA boost as compared with single Ad or Mix immunization.", "In addition, the heterologous prime-boost was shown to improve the durability, polyfunctionality and phenotype of the T-cell response to the dominant epitope [10,13].", "Results of this study reflect the difficulty to induce broad T-cell responses, important for vaccines aiming at inducing broad and long-lasting T-cell responses to variable pathogens such as HIV [20].", "AVSH is named as co-inventor on patents related to recombinant viral vectors for malaria and other indications." ]

[ "Viral-vectored vaccines are expected to induce T-cell responses to sub-dominant epitopes.", "Hierarchy of T-cell response is influenced by the timing of analysis after a single immunization.", "Repeated homologous immunization reduces the breadth of T-cell response.", "Heterologous prime-boost induces a modest increase of the subdominant responses." ]

[ "MEDI" ]

[ "COPD", "biomarker", "emphysema", "serum", "sirtuin-1" ]

[ "2705", "2706", "2740" ]

[ "Commercially available reagents were obtained as follows: Roswell Park Memorial Institute medium (RPMI) medium 1640 (RPMI 1640) (No. 32404-014) and Dulbecco's Modified Eagle Medium (DMEM) (31053-028) were obtained from Life Technologies; fetal bovine serum (FBS), complete protease inhibitor cocktail (11836153001), and rabbit-derived anti-SIRT1 antibody (No. 5322) were obtained from Sigma-Aldrich; anti-β-actin antibody (ab6276) was obtained from Abcam; and goat-derived, peroxidase-conjugated antimouse (P0447) or antirabbit (P0448) secondary antibodies were obtained from DAKO.", "This project was approved by the Ethics Committee of Sismanogleio General Hospital (approval No. 5210-07/03/2012), and written informed consent was obtained from patients and healthy volunteers.", "COPD was defined and categorized according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines.22", "Blood samples were taken from never smoker healthy subjects with normal lung function (NS, 12 subjects), smokers without COPD (SM, 19 subjects), and 26 patients with mild to very severe COPD (stages 1-2, 13 subjects; stages 3-4, 13 subjects) (Table 1).", "All patients with COPD were considered to be clinically stable, because none had required a change in their regular therapy during the 8 weeks preceding the sampling nor had they been treated with systemic corticosteroids or antibiotics.", "Patients with asthma, pneumonia, or lung cancer were excluded from the study.", "The smoking history of each subject was represented by the mean number of pack-years of cigarette consumption by ex-smokers and current smokers.", "All patients with COPD had a history of smoking, but all patients were asked to refrain from smoking for 3 hours before the serum sampling.", "Emphysema was characterized by high-resolution CT (HRCT).31", "The degree of emphysema was determined using a visual emphysema score, as previously described.32", "Briefly, emphysema was identified as areas of hypovascular low attenuation and was graded with a five-point scale based on the percentage of lung involved: 0 = no emphysema, 1 = up to 25% of the lung parenchyma involved, 2 = between 26% and 50% of lung parenchyma involved, 3 = between 51% and 75% of the lung parenchyma involved, and 4 = between 76% and 100% of lung parenchyma involved.", "Grades of the axial images of each lung were added and divided by the number of images evaluated to yield emphysema scores that ranged from 0 to 4.", "Patients with COPD were characterized as having frequent exacerbations if there were two or more severe exacerbations in 1 year.33", "The Medical Research Council (MRC) dyspnea scale,34 Borg scale (dyspnea and fatigue),35 6-min minute walking distance (6MWD),36 BMI, airflow obstruction, dyspnea, exercise capacity (BODE) index,37 and the Charlson index38 were examined according to the original reports.", "We also examined the air trapping by residual volume (RV)/total lung capacity (TLC) and the oxygenation capacity of lung by Pao2/Fio2 or by the alveolar-arterial oxygen difference (Aado2).", "Blood samples were collected in BD Vacutainer Plus Plastic Serum and SST Tubes, which are coated with silicone and micronized silica particles to accelerate clotting.", "Samples were then centrifuged at 1500g for 15 min at room temperature, and supernatants were aliquoted as serum samples and immediately stored at –70 oC until measurement.", "Pulmonary function tests were performed using MasterScreen (Erich Jaeger GmbH) and included postbronchodilator FEV1, FVC, FEV1/FVC ratio, TLC, RV, inspiratory capacity (IC), and diffusing capacity for carbon monoxide (Dlco).", "Dlco and diffusing capacity for carbon monoxide adjusted for alveolar volume (DLCO/VA or KCO) were assessed by the single-breath method with the patient in the sitting position.", "Lung function measurements were expressed as percentage of predicted values.", "Tests were performed according to the American Thoracic Society/European Respiratory Society guidelines by the same technician to ensure consistency of results.", "All lung function data are shown in Table 1.", "Serum samples were diluted in radioimmunoprecipitation assay buffer (Sigma; 150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 50 mM tris(hydroxymethyl)aminomethane, pH 8.0) completed with protease inhibitor, as previously published,39 separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and incubated with anti-SIRT1 antibody or with anti-β-actin antibody overnight.", "The membranes were then incubated with the appropriate peroxidase-conjugated secondary antibodies.", "The bound antibodies were visualized by chemiluminescence (ECL Plus; GE Healthcare).", "The band density in each sample was normalized to the level of standard sample (from one healthy donor), and each SIRT1 protein level was shown as the relative ratio against that in standard sample.", "BEAS-2B cells (SV40-immortalized human airway bronchial epithelial cell line) and A549 cells (human lung adenocarcinoma epithelial cell line) were purchased from the American Culture of Tissue Collection and grown in complete growth medium (RPMI 1640 and DMEM supplemented with heat-inactivated 10% FBS and 1% L-glutamine, respectively) at 37oC/5% CO2.", "Before use, cells were starved in minimum medium (RPMI 1640 or DMEM supplemented with 1% FBS and 1% L-glutamine), and cell culture supernatants were harvested at different time points.", "To eliminate the contamination of supernatant by free-floating cells, the supernatant was centrifuged and the upper half of the medium was taken as the sample.", "Human primary bronchial epithelial cells obtained from three subjects without COPD and three subjects with COPD were cultured as monolayers in LHC-9 media (Invitrogen) on collagen (1% w/v)-coated plates as previously reported.40", "Cells were extracted from lung tissue of patients undergoing lung resection at the Royal Brompton Hospital.", "All subjects gave informed written consent and the study was approved by the National Research Ethics Service London-Chelsea Research Ethics committee, study No. 09/H0801/85.", "All cells were serum starved 16 hours before stimulation.", "Cells were stimulated with 3% cigarette-smoke-conditioned media prepared as previously reported.41", "Data from clinical samples were expressed as mean values ± SD.", "For the analysis of SIRT1, statistical significance was assessed using a nonparametric Kruskal-Wallis test with a Bonferroni multiple comparison procedure to exclude possible interaction between various variables within subgroups (Statcel 2, OMS Publishing Inc.).", "The analysis of correlation between each factor was performed by Spearman correlation coefficient rank sum test.", "All reported P values were two-sided, and P values < .05 were considered to be statistically significant.", "In a previous report, s120S was found to be detectable by Western blot,18 which showed an excellent correlation with the enzyme-linked immunosorbent assay (ELISA).", "As shown in Figure 1, anti-SIRT1 antibody used in this study detected different sizes of SIRT1, including 75, 102, and 120 kDa (the size originally reported) in BEAS-2B cells or A549 cells; therefore, we determined these SIRT1 fractions in serum samples separately.", "Compared with healthy subjects, the patients with COPD showed decreased levels of 120-kDa s120S (SIRT1 ratio in healthy subjects [NS + SM], 0.90 ± 0.34 vs subjects with COPD, 0.68 ± 0.24; P = .014) (Fig 2A), whereas SIRT1 with lower molecular weights (102 kDa and 75 kDa) did not (Figs 2B, 2C, e-Figs 1A, 1B, 1C).", "s120S showed a significant positive correlation with airway obstruction (FEV1/ FVC ratio; r, 0.31; P = .020) (Fig 2D, Table 2) and also with the severity of airway obstruction, measured by FEV1 % predicted (r = 0.29; P = .029) (Fig 2E), suggesting that s120S protein levels decrease with COPD progression (Fig 2F).", "In addition, s120S showed a negative correlation with the amount of cigarette consumption (pack-year; r = –0.33; P = .014) (Fig 3A).", "Patients with a higher degree of emphysema on HRCT had lower levels of s120S (r = –0.40; P = .027) (Fig 3B) when analyzed in all subjects showing some degree of emphysema.", "A good correlation was also observed in all subjects (P = .0048) (Table 2) and COPD subjects only (P = .091) (Table 2).", "In addition, patients with emphysema showed decreased levels of s120S when compared with the patients with normal lungs (SIRT1 ratio in control population, 0.92 ± 0.37 vs 0.71 ± 0.24 in those with emphysema; P = .026) (e-Fig 1D).", "This was confirmed by the significant positive correlation between the s120S SIRT1 and KCO % predicted (r = 0.32; P = .025) (Fig 3C, Table 2).", "The s120S was not correlated with age, probably because of the elderly bias of samples included.", "In contrast, the BMI showed a significant positive correlation with s120S (r = 0.36; P = .0077) (Fig 3D, Table 2).", "In addition, s120S decreased significantly as symptoms (MRC dyspnea score) increased (Fig 3E).", "The severity of hypoxia (Pao2 or desaturation on movement) and oxygenation capacity of lung (Pao2/Fio2 or Aado2) did not show any correlation with s120S (Table 2); however, s120S showed positive correlation with Pao2/Paco2 ratio representing the combined effect on gas exchange42 (r = 0.28; P = .034) (Fig 3F), which suggested that the impairment of aerobic metabolism might contribute to the s120S protein level.", "Other patient background characteristics (Table 2) or subject comorbidities (such as cardiovascular disease or diabetes mellitus) and Charlson index did not show any association with serum levels of SIRT1.", "When we limited analysis to the patients with COPD only, we identified two additional findings.", "First, patients with COPD with frequent exacerbations tend to have lower s120S levels compared with those with stable disease (Fig 4A).", "Second, s120S had a positive correlation not only with the FEV1 % predicted (r = 0.40; P = .046) (Fig 4B) but also with 6MWD (r = 0.45; P = 0.023) (Fig 4C).", "This was also confirmed by the fact that s120S was negatively associated with the MRC dyspnea score (Fig 4D) and with the BODE index (Fig 4E), which is known to be a strong predictor of long-term prognosis in COPD.37", "We further analyzed the correlation with all parameters in the GOLD stage 1-2 population only and the 3-4 population only.", "We did not see any significant correlation except for IC % in stages 1-2 (e-Table 1).", "Regarding the origin of s120S, we were unable to detect any active secretion from lung epithelial cell lines such as BEAS-2B (airway) cells or A549 (parenchymal) cells (Figs 4F, 4G).", "In A549 cells, SIRT1 was detected in supernatant after 72 hours of culturing, but as the housekeeping protein β-actin was also detected, it was likely that this may have been due to increased cell permeability related to loss of function.", "Furthermore, we also investigated SIRT1 protein release in supernatant from non-COPD (n = 3) and COPD (n = 3) primary bronchial epithelial cells.", "We did not find original or degraded SIRT1 protein or β-actin in supernatant in the absence or presence of cigarette-smoke-conditioned media (data not shown).", "Thus, it was unlikely that SIRT1 was excreted from bronchial epithelial cells.", "In the current study, we showed for the first time, to our knowledge, that the protein levels of s120S in its 120-kDa form were significantly decreased in patients with COPD.", "The s120S protein levels were positively correlated with the severity of airway obstruction and showed a strong negative correlation with the amount of cigarette consumption, suggesting that oxidative stress may lead to the reduction of s120S.", "This is contrast to bronchial asthma,21 in which s120S detected by ELISA was increased.", "In addition, we found that s120S was significantly correlated with the severity of emphysema (HRCT reading and KCO % predicted) and functional disability represented by an MRC dyspnea score, 6MWD, and BODE score.", "These results suggest that s120S may be a useful marker for assessing certain disease characteristics in patients with COPD.", "Among the seven sirtuin isozymes,43 SIRT1 is the most widely studied in mammals from the viewpoint of regulation by oxidative stress, which is relevant to cellular senescence and chronic inflammation.2-4", "In fact, dysregulation of SIRT1 has been described not only in aging-associated diseases but also in those associated with long-term cigarette smoking,44,45 and all are characterized by oxidant/antioxidant imbalance.46", "We previously reported a reduction in SIRT1 in the peripheral lungs of patients with COPD.30", "Although reports of reduced SIRT1 relate to intracellular SIRT1 (mRNA or protein), Kumar et al16 first reported that SIRT1 was detectable in the serum.", "In this report, s120S was measured by various methods, including Western blot, ELISA, and surface plasmon resonance, with good correlation with each method, confirming that SIRT1 is a serum protein.", "Interestingly, they also showed significant reduction of s120S protein levels as dementia progressed, suggesting that s120S may be a useful biomarker for assessing cognitive disease.", "This report was surprising, as SIRT1 had originally been described only as a nuclear protein.8,9", "However, recent reports have demonstrated that SIRT1 can shuttle between the nucleus and cytoplasm10-12; therefore, SIRT1 is potentially present in the extracellular component.", "The strength of our study is the selective determination of the fraction of full-length SIRT1 (120 kDa) separate from other truncated SIRT1 proteins by Western blot.", "This is in contrast to previous reports that used ELISA for s120S detection.18-21", "Despite its good quantitative capability, ELISA does not appear to be specific for the full-length functional fraction of SIRT1, because antibodies recognize several fractions with the target motif, irrespective of their function (Fig 1A).", "In previous reports, several bands of SIRT1 protein (original and truncated proteins) were described, indicating different molecular weights by Western blot,47,48 each of which may function differently, although this has not yet been elucidated.", "Therefore, our results are unique, as we were able to analyze only the fully functional fraction of full-length SIRT1 (120-kDa SIRT1) that was separated by Western blot.", "Thus, Western blot should be used for s120S detection.", "In addition, this is the first report to show that s120S is reduced in patients with COPD.", "Compared with healthy subjects, patients with COPD showed decreased levels of s120S, which correlated not only with the airway obstruction but also with its severity, resultant lung emphysema, and decreased diffusion capacity.", "These results were compatible with the previous reports that found that the SIRT1 protein level was decreased in peripheral lung or peripheral mononuclear cells in patients with COPD.30", "Furthermore, we could also detect the association of s120S protein levels with BMI, MRC dyspnea score, and Pao2/Paco2 imbalance, all of which suggested that s120S is not just an indicator of lung damage but is a surrogate marker for oxygen metabolism and systemic metabolic status.", "Interestingly, our result appears to be opposite to that reported in patients with asthma21; therefore, s120S might be a potential biomarker to help to differentiate these two diseases.", "Future studies might be necessary for comparing the s120S levels directly between patients with asthma and COPD populations.", "Since reduced s120S is also reported in association with frailty in elderly people, it may also be useful in understanding the multimorbidity associated with COPD.", "We further analyzed the correlation with all parameters in a GOLD stage 1-2 population only and in a GOLD stage 3-4 population only.", "We did not see any significant correlation in all parameters except for IC % predicted in the stage 1-2 population.", "However, we observed a nearly significant correlation in FEV1/FVC (P = .19), emphysema score (P = .080), KCO % (P = .17), RV % (P = .090), and 6MWD (P = .11) in the stage 1-2 population.", "We did not have enough power to demonstrate the association in the current study, but a future large study will reveal the usefulness of s120S as a potential biomarker to determine the early stage of COPD.", "A limitation of the present study is that we have not identified the precise source of SIRT1 in serum.", "We could not detect any fractions of SIRT1 in the cell culture supernatant in A549 and BEAS-2B epithelial cells, indicating that cellular leakage or active secretion is unlikely.", "In primary bronchial epithelial cells, we could not find full or degraded SIRT1 proteins or β-actin in supernatant in the presence or absence of cigarette-smoke-conditioned media, suggesting that SIRT1 is unlikely secreted by bronchial epithelial cells.", "However, SIRT1 protein was detected in supernatant from A549 cells at a later stage, which was associated with an increase in β-actin expression.", "This suggests that epithelial cells are still a possible source of SIRT1 when cells are damaged.", "Considering that SIRT1 in patients with COPD has been reported to be decreased not only in the lung30 but also in endothelial progenitor cells49 and circulating leukocytes,30,50 decreased s120S might reflect the reduction of SIRT1 in cells as a result of oxidative stress.", "Peripheral blood mononuclear cells or alveolar macrophages might be potential sources of SIRT1.", "It might be necessary in a future study to identify the precise origin of s120S and the factors that modulate s120S levels in COPD and other chronic aging diseases.", "Secondarily, even though there were statistical differences in SIRT1 levels between the comparison groups, there was significant overlap in the values of all groups.", "GOLD stage is defined by lung function (mainly by FEV1 % predicted), but, as discussed earlier, there was good correlation between SIRT1 and certain disease characteristics such as emphysema, MRC dyspnea score, 6MWD, and BODE score.", "Therefore, SIRT1 level is influenced by several factors rather than lung function alone.", "The current study is too small to evaluate further, but we believe that a future large study with more patients will provide a novel approach to classify disease stage based on SIRT1.", "In summary, we report for the first time that s120S was reduced in patients with COPD and that this reduction was correlated with the extent of emphysema and reduced functional measurements that correlate with disease progression.", "Serum SIRT1 might therefore serve as a potential biomarker for COPD." ]

[]

[ "BIOC", "MEDI" ]

[ "Breast cancer", "Clinical trials", "Observational studies", "Randomised trials", "Surrogate markers" ]

[ "1306", "2730" ]

[ "Frustration at the slow rate of progress in trying to develop and improve treatments for early breast cancer is commonly expressed by both patient and medical communities.", "There is an obvious tension between the desire for improved treatments and other technologies and the need for careful evaluation.", "The pathway of progress in the management of this common disease is littered with examples of promising therapies, implemented prematurely and ultimately discarded when shown to be ineffective or unsafe.", "Although large treatment effects may in some circumstances be detected without prospective studies or with small trials, such revolutionary advances are very rare and there are none in the management of early breast cancer.", "In this disease there is a critical need to distinguish reliably between a moderate but worthwhile gain from a new treatment and no worthwhile gain.", "To achieve this, large-scale randomised evidence, interpreted without data-dependent emphasis on particular trials or subgroups, is needed.", "The randomised controlled trial (RCT) is accepted as the most reliable means of providing robust evidence of the efficacy and safety of new treatments, adaptations or withdrawal of existing treatments and new pathways of care.", "Individual patient level meta-analysis represents the ultimate refinement of the biostatistical approach of the collection and analysis of prospectively collected, randomised data and offers greater statistical certainty of effect and may allow the safer exploration of a treatment effect in defined subgroups.", "Unfortunately, deriving a result from a single RCT is costly and time-consuming.", "There are considerable bureaucratic, financial and often perceived ethical hurdles to be overcome before recruitment in a multicentre study can begin and these have worsened over time.", "The prognosis for newly diagnosed patients with early breast cancer has improved and in particular screen-detected disease, with its generally much better prognosis, has become increasingly common.", "‘Event rates’ have correspondingly fallen but this welcome development has meant larger clinical trials with longer follow up are required to reach a statistically secure endpoint.", "There is thus a substantial price to pay in time, cost and the number of trial participants needed, for a robust result that can improve care.", "A recent example is the EORTC trial of regional radiotherapy.", "This critically important trial included 4004 patients and demonstrated a 3% absolute (11% proportional) improvement in disease-free survival.", "This study was published 11 years after the last patient was recruited and 20 years since the trial started [1].", "The long wait for results from RCTs is in contrast to the expressed desire for rapid progress and commercially problematic given the limited patent of newly licensed pharmaceuticals and the life span of other technologies.", "Academic success is also highly dependent on publication in high-profile journals and the long period of time between the awarding of funding for a RCT and the publication of results poses serious problems for academic researchers and their institutions.", "The colossal expense, uncertainty over what should represent ‘best standard care’, large size, increasing emphasis on research governance, burgeoning bureaucracy and apparent slow progress related to the conduct of prospective clinical trials has tended to encourage the research community, funders and policy-makers to seek cheaper and quicker alternatives.", "The use of surrogate markers is one potential approach to try to accelerate progress.", "An endpoint that can be derived relatively quickly (and preferably inexpensively) will (it is hoped) be predictive of longer-term benefit and allow earlier conclusions to be derived.", "This is an active area of clinical research and there are many differing approaches, but at this point in time none are in routine use and their validity in the form of formal testing against clinically established methods of assessment is at an early stage.", "Perhaps the most significant development in the development of surrogate markers in early breast cancer is represented by the use of pathological complete response (pCR) as a possible indicator of future longer-term outcome following the use of neoadjuvant chemotherapy (NACT).", "The CTNeoBC study pooled information from 12 RCTs of NACT and confirmed an improvement in survival following the attainment of a pCR at individual patient level.", "Crucially this correlation could not be confirmed at clinical trial level limiting the ability to compare treatment approaches by a comparison of rates of pCR in groups of patients.", "In addition, the relationship between pCR and disease-free or overall survival at patient level was heavily influenced by biomolecular subtype—being much stronger for HER-2 positive or triple negative disease than for HER-2 negative, ER+ breast cancer [2].", "Within the most recent meta-analysis of cytotoxic chemotherapy conducted by the EBCTCG adjuvant chemotherapy achieved the same proportional reduction in recurrence and mortality risk in ER positive (compared with ER negative) disease [3] despite the findings of much lower pCR rates when this treatment is used as neoadjuvant therapy [4].", "The achievement of pCR clearly has prognostic significance but it seems less useful as a predictive marker of treatment benefit in most circumstances.", "Although the US FDA agreed that the attainment of pCR following NACT could be used as an endpoint to support accelerated drug approval, there remains significant uncertainty of the usefulness and longer-term clinical validity of this approach [5,6].", "Randomised evidence is not available to support the use of all interventions in the management of early breast cancer.", "Reconstructive surgery, newer diagnostic technologies, lymphoedema management and post-treatment surveillance imaging are interventions that are employed without supportive evidence of benefit from RCTs.", "Whether non-randomised evidence can be used to discern the benefits and toxicities of treatments with modest impacts on outcome, particularly commonly used adjuvant therapies, is highly contentious.", "The advantages of observational evidence are the avoidance of the cost and time in setting up and running RCTs, the speed that results can be obtained and the ability to derive very large sample sizes.", "The limitations relate to the inability to predict all of the potential confounding variables that may affect the relationship between a selected treatment and its apparent therapeutic effects and toxicities and the difficulty in quantifying, and accounting for, the effect of confounding variables that can be predicted to exist.", "In 1965, Austin Bradford Hill proposed nine criteria to help assess causality between an exposure such as cancer treatment and an outcome, such as cancer recurrence or cancer-related death or complication.", "These criteria included; strength of association, plausibility, biological gradient, consistency, specificity, temporality, coherence, experimental evidence and analogy with similar factors [7].", "Whilst these criteria succinctly summarise the factors to consider in evaluating a potentially causal association they do not offer a definitive methodology to conclude causation.", "Over the 50 years since his publication, causation has been and is still commonly concluded by the fickle and variable nature of human judgement.", "McGale et al. [8] reported worryingly divergent results between the effects of adjuvant radiotherapy on breast cancer mortality noted within the SEER database and those identified by the meta-analyses of RCTs evaluating the role of adjuvant radiotherapy conducted by the EBCTCG.", "These differences persisted despite detailed adjustment for potential confounding variables.", "SEER data also suggested that the risk of cardiac mortality was lower in patients receiving radiotherapy compared with those who did not receive this treatment, in contrast to data within EBCTCG that confirms that radiotherapy does slightly but significantly increase cardiac mortality.", "Sagara et al. [9] also used SEER data to derive a prognostic score based on tumour size, patient age and nuclear grade for 32,144 patients with DCIS treated with breast conserving surgery.", "Their conclusion that higher risk patients derive a breast cancer and overall mortality benefit from the use of whole-breast radiotherapy is in marked contrast to the randomised evidence from the five individual trials of breast radiotherapy after excision of DCIS and the meta-analysis conducted by the EBCTCG, that suggest no impact on breast cancer–related mortality from the use of radiotherapy [10].", "Perhaps the most prominent example of a treatment mistakenly introduced and used widely in high-risk early breast cancer is myelo-ablative chemotherapy with bone marrow rescue.", "On the basis of retrospective evidence, small uncontrolled studies and a widespread medical belief in superiority, many thousands of women were subjected to a risky and toxic treatment that, following the publication of several RCTs, is now known to be no or only slightly more effective, but unacceptably more toxic, than standard care [11].", "Observational data have a place in the assessment of population-level outcomes following treatment (generalisability), the assessment of longer-term toxicity and can be of use when the characteristics of the population treated vary considerably from the participants of RCTs.", "To determine the presence and size of a modest (but worthwhile) treatment effect requires elimination of the insidious and underappreciated effects of bias.", "Other than in the very unusual circumstance of a treatment with a dramatic effect on outcome, such observational data are unhelpful in determining whether adjuvant treatments are more effective than standard care.", "There are some circumstances where RCTs are not feasible and RCTs have on occasions—because of insufficient size or the over-interpretation of subgroups—provided spurious results.", "However, the use of surrogate markers and the assessment of modest treatment effects by the acquisition of observational data at this point in time are not a substitute for prospective studies and randomised evidence designed to identify the presence of worthwhile benefits of new treatments.", "There is no immediate prospect of any change in this position.", "The last 2–3 decades have seen substantial improvements in breast cancer mortality, falling local recurrence rates and more recently, reduced treatment-related morbidity.", "Improved systemic adjuvant therapies, the introduction of mammographic screening and refinements to locoregional treatments have played the major part in these improvements and all have evolved and been introduced following careful assessment in large randomised trials.", "Simplification, de-escalation of bureaucracy and streamlining of research approval processes to encourage simple large prospective trials should remain a priority.", "Such trials can have optional shorter-term translational components involving for instance radiological, molecular pathological or detailed toxicity assessments in efforts to try to identify reliable surrogate markers that may ultimately obviate the long wait for recurrence and mortality data, but the majority of centres could recruit patients who would have routine follow up care alone.", "‘Lighter touch’ research governance processes to simplify participation, reduce costs and remove much burdensome bureaucracy could be implemented for RCTs where toxicity risks are lower, for instance in duration trials, involving drugs that have been in routine use for many years.", "In future it may also be possible to use routinely collected data in national registries or other data sets to provide endpoint determination provided the accuracy of this approach can be verified.", "Undue reliance on observational data or the premature use of surrogate markers in the contemporary assessment of new treatments or pathways of care in the management of early breast cancer involves a significant risk of embracing ineffective treatments or discarding those that are useful.", "Gurdeep S. Mannu received funding from the National Institute of Health Research Biomedical Centre (Oxford).", "David Dodwell is funded by the Nuffield Department of Population Health, University of Oxford.", "Financial support is provided by Cancer Research UK (grant no C8225/A21133), by a research contract to the University of Oxford under the Department of Health Policy Research Programme (Studies of Ionising Radiation and the Risk of Heart Disease, 091/0203) as well as core funding from Cancer Research UK, the Medical Research Council and the British Heart Foundation to the Oxford University Clinical Trial Service Unit (grant MC_U137686858)." ]

[ "Surrogate markers are yet to be developed and validated in early breast cancer.", "Reliance on observational data to evaluate treatment effects is risky.", "Prospective randomised trials are difficult but remain vital to progress.", "Research approval processes need simplification and streamlining." ]

[ "AGRI", "CHEM" ]

[ "Adulteration", "HPLC-ELSD", "Quantification", "Quillaja", "Saponin", "Steroidal saponin", "Yucca", "Yucca brevifolia", "Yucca schidigera" ]

[ "1106", "1602" ]

[ "Well before their chemical structures were elucidated, Yucca saponins were utilised by American Indians in a series of applications, including soap.", "Multiple uses of a range of compounds originate from different parts of 35–40 Yucca (Agavaceae) species.", "Yucca are mainly distributed in the deserts of Central and Northern America and have evolved over centuries.", "Presently, Yucca schidigera syrup (concentrated juice; Yucca extract), and dried and finely powdered logs (Yucca powder), both abundant in steroidal saponins, are of particular interest to cosmetic, pharmaceutical and beverage industries as well as animal nutrition.", "Those GRAS-labeled products possess foaming features that are of particular interest in soft drinks (root beer), cosmetic (Tanaka, Tamura, Masuda, & Mizutani, 1996), food and feed industries (Fenwick & Oakenfull, 2006).", "The very foaming activity of Yucca extract is due to the abundance of saponins (∼10% dry matter) (Oleszek et al., 2001).", "Such high saponin contents are the reason why, Y. schidigera, together with Quillaja saponaria, are the two major commercial sources of saponins that happen to be misused, one instead of the other, as plant adulterants (Cheeke, 2000).", "The most abundant Y. schidigera stem/bark saponins are steroidal saponins.", "They differ in the structure of their aglycon, according to which, they are classified into spirostane- or furostane-type derivatives.", "Primary saponins are glycosides of three C-25 epimeric pairs of sapogenins: sarsapogenin and smilagenin, markogenin and samogenin, gitogenin and neogotogenin.", "In both, spirostane and furostane derivatives, the C-3 carbohydrate chains are typically branched oligosaccharides with pentapyranosyl and/or hexopyranosyl units.", "As far as furostane bidesmosides are concerned, C-26 linked carbohydrate usually corresponds to a hexopyranose.", "It should be noted that derivatives of other sapogenins occur as minor compounds within Y. schidigera stem/bark.", "The above summarised complexity of Y. schidigera stem/bark steroidal saponins has remained for a longtime an obstacle in their identification and quantification.", "Indeed, the inability of quantifying steroidal saponins from Y. schidigera has been an industrial challenge and constraint, as it directly reflects the incapacity of standardization in steroidal saponins.", "Should such standardization be possible and available in the industry, it would answer an essential question related to the extract quality, as steroidal saponin levels could be directly correlated with features held by these compounds, for instance, with the foaming capacity.", "Classical methods presently used in measurement of steroidal saponins include spectrophotometric measurements (Baccou, Lambert, & Sauvaire, 1977; Uematsu, Hirata, Saito, & Kudo, 2000), foam height measurements (Ross & Miles, 1941) or gravimetric method (Wina, Muetzel, & Becker, 2005).", "Neither of these methods satisfies accuracy or reliability requirements that necessitate industrial quality control.", "For example, a gravimetric technique, which is based on gravimetric determination of butanol fractions, and the spectrophotometric method, both lack specificity, for they measure, besides steroidal saponins, all compounds present in the butanol fraction, including polyphenols and therefore could overestimate steroidal saponin levels.", "Moreover, a poor affinity of butanol for highly polar saponins is another source of imprecision in the estimation of steroidal saponins by this method.", "Furthermore, due to such a lack of specificity, gravimetric or spectrophotometric method cannot distinguish steroidal saponin profiles originating from different plant species.", "Identification of different sapogenins was initiated by Kaneda, Nakanishi, and Staba (1987) and Miyakoshi et al. (2000) and further characterised by Piacente, Pizza, and Oleszek (2005) and Kowalczyk, Pecio, Stochmal, and Oleszek (2011).", "In their last report, Kowalczyk et al. (2011) successfully identified, characterised and quantified certain steroidal saponins present within Y. schidigera concentrated juice using the method of HPLC/MS.", "Although this method is very precise and robust, it remains expensive and time-consuming; therefore it is not used in the food industry.", "The industry remains in need of an analytical method specific to steroidal saponins enabling quantifications at reasonable costs.", "Here we present a rapid, specific, cost- and time-effective HPLC/ELSD method, which accurately measures total Yucca steroidal saponins.", "Although this method has been developed for steroidal saponins from Y. schidigera, it reveals a specific chemical fingerprint profile and therefore distinguishes extracts originating from different saponin-containing species.", "Hence, it can also be used as an investigation tool for steroidal saponin plant adulteration analyses.", "Three samples of Y. schidigera juice were purchased from three suppliers of the Baja California state (samples named A, B, and C).", "Spray drying of sample (B), in the presence of inverted sugar, yielded a Yucca powder D (NATUREX processed).", "Authenticated Yucca trunk raw material was supplied by Compania Agroindustrial Baja (Mexican state) and the shampoo containing Yucca brevifolia root was supplied from Shea Moisture website.", "A standard of Y. schidigera saponin mixture was prepared during this study using Yucca syrup (extract) produced by NATUREX.", "High purity of saponins (>95%) was controlled using multiple test method.", "HPLC-grade solvents were purchased from Biosolv Chimie (Dieuze France); Deionized water was obtained using a Milli-Q Elix system (Merck-Millipore, France).", "All other chemicals were purchased from Sigma-Aldrich.", "Antifoam FD30 K was supplied by Basildon Chemical Company (Abingdon, England).", "The Amberlite FPX68 was purchased from Rhom & Haas Company (Chauny, France).", "SPE AFFINIMIP Phenolic cartridge 100 mg was purchased from Polyintell (Val-de-reuil, France).", "Nitrogen generator using a brezza system was from GENGAZ (Wasquehal, France).", "Most of studies involving saponin quantification by chromatography use a commercial saponin standard (Budan et al., 2014).", "Such commercial standard comprises only one type of saponins, the structure of which is different from the majority of saponins comprised in Yucca extracts.", "Using only one saponin as a standard originating from a distant plant family is thus in most cases inappropriate and insufficient for quantification of a mixture of Yucca saponins.", "Clearly, a need for a standard comprising a mix of relevant saponins remained.", "We explored a new way of quantifying saponins by purifying a mixture containing two types of major saponins of Yucca schidigera, via two distinct purification steps, as described hereafter.", "Crude, Yucca juice concentrate (300 mL, 38°Brix) was repeatedly extracted with 200 mL of ethanol (100%) three times.", "Ethanolic phases were pulled and concentrated to dryness.", "The powder obtained was repeatedly washed with 200 mL of ethyl acetate and 200 mL of hexane under reflux for 30 min yielding a brown viscous residue.", "Subsequently, the residue was partitioned between water phase and 1-butanol phase.", "Following concentration of the organic phase under vacuum, a brown powder (BP_7.5 g) was obtained.", "The obtained powder (5 g) was fractionated using column chromatography (FPX-68; 600 g; 1.5 L of water; 1.5 L of water/ethanol 7:3; 1.5 L water/ethanol 3:7).", "The water/ethanol 7:3 v/v fraction was concentrated in a freeze dryer and yielded 1.2 g of powder.", "All powder samples were subjected to SPE AFFINIMIP.", "After activation of the cartridge with water and methanol, the sample was loaded and washed with water.", "Saponins were recovered by water/acetonitrile (10:90 v/v), and the collected fraction was freeze-dried.", "It resulted in pure saponins, which were precipitated in methanol/ether (20:80 v/v).", "Following the subsequent freeze-drying step, the first pure mixture of saponins was obtained, termed SAPONINS 1 (Supplementary material, Fig. 1).", "A quantity of 500 mg of the brown powder (BP_7.5 g) resulting from the first liquid/liquid extraction was supplemented with 3 mL of water and 800 μL of thus obtained solution was injected onto preparative HPLC system (Agilent, 1200 series equipped with a 126 wells plate fraction collector).", "An Agilent preparative C18 column (30 × 150 mm, 10 μm) was used.", "The mobile phase was composed from two solutions (A and B), where A was 0.1% formic acid (v/v) in water and B 0.1% formic acid in acetonitrile (v/v).", "The gradient profile was the following: 0–10 min linearly from 90% A to 70% A; 10–20 min linearly from 70% A to 20% A.", "The flow rate was set at 25 mL/min, and the temperature of the column was set at 25 °C.", "Fractions containing saponins that were eluted from 15 to 20 min were collected and concentrated before freeze-drying.", "Thus obtained saponins were then dissolved in methanol and precipitated by ethyl ether.", "After repetition of several injections according to the process described above we finally obtained a second pure saponin mixture SAPONINS 2 (Supplementary material, Fig. 1).", "The pure mix called Yucca Saponins Standard (YSS) was obtained by combining two times as much of SAPONINS 1 with SAPONINS 2.", "Mass data enabling YSS characterization were collected on Agilent 6230 accurate-mass TOF_HPLC/MS with an electrospray interface using Agilent Jet stream (AJS) Thermal Focusing technology for a better sensitivity.", "The ionisation conditions were adjusted as follows: a capillary voltage 3500 V and a nebulizer temperature of 300 °C.", "Nebulization was aided with AJS using a Nitrogen sheath gas flow (10 L/min) and sheath gas temperature 300 °C.", "The full scan mass acquisitions in negative mode were performed by scanning from 100 up to 1500 m/z range.", "NMR analysis was conducted on Bruker DRX500 instrument with a Probe head of 5 mm BBI with a dilution in deuterated water.", "Solvent analysis was performed on Agilent 7890A coupled with 5975C Mass detector (simple quad) from Agilent Technologies.", "Water content was determined with coulometric Karl fisher method (KF C20 model from Mettler Toledo).", "The above purified steroidal saponins YSS were weighed in order to produce a standard solution used for the development of a calibration curve in the range 500–3500 μg/mL (total saponins).", "All solutions were diluted with water and filtered through RC 0.45 μm disc filter without excessive agitation in order to avoid foaming.", "The standard solutions prepared could be stored at 4 °C for no longer than two weeks, and used for further calibration assays.", "Y. schidigera juice extracts A, B and C were obtained in several steps of extraction from the raw Yucca trunk material.", "i) Step one: fresh trunks of Yucca were mechanically pressed to produce a first juice.", "ii) Step two: the raw material from the first extraction was extracted again with water and the juice thus obtained was combined with the first juice and adjusted to produce a juice of 13–16° Brix.", "This 13–16° Brix juice was concentrated up to 50°Brix, resulting in Yucca juice sample (A).", "iii) Step three: the Yucca juice from step two (11–13° Brix) was concentrated and clarified to produce a juice of 25–28° Brix.", "Finally the hot juice from step three of clarification was adjusted to pH 3.5–4.0 with phosphoric acid and concentrated either to 38° Brix to produce Yucca juice sample (B) or to 50° Brix to produce Yucca juice sample (C).", "Y. schidigera powder extract (D) was obtained by spray-drying Yucca juice sample (B) on inverted sugar as a carrier.", "An estimated value of saponin content in thus obtained samples, using gravimetric method, ranged between 10 and 13%.", "A blend of Yucca schidigera (trunk)/Quillaja saponaria (root) extract was a mixture (50:50 v/v) of Yucca juice extract (A) with Quillaja saponaria juice extract standardized at 16% total saponins by gravimetric method.", "An approximate level of total saponins of thus obtained blend ranged from 14 to 15% (as measured by gravimetric method).", "Samples were prepared according to the following protocol prior to HPLC/ELSD analysis.", "Y. schidigera juice, powder extract or blend were weighed (∼200–400 mg), dissolved in 20 mL of water containing 0.05% antifoaming FD30 K agent.", "The solution was filtered through 0.45 μm RC disc filter prior to HPLC/ELSD analysis.", "HPLC/ELSD analysis was run according to the following method: an Agilent 1200 series liquid chromatographic system was coupled with an Evaporative Light Scattering Detector (ELSD, G4260).", "An Atlantis® T3 column (3.0 × 150 mm, 3 μm) (Waters, Ireland) was used.", "As above, the mobile phase was composed from two solutions (A and B), where A corresponds to 0.1% formic acid (v/v) in water and B 0.1% formic acid in acetonitrile (v/v).", "However, the gradient profile was the following: 0–25 min linearly from 98% A to 40% A; 25–35 min linearly from 40% A to 20% A.", "The flow rate was set at 0.6 mL/min, and the temperature of the column was set at 25 °C.", "Evaporation and nebulizer temperature of the ELSD were set, respectively, at 50 °C and 40 °C with a gas flow of 1.2 (arbitrary unit).", "The shampoo sample containing steroidal saponins of Yucca brevifolia was analysed by here developed method in order to appreciate the differences in saponin fingerprints between Y. schidigera and Y. brevifolia and therefore to evaluate the applicability of this method in plant/extract adulteration.", "As the shampoo contained very low amounts of saponins, it was necessary to proceed initially by a purification step, prior to implementing the here developed method.", "The shampoo sample was therefore first submitted to liquid/liquid extraction with n-butanol and after concentration of the organic phase, the residue was dissolved in water, filtered and injected onto HPLC.", "Subsequently, the above described method was applied and resulted in a purified sample.", "The analytical method HPLC/ELSD was evaluated for linear response of the calibration curve, precision (intra-day and reproducibility), and accuracy.", "The calibration curve was linear over the range of 500–3200 μg/mL of saponins, which was determined using 6 solutions of the Yucca Saponins Standard (YSS) varying concentrations measured in duplicates.", "A linear regression equation was obtained by plotting the logarithms of peak area responses versus logarithms of concentrations, in μg/mL.", "The precision of the analytic method was determined on Yucca powder sample D.", "The precision (intra-day) was evaluated by calculating the percentage of relative standard deviation of the peak area sum of the saponins from six injections prepared following six different weighings at 100% of the test concentration.", "The reproducibility of the method was conducted on six injections of the Yucca powder sample D, run on a different day, by a different analyst and using a different piece of equipment.", "Accuracy, i.e. agreement of the calculated concentration with the nominal concentration, was examined on three different Yucca extracts at three concentration levels.", "Several methods have been previously used and proposed to evaluate Yucca saponin quantification.", "They are all mainly based either on foaming, gravimetric, spectrophotometric measurements or on HPLC/MS quantification (Skhirtladze et al., 2011).", "These different methods display disadvantages that seriously impact their use, notably at industrial levels.", "Therefore we have developed a new, improved, rapid and cost effective method that alleviates inaccuracies, insufficiencies, inconveniences and inadequacy of the existing techniques.", "In order to appreciate Yucca saponin levels obtained by here developed HPLC/ELSD method, we have compared them with those obtained using classical gravimetric method.", "HPLC/MS was used to control the YSS saponin composition.", "During the ionisation and transfer through a capillary, quasi-molecular ions of steroidal saponins repeatedly lose carbohydrate units from their side chains and may yield in an erroneous structural information.", "This is especially true when analysis is run in the positive mode.", "For this reason, negative ionisation was chosen to get a more important signal and improve the accuracy on [M−H]−.", "Following this method, a more reliable determination of the molecular formula could be used.", "Kowalczyk et al. (2011) identified 19 compounds in crude syrup extract of Yucca schidigera using HPLC/MS.", "YSS analysed by HPLC/MS resulted in the same peaks (Fig. 1) as those previously identified by Kowalczyk et al. (2011).", "Interestingly, in this study, the analysis of YSS revealed additional peaks corresponding to other steroidal saponins.", "Those peaks were identified as saponin peaks based on their molecular formula detected by accurate TOF/MS.", "Future work involving HPLC coupled with triple quadrupole or NMR methods will structurally characterise those remaining steroidal saponins.", "Gravimetric method is one of the first methods that was developed for saponin measurements decades ago (Harbone, 1973).", "It is based on the affinity of saponins for n-butanol.", "The quantification of total saponins using gravimetric method has shown a poor specificity of n-butanol to the most polar saponins (bidesmosidic glycoside and saponins with 3 or 4 branched glycosides), eluted between 12 and 17 min (Fig. 2A).", "Those saponins when extracted by n-butanol could hardly be seen on the chromatogram (Fig. 2B).", "Conversely, aqueous extraction of these polar saponins resulted in clear corresponding HPLC peaks.", "Data clearly show that saponins, notably polar saponins, are poorly extracted by n-butanol, which yields in an imprecision in this method.", "Moreover n-butanol is not specific to certain saponins.", "It extracts other compound classes, including polyphenols, sugars, organic acids, and therefore over-estimates saponin levels.", "Overall, due to its poor specificity and its poor affinity for the extraction solvent of Yucca saponins, the gravimetric method clearly both under- or over-estimates saponins, depending on their polarity, and therefore, does not seem to be utterly adequate for accurate saponin measurements.", "However, despite disadvantages of the gravimetric method, the precision was investigated.", "Relative standard deviation (RSD%) measured on 6 independently prepared samples of Y. schidigera juice extract yielded in 7.2%.", "Relative high RSD value indicates that such imprecision would negatively reflect and impact the manufacturing productivity.", "Moreover, use of gravimetric method is a long process.", "Indeed, the analysis of each sample requires five hours.", "Hence, gravimetric method requires five times as much time as compared to here developed HPLC/ELSD method.", "It is important to note that such time consuming sample preparation is inappropriate for use at the industrial level, more precisely in use for industrial quality control purposes (Table 1).", "Finally, gravimetric method is not considered to be “environmentally friendly” because it requires large amounts (∼400 mL) of petrol-originated organic solvents per each sample.", "In order to alleviate insufficiencies, inadequacy and disadvantages of the existing techniques, and notably of the gravimetric method, we have developed a new, reliable, accurate, rapid and cost effective method for quantitative measurements of Yucca steroidal saponins that could be applied at industrial level.", "This method was applied to various Y. schidigera extracts.", "The liquid chromatography method coupled with ELSD detector is a fast and reliable method for the quantification of non chromophoric molecules, including steroidal saponins (Liang et al., 2007; Libo, Zhihong, & Daofeng, 2012).", "The main advantage of this method as compared to the ultraviolet or mass detector, seems to rely on its response factor, which is less influenced by the molecular structure and the nature of the mobile phase.", "Thanks to this response factor, the developed standard containing a pool of Yucca saponins successfully allows the integration, and therefore the quantification, of all major steroidal saponins, irrespectively of their polarity.", "Hence, this new method does not require the use of an internal standard.", "It should be noted that here presented method alleviates the complexity of the quantification of steroidal saponins from Yucca and successfully measured Yucca extract steroidal saponin compounds that require an appropriate detection mode and adequate markers which set the detection range.", "As Yucca steroidal saponins do not have chromophore moiety, UV detection mode is clearly not suitable for their measurement, while ELSD mode is capable of detecting such compounds.", "A question remained regarding the possibility of having a standard comprising several Yucca steroidal saponins with different polarities.", "The extreme saponins of the standard set the detection range of saponins of different polarity, and therefore the detection limits, within which, a sample of Yucca steroidal saponins could be measured.", "Similar questions were raised regarding Aesculus hippocastanum aescins.", "Like saponins in Yucca, several aescins occur concomitantly within the horse chestnut.", "As those aescins absorb within the UV range, European pharmacopeia suggested HPLC/UV method for their quantification (Monograph ESCOP, 1999).", "As to the integration mode, according to the European pharmacopeia, all peaks within the range delimited by methyl salicylate and the ibuprofen correspond to aescins and are quantified as such.", "Similarly, as ELSD detector does not provide neither spectral nor mass information, it was very important to generate a standard comprising differing Yucca steroidal saponins, the extremes of which would delimit the integration range within which would all steroidal saponins be quantified.", "Similar procedure described by San Martin and Briones (2000) is accepted and used for Quillaja saponin quantification (Food and Agricultural Organisation of the United states FAO 2004, 61st Joint Expert Committee for Food Additives JECFA).", "As structural identification and purification of each single saponin is time-consuming, we generated a standard that contained a majority and, more importantly the extremes, of Y. schidigera saponins.", "Compounds detected by HPLC/ELSD of the purified standard all corresponded to saponins that elute between 12 and 24 min with small impurities detected before 12 min (0.9%, expressed in area percent integration).", "No interfering peaks in UV detection range (200–400 nm) could be observed.", "In order to better characterise the purified YSS we have quantified i) the residual solvent content by GC/MS head space method (<0.05%); ii) the water content by coulometric method (4.1%), and iii) the absence of residual phenolic compounds by 1H NMR determination (focusing on aromatic proton detection range).", "All of these analyses lead to the absolute purity of 95.0% for the new here prepared standard.", "Here developed method presents a slight downside.", "In this method, the aim of having a mixture of saponins within the same standard answers the requirement of the measurement of all Yucca saponins, within a range of detection and retention time defined by the extreme compounds contained within the very standard.", "Because the standard is a mixture of saponins, it cannot specifically quantify each saponin separately.", "The detection limit of the standard mixture is much higher than a detection limit of a single purified saponin.", "Therefore, this method is not applicable to very low contents in saponins.", "However, it should be noted that quantification of saponins in the industry mainly relates to products with significant levels in saponins that approximate 10%.", "Overall, as the standard contains a broad range of Yucca saponins, as confirmed by further recovery data, the overall result yields in an accurate values.", "Moreover, qualitative plant adulteration evaluation seems to be possible at even low concentrations in saponins, provided saponin concentration remains within the detection limit.", "Here developed HPLC/ELSD method was developed using external standardization.", "Because ELSD results in a non-linear response, the calibration curve was plotted in Log/Log scale.", "Linearity of the response is acceptable in the range between 500 and 3200 μg/mL (corresponding to total saponins injected and 6 levels of concentration).", "Linearity in this range was confirmed.", "More precisely, the measure of goodness-of-fit (r2) was higher than 0.995 (Slope of the calibration curve was at 1.671).", "Due to the non-linearity and because all the saponins are not at the same concentration in the here developed standard, difficulties may persist in case of samples containing very low amounts of saponins.", "In such case, estimation of the detection limit and quantification may prove difficult.", "This would be a minor issue as the contents in total saponins generally range from 1% to 20% as exemplified by Yucca juice extracts A, B and C and Yucca powder D.", "To determine repeatability, Y. schidigera powder extract sample (D) that contained ∼6% total saponins was analysed in six replicates and the RSD% value observed for total saponin quantification was at 2.79%.", "Therefore the method is precise for multiple sample injections within the same sequence.", "As far as the intra-day variation is concerned, six replicates corresponding to six weights of samples of the same Yucca powder extract sample (D) were analysed on a different day and by 3 laboratories.", "The inter-day precision and reproducibility RSD% were calculated from the area sum of the total saponins and yielded respectively 3.02% and 4.73%.", "Such RSD% level corresponds to an adequate precision level required for industrial control.", "In order to check the suitability and the applicability of the developed method, we performed the measurement of saponins in three different samples of Yucca schidigera juice extract.", "These measurements resulted in different LC/ELSD profiles (Fig. 3).", "The influence of diluent containing 0.02% of antifoam was investigated by analysing the diluent containing 0.02% of antifoam alone, the sample in water, and the sample with 0.02% antifoam in water.", "No peaks were detected on the LC/ELSD chromatogram of the diluent containing antifoam.", "The difference of data obtained between samples in water and samples with 0.02% antifoam in water remained negligible.", "In conclusion, the antifoam at this concentration has no influence on the overall measurement of Yucca saponins.", "This indication is an advantage as the supplementation with the antifoam facilitates the accurate filling in of laboratory material, including flasks, as it alleviates the formation of the foam while the Yucca sample containing saponins is poured, and therefore enables more precise measurements.", "Meanwhile it is recommended to wash the column with 100% acetonitrile after each sample injection.", "The sample analysis for recovery test was performed in triplicates with a concentration approximating the 100% of the calibration curve.", "Typically, 300–500 mg of Yucca sample that contained ∼10–13% total saponins was dissolved in 25 mL of water containing 0.02% antifoam and analysed.", "To evaluate the influence of the repartition of saponins and validate the method of quantification, we tested the recovery by spiking the Yucca juice samples A, B, and C with known amount of YSS and have shown a recovery in the range of 80–120% (Table 2).", "We concluded that the composition of the Yucca extract has no impact on the overall result and on the response provided by other compounds present within the sample.", "Here developed method enabled accurate quantification of saponins contained within Yucca samples originating from different geographic areas and prepared from plant material harvested during different year and season (Fig. 3).", "However, as mentioned above, here developed method is not applicable to very low contents in saponins.", "Indeed, very low levels in saponin contents in planta could occur in case of (e.g. seasonal changes), as described by Szakiel, Pączkowski, and Henry (2011).", "As reported previously, the gravimetric method could not provide an insight as to the saponin composition of the residue of extraction.", "The newly developed LC/ELSD method answered this issue.", "Thanks to the LC/ELSD saponin profile, it became possible to distinguish saponins originating from different Yucca, or other, taxonomically distant species, and even to check for possible cross-species contaminations.", "We exemplified this by analysing different extracts containing saponins: Y. schidigera juice extracts obtained according to different methods A, B, and C, Y. schidigera dried trunk (identified by TLC), Yucca brevifolia root extract that was contained within a shampoo and a Yucca schidigera (trunk)/Quillaja saponaria (bark) extract blend.", "Following 100% water extraction for 24 h at room temperature of the Y. schidigera dried trunk, the LC/ELSD method showed a typical chromatogram with high concentrations in the most polar steroidal saponins (first two groups Fig. 2A).", "This indicates that the process of aqueous extraction from dry plant material yields in higher amounts of highly polar saponins rather than mechanical pressing of fresh raw material.", "All 5 groups of saponins were detected in all three types of Yucca samples: A, B and C. Indeed, differences in the saponin composition are explained by the distinctions in the extraction or clarification process that may involve hydrolysis, including the use of phosphoric acid to adjust pH with hot Yucca juice concentrate, of the most polar steroidal saponins and the use of differently treated plant material prior to extraction: either fresh or dried trunk.", "Secondly, we showed that spray drying of Yucca juice sample B with inverted sugar had no influence on the saponin repartition (Fig. 3D).", "This is an important finding that guarantees the process and the origin of Yucca even after spray drying and that shows a successful quantification of the ratio of Yucca saponins when blended with saponins originating from another species, including Quillaja.", "Thirdly we analysed the Yucca/Quillaja blend.", "HPLC/ELSD chromatogram (Fig. 4A) clearly shows the presence of Quillaja saponins interfering with the most apolar saponins from Yucca.", "This method clearly can be used for analysing plant adulteration issues of saponins originating from taxonomically close and distant species.", "A range of existing methods, including spectrophotometric, foam height or gravimetric techniques are incapable of addressing thus type of issues.", "Finally, we report the data obtained following the extraction of a shampoo containing Yucca brevifolia root extract.", "This ingredient is used for foaming and as a hair growth promoter.", "The HPLC/ELSD profile showed clearly a different saponin composition and profiles between Y. brevifolia and Y. schidigera (Fig. 4B).", "This method clearly can be used for analysing plant adulteration issues of saponins originating from taxonomically close and distant species.", "A range of existing methods, including spectrophotometric, foam height or gravimetric techniques are incapable of addressing these types of issues.", "Thus, we have developed a new HPLC/ELSD method performed on a reversed-phase C18 column with gradient elution of an aqueous acetonitrile system that rapidly, accurately and reliably quantifies total steroidal saponins in Yucca schidigera extracts.", "Overall, the proposed method exhibited a satisfactory linearity, reliable accuracy (recovery) and adequate reproducibility for quantitative determination of total saponins in Yucca schidigera extracts.", "Compared to the existing techniques, including gravimetric method, time and solvent requirements are respectively divided by 5 and 7 (Table 1) and sample preparation does not need any pre-purification.", "Finally, one of the strengths of the method relates to reliable authentication of species abundant in steroidal saponins that are typically used in commercial extracts.", "Author Mathieu Tenon declares that he has no conflict of interest.", "Author Nicolas Feuillère declares that he has no conflict of interest.", "Author Marc Roller declares that he has no conflict of interest.", "Author Simona Birtić declares that she has no conflict of interest.", "This article does not contain any studies with human participants or animals performed by any of the authors." ]

[ "Yucca steroidal saponins are widely used in food/feed industries.", "HPLC/ELSD is a new technique for measurement of Yucca steroidal saponins.", "Contrary to classical techniques, HPLC/ELSD is rapid, accurate and reliable method.", "HPLC/ELSD is a time- and cost-effective technique suitable for routine analyses.", "HPLC/ELSD can unravel plant adulteration issues." ]

[ "CENG", "CHEM", "ENGI", "ENVI" ]

[ "CO2 reduction", "Ceramic monoliths", "Optical fibres", "Photocatalysis", "Titanium dioxide", "Visible light" ]

[ "1500", "1600", "2209", "2304" ]

[ "The continuous combustion of fossil fuels and depletion of existing resources are intensifying the research and development of alternative future energy options that can directly abate and process ever-increasing carbon dioxide (CO2) emissions.", "Over the past decade, titanium dioxide (TiO2) has been considered to be a promising material for photocatalysis applications due to its chemical stability, nontoxicity and low cost [1,2].", "Currently, the time-consuming and costly process of separating and regenerating powder TiO2 catalyst is a key limitation for slurry reactor designs used in industrial applications.", "Immobilization of TiO2 on fixed substrates eliminates post treatment separation and minimizes light loss caused by scattering and absorption of the resulting solution and reactor [3].", "Selection of appropriate photocatalytic supports that can optimise the interaction between the anchored TiO2 photocatalyst, reactants and light is crucial for the development of efficient fixed bed reactor designs for CO2 photoreduction in both liquid and gas phase applications.", "The use of multichannel monoliths for photocatalysis applications is gaining increased attention over slurry and annular reactors due to its tunable geometry for reactor and catalyst designs and exposed surface area per volume.", "Monolithic substrates usually made of plastic, metal or ceramic materials have uniform parallel channels upon which thick or thin catalyst films can be deposited on the surface or within the substrate.", "The dimensions and amount of channels and wall thickness determine the geometric surface area upon which the catalyst is deposited [4].", "Applications using monolithic structures for photodegradation of organic compounds [5–7] and UV light induced photocatalytic CO2 reduction [8–10] have been reported.", "Monoliths are usually pre-coated with an inorganic oxide prior to coating active catalytic material to ensure good bonding between the monolith and catalyst and prevent catalytic material from being embedded within the macroporous structure of the monolith after calcination.", "Different types of metal oxides and mesoporous materials have been immobilized on TiO2 coated monolithic materials to improve photocatalytic activity and light absorption [8–11].", "For example, Tahir and Amin [12] prepared montmorillonite (MMT) based TiO2 monoliths for CO2 photoreduction with H2O under UV irradiation.", "The addition of MMT into TiO2 matrix was reported to increase surface area with smaller particle size.", "The main drawbacks associated with catalyst immobilized on multichannel monolithic substrates include limited accessibility of the catalytic surface to photons and reactants and reduced active sites of catalysts due to insufficient light penetration [13,14].", "In a model developed by Hossain et al. [15] for influx of UV light within a square channel monolith, half of the incoming light flux was reported to be lost due to light shadowing effect at the entrance of the channel of the monolith wall.", "The UV light flux was also reported to decrease sharply with increasing distance in the monolith channel.", "A strategy for improving light distribution in monolithic structures was originally proposed by Du et al. [6], where non coated side-light emitting fibres were evenly distributed in each TiO2 coated channel to ensure light refracted out of the surface of the fibre could reach the catalyst–reactant interface without attenuation.", "In recent years, studies on CO2 reduction using non coated side-light emitting fibres with geometric notches in the core–cladding system were reported to improve photocatalytic activity [3,8–10].", "Accordingly, the aim of this study is to optimise the visible light photocatalytic activity of TiO2 using metal oxide nanomaterials immobilized onto monolithic structures threaded with side-light emitting fibres for CO2 reduction.", "Transition metal oxides (V-, Cr- and Co) doped TiO2 were chosen for this study due to their ability to enhance visible light absorption and photodegradation of organic compounds [16–19].", "The coated monolithic structures were further characterized in order to evaluate the influence of these metals on physicochemical properties and photocatalytic activity of TiO2.", "SiO2 layer was deposited on cordierite multichannel honeycomb monolith prior to coating with pure TiO2 or TiO2 sol with varying metal concentrations (M–TiO2).", "This was done to ensure catalyst particles were not ingrained in the macroporous walls of the monolith.", "Dip-coating method was applied to immobilize SiO2 sol prepared from a mixture of tetraethyl orthosilicate (Si (OC2H5)4, Acros Organics), ethanol (C2H5OH, Acros Organics) and dilute hydrochloric acid (HCl, Fisher Scientific) in volume ratios of 2, 2 and 0.04, respectively; on the outer and inner walls of the monolith substrate After two repeated coating cycles of 30 min, the coated monoliths were dried and calcined in a furnace at 973 K for 3 h. SiO2-coated monoliths were then subsequently immersed in pure TiO2 or metal based TiO2 sol prepared using n-butanol, acetic acid, titanium (IV) butoxide and fixed amount of chromium (III) nitrate nonahydrate (Cr(NO3)3·9H2O, Acros Organics), vanadium (V) oxide (V2O5, Fisher Scientific) or cobalt (II) acetylacetonate (Co(C5H7O2)2, Acros Organics) as metal precursors.", "The initial loading ratios were chosen arbitrarily and then increasing or decreasing ratios were used based on the photocatalytic activities achieved.", "TiO2/SiO2 or M–TiO2/SiO2 coated monoliths and resulting sols were dried in a furnace (Carbolite, CWF 1100) under airflow at the rate of 3 K/min to 423 K for 3 h and further calcined to burn off organic compounds and complete crystallization at a heating rate of 5 K/min to 773 K for 1 h.", "The resulting nanoparticles were pulverized to obtain metal oxide based TiO2 nanoparticles of varying loading ratios within the range of 0.1–2.0 wt%.", "Side glowing PMMA optical fibres with geometric notches were cut into the desired length of 6 cm and used as light distribution guides within the coated multichannel monolith.", "The crystalline phase and size of the coated monolith samples were examined via powder X-ray diffraction using a Hiltonbrooks X-ray powder diffractometer with Philips PW 1050 goniometer and nickel filtered Cu Kα radiation operating at 20 mA and 40 kV.", "Samples were scanned within the range of 5–65 (2θ) with scan speed of 2° (2θ) per minute and step size of 0.05.", "Surface morphology and quantitative analysis were performed using a Quanta 600 model equipped with energy dispersive X-ray (EDX) system and transmission electron microscopy (TEM) on a JEOL 2100F instrument at an acceleration voltage at 200 kV.", "Pore size distribution of the monoliths were obtained from a mercury (Hg) porosimetry analyser (Micromeritics Autopore IV 9520 V1.05) with Hg pressure in the range of 0.7–275,790 kPa.", "Specific surface area measurements were estimated from N2 adsorption–desorption isotherms at 77 K measured using a ChemBET TPR/TPD analyser connected to a linear mass flow controller/gas blender.", "Chemisorption measurements were conducted with a ChemBET TPR/TPD by injecting pulses of carbon monoxide (CO) gas over the metal coated monoliths to determine metal dispersion.", "The catalysts were degassed in helium (He) gas stream by raising the temperature at the rate of 10 °C/min at 573 K for 30 min, after which they were reduced by flowing gas stream of 5%H2/95%N2 at 673 K for 1 h.", "The samples were purged and cooled to 323 K in He, and pulse titration was performed using CO at that temperature.", "CO uptake was measured during titration, from which the metal dispersion was calculated.", "The elemental ratios and chemical states of the metals contained within the samples were quantified by the Varian Vista MPX ICP-OES (inductively coupled plasma optical emission spectroscopy) system and Kratos AXIS ULTRA with a mono-chromated Al kα X-ray source (1486.6 eV), respectively.", "The high resolution scans were charge corrected to the main C 1s peak of 285 eV and subsequently quantified to determine the amounts of each element present based on the peak areas, using CASAXPS software with Kratos sensitivity factors.", "High resolution and wide survey scans were done on each sample.", "Spectral fitting was performed using CasaXPS software with a line shape based on a Gaussian/Lorentzian mix of 70:30 (GL30).", "Ultraviolet–visible diffuse reflectance spectroscopy was conducted on the monolith samples using Varian Cary 300 spectrophotometer within wavelength range of 200–800 nm.", "The photocatalytic activities of the monolithic structures coated with pure TiO2 or Cr, V or Co–TiO2 were evaluated for CO2 photoreduction under visible light irradiation.", "100 cells per square inch (cpsi) cordierite honeycomb monoliths (Chauger, Taiwan) with dimensions of 40 mm in diameter and 50 mm in length were used in this study.", "As shown in Fig. 1, the coated monoliths were threaded with 177 non-coated side light emitting optical fibres to ensure light distribution within the internal channels of the monolith.", "The reactor was irradiated from the side using a 500 W halogen lamp with wavelength distribution within the range of 400–1100 nm.", "Pressure and temperature were monitored via a pressure gauge and type T thermocouple, respectively.", "The reactor was first purged with helium (He) gas to check for leakage and changed to ultra-pure CO2 (Air Products, 99.9995%) gas saturated with water vapour.", "The flow of CO2 saturated with water vapour was continuous throughout the reaction.", "Gas streams extracted from outlet of the gas-phase photoreactor were analysed using a mass spectrometer (MS, Hiden Analytical) equipped with capillary, quadrupole mass analyser (HAL 201-RC) and Faraday/Secondary electron multiplier (SEM) detectors after 4 h of visible light irradiation.", "Each gas compound was determined by using an assigned unique mass and ionization profile.", "The resulting data were displayed in tabular format as ppm.", "An average of 3 readings was used from 3 experimental runs.", "Blank reactions were performed with and without the light source, He and CO2 in the presence and absence of the catalyst to confirm that reactions were due to CO2 photoreduction.", "The presence of hydrocarbons was not identified in the aforementioned conditions.", "X-ray diffraction patterns of pure TiO2 and V-, Cr-, and Co-based TiO2 monoliths are presented in Fig. 2.", "Diffraction peaks at 2θ = 25.3°, 37.8°, 48.1°, 55.1° and 62.7° are ascribed to the (1 0 1), (0 0 4), (2 0 0), (2 1 1) and (2 0 4) planes of tetragonal anatase TiO2 (JCPDS Card File No. 65-0191), respectively.", "This indicates that anatase was the predominant crystalline phase of the coated monolithic structures after calcination at 500 °C.", "For pure TiO2, diffraction peaks at ca. 27.4° and 36.1° Correspond to (1 1 0) and (1 0 1) plane of tetragonal rutile TiO2 (JCPDS Card File No. 65-0191), respectively.", "This trend is consistent with as-synthesized sol gel samples reported in the literature [20,21].", "Rutile peaks at ca. 27.4° (1 1 0) were further observed in the diffraction patterns of 1.5 wt% V–, 2 wt% V– and 2 wt% Cr–TiO2 monoliths only.", "The introduction of cations with smaller radii and valence lower than 4, e.g. Co2+ or Cr3+, has been reported to accelerate anatase to rutile phase transition due to charge compensation causing the formation of oxygen vacancies that enhance mass transport of atoms in TiO2 anatase structure [22,23].", "Anatase to rutile transformation was promoted at higher metal concentration of Cr and V due to decreased thermal stability of anatase resulting from lattice distortion of anatase by metal incorporation [24,25].", "Increase in V concentration promoted increased crystal growth of rutile from 6.96 nm (1.5 wt% V–TiO2) to 25.12 nm (2 wt% V–TiO2).", "No other diffraction peaks in its metal or oxide phase were detected for Cr or Co–TiO2 monolith samples even at the highest metal concentration of 2 wt%.", "Absence of the crystal phase of Cr and Co could be due to the occurrence of the metals in highly dispersed amorphous phase within TiO2 matrix.", "Absence of the metal or oxide phase in the XRD pattern could be due to their occurrence being in highly dispersed phase within TiO2 matrix or particle size being too small to be measured within the detectable limit of the diffractometer.", "Characteristic peaks at 20.3° (0 0 1) and 31.0° (4 0 0) for orthorhombic vanadium (V) oxide (V2O5) (JCPDS Card File No. 42-0876) and 14.9° (1 1 0) and 29.9° (2 2 0) for tetragonal vanadium (IV) oxide (VO2) (JCPDS Card File No. 42-0876) were observed in the diffraction pattern of the 2 wt% V–TiO2 monolith only.", "This indicates that vanadium species homogeneously nucleate as a separate nano oxide structure with higher metal concentration of 2 wt%.", "V4+ ions can also be easily incorporated into the crystal lattice of TiO2 substitutionally due to the similarity of its tetragonal crystal structure and ionic radius of 0.63 Å with Ti4+ (0.68 Å) [26] when compared to V5+ with an ionic radius of 0.59 Å [27] and an orthorhombic or tetragonal pyramidal crystal structure [28].", "Furthermore, Gu et al. [26] explained that at lower V doping ratios, the majority of the V4+ ions remain in the Ti4+ lattice via substitution due to the thermal stability of Ti–O–V linkages.", "Pure VO2 phase was also reported to be formed from the excess V4+ ions generated from increased metal concentration, while the V2O5 phase was formed from the oxidation of VO2 clusters during calcination [26].", "Vanadium in its metal or oxide phase were not observed here in samples with doping ratios <2 wt% either due to the high dispersion of vanadium species in TiO2 structure or their small particle size [25,29].", "The crystalline size of pure and coated TiO2 monolith samples estimated using the Scherer equation are listed in Table 1.", "The relative intensities and crystalline size of the metal coated monolithic structures decrease with increased metal concentration when compared to pure TiO2 monolith.", "The morphology and elemental composition of TiO2 coating analysed by SEM–EDS is presented in Fig. 3A and B. Agglomerates of different particle sizes were observed from SEM micrograph using magnification of 6000×.", "SEM–EDS micrographs confirmed the presence of TiO2 on the SiO2 layer.", "Al and Mg elements are from the monolith which is made of cordierite (2MgO·2Al2O3·5SiO2).", "Spherical nano-sized particles of varying sizes from 6 to 31 nm were observed from the transmission electron microscopy (TEM) micrographs of pure TiO2, 2 wt% Cr–TiO2 (D), 2 wt% V–TiO2 (E) and 2 wt% Co–TiO2 monoliths (Fig. 3C–F).", "The BET specific surface area of the monoliths were within the range of 62.2–120.2 m2/g (Table 1).", "Table 1 show that a slight increase in specific surface area of the monoliths coated with Cr, Co and V–TiO2 occurs when compared to pure TiO2 monolith (40.7 m2/g).", "This may possibly be linked to crystal size which decreases with higher metal concentration and structural changes resulting from the addition of metals.", "Metal dispersion on the TiO2 based monoliths was determined by CO chemisorption.", "At metal concentrations below 2%, metal oxides of Cr, V and Co were found to be present in a dispersed state (Table 1).", "Dispersion approached almost 90% for Co and V–TiO2 monoliths at low metal loadings of 0.1 wt%.", "The dispersion of metal oxide of Cr was 64.22% at 0.1 wt%.", "Decrease in metal dispersion with higher metal concentration could be due to increasing concentration of metal atoms within TiO2 framework.", "The total elemental composition of metal TiO2 coated monoliths were determined by ICP-OES, while the chemical state and surface elemental composition were evaluated by XPS.", "Table 1 lists the metallic content of the coated monoliths quantified by ICP-OES analysis.", "As shown in Table 1, the surface metal contents for the V and Co–TiO2 monoliths determined by XPS are higher than their total metal contents while the surface metal content of Cr–TiO2 monoliths is lower than the total Cr content derived from ICP-OES.", "This implies that a high concentration of metal ions are primarily located on the surface of V and Co–TiO2 monoliths while a high concentration of Cr dopant probably migrated to the crystal lattice of TiO2 during calcination.", "Table 1 lists the surface elemental composition of the coated monoliths quantified by XPS analysis.", "The high resolution XPS spectra of the monoliths coated with pure TiO2 and 2 wt% of Cr, V and Co–TiO2 are presented in Fig. 4.", "The binding energies of Ti 2p of pure TiO2 are at 458.7 eV and 464.5 eV [30,31].", "Doublet separation between 2p3/2 and 2p1/2 core levels is 5.7 eV.", "These values are consistent with the values reported in the literature for anatase TiO2 [28,30].", "Ti 2p doublets of 2 wt% Co, V and Cr appear broader and are shifted to higher binding energies compared to the reference position of pure TiO2.", "This indicates that chemical state of Ti is influenced by the introduction of metal ions in the TiO2 crystal lattice [25,32].", "Transformation of some Ti ions to higher valence due to the release of electrons in attempt to maintain overall charge might also contribute to shift and broadening of XPS peaks of the metal coated monoliths [33].", "The XPS spectra of the O 1s region shows that oxygen exists in two forms on the sample surface with binding energies of 530 eV and 531.9 eV.", "The main peak appears at 530 eV and can be assigned to bulk oxygen bound onto TiO2.", "The other peak at 531.9 eV can be attributed to surface oxygen from hydroxyl species on the sample surface [34].", "The same phenomenon of peak shift of O 1s to higher binding energies was observed for the metal coated monoliths.", "Fig. 5 shows that the binding energies of the Cr 2p3/2 and Cr 2p1/2 core levels at 576.5 eV and 586.3 eV are characteristic of chromium (III) oxide [30].", "For the 2 wt% V–TiO2 coated monolith, two V 2p1/2 peaks observed at 524.4 eV and 525.1 eV are characteristic of vanadium (IV) oxide and vanadium (V) oxide, respectively (Fig. 4) [35].", "The binding energies of V 2p3/2 at 516.3 eV and 517.4 eV are also characteristic of vanadium (IV) oxide and vanadium (V) oxide, respectively [28,30].", "These values are consistent with the binding energies of V 2p3/2 measured for V doped TiO2 [16].", "The presence of V4+ species in the sol gel derived vanadium doped TiO2 samples were probably due to the reduction of V5+ by the decomposition of organics from starting materials [36,37].", "This is supported by the appearance of both V4+ and V5+ in the XRD pattern of the 2 wt% V–TiO2 monolith.", "Detailed analysis of the relative concentrations of V calculated based on the summation of the corresponding peak areas gave a 2p3/2 and 2p1/2 abundance equal to 56.9%/12.3% for V5+ and 16.3%/14.5% for V4+.", "This indicates that V5+ is the predominate oxidation state with ions mainly present at surface sites at higher concentration compared to V4+ which can preferentially occupy TiO2 lattice sites due to its matching radii.", "Peaks of Co 2p1/2 at 796.9 eV and Co 2p3/2 at 781.2 eV are characteristic of cobalt (II) oxide [30].", "The shake-up satellite peaks of Co 2p3/2 and Co 2p1/2 which are characteristic of the high spin Co2+ are also present at binding energies of 786.1 eV and 802.5 eV, respectively.", "This indicates that Co species exist in the TiO2 lattice sites in the form of Co2+ [38,39].", "UV–Vis absorbance and diffuse reflectance spectra of monoliths coated with pure TiO2 and 2 wt% of V, Cr and Co–TiO2 are presented in Figs. S1 and 5, respectively.", "The absorption edge of pure TiO2 monolith is present at 418 nm and corresponds to the band gap energy of 3.0 eV.", "The band gap energy of the sol gel derived TiO2 differs from the value of anatase TiO2 reported in the literature [1].", "This difference could be influenced by the method of preparation and presence of rutile confirmed from XRD.", "Band gap has been reported to be influenced by defect formation i.e. oxygen vacancies during the dehydration of the sol which results in stoichiometry deficiency of Ti and O during the sol gel process [40].", "Compared to pure TiO2 monolith, all monoliths coated with V, Cr and Co–TiO2 have lower band gaps and exhibit increased light absorption in the visible light region.", "Band gap energy decreased with increasing metal concentration and the highest red shift was observed for the 2 wt% V–TiO2 monolith (1.9 eV).", "Visible light absorption has been reported to be due to charge transfer between TiO2 conduction band and d electrons of the metal dopants [25].", "Since the absorption band of V4+ and V5+ has been reported to be 770 nm and <570 nm [28,37], the formation of an absorption band between 400 and 550 nm for V–TiO2 monoliths in this present study suggests the presence of both V4+ and V5+ which is also due to charge transfer transition from O 2p to V 3d orbital at TiO2 conduction band.", "The incorporation of Cr3+ ions in the TiO2 matrix is responsible for the shift towards the visible light region with band gap energies of 2.2 eV for 2 wt% Cr–TiO2 monolith.", "The tailings observed in the absorption band of Cr–TiO2 samples have been reported to be assigned to Cr loading creating additional energy levels (Cr 2p level) and oxygen vacancies within the band gap of TiO2 [31].", "Broad absorption band from 540 to 800 nm can be attributed to 4A2g → 4T2g d–d transition band of Cr3+ [41].", "Band gap energy of the 2 wt% Co–TiO2 monolith is 2.3 eV.", "Broad absorption shoulders formed at 600–800 nm for the 2 wt% Co–TiO2 monolith is due to the presence of the charge transfer transition between the d orbital of Co 2p to the conduction band of Co2+ and this absorption shoulder also increased with higher Co concentration [42].", "The photocatalytic activities of the monoliths threaded with optical fibres coated with Cr, V and Co–TiO2 were evaluated for CO2 photoreduction after 4 h of visible light irradiation (Fig. 6).", "Trace product formation was observed over pure TiO2 monolith.", "Addition of the metal ions improves the photocatalytic activity under visible light irradiation and the metal based TiO2 monoliths show improved product rates when compared to bare TiO2 monolith.", "Visible light photocatalytic activity of the TiO2 monoliths was influenced by the addition of Cr, V and Co, which is consistent with UV–Vis spectra.", "As shown in Fig. 6, methane and acetaldehyde formation product rates steadily increase with higher metal concentration to give an optimal ratio of 1 wt% Cr–TiO2, 0.5 wt% V–TiO2 and 1.5 wt% Co–TiO2 within the series tested.", "The effect of irradiation time on the product formation of 1 wt% Cr–TiO2, 0.5 wt% V–TiO2 and 1.5 wt% Co–TiO2 coated monoliths is also presented in Fig. 7.", "The product rates of the monoliths coated with 0.1 wt% Cr, V and Co–TiO2 was higher when the monolith threaded with optical fibres was used as a sole support compared to monolithic support without optical fibres.", "Not all immobilised photocatalyst may be activated when the monolith is used as a lone support due to limited light distribution arising from the catalyst coated on the outer surface absorbing most of the light and light intensity decaying rapidly along the opaque channels of the monolith.", "The V–TiO2 monoliths exhibit higher photocatalytic activity for CO2 reduction with methane and acetaldehyde product rate of 4.87 and 11.13 μmol/gcat h in comparison with other metal coated monoliths; while the lowest activity was observed over Cr–TiO2 monoliths.", "The lower product rates can be linked to the dispersion of chromium determined by CO chemisorption.", "The lowest percentage of exposed metal atoms available for reaction was observed by Cr–TiO2 monoliths while the highest dispersion rates were observed over the V–TiO2 monoliths.", "Thus, the enhanced visible light induced activity of the V–TiO2 monoliths can be attributed to increased visible light absorption, surface area and accessible active metal sites arising from the appropriate metal dispersion and loading amount.", "However, increased metal concentration resulted in reduced product rates over subsequent higher loading ratios.", "Rutile formation in 1.5 wt% V–, 2 wt% V– and 2 wt% Cr–TiO2 monoliths can also be a contributing factor in the decline of product formation as the photocatalytic activity of rutile has been reported to be lower than anatase [43].", "It can be seen from Fig. 6 that an optimal loading concentration exists for all the metal based monoliths, after which reduced photocatalytic activities were observed.", "Reduced product rates could be due to the increased level of charge carriers created from the high metal concentration in TiO2 which limits photocatalytic activity and invariably leads to electron/hole recombination.", "Decreased metal dispersion observed in Table 1 with increased metal concentration implies that a large percentage of the metal ions are isolated from the surface such that the probability of the trapped charged carriers being transferred to the surface before recombination is low.", "These results are consistent with the study by Tian et al. [25], where vanadium ions above the optimal loading ratio of 1 wt% resulted in reduced degradation rates of 2, 4-dichlorophenol (2, 4-DCP).", "The decreased degradation rates were attributed to excess V species reducing surface illumination of catalysts and reducing the contact area between TiO2 and the organic pollutant.", "When the loading content of V exceeded 3 wt%, Wang et al. [44] recorded a decrease in photocatalytic oxidation of 1-phenlethnol due to electron hole recombination.", "Visible light induced photocatalysis for the metal based monoliths can also be explained via the metal ions influencing electron hole separation when their energy levels satisfy the necessary requirement for CO2 reduction.", "For V–TiO2 monoliths, increased metal concentration predominantly resulted in the formation of V5+ ions at the surface layer of TiO2.", "An internal electric field was created at the interface of V2O5 and TiO2 during equilibrium due to their p and n-type nature.", "Diffusion of photogenerated charge carries (electrons and holes) in TiO2 and V2O5 will be induced by this internal electric field such that negative acceptor ions and positive donor ions are left on the p and n side, respectively [45].", "Reduced V4+ ions can also function as electron and hole traps and participate in reduction reactions with the surface adsorbed species, and thus, preventing electron–hole recombination.", "Electrons will be transferred from the conduction band of anatase (−0.55 V) to the conduction band of rutile due to the conduction band edge of rutile (−0.35 V) being slightly lower than that of anatase.", "These photogenerated electrons and holes generated from the interaction of metal species also diffuse to the surface where they accumulate for interfacial charge transfer to adsorbed species.", "Adsorbed hydrogen and OH radicals are generated from the reaction of H+ and OH− ions.", "Product formation of methane and acetaldehyde then occurs after the reaction of CO2− radicals and H with holes.", "The conduction band edge for V–TiO2 coated monoliths has been reported to be more negative than the reduction potential of CO2/CH4 (−0.24 V) and CO2/CH4 (−0.33 V) [46], which indicates that product formation can occur over these monoliths.", "Valence band edge of Co2+ nanocrystals has been reported to lie below the oxygen-evolution potential while the conduction-band edge is above the hydrogen evolution potential of −0.41 V [42].", "Band alignment of Co–TiO2 coated monoliths with redox potentials for methane and acetaldehyde evolution explains product formation reported in Fig. 6.", "Results from this study are compared to the study of Liou et al. [10] and Tahir and Amin [11] where monolithic structures were employed as catalyst carriers for CO2 photoreduction.", "Vapour phase CO2 with H2O was reduced to hydrocarbons by NiO/InTaO4 coated monoliths threaded with optical under visible light irradiation.", "Maximum acetaldehyde conversion rate of 0.3 μmol g−1 h−1 was achieved with the 2.6% NiO/InTaO4 by simulated sunlight AM1.5G at 70 °C.", "Recently, In/TiO2 coated monoliths with two different cell densities and channel lengths were tested for photocatalytic reduction of CO2 under UV irradiation.", "The product rates was reported to be influenced by the geometry of the monolith since higher CO and CH4 production was observed over the monoliths with lower cell density of 100 cpsi and channel length of 2 cm.", "Maximum product rates were reported to be primarily influenced by higher illuminated surface area to volume ratio in these monoliths as opposed to structures with higher cell densities and lengths.", "CH4 production rate of 55.4 μmol g−1 h−1 was observed over 10 wt% In/TiO2 monolith with 100cpsi after 10 h of UV irradiation.", "A comparison between previous results and this present study establishes the effect of catalyst carriers in facilitating improved product formation and light distribution.", "Results presented here shows higher acetaldehyde product rate when indirectly compared to the experimental results of Liou et al. [10] derived under visible light irradiation.", "Finally, when comparing this work with the results of Tahir and Amin [11], it should be noted that although both studies chose TiO2 as semiconductor, different metal dopants, light spectrum, wavelength and intensity were used.", "Metal based TiO2 films with varying concentrations of Cr, V and Co were coated on monolithic structures to enhance the photocatalytic reduction of CO2 using H2O under visible light irradiation.", "Compared to pure TiO2 monolith, all monoliths coated with V, Cr and Co–TiO2 exhibit lower band gaps and increased light absorption in the visible light region.", "The V–TiO2 monoliths exhibit maximum photocatalytic activity for CO2 reduction while the lowest activity was observed over Cr–TiO2 monoliths.", "The lower product rates can be linked to the dispersion of chromium determined by CO chemisorption.", "Thus, the enhanced visible light induced activity of the metal coated monoliths can be attributed to increased visible light absorption and accessible active metal sites arising from the appropriate metal dispersion and loading amount." ]

[ "Coated monolithic structures were tested for visible-light-induced CO2 reduction.", "Red shift in absorption edge was observed with increasing metal concentration.", "Fuel production rate is enhanced upon chromium, vanadium or cobalt deposition." ]

[ "ENVI" ]

[ "Fisheries", "Lotka-Volterra", "Overfishing", "System dynamic" ]

[ "2302" ]

[ "Many of the world’s fisheries are showing a decline in the fishing yield, a phenomenon that’s clearly important for the global economy and which is often interpreted in terms of the overexploitation of the fish stock (Pauly, 2009): the fishing industry is consistently depleting the fish stock at a rate higher than the capability of the system to replenish it, as a result of biological reproduction (Lotze and Worm, 2009), (Bailey, 2016).", "This subject is complex and it was explored first in some early studies by Scott Gordon (Gordon, 1954) and Milner Schaefer (Schaefer, 1957).", "In the general field of resource overexploitation, an important influence was the work by Garrett Hardin (Hardin, 1968), known under the name of “The Tragedy of the Commons.”", "Hardin's model was only qualitative, but it established the patterns of overexploitation of any resource that’s exploited at a rate faster than it can reform (Roopnarine, 2013).", "In more recent times, fisheries have been extensively modeled, normally with considerably complex models (Schoener, 1976) and as reviewed, for instance, by Worm et al. (Worm et al., 2009).", "Of course, in modeling fisheries, several factors must be taken into consideration in addition to overfishing including, e.g. the climate change (Perry et al., 2005) and that the models based on trophic chains may be over simplified (Polis and Strong, 1996).", "The problem, here, is to establish exactly which factors are the most important ones in generating the decline of fisheries.", "This is a crucial issue in the management of fisheries; overexploitation can be fought by establishing fishing quotas but we need to quantify whether there are other factors affecting yield declines.", "Although there appears to be a general agreement that overexploitation is an important cause of the decline of many fisheries, its extent is sometimes debated.", "In the present paper, we aim at providing further evidence that overexploitation plays a central role in the collapse of at least some fisheries.", "We demonstrate this point by using a simple system dynamics model that takes into account the coupling of the fish stock and the fishing effort of the industry.", "The fact that the model can provide a good fitting with the historical data is a clear indication that the two factors influence each other in a classic feedback relationship that sees the depletion of the stock being enhanced by the effort of the industry to maintain its production rate.", "The model utilized here is based on the well-known Lotka-Volterra or “prey-predator” model.", "Alfred Lotka (Lotka, 1925) and, independently, Vito Volterra (Volterra, 1928), (Volterra, 1931) were the first to use differential equations in order to describe the dynamics of the predator/prey interaction in biological systems and the structure of the model can be seen as an ancestor of modern “system dynamics” (Forrester, 1989).", "George Gause (Gause, 1932) was probably the first to seek for experimental validation of the Lotka-Volterra (LV) model and he found it, but only for very simple biological systems in the form of two species of yeasts in laboratory conditions.", "In general, the behavior of real biological systems turned out being too complex to be captured by the simple LV model (Hall, 1988).", "However, the model had been originally proposed by Vito Volterra as describing the behavior of human fisheries rather than biological systems, even though, at that time, suitable mathematical tools to fit experimental data were not available (D’Ancona, 1942).", "On the basis of this early idea by Volterra, we developed a simplified version of the model assuming that the fish stock behaves as a non-renewable resource when it is exploited so fast that the reproduction rate becomes a negligible parameter in the system.", "Some data on this approach had been reported in an earlier paper (Bardi et al., 2011) in regard to the mining industry.", "Even though the model we developed does not claim to be able to describe all the complex ecosystem and economics interactions that occur in a fishery, we can report several cases in which it is possible to use the model to describe the historical production patterns of fisheries.", "We believe that this approach can play an important role in helping people to understand the basic mechanisms of fishery management, and in particular of overexploitation.", "Further studies might lead to the model being usable in order to determine the carrying capacity of the system and help in the sustainable management of fisheries.", "The model utilized here is based on the following couple of differential equationsR’ = −k1CRC’ = k2CR − k3Cwhere “R” stands for the resource stock while “C” stands for the capital stock.", "The three constants of the model describe how efficiently fish is caught (k1), how efficiently the fish stock is transformed into capital (k2) and how rapidly capital is dissipated (k3).", "The dimensions of the constants depend on the units used for the capital and resource stocks.", "This model is the same as the well-known Lotka-Volterra, predator-prey model, except in the fact that it lacks the term for the reproduction of the prey (named the resource) in the first equation.", "In the original Lotka Volterra model, the prey is assumed to have an unlimited food supply and to reproduce exponentially unless subject to predation; this exponential growth is represented by a term k0R in the first equation.", "We will show later this term can be neglected in the study of the historical cases reported here, but see also the ‘fleet dynamic model’ reported in the work by Hilborn and Walters (Hilborn and Walters, 1992).", "In the present study, the “resource stock” (or the “prey”) is the fish stock while the “capital stock” (or the “predator”) is a parameter proportional to the capital of the fishing industry in terms of vessels and other resources (including human resources).", "The rate of predation, R’, is assumed to be proportional to the abundance of both the stocks; this is represented above by −k1CR, where the efficiency of the fighting process is described by the k1 coefficient.", "In the second equation, C’ indicates the variation of the capital stock as a function of time or the ‘capital flow’.", "k2 is a constant that describes the rate of growth of the Capital stock, also proportional to the abundance of both the fish and the capital stocks; k3 is a third constant that describes the decline of the capital stock due to asset depreciation.", "The model was implemented using MATLAB® computing language and the associated Simulink toolbox.", "Typical results are reported in Fig. 1.", "In comparing the results of the model to the historical data, the three constants (k1, k2 and k3) were allowed to vary until the best fitting was obtained.", "All the fittings reported here were generated using the unconstrained nonlinear optimization method based on the Nelder-Mead algorithm (termed “fminsearch” in Matlab).", "The objective is to minimize the sum of the square of the residuals (SSE, the sum of squared errors of prediction) generated by the deviations of the LV predicted data from actual empirical values of data.", "The fitting procedure was found to be very sensitive to the initial values of the k1, k2 and k3 parameters, as well as to the initial values of the stocks (Ro and Co).", "The initial guesses for these parameters were provided by the Parameter Estimation tool available in the Simulink® Design Optimization™ toolbox (Fig. 2).", "The Goodness of fit (GOF) was calculated by as the Normalized Mean Square Errors (NMSE) function which measures the discrepancy between the real values and the estimated ones and it was calculated by means of the Matlab Curve Fitting Toolbox.", "An NMSE equal to 1 represents the perfect fit, NMSE equal to zero means that the model is no better than a straight line at fitting the real data.", "For the present study, we collected historical data for several “non-managed” fisheries, that is for fisheries in which neither the catch nor the fishing effort was regulated in terms of quotas or other parameters.", "In other words, these fisheries were managed by operators with the only aim of maximizing profits.", "Data about the actual fish stock during the exploitation cycle are not normally available, but the historical catch data (or “landings”) – that is the flow R’ in the model – are commonly reported for many fisheries.", "A qualitative examination of these data shows that the total fishery production often shows a “bell shaped” trajectory, with production increasing until reaching a maximum and then declines.", "Using the model to fit a single set of data, the landings, is possible but it does not really prove that the system is dominated by a “predator-prey” interaction.", "We need to show that the two entities involved in the cycle, the predator and the prey, are linked to each other in the way described by the simplified LV model.", "Therefore, we need to identify also a ‘predator stock’ or, at least, some parameters that can be assumed to be proportional to the “capital stock” engaged in the fishing activity.", "Data on the capital investments of the fishing industry engaged in a specific fishery are not commonly available.", "However, “proxy” data can be used, such as, for instance, the labor force employed in the fishery industry in terms of the number of persons or in terms of their total salary.", "Another parameter examined was the number of fishing vessels or the tonnage of the fleet.", "In recent statistical surveys, also the data on the investment in currency or as measured by economic indexes of the fishing sector are available.", "All these parameters may be assumed to approximate the “fishing effort” represented by the “C” parameter in the model.", "In the end, the fitting procedure was developed for the two parameters that turned out to be available.", "In all cases, the historical data were normalized before fitting and we found several cases where the", "real world stocks, fish and capital invested in fishing, interact each other in the way described by the model.", "In the following, we show results describing cases of fisheries overexploitation from the 19th Century to recent years.", "This set of data cannot be considered as a complete survey of the historical world’s fisheries, but we believe that the result that we obtained are sufficiently robust to show that overexploitation is an important component of several cases of fishery decline.", "More cases can probably be analyzed in this way if it will be possible to find suitable data for the capital variable.", "The first documented evidence of the overexploitation of a marine resource goes back approximately two centuries ago when the American whaling industry went through an intensive development phase due to the growing demand of whale-derived products as the whale oil, used as fuel for lamps.", "Whales also produced “whale bone,” a ‘stiffener for ladies’ clothing that was described as ‘the plastics of the 19th Century’(Bardi, 2007).", "Here, we examined the data reported in Alexander Starbuck's book about the history of the American whale fisheries from 1807 to 1876 (Starbuck, 1989).", "The sum of the production of oil from ‘right’ and ‘sperm’ whales, the two-main species hunted, is assumed to be proportional to the predation activity.", "The proxy for the predator stock is assumed to be the total tonnage of the whaling fleet.", "These data could be well fitted by the simplified LV model (Fig. 3), as reported in an earlier study (Bardi and Lavacchi, 2009).", "The definitive collapse of the American whaling industry occurred at the beginning of 20th Century, as reported by Walter Tower (Tower, 1907) and more recently by Granville Mawer (Mawer, 2000).", "The Pacific sardine fishery began to operate in central California in the late 1800s and its production grew in response to the increasing demand for food during World War I (Schaefer et al., 1951).", "From the mid-1930s to the mid-1940s this fishery was the largest in the Northern and Southern West Pacific coasts with peak landings of over 790000 tons per year in 1936–1937 and average landings over 600000 tons per year.", "The fishery began to collapse a few years later and catches declined over the following decades to less than 100 tons per year in the 1970s (Radovich, 1982).", "A moratorium was applied to the sardine fishing from 1974 to 1986 (Wolf, 1992), resulting in a new fishery cycle that will not be examined here as we can’t define it as a “non-managed” fishery anymore.", "In Fig. 4, we report the Californian sardine catch as production in comparison to the fishing fleet number of boats as capital, from 1933 up to 1957 and the related fitting obtained by LV modeling.", "The model shows how fishing “capital” in terms of the number of fishing boats reached the peak ten years later than production peak, leading to the fishery collapse.", "Again, we see that the Lotka-Volterra model provides a reasonably good fitting of the experimental data, indicating that the sardine stock was overexploited by the fishing industry.", "The prey-predator dynamic can describe not only the exploitation of a single species; it is also able to illustrate the exploitation tendency of a whole fishing sector of a country.", "Here we report the evolution of the Japanese total fish catch and the National Disbursement for the fishing industry from 1962 to 2002.", "The catch data are expressed as the total landings by weight.", "The disbursement is expressed in currency and it includes expenses for the fisheries’ in terms of wages, fuel, fishing boats capital and replacement and equipment.", "This is a direct measurement of the capital investment in the sector.", "As shown in Fig. 5, the historical data show a decline in the fishery yield starting with approximately 1980.", "The fitted peak of the capital expenditure occurs later, although the oscillations of the data indicate an earlier peak.", "Recent data from the Statistical Handbook of Japan 2015 (OECD, 2015) show that, since 2000, the Japanese fish production trend is still declining.", "The value of the catch is decreasing with a rate of 25% from 2000 to 2014.", "For the same period, the Statistical Handbook of Japan 2015 also reports the number of Enterprises and the number of workers engaged in the Fishery sector.", "The values of such entities, even though they are not expressed in currency, can be reasonable assumed proportional to the capital effort invested in the sector.", "The data show that the trend, for both, is declining: in particular, from 2000 to 2014, the number of enterprises is reduced by 39%, while the number of workers is reduced by 33%.", "Iceland is another region whose economy was historically based on fishing, in particular cod and herrings.", "In the 1970s, the Icelandic fishing zone has been the theater of the so-called ‘cod wars’ which saw the Icelandic fleet engaged in competition against foreign fleets.", "By the early 1970s, the most valuable cod fish stocks had declined to very low levels of catch as a result of overfishing.", "In the same period, the North Atlantic Icelandic spring and summer spawning herring stock collapsed.", "The spring stock has not recovered yet, although the summer stock did (Jakobsson, 1980), (Jakobsson and Stefansson, 1999).", "The herring collapse provoked the drastic fall in the number of Herring Salting industries a few years later (Fig. 6).", "As a response to the depletion of such important stocks, in 1984 Iceland established an individual quota system (ITQs) for fishing vessels that allocates maximum catch on the base of past years’ vessel’s catch performance.", "In recent years the country has adopted a “total allowable catch” (TAC) regulation and other management measures such as area restrictions and fishing gear restrictions.", "Despite these measures, the curve of the total catch of the Iceland from 1905 up to 2014, shows a peak in the late 80 s (Fig. 7) followed by the capital peak, approximately ten years later, this time represented in form of economic index, the volume index at a constant price.", "The scientific literature reports several other documents on historic fisheries collapses, such as the famous case of North Atlantic cod collapse in Newfoundland (Hutchings and Myers, 1994), although it is not always possible to detect a smooth bell-shaped curve.", "Nevertheless, the evidence of overexploitation is still detectable in the fact that the “capital” parameter keeps growing for a period, while the production of the fishery declines.", "A good example is that of the UK trawling fishery that saw a rapid decline in an intensive use of trawlers over 100 years (Thurstan et al., 2010).", "The decline occurred despite the considerable increase of the “fishing power,” something that can be likened to the parameter called here fishing capital (Fig. 8).", "Then, the same behavior can be seen at the planetary level where the LV model appears to be able to qualitatively describe the overexploitation of marine fisheries.", "Statistics from FAO (FishStat) show that the world landings reached a peak in the middle of the 1990s, then started declining; a still ongoing phenomenon.", "Such a behavior can be correlated with the engine power of the global fishing fleet (Fig. 9) (Watson et al., 2013), that is another “proxy” variable to measure the capital invested in fishing at World level.", "The work of Pauly and Zeller, (2016) also shows a similar behavior.", "This set of data cannot be yet fitted with the LV model developed here, but it shows a behavior compatible with the model’s results.", "The problem of overfishing is much debated in the scientific community but it is often difficult to separate the effect of overexploitation from other factors involved in fishing.", "Sometimes, indeed, overexploitation is underestimated or even ignored in the analysis, a tradition that may have started with the early study by Starbuck (Starbuck, 1989) of the American whale fishery, where the decline of the catch was explained by such consideration as “the whales have become shy.”", "However, from the analysis performed here, it appears that overexploitation is, indeed, a major element in determining the behavior of fisheries.", "The dynamical model we used shows that, in many cases, the fishery yield is directly linked to the magnitude of the fish stock and that fishing continues all the way to bringing the yield to nearly zero by increasing the fishing effort despite fish depletion and consistently increased the fishing effort disregarding the risk of the collapse of the fish stock.", "These results evidence that today, despite the necessary efforts already put in the stock preservation, as fishing policy and regulations adopted by the majority of the countries, the fish demand on the market still drives the fishing pressure where fishing remains intensively practiced worldwide, with an increasing fishing power in terms of more sophisticated and expensive fishing techniques or equipment.", "In the history of fisheries, the most common measure adopted to avoid the collapse of the fish stock has been the total moratorium on fishing.", "In some cases, this approach has led to the stock ‘salvation’ as in the case of the Californian sardine (end of 1950), whaling (1985), the Canadian North Atlantic cod (1992), but it was a remedy, not really a solution able to prevent the related fishery industry collapse.", "Moreover, the time lapse necessary for the recovery of stocks has often been quite long in comparison the time-scale of the development of the modern fishing economy, that requires meeting the world demand.", "On this point, it is sufficient to remember that the “right whale” fishery has not yet recovered from the intensive overexploitation it suffered in the 19th Century (Baker and Clapham, 2004).", "Today, in order to prevent the collapse of fisheries, many international, national, and governmental organizations establish quotas for commercial stocks, expressed in terms of “total allowable catch” (TAC).", "This limit is deliberated on the basis of advice from scientific advisory bodies.", "Despite quotas, however, there exist several historical and recent cases where stock collapses still occurred, as shown by the case of the closure of the sardine fishing season off the length of the U.S. West Coast, recommended by the Pacific Fishery Management Council (Decision Summary Document Pacific Fishery Management Council, 2016) in April 2016.", "It was closed mid-season in 2015 due to low stocks, but it has since fallen further.", "Federal rules mandate that the harvest must be closed if adult stocks fall below 150,000 tons, and the government estimates that there are now less than 65,000 tons.", "The innovative approach of the present study is that it describes the quantitative correspondence between a biological resource and a capital by which the dynamic of overfishing can simply be represented.", "We are not just fitting data, but interpreting them with a model that specifies the dynamic interactions within its internal elements, so that obtaining a good fit indicates that the corresponding elements in the real world do behave according to the assumptions made in the model.", "We believe that our results are important in highlighting the fundamental role that overexploitation is playing in the cycle of modern fisheries, confirming the interpretation, for instance, by Daniel Pauly (Pauly, 2009) of the global fishing trends.", "Overexploitation needs to be understood if it is to be avoided by means of appropriate measures such as quotas, restrictions, or sustaining fisheries toward the transition to a circular economy.", "Our model could also be used to provide elements useful to establish fishing quotas, but this will be the objective of further studies.", "The scope of the present study was to evidence how the abstract concept of “overexploitation” can be detected and analyzed by a dynamic model derived from the well-known Lotka-Volterra, “prey/predator” model.", "The innovative approach of this model is that it is a quantitative application of system dynamics that emphasizes the role of depletion and the feedback relationships between the various parameters of the models.", "The history of fisheries provides us with several cases where the behavior of the fishery can be described by a model that assumes that the economic yield of the fishery is directly proportional to the investments in the field.", "With the reduction of the yield generated by overexploitation, the result is a general decline in the landings, followed by a decline of the effort of the fishing industry that comes after a certain time lapse.", "The resulting curves show a typical “bell-shaped” behavior that can be fitted with the model described here that appears to be able to can capture the behavior of a number of cases where the fishing industry operates in an open market framework.", "Of course, from the number of cases examined, we cannot claim that the model describes all the sectors of the fishing industry, nor that overexploitation is the only forcing that affects the system.", "Nevertheless, we believe that it catches some important factors that drive the industry to overexploit the fish stocks.", "This result shows the importance of the problem of overexploitation in the general issue of the management of the “global commons” that include much more stocks of resources other than fisheries." ]

[ "For the first time, the “predator-prey” model is used for the quantitative description of an economic predatory dynamic in real-world fisheries.", "A simple “mind-sized” model highlights the driving forces that lead to the resources’ overexploitation and the collapse of a production system.", "A renewable fish stock can behave as a non-renewable resource when exploited at a speed much higher than its carrying capacity.", "Coupled feedback effects are the origin of overexploitation and collapse in real-world fisheries." ]

[ "ARTS", "SOCI" ]

[ "Globalese", "Globalisation", "Indexicality", "Linguistic landscape", "Writing system mimicry" ]

[ "1203", "3310" ]

[ "The term linguistic landscape, which refers to the visibility of languages in public spaces, was first introduced to linguistics by Landry and Bourhis in 1997.", "In their study, the linguistic landscape of an area is defined by the languages on public road signs, advertisements, and commercial shop signs (Landry and Bourhis, 1997: 25).", "In practice all neighbourhoods, cities, and even entire countries have their own linguistic landscape, because all public entities include some form of written language.", "According to Landry and Bourhis, the linguistic landscape of a territory has two principal functions: an informational function and a symbolic function.", "The signs in a linguistic landscape can serve as a territorial marker for the area of a linguistic community, or may display power dynamics between two languages or dialects used in the area, thus providing an informational function (Landry and Bourhis, 1997: 25–26).", "Meanwhile, both signs including and signs excluding local in-group or minority languages may have a symbolic function, demonstrating public or private attitudes towards certain ethnolinguistic groups (Landry and Bourhis, 1997: 25–26).", "As such, linguistic landscape research is highly relevant for studies in both language policy and ethnolinguistic vitality (Moriarty, 2014: 458).", "The concept of linguistic landscapes has expanded sociolinguistic research from the usual focus on groups of speakers to a focus on physical spaces, and has thus been argued to have immense interdisciplinary value, overlapping research in disciplines such as urban studies, social geography, psychology, and anthropological and sociological diversity (Blommaert, 2013: 1–2; Ben-Rafael et al., 2010: xi).", "The study of linguistic landscapes transcends the official language policies of a region: for instance, you may live in an officially monolingually Dutch city, but in some neighbourhoods or areas you will find that Dutch is used alongside or even occasionally replaced by other languages, such as Turkish, Arabic, or Chinese.", "Types of written language can be professionally produced road signs, street and place names, advertisements or billboards, but may also include shop signs, restaurant menus, or even graffiti (Landry and Bourhis, 1997: 25; Blommaert, 2013: 1).", "For instance, a shop sign in a language differing from the national language can be used to signal to passersby what type of customer is the expected to enter the shop (Papen, 2012: 57).", "As such, linguistic landscape research provides important information about the nature of multilingualism in a community, often showing a more accurate account of the sociolinguistic reality than official language policies can provide (Moriarty, 2014: 457).", "In more recent research, the term linguistic landscape has occasionally been replaced with semiotic landscape.", "Jaworski and Thurlow (2010: 2) argue that referring to a semiotic landscape emphasises “the way written discourse interacts with other discursive modalities: visual images, nonverbal communication, architecture and the built environment.”", "Additionally, the traditional definition of a linguistic landscape as a fixed notion been challenged in recent years, with scholars arguing that linguistic landscapes should be treated as dynamic rather than static locations (e.g. Garvin, 2010: 245–255; Pietikäinen et al., 2011: 280).", "The linguistic landscape today is often defined as a geographical space that should be interpreted in a given moment in time, as the space is constantly changing, and the languages and cultures in it can constantly be repositioned (Garvin, 2010: 245–255; Moriarty, 2014: 457).", "Rather than focusing on signs in a specific local space, this study observes the use of linguistic markers associated with the Nordic countries in the global linguistic landscape.", "The focus of the study is mainly on mobile or “un-fixed” signs (see Sebba, 2010), such as product labels and brand logos, but a few static signs are also discussed.", "Foreign languages are often used in signs or advertising due to stereotypes associated with speakers of those languages, with those uses further enforcing said stereotypical national identities and the concept of “the national Other” (Piller, 2003: 173).", "This study investigates how certain orthographic features are used in products and marketing to index specific stereotypical social or national identities, and, by extension, the values connotated to these identities on a global level.", "The point of interest of the research is the recent rise of stereotypical qualities associated with the Nordic countries, and how linguistic features stemming from Nordic languages are used to evoke these associations in global markets.", "The study explores the use of words or graphemes associated with Nordic languages in public spaces and marketing in several countries, exemplifying and analysing the methods used to convey a sense of “Nordicness” through written language.", "In such situations the consumers are not in-group speakers of any of the Nordic languages, but are speakers of other languages who are expected to be attracted to products, shops or restaurants due to positive ideals associated with the Nordic region.", "Additionally, the study explores how the mimicry of Nordic linguistic features can be used to index globalism and exlusivity in brands with no other discernible Nordic connection.", "It should be noted that in this article the term Nordic is used to refer collectively to the countries of Sweden, Denmark, Norway, Finland, Iceland, and the Faroe Islands, which are usually considered to share common cultural and linguistic history.", "Thus the definition of the Nordics is broader than that of the region of Scandinavia, which traditionally refers only to the countries of Sweden, Denmark, and Norway, although the two terms are often erroneously used interchangeably.", "While traditional linguistic landscape research can still be used to study the language uses and policies of localised physical spaces, a large part of the language we encounter on a daily basis reaches us through some type of advertisement or marketing, which is (usually) not contained within a specific neighbourhood or city.", "This type of sign is purposefully designed to attract our attention, to give us an association, and to sell us a product or idea.", "The language used on a product is a crucial part of its “unique selling proposition” (USP); a partially or wholly symbolic ideal, communicated through the product's label, packaging, and advertising (Kelly-Holmes, 2010: 478).", "Many product labels and brand names are designed to explicitly communicate the purpose, use, or origin of the product.", "However, in some cases it is unimportant whether the name is linguistically relevant, correct, or even comprehensible to the target audience.", "Instead, the associations brought on by the specific language used can be a more powerful marketing tool than the literal meaning.", "In these cases the names of products are not intended to communicate the exact meaning of the word(s) to the consumer, but instead rely on the symbolic value of the language (Kelly-Holmes, 2005: 69).", "Piller (2003) states that the idea behind using a foreign language in advertising is usually to associate the product with the ethno-cultural stereotype about the country where the language is spoken.", "The consumer's connotations between a language and a specific country are such a defining aspect of the marketing tactic that fundamentally the product's actual origin is of minor importance (Piller, 2003: 175).", "Perhaps the most obvious example for the use of a foreign language in advertising is the widespread use of English.", "English is commonly found in brand names and logo designs, even when the target audience is not necessarily expected to understand the language.", "As long as the consumer possesses sufficient knowledge of English in their repertoire to recognise the language (see Blommaert and Backus, 2013: 18–19), English can be used in global marketing to evoke positive associations of modernity and globalisation (Papen, 2012: 66).", "Provided that the consumer identifies the foreign form as belonging to a particular language, they will transfer the ethno-cultural stereotype about the group most frequently associated with that language onto the product (Piller, 2003: 172).", "Consequently, while signs in a given space can provide insights into the local linguistic community, they can also simply symbolise the values or aspirations of the product or business they advertise (Scollon and Scollon, 2003: 119).", "In order to use a foreign language in advertising to sell a product, the language in question has to have positive connotations.", "This concept is closely linked to the idea of language hierarchy.", "In traditional linguistic landscape research, the use of different languages reflects the relative power and status of the languages within that sociolinguistic context, and official signs in these landscapes can provide insight into local language policies and ideologies (Cenoz and Gorter, 2006: 67; Pietikäinen et al., 2011: 278).", "However, when a sign is intended to evoke positive reactions in various consumers from completely different cultural and linguistic backgrounds, the marketing tactic may rely on broad, more stereotypical associations related to the language.", "English, which is spoken as a foreign language or at least recognised in most parts of the world, has a high position in the global hierarchy of language and can be used for product names and advertisements on an international level.", "As long as the consumer audience possesses enough knowledge of English in their linguistic repertoire to recognise when a message is written in English, the language can be used to activate connotations with symbolic values such as cosmopolitanism, success, sophistication or fun (Piller, 2003: 172; Leeman and Modan, 2010: 183; Cenoz and Gorter, 2006: 70).", "Similarly, thanks to French beauty and fashion ideals having been highly regarded throughout history, the French language has a substantial symbolic value and is still favoured on beauty products all over the world (Kelly-Holmes, 2010: 479–480).", "This article proposes that the Nordic languages have acquired significant symbolic value in recent years due to an increase in positive associations relating to Northern Europe, and that this shift is visible in the global language hierarchy and, consequently, in international marketing.", "Furthermore, increasing utilisation of Nordic-language graphemes in international brands may also have entered them into the global consumer register (i.e. globalese, see Jaworski, 2015a).", "As such, Nordic ortographic features may be used in words from other languages in order to suggest a sophisticated or worldly global connection, in addition to a specifically Nordic one.", "Unlike countries such as France and Italy, which have been strongly associated with fashion and food for decades or even centuries, widespread positive associations related to Nordic countries are relatively new.", "In the last century the global perception of the Nordic countries abroad has shifted considerably, from being regarded as a poor and insignificant region in Europe at the start of the 20th century, to being considered one of the most progressive and happy regions in the word today (Musiał and Chacińska, 2013: 289).", "I argue that this shift is visible in the way that Nordic language features are used in marketing abroad, by directly referencing the Nordics or simply creating associations relating to the region.", "Designer furniture from Northern Europe has been marketed globally as “Nordic” since the 1950s, but it has seen a new peak of popularity in recent years with “New Nordic” and Scandinavian retro designs, alongside the general concept of “Nordic living” (Skou and Munch, 2016: 2; Andersen et al., 2019: 215).", "Both Nordic interior design and fashion have come to be associated with positive qualities of simplicity and craftsmanship, and the conceptual aesthetics of “Nordicness” have spread into other domains (Andersen et al., 2019: 216; Østergaard et al., 2014: 254).", "Nordic food has become defined with symbolic values such as ‘purity’ and ‘freshness’, qualities which, over time, have become intrinsic to the idea of “the Nordics” (Hermansen, 2012).", "According to Skou and Munch (2016: 2), this fascination seems to be “fuelled by different global, political, and economic trends and the reactualisation of the Nordic welfare states as historical role models.”", "The Skandinavisk.com website refers to the Nordics using a fictional ideal of a serene, untouched, peaceful place in an otherwise hectic world.", "Exclusivity and luxury is suggested through the imagery of an isolated and ‘natural’ exotic destination (Thurlow and Jaworski, 2010: 193; see also Thurlow and Jaworski, 2012; Jaworski, 2019).", "Skandinavisk perpetuates the idea that ‘silence’ and ‘space’ equal luxury through social exclusion (Thurlow and Jaworski, 2010: 192), and that, by purchasing a product inspired by the idealised Nordic lifestyle, the consumer can become part of the global elite.", "This image of the Nordic Utopia demonstrates the fascination with “Nordic Cool” (see Østergaard et al., 2014), a positive association with exclusivity that can be used to market various products or even acitivites.", "As the Danish word hygge is used to sell candles and lifestyle books, the Swedish word fika, meaning to ‘have a coffee with something (usually sweet) to eat’, has also gained popularity.", "While fika is an insignificant everyday occurrence for many people in Sweden, in international contexts it is often portrayed as a luxurious activity.", "This is demonstrated by an excerpt from the website of Løv Organic, a French company producing organic tea:", "“The logic behind fika is to be present in the moment, in great company, whether with family, friends or colleagues.", "It's a moment of pure relaxation and comfort.", "Participating collectively allows you to take a step back from your activities, decompress, disconnect, and recharge your batteries in a tranquil environment.”", "(‘The perks of fika and taking a break’, Lov-organic.com)", "In recent years the word fika has been employed by restaurateurs across the globe: a quick search on the internet shows that there are cafés named Fika from Benelux to Singapore, from the United Kingdom to Canada.", "Since few establishments attempt to explain the word in as much detail as Lov-organic.com, the positive associations relating to fika, if not the literal meaning of the word, must be relatively salient to people in all of these countries.", "As the popularity and recognisability of Nordicness becomes greater, fewer visual clues are required for people to identify a product as Nordic or Nordic-inspired.", "In some cases, the association can be created by using a full word from a Nordic language, even in situations where the target audience would not understand the word.", "In other situations, the names of products or businesses may be made up words that are simply intended to look like they stem from a Nordic language.", "Quite often, however, the writing system of one or several of the Nordic languages may be mimicked, with orthographic features borrowed into other languages in order to inspire associations of Nordicness or globalism.", "For certain linguistic forms to acquire symbolic value and social meaning, they first need to be noticed as occurring within specific contexts (Jaworski, 2015a: 229, citing Johnstone, 2013a,b).", "The use of a writing system or script is a relatively straightforward way of indexing symbolic values of a certain linguistic identity, if said writing system or script is language-specific.", "In this study I use the term writing system, as defined by Coulmas (2003, 2014), to mean the writing system of an individual language, e.g., the Dutch writing system.", "The writing system of a specific language is thus called orthography, while the term script is used exclusively for the graphic form of the units of a writing system (e.g. Roman vs. Cyrillic letters) (Coulmas, 2003, 2014: 17).", "The focus of this study is the use of writing system mimicry to indicate a specific type of “foreignness.”", "Coulmas (2003, 2014: 17) refers to this as typographic mimicry, and states that it can be compared to accent mimicry, in which speakers who have little or no competence in a foreign language colour their speech with certain sounds and phonetic features identified with that language.", "Like accent mimicry, typographic mimicry involves the use of certain features to indicate a specific foreignness of a text.", "This use can also be referred to as a visual accent (Coulmas, 2003, 2014: 17–18).", "Although Coulmas (2003, 2014) prefers to refer to this type of mimicry as typographic mimicry, this study follows the example of Sutherland (2015: 150), who has renamed it writing system mimicry.", "According to Sutherland, the word ‘typographic’ could be replaced by ‘script’, with typographic mimicry covering examples of mimicry such as faux Arabic or faux Cyrillic.", "However, the term excludes mimicry of languages which share the same script but different orthographies, such as German and English (Sutherland, 2015: 150).", "Therefore, Sutherland (2015: 150) prefers the term writing system mimicry, defining the concept as the imposing of real or imagined features of the mimicked writing system onto a base writing system, “so that the base writing system somewhat resembles the mimicked writing system while retaining legibility.”", "As a broader definition than typographic mimicry, writing system mimicry includes not only the mimicry of other scripts, but can be extended to all linguistic features.", "The term can thus be used to describe situations in which, for instance, product names or texts written in English have been embellished with orthographic features intended to evoke associations relating to another language or culture.", "However, as Jaworski (2015a) argues, the mimicry of features from foreign languages and scripts may not always be indended to index a specific region.", "Instead, it may be the use of globalese, i.e. a commercial register suggesting “typographic-orthographic cosmopolitanism” (Jaworski, 2015a: 232), using innovative orthographic combinations to index worldliness rather than one language or culture.", "When writing system mimicry is used in brand names or logos, the goal is not to be ambiguous about the language in which the messages are written.", "Instead, the focus is on rendering the name in a familiar alphabet or language, while also presenting “the visual equivalent of a foreign accent” (Seargeant, 2012: 192).", "The images displayed in Fig. 2 demonstrate the use of writing system mimicry, such as faux Devanagari (see Chachra, 2014) and faux Cyrillic (see McMichael, 2009).", "All of the signs are clearly written using the Roman alphabet as a base, as the intention is for the target language speakers to be able to read the product names.", "However, the signs are also written in some type of pseudoscript, meant to indicate the thematic identity of the product.", "Image 1 in Fig. 2 is the sign of a restaurant located in Belgium: the French name Le Syrtaki can be clearly read by speakers of French.", "However, even for individuals who do not know that syrtaki refers to a Greek dance, the restaurant can be clearly identified as Greek as a result of the base writing system (i.e. the Roman script) mimicking Greek letters.", "One challenging aspect of linguistic landscape research has always been the question of how to perform sampling (Gorter, 2006: 2–3).", "Although the use of digital cameras allows researchers to take a potentially unlimited number of photographs of languages displayed in a space, it is challenging to pinpoint which signs can be considered representative of a particular linguistic landscape.", "Leeman and Modan (2009: 341) investigated the linguistic landscape of Washington DC's Chinatown, choosing to largely limit their data sampling to signs located in the neighbourhood's two main commercial corridors.", "Similarly, in a study comparing the linguistic landscapes of the minority regions of Friesland (the Netherlands) and the Basque Country (Spain), Cenoz and Gorter (2006: 67) explored signs on a single shopping street in both areas.", "However, even when narrowing down the location from a huge city or an entire neighbourhood to a few streets, it is problematic to establish a sound methodology for quantitative data sampling.", "Defining what does and what does not constitute a sign and how signs should be categorised can be very challenging.", "Consequently, like the studies of previous researchers, such as Jaworski and Yeung (2010), our research focuses on the qualitative rather than the quantitative aspect of signs in the linguistic landscape.", "The examples used in this article were discovered by the researcher by chance in various cities in various countries.", "These examples were photographed where they were encountered and, whenever possible, the websites of the companies were visited.", "While the majority of the signs were identified in the Netherlands, Germany, and Belgium, several of the signs and labels that were noted down or photographed referred to companies or products from other countries.", "The examples were collected during the winter of 2018–2019, and have been defined below according to the methods employed to demonstrate a sense of “Nordicness” or simply “foreignness.”", "The study focuses on three different ways in which Nordic linguistic features are used to suggest Nordicness or globalism in international marketing.", "Some brands make use of actual words stemming from Nordic languages, as in the case of the previously mentioned Hygge candle or the many cafés named Fika.", "Other companies use words or phrases that may originally stem from any of the Nordic languages, but which are intentionally misspelled, exaggerated, or enhanced with additional Nordic or “foreign” features.", "Finally, a wide variety of brands use faux Nordic, i.e. the mimicry of Nordic features and graphemes in foreign language words, to associate themselves either with the Nordic region or to suggest internationalism.", "As the furniture mogul IKEA has shown throughout the years, consumers are quite happy to buy products with incomprehensible and unpronounceable Nordic-language names.", "With the rise of “Nordic Cool” in recent years, progressively more brands have taken to this approach.", "While products such as the Hygge candle and businesses such as the countless Fika cafés capitalise on relatively well-known meanings of these words, many other brands employ lesser-known words or names from various Nordic languages.", "The images below demonstrate a few occurrences of this approach in global marketing.", "Slightly less obscure is the name Distrikt Nørrebro, a Dutch clothing brand clearly inspired by a district in Copenhagen.", "Their logo is pictured as Image 2 in Fig. 3.", "In recent years, the district known as Nørrebro has become well-known for being one of the most hip, fashionable, and multicultural areas of Copenhagen.", "Therefore, it is not difficult to see why a clothing brand would like to be so clearly associated with the district.", "Nørrebro can be compared to Brooklyn in New York City, which likewise has inspired names of countless brands in popular culture due to the reputation of the district, particularly due to its history relating to hip-hop music.", "Although many brands or products make use of actual words from Nordic languages, in many cases these words make little sense in association with the product itself.", "An example of this is German brand of cosmetics called Hej, pictured as Image 4 of Fig. 3.", "The product name Hej stems from the Swedish or Danish word hej, simply meaning ‘Hello’.", "Considering this greeting does not in any way reference or relate to cosmetics, one can only assume that the consumer is intended to associate the Nordic language reference with concepts of health or beauty.", "A similar strategy seems to be employed by the brand Också, pictured in Image 5, the name of which means ‘as well, also’ in Swedish.", "The company states that their cosmetics are intended to double as an interior design feature and that the owners are “passionate about design, brand development and the Scandinavian lifestyle” (Ocksacosmetics.com (2019)).", "Although the company is based in Sweden, the owners are Dutch and South African.", "As the company website is only available in English and Dutch, this suggests that the target consumers are not Swedish (or at least not Swedish-speaking).", "Additionally, both the brand name Också and the name of the soap, Grönt (‘green’), seem intended to draw people in using associations related to the Swedish orthography rather than their meaning.", "In spite of the word ‘green’ possessing connotations of being natural or healthy, this potential meaning of grönt will not necessarily be understood by the non-Swedish-speaking consumer.", "Likewise, the word också (‘as well’, presumably relating to the double use of the product as a cosmetic and a design element) also seems intended to mainly attract people to purchase the product due to its Scandinavian-sounding name.", "While the previous examples have used names and words that have actual meanings in various Nordic languages, some brands prefer to use names that exaggerate the Nordic features.", "Examples of such brands and businesses can be found in Fig. 4.", "A similar near-Finnish name is used by the outdoor clothing brand Napapijri, pictured as Image 2 in Fig. 4.", "Although the brand itself is Italian, the name is a misspelled version from the Finnish word napapiiri, meaning ‘Polar circle’.", "Furthermore, the name Napapijri is often displayed alongside the Norwegian flag.", "The purpose seems to have been to combine linguistic features and imagery from two Nordic countries known for their nature and cold weather as a method of suggesting quality and reliability of the clothing brand.", "The company itself argues that this combination is a demonstration of its intersectionality, of “defying labels, transcending boxes, pursuing the unexpected journey into nature and creativity” (Napapijri.co.uk).", "This qualitative research project explored the use of Nordic linguistic features in international product labels and marketing.", "In many cases companies use Nordic-language words as names, regardless of the actual origin of a product or brand, and sometimes regardless of the connection between the meaning of the word and the product itself.", "Meanwhile, some brands simply use features from one or several of the Nordic languages in order to create or exaggerate the Otherness of the product in question.", "In some cases the incomprehensibility or unreadability of some brand or business names may also be intended to suggest a high-end product or establishment.", "In a study on visible gentrification in Brooklyn, New York, Trinch and Snajdr (2017) demonstrate how up-scale shops and restaurants tend to favour distinction-making signs.", "Brands or establishments seek to distinguish themselves by using names made up of a single word or short phrase, a polysemic or cryptic name, or a name in another language than English (Trinch and Snajdr, 2017: 75).", "By distinguishing the brand with a quirky, weird, or incomprehensible name, the business can be seen as more worldly and thus more attractive to consumers.", "While these types of names seem textually sparce, they often have a rich underlying meaning or association, making those who know the meaning(s) feel exclusive because they are “in-the-know” (Trinch and Snajdr, 2017: 76).", "A name such as Rå can be argued to be distinctive in this way: few Dutch customers would initially be expected to make the linguistic connection between Norwegian/Danish rå and Dutch rauw (‘raw’), but the name is obscure enough to seem exclusive and authentic, and thus to warrant attention.", "Additionally, if or when the customer does find out the meaning of the name, they acquire a feeling of being in on a private joke.", "While distinction-making signs can be used to convey a sense of sophistication in marketing, so can the concept of silence and space, as previously discussed in relation to Skandinavisk.com and Lov-Organic.com.", "The majority of the products or logos in this study seem to strive for a “visual silence,” using simple and understated packaging or logo designs.", "Brands such as Distrikt Nørrebro, Hej, Rå, Töst and Nø use few colours and shy away from intricate texts or images.", "The relative emptiness and simplicity of the product design is reminiscent of the discrete typography used in advertisements for luxury holidays (Jaworski, 2019: 96), suggesting that the tagret consumer is likely at least aspiring upper-middle class.", "The visual silence reflects the consumer perception of a parallel between tranquility and exclusivity, with many brands preferring to use simple black and white designs to index elegance and refinement (Jaworski, 2019: 103; Järlehed et al., 2018: 50; Adami, 2018: 12).", "Although some companies use Nordic linguistic forms simply to be distinctive or to demonstrate a global consumer register, many of the brands analysed in this article seem to purposefully channel positive associations relating specifically to Nordic culture, food, and lifestyle.", "These findings support previous research referencing the idea of “Nordic Cool” as an international marketing tactic (e.g. Andersen et al., 2019; Skou and Munch, 2016; Hermansen, 2012).", "The conscious effort of these brands to associate themselves with what they consider to be Nordic values is also evident from overt declarations found on several websites.", "For instance, the companies Också, Jūttu, and Løv Organic all define their source of inspiration as Nordic or Scandinavian lifestyle or design.", "It is also clear that these companies are specifically targeting a non-Nordic audience, given that most of them are based outside the Nordic countries, and in most cases their websites are not available in any of the Nordic languages.", "The study shows a clear relationship between the linguistic features employed by various businesses and brands selling fashion and cosmetics, and the values they seek to convey.", "With the exception of texts involving pure faux Nordic or globalese, many of the product names stem from existing words in one or several of the Nordic languages.", "Yet in most cases the intended foreignness seems to be evoked through the use of graphemes associated with Nordic languages, rather than through the meanings of the words themselves.", "Indeed, it is not the meaning of the foreign language that is most relevant in advertising, but the identification of this language by the consumer (e.g. Piller, 2003; Blommaert and Backus, 2013: 18–19).", "In some cases simply distinguishing a product or establishment with an unusual name is enough to seem exclusive (see Trinch and Snajdr, 2017).", "For the brand it is unimportant whether the audience understands the meaning of words such as också or løv, or if a name such Jūttu, Töst, or Napapijri is conventionally meaningful in the literal sense.", "The focus is on these names attracting (usually middle- or upper-class) consumers by evoking positive connotations suggesting globalism, sophistication, or the exclusive ideal of the Nordic lifestyle." ]

[ "Positive associations relating to the Nordics increase visibility of Nordic linguistic features.", "International brands use Nordic linguistic features to evoke positive associations in consumers.", "Nordic graphemes may be used as innovative features in globalese.", "The graphemes 〈Ø〉 and 〈Å〉 are particularly visible in brand and product design." ]

[ "AGRI", "EART" ]

[ "Avalonia", "Chitinozoan", "Hirnantian", "Katian", "Ordovician", "Welsh Basin" ]

[ "1105", "1911" ]

[ "The recent development of an integrated Upper Ordovician chitinozoan biozonation in British Avalonia (Vandenbroucke and Vanmeirhaeghe, 2007; Vandenbroucke, 2008a), based on type areas for the British chronostratigraphical scheme (Fortey et al., 1995, 2000), recognises six chitinozoan biozones with two subzones for the upper Katian (Ashgill) and potentially provides a powerful tool for dating Upper Ordovician successions where graptolite preservation is not favourable.", "However, the applicability of the UK scheme, as it currently stands, has not been widely tested outside the type areas on which it was based and there remain problems concerning calibration with graptolite and conodont-based biostratigraphies as well as other regional and global stratigraphical schemes (Vandenbroucke, 2008a).", "Significantly, the development of a UK Avalonia chitinozoan biozonation coincided with the recent British Geological Survey (BGS) re-mapping of the Lower Palaeozoic Welsh Basin and its margins that represent the historic type area for the Ordovician and Silurian systems (e.g. Murchison, 1839; Lapworth, 1879).", "The work reported here links closely with Vandenbroucke et al.'s (2008b) study of Darriwillian (Llanvirn) to mid-Katian (late Caradoc) chitinozoans in the Fishguard–Cardigan area and with work on late Hirnantian and Silurian assemblages in mid-Wales (Vandenbroucke et al., 2008a), from the Type Llandovery succession (Davies et al., 2013) and from Wenlock rocks in the Builth Wells district (Verniers, 1999).", "Here we present a regional biostratigraphical scheme that utilises data on chitinozoan abundance and distribution from the well-constrained upper Katian–Hirnantian Welsh Basin succession (Davies et al., 1997, 2009) and compare it to that proposed by Vandenbroucke (2008a).", "This has allowed the reproducibility of the new British Avalonia chitinozoan biostratigraphical scheme to be tested and, additionally, the influence of sedimentary facies (i.e. basin and shelf) to be assessed.", "The chitinozoan biozonation presented herein also informs correlation of the Cardigan Bay succession (Cardigan to Llangrannog) of west Wales (BGS, 2003, 2006; Davies et al., 2003, 2006) with the easterly successions of the Tywi Anticline, Garth and Type Llandovery areas (Schofield et al., 2004; BGS, 2005, 2008; Schofield et al., 2009; Davies et al., 2013) where graptolites are less common and many chronostratigraphic boundaries are poorly constrained.", "In addition, the mid-southern hemisphere palaeolatitude position of the Welsh Basin during the Late Ordovician makes the area important as a stepping stone between latitudinally restricted polar and tropical chitinozoan provinces that are difficult to correlate (Delabroye and Vecoli, 2010; Vandenbroucke et al., 2010; Ghienne et al., in press).", "The Welsh Basin provides a relatively complete section through Katian (upper Caradoc–lower Ashgill) and Hirnantian (upper Ashgill) rocks formed in turbiditic slope-apron to muddy shelf depositional settings (Davies et al., 1997) (Figs. 1 and 2).", "The line of demarcation between late Ordovician basinal and shelfal settings was located along the eastern flank of the Tywi Anticline where long-lived fractures, including the Llanwyrtyd and Garth fault systems and the Crychan Fault Belt, were influential in controlling facies distribution (e.g. Davies et al., 1997; Schofield et al., 2004; Davies et al., 2009; Schofield et al., 2009).", "As elsewhere, the Katian–Hirnantian Welsh Basin succession records the increasing impact of the late Ordovician glaciation including its mid-Hirnantian acme (e.g. Davies et al., 2009 and references therein).", "The repeated and linked changes in sea level, organic productivity and basin water oxicity that accompanied this event are reflected in marked variations in levels of bioturbation and organic content.", "This allows burrow-mottled and bioturbated oxic facies to be distinguished from typically darker anoxic facies in which burrow-mottling is absent, organic content is high and hemipelagic lamination and graptolites are commonly preserved (e.g. Davies et al., 1997; Challands et al., 2009).", "The oldest basinal rocks assessed here are from the Cardigan area where the lower–middle Katian (upper Caradoc–?", "lower Ashgill) Dinas Island Formation includes the distinctive, dark and anoxic Cwm Degwel Mudstone Member (up to 100 m thick) at its top and where there is a lateral passage into the coeval Cwm-yr-Eglwys Mudstone Formation (Davies et al., 2003; Williams et al., 2003).", "The succeeding middle–upper Katian (lower Ashgill) burrow-mottled Nantmel Mudstones Formation is over 1 km thick along the Cardigan Bay coast where it occupies the cliffs between Cardigan and Penbryn.", "The formation's extensive inland outcrop ranges eastwards as far as the Llandovery and Garth areas and its correlatives are seen in the core of the Plynlimon Inlier and in the Corris area (Cave and Hains, 1986; Pratt et al., 1995).", "Present in the upper part of the Nantmel Mudstones are widely recognised organic-rich, anoxic and graptolitic laminated hemipelagic mudstone units (labelled LH) that together comprise the Red Vein of Pugh's (1923) Corris area succession.", "Up to four anoxic units have been recorded which Challands (2008) and Challands et al. (2009) number LH0 (lowest) to LH3 (highest) (Figs. 1 & 2).", "As at the coast, the dominantly oxic Nantmel Mudstones Formation of the Tywi Anticline succeeds an anoxic lower Katian succession that comprises the St. Cynllo's Church Formation and in which an upper, richly graptolitic, mudstone unit (Sugar Loaf Member) has been recognised.", "The local Red Vein anoxic facies here interleave with the fault-located debrites and shelly turbidites of the Bryn Nicol Formation.", "Further east, lower levels of the Nantmel Mudstones Formation are replaced by a succession of interbedded shelly sandstones and mudstones (Tridwr Formation) that record the transition into a mid- to outer-shelf setting (Schofield et al., 2004).", "Upper parts of the Nantmel Mudstones Formation, above the Cautleyan–lower Rawtheyan shelly debrites of the Llwyncus Member (Schofield et al., 2004), pass similarly into the bioturbated shelfal muddy sandstones of the uppermost Katian (Rawtheyan) Cribarth Formation.", "The Welsh Basin hosts the type section for the original UK Hirnantian Stage at Cwm Hirnant in North Wales (e.g. Fortey et al., 2000).", "Here and along the margins of the basin the distinctive shelly ‘Hirnantia fauna’ first appears above abrupt changes in sedimentary facies that record the impact on sea level and sediment supply of the first stages of an approaching glacial maximum event (e.g. Brenchley and Cullen, 1984).", "In the basin centre, where both shelly and graptolitic faunas are absent, it is the correlative facies contacts that are widely used as a proxy for the base of the UK stage (e.g. Davies et al., 2009) and, by extension, the revised international stage (e.g. Bergström et al., 2009).", "Correlation of the Hirnantian rocks of the shelf and basin has recently been revised by Davies et al. (2009).", "In the southern and central parts of the basin, the lower Hirnantian silty, sandy and commonly slumped turbiditic facies that form the lower part of the Yr Allt Formation are now equated with the Drosgol Formation of the Plynlimon area where the conglomeratic Pencerrigtewion Member records deposition at the time of maximum glacio-eustatic lowstand (Davies et al., 2009).", "The effects of slumping remain widespread in overlying Hirnantian Welsh basin rocks, but the onset of the post glacial-maximum rise in sea level is reflected in the finer grained facies that comprise the upper Yr Allt Formation and, in the Plynlimon area, the equivalent Brynglas Formation.", "It follows that the use here of the terms ‘lower’ and ‘upper’ Yr Allt Formation has specific meaning relating to Hirnantian glacioeustasy.", "The distinctive and widely recognised Mottled Mudstone Member (Cave and Hains, 1986; Temple, 1988), present at the base of the otherwise anoxic Cwmere Formation, records a significant post-glacial re-colonisation event felt throughout the Welsh Basin and along its margins that is linked to the first appearance of late Hirnantian persculptus biozone graptolites in Wales (Davies et al., 2009, 2013).", "The Cwmere Formation mudstones pass eastwards across the Tywi Anticline into the extensively slumped basin margin Twcytta Mudstones Formation with the base of the Silurian lying within these divisions.", "The lithostratigraphy of Hirnantian shelfal facies preserved along the eastern margin of the Welsh Basin is predictably more complicated (e.g. Schofield et al., 2004; Barclay, 2005; Davies et al., 2009; Schofield et al., 2009).", "To the west of the Crychan Fault Belt, the lower Hirnantian burrowed sandstones and silty mudstones of the Ciliau Formation pass into wave-rippled Cwmcringlyn Formation sandstones.", "Transgressive later Hirnantian facies are represented by the interbedded thin sandstones and smooth mudstones of the trace fossil-rich Garth House Formation (Fig. 2).", "However, when traced eastwards upper Katian (Rawtheyan) and lower Hirnantian strata are truncated by an unconformity that records emergence and erosion during the glacioeustatic lowstand; and here conglomerate and sandstone bodies (Cwm Clyd Sandstone and Glassallt-Fawr Sandstone formations) present below the base of the Garth House Formation record the subsequent late Hirnantian marine transgression.", "Within the overlying uppermost Hirnantian succession, weakly burrowed silty mudstones (Chwefri Formation) pass into thoroughly bioturbated sandy mudstones (Bronydd Formation) as part of a distal to proximal facies transition.", "Davies et al. (2009) correlate the burrow-mottled Ystradwalter Member at the base of the Chwefri Formation with the basinal Mottled Mudstone Member (see below).", "Though absent in lower Hirnantian (upper Ashgill) rocks of south-mid Wales, biostratigraphically significant graptolite assemblages are locally abundant in both the underlying Katian and overlying uppermost Hirnantian successions.", "Together these assemblages provide a key framework that allows the regional Welsh Basin chitinozoan biostratigraphy developed here to be compared with the British Avalonian scheme of Vandenbroucke (2008a).", "Graptolites from the Dinas Island Formation and from the Sugar Loaf Member confirm the lower Katian clingani biozone.", "However, from the Cwm Degwel Mudstone Member at Frongoch (SN 076 410), Williams et al. (2003) record probable Climacograptus tubuliferus, a proxy for the middle Katian Pleurograptus linearis biozone, though P. linearis itself is not present.", "In the succeeding Nantmel Mudstones Formation graptolites have only been recovered from the ‘Red Vein’ anoxic units, but these confirm the anceps Biozone of late Katian age.", "Pugh (1923) records the index taxon Dicellograptus anceps along with Orthograptus truncatus var.", "abbreviatus and Climacograptus scalaris var.", "miserabilis in the Corris area assemblages.", "Those from the Cardigan–Llangrannog section lack the index fossil, but are dominated by Orthograptus abbreviatus with rarer ‘Climacograptus’ cf. supernus, Normalograptus miserablis and fragmentary dicellograptids (Williams, 2001a, 2001b) and compare with similar assemblages recovered from the east of the basin (Davies et al., 1997).", "O. abbreviatus is present in the broadly coeval Sholeshook Limestone of the Whitland area of south Wales (Zalasiewicz et al., 1995).", "In the Llandovery area, Cocks et al. (1984) report long ranging Upper Ordovician graptolites associated with a Rawtheyan shelly fauna from the upper part of the Tridwr Formation.", "Graptolites collected from the Cribarth Formation during this study include O. abbreviatus (sample TJC 09-06-01) from Garth Bank [SN 942 499] and Normalograptus normalis from Glasallt Farm, south of Llandovery, both consistent with the anceps biozone (Zalasiewicz et al., 2009).", "The lower Hirnantian extraordinarius biozone has not been recognised in Wales, but the base of the upper Hirnantian Normalograptus persculptus biozone within the Mottled Mudstone Member provides an important marker horizon throughout the Welsh Basin (e.g. Jones, 1909; Pugh, 1923; Hendriks, 1926; Temple, 1988; Davies et al., 2013).", "Critically, N. persculptus biozone assemblages persist for some distance into the succeeding Cwmere Formation and have allowed Blackett et al. (2009) to chart progressive changes in the morphology of Normalograptus?", "parvulus for example at Cerrig Gwinion Quarry [SN 973 656] and Lynn Brianne [SN 816 493].", "It is the morphotypes of this taxon present in lower levels of the Llandovery area Chwefri Formation that support the correlation of the underlying Ystradwalter Member with the Mottled Mudstone Member (Davies et al., 2009).", "In addition, Cocks et al. (1984) report a form of Climacograptus normalis from a low level in the Bronydd Formation consistent with the persculptus biozone, but certainly no higher than the basal Silurian acuminatus biozone (now ascensus-acuminatus Biozone; see Zalasiewicz et al., 2009).", "A shelly fauna collected from the Pen Derlwyn facies of the Bryn Nicol Formation has yielded a diverse fauna suggestive of a Rawtheyan age (Rushton, 1994).", "The diverse, reworked shelly fauna recovered from the Llwyncus debrite of the Garth section is of Cautleyan to early Rawtheyan aspect (Schofield et al., 2004) and a shelly fauna recovered from the uppermost Cribarth Formation at Glassallt Fawr includes the trilobite Brongniartella cf. robusta which, according to Williams and Wright (1981, p. 8), indicates a “very high Rawtheyan age”.", "From the Garth area, Williams and Wright (1981) record the distinctive cool water ‘Hirnantia fauna’ from rocks now included in the Ciliau Formation and Cocks et al. (1984; also Woodcock and Smallwood, 1987) report similar Hirnantian shelly assemblages from levels within the former Scrach Formation that are now viewed as part of the Cwmcringlyn Formation (see discussion by Davies et al., 2009).", "Vandenbroucke (2008a) has developed a chitinozoan biostratigraphy for the upper Katian and Hirnantian (Ashgill) from historical type sections in the Anglo-Welsh area, including the Cautley District, Pus Gill (Vandenbroucke et al., 2005), Greenscoe (Van Nieuwenhove et al., 2006), Whitland (Vandenbroucke et al., 2008b), Cwm Hirnant and the Wye Valley (Vandenbroucke et al., 2008a).", "Six chitinozoan biozones, with two subzones, were recognised by Vandenbroucke (2008a) (Fig. 3) who provided the following criteria for their definition:", "The Fungochitina spinifera biozone corresponds to the total range of the index fossil.", "In the Cardigan–Llangrannog section it has tentatively been recognised in the Dicranograptus clingani biozone (Vandenbroucke et al., 2008b).", "In N. England, the F. spinifera biozone straddles the Caradoc–Ashgill boundary and is dated as mid Katian (Onnian to Pusgillian) of the British scheme (Fortey et al., 2000, time slice 5d–6a of Webby et al., 2004).", "In Avalonia, Saharochitina fungiformis is taken as a proxy for the F. spinifera biozone.", "Other important associated taxa corresponding to this biozone are Lagenochitina baltica, Lagenochitina prussica, Belonechitina robusta, Spinachitina ?", "coronata and Conochitina ?", "incerta.", "The Armoricochitina reticulifera subzone, defined by Nõlvak and Grahn (1993) in Baltoscandia, of the F. spinifera biozone has been recognised in the Cardigan area associated with graptolites of the Dicellograptus morrisi subzone of the D. clingani biozone.", "The Tanuchitina bergstroemi biozone in Avalonia is defined as corresponding to the first appearance datum (FAD) of T. bergstroemi to the first occurrence of Conochitina rugata, the index fossil of the overlying biozone.", "Belonechitina americana is an associated taxon in this biozone.", "In the aforementioned Frongoch locality of the Cardigan area, numerous fragments cautiously attributed to the biozone index fossil (which is a very fragile species) correspond to the middle Katian P. linearis biozone (Vandenbroucke et al., 2008b).", "Vandenbroucke et al. (2005) viewed the T. bergstroemi biozone as mid-late Katian (~ Cautleyan) in age, but on recognising the implications of this for the correlation of the Welsh Basin succession, Vandenbroucke et al. (2008b, p. 211) have subsequently advised that “confirmation of the presence of T. bergstroemi at Frongoch, and in equivalent strata elsewhere, should be viewed as an urgent requirement”.", "The C. rugata biozone in Avalonia is defined as the total range of the index fossil.", "It corresponds to the C. rugata biozone in Baltoscandia (Nõlvak and Grahn, 1993) and is dated late mid to late Katian (late Cautleyan to early Rawtheyan).", "The Spinachitina fossensis biozone is a partial range biozone defined by the first appearance of S. fossensis to the first appearance of Bursachitina umbilicata, the overlying biozone's index taxon.", "In the Cautley district of northern England, this biozone occurs in the middle of the shelly fauna Zone 6 of Ingham (1966).", "Rickards (2002) assigned associated graptolites to the P. linearis biozone, although the upward revision of biozonal boundaries implied by this has not been generally accepted (e.g. Zalasiewicz et al., 2009; Vandenbroucke et al., 2013).", "Associated taxa include Belonechitina sp. 8 Vandenbroucke, 2008b and Spinachitina sp. 4 Vandenbroucke, 2008b.", "This biozone has been recorded from the Condroz inlier of Belgium (Vanmeirhaeghe and Verniers, 2004) but is not known outside Avalonia.", "The B. umbilicata biozone is defined as the total range of the index taxon.", "It has also been recorded from Condroz inlier of Belgium (Vanmeirhaeghe and Verniers, 2004) and from the Kaugatuma core in Estonia (Baltica) by Kaljo et al. (2008).", "It is of late Katian (mid- to late-Rawtheyan) age.", "Vandenbroucke (2008a) did not recognise the Laurentian and Baltoscandian Hercochitina gamachiana biozone in Avalonia, but discoveries made as part of this study suggest it may be represented in the Welsh Basin.", "However, its age is disputed as being either exclusively Hirnantian (Achab et al., 2011, 2013) or as partially Katian and Hirnantian (Hints et al., 2004; Kaljo et al., 2008).", "It is defined in Laurentia by Achab (1989) as the partial range of B. gamachiana between its first appearance and that of the index of the succeeding biozone, Spinachitina taugourdeaui.", "In Baltoscandia, Nõlvak (1999) defines it as corresponding to an interval between the C. rugata and S. taugourdeaui biozones.", "The Ancyrochitina merga biozone, is defined in Avalonia as corresponding to the total range of the index fossil and is of late Katian age (Vandenbroucke, 2008a).", "This is the same definition as for the A. merga subzone in northern Gondwana where it was first defined (Paris, 1990).", "Euconochitina lepta is an associated chitinozoan species.", "It is of late Katian (late Rawtheyan) age.", "The S. taugourdeaui biozone is a partial range biozone.", "Its base in Baltoscandia (Nõlvak and Grahn, 1993), is defined by the first appearance of S. taugourdeaui and its top by the first appearance of Conochitina scabra.", "The index taxon was first recognised in Avalonia in North Wales where it was restricted to the Hirnant Limestone (Vandenbroucke et al., 2008a).", "Because of its co-occurrence there, Belonechitina sp. 11 (Vandenbroucke, 2008a, 2008b) was considered to serve as a correlative proxy taxon for the S. taugourdeaui biozone in Wales (Vandenbroucke, 2008a), Here we define Belonechitina sp.11 as a new species and erect a new biozone from its partial range, the Belonechitina llangrannogensis biozone.", "A total of 64 samples were collected from localities representing a shelf to basin transect from the middle–upper Katian (uppermost Caradoc–Ashgill).", "Localities were chosen on completeness of section (lack of faulting), exposure and accessibility.", "Twenty-seven samples representative of basinal facies came from the Cardigan Bay region, from the Cwm Degwel Mudstone Member (oldest), the Nantmel Mudstones (including the ‘Red Vein’), both the lower and upper Yr Allt Formation and the Cwmere Formation (youngest) (Fig. 3).", "This sampling programme complements that of Vandenbroucke et al. (2008b) who collected from the underlying early Katian (Caradoc) succession up to and including the Cwm Degwel Mudstone Member.", "The remaining 37 samples were obtained from seven sections (Figs. 4, 5, 6 and 7) located in the east of the basin and along its marginal shelf.", "These included surface exposures in basin facies (Sugar Loaf Member and Nantmel Mudstones Formation), basin margin facies (Pen Derlwyn and Coed Ifan facies of the Bryn Nicol Formation) and the shelfal successions of the Garth and Type Llandovery areas (Tridwr, Cribarth, Ciliau, Cwmcringlyn, Cwm Clyd Sandstone, Glasallt-Fawr Sandstone, Garth House and Bronydd formations; Fig. 4).", "In addition, samples from the basinal Yr Allt and Cwmere formations were obtained from the Dolaucothi M8 Borehole, Pumsaint.", "The sampling in the Llandovery area compliments the study by Davies et al. (2013) that focused principally on Silurian strata.", "All sample localities are listed in Appendix II.", "Extraction of organic residues followed the procedure outlined by Paris (1981).", "A total of 678 chitinozoans were recovered from 27 samples from the coast section between Cwm Degwel, Cardigan and Traeth-yr-Ynys Lochtyn, Llangrannog (Fig. 1).", "Abundance data for the Cardigan Bay region is given in Table 1 and the range of recovered taxa is presented in Fig. 8.", "The preservation of chitinozoans from the Cardigan–Llangrannog coast section varies from poor, crushed and broken specimens, as typically recovered from oxic facies, to slightly worn 3-dimensional vesicles maintaining diagnostic features.", "The best material was recovered from the anoxic units of the Red Vein (samples TJC D979, TJC D1015) and dark mudstones of the lower Yr Allt Formation at Llangrannog (samples TJC D946 and TJC D1005).", "Poor chitinozoan recovery may possibly be attributed to high sedimentation rate and oxic palaeoenvironment in the Nantmel Mudstones Formation and the Hirnantian sediments accounting for the best yields coming from the anoxic units of the Katian Red Vein (LH0–LH3 of Challands et al., 2009).", "A total of 736 chitinozoans were obtained from the 37 samples (including the Bryn Nicol section and Dolaucothi M8 core) collected in this region.", "The numerical results are presented in Tables 2 and 3 and the chitinozoan ranges are plotted on a composite range chart (Fig. 9).", "Chitinozoan preservation is variable from section to section in this region.", "Multiple samples taken from the same formation along strike were noted to vary considerably in chitinozoan abundance and state of preservation.", "For example, in the Garth House Formation the majority of specimens from the Y-Grug - A40 road section are flattened (TJC D971, TJC D974) whereas from the formation's type locality on Garth Bank (TJC D976) numerous 3-dimensionally preserved chitinozoans were recovered.", "Only one sample (sample TJC D951) from the Nantmel Mudstones Formation of the Sugar Loaf section (Section A, Fig. 4) was barren.", "Five Ordovician biozones can be recognised in the Cardigan Bay–Garth/Llandovery transect: four formerly known Avalonian biozones and one Laurentian biozone not previously recorded from Avalonia.", "The lowest recognised biozone is the T. bergstroemi biozone, originally defined by Nõlvak and Grahn (1993) in Baltoscandia as corresponding to the total range of the index fossil.", "T. bergstroemi is recorded in the Cardigan area from samples TJC D946 (Gwbert) to TJC D943 (Pen y Craig).", "Samples stratigraphically below these levels did not yield any biostratigraphically useful chitinozoans.", "The associated taxon B. americana was recovered within the range of T. bergstroemi at Mwnt (sample TJC D941) and also much higher stratigraphically in the Yr Allt Formation (sample TJC D946).", "Neither T. bergstroemi nor the associated B. americana were recovered in the Llandovery area.", "A single occurrence of the index for the C. rugata biozone was recorded in LH0 (sample TJC D1015), but occurs above the base of the S. fossensis Biozone and is coincident with the FAD of B. umbilicata.", "C. ?", "rugata has also been recorded alongside B. umbilicata in the Cautley district of northern England by Vandenbroucke et al. (2005), and Kaljo et al. (2008) record B. umbilicata from the rugata Biozone from the Kaugatuma core.", "From these observations, we suggest the C. rugata biozone in Avalonia, and possibly Baltoscandia, should be redefined as a partial range biozone between the first appearance of the index fossil and the first appearance of S. fossensis; with C. rugata extending through the S. fossensis and into the B. umbilicata zone.", "This biozone is recognised in both the Cardigan Bay and Tywi Anticline areas.", "In the former area the base is marked by the first appearance of S. fossensis in sample TJC D943 (Pen y Craig) and the top is marked by the first appearance of B. umbilicata from sample TJC D1013 (LH0), the index taxon of the overlying biozone.", "In the Tywi Anticline area, the first occurrence of S. fossensis is in the Nantmel Mudstones Formation at Bryn Nicol (sample TJC D958).", "This biozone is defined by the total range of the index taxon which in the Cardigan Bay region is from sample TJC D1013 to TJC D1015 and is restricted to LH0.", "Taxa consistent with this biozone were not recorded in the Garth–Llandovery area.", "The original B. gamachiana biozone erected in Laurentia by Achab (1989) and subsequently recognised in Baltoscandia (Nõlvak, 1999), corresponds to the partial range of B. gamachiana between its first appearance and the appearance of S. taugourdeaui, the index species of the succeeding biozone.", "In Baltoscandia, this corresponds to upper Pirgu (uppermost Rawtheyan, uppermost Katian) (Nõlvak, 1999) to lowest Hirnantian.", "The original B. gamachiana biozone was not recorded in Avalonia by Vandenbroucke (2008a).", "However, he records specimens of H. cf. gamachiana from the overlying S. taugourdeaui biozone in Cwm Hirnant and specimens doubtfully identified as H. aff. gamachiana from Hirnantian rocks in the Claerwen Valley (Vandenbroucke et al., 2008a).", "In the present study, Belonechitina cf. gamachiana was recovered in both the Cardigan and Llandovery areas.", "In the Cardigan area its FAD was in sample TJC D979 (Nantmel Mudstones Formation, LH1) and in eastern sections in sample TJC D1023 (Cribarth Formation).", "This warrants tentative recognition of the biozone in Wales.", "However, pending the recovery of the global index taxon, here we erect a local Belonechitina cf. gamachiana Biozone defined by the partial range of B. cf. gamachiana, from its first appearance to the FAD of B. llangrannogensis n. sp., the index species of the succeeding newly erected biozone (but see discussion below).", "How this biozone relates to the original B. gamachiana biozone is to be determined.", "This biozone is recognised in the Garth area by the first appearance of S. cf. taugourdeaui in sample TJC D976 (Garth House Formation, Garth House).", "The same taxon occurs stratigraphically higher in the Garth House Formation in the Llandovery area (Crychan Forest sample TJC D1026), 2 m below the base of the overlying Bronydd Formation and, from the same section, Davies et al. (2013) report poorly preserved chitinozoans from the base of the Garth House Formation which they too assign to this biozone.", "These occurrences in post-dating the initial post-glacial maximum sea level rise to affect the Welsh Basin and pre-dating the FAD of persculptus biozone graptolites in Wales, imply a late Hirnantian (upper extraordinarius biozone) age.", "However, the upper boundary of the Spinachitina cf. taugourdeaui biozone is currently poorly constrained in Wales.", "Here the upper Hirnantian Baltoscandian C. scabra biozone is not recognised and the anticipated lowermost Silurian fragilis and postrobusta biozones of Verniers et al. (1995) remain unproven below a succession of younger Llandovery and Wenlock biozones (Verniers, 1999; Davies et al., 2013).", "The same cautionary comments apply here as for the B. cf. gamachiana biozone, but the implications of better preserved and positively identified specimens of S. taugourdeaui found elsewhere in the Welsh Basin are assessed below.", "The six mid-Katian to Hirnantian chitinozoan biozones recognised in this study permit the more precise correlation of the succession developed in the west of the Welsh Basin, as seen along the Cardigan Bay coast, with that present in the east of the basin and along its marginal shelf (Tywi Anticline, Garth and Llandovery areas) (Fig. 10).", "In the Cardigan Bay area, the index taxon for the Saharochitina spinifera biozone as well as the taxa normally associated with this middle Katian chitinozoan biozone are absent from the strata sampled as part of this study.", "Whereas the possible presence of T. bergstroemi at Frongoch (Vandenbroucke et al., 2008b) and its certain occurrence at Gwbert implies that the bergstroemi biozone may indeed extend downwards from Nantmel Mudstones Formation into the underlying Cwm Yr Eglwys Mudstone Formation as Vandenbroucke et al. (2008b) suggest.", "It follows, in accord with Vandenbroucke et al. (2005) and contrary to Fortey et al. (2000; see also Davies et al., 1997, 2003), that the base of the Pusgillian and possibly the Cautleyan may lie well below the base of the Nantmel Mudstones Formation throughout mid Wales.", "Unfortunately the long ranging chitinozoans obtained from comparable statigraphical levels in the east of the basin (Fig. 9) fail to aid regional correlation.", "The FAD of the index taxon for the S. fossensis biozone lies below the lowest Red Vein anoxic unit exposed on the Cardigan Bay coast (below LH0) but its FAD in the east, in the Bryn Nicol Formation type section, occurs above one such anoxic (LH2).", "At face value this points either to some form of ecological control, or it implies that the Red Vein anoxic units are diachronous and cannot be correlated across the basin.", "In addition, Spinachitina ?", "bulmani sensu Achab (1977a), considered a key associated taxon of the S. fossensis biozone by Vandenbroucke et al. (2005), enters alongside S. fossensis in the west, but is absent from eastern sections; and the distinctive Hercochitina aff. normalis enters alongside S. fossensis in the east, but first appears on the coast in a sample from LH1.", "Further sampling of this section of stratigraphy appears necessary before these inconsistencies can be resolved or fully understood.", "The chitinozoans present in the lower part of the Cribarth Formation include taxa found in the S. fossensis and B. umbilicata biozones of the Cardigan Bay section and in the Tywi Anticline Bryn Nicol section such as Spinachitina coronata and Hercochitina cf. longi.", "Other taxa are recorded for the first time in the Welsh Basin such as Hercochitina sp.", "A sensu Vanmeirhaeghe (2006) and Belonechitina capitata.", "The base of the Belonechitina cf. gamachiana biozone is marked by the appearance of the index taxon in the upper part of the Cribarth Formation at Glasallt Fawr in the Llandovery area where the trilobite B. cf. robusta confirms its Rawtheyan age (see above).", "Along the Cardigan Bay coast the taxon's first appearance in the second Red Vein anoxic (LH1) is associated with anceps Biozone graptolites.", "These findings clearly show that Belonechitina cf. gamachiana enters well below the local base of Hirnantian strata.", "The base of the newly-defined local B. llangrannogensis n. sp. biozone is recorded from the base of the lower Yr Allt Formation in both the Cardigan Bay and eastern sections allowing good correlation and offering confirmation of the early Hirnantian age for this formation", "S. taugourdeaui is recorded in the Hirnant Limestone in the historic type area of the UK Hirnantian Stage (Vandenbroucke et al., 2008a).", "This distinctive formation is widely viewed as deposited during a Hirnatian glacial maximum (e.g. Brenchley and Cullen, 1984).", "However, all records of Spinachitina cf. taugourdeaui in the Garth and Llandovery areas (this study; also Davies et al., 2013) are from post-glacial transgressive facies (Garth House Formation) that pre-date the FAD of persculptus biozone graptolites in Wales (Davies et al., 2009).", "Pending either a re-interpretation of the depositional history of Hirnant Limestone, or the discovery of S. taugourdeaui from strata acknowledged as transgressive, the possibility exists that the S. taugourdeaui Biozone established in Cwm Hirnant pre-dates the Spinachitina cf. taugourdeaui Biozone of this study.", "The absence of both S. taugourdeaui and Spinachitina cf. taugourdeaui precludes any chitinozoan-based correlation with the Cardigan Bay area.", "Recognition of the T. bergstroemi, S. fossensis, and B. umbilicata biozones allow direct correlation with other Avalonian sections in the Cautley District and in Belgium, where, the T. bergstroemi, S. fossensis and B. umbilicata biozones have also been recognised (Vanmeirhaeghe and Verniers, 2004; Vanmeirhaeghe, 2006).", "Outside Avalonia, the presence of the T. bergstroemi and B. umbilicata biozones provide correlation with Baltoscandia whereas the B. cf. gamachiana and S. cf. taugourdeaui biozones carry the potential of correlation with Laurentia and Baltoscandia.", "The B. cf. gamachiana biozone in Wales may also demonstrate further similarities of the Avalonian chitinozoan composition with that of Laurentia despite Vandenbroucke (2008a) considering a Baltoscandian signature to be dominant.", "Furthermore, Vandenbroucke (2008a, 2008b) also records H. cf. gamachiana and H. aff. gamachiana from British Avalonia in Cwm Hirnant and Rhayader but until unambiguous material is recovered from Anglo-Welsh sections in Avalonia, this suggestion remains open to speculation.", "The C. rugata biozone has not been identified in the Welsh Basin in the current study, but has been recognised elsewhere in Avalonia, from the Cautley district in northern England (Vandenbroucke et al., 2005) and the Condroz Inlier in Belgium (Vanmeirhaeghe and Verniers, 2004) where it occurs below the S. fossensis biozone (Vandenbroucke, 2008a).", "The failure of the current study to confirm this biozone may reflect the low sampling resolution below the base of the S. fossensis Biozone in the Nantmel Mudstones Formation.", "However, Kaljo et al. (2008) record B. umbilicata from the rugata biozone from the Kaugatuma core implying correlation of the Welsh Basin B. umbilicata biozone with the upper part of the Baltica C. rugata biozone.", "Hercochitina cf. longi, recorded from the Nantmel Mudstones Formation in the B. umbilicata biozone in the Cardigan area and from the S. fossensis biozone in the Llandovery area provides further support for correlation with the Avalonian S. fossensis biozone and with the upper Katian (upper Rawtheyan) Hercochitina florentini–Conochitina concava biozone of North America (Laurentia, Achab et al., 2013).", "Hercochitina cf. longi occurs below the first appearance of B. cf. gamachiana in keeping with the Herochitina florentini–Conochitina concava biozone as defined by Achab et al. (2013).", "Hercochitina minuta, recorded in this study in the fossensis biozone, is also mentioned as being an associated taxon of the Tanuchitina anticostiensis biozone, which underlies the Hercochitina crickmayi biozone (Achab, 1977b).", "Within the T. anticostensis biozone, H. minuta is succeeded by Hercochitina normalis, each taxon defining a subzone.", "In the Cardigan–Llangrannog region, H. cf. normalis is reported above H. minuta in the umbilicata biozone implying that, if the presence of these two taxa does represent a direct correlation with Laurentian zonation scheme, then the T. anticostiensis and H. florentini–C. concava zones may represent the correlative equivalents of the fossensis–umbilicata zones in Avalonia.", "The upper Katian (upper Rawtheyan) A. merga biozone, reported from Laurentia, Gondwana, and Avalonia, is not recognised in the Welsh Basin but has been recorded from Avalonia in the Cautley District in England (Vandenbroucke, 2008a).", "Correlation of the lower Hirnantian within Avalonia and to other palaeocontinents has previously been inhibited by the lack of a consistent discrete range-defining taxon.", "Vandenbroucke et al. (2008a) and Vandenbroucke and Vanmeirhaeghe (2007) recognised that Cyathochitina spp. and Ancyrochitina spp. become abundant in the uppermost Ordovician and lower Silurian and demonstrated this from analysis of samples taken from Cerrig Gwinion Quarry.", "This pattern is also reported from the Llandovery sections in this study (Fig. 9, Table 3), but these taxa are either long-ranging (Cyathochitina spp.) or too poorly preserved (Ancyrochitina spp.) to be utilised for biostratigraphical purposes.", "Following his study of the historic type area for the Hirnantian at Cwm Hirnant, Vandenbroucke et al. (2008a) recognised that Belonchitina sp. 11 (Belonchitina llangrannogensis n. sp. of this account) may prove a worthy regional zone fossil if more assemblages could be recovered from other Hirnantian horizons.", "This species, commonly in association with S. taugourdeaui, was recovered by Vandenbroucke (2008b) and Vandenbroucke et al. (2005) from Hirnantian sections in the Cautley district and the Welsh Basin (Hirnant Limestone) (Vandenbroucke et al., 2008a) and from the Brabant Massif in Belgium (Vanmeirhaeghe, 2006).", "These studies appear to reinforce a restricted stratigraphical and palaeogeographical range of B. llangrannogensis n. sp., which, at the time of writing, is unknown outside Avalonia.", "H. crickmayi was considered to define a biozone in Laurentia until Achab et al. (2013) recognised three new Hercochitina species Hercochitina florentini, Hercochitina longi and Hercochitina changi and erected the Hercochitina florentini–Clathrochitina concinna biozone defined by the coexistence of the two taxa.", "Achab et al. (2013) erected the zone as an assemblage zone to circumvent the difficulty in identifying species of Hercochitina, especially when they are poorly preserved.", "The Welsh Basin Hercochitina material is poorly preserved and identification of hercochitinids is made here on shape of test, size range and on what ornamentation is present (see Appendix I).", "This has regrettably necessitated placing several Hercochitina taxa in open nomenclature thus reducing the certainty of correlation outside of the study area, for instance correlating the Belonechitina cf. gamachiana Biozone with the H. gamachiana biozone in Laurentia and Baltica.", "This is unfortunate given that the chronostratigraphic position of the base of the B. gamachiana biozone is disputed.", "Achab et al. (2011, 2013) infer that the base of the B. gamachiana biozone lies within the Hirnantian.", "Vandenbroucke (2008a) also records H. cf. gamachiana from the Hirnant Limestone Member of the Foel-y-Ddinas Mudstone Formation in Wales which is unequivocally of Hirnantian age.", "However, in Baltoscandia Kaljo et al. (2008) and Hints et al. (2004) consider it to lie in the upper Katian Pirgu Stage.", "In the Cardigan Bay section, B. cf. gamachiana occurs in Red Vein anoxics LH1 and LH3 in association with graptolite assemblages of the Rawtheyan anceps biozone (Williams, 2001a, 2001b).", "Near Llandovery B. cf. gamachiana is present in the Cribarth Formation alongside the graptolite O. abbreviatus and associated with the trilobite B. cf. robusta (see above); co-occurrences that are again consistent with a pre-Hirnantian, late Katian (late Rawtheyan) age for the local base of the B. cf. gamachiana biozone.", "This can be seen either to support a late Katian age for the base of the Laurentian–Baltoscandia H. gamachiana biozone (e.g. Nõlvak, 1999; Hints et al., 2004; Kaljo et al., 2008) contrary to Achab et al. (2011, 2013), or, alternatively, to suggest that the FAD of a precursor to the index taxon allows the underlying S. fossenis biozone to be subdivided.", "The latter interpretation implies the coincidence of the Laurentian–Baltoscandia B. gamachiana biozone with the Avalonian B. llangrannogensis n. sp. biozone here erected.", "Until further investigations have documented the stratigaphic and taxonomic relationship of B. cf. gamachiana to B. gamachiana, this remains speculative.", "Similarly, further study in Wales is needed to assess the taxonomic and stratigraphic relationships between S. taugourdeaui (from the Cwm Hirnant section by Vandenbroucke et al., 2008a) and Spinachitina cf. taugourdeaui, and to assess the possibility of the latter occurring in slightly younger rocks than the former.", "Nevertheless, pending further sampling and taxonomy, the Welsh Basin is one of the rare areas where we can (realistically) expect to find better preserved specimens of B. (cf.) gamachiana and S. (cf.) taugourdeaui to study in sections with graptolite control.", "Several taxa appear to occur anomalously high in the Nantmel Mudstones Formation in the Cardigan Bay section, for example Cyathochitina cf. reticulifera (sample TJC D941) and Saharochitina cf. fungiformis (samples TJC D941, TJC D957).", "These out of context taxa are likely to have been reworked from older sediments and this is consistent with the turbiditic origin of the bulk of the Welsh basin succession (Davies et al., 1997, 2003; Schofield et al., 2004).", "Reworking is potentially more of an issue in the Hirnantian (Paris et al., 2007).", "In Wales, the deep erosion of sediment during the associated glacioeustatic lowstand is dramatically indicated by the presence of a pronounced unconformity at the base of the Cwm Clyd Sandstone Formation in Crychan Forest (Davies et al., 2009).", "Three taxa, Eisenackitina inconspicua, Desmochitina juglandiformis and Lagenochitina cf. ponceti, are notably out of place in Hirnantian rocks of the Llandovery region and in the Cardigan Bay section Desmochitina cocca is also out of stratigraphic context.", "A regional chitinozoan biozonation scheme has been developed that provides confirmation and allows refinement of the recently constructed Avalonian chitinozoan biozonation scheme of Vandenbroucke (2008a) (Fig. 11).", "It identifies the Avalonian T. bergstroemi, S. fossensis and B. umbilicata biozones in the middle–upper Katian of the Welsh Basin.", "The lowest biozone reported in this study is the T. bergstroemi biozone.", "The underlying F. spinifera biozone is not identified in the Nantmel Mudstones Formation in either the Cardigan Bay or more eastern sections.", "This implies, as proposed by Vandenbroucke et al. (2008b), that the base of the Ashgill (mid-Katian) is lower than the lithostratigraphic boundary that marks the onset of basin oxygenation (base Nantmel Mudstones Formation).", "The C. rugata biozone, which elsewhere overlies the T. bergstroemi biozone, is not recognised in the Welsh Basin in this study although the index taxon has been identified.", "At a higher level in the stratigraphy, the presence of the B. cf. gamachiana and S. cf. taugourdeaui biozones implies a link to Laurentian and Baltoscandian chitinozoan provinces.", "However, the base of the B. cf. gamachiana biozone within the upper Nantmel Mudstones Formation on the Cardigan Bay coast and at a comparable level (upper Cribarth Formation) in the Llandovery area is associated with late Katian (Rawtheyan) graptolites (anceps biozone) and trilobites.", "Further sampling is recommended to validate the presence and lithostratigraphic position of B. gamachiana in the Welsh Basin.", "A new lower to middle Hirnantian regional biozone, the B. llangrannogensis n. sp. biozone, has been defined and typifies rocks that record the local impact of the coeval glacioeustatic regression.", "This new biozone enables correlation between other Avalonian sections in the Anglo-Welsh region and in Belgium.", "This study has found Spinachitina cf. taugourdeaui within upper Hirnantian strata deposited during a post-glacial maximum rise in sea level and pre-dating the entry of the persculptus biozone graptolites in Wales.", "These occurrences appear to post-date assemblages with S. taugourdeaui recognised in the historic type section for the UK Hirnantian Stage.", "They point to the need to confirm the sedimentary interpretation of this section and to establish the relationships between S. taugourdeaui and Spinachitina cf. taugourdeaui within the Welsh Basin.", "Chitinozoan assemblages have contributed to the improved correlation of the basin centre Cardigan Bay succession with sections in the east that record deposition at the basin margin and on its shelf.", "This demonstrates the growing effectiveness of Lower Palaeozoic chitinozoan biozonal schemes as correlative tools across a range of palaeogeographical and palaeoecological settings.", "However, distinct differences in the FADs of some taxa are apparent, notably the index taxa for the S. fossensis and S. cf. taugourdeaui biozones.", "In implying some palaeoecological constraint on their distribution, this urges caution in the selection of the taxa employed in the erection and definition of the evolving Avalonian chitinozoan biozonal scheme.", "In the Welsh Basin B. llangrannogensis n. sp. (as defined herein) appears to have a restricted stratigraphical range.", "An eponymous biozone was not recognised in Avalonia by Vandenbroucke (2008a), but the taxon (Belonechitina sp. 11 of Vandenbroucke, 2008a, 2008b) was noted to have the potential for biostratigraphical use.", "At Hirnant Quarry in the north Wales it occurs with S. taugourdeaui and in Cerrig Gwinion Quarry (Tywi Anticline), it occurs in the upper Yr Allt Formation below the Mottled Mudstone Member FAD of persculptus biozone graptolites (Vandenbroucke et al., 2008b).", "In the Cardigan Bay area it ranges from the lowermost Yr Allt Formation at Traeth Penbryn (sample TJC D1005), where it enters above the local last appearance of B. cf. gamachiana, into the upper Yr Allt Formation at Llangrannog (sample TJC D946).", "It appears to have a more restricted range in eastern sections first appearing in the lower Yr Allt Formation (sample TJC D980, Dolaucothi M8 Borehole) and ranging no higher than the Glasallt-Fawr Sandstone Formation in Glasallt Fawr Wood (sample TJC D978).", "Within the Welsh Basin, the partial range of B. llangrannogensis n. sp. defines a local range biozone corresponding to the FAD of B. llangrannogensis n. sp. to the FAD of Spinachitina cf. taugourdeaui, the index taxon of the overlying biozone.", "Its occurrence between the B. cf. gamachiana and S. cf. taugourdeaui biozones implies an early Hirnantian age for this newly recognised biozone." ]

[ "A new composite Katian-Hirnantian chitinozoan biozonation for the Welsh Basin, UK.", "Three new species of chitinozoan are defined.", "The Welsh Basin chitinozoan assemblage implies Laurentian-Avalonian faunal links.", "Belonchitina cf. gamachiana occurs in the anceps Biozone." ]

[ "MEDI" ]

[]

[ "2731" ]

[ "The Blue Mountains Eye Study was approved by the Human Research Ethics Committee of the University of Sydney for investigation of the epidemiology and genetics of ocular disease.", "The BMES has been described previously.10", "Briefly, the BMES is a population-based study of individuals living in the Blue Mountains region west of Sydney, Australia.", "Any permanent, noninstitutionalized resident of the defined geographic region born before January 1, 1943 (aged over 49 years at time of recruitment) and able to give written informed consent was eligible for enrollment in BMES and was contacted by door-to-door canvassing.", "Participants underwent a baseline visit, with follow-up at 5 years and at 10 years.", "At baseline, all participants received a detailed eye examination, including applanation tonometry, suprathreshold automated perimetry (Humphrey 76-point test, followed by 30-2 fields [Humphrey Visual Field Analyser 630 with StatPac 2, Humphrey Instruments, Inc, San Leandro, California, USA]), and stereoscopic optic disc photography (Carl Zeiss Australia, Sydney, New South Wales, Australia).", "The current sub-study consisted of a case-control design from within the BMES cohort study.", "Participants with normal threshold or suprathreshold field tests and no sign of glaucoma at the baseline visit were included in the current study.", "Participants with OAG at baseline (prevalent OAG) were excluded.", "As previously reported,11 incident OAG cases were defined as participants free of OAG at baseline who showed glaucomatous field loss on full-threshold perimetry (Humphrey 24-2 or 30-2), which matched the optic disc appearance, at either the 5-year or 10-year follow-up visit, without reference to intraocular pressure.", "Patients with pseudoexfoliation syndrome were not excluded (n = 7).", "DNA was extracted from peripheral whole blood using standard techniques.", "Genotyping was performed on the HumanHap670 array (Illumina, San Diego, California, USA) as part of the Wellcome Trust Case Control Cohort 2 Genome-Wide Association Study.", "Data were cleaned and genotypes called as previously described.12", "No significant population stratification was detected in this population.12", "Single nucleotide polymorphisms (SNPs) were selected for analysis if they had been previously reported to be associated with OAG (including normal tension glaucoma) at genome-wide significance in white populations.", "The reported SNPs with the smallest P values at each locus were chosen for this analysis.", "In the case of the 9p21 locus reported independently in 2 papers,7,9 the top SNP from each paper was chosen, as well as a third SNP at genome-wide significance in the replication cohorts of Burdon and associates (rs1412829).7", "We hypothesize that if this SNP had been typed in the discovery cohort for this study, it would likely have been the top-ranked SNP at this locus.", "Seven SNPs at 5 loci were chosen for analysis in total.", "Power calculations were conducted with the Genetic Power Calculator.13", "This study had 77% power to detect an association at a SNP with an allele frequency of 30% and an odds ratio of 1.6 under an additive model at a P value of .007, assuming a population disease prevalence of 5.67%.14", "These parameters are similar to those reported for most of these loci in cross-sectional studies of OAG genetics.", "Differences in the demographics of the available cohort were assessed using IBM SPSS Statistics V20.", "Association analysis was conducted under a univariate allelic model and also using logistic regression under an additive model adjusted for baseline measurements of age, sex, mean IOP of both eyes, mean cup-to-disc ratio of both eyes, mean disc diameter of both eyes, and systolic and diastolic blood pressure using Plink.15", "Statistical significance was set to P < .007 under a Bonferroni correction, to account for the 7 SNPs tested.", "One associated SNP from each significant or nominally significant locus and the clinical variables were included in a logistic regression model using IBM SPSS Statistics V20.", "SNPs were coded to the number of OAG risk alleles carried by each participant at each SNP (0, 1, or 2).", "Collinearity between variables in the model were assessed by calculating the tolerance and the variance inflation factor (VIF).", "No collinearity was detected (no VIF >2).", "The rank importance of each model component was also assessed using a large population of neural networks (produced using Matlab; The MathWorks, Inc, Natick, Massachusetts, USA).", "A neural network can be thought of as a small machine capable of learning.", "It is trained by exposure to a dataset comprising inputs (for example, the characteristics of horses in a race) and outputs (the winning horse).", "After each round of training, the link strengths within the network are changed, and further training is undertaken until its predictive performance on a previously unseen “validation” dataset no longer improves.", "The resulting network's performance is then measured using a final, also unseen “test” dataset.", "In this study, each neural network drew its inputs from unique subset of 7 SNPs and 7 clinical variables (age, sex, diastolic and systolic blood pressure, cup-to-disc ratio, IOP, and disc diameter).", "To cover all possible permutations of these 14 inputs, 16 383 neural networks were required.", "Each neural network was trained and tested with a cohort comprising glaucoma patients (n = 67) and an equal number of randomly selected controls: 70% of the cohort was used to train the network, 15% to validate its performance during training, and the remaining 15% were unseen during training and were used to test the final performance of each network.", "Each neural network was trained and tested 20 times.", "In separate analyses, controls were either age matched to within 2 years of incident cases or not age matched.", "An error score was calculated to describe the performance of each neural network: the error score was the proportion of cases incorrectly classified (as glaucoma or as not glaucoma) by the network.", "Ranking of the importance of input variables (clinical parameters and SNPs) was achieved by ranking their influence on neural network error score.", "If the presence of a particular SNP or clinical variable (among the neural network's input variables) reduced the error score, that SNP or variable can be considered to make a positive contribution to the performance of the network (ie, it is of useful predictive value).", "The BMES cohort consisted of 1986 individuals with follow-up phenotype data at either the 5-year, 10-year, or both visits with genotypes available (Table 2).", "Of the 1986 participants, there were 67 incident OAG cases over the full 10-year follow-up period.", "At baseline, the incident OAG cases were significantly older than controls (P < .001) and had a higher proportion of female subjects (P = .009).", "IOP and VCDR at the baseline visit were also significantly different between those who later developed OAG and those who did not (Table 2), as was systolic blood pressure.", "These features of this cohort have been previously reported.11", "Association analysis indicates that incident OAG was associated with SNPs at 3 of the 5 loci tested (Table 3).", "Significant association under an allelic test was seen at rs1412892 (P = .006) at the 9p21 locus as well as rs10483727 (P = .004) at the SIX1/SIX6 locus.", "Additional SNPs at 9p21 and also at TMCO1 were nominally significant but did not survive after correction for multiple comparisons.", "The SNPs at the 8q22 and CAV1/CAV2 loci did not show association with incident glaucoma.", "Adjustment for covariates under an additive genetic model showed association at the same SNPs, although only SIX1/SIX6 remained significant after correction for testing 7 SNPs (P ≤ .007) (Table 3).", "When all covariates and the 3 associated loci (TMCO1, 9p21, and SIX1/SIX6) were included in a single regression model, all variables except blood pressure contributed significantly to the model (Table 4).", "The population of neural networks was used to compare the rank importance of variables in the predictive model both with and without age matching between controls and incident cases (Table 5).", "As expected, when not age matched, vertical cup-to-disc ratio, age, and intraocular pressure rank the highest for predicting incident OAG.", "The top-ranked SNP in this analysis is at the SIX1/SIX6 locus, which also showed the strongest genetic association.", "When cases and controls were closely age matched the rank order of variables changed, likely indicating an interaction between age and the other variable, although vertical cup-to-disc ratio and intraocular pressure are still the most predictive variables.", "In this situation the SNP at the TMCO1 locus was most predictive.", "Of note, in both analyses, all SNPs significantly associated with incident OAG under the traditional statistics contribute positively to the neural network and improve its ability to predict incident OAG.", "The selected loci have been discovered through genome-wide association studies for OAG (SIX1/SIX6, CAV1/CAV2),8,9 advanced OAG (9p21, TMCO1),7 or normal tension glaucoma (9p21, 8q22).9", "In addition, variation at TMCO1 has been associated with intraocular pressure,16 while 9p21 and SIX1/SIX6 are associated with cup-to-disc ratio17 in normal individuals.", "We provide evidence for association at SIX1/SIX6, 9p21, and nominally at TMCO1 with incident OAG.", "Thus, loci associated with advanced glaucoma and relevant biometric traits are also associated with the initial onset of OAG (incidence).", "Those SNPs discovered in previous cohorts with typical (nonadvanced) OAG are not found to be associated with OAG incidence in our cohort, although power to detect weaker associations or those at rarer SNPs is limited.", "The association of sex with incident OAG in the cohort has been previously reported,11 as has the higher-than-expected level of hypertension in the BMES cohort.18,19", "The current cohort was sufficiently powered to detect an odds ratio of ∼1.6.", "This is larger than those observed in the original discovery cohorts of cross-sectional (prevalent OAG) patient recruitment, although significant effects were still observed in this study, suggesting that the SNPs may be more important in predicting disease onset than progression, or that the true effect size is larger than previously reported.", "However, larger prospective cohorts will be needed to properly assess the 8q22 and CAV1/CAV2 loci in particular.", "A nominal association was observed at TMCO1.", "This SNP has a lower allele frequency than others in the study (11% in controls) and the finding did not reach significance here in the context of multiple testing, owing to the lower power of this study (∼36%) to detect an effect at the minor allele frequency of 11%.", "We have previously reported an association of this locus with prevalent OAG in the BMES cohort with odds ratio (OR) = 1.57, P = .022.7", "The odds ratio for incident OAG reported in the current study was larger (OR = 1.74, P = .013) despite the smaller sample size.", "We thus conclude that TMCO1 is also confirmed to be associated with incident OAG.", "The current study shows that OAG loci that are associated with OAG-relevant ocular parameters (cup-to-disc ratio and intraocular pressure) are specifically associated with OAG incidence independently of other known risk factors.", "This suggests that these loci are responsible at least in part for the initiation of OAG, consistent with their role in determination of these risk factor traits, which are themselves predictive for OAG development.", "We show also that the loci specifically associated with advanced glaucoma may also be important in initiation of OAG, and thus could be important in risk stratification among glaucoma suspect and early glaucoma patients.", "The multivariate logistic regression model indicates that mean vertical cup-to-disc ratio at baseline is by far the most significant predictor of incident glaucoma in this cohort, while in the context of other parameters blood pressure contributes almost nothing to this model.", "Although vertical cup-to-disc ratio is a well-recognized parameter in the prediction of OAG risk, the accuracy of prediction based solely on this parameter is poor owing to disc appearance in preclinical and early glaucomatous damage overlapping with the normal range of this trait.", "Predictive accuracy for the individual patient should be improved by the inclusion of other variables, including genetics.", "With the genetics tools available at this time, discriminatory power above and beyond that achievable with clinical risk factors is minimal; however, ongoing research uncovering the genetic basis of OAG is likely to lead to better risk prediction models.", "Neural networks allow an alternative approach to estimating the usefulness of clinical and genetic variables in predicting incident glaucoma.", "Input variables that are predictive of incident glaucoma naturally benefit the performance of the network.", "However, we see that those variables of trivial or no predictive value negatively affect the performance of the network: their inclusion necessarily makes the network structure more complex, which will lead to increased noise in the network.", "Neural networks are therefore helpful in distinguishing those patient characteristics that might help the clinician to predict glaucoma incidence and those that will merely overload him or her with unhelpful information.", "This approach could easily be expanded to larger datasets where specific combinations of variables that are particularly beneficial might become apparent.", "The matching of age (an important OAG risk factor) between cases and controls in the neural network analysis resulted in the TMCO1 SNP, rs4656461, becoming the highest-ranked genetic variable.", "This is consistent with a previously reported finding of the association of this SNP with age of onset of OAG.20", "Each of the associated SNPs in the logistic regression model also contributed positively in the neural network.", "Thus, the combination of IOP, disc parameters, and genotype at-risk SNPs could improve the accuracy of OAG risk prediction, which in turn will inform early treatment decisions for those most likely to develop this blinding disease." ]

[]

[ "MULT" ]

[ "Chicken", "Peptidome", "Sperm", "Top-Down HRMS" ]

[ "1000" ]

[ "This dataset consists of a compendium of peptidoforms and small proteoforms extracted from chicken ejaculated sperm, that were pre-fractionated using gel filtration or reverse phase chromatography and identified through a Top-Down mass spectrometry analysis.", "Hence, this set includes data regarding the identity of chicken sperm intact peptides and proteins as well as some structural biologically relevant information like N-terminal amino acids or post-translational modifications.", "See Figs. 1 and 2 and Supplementary Table S1.", "This dataset was produced with the objective of identifying by Top-Down mass spectrometry the peptides and small proteins contained in chicken sperm cells [1].", "Sperm cells (200 μL) protein extraction was performed by sonication in 400 µL of 6 M Urea 50 mM Tris–HCl pH 8.8 buffer containing protease inhibitor cocktail (Roche, Switzerland).", "Samples were centrifuged (45 min at 13,000 rpm and 4 °C) and supernatants containing the extracted proteins were kept for further analysis.", "The protein content was determined using a Bradford assay (BioRad, Marnes-la-Coquette, France).", "One mg of the extracted peptides/proteins were subjected to fractionation through chromatographic separation on an UltiMate 3000 RSLC system controlled by Chromeleon version 6.80 SR13 software (Thermo Scientific Dionex, Sunnyvale, USA) using reversed phase (RP) and gel filtration (GF) chromatography as described elsewhere [1].", "A total of 35 and 65 fractions were obtained after RP and GF chromatography, respectively.", "All fractions obtained after fractionation were then analyzed by on-line micro-liquid chromatography tandem mass spectrometry (µLC-MS/MS) on a dual linear ion trap Fourier Transform Mass Spectrometer (FT-MS) LTQ Orbitrap Velos (Thermo Fisher Scientific, Darmstadt, Germany) coupled to an Ultimate® 3000 RSLC Ultra High Pressure Liquid Chromatographer (Thermo Scientific Dionex, Sunnyvale, USA) controlled by Chromeleon Software (version 6.8 SR11; Thermo Scientific Dionex, Sunnyvale, USA).", "Proteo/peptidoform identification and structural characterization were performed using ProSight PC software v 3.0 SP1 (Thermo Fisher Scientific, Darmstadt, Germany).", "The detailed procedure followed for this analysis is described elsewhere [1].", "Raw data derived from Top-Down analysis have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002768.", "In total, 1038 intact or fragment protein masses were detected by Top-Down mass spectrometry (Supplementary data Table S1).", "A total of 447 biomolecules were detected in RP-derived fractions and 591 biomolecules in GF-derived fractions.", "From all the identified biomolecules, 65 were identified using both fractionation methods, while the rest was identified uniquely after RP or GF chromatography separation.", "The distribution of molecular weight and isoelectric point of the identified masses after each pre-fractionation method is represented in Fig. 1A and B, respectively.", "In order to identify which cell compartments/functions/pathways were mainly represented by the data set, a systems biology analysis was performed.", "In brief, the UniProtKB accession numbers from all m/z masses that were confidently identified through Top-Down analysis were recovered and listed.", "From these, the official human gene symbols (HuGO Gene Nomenclature Committee) were retrieved from Gallus gallus annotated proteins, whereas uncharacterized proteins were mapped to the corresponding Homo sapiens orthologs by identifying the reciprocal-best-BLAST hits.", "When manual annotation was needed (i.e. for deleted entries), this was performed by BLASTp (E value ≤e−04), together with Gene Ontology (GO) functional annotation.", "Because far more human genes are annotated and more information in databases is available for humans than for chicken, the human background was employed when possible.", "The functional classification tool from the Database for Annotation, Visualization and Integrated Discovery (DAVID version 6.7) website (https://david.ncifcrf.gov/home.jsp) was employed to group proteins contained in our dataset based on functional similarity (Supplementary Data Table S2).", "Functional analysis was also performed using the Panther Functional Classification System (http://pantherdb.org/) to evidence the most represented molecular functions (Fig. 2A) and biological processes (Fig. 2B).", "The functional analysis was completed using the “Set Distiller” module of GeneDecks (http://genecards.weizmann.ac.il/v3/index.php?path=GeneDecks; Supplementary Data Table S3).", "The data presented here consist in a list of intact and unmodified (by chemical treatment) endogenous low molecular weight biomolecules (<15 kDa) present in chicken sperm.", "This dataset can be further employed as reference for other studies focused on the research of sperm cells." ]

[]

[ "NURS" ]

[ "Cognitive impairments", "Dementia", "Hospitalization", "Length of stay", "Mortality", "Older adults", "Patient readmission" ]

[ "2900" ]

[ "Emergency hospital admissions of older people are increasing globally, and are likely to continue to rise given current demographic trends.", "Older adults now comprise around two-thirds of hospital inpatients, and up to 50% of these patients have some form of cognitive impairment, including that related to dementia (Fogg et al., 2017; Goldberg et al., 2012; Jackson et al., 2016; Reynish et al., 2017; Sampson et al., 2009).", "This epidemiologic transition needs to be reflected in improved knowledge about hospital outcomes for this group, and subsequently in the evidence base of innovations for appropriate and effective care.", "Older adults with a diagnosis of dementia are more likely to die during an emergency hospital admission (Hsiao et al., 2015; Marengoni et al., 2013; Oreja-Guevara et al., 2012; Sampson et al., 2009).", "However, it is not clear whether dementia is an independent factor leading to increased mortality, or if other aspects of clinical presentation, patient characteristics or care pathways are responsible for the observed outcomes.", "An integrative review found that known predictors for deterioration and mortality, for example severity of illness, multimorbidity or risk of malnutrition (Henderson et al., 2008; Smith et al., 2013; Steventon et al., 2018; Stratton et al., 2006), were not consistently adjusted for in observational studies exploring this relationship (Fogg et al., 2018).", "There is also limited and discrepant information regarding the impact of cognitive impairment in the absence of a diagnosis of dementia on outcomes of hospitalisation.", "Several large cohorts have described similar frequencies of hospital mortality and lengths of stay in patients with cognitive impairment compared to patients with a diagnosis of dementia, but the role of cognitive impairment as an independent risk factor has not yet been explored (Connolly and O’Shea, 2015; Fogg et al., 2017; Reynish et al., 2017).", "Additionally, mortality in the post-discharge period and early re-admissions are important outcomes for this population, as these may be influenced by increasing pressure for hospitals to shorten patient stays, and the variability of care available outside acute hospitals.", "Understanding whether cognitive impairment is an independent risk factor for poor hospital outcomes could encourage the timely identification of these patients, thereby enabling planning of appropriate care within and outside hospital.", "Systematic screening to identify cognitive impairment in older hospitalised patients is becoming more widespread in the UK and elsewhere, with the aim of identifying people with potentially undiagnosed dementia, improving rates of dementia diagnosis, planning appropriate post-hospital care and detecting delirium (Alzheimer’s Australia, 2014; National Institute for Health and Care Excellence, 2010; NHS England, 2014).", "If cognitive impairment in the absence of a dementia diagnosis also contributes directly to poor hospital outcomes, appropriate care solutions need to be devised and applied.", "This study aimed to identify whether, after adjustment for known risk factors for poor outcomes, including primary diagnosis, severity of illness, age, comorbidities and risk of malnutrition, there are independent associations between cognitive impairment (with or without a diagnosis of dementia) and each of: mortality (in-hospital or within 30 days of discharge), days until hospital discharge, and readmission to hospital within 30 days of discharge.", "A retrospective cohort study using pseudonymised, routinely collected electronic healthcare records.", "A district general hospital in England, with a catchment of approximately 675,000 people.", "Acute, non-elective incident admissions of people aged ≥75 with at least one cognitive screening performed (>80% coverage), who were admitted and discharged between 29th January 2014 and 31st March 2017 inclusive.", "Patients aged ≥75 with unscheduled admissions were routinely screened for cognitive impairment as part of clinical care by trained staff, and results recorded using an electronic tool (CareFlow Vitals, System C, London).", "Patients with an existing diagnosis of dementia were identified from their medical history.", "In the absence of a diagnosis of dementia, the following screening questions were completed, based on clinical assessment, history from the patient, the patient’s carers or relatives, or from accessing medical notes: (1) “Is the patient exhibiting disturbed behaviour?”", "(2) “Has the patient been increasingly forgetful over the last 12 months so that it has had an impact on their daily life?”", "If the answer to one or both questions was ‘yes’, an Abbreviated Mental Test Score (AMTS) was performed.", "If the patient was exhibiting disturbed behaviour, delirium was assessed and recorded.", "Demographic data, details of admission and discharge (dates, route, primary diagnosis, specialty, ward), ward transfers, date of death (up to 30 days after discharge) and diagnoses (International Classification of Disease 10) were recorded in the Patient Administration System.", "Vital signs and other clinical assessments were recorded electronically on CareFlow Vitals, which generated a National Early Warning Score (NEWS) value indicating severity of illness (Royal College of Physicians, 2012).", "Pseudonymised electronic records were extracted from operational databases and stored in Microsoft Access.", "Stata MP version 15.1 (StataCorp, College Station, Texas) was used to link datasets on an anonymous identifier and perform analyses.", "The primary explanatory variable was derived from cognitive screening data, categorising admissions into 3 groups: (1) ‘dementia’: a known diagnosis of dementia, (2) ‘cognitive impairment’: a positive response to one/both screening questions (i. disturbed behaviour, ii.", "forgetful in last 12 months) and an Abbreviated Mental Test Score of 8 or below with no known diagnosis of dementia, (3) ‘no cognitive impairment’: a negative response to both screening questions, or a positive response to one/both screening questions and an Abbreviated Mental Test Score of 9 or 10.", "Potential confounders that were controlled for in analyses are described in Table 1.", "Throughout the remainder of this paper, the term “patients with cognitive impairment” refers to patients with cognitive impairment without a diagnosis of dementia.", "Data completeness for covariates was above 99%, apart from the Malnutrition Universal Screening Tool score, missing in 31.3% (n = 6688) of admissions.", "To maintain sample size and reduce selection bias, the primary analysis included a dummy category to represent missing Malnutrition Universal Screening Tool values, and a sensitivity analysis using only records with available Malnutrition Universal Screening Tool data was performed.", "Binary variables were analysed using logistic regression, i.e. death within hospital, death within hospital or within 30 days of discharge, and re-admission to hospital within 30 days of discharge.", "Categorical variables with similar levels of association in several categories in univariable analysis, such as discharge specialty, were re-grouped for multivariable analysis.", "A forward stepwise regression approach was used for multivariable analysis, first entering variables most significant in univariable analysis, and utilising the Akaike and Bayesian information criteria (AIC, BIC) to assess variable contribution to model fit by only maintaining inclusion of variables which decreased the Information Criteria in both cases.", "Interactions between the primary explanatory variable (cognitive impairment category) and covariates (age, severity of illness (National Early Warning Score value), Malnutrition Universal Screening Tool and comorbidity) were explored by comparing the model with both covariates with a nested model including the interaction term (or terms when considering three-way interactions) and applying the Likelihood Ratio test to determine if interactions were candidates for inclusion.", "However, the same inclusion criteria of improving model fit as measure by AIC/BIC were then applied.", "The Area Under the Receiver Operating Characteristic curve (AUROC) was calculated for final models to provide a measure of usefulness of the model in predicting the outcome.", "The association between cognitive impairment and length of hospital stay was explored using a Fine and Gray model for competing risk analysis for the ‘time-to-discharge’ from hospital, with in-hospital death as the competing risk, using Akaike and Bayesian Information Criteria to assess model fit (Hutchings et al., 2018).", "As the majority of deaths and discharges occurred in the first three months of a hospital stay, follow-up duration was censored at 91 days.", "Regression modelling of the effect of covariates on the cumulative incidence frequency (CIF) were performed with the Fine and Gray semiparametric proportional hazard model for the subdistribution hazards.", "Proportionality of hazards was assessed by examining Schoenfeld residuals.", "A group of patients and carers with recent experience of hospitalisation informed the selection of descriptive characteristics and outcomes, including ward transfers and indications of end-of-life.", "They will be involved in writing a lay summary of the results and identifying avenues for dissemination to patients and the public.", "Ethical approval was obtained from the Isle of Wight, Portsmouth and South East Hampshire Research Ethics Committee, reference 08/02/1394.", "Consent from participants was not required for this study.", "Demographic, clinical and health service characteristics of the 21,399 eligible incident admissions are described in Table 2.", "Twenty-seven percent of patients (5,774/21,399) were found to be cognitively impaired at screening, of which 61.5% (n = 3547) had an existing diagnosis of dementia and 38.5% (n = 2227) did not.", "The mean age of the cohort was 84 years; 56.9% of patients were female.", "The Malnutrition Universal Screening Tool score was medium or high in 29.5% of the cohort, 54.7% had a Charlson score of 6 or more and 13.3% had a National Early Warning Score value in the medium or high risk categories on the first set of recorded vital signs.", "Most patients (87.6%) were admitted to medical wards, including 21.6% to Medicine for Older People, Rehabilitation and Stroke (MOPRS) wards.", "Of the cohort, 6.3% (n = 1343) were placed on end-of-life (EOL) pathways during admission, such that subsequent vital signs observations were not mandated.", "Patients with dementia were the least likely to have two or more ward transfers during their admission (35.4%), whereas those with cognitive impairment were the most likely (48.0%).", "There were 1704 deaths in hospital (8.0% of the cohort) (Table 3).", "Patients with cognitive impairment had the highest in-hospital mortality (12.6%), the longest lengths of stay (median 12 days) and the highest readmission rates (10.3%).", "Patients with a diagnosis of dementia had the highest mortality in the period covering hospitalisation and 30 days following discharge (20.8%).", "Patients with cognitive impairment and those with a diagnosis of dementia had significantly increased risks of mortality both during hospitalisation and within 30 days of discharge in univariable (Supplementary Table 1) and multivariable analyses (Table 4).", "Interactions between the cognitive impairment category and both the Malnutrition Universal Screening Tool score and Charlson scores were present (all likelihood ratios significant at p ≤ 0.005).", "However, including interaction terms in the stepwise regression did not improve the parsimony of the multivariable model (death in hospital AIC 10288 vs 10286, BIC 10511 vs 10732, for full model vs full model with interactions respectively; death within 30 days AIC 13754 vs 13761, BIC 13993 vs 14222), and so were excluded from the model.", "For both categories of cognitive impairment and dementia, the odds of mortality increased with increasing severity of Malnutrition Universal Screening Tool and increasing levels of co-morbidity.", "The Area Under the Receiver Operating Characteristic curve for the model for death within hospital was 0.76, and 0.77 for death in hospital or within 30 days, indicating moderate ability of the model to predict patient mortality in this population.", "The sensitivity analysis including only patients with a Malnutrition Universal Screening Tool score available showed only minor differences in the adjusted Odds Ratios.", "Patients with cognitive impairment or a dementia diagnosis had a significantly higher risk of readmission within 30 days of discharge in univariable (Supplementary Table 1) and multivariable analyses (Table 4).", "The Area Under the Receiver Operating Characteristic curve for the multivariable model was 0.58, indicating low of ability of the model to predict readmission.", "In the univariable Fine and Gray model, the cumulative incidence function of time to discharge, with death in hospital as a competing risk, was significantly lower (i.e. longer time to discharge) for patients in the cognitive impairment and dementia groups than those with no cognitive impairment (Gray test p-value <0.001).", "This relationship was maintained after adjustment in the multivariable model for patients with cognitive impairment (subhazard ratio 0.66, 95% confidence interval 0.63-0.69) and dementia (subhazard ratio 0.80, 95% confidence interval 0.76-0.83) (Table 5).", "This study aimed to assess whether older patients admitted to hospital with cognitive impairment are at higher risk from poor outcomes than those with normal cognition, after adjustment for known risk factors.", "We found independent associations between cognitive impairment and increased mortality, both within hospital and including 30 days after discharge, increased time until discharge from hospital, and increased readmission within 30 days of discharge.", "This was also the case for patients with a diagnosis of dementia.", "Additionally, the risks of mortality in hospital, re-admission and extended hospital stay were higher in patients with cognitive impairment alone than in those with a diagnosis of dementia.", "In previous research, an association between cognitive impairment and poor outcomes could not be ascertained due to key missing covariates and small sample sizes.", "This study highlights that patients with cognitive impairment are at considerably greater risk than those with normal cognition, taking into account other key factors which influence these outcomes.", "Possible mechanisms for the increased risks observed in patients with cognitive impairment include differences in effective care for people with cognitive impairment in hospital, and intrinsic mechanisms which place these patients at higher risk of deterioration.", "Variations in care that may disadvantage patients with dementia have been described in the literature for patients with a known diagnosis of dementia include reduction in routine monitoring for deterioration (Hope et al., 2017), increased time waiting for investigations (Griffiths et al., 2014), and under-treatment of concurrent conditions such as chronic obstructive pulmonary disease (Frohnhofen et al., 2011).", "It may be the case that such variations also apply to the wider group of cognitively impaired patients, especially given that these patients appeared to have even worse outcomes than those already known to have dementia.", "Higher rates of potentially preventable complications, including urinary tract infections, pressure ulcers, pneumonia and delirium, have been identified in hospitalised patients with dementia, and may provide areas of focus for nursing care for all patients with cognitive impairment (Bail et al., 2013).", "Our study also highlighted that patients with cognitive impairment were the most likely to have two or more ward moves during their hospital stay, whereas those with dementia had the least moves, possibly because staff may have endeavoured not to further disorientate them and risk onset of delirium or other behavioural issues.", "It is possible that knowing patients had a diagnosis of dementia also impacted on other areas of care in a positive manner, which may have contributed to our finding that patients in the cognitive impairment group had worse outcomes than patients with a diagnosis of dementia, as the cognitive impairment group is not yet recognised as a ‘high-risk’ group of patients.", "The elongated stays in hospital and higher rate of readmissions experienced by patients with cognitive impairment and no diagnosis of dementia are key areas for future examination, as they lead to further deconditioning and dependence for patients, and increase hospital costs.", "Using an instrument such as Comprehensive Geriatric Assessment during admission, and monitoring rates of functional and cognitive decline as hospital performance indicators may be useful to better understand their care needs and how we can best assist maintenance of their function and independence whilst in hospital and support appropriate holistic discharge planning (Ellis et al., 2011).", "It is interesting that patients with a diagnosis of dementia seemed to fare better in relation to these outcomes, possibly due to their diagnosis enabling focussed assessment and advanced care planning prior to or during hospitalisation, enabling quicker discharge and appropriate community care provision.", "However, the increased risk of mortality when including the 30 days after discharge was very similar for all cognitively impaired patients, suggesting that although care pathways may differ according to known diagnoses, the impact of acute illness and hospitalisation on these patients is equally significant.", "The current emphasis on screening older patients for cognitive impairment in hospital to detect possible cases of dementia and refer to memory assessment clinics (National Collaborating Centre for Mental Health, 2018; NHS England, 2014) may need to be bolstered by clear guidelines on additional assessments and hospital care for those that are identified.", "These could explicitly include patients with cognitive impairment, who have not yet been assessed for dementia.", "What this would actually comprise is currently uncertain – although ‘best practice’ guidelines for patients with dementia, including prevention of delirium, may be considered (Alzheimer’s UK, 2016; National Institute for Health and Care Excellence, 2010), there is limited evidence that these are effective in improving outcomes such as mortality (Goldberg et al., 2013).", "People with a known diagnosis of dementia have lower conveyance rates to hospital (Pocock et al., 2018), and although admission avoidance is not appropriate in all cases, assessment of cognitive impairment in the community is important to identify people who may benefit from community-based care where possible.", "Achieving this may require additional support for people with cognitive impairment in the community, who, without a diagnosis of dementia, would not benefit from the services of Dementia Care Co-ordinators or other interventions (National Collaborating Centre for Mental Health, 2018).", "Although hospitals do refer patients with cognitive impairment for monitoring in General Practice within six weeks of discharge, it is unknown how often this referral is acted upon, and the high rate of re-admissions in this group suggests that this may be too long an interval prior to General Practitioner re-assessment, both for cognitive issues and for concurrent medical conditions.", "This study utilised a large dataset reflective of routine care in a general district hospital, which has comparative mortality and performance statistics representative of similar institutions in NHS England.", "The demonstration of known predictors of mortality in the final models increase confidence in the validity of the data and the study conclusions as regards the additional independent risk posed by presence of cognitive impairment and dementia.", "In contrast to previous literature, the use of a competing risks model to evaluate length of stay removes biases introduced by considering death as a form of discharge.", "Data on frailty or function, which have also been related to poor outcomes (Dani et al., 2018; Kojima et al., 2018), were not available as these are not currently measured systematically or electronically as part of routine care.", "Missing Malnutrition Universal Screening Tool score data was unavoidable, as this assessment should be performed within 24 h of admission, thus missing those with early mortality and shorter lengths of stay, as illustrated by the ‘missing’ category.", "However, the sensitivity analysis showed that when the same covariates were included in the final model, Odds Ratios and Area Under the Receiver Operating Characteristic Curve were very similar and these results did not alter the main conclusions.", "The follow-up period was limited to 30 days, but this is more reflective of outcomes of acute care, and outcomes are therefore not overly diluted by differing community care pathways.", "Although the cohorts were grouped based on characteristics at admission (as in the majority of other studies), occurrence of delirium during the admission would be an important mediator to assess in future work.", "Patients with hypoactive delirium may possibly have been misclassified as non-cognitively impaired in this study as this is often undetected in routine practice, and, as these patients are known to have worse outcomes, may have reduced the effect sizes between groups.", "Due to the broad nature of the cognitive screening pathway and its “real-life” implementation in routine clinical practice, other sources of misclassification between exposure groups are possible, for example patients with undiagnosed non-amnestic dementia may have been classified as non-cognitively impaired, and systematic differences in response to the question regarding memory loss over the last 12 months associated with the responder (patients vs carer/relative).", "However, the purpose of the screening process is to identify those with a current diagnosis of dementia, and to provide a broad screen to identify other patients who may be undiagnosed or benefit from further assessment.", "The results of the study do suggest that the screening process is indeed identifying a large cohort of older patients with additional vulnerability to poor hospital outcomes.", "This study demonstrated an independent association of cognitive impairment and dementia with poorer outcomes for older adults in hospital.", "Furthermore, patients with cognitive impairment and no diagnosis of dementia experienced poorer outcomes than those with dementia, thus representing a high-risk patient group with high costs.", "Whilst increased risks of hospitalisation for people with dementia are widely recognised and consequently can be acted upon, older patients with cognitive impairment may be largely undetected unless routine screening is in place.", "There is now a need to understand more about the mechanisms leading to these outcomes, including the relative contributions of intrinsic pathological pathways of deterioration, and extrinsic factors relating to context of care received, such as workforce arrangements, transfers of care, availability and content of care at home.", "Combined with systematic, enhanced recognition of cognitive impairment, future research may enable development of informed interventions for modification of care in hospital to improve care for this vulnerable group of older people.", "CF conceptualised the paper, performed the analyses and drafted the paper.", "PM acquired the data.", "DC, PM and PG provided statistical input.", "JB, PG and CS provided critical revision with clinical context.", "All authors contributed to the design of the study, interpretation of the results and revising the paper.", "All authors approve the final submitted version.", "The research was supported by the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care (CLAHRC) Wessex at Southampton NHS Hospitals Foundation Trust and Portsmouth Hospitals Trust.", "The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care.", "The ethics and governance permissions for this study do not include sharing of the dataset." ]

[]

[ "MATE" ]

[ "Atom probe tomography", "Ion irradiation", "Nanoindentation", "Nuclear fusion", "Tungsten rhenium osmium alloys" ]

[ "2503", "2504", "2506", "2507" ]

[ "Tungsten is the prime candidate for plasma facing applications in future nuclear fusion reactors, being one of only a handful of materials that are capable of withstanding the harsh operating conditions of up to ∼1300 K, power densities of 1–20 MW m−2 and irradiation from both 14 MeV neutrons and 2 MeV alpha particles [1,2].", "As well as causing large degrees of cascade damage (up to 20 displacements per atom per full-power year) [3], the neutron irradiation will cause pure tungsten to transmute into Re and subsequently Os, yielding potential solute contents as high as 3.8 at.", "% Re and 1.38 at.", "% Os after 5 years of reactor operation [4].", "Although the equilibrium W–Re–Os phase diagram predicts complete solubility at this composition [5], the mechanical properties of components could be severely degraded due to the formation of irradiation-induced solute-rich clusters as the precursors to sigma and chi precipitates [6,7].", "Previous studies of the irradiation responses of these systems are sparse and limited to compositions with more than 3 at.", "% rhenium and/or osmium present [8–11].", "Tanno et al. [8] irradiated W–3Os, W–5Re and W–5Re–3Os in the JOYO Japanese nuclear research reactor.", "Over a one-year period the alloys were exposed to a neutron dose (>0.1 MeV) of 1.2 × 1026 nm−2, corresponding to 1.54 dpa at ∼1032 K, conditions designed to mimic those of the ITER environment [12].", "Needle-shaped precipitates were identified by transmission electron microscopy (TEM).", "Osmium appeared to promote nucleation of precipitates, while Re encouraged their growth [11].", "Where Os and Re coexisted in the ternary W–5Re–3Os alloy, Os had a dominant effect, and the precipitation characteristics closely mirrored that of the W–3Os alloy.", "However, the precipitates were too small to accurately ascertain the compositions and full structures by TEM.", "Microhardness tests showed increases in hardness in all irradiated samples, with osmium-containing alloys (either binary or ternary) showing a larger increase (up to 8 GPa at 1.54 dpa) than W–Re alloys [11] (up to 5 GPa at 1.54 dpa).", "The most detailed chemical characterization of irradiation-induced precipitates in W–Re alloys has been conducted by Herschitz and Seidman [13,14].", "Their work used field ion microscopy to image the platelets formed in W–Re alloys post-neutron irradiation.", "They were able to determine, using 1-D atom probe mass spectrometry, that the precipitates contained either ∼50 or ∼75 at.", "% Re.", "However, the alloys they considered were of much higher rhenium content (10 and 25 at.", "%) than are currently of interest for fusion applications.", "Tungsten ion implantation experiments were performed on a commercial W–5 at.", "% Re alloy by Armstrong et al. [15] from 0.04 to 33 dpa damage levels at 300 °C.", "Atom probe tomography (APT) showed that a large increase in hardness above the 13 dpa damage level was associated with the formation of rhenium clusters ∼3 nm in diameter, though no study of the composition or density of these clusters was performed.", "In this study, ion irradiation was used as an analogue for neutron damage in W–Re–Os binary and ternary alloys.", "APT was used to study clustering and nanoindentation to measure the mechanical properties of the ion-irradiated layer.", "Alloys of W–2 at.", "% Re and W–1 at.", "% Re–1 at.", "% Os and were produced using vacuum arc melting of high-purity powders (Sigma Aldrich and AEE), as described in a previous study [16].", "Both alloys were found to have large grain sizes (0.5–2 mm measured via optical microscopy) and to be of high purity (>99.99 at.", "%, with trace impurities of Al, Si and Ca), as measured using APT and electron probe microanalysis.", "The ingots were cut into 1 mm thick slices, which were ground and polished with SiC paper and diamond paste.", "A final colloidal silica (40 nm) finish was used to produce a damage-free surface suitable for implantation and subsequent analysis.", "Ion irradiation was performed at the National Ion Beam Centre, University of Surrey, UK.", "Samples were mounted on a stainless steel temperature-controlled stage (held at either 573 or 773 K) under a vacuum pressure <10−6 Pa; for full details see Armstrong et al. [16].", "These temperatures were chosen to lie on either side of the stage III vacancy recovery temperature for W of 743 K [17].", "A tandem accelerator was used to irradiate the samples with 2 MeV W+ ions to a fluence of 2.64 × 1015 ions cm−2 at a flux of 1.96 × 1010 ions cm−2 s−1.", "A SRIM (stopping range of ions in matter) calculation predicts a damage profile extending to 300 nm below the surface, yielding an equivalent peak damage level of 33 dpa (calculated using the full cascade damage model with a binding energy of 68 eV) [15,16,18].", "A dual-beam focused ion beam system (Zeiss Nvision) was used to manufacture lift-out samples for APT analysis pre- and post-irradiation, following procedures described elsewhere [19,20].", "Materials were analysed using a Cameca LEAP3000X HR (37% total detection efficiency) in laser mode, running at pulse energies of 0.6–0.8 nJ and a sample stage temperature of 50 K.", "A minimum of three samples were studied for each alloy and each condition, the data from which were reconstructed using Cameca’s IVAS (3.6.6) software.", "Nanohardness measurements were made using a Nanoindenter XP (MTS, TN, USA) with a Berkovich diamond indenter calibrated against fused silica.", "The continuous stiffness measurement mode was used, allowing the hardness to be calculated as a function of depth [21].", "The indents were made to 1 μm final depth at a target indentation strain rate of 0.05 s−1, significantly deeper than the 300 nm deep damage layer.", "Grids of 16 indents were made on each of three different grains at room temperature for each sample and condition.", "There are difficulties with extracting hardness values from such thin irradiated layers where there will be significant penetration of the plastic zone into the unirradiated material.", "The method described by Armstrong et al. [15] was used to identify the region in which the hardness of the implanted layer dominates over the unimplanted layer, thus reported hardness values are taken at 125 nm indenter penetration.", "In the unirradiated materials, rhenium and osmium atoms are seen to be homogeneously distributed within the tungsten (see Supporting information for atom maps).", "The measured solute composition is 1.90 at.", "% Re in the binary alloy and 1.06 at.", "% Re/1.11 at.", "% Os in the ternary (wt.", "% equivalent to two significant figures).", "Following ion irradiation, solute clustering was observed in both alloys at both irradiation temperatures, and the level of clustering was seen to vary with implantation depth.", "This is apparent in Fig. 1a, which shows 4 nm thick sections through an atom map of the W–2Re sample following 33 dpa irradiation at 773 K.", "The outer surface of the alloy is marked by the carbon layer visible on the left-hand side of the map, below which a region of approximately 200 nm has prominent clustering of Re atoms; at greater depths, the structure returns to being a homogeneous solid solution.", "Fig. 1b shows the calculated average cluster diameter and volume fraction determined within 10 nm thick bins of the dataset (see below for further cluster analysis details).", "The cluster volume fraction profile strongly correlates with the damage profile from the SRIM calculation for tungsten at 33 dpa.", "To compare the effects of different irradiation conditions/alloy compositions on the microstructures, Fig. 2 shows further 4 nm thick slices over the full set of irradiation conditions and samples, confining the analyses to a depth range of 50–200 nm below the implanted surface, where the peak dose would correspond to 33 dpa.", "Fig. 2a shows that the irradiation has induced the formation of a high volume fraction of Re-rich clusters in the binary alloy, approximately 5 nm in diameter.", "Increasing the temperature to 773 K (Fig. 2b) yields a lower number density of clusters but of larger size, up to 15 nm in diameter.", "However, this is significantly altered when 1 at.", "% Os is added to the alloy; at 573 K only slight clustering is noticeable in W–1Re–1Os (Fig. 2c), suggesting that Os is suppressing cluster formation.", "However, following the 773 K irradiation (Fig. 2d), a high number density of very small (<4 nm dia.)", "precipitates is formed, enriched in both Re and Os.", "To quantify the size, chemical composition and density of clusters observed the maximum separation method algorithm [20] was used to identify and separate any clusters present in the 50–200 nm depth range of each dataset.", "The method classifies two solute atoms as part of the same cluster if the inter-solute distance falls below a user-defined dmax value.", "Clusters containing fewer solute atoms than a user-specified term Nmin are rejected to eliminate any small random clustering.", "An optimum dmax value was selected for each irradiation condition/alloy combination based on the procedure proposed by Chen et al. [22] and by Williams et al. [23].", "This method relies on the generation of a nearest neighbour distribution, comparing distances between the clustering species (Re in this case) with those from a randomized data set; dmax is selected at a threshold value that excludes randomized clusters from the analysis.", "Typical dmax values used were within the range of 0.9–1.3 nm.", "As a complementary method of identifying and isolating clusters, iso-concentration surface analyses were also carried out, with the aim of ensuring that the maximum separation and iso-concentration surface methods yielded similar values for cluster number densities and compositions.", "The values used for the iso-concentration analyses were determined by picking a level at which the cluster number densities, volume fractions and diameters begin to plateau (6.5 at.", "% Re, in the case of W–2 at.", "% Re).", "Finally, for the cluster analysis method, an Nmin value selected using the established protocol of Styman et al. [24].", "In this, for the chosen dmax value, a cluster size distribution is generated and compared with that from a randomized dataset with the same overall chemical composition.", "A minimum cluster size (Nmin) can then be determined by setting a threshold sufficiently high to exclude any wrongly identified random clusters, which in the cases studied here corresponded to a threshold size of Nmin = 20 detected atoms.", "Using these optimized values, the resulting figures for cluster compositions, sizes and volume fractions are given in Table 1.", "The data in each column were averaged over two or three runs under the same alloy/exposure conditions.", "At 573 K, the data show a strong segregation of Re in the clusters of the binary alloy, with an average of 8 at.", "% Re, compared to a bulk value of 2 at.", "% Re.", "In the ternary alloy there is no segregation of Re, but instead the clusters are enriched in Os.", "On raising the irradiation temperature to 773 K, the Re content in the clusters in the binary alloy rises further to nearly 13 at.", "%.", "In the ternary alloy there is still little Re segregation to the clusters, while the level of Os rises to nearly 7 at.", "%.", "In all cases solute enrichment is accompanied by a slight depletion of the solutes within the matrix, increasing with temperature as cluster formation becomes more marked.", "Along with differences in composition, there are also differences in the cluster size distributions with temperature.", "At 573 K both alloys show similar average cluster radii (2.4 nm in the W–Re alloy, 2.8 nm in the W–Re–Os alloy), but the number densities and volume fractions are higher in the W–2Re alloy.", "At 773 K significant changes in the cluster characteristics for both alloys are apparent.", "In W–2Re the average size increases to 3.3 nm, while the number density halves compared to 573 K.", "In the ternary alloy the size distribution broadens at 773 K, accompanied by a great increase in the number density of the clusters.", "For both alloys the cluster volume fractions (determined from the cluster average size and number density divided by the reconstructed volume) increase slightly with irradiation temperature, and the ternary alloy has a lower volume fraction.", "While the average cluster sizes allow simple comparisons between alloys and irradiation conditions, examining the distribution of sizes obtained gives further insights into the alloy irradiation response.", "Fig. 3 shows cluster distribution plots for the W–2Re and W–1Re–1Os alloys, with both irradiation temperatures plotted in each.", "For W–2Re, raising the irradiation temperature reduces the cluster number density; however, Fig. 3) also shows that the similar average cluster sizes reported in Table 1 for W–2Re for both irradiation conditions masks a broadening of the size distribution for irradiation at 773 K.", "The higher temperature cluster size profile also appears to have a bimodal distribution.", "W–1Re–1Os responds very differently, however, with a more narrow size distribution that changes only slightly with temperature, as shown in Fig. 3b.", "The main effect of irradiation temperature is the dramatic rise in cluster number density at the higher temperature (as seen in Table 1).", "Proxigram analyses were used to explore the internal chemistry of the clusters as a function of distance from the cores.", "For each composition and irradiation condition, several similarly sized clusters were isolated using iso-concentration surfaces (set at half the measured cluster compositions reported by the cluster analysis in Table 1).", "The Re iso-concentration was used for identifying clusters in the binary alloy, whilst the Os iso-concentration was used for the ternary alloy.", "Averaged proxigrams were then generated for the individual cluster elements, and these are presented in Fig. 4.", "They show that the Re content of the clusters is significantly higher in the cluster cores than near the shells in W–2Re, particularly so for the alloys irradiated at 773 K. Likewise, the cluster chemistry is non-uniform in the W–1Re–1Os alloy, but in this alloy Os is strongly enriched in the centre of the clusters, again most strikingly at 773 K.", "The level of Re in the ternary alloy remains low and is consistently within errors across the full cluster profile.", "At the higher irradiation temperature in the binary alloy there is evidence for cluster growth, although the full mechanism of the irradiation response needs further detailed study.", "This is demonstrated in more detail by the change in the shape of the size distributions, where a single peak is observed at 573 K and two peaks are seen at 773 K.", "The ternary alloy has a slower cluster nucleation rate at 573 K, evident from the lower levels of Os present in the proximity histograms.", "This nucleation rate increases significantly at 773 K.", "We suggest that the double peak observed in 33 dpa 773 K W–2 at.", "% Re is most likely a result of some clusters being dissolved by irradiation and the solute atoms then renucleating as clusters, thus producing the smaller peak in this distribution.", "The mechanism of radiation-induced dissolution of precipitates and the reprecipitation of fine sub-particles has been studied analytically, e.g. by Frost and Russell [25] and later by Heinig et al. [26], who also used kinetic Monte Carlo methods.", "This mechanism is predicted to lead to a double peak in the particle size distribution under certain conditions.", "Experimentally, duplex distributions of coarse and fine precipitates have been observed previously, e.g. by Heinig et al. [26] during the ion irradiation of gold particles in silica and by Monnet et al. [27] in ion-irradiated oxide-dispersion-strengthened ferritic steels.", "To explore the kinetics of the clustering in both alloys, the diffusion coefficients for Os and Re in W have been calculated using the Arrhenius equation with D0 and activation energies values from Ref. [28], which provides values from methods such as the activity of radioactive isotopic solutes diffusing in single crystal tungsten [29].", "For Os in W, D0 = 0.64 × 10−4 m2 s−1 and Ea = 5.58 eV; for Re in W, D0 = 4.00 × 10−4 m2 s−1 and Ea = 6.18 eV [28].", "These yield D values of 1.8 × 10−58 m2 s−1 (Re) and 5.3 × 10−54 m2 s−1 (Os) at 573 K, which increase to 2.0 × 10−44 m2 s−1 (Re) and 2.7 × 10−41 m2 s−1 (Os) at 773 K.", "These values indicate that diffusion of either alloying addition would be extremely slow if driven by temperature alone.", "The tungsten ion implantation enhances diffusion via the large number of vacancies produced.", "Vacancy clusters may also act as nucleation sites for the solute clusters [30–33].", "To quantify the extent of diffusion enhancement, we used the methodology of Lescoat et al. [31], which assumes that the particles behave as perfect sinks for point defects to compare these diffusion coefficients.", "The full mathematical models can be found in the aforementioned reference and are used to derive the irradiation enhanced diffusion coefficients for W–2Re (Re) and W–1Re–1Os (Os) at 33 dpa/573 K, which are 3.24 × 10−29 and 1.40 × 10−24 m2 s−1, respectively (we consider Os in the ternary alloy as it is the major clustering species); these values are both nearly 30 orders of magnitude higher than those determined above for thermal-vacancy-mediated diffusion at 573 K.", "At 773 K, the derived diffusion coefficients are even higher, at 4.99 × 10−26 and 3.69 × 10−23 m2 s−1, respectively, for W–2Re (Re) and W–1Re–1Os (Os), which correspond to an enhancement of 18 orders of magnitude compared to thermal-vacancy-mediated diffusion.", "There is further evidence in the literature supporting the prominent role of irradiation-produced vacancies in the alloy’s response to irradiation.", "Becquart and Domain [34] used density functional theory to calculate the Re–vacancy and Os–vacancy binding energies and found them to be 0.2 and 0.35 eV, respectively, indicating that vacancies are more attractive to Os.", "First-principles calculations by Kong et al. [35] have produced similar values for the binding energies.", "A possible mechanism therefore is that, for the binary alloy, Re atoms bind to vacancies, which then leads to Re-rich cluster growth as more Re diffuses.", "This diffusion is likely to be enhanced by local thermal spikes from cascades and the large vacancy population present in the irradiated alloy.", "In the ternary alloy, Os has a stronger binding energy to vacancies than Re, therefore Os-rich clusters form and subsequently grow, while Re cluster formation is competitively suppressed.", "In order to discount the possibility that the lack of Re–rich clusters in the ternary alloy was due to the lower Re content, a W–1 at.", "% Re alloy that had been implanted at 773 K with 2 MeV W+ ions to 3.6 dpa damage for a separate APT study was also examined.", "Despite the peak damage experienced being an order of magnitude lower than for the W–1Re–1Os alloy studied here, there is clear formation of Re clusters shown by the Re atom maps in Fig. 6a.", "These clusters contain an average Re content of 6 at.", "% and a measured volume fraction of 7.29%.", "Therefore, a bulk Re content of 1 at.", "% is adequate to form Re-rich clusters, but the co-presence of 1 at.", "% Os is sufficient to suppress Re cluster formation, with smaller Os-rich clusters forming instead.", "Nanoindentation hardness measurements taken at an indenter penetration of 125 nm are shown in Fig. 5.", "The average hardness of the unirradiated ternary alloy is higher (7.04 GPa) than that of the binary alloy (5.85 GPa).", "After irradiation at 573 K both alloys show an increase in hardness, of 3.22 GPa in the ternary alloy and 3.27 GPa in the binary alloy, to absolute values of 10.26 and 9.12 GPa, respectively.", "Following irradiation at 773 K, whilst there is a further increase in hardness in both alloys, it is significantly larger in the ternary alloy, increasing by 5.39 GPa to an absolute value of 12.43 GPa, compared to an increase of 3.84 GPa in the binary alloy to 9.69 GPa.", "The dramatic rise in the cluster number density seen in W–1Re–1Os following 773 K irradiation is therefore also mirrored in the most pronounced hardness increase being for this alloy.", "The critical breakaway angle is found to be ∼86o in all alloys (Table 2).", "This high value indicates that individual clusters are weak obstacles to dislocation motion; the considerable hardening observed is therefore a result of their high density (and thus small spacing, λ).", "As such, it is likely that dislocations pass individual clusters relatively easily at room temperature by cutting the clusters, rather than by dislocations bypassing the smaller clusters by climb or cross slip.", "Such a mechanism is promoted by the coherent nature of the clusters with the matrix (see Fig. 6b).", "It is also possible that vacancies or vacancy clusters that are not associated with clustering could have a hardening effect.", "The detection of such vacancies or clusters is not possible in APT experiments; however, recent modelling work by Kong et al. [35] suggests that their formation and thus hardening effect should be minimal.", "There have been no previous reported results for the irradiation response of such dilute binary or ternary W–Re–Os alloys to such damage levels.", "However, Tanno et al. [8,11] have reported the effect of neutron irradiation to 1.54 dpa at 673 and 1024 K in W–5 at.", "% Re and W–5 at.", "% Re–3 at.", "% Os alloys.", "Although the irradiation conditions were different from the current study, they observed similar cluster densities (2.2 × 106 μm−3 in W–Re and 6.7 × 106 μm−3 in W–Re–Os) and radii (1.6 nm in W–Re and 3.4 nm in W–Re–Os) to those observed here.", "The nanoindentation hardness results presented here are also comparable to the microhardness results obtained by Tanno et al. [11].", "They measured an ∼11.8 GPa hardness for a W–3 at.", "% Os alloy neutron-irradiated to 1.5 dpa at 1023 K. Similarly, the W–2 at.", "% Re exhibited a hardness of 9.34 GPa at 773 K after 33 dpa ion implantation, while in W–5 at.", "% Re at 1023 K this approached ∼9.86 GPa following neutron irradiation.", "The fact that the cluster size distributions and hardness values are similar despite the significantly higher damage level in the ion-implanted material suggests that the saturation level for the growth of such Re- and Os-rich clusters is at a relatively low damage level.", "The different time scales of ion and neutron irradiation may also be also important.", "Firstly, the higher ion damage dose rate may lead to significantly faster accumulation of damage by ions compared to neutrons, altering the nucleation and growth of the clusters.", "Such effects have been observed in binary FeCr alloys [39].", "Secondly, the total irradiation time in the neutron irradiation is significantly longer, thus allowing more time for diffusion to operate and promoting enrichment of clusters into discrete precipitates.", "Robust methods to cross-calibrate ion and neutron irradiation experiments are required if ion irradiations are to usefully inform the design and development of future nuclear systems.", "In this study, atom probe tomography and nanohardness measurements have offered fresh insights into the phenomenon of irradiation-induced precipitation in W–Re and W–Re–Os alloys.", "The following major conclusions can be drawn:", "The microstructures developed after ion irradiation in W–Re and W–Re–Os are similar to those produced previously using neutron irradiation.", "Os has a profound effect on the alloy clustering characteristics, almost completely suppressing the formation of rhenium clusters after irradiation to 33 dpa.", "This is most likely due to Os atoms being more strongly bound than Re atoms to vacancies.", "Individual nanoscale clusters produced are relatively weak obstacles to dislocation motion.", "Their significant contribution to the hardening in both binary and ternary alloys is due to the large volume fraction present.", "The presence of Os in the ternary W–Re–Os alloy hardens the material significantly after ion irradiation compared to the binary W–Re alloy.", "The trend matches that of prior neutron irradiation studies.", "When predicting the mechanical behaviour of tungsten after transmutation in a fusion neutron spectrum, all transmutation products must be accounted for.", "Using binary W–Re alloys as a reference will cause the likely irradiation hardening to be substantially underestimated." ]

[]

[ "COMP", "SOCI" ]

[ "Digital platform", "Digitalization", "Gig economy", "Lyft", "Platform economy", "Ride-hailing", "Sharing economy", "Taxi", "Uber", "Work" ]

[ "1705", "1710", "3309" ]

[ "Digital platforms such as Amazon MTurk, Uber, and AirBnB have enabled the emergence of various new forms of work (Colbert, Yee, & George, 2016; Gandini, 2019; Howcroft & Bergvall-Kåreborn, 2019; Kuhn & Maleki, 2017).", "In the context of platform-enabled work, we define a digital platform as the digital infrastructure, rules, and processes that enable resource exchange between external producers and consumers (cf. de Reuver, Sørensen, & Basole, 2018; Howcroft & Bergvall-Kåreborn, 2019).", "An increasing number of people globally obtain their primary or supplementary income from platform-enabled work.", "For example, the number of rides completed with Uber increased from five billion to ten billion between June 2017 and June 20181 2 .", "This increase was made possible by advances in information technology (IT) that have reduced transaction costs, thereby increasing the relative competitiveness of the market-based approach to organizing work compared to traditional organizational hierarchies (Parker, Van Alstyne, & Choudary, 2016; cf. Williamson, 1980).", "From workers’ perspective, digital platforms provide a means of employment and a source of income for many people who would otherwise be unable to participate in the job market.", "Simultaneously, the working conditions and sustainability of work that digital platforms enable have become a controversial issue (Malhotra & Van Alstyne, 2014; Van Doorn, 2017).", "For example, while ride-hailing services such as Uber and Lyft offer income for a large number of drivers, many drivers struggle to reach even a minimum income from such work.", "In sum, digital platforms carry profound economic and social implications as they challenge traditional business models (Cramer & Krueger, 2016; Parker et al., 2016: de Reuver et al., 2018) and the common structure of the “employer–employee” relation (Aloisi, 2016; Colbert et al., 2016).", "One of the dilemmas arising from platform-based work structures is whether digital-platform workers should be classified as employees or self-employed (Cherry, 2016; Todoli-Signes, 2017; Schor & Fitzmaurice, 2015), as exemplified by the legal disputes on the status of drivers for drive-hailing services.", "Due to the increase of independent contracting, self-employment, part-time, temporary, and remote work, different forms of nonstandard work have become important research areas for scholars who study work and employment relations (Ashford, George, & Batt, 2007; Kalleberg, 2000).", "However, despite the emerging scholarly interest in the work done for digital platforms (Gandini, 2019; Howcroft & Bergvall-Kåreborn, 2019; Kuhn & Maleki, 2017), there is nascent understanding of the nature of work relations in the context of digital platforms (Cappelli & Keller, 2013; Forman, King, & Lyytinen, 2014).", "In particular, the role of the digital platform in shaping the nature of platform-enabled work is not well understood.", "Against this contextual and theoretical backdrop, we explore how the presence of the digital platform manifests itself in workers’ perceptions of their work in the context of ride-hailing.", "We conducted 39 semi-structured interviews of drivers who work for Uber and Lyft in Boston.", "We subsequently analyzed the data using the Gioia method (Gioia, Corley, & Hamilton, 2013) and identified six key mechanisms of platform-based work, namely self-employment, time control, income, control, pricing, and rating.", "We further show that from workers’ perspective, the flexibility characterizing the work arrangement is a key positive element of platform-enabled work, whereas the power disparity between workers and the platform is a major source of discontent.", "As a result, by drawing on the concept of work relations (e.g., Pfeffer & Baron, 1988; Edwards & Rothbard, 2000; Kalleberg, 2000; Abbott, 2006), we reconceptualize the dimensions of work relations to capture the nature of platform-based work.", "We name these dimensions digital temporality and algorithmic administrativity.", "Our study extends nascent research on the nature of work relations in digital platforms (cf. Baiyere & Salmela, 2015; Hamari, Sjöklint, & Ukkonen, 2015; Sutherland & Jarrahi, 2018; Van Doorn, 2017).", "Specifically, we advance research on the dark side of digital-platform-enabled work (Gandini, 2019; Malhotra & Van Alstyne, 2014) by elaborating on the positive and negative elements that drivers associate with their work for ride-hailing platforms and subsequently theorizing on the resulting changes in the work relations.", "In doing so, this paper contributes to the IS literature on the dualities, i.e., mutually connected positive and negative aspects of information technology (IT) (Turel & Serenko, 2012; Mäntymäki & Islam, 2016; Islam, Mäntymäki, & Benbasat, 2019; Baiyere, Islam, & Mäntymäki, 2019) in the context of digital work intermediaries.", "Our findings can help governmental decision-makers and authorities better understand the societal implications of platform-enabled work.", "Furthermore, our study holds relevance for organizations considering how to utilize elements of the platform and sharing economies in their business operations.", "The article proceeds as follows.", "The next section describes the theoretical background of the research.", "The third section covers empirical research, followed by the study’s findings in the fourth section.", "In the fifth section, we discuss the study’s implications and limitations and offer ideas for future research.", "Working for ride-hailing platforms is a subset of platform-enabled work that the prior literature has placed under umbrella terms such as “gig economy” (Gandini, 2019) and “crowdwork” (Howcroft & Bergvall-Kåreborn, 2019).", "According to Gandini (2019), p. 1040) the term gig economy denotes “the establishment of a capital–labor relationship between a worker and a digital platform, that mediates workers’ supply and consumer or professional demand for the completion of a small task or ‘gig’ and operates at once as a market intermediary”.", "Crowdwork in turn “functions as a marketplace for the mediation of both physical as well as digital services and tasks” (Howcroft & Bergvall-Kåreborn, 2019, p. 23).", "For the persons doing the work, the digital platform is the primary contact point that replaces interactions with an employer or employer representative.", "The omnipresence of the digital platform alonside the physical absence of an employer or employer representative arguably plays a focal role in shaping workers perceptions of their work and affects many aspects of the work relationships in platform-enabled work.", "As a result, we draw on the work relationship literature (Ashford et al., 2007; Kalleberg, 2000; Pfeffer & Baron, 1988) to scrutinize the nature of platform-enabled work from workers’ perspective.", "The genesis of different types of platform-enabled work (Howcroft & Bergvall-Kåreborn, 2019; Kuhn & Maleki, 2017) can be viewed as a part of a larger societal trend of increasing nonstandard work (e.g. Gandini, 2019).", "The notion of nonstandard work stems from the concept of standard work, in which work is full-time by default, continues indefinitely, and is performed at the employer's physical premises under the employer's direct control (Kalleberg, 2000).", "Standard work was the norm in many industrialized nations for much of the 20th century and has been the basis on which labor law, collective bargaining, and social security systems were developed (Kalleberg, 2000).", "Nonstandard work, on the other hand, was conceptualized by Pfeffer and Baron (1988) using three dimensions––temporal, physical, and administrative attachment––that capture the nature of the work relationship between the employee and the employer.", "According to Pfeffer and Baron (1988), these three work-relationship dimensions differentiate nonstandard workers from standard workers who have fixed work hours and work on indefinite contracts at fixed locations under employers’ direct administrative control (cf. Kalleberg, 2000; Ashford et al., 2007).", "First, limited temporal attachment refers to, for example, temporary and part-time workers whose working hours depart from what is considered full-time employment.", "Second, limited physical attachment refers to, for example, teleworkers and those who work from home, thus capturing the spatial distance between the employee and employer.", "Third, limited administrative attachment refers to the extent to which workers are under an organization’s direct administrative control.", "For example, workers who work through intermediaries or as self-employment contractors have limited administrative attachment to the organizations for which they work.", "Ashford et al. (2007) built on Pfeffer and Baron (1988) in their study on nonstandard work, concluding that when an employment duration is limited, the organization has limited administrative control over the employee, and/or workers are not physically in proximity to the organization, work is more nonstandard.", "Polivka and Nardone (1989), in turn, focused on the temporal dimension, defining “contingent work” as any work arrangement that does not contain an explicit or implicit commitment between employee and employer for long-term employment.", "Extant research has also used these three dimensions to predict the outcomes for different kinds of work relations.", "These outcomes include the development of shared cognition (Levesque, Wilson, & Wholey, 2001), organizational identification (George & Chattopadhyay, 2005), organizational citizenship behavior (Pearce, 1993), and social relationships at work (Broschak & Davis-Blake, 2006).", "The nonstandard work category encompasses various forms of employment relations that have become increasingly prominent in organizing work (Kalleberg, 2000).", "Such forms (Casey, 1991; Goldthorpe, 1984; Green, Krahn, & Sung, 1993; Kalleberg, 2000) include alternative work arrangements (Polivka, 1996; Sherer, 1996), market-mediated arrangements (Abraham, 1990), nontraditional employment relations (Ferber & Waldfogel, 1998), flexible staffing arrangements (Houseman & Osawa, 1998), flexible working practices (Brewster et al., 1994), atypical employment (Córdova, 1986; De Grip, Hoevenberg, & Willems, 1997), vagrant or peripheral employment (Summers, 1997), vulnerable work (Tregaskis, Brewster, Mayne, & Hegewisch, 1998), precarious employment (Treu, 1992), disposable work (Gordon, 1996), and contingent work (e.g., Polivka & Nardone, 1989).", "Due to the proliferation of nonstandard employment relations, this research realm has moved toward self-employment, contract-work relationships, part-time work, and remote work, among many other forms (Edwards & Rothbard, 2000).", "Some researchers have argued that the growth in nonstandard employment has contributed to workers’ increased perceived job insecurity and greater economic inequality (cf. Kalleberg, 2009).", "However, from employers’ vantage point, nonstandard forms of employment can be advantageous, as temporary employment can allow the employer to screen potential employees before committing to a permanent employment (Engellandt & Riphahn, 2005).", "Furthermore, the literature suggests that temporary workers are considerably more likely to work unpaid overtime than employees in permanent employment (Engellandt & Riphahn, 2005).", "Nonstandard forms of employment may also help employees to gain a more stable position in the labor market.", "There is evidence that temporary contracts may serve as stepping stones into more attractive employment even with organizations other than the current employer (Booth, Francesconi, & Frank, 2002).", "However, it is questionable if for example work for ride-hailing platforms can help to improve worker’s position in the labor market (Gandini, 2019).", "Studies have shown that the proliferation of IT in recent decades has been shaping and redefining the concept of work (Dillahunt & Malone, 2015), as IT enables new types of work relations (Kalleberg, 2009).", "More specifically, advances in digital technology, such as the emergence of digital platforms like Uber and Amazon MTurk, which are also referred to as digital labor intermediaries (Peck & Theodore, 2013) or labor platforms (Kuhn & Maleki, 2017), have been changing the structure of work (Howcroft & Bergvall-Kåreborn, 2019; Kuhn & Maleki, 2017).", "This in turn has significant implications for the conceptualization of work as well as the individual, managerial, and organizational outcomes attributed to work (Gandini, 2019; Kuhn & Maleki, 2017; cf. Ashford et al., 2007; Grant & Parker, 2009).", "Our research context is digital ride-hailing platforms, specifically Uber and Lyft.", "Since driving for ride-hailing platforms represents one of the most common examples of platform-based work.", "Moreover due to controversies and the associated lawsuits and legislation initiatives related to drivers’ legal status (Kuhn & Maleki, 2017), they provide a representative context in which to investigate how workers perceive their work and how work relations manifest themselves in platform-enabled work.", "In essence, ride-hailing platforms connect people who need transportation with drivers willing to provide transportation.", "In doing so, the platforms provide a means to fulfill people’s needs for mobility while allowing those with the necessary resources, i.e., time and a car, to earn money by providing those needed resources (Van Alstyne et al., 2016).", "To this end, the platforms obtain location data from both drivers’ and riders’ mobile devices, employ feedback mechanisms that allow for both drivers and riders rate each other alongside other parameters in the algorithm that ultimately connects drivers with riders.", "For both drivers and riders this materializes in the smartphone app that is the user interface.", "To start working for these platforms, an aspiring driver need to register him/herself and undergo a background check conducted by the platform (Kuhn & Maleki, 2017).", "The purpose of the background checks, alongside the insurance coverage provided by the platforms is to reinforce trust that is another key prerequisite for the successful employment of the platform-based business model (Van Alstyne et al., 2016).", "Furthermore, to achieve the scalability that running a platform-based business model requires (Van Alstyne et al., 2016), ride-hailing platforms utilize cloud computing.", "Besides its traditional purpose, to transport people, digital ride-hailing services have gradually been evolving into platforms with flexible capabilities that enable their use cases to be extended in new ways.", "For instance, the Uber platform into a goods-delivery platform (e.g., food delivery), with the company experimenting with campaigns and business models such as “#UberEATS” and “Uber for Business,” among others.", "In addition, Uber has “Uber for developers,” enabling third-party developers to extend the digital platform with applications such as Google Maps, HotelTonight, Starbucks, and TripAdvisor through the Uber Application Programming Interface (API).", "Our empirical data comprise 39 semi-structured interviews with drivers working for Uber and Lyft in Boston.", "We decided to focus on Boston because of its reportedly high concentration of ride-hailing activity, including a large number of drivers.", "More broadly, the State of Massachusetts rates among the top 10 in terms of driver populations and ride-hailing traffic in the US (Hall & Krueger, 2015).", "Since this study’s premise concerns work relations, we consider drivers to be the most appropriate respondents for our inquiry.", "Finally, we chose the semi-structured interview approach, as it provides us with the freedom to adapt interview questions as new knowledge emerges from interviews (Gillham, 2005).", "Each interview lasted approximately 30 min, and we recorded all the interviews after securing interviewees’ permission.", "The interviews were transcribed to enable further analysis, and we also took notes during the interviews to use in the data analysis.", "We employed the Gioia method (Gioia et al., 2013) to guide the analysis of interview data.", "As pointed out by Gioia et al. (2013), novel insights often can be yielded by carefully investigating how different actors experience events.", "Gioia et al. (2013) further suggested certain practices that bring qualitative rigor, such as creating a data structure to visualize the analysis process.", "As is typical of inductive research, our analytical process was iterative and partially overlapped with data collection.", "Nevertheless, certain phases, as outlined below, in the analytical process can be recognized wherein we iterated and refined inferences of theoretical mechanisms from the empirical material.", "We started our analysis with open coding (Strauss & Corbin, 1998).", "The first stage of the analysis process included reading the interview transcripts and marking certain areas with codes to describe interview content (Miles & Huberman, 1994).", "We used the research question to guide the first round of coding.", "We also identified differences and similarities among different segments of empirical data.", "This practice resembled constant comparisons in grounded theory research (Strauss & Corbin, 1998).", "Table 1 provides a summary of the codes relevant to the present study and their related content.", "During the second stage of the analysis process, we further categorized the first-order codes into more abstract second-order concepts (Charmaz, 1990; Van Maanen, 1979).", "The second-order concepts were established by systematically combining first-order codes with similar content (cf. Dubois & Gadde, 2002).", "During the analysis process, we wrote numerous brief notes and memos to document the choices made and to further develop our insights.", "These notes and memos totaled up to 30 pages.", "As is typical for an iterative research process, we refined our coding procedures according to our evolving understanding (Strauss & Corbin, 1998).", "As a result of the second stage of the analysis, we came up with six categories describing the mechanisms of the digital platform and two aggregate themes covering the temporal and administrative dimensions of work relations.", "During the third stage of the analysis process, we incorporated the work relations perspective (Pfeffer & Baron, 1988) into the analysis and specifically looked at how the dimensions of work relations manifest themselves in the categories identified in the previous stage.", "This led to the emergence of two theory-guided aggregate themes, namely temporal and administrative, that comprise three second-order categories each.", "The temporal dimension of the work relation (Pfeffer & Baron, 1988) was manifested in the drivers’ narratives describing the flexibility and freedom in determining their working hours.", "A common theme in drivers’ descriptions of the positive sides of working for ride-hailing platforms was the temporal nature of work that comprises self-employment, the ability to control one’s working hours, and source of income.", "Most of the drivers whom we interviewed emphasized that they particularly appreciate the flexibility to work without constant supervision.", "This was considered positive and desirable.", "In particular, drivers who were retired, single parents, or were taking care of family members with severe illnesses provided rich descriptions of how they valued the flexibility associated with driving for Uber and Lyft.", "Furthermore, the platforms allow drivers to decide whether to accept or reject ride requests, which was considered an important positive aspect.", "Some drivers stated that they specifically valued the freedom to be able to stay home to care for a sick family member, rather than be obligated to go to work under a rigid schedule.", "In light of these experiences, it is not surprising that respondents appreciated the temporal nature of working as a ride-hailing driver.", "The category labelled as self-employed status refers to the informants’ views that being legally considered self-employed was viewed as an advantage of platform-enabled work.", "Some of the drivers we interviewed explicitly referred to a lawsuit against Uber that aimed to require the company to treat drivers like employees instead of as independent contractors under labor laws.", "While acknowledging the benefits that come with such a classification, the drivers we interviewed frowned at the prospect of losing the flexibility associated with being self-employed.", "Some even viewed the work arrangement enabled by the platform as a means of carrying out part-time work and feared that being classified as employees potentially would render them unable to work for the platforms.", "For some of the drivers, the value of being self-employed has even motivated them to quit full-time jobs and start driving for Uber and Lyft exclusively.", "The following interview quote describes these viewpoints:", "“The thing I really value in this…[driving Uber] is that I can be my own boss.", "I’m like the CEO of my own company.”", "Second, one of the most frequently and clearly highlighted desirable outcomes of platform-based work is related to time management, that is, the control that drivers have over when they work.", "The driver interface, i.e., the smartphone app, has been designed so that workers only need to turn it on when they are available and are interested in working.", "This temporal flexibility enables workers to adjust their work schedules to fit their personal life activities.", "Some of the interviewed drivers specifically noted this flexibility for the option to drive at night, while others leveraged the temporal flexibility by driving while their children are at school.", "Taken together, platform-based work’s on/off nature was viewed as enabling different configurations of work and free time.", "One of the interviewees describes his experiences in the following way:", "“[I drive], like, 30 h [or] so [per week], not really full-time.", "Not really part-time either.", "…I pick whenever I want.", "It’s nice.”", "The third category of data falling under the temporal dimension of work relations, source of income, refers specifically to the flexible income mechanisms that working for the platforms afford.", "In fact, most of the interviewed drivers stated that they began working for Uber and Lyft because of the income possibilities.", "In addition to providing a means of employment for many who are otherwise unemployed, working for digital platforms enables people with full-time jobs to generate additional income whenever they choose.", "One of the respondents used a faucet analogy to describe driving for Uber, stating it is like “turning on the income tap.”", "The respondent mentioned that whenever he needs extra funds, he just schedules a driving plan for Uber in his free time:", "“I am a business owner.", "I have a business that I run during the day, and this is just extra income.", "I just [do it] at night for a few hours usually.", "And the reason why I’m doing it is because I have a German shepherd dog, [a] puppy, and I have to send him to a specialty school, one of the best in the world, that is in Massachusetts.", "And it costs about $7000 a month.", "So I’m trying to just supplement my income specifically for that.”", "While the temporal dimension of the work relation was predominantly attributed to the positive aspects of working for ride-hailing platforms, the data describing the administrative dimension contained considerably more critical voices.", "In particular, interviewees heavily criticized how the platforms exercise control and power over the drivers, particularly describing various rich narratives about how the platform exercises superior bargaining power over drivers through controlling in the information that is provided to the drivers as well as the pricing mechanism and the rating mechanism.", "This administrative control ultimately results from the fact that the platform operators control and use the data produced within the platform.", "The following respondent further explained the nature of the administrative control:", "“They [platforms] have their own policies, and sometimes they change their policies.", "Like last year, it was very good.", "The money that I was making last year was much more than what I’m [making] this year because last year, the rate was high, but this year, they rode down the rates, and they raised some other expenses.", "And it kind of sucks.", "But it is what it is… No matter what, they get their own percentage.", "And if there are more people who use Uber, they get more and more percentage.”", "Lack of information was a major source of complaints attributed to the administrative dimension of work relations.", "The principal contact point in the work relationship is the mobile application that connects passengers with drivers.", "The platforms control the app, so drivers have no control of the information that the app provides, or how the data are used.", "For many drivers, a more transparent view of certain aspects of work-related information would be valuable in helping them make better decisions regarding their work options.", "For example, Uber and Lyft have designed their apps such that they withhold passengers’ destinations until the driver picks up each passenger.", "Some respondents stated that having an idea about the direction a passenger is heading would help them decide whether it is where they are heading at a particular time.", "This is particularly important in cases when they are considering shutting down for the day and would appreciate having the option of driving a passenger to his or her destination on the way home or wherever they are heading after driving.", "One of the drivers described his frustration in this regard:", "“Probably the only thing I don’t like is I can’t see where the person is going until I start the trip.", "Sometimes you are taking, like, 10 min to pick someone up. [", "You] drive 10 min to wherever they are, and then they only go like two blocks.", "So, I did, like, 15 min of work, and they’re only gonna pay, like, $5.”", "The second category of data related to the administrative dimension of work relations was related to the pricing mechanism employed by the platforms that often manifests itself in price reductions.", "Drivers cited price reduction as perhaps one of the most undesirable aspects of the platforms’ control over the apps.", "Almost all the interviewees mentioned Uber and Lyft’s unilateral price reductions for passengers, which require drivers to work more hours if they want to sustain their income levels.", "For example, a driver who decided to quit his or her old job based on the comparable potential income from driving may soon realize that their original calculations no longer make ends meet if the company reduces prices for passengers.", "This means drivers who make a certain amount of income upon joining the platform would only realize after each price-reduction move made by the company that maintaining the same level of income would require allocating more time to driving for the platform.", "Furthermore, since the platform owners have the right to determine the price of transactions and the allocated share to the driver, the drivers are at the mercy of the platform owner’s decisions on fares at any time.", "This leads to a “Red Queen’s race” situation in which the drivers work harder to earn the same amount.", "A respondent who was a taxi driver and was experimenting with Uber with his private car put it this way:", "“I tried Uber because back then, you can easily in a weekend make $300-$400.", "They have a surge basically every time….", "So it’s quick, and it’s good.", "But later, because a lot of drivers get into it, then you don’t do that anymore [because] It’s not [worth it] anymore.", "… Now it’s up and down; it’s not something you can trust.", "… If you wanna make money with UberX, you have to work [a lot].", "Let’s say I do 40 h; you have to make 80 h working UberX, [and yet] you make barely [the] same as [a] taxi.", "It’s too much.”", "The third manifestation of the administrative dimension of work relations was the rating mechanism.", "Uber and Lyft’s digital platforms are structured not only to facilitate the work process, but also to serve the purpose of collecting feedback in the form of ratings on each transaction.", "The platform enables both the driver and passenger to rate each other after a trip.", "The rating is structured from one to five stars, with five being the best rating possible.", "The undesirability element of this comes from how ride-hailing platforms use this data.", "They set a threshold value (reportedly 4.5 for Uber) as the minimum rating that a driver can have before being terminated from working on the platform.", "Considering that every human (or specifically passenger) rates differently and weighs his or her rating criteria in a variety of ways, drivers have little control over the ratings that passengers give them.", "The following interview quote illustrates the issue:", "“They have this rating thing, some people might misunderstand, so they get [a] low rating a couple of times, and that person buys a new car just to use it for Uber.", "It never happened to me, but I’ve seen a lot of people that have very new cars, stuck in the middle….", "If their rating goes down, they might get suspended for a little bit… [Imagine] you deactivate his account, but he has a new car that he has to pay for!”", "Most of the interviewed drivers acknowledged that they do not control many terms of their work relationship.", "In fact, some said the terms seem to be a one-way street, in which one party makes the terms and the other party has the option of agreeing or simply quitting.", "There is no option to negotiate in the terms’ formulation, making drivers feel vulnerable to the company’s decisions.", "For example, the insurance used by the platforms may cover only accidents that take place when there is a passenger in the car, which leaves a gap in coverage for the drivers that they may not have prepared for.", "If they were to get into an uncovered accident, that leaves them responsible for the full cost of the damage or bodily injury.", "The three analysis stages and the respective data-structure processes are summarized in Fig. 1.", "Our results highlight two dimensions of work relations pertinent to digitally intermediated ride-hailing: temporal and administrative.", "Compared with Pfeffer and Baron’s (1988) three-dimensional conceptualization (temporal, physical, and administrative), the temporal and administrative dimensions particularly emerged from our data.", "Furthermore, these two dimensions manifest themselves quite differently than in Pfeffer and Baron’s (1988) conceptualization.", "Therefore, we reconceptualize the temporal and administrative dimensions of work relations into digital temporality and algorithmic administrativity.", "Table 2 summarizes our findings in compare to Pfeffer and Baron (1988).", "First, with respect to the temporal dimension, our analysis revealed a number of temporal patterns related to work enabled by digital platforms.", "Work for digital platforms like Uber and Lyft is seldom the result of a long-term career-planning process.", "Our results suggest that the flexibility of Uber and Lyft work allows for adjusting work to fit the driver’s life situation.", "Thus, while the typical work relationship’s temporal dimension carries the expectation that the relationship will last over extended periods of time, in the case of platform-enabled work, the temporal dimension reflects the amount of time that the worker intends to allocate for the work.", "In fact, due to this temporal nature of work, many people try working for the service, but only a subset of workers continue for extended periods of time.", "Some can work for digital platforms when they are between jobs.", "For others, working for platforms may provide them with the kind of flexible work schedule and income source that they have been looking for, leading to a long-term work relationship.", "Therefore, we extend the notion of the temporal dimension of work relation (Pfeffer & Baron, 1988) from a long-term vs. short-term focus to digital temporality, highlighting specifically when the work takes place and the high degree of fluidity between work and free time.", "This fluidity stems from the digital platform’s resource-connecting functionalities that enable very fast changes between the on-and-off status of work based on workers’ context and the availability of work.", "Second, with respect to the administrative dimension, our results show that the firms behind the ride-hailing platforms exercise significant administrative control over drivers.", "However, instead of management control, organizational hierarchies, and physical proximity––which characterize traditional organizations (Ashford et al., 2007; Pfeffer & Baron, 1988)––ride-hailing platforms rely on complex, invisible algorithms for both drivers and clients that allow them to employ a market mechanism, driver ratings, and online surveillance to exert administrative power over drivers.", "Through these algorithmic processes and the market mechanism, the platforms increase prices for rides during peak hours, in busy areas, or during bad weather to ensure capacity and capitalize on demand.", "The market-driven pricing of rides is a means of ensuring that a sufficient number of cars is available when demand rises.", "However, this also means that drivers’ incomes depend not only on how much they work, but also on the platform’s pricing algorithm.", "Furthermore, customer ratings are a mechanism designed to ensure service quality and to exercise power over drivers, as customer feedback may even lead to deactivating a driver from the system.", "As a result, we shift the notion of the administrative dimension (Pfeffer & Baron, 1988) from management and organizational hierarchies to a digital administrative dimension, which highlights the use of the mediating platform’s digital properties to exercise administrative control over workers (cf. Gandini, 2019).", "Consequently, we refer to this dimension of the work relationship as algorithmic administrativity.", "Finally, instead of physical proximity to the organization or its physical premises, ride-hailing workers operate in virtual proximity to the organizations behind the platforms.", "Thus, we suggest that virtual proximity replaces physical proximity in the context of digital platform-enabled work.", "For example, Uber and Lyft monitor cars’ locations and collect information about routes used and ride durations.", "Uber also tracks risky driving behaviors, such as speeding, harsh braking, and even using phones while driving.", "In addition, the Uber app sends drivers reminders to take breaks from driving, messages that prompt drivers to mount their phones on the dashboard, and reports that show how a driver’s behavior compares with that of other drivers in the same city.", "This is expected not only to contribute to safer driving patterns, but also to help many drivers receive better customer feedback ratings.", "On the other hand, this type of digital surveillance on drivers’ behavior also enables various forms of privacy intrusions.", "Taken together, we view the physical dimension of the work relationship (Pfeffer & Baron, 1988) as being essentially embedded in both the temporal and administrative dimensions, with an emphasis on the virtual proximity of the work relationship as facilitated by the digital platform’s omnipresent nature.", "This study aimed to explore how the presence of the digital platform characterizes work relations in the context of digitally intermediated ride-hailing.", "In essence, our study provides a workers’ account of the controversies surrounding this type of platform-enabled work.", "Our findings advance the understanding of the nature of work done for digital platforms (Gandini, 2019; Howcroft & Bergvall-Kåreborn, 2019; Kuhn & Maleki, 2017; Sundararajan, 2014; Leighton, 2016; Todoli-Signes, 2017) by analyzing the mechanisms embedded in the digital ride-hailing platform from the drivers’ vantage point and by providing a fine-grained analysis of work relations in the context of platform-enabled work.", "Finally, beyond the ride-hailing context, our findings advance the understanding of contributors’ value-creation processes and motivations (Mäntymäki & Riemer, 2016) and the underlying mechanisms that foster contributors’ engagement with activities that they undertake in digital platforms (Pacauskas, Rajala, Westerlund, & Mäntymäki, 2018).", "As a result, we highlight three specific implications of the study.", "First, in our data, the self-employed status of the driver was considered a specific advantage of working for ride-hailing platforms.", "This is particularly interesting, since the ride-hailing platforms represent platforms where the worker’s relationship with the platform is generally characterized by low autonomy and high dependence on the firm (Kuhn & Maleki, 2017).", "In fact, drivers’ critical remarks related to the administrative dimension of the work relation essentially relate to the lack of autonomy, dependence on the firms behind the platform, and certain means the firms behind the platforms use to exercise control and power over the workers.", "Second, as our analysis shows, ride-hailing platforms exercise control and power by determining prices for the rides in a demand-driven manner and by using clients’ ratings to evaluate drivers, consequently exerting algorithmic administrative control that, for the drivers, materializes via the app (algorithmic administrativity).", "While these manifestations of administrative control and power were a major source of discontent, our informants did not even mention the background checks conducted for new drivers, or code-of-conduct instructions that are also common forms of administrative control among ride-hailing services (De Stefano, 2015; Lee, 2016).", "Third, considering the rapid growth of ride-hailing platforms, the mechanisms underlying digital temporality that allow the type of flexibility that can meet drivers’ unique conditions and preferences and the high degree of fluidity between work and free time can potentially be viewed as the unique characteristics of these platforms as IT artefacts.", "Future IS research may build on our findings and operationalize these artefact characteristics in order to test their effects on both positive and negative outcomes of ride-hailing from workers’ perspective.", "Fourth, our results show that the mechanisms contributing to workers’ discontent, such as the rating mechanism and dynamic pricing, are nevertheless instrumental for the platforms’ current business models.", "Hence, this study can be viewed as a starting point for a discussion on the role of the IT artefact characteristics in the parallel manifestation of the positive and negative aspects of platform-enabled work from workers’ perspective.", "As a result, our findings contribute to the IS literature on the dualities of IT (Islam et al., 2019; Mäntymäki & Islam, 2016; Turel & Serenko, 2012) in the context of digital work intermediaries.", "To this end, our findings elucidate the controversies in the employer-employee relationships resulting from certain characteristics of ride-hailing platforms as IT artefacts.", "As with any other study, the results are subjective to several limitations.", "However, the limitations of the present study simultaneously are avenues for future research.", "First, we focused on ride-hailing platforms; thus, a natural avenue for future research is to broaden this study’s contextual scope to other types of platform-enabled work (Gandini, 2019; Howcroft & Bergvall-Kåreborn, 2019; Kuhn & Maleki, 2017).", "For example, AirBnB is a dominant digital platform in the travel-accommodation industry and an income source for many people globally.", "Second, in this study, we adopted work relations and non-standard work as the theoretical foundations, but it is also possible to investigate ride-hailing work as a business relationship between a micro-size enterprise and a multinational corporation and employ agency theory (Eisenhardt, 1989), transaction-cost theory (Williamson, 1993), or contract theory (Bolton & Dewatripont, 2005), among others, as theoretical lenses to guide the inquiry.", "For example, future research could specifically examine issues related to security and liability from these theoretical vantage points.", "Third, Uber and Lyft have been changing their business models and strategies to adapt to local markets.", "For example, in some countries like South Africa, passengers have the option to pay in cash.", "Therefore, the positive and negative outcomes identified in this study by no means are an exhaustive presentation of the complexities and challenges surrounding work done for digital platforms such as Uber and Lyft.", "Thus, future research could focus on more in-depth inquiries in a particular region or country to provide contextualized insights on the influence of ride sharing on work relations.", "Finally, the business model behind intermediary-based digital platforms like Uber and Lyft stems from the platform’s control over the transactions and data generated.", "The data-control mechanism is perhaps a key element of the administrative control that drivers experience.", "Decentralized technology, such as blockchain (Hughes et al., 2019; Tapscott & Tapscott, 2016), offers a technical foundation for peer-to-peer transactions and may disrupt the current landscape of intermediary-based digital platforms significantly.", "This may reduce the power disparity between drivers and the platform owner.", "For example, Arcade City is a peer-to-peer rideshare network and marketplace operated by drivers where clients connect directly with drivers using a blockchain-based IT architecture3 .", "Thus, future research could investigate to what extent a power disparity could be mitigated through the use of blockchain-based digital platforms." ]

[ "We identify six key mechanisms of platform-enabled work, namely self-employment, time management, income, information control, pricing, and rating.", "From workers’ perspective, flexibility in work relationships is a key positive element of platform-enabled work.", "The stark power disparity between workers and the platform is, in turn, a major source of discontent among workers.", "We put forward two key dimensions of work relations in the context of platform-enabled work: digital temporality and digital administrativity.", "We identify six key mechanisms of platform-enabled work: self-employment, time management, income, information control, pricing, and rating." ]

[ "NEUR" ]

[]

[ "2805", "2808" ]

[ "Mental imagery – the ability to generate internal representations that preserve the core features of a perceptual experience – relies on similar neural mechanisms as those of actual perception (Kosslyn et al., 2001; McNorgan, 2012).", "This mechanism of shared representation between imagery and perception is common across sensory modalities, including the tactile domain (Gallace, 2013; Olivetti Belardinelli et al., 2009; Schmidt et al., 2014; Yoo et al., 2003).", "However, the possibility for imagery to engage representations of painful percepts is unknown.", "Indirect evidence of the interplay between mental imagery and pain perception is provided by clinical investigations of the therapeutic efficacy of motor imagery and spontaneous imagery in chronic pain patients.", "For example, imagining the movement of an affected limb can temporarily reduce pain symptoms in patients with chronic complex regional pain syndrome and phantom limb pain (e.g., MacIver et al., 2008; Moseley, 2004, 2006).", "Similarly, clinical investigations showed that patients with chronic pain spontaneously experience distressing mental images that contribute to a negative feedback loop maintaining and exacerbating pain (Berna et al., 2011, 2012; Gosden et al., 2013).", "The use of coping images has also been found to facilitate control over symptoms (Berna et al., 2011, 2012).", "However, while the relevance of mental imagery as either a strategy for pain reduction or therapeutic target (as in the case of spontaneous negative images) is well established (Berna et al., 2011), the cognitive and neural mechanisms responsible for imagery-driven modulatory effects on pain perception remain unclear.", "For instance, it is presently unknown whether pain-related mental images influence perception by merely redirecting attention away from the source of pain towards an internal mental image or rather by acting specifically on sensory or affective pain-related processing.", "We thus tested whether pain imagery produces specific directional (e.g., hypoalgesic and hyperalgesic) effects, or rather only interferes with pain processing regardless the imagined content.", "To address these issues, we assessed the influence of inhibitory and facilitatory mental imagery on pain intensity and unpleasantness using subjective ratings, signal detection measures, pain-related electrophysiological potentials (ERPs) and source reconstruction.", "Pain subjective ratings provided a measure of the participants' efficacy using imagery to modulate sensory and emotional aspects of pain.", "The use of signal detection theory allowed us to establish whether the imagery-driven alteration of pain sensations was linked to changes in stimulus discriminability (d-prime) and response bias (criterion) (Macmillan and Creelman, 2004).", "Further, we applied a mass-univariate approach to identify which of the temporally distinct pain-related ERP components (N2 and/or P2 potentials) reflected the imagery effects in a spatio-temporally unbiased analysis; see Kilner (2013).", "Finally, we applied a minimum norm source reconstruction method, in order to delineate possible neural origins of the ERP effects associated with the two modulatory conditions.", "Specifically, to illuminate the role of mental imagery in modulating pain, we directly contrasted two mechanistic hypotheses.", "First, we reasoned that if mental imagery primarily depends upon cognitive demand in modulating the upcoming stimuli, we should observe a general attenuating effect of imagery vs. baseline coupled with no difference between pain-inhibitory and facilitatory imagery (“a-directional hypothesis”).", "In such a case, the effort of generating and maintaining a mental image may serve to distract from or interfere with pain processing, regardless of the imagined content.", "This hypothesis proposes that hyperalgesic imagery should have a minimal effect on pain exacerbation and that both hypoalgesic and hyperalgesic imagery should correspond to similar amplitudes of pain-related ERPs, as well as similar underlying cortical sources.", "Alternatively, if mental imagery exerts distinct effects on the processing of stimulus features or affective responses related to upcoming stimuli, we expected content-specific differences when contrasting hypoalgesic and hyperalgesic imagery (“directional hypothesis”).", "This latter hypothesis would predict similar effects for both hypoalgesic and hyperalgesic effects, albeit in opposite directions, across the subjective, behavioral, scalp and source-level ERP measures.", "The clarification of this mechanism offers important insights on the role of top-down mental imagery on pain regulation.", "The present study investigated the neural mechanisms by which mental imagery produces hypoalgesia and hyperalgesia in pain perception.", "Participants utilized mental images of a glove or a wounded forearm as strategies to attenuate or exacerbate their pain perception.", "We considered two main hypotheses to dissociate whether modulatory imagery effects were primarily driven by the redirection of attention away from upcoming sensations (a-directional hypothesis) or by altered processing of sensory and affective features embedded within each modulation condition (directional hypothesis).", "We found robust support for the latter; the participants' ability to modulate their subjective pain experience was paralleled by content-specific alterations of the signal discrimination performance, reaction times and the N2 pain-related component between 122 and 180 ms.", "In accordance with the directional hypothesis, both inhibitory and facilitatory instructions moved the experience of pain intensity and unpleasantness towards imagined outcomes.", "When participants actively inhibited pain, not only did subjective pain perception decrease, but the detection of high-intensity stimuli as painful was also delayed.", "Moreover, high-intensity stimuli were more frequently categorized as non-painful and participants were less willing to report pain under inhibition.", "Congruently, when participants actively facilitated pain, the increase in perceived pain intensity and unpleasantness was associated with a delayed judgment of low-intensity stimuli as non-painful.", "Further, participants showed increased classification of low-intensity stimuli as painful and were more willing to report pain under facilitation.", "We also found evidence for changes in amplitudes of the pain-related N2 potentials by the content of instruction at 122–180 ms. However, no intensity by instruction interaction was observed in the time range of the N2, suggesting that the observed neural mechanisms underlying the modulation of pain are not pain-specific, but rather reflect modulatory mechanisms common to both pain and somatosensory-related processing (Bromm and Scharein, 1982).", "With respect to the N2 amplitudes, the two modulatory conditions diverged, with inhibition and facilitation respectively increasing or decreasing the magnitude of these negative potentials with respect to baseline.", "We note that given the larger N2 amplitudes for high vs. low stimulus intensity, one may have expected N2 amplitudes to decrease under inhibition vs. baseline (Bromm and Lorenz, 1998; Dowman, 1994).", "Surprisingly, here we observed the opposite effect; participants reported reduced pain under inhibition, but pain inhibition elicited greater N2 amplitudes.", "This finding contrasts with the interpretation of these potentials as objective pain intensity markers, suggesting that the magnitude of the N2 response cannot be considered a direct read-out of pain intensity (Bromm and Lorenz, 1998), but rather reflects a complex pain-related process integrating stimulus salience and cognitive expectations (e.g., Legrain et al., 2011).", "This pattern of N2 potentials fits with recent evidence that both sensory prediction errors conveyed by forward connections and top-down predictions conveyed by backward connections are implicated in the generation of late cortical ERPs (> 100 ms) (Garrido et al., 2007).", "For example, when a subject expects to have less pain (inhibitory mental imagery), but actually receive a painful stimulus, this would produce a net positive pain prediction error.", "In contrast, when a participant expects to feel more pain (facilitatory mental imagery), but actually receives less pain, the relative pain prediction error is negative.", "Thus, the directed alterations of the N2 potentials observed here may reflect the interaction between the sensory expectations encoded in the mental imagery and the actual sensory input.", "Future work would benefit from directly manipulating expectations to better determine the role of hypoalgesic and hyperalgesic prediction errors in influencing the amplitude of pain-related potentials.", "Within the 122–180 ms time window corresponding to the N2 instruction effect, neural activity associated with inhibitory vs. facilitatory mental images was localized in the right inferior frontal and temporal regions (Fig. 5).", "These right-lateralized sources are consistent with evidence of right-hemisphere dominance in pain processing (Symonds et al., 2006) and inhibitory control (Garavan et al., 1999).", "Specifically, the right inferior frontal gyrus is part of a fronto-parietal network of brain regions involved in inhibitory control across a wide range of cognitive tasks (Hampshire et al., 2010).", "Further, previous studies showed that rIFG interacts with temporal brain regions during the retrieval of semantic information from long-term memory (Aron et al., 2004) and suppressing intrusive thoughts (Anderson et al., 2004).", "Our results are in agreement with the general role of right IFG in “reconfiguring a representation of a currently attended input” (Hampshire et al., 2010) and in generating inhibitory outputs.", "In the present experiment, right inferior frontal and temporal regions are likely involved in retrieving an alternative inhibitory content driven by the voluntarily generation of a mental image.", "Instead, sources of facilitatory vs. inhibitory mental imagery were identified in left insular, inferior frontal, middle frontal, precentral and supplementary motor areas.", "Sources in the left hemisphere are consistent with the sensory stimulation of the right hand.", "In particular, these regions partially overlap with a network of cortical regions involved in salience processing and cognitive emotion regulation (Buhle et al., 2014; Kalisch, 2009; Kohn et al., 2014).", "Consistent with these findings, cognitive up-regulation and down-regulation of negative emotions have been shown to rely on both shared and distinct prefrontal activations.", "In particular, up-regulation (i.e., facilitation) has been uniquely linked to the recruitment of left medial prefrontal regions likely involved the retrieval of emotional contents; whereas down-regulation (i.e., inhibition) specifically recruited right lateral prefrontal regions involved in inhibitory cognitive control (Ochsner et al., 2004).", "Although these findings strongly support a directional effect, the precise regulatory mechanism underlying hypoalgesic and hyperalgesic mental imagery effects can be related to a variety of explanations; for instance, expectation, placebo–nocebo and cognitive reappraisal of negative affect.", "In conclusion, our results converge towards an interpretation of mental imagery as a flexible tool to alter pain and somatosensory sensations according to specific contextual expectations.", "We provided novel evidence for content-specific hypoalgesic and hyperalgesic imagery in modulating perception in opposite directions, biasing the perceptual boundary between high and low-intensity stimuli towards the instructed outcome, and modulating pain-related cortical responses at 122–180 ms. More specifically, inhibitory effects were associated with the recruitment of right lateralized brain regions commonly activated during cognitive control, whereas facilitatory pain effects were related to increased activity within salience and affective cortical regions.", "An important implication is that mental imagery may be a particularly relevant strategy for advancing clinical understanding and management of pain, such as in chronic pain and post-operative recovery, as well as further elucidating the neurocognitive mechanisms underlying pain regulation.", "25 healthy volunteers were recruited from Aarhus University and the local community.", "All participants were proficient Danish speakers, right-handed, with normal or corrected-to-normal vision.", "No participants reported a history of pain disorders, neurological or psychiatric illness, or use of analgesics.", "All participants gave informed consent before participation and received a reimbursement of 300 DKK (~ 40EUR).", "Data from two participants were not included in any analyses due to incomplete data collection (one participant did not complete the experiment, and another had missing behavioral data in most blocks).", "Two other participants were excluded from statistical analysis on account of excessive EEG artifacts.", "The final sample included 21 participants (9 female; mean age = 24.5 years; range = 21–36 years).", "The study was approved by the Ethical Committee of Central Region Denmark and conducted in accordance with the Declaration of Helsinki.", "Participants were asked to (1) modulate the upcoming sensory stimulation through script-driven mental imagery, (2) identify each stimulus as either painful or non-painful (pain judgment task), and (3) evaluate their pain experience (pain intensity, pain unpleasantness, and efficacy ratings), see Fig. 1.", "At the beginning of each block of trials, participants were required to imagine the content of a verbal script and to use the suggested mental images to either inhibit or facilitate the triggered pain responses or to experience pain without modulation (baseline).", "In the inhibition and facilitation conditions, instructions suggested that participants generate and maintain the mental image of either a glove or a wound on the right forearm to attenuate or to exacerbate pain sensations, respectively.", "In the baseline condition, instructions were to simply imagine the skin of the right forearm, without any pain modulation.", "The instruction for inhibition was chosen according to a previous study (De Pascalis et al., 1999), whereas the instruction for facilitation was specifically designed to mirror the inhibition condition, altering the content of the image (wound instead of a glove) and the directionality of the modulation (amplification instead of attenuation).", "Within each block of trials, participants were required to judge each stimulus as either painful or non-painful as quickly and accurately as possible by pressing two possible keyboard buttons, counterbalanced across participants (Fig. 1).", "Finally, at the end of each block of trials, participants were invited to rate the worst pain intensity and unpleasantness felt in the previous block and to judge their ability to influence the triggered responses accordingly to the given instruction (efficacy ratings; Fig. 1).", "In a single experimental session, participants performed separate pain and empathy-for-pain tasks in a counterbalanced order.", "The rationale of the empathy-for-pain task was very similar to the pain task explained here, with instructions to either decrease or increase empathy, while viewing pictures of faces with neutral or painful expression.", "The pain and empathy-for-pain tasks were separated by a pause of 15–20 min.", "Here, only the pain task is reported.", "The pain session started with a calibration task identifying the intensity of non-painful and painful stimulation suitable for each participant.", "Using a bipolar electrode placed on the right forearm over the medial nerve, stimulus trains of increasing intensity (starting intensity = 0.39 mA; step = max 0.39 mA) were delivered via a Digitimer DS5A stimulator (Digitimer, Hertfordshire, UK).", "Participants rated the intensity of each stimulus on a horizontal visual-analogue scale (VAS; range = 0–10, where 0 equals “no pain sensation”, 1 “just noticeable pain” and 10 “worst imaginable pain”).", "The calibration terminated when the participant rated intensities with a score greater than 8.", "Hence, intensities corresponding to 0.8 and 8 ratings on the VAS were chosen for the experimental task.", "The calibration task was followed by written and oral instructions describing the experimental task and a brief training session consisting of three blocks, one for each condition (i.e., inhibition, baseline, and facilitation).", "The training always began with the baseline condition, whereas the second block could be either inhibition or facilitation in a counterbalanced order.", "All participants reported that the three blocks were sufficient for understanding the task.", "The experimental task consisted of 24 blocks, 8 for each condition.", "Each block began with a display of a verbal script informing the participant of the upcoming block condition (i.e., inhibition, baseline, or facilitation; Fig. 1A, “instruction”).", "Each instruction was presented for 15 s, followed by a 2 s inter-stimulus interval and a random set of 12 stimuli of two fixed intensities (low-intensity and high-intensity; Fig. 1A, “stimulation”).", "The duration of each stimulus was 5 ms. Importantly, participants were not informed that only two electrical intensities were delivered and were instead instructed that the stimuli could have any intensity corresponding to the range between 0.8 and 8 in the VAS, according to their ratings in the calibration task.", "Participants were asked to maintain the mental image throughout each block while performing a pain judgment task.", "After each stimulus, participants pressed a button with either the middle or the index finger of the left hand to indicate whether the stimulus was perceived as painful or not (Fig. 1B).", "If no button was pressed within 1000 ms of the stimulation, the response was marked as missing.", "Buttons identifying perception of painful and non-painful stimuli were counterbalanced across participants.", "The inter-stimulus interval (ISI) corresponded to 1200–1800 ms after participants' response.", "Following each set of stimuli, participants were asked to recall the most painful stimulus felt and to provide ratings on three VASs (Fig. 1A, “rating scales”).", "On the first scale, participants rated the highest painful intensity they felt (“How much pain did you feel?”", "; 0 = no pain, 10 = the worst imaginable pain).", "On the second scale, they were asked to rate the highest unpleasantness (“How unpleasant did you feel?”", "; 0 = no unpleasantness, 10 = the worst imaginable unpleasantness).", "Finally, in inhibition and facilitation blocks only, they were asked to rate their ability to influence their pain experience (“How efficient were you in influencing your sensations?”", "; 0 = no control, 10 = perfect control).", "After each rating session, 5-s intervals separated contiguous blocks.", "The blocks were presented with two possible pseudo-randomized orders (direct or reverse) to counterbalance order effects.", "The direct order was the following: B, B, F, F, I, I, B, B, I, I, F, F, I, I, F, F, B, B, F, F, I, I, B, and B (where B corresponds to baseline, F to facilitation, I to inhibition).", "Before and after the pain and empathy tasks, 5-min resting EEG sessions were recorded, while participants were sitting relaxed on the chair with their eyes open.", "Between the second and the third session (i.e., in between the two tasks), participants completed a computerized form of the Interpersonal Reactivity Index (Davis, 1980) and a short version of the Tellegen's Absorption Scale (Tellegen and Aktinson, 1974), for an assessment of individual differences in empathy and absorption, namely the ability to be engaged by thoughts, feelings and mental images.", "Data of the empathy task and IRI are not reported here.", "We calculated modulatory intensity and unpleasantness indices for each participant to highlight the rate of change (Δ) in terms of points in the VAS scale, by subtracting the mean rating at baseline from the mean rating at modulation (e.g., pain inhibition modulatory index corresponds to ΔI = mean I rating − mean B rating, whereas pain facilitation modulatory index corresponds to ΔF = mean F rating − mean B rating).", "These indexes were then transformed into percentages.", "Thus, modulatory indexes estimated the degree to which pain perception varied during each imagery condition.", "The participants' performance on the pain judgment task was assessed by signal-theoretic measures (i.e., d-prime and criterion) and reaction times.", "Signal detection theory (SDT) estimates participants' sensitivity (d-prime or d′) and response bias (criterion) while either detecting the presence of a stimulus or judging stimuli of two different intensities (Macmillan and Creelman, 2004).", "The measure d′ indicates the degree of stimulus sensitivity or discriminability, whereas the criterion reflects the participants' decision-making in terms of a more conservative or liberal decision bias in responding whether a certain stimulus has occurred.", "STD was thus used to identify whether changes in performance were driven by either alteration of d′, criterion or a combination of the two as a result of our conditional manipulation.", "In the present experiment, d′ and criterion were measured in relation to a binary decision, namely “painful” or “non-painful” responses with respect to high and low-intensity stimuli.", "Participants' responses were classified on the basis of an ideal performance, with high-intensity stimuli judged as painful and low-intensity stimuli as non-painful.", "Thus, “hits” corresponded to the classification of high-intensity stimuli as painful; “misses” to high-intensity stimuli classified as non-painful; “false alarms” to low-intensity stimuli classified as painful, and finally “correct rejections” to low-intensity stimuli classified as non-painful.", "As only two outcomes provide independent information about the participant's performance, d′ and criterion are computed using the proportion of hit (hit rate; H) and false-alarm trials (false alarm rate; F).", "If a hit and false alarm pair corresponded to perfect performance (i.e., H = 1 and F = 0), we converted F = 0 to F = .01 using the formula 1 / (2N) and H = 1 to H = .99 using the formula 1 − 1 / (2N) (Macmillan and Creelman, 2004).", "The sensitivity measure d′ was calculated as the normalized difference between hit and false alarm rates for each instruction (i.e., inhibition, baseline and facilitation).", "The formula corresponded to d′ = z(H) − z(F).", "Specifically, d′ measured the ability to categorize the two classes of stimuli across the three different instruction conditions.", "Higher sensitivity values were obtained when the participant's responses were closer to the ideal performance (high-intensity = “pain”; low-intensity = “no-pain”).", "Instead, lower sensitivity values corresponded to an increased mismatch between the ideal and the actual classification.", "Ceiling effect corresponded to d′ = 4.65.", "Alternatively, criterion was calculated as the normalized sum of hit and false alarm rates multiplied by − .5 for each instruction (i.e., inhibition, baseline and facilitation).", "The formula corresponded to c = [z(H) + z(F)] ∗ − .5.", "The criterion provided information about the participants' response bias in judging the stimuli as painful with a more conservative or liberal bias across conditions.", "A more conservative bias corresponds to positive criterion values.", "Instead, a more liberal bias corresponds to negative criterion values.", "Unbiased responses are obtained when the criterion value is close to zero.", "As changes in sensitivity (d′) and response bias (criterion) are independent, signal detection theory distinguishes between discriminative and motivational components in perceptual decision-making.", "Our application of SDT to pain differed from previous studies; for reviews and also a critique, see Lloyd and Appel (1976) and Rollman (1976, 1977).", "Here, we used SDT not to identify pain threshold or tolerance, but to determine mental imagery-driven changes in sensitivity and response bias across conditions that were identical in terms of stimulus intensities (50% low-intensity; 50% high-intensity stimuli).", "Importantly, the two levels of intensity were calibrated for each participant to reflect non-painful and painful perception in a neutral condition before the beginning of the experimental task.", "Reaction times measured the participants' speed in judging the high and low-intensity stimuli.", "Average reaction times (RTs) were calculated for each stimulus intensity and instruction condition, excluding false alarms (low-intensity stimuli judged as painful) and misses (high-intensity stimuli judged as non-painful).", "This exclusion was based on the assumption that response times for hits and correct rejections (matching trials) vs. false alarms and misses (non-matching trials) might reflect different underlying cognitive processes and as such the inclusion of all trials is likely to prevent correct identification of which conditions are driving our experimental effects of interest.", "Hence, we focused only on the neural correlates of matching trials both to ensure reliable conditional estimates and to facilitate a clear interpretation of our results.", "The average RTs for miss and false alarm trials were not analyzed given the low overall rate of these trials (average ± SE: false alarms = 5.57 ± 1.03 trials; misses = 7.81 ± 1.04 trials).", "Subjective ratings of perceived pain intensity and unpleasantness, as well as signal detection measures (d′ and c) were analyzed using one-way repeated measures ANOVAs, with the within-subject factor instruction (3 levels: inhibition, baseline, facilitation).", "Reaction times were analyzed using two-way repeated measures ANOVAs.", "The two within-subject factors were stimulus intensity (2 levels: high, low) and instruction (3 levels: inhibition, baseline, facilitation).", "Statistical significance was set at p < .05 and effect sizes were calculated using the partial η2.", "When the assumption of sphericity was violated, the p-values were adjusted according to the Huynh–Feldt correction (HF ε), and in case of significant effects, the Tukey HSD test was applied for post-hoc comparisons.", "Paired sample t-tests were applied to determine mean differences in subjective efficacy ratings, as well as intensity and unpleasantness indices across inhibitory and facilitatory conditions.", "Finally, to investigate factors driving pain control, we estimated a Pearson's correlation exploring the relationship of inhibition and facilitation efficacy ratings.", "All subjective and behavioral analyses were performed using the data analysis software system STATISTICA (version 8.0, www.statsoft.com).", "The E-Prime v.2.0 (PST, Inc.) software package was used for instructions, stimulation and presentation of VASs.", "Continuous EEG data were recorded with a 64-electrode active cap (actiCAP) and amplified (BrainAmp MR plus amplifiers) using the Brain Vision Recorder software (Brain Products, Munich, Germany).", "Two electrodes (i.e., PO9 and PO10) were removed from the cap and placed on the superior orbit and on the outer canthus of the right eye to detect vertical and horizontal eyes movements.", "The EEG was referenced to the FCz electrode, grounded at AFz, and sampled at 1000 Hz.", "The impedance was kept below 25 kΩ.", "EEG data were processed using EEGLAB (Delorme and Makeig, 2004) and SPM8 (Statistical Parametric Mapping 8, http://www.fil.ion.ucl.ac.uk/spm).", "Using EEGLAB, the continuous EEG was downsampled to 500 Hz, band-pass filtered (0.1–30 Hz) and segmented into 600-ms stimulus time-locked epochs (− 100/+ 500 ms).", "An independent component analysis (ICA) algorithm was applied to identify, select, and discard components representative of eye movements and an electrical artifact induced by the stimulation.", "The segments were then baseline-corrected using the average pre-stimulus activity (− 100/0 ms).", "Data were re-referenced to the algebraic mean of the left and right mastoids for scalp analysis of the N2 and P2 potentials and to the mean of all electrodes (i.e., average reference) for source reconstruction of the ERP effects.", "The following steps were identical for both datasets.", "Then, the two electrodes used to detect eye movements were removed from subsequent processing and the original reference activity (FCz) was reconstructed.", "Following downsampling and ICA, single-trial epoched data corresponding to high-intensity stimuli judged as painful and low-intensity stimuli classified as non-painful were imported to SPM8 for final preprocessing and statistical analysis.", "The exclusion of epochs corresponding to stimulus intensities deviating from ideal performance (i.e., false alarms and misses) resulted in 14% of the data being discarded.", "The rationale for the exclusion was the same previously applied for RTs.", "Further, epochs with extreme values exceeding a threshold of 150 mV were automatically detected, leading to an additional 1.8% of data discarded.", "Moreover, bad channels were visually identified, marked for removal and then interpolated.", "Robust averaging was applied for further artifact removal (Wager et al., 2005).", "This averaging method down-weights the contribution of outliers, canceling out extreme values, e.g., the portion of signal contaminated by artifacts, providing a good alternative to classical averaging, if artifacts do not regularly occur at the same time points across trials.", "A visual inspection of the data confirmed that this assumption was not violated.", "Thus, epochs were averaged separately for each condition, leading to six average waveforms corresponding to high-intensity inhibition, high-intensity baseline, high-intensity facilitation, low-intensity inhibition, low-intensity baseline, and low-intensity facilitation.", "To perform statistical analysis on ERPs across subjects, we converted the ERP amplitudes for each condition into three-dimensional scalp maps including two-dimensional sensor-space (x, y) and time (z) (Kilner and Friston, 2010; Litvak et al., 2011).", "For each participant, each time point of the averaged conditions was transformed into a two dimensional 64 × 64 pixel scalp map using linear interpolation and concatenated over the interval from 0 to 500 ms. The obtained 3D scalp map volumes (i.e., 6 images for each participant) were smoothed with a low-pass kernel (6 mm × 6 mm × 6 ms; full width at half maximum, FWHM) and entered into a general linear model (GLM) analysis.", "The time interval of interest for the statistical analysis did not include the baseline (− 100/0 ms), which by definition cannot differ across conditions and across subjects.", "Averaged referenced data were used for source reconstruction.", "As individual MRIs were not available, we used a template brain.", "A three-shell sphere (skin–skull–brain) was used to compute independent identically distributed (IID) solutions, as implemented in SPM8 (Litvak et al., 2011).", "This inversion method, equivalent to classical minimum-norm, determines the combination of sources that have the minimum total power (Hämäläinen and Ilmoniemi, 1994).", "The choice of the method was grounded on the assumptions of few distributed neural sources underlying the ERP effects.", "Further, compared with dipole-based methods, minimum norm estimates do not need a priori hypotheses regarding the distribution of active sources.", "The time window was restricted to the period in which significant instruction effects were present as revealed by scalp-level statistics.", "As for the scalp map volumes, the 3D current density maps were smoothed with a low-pass kernel (6 mm × 6 mm × 6 ms; full width at half maximum, FWHM) and entered into a general linear model (GLM) analysis.", "We first conducted a general linear model (GLM) analysis of stimulus intensity by instruction interactions, as well as instruction and intensity main effects, across temporal and spatial dimensions on scalp maps.", "Each individual conditional average was modeled in a flexible factorial design with the factors subject (21 levels), stimulus intensity (2 levels: high, low), and instruction (3 levels: inhibition, baseline, facilitation).", "The analysis was performed on the time window from 0 to 500 ms, to capture two well-known pain-related ERPs, namely the N2 (around 100–200 ms) and P2 potentials (around 200–400 ms) (Bromm and Lorenz, 1998).", "Uni-directional t-contrasts were used to evaluate the overall effect of stimulus intensity (high and low), and our two alternative hypotheses (a-directional and directional).", "To assess differences in amplitudes related to stimulus intensity, we conducted paired t-contrasts for the stimulus intensity main effect, collapsing the three instruction conditions (high > or < low).", "Second, to test the a-directional hypothesis, we contrasted the modulation conditions inhibition and facilitation vs. baseline (inhibition + facilitation > or < baseline).", "Finally, to address the directional hypothesis, we contrasted inhibition vs. facilitation, i.e., tested for responses driven by their specific modulatory content (inhibition > or < facilitation); and each imagery condition vs. baseline (inhibition > or < baseline; facilitation > or < baseline).", "Significance thresholds were corrected for multiple comparisons, across sensors and time points, using Gaussian random field theory to adjust the threshold for null hypothesis rejection (Kilner and Friston, 2010; Litvak et al., 2011).", "The statistical parametric maps were thresholded at pUNC < .001 (peak-level, uncorrected) and corrected for multiple comparisons based on cluster size using a family-wise error rate set at pFWE < .05.", "Mass univariate statistics, in conjunction with appropriate multiple comparison corrections, enable identification of conditional differences across electrodes and time windows, avoiding the use of a priori spatial or temporal regions of interest which may inflate the risk of false positive (type-II) errors when used inappropriately (Crowley et al., 2012; Lage-Castellanos et al., 2010).", "This approach reduces the risk of biasing statistical results, and offers the opportunity to maximize the information obtained when recording from a large electrodes array.", "Indeed, if analyses are guided by a posteriori observations of where or when the effects appear, type-II errors rates can be grossly inflated (Kilner, 2013; Kriegeskorte et al., 2009).", "On the other hand, if time windows and electrodes are a-priori selected on the basis of previous studies, it is possible that important effects may remain undiscovered.", "Thus, the mass-univariate approach provides a balance of sensitivity and specificity in large-array EEG investigations.", "We then conducted a general linear model (GLM) analysis of the instruction main effects at the source level.", "As for the scalp maps, each individual conditional average was modeled in a flexible factorial design with the factors subject (21 levels), stimulus intensity (2 levels: high, low), and instruction (3 levels: inhibition, baseline, facilitation).", "The analysis was performed on the time window from 122 to 180 ms, as this period was associated with significant instruction effects at the scalp level.", "Uni-directional t-contrasts were used to evaluate the overall effect of inhibition vs. facilitation, inhibition vs. baseline, facilitation vs. baseline, and modulation (inhibition and facilitation) vs. baseline.", "Significant effects were thresholded at pFWE < .05 corrected for both peak- and cluster-level, with a minimum cluster size of 10 voxels.", "Labels for each significant source location were determined using cytoarchitectonic probabilistic maps from SPM Anatomy Toolbox (Eickhoff et al., 2005).", "Participants reported significant differences in the perceived pain intensity and unpleasantness experience according to the given instruction.", "Compared to baseline, inhibition and facilitation blocks were associated with significantly decreased or increased intensity and unpleasantness ratings, respectively.", "The instruction main effect for subjective intensity ratings corresponded to F(2,40) = 22.29, partial ƞ2 = 0.52, p < .001 (Fig. 2A).", "Further, the instruction main effect for subjective unpleasantness ratings was F(2,40) = 32.61, partial ƞ2 = 0.62, p < .001 (Fig. 2B).", "To clarify the extent to which participants reported effective modulation of intensity and unpleasantness, we calculated four modulatory indices corresponding to the difference between the ratings under each instruction condition minus the corresponding rating at baseline.", "The four resulting indices were ΔI Intensity, ΔF Intensity, ΔI Unpleasantness, ΔF Unpleasantness.", "The intensity modulation indexes showed considerable variability across participants, ranging from no modulation at all to decrements of 27% (mean ± SE = 6.92 ± 1.93%) and increments of 21% (mean ± SE = 7.52 ± 1.62%).", "Similarly, the unpleasantness modulation indexes varied from no modulation at all to decrements of 33% (mean ± SE = 6.1 ± 1.71%) and increments of 25% in the ratings (mean ± SE = 9.64 ± 1.74%).", "The inhibition vs. facilitation modulation indexes did not differ for both the intensity (t(20) = 1.03, p = n.s.) and the unpleasantness ratings (t(20) = − 1.39, p = n.s.).", "The mean value of intensity and unpleasantness modulation for each participant is represented in Figs. 2A and B.", "Crucially, participants were asked to rate the degree to which they were able to modulate pain sensations in either direction after each block.", "The results showed that the perceived efficacy ratings for pain inhibition (mean rating ± SE = 4.13 ± 0.56) and pain facilitation (mean rating ± SE = 3.93 ± 0.56) did not differ (t(20) = 1.09, p = n.s.); participants reported feeling equally capable of inhibiting or facilitating pain.", "The mean values of the efficacy ratings for each participant are represented in Fig. 2C. Interestingly, the efficacy ratings under inhibition and facilitation were strongly correlated (r = .95, p < .001; Fig. 2C).", "The behavioral performance in the pain judgment task was evaluated in terms of the signal theoretic measures d′ and criterion, which are two measures depending upon the rate of hits or correct responses (i.e., high-intensity stimuli classified as painful) and false alarms (i.e., low-intensity stimuli classified as painful).", "Two separate ANOVAs performed on the hit and false-alarm rates showed instruction main effects for both measures (hit rate: F(2,40) = 15.34, partial ƞ2 = 0.43, p < .001, Fig. 3A; false-alarm rate: F(2,40) = 14.00, partial ƞ2 = 0.41, p < .001, Fig. 3B).", "With respect to baseline, the hit rate was significantly lower when inhibiting pain, while the false alarm rate was significantly higher when facilitating pain.", "During inhibition, 36.14 ± 2.46 high-intensity trials and 1.52 ± 0.64 low-intensity trials were classified as painful.", "During baseline, 44.62 ± 0.87 high-intensity trials and 3.14 ± 0.93 low-intensity trials were classified as painful.", "Finally, during facilitation, 46.52 ± 0.35 high-intensity trials and 9.81 ± 2.16 low-intensity trials were classified as painful.", "The sensitivity measure d′ described the participants' performance in classifying high and low-intensity stimuli as “painful” and “non-painful” during the three different instruction conditions.", "The ANOVA on d′ showed a significant instruction main effect (d′: F(2,40) = 5.70, partial ƞ2 = 0.22, p < .01; Fig. 3C).", "These results demonstrated that participants judged the two classes of stimuli differently, in accordance with the given instruction.", "The average participants' response during baseline was closer to the ideal performance with respect to inhibition (B > I, p < .01) and facilitation (B > F, p = .06).", "No difference in d′ was found for the contrast inhibition vs. facilitation (I > F, p = .62).", "This null effect was due to a similar difference between hit and false alarms rates within the two instruction conditions, although hit and false alarm rates significantly differed when inhibiting and facilitating pain.", "Further, response criterion (c) represented a measure of how biased participants were in judging the stimuli as painful.", "The ANOVA on c found a significant instruction main effect (F(2,40) = 32.76, partial ƞ2 = .62, p < .001; Fig. 3D).", "Participants showed unbiased judgment at baseline, but a more conservative bias during inhibition (I > B, p < .001) and a more liberal bias during facilitation (B > F, p = .001).", "Participants were less willing to report pain when instructed to inhibit the upcoming sensations, whereas they more willing to report painful sensations following the facilitation instruction.", "In the pain judgment task, behavioral performance was evaluated in terms of how quickly participants classified high and low-intensity stimuli as painful and non-painful in the three different conditions.", "The ANOVA on RTs showed significant stimulus intensity by instruction interaction (F(2,40) = 39.25, partial ƞ2 = 0.66, HF ε = .63, p < .001; Figs. 3E and F), stimulus intensity main effect (F(1,20) = 35.63, partial ƞ2 = 0.64, p < .001) and instruction main effect (RTs: F(2,40) = 7.29, partial ƞ2 = 0.27, p < .01).", "The results revealed a differential modulation of participants' speed in judging the stimuli as either painful or non-painful, in accordance with the given instruction.", "Indeed, participants classified high-intensity stimuli as painful with slower RTs under inhibition (p < .001) and faster RTs under facilitation (p < .05), as compared to pain baseline.", "The opposite pattern was found for low-intensity stimuli, where participants judged them as non-painful with slower RTs under facilitation (p < .05), compared to the no-pain baseline.", "No statistical between-condition differences emerged for inhibition vs. baseline when categorizing low-intensity stimuli.", "The stimulus intensity main effect indicated that overall, high-intensity stimuli were detected faster than low-intensity ones.", "Accordingly, post-hoc comparisons confirmed that participants were significantly faster in responding to high vs. low-intensity stimuli at baseline (p < .001, mean difference of 70 ± 55 ms) and under facilitation (p < .001, mean difference of 170 ± 91 ms).", "In contrast, participants responded similarly to the two levels of stimulus intensity under inhibition (mean difference of − 29 ± 90 ms, p = n.s.).", "The accelerating effect of high vs. low-intensity observed in the baseline condition was thus greatly increased during facilitation, but abolished during inhibition.", "Stimulus intensity-related information was thus differentially conveyed to the motor system in accordance with the instruction condition.", "According to the evaluations at the 12-items Tellegen's Absorption Scale (TAS), the participants can be considered ‘average imagers’ (mean ± SE = 6.76 ± 0.39, min = 3, max = 9).", "To explore the relationship between absorption and efficacy in pain modulation, we carried out a regression analysis with TAS scores as predictor and the efficacy ratings (associated with inhibition and facilitation) as dependent variables.", "The TAS scores failed to predict both the efficacy ratings of pain inhibition and facilitation (r = .05, r = .01, respectively).", "Stimulus intensity main effects were found at 102–130 ms post-stimulus, over an anterior-central cluster (peak-level Tmax = 5.88; cluster-level pFWE < .001; Fig. 4 and Table 1).", "This effect corresponded to increased N2 amplitudes elicited by high vs. low-intensity stimuli.", "Additional stimulus intensity main effects, corresponding to increased P2 amplitudes elicited by high vs. low-intensity stimuli, were found in a central cluster at 202–414 ms (peak-level Tmax = 10.56; cluster-level pFWE < .001; Fig. 4 and Table 1).", "These results show that the classic pain-related N2 and P2 components were distinctly modulated by stimulus intensity.", "To assess whether ERP pain-related responses are influenced by hypoalgesic and hyperalgesic mental imagery in terms of similar mechanisms, we collapsed the two modulation conditions and compared them to baseline.", "Neither the intensity by instruction interaction, nor the instruction main effect revealed any result surviving FWE cluster corrections.", "To evaluate whether ERP amplitudes are specifically modulated by the content of the mental imagery, we compared inhibition vs. facilitation conditions with two unidirectional t contrasts (I > F and I < F).", "No significant intensity × instruction interaction was found.", "However, a significant instruction main effect was observed at 122–180 ms for the contrast I < F over an anterior right cluster (Fig. 5 and Table 2).", "The stimuli elicited greater negative amplitudes under inhibition compared to facilitation, regardless of stimulus intensity (peak-level Tmax = 5.00; cluster-level pFWE < .001; Fig. 5 and Table 2).", "We thus performed planned follow-up contrasts to determine whether either condition diverged from baseline; i.e., I < B and B < F.", "In the inhibition vs. baseline follow-up contrast, a significant instruction effects survived cluster correction at 156–168 ms (peak-level Tmax = 4.21; cluster-level pFWE = .003; Fig. 5 and Table 2).", "Finally, in the facilitation vs. baseline follow-up no significant intensity by instruction interaction or instruction main effect survived the cluster correction.", "However, cluster-level uncorrected results revealed an instruction main effect at 160–162 ms (peak-level Tmax = 3.47; cluster-level pFWE = n.s.).", "Source reconstruction of the instruction effect was performed for the interval between 122 and 180 ms, corresponding to the significant instruction scalp effect.", "Following IID source reconstruction, statistical parametric maps were thresholded at pFWE < .05 at both cluster and peak-level.", "All MNI coordinates of significant effects are reported in Table 2.", "The inhibition vs. facilitation contrast (Fig. 5) revealed sources in right inferior frontal gyrus (peak-level Tmax = 5.25; cluster-level pFWE = .006) and right inferior temporal gyrus (peak-level Tmax = 4.55; cluster-level pFWE = .03).", "Instead, the facilitation vs. inhibition contrast (Fig. 5) was associated with sources in left insula (peak-level Tmax = 8.24; cluster-level pFWE = .001), left inferior frontal gyrus (peak-level Tmax = 6.94; cluster-level pFWE = .007), left middle frontal gyrus (peak-level Tmax = 6.43; cluster-level pFWE = .004), left supplementary motor area (peak-level Tmax = 5.87; cluster-level pFWE = .001), as well as bilateral precentral gyri (peak-level Tmax = 5.48; cluster-level pFWE = .005).", "Similar patterns of sources were identified for the contrasts inhibition vs. facilitation and inhibition vs. baseline, as well as for contrasts facilitation vs. inhibition and facilitation vs. baseline (Table 2).", "No significant effect was found for the two modulation conditions (inhibition and facilitation) vs. baseline.", "Finally, we carried on an explorative analysis as suggested by a reviewer to elucidate potential ERP differences between subjective pain perception and objective physical intensity.", "We thus analyzed two additional contrasts at the scalp-level; i.e., low-intensity stimuli classified as non-painful vs. painful and high-intensity stimuli categorized as painful vs. non-painful, regardless the instruction conditions.", "We found that low-intensity stimuli judged as non-painful elicited increased positive amplitudes at 396–416 ms over occipito-parietal electrodes with respect to stimuli of identical physical intensity but classified as painful (i.e., peak-level Tmax = 4.27; cluster-level pFWE = .01).", "Further, uncorrected results suggested that high-intensity stimuli judged as painful vs. non-painful could evoke different neural activity at 26–52 ms and 90–104 ms; however these results were not significant after cluster correction.", "It is important to note that this analysis is underpowered given that lower amount of trials for the critical conditions (high-intensity stimuli perceived as non-painful and low-intensity stimuli perceived as painful).", "The directional pain modulation driven by mental-imagery supported by our results might be differentially attributed to expectation, placebo, and reappraisal (Ochsner and Gross, 2005; Tracey, 2010; Wiech et al., 2008).", "We examine each interpretation in turn.", "First, the modulatory effects can be related both to expectations about pain itself and expectations about mental imagery.", "An expectation of reduced or increased pain is sufficient to alter how events are attended and thus experienced (Atlas et al., 2010; Keltner et al., 2006; Koyama and McHaffie, 2005).", "Alternatively, expectations embedded in mental imagery can be conceived as a placebo- or a nocebo-like effect.", "Indeed, imaginative suggestions include elements of both verbal suggestions and conditioning, by virtue of the associative content of a protective glove and excruciating wound with pain relief and pain exacerbation, respectively.", "Interestingly, placebo effects have been postulated to rely upon patients' mental imagery (Kojo, 1988).", "A final possibility is that mental imagery sustains reappraisal, which operates through cognitive reshaping of the stimulus meaning (Gross, 2001).", "In the context of pain, the perceived controllability of pain is thought to change the meaning of the upcoming stimulation (i.e., reappraisal), thereby reducing pain perception (Wiech et al., 2006, 2008).", "Expectation, placebo and reappraisal can be unified in terms of a common mechanism, in terms of integrating between bottom-up sensory signals and top-down predictions (e.g., Frith and Dolan, 1997).", "These differential interpretations are ripe for future experimental research into the underlying mechanisms driving the potent content-specific pain modulations observed here.", "Our findings supporting the role of mental imagery in altering pain-related cognitive and emotional processing may shed light on the putative mechanisms underlying the effects of spontaneous mental images in patients suffering of chronic pain.", "For example, Philips (2011) showed that chronic pain patients frequently experience intrusive negative images linked to pain and one of the common theme of such images are physical and anatomical details of the pain or injury.", "Moreover, Berna et al. (2011, 2012) reported that some patients suffering chronic pelvic pain described not only negative imagery, but also coping images such as “imagery of treatment applied to the body” to reduce pain sensations.", "It is thus possible that the neurocognitive mechanisms observed in the present study are involved in spontaneous imagery reported in clinical settings.", "A potential limitation of the present study is the lack of subjective ratings for non-painful stimuli.", "This limitation partially precludes us from determining whether the facilitation of non-painful stimuli corresponded to an allodynic effect at the perceptual level.", "A related worry is that pain ratings may have somehow biased responses to painful versus non-painful stimuli.", "While these issues should be raised in future studies, we note that the subjective effects were well mirrored behaviorally, with mental imagery consistently modulating reaction times, stimulus judgments and response biases.", "The consistency of our findings across behavioral, subjective, and electrophysiological measures, coupled with the fact that pain ratings were collected for “the most painful stimuli” across blocks of both painful and non-painful conditions thus suggests that these effects depended upon mental-imagery regulation rather than non-specific effects of ratings.", "However, future research may benefit from collecting ratings following individual stimulus presentations to better elucidate this issue." ]

[ "Specific mental imagery contents bias pain perception towards hypoalgesic and hyperalgesic outcomes", "Mental imagery influences pain-related perceptual decision-making in terms of stimulus discriminability and response bias", "Pain-related N2 amplitudes are modulated by mental-imagery content", "Cortical activity associated with pain inhibition likely reflects cognitive inhibitory control", "Cortical activity associated with pain facilitation likely reflects salience and emotional processing" ]

[ "MULT" ]

[ "Diabetes", "Methylglyoxal", "Osteoclast", "Osteoporosis", "Pathways" ]

[ "1000" ]

[ "The band densities quantification of Western Blotting was shown in the graph below.", "In Fig. 1a, Representative blots of p-p38, p-p44/42, p44/42, and beta-actin of the cells after exposing to MG (400 μM) after the treatment time intervals between 1 h and 24 h. Phosphorylation of p38 and p44/42 were induced by MG in RAW264.7 cells.", "Endogenous p44/42 and beat-actin were used as the internal control.", "Data shown was corresponding to 5 repeated experiments.", "In Fig. 1b, the mRNA expression of osteoclast biomarkers: TRACP5, OSCAR, and CTSK still increased in MG group and the effects of MG did not countered by the transfection.", "Before 400 μM MG treatment, RAW264.7 cells underwent transfection under treatment of 10 nM siRNA for 24 h.", "The cells were collected after 24 h MG treatment.", "However, the mRNA expression of the osteoclast bone biomarkers persisted to increase under p38 and p44/42 inhibition in MG-treated macrophages.", "Beta-actin acted as mRNA internal control.", "Data shown was corresponding to 5 repeated experiments.", "These data implied that MG activated the p38 and p44/42, which was reported to regulate proliferation and differentiation of osteoclast.", "However, the decreasing MAPK though siRNA knockdown did not change expression of those target markers, TRACP5, OSCAR, and CTSK, in mRNA level.", "The effects of MG to other osteoclast markers through p38 and p44/42 would be worth to be investigated.", "Methylglyoxal was purchased from Sigma (St. Louis, MO, USA).", "Anti-β-actin antibody was purchased from Sigma (St. Louis, MO, USA).", "Lipofectamine® 2000 Transfection Reagent and siRNA were both obtained from Invitrogen (Carlsbad, CA, USA).", "Remaining antibodies that were used in this study were ordered from Cell Signaling Technology (Beverly, MA, USA).", "Other chemicals of reagent-grade quality were obtained from Sigma (St. Louis, MO, USA).", "In the study of the cell model, a transfect-able macrophage cell line with the capacity to form osteoclast-like cells, RAW264.7 macrophages (ATCC, TIB-71), was employed.", "RAW264.7 were cultured in DMEM supplemented with 10% fetal bovine serum, and 1% antibiotic-antimycotic (Invitrogen, Carlsbad, CA, USA) at a humidified atmosphere with 5% CO2 at 37 °C.", "Cells were seeded on 96 and 6 well plates respectively for experimental use.", "To conduct MTT assay, 24 hours after cell seeding on 96 well plates, cells were treated with MG or vehicle control for 24 hours.", "For the 6 wells plates, cells were treated with MG and collected at different time intervals (1 h, 3 h, 6 h and 24 h) after the MG treatment.", "After that, proteins and mRNA of cells were collected for further studies.", "To conduct experiments of inhibiting p38 and p44/42, cells were treated with 10 nM p38 (MAPK14) and p44/42 (MAPK3) siRNA in Opti-MEM reduced serum culture medium respectively for 24 h before MG treatment.", "For the negative control, siRNA was added onto cells in Opti-MEM reduced serum culture medium for 24 h before MG treatment.", "After transfection, cells were treated in MG (400 μM) for 24 h.", "After that, mRNA of cells was extracted for further studies.", "Proteins from the cells were extracted with radioimmunoprecipitation assay buffer (RIPA) with protease inhibitors containing Complete Mini™ protease inhibitor cocktail (Roche), 1 mM PMSF, and 1 mM Na2VO4.", "Protein samples were then separated by SDS-PAGE gel and transferred to the PVDF Western Blotting membrane.", "The membranes were later probed with respective primary antibodies overnight at 4 °C, and then respective HRP-conjugated secondary antibodies for 1 h.", "The membranes were incubated with ECL reagents and stained protein bands were exposed and captured using Bio-Rad ChemiDoc Touch Imaging System.", "The intensities of the protein bands were quantified using Image J, an image-processing program.", "The endogenous mRNA in cell samples was extracted, using Trizol (Invitrogen).", "The mRNA was then converted into complementary DNA (cDNA) under reverse transcription.", "Expressions of the osteoclast biomarkers were assessed by real-time PCR with respective primers:", "Mouse CTSK (F: CCCTTAGTCTTCCGCTCACA, R: TCCTCCGGAGACAGAGCAAA);", "Mouse OSCAR (F: GTCCTGTCGCTGATACTCCAG, R: CCCAGTCTGTCTTGCGGTAG);", "Mouse TRACP5 (F: GCAGCTCCCTAGAAGATGGAT, R: AACACGTCCTCAAAGGTCTCC);", "Mouse β-Actin (F: TAGGCGGACTGTTACTGAGC, R: TGCTCCAACCAACTGCTGTC).", "Results were shown as means ± standard deviation (SD).", "The data were processed using Student's t-test and one-way analysis of variance (ANOVA) followed by Tukey's range test.", "While P < 0.05 was considered statistically significant (*p < 0.05 in comparison to the control.", "**p < 0.01 in comparison to the control)." ]

[]

[ "AGRI", "ECON", "ENVI", "SOCI" ]

[ "Aquaculture", "Economic modeling", "Fish aggregating devices", "Natural resource management", "Timor-Leste", "Vanuatu" ]

[ "1104", "2002", "2300", "2308", "3308" ]

[ "Throughout the tropical Pacific where the majority of rural people still live and depend on subsistence agriculture, the fisheries sector (including aquaculture) is vital to economic development, livelihood, and food and nutrition security.", "Vanuatu has a long tradition tied to inshore and offshore fisheries; the sector supplies the main source of income and protein for many island inhabitants and makes up the country's biggest export.", "On the other hand, Timor-Leste has historically depended on the rural interior for the bulk of its calories and national revenue, despite its sea surroundings.", "Since gaining sovereignty over a decade ago, efforts to stimulate sustainable fisheries development in Timor are increasingly in the spotlight.", "Like other maritime islands and nations in the region, Vanuatu and Timor-Leste are particularly susceptible to climate change because of their geographical location, socioeconomic status, and political instability.", "Nonetheless, adaptive strategies are available to alleviate vulnerability in the region's fisheries sector to the damaging effects of climate change.", "Using economic modeling, this paper estimates the potential impact of climate change and climate adaptation strategies for the fisheries sector in Vanuatu and Timor-Leste.", "Among the Melanesian countries, Vanuatu has the smallest total water area and exclusive economic zone (EEZ) of 680,000 square kilometers (km2) [1].", "Fisheries is fundamentally important to the nation's economy and food security, and generates income for many of its inhabitants; approximately 72% of the country's rural households are involved in some form of fishing [2].", "Vanuatu's fisheries resources can be categorized into six groups: coastal commercial fisheries, coastal subsistence fisheries, offshore foreign-based fisheries, offshore locally based or domestic fisheries, freshwater fisheries, and aquaculture [3].", "Unlike Fiji and Solomon Islands, coastal subsistence fisheries produce the highest value of fish harvest in Vanuatu.", "The four major commercial species of tuna (bigeye, yellowfin, skipjack, and albacore) dominate fish exports within Vanuatu's EEZ [2].", "In contrast to Vanuatu, Solomon Islands and Fiji, which have numerous islands, Timor-Leste has only two: the 144-km2 Atauro Island and the 8-km2 Jaco Island [4].", "Fisheries supplements income and protein for many of Timor-Leste's coastal and inland communities, though at smaller scale than Vanuatu.", "An estimated 7600 of its inhabitants were employed in the fisheries sector in 2008 [5].", "Since the country only gained independence in 2002, historical information on the fisheries sector is scant and effective data systems (and policies) have been slow to develop.", "Timor-Leste currently lacks a domestic commercial fishing fleet and motorized vessels were largely destroyed during the struggle for independence.", "This limited offshore fishing and concentrated the industry to mostly small-scale fisheries near the coast.", "In the mid-2000s the government signed bilateral agreements allowing foreign fishing vessels limited access to the country's deep-sea fishing grounds, under conditions that fish landings and shipment be channeled through Timor's ports [5].", "However, some of these agreements have expired and others have suffered from noncompliance.", "Freshwater fishing in Vanuatu is mostly subsistence and restricted during monsoon season.", "The government has identified aquaculture as a means to improve food and nutrition security and generate income for both inland and coastal communities, although little progress has been made since aquaculture is costly and requires a heavy governmental hand and infrastructure.", "Despite current status, the fisheries sector in Timor-Leste is at a pivotal time as the government grapples with new policies, infrastructure and strategies that aim to sustainably develop the sector for improved food security and welfare.", "In 2012, for example, the National Coral Triangle Initiative Coordinating Committee of Timor-Leste reported the preparation of an integrated fisheries policy: The Future of Fisheries: A Policy and Strategy for the Responsible Development and Management of Fisheries in Timor-Leste.", "This policy encompasses optimal use and management of living resources, habitat conservation and development of fishing and aquaculture industries and fisheries institutions [6].", "Though several studies have analyzed the vulnerability of Asia-Pacific fisheries (including aquaculture) to climate change and adaptation scenarios (see, for example [7–10]), this is the first study to evaluate economic impact of climate change adaptation strategies in the region.", "Through modeling applications, trends in fish production, consumption, and trade for medium-term (2035) and long-term (2050) can be projected with and without adaptation strategies.", "These scenarios provide important information in developing and supporting national strategies and policies to ensure food security, nutrition, assured income and improve economic conditions, particularly for the coastal communities of Vanuatu and Timor-Leste.", "While the climate adaptation strategies analyzed represent only a few of the potential alternatives for an inherently uncertain future, the developed model and the analyses presented in this paper are robust for the scenarios implemented.", "Sensitivity analyses of a range of demand and supply elasticities around best estimates show that orders of magnitude in the results are stable with respect to the range of plausible elasticity estimates.", "A companion article in this special section [6] discusses the study's methodologies in greater detail including data collection, assumptions and modeling.", "The rest of the paper is divided into modeling scenarios, results and discussion of country case studies, and summary of conclusions.", "Vanuatu has adopted four main climate change adaptation strategies: (1) aquaculture (coastal and freshwater [AQ]); (2) regulations concerning habitat protection in marine protected areas (MPAs), with seasonal closure, traditional management of natural resources, control of critical species (sea cucumber, Trochus, parrot fish, giant clam), and selective control of fishing methods and gears; and (3) low-cost fish aggregating devices (FADs).", "AQ was introduced to Pacific Island countries as early as the 1920s, specifically in Vanuatu with the introduction of Pacific oysters [11].", "This was followed by the short-lived culture of Macrobrachium in Santo Island from 1978 to 1983, tilapia from New Caledonia in Efate Island in the early 1980s, Trochus in the late 1980s, and the ongoing Teouma prawn hatchery in Efate at commercial-scale.", "MPAs in Vanuatu have an estimated size of 58 km2 [12].", "Marine conservation activities implemented by the communities have been ongoing for several years, starting with the assistance of the Vanuatu Department of Fisheries (VDOF) in the 1990s [13].", "VDOF persuaded the North Efate communities to close certain marine areas for Trochus recovery.", "This was followed by turtle monitoring spearheaded by the Vanuatu Wan Smolbag to conserve turtles in Nguna-Pele Islands.", "In 2003 the Nguna-Pele MPA established its network, comprised of local and indigenous non-government organizations (NGOs) of 16 communities from the two islands, with the main objective of sustainable use and long-term existence of marine and terrestrial resources.", "In 2007 Nguna-Pele joined the locally marine managed area (LMMA) network, and is currently in the initial phase of establishing it countrywide [14].", "Pascal, Seidi and Tiwok [15] analyzed the economic benefit of MPAs in Vanuatu and demonstrated their positive effects on marine resource productivity, coastal environment, as well as income growth through tourism revenue.", "Previous studies suggest climate change is likely beneficial for tuna and oceanic fish production [16] but detrimental for coastal fish production [17] in Vanuatu.", "Furthermore, the likely impact of various climate change adaptation strategies on fish production indicate favorable responses: use of FADs is expected to increase tuna and other oceanic fish catch in coastal waters; and various natural resource management (NRM) strategies (such as MPAs and LLMAs) are likely to mitigate some of climate change's negative effects on coastal fisheries.", "Therefore, NRM and FAD strategies are anticipated to expand supply of all four capture fisheries groups (tuna, other oceanic species, coastal finfish and coastal invertebrates).", "Given that many of these strategies are still in the formative stage, three climate change adaptation scenarios were modeled.", "Scenario 1 (AQ development involves improvements in the productivity of freshwater (both finfish and invertebrate) aquaculture.", "Scenario 2 (NRM+FAD) addresses changes in production and productivity in coastal and oceanic capture fisheries resulting from FADs, various management regime shifts, and adoption of resource enhancement practices.", "Scenario 3 is a combination of scenarios 1 and 2.", "Estimated assumptions about the production impact of these strategies are described in Dey et al. [6] and given in Appendix Tables A.1 and A.2.", "Over the period 2009–2013, real per capita income in Vanuatu essentially stagnated.", "A modest recovery in income growth over the longer run was assumed.", "For both medium- (2035) and long-term (2050), two baseline (most plausible) scenarios were implemented representing two annual growth rates of real per capita income: medium (1.5% per year) and high (2.5% per year).", "Populations were assumed at 424,122 in 2035 and 538,707 in 2050 [18].", "Validated baseline data (production, consumption, trade, and price) used in the Vanuatu model are presented in Appendix Table A.1.", "Supply quantities reported in Table A.1 do not include catch by foreign fleets, which is small in Vanuatu.", "Supply volumes reported in Table A.1 and used in the analysis include catch by national fleets in both national and international waters.", "Appendix Table A.2 reports the coefficients of supply shifters used in the Vanuatu model to represent climate change and climate change adaptation strategies.", "The coefficients for climate change, as reported in column 2 and column 6 of Appendix Table A.2, were taken or modified from Bell et al. [19], Gehrke et al. [20], Lehodey et al. [16], Pickering et al. [21], and Prachett et al. [17] 2.", "The likely effects of various climate change adaptation strategies on fish production, as reported in columns 3, 4, 5, 7, 8, and 9 of Appendix Table A.2, were collected through expert opinion survey (EOS) and focus group discussion (FGD).", "As in the Vanuatu model, secondary data and FGDs were utilized to develop the parameters for the Timor-Leste model.", "For each time period (2035 and 2050), two baseline (most plausible) scenarios were implemented representing two annual growth rates of real per capita income: medium (2% per year) and high (3% per year).", "Populations of 1,724,683 in 2035 and 2,040,271 in 2050 were assumed for Timor-Leste [18].", "Similar to Vanuatu, three climate change adaptation scenarios namely, AQ, NRM, and a combination of AQ+NRM were considered for Timor-Leste.", "MPA in Timor-Leste has an estimated area of 7 km2 in 2010 [22].", "The Coral Triangle Support Partnership requested the international LMMA Network to visit Timor-Leste to encourage coastal and small island management.", "During this visit, three sites—Tutuala, Batugade, and Atauro Island—were invited to develop LMMA activities in 2010 [14].", "Scenario 1 (AQ) involves improvements in the productivity of freshwater (both finfish and invertebrate) aquaculture.", "Scenario 2 (NRM) addresses the changes in production and productivity of coastal and oceanic capture fisheries resulting from management regime shifts and adoption of resource enhancement practices.", "Scenario 3 combines AQ and NRM.", "Baseline data (production, consumption, trade and price) used in the fish sector model for Timor-Leste are given in Appendix Table A.3.", "Similar to other countries under study in this special section of Marine Policy, supply volumes reported in Appendix Table A.3 and used in the analysis include catch by national fleets in both national and international waters, but do not include catch by foreign fleets in national waters.", "The overall shifts in the supply curve resulting from climate change and various climate change adaptation strategies used in Timor-Leste model are reported in Appendix Table A.4.", "The Spatial Ecosystem and Population Dynamics Model (SEAPODYM) [23] was used for the projection of the likely effects of climate change on tuna catch, which show tuna stock likely shifting to the eastern Pacific Ocean, with increased production due to climate change.", "Unlike for the other country case studies, the assumption was made that that tuna stock and catch will not increase in the more westward Timor-Leste because of climate change.", "However, our model uses the baseline projection of declining catch of coastal fisheries in Timor-Leste due to climate change.", "Various NRM strategies are likely to mitigate some of the negative effects resulting from climate change on coastal fisheries and shift supply curves for coastal species to the right.", "Likely effects of climate change adaptation strategies on fish production are reported in columns 3, 4, 5, 7, 8, and 9 of Appendix Table A.4.", "Tables 1–4 present the projected real prices of several fish types in 2035 and 2050 under different scenarios, that is 1.5% annual growth per capita real income to 2035 and 2050 (Tables 1 and 2 respectively), and 2.5% annual growth per capita real income to 2035 and 2050 (Tables 3 and 4, respectively).", "The model predicts real price of most fish species (including coastal finfish, which contribute about 54% of current fish consumption) will increase in 2035 and 2050 under baseline scenarios.", "Adoption of NRM strategies and FADs is expected to decrease the prices of tuna, coastal finfish, coastal invertebrates, and freshwater finfish in 2035.", "Coastal finfish and tuna are the two most important sources of fish and seafood in Vanuatu, contributing about 77% of current consumption; the former is widely consumed by poorer households.", "Demand growth considerably outpaces production growth under the baseline scenario with climate change.", "Despite the disparity in growth, Vanuatu is projected to remain a net exporter of fish in the long-term, although the level of exports will decline (Fig. 1 below).", "The scenarios also show the FAD+NRM adaptation strategy is likely to have positive impact on poorer consumers.", "Furthermore, adoption of the FAD+NRM strategy will have the highest positive impact on oceanic fish supply.", "This strategy is expected to increase supply by about 14% from the present (2006–2009) level in 2035 and by about 19–20% in 2050.", "This indicates the potential of Vanuatu to be exporter of fish and seafood in the region.", "However, with its current pace of implementation, this adaptation strategy will not be able to halt the rise of fish prices in the long-term (2050).", "Among the adaptation strategies examined, adoption of FAD+NRM strategy is projected to have the highest positive impact on oceanic fish supply (Tables 1–4).", "Modeling results reveal that this strategy is expected to improve current supply of oceanic fish by about 14% in 2035 and about 19–20% in 2050.", "Projected rate of expansion in national tuna production due to rising investment in FADs is well within the sustainable tuna catch [16, 24].", "Freshwater and marine aquaculture sector is a minor supplier of fish in Vanuatu.", "The Government of Vanuatu has placed high priority on developing the aquaculture sector (both freshwater and marine), and has taken various initiatives for improvement.", "To assess the potential for aggressive adaptation policies, highly optimistic productivity growth scenarios for aquaculture were applied in the model: 75% growth in productivity from 2009 to 2035, and 125% growth from 2009 to 2050.", "The model projects that with this high growth, production from freshwater aquaculture is likely to improve by about 20 times in the medium-term and about 30 times in the long-term (Tables 1–4).", "Even with these very high growth rates, the total production of freshwater aquaculture is projected to be 1053 to 1278 mt in 2050, lower than the 3024 to 3243 mt from coastal fisheries in 2050 under two income growth scenarios, because base year production is extremely low.", "Modeling results show that aggressive aquaculture development strategy without any accompanying NRM strategies is likely to reduce catch from oceanic resources.", "This phenomenon is plausible due to reduction in per unit cost of production in aquaculture relative to other fishing sectors (in other words, shift in the supply curve for aquaculture without changes in other fisheries supply curves) and the resulting resource transfer to aquaculture from other sectors.", "These results highlight the importance of having a balanced approach with multiple climate change adaptation strategies in Vanuatu.", "Modeling estimates show that fish demand will rise over time under baseline (Tables 1–4), and level of increase will be higher, with faster growth in per capita real income.", "Among various fish categories, tuna and other oceanic fish are expected to experience highest increase in demand.", "With growth in population and income, oceanic fish demand may intensify from current levels (2006–2009) by about 3 times in 2035 and about 5 times in 2050.", "Among the adaptation strategies, expanding aquaculture is likely to encourage demand for tuna and other oceanic fish, mainly as a result of higher income from aquaculture growth.", "The model predicts that NRM+FAD strategy will increase fish demand in the long-term and for coastal and freshwater fish in the medium-term.", "The NRM+FAD strategy is likely to promote consumption through expected reduction in real fish price.", "Fig. 1 presents the projected effects of different climate change adaptation strategies on fish trade in Vanuatu in 2035 and 2050.", "Though Vanuatu is expected to remain a net exporter of fish in the long-term, the volume of net exports (export minus import) is likely to decrease over time under the baseline scenario.", "Income growth outstrips production growth in the baseline, resulting in a reduction in export volume over time.", "Thus, the volume of net imports is expected to increase with higher growth in real income.", "AQ results in further reduction in exports relative to the baseline because the income generated under AQ boosts demand across fish species by an amount greater than the increase in freshwater species.", "The other strategies increase exports relative to the baseline.", "Table 5 reports the estimated national-level net economic gains to both consumers and producers from adopting these adaptation strategies in Vanuatu.", "Projections show that substantial net income gains are likely to be generated where aquaculture and NRM+FAD strategies are expected to generate several million dollars in annual net incomes in 2035 and 2050.", "Cost of various planned adaptation strategies were collected from secondary literature and validated through expert consultation.", "Even under very generous estimates of costs and conservative estimates of returns, the yearly net returns from these adaptation strategies are likely to be at least 20- to 50-fold higher than their yearly investment costs3.", "Tables 6–9 show the projected real prices of several fish types in Timor-Leste in 2035 and 2050 under different scenarios.", "The real price of most fish categories is projected to increase only marginally during 2009–2050 under the baseline scenarios, because high fish imports are likely to keep real fish prices under check.", "The real price of freshwater fish is expected to decrease over time under the baseline scenarios, mainly because of higher supply within the sector.", "Among various climate change adaptation strategies analyzed, aquaculture development is expected to reduce the real price of freshwater fish, and is likely to raise the real price of other fish categories, mainly because of the higher incomes associated with aquaculture development and lower supply of other species.", "As reported earlier for Vanuatu, higher productivity in aquaculture without any accompanying technical change in other fisheries sectors is likely to draw resource away from other sectors, and thereby reduce their supply and increase price.", "Adoption of NRM strategy is likely to have positive impact on coastal fish production in 2035 and 2050 (Tables 6–9).", "Aquaculture development is expected to affect freshwater fish production substantially, by about 100% in 2035 and by about 150% in 2050, from a low base level.", "Even this large percentage increase adds a relatively small increase to total fish production in volume terms.", "The model projects that combined AQ+NRM strategy may not have any added advantage (i.e., additive effects) on freshwater fish supply in Timor-Leste.", "This is mainly because NRM is expected to have positive effect on coastal fish production, which may take away some production resources from the freshwater system.", "On the other hand, aquaculture growth is projected to increase income, resulting in higher demand for coastal fish and seafood relative to freshwater fish4.", "These effects together may neutralize some positive impacts of the combined AQ+NRM strategy on freshwater production.", "Given that freshwater ecosystem supplies fish for domestic consumption only, which was reflected in the closed economy model chosen for this species group, the AQ+NRM strategy may not have any additive effects on freshwater fish consumption.", "Aggressive aquaculture development is projected to have a substantial positive impact on consumption of freshwater fish (Tables 6–9), although again, the increased on fish consumption in per capita terms is relatively modest (around 9.6 kg/capita/year) to 2050.", "Implementation of the aquaculture development strategy is expected to have larger positive impact over time.", "The likely increases in fish consumption from current levels under the baseline and under NRM strategy are relatively small, implying that NRM strategy is not expected to have any substantial impact on fish consumption in Timor-Leste.", "Unlike the other countries studied, Timor-Leste is a net importer of fish and seafood.", "Total imports are 1496 mt in 2006–2009, and are projected to increase to 10,162 mt in 2035 and 14,259 mt in 2050 with 3% annual growth of real per capita income.", "Model estimates that the country will remain a net fish importer in the long-run under the baseline and with climate change adaptation scenarios.", "Fig. 2 show the projected effects of these strategies on net fish imports in Timor-Leste in between 2035 and 2050.", "Results indicate that both aquaculture and NRM strategies will reduce net fish imports and if Government of Timor-Leste placed more emphasis on aquaculture development, as reflected in the modeling exercise, aquaculture development will have a stronger effect on import reduction.", "Increased investment in NRM activities is likely to have similar effects.", "The reductions in imports are relatively small across the scenarios, because the adaptation strategies result in increased income and consumption, partly offsetting the higher production.", "Table 10 provides the estimated national-level net economic gains to consumers and producers resulting from various adaptation strategies in Timor-Leste.", "Aquaculture is the main adaptation strategy where several ongoing initiatives are being implemented with regional/international agencies in the country.", "Compared with other case study countries, the potential increase in net income from NRM strategy is less since it is in the early stage of developing a comprehensive strategy for coastal and marine environments.", "Vanuatu is currently a net exporter of fish and seafood, with domestic production far exceeding domestic consumption.", "Under the baseline scenario, projected aggregate fish consumption is expected to rise substantially in 2035 and 2050; yet the country will remain a net fish exporter by 2050 due to production and export of oceanic fish, predominately tuna.", "Simulations show an increase in production of oceanic fish and a decline in coastal fish during 2010–2050.", "Though consumption of oceanic fish is anticipated to rise at a faster rate than any other sector, this subsector is projected to remain a net exporter, although at a lower level.", "However, Vanuatu will most likely have to import coastal fish to meet expanding demand from population and income growth.", "Given that many of Vanuatu's poorer households rely on coastal fisheries for their consumption needs, this projected scenario has serious food security implications.", "Cost-effective expansion of coastal fisheries would provide income and calories while reducing imports.", "It is also projected that freshwater fish consumption will exceed domestic production in 2035 and 2050, and Vanuatu will also need to rely on imports to meet this demand.", "The real price of most fish types—including coastal finfish, which contribute about 54% of current fish consumption—will escalate in 2035 and 2050 under baseline scenarios.", "Adoption of FADs and NRMs will likely reduce prices of coastal finfish, tuna, coastal invertebrates, and freshwater finfish in 2035.", "Coastal finfish and tuna contribute about 77% of current consumption and because coastal finfish are widely consumed by Vanuatu's poorer households, FAD+NRM is likely to have positive impact on consumers.", "This strategy will also favorably affect oceanic fish supply; from present (2006–2009) level an increase of about 14% in 2035 and 19–20% in 2050 were anticipated.", "Fish demand is projected to increase for coastal and freshwater fish in 2035 through expected real price reduction of these fish categories.", "But with its current pace of implementation, FADs and NRMs will not halt the increase of fish prices in 2050 unless more aggressive investments are put in place.", "Tuna and other oceanic fish are expected to experience the highest rise in demand, about three-five times in 2035 and 2050, respectively, due to growth in population and projected improvement in income from aquaculture development.", "With higher income from the aquaculture development scenario, Vanuatu's net export is predicted to decrease.", "FAD+NRM strategy is projected to reduce the country's fish imports and increase its net exports.", "Calculated values of national-level economic gains are US $4 million for aquaculture, US $18 million for NRM+FAD, and US $22 million for AQ+NRM+FAD (in 2009 US$ constant price) in 2035; and US $4.5 million for aquaculture, US $35 million for NRM+FAD, and US $38 million for AQ+NRM+FAD (in 2009 US$ constant price) in 2050.", "Unlike Vanuatu, Timor-Leste is a net importer of fish and seafood, and net imports are projected to increase in the long-run under baseline and various climate change adaptation scenarios.", "Under baseline scenario, total fish production will increase only marginally in 2035 and 2050.", "Aggregate fish demand is expected to rise substantially due to growth in population and real per capita income, thus increasing fish import to fill this escalating deficit in the domestic fish supply.", "Fish supplies from oceanic and coastal ecosystems are projected to decrease during 2010–2050 due to climate change (please see Appendix Table A.4 for detailed assumptions).", "Only freshwater ecosystems will be able to supply more fish in the future.", "Given that oceanic and coastal fisheries supply about 94% of current fish consumption in Timor-Leste, this projected fish supply scenario has serious food security implications for the country.", "Based on the modeling outcome, adoption of climate change adaptation strategies will likely result in a marginal increase in real fish price over 2009–2050, because increased fish imports are likely to keep real prices under check.", "Real price of freshwater fish is expected to decrease over time, mainly because of higher supply within the sector.", "Among various climate change adaptation strategies analyzed, aquaculture development is expected to reduce the real price of freshwater fish, but likely to raise the real price of other fish categories, mainly because of the higher incomes associated with aquaculture development.", "Aquaculture is also expected to increase freshwater fish production substantially, by about 100% in 2035 and by about 150% in 2050.", "Aquaculture development will have significant and positive impact on fish consumption in Timor-Leste, mainly through increased consumption of freshwater fish.", "Continuation of the aquaculture development strategy is expected to have a larger positive impact as time goes by.", "The likely increases in fish consumption from current levels under the baseline scenario and the NRM strategy are predicted to be very similar, implying that NRM strategy is not expected to have significant impact on fish consumption in the country, but rather will reduce fish imports.", "Combined AQ and NRM strategies may not produce any added advantage (i.e., additive effects) for freshwater fish supply in Timor-Leste based on the modeling results.", "Aquaculture growth increases income, leading to higher demand for coastal fish and seafood and lower demand for freshwater fish.", "These effects together may neutralize some positive impacts of the combined AQ+NRM strategy on freshwater production.", "Given that the freshwater ecosystem supplies fish for domestic consumption only, as reflected in the close economy model chosen for this species group, the AQ+NRM strategy may not have any additive effect on freshwater fish consumption.", "Estimated national-level annual economic gain resulting from the adoption of climate change adaptation strategies range from US $0.65 million for aquaculture, to US $0.4 million for NRM, and to US $1 million for combined aquaculture+NRM (US$ in 2009 constant prices) in 2035.", "Interestingly, economic gain from NRM remained the same at US $0.4 million (US$ in 2009 constant prices) in 2050, whereas the annual benefits from aquaculture expansion increased US $1.2 million in 2050 and the annual benefits aquaculture+NRM increased to US $1.6 million in 2050.", "Three main adaptation strategies to the effects of climate change are presented and applied in the modeling exercises for the Pacific coral triangle countries, in this case, Vanuatu and Timor-Leste.", "These strategies are aquaculture development, expansion of NRMs, and establishment of FADs which all indicated favorable effects on the fisheries sector in these two countries.", "Very importantly, substantial results of the modeling application demonstrate national-level economic gains for the adaptation strategies, with significantly higher estimated economic gains in Vanuatu.", "Finally, it is critical to note that the current scales of adaptation strategies in these Pacific countries are relatively too small to have the necessary impacts in the future.", "There is a need for an aggressive, but judicious, increase in investment in aquaculture, low-cost FADs, and NRM technologies and management strategies to adapt to climate change and meet growing fish demand.", "These adaptation strategies need to be location-specific.", "Aquaculture, FADs, and NRM affect diverse products and ecosystems differently.", "Therefore, it is essential to tailor these climate change adaptation strategies and policies to the conditions in each of the countries and locations." ]

[ "Analyzed economic impact of adaptation strategies in Vanuatu and Timor-Leste.", "Strategies: aquaculture, natural resource management, and inshore fish aggregating devices.", "Positive impact of strategies, but current scales of adaptation are too small.", "Need aggressive, but judicious, increases in investments in adaptation strategies.", "Essential to tailor these strategies and policies to conditions in various locations." ]

[ "BIOC", "CHEM" ]

[ "Computational fluid dynamics", "Extra column volume", "Film mass transfer", "Hydrodynamic dispersion", "Mass transfer mechanism", "Peak broadening effects", "Pore diffusion", "Scalability" ]

[ "1303", "1602", "1605" ]

[ "Peak broadening under non-binding conditions is caused by extra column dispersion, hydrodynamic dispersion in the column and mass transfer resistances [1].", "The (relative) contribution of these mechanisms varies at different scales.", "For instance, in process development, virus clearance studies and exploration of the design space are done at very small scale [2,3].", "Here we observed completely different dispersion mechanism compared to pilot and full scale operation [4,5].", "Thus, knowledge about the performance at each scale is of utmost importance in preparative and industrial chromatography of proteins, because during scale up and scale down we may over or underestimate the separation power of a column with severe consequences.", "Despite the tremendous importance of chromatographic separation in downstream processing, a reliable quantitative understanding of the dispersion mechanisms involved is currently lacking [6].", "General (theoretical) descriptions of dispersion mechanisms have often been made by neglecting extra column dispersion [7,8].", "This assumption is valid when the column is sufficiently large and extra column dispersion can be neglected [9–12].", "In protein chromatography, however, it is not practicable to use such columns as the costs of material consumption for these experiments are prohibitively large.", "Moreover, the effect of extra column dispersion is difficult to assess experimentally [9,13,14].", "Attempts have been made to extrapolate from total peak dispersion of columns with different sizes to the extra column dispersion.", "This approach is time consuming, because several experiments must be conducted with columns of different length but same column inlet an outlet.", "While the extra column dispersion remains identical in this experimental design the dispersion in the column increases [12]", "To overcome theses problems, we utilize computational fluid dynamics (CFD) to analyze the flow properties of chromatography columns.", "In fact, CFD simulations have the potential to provide deeper insight into the different dispersion processes [15,16] and predict the (relative) contribution of each effect [17,18].", "It was shown that CFD modelling with resolved extra-particle channels in chromatography columns can quantify components of hydrodynamic dispersion in packed beds on different length and time scales [19].", "Moreover, such micro-scale simulations in combination with mass-transfer models are able to mimic adsorption [20] as well as adsorption and diffusion in the beads [21].", "To reduce the computational burden, we performed simplified macro-scale simulations that do not resolve extra-particle channels.", "Rather packed beds are modelled as medium with uniform porosity.", "Here we demonstrate that a properly calibrated CFD simulation is not only able to properly dissect the impact of various dispersion mechanisms, but is also able to accurately extrapolate the qualities of a chromatography column.", "Specifically, we experimentally characterized a large chromatography column with three different solutes, reconstructed and calibrated the experimental setup in silico, and (quantitatively correctly) predicted the contributions of individual peak broadening mechanisms in an identically constructed smaller column.", "All experiments were made on an ÄKTA pure 25 M2 chromatography system (GE Healthcare), schematically shown in Fig. 1 and detailed in supplementary Table S1.", "The total volume of all of the parts of the workstation from the middle of the injection loop to the middle of the UV detector (without the columns) is the extra column volume = 0.208 mL.", "The used parts of the workstation (injection loop, valves, tubing and detectors) are simulated by CFD simulations in their respective dimensions.", "Tris, sodium chloride and disodium hydrogen phosphate dehydrate were purchased from Merck Millipore.", "Acetone was obtained from VWR.", "Silica nanoparticles (1000 nm diameter, surface plain) were purchased from Kisker Biotech.", "Lysozyme from chicken egg white was obtained from Sigma Aldrich.", "Two pre-packed MiniChrom columns (Repligen) were used for the experiments and reconstructed in silico, see Fig. 2.", "The filters of both columns were made out of polypropylene/ polyethylene fibers and had a thickness of 0.42 mm.", "Assuming a 50:50 mixture of the two polymers, the density and the weight of the membrane were used to calculate a porosity of the filters of 0.51.", "The columns were packed with Toyopearl Gigacap S-650M (Tosoh).", "This strong cation exchange medium has a particle diameter of dp = 75 μm and a pore size of 100 nm.", "The main parameters of the two evaluated columns, referred to as “8–100” and “5–10”, are summarized in Table 1.", "Other dimensions are listed in Supplementary Table S2.", "Silica nanoparticles were used to characterize the flow behaviour outside of the beads since their diameter was too large to penetrate the pores.", "10 μL of mixture was injected into the column; HQ-H2O was used as mobile phase.", "The extra particle porosity ϵ was calculated based on the peak maximum of the peak profile measured at the UV detector at a wavelength of 280 nm and was equal for all the further discussed solutions.", "Acetone was used as a small non-interacting tracer.", "Due to its small molecular size, it is able to diffuse into the interior of the beads.", "Acetone was injected at a concentration of 1% (v/v) at a volume of 10 μL to the column.", "The mobile phase was 50 mM Tris, 0.9% (w/v) sodium chloride, pH 8.0 (pH adjusted with HCl).", "The total porosity ϵt of the packed bed was calculated from the first peak moment corrected by the extra column volume (determined by pulses through the workstation alone [22,9]) of the runs through the 8–100 column at a superficial velocity of 150 cm/h from triplicate measurements.", "Lysozyme was injected at non-binding conditions to the column in order to evaluate hindered diffusion of large molecules inside the pores.", "10 μL lysozyme at a concentration of 5 mg/mL was injected in running buffer (25 mM Na2PO4, 1 M NaCl, pH 6.5) to the column.", "The total porosity ϵt of the 8–100 column was calculated in the same way as for acetone; it is smaller than for acetone since not the whole pore space is accessible to lysozyme.", "The effective diffusivity of lysozyme inside the pores was determined by direct numerical integration of the peaks at velocities from 60 to 150 cm/h and calculated as described in Section 3.", "Parameters of the solutes used are summarized in Table 2.", "The program used for CFD simulations is STAR-CCM+® (Siemens PLM Software, Plano, TX, USA).", "Eq. (10) was already present in the software.", "The source term on the right hand side of Eq. (9) as well as Eqs. (11)–(16) are incorporated into the software.", "The coefficient of hydrodynamic dispersion is computed by Eq. (S.4.2).", "The flow of the solutes is modelled as passive scalar transport, i.e., flow properties do not depend on the solute concentration.", "Indeed, concentrations of the solutes are so low that their influence on the thermophysical parameters are negligible [23].", "Two types of axisymmetric model setups were used to show the influence of the extra column effects.", "This setup covers only the chromatography column (parts No. 8–16 in Supplementary Table S2) with short tubes (radius 0.125 mm, length 10 mm) added before and after the column.", "A numerical quadrilateral mesh consisting of 26,493 and 11,395 cells was generated in this setup for the 8–100 and 5–10 column, respectively.", "Computational time of each run on 2 cores of Intel® Xeon® Processor E5-2643 v4 was 8 and 3.5 h for the larger and the smaller column, respectively.", "Value of the passive scalar at the inlet boundary is 1.", "When the volume of the Injection Loop (see Fig. 1 and Table S1) has entered the setup, this boundary condition changes to 0 value.", "The reported peaks are postponed by extra column volume value normalized by column volume, since the extra column volume is absent in this setup.", "This setup contains not only the column but all parts listed in Supplementary Tables S1 and S2.", "The numerical mesh contained 164,513 and 149,269 cells for the 8–100 and 5–10 column, respectively (see Supplementary Fig. S1).", "Computational time of each run on 8 cores of Intel® Xeon® Processor E5-2643 v4 was 29 and 26 h for the larger and the smaller column respectively.", "In order to verify that the resolution of the used mesh was sufficient, several runs with a finer mesh with 378,300 cells (about 2.53 times more than the rough mesh) were performed in the smaller column, which resulted in a difference of less than 0.7% in terms of normalized root mean square deviation (NRMSD), see Eq. (3).", "The initial condition of the simulations imitates the one of the experiment – the value of the passive scalar is equal to 1 in the Injection Loop, and to 0 elsewhere.", "We modelled the pulse response experiments (see Section 2.4) of the three solutes acetone, lysozyme, and silica nanoparticles.", "The solutes vary by their ability to diffuse into the beads.", "Thus different transport equations are used.", "Silica nanoparticles do not enter into the beads, thus Eq. (10) is used to simulate their transport through the chromatography column.", "Since both acetone and lysozyme can diffuse into the beads, transport Eq. (9) is employed.", "Additionally, when acetone and lysozyme diffusion into the beads pores is assumed to be infinitely fast, the simplified transport Eq. (17) is used.", "Two columns described in Section 2.3 are used to model silica nanoparticles, acetone and protein pulse propagation.", "Both numerical setup types – with and without extra column volume – are used to model transport of the solutes.", "The diffusion–dispersion coefficient is calculated by Eq. (18) only for the packed bed region; elsewhere it is equal to the diffusion coefficient and dispersion is modelled explicitly.", "In this work, the concentrations are normalized by their maximum values; the volume passed through the setup and moments of the distributions are normalized by the respective column volume.", "There are three distinct conditions of the solute transport through the chromatography column packed with porous beads", "The solute is larger than the size of the bead's pores.", "Thus the solute cannot penetrate into the beads and the column can be treated as a column packed with non-porous beads.", "The solute is slightly smaller than the size of the bead's pores.", "Thus the solute penetrates into the beads with finite speed and the column can only be treated with a descriptive model of the mass transfer.", "The solute is much smaller than size of the bead's pores that the mass transfer of the solute between the mobile and solid phase can be model as infinite fast.", "In the following we first calibrated our approach on the larger column and then validated it on the smaller column.", "This strategy was adopted as extra column volume effects are typically more prominent in smaller columns [9] and a scale-down therefore presents a more selective test than a scale-up.", "All parameters used and simulations performed in this work are summarized in Tables 2 and 3 , respectively.", "In general, we noticed that all computed solute distributions showed noticeable radial inhomogeneity.", "The concentration of solutes across the columns are illustrated in Supplementary Fig. S2.", "These inhomogeneities underline the relevance of an accurate description of the real, three dimensional geometry of the columns also including the extra column volume.", "First, the model of the hydrodynamic dispersion in the packed bed and the mass transfer model are validated using experiments performed on the 8–100 column.", "We simulated the flow of silica nanoparticles through the larger, 8–100 column with and without extra column volume (simulation S2 and S1 in Table 3, respectively).", "Excellent (overall) agreement between experiment and simulation was found, when the extra column volume was taken into account (NRMSD = 2.4% versus NRMSD = 12.9% without extra column volume, see Supplementary Fig. S3).", "Moreover, compared to the simulation without extra column volume, the run with extra column volume strongly reduced the estimation error in the pulse parameters σ2, γ1, and h, while it modestly increased in μ, see Table 4.", "This is explained by the very low diffusion of the silica nanoparticle in the water and the resulting strong effect of the longitudinal dispersion of the silica nanoparticle in the extra column volume components.", "Low diffusion of the silica nanoparticles in water (see Table 2) results in the strong hydrodynamic dispersion of the silica nanoparticles in the extra column volume components.", "Thus, simulation S1 that does not account for the extra column volume, strongly disagrees with the experiment.", "According to the decomposition analysis presented in Section 3.6, extra column volume accounts for 65.9% of the signal broadening, meanwhile hydrodynamic dispersion in the column is responsible only for 34.1% of the broadening.", "In contrast to silica nanoparticles, acetone (and lyzosome) are small enough to penetrate the beads.", "Thus, in the following simulations in-beads diffusion was considered according to the MTL-model in Eq. (9).", "Again, all parameters used are listed in Table 2.", "To investigate whether or not in-bead diffusion is essentially an instantaneous process, we repeated the simulations described above, but use of the LEA, see Eq. (17), rather than the MTL model to describe mass transfer.", "The extra column volume was considered, too, and this new set of simulations is referred to as S5, see Table 3.", "However, with increasing flow velocity usage of the LEA worsened the agreement with experiments.", "Average NRMSD for this simulation set is 4.9%.", "Thus, we conclude that acetone slowly diffuses into the beads while propagating through the column at high speed.", "We simulated the flow of lysozyme through the larger, 8–100 column as summarized in Table 3.", "Like acetone, lysozyme are able to diffuse into beads.", "With our parameters, see Table 2, lysozyme probe the space at larger reduced velocities that were not accessible with acetone.", "Thus, a larger reduced velocity range is covered for each column.", "In the two preceding sections we developed and validated a hydrodynamic model of a large chromatography column (8–100).", "Here, we predict the performance parameters of pulse response experiments of an identically constructed, smaller column (5–10).", "As the columns were constructed in the same way, we assumed that porosity values, dispersion coefficients and mass transfer coefficients are identical in both columns.", "We predicted the flow of silica nanoparticles, acetone and lysozyme through the small 5–10 column.", "We ran simulations with and without extra column volume and modeled in-bead diffusion employing LEA and the MTL model.", "An overview of all sets of simulations is listed in Table 3.", "Subsequently we experimentally verified our predictions for silica nanoparticles and acetone.", "We found that the predicted pulse shapes at the UV detector agreed reasonably well with the verification experiments (see Fig. 7 and S10) when effects of the extra column volume were included (average NRMSD = 4.3% and 5.0% for silica nanoparticles, and acetone, respectively; also see Table 5 as well as Fig. 6 and supplementary Fig. S6 for the corresponding chromatograms).", "However, in contrast to our simulations, the experiments with silica nanoparticles showed a second peak at around 6 column volume, see Fig. 6.", "Its origin is not yet clear and scope of further investigation.", "The dominance of effects due to the extra column volume is reasonable and expectable as the extra column volume is about the same as the volume of the 5–10 column while it makes only 5% of the volume of the larger 8–100 column.", "Here we used 2D computational fluid dynamics (CFD) coupled with mechanistic mass transfer models to investigate and separate effects of peak broadening in chromatography columns.", "2D CFD simulations naturally capture the anisotropic propagation of the solute in extra column volume and in packed bed and do not require to employ dispersed plug flow reactors (DPFRs) and continuous stirred tank reactors (CSTRs) as it is the case of 1D models [35,36].", "Several benchmark cases of non-binding solute transport in two differently sized chromatography columns were reported.", "The solutes (silica nanoparticles, acetone, and lysozyme) differed (i) by their ability to diffuse into the pores of the beads and (ii) by their diffusion-dispersion behaviour as characterized by the coefficient CL.", "The different properties of the solutes allowed us to probe a wide range of reduced velocities (from 10 to 10,000).", "The good agreement between experiments and simulations supports not only the validity of the CFD approach but also illustrates the good scalability of the studied columns, which – in turn – indicates a good quality of the bed packing.", "Our simulations confirm the dynamic variability of dispersion mechanism of across scales (see Fig. 9).", "Even in the large column, where the extra column volume makes less than 5% of the total column volume, the extra column volume is the dominating peak broadening mechanism, when the column is operated with silica nanoparticles.", "That impact decreases to less than 10% when the column is used together with the other solutes.", "In contrast, extra column volume plays the dominant role for all the studied flow rates of acetone and low and medium flow rates of lysozyme for the smaller column.", "Only at high flow rates of lysozyme, extra column volume and mass transfer are almost equally important.", "This implies that low-diffusive solutes at high flow rates characterise the column rather than extra column volume.", "In order to adapt our approach to an uncharacterised column one (i) needs to identify the parameters and (ii) differentiate the contributions by means of CFD.", "We need to conduct at least one experiments with a non-penetrating solute to determine the extra-particle porosity ϵ and at least two more at different flow rates for each additional penetrating solute to determine intra-particle porosity ϵp and effective diffusivity De.", "These parameters can be used for the analysis of the same column or extrapolated to another column with the same packing resin.", "Two CFD simulations have to be run at the flow rate of interest to estimate the contribution of extra column volume.", "If the solute is able to diffuse into the beads, one additional simulation should be run to differentiate between MTL and hydrodynamic dispersion effects." ]

[ "Mass transfer coupled computational fluid dynamics model is used to analyze columns.", "Peak broadening mechanisms incl. extra column effects are quantitatively assessed.", "Band broadening under non-binding condition is accurately predicted across scales." ]

[ "BIOC" ]

[ "Axonal regeneration", "Cell-cell fusion", "Dendrite remodelling", "Fusogen", "Heterokaryon", "Neuron theory" ]

[ "1307", "1309" ]

[ "The neuron theory proposed by Ramón y Cajal, according to which neurons exist as individual cells, has been the foundation of modern neuroscience and has paved the way for our current understanding of how the nervous system develops and functions.", "Neurons are highly polarized cells that extend two functionally and morphologically different compartments from the soma: dendrites and an axon.", "Dendrites are specialized to receive and process electrochemical inputs, whereas the axon transmits the electrochemical messages to other neurons or a target tissue via chemical or electrical synapses.", "Although a number of other tissues normally develop through cell-cell fusion, thereby generating multinucleate syncytia (placenta, muscle, osteoclasts, macrophages), this cellular mechanism was thought to be absent from the nervous system.", "However, several reports suggest that neurons, similar to other cells, have the capacity to fuse their plasma membranes, and that these events occur under both physiological and pathological conditions.", "This raises a number of intriguing questions.", "Is it possible that the true extent of neuronal fusion across species has been overlooked due to the complexity of the nervous system?", "What consequences do these cellular events have for the identity and function of the neurons involved, and for the circuits they form?", "Could neuronal fusion be part of the etiology underlying certain neurological diseases?", "What is the molecular machinery that governs neuronal fusion?", "This review aims to present the current evidence in the field, and to highlight the potential impact of recent advances on our understanding of the nervous system.", "One of the simplest examples of the capacity of neurons to fuse their membranes is observed during neuronal self-fusion.", "This term defines the ability of a neuron to fuse sections of its own neuronal processes (dendrites or axon), in order to remodel them during development or repair them following injury (Fig. 1 and Table 1).", "The PVDs (left and right) are a bilateral pair of mechansensory neurons in Caenorhabditis elegans, each of which extends anteriorly and posteriorly directed dendrites, and a ventrally directed axon.", "Each dendrite extends several multibranched units (named menorahs based on their shape) both ventrally and dorsally, covering almost the full body of the animal (Fig. 1A).", "This highly branched and stereotypical dendritic arbor has made PVD one of the best-characterized neurons in terms of dendrite development and repair [1–7].", "Using this cell as a model system, Oren-Suissa and colleagues have described a new mechanism for dendritic arbor development involving membrane fusion [7].", "They have elegantly shown that, during development, the PVD dendritic arbor is pruned and shaped through branch retraction and, most interestingly, through loop formation by neurite self-fusion.", "Both these processes were shown to be mediated by the nematode-specific fusogen Epithelial Fusion Failure-1 (EFF-1), a bona fide fusogen previously shown to mediate cell-cell fusion during development in other C. elegans tissues [8].", "The authors proposed that assembly of EFF-1 complexes in cis causes membrane curvature and retraction, whereas interactions between EFF-1 molecules in trans across closely apposed membranes causes dendrite fusion.", "EFF-1 sculpts these neurons in a dose-dependent manner to maintain the angle of neurites at branching points and avoid overlapping branches.", "This process could be compared to the self-contact elimination process described decades ago for the development of neuronal growth cones in vitro [9].", "A similar self-contact elimination by membrane self-fusion has recently been characterized in epithelial cells [10] and in the vascular endothelial cells of zebrafish embryos [11], raising the possibility that it might be a common mechanism to shape cellular processes (Fig. 1B).", "These findings show that some neurons express a functional fusogen, and that dendrites are capable of membrane fusion.", "Another example of neuronal self-fusion is the process of axonal fusion observed during axonal regeneration.", "In this case, following transection of the axon, the proximal axonal fragment that is still attached to the cell body regrows toward and fuses with its own separated axonal fragment in an end-to-end or end-to-side configuration (Fig. 1C), re-establishing membrane and cytoplasmic continuity and therefore the original axonal tract.", "This process has been recognized for more than 50 years, and has been described in the motor neurons of crayfish [12], sensory neurons of the leech [13,14], giant axons of the earthworm [15], dissociated Aplysia sensory neurons in vitro [16] and, more recently, in the mechanosensory neurons of the nematode C. elegans [17,18].", "In these studies, cytoplasmic continuity after rejoining of the two separated fragments was confirmed by electron microcopy [13,15,17,18], by injection of high molecular weight dyes (such as horseradish peroxidase) into the soma [14], or by expressing genetically encoded photoconvertible fluorophores such as Kaede [18], which were able to diffuse through the fusion site, from the soma to the distal axonal fragment.", "In some models, neuronal function has also been shown to recover fully at the electrophysiological [12,13,15,16] and behavioral levels [12,15].", "Although the process of neuronal self-fusion during axonal regeneration has been well characterized at the morphological level, it was not until recent studies in C. elegans that the molecular mediators of this fusion process were identified [17,19].", "In the C. elegans mechanosensory neurons, membrane fusion of the rejoining axonal fragments is mediated by the nematode fusogen EFF-1 [17,19].", "In this process, EFF-1 is the final effector of a pathway involving changes in membrane lipid composition, which mediates the recognition of the separated distal fragment by the regrowing proximal fragment.", "In particular, these studies revealed that, following axonal transection, the lipid phosphatidylserine (PS) becomes exposed on the outer leaflet of the plasma membrane of the distal axonal fragment.", "Exposed PS itself, or PS bound by specific secreted ligands (such as the transthyretin TTR-52 or the lipid-binding protein NRF-5), is detected by transmembrane receptors present on the regrowing fragment (such as the PS receptor PSR-1, and possibly the TTR-52-binding receptor CED-1), thereby mediating recognition between the two separated axonal fragments prior to specific membrane fusion [19].", "It is not known whether the role of these molecules in mediating axonal self-fusion during regeneration is conserved among species, but it is likely that similar molecular pathways are involved in other organisms, given that membrane fusion is an active process that requires specialized molecular players.", "PS exposure and recognition by cell surface receptors is a common mechanism for many cell-cell fusion events, and has been implicated in the fusion of myoblasts [20,21], syncytiotrophoblast cells in the placenta [22], and macrophages [23,24], as well as in the fusion that mediates the entry of some viruses into host cells [25,26].", "Finally, it is likely that species-specific fusogens act as the last effectors in the mediation of membrane fusion.", "Taken together, these findings demonstrate that neurons of different classes and from different invertebrate species likely express functional fusogens and can fuse their membranes as a mechanism of repair.", "It is also possible for neurons to undergo fusion with other neurons or with other cell types, a phenomenon that we will refer to as neuronal cell-cell fusion.", "A select number of studies have shown that neuronal cell-cell fusion may occur spontaneously.", "For example, the giant axons of the squid are generated by complete fusion of multiple neurons (third order giant fibers) and the syncytial axon is enclosed in a single sheath cell [27].", "In vertebrates, some studies using electron microscopy and brightfield microscopy have reported the formation of neuronal syncytia in vitro and in vivo [28–32]; unfortunately, however, these results were not quantified, making it difficult to determine their general relevance.", "Overall, the vast majority of neuronal cell-cell fusion events in different species have been described in the context of specific conditions or insults, which include viral infection, axonal injury, or the presence of stem or precursor cells (Fig. 2 and Table 1).", "It is well established that some viruses cause the formation of syncytia in infected tissues as a means of propagation and spreading.", "This ability to mediate cell-cell fusion of infected cells was used in early experiments as a technique to study gene expression and nuclear reprogramming in different cell types, including neurons.", "Using inactivated Sendai virus (one of the Para-influenza I group of Myxoviruses) [33], neurons were fused for the first time with undifferentiated green monkey kidney fibroblasts in vitro to form heterokarya [34,35].", "Recent evidence shows that viral-mediated cell-cell fusion also occurs in vivo in the nervous system, with infected neurons forming viable syncytia (with either other neurons or different cell types).", "Currently, the mechanisms of viral-mediated neuronal cell-cell fusion, and the effects of these fusion events on the function of the nervous system, are just beginning to be elucidated.", "A number of neuroinvasive viruses from the Herpesviridae family have been shown to induce neuronal cell-cell fusion in vitro and in vivo.", "Pseudorabies virus (PrV) was first shown to induce fusion between pre-and post-synaptic membranes of neurons in infected rat sympathetic and sensory ganglia, as evidenced by the observation of fusion pores at the electron microscopy level [36].", "Later studies confirmed that PrV infection was able to induce fusion between infected sympathetic neurons in vitro, based on the diffusion of high molecular weight dyes from the soma of an injected neuron to neighboring neurons [37].", "More recently, neuronal cell-cell fusion caused by PrV infection has been observed in vivo in the autonomic ganglia of infected mice [38].", "Similarly, varicella-zoster virus (VZV) has been shown to induce fusion between neurons and satellite cells in human dorsal root ganglia (DRG) xenographs in mice.", "Half of the neuron-satellite cell complexes analyzed showed signs of cell-cell fusion, which is consistent with the ability of VZV to induce cell-cell fusion in skin lesions [39].", "VZV can also induce fusion of DRG neurons with other non-neuronal cells that are in contact with the DRG axons, and potentially generates syncytia containing neurons and keratinocytes [40].", "Finally, herpes simplex virus type 1 (HSV1) is capable of fusing DRG neurons with non-neuronal cells [40], although unlike VZV, it has not been found to mediate fusion with satellite cells [41].", "Another example of viral-mediated neuronal cell-cell fusion comes from an experimental setting designed to label dividing cells in the mouse brain.", "Replication-incompetent murine retrovirus type-C, from the Retroviridae family, was found to induce fusion between neurons and microglia in the mouse neocortex [42].", "Taken together, these lines of evidence show that some viral infections can generate viable neuronal syncytia or heterokarya, presenting a paradigm to study neuronal cell-cell fusion and, most importantly, the consequences of this fusion in terms of nervous system function.", "In order to understand the mechanisms of cell-cell fusion between infected neurons, McCarthy and colleagues investigated the temporal and spatial formation of the fusion pores between neurons in vitro [37].", "Using a combination of dyes with different molecular weights, they found that PrV infection first induced the formation of small pores, which allowed the diffusion of only low molecular weight dyes between neurons, followed by the formation of larger fusion pores later in the course of infection, as evidenced by the diffusion of high molecular weight dyes.", "As infection progressed, signs of membrane fusion became apparent at the level of the soma, with multi-nucleated syncytia clearly visible.", "Interestingly, viral-induced cell-cell fusion might occur in a specific compartment of the neuron.", "McCarthy and colleagues observed that in vitro PrV-infected neurons underwent fusion at the level of the soma, whereas PrV infection in vivo led to neuronal fusion at the axonal level (unmyelinated axons).", "Indeed, a PrV strain defective for the anterograde transport of virions and viral proteins into the axon was not able to induce the formation of neuronal syncytia [38].", "VZV-induced fusion observed between DRG neurons and infecting cells occurred at either the axon or the soma [40].", "In contrast, HSV1 was able to induce the fusion of DRG neurons with cells contacting the axon, but not with satellite cells, which are in contact with the soma.", "It has been proposed that the HSV1 virion, similar to PrV, may require transport into the axon to be functional, and therefore might only mediate fusion in this compartment [40].", "Lastly, during fusion of neurons and microglia in murine C-type retrovirus-infected mouse brains, Ackman and colleagues observed a consistent location of fusion between the apical dendrites of neurons and the processes of the associated microglia [42].", "This, together with the absence of other cell-cell fusion partners, pointed to a unique interaction between microglia and neurons that is favorable to membrane fusion.", "Taken together, these results indicate that viruses are able to hijack a neuron and make it transition into a fusion-competent cell, possibly in a spatially controlled manner to target particular neuronal compartments (Fig. 3).", "Although the molecular mechanisms of this transition are still to be identified, a likely prediction is that infected neurons are forced to express fusogens on their membranes.", "Some evidence does suggest that fusion is mediated by the same viral membrane fusion machinery that mediates the entry of the virus into the host cell.", "A mutated version of PrV lacking a functional viral fusion glycoprotein B (gB) (necessary for cell infection by the virus and its propagation to other cells), is unable to mediate the formation of neuronal syncytia following infection [37,38,43].", "An important question that arises from these discoveries is what are the physiological and behavioral consequences of the formation of neuronal syncytia?", "Remarkably, it has been shown that PrV-induced syncytia in cultured neurons have altered electrophysiological activity, as fused neurons display synchronous electrical activity, as well as elevated rates of spontaneous action potential firing [37].", "Similar results have been found in vivo, with neurons in the ganglia of PrV-infected mice showing a synchronous and cyclical calcium pattern [38].", "This spontaneous and synchronous cyclic activity had already been reported decades ago in neuronal tissues infected by PrV virus [36,44,45].", "Abnormal electrical activity is hypothesized to be the cause of the characteristic symptoms caused by alpha-herpesviruses, i.e. numbness and tingling, and the sensations of itching and pain.", "At the cellular level, the formation of neuronal syncytia was also shown to affect mitochondrial dynamics in vitro [43].", "After PrV infection, the formation of syncytia was followed by an increase in the intracellular calcium level, which in turn disrupted the association of the mitochondrion membrane anchored-Miro1 with Kinesin-1 through its calcium binding sites; this resulted in disruption of recruitment of mitochondria to Kinesin-1 and, as a consequence, altered mitochondrial transport.", "It is important to note that such impairment of mitochondrial dynamics could lead to neuronal degeneration.", "Overall, viral-induced neuronal syncytia are viable, but their formation alters the normal electrophysiological properties of the neurons, which in turn could result in an altered “behavioral” output.", "Moreover, cell-cell fusion also alters cellular processes required for maintaining neuronal homeostasis.", "Fusion between individual neurons has also been reported to occur during axonal repair after injury, first in the leech several decades ago [14] and more recently in C. elegans [18,19].", "Although the fusion between the regrowing proximal fragment and its own distal fragment is generally specific, it has been reported that when two or more fasciculating axons are simultaneously transected, the regrowing proximal axonal fragment of one neuron can fuse with the proximal or distal axonal fragment of the nearby injured neuron [14,18].", "In C. elegans, this event has been well characterized in two pairs of tightly associated neurons: PLM-PLN and ALM-ALN [18].", "The axons of PLM and PLN extend from their respective cell bodies on the posterolateral side of the animal, and run in close association under the epidermis toward the midbody.", "Similarly, the axons of ALM and ALN run in close association on the anterolateral side under the epidermis, toward the head of the animal.", "When both axons in each pair were transected, the transfer of fluorophore from one cell to the other revealed fusion between PLM and PLN in ∼10% of cases, and between ALM and ALN in 13% of cases [18].", "Remarkably, PLM-PLN neuronal fusion has also been observed in a different model of axonal injury, induced by the lack of the cytoskeletal component UNC-70/β-Spectrin that causes axonal fragility [19,46].", "Importantly, it has been demonstrated that PLM-PLN fusion depends on the same molecular machinery that mediates self-fusion between the PLM proximal and distal axonal fragments, requiring the fusogen EFF-1 [19].", "It remains unclear whether this type of cell-cell fusion occurs by accident, or if it plays a physiological role.", "It is also unknown whether it has consequences in terms of the identity and function of the neurons involved.", "However, the very low frequency at which such fusion is observed makes it tempting to speculate that a specific molecular mechanism favors specific self-fusion repair and prevents cell-cell fusion.", "As briefly mentioned above, cell-cell fusion events involving neurons may occur with a glial partner.", "VZV infection causes fusion between neurons and the support cells of the DRG [39], and replication-incompetent C-type retrovirus injected in the mouse neocortex causes very specific fusion between neurons and microglia [42].", "Fusion has also been shown to occur between microglia and neuronal stem cells [47].", "However, one of the clearest examples of developmentally regulated glial fusion occurs in the major sensory organs of C. elegans, the amphids, which are located on the left and right sides of the nematode’s head.", "Two support cells, the amphid sheath (AMsh) and socket (AMso) cells, provide the structural support and a channel for the sensory cilia of 12 neurons that are exposed to the environment and detect external stimuli.", "In harsh environmental conditions, such as the absence of food, high temperature, or high population density, C. elegans enter a diapause stage (Dauer stage), which is characterized by a dramatic remodeling of body structures, including the amphids [48,49].", "Cell-cell fusion occurs between the left and right AMsh glial cells, and this event is required for the correct morphological and functional changes to the ciliated receptive endings of the chemosensory neurons.", "This AMsh glial fusion event is mediated by the second known bona fide C. elegans fusogen, Anchor cell Fusion Failure-1 (AFF-1) [48].", "These examples reveal that not only neurons but also glial cells can engage in cell-cell fusion events.", "In the past decade, it has become clear that cell-cell fusion is a key mechanism through which stem or progenitor cells of one tissue give rise to differentiated cells of another tissue.", "In 2002, Ying and colleagues demonstrated that progenitor cells of the central nervous system were able to fuse with embryonic stem cells, giving rise to pluripotent hybrid cells [50].", "Other milestone discoveries revealed that bone marrow cells were able to fuse spontaneously with neuronal progenitors in vitro [51], and that following bone marrow transplantation, bone marrow-derived cells fused with Purkinje neurons in vivo, forming binucleated heterokarya [51–53].", "This phenomenon has been demonstrated in both humans and mice, and can also occur between cells of different species [54].", "Interestingly, these binucleated heterokarya retain the Purkinje neuron cell fate, as the nucleus of the stem cell (usually round with condensed chromatin) acquires a Purkinje neuron-like nucleus shape (with dispersed chromatin), and also expresses some Purkinje neuron-specific genes [51,55,56].", "Most importantly, these heterokarya have been shown to act as electrically active Purkinje neurons [57], suggesting that in this case fusion is not detrimental to the neuron.", "A series of important subsequent studies further characterized the formation of these heterokarya and their possible physiological role (reviewed in [58]).", "The frequency of heterokaryon formation in both humans and rodents is very low, and ranges between ∼0.1% and 0.4% of the total Purkinje neuron population [51–54,59].", "However, several studies indicate that this rate of fusion increases (10–100 fold) with damage [60–62], inflammation [56,61,63], neurodegenerative disease [54,56,59,64] or chemically induced degeneration [65].", "Although some authors defend the hypothesis that fusion is almost exclusively a consequence of damage and irradiation [61], others report that fusion is not caused by the irradiation and transplantation procedure itself, as blood chimerism obtained by surgically joining two mice (parabionts) also leads to the formation of heterokarya between the hematopoietic cells of one mouse and the Purkinje neurons of the other mouse [56].", "Most importantly, other studies have revealed that neuronal fusion might also occur during normal development and during aging in non-manipulated mice [60,65].", "An increase in fusion rate with aging has also been found in irradiated models [53,60], supporting the idea that aging somehow favors the acquisition of fusion competence by neurons.", "The contribution of hematopoietic cells to Purkinje neurons can occur in physiological conditions; however, these cells were found to be mononucleated, suggesting that fusion might be a transient event or that another mechanism is in place [63].", "Despite considerable evidence of heterokaryon formation accumulated during the past decade, it is still unclear if this process plays a specific physiological role.", "As noted above, the heterokarya remain functional Purkinje neurons, and a study by Bae and colleagues found that the degeneration of Purkinje neurons in a mouse model of Niemann-Pick disease type C1 (a disease that affects the function of the cerebellum leading to impaired motor function) was alleviated after transplantation of bone marrow-derived mesenchymal stem cells [64].", "Similar results were obtained in a mouse model of cerebellar ataxia, although the exact role of cell fusion in this functional recovery was not clear [66].", "Overall, it has been hypothesized that the fusion of progenitor or stem cells with damaged neurons provides a healthy nucleus that is able to rescue these highly complex neurons that would otherwise be impossible to replace.", "This is similar to the regenerative role of cell-cell fusion and heterokaryon formation first described in skeletal muscle [67] and liver [68,69] (for a review see [70]).", "Fusion in the nervous system has also been observed outside the cerebellum, with reports that bone marrow-derived stem cells can fuse with cortical neurons [55] and spinal motor neurons [71] following transplantation.", "Fusion has also been described between embryonic stem cell-derived neuronal stem cells and microglia in vitro, and between neuronal stem cells and mature pyramidal neurons in the neocortex of mouse and rats in vivo [47].", "Another important study has revealed that injured retinal neurons can undergo fusion with transplanted hematopoietic stem and progenitor cells [72].", "Remarkably, following activation of the Wnt/beta-catenin signaling pathway, these hybrids are programmed to a precursor stage, proliferate and develop into differentiated neurons, providing partial regeneration of the damaged retina and functional rescue.", "However, lack of activation of the Wnt/beta-catenin signaling pathway leads to apoptosis of the newly formed heterokarya [72].", "It is important to note that some neuronal types seem to be refractory to fusion, even under the same conditions in which Purkinje neurons form heterokarya [61,73].", "This raises the possibility that some neurons may be more prone to fusion than others.", "It also suggests that there might be an inherent resistance to cell-cell fusion, and that neuronal fusion is potentially harmful in some contexts.", "Consistent with this, fusion of bone marrow-derived cells with DRG neurons has been implicated in the pathogenesis of diabetic neuropathy.", "In a mouse model of diabetes, cells thought to arise from these fusion events following bone marrow transplantation displayed abnormal calcium homeostasis and accelerated apoptosis [74].", "In summary, the presence of progenitor cells or stem cells is able to facilitate neuronal fusion under certain circumstances, an effect that can be enhanced by different types of neuronal insults as well as aging.", "However, the cellular and molecular mechanisms underpinning these fusion events are still unknown.", "For example, it is not known whether a specific fusogen becomes expressed on the surface of progenitor cells or neurons, making them more prone to fusion, or if specific neuronal types have a particular proteolipidic membrane composition that makes them fusion competent.", "Moreover, it is still controversial whether these cell-cell fusion events are beneficial or detrimental to the neurons involved.", "Answers to these questions might allow us to control this biological process and potentially expand it for beneficial medical purposes, a concept already under consideration [58,75,76].", "In 2004, a seminal study revealed the existence of specific nanotubular membrane structures that formed de novo between neuroendocrine cells, thereby allowing the transfer of vesicles, molecules, and organelles, and providing a new route for long-distance cell-to cell-communication [77].", "These peculiar structures, named tunnelling nanotubes or TNTs, have a diameter of 50–200 μm and a length of several cell diameters [78].", "In addition to their role in HIV-1 transmission in T-cells [79], an important study has shown that TNTs represent an efficient route for spreading of prions between neurons in the central nervous system, as well as between immune cells and neurons [80].", "Importantly, TNTs formed between neurons and distant astrocytes have also been shown to facilitate electrical coupling and calcium signaling [81].", "Thus, TNTs represent a de facto neuron-neuron or neuron-glia membrane fusion event, albeit they are mostly temporary, they transport only selected cargo, and they do not form true syncytia.", "It is still unclear, and of the utmost interest, what molecular elements regulate the formation of TNTs, how the recognition proceeds between the cells involved, and most importantly what fusogenic elements facilitate the actual fusion of the two distinct membranes and what limits it to a transient process.", "Most of the studies on neuronal or glial cell-cell fusion report these events at the cellular level, studying the circumstances and consequences of fusion; however, only a few have explored the underlying molecular mechanisms.", "Membrane fusion is an active process and therefore requires specific protein and lipid effectors.", "Viral-induced neuronal cell-cell fusion is likely to be mediated by the viral fusogens expressed by infected neurons [37,38,43].", "Studies on neuronal fusion in C. elegans have identified the nematode fusogen EFF-1 as the main effector of self-fusion during PVD dendrite development [7] and axonal regeneration of PLM neurons [17,19].", "EFF-1 is also responsible for PLM-PLN cell-cell fusion following axonal injury [19], and the second nematode fusogen AFF-1 has been identified as the main effector of AMsh glial cell-cell fusion [48].", "Interestingly, when EFF-1 and AFF-1 were first discovered in C. elegans, both fusogens appeared to be expressed in neurons and glia [8,82].", "However, this was a puzzling discovery, given that at the time, these cells were not known to engage in fusion events.", "Only recently has it been shown that these fusogens are active in neurons and glia, providing evidence that the C. elegans nervous system has the molecular machinery necessary for membrane fusion.", "With this in mind, could the expression pattern of fusogens in vertebrates be used to predict if these neurons are also fusion competent?", "In humans, two highly fusogenic proteins of retroviral origin have been identified: Syncytin-1 (encoded by the HERV-W envelope gene located in chromosome 7q21.2) [83,84] and Syncytin-2 (encoded by the HERV-FRD envelope gene located in chromosome 6p24.1) [85].", "Both fusogens are almost exclusively expressed in the placenta, and Syncytin-1 and its receptors (the neutral amino acid transporter or type D mammalian retrovirus receptor ASCT 2 and the related protein ASCT 1) are responsible for mediating the fusion events involved in the formation of the syncytiotrophoblast, the syncytial outer layer of the placenta.", "Interestingly, although expression of these fusogens has not been detected in the nervous system of healthy patients, expression of Syncytin-1 has been found to be upregulated in the brain of patients with multiple sclerosis [86,87], and envelope genes of the HERV-W family have been found in the cerebrospinal fluid of patients with schizophrenia and bipolar disorder [88–90] (for a review on this topic see [91]).", "How the expression of fusogens might contribute to the etiology of neurological disorders is just beginning to be explored.", "In multiple sclerosis, the expression of Syncytin-1 in astrocytes and microglia mediates neurotoxicity by triggering a robust inflammatory response that is toxic to oligodendrocytes and leads to demyelination, which explains at least in part the deleterious effect of this fusogen [86].", "Whether cell-cell fusion events also contribute to this pathology has not yet been tested, but it is tempting to hypothesize that the expression of Syncytin-1 in the nervous system could lead to cell-cell fusion.", "Finally, in mice, Syncytin-A and Syncytin-B proteins have been identified as functional fusogens that are also involved in the formation of the syncytiotrophoblast layer of the placenta [92].", "These genes are essentially expressed in this tissue; however, Syncytin-A transcript expression is also detected in the brain above background levels [92].", "Ultimately, there is evidence that fusogens are expressed in the nervous systems of multiple species; whether this expression occurs in health or disease, it supports the idea that neurons and glia may acquire fusion competence under certain circumstances, and suggests that unknown self-fusion or cell-cell fusion events remain to be discovered.", "The concept that neurons can form syncytia or heterokarya is still relatively novel, and the evidence of neuronal fusion discussed here represents the tip of the iceberg.", "A range of studies addressing different questions in biology (viral infection, tissue regeneration, neuronal development, and more recently the discovery of nanotubes) has converged on the concept of neuronal fusion; however, much remains to be explored in this fascinating field.", "Are all classes of neurons fusion competent?", "Under which circumstances do neurons fuse?", "What are the ultimate consequences of neuronal fusion?", "There are a number of factors unique to the nervous system that make in vivo studies of cell-cell fusion difficult or impossible to conduct.", "Firstly, the sheer complexity of the central nervous system, in terms of cellular number, architecture and density, makes observations of neuronal fusion technically challenging.", "Secondly, neuronal fusion can occur not only at the level of the soma, but also at the level of axons or dendrites, and cellular connections in such a spatially restricted area may be impossible to discern with the resolution of current imaging techniques.", "Finally, identifying cell-cell fusion in neurons may be further confounded by the fact that such fusion can be transient and result in mono-nucleated diploid cells [63].", "There are clearly challenges to address, and progress is most likely to be achieved through a combination of different experimental paradigms in diverse model systems.", "We have begun to elucidate some of the molecular mechanisms that mediate neuronal fusion, starting with the EFF-1-containing pathways for neuronal self-fusion and cell-cell fusion in C. elegans.", "The molecules that mediate neuronal fusion in vertebrates, and the role for as-yet-undiscovered fusogens in these systems, are currently unknown.", "It also remains to be determined whether membrane-exposed PS, an essential requirement for neuronal self-fusion during regeneration, has a common role in neuronal fusion.", "Another important step will be to determine how the molecular machinery for neuronal fusion is regulated.", "The current evidence suggests that the circumstances of neuronal fusion are highly specific.", "Although very little is known about the physiological advantage or possible detrimental effect of neuronal cell-cell fusion, it is clear that it can have consequences for the neuron at both the subcellular and electrophysiological levels.", "Exactly how the balance is established between healthy, developmental self-fusion, and protection from ectopic cell-cell fusion, is still to be determined.", "One hypothesis is that it involves subcellular regulation of cell-specific fusogens, with changes in expression and localization of these molecules occurring in response to specific stimuli or insults.", "The finding that neurons can form syncytia or heterokarya might be seen as a challenge to Ramón y Cajal’s neuron theory.", "Conversely, our current understanding of neuronal fusion may instead strengthen this theory, as neurons appear to fuse only under very specific circumstances, and it is likely that mechanisms exist to prevent uncontrolled cell-cell fusion, thereby maintaining neurons (for the most part) as individual units." ]

[ "The neuron theory of Ramón y Cajal states that neurons are individual cells.", "Despite the neuron theory, neurons and glia are able to fuse their plasma membranes.", "Some neurons undergo self-fusion during development and repair.", "Neurons and glia are able to form multinucleated cells." ]

[ "BIOC", "CENG", "ENGI", "MATE" ]

[ "3D Cell culture", "Cell differentiation", "Hepatocyte", "Multicellular spheroids", "Nanocellulose", "Organotypic" ]

[ "1304", "1502", "2211", "2502", "2503" ]

[ "Pharmaceutical industry, regulatory authorities, and academic investigators need liver cell cultures to predict and estimate metabolism, excretion and toxicity of drugs and other chemicals in the human liver.", "Due to the inter-species differences animals and animal cells lead frequently to misleading, and sometimes hazardous, estimates of pharmacokinetics and toxicity in humans.", "Therefore, Food and Drug Administration of the United States has emphasized the need for improved preclinical cell models for drug development in their Critical Path Initiative.", "Human liver microsomes are used to study xenobiotic metabolism, but the microsomes do not have drug transporters or transcription machinery.", "This limits seriously their usefulness in pharmacokinetics and toxicology.", "Primary human hepatocyte cultures in 2D are the gold standard in in vitro evaluation of hepatic metabolism and toxicity.", "However, the availability of human primary hepatocytes is limited, they show substantial functional variability and restricted lifespan, and their drug transporter activity is low unless they are ‘sandwich-cultured’ [1,2].", "Immortal human liver cell lines, such as HepG2 and HepaRG are widely used in in vitro studies [3,4].", "Compared to HepG2 cells, HepaRG liver progenitor cells generate improved hepatic phenotype in culture and this continuous cell line has been successfully applied in the evaluation of chemicals and drug candidates [4–8].", "However, 2D format of the current HepaRG cultures clearly deviates from the vectorial 3D morphology of the hepatocytes in the liver.", "Overall, the existing liver cell models are not satisfactory and more representative cell models are needed for biological research and drug and chemical testing.", "Cellular phenotype can be tuned with the culture environment, particularly with extracellular matrix mimicking biomaterials.", "Preferably the biomaterials should provide fibrillar structures, extracellular matrix mimicking stiffness and hydrous environment with unrestricted permeation of nutrients and endogenous factors [9–13].", "We investigated hydrogels of native wood-derived nanofibrillar cellulose (NFC) and hyaluronan-gelatin (HG) as supporting materials in the 3D culture of HepaRG liver progenitor cells.", "Growdex™ nanofibrillar cellulose (NFC) hydrogel was obtained from UPM Corporation, Finland.", "The preparation and properties of NFC hydrogel have been described in detail earlier [12].", "The NFC concentration of the hydrogel was 1.7 wt% and the product was sterile.", "Due to the used raw material, the NFC hydrogel contains also substantial amounts of hemicellulose, mainly xylene (25%), which generates a slightly anionic surface charge (−2 mV) on the fibrils.", "Extracel® (HG) hydrogel is based on thiol-modified hyaluronan, thiol-modified gelatin and crosslinker (polyethylene glycol diacrylate) (PEGDA) [21,22].", "This material was obtained from Glycosan Biosystems, USA.", "Genomic DNA was quantitated with Quant-iT™ PicoGreen dsDNA assay kit (Molecular probes, Invitrogen).", "At first, the cell cultures were washed with dPBS.", "Hydrogel cultures were washed and ruptured by pipetting up and down with dPBS followed by centrifugation (2500 G 5 min).", "After dPBS removal the cultures were lysed in RLT buffer (RNeasy Mini kit, Qiagen).", "Additionally hydrogel cultures were disrupted with TissueRuptor (Qiagen) to release the nucleotides from the gel and centrifuged (3000 G 5 min) to remove the hydrogel.", "Then, the RLT cell lysates were diluted 1:20 with autoclaved milliQ water, 5 μl of each diluted sample and standard were pipetted into DNA-free black 96-well plate (Greiner bio-one), and 100 μl of PicoGreen (diluted 1:100) was added to the wells.", "The final buffer concentration of 1:400 in the analysis is known to be compatible with the PicoGreen signal [31].", "After 5 min incubation at RT the emission of dsDNA samples at 520 nm was recorded with fluorometer (Varioskan Flash, Thermo).", "Readings from samples were compared to 1–500 ng/ml of control dsDNA provided with kit.", "Samples from three independent experiments of both low-density and high-density experiments were analyzed.", "Each lysate was analyzed as triplicate.", "Structural polarity of the HepaRG cultures was analyzed by fixing the cell cultures in 4% paraformaldehyde for 15 min at 4 °C.", "After washing with PBS, the cells were permeabilized with 0.1% Triton X-100 for 15 min and subsequently incubated overnight with a blocking buffer (10% goat serum and 0.2% BSA in PBS) at 4 °C.", "Then, the cultures were incubated with a primary antibody, anti-MDR1 (1:100, Sigma P7965) or anti-MRP2 (1:300, Abcam ab3373) (diluted in 5% goat serum in 1 × PBS buffer) overnight at 4 °C in a humidified chamber.", "After washing unbound primary antibody, samples were incubated with goat anti-mouse or goat anti-rabbit antibody conjugated with Alexa Fluor 488 (Invitrogen, diluted 1:200 in 5% goat serum in PBS buffer) for 4 h at RT in a humidified chamber.", "The cultures were washed with 1 × PBS (four times for an hour) and 1 μg/ml Hoechst 33258 (Sigma–Aldrich, bisbenzimide) together with Alexa Fluor 594-labelled phalloidin (Invitrogen, diluted 1:100 or 1:50 in 1 × PBS buffer) was placed and held for overnight to visualize the nuclei and cellular distribution of the filamentous actin (F-actin) cytoskeleton, respectively.", "For immunostaining of the cells in HG hydrogel, the cultures were fixed in 4% paraformaldehyde overnight at 4 °C, dehydrated by passing the hydrogel through a series of increasing alcohol concentrations (50% overnight at 4 °C, 70% for 30 min at RT, 94% for 30 min at RT, 99.5% for 15 min at RT), treated with xylene for 60 min at RT, and filled with paraffin by embedding in hot paraffin for 60 min in Tissue-Tek® TEC™ tissue embedding center (Sakura Finetek Europe).", "After paraffin was cooled to become solid, the culture was cut in 5–20 μm sections by microtome.", "The sections on objective glasses were deparaffinized with xylene washes (6 × 10 min) and decreasing alcohol concentrations (99.5% for 3 × 3 min, 94% for 2 × 3 min, 70% for 1 × 3 min, and 50% for 1 × 3 min, all at RT).", "After cold water wash, antigens were retrieved by boiling in sodium citrate buffer for 5–20 min.", "The culture sections were washed in TBS buffer with 0.025% Triton X-100, and blocked in 10% goat serum with 0.2% bovine serum albumin in TBS buffer for 2 h or overnight.", "Staining of proteins and imaging was performed as for the whole mount cell cultures.", "As exception the actin filaments were stained in uncut HG cultures since phalloidin is not compatible with paraffin embedding.", "Stained samples were protected with an antifade reagent (Prolong gold, Invitrogen) and analyzed with a confocal microscope (Leica TCS SP5 with Leica DM5000 upright microscope, HCX APO 63×/1.30 (glycerol) objective and TD 488/561/633 beam splitter).", "Images were acquired via three channels (430–525 nm, 500–550 nm and 600–700 nm) excited with laser diode 405 nm/50 mW, laser OPSL 488 nm/270 mW, and DPSS 561 nm/20 mW.", "The confocal images were analyzed with Imaris program (Bitplane) and either slice or surpass images were constructed.", "The possible deconvolution was done with AutoQuant X program (MediumCybernetics).", "Hepatobiliary transport was investigated with fluorescein diacetate, a marker of efflux transport in hepatocytes [32,33].", "Fluorescein diacetate enters the cells passively and undergoes esterase-mediated hydrolysis.", "Polar fluorescent metabolite, fluorescein, will be entrapped in the cells unless it is actively transported from the cells into the canalicular space.", "The HepaRG cultures in Nunc™ chambered coverglass slides were exposed to fluorescein diacetate at a final concentration of 10 μm for 5 min at 37 °C in 5% CO2 and washed with HBSS.", "Cell-permeable DNA probe Draq5™ (Biostatus Limited) was placed to cultures in HBSS buffer at 30–60 μm final concentrations.", "The cells were incubated 30 min at 37 °C in 5% CO2 to get nuclei stained after which observed with a Leica TCS SP5 II HCS A confocal laser scanning system with Leica DMI6000 B inverted microscope, HCX PL APO 20×/0,7 (water) objective, QD 405/488/561/635 beam splitter and incubator box with air heater system (Life Imaging Services).", "The fluorophores were excited with lasers Argon 488 nm/35 mW and HeNe 633 nm/12 mW.", "Emission was acquired at 500–550 nm (fluorescein) and 650–750 nm (Draq5 ™).", "The transmitted light was also collected.", "The confocal images were analyzed with Imaris 7.4 program (Bitplane) and either slice or surpass images were constructed.", "Human liver tissue was obtained from harvested organs for liver transplantation in the Transplantation and Liver Surgery Clinic (Helsinki, Finland).", "Donor livers were from brain dead male (age 41) and female (ages of 58, 13 and five) subjects with beating heart, normal liver function, negative hepatitis serology, and non-pathologic liver histology.", "The livers were flushed in situ with University of Wisconsin solution and kept at +4 °C until resection.", "Liver segments that were redundant for liver transplantations were sliced and stored at −70 °C in TRI reagent (Sigma–Aldrich) or at −20 °C in RNAlater (Qiagen).", "RNA was extracted (see paragraph for real time polymerase chain reaction).", "The research was authorized by National Supervisory Authority for Welfare and Health and by the Hospital District of Helsinki and Uusimaa Ethics Committee Department of Surgery.", "HepaRG cells have been derived from a liver tumor of a female patient who suffered from hepatitis C virus and hepatocarcinoma [23].", "HepaRG cells are capable of differentiating into biliary-like epithelial cells (cholangiocyte-like cells) and hepatocyte-like cells.", "At low culture density, the cells express markers of early liver progenitors, and at confluence the cells will be committed to hepatocyte-like differentiation [24].", "The differentiation into mature hepatocyte-like cells is potentialized by dimethyl sulfoxide (DMSO) treatment.", "Subcultivation at low-density induces dedifferentiation of differentiated cells into early liver progenitors.", "The 2D HepaRG cultures were used as a benchmark to compare with the 3D hydrogel cell cultures.", "HepaRG cells were cultured in 2D format as described previously [23,24].", "Shortly, HepaRG cells were plated either at progenitor state to study the differentiation process or at differentiated state (obtained with DMSO treatment) to study the maintenance of the differentiation.", "Undifferentiated progenitor cells were seeded at low-density (2.6 × 104 cells/cm2) and differentiated at high-density (45 × 104 cells/cm2).", "The cell cultures were placed to the different dishes depending on the end-point analyses (Table 1).", "The medium volume was set to be 400 μl/cm2 in low-density cultures and 600 μl/cm2 in high-density cultures, hereby representing the equal total volumes with 3D hydrogel cultures.", "The HepaRG cells were embedded in 3D NFC and HG hydrogels.", "Undifferentiated cells were seeded at low-density (one million cells/ml of hydrogel) and differentiated at high-density (nine million cells/ml of hydrogel).", "NFC hydrogel-based 3D cell cultures were prepared by mixing the HepaRG cell suspension with 1.7 wt% NCF hydrogel to achieve 1 wt% hydrogels with desired cell density.", "The mixing was performed by pipetting up and down with low-retention pipette tips (TipOne®, Starlab Group).", "HG hydrogel-based 3D cell cultures were prepared according to the manufacturer's instructions (Glycosan Biosystems).", "Briefly, the cells were mixed with HG solutions followed by the gelification at 37 °C in an atmosphere containing 5% CO2.", "The cell cultures were placed to the different dishes depending on the end-points analysis (Table 1).", "The volume of the hydrogel cell cultures was set to be 200 μl/cm2 in each dish.", "The medium volume (on the top of the hydrogel) was set to be 200 μl/cm2 for low-density cultures and 400 μl/cm2 for high-density cultures, hereby representing the equal total volumes with 2D cultures.", "The low-density cultures were maintained with the standard HepaRG growth medium and the high-density cultures with HepaRG differentiation medium supplemented with DMSO [23].", "The equal volume of the medium was renewed daily both from the 2D and 3D hydrogel cultures (2/3 of the medium volume in the hydrogel cultures).", "The cell cultures were maintained at 37 °C in an atmosphere containing 5% CO2.", "Cell cultures were monitored by phase contrast microscopy (Leica DM IL LED) and images were taken over the time.", "The average diameter of spheroids was defined from the phase contrast images with LAS EZ software (Leica Microsystems) using the distance line facility.", "Minimum of 10 spheroids were measured to attain the average size.", "The mitochondrial metabolic activity of the cells was determined using oxidation-reduction indicator, resazurin (alamarBlue® Cell Viability Reagent, Invitrogen), 1/10 of the co-volume of medium and hydrogel.", "To ensure the mixing of the indicator in hydrogels the culture plates were gently shaken (150 rpm) for 10 min both in the beginning and the end of the incubation (Heidolph incubator 1000 equipped with Titramax 1000 shaker).", "After exposure of 3 h to resazurin at 37 °C in 5% CO2, 50 μl of medium was transferred from each culture well to another 96-well plate and the fluorescent metabolite of resazurin (resorufin) was recorded with a plate reader (Varioskan Flash, Thermo Fisher) using excitation at 560 nm and emission at 590 nm.", "Three independent experiments of both low-density and high-density cultures were carried out.", "The mitochondrial metabolic activity of the cells was examined within the same wells as CYP3A4 activity and RT-PCR.", "Cell viability was analyzed using LIVE/DEAD® viability/cytotoxicity kit (Molecular Probes™).", "In this case, 0.5 μm calcein-AM and 2 μm ethidium homodimer-1 were added to the cultured cells in Hank's balanced salt solution (HBSS).", "The conversion of non-fluorescent calcein-AM into fluorescent calcein and the binding of ethidium homodimer-1 (EthD-1) to DNA were followed with Leica TCS SP5 II HCS A confocal microscope equipped with HCX PL APO 20×/0.7 Imm Corr (water) objective and QD 405/488/561/635 beam splitter.", "Within 30 min after addition of the reagents, the fluorophores were excited with a 488 nm/35 mW argon laser, and emission was acquired with HyD detectors at 500–550 nm (calcein) and 620–700 nm (EthD-1).", "The confocal images were analyzed with Imaris 7.4 program (Bitplane) and either slice or surpass images were constructed.", "Minimum of three independent low-density and high-density cultures were analyzed.", "The transcript levels of regulative and functional genes in 3D hydrogel cultures and standard 2D cultures were determined at various times after cell seeding.", "The expression levels were compared to the human liver tissue.", "Hydrogel cultures and liver tissues were disrupted with TissueRuptor (Qiagen).", "Total RNA was extracted from liver samples with TRI reagent (Sigma) or RNAlater (Qiagen), and treated with DNase I (Fermentas) according to the manufacturer's instructions.", "RNA extraction from the 2D and 3D cell cultures was performed with RLT buffer and spin technology (RNeasy Mini kit, Qiagen).", "RNA extraction was performed from the same RLT lysates as DNA measurements (see Paragraph 2.8.).", "The concentration and purity of aqueous RNA solutions was analyzed with NanoDrop™ spectrophotometer (ND-1000 V3.7.0, Thermo Scientific).", "Additionally, quality of RNA from human liver tissues was analyzed by determining the RNA Integrity Number (RIN) with RNA 6000 nano kit and 2100 Bioanalyzer (Agilent).", "The samples with RINs greater than five were selected for reverse transcription.", "cDNA was prepared with RevertAid H minus first strand cDNA synthesis kit (Fermentas, Life technologies) according to the manufacturer's RT-PCR protocol in 20 μl volumes using both oligo dT18 primers (1 μl/reaction) and random hexamer primers (0.2 μl/reaction).", "Comparative CT experiment was performed in 20 μl reaction volume containing Fast SYBR® Green master mix (Applied Biosystems, Life Technologies), 5 ng or 10 ng of cDNA and 200 nM of each primer set.", "CYP3A4 primers were designed with Primer3-BLAST [25] and KRT19 with Primer Express® (Applied Biosystems, Life Technologies) (Supplementary Table 1).", "CycloG and HNF4A [26], ALB [27], MDR1 and MRP2 [28] primers have been designed and published previously (Supplementary Table 1).", "The primers were synthesized by solid phase phosphoramidite (PA) chemistry followed by reversed phase chromatography (RPC) purification (Oligomer Oy, Helsinki, Finland).", "The relative quantity of target nucleic acid sequence in samples of three independent experiments was determined with StepOnePlus™ instrument (Applied Biosystems) using Fast mode (95 °C for 20 s, followed by 40 cycles of 3 s at 95 °C, and 30 s at 60 °C).", "The specificity of amplified targets was validated by melting curve analysis (15 s at 95 °C, 60 s at 60 °C, and 15 s at 95 °C).", "To determine ΔCt (CT target − CT reference), the expression of the target gene was normalized to expression levels of reference gene (cyclophilin G) within the same sample.", "Then ΔCt Expression was calculated by using 2–ΔCt method and the actual amplification efficiency of each primer pair.", "Finally to compute the comparative expression of each gene in HepaRG cultures, the ΔCt expression was normalized to the ΔCt expression in the human liver samples (cDNA mixture from four human donors).", "The activity of the most abundant cytochrome P450 isoform, CYP3A4, in HepaRG cells was studied with P450-Glo™ CYP3A4 assays (Promega) containing luciferin isopropyl acetate (luciferin-IPA).", "Luciferin-IPA is metabolized by CYP3A4 to release luciferin [29], which can be quantified by luminescence.", "Water volume of the hydrogels was taken into account when the cell cultures were exposed at 3 μm luciferin-IPA for 60 min at 37 °C in 5% CO2.", "To ensure the mix in hydrogels the culture plates were shaken gently (150 rpm) during the first and the last 10 min periods of the incubation (Heidolph incubator 1000 equipped with Titramax 1000 shaker).", "At the end of incubation period, 50 μl of substrate solution from each well was removed, placed into a white 96-well plate, and 50 μl of firefly luciferase (Luciferin Detection Reagent, Promega) was added to induce luminescence production.", "After 20 min at RT, the luminescence was recorded with a plate reader (Varioskan Flash, Thermo Fisher Scientific).", "The luminescence intensity and hereby the CYP3A activity were normalized to the genomic DNA content of each culture.", "Three independent experiments of both low-density and high-density cultures were carried out.", "After CYP3A4 activity analysis the same culture wells were examined for mitochondrial activity and RT-PCR.", "In order to determine influence of 3D culture conditions on the regulatory pathways involved in HepaRG cells, the cells were exposed to prototypic CYP inducers.", "The activity of the CYP3A4 was assayed.", "Differentiated HepaRG cells were seeded in NFC and HG hydrogels at 9 million cells/ml of hydrogel and the standard 2D high-density cultures were created by seeding at 0.45 × 106 cells/cm2 [23,30].", "During the first 24 h, the cell cultures contained differentiation medium that was changed to DMSO-free medium for the following 72 h.", "This was done to remove the induction effects of DMSO.", "On the fourth culture day, dexamethasone (100 μm and 250 μm), phenobarbital (0.2 mM and 1 mM), rifampicin (5 μm and 20 μm) and DMSO (0.5% and 2%) were introduced to the cultures and 0.5% DMSO was used as a vehicle control.", "The medium with inductor (above the hydrogel) was renewed daily during the 48 h of induction.", "During the sixth culture day, CYP3A4 activity was analyzed (see CYP3A4 activity chapter).", "Prior to the analysis, the cell cultures were washed once with 1 × dPBS buffer to remove inductor before adding the luciferin-IPA.", "The viability of the cultures was analyzed with resazurin (see paragraph Cell viability).", "Three independent experiments were performed.", "Each experiment contained triplicate wells per each inducer concentration.", "HepaRG cells formed multicellular spheroids in NFC and HG hydrogels during the first culturing days (Fig. 1A and B).", "The number of spheroids in NFC and HG hydrogel images cannot be directly compared due to the differences in the optical and structural properties of the hydrogels (Fig. 1A and B).", "The size of the spheroids exceeded the size of a single HepaRG cell (circa 13 μm in diameter) already during the first day (Fig. 1A and B), and they reached the full size (circa 40 μm in diameter) within a week.", "NFC hydrogel cultures of progenitor state (low-density) and differentiated (high-density) HepaRG cells resulted in similar spheroids sizes (about 40 μm in diameter).", "The spheroids of the high-density NFC cultures appeared to be less organized (Fig. 1B) when compared to the low-density NFC cultures (Fig. 1A).", "The spheroids in high-density HG retained the same size during the six days of culture (Fig. 1B) and were smaller than in the low-density HG cultures (Fig. 1A).", "Based on the DNA quantification and mitochondrial activity, the low-density HepaRG cells exhibited slow growth within the hydrogels (Figs. 2A and C).", "No proliferation was seen in the high-density cultures and the content of DNA in fact decreased in NFC and 2D cultures.", "On the first culture day, the different DNA content between the 2D, NFC and HG cultures results from the different numbers of the seeded cells (Fig. 2A and B).", "Overall, the mitochondrial activity was lower in high-density than in low-density cultures (Fig. 2C and D).", "Mitochondrial activity was higher in 2D and NFC cultures than in the HG cultures (Fig. 2C and D).", "Nonetheless, the live/dead staining studies indicated that all the cultures were viable at both seeding densities (Fig. 3).", "HepaRG spheroids were viable for at least two weeks (Fig. 3).", "The dead cells were mainly the singular cells outside of the spheroids (Figs. 1 and 3).", "Expression levels of hepatic nuclear factor 4α (HNF4A), albumin (ALB), CYP3A4 and keratin 19 (KRT19) transcripts were studied to assess the differentiation of the HepaRG cells towards hepatocytes or cholangiocytes.", "Additionally efflux transporters MDR1 (P-gp, ABCB1) and MRP2 (ABCC2) were analyzed as markers of hepatic differentiation and drug transport capacity.", "HepaRG showed differentiation towards hepatocyte phenotype in all tested microenvironments after low-density seeding (Fig. 4A).", "The upregulation of HNF4A, ALB and CYP3A4 demonstrates the differentiation to hepatocyte direction.", "Furthermore, downregulation of the cholangiocyte marker KRT19 in low-density NFC and 2D cultures shows that the differentiation proceeds towards hepatocyte phenotype, not cholangiocyte (Fig. 4A).", "No downregulation of KRT19 in HG cultures was detected, but the initial expression level of KRT19 in HG cultures was lower than in 2D and NFC cultures.", "Expression of HNF4A was higher in HG cultures than in 2D and NFC cultures suggesting higher differentiation state of the cells (Fig. 4A).", "The transcript of CYP3A4 was not detectable in standard 2D cultures during the first week of culture.", "However, in NFC and HG hydrogel cultures the mRNA of CYP3A4 was seen after culture day one and the expression levels of human liver were reached within a week of cell culture.", "When differentiated HepaRG cells were seeded in NFC at high-density, KRT19 was upregulated and the hepatic differentiation markers; CYP3A4, HNF4A, ALB, MDR1 and MRP2 were downregulated when compared to HG and 2D cultures (Fig. 4B).", "Expression of KRT19 mRNA was lower in 2D and HG microenvironments than in NFC, which showed high KRT19 expression during the whole culturing time.", "Furthermore, the expression of KRT19 was remarkably higher in HepaRG cells than in human liver.", "CYP3A4 mRNA expression in high-density 2D and HG cultures was comparable to human liver tissue or even higher (Fig. 4B).", "Instead, high-density NFC cultures expressed less CYP3A4 mRNA than the human liver.", "Expression of the efflux transporters, MDR1 and MRP2, was higher in all the HepaRG cultures (both 2D and 3D cultures) than in the human liver tissue (Fig. 4).", "In low-density cultures, HG hydrogels increased the expression of MDR1 and MRP2 efflux transporter transcripts.", "In the case of high-density cultures, the highest expression of efflux transporters was seen in the 2D cultures.", "CYP3A4 enzyme activity of the HepaRG cells was higher in the NFC embedded cells than in the HG and 2D cultures (Fig. 5A).", "In the beginning of the culture the activity was equal in NFC and 2D cultures, but the activity of the NFC cultures increased in average 4-fold during the two weeks of culture while the activity of 2D cultures remained at the same level.", "The 4-fold increase took place also in HG hydrogel but the activity in the HG cultures was very low.", "CYP activity of differentiated cells (Fig. 5B) was higher in the 2D environment than in the hydrogels, and the activity was circa 100-fold higher in NFC than in HG cultures.", "CYP3A4 activity was inducible in HepaRG cells cultured in NFC and HG hydrogels and 2D environment (Supplement Fig. 1).", "The relative levels of enzyme induction were similar in the 3D hydrogel-based cell cultures and in the 2D cell cultures.", "The prototypical CYP3A4 inducers rifampin and phenobarbital and non-specific gene inducer DMSO induced the CYP activity, but dexamethasone did not.", "The characteristic markers of the canalicular plasma membrane were studied by staining the F-actin and the canalicular resident membrane transporters, MRP2 and MDR1 (Fig. 6).", "The accumulation of F-actin and the localization of MRP2 and MDR1 on the canalicular membrane were seen in all low-density HepaRG cultures (Fig. 6A).", "All spheroids in the NFC or HG hydrogels showed either several canalicular structures (Fig. 6A) or the canaliculi that stretched through the spheroid (Supplementary Fig. 2).", "Again, in the 2D cultures, the stained canalicular structures were located in the hepatocyte phenotype areas.", "Canalicular structures were also formed in the high-density cultures (Fig. 6B).", "MRP2 was localized at canalicular membranes in all high-density cultures, but MDR1 localization was not detected in the high-density spheroids.", "Accumulation of fluorescein into the round vesicle-like compartments between adjacent cells was evident in the 3D low-density spheroid cultures and 2D cell cultures (Fig. 7A, Supplementary Animation 1).", "In the standard 2D cultures, fluorescence accumulated in the areas that show hepatocyte-like phenotype.", "Flat biliary-like epithelial cells remained green longer time than the granular hepatocyte-like cells and they did not form vesicle structures.", "High-density cultures presented less vesicle structures than low-density cultures (Fig. 7B).", "Efflux of fluorescein was not seen in the single cells, outside of the spheroids.", "These single cells remained green in the NFC cultures, demonstrating that the efflux transport was associated with the formation of intercellular vesicle compartments (Fig. 7B).", "The following are the Supplementary data related to this article:", "The majority of the HepaRG spheroids is highly polarized and expresses intercellular bile canaliculi-like structures (green).", "3D-animation shows the z-stacks throughout the whole thickness of a HepaRG cell culture in hyaluronan-gelatin hydrogel.", "Cells were cultured in the hydrogel for seven days, followed by incubation with fluorescein diacetate and Draq5™ nuclear stain (red).", "Fluorescein diacetate that enters the HepaRG cells, is cleaved by intracellular esterases to fluorescein (green), and is excreted into the intercellular bile canaliculi-like structures by hepatobiliary transporters.", "Accumulation of fluorescein (green) in canalicular vesicles demonstrates formation of bile canaliculi-like structures, and vectorial function of MRP2 hepatobiliary transporter.", "Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2014.03.020", "Canalicular domain activity was further confirmed with calcein, a MRP2 substrate [34].", "This was evident in the live/dead analyses of the cultures.", "Green calcein staining is normally retained in the cytoplasm of live cells but in this study we saw time-dependent accumulation of calcein in the intercellular canalicular vesicles (Supplementary Fig. 3).", "After 20–30 min from calcein introduction to the cultures, the green color in the cytosols became lighter and the fluorescence in the vesicles emerged.", "The fluorescent vesicle structures were identified both in low-density and high-density cultures, in both 3D hydrogel and 2D cultures.", "The data (Supplementary Fig. 3) suggests that there are more bile canaliculi-like structures in HG than in NFC cultures.", "New organotypic liver cell culture systems are developed with different combinations of tissue engineering techniques, biomaterials and cell types [35].", "Continuous cell lines are more practical than primary cells in drug and chemical testing, because they can be frozen and cultured more conveniently.", "Among continuous cell lines, HepaRG more closely resembles human hepatocytes than HepG2 and many other liver cell lines [6,36], and have been successfully used to predict hepatic pharmacokinetics in humans [6–8] and to analyze toxicity of chemicals [4,5].", "However, HepaRG has never been cultured in 3D format in biomaterials.", "Prior 3D cultures of HepaRG have been generated using bioreactor [8] and hanging-drop technique [37].", "Herein, the plasticity of HepaRG liver cells was exploited to evaluate the impact of naturally derived hydrogels, native nanofibrillar cellulose (NFC) and hyaluronan-gelatin (HG), on the phenotype of both undifferentiated HepaRG progenitors and differentiated HepaRG cells.", "Wood-derived NFC is a promising new material for 3D cell culture.", "In our previous study [12], NFC and HG were the most promising biomaterials for 3D cell culture and, therefore, they were chosen to this study.", "HepaRG cells formed spheroids in both NFC and HG hydrogels.", "The spheroids of low-density progenitor cells had smooth surfaces suggesting that they are better organized than the spheroids of high-density cell cultures.", "The spheroids were smaller than the HepaRG spheroids that were previously generated in hanging-drops [37].", "The high cell viability in hydrogels is in line with previous publications on cell studies in fibrillated cellulose [14,38] and hyaluronan-gelatin gels [21].", "The decreased proliferation is typical for the cells in 3D hydrogels, and it may be due to the effects of the mechanical strength and hydrogel chemistry on the cell phenotype [39,40].", "Interestingly, HG hydrogel hosts similar biochemical signals than the ECM in the liver but NFC is a polysaccharide from plants without any human ECM components.", "Also the internal structure of these hydrogels is different: HG is a strong hydrogel with covalent cross-links, while the NFC hydrogels are physically cross-linked by hydrogen bonds between nanofiber flocs [41].", "The viscoelastic properties of NFC and HG hydrogels resemble each other: the shear elastic modulus of 0.4% HG hydrogel is 70–95 Pa [22] while the storage modulus G′ of 1% NFC hydrogel is close to 100 Pa [42].", "Not surprisingly, the cell proliferation was not seen in the high-density cell cultures.", "The high cell number and the hydrogel environment limit the proliferation [24,39,40].", "RT-PCR assay demonstrates that HepaRG differentiates in the NFC and HG hydrogels since the mRNA levels of CYP3A4 and ALB increased during the two weeks of culture.", "The elevated HNF4A, ALB, CYP3A4, MDR1 and MRP2 expression in 3D HG cultures suggests more prominent hepatocyte-like phenotype compared to the 2D cultures.", "NFC hydrogels induced the expression of CYP3A4 and ALB but also the upregulation of KRT19 transcript was seen.", "Interestingly, CYP3A4 expression was remarkably higher in low-density 3D hydrogel cultures than in the 2D cultures, and was nearly the level of human liver.", "The expression of CYP3A4 mRNA was not even detectable during the first week of the 2D cultures that is in line with the previous data [30].", "RT-PCR data on HNF4A implies that the differentiation level of differentiated high-density HepaRG cell cultures decreased after seeding in the hydrogels.", "The loss of the hepatocyte differentiation markers was milder in the HG than in the NFC cultures.", "The mRNA expression of CYP3A4 in high-density 2D and HG cell cultures was comparable to the human liver, whereas in NFC the expression was clearly lower.", "The expression of KRT19 transcript was higher in HepaRG cells, both in 2D and 3D hydrogel cultures, than in the human liver.", "This shows the bipotent character of HepaRG and indicates increased presence of cholangiocyte-like cells in the HepaRG cultures compared to the human liver.", "The higher CYP3A4 activity in the hydrogel cultures than in the standard 2D cultures shows that both NFC and HG hydrogels expedite the differentiation of HepaRG progenitor cells.", "We propose that the hydrogel microenvironments inhibit the proliferative phase [39,40], but the hydrogel culture accelerates cell differentiation possibly by facilitation the cell–cell contacts.", "On the contrary, the high-density cell cultures with differentiated HepaRG cultures showed higher CYP3A4 activity in 2D environment than in 3D NFC and HG hydrogels.", "Nevertheless, the CYP activity of high cell density NFC cultures was near the activity of high cell density 2D cultures and reached the level of low cell density NFC cultures.", "The study on CYP3A4 inducibility demonstrated that induction was in the same range in both the hydrogel and 2D cultures.", "Thus, 3D culturing in NFC and HG hydrogels did not violate the sensitivity of HepaRG cells to the prototypical CYP inducers.", "The induction of luciferin-IPA metabolism by rifampicin and phenobarbital was consistent with the induction measured earlier in HepaRG cultures [4,8,43].", "In this study, dexamethasone induction of CYP3A4 activity was detected neither in 2D nor 3D hydrogel HepaRG cultures, but earlier study with 2D HepaRG culture has reported 2-fold induction at mRNA when treated with dexamethasone [7].", "Although CYP activity of HG cultures was low the CYP3A4 induction was in same range as seen in 2D and NFC cultures.", "In low-density NFC and 2D cultures, the expression of MDR1 and MRP2 transcripts were in the same range, whereas the expression was higher in the HG cultures than in the NFC and 2D cultures.", "On the contrary, high-density 2D cultures expressed transporters at higher level than the NFC and HG cultures.", "Altogether, the expression of MDR1 transcript was higher in HepaRG cells, both in 2D and hydrogel cultures, than in the human liver, which is in agreement with the previous findings [36,44].", "The expression of another efflux transporter, MRP2, was either at same level or higher than in the human liver, which agrees with a previous study [36] but contrasts with some other findings [5,44].", "The canalicular accumulation of F-actin in the spheroids of both low-density and high-density cultures indicates in vivo-like polarity and formation of bile canaliculus.", "Bile canaliculus formation was confirmed in the expression and functional studies.", "The canalicular localization of MRP2 and MDR1 resembles the situation in vivo and suggests that the cells have hepatocyte-like phenotype in the spheroids.", "In the 2D cultures, MRP2 and MDR1 were located solely in the granular hepatocyte-like cells as previously published [8,37].", "Transport of fluorescein and calcein into the canalicular vesicles suggests that the MRP2 is functionally active in the bile canalicular structures of HepaRG spheroids.", "Fluorescein is a substrate for canalicular efflux transporter MRP2 [32,33] and bile salt export pump (BSEP) [45], whereas calcein is a substrate for MRP2 [34], but not for the other liver canalicular efflux transporters MDR1 [46] and BCRP [47].", "Previously, MRP2 activity in HepaRG cells has been discovered with fluorescein, 5 (and 6)-carboxy-2′,7′-dichlorofluorescein, and glutathione-methylfluorescein [5,24,37].", "Both infrequent bile canaliculi structures and lack of MDR1 staining in high-density spheroids suggest that the differentiated cells are less polarized than the low-density spheroids.", "Table 2 summarizes the results of this study.", "Compared to high-density cultures of pre-differentiated cells, the low-density cultures result in improved organotypic cell differentiation.", "Among low-density cultures, the 3D format typically results in improved albumin expression and activity and expression of CYP3A4 and efflux transporters.", "Overall, NFC and HG showed similar performance as 3D matrices for hepatic progenitor differentiation.", "This study proves that culturing HepaRG cells in hydrogels allows formation of 3D structures toward more organotypic liver tissue model.", "The improved CYP3A4 activity together with the apicobasal polarity and efflux transporter activity makes HepaRG spheroids interesting for drug and chemical testing.", "The 3D HepaRG spheroids may be of great value in assessing drug excretion to the bile, efflux transporter-mediated drug–drug interactions and toxicity of chemical compounds.", "The current 2D hepatocyte cultures do not reach the apicobasal polarity and only the sandwich-cultured hepatocytes are appropriate for the prediction of in vivo bile excretion [1,2].", "Improved cell culture models of hepatocytes are needed in the drug discovery, drug development, and chemical testing.", "Biomaterial (wood-derived nanofibrillar cellulose and hyaluronan – gelatin) hydrogels induced differentiation of continuous HepaRG progenitor cell line to organotypic 3D spheroids with bile duct compartment in the core.", "The spheroids showed expression of hepatocyte markers, metabolic activity and vectorial molecular transport towards bile duct compartment.", "Wood-derived nanofibrillar cellulose and hyaluronan-gelatin hydrogels are powerful matrices for 3D hepatocyte spheroid formation.", "Generation of organotypic hepatocyte cultures from continuous progenitor cell line paves way to improved hepatocyte cultures for drug and chemical testing." ]

[]

[ "MEDI", "PHAR" ]

[ "Chronic obstructive pulmonary disease (COPD)", "Comorbidities", "Pulmonary rehabilitation", "Skeletal muscle", "Therapeutic strategies", "Wasting" ]

[ "2736", "3004" ]

[ "Chronic obstructive pulmonary disease (COPD) is a progressive lung disease caused by excessive inflammation leading to irreversible damage to the airways and lung tissue.", "Symptoms include cough and shortness of breath, relating to underlying small airways disease with or without chronic bronchitis, and emphysema (an enlargement of the airspaces caused by destruction of the alveoli).", "COPD is a major global health burden and is currently the 3rd leading cause of death worldwide (Lozano et al., 2012).", "A major cause of COPD is cigarette smoking; however, COPD may also occur in non-smokers.", "Other important risk factors for COPD include inhalation of noxious substances such as particulate matter in wood smoke, air pollution or dust (Salvi & Barnes, 2009), asthma, and genetic factors.", "The severity of airflow limitation in COPD is graded based on spirometric measurements of lung function according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria, with the mildest airflow limitation classed as GOLD 1, progressing to the most severe airflow limitation at GOLD 4 (GOLD, 2016).", "COPD is increasingly being recognized as a systemic disease, and additional metrics such as body mass index (BMI), degree of shortness of breath (dyspnea), and physical capacity (6-minute walk distance, 6MWD) are also predictive of patient outcomes (Celli et al., 2015).", "The pulmonary pathology of COPD arises due to remodeling and narrowing of the airways, and damage to the lung parenchyma that leads to alveolar damage and emphysema (Barnes, 2014).", "Development and progression of the disease is driven by chronic inflammation and oxidative stress within the lungs, initiated in response to inhalation of noxious substances.", "Inflammation is perpetuated by immune cells such as neutrophils and macrophages recruited to the lungs as part of the inflammatory response.", "Once the process is initiated, the disease progresses due to persistent inflammation and the production of oxidants from endogenous cellular sources, even in the absence of external stimuli such as cigarette smoke.", "In addition to chronic inflammation and oxidative stress in stable COPD, patients are also more susceptible to respiratory infections, which are the predominant cause of acute exacerbations of COPD (AECOPD).", "During an exacerbation, patients experience a worsening of their symptoms, including increased sputum production and dyspnea, necessitating a change to their regular medication and often requiring hospitalization for additional treatment.", "Acute exacerbations can accelerate the decline in lung function in COPD patients, through the generation of persistent inflammation within the lungs (Wedzicha et al., 2014).", "The pathology of COPD extends beyond the lung environment, giving rise to systemic effects and the development of a number of comorbidities including skeletal muscle wasting, cardiovascular disease, and osteoporosis (Barnes & Celli, 2009).", "Skeletal muscle wasting and dysfunction is experienced by up to 40% of COPD patients (Schols et al., 2005; Sergi et al., 2006; Vestbo et al., 2006) and loss of muscle mass is commonly assessed by measuring the patients' fat-free mass index (FFMI).", "Some studies have shown that the incidence of patients with a low FFMI increases with disease severity, reaching 50% in patients with GOLD stage 4 COPD (Vestbo et al., 2006), reflecting an association between muscle mass and the severity of the lung pathology.", "Muscle dysfunction is, however, observed across the full spectrum of COPD severities—an important observation suggesting that muscle is affected from an early stage in the progression of COPD and could be effectively targeted therapeutically before it becomes a debilitating condition.", "COPD develops slowly but inexorably with most COPD patients being diagnosed at age 45 or older.", "While it is normal for people to experience a decline in muscle mass with increasing age, COPD patients in one study experienced a decline in muscle mass of up to 4.3% per year (Hopkinson et al., 2007), approximately double the rate of decline associated with ageing (Frontera et al., 2000).", "The clinical impact of muscle wasting in COPD patients is significant, resulting in not only reduction in quality of life, independence, and exercise capacity, but also overall survival.", "Patients with severe COPD who have reduced mid-thigh cross-sectional area (less than 70cm2) have an approximately 4 times higher odds ratio for mortality than similar patients with a similar degree of airflow limitation but with preserved muscle size (Marquis et al., 2002).", "Low FFMI and reduced quadriceps strength have been identified as predictors of COPD mortality, independent of lung function decline (Schols et al., 2005; Swallow et al., 2007), highlighting the importance of muscle mass and function in the overall pathology of COPD.", "Concomitant with a reduction in muscle mass is a loss of strength in the limb muscles.", "Interestingly, it appears that the lower limbs are more susceptible to wasting than the upper limb muscles (Caron et al., 2009), a feature that is similar to the pattern of wasting seen in disuse.", "While this suggests that disuse plays a significant role in wasting in COPD, it is likely that other mechanisms also contribute and therapies could be combined with exercise training to yield overall improvements in muscle strength and function.", "The maximum voluntary contraction of the quadriceps muscles is reduced by as much as 40% in COPD patients (Bernard et al., 1998; Plant et al., 2010; Seymour et al., 2010).", "Some studies have demonstrated an increase in the prevalence of quadriceps weakness with increasing COPD severity (Kharbanda et al., 2015) and a positive correlation between mid-thigh cross-sectional area and severity of airflow limitation (Bernard et al., 1998), indicating a link between the severity of the lung pathology and the extent of muscle impairment.", "However, significant muscle weakness has also been detected in patients with mild COPD (GOLD stages 1–2) or mild dyspnea (Seymour et al., 2010) and even in smokers without COPD compared to healthy controls (Seymour et al., 2010; Kok et al., 2012), suggesting that skeletal muscle dysfunction may occur at an early stage even prior to the onset of respiratory symptoms.", "In addition to loss of muscle mass and strength, phenotypic changes also occur in the muscle of COPD patients.", "A shift in the fiber types has been observed, with an increase in the proportion of fast glycolytic Type II fibers and a reduction in slow, oxidative Type I fibers (Jobin et al., 1998; Whittom et al., 1998; Debigare et al., 2003; Gosker et al., 2007; Vogiatzis et al., 2011).", "Skeletal muscle mass is adversely affected both during and following an acute exacerbation; quadriceps strength declines in hospitalized patients during an AECOPD and only partially recovers even up to 90 days following an exacerbation in the absence of interventions such as pulmonary rehabilitation (Spruit et al., 2003).", "More frequent exacerbations are associated with a more rapid decline in strength (Ansari et al., 2012), further suggesting a link between AECOPD and muscle health.", "Weight loss and weakness have long been observed in patients with COPD, but it is only in recent decades that we have begun to understand at the molecular level the processes contributing to wasting.", "To date, therapeutic options to restore muscle mass have been limited.", "In 2006, we reviewed the development of therapies for muscle wasting in COPD (Hansen et al., 2006).", "Here we provide an update on the current state of therapy targeting COPD-associated muscle dysfunction, discussing some of the mechanisms of muscle wasting as well as the advancement of therapeutic options in the last decade.", "We also discuss emerging therapies in development and clinical trials to treat muscle wasting in other conditions such as cancer cachexia and sarcopenia that share some similarities with the wasting observed in COPD, as these may be of relevance for the treatment of COPD patients to restore muscle mass and function.", "Broadly speaking, overall muscle mass is regulated by the balance between protein synthesis and protein degradation, with additional contributions from regenerative processes and satellite cells.", "In cases of atrophy in response to disuse, immobilization, or pathological conditions such as cancer cachexia, the balance shifts toward an increase in protein degradation and a decrease in protein synthesis.", "The overall aim in developing therapeutic approaches to tackle muscle wasting is to push the balance in the opposite direction, increasing protein synthesis and decreasing protein breakdown to lead to an overall increase in muscle mass.", "The mechanisms and signaling pathways regulating muscle anabolic and catabolic processes are complex (Fig. 1).", "These intracellular pathways are controlled by a wide range of extracellular factors including inflammatory mediators, oxidative stress, circulating hormones, nutritional and exercise factors.", "In addition, chronic use of anti-inflammatory corticosteroids such as prednisolone is known to cause muscle weakness and atrophy, through their effects on various signaling pathways regulating muscle mass (Decramer et al., 1994).", "The major pathway leading to muscle growth (hypertrophy) is through the insulin-like growth factor (IGF-1)/Akt/mTOR pathway, which promotes protein synthesis through stimulation of protein translation via activation of positive regulators of translation (p70S6 kinase) and inhibition of the negative regulator 4E-BP1 (Glass, 2003).", "Akt signaling also inhibits the FoxO transcription factors, leading to inhibition of the ubiquitin proteasome pathway and decreasing muscle proteolysis (Stitt et al., 2004).", "Muscle mass can also be increased through regenerative pathways following the activation of the muscle-resident stem cells, called satellite cells (Charge & Rudnicki, 2004).", "Opposing the hypertrophy pathway are mechanisms that lead to atrophy.", "Muscle atrophy is largely driven by increased proteolytic breakdown of muscle proteins via the ubiquitin–proteasome system.", "Proteins are targeted for degradation by the proteasome by the covalent attachment of multiple ubiquitin molecules by ubiquitin-ligase proteins.", "A number of muscle-specific ubiquitin ligases are expressed in skeletal muscle, including atrogin-1/Fbxo32 and muscle ring finger 1 (MuRF-1/Trim63), and expression of these proteins is often elevated in conditions of muscle atrophy (Bodine et al., 2001; Gomes et al., 2001; Bodine & Baehr, 2014; Yuan et al., 2015), reflecting the central role of the proteasome in regulating protein catabolism in atrophic states.", "An additional pathway of protein breakdown is the autophagy pathway that is responsible for degradation of organelles and other cellular constituents through the lysosomal degradation process (Hussain & Sandri, 2013).", "Alterations in many of these pathways have been characterized in the muscle of COPD patients and are associated with loss of muscle mass in these patients.", "Increased expression of atrogenes – genes that encode proteins involved in muscle atrophy pathways – has been measured in COPD.", "The muscle-specific E3 ligases atrogin-1 and MuRF-1 are upregulated in COPD muscles (Doucet et al., 2007; Plant et al., 2010), suggesting an upregulation of the Ub-proteasome system and increased proteolysis.", "Indeed, increased muscle protein breakdown occurs in cachectic COPD patients compared to COPD patients with normal FFM and healthy controls (Rutten et al., 2006).", "There is little information on the contribution of the autophagy pathway to muscle protein breakdown; however, Plant et al. detected no change in the levels of autophagy proteins beclin-1 and LC3, suggesting that autophagy was not upregulated in the muscle from COPD patients in that cohort (Plant et al., 2010).", "While reduced IGF-1 expression has been observed in some COPD patients (Plant et al., 2010), consistent with a decrease in protein synthesis, others have reported upregulated phosphorylated Akt and other components of the Akt/mTOR pathway in muscle from COPD patients (Doucet et al., 2007).", "It has been suggested that in the presence of strong atrophy signaling, the Akt pathway, may be upregulated in an effort to normalize muscle mass (Doucet et al., 2007); however, the mechanisms underlying these findings are yet to be fully explored.", "Satellite cells in the muscle of COPD patients show evidence of senescence (Theriault et al., 2012), and COPD patients exhibit reduced muscle regenerative capacity (Theriault et al., 2014).", "Isolated satellite cells from COPD patients show reduced myotube diameter and increased atrophy signaling and oxidative stress in vitro (Pomies et al., 2015), suggesting an inherent alteration in the satellite cells themselves could be contributing to the reduced regenerative ability in COPD patients.", "The exact role of satellite cells and their ability to contribute to muscle regeneration in COPD is not yet fully understood and further investigation into this area is needed.", "The link between inflammation and skeletal muscle health is well established.", "A number of inflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-6, are known to induce muscle atrophy in animal models and healthy human volunteers (Goodman, 1991, 1994; Haddad et al., 2005; Bach et al., 2013; Zhou et al., 2016).", "Pro-inflammatory mediators including TNF-α, IL-6 and C-reactive protein (CRP) are elevated in COPD patients (Yende et al., 2006; Vogiatzis et al., 2007) and are associated with reduced muscle function.", "Elevated IL-6 levels have been associated with reduced lung function and increased muscle wasting in COPD patients compared to healthy elderly controls (Yende et al., 2006).", "TNF-α is a key cytokine in the regulation of muscle mass signaling via a number of pathways to reduce muscle protein content and satellite cell differentiation.", "Through activation of the transcription factor NF-κB, TNF-α induces expression of MuRF-1 and increases activity of the ubiquitin–proteasome pathway leading to muscle protein degradation (Llovera et al., 1997).", "TNF-α-induced activation of the p38 mitogen-activated protein kinase (MAPK) pathway stimulates expression of atrogin-1 (Li et al., 2005), and this is proposed to be through the transcription factor FoxO4 (Moylan et al., 2008).", "In addition, TNF-α reduces muscle regeneration via inhibition of the myogenic regulatory factor MyoD (Guttridge et al., 2000; Langen et al., 2001).", "Given the established effects of TNF-α in muscle atrophy, and the association between elevated TNF-α levels and reduced muscle strength in COPD, targeting this cytokine therapeutically is an attractive prospect.", "This notion is supported by evidence from mouse cancer models, where inhibiting TNF-α signaling through administration of a soluble TNF-α receptor resulted in both increased bodyweight and increased appetite in tumor-bearing mice (Torelli et al., 1999).", "Despite the important role of TNF-α in mediating muscle wasting and encouraging preclinical data supporting the use of anti-TNF-α agents for muscle wasting, the results of clinical trials for both muscle wasting in a range of conditions and the lung pathology of COPD have been somewhat disappointing (See Table 1).", "Treatment with the anti-TNF-α agent etanercept in cancer patients showed no significant improvement in bodyweight compared to those treated with the placebo (Jatoi et al., 2007).", "Similar results were seen in a later trial where patients were treated with the anti-TNF-α monoclonal antibody infliximab, and patients reported increased fatigue and an overall reduction in quality of life following infliximab treatment (Jatoi et al., 2010).", "In this study, no patients reached the primary endpoint of 10% or greater weight gain, and the trial was terminated early due to the lack of efficacy (Jatoi et al., 2010).", "It is worthwhile noting that while cancer cachexia and the wasting associated with COPD may be initiated by differing pathological conditions, the underlying molecular changes and mechanisms have been shown to be similar.", "For example, similar alterations in levels of oxidative stress and systemic inflammation, signaling pathways and muscle architecture have been observed in the muscles of patients with both lung cancer and COPD (Puig-Vilanova et al., 2015).", "Therapies targeting cachexia in cancer may prove to be of value to treat muscle wasting in COPD.", "There has been considerable interest in targeting TNF-α in COPD as preclinical studies using TNF knockout mice display a marked reduction in lung inflammation and emphysema in experimental cigarette smoke models (Churg et al., 2004).", "However, a clinical study using anti-TNF therapy (Infliximab) failed to improve outcomes for COPD patients including symptom score, lung function, exercise capacity, dyspnea score, health status, and rate of acute exacerbations (Rennard et al., 2007).", "Anti-TNF-α therapies have been similarly ineffective at reducing local and systemic inflammation in COPD patients (Loza et al., 2012) and to date have not resulted in any overall clinical benefit in terms of the lung pathology (van der Vaart et al., 2005; Rennard et al., 2007).", "The reasons for this are unclear; however, there are important features that contribute to the differential response to blocking the function of TNF-α in preclinical experimental models versus human clinical trials.", "It is well established that the cigarette smoke exposure mouse model represents a mild stage of COPD where there is modest airspace enlargement and airway remodeling.", "In these models, therapies are either applied prophylactically to prevent development of disease or the gene of interest is knocked out prior to exposure.", "This is in contrast to the clinical scenario, where anti-TNF trials have been performed on COPD patients with severe or very severe COPD.", "Hence, therapies would need to reverse established disease pathology including muscle wasting in order for the patient to obtain benefit from the treatment.", "An alternative approach would be to perform clinical trials on early-stage or mild COPD patients and determine whether anti-TNF-α therapy prevents or slows progression of disease as seen in experimental models; however, such an approach is cost prohibitive at this stage and chronic administration of TNF-α blockers have been linked to an increase risk of clinically diagnosed pneumonia and lung malignancies (Rennard et al., 2007).", "It is increasingly recognized that therapies will continue to fail in large COPD trials that include a broad spectrum of clinical and molecular phenotypes, which fall within a defined group based on lung function.", "Novel therapies targeted toward improving muscle function in advanced COPD patients should first be assessed for their ability to reverse established or severe disease in novel preclinical models.", "A better approach would be to prevent progression of disease and muscle wasting, where preclinical models can be informative; however, the application of long-term trials in early-stage disease remains a challenging prospect for industry.", "Another important limitation of targeting TNF-α is the multifactorial and multi-cytokine nature of COPD and skeletal muscle wasting; it is likely that the inhibition of a single cytokine is not sufficient to overcome the complex wasting pathways and disease pathology and lead to detectable clinical improvement.", "While not currently used for treatment of muscle wasting in COPD, anti-IL-6 therapies have shown promise in the treatment of cancer cachexia.", "The humanized anti-IL-6 monoclonal antibody ALD518 has a high affinity for binding IL-6, and clinical trials in non-small-cell lung cancer have yielded some positive results, including a significant reduction in the proportion of patients losing more than 5% lean body mass as well as improvements in overall scores of fatigue (Bayliss et al., 2011).", "Tocilizumab, an IL-6 receptor antibody, has been reported to improve cachexia in two patients (Ando et al., 2013) and also in a mouse cachexia model (Ando et al., 2014), but has yet to be tested on a larger scale in human studies.", "Similarly to other cytokines, signaling through the IL-1 receptor stimulates muscle atrophy via upregulation of atrogene expression and inhibition of IGF-1 signaling (Benbassat et al., 1999; Li et al., 2009).", "Both IL-1β, and more recently, IL-1α, have been shown to be elevated in the lungs of COPD patients compared to never smokers (Botelho et al., 2011; Pauwels et al., 2011), suggesting a role for these cytokines in the pathology of COPD.", "Initial clinical trials of MABp1, a monoclonal anti-IL-1α antibody, demonstrated an increase in lean body mass in 70% of MABp1-treated cancer patients, along with improvements in fatigue and general quality of life scores (Hong et al., 2014).", "Although the trial was small with only 30 patients being assessed for body mass by dual energy X-ray absorptiometry (DEXA) scan, it is of interest that the patients who experienced a gain in lean body mass also saw a reduction in fat mass, suggesting a role for IL-1α in regulation of metabolism and body composition.", "Such an effect could be of benefit in COPD patients, where body composition is often altered such that patients display increased abdominal adiposity while simultaneously losing muscle mass in their limbs (Schols et al., 2005).", "During an exacerbation, inflammation is elevated above the levels seen in stable COPD patients.", "In addition, the acute phase protein Serum Amyloid A (SAA) rises dramatically, up to 1000-fold, and SAA has been identified as a biomarker for the severity of the exacerbation (Bozinovski et al., 2008).", "We have recently shown that SAA has direct effects on muscle cells, inducing both the expression of pro-inflammatory cytokines and an atrophy response (Passey et al., 2016).", "These findings suggest that acute phase proteins elevated in COPD or during an exacerbation could be contributing to the decline in muscle function observed during and following an exacerbation.", "The regulation of SAA in the circulation is complex, and SAA is known to interact with a number of cell surface receptors.", "As yet there are no therapeutics targeting SAA-mediated inflammation in the clinic; however, approaches aimed at promoting the resolution of inflammation through the administration of mediators that oppose the actions of SAA could hold promise (Bozinovski et al., 2014).", "Oxidative stress is emerging as an important contributor to both the lung and systemic pathologies of COPD.", "Recent detailed reviews describe the mechanisms by which oxidative stress contributes to COPD pathology in the lung (Rahman & Kinnula, 2012) and to the development of systemic comorbidities (Bernardo et al., 2015), and readers are directed to these extensive reviews for more detailed information on this topic.", "Oxidative stress and damage occurs when there is an imbalance between the levels of oxidants and antioxidants, leading to an excess of highly reactive oxygen species (ROS) including superoxide, hydroxyl radicals and hydrogen peroxide, and reactive nitrogen species (RNS) such as nitric oxide and peroxynitrite (Bernardo et al., 2015).", "This imbalance can arise due to reduced antioxidant defenses or increased oxidant production.", "In the context of COPD, there is a high oxidant burden within the lungs arising both as a result of the oxidants found within cigarette smoke and also due to endogenous oxidants produced by leukocytes recruited to the lung as part of the inflammatory response (Rahman & Kinnula, 2012).", "Oxidative stress is also acutely elevated following exercise in COPD patients (Van Helvoort et al., 2006; Fisher-Wellman et al., 2009; Mercken et al., 2009).", "In healthy populations, such acute increases act as a stimulus for increased antioxidant defenses, resulting in a lower overall oxidative stress over time.", "These responses appear to be partially blunted in COPD patients with low muscle mass (Fisher-Wellman et al., 2009).", "There is an increased concentration of hydrogen peroxide (H2O2) in exhaled breath condensate of COPD patients and this is further elevated during AECOPD (Dekhuijzen et al., 1996; Nowak et al., 1996).", "Increased levels of the oxidation product malondialdehyde (MDA) are present in both the sputum and plasma of COPD patients (Zeng et al., 2013), and increased lipid peroxidation and protein carbonylation has also been measured in plasma from COPD patients compared to healthy controls (Santos et al., 2004; Nadeem et al., 2005), indicating an increase in systemic oxidative stress in COPD.", "The level of local lung oxidative stress correlates with systemic (plasma) levels of oxidative stress (Zeng et al., 2013), lending support to the ‘spillover’ hypothesis that oxidants and inflammation within the lungs can extend into the systemic circulation leading to the development of systemic comorbidities of COPD.", "Antioxidant defenses including superoxide dismutase (SOD), glutathione peroxidase (Gpx) and glutathione levels have been found to be decreased in COPD and AECOPD patients compared to smokers and healthy controls (Nadeem et al., 2005; Zeng et al., 2013), further indicating that an imbalance in antioxidant/oxidant status exists in patients with COPD.", "In addition to oxidative damage in the lungs and plasma, total protein carbonylation is elevated in the vastus lateralis muscle of COPD patients compared to healthy controls (Barreiro et al., 2008), and levels of protein carbonylation correlate inversely with quadriceps strength (Barreiro et al., 2010).", "MDA-protein adducts and tyrosine nitration is also increased in muscle from COPD patients, and in cigarette smoke-exposed rodent models (Barreiro et al., 2010).", "A number of abundant proteins with important functions in glycolysis, energy production and muscle contraction show increased levels of carbonylation in both COPD patients and in cigarette smoke-exposed guinea pigs compared to controls (Barreiro et al., 2010).", "Interestingly, oxidative changes have also been detected in muscle from non-COPD smokers and in animal models before the onset of overt lung pathology (Barreiro et al., 2010), indicating that cigarette smoke itself can exert an oxidative burden on muscle that could contribute to the development of muscle dysfunction at early stages of COPD development.", "The function of skeletal muscle is regulated in part by endogenous ROS and RNS, which are involved in processes such as contractile function, glucose uptake, and regenerative processes (Espinosa et al., 2016).", "ROS and RNS production increases during muscle contraction, and hence muscle has a high level of endogenous antioxidant defenses to prevent excess oxidative damage from free radicals generated during exercise and daily movement.", "Endogenous antioxidant defenses are often upregulated in the muscle of COPD patients (Gosker et al., 2005; Barreiro et al., 2009; Rodriguez et al., 2012) and the clinical benefit of antioxidant therapies to ameliorate muscle dysfunction in COPD is not yet clear.", "However, the elevated levels of oxidative damage observed in the muscle of COPD patients and the link between oxidative stress and muscle strength suggest that in the context of COPD, the levels of oxidative stress overwhelm the endogenous defenses.", "Thus, therapy aimed at reducing oxidative stress in muscle could be of benefit for improving the muscle pathology in COPD.", "Therapeutic strategies to target oxidative stress include the use of scavenging molecules to ‘mop up’ excess oxidants and also the administration of molecules that mimic or increase the activity of endogenous antioxidant proteins such as SOD and Gpx.", "Many antioxidant compounds are naturally occurring and can be obtained from dietary sources or through dietary supplementation, including vitamins E and C, carotenes, and resveratrol.", "Clinical trials of dietary antioxidants have yielded mixed results.", "Oral administration of vitamins E and C has been shown to be effective in reducing markers of oxidative stress but does not improve measures of fatigue in healthy people (Bentley et al., 2015) or COPD patients (Rossman et al., 2013).", "A recently completed trial (see ClinicalTrials.gov identifier: NCT01942889) investigated the effect of combined antioxidant (vitamins C and E, selenomethionine, and zinc gluconate) with pulmonary rehabilitation therapy to determine if dietary antioxidants could enhance the physiological effects of exercise training; however, the results are not yet published.", "Reseveratrol, a polyphenol found most commonly in red wine, was able to reduce some markers of oxidative stress in mouse models of sarcopenia and disuse atrophy (Jackson et al., 2010, 2011).", "Resveratrol treatment was not able to attenuate sarcopenia in ageing mice (Jackson et al., 2011); however, it did reduce disuse atrophy in a hindlimb suspension model (Jackson et al., 2010) and has recently shown some promising effects in preventing cancer cachexia in the mouse C26 colon cancer cachexia model (Shadfar et al., 2011).", "Currently, there are no published data on the effect of resveratrol on muscle mass in COPD; however, a trial is currently underway (see ClinicalTrials.gov identifier: NCT02245932) investigating the effects of resveratrol in COPD patients and includes muscle volume and body composition as secondary outcomes, so further information on resveratrol as a therapeutic option for muscle wasting in COPD may be available in coming years.", "Glutathione is the most abundant non-protein thiol in muscle cells and is a strong determinant of the intracellular redox status (Schafer & Buettner, 2001).", "N-acetyl cysteine (NAC) is a small molecule derivative of the amino acid cysteine and acts as a strong reducing agent (Rahman & Kinnula, 2012) that can act directly with ROS and RNS as well as supporting glutathione synthesis through acting as a cysteine donor.", "Initial results of trials in the use of NAC were mixed (Rahman & Kinnula, 2012); however, high doses of NAC have been shown to be effective in reducing mucus hypersecretion and the frequency of acute exacerbations of COPD, and NAC treatment is now included in the American College of Chest Physicians and the Canadian Thoracic Society guidelines for preventing acute exacerbations of COPD (Criner et al., 2015).", "It is also included in the recent COPD-X guidelines in Australia and New Zealand (Abramson et al., 2015).", "Increasing levels of glutathione through the administration of glutathione itself or NAC and other thiol donors improves measures of muscle fatigue and reduces oxidative stress both in animal models and in humans (Lands et al., 1999; Ferreira & Reid, 2008), including in COPD patients (Koechlin et al., 2004).", "Recently, a novel glutathione precursor, named FT061452 or F1, has shown some efficacy in reducing muscle loss in ageing mice (Sinha-Hikim et al., 2013).", "Treatment with F1 resulted in reduced lipid peroxidation, improved GSH/GSSG ratio, reduced IL-6, and increased phosphorylation of Akt suggesting increased protein synthesis signaling through the Akt/mTOR pathway (Sinha-Hikim et al., 2013).", "Data on this formulation are only available from preclinical animal models at this time, and to date, no clinical studies have been conducted.", "Preclinical studies using antioxidants in cigarette smoke-exposed mouse models have shown that the nox2 inhibitor apocynin is effective in reducing lung inflammation in influenza A-infected smoke-exposed mice (Vlahos et al., 2011; Oostwoud et al., 2016).", "Apocynin reduces the levels of hydrogen peroxide and nitric oxide in exhaled breath condensate of asthmatic patients (Stefanska et al., 2012), suggesting therapeutic potential for apocynin in the treatment of lung pathology in COPD patients.", "Daily administration of apocynin in a mouse model of chronic cigarette smoke exposure (8 weeks) resulted in improvements in both lung inflammation and muscle mass (Vlahos, R. unpublished observations), offering promise that the therapeutic targeting of oxidative enzymes could be a useful therapy for COPD and muscle wasting.", "Whether these findings will translate into clinical benefit in COPD patients warrants further investigation.", "Drugs that mimic the effect of endogenous antioxidant enzymes have been developed with the aim of raising the levels of antioxidant defenses and reducing oxidant burden.", "SOD mimetics can be classified into three major classes—macrocyclic ligands, manganese metalloporphyrins, and ‘salens’ (manganese-based SOD mimetics) (Rahman & Kinnula, 2012).", "While SOD mimetics have shown some promise in animal models of lung inflammation (Rahman & Kinnula, 2012), they have not yet been clinically tested for efficacy in COPD or muscle wasting contexts.", "Glutathione peroxidases are selenium-dependent antioxidant enzymes that catalyze the breakdown of reactive hydrogen peroxide to water and oxygen (Vlahos & Bozinovski, 2013).", "Levels of systemic Gpx have been shown to be depleted in COPD patients (Santos et al., 2004; Vibhuti et al., 2007).", "The Gpx mimetic ebselen reduces lung inflammation in response to cigarette smoke (Duong et al., 2010) and influenza infection (Yatmaz et al., 2013; Oostwoud et al., 2016) in mouse models, suggesting that increasing the levels of Gpx activity could be a potential therapeutic for COPD.", "The effects of Gpx mimetics on skeletal muscle are not well characterized, particularly in the context of COPD.", "To the best of our knowledge, no clinical trials have yet been reported for the use of ebselen to treat muscle wasting in COPD, cancer cachexia, or sarcopenia.", "The expression of many antioxidant enzymes, including Gpx, is regulated by signaling through the transcription factor Nrf2 (Rahman & Kinnula, 2012).", "Disruption of Nrf2 in skeletal muscle impacts on muscle cell differentiation and regeneration and increasing the activity of Nrf2 by administration of sulforaphane has shown some efficacy in mouse muscular dystrophy models (Sun et al., 2015, 2016).", "However, there are no published data on the expression and activity of Nrf2 in the muscles of COPD patients, and further investigation is warranted to fully characterize the antioxidant defenses and redox balance at the molecular level in muscle from COPD patients and preclinical COPD models.", "There are currently few therapeutic options to target oxidative stress in muscle wasting (Table 2), and the full effect of reducing the oxidant burden in COPD is not completely understood.", "Given the evidence of elevated oxidative burden and oxidative damage in lungs, systemic circulation and muscles of COPD and the known effects of oxidative damage on tissue function, further investigation into the area is important.", "(See Table 1.)", "The transforming growth factor (TGF)-β family member myostatin has been identified as a key negative regulator of muscle mass (McPherron et al., 1997), acting via both upregulation of atrophy genes and inhibition of Akt signaling to promote proteolysis and reduce protein synthesis.", "Deletion or mutation of the myostatin protein results in dramatically increased muscle growth in mice (McPherron et al., 1997), cattle (Grobet et al., 1997; McPherron & Lee, 1997), and humans (Schuelke et al., 2004).", "Myostatin is also known to have other effects on muscle physiology, including regulating muscle ROS production (Sriram et al., 2011) and also fiber type (Girgenrath et al., 2005).", "Myostatin signals through the activin receptor type IIB (ActRIIB), a receptor that is shared with other TGF-β family member proteins activin and GDF11, which leads to phosphorylation of the transcription factors Smad2/3.", "Signaling through the Smads 2/3 pathway regulates muscle balance through inhibition of Akt and consequent activation of FOXO transcription factors leading to upregulated expression of atrogenes, and through direct effects of Smad on gene expression.", "In addition, it is becoming increasingly recognized that the Smad pathway has extensive cross-talk with other signaling pathways such as the mitogen-activated protein kinase pathway, inducing other cellular effects that regulate muscle atrophy and hypertrophy.", "Consistent with its role in regulating muscle mass, myostatin expression is elevated in the vastus lateralis muscle from COPD patients compared to healthy controls (Man et al., 2010; Plant et al., 2010).", "Circulating myostatin protein is also elevated in serum from COPD patients and correlates with reduced muscle mass in male COPD patients (Ju & Chen, 2012).", "A number of molecules targeting the myostatin/ActRIIB pathway have been, or are currently being, tested for efficacy in muscle wasting, in both COPD and other cachectic conditions (Table 3).", "Given the dramatic effects on muscle mass arising from myostatin deletion or loss of function, inhibition of myostatin itself using neutralizing antibodies is an obvious therapeutic approach.", "This notion is supported by a number of in vivo studies that have used antibodies directed against myostatin to treat muscle wasting in mouse models of cancer, disuse atrophy, sarcopenia, and glucocorticoid-induced wasting (Siriett et al., 2007; Murphy et al., 2010, 2011; Latres et al., 2015; Smith et al., 2015).", "Preclinical studies using the monoclonal antimyostatin antibody LSN2478185 in naïve mice demonstrated an increase in muscle mass (Smith et al., 2015).", "Testing of the humanized derivative of this antibody, LY2495655, in the mouse C26 tumor model and the PC3 tumor model also demonstrated increased muscle mass and improvements in grip strength compared to the isotype control antibody (Smith et al., 2015).", "Interestingly, these antibodies also caused an increase in muscle mass even in the context of caloric restriction, a finding that may be of importance in the treatment of muscle wasting in patients with reduced appetite and calorie intake as is often the case in COPD.", "The humanized antimyostatin antibody LY2495655 (Landogrozumab) has since progressed to clinical trials and has demonstrated some promising, if modest, effects on muscle size in humans.", "In elderly patients with a history of falls, LY2495655 treatment resulted in a 2.5% increase in appendicular lean body mass after 24 weeks and showed a corresponding increase in total lean body mass and a reduction in fat mass (Becker et al., 2015).", "Treatment with LY2495655 also resulted in general increases in physical performance in tests of fast gait speed, stair climbing, and chair rising, although improvements in hand grip strength were not significant and no improvement was seen in isometric leg extension.", "Clinical trials for the use of LY2495655 to treat muscle atrophy in patients undergoing a total hip arthroplasty have revealed some efficacy in increasing muscle mass at 105 and 315 mg; however, the threshold of the primary objective of the trial was not reached (Woodhouse et al., 2016).", "The antibody has also been tested for use in cancer cachexia in patients with pancreatic cancer (see ClinicalTrials.gov identifier: NCT01505530), although results are not yet available on the outcome of this trial.", "REGN1033 is a fully humanized, antimyostatin monoclonal antibody developed by Regeneron, that shows high specificity for binding myostatin and no detectable binding of either GDF11 or activin (Latres et al., 2015).", "In vitro reporter gene assays confirmed that the antibody specifically blocks Smad phosphorylation and activation following myostatin treatment but not after treatment with activin, further confirming the specificity of the antibody against myostatin alone.", "In naïve mice, REGN1033 caused a significant 19–25% increase in the weight of the gasctrocnemius following 28 days of treatment, with a similar magnitude effect in the tibialis anterior.", "The increased muscle size was accompanied by an increase in the isometric force production of the tibialis anterior, with maximal tetanic force increasing by 16.7%, indicating that muscle functional performance is also improved following treatment.", "In models of disuse atrophy and glucocorticoid-induced atrophy, REGN1033 prevented loss of both muscle mass and function.", "Following these promising preclinical results, REGN1033 is now in clinical testing.", "Initial clinical trials have been completed for safety and bioeffect (see ClinicalTrials.gov identifier: NCT01910220) and more recently for safety and efficacy in patients with sarcopenia (see ClinicalTrials.gov identifier: NCT01963598).", "A slightly different approach to inhibition of myostatin has been developed in the form of AMG-745, a peptibody against myostatin.", "Peptibodies are fusion proteins consisting of an active peptide fused to the Fc portion of an antibody and offer a number of advantages over individual peptides and antibodies as therapeutic agents, including improved longevity in vivo (Shimamoto et al., 2012).", "AMG-745 contains a fusion protein with a human Fc at the N-terminus, and a C-terminal bioactive peptide that neutralizes myostatin activity (Padhi et al., 2014).", "Initial clinical trials have shown results similar to those seen with monoclonal antibodies; in patients receiving androgen deprivation therapy for prostate cancer, AMG-745 treatment resulted in a 2.2% increase in lean body mass compared to placebo, and an overall decrease in fat mass of around 2.5% at 1 month follow up after a 28 day treatment protocol (Padhi et al., 2014).", "Similar to the findings with LY2495655, no improvement in leg strength was seen following AMG-745 treatment, suggesting either that a greater level of myostatin inhibition is needed or that inhibiting myostatin alone is not sufficient to translate into functional improvement.", "In addition to myostatin, other TGF-β family members such as activin also exert effects on muscle mass via ActRIIB suggesting a certain level of redundancy within this pathway that requires important consideration for the development of therapeutics targeting myostatin and its signaling pathway.", "An alternative strategy to inhibiting or neutralizing myostatin itself is to develop receptor antagonists that prevent myostatin from binding to ActRIIB and initiating downstream signaling.", "This strategy has worked well in cell culture and mouse models, where the ActRIIB antibody Bimagrumab (BYM338) effectively prevented the atrophic effects of myostatin and activin on cultured myotubes and caused muscle hypertrophy in mice (Lach-Trifilieff et al., 2014).", "Bimagrumab also inhibited the muscle wasting in response to glucocorticoid treatment in mice, a finding that may be of particular interest in the context of COPD where many patients are treated with glucocorticoids that are known to negatively impact on muscle mass and health.", "Interestingly, Bimagrumab also increased muscle mass in a mouse carrying a loss of function mutation in the myostatin gene, suggesting additional ligands such as activin play important roles in the regulation of muscle mass.", "ActRIIB blockade with Bimagrumab was around twice as effective as treatment with the myostatin-specific inhibitor propeptide D76A, further emphasizing the overlap in function between different TGF-β family members.", "This indicates that blockade at the point of receptor signaling is likely a more effective strategy than targeting ActRIIB ligands individually.", "Together these findings provide ample support for further testing into possible clinical uses of Bimagrumab in muscle wasting conditions.", "Indeed clinical trials have recently been completed to test the efficacy of Bimagrumab in both sarcopenia (see ClinicalTrials.gov identifier: NCT01669174) and muscle wasting in COPD patients (see ClinicalTrials.gov identifier: NCT01601600), results have not yet been made available.", "Body composition in COPD patients is complex, and loss of muscle mass can occur with or without accompanying changes in fat mass, resulting in a population of patients with underlying muscle dysfunction but varying levels of underweight or obesity (Schols et al., 2005; Steuten et al., 2006).", "While higher body weight is generally associated with a better prognosis for COPD patients, excess body fat and obesity are associated with additional health risks.", "Anabolic therapies are aimed at increasing muscle mass through stimulation of protein synthesis and reduction of protein breakdown, while preferably avoiding excess fat gain, so promoting a favorable body composition to improve overall health.", "IGF-1 appears to be an attractive therapeutic target due to its positive effects on muscle hypertrophy; however, previous trials administering recombinant human IGF-1 have yielded mixed results in human patients with a range of muscle conditions including sarcopenia and cancer cachexia (Philippou & Barton, 2014).", "The biology of IGF-1 is complex—there are several isoforms of the protein and the protein is subject to post-translational processing—and several issues make therapeutic administration of IGF-1 problematic.", "The IGF-1 receptor is ubiquitously expressed and IGF-1 is a potent growth factor affecting cell proliferation and survival in many tissues, therefore IGF-1 administration could represent an increased cancer risk; a concern that has limited the doses and timeframes of IGF-1 administration in clinical trials to date (Philippou & Barton, 2014).", "Therapeutic methods aimed at increasing the local expression of IGF-1 within the muscle tissue may be more effective in promoting muscle hypertrophy and may help to avoid unwanted systemic effects.", "Gene transfer or virus-mediated expression of IGF-1 within muscle tissue has been shown to be effective in increasing IGF-1 and promoting muscle repair and growth in animal models (Barton-Davis et al., 1998; Schertzer & Lynch, 2006) but has yet to be transferred to human studies.", "Although there are currently some trials investigating the use of IGF-1 in the treatment of muscular dystrophy, at present, to the best of our knowledge, no trials are in progress for the use of IGF-1 to treat muscle wasting in COPD.", "A number of other anabolic hormones have been tested for efficacy in COPD patients, including testosterone, ghrelin, and growth hormone analogues and offer promise for future therapeutic use.", "Perhaps the most widely known anabolic compound is the anabolic hormone testosterone, which has potent anabolic activity in muscle cells along with a lipolytic effect resulting in increased muscle mass and reduced fat mass (Dubois et al., 2012).", "Testosterone signals both through the androgen receptor and also via crosstalk with a number of intracellular signaling pathways to regulate protein synthesis and satellite cell differentiation, leading to an increase in muscle mass (Dubois et al., 2012).", "Testosterone administration is known to increase the size of muscle fibers in a similar manner to that seen with resistance training.", "Testosterone itself must be administered by injection, or through the skin via a gel or patch; however, a number of testosterone analogues have been developed that can be taken orally.", "Testosterone analogues have shown some promising results in COPD, with patients in a number of clinical studies showing increases in muscle mass following treatment (Schols et al., 1995; Creutzberg et al., 2003; Casaburi et al., 2004).", "To date, however, these changes have not consistently translated into other measurable patient-centered outcomes such as exercise capacity and lung function (Velema et al., 2012; Pan et al., 2014b).", "Testosterone exerts both anabolic and androgenic effects, and the administration of testosterone and its analogues is associated with a number of side effects, including increased risk of cardiovascular events and dermatological disorders (Basaria et al., 2010), that have limited its uptake in clinical practice.", "In recent years, a new class of molecules known as selective androgen receptor modulators (SARMs) have been developed that interact with the androgen receptor and can mimic some of the effects of testosterone while avoiding some of the side effects (Mohler et al., 2009).", "These molecules offer some promise for the therapeutic treatment of muscle wasting in COPD and have recently been tested in clinical trials (See Table 2).", "The nonsteroidal SARM Enobosarm (GTx-024) is reported to increase muscle mass and bone density in animal models and in healthy human males (Dalton et al., 2011).", "In elderly men and postmenopausal women, Enobosarm at 3 mg per day for 86 days caused a significant 1.3 kg increase in lean body mass, which was accompanied by a significant decrease in fat mass (Dalton et al., 2011).", "This increase also appeared to translate into improved function, with patients showing a decrease in the time to climb 12 stairs and an increase in stair climb power (Dalton et al., 2011).", "A more recent trial investigating the efficacy of Enobosarm in cancer patients also demonstrated a significant improvement in lean body mass and reduced fat mass (Dobs et al., 2013).", "No significant improvement was seen in 6-minute walk test or hand grip strength; however, Enobosarm-treated patients showed a decrease in stair climb time and increased stair climb power compared to placebo-treated patients, suggesting some improvement in muscle function following Enobosarm treatment (Dobs et al., 2013).", "At this time, no trials are currently registered for the investigation of the effectiveness of SARMs in treating muscle wasting underlying COPD; however, this avenue offers promise, particularly in combination with other therapeutic approaches such as the combination of nutritional intervention and exercise training, which has been demonstrated to be an effective multimodal treatment approach in previous studies (Casaburi et al., 2004).", "Ghrelin is a peptide hormone of 28 amino acids that regulates growth hormone (GH) secretion from the anterior pituitary gland and has a number of effects with potential benefit for treating muscle wasting in COPD.", "Ghrelin is a potent orexigenic agent, stimulating appetite and feeding (Wren et al., 2001a, 2001b).", "Such effects could be beneficial in COPD patients who have reduced appetite and calorie intake.", "In addition, ghrelin can indirectly stimulate protein synthesis through stimulation of the GH-IGF-1 axis, promoting muscle anabolic processes.", "It also stimulates adiposity through inhibition of fat oxidation (Tschop et al., 2000), implying that caution may be needed in administering ghrelin to patients with higher fat mass.", "Ghrelin levels appear to be higher in underweight patients with COPD compared to healthy controls (Itoh et al., 2004; Uzum et al., 2014) and normal weight COPD patients (Itoh et al., 2004), a phenomenon that has been observed in other cachectic states (Nagaya et al., 2001; Shimizu et al., 2003).", "Another study found reduced ghrelin levels in COPD patients (mean BMI of 18.5) compared to healthy controls (mean BMI 22) (Luo et al., 2005).", "The reason behind this discrepancy is unclear but may reflect differences in the COPD populations sampled (e.g., current smoking status, different inclusion criteria) and methodological differences in the isoforms of ghrelin measured in each study.", "Clinical studies administering human ghrelin to COPD patients resulted in increased bodyweight and lean body mass and increases in both limb and respiratory muscle strength (Nagaya et al., 2005).", "Functional improvements have also been reported following ghrelin administration, with improved 6-minute walk distance reported in two studies (Nagaya et al., 2005; Miki et al., 2012).", "A recent study using the synthetic ghrelin peptide SUN11031 similarly resulted in increased bodyweight and lean body mass, accompanied by increased levels of IGF-1 (Levinson & Gertner, 2012).", "This study did not observe significant increases in 6-minute walk distance or handgrip strength in the overall trial population following SUN11031 treatment.", "However, in a post-hoc analysis of a subpopulation of patients with advanced muscle wasting at baseline then treated with the highest dose of SUN11031 had a significant increase in 6MWD and handgrip strength compared to placebo-treated patients.", "The ghrelin receptor agonist anamorelin has not yet been tested in the context of COPD-associated muscle wasting.", "It has shown positive results on lean body mass in clinical studies in cancer cachexia; however, no increase in handgrip strength was observed despite increases in IGF-1 (Garcia et al., 2015; Takayama et al., 2016; Temel et al., 2016).", "Despite evidence of increased bodyweight and lean body mass following treatment targeting the ghrelin pathway in cachectic patients with both COPD and other conditions, the translation of increased mass to improvements in strength and physical function has not been consistently demonstrated.", "Despite this, therapies targeting the ghrelin-GH pathway may still hold promise and, in combination with effective pulmonary rehabilitation programs, could result in beneficial increases in both muscle mass and function (See Table 2).", "Numerous studies have shown that COPD patients are less physically active than healthy controls (Watz et al., 2009; Troosters et al., 2010; Vorrink et al., 2011; Watz et al., 2014), and physical inactivity is seen as a major contributor to the muscle wasting and loss of strength seen in COPD patients.", "As with healthy people, physical activity and exercise have numerous health benefits in patients with COPD and exercise training through pulmonary rehabilitation programs is currently the gold standard for improving muscle strength and function (Spruit et al., 2013).", "Pulmonary rehabilitation incorporates a range of interventions including education, behavioral change, and exercise training with the aim of improving the physical and psychological condition of people with COPD (Spruit et al., 2013).", "The beneficial effects of exercise training in pulmonary rehabilitation are well established and a recent systematic review of 65 randomized controlled trials revealed that pulmonary rehabilitation leads to improvements in exercise capacity as well as in quality of life scores including symptoms of fatigue and dyspnea (McCarthy et al., 2015).", "COPD patients often exhibit exercise intolerance due to a variety of factors including dyspnea, ventilatory limitation, fatigue, and peripheral muscle dysfunction (Spruit et al., 2013).", "Although exercise training in COPD patients is an effective therapeutic approach, the training modalities of pulmonary rehabilitation programs vary and there is still some debate about the most effective training protocols for the heterogeneous COPD population and a need to develop tailored training programs to accommodate differing patient abilities and needs.", "Commonly used training modes include endurance/aerobic training and resistance/strength training, and protocols and training programs often differ even within individual training modalities.", "Endurance training, commonly through stationary cycling or walking activities, aims to condition the ambulatory muscles and improve cardiovascular fitness (Spruit et al., 2013).", "Factors such as duration, frequency, and intensity are important determinants of the physiological adaptations to exercise training.", "Although the optimal duration for a pulmonary rehabilitation is unclear, evidence of benefit in exercise capacity and quality of life is demonstrable if participants attend a minimum of 12 sessions during a 2 or 3 day per week 6–12 week program (Bolton et al., 2013).", "In addition to the supervised exercise, patients are encouraged to continue exercising at home during the period of the program, following standard exercise prescription guidelines such as those of the American College of Sports Medicine, with exercise sessions of 20–60 min, 3–5 times per week at > 60% of the patient's maximal work rate (Garber et al., 2011).", "Endurance training leads to improved exercise capacity (Whittom et al., 1998; Troosters et al., 2000; Ortega et al., 2002; Vogiatzis et al., 2007, 2011).", "Additional increases in muscle cross-sectional area, IGF-1 expression, and a shift toward more oxidative type I fibers have also been reported (Vogiatzis et al., 2007, 2011).", "Resistance or strength training involves lifting heavy weights for a prescribed number of repetitions, to build strength and increase muscle mass in individual muscles or muscle groups through upregulation of the mTOR signaling pathway (Terzis et al., 2008; Walker et al., 2011).", "Numerous randomized controlled trials have confirmed that resistance training protocols increase arm and leg strength, exercise capacity and muscle mass in COPD patients (O'Shea et al., 2009; Vonbank et al., 2012; Constantin et al., 2013).", "Similar changes in muscle size and isometric strength were observed in healthy controls and COPD patients after an 8 week resistance protocol, confirming that the muscles of COPD patients are still able to respond to strength training stimuli to the same extent as healthy controls, despite starting from a depleted baseline (Constantin et al., 2013).", "While resistance training is well accepted as a method for improving strength, its effects on aerobic fitness and exercise endurance are less apparent.", "Resistance training results in some improvement in walking distance and cycling endurance compared to no exercise; however, it is much less effective than endurance/aerobic training at improving performance in these areas (O'Shea et al., 2009).", "It is likely that a combined training program including both resistance and endurance exercise modalities would be optimal to increase muscle mass, strength, and endurance in COPD patients, a hypothesis that has been supported by several studies (Ortega et al., 2002; Vonbank et al., 2012; Zambom-Ferraresi et al., 2015), and indeed most pulmonary rehabilitation programs include aspects of both endurance and resistance exercise to maximize gains from both modalities.", "Despite the accepted benefits of exercise training for improving muscle mass and function in COPD patients, a number of therapeutic challenges still remain.", "Within the COPD population, there are ‘non-responder’ patients who do not show improvement with exercise interventions, largely due to the presence of more severe airflow limitation or comorbidities (Hill & Holland, 2014).", "It is estimated that around 50% of patients referred for pulmonary rehabilitation present with comorbidities that affect their ability to exercise, the most common of which are metabolic disorders (hypertension, diabetes, and dyslipidaemia) and heart diseases (chronic heart failure and coronary heart disease) (Crisafulli et al., 2008).", "Patients with comorbidities were still able to respond to exercise training and improve their physical capacity (Crisafulli et al., 2008); however, tailored and multimodal training programs are needed to accommodate the needs and abilities of different COPD patients, and a number of additional interventions have been proposed (Hill & Holland, 2014).", "Interval training involves performing short periods of intense exercise interspersed with periods of rest or lower intensity exercise.", "Although used for years in the training of athletes, interval training has emerged as an option for patient rehabilitation only recently.", "Interval training is generally well tolerated and offers some benefits for those patients who are unable to tolerate long periods of continuous aerobic exercise or are unable to achieve the target exercise intensity or duration to provide a sufficient training stimulus for improvement (Spruit et al., 2013).", "A number of trials have confirmed the efficacy of interval training methods in COPD if the same total work is performed as an endurance protocol (Puhan et al., 2006; Beauchamp et al., 2010; Zainuldin et al., 2011).", "Interval training is generally well tolerated and results in lower symptom scores than continuous endurance exercise protocols, despite similar overall work (Beauchamp et al., 2010).", "Neuromuscular electrical stimulation (NMES) involves external electrical stimulation of isolated muscles via electrodes placed on the skin to evoke an action potential within the muscle and cause involuntary contraction (Maffiuletti, 2010).", "NMES offers a number of advantages for training of COPD patients; (1) the equipment used is portable and easy to use, allowing patients to use the equipment in a home setting for regular treatment; (2) NMES is generally well tolerated and has few side effects; (3) the exercise is passive, involving a low ventilatory load, which may be useful for patients who are not able to exercise due to respiratory symptoms; and (4) it can be used in bedbound or even unconscious subjects, making it potentially useful for maintaining muscle mass in hospitalized patients who cannot exercise (Spruit et al., 2013).", "Trials in the use of NMES have reported consistent benefits in terms of improved muscle size and function, exercise endurance and capacity, improved lung function, and reduced dyspnea (Neder et al., 2002; Abdellaoui et al., 2011; Giavedoni et al., 2012; Vivodtzev et al., 2012; Sillen et al., 2014; Vieira et al., 2014; Maddocks et al., 2016)(see Table 3).", "In a direct comparison of high-frequency NMES with a resistance training protocol over 8 weeks, the increase in isokinetic quadriceps strength was similar in the two training groups, indicating that NMES can be as effective as traditional strength training (Sillen et al., 2014) and supporting the notion that it may be a particularly useful training modality to restore or maintain muscle mass in COPD patients who have difficulty exercising, for example, during hospitalization for an acute exacerbation (Zanotti et al., 2003; Vivodtzev et al., 2006).", "In addition to improved muscle size and function, changes at the level of muscle architecture and biochemistry have been reported following NMES in COPD patients, including decreased expression of the atrophy-related gene atrogin-1 and increased phosphorylation (and hence activity) of P70S6K, a protein involved in the anabolic protein synthesis pathway (Vivodtzev et al., 2012).", "An increase in the proportion of Type I and Type IIa/x fibers has also been observed following NMES, suggesting a fast-to-slow shift in fiber type toward a more oxidative phenotype.", "Another study reported a decrease in Type I fibers, albeit in a population with less severe COPD and with variations in the stimulation protocol (Dal Corso et al., 2007).", "Other molecular changes reported include a reduction in markers of oxidative stress in the muscle of patients treated with NMES following acute exacerbation (Abdellaoui et al., 2011), and a reduction in serum TNF-α levels (Vieira et al., 2014), suggesting that NMES could affect levels of systemic oxidative stress and inflammation in addition to its direct effects on muscle strength and function.", "While these observations are promising for the use of NMES to treat muscle wasting in COPD, they must be interpreted with caution due to the small size of many trials, the highly selected patients who were included and wide variation in treatment protocols used (Sillen et al., 2013; Pan et al., 2014a).", "Further investigation and large scale trials are needed to fully optimize treatment protocols and evaluate efficacy of NMES for the treatment of muscle wasting in COPD, and indeed a number of such trials are either recently completed or currently recruiting patients (see ClinicalTrials.gov identifiers: NCT02321163, NCT01799330, ISRCTN87439020, NCT02171377).", "Many COPD patients are not able to exercise at high intensity due to ventilatory constraints, which may limit the extent to which muscular adaptations can be achieved through traditional endurance exercise training.", "An alternative method is to partition or limit the exercise to a smaller subset of muscle groups in order to reduce the ventilatory load on the patient and allow them to exercise that muscle group at higher intensity than they could if using larger muscle groups (Richardson et al., 1999).", "An example of this is one-legged cycling, which has emerged as a potential training modality for COPD patients (Dolmage & Goldstein, 2006), and has been shown to improve peak muscle power and oxygen uptake while resulting in lower dyspnea than two-legged cycling at the same workload (Dolmage & Goldstein, 2008; Bjorgen et al., 2009).", "To date, only a few small trials have been conducted using this training modality in this area, and none have looked specifically at molecular or phenotypic changes in muscle tissue.", "The method is currently being expanded in a trial using single and double leg elastic band exercises (see ClinicalTrials.gov identifier: NCT02283580), that includes measurement of anabolic/catabolic signaling pathways, fiber types, and enzyme activities in the quadriceps muscles as secondary outcomes (Nyberg et al., 2015).", "Partitioning exercise approaches remain a very promising therapeutic alternative to traditional exercise training and could be readily translated into clinical practice (Evans et al., 2015).", "Additional challenges in pulmonary rehabilitation include the problem of waning effects over the 12 months following completion of an exercise training program, and improving access to enable patients who have difficulty attending an outpatient rehabilitation program to participate in structured and supervised exercise training and improve their physical capabilities.", "It is estimated that up to 50% of patients who are referred for pulmonary rehabilitation will never attend, and dropout rates are estimated at 10–32% (Keating et al., 2011).", "The relatively new and expanding field of telehealth offers opportunities for patients to participate in exercise programs in their own home or within the community while still under the remote supervision of trained medical staff and exercise therapists.", "Pilot studies using telehealth approaches have shown some promise.", "Recent studies have demonstrated improvements in 6MWD and dyspnea scores (Holland et al., 2013a; Wang et al., 2014; Zanaboni et al., 2016); however, patient numbers in each trial are still small.", "A meta-analysis conducted on 9 trials from 1996 to 2013 reported that telemedicine approaches did not improve physical exercise capacity or dyspnea but may have an effect on physical activity (Lundell et al., 2015); however, comparisons between trials were hampered by large variability in telehealth protocols and outcome measures.", "Home- or community-based telerehabilitation programs have been shown to be well tolerated by participants, especially those living in remote areas for whom travelling to attend clinics is problematic (Burkow et al., 2015).", "The cost of delivering such programs is variable depending on the nature of the program and the equipment provided to participants but has been shown to be comparable or lower than the cost of attending an outpatient clinic (Burkow et al., 2015).", "Current and upcoming studies aimed at more directly comparing home-based programs with outpatient clinic rehabilitation will be valuable in addressing the direct and indirect costs associated with each mode of therapy (see ClinicalTrials.gov identifier: NCT01423227) (Holland et al., 2013b).", "Some recent trials have further explored the use of modern technology for the delivery of home-base rehabilitation, with some exciting results.", "Interesting studies from Wang and colleagues involved a home-based walking program for COPD patients, which asked patients to walk in time to the speed of music played on their mobile phones (Liu et al., 2008; Wang et al., 2014).", "The tempo of the music was calculated to match a measured walking speed of 80% of maximal walking capacity based on exercise testing, allowing a fully personalized intervention to encourage patients to exercise at the required intensity.", "Patients using the music app showed clinically significant increases in walking distance in the Incremental Shuttle Walk Test (ISWT) of 49 and 58 m at 3 and 6 months, respectively, compared to baseline testing, which is above the minimum important difference of 47.5 m (Singh et al., 2008) and comparable to results typically seen following in-patient rehabilitation programs (Spruit et al., 2013).", "Strength was also improved after 6 months in patients using the music app, whereas control patients given the same walking program but not using the app showed a decline in walking distance over the same time period and no changes in strength (Wang et al., 2014).", "Measures of inflammation (CRP, IL-8, TNFα, and IL-6) all decreased in patients using the app compared to controls (Wang et al., 2014).", "In the last few decades, as skeletal muscle wasting has become more recognized as a serious and prevalent comorbidity of COPD, our understanding of the molecular pathways leading to muscle wasting and the complex interplay between the lung and systemic pathologies of COPD has improved dramatically.", "As our knowledge of molecular mechanisms leading to muscle wasting increases, so promising new pharmacological targets are identified that could be targeted to reduce muscle wasting or delay disease progression.", "Despite this, exercise training through pulmonary rehabilitation remains the cornerstone of COPD management with proven effectiveness in improving lean body mass, physical capability, and lung symptoms.", "Currently, there are no clinically proven licensed pharmacological therapies for muscle wasting in COPD.", "While exercise training is effective, many patients are limited in the amount and/or type of exercise they can do or are not able to access pulmonary rehabilitation services.", "There is a clinical need to develop effective pharmacological agents and therapeutic approaches to target muscle wasting processes in COPD, acting either alone or in conjunction with pulmonary rehabilitation programs.", "The mechanisms of wasting in COPD are complex, including contributions from physical activity, systemic inflammation, lack of appetite, hormone signaling, and oxidative stress.", "It is likely that wasting may be worsened in patients for whom multiple factors are involved, for example, those who show elevated systemic inflammation, lack of appetite, and reduced physical activity.", "The signaling pathways leading to wasting may also interact with each other, working synergistically to increase atrophy.", "While it is unlikely that targeting a single aspect will be an effective approach to treating such a complex condition, it may be that even reducing the impact of a single factor could lead to clinically meaningful improvements in muscle mass and strength.", "However, the most promising outcome is likely to be achieved from combining therapeutic approaches to tackle multiple factors simultaneously.", "The authors declare that there are no conflicts of interest" ]

[]

[ "NEUR", "PSYC" ]

[ "Adaptation", "Addiction", "BDNF", "CRH", "Cognition", "Coping", "Depression", "Dopamine", "Glucocorticoids", "Habituation", "Mesocorticolimbic system", "Microglia", "Neurodegeneration", "Opioids", "Stress", "Ventral tegmental area" ]

[ "2802", "2805", "3206" ]

[ "Although the stress response is essential for adaptation and survival, the term ‘stress’ is today generally associated with a negative experience (McEwen, 2013a,b).", "Work-related stress is common and chronic exposure to stress is linked to various neuropsychiatric disorders, such as major depressive disorder (Calabrese et al., 2009; Kessler, 1997).", "However, the underlying causative mechanism of these stress-related disorders remains poorly understood and the etiologic knowledge is limited to risk factors and vulnerability (Chrousos, 2009; Nestler et al., 2002).", "Therefore, the understanding of the neurobiology of stress may help the prevention and treatment of mental disorders.", "One of the neurotransmitters that is profoundly affected by stress is dopamine (DA), which is classically associated with sensorimotor functions, incentive motivation, reward processing, and reinforcement learning (Arias-Carrión et al., 2010; Cabib and Puglisi-Allegra, 2012; Ljungberg et al., 1992; Romo and Schultz, 1990; Schultz et al., 1993, 1997).", "Of crucial importance in this respect is the mesocorticolimbic DA circuitry, which originates from the ventral tegmental area (VTA) neurons (Holly and Miczek, 2016; Trainor, 2011; Tye et al., 2013; Valenti et al., 2012) and has a high grade of neuronal connectivity with limbic structures and the medial prefrontal cortex (mPFC) (Beier et al., 2015; Watabe-Uchida et al., 2012).", "This VTA network is important for processing the linkage of internal states with appraisal of environmental stimuli, thereby forming emotional-motivational valuations (Burke et al., 2014; Lloyd and Dayan, 2016; Papageorgiou et al., 2016; Salamone and Correa, 2012).", "The valuations allow an organism to prioritize its goals and to adjust its choice of behavioral response to cope with a challenge (Burke et al., 2014; Moore and Depue, 2016; Papageorgiou et al., 2016).", "For instance, if a stimulus is considered rewarding, the individual will approach the reward rather than avoid it (Burke et al., 2014; Dayan and Berridge, 2014).", "Such valuations may change rapidly, however, when the rewarding stimulus turns into an adverse one, and results in a switch from approach into avoidance behavior (Berridge et al., 1984; Burke et al., 2014; Dayan and Berridge, 2014; Rangel et al., 2008; Robinson and Berridge, 2013; Tindell et al., 2009).", "This conflict between approach and avoidance also may occur if (anticipation of) reward presentation is compromised by a potential threat or stressor (Stanton et al., 2019).", "Mesocorticolimbic DA neurotransmission may be involved, therefore, in the pathophysiology of mood, anxiety, and addiction disorders, of which the deficit in emotional-motivational activation of reward-seeking is a core symptom (Grace, 2016; Nestler and Carlezon, 2006; Polter and Kauer, 2014).", "Excellent reviews on the theme of stress and VTA-DA function have appeared (Belujon and Grace, 2015, 2017; Chrousos, 2009; Hollon et al., 2015; Ironside et al., 2018; Kalafatakis et al., 2016; Koob and Volkow, 2016; Kwako and Koob, 2017; Lammel et al., 2014a,b; Le Moal and Simon, 1991; Nestler and Carlezon, 2006; Polter and Kauer, 2014; Russo and Nestler, 2013; Stanton et al., 2019; Weger and Sandi, 2018; Wise, 2004).", "Here, we will delineate how plasticity and connectivity of VTA-DA neurons may change in response to acute and chronic stressors.", "In order to understand how such stressors may act, we focus on the hormones of the hypothalamus-pituitary-adrenal (HPA) axis (de Kloet et al., 2005; McEwen, 2007; Ulrich-Lai and Herman, 2009).", "This includes the action of corticotropin-releasing hormone (CRH), which orchestrates the HPA-axis response to stress in the paraventricular nucleus (PVN) and also acts as neuropeptide produced in e.g. the extended amygdala, a region that consists of the amygdala and bed nucleus striae terminalis (BNST).", "Also opioids are briefly discussed as part of the stress response system and their inherent addictive properties.", "We will focus in particular on the master regulator of stress-adaptation, the adrenal glucocorticoid hormone, and its downstream targets such as the locally produced brain-derived neurotrophic factor (BDNF).", "The review is concluded with a conceptual framework of the function of the VTA-DA system in stress-coping and adaptation.", "The brain coordinates the response to stressors, decides on the selection of an appropriate coping strategy, and promotes adaptation (McEwen, 2016).", "If something novel happens, first the perception of salient sensory information triggers an alarm resulting in increased arousal, alertness, vigilance, and focused attention with the goal to instantaneously defend the ‘self’.", "The stressor can be physical such as pain, blood loss, or tissue damage, and these reflexive autonomic reactions are triggered by brain stem nuclei that convey visceral information via ascending aminergic pathways to frontal brain regions including the PVN (McCarty and Gold, 1996).", "If the stressor is psychogenic the alarm results in rapid activation of the locus coeruleus noradrenergic network and the sympathetic nervous system including the adrenomedullary release of adrenaline (Reyes et al., 2015).", "Psychogenic stressors have a strong anticipatory component implying that the stress reaction may start even by imagination without an environmental trigger (Herman et al., 2003; Ulrich-Lai and Herman, 2009; Cabib and Puglisi-Allegra, 2012).", "All real and anticipated stressors activate the PVN-CRH/vasopressin neurons which orchestrate control of the autonomic and pituitary adrenocorticotrophic hormone (ACTH) reactivity resulting in increased secretion of the glucocorticoids cortisol and corticosterone, the latter steroid only in rodents (Spencer and Deak, 2017).", "Besides triggering an immediate alarm reaction, stressors are subject to appraisal.", "Alarm reactions and appraisal processes interact and produce the selection of a coping strategy (de Kloet et al., 2019).", "The circuitry underlying appraisal and choice of coping style is organized by the mPFC, which exerts top-down its executive control over limbic, striatal, midbrain, and hypothalamic neurons (Ulrich-Lai and Herman, 2009).", "These neurons are also a target for the glucocorticoids secreted by the adrenal cortex.", "Glucocorticoids provide energy substrates by gluconeogenesis and allocate these substrates to cells and tissues in need, exert anti-inflammatory and immunosuppressive actions, and have profound actions on brain and behavior.", "Within the brain the glucocorticoids control numerous processes involved in processing of stressful information.", "The hormones exert a negative feedback action on the synthesis and release of CRH in the PVN, and on pituitary pro-opiomelanocortin (POMC) synthesis and ACTH release.", "At other sites glucocorticoids promote the synthesis of CRH.", "One of these sites is the central amygdala nucleus and BNST, which is a hub for processing of stress, fear, and anxiety (Makino et al., 1994, 1995; Zalachoras et al., 2016).", "Finally, glucocorticoids promote the storage of memory and of energy substrates in fat and liver in preparation of future challenges.", "The actions exerted by the glucocorticoids are mediated by two closely related receptor types: the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) (Reul and de Kloet, 1985).", "MR binds the naturally occurring glucocorticoids with high affinity and are expressed abundantly in neurons of the hippocampus, lateral septum, and amygdala as well as striatal and cortical regions (Reul and De Kloet, 1986; Ahima et al., 1991; Diorio et al., 1993; Brinks et al., 2007); MRs also occur as aldosterone-selective receptors in the nucleus tractus solitarii (NTS) (Gasparini et al., 2018).", "GRs are widely expressed in the brain and display a lower affinity to cortisol and corticosterone, implying that these receptors become only substantially occupied at high hormone levels after stress and at the circadian peak (Reul and de Kloet, 1985).", "MR and GR are nuclear receptors engaged in regulation of gene expression, but recently also rapid non-genomic actions were identified in brain (Di et al., 2003; Karst et al., 2005).", "MRand GR mediate in complementary fashion the action of glucocorticoids: MR activation may modulate the activity of neuronal circuits underlying appraisal processes and decision-making.", "Via GR the glucocorticoids regulate energy allocation, and act on brain circuits to promote motivation, emotional expressions, cognitive performance, behavioral adaptation and memory performance.", "These actions exerted by the glucocorticoids are context-dependent and procede in concert with neurotransmitters, neuropeptides, and growth factors over different time domains (de Kloet et al., 2005; McEwen, 2007; Joëls et al., 2007).", "Thus, the stress response is the body’s signaling system to coordinate defense, coping, and adaptation.", "Appraisal processes direct the decision how to cope with stress.", "For this purpose the mPFC, limbic structures, the VTA-DA circuitry, and the stress system are of crucial importance.", "Repeated failure to cope properly may cause a condition of chronic stress, which is characterized by lack of control, uncertainty, and fear, while motivation and reward processing regulated by the VTA-DA circuitry become compromised.", "In the next sections, the function, plasticity, and connectivity of the VTA-DA circuit is discussed in response to acute and chronic stressors.", "Validated animal models, used for these studies, are presented in Box 1.", "Over the past decade, the concept of the VTA changed from a simple homogeneous group of neurons – sharing analogous biomolecular, electrophysiological, and functional characteristics – to a complex heterogeneous structure containing neuronal subtypes, which are often associated with distinct molecular and synaptic characteristics (See Box 2 Beier et al., 2015; Holly and Miczek, 2016; Ikemoto, 2007; Lammel et al., 2008, 2011, 2012, Margolis et al., 2006, 2008; Morales and Margolis, 2017; Watabe-Uchida et al., 2012; Parker et al., 2019).", "Specifically, its main neuronal population, the DA neurons, respond to a broad array of environmental stimuli, these responses are context-dependent, and can ultimately lead to the modulation of distinct behavioral patterns (Bromberg-Martin et al., 2010; Kessler et al., 2003; Lammel et al., 2012; Matsumoto and Takada, 2013; Zweifel et al., 2011; Belujon and Grace, 2015; Grace, 2016; Lammel et al., 2014a,b; Moore and Depue, 2016).", "Given their unique characteristics – and especially their high degree of neuronal connectivity (see Fig. 1; Beier et al., 2015; Juarez and Han, 2016; Morales and Margolis, 2017; van den Heuvel and Sporns, 2013; Watabe-Uchida et al., 2012) – VTA-DA neurons in the mesocorticolimbic circuitry can be considered as a hub linking circuits involved in emotional-motivational appraisal of salient information with networks underlying executive functioning (Juarez and Han, 2016; Melis and Pistis, 2012; Polter and Kauer, 2014).", "Besides rewarding stimuli, the appraisal processes concern aversive stimuli as well.", "The VTA-DA circuitry, thus, plays a significant role in learning and memory processes that contribute to an animal’s ability to differentiate between “good and bad”.", "Accordingly, many behavioral and cognitive changes that are influenced by VTA-DA modulation are at least partially beneficial and promote adaptive responses on the basis of these learned associations (Bromberg-Martin et al., 2010; Cabib and Puglisi-Allegra, 2012; Cools, 2016; Hauser et al., 2017; Lloyd and Dayan, 2016; Pignatelli and Bonci, 2015).", "The VTA-DA circuitry is thus of crucial importance in effort- and value-based decision-making and provides a mechanism to determine the value of different options in order to fulfill goal-directed behavior, which in the case of stressors is directly related to the animal’s well-being (Lloyd and Dayan, 2016).", "Therefore, key functions where VTA-DA stress responses are involved include updating the assignment of expected value of behaviorally relevant stimuli and behavioral outcomes and, additionally, the modulation of coping strategies in order to efficiently deal with the specific stressful situation (Burke et al., 2014; Lloyd and Dayan, 2016; Salamone and Correa, 2012).", "Survival of most animal species depends on behavioral patterns that maximize contact with beneficial stimuli while contact with harmful stimuli is preferably avoided (Burke et al., 2014).", "The ability to make such differentiations evolves over lifetime and are thought to largely depend on processes of VTA-DA neuron-dependent reinforcement learning (Lloyd and Dayan, 2016; Salamone and Correa, 2012).", "During reinforcement learning processes, formerly neutral stimuli can reinforce or strengthen behaviors through the formation of associations with certain cues (Koob and Volkow, 2016; Lloyd and Dayan, 2016).", "Consequently, in the face of adversity, this stimulus-dependent learning mechanism combines past experiences with information about the current state of the environment, leading to learning of both danger- and safety-predicting conditioned stimuli (Grupe and Nitschke, 2013; Lloyd and Dayan, 2016).", "This VTA-DA-dependent learning increases the predictability of future events, and enables an organism to choose and execute behavioral actions more precisely and with the appropriate behavioral vigor (Lloyd and Dayan, 2016; Salamone and Correa, 2012).", "Such behavioral decisions are, however, not rigid but state-dependent, (Burke et al., 2014; Papageorgiou et al., 2016).", "This is illustrated, for instance, by salt-appetite (Berridge et al., 1984; Robinson and Berridge, 2013; Tindell et al., 2009).", "In normal situations, rats usually do not ingest extremely salty solutions and avoid cues that predict them.", "In salt-deficient situations, however, rats do ingest salt.", "This example also describes that a previously disgusting taste can become rewarding to approach, demonstrating that internal states can have profound effects on motivational processes for behavioral activation (Dayan and Berridge, 2014; Papageorgiou et al., 2016).", "These often subjective state-dependent value representations allow an animal to prioritize its goals, and lead to the generation of both proximal (direct approach or avoidance) and distal (allocating resources for effortful approach or avoidance of a goal stimulus) motivated behaviors (Abraham et al., 2014; Salamone and Correa, 2012).", "Thus, by combining past experiences with information about the current internal and external states, relevant behavioral decisions can be made.", "A mismatch between such predicting stimuli and reality generates a prediction error signal of any kind and it is well known that in this context dopamine signals the prediction of a reward (den Ouden et al., 2012; Schultz, 2016).", "As was recently proposed, stress can be conceptualized as a cumulative state of prediction errors (Trapp et al., 2018).", "Or, similarly, Peters et al. (2017) defined stress as “the individual state of uncertainty about what needs to be done to safeguard physical, mental or social well-being” (Peters et al., 2017).", "These views align with the notion that loss of control and lack of information are amongst the most potent stressors that evoke a profound sympathetic and HPA-axis response (Mason, 1971; Levine, 2005; Koolhaas et al., 2011; McEwen et al., 2015; Reyes et al., 2015).", "Since perception of sensory information is based on expectations, the cumulative prediction errors may cause a stress response aimed to promote adaptation to these repeated expectations.", "Therefore, it was proposed recently that repeated failures to precisely predict upcoming events can modify the way one will perceive the world through reinforcement learning processes (Trapp et al., 2018).", "Furthermore, when an initially selected coping strategy defies expectancies about response-outcome relationships, the “ability to switch behaviors when contexts change, or to switch from an ongoing behavior to a new one”, commonly referred to as behavioral flexibility (Baker and Mizumori, 2017), is an essential adaptive mechanism in the attempt to successfully deal with the situation.", "Specifically, in the case of novel unescapable or uncontrollable stressful situations, the behavior that is initially selected is generally aimed to remove, avoid, or control the source of the stressor.", "This is indicated as an active coping strategy, which is characterized by behavioral responses that act upon the source of the stressor and are directed to defend the “self” (Carroll, 2013a; Wood and Bhatnagar, 2015).", "If, however, an animal’s experience of stress cannot be terminated by using its own resources, which will depend on the interaction between genetic predisposition and life-experience (Daskalakis et al., 2013), animals tend to switch to conservation withdrawal (Henry and Stephens, 1977), which is a more passive coping strategy (Cabib and Puglisi-Allegra, 2012).", "Passive behaviors support more altruistic solutions and thereby rely more on others to solve or terminate the emotional aspects of the stressful experience (Carroll, 2013b; Wood and Bhatnagar, 2015).", "Therefore, whether the stressful situation is appraised as “controllable” is of crucial importance to the increased expression of a particular behavioral coping strategy.", "Accordingly, behavioral flexibility may support coping when the situation goes beyond the animal’s initial own abilities (Cabib and Puglisi-Allegra, 2012; de Kloet and Molendijk, 2016; Tye et al., 2013).", "In conclusion, the VTA-DA circuitry supports adaptive behavior by assigning prediction and valence to a salient stimulus.", "Positive valence is linked to a reward, but fear and uncertainty are examples of negative valence.", "Coping with a stressor – either passive or active – can be considered a rewarding experience as opposed to failure to cope that may result ultimately in chronic or ‘toxic’ stress (McEwen et al., 2015).", "Behavioral flexibility or the ability to rapidly adjust coping with a changing context and valence is essential for adaptation to stress and involves the VTA-DA circuit.", "The contribution of VTA-DA neurons to such adaptive behavior in stressful situations is strongly dependent on the type of activity these neurons display.", "VTA-DA neurons can be in either an inactive or active state.", "Active VTA-DA neurons can switch between two neural firing patterns: either asynchronous low-frequency (2–4 Hz) tonic firing with a single action potential discharge or a transient high frequency phasic activity (>15 Hz), known as burst firing (Fig. 2A) (Cao et al., 2010; Grace and Bunney, 1984; Grace and Onn, 1989; Juarez and Han, 2016; Walsh and Han, 2014).", "The regulation of tonic and phasic VTA-DA neuron activity is, however, complex (Fig. 2B).", "Dopaminergic firing patterns are facilitated through presynaptic interactions, with γ-aminobutyric acid (GABA)-ergic and glutamatergic input as main regulators (Bariselli et al., 2016; Belujon and Grace, 2015; Grace and Onn, 1989).", "Inhibitory control from GABA-ergic neurons can keep VTA-DA neurons in a hyperpolarized, inactive state, while input from excitatory glutamatergic neurons can induce phasic burst-firing patterns.", "The balance between inhibitory and excitatory input therefore determines the neuronal state of a VTA-DA neuron and its ability to respond to behaviorally relevant stimuli.", "Importantly, phasic activity can only occur in VTA-DA neurons where tonic activity is present.", "Thus, the amount of neurons that express tonic firing represents the degree of potential DA signaling amplification (Belujon and Grace, 2015; Grace, 2016).", "The tonic neural firing profile sets a low background pacemaker DA activity in a subset of VTA-DA neurons and the number of these spontaneously active DA neurons can be defined as the population activity (Belujon and Grace, 2015).", "In benign, neutral environmental contexts, the number of responding VTA-DA neurons is kept low – approximately half of the neurons in the VTA do not fire.", "Thus, since phasic burst firing can only occur in tonic firing neurons, the effects of excitatory input from brain regions which actually can initiate VTA-DA phasic activity are restricted to the fraction of responding, tonically firing VTA-DA neurons.", "In perceived deviant emotional-motivational charged environmental conditions, appraisal processes may alter the population activity (Fig. 2C).", "Therefore, the responsiveness of VTA-DA neurons to context-dependent salient stimuli can be affected, and thus the way VTA-DA neurons are modulating behavior.", "This suggests that the regulation from an inactive to an active tonic VTA-DA neuronal state (and vice versa) attaches a certain emotional-motivational load to the role of these neurons in coping with specific environmental situations.", "These transitions in VTA-DA neuronal activity are also linked to the type of behavioral strategy animals use in order to cope with acute stressful situations.", "Specifically, an increased tonic firing population activity is linked to the enhanced use of more active coping strategies, whereas a decrease in the VTA-DA population activity is associated to the use of more passive ones (see Fig. 2C; Cabib and Puglisi-Allegra, 2012; Rincón-Cortés and Grace, 2017; Tye et al., 2013).", "The direct involvement of VTA-DA neurons in switching between different behavioral coping styles is demonstrated by the use of optogenetic manipulation of VTA-DA neurons (Lammel et al., 2014a,b; Tye et al., 2013).", "Tye et al. (2013) demonstrated that the inhibition of putative VTA-DA neurons during an acute stressful situation led to the increased use of passive coping strategies, as demonstrated by an increased immobility in a forced swim stress test (Tye et al., 2013).", "They additionally observed a robust increase in the use of active coping when VTA-DA neurons were optogenetically activated (Tye et al., 2013).", "Given that these behavioral responses were not time-locked to the administration of the light pulses, these transitions of active to passive coping with stress are most likely supported by changes in the tonic firing VTA-DA neuronal population activity, as confirmed electrophysiologically (Rincón-Cortés and Grace, 2017).", "Therefore, by supporting the switch between active and passive coping strategies, tonic VTA-DA neuronal activity is involved in behavioral decision-making.", "In conclusion, VTA-DA neurons are responsive to a broad array of behaviorally relevant stimuli, including stimuli related to the seemingly opposite experiences of reward and aversion.", "These behaviorally relevant stimuli are subjected to appraisal processes and contribute to the complex integration of excitatory and inhibitory input onto VTA-DA neurons.", "As will be discussed in Sections 4 and 5, the characteristic VTA-DA firing patterns are modulated through these afferent inputs, which are in particular orchestrated by hippocampal and amygdala circuits (Belujon and Grace, 2017).", "Since these modulating effects are state-dependent and the eventual behavioral outcomes can affect future appraisal processes, the VTA-DA mesocorticolimbic circuitry likely supports adaptive coping strategies.", "In response to acute stressors robust increases of extracellular dopamine and its metabolites were found with microdialysis in the most prominent VTA-DA neuron target areas, the N. accumbens (NAc) and the mPFC (Fig. 1) (see Holly and Miczek, 2016).", "The research by Cabib’s group has shown that stress-induced norepinephrine (NE) release in the mPFC corresponds to an increased DA release in the NAc and that increased mPFC DA is causally related to a decreased NAc DA efflux (Pascucci et al., 2007; Fiore et al., 2015).", "High DA in NAc supports active coping strategies, goal-directed behavior, and motivational arousal, while a diminished stress-induced DA is linked to passive coping in situations that are uncontrollable (Cabib and Puglisi-Allegra, 2012; Fiore et al., 2015).", "In agreement with these microdialysis studies stress-induced increases in tonic firing population activity were observed which facilitated phasic VTA-DA activity (Belujon and Grace, 2015).", "On the basis of differential responses, other authors proposed two rather than one distinct VTA-DA neuron subpopulations (Lammel et al., 2014a,b; Ungless et al., 2010).", "The dorsolateral VTA-DA neurons were mainly inhibited by acute stress (Guarraci and Kapp, 1999; Mantz et al., 1989; Mirenowicz and Schultz, 1996; Schultz and Romo, 1987; Ungless, 2004), and most of these neurons showed phasic excitation upon termination of the stressor (Brischoux et al., 2009; Navratilova et al., 2012; Tanimoto et al., 2004).", "In contrast, rapid and potent phasic excitations at the onset of stressor exposure were discovered in the ventromedially located “non-conventional” VTA-DA neurons (Anstrom et al., 2009; Anstrom and Woodward, 2005; Cohen et al., 2012; Lammel et al., 2014a,b; Zweifel et al., 2011).", "Thus, VTA-DA neurons are responsive to acute stress, but phasic activity is differentially expressed in VTA-DA neuron subpopulations at onset and termination of the stressor.", "Other studies have shown that the activation of VTA-DA neurons upon acute stress exposure can alter VTA-DA activity responses to later stimulation (Holly and Miczek, 2016; Valenti et al., 2012).", "Importantly, these alterations in VTA-DA neurons are shown in both tonic and phasic firing patterns, but appear to depend on the experimental conditions.", "Mild or intermittent stress protocols generally tend to increase VTA-DA population activity, while exposure to prolonged, more severe and uncontrollable/inescapable stress paradigms tend to blunt tonic firing in VTA-DA neurons (Chang and Grace, 2014; Rincón-Cortés and Grace, 2017; Kaufling, 2019).", "These differences in tonic firing may lead to transitions in the use of active and passive coping strategies in response to acute stressors (Cabib and Puglisi-Allegra, 2012; Lloyd and Dayan, 2016; Tye et al., 2013).", "Furthermore, when an animal is subsequently exposed to a heterotypic stressor – thus of a different nature than the previous stressor(s) used in the stress paradigm – the phasic responses are generally sensitized and/or amplified (Cuadra, 2001; Cuadra et al., 1999; Di Chiara et al., 1999; Finlay et al., 1995; Gresch et al., 2002; Murphy et al., 2003; Tidey and Miczek, 1997, 1996; Watt et al., 2014).", "Thus, VTA-DA neurons are responsive to stressors, and these responses may show spatiotemporal variation.", "Furthermore, although different stressors can result in diverse VTA-DA neuron activity responses, it is clear that stressor pre-exposure can alter future VTA-DA activity responses to stressful behaviorally relevant stimuli.", "However, discrepancies between experimental findings make it currently a challenge to determine the exact link between VTA-DA neuron activity and specific behavior modulating effects.", "A further complication in interpreting data on VTA-DA neuron stress responses is the divergence in methodological procedures; the nature, schedule, and intensity of the stressor matters, and stressors can be novel and may be promoted by conditioned or unconditioned stimuli (Cabib and Puglisi-Allegra, 2012; Holly and Miczek, 2016).", "Consequently, in response to stressors both increases and decreases in VTA-DA neuron activity have been observed (see Holly and Miczek, 2016).", "A recent series of experiments place GABA-ergic control of VTA-DA neurons in a novel perspective.", "It was discovered that the local infusion of the benzodiazepine diazepam, a GABAA receptor (GABAAR) agonist, in the VTA exerted anxiolytic activity in rats and, surprisingly, also increased the rewarding experience of social competitiveness (van der Kooij et al., 2018a,b).", "Further study showed that intra-VTA diazepam administration increased DA release selectively from its NAc terminals, and not from innervations in the mPFC, hippocampus, and amygdala.", "Upon stimulated DA release c-fos protein was induced in NAc target neurons that expressed D1 rather than D2 receptors.", "Accordingly, social dominance was also promoted by local administration of a D1 agonist in the NAc.", "In agreement with a previous observation by the same group (Hollis et al., 2015) mitochondrial activation in the NAc target neurons appeared causal to improved social competence.", "Intra-VTA diazepam infusion increased NAc adenosine triphosphate (ATP) production and post-mortem mitochondrial O2 consumption.", "Blockade of mitochondrial respiration and function with rotenone reversed the VTA-DA effects on anxiety and social competitiveness (van der Kooij et al., 2018a,b).", "So, social interaction depends on VTA-DA activity.", "The findings are supported by a companion paper of the same authors that demonstrated the engagement of GABA-α2 rather than the GABA-α1 receptor subunits linked to addiction, by using selective benzodiazepine agonists for each receptor type (van der Kooij et al., 2018a).", "Recently, Rincón-Cortés et al. (2018) showed that a low systemic dose of diazepam could alleviate the negative symptoms of amphetamine withdrawal by ameliorating anxiety-like and social behavior (Rincón-Cortés et al., 2018).", "This treatment attenuated inhibitory GABA-ergic control, thereby increasing the number of spontaneously firing VTA neurons.", "As has been examined in detail by Antony Grace’s group, the number of cells showing this firing pattern represents tonic activity of the VTA neurons as a determinant of the gain of phasic DA bursts during amphetamine withdrawal (Belujon and Grace, 2017).", "The discovery has important implications for the potential of benzodiazepine analogs to increase social competence and to attenuate anhedonic symptoms in patients suffering from anxiety- and depressive disorders (Soria et al., 2018).", "In conclusion, the findings rekindle the scheme by Belujon and Grace on the prefrontal-limbic-VTA circuitry with its activating hippocampal and inhibiting amygdala inputs.", "The new experiments suggest that local manipulation of GABA-ergic interneurons in the VTA as well as the utilization of energy substrates in the NAc has profound consequences for mesolimbic DA activity, and its function in the rewarding, emotional, and motivational aspects of social interaction.", "Interestingly, a recent study demonstrated a role for CRH in the modulation of social interaction in GABA-ergic terminals (Dedic et al., 2019).", "However, the underlying mechanism of GABA-ergic control of VTA-DA excitability requires further study.", "For instance, it is unclear to what extent the GABA-dependent suppression of the inhibitory afferent input versus potentiation of the GABA-ergic interneuron input is involved.", "Acute stress responses trigger powerful adaptive reactions in order to protect and to prepare body and brain for coping with similar environmental challenges in the future.", "In this section, the effects of chronic stress on excitatory and inhibitory transmission in the VTA-DA circuit will be discussed (see Tables 1 and 2; Fig. 3).", "It appears that neurons in the VTA may be lost and we highlight the role of microglia in this neurodegenerative process.", "Chronic stress exposure can induce morphohological changes in VTA-DA neurons, as demonstrated in a study by Kaska et al. (2017) who reported that in mice susceptible to chronic social defeat stress (CSDS) the VTA-DA neuron soma size decreased (Kaska et al., 2017).", "In this study, western blot analysis on micro-dissected VTA tissue revealed a decreased level of phosphorylated cofilin – a protein which can disassemble cytoskeletal actin filaments.", "Therefore, the authors hypothesize that chronic stress may alter – amongst many other effects – the cytoskeleton of VTA-DA neurons.", "Moreover, the chronic stress-induced shrinkage of neuronal soma sizes may be related to the diminished availability of neurotrophic factors (Chu et al., 2007; Stockmeier et al., 2004).", "Furthermore, chronic stress exposure can additionally result in excessive loss of dopaminergic neurons in the VTA (Sugama and Kakinuma, 2016).", "By using immunohistochemical and in situ hybridization techniques, Sugama and Kakinuma (2016) showed that a 16-week chronic restraint stress (CRS) paradigm can induce dopaminergic neurodegeneration in male Wistar rats (Sugama and Kakinuma, 2016).", "They demonstrated that the number of tyrosine hydroxylase (TH)-immunoreactive and mRNA expressing neurons in the VTA were decreased down to 40 percent as compared to control animals after 16 weeks of CRS.", "The TH positive cells were also positive for the neurodegeneration marker Fluoro-Jade B in the CRS exposed animals (Sugama et al., 2016).", "Together, these studies imply that chronic stress exposure can induce profound morphological changes and, if extended to a longer time, may cause loss of VTA-DA dopaminergic neurons.", "The stress-induced loss of dopaminergic neurons awaits further confirmation, however.", "In support, male Wistar rats exposed daily for 3.5 h to a combination of chronic unpredictable stressors during three weeks showed in vivo under urethane anesthesia with magnetic resonance imaging (MRI) a decreased volume of VTA, NAc, hippocampal, and cortical regions.", "This decrease in VTA volume was accounted for by rats that were highly responsive to the stressor.", "However, connectivity was found increased between VTA and the ventral subiculum of the hippocampus, with an inflection at day 7 of stress exposure (Magalhães et al., 2017).", "The findings on chronic-stress-induced VTA degeneration align with parallel decreases in population activity (see Belujon and Grace, 2015; Grace, 2016); the reduced amount of tonic firing neurons can be explained by the possible VTA-DA neuronal cell loss.", "In contrast, using 10-day CSDS the “susceptible” (the defeated) rather than the resilient mice showed increased spine density of VTA and NAc neurons, as opposed to decreased spine density in the prelimbic PFC and hippocampal CA3 and DG neurons.", "In the same mouse CSDS paradigm, using ex vivo structural imaging, the volume of the VTA was found increased, while that of the NAc was decreased in correlation with the severity of social avoidance used as a criterion for susceptibility (Anacker et al., 2016; Chaudhury et al., 2012).", "It cannot be excluded therefore that the presumed neuronal loss is preceded by an overexcitation of VTA-DA neurons, however, because of the transiently increased spine density (Hausknecht et al., 2013; Christoffel et al., 2011; Qu et al., 2017), the enhanced firing of the ventral subiculum-mesolimbic DA connection, and the increased BDNF expression (Krishnan et al., 2007) in the CSDS susceptible animals (see also Sections 2.2, 2.5, 4.1,", "and 5).", "If this increase in spine density also occurs in the face of neuronal loss during prolonged stress, this may indicate that the connectivity patterns in the remaining VTA-DA neurons are strengthened which, in turn, may have consequences for VTA-DA dependent modulation of behavioral patterns (Magalhães et al., 2017).", "However, it is unclear to what extent these structural changes are linked to pathology or whether they reflect an adaptive response.", "In the next sections, the role of excitatory and inhibitory transmitters will be examined.", "In addition to structural plasticity of VTA-DA neurons, chronic stress exposure can promote long-lasting functional changes at the synaptic level.", "These include changes at excitatory synapses and, since excitatory input guides the switch from tonic firing to phasic firing activity, these synapses largely determine the eventual dopaminergic output.", "The main source of excitatory input onto VTA-DA neurons derives from glutamatergic innervation of the pedunculupontine tegmentum (PPTg) and habenular nucleus, targeting the ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and N-methyl D-aspartate receptors (NMDARs).", "Only few studies have addressed the effects of chronic stress exposure on the modulation of glutamatergic synaptic transmission.", "Yet, even though diverse stress paradigms were used, the results of these studies are fairly in harmony and are implying that chronic stress exposure induces an enhanced excitability of VTA-DA neurons.", "This is demonstrated by early work showing that both 10 days of chronic unpredictable stress (CUS) or CRS increased the global expression of GluR1 (an AMPAR subunit) and NMDAR1 (a NMDAR subunit) in the VTA (Fitzgerald et al., 1996), but see Toth et al. (2008), who did not see such changes after 4 weeks of CUS.", "In addition, other studies, using CSDS and social isolation stress paradigms, demonstrated chronic stress-enhanced long-term potentiation (LTP) of NMDAR-mediated glutamatergic synaptic plasticity in the VTA, which was also shown in animals with a history of social isolation (Stelly et al., 2016; Whitaker et al., 2013).", "NMDAR LTP is facilitated by protein kinase A (PKA)-dependent and metabotropic glutamate receptor (mGluR)-mediated inositol 1,4,5-triphosphate (IP3)-induced Ca2+ signaling (Stelly et al., 2016): mGluRs mediate the generation of IP3, leading to IP3-induced Ca2+ signaling.", "This is gated by phosphorylation of the IP3 receptor (IP3R) by PKA, which increases the IP3 sensitivity of this receptor.", "In the same study, however, Stelly et al. (2016) found, in contrast to Fitzgerald et al. (1996), no CSDS-induced changes in global NMDAR-mediated excitation.", "In another study, GluR1 subunit protein expression levels in the VTA were increased 10 days after the final defeat of an intermittent social defeat stress (ISDS) paradigm, while NMDAR1 expression levels were unaffected (Covington et al., 2008).", "Interestingly, enhanced afferent glutamatergic innervation from the ventral hippocampus to the NAc determined the phenotype of the susceptible animals in the 10-day CSDS model.", "The phenotype is reversed by evoking long-term depression (LTD) to attenuate the excitatory input.", "This effect is specific for the hippocampal input, since opposite effects are observed after optogenetic stimulation of mPFC and basolateral amygdala (BLA) (Bagot et al., 2015).", "The data support the finding that in the 10-day CSDS model phasic rather than tonic optogenetic stimulation of the VTA-DA neurons rapidly induced a susceptible phenotype characterized by reduced sucrose preference and social avoidance.", "The effect of optogenetic stimulation was mediated by the mesolimbic- rather than the mesocortical DA pathway (Chaudhury et al., 2012) (see Sections 2.1, 2.5 and 5).", "Overall, only few studies have addressed how exposure to chronic stress affects glutamatergic synaptic plasticity onto VTA-DA neurons.", "These studies, however, are relatively congruent and imply that chronic stress exposure may result in a transient increased excitability of VTA-DA neurons.", "However, in one CUS stress paradigm no changes in GluR1 and NMDAR1 expressions were found (Toth et al., 2008).", "This paradigm of Toth et al. (2008) lasted four weeks, while in all other studies stress exposure lasted for a maximum of 10-12 days (Covington et al., 2008; Fitzgerald et al., 1996; Stelly et al., 2016; Whitaker et al., 2013).", "Inhibitory synaptic transmission in the VTA is also susceptible to undergo plasticity (Xin et al., 2016).", "While excitatory input is essential for the transition from tonic to phasic firing activity, GABA-ergic inhibition can keep VTA-DA neurons in a hyperpolarized, inactive state, thereby silencing these neurons.", "Moreover, this inhibitory synaptic transmission is thought to underlie extinction of learned behaviors and to moderate excessive VTA-DA neuron excitability (Xin et al., 2016).", "Inhibitory input onto VTA-DA neurons is mediated by GABAARs and GABAB receptors (GABABRs).", "While the postsynaptic ionotropic GABAARs act by fast synaptic transmission, presynaptic GABABRs are slower metabotropic channels linked to G-protein coupled inwardly rectifying potassium channels (GIRKs) (Lüscher et al., 1997).", "In addition to excitatory synaptic plasticity, the importance and prevalence of inhibitory plasticity is becoming increasingly apparent (Xin et al., 2016).", "The mechanisms by which inhibitory synapses on VTA-DA neurons can strengthen or weaken activity are mainly derived from studies in the field of addiction.", "Increase in inhibitory synaptic strength is a result of LTP of GABAA currents.", "While for LTP of AMPA currents AMPAR activation is required, activation of GABAARs is not required for LTP of GABAA currents (Nugent et al., 2007; Nugent and Kauer, 2008).", "Instead, as demonstrated in ex vivo experiments in tissue slices, GABA-ergic synaptic plasticity derives from glutamatergic terminals, and is driven by NMDARs located on the VTA-DA neurons.", "Upon NMDAR activation, the induced rise in postsynaptic calcium levels results in the Ca2+/calmodulin-dependent protein kinase (CAMK)-mediated activation of nitric oxide synthase (NOS), thereby catalyzing the synthesis of nitric oxide (NO).", "In turn, by traveling retrogradely to the presynaptic GABA-ergic neuron, where it increases the levels of cyclic guanosine monophosphate (cGMP) by activating soluble guanylyl cyclase (sGC), NO is able to mediate long-term enhancement of GABA release onto dopamine neurons (Nugent et al., 2007; Nugent and Kauer, 2008).", "Thus, this enhancement of GABA-ergic input can decrease the excitability of VTA-DA neurons.", "Similar to LTD in excitatory glutamatergic synapses, inhibitory synaptic strength can decline.", "This is also the case for VTA-DA neurons (Xin et al., 2016).", "Repeated cocaine administration can result in a decreased GABAAR-mediated inhibition of VTA-DA neurons, thereby facilitating LTP at glutamatergic synapses onto these neurons, as demonstrated in tissue slices (Liu et al., 2005).", "In the decrease of GABA-ergic postsynaptic responses the activation of group I mGluRs on the postsynaptic VTA-DA neurons may be involved (Quraishi and Paladini, 2017).", "The activation of these mGluRs leads to the synthesis of endocannabinoids, which bind to the G protein-coupled cannabinoid receptor type 1 (CB1) located on presynaptic GABA-ergic neurons (Chevaleyre and Castillo, 2004, 2003).", "Therefore, endocannabinoids show trans-synaptic retrograde signaling in a similar manner as NO in GABA LTP processes, causing a decrease of GABA-ergic input to VTA-DA neurons.", "Moreover, endocannabinoid signaling interacts with opioids in the modulation of the VTA circuit underlying reward processing and reinforcement behavior (Wenzel and Cheer, 2017).", "However, this interaction is very complex and to date there is very little work on linking these three signaling systems, let alone linking stress to their interaction.", "The role of the endogenous opioid system in (stress-related) VTA-DA neuron activity is discussed in more detail in Section 4.3.", "Nevertheless, endogenous opioid and endocannabinoid signals are predominantly involved in the suppression of presynaptic GABA-ergic input to VTA-DA neurons, which results in enhanced excitability of the VTA-DA neurons (Van’t Veer and Carlezon, 2013; Polter and Kauer, 2014; Covey et al., 2017).", "Only few studies have addressed chronic stress-induced changes in GABA-ergic synaptic strength.", "Krishnan et al. (2008a, 2008b) used a 10-day CSDS paradigm and found significant decreases in Akt (thymoma viral proto-oncogene) phosphorylation in the VTA of susceptible rats (Krishnan et al., 2008b).", "Since Akt can increase membrane insertion of GABAARs by β2-subunit phosphorylation, the inhibition of Akt is sufficient to increase VTA-DA neuron excitability through GABAA-dependent mechanisms (Krishnan et al., 2008a,b; Wang et al., 2003).", "In addition, Akt is involved in the phosphatidylinositol 3 kinase (PI3K)/Akt/glycogen synthase kinase 3 (GSK3)/mammalian target of rapamycin (mTOR) pathway in cell signaling.", "The PI3K/Akt/GSK3/mTOR signaling pathway is important in subcellular integration of synaptic neurotransmission, and its function is linked to the regulation of metabolism, cell growth, cell survival, and thus neuronal excitability (Abelaira et al., 2014; Kitagishi et al., 2012; Polman et al., 2012).", "Together, these results are in line with a chronic stress-induced increased excitability of VTA-DA neurons.", "To our knowledge, no direct evidence for strengthened GABA-ergic synapses on VTA-DA neurons upon chronic stress-exposure has been reported.", "Interestingly, however, Warren et al. (2013) found with a 10-day CSDS paradigm a long-lasting significant decrease in δ-GABAAR expression in the VTA (Warren et al., 2013).", "δ-GABAARs mediate extrasynaptic tonic inhibition and are strongly regulated by neurosteroids – which are synthesized de novo in the brain from progesterone or deoxycorticosterone precursors (Reddy, 2010; Brickley and Mody, 2012).", "While one study implies an indirect mechanism that may increase inhibitory synaptic transmission, most studies hint that exposure to chronic stress weakens inhibitory synaptic strength onto VTA-DA neurons, thereby contributing to an increased excitability of these neurons.", "In order to form a proper behavioral response to stress and to prioritize metabolic needs, neural, immune, and endocrine products of the stress response need to be integrated.", "Microglia, the main cellular actors of the brain’s immune defense network (Ransohoff and Cardona, 2010), have the necessary repertoire of receptors to serve such an integrative role on a local neuronal level (Frank et al., 2019).", "Moreover, by secretion of cytokines, prostaglandins, and growth factors, microglia can shape neuronal networks and contribute to modulation of synaptic function including that of the VTA-DA neurons (Delpech et al., 2015).", "The midbrain contains 4.5 times more microglial cells than other brain regions (Kim et al., 2000; Sugama and Kakinuma, 2016) and DA neurons show higher susceptibility to neuroinflammatory signals than other brain cells (Block and Hong, 2007; Gao et al., 2003).", "While most of glial functions are health-promoting, their neuroinflammatory reaction to insults may overshoot and cause exacerbation of neuronal damage (Block and Hong, 2007).", "Various studies have demonstrated that acute and chronic stress exposure can affect inflammatory processes (Dowlati et al., 2010; Gilman et al., 2013; Kendler et al., 1999; McLaughlin et al., 2010; Miller et al., 2008; Frank et al., 2019).", "Chronic stress exposure showed profound effects on microglia in the VTA, both in structural and functional ways (Table 2).", "In the previously described study where a 16-week CRS paradigm induced VTA-DA neuronal cell loss, it was found that stress increased intra-VTA microglial cell body sizes significantly (Sugama and Kakinuma, 2016).", "Enlarged microglial body sizes imply an activated status, and these enlargements were observed both after acute prolonged (8 h session) stress and, although relatively lesser, after chronic (16-weeks) stress.", "Furthermore, Tanaka et al. (2012) showed a CSDS-induced increase in reactivity of ionized calcium-binding adaptor protein-1 (Iba1), which is a marker of morphological changes in activated microglia (Tanaka et al., 2012; but see Tynan et al., 2010).", "In the study by Sugama and Kakinuma (2016), the possible comorbidity between neuronal cell loss and increased microglial activation may imply a role for microglial neuroinflammatory mechanisms in chronic stress-induced adaptations of VTA-DA neurons (Sugama and Kakinuma, 2016).", "Moreover, since microglial-induced inflammatory mediators, including cytokines, can orchestrate physiological and behavioral responses (Dantzer et al., 2008), these modulations may have consequences for stress-induced behavior during health and disease.", "Various studies, indeed, have implicated elevated cytokines in stress-related disorders or their symptoms (Dowlati et al., 2010; Felger and Miller, 2012; Dantzer et al., 2008).", "Moreover, chronic stress-induced alterations in microglial neuro-inflammatory functionality is implicated in neurodegenerative diseases (Lull and Block, 2010; Réus et al., 2015).", "In this link between microglia and the vitality of (dopaminergic) neurons, the involvement of microglia in regulating homeostatic processes of local brain environments may be of crucial importance (Colonna and Butovsky, 2017).", "As discussed in Section 3.2, mGluR/IP3-enhanced promotion of intracellular Ca2+-release is involved in synaptic plasticity processes (Stelly et al., 2016; Whitaker et al., 2013).", "Increased intracellular Ca2+ concentrations are featured in pathological cellular states associated with oxidative stress and excitotoxicity, processes known to promote apoptosis (Radley and Morrison, 2005; Salido, 2009; Dantzer and Walker, 2014; Pan et al., 2017).", "Thus, the enhanced neuronal reactivity may occur at the expense of intra-cellular mechanisms, putting their homeostatic limits to the test and ultimately may result in neuronal degeneration.", "Consequently, this may lead to a structural decrease in tonic firing population activity, and therefore produce a structural decrease in vigor of VTA-DA neuron behavior modulating effects.", "Accordingly, the degenerated VTA-DA neuronal network may be causal to depressive pathology, if translated to humans (Belujon and Grace, 2017; Cabib and Puglisi-Allegra, 2012; Tye et al., 2013; Wood and Bhatnagar, 2015).", "Interestingly, glucocorticoids regulate neuroinflammation controlled by microglial cells in a sequential pattern.", "First, via MR the hormone acts initially pro-inflammatory (Brocca et al., 2017; de Kloet et al., 2018).", "Next, via GR the well-known anti-inflammatory and recuperative phase occurs.", "Finally, microglial cells can be primed in their response by the inflammatory experience (Frank et al., 2015).", "Specifically, during inflammation the recuperative anti-inflammatory action of glucocorticoids via GR can leave a footprint, the so-called inflammasome.", "This ‘memory’ of the brain’s immune response ensures that microglia are prepared for a next time and can support an immediate pro-inflammatory response to damage and infections in the future (Frank et al., 2015).", "Such adaptive changes of microglial neuroinflammatory activity support optimal allocation of energy resources when faced with stressful environmental challenges (McNamara, 2005; Rauw, 2012).", "In conclusion, a role of microglial activation and cytokines in VTA-DA function during chronic stress cannot be excluded and requires further study.", "It should be noted, however, that besides the locally produced inflammatory signals, peripheral inflammatory responses can exert profound effects on (dopamine-related) motivated behaviors as well (Felger and Treadway, 2017; Stanton et al., 2019).", "The response of the VTA-DA neurons to stressors is context-dependent and is generally implicated in emotional-motivational appraisal processes for assessment of valence.", "The activity of the VTA-DA neurons can guide and modulate various coping responses and adaptive behaviors, including reinforcement learning and behavioral flexibility.", "Given that VTA-DA neurons are highly innervated by afferents from various brain regions, particularly of mPFC and limbic origin (Belujon and Grace, 2017), the VTA-DA-mediated behavioral effects are dependent on the balance of specific excitatory, inhibitory, and modulatory inputs.", "In response to acute stressors, such an integration of presynaptic inputs leads eventually to fluctuations in both tonic and phasic VTA-DA activity.", "In general, tonic firing population activity reflects the state and can be affected by acute stress, while phasic responses, on the other hand, are more spatiotemporal specific and may vary over different stressors and stressor intensities.", "These VTA-DA responses to stress are thought to result in increased vigilance and to support behavioral activation, with the goal to attain control over the stressor by using one’s own resources.", "Chronic exposure to stress, however, can lead to profound changes in VTA-DA neuron activity, not only in response to the stressors, but also to other behaviorally relevant stimuli.", "Such chronic stress-induced changes in VTA-DA reactivity vary over methodological procedures, especially with respect to the tonic firing population activity.", "When an animal has been exposed to chronic stress and is subsequently facing a stressor of a different nature, phasic responses generally appear sensitized.", "These changes in VTA-DA activity are promoted by plasticity induced by glutamatergic excitatory and GABA-ergic inhibitory synaptic input.", "The reported synaptic adaptations show that chronic stress exposure mainly alleviates the GABA-ergic inhibitory input onto VTA-DA neurons and facilitates excitatory synaptic transmission, which is in line with the sensitized VTA-DA activity responses.", "The chronic stress-induced alterations in VTA-DA neurons rather reflect vulnerability.", "However, although current knowledge is far from complete, one can conclude that chronic exposure to stress is capable of causing long-lasting changes, and may lead, after possibly an initial transient activation (Krishnan et al., 2007; Chaudhury et al., 2012; Magalhães et al., 2017) over time to degeneration or perhaps even loss of VTA-DA neurons, a process in which microglia activation participates (Tables 1 and 2, and Fig. 3).", "These neuroadaptations involve both functional changes, predominantly leading to a transiently increased VTA-DA neuron excitability, morphological changes, and altered connectivity, potentially increasing the risk for stress-related and neurodegenerative diseases (Abelaira et al., 2014; Ceretta et al., 2012; Hoyo-Becerra et al., 2014; Iseme et al., 2014; McInnis et al., 2014).", "The exact link between increased VTA-DA neuron excitability, neuroinflammatory responses, and neurodegeneration is, however, not yet well understood.", "The excitation-inhibition balance is crucial for the function of the VTA-DA circuit and can be modulated by a variety of local and systemic factors that are induced by stress.", "While these stress-induced factors are essential in neuroadaptive processes, each one by itself is believed to be insufficient to cause the behavioral changes seen after (chronic) stress exposure.", "Moreover, these stress mediators act conditionally and presumably rely on the nature and integration of excitatory input (Joëls and Baram, 2009).", "We will discuss four classes of mediators: BDNF, as an example of a locally acting trophic factor, the CRH neuropeptide, as the central organizer of the stress response, the endogenous opioid system, having addictive properties while also released as a consequence of stressors, and the glucocorticoid hormones.", "The latter will be addressed in a separate section (Section 5).", "The structure and function of VTA-DA neurons is affected by neurotrophins, which are key mediators of growth and neuronal plasticity (de Azevedo Cardoso et al., 2014; Russo et al., 2009).", "Although there are four neurotrophin family members (nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and BDNF) expressed in the brain, we focus on the action of BDNF (Nikulina et al., 2014), which is best investigated in the hippocampus and the VTA-DA system.", "The expression of BDNF is activity-dependent and is affected by stress and glucocorticoids.", "For instance, if coping is successful, glucocorticoids are permissive and hippocampal BDNF expression is increased, but downregulated after exogenous glucocorticoids or chronic stress (Schaaf et al., 1998, 2000).", "Its downregulation in hippocampus has been implicated in stress-related mood disorders, and many current antidepressant drugs, that are used to treat these disorders, promote BDNF expression and signaling (Calabrese et al., 2009; Hashimoto et al., 2004; Post, 2007).", "BDNF binds to the tropomyosin-receptor kinase B (TrkB) receptor and TrkB signaling leads to the activation of multiple intracellular signaling cascades, including PI3K/Akt/GSK3/mTOR, Ras/extracellular signal-regulated kinase (ERK), and phospholipase C-γ (PLCγ) (Nikulina et al., 2014).", "Chronic stress-induced alterations in BDNF activity vary over stress paradigms and show regionally different response patterns in the brain.", "After ISDS (Fanous et al., 2010), BDNF expression was decreased in hippocampus.", "Using CSDS, BDNF was also decreased in hippocampus, but increased in the VTA and NAc of rats and mice (Krishnan et al., 2007).", "In the latter study a 10-day CSDS paradigm was used to differentiate male C57Bl6 mice in susceptible and resistant phenotypes as judged from their performance in a social interaction test (Krishnan et al., 2007; Berton et al., 2006).", "The susceptible mice showed social avoidance towards a conspecific control, a passive coping style in the forced swim test, and reduced preference for sucrose.", "In susceptible mice, BDNF expression is increased in the VTA-NAc circuit, rather than the decrease commonly observed in hippocampus.", "The susceptible phenotype could be reversed to a resistant variant with deletion or suppression of BDNF expression in the VTA (or alternatively by stimulating its overexpression in hippocampus) (Berton et al., 2006).", "In the studies by Miczek et al. (2011) a 10-day ISDS also produced increased DA release and BDNF expression in the VTA-NAc system (Miczek et al., 2011), while a continuous CSDS paradigm (36 days) showed the opposite down-regulatory effect.", "In contrast to the various social defeat stress paradigms, a four-week as well as a 38-day lasting CUS paradigm left BDNF expression levels in the VTA unchanged, however (Gersner et al., 2010; Toth et al., 2008).", "BDNF is considered as critical for survival and function of VTA-DA neurons, but the effects of chronic stress exposure are diverse and vary over stress procedure and susceptibility of the animals.", "While after stress BDNF expressions levels are initially rising, there is some evidence that prolonged stress exposure may curtail the BDNF increase.", "Moreover, BDNF increases seem to be dependent on disinhibition of VTA-DA neurons, as VTA μ-opioid receptor (MOR), see Section 4.3, knockdown blocks induction of VTA BDNF expression and prevents social defeat-induced cross-sensitization to amphetamine (Johnston et al., 2015).", "Furthermore, since BDNF activity is dependent on inhibitory control (Johnston et al., 2015) and strongly associates with enhanced (ERK activity dependent) stimulus-response behavior (Grimm et al., 2001; Lu et al., 2009, 2004; Neisewander et al., 2000), the effects of BDNF on VTA-DA neuron excitability seem to be stimulus-dependent and rely on the integration of excitatory input (Leonard et al., 2017).", "Nevertheless, although the effects of chronic stress exposure on BDNF-mediated signaling are complex, the growth factor appears to be an important permissive mediator of neuronal remodeling.", "High VTA and low hippocampal BDNF expression in post-mortem tissue of depressed patients matched the opposite BDNF levels in these two brain regions of the male susceptible CSDS mice (Krishnan et al., 2007).", "Moreover, the Bdnf G196A (Val66Met) single nucleotide polymorphism (SNP) impairs BDNF signaling and contextual learning, and is associated with psychopathology (Chen et al., 2014), a finding that probably refers to hippocampal BDNF expression.", "In translational studies with these SNPs, transgenic BDNF Met/Met mutants appeared resilient to CSDS and expressed 50 % of the BDNF found in the ventral striatum of the Val/Val mutants (Krishnan et al., 2007).", "Interestingly, a similar CSDS-induced pattern was recently found for the induction by NGF of the Vgf gene and its C-terminal AQEE-30 and TLQP-62 neuropeptide (Lin et al., 2014; Mo et al., 2015).", "Although these data are very interesting, they add to the conundrum that increased bio-availability of the growth factors and enhanced VTA-DA activity would predispose for susceptibility in the CSDS model, while exposure to chronic unpredictable stressors produces a similar susceptible phenotype, but is characterized by decreased VTA-DA function.", "In conclusion, BDNF is an important regulator of VTA-DA function; its effects are conditional and seem related to the extent of controllability, duration, and severity of the stressor.", "CRH orchestrates the autonomic and neuroendocrine response to stress and coordinates the various emotional expressions as part of the behavioral adaptation.", "These actions exerted by CRH occur widespread in the brain and are mediated by CRH-R1 and the lower affinity CRH-R2 receptors; the latter receptors actually respond predominantly to urocortin (Hsu and Hsueh, 2001; Reyes et al., 2001).", "Using in situ hybridization of mRNA expression or autoradiography of radioligand binding, CRH-R1, but not CRH-R2, was found co-localized with DA markers in neurons of the VTA (Van Pett et al., 2000; Tan et al., 2017; Kelly and Fudge, 2018).", "Yet, acute activation of VTA-CRH-R2 receptors amplified NMDA excitatory currents (Ungless et al., 2010).", "Furthermore, CRH strongly affects the excitability of VTA-DA neurons upon stress exposure (Holly et al., 2016; Korotkova et al., 2006; Leonard et al., 2017; Wang, 2005).", "With in vivo microdialysis, phasic release of CRH was demonstrated in the posterior VTA during the acute stress of social defeat, but when stress exposure is repeated CRH is also recruited in the anterior VTA (Holly et al., 2016).", "This effect of CRH exerted on VTA-DA neuronal firing involves protein kinase C (PKC)- rather than cAMP–protein kinase A (PKA)-dependent enhancement of Ih currents (Korotkova et al., 2006; Wanat et al., 2008).", "The chronic stress paradigm also increased cocaine self-administration, which could be prevented by antagonism of CRH-R1 in the posterior VTA and of CRH-R2 in the anterior VTA prior to each stress session (Holly et al., 2016).", "Leonard et al. (2017) studied cocaine-taking behavior and compared the effects of repeated CRH microinjections in the VTA of male rats with a CSDS paradigm (Leonard et al., 2017).", "They found that both protocols produced intense, but slightly different, patterns of drug taking behavior.", "The authors of this recent study suggest that the effects of CRH in the VTA may be stimulus-dependent, and that the chronic stress-induced increase in drug bingeing behavior is independent of reward valuation.", "Instead, chronic stress exposure impairs inhibitory control and therefore empowers the stimulus-dependent effects of CRH (Leonard et al., 2017).", "Besides effects on addiction, reward, and locomotion (Wang, 2005; Kalivas, 2009), the best known action of CRH is on fear-motivated behavior.", "This effect on fear expression involves the recently discovered GABA-ergic neurons that co-express CRH in the extended amygdala – comprising the BNST and central amygdala – that has mono-synaptic projections to the VTA (Dedic et al., 2018).", "Stimulation of this subpopulation of CRH neurons appeared to increase dopamine release from VTA-DA neurons and to exert anxiolytic effects (Dedic et al., 2018).", "Under stressful conditions and during the withdrawal phase of addictive behavior, CRH, however, aggravates anxiety (Lemos et al., 2012; Wanat et al., 2013).", "Recent research has demonstrated that trait anxiety is actually a determinant in the outcome of VTA-CRH-R1 stimulation by either stress exposure or exogenous local CRH application.", "In rats selected for low anxiety these manipulations improved motivation to collect a reward, while behavior of the high anxious rat was impaired.", "Intra-VTA infusion of CRH in the low anxious rats evoked a larger DA response than in their high anxious littermates (Zalachoras et al., 2018).", "It is noteworthy that Lemos et al. (2012) revealed in male mice the importance of CRH in stress responses at the terminal level of VTA-DA transmission (Lemos et al., 2012).", "While CRH results in increased VTA-DA release in the NAc, a two day repeated forced swim stress paradigm abolished this effect for at least 3 months.", "Furthermore, in naïve animals (pre-stress) intra-NAc microinjections with CRH promoted conditioned place preference behavior (indicated as appetitive), while after the swim stressor the CRH effect switched to conditioned place aversion.", "Interestingly, treatment with the GR antagonist prior to the forced swim reinstated the appetitive CRH effect (Lemos et al., 2012).", "Although the effect of CRH seemed to involve co-activation of both CRH-R1 and CRH-R2 receptors in the NAc, the precise mechanism underlying this stress-induced switch in function of CRH has yet to be fully elucidated.", "It might be linked to the stress-induced increase in use of habitual (stimulus-response) instead of cognitive behavioral strategies, which may occur after blockade of GR when MR activation is privileged (Dias-Ferreira et al., 2009; Leonard et al., 2017; Wanat et al., 2013; Schwabe et al., 2013) (see Section 5.3).", "In conclusion, the effects of CRH on VTA-DA neuron excitability seem to be conditional and, therefore, stimulus-dependent (Leonard et al., 2017; Wanat et al., 2013).", "Although these CRH-DA interactions are best studied in locomotion, addiction, and reward, recent research highlights emotionally valenced social behaviors driven by CRH expressing GABA-ergic projections from the extended amygdala.", "Since glucocorticoids induce amygdala CRH and can modulate VTA-DA function it is of interest to examine how these hormones are implicated in control of VTA-DA function (see Section 5).", "Besides BDNF and CRH, we also wish to highlight the role of the endogenous opioid system in VTA control.", "While this neuromodulatory system, which is famously known for its abuse liability, is involved in widespread physiological processes, including pain modulation, respiratory function, and gastrointestinal transit, the endogenous opioids are also linked to motivated behavior and, specifically, the modulation of VTA-DA activity in stressful situations (Miller et al., 1984; Kalivas and Abhold, 1987; Cabib et al., 1989; Latagliata et al., 2014; Margolis and Karkhanis, 2019).", "Moreover, endogenous opioids are massively released during stressful situations (Latagliata et al., 2014).", "We will not elaborate on the endogenous opioid system in full detail, since excellent articles have appeared, including their interaction with glucocorticoids (Witkin et al., 2014; Benarroch, 2012; Henry et al., 2017; Szklarczyk et al., 2016).", "Briefly, the endogenous opioid system consists of four major subtypes of G protein-coupled opioid receptors, the MOR, the δ-opioid receptor (DOR), the κ-opioid receptor (KOR), and the nociceptin receptor (NOPR), which can be targeted by four families of opioid peptides, viz. β-endorphin, enkephalins, dynorphins, and nociceptin/orphanin FQ (N/OFQ).", "These opioid peptides derive from four different precursor proteins: POMC, which is the same precursor protein as for ACTH, proenkephalin, prodynorphin, and prepronociceptin, respectively.", "Current literature suggests that DA and opioid stimulation have similar behavioral effects (Callaghan et al., 2018).", "While acting on various intracellular signaling cascades (Al-Hasani and Bruchas, 2011), all opioid receptors share that they inhibit cAMP formation upon receptor agonist stimulation.", "While opioids may act on VTA-DA neurons directly, at the cell bodies within the VTA, or at terminals at projection areas, the most profound effects of the endogenous opioid system on VTA-DA dynamics are pathway specific and act indirectly (Langlois and Nugent, 2017; Thomas et al., 2018; Callaghan et al., 2018).", "Stress-induced alterations within the endogenous opioid system may contribute to changes in VTA-DA dynamics, ultimately supporting the development of maladaptive behavior, but stress-induced changes in VTA-DA dynamics can also be modulated by opioids (Callaghan et al., 2018; Przewlocki and Almeida, 2017).", "In this section we will describe the effects of the endogenous opioid system on VTA-DA neuron dynamics and discuss how stress can alter these dynamics.", "Various studies show that stress-induced alterations in dopamine activity and behavioral outcome can be modulated by opioids (Kalivas and Abhold, 1987; Latagliata et al., 2014; Callaghan et al., 2018; Tejeda and Bonci, 2019; Kalivas et al., 1988; Przewlocki and Almeida, 2017).", "For example, daily exposure of mild footshock stress enhances the motor stimulatory effect of intra-VTA administration of an enkephalin analog, and daily intra-VTA administration of this opioid receptor agonist led to an elevated dopaminergic response in the NAc.", "Interestingly, pre-stress treatment with naltrexone, a competitive antagonist for opioid receptors, augmented these dopaminergic responses (Kalivas et al., 1988).", "More specifically, MOR activation is linked to changes in VTA-DA responses to stress (Latagliata et al., 2014; Nikulina et al., 2008).", "In a study where an acute restraint stress paradigm was combined with microdialyis, Latagliata et al. (2014) showed that MOR activation can reduce NAc DA tone via enhancement of DA transmission in the mPFC (Latagliata et al., 2014).", "In studies from Nikulina et al. (2005, 2008), 5 days of CSDS exposure increases mRNA expression of MOR in the VTA for up to 14 days after the last episode of stress (Nikulina et al., 2005, 2008).", "Predominantly, the effects of MOR activation on VTA-DA neuron activity are indirect, as they act presynaptically on GABA-ergic afferents derived from the ventral pallidum and RMTg, and result in tonic inhibition of this input onto VTA-DA neurons (Steffensen et al., 2006; Chieng et al., 2011; Hjelmstad et al., 2013; Matsui et al., 2014; but see Margolis et al., 2014).", "However, NAc-derived MOR sensitive GABA-ergic afferents to the VTA can have opposing actions on VTA-DA neurons, as NAc-derived GABA-ergic input primarily synapse to non-dopaminergic neurons in the VTA (Xia et al., 2011).", "Thus, while intra-VTA treatment with MOR agonists can inhibit GABA-ergic input directly synapsing onto VTA-DA neurons, it can also disinhibit the same GABA-ergic neurons via NAc afferent MOR activation.", "Consequently, MOR agonist studies will predominantly address the most prominent MOR sensitive inhibitory neuronal populations to the VTA, presumably the RMTg, thereby overshadowing projection-specific and subtle MOR-induced modulation of VTA-DA neuron activity (Thomas et al., 2018).", "Moreover, given the indirect nature of VTA-DA modulation via MOR, eventual dopaminergic and behavioral output relies on integration of excitatory input.", "In addition, stressors potently activate KORs within the VTA.", "KORs inhibit excitatory and inhibitory transmission onto VTA-DA neurons, somatodendritic dopamine release, and dopamine release in dopaminergic terminals at the NAc and mPFC (Margolis et al., 2003, 2005; Margolis et al., 2006; Ford, 2006; Tejeda and Bonci, 2019).", "However, VTA-DA modulating effects of VTA KOR can be species specific as in rats KOR agonists do hyperpolarize VTA-DA neurons that project to mPFC but not NAc, while in mice KOR agonists were able to inhibit dopaminergic input onto the NAc as well (Ford, 2006; Baimel et al., 2017).", "However, a KOR agonist in striatal slices reduced DA release via blockade of presynaptic nicotinic receptors (nAChRs) by inhibiting striatal cholinergic interneurons (Mamaligas and Ford, 2016), which can explain the ability of KOR to inhibit DA transmission at dopaminergic terminals in the NAc.", "Sex differences may complicate the above described findings, however, since female mice show relatively less KOR inhibition of dopamine release (Abraham et al., 2018).", "Nevertheless, KOR and stress are linked and stress can alter the way KORs modulate dopamine release (Bruchas et al., 2010; Van’t Veer and Carlezon, 2013; Karkhanis et al., 2016; Polter et al., 2017).", "This is demonstrated by Graziane et al. (2013), showing that acute stress blocks synaptic plasticity at GABA-ergic input onto VTA-DA neurons (Graziane et al., 2013), and post-stress treatment with a KOR antagonist can rescue stress-induced behavioral effects (Polter and Kauer, 2014).", "Moreover, a single exposure to a brief cold-water swim stress induces prolonged KOR activation in VTA-GABA neurons synapsing on VTA-DA neurons (Polter et al., 2017).", "While the link between DORs and dopamine dynamics is less studied than the above described opioid receptors, the most recently discovered member of the opioid receptor superfamily, the NOPR, can regulate DA transmission and is highly linked to stress (Driscoll et al., 2019; Khan et al., 2018).", "The VTA expresses NOPRs in a high density (Ikeda et al., 1998) and, additionally, high levels of TH-containing neurons in the VTA co-express NOPR mRNA, but not N/OFQ mRNA (Norton et al., 2002).", "Although this nociceptin-ergic DA modulation derives from various sources (Khan et al., 2018), it is notable to highlight that in a recent paper by Parker et al. (2019) a nociceptin-ergic population of neurons within the VTA is identified – thereby emphasizing the heterogeneity of the midbrain once again.", "These neurons contain the precursor peptide for N/OFQ, the protein called prepronociceptin, they project directly to VTA-DA neurons, and are linked to motivation in a reward-seeking behavioral paradigm (Parker et al., 2019).", "Moreover, their data suggest that the input of these neurons constrain VTA-DA neuron activity and that the NOPR is at least partially responsible for this dopaminergic modulation.", "Nevertheless, the effects of N/OFQ on dopaminergic transmission are evident, as N/OFQ inhibits dopamine transmission in striatal brain slices (Flau et al., 2002), antagonism of NOPRs demonstrates enhancement of dopaminergic transmission (Marti, 2004, 2005), and downregulation of NOPR has neuroprotective effects on dopamine neurons (Arcuri et al., 2016).", "In addition, Olianas et al. (2008) found that striatal NOPR activation inhibits TH phosphorylation, thereby limiting its potency in dopamine synthesis (Olianas et al., 2008).", "Besides these presynaptic effects, the same authors found that postsynaptically D1R signaling was inhibited as well.", "Furthermore, NOPRs have a modulatory role on the HPA-axis, suggesting that N/OFQ signaling may play a role in HPA-axis feedback mechanisms (Khan et al., 2018).", "In a study by Der-Avakian et al. (2017) NOPR mRNA was found increased in the striatum and Fos mRNA, a molecular marker for neuronal activity, decreased in the VTA of rats after exposure to a 3-day SDS paradigm (Der-Avakian et al., 2017).", "Thus, although the link with stress requires further elucidation, literature suggest a role for N/OFQ signaling in dopaminergic regulation, with NOPR activation generally inhibiting dopaminergic transmission.", "In conclusion, various studies show that stress-induced alterations in dopamine activity and dopamine-related behavioral outcome can be modulated by opioids.", "However, due to a lack of studies it is currently unclear how – especially after chronic stress exposure – the post-stress dynamics in levels of endogenous opioids affect VTA-DA neuronal excitability and plasticity.", "The above described dopamine neurotransmission modulatory role of the endogenous opioid system, however, suggest that the most prominent effects by MOR and KOR on VTA-DA dynamics are regulated in an indirect manner in inhibitory presynaptic input or at dopaminergic terminals.", "This would suggest that in the case of opioid regulation of dopaminergic terminals, activity of VTA-DA neurons would remain unperturbed and, although effects can be significant, local control is the main influence of the opioid system.", "The described N/OFQ modulation of dopaminergic neurons suggests a more direct influence on dopaminergic transmission.", "The link with stress, however, requires more investigation.", "It is well-established that glucocorticoids increase dopamine-dependent motivation and psychostimulant intake and, in excess, may cause psychotic and depression symptoms (Schatzberg et al., 1985; Marinelli and Piazza, 2002), while these effects can be attenuated by GR antagonists (DeBattista and Belanoff, 2006; Block et al., 2018) (see Section 2.1).", "In the first section we will review the role of the glucocorticoid hormone and its receptors, MR and GR, in the function and regulation of the VTA-DA circuit.", "Then, the action mediated by these receptors in limbic afferents to the VTA circuit is discussed.", "In the third section, glucocorticoid action is highlighted in the coordination of VTA-DA and limbic-prefrontocortical circuitry during stress-coping and adaptation.", "The hormone feeds back for this purpose on the very same brain circuitry that initially produced the stress response and thus attenuates its own secretion.", "Best known are the genomic actions mediated via MR and GR.", "Recently non-genomic MR- and GR-mediated actions have been discovered and identified in limbic, hypothalamic and prefrontal cortex, the immune system and other peripheral target tissues (Lösel and Wehling, 2003; Groeneweg et al., 2012), but need to be demonstrated still in VTA-DA neurons.", "Strong GR-immunoreactivity was found in the medial parts (nucleus interfascicularis and nucleus linearis caudalis; see Holly and Miczek, 2016) of the VTA along its entire rostro-caudal extent in 61 ± 9 % of all TH-immunoreactive cells (Härfstrand et al., 1986).", "This medial part of the VTA projects to the mPFC and is involved in cognitive aspects of reward processing and strongly responds to stress (Thierry et al., 1977) .", "Besides GR, the VTA circuit and its NAc and mPFC target regions do express MR as well, but in lower abundance than GR.", "MR and GR occur both abundantly in limbic afferents of the hippocampus, amygdala, and lateral septum (Arriza et al., 1988; van Eekelen et al., 1991; Ahima et al., 1991; Diorio et al., 1993; Brinks et al., 2007; Caudal et al., 2014).", "Only few experiments were directed to explore the role of MR and GR within the VTA-DA system.", "Firstly, pharmacological experiments in urethane anesthetized rats showed that under basal conditions MR and GR cooperate in sustaining a circadian pattern in release of dopamine from the NAc core (Tye et al., 2009).", "The authors conclude that this MR/GR cooperation stabilizes tonic meso-accumbens dopaminergic transmission and ensures maintenance of NAc dopamine activity in the face of changing glucocorticoid levels over the circadian cycle.", "Other experiments suggested that the corticosterone action becomes apparent in the VTA when glutamatergic afferents are activated (Overton et al., 1996).", "In addition, intra-VTA, but not intra-BLA, infusion of the MR antagonist spironolactone attenuated a conditioned fear response but did not affect locomotion and exploration in an open field test condition.", "This intra-VTA treatment with the MR antagonist decreased DA release within the BLA.", "In these experiments the GR antagonist neither was anxiolytic nor prevented dopamine release from the VTA efferent projections to the amygdala (de Oliveira et al., 2014).", "Thus, the glucocorticoid action is conditional, and MR- and GR-mediated actions cooperate with the goal to maintain VTA-DA activity.", "During acute stress, a GR-mediated action activates the VTA-DA neurons.", "Hence, the effect of stress exposure on dopamine can be altered by antagonizing GR function (Chen et al., 2016; DeBattista and Belanoff, 2006; Goshen et al., 2008; Gourley and Taylor, 2009; Kvarta et al., 2015; Wu et al., 2007).", "On the cellular level, CSDS-induced facilitation of LTP of NMDAR-mediated transmission in VTA-DA neurons requires GR activation (Stelly et al., 2016).", "The GR antagonist mifepristone administered prior to each defeat session prevented inositol IP3R sensitization – which was otherwise necessary to increase VTA-DA neuron excitability.", "Interestingly, various studies suggest that this glucocorticoid-mediated regulation of VTA-DA neuron excitability is not regulated by GR expressed in VTA-DA neurons, but by GR expressed in the VTA’s main projection areas, i.e. the dopaminoceptive neurons in prefrontal cortical areas and ventral striatum (Ambroggi et al., 2009; Barik et al., 2013; Butts et al., 2011).", "In the study by Barik et al. (2013), adult mice exposed to three weeks CSDS showed symptoms of anxiety and social aversion (Barik et al., 2013).", "Genetic deletion of the GR in the dopaminoceptive neurons rather than in the DA neurons per se obliterated stress-induced social aversion – but not anxiety – and a similar result was obtained when mifepristone was given during the repeated defeat experience.", "Moreover, blockade of the GR in dopaminoceptive neurons of the mPFC attenuated the glutamatergic excitatory feedforward drive to the VTA-DA neurons (Butts and Phillips, 2013).", "Their finding is in support of a positive feedback loop under control of these postsynaptic GRs that maintains the firing rate of the VTA during stressful conditions.", "Interestingly, the dopaminoceptive neurons in the NAc mediate the precipitation of stress-induced addictive behavior (Ambroggi et al., 2009; McEwen, 2013a,b).", "Niwa et al. (2013) reported that glucocorticoids also can exert long-term control via presumed epigenetic programming.", "After exposure to three weeks of isolation stress during adolescence, adult mice showed a preferred passive coping response in the forced swim test (Niwa et al., 2013).", "However, this response was displayed only in animals that carried a DISC1 allele, of which variants can precipitate a schizophrenia-like phenotype.", "Chronic stress reduced DA activity in the VTA projection to the frontal cortex, but DA activity was not changed in the NAc projection.", "The programming of this ‘low VTA-DA passive phenotype’ by chronic stress during adolescence was prevented if stressor exposure was experienced in the presence of the anti-glucocorticoid mifepristone.", "In animals subjected to CUS the glucocorticoid response to an acute stressor is prolonged, but the VTA-DA release is diminished.", "This failure of elevated glucocorticoids to increase DA release in NAc and mPFC during CUS may be the reason for lack of motivation, lack of pleasure, and depressed mood (Chen et al., 2017).", "Interestingly, chronic exercise also increases circulating glucocorticoid concentrations, but now DA release is increased and the VTA-DA system is functional.", "Moreover, adrenalectomy (ADX) attenuates the exercise-induced rise in DA, which is reinstated with corticosterone replacement (Chen et al., 2017).", "There is no answer yet on the mechanism that underlies this glucocorticoid paradox in stimulation versus suppression of DA release during either exercise or CUS exposure, respectively.", "However, it likely refers to the rewarding experience of exercise, which is a stimulus leading to DA release, and associated reinforcement learning.", "In sum, GR-mediated glucocorticoid actions sustain VTA-DA activation during stress via stimulation of TH in their cell bodies, while promoting motivational arousal particularly under rewarding circumstances of successful coping, winning a contest, and exercise.", "VTA-DA activity is increased during such acute challenges by a GR-dependent positive glutamatergic feedback loop, but reduced with a prolonged stress experience during failure to cope in the face of elevated circulating glucocorticoids.", "The few data available suggest that under basal conditions MR and GR seem to be involved in spontaneous firing and thus tonic activity of the VTA-DA system and possibly in promoting DA release from its efferents innervating limbic and cortical regions.", "Limbic afferents, e.g. from hippocampus and amygdala, are considered to be the main determinants of tonic VTA-DA neuronal activity (Belujon and Grace, 2017).", "It is therefore of interest to briefly summarize the seminal studies with rat hippocampal neurons showing that in these hippocampal cells “MR-mediated steroid actions enhance cellular excitability, whereas activated GRcan suppress temporarily raised neuronal activity.”", "(Joëls and de Kloet, 1992).", "The results revealed in hippocampal CA1 neurons that these opposing MR- and GR-mediated actions actually produced a U-shaped dose response in a number of cellular responses.", "The steroid actions, that developed with a delay of 15–30 min, required GR dimer formation and protein synthesis (Karst et al., 2000).", "Thus, in CA1 neurons MR occupation with low glucocorticoid concentrations caused small Ca2+-currents through L-type Ca2+-channels as a result of transcriptional regulation of the Ca2+-channel β4 subunit (Chameau et al., 2007).", "Hence, in the pyramidal neurons the slow Ca2+-dependent K+-conductance (after-hyperpolarization), NE- and current-induced cell firing frequency accommodation, and 5HT1A K+-hyperpolarization response were all attenuated by MR activation (Joëls and de Kloet, 1989, 1990; Joëls et al., 1991).", "These currents were increased in absence of steroid by adrenalectomy (ADX) as well as during excess stimulation of GR.", "The most stable condition occurred with mostly MR and little GR activation, which represents the average receptor occupancy under basal HPA axis conditions throughout the day.", "However, glucocorticoids also exert non-genomic actions in hippocampus and BLA via MRs and GRs (Groeneweg et al., 2011, 2012).", "Membrane GRs control the release of endocannabinoids which exert an inhibitory presynaptic action on transmitter release and have effects on appetite and mood (Hill and Tasker, 2012).", "MRs were found in the hippocampus to promote rapidly the release probability of glutamate from single vesicles.", "The mechanism involved a fast MAP kinase pathway, which is measured postsynaptically as an increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs) (Karst et al., 2005; Pietranera et al., 2006; Olijslagers et al., 2008).", "This increased excitability is in hippocampus rapidly suppressed via GR.", "A similar increased frequency of mEPSCs was observed in the BLA, but this effect persisted because it was sustained rather than inhibited by a GR-mediated genomic effect as observed in hippocampus.", "This cooperativity of non-genomic MR and genomic GR was termed ‘corticosterone metaplasticity’ (Joëls et al., 2010).", "In another series of experiments the neurons of the amygdala were first excited by isoproterenol (a β-agonist) followed 20 min later by exposure to corticosterone in order to mimic in vitro the release of the agents under in vivo stress conditions.", "NE-induced mEPSCs were suppressed by moderate concentrations of corticosterone, but with very high concentrations of both hormones, mimicking severe stress, the mEPSCs were enhanced in a long-term manner (Karst and Joëls, 2016).", "This coordinated glucocorticoid-NE mechanism may explain why emotions are so strongly remembered.", "The studies on genomic and non-genomic actions by Marian Joëls and coworkers (Joëls et al., 2012) were performed with slices of the dorsal hippocampus, a region primarily involved in spatial learning and cognitive processes.", "The ventral hippocampus (vHipp), however, processes information on the stress- and fear-related emotional state of the individual rather than cognitive processes (Moser and Moser, 1998), but here MR activation did not activate EPSCs.", "Instead, corticosterone acting via MR was found to reduce the frequency of inhibitory postsynaptic currents (IPSCs) and suppressed paired pulse of evoked synaptic currents in the vHipp, and this action did not occur in the dorsal part (Maggio and Segal, 2007, 2009).", "Therefore, in response to MR stimulation, both the increased mEPSCs in the dorsal and the reduced mIPSCs in the ventral part of the hippocampus promote excitability, and thus can enhance excitatory outflow of the hippocampus to the VTA-NAc-DA system, albeit via different pathways related to either emotion in the ventral or cognition in the dorsal part.", "Recently, a hippocampal-NAc pathway was firmly established using an electrophysiological approach.", "It was found that LTP induced in the hippocampal-NAc pathway was required for reward-motivated conditioned place preference.", "Chronic stress attenuated the hippocampal input and was accompanied by anhedonia.", "This finding documented that the activity-dependent hippocampal-NAc input can be modulated by contextual aspects of the anticipation or presentation of rewarding stimuli.", "This suggests the presence of a population of reward associated cells in the hippocampus that is linked to goal-directed reward processing in the VTA circuit (LeGates et al., 2018).", "Collectively, these data suggest that MR and/or GR modulation of the hippocampal and amygdala afferent inputs may be a determinant of VTA-DA neuronal firing (Belujon and Grace, 2017).", "Although speculative, predominant MR activation of excitatory hippocampal outflow could activate via the NAc trans-synaptically the gain of the VTA-DA system, which then can be suppressed in an hour or so by subsequent GR activation in this structure.", "The hippocampal input possibly can be overridden during severe stress by the amygdala upon synergistic MR, GR, and β-adrenergic activation.", "When a threat is detected, an alarm triggers an immediate defense reaction by activating the sympathetic nervous system and the HPA-axis.", "At the same time appraisal processes and retrieval of previous experiences are activated, emotions are generated, and decisions are made how to cope with the stressor (Henry and Stephens, 1977).", "By combining data of human brain imaging and animal experiments, Hermans et al. (2014) identified a frontocortical salience network as substrate for the integration of sensory information with the immediate stress reaction.", "This salience network innervates the hypothalamus and amygdala for emotional and neuroendocrine responses.", "The locus coeruleus-based norepinephrine circuit is activated for vigilance and attention.", "Subsequently, resources are gradually shifted to the executive network which supports contextualization and rationalization of coping with the stressful experience, and ultimately the selected coping style is stored in the memory for future use (Hermans et al., 2014).", "Exposure to inescapable stressors such as experienced by animals in the Porsolt swim stress or tail suspension test (see for validity of these tests Molendijk and de Kloet, 2015; de Kloet and Molendijk, 2016; Molendijk and de Kloet, 2019), recent studies have revealed how the mPFC controls top-down peri-aquaductal grey (PAG)-mediated behavioral coping.", "Using optogenetic stimulation or inhibition combined with neuro-anatomical tracing techniques, it was found that activation of the excitatory projection from mPFC neuronal ensembles stimulate GABA-ergic neurons in the anteroventral bed nucleus stria terminalis (avBNST) (Johnson et al., 2016).", "Then, these GABA-ergic neurons project to the ventrolateral-PAG, which upon activation promotes a passive coping response (Johnson et al., 2016, 2018).", "Of interest, stimulation of the dorsolateral/lateral-PAG (Keay and Bandler, 2001) evoked an active coping response.", "Other branches of the avBNST innervate the PVN where – at distinct PVN neurons – stimulation of the GABA-ergic input inhibited either stress-induced HPA axis activity or sympathetic activity (Radley and Johnson, 2018) (Fig. 4).", "Although not demonstrated, the avBNST likely also conveys information to the VTA.", "Studies using a fear conditioning paradigm revealed that the prelimbic PFC activation was linked to expression of fear as shown by increased ‘freezing’ (Milad and Quirk, 2012), while such downstream emotional expressions were suppressed from the infralimbic PFC. Other studies demonstrated that the switch from prelimbic-dorsomedial striatum to infralimbic-dorsolateral striatum neuronal ensembles actually serves to attenuate overactivation of the salience network (Fiore et al., 2015) thereby acting in the same direction as the dampening effects of glucocorticoids.", "Glucocorticoids modulate bottom-up the large scale dynamics in the shift from the salience to the executive neuronal networks (de Kloet et al., 2019).", "The hormones support the reallocation of energy resources and coordinate in a conditional and time-dependent manner the central and peripheral demands via MR and GR.", "Thus, the high affinity genomic MRs are occupied by basal glucocorticoid levels and signal a tonic influence over brain functions which is as a determinant in the threshold and responsivity of the neuroendocrine, autonomous, and behavioral stress response.", "Next with rising hormone levels, the non-genomic MRs are involved in regulation of attention, vigilance and appraisal processes, retrieval of previous experiences from memory, and the selection of an appropriate coping response followed by encoding of the experience for learning (Schwabe et al., 2010; Joëls et al., 2012; Schwabe et al., 2013; Vogel et al., 2016).", "fMRI and EEG measurements confirmed that MR activation promoted the switch towards amygdala-dorsolateral striatum (DLS) at the expense of amygdala-hippocampal connectivity (Schwabe et al., 2013).", "The bias towards habit learning was also privileged by a gain in function polymorphism of the MR (Wirz et al., 2017, 2018).", "Moreover, MR antagonists could block the amygdala switch, and re-introduce hippocampal declarative learning, but performance was found diminished, though MR manipulation affected risk assessment and choice of coping style in a Morris maze, during object recognition, and in an olfactory fear conditioning task (Oitzl and de Kloet, 1992; Souza et al., 2014; ter Horst et al., 2013; Harris et al., 2013).", "Fear and aggression is amplified by MR activation and can be attenuated by MR (Korte et al., 1995; Kruk et al., 2013).", "Again, how the VTA-DA circuit is integrated in the MR-mediated selection of coping style needs further investigation.", "The rapid membrane GR-endocannabinoid mechanism attenuates the initial stress reactions.", "Membrane GRs are activated to attenuate the immediate stress reactions (Hill and Tasker, 2012).", "Then, after ten minutes the rising glucocorticoid concentrations progressively activate the lower affinity genomic GR.", "This action is aimed to dampen stress-induced cascades, including those in the salient network, which are essential for the initial defense reactions, but may become harmful if not controlled via GR.", "(Munck et al., 1984; Sapolsky et al., 2000; McKlveen et al., 2013).", "The stressful experience is contextualized by recruitment of specific co-regulator cocktails with the GRs (Meijer et al., 2018).", "Transactivation of GR promotes subsequently the memory storage of the experience (De Kloet et al., 1998; Maier and Watkins, 2010; Frank et al., 2015; Harris et al., 2013).", "The GR-dependent actions in the VTA mesocortico-DA system supporting reward processing and motivation also are under genomic control by glucocorticoids (Barik et al., 2013; Piazza and Le Moal, 1996), but see Imperato et al. (1991) who did not observe effects of glucocorticoids on striatal DA dopamine release (Imperato et al., 1991).", "The pattern of glucocorticoid secretion reflects how the individual has managed to cope.", "Thus, a rapid rise in circulating glucocorticoid is a sign of a resilient individual in coping with stress as long as its secretion is also readily terminated.", "It follows that if coping is less successful this can be read from the slower rate the stress reaction is terminated: glucocorticoid secretion remains elevated for a prolonged period of time.", "The selected coping style is meant to gain control over the stressor.", "This is a rewarding experience, produces incentive motivation, and increases social competitiveness.", "In social competition the active coping style is often termed pro-active to indicate a phenotype that is inclined to take control before the confrontation evolves.", "The “losing” opponent is labeled with a reactive phenotype whose response characteristics are driven by environmental stimuli.", "The two phenotypes show extreme differences in physiological and behavioral response patterns (Koolhaas et al., 1999, 2010; de Boer et al., 2017).", "The pro-active dominant individual is characterized by a high sympathetic tone and low HPA-axis activity.", "Interestingly, the high sympathetic tone corresponds to high noradrenergic activity of the locus coeruleus driven by a CRH input from the central amygdala.", "The re-active subordinate individual displays the opposite phenotype showing high HPA-axis activity (de Boer et al., 2017).", "In a similar protocol Reyes et al. (2015) distinguished rats based on time to defeat.", "The authors reported that – in both male asfemale rats – this criterion for social competition revealed that after a single challenge the rapidly defeated animal showed a higher locus coeruleus noradrenergic activity driven by a central amygdala CRH input, which was counteracted by an activated enkephalin afferent from the nucleus gigantocellularis.", "Upon repeated defeat the CRH-induced locus coeruleus activity persisted.", "The findings led to the recognition of the CRH/enkephalin balance at the locus coeruleus as a determinant in the neurobiology of social competition, and thus are relevant for VTA function.", "There are no sex differences identified (Reyes et al., 2015, 2019).", "A striking example of the role of GR in expressing VTA-DA-dependent social competition was recently uncovered by Carmen Sandi’s laboratory in rats that were selected for high and low corticosterone levels during puberty.", "They found large differences in aggressiveness at adulthood, which could be prevented not only by treatment with anti-glucocorticoids at puberty, but also by anti-glucocorticoid treatment at a time outside the context of the social interaction test (Papilloud et al., 2018).", "This finding adds to a growing database showing that anti-glucocorticoids can reset the activity of the stress system (Dalm et al., 2018) with consequences for VTA-DA function.", "In conclusion, there is evidence that MR and GR coordinate in complementary fashion during stress the action of glucocorticoids from modulation of appraisal processes and selection of coping style to facilitation of memory storage of the experience, respectively.", "MR activation causes a bias towards activation of amygdala-striatal pathway associated with habit learning and active coping, while MR blockade rather promotes hippocampal passive coping albeit with poorer performance.", "The role of MR activation in VTA-DA function is still poorly understood, but likely involves potentiation of limbic inputs.", "GR activation promotes memory storage and boosts VTA-DA function as can be learned from the increased motivation and reward processing under a variety of conditions (Fig. 4).", "As is previously highlighted in this review, VTA-DA activity in the mesocorticolimbic DA circuitry is involved in the formation of subjective emotional-motivational values for particular behavioral options – rather than objective properties about distinctions between “good and bad” (Burke et al., 2014; Lak et al., 2014; Papageorgiou et al., 2016).", "These valuations, which are based on the integration of internal and external states, are experience-dependent and lead, depending on an animal’s specific situation and associated needs, to subjective preferences for the selection of specific behavioral choices (Burke et al., 2014; Papageorgiou et al., 2016).", "Mesolimbic DA responds to rewards, contributes to stress-coping, and promotes social interactions.", "The release of DA promotes reinforcement learning, increases motivation to acquire anticipated rewards, and the drive to succeed in stress-coping.", "VTA-DA activity-dependent reinforcement learning processes may promote repetition of behavioral preferences during goal-directed behaviors, thereby contributing to the formation of habitual behaviors in particular situations with associated needs.", "The mesocortical branch rather mediates computational aspects of the cost and benefit, thereby investing in effort and motivation to acquire a ‘feel good’ sensation as expressed in planning and decision-making during anticipation of rewards versus facing the challenge to cope with stress (Ironside et al., 2018; Weger and Sandi, 2018; Stanton et al., 2019; Hauser et al., 2017).", "Acute stress enhances attention and vigilance.", "The sensitivity to perceive rewards is augmented and motivational arousal is increased.", "At the same time the stressor may signal danger and evoke fear and emotion.", "It is the role of the mPFC to guide decision-making in the trade-off between a threat and the pursuit of a reward.", "Glucocorticoids act in such circumstances as a double edged sword.", "They increase motivation to win a social competition or collect a reward via VTA-DA GR, while on the other hand, and also via GR, fear-motivated behavior is enhanced by increased e.g. amygdala CRH expression involving the VTA.", "This contrast is most striking when it concerns the need for life-sustaining food versus coping with a life threatening situation.", "There are surprisingly few studies available on the topic except for excellent books and overviews (Sapolsky, 1994; Denton, 1984; Krause and Sakai, 2007; de Kloet and Herman, 2018).", "Thus, these VTA-DA neuron-dependent processes are essential for adaptive behavior in an ever-changing environment (Lloyd and Dayan, 2016).", "In this review we have reported evidence that a chronic stress experience may compromise the role of the VTA-NAc circuit to pursue rewards and social interactions as well as the ability of the VTA-mPFC to cope cognitively with flexible adjustments to environmental demands.", "Fundamental to the behavioral consequences of chronic stress-induced neuroadaptive changes is the bias towards more habitual control of instrumental rather than goal-directed behavior (Dias-Ferreira et al., 2009; Schwabe et al., 2011; Schwabe and Wolf, 2013) as is applicable to both addictive (Section 6.1) and low-risk/low-effort (Section 6.2) behaviors.", "In the case of addictive behaviors, this VTA-DA neuron-dependent bias may contribute to an acceleration in the switch from initial voluntary use to habitual-like addictive behaviors.", "In the case of behavioral changes towards low-risk and low-effort, which are in animal studies frequently referred to as “depression-like behaviors”, the habitual bias contributes to “the generalization between environments”, as argued by Dayan and colleagues (Huys and Dayan, 2009; Lloyd and Dayan, 2016).", "In both cases, when environmental challenges are appraised as such, the initial responses may rapidly shift towards experience-based habitual behavioral patterns in a manner that favors low-risk and/or low-effort behavioral solutions and without re-updating or devaluating learned associations between contextual stimuli and responses.", "This rapid switching towards habitual behaviors increases an animal’s behavioral efficiency in certain environmental situations, while it may come at the expense of its behavioral flexibility in fluctuating, natural environments (Dias-Ferreira et al., 2009; Sousa, 2016).", "Therefore, the essential issue in the contribution of chronic stress-induced VTA-DA neuronal plasticity to the etiology of stress-related disorders is whether they affect an animal’s ability to change behaviors when contexts change.", "Hence, the VTA-DA neurons may contribute to maladaptive modulation of emotional-motivational behavioral patterns of which we will address below a few specific manifestations.", "VTA-DA neuron-dependent reinforcement learning processes are involved in facilitating the development and consolidation of habitual behaviors (Schwabe et al., 2011; Lloyd and Dayan, 2016) and a chronic stress-enhanced influence of excitatory input onto VTA-DA neurons may contribute to alterations in an animal’s behavioral response towards addictive substances.", "Indeed, the link between (chronic) stress exposure and addictive behaviors is evident (Chen et al., 2010; Koob and Volkow, 2016) and “the VTA is required for stress-induced reinstatement of drug-seeking behaviors” (Polter and Kauer, 2014).", "Furthermore, VTA-DA dependent development and consolidation of addictive behaviors are associated with the well-described chronic stress-induced bias towards more habitual control of instrumental behavior (Dias-Ferreira et al., 2009; Schwabe et al., 2011).", "This link between chronic stress, VTA-DA adaptation, and addictive behavior has received substantial support (Table 1).", "The studies demonstrate increases in stress- and drug-induced conditioned place preference (Stelly et al., 2016; Whitaker et al., 2013), locomotor activity (Nikulina et al., 2005, 2008; Miczek et al., 2011), and cocaine self-administration (Covington et al., 2008; Holly and Miczek, 2016).", "Moreover, these processes may be modulated by CRH and BDNF (Holly and Miczek, 2016).", "In addition, the pioneering research by Piervi Piazza and colleagues in the 1990s (Piazza and Le Moal, 1996) has demonstrated in rodent studies that the enhanced VTA-DA activity induced via GR by glucocorticoids and stress can increase dopamine release particularly from the NAc shell.", "This action of the glucocorticoid hormone has a facilitatory role in the behavioral effects of psychostimulants on locomotor activity, self- administration, and relapse, and can be blocked by GR antagonists (Marinelli and Piazza, 2002).", "GR rather than MR activation by corticosterone replacement of ADX animals enhanced amphetamine sensitization (Rivet et al., 1989).", "Using cocaine, it appeared to be the initiation rather than the expression of psychostimulant-induced sensitization of the locomotor response in the ADX-corticosterone replaced rats, although co-administration of epinephrine was required (De Jong et al., 2009).", "Likewise, mifepristone given to intact animals blocked psychostimulant sensitization only in the initiation phase (van der Veen et al., 2013).", "Glucocorticoid-dependent sensitization was strain-dependent and occurred in DBA, and not in C57Bl mice (de Jong et al., 2008).", "Moreover, for cocaine intake at adulthood, maternal environment experienced as pups appeared crucial for these DBAs (van der Veen et al., 2008).", "Differential sensitivity to psychostimulants such as amphetamine is often used as an indication of psychosis susceptibility in humans.", "In the search for the involved common genes and pathways, DBA2 mice were divided by their locomotion response to amphetamine in sensitized and resistant animals.", "Subsequently, transcriptome analysis of the NAc, prefrontal cortex, and the hippocampal CA1 regions revealed a large number of uniquely responsive gene patterns in each region.", "By far the most profound and reproducible differences between the sensitized and resistant animals were observed in the hippocampus: many of the validated genes in CA1 are members of the cAMP response element (CRE) family and targets of GR and myocyte enhancer factor 2 (Mef2) transcription factors.", "Hence, these genes were postulated to contribute to susceptibility to amphetamine-induced psychotic symptoms (Datson et al., 2011).", "Together, these studies support the notion that chronic stress exposure may contribute to addictive-like behaviors by adaptive changes of VTA-DA neurons.", "The studies also show the complexity of glucocorticoid involvement.", "The hormones were found to potentiate particularly the initial phases of drug addiction implying a role in sensitization of the VTA-DA reward and motivation circuitry.", "Chronic stress-induced changes in VTA-DA activity may lead to changes in effort- and value-based decision-making in a manner that behavior which minimizes contact with aversive environmental input is prioritized.", "Accordingly, counterproductive behavioral strategies are usually avoided.", "Hence, chronic stress may compromise the involvement of the VTA neurons in translation of emotional-motivational valuations to approach – or withdrawal – as well as explorative or exploitative behaviors (Lloyd and Dayan, 2016; Pizzagalli, 2014).", "Specifically, the stress-induced enhancement of excitatory input of VTA-DA neurons may facilitate reinforcement learning of value representations, and therefore support such shifts in behavioral prioritization.", "This is well-supported by symptomatology of major depressive disorder, such as e.g. anhedonia, which represents devaluation of rewarding substances, and reduced motivation for explorative behaviors (Der-Avakian and Markou, 2012; Treadway and Zald, 2011; Venzala et al., 2013).", "The effects of chronic stress on shifts in behavioral prioritization involving VTA-DA are listed in Table 1.", "CSDS exposure resulted in an increased social avoidance, decreased sucrose preference and decreased explorative behavior (Krishnan and Nestler, 2011; Warren et al., 2013; Qu et al., 2017).", "Additionally, an animal’s history of repeated exposure to uncontrollable stress, where the use of active coping strategies repeatedly have failed to control the environmental challenges, may facilitate a switch in coping strategy towards more passive ones as is supported by an increased immobility in the forced swim stress test upon chronic stress exposure (Krishnan et al., 2008a; Cabib and Puglisi-Allegra, 2012; de Kloet and Molendijk, 2016).", "The influence of prolonged stressors appears variable, however.", "A predictable stressor such as studied in the CSDS model was shown to enhance the phasic firing rate of the VTA-DA system in the defeated animals (Krishnan et al., 2007; Barik et al., 2013).", "Alternatively, chronic stress leads to a reduced spontaneous firing and atrophy of the VTA-DA system (Chang and Grace, 2014; Sugama and Kakinuma, 2016; Belujon and Grace, 2017).", "This reduced spontaneous (tonic) firing may be due to the high DA activity in the mPFC which feeds back on the NAc as was demonstrated by a series of highly interesting studies of Simona Cabib’s group (Pascucci et al., 2007; Fiore et al., 2015).", "Yet, both the increased as well as the decreased VTA-DA activity displayed a phenotype characterized by social withdrawal, anxiety, and decreased sucrose appetite, that was investigated mechanistically in great detail (see Section 3).", "Moreover, optogenetic manipulation of the DA neurons could reverse these phenotypes either produced by social defeat or CMS (Tye et al., 2013; Chaudhury et al., 2012).", "To explain this conundrum of opposing outcomes by exposure to different repeated stressors a role fort the Ih current was suggested (Friedman et al., 2014).", "Also the nature of the stressor, the time of measurement during the circadian cycle, and the heterogeneity of the VTA-DA cells involved in the various stressors may play a role and of course the characterization of either tonic versus phasic firing in each model.", "Nevertheless, the involvement of VTA-DA neurons in the symptomatology of major depressive disorder is evident.", "Causality, however, may be linked to the higher mPFC top-down control of stress-coping and behavior as reviewed in more detail in Section 5.3.", "Eventually, degeneration of hippocampal, VTA-DA, and pre-frontal cortex circuits may occur after chronic stress exposure as is demonstrated by MRI (Magalhães et al., 2017).", "This breakdown of the stress-coping network is facilitated by the elevated glucocorticoids linked to chronic stress, in a manner that involves increased emotional expression generated by the hypertrophied extended amygdala (McEwen et al., 2016).", "Most studies on the plasticity and functioning of the VTA have been performed with males.", "However, the relatively few animal studies available point to a sex differences in VTA-DA dynamics and function (Russo et al., 2003; Shansky et al., 2010; Bale and Epperson, 2015; Gillies et al., 2014).", "Females show higher VTA-DA turnover and release than males, particularly in response to psychoactive drugs such as amphetamine (Becker and Chartoff, 2019).", "These differences may be due to sex steroids.", "Estradiol was found to stimulate dopamine function and estrogen receptors are expressed in dopaminergic neurons of the VTA that project to the NAc (Kritzer and Creutz, 2008).", "However, androgen receptors occur in dopaminergic neurons that project to the mPFC (Kritzer and Creutz, 2008).", "Such hormonal effects are achieved either by genomic actions, although also recently discovered rapid membrane effects cannot be excluded (Gillies et al., 2014).", "In addition to these direct effects of the steroids also the afferents to the VTA are sexual dimorphic and thus may contribute to the sex difference in dopamine function (Bale and Epperson, 2015).", "This is exemplified by the inputs from the mPFC and limbic regions which underly the very different strategies males and females use to cope with stress.", "The preferred coping strategy of males is the well-known ‘fight-or-flight’ response in attempts to gain control.", "As pointed out previously social defeat increases dopamine turnover in the ventral striatum.", "In contrast, females rather rely on a more passive strategy that can be characterized by ‘tend-and-befriend’ (Taylor et al., 2000), which relies on social support involving the mesolimbic dopamine system (Weger and Sandi, 2018).", "The above ‘activational’ actions of the steroids are superimposed on their ‘organizational’ effects during perinatal life.", "It is well established that during early-life testosterone masculinizes the brain (Bangasser and Shors, 2008).", "Subsequently, at puberty these masculinizing effects are further potentiated by the androgens (Ahmed et al., 2008).", "The female brain becomes around puberty responsive to estrogens and progestins (Becker, 2009).", "Accordingly, given the profound differences in brain organization, it is perhaps not so surprising that sexual dimorphisms of the VTA system has evolved.", "Superimposed on the these perinatal organizational actions of the sex steroids are the programming effects of adverse early-life experiences.", "It is well established that early-life adversity, lack of care, immune activation by infections, and emotional neglect have profound effects on the organization of limbic and cortical brain circuits underlying stress-coping and adaptation, including the dopamine circuitry (Bilbo and Schwarz, 2012; Daskalakis et al., 2013).", "The studies generally agree that a history of early-life adversity increases emotional responding – generated by a hypertrophied amygdala – and impairs cognitive performance as reflected by reduced dendritic branching of hippocampal and mPFC neurons.", "Early-life adversity reduces VTA-DA mesocorticolimbic function and causes a bias towards an anhedonic phenotype (Moriceau, 2009; Marusak et al., 2017).", "However, whether such programming effects reflect maladaptive or adaptive value should be discussed with caution.", "The outcome of these effects may affect vulnerability and resilience differentially for better or worse.", "It provides an advantage in certain contexts, but in others it may contribute to the etiology of neuropsychiatric disorders (Champagne et al., 2008; Ellis et al., 2011; Nederhof and Schmidt, 2012; Daskalakis et al., 2013; Nesse et al., 2016).", "Relevant for this review is that in rats genetically selected for a high striatal DA responsiveness, exposure to stressors in early as well pubertal life had a particular negative outcome.", "This combination of genetic load and early-life adversity is the basis of the ‘three-hit hypothesis’ for the study of vulnerability to stress-related disorders (see Daskalakis et al., 2013).", "In an intriguing series of experiments, Soares-Cunha et al., 2014 showed that intra-uterine treatment of the rat fetus with dexamethasone impaired dopamine function in the offspring’s later life (Soares-Cunha et al., 2014).", "This was shown in behaviors interpreted as impairment of salient incentives and motivational drive.", "Gillies et al. (2016) demonstrated sexual dimorphic effects of perinatal treatment with the potent synthetic glucocorticoid dexamethasone.", "This treatment is known to affect cognitive functions and behavior in later life, although the outcome is dependent on context.", "Antenatal dexamethasone profoundly increased DAT expression in male offspring, while it was decreased in females.", "This observation was confirmed with amphetamine, which increased dopaminergic function in males and decreased it in females.", "Behavioral studies provided support.", "The male antenatal dexamethasone rats displayed a profound pre-pulse inhibition of a startle response.", "The females showed a diminished locomotor response to amphetamine stimulation (Virdee et al., 2014; Gillies et al., 2016).", "These findings led to the hypothesis that sexual dimorphic responses of the mesocorticolimbic system to stress and stress hormones, in particular glucocorticoids, during adulthood or development represents a mechanism which may contribute to sex biases in common DA-dependent-associated disorders (Gillies et al., 2014).", "These include deficits in decision-making (Georgiou et al., 2018), risk assessment, and resilience (Wellman et al., 2018).", "Stress- and dopamine-related brain disorders are female prevalent as is the case in addictive behaviors, anxiety, and depression (Bale and Epperson, 2015, 2017; Kessler et al., 2003; Bangasser and Valentino, 2014).", "If patients suffering from these disorders are subjected to stressors, males and females show profound differences in HPA-axis reactivity, with males having a higher cortisol secretion in anxiety and depression.", "In healthy individuals androgens are known to stimulate and estrogens to inhibit HPA-axis activity, and this difference is sustained and apparently amplified during depression.", "This finding suggests that there is a sex difference in glucocorticoid feedback (Kudielka and Kirschbaum, 2005; Goel et al., 2014; Zorn et al., 2017).", "Interestingly, glucocorticoid action is sexual dimorphic.", "Duma et al. (2010) showed profound sex differences in genome wide transcriptional response to dexamethasone in the liver.", "Glucocorticoid responsive genes were overrepresented in males versus females suggesting that males are more susceptible to e.g. the anti-inflammatory actions of dexamethasone.", "In fact, according to Cidlowski (personal communication) such profound sexual dimorphism in glucocorticoid action is a common phenomenon, and is also observed in brain (Duma et al., 2010).", "In conclusion, the VTA-DA and the stress response systems are sexual dimorphic.", "The sex differences originate from genetic background and organizational actions of sex hormones.", "Glucocorticoid actions are sexual dimorphic.", "These findings warrant further research into the sexual dimorphic function of the VTA during stress.", "One of the messages conveyed in this review is that the VTA circuit harbors a mechanism that enables an individual to predict outcome valuation of a salient experience or a social interaction, while taking emotional and contextual inputs into account.", "Predictability manages a sense of control, which is the determinant of the severity of a psychogenic stressor.", "In first instance, the stressor triggers immediate behavioral, autonomous, and neuroendocrine defense reactions.", "At the same time, appraisal processes, memory retrieval, and emotional aspects bias decision-making how to cope with the stressor in order to prevent the initial defense reactions from overshooting.", "The substrate for decision-making is in mPFC ensembles that inform via the BNST either the dorsal or ventrolateral PAG divisions about the selected behavioral coping strategy and the hypothalamus about control of the autonomic and neuroendocrine response patterns (Keay and Bandler, 2001; Mcklveen et al., 2015; Johnson et al., 2018).", "How the VTA-DA circuit is embedded in this stress-coping circuitry is not precisely known.", "In this review we focused on the role of the excitation-inhibition balance in control of VTA-DA function as it was modulated by BDNF, CRH, opioids, and in particular the glucocorticoid master regulator.", "Fig. 4A provides a schematic overview of the glucocorticoid action on VTA-DA and its limbic-prefrontocortical afferents that is mediated in a complementary fashion by MR and GR.", "Fig. 4B shows how these same regions are implicated in the stress-coping circuit.", "In the representation it is understood that acute stressors first cause a state of alarm that energizes immediate coping responses.", "Next, the stressful experience is rationalized and contextualized as a prelude to executive functions that serves recovery and behavioral adaptation.", "Finally, the experience is stored in memory for future use.", "Outstanding questions are e.g. how the VTA-DA circuit links valence assessment with coping style and how VTA-DA-dependent functioning contributes to psychosocial resilience.", "Actually, a recent study in rats demonstrated that mesolimbic DA circuitry links the position in a social hierarchy with alertness in response-reward behavior (Lozano-Montes et al., 2019).", "A second message concerns the role of the VTA during chronic stress.", "Initially, repeated exposure to stressors generates a state of resistance, which requires additional resources (allostatic load) for maintenance of an altered homeostatic state (allostasis) (Selye, 1946; McEwen and Wingfield, 2010).", "From the VTA-DA perspective motivation is enhanced to either pursue rewards and social relationships or to withdraw and conserve energy resources in a process that depends on mPFC-based computation of cost and benefit that is affected by the mesocortical dopaminergic input (Ulrich-Lai and Herman, 2009; Radley and Johnson, 2018; Weger and Sandi, 2018).", "Fundamental for these processes are the number of spontaneously active (tonic firing) VTA-DA neurons that depend on limbic input as a determinant of the magnitude of the phasic bursts in response to excitatory inputs (Belujon and Grace, 2015, 2017).", "A future challenge is how precisely the excitation-inhibition balance in the VTA is regulated.", "For instance, is the enkephalin-CRH balance implicated as it is in the regulation of locus coeruleus neurons (Reyes et al., 2015; Hupalo et al., 2019) or for that matter to what extent is the tonic and phasic VTA-DA activity under control of this CRH-driven locus coeruleus mechanism or how does MR/GR control of limbic afferents contribute?", "Finally, with continuous chronic stress, the energy resources that drive coping and adaptation may become exhausted.", "The mPFC, hippocampal circuits, and VTA circuits show atrophy versus the hypertrophy of the extended amygdala (Sousa, 2016; Magalhães et al., 2017).", "mPFC circuits meant to provide flexibility are becoming ‘locked’ (McEwen et al., 2015).", "The role of the microglia is increasingly recognized in this process (Frank et al., 2019).", "However, the nature of the stressor is important.", "For instance, the outcome of CUS and CSDS is opposite for the VTA-DA activity, while anhedonia and passive coping styles are the outcome in both models.", "Various explanations for this conundrum have been offered including the involvement of mesocortical- and/or mesolimbic pathways, the day or night phase the experiments were performed, and, of course, the nature and severity of stressor, particularly in view of the ‘predictability’ in social interactions of the animals in the CSDS model (Muir et al., 2019).", "The three stage process of alarm, resistance, and exhaustion is recapitulated during addiction with reference to the VTA-DA circuit (Koob, 2015; Koob and Volkow, 2016; Kwako and Koob, 2017).", "Briefly, the binge phase depends on the VTA-DA system regarding positive reinforcement of impulsive drug intake.", "The withdrawal phase causes a dysfunctional VTA-DA system that impose a dysphoric stressed-out phenotype.", "And the craving phase is characterized by a dramatic reorganization of the salience and executive network with profound consequences for the VTA-DA circuitry.", "The mPFC and hippocampal inputs are uncoupled, implying a severe deficit in decision-making, planning, cognitive flexibility, and declarative/spatial cognitive function.", "Compulsivity is the main driver of habit behavior, and the functioning and plasticity of mPFC control over the emotional extended amygdala is compromised.", "The features of drug addiction are reminiscent to VTA-DA involvement in the pathogenesis observed in increased vulnerability to obsessive compulsive disorder, obesity, major depressive disorder, and post-traumatic stress disorder.", "With our current understanding of the mechanisms and signals involved in mental states, the question can be raised whether there are options for preventive or curative treatment.", "CRH antagonists have met success as anxiolytics and anti-depressant in animal studies and probably involve a VTA-DA component as well in their action (Holsboer and Ising, 2010).", "MR antagonists have not been tested yet, but the reports with GR antagonist and modulators are encouraging.", "In experiments in animals and humans GR antagonists were effective in blocking the withdrawal/negative affect phase of alcohol dependence (Vendruscolo et al., 2015), likely by interfering in the CRH-VTA-DA cascade.", "Local manipulation of GABA-ergic interneurons and energy metabolism in the VTA circuitry also holds promise because of the positive outcome for social competence and resilience (Ghosal et al., 2019).", "Currently, chemogenetic and optogenetic technology combined with live-imaging methods offer an unprecedented opportunity to examine plasticity and connectivity of the limbic-VTA-striatal-cortical stress and reward network (Muir et al., 2019).", "The single cell RNA sequencing is beginning to provide the precise topography and diversity of the VTA neurons (Tiklová et al., 2019).", "If combined with tracing techniques, Allen Brain Connectivity Atlas data-mining will allow to combine cytoarchitectonic data and unique molecular-genetic blueprints with circuits dedicated to VTA function.", "The coordinating and integrating role of e.g. BDNF, CRH, opioids, and glucocorticoids from gene to behavior provides in this respect an excellent opportunity towards understanding the functioning of this VTA-DA circuit in limbic-forebrain management of stress-coping and adaptation.", "Moreover, how VTA-DA function is being affected by early-life adversity is also an important topic for further research.", "A sobering thought is, however, that the sexual dimorphism of the stress-reward system is still poorly documented.", "This lack of knowledge of VTA-DA sex differences hampers progress in the translation to clinical understanding of female prevalence in vulnerability to stress-related brain disorders." ]

[ "mPFC coordinates VTA-linked emotional-motivational valuation with stress-coping.", "CRH reduces, while opioids and glucocorticoids increase tonic VTA excitability.", "VTA-DA neurons degenerate during chronic stress after a transient rise in excitability.", "VTA-DA neurons are sexual dimorphic.", "VTA-DA dysfunction is a common substrate for obesity, depression, OCD, and addiction." ]

[ "EART", "PHYS" ]

[ "Charon", "Cryovolcanism", "Geological processes", "Image processing", "Pluto" ]

[ "1912", "3103" ]

[ "New Horizons has provided the first geologically useful images of Pluto's moon Charon (Stern et al., 2015), which have revealed that its geology is strikingly diverse (Moore et al., 2016).", "The Pluto-facing hemisphere, which was seen in the most detail by New Horizons during its flyby, is divided into two primary regions: Oz Terra and Vulcan Planitia.1", "Oz Terra consists of tectonically disrupted terrain that extends to the north pole and beyond onto the anti-Pluto hemisphere (Beyer et al., 2017).", "A prominent series of scarps near the equator separates Oz Terra from a topographically low plains unit, Vulcan Planitia, that occupies at least 400,000 km2 on the visible encounter hemisphere, and extends an unknown distance beyond the terminator (Fig. 1 and units Sm1, Mt, and Rt in Fig. 2).", "Vulcan Planitia contains craters, rilles, mountains, and other topographic features (Figs. 2, 3, 4, and Robbins et al., 2018).", "This region exhibits a variety of textures: a smooth, lobate texture, a ridged/lineated texture, and mottled textures.", "We present observations of these plains from the New Horizons Long-Range Reconnaissance Imager (LORRI, Cheng et al., 2008) and the Multi-spectral Visible Imaging Camera (MVIC) on the Ralph instrument (Reuter et al., 2008).", "This work analyzes these observations and examines possible analogs in the solar system, proposes possible mechanisms for a resurfacing cryoflow, estimates flow rheology, measures the orientation of tectonic features, and concludes that Vulcan Planitia is a unit emplaced by flow of icy material.", "We define longitude and latitude on Charon according to the right hand rule and follow the recommendations of Zangari (2015).", "Charon's positive latitude pole points in the direction of the angular momentum vector and longitudes increase to the east.", "Charon's prime meridian is the sub-Pluto longitude.", "Pluto and Charon's poles are defined by Archinal et al. (2011a,b).", "Latitudes on Charon, following the current scheme for dwarf planets, are simply positive or negative.", "Charon exhibits a visually prominent belt of fractures and scarps that define the boundary between Oz Terra and Vulcan Planitia.", "These fractures are highlighted by the solar illumination angle at the middle latitudes where they occur.", "The morphologic features in Vulcan Planitia are also evident due to the lower solar illumination angle.", "There is no information on structures southward of roughly − 35° due to the inclination of the Pluto system to its orbit around the Sun.", "All of that territory was in darkness during the New Horizons flyby.", "New Horizons obtained extensive stereo coverage of the encounter hemisphere, and those data were processed to derive elevation data from the images (Fig. 4 and Schenk et al., 2018) upon which our topographic interpretations are based.", "This terrain model has a ground scale of 300 m/pixel, and stereo height accuracy from ∼1.4 km to as good as 100 m in some places.", "The mean elevation of Vulcan Planitia is one to two kilometers below that of Oz Terra, and the amplitude of topographic variations is much smaller across the plains than in Oz Terra (Schenk et al., 2018).", "There are a host of possible explanations for these topographic amplitude differences, which could include regolith infilling the lows, or viscous relaxation affecting the landscape.", "However, little evidence has been identified for these two hypotheses.", "While there are landslides that have been identified within Serenity Chasma (Beddingfield et al., 2018, and unit La1 in Fig. 2 and Robbins et al., 2018), there are no observable geomorphic features that indicate infilling or movement of substantial additional amounts of loose regolith on Vulcan Planitia.", "If viscous relaxation were responsible, then we would expect other viscous relaxation signatures (such as up-bowed crater floors) that would be present in Vulcan and not in Oz, especially in similarly-sized craters in the two regions, and we do not.", "Therefore, we hypothesize that the lower topographic amplitude found on Vulcan Planitia is due to the plains-forming material itself infilling previously existing topographic lows.", "Supporting this interpretation, the lack of high-standing topography probably implies extensive subsidence of the original ice lithosphere prior to, and perhaps associated with, emplacement of the plains material.", "Most of the highest-resolution images presented here were obtained with the New Horizons LORRI camera.", "The LORRI point spread function has a full width at half maximum (FWHM) of ∼2 pixels, which meets its specifications (Cheng et al., 2008), but can cause noticeable blurring.", "This blurring can be reduced through Lucy (1974)-Richardson (1972) deconvolution, and the images that this has been applied to are noted in the figure captions.", "The surface of Vulcan Planitia is smooth on 100-km scales (Schenk et al., 2018), but its surface is marked with many smaller-scale features (Fig. 2).", "Numerous low hills, several kilometers across and several hundred meters high, occur on the planitia, often in clusters (Fig. 5).", "There are also broader domes and swells that produce a gently undulating surface over most of the planitia.", "In several places, particularly near the southernmost visible part of the planitia, some of these swells form convex sinuous features (‘broad warp’ features in Fig. 2) associated with depressions (Fig. 6 and units Dm1 in Fig. 2): these convex margins appear similar to the convex margins of the montes (units Mob and Dm2 in Fig. 2 and detailed below in Section 2.3) and may similarly represent converging flow fronts as suggested by Schenk et al. (2018).", "Some parts of the planitia are densely covered in mottled terrain with a locally-varying characteristic wavelength of about a kilometer (Fig. 5 and unit Mt in Fig. 2).", "In some places there are sharp boundaries between these mottled plains and nearby unmottled plains, although the nature of the contact is uncertain (Robbins et al., 2018).", "Vulcan Planitia is criss-crossed with a pattern of rilles, identified as small grooves (Stern et al., 2015; Moore et al., 2016 and the ‘groove’ feature in Fig. 2 and in Robbins et al., 2018), which range from less than a kilometer to 3 km in width (Fig. 7).", "These rilles range from a few kilometers to hundreds of kilometers in length, and in some locations show an arcuate or sinuous pattern.", "The resolutions of available images are insufficient to determine rille cross-sectional profiles in detail, although some appear to be simple graben, due to their inward facing walls, of meters deep.", "In some locations, these features display an en echelon pattern and exhibit Y-shaped intersections, consistent with a graben interpretation.", "These rilles appear to cut, and thus post-date, many of the broad domes and swells.", "However, the available images do not display unambiguous examples of grooves cutting any of the numerous impact craters that are superposed on the plains unit, or their ejecta.", "Therefore, the grooves likely formed soon after plains emplacement, before subsequent cratering events.", "Stern et al. (2015) and Moore et al. (2016) described Vulcan Planitia's mountains.", "There are two large solitary massifs (Butler and Kubrick Mons) and one cluster of smaller peaks (Clarke Montes).", "Vulcan Planitia does not cleanly onlap the toe of these mountains, but instead displays a convex, depressed margin that surrounds them (e.g. Figs. 8 and 9), engendering the term ‘mountain in a moat’ for these features.", "Butler Mons is the largest massif (Fig. 10), but by the time of the closest approach it had set below the terminator.", "Butler can be seen in New Horizons approach images with substantial scarps to its east and south.", "These scarps may indicate a hitherto unknown southern boundary to Vulcan Planitia that might mirror the Oz Terra scarps to its north.", "Kubrick Mons is also a single massif that measures 30 by 20 km in plan form, and rises 3 km above the Vulcan Planitia surface with a 1 km deep encircling moat (Figs. 8 and 9).", "Kubrick displays a ridge crest and other sharp-looking ridges that run down its flanks.", "This morphology on Kubrick (and to a lesser extent on Clarke) is similar to morphologies observed in Oz Terra, and not similar to the surrounding rounded lobate shapes of Vulcan Planitia.", "This morphology implies that Kubrick (and other Mob units in Fig. 2) is older than the Vulcan Planitia surface.", "There are also at least two other, smaller features that share a strong resemblance to Kubrick.", "They have steep sides, and a depressed margin (Fig. 11 and other unit Mob outcrops in Fig. 2).", "The Clarke Montes is a cluster of peaks that sit together in a single, variable margin depression (Fig. 12).", "They stand ∼1 km above the surrounding plains.", "The northern margin exhibits a 2 km drop from Vulcan Planitia, but the southwestern margin exhibits only a 1 km drop.", "The east margin does not have a distinct lobate margin, but appears to smoothly grade out from the floor of the depression up to the plains (Fig. 13).", "Two hypotheses have been proposed for the moats which surround these montes (Moore et al., 2016): (1) downward flexure of pre-existing plains material from loading by younger mountain material (formed for example by extrusive cryoflow), or (2) incomplete embayment of pre-existing mountains by the plains material.", "The downward flexure hypothesis was explored by Desch and Neveu (2017) and they came to a conclusion similar to one discussed internally by the New Horizons team that the thickness of the elastic lithosphere, Te, would need to be very thin (less than 2.3 km).", "Desch and Neveu (2017) hypothesized that the warm icy material that erupted onto the surface was from a local hotspot that was warming the subsurface in this region, and thinning the elastic lithosphere in the area near Kubrick.", "The Desch and Neveu (2017) hypothesis is consistent with the rift flank Te estimates obtained by Beyer et al. (2017), but we concluded that such a low Te was unlikely to be correct given the fact that large craters do not display evidence of viscoelastic relaxation, e.g., up-bowed floors.", "Additionally, the morphology of Kubrick doesn’t resemble what might be expected of a constructional cryovolcano, for example, central depressions consistent with a caldera.", "The embayment hypothesis is supported by the presence of similar convex scarps elsewhere on Vulcan Planitia surrounding depressions without mountains, as noted by Moore et al. (2016).", "Furthermore, the arrangement of Clarke Montes and the presence of a break in slope at the base of its enclosing scarp, which is in some locations several kilometers from the massifs, would be consistent if the scarp was a flow front, but not a flexural bending of the lithosphere.", "It is not clear, however, why the flow would have stopped close to the mountains but not at their base.", "Perhaps the base of the Clarke Montes changes from the steeper slope of the montes and shallows, but still slopes away from the mountains, causing an oncoming flow to stall before reaching the mountains themselves.", "Also, the local hotspot hypothesis does not explain the depressed margin around the Vulcan Planitia boundary, but the embayment hypothesis does.", "Along the south-facing boundary scarps of Oz Terra (Beyer et al., 2017) that define Vulcan Planitia's northern boundary, the surface of the planitia is depressed by a kilometer or more in elevation within about 20 km distance of the boundary (Figs. 4, 14, and 15).", "Where Vulcan Planitia meets the scarps of Oz Terra, the Vulcan Planitia units appear to onlap the scarps, consistent with a surface that was emplaced after the scarps formed.", "In the portion of Vulcan Planitia between Spock crater and where Serenity Chasma's eastern end transitions from a graben to a south-facing scarp, there is a well-resolved pattern of closely spaced and parallel rilles and ridges, which parallel the boundary.", "This surface texture only appears at distances greater than 15 km from the bounding scarp where the planitia materials are presumably thicker, implying a pile-up of flow material where it was thick enough to do so.", "Borrelli and Collins (2018) examined the shapes of the margin depressions along the Oz/Vulcan boundary, and tested a variety of viscous flow and plate bending models.", "Their calculations did not strongly favor any mechanism, nor were any disallowed.", "They conclude that the morphological evidence better supports the advancement of a spreading viscous cryoflow instead of a plate bending model.", "Charon is comparable in size and density to several icy satellites of the giant planets, some of which also show resurfacing in the form of plains (although to different extents).", "Fig. 16 compares the masses and densities of these satellites, indicating whether or not resurfacing is observed on their surfaces.", "In general, higher satellite densities and masses appear to be associated with resurfacing, although not uniquely.", "We interpret the lobate, rounded features on Vulcan Planitia to be the result of flow emplacement of the surface.", "The lobate margins, which encircle the mountains, appear to be the result of viscous flow that has encountered an obstacle.", "The depressed margins along the Oz Terra scarps also imply a viscous flow, but the difference in width between the moats around the mountains and the depressed margin along the scarps may be due to the different depths of the viscous fluid in the center of Vulcan Planitia versus the potentially shallower depths at the margins.", "To estimate a viscosity for such a flow in Vulcan Planitia (although we envision many happening simultaneously), we will make a number of simplifying assumptions: that the flow was emplaced from a single eruption event, that the viscosity was high enough that inertia can be neglected, and that the pre-emplacement surface was horizontal (although we suspect that it was not).", "Under these assumptions, the emplaced fluid spreads under its own weight and the equations of Huppert (1982) can be used to analyze its motion.", "Here ηN is a dimensionless constant which is equal to 1.411 for a flow that is emplaced instantaneously (Pattle, 1959), ρ is the density, h is the flow thickness, L its length, g the acceleration due to gravity, and t the time that the flow was in motion.", "The approximation results from taking the cross-sectional area of the flow to be hL.", "Thinner or faster-moving flows imply lower viscosities.", "There are other possible compositions for cryo-fluids.", "Methanol (CH3OH) water compositions (Kargel et al., 1991; Zhong et al., 2009), for example, would also lower the freezing point and increase the viscosity.", "As an example, assume a flow with h = 2 km, L = 30 km, and g = 0.288 ms−2.", "The lobate features interpreted as flow fronts along the Vulcan Planitia margin and the moats around the montes have a 1 km elevation, we assume 2 km here to account for a slightly deeper pre-Vulcan surface.", "We assume a 30 km length because we don’t see morphologic evidence for long distinct flows, but this parameter could assume a number of values.", "Here we take T0 to be set by the melting point of the substance, Tmelt, and the results are given in Table 2.", "The viscosities derived assuming radiative cooling (∼ 1013–10 15 Pa s) are much higher than likely liquid or slush viscosities, but reasonable for glacial ice on the Earth (which is at the low end of that range) and viscosities derived by Jankowski and Squyres (1988) for flows on Ariel (at the upper end of that range).", "The viscosities derived assuming conductive cooling (∼ 1016–10 17 Pa s) are similar to those derived for solid creeping water-ice, but unreasonably high for N2 and CO so close to their melting points (which rules them out as likely cryo-materials in this scenario).", "Due to the much higher latent heat of fusion for likely cryoflow material candidates vs typical silicate materials, κ in Eq. (2) might need to be replaced by a value that is effectively 10 to 20 times less than κ, which would increase the values in Table 2 by an order of magnitude (O. Umurhan, pers.", "comm.).", "In any case, such high viscosities suggest that very little vertical mixing will occur, and thus that conductive cooling is an internally self-consistent assumption.", "Whether other assumptions (e.g. emplacement as a single flow) are correct is currently unclear.", "The rilles within Vulcan Planitia formed either contemporaneously with or following plains emplacement.", "The morphology of the rilles in Vulcan Planitia is consistent with a tectonic origin, due to their regular spacing, mostly linear character, and negative topography.", "In addition, the en echelon arrangement of these rilles are consistent with extensional graben (Moore et al., 2016).", "If the rilles are extensional graben, then their orientations are determined by the stress patterns present during their formation.", "Consistent rille orientations across Vulcan Planitia would reflect a consistent and large-scale stress field.", "Charon likely underwent extensional stresses following its formation (e.g. Beyer et al., 2017).", "Rilles present within Vulcan Planitia appear to exhibit consistent orientations in adjacent regions.", "However, additional rilles, with more complex orientations, are present in regions farther from the Oz Terra boundary.", "Here, we compare rille orientations across Vulcan Planitia to quantify their spatial variations in azimuth (see the appendix for more information).", "To conduct this analysis, we measure and compare the distribution of rille orientations within various regions (Fig. 17).", "Nearly all analyzed areas exhibit prominent rille azimuths that parallel the orientation of Serenity Chasma.", "Eleven out of the 14 analyzed areas exhibit prominent E-W rille orientations, and this orientation is the most prominent across Vulcan Planitia.", "Many areas exhibit rille azimuth distributions similar to their proximal neighbors (Fig. 17).", "For example, Areas 4, 6, and 8 exhibit rilles more similar in distribution to each other than with those near Serenity Chasma, with a prominent N-S orientation.", "The variation in Vulcan Planitia rille orientations with distance from Serenity Chasma suggests that another stress mechanism, in addition to global expansion, has acted on the lithosphere in these regions during rille formation.", "It is worth noting that there seems to be an absence of rilles in the areas surrounding Kubrick and Clarke Montes (also noted by Robbins et al., 2018).", "This lack of rilles may be the consequence of local stresses associated with these peaks, or from stresses related to the resurfacing cryoflow during emplacement or cooling.", "We estimate the total strain contributed to Vulcan Planitia from rille formation.", "Because the rille widths are too small to accurately measure using available images, the strain for each rille was estimated based on their lengths.", "The maximum displacement-to-length ratio for extensional faults is 0.02, and this ratio has been measured on icy satellites, including Europa (Nimmo and Schenk, 2006) and Charon (Beyer et al., 2017).", "The increase in surface area for each rille is the sum of the two inward facing exposed fault scarp areas that make up the graben walls, scaled by the cosine of the dip angle.", "The average maximum rille displacement is ∼ 1 km, assuming the rille walls exhibit 60° dips.", "The total exposed new rille planform area is estimated to be 6680 km2.", "For an estimated Vulcan Planitia area of 400,000 km2 to 500,000 km2, this areal expansion corresponds to an estimated horizontal strain range of 1.4% to 1.7%.", "Our observations and analysis above indicate that the area of Vulcan Planitia was resurfaced as compared to the more ‘primary’ surface in Oz Terra.", "However, the manner of this resurfacing is difficult to ascertain.", "We present three hypotheses below: (1) outright melting and refreezing of the lithosphere in place (Section 3.3.1), (2) lunar maria-style flood volcanic cryoflows (Section 3.3.2), and (3) foundering of lithospheric blocks into a viscous mantle (Section 3.3.3).", "We favor the latter two hypotheses because the presence of embayed or submerged, intact blocks supports some of the observed features that melting does not, and the melting-in-place hypothesis has some thermodynamic and energetic challenges that would need to be resolved.", "The cryoflow (Section 3.1) and rille (Section 3.2) investigations discussed above are independent of these mechanisms, and mostly explore what these surface features can tell us about the character of the material that resurfaced Vulcan Planitia, regardless of what mechanism was responsible for causing it.", "The tectonic structures in Oz Terra are fairly ancient as they cut few craters, yet are preserved via the lack of saturation impact cratering (Robbins et al., 2017).", "The transition between Oz Terra and Vulcan Planitia appears abrupt in available New Horizons images, but we propose that these two terrains are natural terrain end-members.", "Between those two end-members are the tilted surfaces of the lithospheric blocks in Oz Terra that directly border Vulcan Planitia (Fig. 4).", "Similar tilted blocks are observed on Ariel (Schenk, 1991; Schenk et al., 2018).", "These blocks on Charon represent a point on the continuum between Oz Terra lithospheric blocks that are not tilted, but translated away from each other, and the blocks that we think existed on the pre-Vulcan surface before the cryoflows.", "The one to two kilometer difference in average elevation between Vulcan Planitia and Oz Terra (Schenk et al., 2018) is difficult to explain.", "Malamud et al. (2017) without the benefit of knowing that Vulcan had a lower elevation proposed that perhaps Vulcan was a giant impact basin.", "That could be an explanation, but there doesn’t seem to be any significant morphological evidence that it is the case.", "Alternately, one could imagine that there was a subsurface difference in properties that could have contributed to this variation.", "As the ice shell was freezing, it would be a thickening spherical shell, over a spherical ocean, over a cooling core (Bierson et al., 2018).", "If the poles were colder than the equatorial regions, perhaps due to insolation or early tidal dissipation energy still being deposited in Charon, this might cause the ocean to transition from spherical to a band beneath the surface as the last warm spots were centered on the equator.", "As the ocean froze, pressure could have built up in the remaining liquid (Manga and Wang, 2007).", "In this situation, when the ice shell cracked and fractured from the extension, the poles would have a strong ‘support’ but the equator would be weak, and lithospheric blocks would more easily sink or subside in that area.", "Furthermore, the pressurization may have helped push the remaining buoyant liquid upwards but once it erupted, the pressure was released and erupting material became harder to mobilize, leaving the Vulcan Planitia area just a little lower in elevation than Oz Terra.", "Unfortunately, there are currently no data that might suggest this situation.", "Insolation models of Charon based on current day inclination indicate that the opposite is true: that the poles receive more solar energy than the equator integrated over millions of years (Binzel et al., 2017; Earle et al., 2017).", "However, those models don’t reach into the first billion years of solar system history.", "The resurfacing of Vulcan Planitia could have been the result of a large-scale thermal event centered on the equator in this region of Charon.", "This event could have caused the existing lithosphere to melt in place at the time of crustal expansion.", "Such a hypothesis would indicate that Vulcan Planitia is simply the melted and refrozen remains of the pre-existing lithosphere.", "In this scenario, the montes present on Vulcan Planitia could still be explained in one of two ways.", "They could be blocks that were not melted completely and simply bobbed and rotated into their present position and then were frozen in place, implying a mostly water-ice composition such that the lithospheric blocks float on the melted material.", "Alternately in this scenario, their explanation as constructional features over local hotspots might be strengthened and explained as these hotspots being the last remnants of this large thermal event.", "The tilted blocks at the Oz boundary could be the expression of the edge of this thermal melting event, such that these blocks did not melt or sink, but with disruption at depth in this boundary zone sufficient to severely tilt them.", "A similar hypothesis was put forward for chaos formation on Europa (O’Brien et al., 2002).", "It required a thin ice shell and tidal heating supplied by Europa's orbit around Jupiter.", "Charon is unlikely to have had a shell so thin at the time of resurfacing, and probably lacked the strong input of tidal energy.", "Finding a mechanism to provide a regional thermal pulse is challenging, and so is the amount of energy needed to melt so much near-surface material that is near 50 K today (or lower earlier in solar system history) and can radiate unimpeded to space.", "Voluminous eruptions of cryo-materials are to be expected on midsize icy satellites, due to the low driving stresses due to buoyancy (low gravity) and the presence of NH3 (ammonia) hydrates or other molecules which could provide the needed density relationships between solid and liquid (Stevenson, 1982).", "This hypothesis advances that cryo-material was erupted onto the low, pre-Vulcan surface from the interior and cryoflows flooded the area, emplacing the Vulcan Planitia we observe today (Fig. 18).", "The icy lithosphere of Charon broke into large polygonal blocks in response to global scale tensional stresses (Beyer et al., 2017).", "We envision that this breakup was global and also extended to the area that was to become Vulcan Planitia.", "It is possible that this breakup was more severe in this area than in Oz Terra, or that whatever caused the pre-Vulcan area to be topographically lower promoted significant lithospheric weakening in this area that permitted cryo-material to rise up and erupt onto the surface.", "The traditional difficulty to overcome with cryovolcanism is how to develop a density inversion on an icy world that would allow cryo-material to rise and erupt.", "The density properties of pure water ice would cause icy lithosphere to float on an icy mantle, even if it were physically disrupted.", "Johnson et al. (2017) suggest that salt content and porosity differences may provide such a density inversion.", "For Charon, we propose that the presence of an NH3 hydrate mixture in Charon's lithosphere and mantle is the solution for getting a density inversion (Kargel et al., 1991; Schenk, 1991; Kargel, 1992; Hogenboom et al., 1997; Cook et al., 2007).", "It should be noted that the density differences between an NH3 hydrate liquid and its solid (or even pure water ice) are not very large and an NH3-H2O peritectic liquid has a lower density than solid ammonia hydrate, but slightly greater than pure water ice (Hogenboom et al., 1997), but all of these densities are very close to one another, and small variations may allow extrusion of NH3 hydrate even through a water ice layer.", "The broad surface of Charon is primarily water ice, but Grundy et al. (2016) and Dalle Ore et al. (2018) note that at least a few craters appear to excavate NH3 hydrates from the subsurface.", "Cook et al. (2017) also find evidence for NH3 on Pluto's satellites Nyx and Hydra.", "Based on these observations and considering the likely incorporation of NH3 in Kuiper Belt objects (McKinnon et al., 2008), we have an expectation of a bulk abundance in the Pluto-Charon system of about 1% compared to water.", "However, ammonia would concentrate in any primary melt or residual ocean subject to freezing.", "We hypothesize a possible case involving NH3 hydrates that would achieve a density inversion and support the observations.", "We assume that beneath the ancient surface of Charon, a subsurface ocean formed and then refroze (Bierson et al., 2018).", "In this ocean, water would freeze out of the solution first, enriching the remaining ‘ocean’ in NH3 as freezing progressed (Kargel, 1992).", "This process would leave a mostly original bulk composition (mostly water ice, but with rock and other contaminants) lithosphere overlying a water-NH3 ocean, and would also concentrate NH3 in that ocean, even from relatively small original bulk amounts.", "If the lithosphere were pure water ice, it would still float on a water-NH3 ocean.", "However, as mentioned above, the densities are very similar, and if the lithosphere were not pure water ice but was an NH3 hydrate that contained even a small fraction of rocky material, that might enable a density inversion.", "When the lithosphere expands and is disrupted, the water-NH3 ocean material would rise up above the mostly-water-ice lithospheric blocks.", "This cryo-material wells up, resurfaces the area and then proceeds to freeze.", "This newly-emplaced unit should preserve the bulk composition of the ‘ocean’ that it came from, because it would have cooled much faster than the slow fractional crystallization described above.", "With such a high ammonia concentration, one might have expected the spectrometers on New Horizons to have detected it, but photolysis and proton bombardment act to remove this constituent from the surface over a few tens of millions of years (Cooper et al., 2003; Moore et al., 2007; Cassidy et al., 2010).", "Therefore, spectrometers on New Horizons would not have detected NH3 hydrates in substantial amounts on the surface of Charon in this case, even though they are probably present not far below the surface.", "However, the fact that this spectral feature is seen in spectra of the ancient surfaces of Nix and Hydra (Cook et al., 2017) casts some doubt on the anticipated destruction rate, or perhaps the identification of the feature with NH3 or NH3 hydrate.", "It is important to note that there are different ‘styles' of large igneous provinces in basaltic volcanism, and under this analogous hypothesis for Charon we are advocating a style that is similar to the volcanism of the lunar maria where there is an absence of clear vents or feeder dike systems, and it took very-high-resolution gravity data to reveal the volcanic plumbing of the maria (Andrews-Hanna et al., 2014).", "The other ‘style’ of large igneous province are the large sheet-flow flood basalts on the Earth like the Deccan Traps or the Columbia River Basalts.", "These mechanisms have a few discrete volcanic centers as sources, and have lavas that flow out over very large areas.", "Application of this style to Charon would require only a few discrete sources for the cryoflow, and then a large volume of cryo-material needs to stay mobile long enough to cover a large area, which seems thermodynamically challenging.", "The lunar maria style allows for many ‘local’ sources of cryo-material all across the region, and also explains the lack of large, obvious flows.", "Moreover, the surface swells that form convex sinuous depressions (unit Dm1 and the ‘broad warp’ features in Fig. 2), could mark eruptive centers, if late-stage cryo-materials were withdrawn.", "In this hypothesis, the montes on Vulcan Planitia are simply pre-existing areas of high elevation that were embayed by the cryoflows like kipukas or nunataks.", "These montes are not significantly different in elevation than the high terrain in Oz Terra and could simply be remnants of higher-standing topography in this area.", "The tilted blocks at the Oz boundary could be tilted due to mechanisms that acted in the pre-Vulcan area related to the subsidence of the pre-Vulcan surface and not related to the flows, or they could have been tilted due to subsidence when the cryo-material erupted from depth.", "Charon need not have undergone extensive internal melting of its ices, but an ocean earlier in geologic time is plausible (Bierson et al., 2018), and it is a natural location for NH3 to be sequestered.", "Even if non-buoyant compared with the porous icy lithosphere above, internal pressurization (Manga and Wang, 2007) may have led to the eruption of cryo-materials.", "We estimate that Charon should have accreted sufficient nitrogen (in the form of ammonia, or else converted to ammonia via subsequent chemical processing) to explain such an extensive cryovolcanic unit as Vulcan Planitia.", "For example, a 400,000 km2 plains unit 2 km thick and of approximately dihydrate composition would require a bulk NH3 abundance of ∼ 2 × 104, about 20 times less than Charon's likely accreted NH3-ice (McKinnon et al., 2008, 2017).", "Sequestration in a global residual ocean of dihydrate composition (32 wt.", "% NH3) would only yield an approximately 15 km thick basal layer, however, unless there is another source of NH3 (i.e., core organics) or other substances (salts, methanol) are involved (McKinnon et al., 2008).", "The above hypothesis assumes a moderate disruption which fractures the pre-Vulcan landscape enough for cryo-materials to erupt.", "This new hypothesis is a modification of the previous hypothesis in which the disruption is so great, that the individual blocks of the pre-Vulcan surface lose their support and founder into the interior followed by the mantle material rising up under the same density inversion and chemistry arguments as above and resurfacing Vulcan Planitia (Fig. 18).", "Kattenhorn and Prockter (2014) outline a subduction mechanism where large plates on the surface of Europa are subducted under one another driven by a plate-tectonic mechanism, supported by further work by Johnson et al. (2017).", "What we are proposing here is different from plate-tectonic subduction and is a foundering block mechanism (first proposed by Schenk et al., 2018) similar to terrestrial magmatic stoping.", "The lithosphere is extensively fractured, and the resulting blocks sink due to a density inversion and potentially rotate into the underlying material that wells upwards and envelopes those blocks.", "The erupted cryo-material flows across the surface of these submerged blocks and then freezes in place creating a new lithospheric surface.", "The isolated massifs identified above would in this case be the tips of such tilted polygonal lithospheric blocks, which stand above the plains of Vulcan Planitia like kipukas or nunataks, as the above.", "The tilted blocks on the border between Oz Terra and Vulcan Planitia under this hypothesis are explained as transitional between the Oz Terra blocks that are just translated away from each other, and the blocks that foundered, sunk, and were covered by Vulcan Planitia plains material.", "Of course, in this density inversion situation, when the solid is denser than the liquid, the resulting lithospheric forces will be compressive, and not extensional at the surface, which may seem to contradict the global expansion hypothesis, but this density inversion situation would only happen late in the freezing process, still allowing for global expansion, but may also point to incomplete ocean freezing.", "Irrespective of the exact mechanism, the emplacement of warm flows on the surface would result in subsequent cooling and contraction of those flows.", "As an example, a thin sheet of water ice cooling from 240 K to 40 K has a mean linear thermal expansion coefficient of about 2 × 10−5 K−1 (Röttger et al., 1994).", "The areal strain resulting from the expansion of this cooling would then be about 0.8%.", "Water ice expands, but NH3 hydrate will contract, and Croft et al. (1988) indicate that for ammonia water liquid, the volumetric thermal expansion coefficient is about 5 × 10−4 K−1.", "The areal strain resulting from the contraction of cooling from 240 K to freezing at 175 K is about 2%.", "This can be compared with the areal strain of ∼ 1.5% across Vulcan Planitia estimated in Section 3.2.", "On the other hand, if the cryo-material flow is not a thin sheet, but is thicker, then there would also be vertical contraction of the emplaced material, perhaps up to several hundred meters.", "Given the substantial uncertainties in the properties of the emplaced material and in the inferred strain, this apparent agreement is quite encouraging.", "Cratering on Vulcan Planitia and other terrains on Charon is discussed in Robbins et al. (2017).", "This abundance of craters is consistent with an old age of ∼4 Ga found for Charon's northern and southern terrains (Moore et al., 2016; Singer et al., 2016).", "This age estimate is based on crater distributions and modeled impact rates (Greenstreet et al., 2015, 2016).", "It is notable that within Vulcan Planitia there are no unambiguous examples of fractures cutting craters, or other indication of endogenic modification or infilling of craters, suggesting that the resurfacing and further endogenic modification of Vulcan Planitia occurred rapidly relative to cratering timescales.", "Vulcan Planitia appears to embay Oz Terra from the south, and has at least one possible outlier within Oz Terra (Beyer et al., 2017 and unit Sm2 in Fig. 2 and in Robbins et al., 2018).", "Vulcan Planitia is somewhat younger than the terrains of Oz Terra (Moore et al., 2016; Robbins et al., 2017, 2018), and we lay out a hypothesized relative timing of features and events based on the cryovolcanism and foundered lithospheric block hypotheses in Table 3.", "That sequence assumes a single ‘ocean freezing’ in Charon's history to drive this activity.", "This is consistent with the possibility of a subsurface ocean (Bierson et al., 2018) or intense serpentinization (Malamud et al., 2017) resulting in a large expansion event at approximately 1 Ga after formation.", "Desch and Neveu (2017) have hypothesized that there might be two ‘ocean’ episodes in Charon's history.", "Their proposed long-lived initial ocean doesn’t seem to agree with Vulcan Planitia formation early in Charon's history, which is constrained by the cratering record, but it does raise the possibility of different episodes in the expansion story above.", "The Malamud et al. (2017) modeling also indicates the possibility for a secondary, smaller period of expansion.", "It is possible that there was an initial episode that broke up Charon's lithosphere globally into large blocks.", "Then perhaps there was a significant pause after that initial disruption, and then a second episode (perhaps more localized to the equator or just this part of Charon) occurred and caused the cryo-material eruption or block foundering.", "Of course, these episodes can’t be too far apart, or last too long, because Charon's cratering record shows that Oz Terra and Vulcan Planitia have very similar ages.", "Large icy worlds in the solar system exhibit resurfacing to different degrees.", "Other objects for which resurfacing is observed either have limited areas of resurfacing, later activity that overprints the resurfacing, or just poor data resolution, such that developing and then testing hypotheses are difficult.", "We argue that the global expansion of Charon's surface (Beyer et al., 2017) and subsequent resurfacing were both the result of the progressive freezing of a subsurface ocean.", "This provided a surface that stands as a witness plate to this geologic resurfacing, without much overprinting by other processes, which was excellently observed by New Horizons (Stern et al., 2015).", "We hypothesize that the resurfacing of the plains is not the result of a singular eruptive or effusive center from which cryoflows spread out across the more than 400,000 km2 of Vulcan Planitia.", "Similarly, the melting-in-place hypothesis is difficult to validate due to energetic considerations and does not account for all of the observations.", "Instead, we hypothesize that the resurfacing was the result of ammonia-rich cryo-material from the last stages of ocean freezing either buoyantly rising and flowing out on to the pre-Vulcan lowlands, or as a result of more severe disruption that resulted in lithospheric blocks foundering (as suggested by Schenk et al., 2018), and the buoyant, viscous cryo-material under those blocks rising up and spreading out.", "Under these hypotheses, there would be no singular effusive center, but the sources of the plains material would be in many places across the region, and as the material flowed across the pre-Vulcan lowlands or enveloped the foundering blocks, it would create an extensive plains unit (Fig. 18).", "These observations, and our analysis of them, support the conclusion that the extensive plains on Charon are a vast cryoflow emplaced unit (Stern et al., 2015; Moore et al., 2016; Spencer et al., 2016), similar to those seen on Ariel and Miranda (Jankowski and Squyres, 1988; Schenk, 1991) and possibly other icy worlds in the solar system.", "Charon fits into the panoply of icy satellites which display evidence for the movement of cryoflows and resurfacing." ]

[ "The extensive plains on Charon are a result of global expansion and an early subsurface ocean that flowed as a cryoflow and resurfaced a large area.", "The cryoflow most likely was composed of ammonia-rich water ice.", "The cryoflow was similar to Lunar maria emplacement or via a mechanism where lithospheric blocks were severely disrupted." ]

[ "BIOC" ]

[ "Protein stability", "Stability changing mutations", "Stabilization centers", "Thermal stability", "Thermophiles" ]

[ "1303", "1304", "1307", "1312" ]

[ "We introduced the concept of stabilization centers (SCs) to solve the problem raised by the fact that the three-dimensional structure of a protein cannot be calculated from amino acid sequence considering only local (short range) interactions [1,2].", "The idea of stabilization centers came from the fact that the functional sites of proteins are usually composed only of a couple of residues.", "According to our hypothesis there might be a few additional residues which might be responsible for the stability of three-dimensional structure of proteins.", "The definition of stabilization centers is based on the contact map of a protein with known three-dimensional structure [1] in which residue contacts are identified with pairs of heavy atom distances.", "If there is at least one pair of heavy atoms with a distance less than the sum of the van der Waals radii of the two atoms plus 1.0 Å, the corresponding residues are considered to be in contact.", "Long-range contacts are defined as contacts between residues, which are separated by at least 10 residues or belong to different polypeptide chains.", "Two residues are stabilization center elements (SCEs) and form a SC together if they are involved in long-range contacts and at least one supporting residue can be found in both flanking tetra-peptides of these residues in a special way that at least seven out of the possible nine interactions are actually found between the two triplets.", "Stabilization centers can be identified with the SCide public server (www.enzim.hu/scide) [3].", "It is important to note that a SCE can participate in more than one SC simultaneously.", "The role of SCs on protein thermal stability was not considered when the concept was introduced and has not yet been explored in detail.", "There were some efforts to reveal a possible connection between stabilization center content and proteins stability [4] and we investigated our hypothesis that some residues determine function, while others are responsible for stability and found that despite our expectations there is an overlap between residues responsible for protein function and stability [5].", "However, this is the first comprehensive study until now, which deals with possible connections of stabilization centers and protein thermal stability in detail.", "Long range cross-links help maintaining the native structure of proteins [6].", "These cross-links can be either covalent, like disulfide bonds, or noncovalent like SCs.", "It is reasonable to consider that both types of crosslinks stabilize the folded structure.", "While covalent crosslinks do it by reducing the entropy of the unfolded state, thereby they increase the refolding rate, noncovalent crosslinks disappear during denaturation thus only slowing down this process, therefore they stabilize the folded state by reducing the unfolding rate [7].", "The relationship of SCs with secondary structural elements as well as functionally important regions of proteins were already presented [1,5,8] to show a detailed picture of these clusters.", "Stabilization centers incorporate clusters of cooperative long-range interactions.", "Although the Gibbs free energy contribution of an individual noncovalent bond is generally marginal due to the entropy loss, it can become significant when several inter-residue interactions act together [9], thus we believe that SCs might be important from the viewpoint of stability.", "To check the putative role of SCs in thermal stabilization of proteins a survey was done on the ProTherm database [10].", "The interplay between SCEs and residues which influence the thermal stability has been investigated with multiple approaches.", "First we have analyzed the relationship between melting temperature or Gibbs free energy of unfolding in water and the SCE content of wild type proteins.", "Subsequently, we have explored the possible correlation between protein stability change upon mutations and SCEs using thermodynamic parameters in the ProTherm database.", "We have used our in-house computer programs for identifying the stabilization center elements.", "In our protocol, we have not only considered the intra-chain stabilization centers, where both SC forming residues come from the same polypeptide chain, but also inter-chain SCs.", "We used the transformation matrix found in the PDB files to generate the quaternary structure of the proteins.", "To get an idea of the average SCE content of proteins we checked the PDBselect [11] database.", "We took all protein chains in the 25% list from November 2012, obtained from http://bioinfo.mni.th-mh.de/pdbselect/recent.pdb_select25.nsigma3.0 and identified intra- and interchain SCs.", "We investigated a total of 3096 protein chains.", "The thermal stability data for proteins and mutants have been obtained from ProTherm database, which is a collection of a large number of experimental data on protein stability such as unfolding Gibbs free energy change, enthalpy change, heat capacity change and transition temperature.", "ProTherm is freely available at http://www.abren.net/protherm/, which has been effectively used to understand the factors influencing the stability of protein mutants and developing methods for predicting the stability upon mutations [12–14].", "Two different databases were constructed from the original Protherm database.", "First a wild type stability database was set up to check if there is a correlation between the thermal stability and the SCE content of a protein, called the wild type database.", "In order to investigate broader aspects of stability two datasets were set up within this database.", "In the first dataset entries with data from thermal denaturation experiments, while in the second dataset entries with data from chemical denaturation experiments were included.", "We were using the following filtering criteria:", "Only entries with known 3D structure of wild type proteins were taken into account.", "In the first wild type dataset only entries with Tm melting temperature data were included.", "In order to get a uniform database only Tm values obtained by Circular Dichroism (CD) or Differential Scanning Calorimetry (DSC) measurement methods were used.", "In the second wild type dataset only entries with ΔG_H2O (Gibbs free energy of unfolding in water) data were included.", "Since we are interested in stability of the native like protein structure entries with measurements under extreme conditions were discarded.", "Only entries with 5 ≤ pH ≤ 9 experimental conditions were kept.", "Furthermore we used the Tm ≤ 105 °C filtering criteria for the first dataset.", "In the second dataset entries with the most extreme ΔG_H2O values (>25.5) were discarded.", "After applying these filters in many cases more than one Tm value was present in the first dataset.", "We discarded proteins where difference between the lowest and highest Tm value was greater than 10°.", "In the remaining cases the average of the different Tm values was used.", "In the second dataset proteins, where differences in the Gibbs free energy of unfolding in water values were bigger than 1.5 kcal/mol, were discarded.", "For proteins with smaller differences average values were used.", "A second database was set up to investigate if stability altering amino acid substitutions overlap with SCEs or not.", "This is called the mutant database.", "We used the following filtering criteria:", "Only entries of single point mutants were taken into account.", "Wild type and multiple mutation entries were discarded.", "In the latter case we would not be able to distinguish, which mutation has high contribution to the cumulative effect of mutations.", "This is not a serious restriction since more than 80% of data in the Protherm database is for single point mutations.", "One of the following experimental thermodynamic data had to be available, ΔTm: melting temperature change upon mutation, ΔΔG: change in Gibbs free energy of unfolding obtained by thermal denaturation or ΔΔG_H2O: change in Gibbs free energy of unfolding in water, determined by chemical denaturation.", "These measures were collected in different datasets because of the different nature of these measurements.", "The values within one datasets can originate from different type of measurement methods.", "In order to get reliable and comparable data, only entries obtained by CD or DSC methods were kept for the ΔTm and ΔΔG datasets and only fluorescence or CD methods in the case of ΔΔG_H2O dataset [15].", "Since an atomic resolution structure is needed for the identification of the stabilization centers, all remaining entries had to have an available PDB structure.", "Ideally this would be the structure of the mutant, but since the number of mutated protein structures in the Protherm database is very low compared to the wild type structures, the wild type structures were used in all cases.", "Like in the wild type case, we have considered the stability of the native like protein structures and the entries with measurements under extreme conditions were discarded.", "In all three datasets negative values mean a decrease in stability, while positive ones denote increase in stability.", "Since we are interested in stability changes related to an amino acid position rather than the amino acid itself, we were using the maximum value for stabilizing and the minimum value for destabilizing mutations if measurements for several different mutations are available for the same position, rather than an average value.", "An average value would be unadvisable because of the asymmetrical nature of the database composition, there are a lot more destabilizing mutations, than stabilizing ones.", "We calculated the average SCE content of the protein chains from the PDBselect database.", "The number of stabilization center elements was 69,706, while the total number of residues was 351,385, giving an average SCE content of 19.84%, which is used as a reference value hereafter.", "We were interested if there is any general correlation between the thermal stability of a protein and its stabilization center element content.", "First we identified the SCEs in the proteins of first dataset of the wild type database and calculated the average SCE content for all proteins.", "There were a total of 181 Tm measurements in the range of 25 and 105 °C.", "We averaged the SCE content of proteins within a Tm window of 20°, i.e. the data at temperature T is the average SCE value of proteins with a Tm value within the T ± 10 interval.", "Since there were only a few points in the highest temperature range, the entries with the next two highest Tm value (glutamate dehydrogenase from Thermococcus litoralis and Pyrococcus furiosus; 1BVU, Tm = 109 °C and 1GTM, Tm = 114 °C) [16,17] were added to this dataset, which were originally excluded from the database.", "One entry (1BGL) was deleted from the dataset, because our program could not process this structure.", "This is not a significant problem, because this structure with Tm value of 49.8 °C fall into a region, where relatively many data can be found.", "There were a couple of PDB structures in which no SCs could be identified.", "Most of these protein structures were solution NMR structures.", "Since the definition of SCs is very sensitive to the quality of the structure due to the hard limit of heavy atom distances, we prefer high resolution X-ray structures.", "We searched the PDB database for X-ray structures of the same proteins.", "We used the PDB clustering available at http://resources.rcsb.org/sequence/clusters/bc-100.out to identify X-ray equivalents.", "We managed to find an appropriate structure in the cases listed in Table 1.", "SCE content was recalculated using these alternate X-ray structures.", "Results can be seen in Fig. 1.", "It can be stated that the SCE content is increasing until 80 °C.", "Above this temperature, the average SCE content goes into saturation or even starts to decrease.", "However, standard deviations are quite large, and there is a low number of data points in the Tm > 80 region, this temperature range coincides well with the temperature range in which organisms are classified as hyperthermophiles.", "There are several reviews which indicate that thermophiles and hyperthermophiles apply different strategies (like optimizing cavities) in the optimization of their protein structures to reach increased thermal stability needed at their physiological temperature [18,19].", "This finding might explain the change in the SCE content at extreme temperatures.", "Gibbs free energy of unfolding in water ΔG_H2O values obtained by chemical denaturation measurements represent a different aspect of protein stability.", "The second dataset of the wild type database includes entries with ΔG_H2O measurements.", "There were 170 entries within the range of 0 and 25.5 kcal/mol.", "We created 8 intervals as in the previous Tm wild type dataset and used a window size of 6 kcal/mol for averaging the SCE content for all proteins of the dataset within the given range.", "Results can be seen in Fig. 2.", "Since there is an overlap between entries belonging to adjacent intervals, a relatively smooth curve is expected even for an uncorrelated case.", "From the S-shape of the curve we can conclude that there is no correlation between the SCE content and Gibbs free energy of unfolding in water of proteins.", "Thermodynamic data from the Protherm derived mutant datasets were analyzed in the following way.", "The ΔTm, ΔΔG and ΔΔG_H2O datasets were handled separately because of the different nature of the measurements.", "In all three cases data were represented with histograms.", "For every N whole number of ΔTm, ΔΔG and ΔΔG_H2O values we counted the number of entries within the [N−0.5,N+0.5[ intervals.", "For example the 0 point in Fig. 3 gives the number of mutation which have a −0.5≤ΔTm<0.5 value.", "We fitted the sum of two Gaussians to these data points.", "By means of the equation of the fitted curve, the two points of the curve at the half maximum were determined and rounded to one decimal precision.", "These two points defined 3 regions (destabilizing, neutral and stabilizing), which were different for the three datasets.", "The average stabilization center element content was 20.10%, 19.50% and 20.50% for the ΔTm, ΔΔG and ΔΔG_H2O datasets, respectively.", "We identified the stabilization center elements in the wild type PDB structures assigned to the mutations.", "In every entry we checked if the position of the mutation is a stabilization center element or not.", "We calculated the number of overlaps of mutations with stabilization center elements for all the three (destabilizing, neutral and stabilizing) regions in the different datasets.", "Results can be seen in Table 2.", "Mutations in Range2 are considered neutral in terms of thermal stability.", "Mutations in Range1 decrease stability and mutations in Range3 increase thermal stability.", "According to the results, it can be stated that for all the three measurements, in the destabilizing range (1) the ratio of stabilization centers among the mutated positions is higher than the average SCE content, thus there is an overlap between the SCEs and the positions of mutations.", "In the neutral range (2) we can see segregation of SCEs and mutations in all three datasets.", "In the stabilizing range (3) only in the ΔΔG dataset is there a significant overlap.", "Since this dataset has the lowest number of data, results for this range do not provide a strong evidence.", "The results for the first two ranges, that SCE overlap with destabilizing mutation and segregate from neutral mutation positions strengthen our hypothesis that SCs are important in maintaining protein stability, mutations of SCE residues usually destabilize the structure.", "The result for Range3 that the mutation of SCEs which already take part in the stabilization of a proteins structure do not improve stability further is not surprising either.", "Since the geometric definition of stabilization centers might prefer buried residues over solvent exposed ones, we investigated if this might play any role in our findings.", "First we investigated if SC elements are biased to favor buried positions.", "Solvent accessibility was calculated using the Naccess 2.1.1 software [20].", "Before solvent accessibility calculation the quaternary structure of all proteins were created based on the BIOMT transformation matrices.", "Based on the relative surface area (RSA) all protein residues were divided into three categories:", "Buried residues (RSA < 0.1)", "Partially buried residues (0.1 < RSA ≤ 0.5)", "Exposed residues (0.5 < RSA)", "In the PDBselect database 31.9% of all residues were classified as buried, 40.97% were partially exposed and 27.13% were exposed.", "We investigated the distribution of SC elements among these three categories, as well.", "In the PDBselect database 52.87% of SC elements were buried, 39.2% were partially buried and only 7.93% were exposed, thus SC elements are more frequent among buried residues.", "Further we found that 33.24% of buried residues are SCEs, while in the partially buried and exposed categories this ratio is 19.19% and 5.86%, respectively.", "Compared to the average SCE content of about 19% we can see that buried residues are really overrepresented among SCEs.", "This is probably because of the geometric criterion in the SC definition which can be much easier fulfilled in the inner part of a protein structure than on the surface.", "Taking this observation into account exposed SCEs might be even more important in stabilizing protein structures.", "We analyzed the largest subset of the Protherm database, the destabilizing mutation of Range1 of the ΔTm dataset.", "We found in this dataset the strongest correlation between SCE content and stability change.", "We calculated how many residues of the three classes are SCEs in this dataset: 33.71% and 17.91% of buried and partially buried residues were SCEs, respectively.", "This ratios are almost the same as the values obtained for the PDBselect database.", "However for exposed residues in this dataset 14.04% of the residues were SCEs, while for the PDBselect database the corresponding value is only 5.86%.", "This finding underlines the role of exposed SCs in the thermal stabilization of protein structures.", "In virtue of this result we checked if solvent exposed SCE content shows any correlation with thermal stability using the wild type database.", "The result can be seen in Fig. 4, where the exposed SCE content is plotted as function of melting temperature.", "As previously seen in Fig. 1, we can see that protein structures with higher thermal stability contain more solvent exposed SCE residues.", "Although the number of 3D structures for mutated proteins is low in the Protherm database, we managed to find a couple of examples, where the effects of a mutation at an SCE position can be verified by the structure of the mutated protein.", "In Table 3 [21–23] we can see a couple of destabilizing mutations, where the wild type residue is a SCE, but in the mutated structure the corresponding residue is not a SCE, any more.", "We even found a special case, where a stabilizing mutation resulted in the formation of a SC, which was not present in the wild type structure.", "It is the Staphylococcus aureus nuclease; 1STN, where the proline 117 residue was mutated [24–26].", "1SYE, P117T, ΔTm = +0.6 °C [24] and ΔΔG_H2O = +1.10 kcal/mol [25]; 1SNP, P117G, ΔTm = +5 °C [24] and ΔΔG_H2O = +1.60 kcal/mol [25]; 1SYG, P117A, ΔΔG_H2O = +0.8 kcal/mol [26].", "Stabilization centers are thought to have a role in “maintenance” of the three-dimensional structure of proteins by preventing their decay due to their cooperative long range interactions.", "Our results coincide well into this picture that proteins with higher melting temperature have a higher average number of SCEs, but there is no correlation between Gibbs free energy of unfolding in water and SCE content.", "Mutations which do not influence protein stability are not overlapping with SCs, while mutations which change stability in either way are more frequently found at SC residues.", "The investigation of the wild type protein structures showed that structures with higher denaturation temperatures up to 85 °C have a higher number of SCEs.", "However, the trend is not true in the temperature range of hyperthermophilic proteins.", "This seems even more clearly in the case of solvent exposed SCs, where the trend is even true for hyperthermophilic proteins.", "These findings strengthen our hypothesis that SCs are important for the maintenance of a stable protein structure.", "Thus we can conclude that stabilization centers are deeply involved in thermal stabilization of protein structures." ]

[ "Stabilization centers contribute to thermal stabilization of protein structures.", "Stabilization center content correlates with melting temperature of proteins.", "Exposed stabilization center content correlates with stability even in hyperthermophiles.", "Stability changing mutations are frequently found at stabilization centers." ]

[ "ECON" ]

[ "Anomaly", "Bitcoin", "Cryptocurrency", "Day of the week effect", "Efficient Market Hypothesis", "Trading strategy" ]

[ "2003" ]

[ "There exists a vast literature analyzing calendar anomalies (the day of the week effect, the Turn of the Month Effect, the Month of the Year Effect, the January Effect, the Holiday Effect, the Halloween Effect etc.), and whether or not these can be seen as evidence against the Efficient Market Hypothesis (EMH – see, e.g., Fama, 1965; Samuelson, 1965; Jensen, 1978).", "However, with one exception (Kurihara and Fukushima, 2017) to date no study has analysed such issues in the context of the cryptocurrency market – this being a newly developed market, it might still be relatively inefficient and it might offer more opportunities for making abnormal profits by adopting trading strategies exploiting calendar anomalies.", "We focus in particular on the day of the week effect, and for robustness purposes apply a variety of statistical methods (average analysis, Student's t-test, ANOVA, the Kruskal–Wallis and Mann–Whitney tests, and regression analysis with dummy variables) as well as a trading robot approach that replicates the actions of traders to examine whether or not such an anomaly gives rise to exploitable profit opportunities.", "The paper is structured as follows: Section 2 briefly reviews the literature on the day of the week effect; Section 3 outlines the empirical methodology; Section 4 presents the empirical results; Section 5 offers some concluding remarks.", "The day of the week effect (concerning statistically significant differences between returns on different days of the week) was one of the first calendar anomalies to be examined.", "Fields (1931) showed that the best trading day of the week is Saturday.", "Cross (1973) provided evidence of statistical differences in Friday–Monday data in the US stock market.", "French (1980) reported negative returns on Mondays.", "Further studies found evidence of a positive Friday/negative Monday pattern (see Gibbons and Hess, 1981; Rogalski, 1984; Smirlock and Starks, 1986, etc.).", "Other studies on the stock market include Sias and Starks (1995), Hsaio and Solt (2004), and Caporale et al. (2016), whilst commodity markets were analysed by Singal and Tayal (2014), and the FOREX by Caporale et al. (2017).", "Ariel (1990), Fortune (1998) and Schwert (2003) all reported evidence against the Monday effect in developed markets, but this anomaly still appears to exist in many emerging markets (Caporale and Plastun, 2017).", "The cryptocurrency market is rather young but sufficient data are now available to examine its properties.", "Dwyer (2014), Cheung et al. (2015) and Carrick (2016) show that it is much more volatile than other markets.", "Brown (2014) provides evidence of short-term price predictability of the BitCoin.", "The inefficiency of the BitCoinmarket is also documented by Urquhart (2016), whilst Bartos (2015) reports that this market immediately reacts to the arrival of new information and can therefore be characterised as efficient.", "Halaburda and Gandal (2014) analyse correlations in daily closing prices.", "However, so far the only study examining anomalies in this market is due to Kurihara and Fukushima (2017), who focus exclusively on the BitCoin, which is not necessarily representative of the cryptocurrency market as a whole.", "The present paper aims to fill this gap in the literature by providing much more extensive evidence on the day of the week effect in this market.", "We examine daily data for 4 cryptocurrencies, choosing those with the highest market capitalization and the longest data span (2013–2017), namely BitCoin, LiteCoin, Ripple and Dash.", "The data source is CoinMarketCap (https://coinmarketcap.com/coins/).", "More information on the cryptocurrency market is provided in Table 1 below.", "Average analysis provides preliminary evidence on whether there are differences between returns for the different days of the week.", "Both parametric and non-parametric tests are carried out given the evidence of fat tails and kurtosis in returns.", "The Null Hypothesis (H0) in each case is that the data belong to the same population, a rejection of the null suggesting the presence of an anomaly.", "We carry out Student's t-test, ANOVA, Kruskal–Wallis and Mann–Whitney tests for the whole sample, and also for sub-samples in order to make comparisons between periods that might be characterised by an anomaly and the others.", "Student's t-tests are carried out for the null hypothesis that returns on all days of the week belong to the same population; a rejection of the null implies a statistical anomaly in the price behaviour on a specific day of the week.", "Given the size of our dataset, it is legitimate to argue that normality holds, and therefore these are valid statistical tests.", "To provide additional evidence one more method is used, namely ANOVA analysis.", "The main advantages of these methods are their simple interpretation, robustness and overall ease of use.", "Their main disadvantage is that they do not consider the possibility of non-normal distributions of the data.", "To take this into account, a number of additional non-parametric tests can be used, such as the Kruskal–Wallis and Mann–Whitney tests.", "Their key advantage is that they do not require any assumptions about the distribution of the population.", "The main limitation of the Mann–Whitney test is that it can only be used for 2 groups.", "Therefore we also carry out the Kruskal–Wallis tests that also allow testing for 3 groups or more.", "The reason for carrying out both non-parametric and parametric tests is to check for robustness.", "The size, sign and statistical significance of the dummy coefficients provide information about possible anomalies.", "If an anomaly is detected we then apply a trading robot approach that simulates the actions of a trader according to an algorithm (trading strategy) with the aim of establishing whether or not that anomaly gives rise to exploitable profit opportunities, which could be seen as evidence against market efficiency.", "This is a programme in the MetaTrader terminal that has been developed in MetaQuotes Language 4 (MQL4) and used for the automation of analytical and trading processes.", "Trading robots (called experts in MetaTrader) allow to analyse price data and manage trading activities on the basis of the signals received.", "If a strategy results in the number of profitable trades > 50% and/or total profits from trading are > 0, then we conclude that there is a market anomaly.", "The results are presented in the \"Report\" in Appendix A.", "The most important indicators given in the “Report” are:", "Total net profit — financial result of all trades.", "This parameter represents the difference between “Gross profit” and “Gross loss”.", "Expected payoff — mathematical expectation of a win.", "This parameter represents the average profit/loss for one trade.", "It also shows the expected profitability/unprofitability of the next trade.", "Total trades — total number of trade positions.", "Bars in test – the number of observations used for testing.", "The findings are summarised in the “Graph” section of the “Report”: this represents the account balance and general account status considering open positions.", "The “Report” also provides full information about all the simulated transactions and their financial results.", "To make sure that the results we obtain are statistically different from the random trading ones we carry out t-tests.", "We chose this approach instead of carrying out z-tests because the sample size is less than 100.", "A t-test compares the means from two samples to see whether they come from the same population.", "In our case the first is the average profit/loss factor of one trade applying the trading strategy, and the second is equal to zero because random trading (without transaction costs) should generate zero profit.", "The null hypothesis (H0) is that the mean is the same in both samples, and the alternative (H1) that it is not.", "The computed values of the t-test are compared with the critical one at the 5% significance level.", "Failure to reject H0 implies that there are no advantages from exploiting the trading strategy being considered, whilst a rejection suggests that the adopted strategy can generate abnormal profits.", "An example of the t-test is presented in Table 2.", "As can be seen there is no evidence of statistically significant difference in terms of total net profits relative to the random trading case, and therefore no market inefficiency is detected.", "The complete set of results can be found in Appendix B.", "The average analysis (Figs. B.1–B.4) provides preliminary evidences in favor of a day of the week anomaly in the dynamics of BitCoin and LiteCoin, whilst in the cases of Ripple and Dash it is unclear whether or not this is present.", "The results of the parametric and non-parametric tests are reported in Appendices C–F) and summarized in Table 3.", "There is clear evidence of an anomaly only in the case of BitCoin.", "The next step is to apply a trading simulation approach.", "First we design appropriate trading rules for the days when long or short positions, respectively, should be opened (see Table 4 for details).", "As can be seen most of the tests above provide evidence in favour of the presence of an anomaly in Monday returns for BitCoin.", "To make sure that this is not the result of the base effect we extend the analysis in order to be able to compare provide returns on Mondays with average returns on all other days of the week except Mondays and Sundays.", "The results are presented in Appendix G.", "Most of them (average analysis, t-tests and ANOVA analysis, regression analysis with dummy variables) confirm the existence of statistically significant differences between the two sets of returns, more specifically they indicate the presence of abnormally high returns on Mondays, i.e. of a day of the week effect in the case of Bitcoin.", "Since the anomaly occurs on Mondays (when returns are much higher than on the other days of the week) the trading strategy will be the following: open long positions on Monday and close them at the end of this day.", "The trading simulation results are reported in Table 5.", "In general this strategy is profitable, both for the full sample and for individual years, but in most cases the results are not statistically different from the random trading case, and therefore they do not represent evidence of market inefficiency.", "This paper examines the day of the week effect in the cryptocurrency market focusing on BitCoin, LiteCoin, Ripple and Dash.", "Applying both parametric and non-parametric methods we find evidence of an anomaly (abnormal positive returns on Mondays) only in the case of BitCoin.", "Further, using a trading simulation approach we show that a trading strategy based on this anomaly is profitable for the whole sample (2013–2017): it generates net profit with probability 60% and these results significantly differ from the random ones.", "However, in the case of individual years the opposite conclusions are reached.", "On the whole, there is no conclusive evidence that the cryptocurrency market is inefficient." ]

[ "This paper examines the day of the week effect in the cryptocurrency market.", "It using a variety of statistical techniques – as well as a trading simulation approach.", "Most crypto currencies (LiteCoin, Ripple, Dash) are found not to exhibit this anomaly.", "The only exception is BitCoin, for which returns on Mondays are significantly higher than those on the other days of the week.", "Trading simulation analysis shows that there exist exploitable profit opportunities; however, most of these results are not significantly different from the random ones and therefore cannot be seen as conclusive evidence against market efficiency." ]

[ "AGRI" ]

[ "CCM", "CO2 uptake", "HCO3- uptake", "Leakage" ]

[ "1104", "1105" ]

[ "During photosynthetic carbon fixation, the lighter carbon isotope 12C is preferred over the heavier carbon isotope 13C, thereby causing carbon isotope fractionation (εp) between the inorganic carbon (Ci) source and the organic carbon.", "Values for εp of marine phytoplankton have been shown to be CO2-sensitive (e.g. Degens et al., 1968), and thus were discussed to serve as a proxy for past CO2 concentrations (Jasper and Hayes, 1990; Pagani, 2014; Van de Waal et al., 2013; Hoins et al., 2015).", "Large species-specific differences in εp have been described, which are yet poorly understood (e.g. Hinga et al., 1994; Burkhardt et al., 1999).", "Moreover, irrespective of the phytoplankton species investigated, most of these studies have solely described the relationship between εp and CO2, and only few have investigated the underlying physiological processes.", "Such mechanistic understanding is, however, needed to identify the reasons of the CO2-dependency of εp.", "Carbon isotope fractionation of phytoplankton is primarily driven by the enzyme ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RubisCO), which is responsible for the fixation of CO2 into organic compounds.", "The intrinsic fractionation associated with RubisCO (εf) has been estimated to range between ~ 22 and 30‰ (e.g. Roeske and O'Leary, 1984; Guy et al., 1993; Scott et al., 2007), even though a recent study obtained values as low as 11‰ for the RubisCO of the coccolithophore Emiliania huxleyi (Boller et al., 2011).", "While RubisCO principally sets the upper limit of fractionation, other processes strongly determine the degree to which RubisCO can express its fractionation (Sharkey and Berry, 1985; Burkhardt et al., 1999; Rost et al., 2002).", "First, there is leakage, i.e. the amount of CO2 diffusing out of the cell in relation to Ci uptake.", "With higher leakage, the intracellular Ci pool is ‘refreshed’, thereby preventing accumulation of 13C and allowing RubisCO to approach its upper fractionation values.", "Second, the relative contribution of bicarbonate (HCO3−) to total Ci uptake plays a role, as HCO3− is enriched in 13C by ~ 10‰ relative to CO2 (Mook et al., 1974).", "An increasing HCO3− contribution thus lowers εp.", "The enzyme carbonic anhydrase (CA), which accelerates the otherwise slow interconversion between CO2 and HCO3−, can also influence εp under certain conditions, e.g. by influencing leakage as well as the relative HCO3− contribution.", "All these processes play a role in the CO2-concentrating mechanisms (CCMs) of phytoplankton.", "Assessing the mode of CCMs may therefore help to understand the reasons for CO2-dependent changes in εp and species-specific differences therein.", "Dinoflagellates are cosmopolitan unicellular algae that occur in many different environments, including eutrophic coastal regions and oligotrophic open oceans.", "In this study, we investigated whether the CO2-dependency of εp, which was found in the dinoflagellate species Alexandrium fundyense, Gonyaulax spinifera, Protoceratium reticulatum and Scrippsiella trochoidea (Burkhardt et al., 1999; Hoins et al., 2015), can be explained by changes in their Ci fluxes.", "Characteristics of CCMs in the tested species, including their CA activities and Ci fluxes, were measured by means of membrane-inlet mass spectrometry (MIMS).", "Results were fed into a single-compartment model that considers cellular leakage, the relative HCO3− contribution as well as the carbon isotope fractionation of RubisCO (Sharkey and Berry, 1985; Burkhardt et al., 1999).", "The calculated carbon fractionation (εp-mod) was then compared to the measured carbon fractionation (εp-meas).", "Cultures of the dinoflagellate species A. fundyense (formerly Alexandrium tamarense strain Alex5; John et al., 2014), S. trochoidea (strain GeoB267; culture collection of the University of Bremen), G. spinifera (strain CCMP 409) and P. reticulatum (strain CCMP 1889) were grown in 0.2 μm filtered North Sea water (salinity 34), which was enriched with 100 μmol L− 1 nitrate and 6.25 μmol L− 1 phosphate.", "Metals and vitamins were added according to f/2 medium (Guillard and Ryther, 1962), except for FeCl3 (1.9 μmol L− 1), H2SeO3 (10 nmol L− 1) and NiCl2 (6.3 nmol L− 1) that were added according to K medium (Keller et al., 1987).", "Each of the strains was grown in 2.4 L air-tight borosilicate bottles at 15 °C and 250 ± 25 μmol photons m− 2 s− 1 at a 16:8 h light:dark cycle.", "Bottles were placed on roller tables in order to avoid sedimentation.", "Dissolved CO2 concentrations ranged from ~ 5–50 μmol L− 1 and were reached by pre-aerating culture medium with air containing 180, 380, 800 and 1200 μatm pCO2.", "The carbonate chemistry was calculated based on pH and total alkalinity (TA), using the program CO2sys (Pierrot et al., 2006).", "pH values were measured using a WTW 3110 pH meter equipped with a SenTix 41 Plus pH electrode (WTW, Weilheim, Germany), which was calibrated prior to measurements to the National Bureau of Standards (NBS) scale.", "An automated TitroLine burette system (SI Analytics, Mainz, Germany) was used to determine TA.", "Dissolved inorganic carbon (DIC) was determined colorimetrically using a QuAAtro autoanalyser (Seal Analytical, Mequon, USA).", "For more details on the carbonate chemistry in the acclimations, please refer to Eberlein et al. (2014) for A. fundyense and S. trochoidea and to Hoins et al. (2015) for G. spinifera and P. reticulatum.", "To determine εp values, the isotopic composition of the organic material was measured using an Automated Nitrogen Carbon Analyser mass spectrometer (ANCA-SL 20–20, SerCon Ltd., Crewe, UK), and the isotopic composition of the DIC in growth medium was measured using a GasBench-II coupled to a Thermo Delta-V advantage isotope ratio mass spectrometer (see Hoins et al., 2015 for details on isotope analysis).", "Prior to assays, cells were acclimated to the different CO2 concentrations for at least 7 generations (i.e. > 21 days).", "To prevent changes in the carbonate chemistry, i.e. keeping drawdown of DIC < 3%, incubations were terminated at low cell densities (< 400 cells mL− 1).", "A custom-made membrane-inlet mass spectrometer (MIMS; Isoprime, GV Instruments, Manchester, UK; see Rost et al., 2007 for details) was used to determine CA activities and Ci fluxes of A. fundyense and S. trochoidea acclimated to four different pCO2 (i.e. 180, 380, 800 and 1200 μatm; Eberlein et al., 2014), and of G. spinifera and P. reticulatum acclimated to a low and high pCO2 (i.e. 180 and 800 μatm).", "Assays were performed in an 8 mL temperature-controlled cuvette, equipped with a stirrer.", "Assay tests over ~ 1 h confirmed that conditions during the assay do not cause physiological stress (i.e. no decline in O2 production rates), and subsequent microscopic inspection did not reveal any visual effects on cell morphologies.", "Prior to the measurements, acclimated cells were concentrated using a 10 μm membrane filter (Millipore, Billerica, MA) by gentle vacuum filtration (< 200 mbar) and stepwise transferred into Ci-free medium buffered with a 4-(2-hydroxylethyl)-1-piperazine-ethanesulfonic acid (50 mmol− 1 HEPES) solution at 15 ± 0.3 °C and a pH of 8.0 ± 0.1.", "Chlorophyll a (Chl-a) concentrations were determined fluorometrically by using a TD-700 Fluorometer (Turner Designs, Sunnyvale, CA, USA) and ranged between 0.15 and 1.70 μg mL− 1 during the assays.", "To quantify activities of extracellular CA (eCA), the 18O depletion rate of doubly labeled 13C18O2 in seawater was determined by measuring the transient changes in 13C18O18O (m/z = 49), 13C18O16O (m/z = 47) and 13C16O16O (m/z = 45) in the dark, following the approach of Silvermann (1982).", "If cells possess eCA, exchange rates of 18O are accelerated relative to the spontaneous rate.", "To monitor the spontaneous rate, NaH13C18O3 label was injected to the cuvette, waiting until the m/z = 49 signal reached a steady-state decline.", "This rate was then compared to the steady-state decline after cells were added.", "Following Badger and Price (1989), eCA activity is expressed as percentage decrease in 18O-atom fraction upon the addition of cells, normalized to Chl-a.", "Consequently, 100 units (U) correspond to a doubling in the rate of interconversion between CO2 and HCO3− per μg Chl-a.", "Photosynthetic O2 and Ci fluxes were determined following Badger et al. (1994).", "Making use of the chemical disequilibrium, this approach estimates CO2 and HCO3− fluxes during steady-state photosynthesis.", "It is based on the simultaneous measurements of O2 and CO2 concentrations during consecutive light and dark intervals with increasing amounts of DIC.", "Oxygen fluxes in the dark and light are converted into Ci fluxes by applying a respiratory quotient of 1.0 and a photosynthetic quotient of 1.1 (Burkhardt et al., 2001; Rost et al., 2003).", "The light intensity in the cuvette was adjusted to the acclimation conditions (i.e. 250 ± 25 μmol photons m− 2 s− 1).", "Net CO2 uptake was calculated from the steady-state decline in CO2 concentration at the end of the light period, corrected for the interconversion between CO2 and HCO3−.", "The uptake of HCO3− was calculated by subtracting net CO2 uptake from net Ci uptake, and the CO2 efflux from the cells was estimated from the initial slope after turning off the light.", "Rate constants k1 and k2 were determined based on temperature, salinity and pH (Zeebe and Wolf-Gladrow, 2001; Schulz et al., 2006), yielding mean values of 0.9241 (± 0.0506) min− 1 and 0.0085 (± 0.0008) min− 1, respectively.", "To eliminate any eCA activity, a prerequisite to apply the rate constants, we added dextran-bound sulfonamide (DBS; 50 μmol L− 1) to the cuvette.", "For more details on the calculations, please refer to Badger et al. (1994) and Schulz et al. (2007).", "Shapiro–Wilk tests confirmed normality of the data.", "Significant differences between CO2 treatments were confirmed by a one-way ANOVA followed by post hoc comparison of the means using the Tukey HSD (α = 0.05; Table 1).", "In A. fundyense and P. reticulatum, eCA activities were low with maximum activities of 156 U (μg Chl-a)− 1 and 44 U (μg Chl-a)− 1, respectively.", "In S. trochoidea and G. spinifera, eCA activities were comparably high with up to 1600 U (μg Chl-a)− 1 and 1100 U (μg Chl-a)− 1, respectively.", "In neither of the species, eCA activities were responding to changes in pCO2.", "Please note that for G. spinifera and P. reticulatum no statistics could be applied due to the lack of replication.", "Relative HCO3− contribution was around 0.2 in A. fundyense and G. spinifera (Figs. 1A and 3A; Table 1), whereas S. trochoidea and P. reticulatum showed higher values of ~ 0.5 (Figs. 2A and 4A; Table 1).", "In other words, in A. fundyense and G. spinifera approximately 80% of the Ci taken up is in the form of CO2, whereas in S. trochoidea and P. reticulatum this was 50%.", "There was a significant decrease in HCO3− contribution with increasing CO2 concentration in S. trochoidea, while no such CO2-dependency was observed in any of the other tested species.", "Leakage differed significantly between the tested species, with values of up to 0.7 at 800 μatm pCO2 in G. spinifera (Fig. 3A; Table 1) and lowest average values of ~ 0.5 in S. trochoidea and P. reticulatum, respectively (Figs. 2A and 4A; Table 1).", "Only in A. fundyense, leakage was significantly CO2-dependent and increased from 0.44 to 0.63 (Fig. 1A; Table 1).", "For details on the kinetics of O2, CO2 and HCO3− fluxes in A. fundyense and S. trochoidea, please refer to Eberlein et al. (2014).", "Estimates for εp-mod are in the same range as εp-meas in A. fundyense and P. reticulatum (Figs. 1B and 4B; Table 1), while the model overestimated the fractionation by up to 5‰ and 8‰ in S. trochoidea and G. spinifera, respectively (Figs. 2B and 3B; Table 1).", "Except for S. trochoidea, εp-mod generally matches trends observed in εp-meas.", "In A. fundyense, for instance, εp-mod increased significantly from 10.1 to 15.3‰, thereby closely matching εp-meas values (Fig. 1B; Table 1).", "Also in G. spinifera, εp-mod matches trends observed in εp-meas, if the highest pCO2 treatment of εp-meas is excluded.", "In this treatment, carbon isotope fractionation dropped significantly, most likely due to 2.5-fold increased cellular carbon contents (see discussion in Hoins et al., 2015).", "In S. trochoidea, neither the relative HCO3− contribution nor leakage showed a CO2-dependency; hence εp-mod did not match the increase in εp-meas with increasing CO2 concentration (Fig. 2B; Table 1).", "By expressing CA, many marine algae species accelerate the otherwise slow interconversion between CO2 and HCO3−, thereby possibly facilitating the Ci uptake and internal Ci fluxes.", "In line with previous studies on dinoflagellates (Rost et al., 2006; Ratti et al., 2007), A. fundyense and P. reticulatum exhibit rather low eCA activities, even under low CO2 concentrations.", "In view of this, eCA is not expected to significantly influence Ci fluxes or εp in these species.", "In S. trochoidea and G. spinifera, however, eCA activities were high at all tested CO2 concentrations, comparable to values observed for temperate diatoms (Trimborn et al., 2009).", "Hence, the inhibition of eCA by the inhibitor DBS during the MIMS assay might have biased the Ci fluxes, i.e. underestimated CO2 uptake (Rost et al., 2003), thereby potentially causing an underestimation of εp.", "As these were the species for which the model overestimated εp values, however, it can be concluded that eCA (and its inhibition by DBS during the Ci flux measurements) did not influence the Ci fluxes much.", "The HCO3− contribution differed considerably between the tested species.", "While A. fundyense and G. spinifera showed a strong preference for CO2, S. trochoidea and P. reticulatum used CO2 and HCO3− in equal proportions.", "The latter contradicts with the findings of an endpoint pH-drift experiment, suggesting that P. reticulatum is not able to efficiently use HCO3− (Ratti et al., 2007).", "Testing other dinoflagellates with a modified pH-drift method, including the genus Protoceratium, demonstrated that the high pH itself can affect growth and thus interpretations about the used Ci source based on pH-drift experiments must be considered with caution (Hansen et al., 2007).", "From an energetic point of view, high CO2 usage would be of advantage as CO2 can be taken up passively by diffusion, while HCO3− is charged and thus has to be taken up by active uptake.", "And yet, the tested species covered a large part of their Ci demand by HCO3−, as observed in S. trochoidea and P. reticulatum (Figs. 2A and 4A).", "Similarly high HCO3− contributions were observed for other dinoflagellate species (Rost et al., 2006) and cyanobacteria (Price et al., 2008; Kranz et al., 2011).", "This preference for HCO3− has been associated with the very low CO2-affinity of RubisCO type IB, which is expressed in cyanobacteria, and RubisCO type II expressed in dinoflagellates (Badger et al., 1998; Whitney and Andrews, 1998).", "To compensate for this low affinity, high amounts of Ci have to be accumulated, which in seawater can more easily be accomplished with the abundant HCO3− ion rather than with CO2.", "In addition, HCO3− is about 1000-fold less permeable to lipid membranes than CO2, making it the preferred Ci form to be accumulated within the cell (Price et al., 2008).", "In the case where species covered the majority of their Ci demand by CO2, as observed in A. fundyense and G. spinifera (Figs. 1A and 3A), one could therefore speculate about chloroplast-based Ci accumulation rather than pumping of HCO3− at the cell wall (Badger et al., 1998).", "The observed differences in the preferred Ci source and likely consequences for internal Ci fluxes may also affect the overall leakage of cells.", "Also leakage differed considerably among the species.", "A. fundyense showed a relatively low leakage of 0.44 at 180 μatm pCO2, which increased to 0.63 at 1200 μatm pCO2.", "Leakage in G. spinifera varied between 0.60 at 180 μatm pCO2 and 0.70 at 800 μatm pCO2.", "Leakage estimates in S. trochoidea and P. reticulatum were lower with ~ 0.50 and remained constant over the applied range of pCO2.", "These differences may be caused by different membrane permeabilities, which again potentially relate to the preferred Ci source.", "In fact, A. fundyense and G. spinifera both preferred CO2 over HCO3− and likewise showed the highest degrees of leakage, thereby suggesting highly permeable membranes with respect to CO2.", "In these species, also CO2-related changes in the membrane permeability are indicated as they show significantly increased leakage under higher pCO2 (see also Eberlein et al., 2014 for A. fundyense, formerly A. tamarense).", "Using results for HCO3− contribution and leakage obtained in this study, carbon isotope fractionation was calculated and compared to previous measurements (Figs. 1B–4B; see also Hoins et al., 2015).", "Generally, there is a good agreement as εp-mod and εp-meas values were in the same range (A. fundyense, S. trochoidea, P. reticulatum) and/or followed the same trend (A. fundyense, G. spinifera, P. reticulatum; Figs. 1B-4B).", "Despite the overall agreement between flux-based estimates and directly measured carbon isotope fractionation, εp-mod was overestimated in S. trochoidea and G. spinifera.", "Such offsets could principally be attributed to biases in the Ci flux measurements, i.e. uncertainties in the estimation of HCO3− contribution and/or leakage.", "It has been argued, however, that the MIMS approach tends to overestimate the HCO3− contribution (due to the constant pH of 8.0 during the assay, see Burkhardt et al., 2001), and rather underestimates cellular leakage (due to fact that CO2 fixation does not cease instantly upon darkening, see Badger et al., 1994).", "Hence, by correcting for these potential biases, i.e. assuming slightly lower HCO3− contribution and higher leakage values, we would actually overestimate the fractionation even more for S. trochoidea and G. spinifera.", "An alternative explanation for the overestimation by the model may be attributed to the fractionation factor of RubisCO, which we assumed to be 28‰ (Raven and Johnston, 1991).", "Recent studies have found lower values, even as low as 11‰ as in the case of the coccolithophore Emiliania huxleyi (Boller et al., 2011).", "Even though there are no indications for such low fractionation values in the highly conserved type II RubisCO, a lower fractionation would bring modeled and measured εp values closer in S. trochoidea and G. spinifera.", "In A. fundyense and P. reticulatum, however, it would lead to underestimated εp-mod.", "Hence, we would refrain from assuming much lower fractionation values for RubisCO type II in dinoflagellates in our calculations.", "Lastly, the fact that internal Ci fluxes were not taken into account might have also contributed to the offsets between εp-mod and εp-meas.", "Models that incorporate internal Ci cycling have, however, caused even higher εp-mod, as these processes work against the 13C accumulation within the chloroplasts (Cassar et al., 2006; Schulz et al., 2007) or the carboxysome (Eichner et al., 2015).", "Therefore, although the values and trends in carbon isotope fractionation are relatively well understood based on our physiological experiments, differences between theory and measurements are at present not fully resolved.", "Our study demonstrates that carbon isotope fractionation in dinoflagellates can, to a large degree, be explained by considering their Ci fluxes.", "Relative HCO3− contribution and/or leakage were CO2-dependent in A. fundyense, S. trochoidea and G. spinifera, which in turn can explain the CO2-dependency of their εp observed in previous studies (Hoins et al., 2015).", "To further advance our understanding of the εp patterns in dinoflagellates, Ci fluxes measurements should be performed at in situ pH (Kottmeier et al., 2014, 2016) and ideally differentiate between 13C and 12C fluxes (McNevin et al., 2006)." ]

[ "To understand 13C fractionation in dinoflagellates, inorganic carbon fluxes were measured by MIMS and used for modeling.", "Changes in cellular carbon fluxes, i.e. HCO3− contribution and CO2 leakage were CO2-dependent.", "These CO2-dependencies could largely explain the CO2-dependent fractionation patterns observed in the four tested species." ]

[ "AGRI" ]

[ "Characterization", "Expression analysis", "Mannose-binding lectin", "Mulberry", "Prokaryotic expression" ]

[ "1110" ]

[ "Lectin is a kind of proteins possessing at least one noncatalytic domain, which binds reversibly to specific mono- or oligo- saccharides (Peumans and Van Damme 1995).", "It has the role of agglutinating cells and precipitating glycan or sugar compounds (Agard and Bertozzi 2009).", "Mannose-binding lectin (MBL) is a kind of protein that can combine with mannose specifically and belongs to the monocot mannose-binding (GNA related) lectin family (Adamek et al. 2013).", "MBL is important body natural immune molecules and can remove pathogens by activating complement and opsonophagocytosis.", "It plays an important role in defensive self-justification in plant (Peumans and Van Damme 1995) and resistance malignant tumor cell proliferation in human (Balzarini et al. 1991; Xu et al. 2007).", "Mannose distribution is very little in the plant, but it's more found in fungi, viruses and insects.", "MBL can affect normal metabolism of cells, because it can combine specifically with the mannose and other sugars on the microorganism's surface and interferes with the synthesis of the cell wall or the lectin strongly bound to larval brush border and peritrophic membrane detected by immunostaining (Ohizumi et al. 2009).", "Recently, a lectin receptor kinase 1.9 has been identified as a critical receptor for extracellular ATP (Choi et al. 2014).", "So MBL involves in diverse responses and plays an important role in resist insects, pathogenic microorganisms and plant-eating animals of plant.", "Now, there are more study on snow lotus herb (Liu and Zhang 2009), Crinum asiaticum (Chai et al. 2003), rice (He et al. 2016), wheat (Ma et al. 2010), pinellia (Lin et al. 2008), ginger (Chen et al. 2005) and other plants.", "The resistance of galanthus nivalis agglutinin (GNA) on aphid homoptera and nematode has been verified (Bhat et al. 2010).", "But the study of MBL on abiotic stress is less, in which the MBL can response to salt stress in rice (Zhang et al. 2000; He et al. 2016).", "As defense proteins, the relationship between MBL and abscisic acid (ABA) induction, low temperature stress and the mechanical damage stress is unclear.", "Mulberry (Morus multicaulis), a perennial tree or shrub, is an economically important plant used not only in sericulture, as the sole plant food for the domesticated silkworm, but also for a variety of other purposes, such as the production of edible fruits, useful timber, medicinal substances, ecological management and ornamental objects (Rai et al. 2009; Natić et al. 2015; Liu et al. 2016).", "The growth and productivity of mulberry is adversely affected by abiotic and biotic stresses.", "Higher plants always adapt to environmental stresses by the activation of cascades of molecular networks involved in stress perception, signal transduction, and expression of specific stress-related genes (Zheng et al. 2015).", "Despite several studies have investigated the stress response role of MBL in mulberry and other plants (Gu et al. 2009; Zhang et al. 2011; Saeed et al. 2016), the exact functions of MBL genes and encoded proteins in the stress response is still not fully understood in mulberry and their functions need to be defined essentially.", "In this paper, the cDNA of the MMBL gene was cloned from the leaves of mulberry (Morus multicaulis) based on mulberry expressed sequence tags (ESTs) (Zhao 2008) and homologous cloning technology, using reverse transcriptase polymerase chain reaction (RT-PCR).", "The structure of MMBL protein was analyzed.", "Fluorescent quantitative real time PCR was performed to analyze the expression characteristics of MMBL in the leaf, bud, fruit, stem, phloem and xylem.", "Furthermore, changes in the transcription levels of MMBL under drought, salt, cold and mechanical damage stresses were investigated by qRT-PCR (quantitative real-time PCR) to explore the relationship between MMBL genes and mulberry's resistance to adversity.", "The recombinant protein of MMBL was expressed in prokaryotic system by inserting an open reading frame (ORF) segment of the MMBL into the expression plasmid pET-28a(+).", "Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot results revealed that His-tagged fusion protein was successfully expressed.", "The results should not only lay a good foundation for understanding of the complicated signal transduction mechanism underlying stress responses, but also provide a basis in molecular biology for the improvement of mulberry resistance by gene regulation technology and the breeding of resistant varieties.", "To analyze the gene expression under different conditions, mulberry (Morus multicaulis) variety ‘Yu711’ was grown under standard conditions at the National Mulberry Gene Bank of the Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, P. R. China.", "The mulberry shoots were grafted to mulberry stock.", "The grafted seedlings were planted in 35 cm diameter pots, each pot containing one seedling, and were grown in an incubator with a 16 h photoperiod at 25 °C/22 °C (day/night, respectively).", "After approx. 2 months, when the shoots had reached approx. 20 cm in length, the seedlings were subjected to drought, salt, cold and mechanical damage stress treatments.", "To simulate drought stress, mulberry seedlings were treated without water for 0 d, 1 d, 3 d, 5 d, 10 d, and 15 d.", "Salt stress was imposed by irrigating the mulberry with approx. 100 ml of 300 mM NaCl for 0 d, 1 d, 2 d, 4 d, 7 d, and 10 d.", "To simulate cold stress, mulberry seedlings were treated under 4 °C for 0 d, 12 h, 1 d, 3 d, 6 d, and 10 d and then grown under the normal conditions as mentioned above for 2 d and 5 d to renaturation.", "Mechanical damage stress was imposed by scratching three times on the surface of leaves for 0 h, 1 h, 3 h, 5 h, 8 h, and 12 h.", "For the relative transcription analyses, different tissues (leaf, bud, fruit, stem, phloem and xylem) of mulberry seedlings were collected.", "Leaves and other tissues were collected at different time points, immediately frozen in liquid nitrogen and stored at − 80 °C.", "They were subsequently used for total RNA extraction.", "Total RNA was extracted from browses (net weight approx. 100 mg) of grafted mulberry seedlings using RNAiso Plus reagent (Takara, China)according to the giving protocol, treated with Deoxyribonuclease I, then resuspended in 0.1% (v/v) diethylpyrocarbonate (DEPC)-treated water and stored at − 80 °C.", "The quality of total RNA was determined using an ultraviolet spectrophotometer combined with 1.0% (w/v) agarose gel electrophoresis.", "Then the first strand cDNA was synthesized from total RNA using the RNase H-Reverse Transcriptase M-MLV kit (Takara, China) following the manufacturer's instructions.", "The first-strand cDNA was used as the template for PCR in gene cloning.", "Forward primer 1 (MMBL-F1: 5′-AAACATAACCTAATTGTGCTC-3′), reverse primer 1 (MMBL-R1: 5′-CTGGATTTTATTTAATTCGCTCT-3′), forward primer 2 (MMBL-F2: 5′-AAATCAATCTTTCTCGTGGT-3′) and reverse primer 2 (MMBL-R2: 5′-AAAATATTAAACATTCTTGCTGGAT-3′) were designed according to the EST with the inference function from mulberry cDNA library (Zhao 2008) and the homology sequences in the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) databases using the BLAST program.", "The amplification conditions were used for PCR as follows: initial denaturation at 94 °C for 5 min; followed by 36 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 35 s, and elongation at 72 °C for 45 s; with a final extension at 72 °C for 7 min.", "The PCR products were separated on 1.0% agarose gels and sequenced by Sangon Biotech (Shanghai, China).", "Thus, a cDNA sequence of the MMBL was obtained according to fragment assembly.", "The nucleotide sequence was submitted to GenBank with the accession number KY348866.", "The sequence encoding MMBL was analyzed for identity and similarity using the NCBI online Search Tool (BLASTX) and ORF of MMBL was found using the ORF finder program at NCBI (http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi).", "The MMBL protein structure and functional domains were predicted using the prosite tools of ExPASy (http://prosite.expasy.org/prosite.html/) and SMART (http://smart.embl-heidelberg.de/) respectively.", "SWISS-MODEL (http://swissmodel.expasy.org/) was used to analyze the molecular modeling of the MMBL protein (Zhao et al. 2010).", "The molecular weight and theoretical isoelectric point (pI) of the protein were predicted using the ProtParam tool of ExPASy (http://web.expasy.org/protparam/).", "Subsequently, the homologous sequences were downloaded from the NCBI database, and multiple sequence alignments were performed of amino acid sequences of MMBL sequences from different species using the ClustalX 2.0 program and DNASTAR software (Thompson et al. 1997).", "Finally, to determine the relationship between MMBL and other MBL genes, phylogenetic analysis was carried out using the MEGA 6.0 and ClustalX 2.0 program (Tamura et al. 2007).", "The phylogenetic tree of the MMBL proteins from different species was constructed according to the minimum-evolution test method.", "The reliability of the tree was accessed by a bootstrap analysis with 1000 replications.", "The qRT-PCR method was used to detect the expression level of MMBL in mulberry between different tissues.", "Total RNAs of different tissues were isolated respectively from leaf, bud, fruit, stem, phloem and xylem (approx. 1.0 g) using RNAiso Plus reagent (Takara, China) according to the manufacture's procedures and then quantified by measuring the absorbance at 260 nm 1 μg of each RNA sample was used as a template for the synthesis of the cDNAs by the same protocol mentioned above.", "The resultant was then diluted 12-fold and 4 μl cDNA was used as the template to perform the qRT-PCR.", "The SYBR Green RT-PCR assay was performed in LightCycler® 96 Real-Time PCR System (Roche, USA) by three-step real-time qPCR according to the FastStart Universal SYBR Green Master Mix kit (Roche, USA) protocol.", "The thermal cycling parameters for qRT-PCR were 95 °C for 600 s followed by 45 cycles of 95 °C for 10 s, 57 °C for 10 s and 70 °C for 10 s using primers MMBL-1-F (5′-ACACATCCAGGAAACGAACC-3′) and MMBL-1-R(5′-TACCCACTCACGCTCACTAA-3′).", "During amplification, melting curves were constructed.", "The internal housekeeping gene β-actin using primers β-actin-F (5′- AGCAACTGGGATGACATGGAGA-3′) and β-actin-R (5′-CGACCACTGGCGTAAAGGGA-3′) was used as a normalization control for each starting quantity of cDNA templates (Jiang et al. 2015).", "All reactions were assayed in three biological replicates with three technical replicates.", "The relative expression differences of mRNAs in different tissues were calculated by 2− ΔΔCt method (Schmittgen and Livak 2008).", "Standard errors and standard deviations were calculated from replicates and statistical significance was measured through One-Way ANOVA Duncan's multiple rang test at the level of 0.01 < p ≤ 0.05 and p ≤ 0.01using SPSS 19.0 software.", "Expression level of the MMBL mRNA under abiotic stresses (drought, salt, cold and mechanical damage) in mulberry were measured by qRT-PCR.", "Total RNA was isolated from the young leaves (approx. 1.0 g) of mulberry shoots under the either stress or control conditions at different time points.", "Every template cDNA for qRT-PCR was gotten with the same method mentioned above.", "The two pairs of qRT-PCR primers (mentioned above) were used for the quantitative real-time PCR to check the relative expression value.", "The reaction conditions were the same as those mentioned above.", "Total RNA was extracted from leaves of mulberry using RNAiso Plus reagent (Takara, China) according to the given protocol and the first strand cDNA was synthesized using PrimeScript™ RT Reagent Kit (TaKaRa) according to the instructions of the manufacturer.", "The ORF of MMBL(KY348866)with BamH Ι and Sal Ι restriction sites was amplified from the cDNA using the primers MMBL-2-F (5′-TTAGGATCCATGGCGGGCACAAGCACGAA-3′) and MMBL-2-R (5′-GCCGTCGACTTAAAGTGACAAGTGGAAGC-3′) by PCR following: initial denaturation at 94 °C for 5 min; followed by 32 cycles of denaturation at 94 °C for 30 s, annealing at 64 °C for 35 s, and elongation at 72 °C for 40 s; with a final extension at 72 °C for 7 min.", "The amplified products were gel-purified and cloned into pMD20-T vector (Takara, China).", "After being transformed into E. coli Top10, the positive clones were identified by PCR and sequenced.", "Then the fragment of MMBL and the expression plasmid vector pET-28a(+) were digested with BamH Ι and Sal Ι respectively and then ligated together with T4 DNA Ligase (Takara, China).", "The resulting recombinant plasmid pET-28a(+)-MMBL was identified by PCR, enzyme cleavage and sequencing to confirm the correct insert of MMBL gene.", "The resulted recombinant plasmid pET-28a(+)-MMBL was transformed into E. coli BL21.", "The positive clone of E. coli BL21-pET-28a(+)-MMBL was grown in Luria-Bertani (LB) broth containing 50 μg/ml kanamycin overnight(about 12 h) at 37 °C under shaking (200 rpm).", "A 100 μl aliquot of the overnight culture was added into 3 ml fresh LB medium containing kanamycin, and the latter was incubated at 37 °C on 200 rpm shaker until an OD600 of approximately 0.6 was obtained.", "Then, isopropyl β-D-thiogalactoside (IPTG) was added to the culture to a final concentration of 0.5 mM for the induced expression of the recombinant protein for 3.5 h at 37 °C.", "The bacterial cells were harvested by centrifugation at 8000 r/min for 5 min.", "The cell pellet was washed by 1 × phosphate buffer saline (PBS) (pH 7.4) three times and subjected to sonication at 500 W for 6 times with 5 s working and 5 s free on ice.", "After centrifugation at 10,000 r/min for 5 min at 4 °C, the supernatant was preserved directly at − 20 °C and the precipitate was also preserved at − 20 °C after resuspended by1 × PBS.", "The recombinant protein was analyzed by SDS-PAGE according to Tricine-SDS-PAGE protocol (Schagger 2006).", "The protein samples of the supernatant and precipitate were mixed with 1 × SDS-PAGE loading buffer (0.05 M Tris–HCl, pH 6.8, 10% glycerol, 2% SDS, 0.01% bromophenol blue, 0.1 M β-ME) and boiled for 5 min at 95 °C.", "After centrifugation at 12,000 r/min for 3 min, the supernatant and aliquots of 10 μl of the non-induced, induced hollow plasmid and induced hollow cell samples were loaded on a 4% stacking gel-12% separating gel, and the gel running was performed according to the manufacturer's instructions (BIO-RAD).", "Electrophoresis was performed by 80 V–150 V voltage.", "After, the protein samples were detected using coomassie brilliant blue R-250 staining for 1 h with 45 rpm and discoloration for 3 h.", "For protein western blot analysis, the non-stained SDS-PAGE gels were transferred onto nitrocellulose membrane by electroblotting using Mini Trans-Blots Electrophoretic Transfer Cell System (BIO-RAD).", "The membrane was blocked in TBST (0.01 M Tris-HCl, pH 7.5 + 0.15 M NaCl + 0.1% Tween-20) containing 5% (v/v) skimmed milk powder for 2 h at 37 °C by shaking.", "After being washed with TBST three times of 10 min intervals, the membrane was incubated with mouse anti-His tag primary antibody at 1:1000 dilution in TBST for 2 h in darkness at 37 °C with shaking.", "The membrane was then washed three times with TBST and incubated with horseradish peroxidase (HRP) conjugated goat anti-mouse antibody at 1:2000 dilution in TBST for 1 h in darkness at 37 °C with shaking.", "Immunoreactivity was detected by using a chromogenic substrate of diaminobenzidine (DAB).", "Based on the EST with the inference function from mulberry cDNA library and sequence of the conserved regions of known MBL genes from other plant species, the cDNA of the mulberry MMBL gene (GenBank accession: KY348866)was cloned by RT-PCR and then sequenced.", "The obtained cDNA of MMBL gene is 978 bp long and contains a 189 bp incomplete 5′-UTR, a 303 bp complete 3′-UTR and a 486 bp ORF.", "The ORF encodes 161 amino acids with a predicted molecular weight of 16.97 kD and a theoretical isoelectric point of 6.07 (Fig. 1A).", "The MMBL had Jacalin domain and six sugar binding sites, and belonged to Jacalin-like superfamily as shown in Fig. 1B.To better understand the molecular modeling of MMBL, the protein tertiary structure of mulberry MMBL was performed online with Swiss-Model software.", "The intricate spatial architecture showed that, there were 1 α-helix, 12 β-sheet and 14 β-turn in the three-dimensional structure of MMBL (Fig. 2).", "To better understand the structure of the deduced amino acid sequence encoded by mulberry MMBL gene, a homologous multiple protein sequence alignment was carried out using ClustalX 2.0 software between the amino acid sequences of MMBL and MBL of ten other species selected from the NCBI database.", "The results revealed that the primary structure of the mulberry MMBL showed high levels of similarity to MBL of ten other plants and all the proteins above appear to be rather conserved in structure (Fig. 3): the amino acid sequence encoded by the MBL gene shared very high similarity with Morus notabilis (92%), and shared high similarity with Vitis viniferaand Theobroma cacao (54%), and with Capsicum annuum (53%), and with Citrus clementine and Gossypium hirsutum (52%).", "Homology analysis revealed that MBL was highly conserved among the species, but exhibited some differences.", "To investigate the evolutionary relationships, a phylogenetic tree was constructed with MEGA 6.0 software based on the deduced amino acid sequences encoded by the MMBL gene from mulberry and 23 other species using the minimum-evolution test method.", "The results revealed that Morus multicaulis (KY348866), Morus notabilis (XP_010092355.1), Ipomoea nil (XP_019198333.1), Capsicum annuum (XP_016562621.1), Solanum lycopersicum (XP_004235622.1) and Solanum tuberosum (XP_006343016.1) all had a relatively close relationship.", "The MMBL of mulberry was somewhat close to that of Erythranthe guttata (XP_012835090.1), Sesamum indicum (XP_011081586.1), Eucalyptus grandis (XP_010062570.1), Malus domestica (XP_008348486.1), Prunus mume (XP_008244753.1) and Prunus persica (XP_007215056.1), but was slightly distant from that of other species as shown in Fig. 4.", "Nonetheless, MMBL showed relationships with all the MBL with respect to length and structure, as compared above, which revealed that the amino acid residues of MBL were highly conserved.", "qRT-PCR analysis of the mRNA from different tissues of mulberry revealed that the gene of MMBL was expressed in all the six tested tissues, namely leaf, bud, fruit, stem, phloem and xylem (Fig. 5A).", "The highest transcript level was found in the bud, followed by that in the leaf and the lowest transcript level in the stem as compared to other tissues.", "And the relative expression of MMBL gene in bud was approximately 1.46, 42.83, 16.51,11.04 and 3.40 fold higher than that in leaf, fruit, stem, phloem and xylem respectively (p < 0.01), which illustrated that although the gene of MMBL could be expressed in all the six tested tissues, but the expression difference was great.", "In order to further investigate whether the expression of MMBL gene was induced by abiotic stresses, the mRNA transcript level of the MMBL gene under different abiotic stress treatments, including salt, drought, cold, mechanical damage, were measured at different time points by qRT-PCR analysis (Fig. 5B–E).", "Fig. 5B illustrated the effect of salt stress on mRNA expression of MMBL.", "MMBL gene expression under salt stress sharply dropped with about 0.03 times of the control on the first day and straightly increased to about 0.27 times of the control on the second day and then decreased on the fourth and seventh day.", "The expression of MMBL rose to 0.27 times of the control under continuous high salt stress to the tenth day.", "We can see that the salt stress inhibited the expression of MMBL (p < 0.01).", "To study the role of drought on regulating the expression of MMBL gene, we measured expression at 1 d to 15 d drought stress relative to the control.", "As shown in Fig. 5C, the expression level of the MMBL gene transcripts under drought stress slightly increased to the value of about 1.76 times of the control on the first day, slightly decreased on the third day and then gradually rose to the maximum (about 2.69 times of the control) (p < 0.01) on the fifteenth day.", "In Fig. 5D, we can see that MMBL gene expression under cold stress gradually rose to the maximum with 9.52 fold (p < 0.01) of the control on the sixth day and then straightly decreased to the value of about 0.49 times of the control on the tenth day.", "There were no obvious changes of MMBL gene expression between the tenth day and renaturation for five days under indoor temperature (25 °C) (p > 0.05).", "Under mechanical damage stress, the expression level of MMBL gene had great changes on the time points of 1 h, 3 h, 5 h, 8 h and 12 h (Fig. 5E).", "The expression level of the MMBL transcripts at 0 h had no change compared with the control.", "Following, MMBL gene expression under mechanical damage stress had an obvious fluctuation state.", "Reduced first (about 0.36 times of the control) (p < 0.05), then, increased dramatically with about 1.18 times of the control at 3 h, and then dropped again.", "It then increased to the maximum with 4.83 fold (p < 0.01) of the control on 12 h.", "Overall, these results revealed that MMBL gene was constitutively expressed under non-stress conditions, while its expression level changed by different degrees when the mulberry was under the abiotic stresses, including salt, drought, cold and mechanical damage stresses.", "All these results showed a better understanding of the molecular basis for signal transduction mechanism during the stress responses in mulberry trees and these suggest that the proteins encoded by MMBL gene may be involved in multiple mechanisms of abiotic stress tolerance in mulberry.", "To clone the sequence encoding mature MMBL, two specific primers MMBL-2-F and MMBL-2-R which contained BamH I and Sal I restriction enzyme site were used to amplify ORF of MMBL gene by PCR.", "After digestion with BamH I and Sal I, the amplified fragment was ligated into the pQE-28a(+) vector to generate the recombinant plasmid pET-28a(+)-MMBL (Fig. 6).", "The proper construction of the expression plasmid pET-28a(+)-MMBL was identified by PCR, restriction enzyme digestion and nucleotide sequencing.", "The sequence contained an initiating ATG, His-tag and lac operator provided by express pQE-28a(+) vector at its N-terminal.", "The recombinant plasmid pET-28a(+)-MMBL was transformed into E. coli by freeze thawing method for expression of MMBL.", "After induction by IPTG for 3.5 h, the recombinant protein of MMBL was highly and effectively expressed in E. coli BL21 as a major protein product in the total cellular protein compared with the negative control.", "As for the weight of MMBL (without signal peptide) protein, the predicted molecular weight was16.97 kDa, and the N-terminal fusion protein with His labeling of amino acids was about 5 kDa, a total of about 21.97 kDa.", "Expressed recombinant proteins was in precipitation as a form of inclusion bodies by SDS-PAGE detection and the fusion protein band's corresponding size was consistent with the expected value in SDS-PAGE (Fig. 7).", "Recombinant protein induced by IPTG was transferred to nitrocellulose membrane after separation by SDS-PAGE, and was detected by western blot with mouse anti-His tag primary antibody and horseradish peroxidase (HRP) conjugated goat anti-mouse antibody.", "As shown in Fig. 8, an obvious protein band was detected by western blot and estimated to be 21.97 KDa in molecular weight.", "In this study, a MMBL gene encoding mannose-binding lectin from a mulberry variety ‘Yu711’ was obtained for the first time.", "MMBL was a small gene, which contained a 486 bp ORF encoding a protein of 161 amino acids with a predicted molecular weight of 16.97 kD and a theoretical isoelectric point of 6.07.", "MMBL lacked any known signal peptide sequences and transmembrane regions.", "However, MMBL contained a highly conservative Jacalin domain, four subunits and six carbohydrate-binding sites (Sankaranarayanan et al. 1996; Bourne et al. 2002; Abhinav et al. 2015), belonging to the protein--Jacalin-like superfamily that was a kind of plant lectins (Van Dammeet al. 2002; Song et al. 2014).", "Jacalin also showed significant affinity for Core3 and sialyl-T (ST)-attached peptides, but (2) Jacalin could not bind to Core2, Core6, and sialyl-Tn (STn)-attached peptides.", "The results were also confirmed by FAC using p-nitrophenyl (pNP)-derivatized saccharides.", "Jacalin binds to a GalNAcalpha1-peptide, in which C6-OH of alphaGalNAc is free (i.e., Core1, Tn, Core3, and ST), whereas it cannot recognize a GalNAcalpha1-peptide with a substitution at the C6 position (i.e., Core2, Core6, and STn) (Tachibana et al. 2006).", "Homology analysis revealed that MBL was highly conserved among species, but exhibited some differences.", "Meanwhile, phylogenetic analysis based on MBL gene with other species showed that Morus multicaulis, Morus notabilis, Ipomoea nil, Capsicum annuum, Solanum lycopersicum and Solanum tuberosum had a relatively close relationship.", "The ability of recognition cell surface carbohydrates with high specificity of plant lectin plays an important role in gene transcription regulation, and can affect a variety of biological processes, such as cellular, stimulating response, signal transduction, cell growth, cancer and spectrum resistance on plant pathogens, viral pathogens, and fungi (Raval et al. 2004).", "The results of qRT-PCR analysis shows that the MMBL transcript could be detected in all of the tested tissues, including leaf, bud, fruit, stem, phloem and xylem of mulberry, but highest expression was detected in the bud and leaf.", "Preferential accumulation of MMBL in the bud and leaf suggests a crucial role as a defense related storage protein.", "Like the other MBL genes, the expression of MMBL gene had obvious tissue specificity (Van Damme et al. 1998).", "Recently, galactose-binding lectins have also been found to accumulate in mulberry laticifers, which is suggestive of a defense related role (Kitajima et al. 2013).", "This might suggest functional diversification and specialization of MMBL in different tissues.", "The defense function of MMBL was embodied in different growth stages of plants and protected plants from damage of diseases and pests in different ways.", "The expression patterns of MMBL can aid in understanding their function by identifying the probable tissues in which they are expressed.", "Song et al. (2014) have found that the expression of wheat JRL genes was largely tissue specific, typically low, and mostly inducible by biotic and abiotic stresses and stress hormones.", "Atalah et al. (2013) studied the promoter activity for three types of Euonymus -related lectins (EUL) from rice and found that the activity of OrysaEULS3 and OrysaEULD1A promoters was increased after ABA and mannitol treatments but decreased after NaCl treatment, and they hypothesized that the Euonymus -related rice proteins have a role in sensing and responding to external stresses as well as in the growth of the plant.", "Mulberry belongs to the perennial woody plants and its genetic background is very complex.", "Mulberry has a long time of child-tree and be more vulnerable to certain effects of various environmental stresses in time and space compared with the common herbs.", "Zhang et al. (2000) also identified the rice lectin as a stress protein and highlighted the possible importance of protein-carbohydrate interactions in stress responses in plants.", "The results of qRT-PCR analysis showed that the transcriptional level of MMBL mRNA significantly changed under the conditions of drought, salt, cold and mechanical damage stresses compared to the normal growth environment.", "The salt stress inhibited the expression of MMBL and the expression level of the MMBL transcripts under stress decreased to the minimum on the fourth day.", "In drought stresses, the transcriptional level of MMBL mRNA was in a fluctuation condition.", "It slightly increased to the value of about 1.76 times of the control on the first day and slightly decreased on the third day and then gradually rose to the maximum on the fifteenth day.", "The cold stress positively regulated the expression level of MMBL transcripts.", "MMBL gene expression under cold stress gradually rose to the maximum on the sixth day and then straightly decreased on the tenth day.", "There were no obvious changes about MMBL gene expression between the tenth day and renaturation for five days under indoor temperature (25 °C).", "The expression level of MMBL gene under mechanical damage stress also had an obvious fluctuation state.", "Changes were stable in the first few time points, and then reached the maximum amount on 12 h, which might be suggestive of a specific role of MMBL under wounding, such as insect attack and other animals injury.", "Together, these results showed that MMBL most likely acts as an important common component that responds to different abiotic stresses in mulberry.", "With the rapidly development of recombinant DNA technology, numerous systems have been utilized for the overexpression of varieties of proteins.", "E. coli was the most common foreign gene expression host which could provide large quantities of the protein of interest in a relatively short period of time.", "As in some plant, lectins have shown entomotoxic effects towards a wide range of insects, fungi and viruses (Huesing et al. 1991; Powell et al. 1993; Peumans and Van Damme 1995; Zhao et al. 2006), various efforts have been made on the expression of plant lectins in E. coli.", "Up to now, there are, nevertheless, few reports on the successful expression of active plant lectins in E. coli (Wang et al. 2001; Nogueira et al. 2002; Lin et al. 2009).", "By using a similar method, we successfully strongly expressed recombinant MMBL in E. coli.", "By cloning the ORF of MMBL gene encoding the mature protein into the expression vector, pET-28a, underT7 promoter and an inducible lac operator, we successfully expressed the mature MMBL in E. coli BL21.", "The recombinant protein is approximately 22 kDa detected by SDS-PACE and Western blot, which was consistent with the deduced protein molecular mass of MMBL.", "The yield of recombinant MMBL expression in the inclusion bodies was high.", "In summary, we obtained useful information of MMBL gene and our analysis confirmed the upregulation of MMBL in response to abiotic and biotic stress treatments except under salt stress.", "Together, these findings suggest a crucial role of MMBL under different stress-responsive pathways in mulberry.", "These make a foundation to the breeding of new mulberry varieties with excellent quality and high-level resistant.", "We also successfully strongly expressed recombinant MMBL in E. coli.", "The study of prokaryotic expression of MMBL gene will facilitate subsequent purification and examination of the controlling effect.", "Further study about the exact function of MMBL gene and molecular adaptation mechanisms to stresses in mulberry should be taken into consideration.", "The authors declare that there is no conflict of interest regarding the publication of this paper." ]

[ "MMBL gene was cloned from the leaves of mulberry (Morus multicaulis) that had an ORF of 486 bp encoding a protein of 161 amino acids.", "The estimated molecular weight and pI of the putative protein were16.97 kDa and 6.07, respectively.", "MMBL was expressed in all the tissues tested, including leaf, bud, fruit, stem, phloem and xylem in mulberry.", "The expression level of the mRNA has significantly change under abiotic stress conditions.", "We also successfully expressed recombinant MMBL in E. coli." ]

[ "CENG" ]

[ "Phosphorescence", "Photophysics", "RT-DFP", "RTP", "Thianthrene" ]

[ "1500", "1508" ]

[ "Thianthrene is a heterocyclic analog of anthracene but with two sulfur atoms substituting carbon atoms at the 9,10-positions.", "This structure leads to a bent, non-aromatic geometry of the central thianthrene ring [1–3].", "Thianthrenes are commonly known due to their interesting electrochemical properties [3–7] and quite well studied chemistry [2,3,6,8].", "Thianthrene being an electron-donor with a stable mono- and bicationic forms [4,7] has been used in several materials, including small molecules [2,6,8–11] and polymers [12,13].", "Although the chemistry and electrochemistry of thianthrenes are well known, the photophysics has not been performed extensively.", "In particular, phosphorescent properties of thianthrene crystals have already been demonstrated [1], but no study has been performed on its derivatives.", "Of particular interest are the room temperature phosphorescent (RTP) properties of thianthrene, which suggests this group can be used to promote dual fluorescence-phosphorescence at room temperature in its derivatives, which brings additional interest on thianthrene based compounds.", "The aim of controlling and tuning luminescence of organic materials in the solid state is an attractive topic for both fundamental research, and for practical applications in several fields.", "For example, the observation of dual luminescence [14] e.g. dual fluorescence-phosphorescence emissions at room temperature, (RT-DFP), enhances the possibility to achieve tunable luminescent characteristics under external stimuli, which expands the potential of these compounds in photonic and optoelectronic applications.", "Room temperature phosphorescence in purely organic, non-heavy-metal-containing molecules has been investigated for many years [1,15–23], and continues to be the topic of great interest, as confirmed by many recent publications, where RTP and RT-DFP are reported in several different systems [24–30].", "Many of these molecules contain a non-metal d-electron element such as bromine, phosphorus, sulfur, tellurium or iodine, which enhances intersystem crossing from the emissive singlet state to the triplet state [15–23,31].", "However, RTP or RT-DFP are observed usually in the crystalline [25] state, as powder, or in solid state blends, with the emitter dispersed in inert glassy host matrices made of amorphous polymers, such as PMMA, Zeonex® or in β-estradiol films that are non-permeable by oxygen [32].", "Hosts are used to restrict the competitive thermal non-radiative decay pathways and facilitate the radiative decay of the triplet state.", "However, there are a few examples where surprisingly RTP has also been reported in solution [33].", "While the strategies that have been implemented to restrict vibrations and induce RTP have gathered some success, further work is necessary to understand the mechanism driving RTP, and achieving design rules to obtain dual emitters is even a major challenge.", "It is still not clear why some emitters give origin to RT-DFP and others just to fluorescence, sometimes with very similar molecular structures and in the same host [34].", "The main advantage of organic materials showing RT-DFP is the possibility to achieve ratiometric measurements due to the presence of fluorescence and phosphorescence bands that are observed simultaneously.", "This expands the dynamic range of the emission of these compounds and introduces an internal calibration, allowing their use in applications as optical thermometry [35–37], money anti-counterfeiting techniques [38], oxygen sensors [39], analytical chemistry [40], mechanochromic materials [30], and bioimaging [41], among others.", "Optimization of RT-DFP emitters involves enhancing the total luminescence yield, and achieving a large separation of the fluorescent and phosphorescent bands, which expands the colorful emission switching under external stimuli.", "However, increasing the separation of the two emission bands, implies achieving a large energy gap between the singlet (S1) and triplet (T1) excited states which negatively impacts the triplet formation yield, due to less favorable intersystem crossing, and thus giving weaker phosphorescence yield [26].", "Therefore RT-DFP molecules need to be designed in a way that the intersystem crossing is enhanced, while the singlet-triplet energy is tuned to give well separated emission bands.", "In this study we present a group of thianthrene derivatives (Scheme 1) examining the effect of both the substitution positions and the type of substituent on their room temperature phosphorescent properties.", "The aim of the study is to understand structural and electronic effects, such as produced steric hindrance or donor/acceptor properties, of popular substituents on thianthrene.", "We further examine the effect of ππ* states on phosphorescence lifetime.", "Our results enabled us to understand the structure-property relationships in the RTP and RT-DFP systems based on thianthrene to further optimize the efficiency and performance of these emitters in future applications.", "Interestingly, the phosphorescence in these thianthrenes, when dispersed in Zeonex® matrix, shows broad, featureless spectrum at room temperature.", "However, while compounds 1a to 1c are mainly RTP emitters, compounds 2a to 2d are dual emitters but with different fluorescence-phosphorescence ratios, with compound 2a showing mainly phosphorescence, 2b and 2c showing dual fluorescence-phosphorescence bands, and 2d showing mainly fluorescence.", "However, while 2b, 2c and 2d have very low luminescence yields, 2a is a strong emitter with the luminescence yield close to 40%.", "This is a significantly strong phosphorescence yield at RT for a metal-free pure organic emitter.", "Thianthrene derivatives were synthesized as described in previous work [3].", "Absorption and fluorescence spectra were collected using a UV-3600 double beam spectrophotometer (Shimadzu), and a FluoroLog or FluoroMax-3 fluorescence spectrometer (Jobin Yvon).", "Low and room temperature measurements were acquired using a model liquid nitrogen cryostat (Janis Research) coupled with a rotary vacuum pump.", "Phosphorescence (PH) and prompt fluorescence (PF) spectra and decays were recorded using nanosecond gated luminescence and lifetime measurements (from 400 ps to 1 s) using a high energy pulsed Nd:YAG laser emitting at 355 (3rd harmonics) and 266 nm (4th harmonics) (EKSPLA).", "Emission was focused onto a spectrograph and detected on a sensitive gated iCCD camera (Stanford Computer Optics) having sub-nanosecond resolution.", "PF/PH time resolved measurements were performed by exponentially increasing gate and delay times.", "Photoluminescence quantum yield of solid films was done according to the procedure described elsewhere [42].", "Blends of all materials in Zeonex® were made at 0.5–1% w/w concentration.", "Measurements taken at room temperature (295 ± 1 K) at all time unless stated otherwise.", "Fluorescence spectra in solution were recorded after degassing by 3 freeze/thaw cycles.", "All measurements performed in 0.1 M Bu4NBF4 (99%, Sigma Aldrich) in dichloromethane (CHROMASOLV®, 99.9% Sigma Aldrich).", "Nitrogen bubbling was performed before measurement.", "Electrodes: working (Pt disc of 1 mm2 area), counter (Pt wire), reference (Ag/AgCl calibrated against ferrocene).", "All cyclic voltammetry measurements conducted at room temperature with scan rate of 50 mV s−1.", "DFT calculations of MO surfaces have been carried out using the B3LYP [43–45] hybrid functional combined with a 6-31G(d,p) [46–48] basis set.", "For all investigated compounds ground state geometries were optimized.", "All calculations have been carried out with Jaguar [49] version 9.1 in Maestro Materials Science 2.1 from Maestro Materials Suite 2016-1 software package [50].", "All alkyl chains have been reduced to methyl groups to decrease calculation complexity.", "The investigated materials were divided in two groups.", "The first one formed by compounds 1a-1c, consists of molecules with two thianthrene moieties connected by a bridge, whereas the second group, compounds 2a-2d, represents bisubstituted derivatives of thianthrene.", "All the compounds give blue fluorescence when dissolved in a non-polar solvent (Fig. 1).", "Interestingly, the emission spectrum within each group of materials is very similar.", "The fluorescence spectrum of 1a-1c derivatives resembles thianthrene emission with the onset being only slightly red-shifted.", "Molecules 2a, 2c and 2d show well-resolved fluorescence spectrum, but 2b shows emission spectrum resembling thianthrene.", "Moreover, the fluorescence from compounds 2a-2d appears clearly at lower energies than fluorescence of 1a-1c as indicated by the onset of the fluorescence spectrum.", "Before discussing the absorption spectra in Fig. 1, it is essential to note the key features of thianthrene absorption spectrum (Fig. 1).", "The reader can also refer to supporting information (Figs. S1 and S7).", "The UV-Vis spectrum of thianthrene in the presented wavelength range consists of two bands: 1) low energy band that forms a shoulder at λ = 270–340 nm with ε < 3.5 × 103 M−1cm−1 which clearly indicates a forbidden transition such as an n-π∗ transition involving the non-bonding electrons of sulfur; 2) the high energy band at λmax = 257 nm with ε ≈ 4 × 104 M−1cm−1 that indicates a π-π∗ transition.", "In contrast, the absorption spectra of derivatives 1a-1c consist of several absorption bands in the region of 270–360 nm where the thianthrene absorption is negligible.", "It is remarkable and interesting that the π-π∗ absorption band of thianthrene λmax = 257 nm can be clearly identified in 1a-1c derivatives (λmax = 261–264 nm, ε ≈ 0.4–1x105 M−1cm−1).", "This is a strong indication that the thianthrene moiety in this case is not well conjugated with the bridge unit.", "The absorption of 1a consists of a π-π∗ absorption band at λmax = 314 nm (ε ≈ 2 × 104 M−1cm−1) with a shoulder, presumably of a forbidden transition, most likely n-π∗ of phenothiazine.", "There is also another band at λmax = 290 nm.", "In 1b the well-resolved band at 330–360 nm (ε ≈ 3–4x103 M−1cm−1) is associated with n-π∗ transition of carbazole.", "This is followed by an absorption band at λmax = 290 nm.", "For 1c the energetically lowest absorption band is clearly of π-π∗ character λmax = 319 nm (ε ≈ 7 × 104 M−1cm−1).", "This band is followed by absorption at λmax = 298 nm.", "Absorption spectra of 2a-2d derivatives are very similar to each other.", "The absorption maximum (ε ≈ 3–4x104 M−1cm−1) observed shifts gradually from 2a (λmax = 277 nm) through 2b (λmax = 286 nm) and 2c (λmax = 287 nm) to 2d (λmax = 296 nm).", "These absorption bands are associated with π-π∗ transitions.", "It is worth to note that in this case the π-π∗ absorption band of thianthrene is no longer observed.", "This might suggest that thianthrene is in this case a part of a larger conjugated system.", "A shoulder is also observed at 320–380 nm in the absorption spectra of molecules 2a-2d, which is particularly evident in 2a, and similar to pure thianthrene, this shoulder can be associated to a n-π* transition.", "It is worth to note that overlap between those n-π* and π-π* absorption bands increases gradually from 2a to 2d which is expected to induce mixing of the nπ* and ππ* excited states thus formed.", "The conclusions from the analysis of the UV-Vis spectra of the presented thianthrenes are consistent with the calculated HOMO and LUMO surfaces (Fig. 2 and Table S2).", "The thianthrene moiety shows a high contribution of non-bonding electrons of sulfur to the HOMO, which makes the HOMO → LUMO transition to be of n-π* character.", "The same conclusion can be drawn from the observation of the HOMO in 2a-2d, where the contribution of the non-bonding electrons of sulfur is particularly evident.", "In case of 1a-1c the HOMO → LUMO transition is dominated by the bridge with some contribution of thianthrene to the LUMO.", "In 1a the nitrogen and sulfur atoms of phenothiazine bridge clearly contribute to the HOMO with their non-bonding electrons, making the HOMO → LUMO transition to be n-π*.", "In 1b the nitrogen atom of carbazole contributes to the HOMO giving somehow n-π* character to the energetically lowest transition.", "In 1c the HOMO is clearly dominated by π-conjugated system of fluorene which indicates π-π* character of HOMO → LUMO transition.", "All the molecules studied here show phosphorescence at room temperature that is comparable in intensity or much more intense than fluorescence (Fig. 3), when dispersed in a commercially available amorphous polymer Zeonex® 480 with high tg = 138 °C [51] and noticeable air-permeability.", "The difference between emission recorded in air and in vacuum is due to the quenching of long-lived triplet states by oxygen.", "Therefore, by comparing the spectra in Fig. 3 in air and in vacuum it is possible to quantify the relative contributions of fluorescence and phosphorescence to the overall emission, with the assumption that in vacuum no triplet oxygen quenching effect is present.", "Fig. 3 also shows that in case of derivatives 1a-1c and 2a the contribution of phosphorescence is very high and exceeds 90% of the total photoluminescence at room temperature.", "This is more than for pure thianthrene.", "The high photoluminescent quantum yield in these compounds and the large contribution of phosphorescence to the total luminescence, see Table 1, indicates that the phosphorescence properties of thianthrenes 1a-1c and 2a are induced by a high triplet formation yield.", "This is caused by the presence of two sulfur atoms in the thianthrene core giving origin to an enhanced spin orbit coupling due to the presence of lone pairs in the sulfur and heavy atom effect.", "However, in the case of derivatives 2b-2d the electron-donating effect (see below) of substituents causes reduction of the phosphorescence contribution to ≈50% in case of 2b and 2c and to only ≈25% in case of 2d.", "In these compounds, the reduction in the phosphorescence contribution is accompanied by the observation of a weak fluorescence band at high energies.", "This indicates that in compounds 2b-2d the radiative decay of the singlet excited state is able to compete with the slow intersystem crossing, and so less triplets are formed in these compounds (also see Table S1).", "Dual luminescence, i.e. RT-DFP, is thus achieved.", "However, the very low luminescence (fluorescence + phosphorescence) quantum yield of these compounds in solid state, roughly Φ < 0.1, without the presence of oxygen (Table 1) suggests that these thianthrenes are affected also by significant internal conversion.", "The presented materials show strong dependance of emission intensity and photoluminescence color upon oxygen concentration.", "This is because the blue fluorescence is almost not affected by oxygen, while the phosphorescence is strongly suppressed by oxygen, therefore the fluorescence-phosphorescence ratio at a certain temperature is itself an indication of total amount of oxygen in the surrounding atmosphere.", "Cyclic voltammetry (CV) was chosen to investigate the first oxidation potential of thianthrenes to explain the effects of substituents on the electron-donating properties of the compounds.", "Withdrawal of the electron from the compound in a redox reaction is performed from its HOMO, therefore CV gives an insight into the properties of the highest occupied molecular orbital.", "Oxidation of thianthrene is reversible (Fig. 4) as reported in the literature [5,7].", "All presented thianthrenes, except 2d, show reversible process as well.", "The oxidation onset potential of thianthrene 0.74 V is very close to the oxidation potentials of derivatives 1a-1c and 2a: 0.71–0.75 V.", "This behavior is especially surprising for 1a, where electron-rich phenothiazine is expected to oxidize at lower potential (Eox = 0.1 V vs. Fc/Fc+) than thianthrene does [52].", "In case of derivatives 2a-2d the oxidation potential lowers with the increase of electron-donating properties of substituent in the order from electron-deficient pyridine in 2a to the most electron-rich ethylenedioxythiophene (EDOT) in 2d.", "The electron-donating impact of the substituent in conjugated molecules 2b-2d moves the HOMO towards the electron-rich groups (Table S2 in supporting information), therefore reducing the contribution of non-bonding electrons to the HOMO.", "In the most electron-rich 2d the oxidation is centered mainly on the EDOT group which as a result gives an irreversible oxidation.", "EDOT and EDOT-containing molecules are known of being able to electropolymerize [53,54] and this is the most probable reason why 2d shows irreversible oxidation.", "Film formation on the electrode has been observed as a consequence of electrooxidation of 2d which confirms this statement.", "Interestingly, the electrochemical oxidation of thianthrenes correlates with their photophysical behavior.", "In particular, the derivatives 1a-1c and 2a show large contribution of phosphorescence in total emission (>90%) (Table 1) – the same compounds for which the oxidation potential differs only ±0.04 V from the thianthrene.", "In case of 2b-2d where the electron-donating effect of substituent starts to be important, the contribution of phosphorescence decreases dramatically to reach only ≈25% for 2d.", "Moreover, with decreasing intensity of phosphorescence, structure of the 2d spectrum is enhanced, which is particularly evident at low temperature.", "Fig. 5 shows that the phosphorescence spectra of derivatives 1a-1c at room temperature are almost identical to the phosphorescence spectrum of thianthrene under the same conditions, whereas derivatives 2a-2d show a red-shifted spectrum.", "The difference between the 1 and 2-series of thianthrenes is the position where thianthrene is substituted.", "It was shown earlier in text that 2a-2d compounds are well conjugated, whereas in 1a-1c, due to steric hindrance, the conjugation between thianthrene and the bridge unit is broken.", "This is also consistent with MO surfaces and simulated molecular geometry (see Table S2).", "It is also worth noting that the phosphorescence spectra of compounds 1a-1c and 2a are featureless, both at RT and 80 K, with the same effect being observed for pure thianthrene.", "However, low temperature spectra of 1a, 1b and 2a are all blue-shifted in the same manner, with the exception of compound 1c, for which the phosphorescence is not blue shifted.", "This shows that at higher temperatures the triplet state of these compounds is able to relax, probably due to the molecule adopting a new conformation and affecting the excited state of these thianthrenes derivatives.", "Remarkably, compounds 2b-2d behave differently – their low temperature phosphorescence spectrum has a clear vibronic structure, and the onset of their phosphorescence spectrum does not shift with temperature, unlike that of 2a, 1a, and 1b.", "The lack of structure in the phosphorescence spectrum, even at low temperature, can be a result of interconverting excited state conformers.", "Indeed, the thianthrene moiety is expected to wobble by the inversion of configuration at the sulfur atoms.", "Moreover, in the 2a-2d series the electron-donating/accepting properties of the substituents strongly affect the phosphorescence contribution to the overall luminescence and cause a significant effect on the phosphorescence spectrum.", "This suggests that the five-membered heterocyclic ring affects the electronic structure of thianthrene through conjugation and the effect escalates with electron-donating properties of the substituent (2d).", "In this case pyridine moieties in 2a, that are acceptors, can be treated as the least electron-donating substituents.", "This is supported by the computational study (Table S3) that shows which in this series the dihedral angle between the substituent and respective thianthrene phenyl ring is small enough to allow conjugation.", "As a result of conjugation the electrochemical oxidation potential of derivatives 2a-2d correlates with the results of the photophysical studies.", "It is particularly clear that the behavior of 2a-2d, is a consequence of their chemical structure and is closely related to their electronic structure which is expressed i.e. through electrochemical and calculation results.", "On the other hand, the mechanism causing the reduction in phosphorescence intensity in the series from 2a to 2d is related to an increase of both fluorescence and internal conversion rate constants.", "Analysis of time-resolved emission spectra of 2a reveals that a weak delayed fluorescence is also present (Fig. S5).", "However, power dependence studies, show that this delayed emission is due to triplet-triplet annihilation rather than thermally activated delayed fluorescence, moreover the singlet-triplet energy difference in 2a, ΔEST = 0.46 eV, is also too large for an efficient TADF to be observed.", "Finally, all these thianthrenes derivatives show long phosphorescence lifetimes at room temperature, ranging from 6 to 88 ms (Fig. 6, Table 1).", "Moreover, the phosphorescence decays monoexponentially in almost all cases, with exception of compound 1a.", "The biexponential behavior of the phosphorescence decay in 1a is accompanied by the change of the phosphorescence spectrum with time – a small blue-shift of the spectrum is observed at room temperature (Fig. S3).", "A similar, but less significant shift is observed in the case of compound 1c, but this compound shows rather monoexponential decay.", "This behavior clearly suggests involvement of two emissive species in 1a and 1c.", "These species can either be two excited state conformers or two simultaneously emitting local triplet states.", "In the latter case the emissive states can be associated with thianthrene side groups and the bridge, whereas interconversion between the states is slow.", "In case of 1b the carbazole has much higher triplet energy than thianthrene (≈3 eV) [55,56] thus all energy is transferred to the lowest triplet state of thianthrene by internal conversion.", "This does not apply to 2a-2d where the modifying group is conjugated with thianthrene resulting one excited state.", "Thianthrene derivatives with different substituents and substitution positions have been shown as purely organic room temperature phosphorescent emitters with large (>90%) or moderate phosphorescence contribution to the total photoluminescence.", "Molecules showing green phosphorescence of lifetime τ = 88 ± 6 ms and strong (ϕ ≈ 0.4) yellow triplet emission have been presented.", "It has been shown that substituents of thianthrene may tune the lifetime of phosphorescence and fluorescence-phosphorescence ratio, thus giving a variety of RTP and DFP-RT emitters.", "On the other hand, tuning of the phosphorescence color is performed mainly by the extension of their conjugation.", "By substituting thianthrene at position 1- the substituents are weakly conjugated due to a steric hindrance – this results in green phosphorescence similar to that of thianthrene - whereas at 2,8- positions steric hindrance is reduced – conjugated systems show yellow triplet emission.", "It has been observed that conjugated electron-rich substituents reduce the RTP intensity of thianthrene, they also have strong influence on the phosphorescence spectrum.", "This is due to depleting the n-π* character of the excited state by conjugated π-donor furan/thiophene-based systems.", "Such a substituent reduces the triplet formation yield of molecule by inducing larger radiative and internal conversion rate constants of a singlet state.", "This phenomenon is well correlated with electrochemical onset oxidation potential and escalates with decrease of its value, so with increase in electron-donating effect of the substituent.", "It has been presented that thianthrene may act as a RTP-promoting unit, which induces efficient intersystem crossing, whose phosphorescent properties may be tuned by substituents.", "Although thianthrene itself presents reasonable RTP properties, its absorption >280 nm is very weak which limits the possible application as an optical ratiometric sensor.", "Substitution of the thianthrene moiety with popular chromophores is therefore crucial to pave the way for possible applications due to introduction of strong π-π* absorption bands in the region of 280–380 nm, which allows the molecules to be excited by popular light sources, such as nitrogen or Nd:YAG lasers and laboratory UV-lamps.", "This opens a new route of designing molecules for applications as room temperature phosphorescent agents.", "Numbers of possible modifications of thianthrene [2] show the possible pathways in future studies that also include their application as phosphorescent dyes with tunable phosphorescence color or metal-free OLED emitters." ]

[ "Thianthrene-based purely organic efficient room temperature phosphors are introduced.", "Variable phosphorescence color, phosphorescence lifetime and fluorescence to phosphorescence ratio are observed.", "Molecules are characterized using photophysical methods to explain the diversity of properties and substituent effects." ]

[ "ENGI", "ENVI", "SOCI" ]

[ "Fuel efficiency", "Peak car", "Rebound effect" ]

[ "2205", "2300", "3313" ]

[ "Per capita car travel reached a plateau or began to decline in several OECD countries after the millennium, following more than half a century of continuous growth (Schipper, 2011; Van Dender and Clever, 2013).", "In Great Britain (GB), per capita car travel reached a peak in 2002 and fell by 9% over the subsequent decade.", "Although the 2008 financial crisis accelerated this trend, it was clearly established several years before.", "There has been much debate about the causes of this so-called ‘peak car’ phenomenon and the extent to which it represents a permanent or merely a temporary break with historic trends (Goodwin, 2012; Goodwin and Van Dender, 2013; Millard-Ball and Schipper, 2011; Newman and Kenworthy, 2011; Puentes and Tomer, 2008).", "Some authors, such as Bastian et al. (2016), argue that simple economic models based solely on changes in income and fuel prices “… are able to predict the plateau and decrease of car travel with quite remarkable accuracy…” (Bastian et al., 2016).", "Others consider these economic factors to be insufficient and focus instead on changes in demographics, spatial patterns, social norms and other variables (Garikapati et al., 2016; Metz, 2013; Wee, 2015).", "For example, Goodwin and Van Dender (2013) argue that: “…an aggregate model focusing on GDP effects and fuel prices is too crude to catch the diversity and dynamics underlying aggregate car travel demand and how it changes…”.", "Bastian et al. (2016) emphasise that their conclusions do not rule out the existence of alternative explanations, or imply that there been no changes in other variables such as lifestyle and attitudes, or demonstrate that those variables have no effect on travel patterns.", "Instead, they simply argue that there is no “…compelling evidence that one needs to assume something else than fuel price and GDP to explain the aggregate VKT development after 2003…”.", "Wadud and Baierl (2017) question Bastian et al's findings, arguing that their use of ‘out of sample’ forecasts is invalid.", "But in response, Bastian et al. (2017) argue that a longer time series is preferred for estimation and that a model estimated with a shorter time period nevertheless gives consistent results.", "Over the last few years a remarkably wide range of factors have been cited as contributing towards ‘peak car’, although some have received more attention than others (DfT, 2015e; Goodwin, 2012; Newman and Kenworthy, 2011; Van Dender and Clever, 2013).", "They include, for example: increasing income inequality and the worsening economic situation of young people (Klein and Smart, 2017); the increased take-up of higher education opportunities amongst young people, thereby delaying their access to cars (DfT, 2015e); the changing age structure of the population, with a growing proportion of older people who tend to drive less (Goodwin, 2012); relative increases in the non-fuel costs of car ownership and use (e.g. insurance and parking) (DfT, 2015e; Le Vine and Jones, 2012; Rohr and Fox, 2015); the approach towards saturation levels of both car and driving license ownership (Delbosc, 2016; Goodwin, 2012; Le Vine and Jones, 2012); changes in company car taxation leading to reductions in the amount of subsidised car travel (Le Vine et al., 2013); the substitution of car transport by electronic communication, together with the growth of e-commerce, home-working and online shopping (McDonald, 2015; Metz, 2013; Wee, 2015); changing preferences regarding the ownership and use of cars relative to other goods and services (McDonald, 2015); the growing trend towards urbanisation, reversing the historic ‘flight to the suburbs’ (Headicar, 2013); increased congestion, especially on urban roads (DfT, 2015a); modal shifts encouraged by improvements in public transport, cycling and walking infrastructure (DfT, 2015e; Goodwin, 2012); the declining marginal utility of increasing average trip length (Metz, 2013); the levelling off of door-to-door car speeds coupled with relatively stable travel time-budgets (Metz, 2013); and the high rate of net immigration in the first decade of the 21st century, coupled with a lower propensity to drive amongst immigrant communities (Headicar, 2013).", "Identifying the relative importance of these factors is very challenging, partly because the required data is often lacking but also because the different factors are highly interdependent.", "For example, substitution of car transport by ICT is more likely amongst young people, but these also face some of the biggest economic difficulties and are more likely to live in urban areas.", "Hence, while there is increasing amount of research on ‘peak car’, a consensus on the explanations of the phenomenon remains elusive.", "Moreover, the most recent data from the US and the UK suggests that car travel may be on the rise again – perhaps encouraged by an improving economic situation and falling oil prices (DfT, 2015d, 2016).", "If this trend continues, it would reinforce the argument that economic factors remain dominant.", "Dimitropoulos et al. (2016) conducted a meta-analysis of the results from 76 primary studies in this area and found a mean long-run rebound effect of 32%.", "However, most of these estimates were from the US, with only Stapleton et al. (2016) providing estimates for GB.", "Also, most of the studies use a double log functional form that constrains the elasticities to be constant.", "In practice, the rebound effect may change over time or with increasing incomes, but few studies have investigated this.", "Fouquet (2012) provides a very long-run perspective and estimates that the own price elasticity of UK passenger transport demand fell from −1.5 in 1850 to −0.6 in 2010, while Small and Van Dender (2007) estimate that long-run rebound effect in the US was around 22% over the period 1960–2001, but fell to only 10.7% over the period 1997–2001.", "Greene (2012) confirmed Small and van Dender’s estimate using aggregate time-series data, but a study by Hymel and Small (2015) found that the rebound effect had increased after 2000 – perhaps as a consequence of fuel price volatility and media coverage of rising fuel prices.", "In sum, there is strong evidence that per capita car travel has declined in several OECD countries over the 10–15 years, together with strong evidence that improvements in vehicle fuel efficiency have encouraged more car travel.", "But there is a lack of consensus on the causes of the former and on whether and how the rebound effect has changed over time.", "This paper therefore seeks to contribute to this literature in two ways: first, by investigating how different factors have influenced the demand for car travel in GB since 1970; and second, by investigating how the rebound effect has changed over that period.", "Our approach involves the econometric analysis of aggregate, time-series data on travel patterns, fuel consumption and other variables in GB over the period 1970–2012.2", "We estimate a number of models with different specifications, systematically evaluate and compare the statistical robustness of these models and base our conclusions on the ‘best performing’ models.", "Our results suggest that changes in income, the fuel cost of driving and the level of urbanisation largely explain travel trends over this period.", "We also estimate a mean rebound effect of 26% and find some evidence that this effect has increased over time.", "However, the limited number of data points constrains the number of variables we can test and limits the confidence we can have in our results.", "The following section describes our methodology, including the specification of the econometric models and the robustness tests used to select between them.", "Section 3 summarises the data sources and discusses the trends in the relevant variables.", "Section 4 presents the results, including the significance of different variables in explaining ‘peak car, the estimated rebound effect and how that effect has changed over time.", "Section 5 concludes by highlighting the limitations of the current approach and the priorities for future research.", "Our approach involves estimating a total of 17 econometric models - 9 of which have a static specification and the remainder a dynamic specification.", "Each model includes a different combination of explanatory variables, and we use a comprehensive series of robustness tests to select the ‘best performing’ models.", "Previous studies have not been consistent in their specification of distance travelled, either measuring it in absolute terms or normalising it to population, the number of adults or the number of licensed drivers (Sorrell and Dimitropoulos, 2007b).", "Changes in total population, the age structure of the population, the propensity of young people to learn to drive and/or the proportion of female drivers may have different effects on the explained variable depending upon the normalisation used.", "Again, Stapleton et al. (2016) found this choice made relatively little difference to the estimated price and income elasticities.", "Here we normalise to the number of adults: partly because this is the most common specification in the literature, and partly because the proportion of licensed drivers is one of the variables that we want to test.", "Below we explain the specification of our two base models, together with fifteen variants of those models, the robustness tests used to select between them and the sequence of model testing (see Table 1).", "We then estimate a number of variants of these models, using combinations of the additional explanatory variables indicated in Table 2.", "The rationale for each variant is summarised below.", "Income inequality has grown in Great Britain since 1979, with the income distribution becoming more positively skewed.", "If the incomes of an increasing part of the population are growing more slowly than GDP, the rate of growth of car travel may become progressively decoupled from GDP.", "To test for this, we substitute median for mean equivalised household income, since the former has grown more slowly and is correlated with both total income and income inequality.", "Urban areas provide high-density living arrangements, easy access to desired destinations and good public transport alternatives to the private car.", "As a consequence, car ownership and use tends to be significantly lower in urban areas: for example, vehicle kilometres per person in inner London are only one quarter of those in rural areas (Headicar, 2013).", "While the growth in car travel during the late 20th century was correlated with the trend towards suburban living, this trend has reversed over the past 15 years – stimulated in part by urban regeneration initiatives and a policy of locating new housing developments on urban brownfield sites.", "This trend may have restrained the growth in car travel, and may also have been reinforced by other factors such as increasing restrictions on urban parking, significant investment in public transport infrastructure (especially within the major cities), growing congestion on urban roads and the increasing effectiveness of land use planning in restricting traffic growth (Bastian and Börjesson, 2015; Goodwin and Van Dender, 2013; Jahanshahi et al., 2015; Le Vine and Jones, 2012; Metz, 2013).", "For example, data from the English travel survey (DfT, 2015a) suggests that the largest reduction in traffic since 2000 has occurred on urban roads and that travel trends in London are increasingly different from those in the rest of the country.", "To test for this, we include a variable representing the proportion of the population living in the five largest British cities, namely London, Birmingham, Leeds, Glasgow and Sheffield.4", "Rising incomes were associated with increasing car travel during the latter half of the 20th century, but these two variables have become increasingly decoupled.", "This is despite household car ownership continuing to rise and remaining some way short of projected saturation levels (DfT, 2015c).", "Travel survey data (DfT, 2015a) suggests that both the average time spent travelling and the share of transport in total household expenditure have remained broadly stable since 1970, while the average trip distance has remained stable since the mid-1990s.5", "Metz (2013) argues that increases in average trip length now provide only limited gains in utility since current trip lengths allow much of the UK population to have good access to desired destinations.", "In addition, in the absence of an increase in average speed, any increase in utility from accessing more destinations will be offset by the decrease in utility from the additional time spent travelling.", "But while these arguments appear plausible, they do not explain the ∼12% fall in the number of car trips since 1995/97 (DfT, 2015a).", "Changes in the proportion of licensed drivers in the population may also contribute to changes in per capita car travel.", "These changes may in turn be driven by urbanisation, changing demographics, the economic constraints faced by certain groups (e.g. the cost of insurance and tuition has grown faster than young people's incomes) and other factors.", "License ownership may be saturating, with ∼74% of GB adults now holding a driving licence compared to 48% in 1975 (DfT, 2015a).", "There has been a decline in the proportion of young people holding a licence (possibly linked to changing norms about car ownership (McDonald, 2015)),6 but this has been offset by a countervailing trend towards more retired people holding licences (DfT, 2015a).7", "The impact of these trends is complicated by the variation in driving patterns between age groups (e.g. men drive nearly twice as far as women, while average driving distance declines from age 50 onwards (DfT, 2015a)), as well as over time (e.g. female car owners now drive further than they did in the past, while male car owners drive less (Le Vine et al., 2013)).", "Again, it is not possible to capture all these complexities within an aggregate model of this type.", "Instead, we simply test a variant that includes a variable representing the proportion of adults in GB who hold a driving licence.", "Several authors have highlighted the coincidence between the peak in per capita car travel and the explosion in the use of information and communication technologies (ICT).", "Survey data suggest a modest growth in home working since 2000 and a significant growth in internet shopping – which could have contributed to the reduction in the number of trips per household (McDonald, 2015; Metz, 2013; Wee, 2015).", "High-speed networks also allow people to use their smart devices in transit which could make public transport more attractive (Lyons and Urry, 2005; Wee, 2015).", "However, the evidence in this area is ambiguous, with some studies indicating substitutability between telecommunications and travel and others indicating complementarity (Choo et al., 2007a,b; Choo and Mokhtarian, 2007; Choo et al., 2005; Mokhtarian, 2009).", "Given limited data and degrees of freedom, we again test for this in a rather crude fashion by including a variable indicating the proportion of GB households with an internet connection.", "This acts as a proxy for the broader diffusion of ICT within the GB population.", "Several of the above variables are co-linear and their inclusion restricts the degrees of freedom within our small dataset We therefore investigate removing variables that are individually and jointly insignificant at the 5% level – thereby optimising the trade-off between goodness of fit and complexity.", "With time series data it is common for one or more of the variables to be non-stationary, creating the risk of spurious regressions.8", "While this may be avoided by differencing the data, this would prevent the estimation of the long-run relationships we are interested in.", "But it is possible for two or more non-stationary variables to be co-integrated, meaning that certain linear combinations of these variables are stationary and that there is a stable, long-run relationship between them.", "We therefore test the time series and residuals of our models for unit roots.", "If the variables are found to be non-stationary and co-integrated, this can be interpreted as an equilibrium relationship (Basso and Oum, 2007).", "In this circumstance, we re-estimate the ‘best performing’ static model using a specialised technique (‘canonical cointegrating regression – CCR) (Park, 1992).", "Econometric studies vary widely in their inclusion or otherwise of different diagnostic tests.", "Here we conduct a comprehensive set of 13 diagnostic tests on each of our models and use the results to form an overall robustness score (0–100%) for each model – with higher scores indicating ‘better’ models.", "The relevant tests and the associated scores are summarised in Table 3.", "We use two different weighting rules: the first based on our judgement of the ‘relative importance’ of each test, and a second giving equal weighting to each test – although the ranking of models is broadly the same in each case.", "For the CCR technique in Stage 9, we use the more limited set of six diagnostic tests summarised in Table 4.9", "We estimate a total of 17 models (9 dynamic and 8 static), in the sequence summarised in Table 5.", "The selection of models relies upon the results of the diagnostic tests, with the ‘best’ model being carried forward at each stage.", "For example, if the inclusion of an urban population variable in Stage 3 leads to a model with a higher (lower) robustness score than in Stage 2, then the urban population variable is retained (omitted) in subsequent stages.", "Stage 8 takes the best performing model and removes variables that are individually or jointly insignificant, while Stage 9 re-estimates the best performing static model using the CCR technique.10", "We take data on mean and median equivalised real household income (Yt) from IFS (2014), data on licensed drivers from DfT (2014, 2015d) and data on population and the number of households with internet access from ONS (2016).", "Where necessary, we use linear interpolation to adjust these series to end-of-year values.", "The use of equivalised incomes adjusts for changes in average family size and composition.", "Mean equivalised real household income doubled between 1970 and 2009, but then fell slightly following the financial crisis (Fig. 2).", "Median income has grown more slowly than mean income since 1983, with the gap increasing over time.", "The proportion of adults with driving licences rose steadily between 1970 and 1988 (driven in part by more women acquiring licences), but has now reached a plateau (Fig. 3).", "The proportion of adults living in the largest five cities fell between 1970 and 1997, but has since increased, reaching 18.1% in 2012 (Fig. 3).", "Finally, the proportion of households with an internet connection increased very rapidly after 1992 and had reached 83% by 2012 (Fig. 4).", "We first consider the performance of the different models against the various diagnostic tests.", "Table 6 indicates the aggregate ‘robustness score’ of each static model, together with the variables included at each stage, while Table 7 presents the detailed results of the diagnostic tests on those models.", "Tables 8–10 do the same for the dynamic models.", "Looking first at the static models, we make three observations.", "First, the overall robustness scores are relatively modest, with the ‘best’ specification (stages 6 and 8) scoring only 55% with the unequally weighted criteria and 62% with the equally weighted.", "All the static models suffer from serial correlation, which suggests the standard errors of the estimates may be underestimated – although the estimates should be unbiased.", "There is also evidence of collinearity between the variables, and the results of the RESET tests suggest potential misspecification.", "Second, three of the variables tested – namely median rather than mean income, ICT access and quadratic income – failed to improve the performance of the models against the diagnostic tests and were therefore omitted in subsequent stages.", "In contrast, both the urbanisation variable and the proportion of licensed drivers improved model performance, so were included in subsequent stages.", "Third, the highest scoring model under both weighting schemes (Stage 8) included mean income, fuel costs, urbanisation, the proportion of licensed drivers and the interaction term.", "This model was re-estimated with the CCR technique (Stage 9), but the results were very similar.", "The robustness score for Stage 9 is not directly comparable as it is based upon a different set of tests (Table 4).", "The overall results from the static models suggest that changes in the level of urbanisation and the proportion of licensed drivers have influenced travel trends in GB, but that changes in income inequality and the diffusion of ICT technology have not played a significant role.", "The results also suggest that, after controlling for fuel cost, the level of urbanisation and the proportion of licensed drivers, there is little evidence that traffic growth has become decoupled from mean equivalised income.", "The interaction term also improves model performance; suggesting that the impact of fuel costs on driving patterns has varied with income – and vice versa.", "Looking next at the dynamic models (Tables 9 and 10), we make two further observations.", "First, these models score significantly better against the diagnostic tests than do the static models, with the best’ specification (stage 3) scoring 95% against the unequally weighted criteria and 92% against the equally weighted.", "The results suggest that the inclusion of the lagged dependent variable reduces problems of both serial correlation and misspecification.", "Second, the inclusion of the urbanisation variable improves model performance, but (in contrast to the static models) the inclusion of the licensed drivers variable does not.", "Again, the results provide little evidence that income inequality and ICT access have influenced travel trends.", "The best fitting dynamic model (Stage 3) explains travel trends using mean income, fuel costs, urbanisation, the oil shock dummies and the lagged dependent variable.", "In contrast to the static models, the interaction term between mean income and fuel costs does not improve model performance.", "It is also important to consider the stationarity properties of the models.", "Table 10 summarises the results of two types of unit root tests on the residuals from the static models.", "The results are ambiguous, and should be interpreted with caution since the tests have only limited power with the number of observations used here.", "The results suggest that the variables in the ‘preferred’ static model (Stage 6) are co-integrated, thereby justifying the use of the CCR technique – and partly counterbalancing the low robustness score of this model relative to the dynamic models.", "The results of the diagnostic tests for Stage 9 are summarised in Table 11.", "As Table 12 indicates, 13 of the 17 models produced statistically significant estimates of the long-run income elasticity of distance travelled.", "Overall, the results suggest that, ceteris paribus a 1% increase in equivalised real household income was associated with a 0.55% increase in vehicle kilometres over this period.", "The ‘best performing’ static model suggested a value of 0.40%, and the best performing dynamic model a value of 0.49%.", "The mean income elasticity from the static models was 20% higher than that from the dynamic models – which is arguably consistent with the interpretation that static models provide long-run equilibrium estimates, while dynamic models provide intermediate-run estimates (Basso and Oum, 2007).", "For comparison, a review of international studies by Goodwin et al. (2004) found a mean income elasticity estimate of 0.5 from static models and 0.3 from dynamic models.", "In contrast to our previous work (Stapleton et al., 2016), only one model (dynamic model 4) produced a coefficient estimate (of −0.03) for the price shock dummy that was statistically significant at the 5% level.", "However, many of the estimates were significant at the 10% level and the inclusion of this variable improved model performance against the diagnostic tests.", "The urbanisation variable was found to be highly significant within all relevant models (Table 13).", "Overall, the results suggest that a 1% increase (decrease) in the proportion of the GB population living in the five largest cities was associated with a 1.7% decrease (increase) in distance travelled over the period.", "The ‘best performing’ static model suggested a value of -1.3%, and the best performing dynamic a value of 2.0%.", "These estimates appear surprisingly high, even allowing for the much lower levels of car ownership and use within the major cities.", "A possible explanation is that the urbanisation variable is co-linear with other, non-included variables that also affect distance travelled.", "There may also be an asymmetric relationship between urbanisation and distance travelled that we have not tested for (see Table 14).", "The variable representing the proportion of licensed drivers was found to be significant in the four static models in which it was included, but not in the dynamic models (Table 15).", "Since the latter performed better against our diagnostic tests, the evidence for the significance of licensed drivers appears weaker than that for urbanisation.", "On average, the results suggest that a 1% increase in the proportion of licensed drivers was associated with a 0.5% increase in distance travelled over this period.", "The static models produced estimates of the rebound effect that were on average 25% smaller than those from the dynamic models.", "This finding is consistent with the results of a meta-analysis of traffic and fuel price elasticities by Goodwin et al. (2004), who found exactly the same percentage difference between the results of the two types of models.", "Goodwin et al. further observe that transport modellers have tended to base their elasticity assumptions on the results of dynamic rather than static models and hence may be underestimating the income elasticity of distance travelled and fuel consumption, while at the same time overestimating the price elasticity.", "Finally, the interaction term between income and fuel costs was found to be individually significant in the three static models in which it was included (together with the CCR model) and jointly significant in the single dynamic model in which it was included.", "The dynamic models score better against the diagnostic tests than the static models.", "However, since the inclusion of the interaction term in the dynamic models did not improve model performance, this gives us only medium confidence that the rebound effect varies with income.", "Moreover, the coefficient on the interaction term is negative, suggesting that the rebound effect increases with income - the opposite of that found for the US by Greene (2012) and Small and Van Dender (2007).", "The static models suggest that a 1% increase in equivalised income was associated with 0.3% increase in the rebound effect over this period – with the CCR model suggesting a stronger relationship and the dynamic model a weaker one (Fig. 5).", "This paper has explored how different factors have influenced the demand for car travel in Great Britain since 1970 and how the rebound effect has changed over that time.", "Our results suggest that changes in income, the fuel cost of driving and the level of urbanisation largely explain travel trends over this period – with peak car being driven by a combination of the rising fuel cost of driving, increased urbanisation and the economic difficulties created by the 2009 recession.", "We find some evidence that the proportion of licensed drivers has influenced aggregate travel trends, but no evidence that growing income inequality and the diffusion of ICT technology have played a role.", "While our results do not wholly support Bastian et al's contention that “… GDP per capita and fuel price are able to explain most of the trends in vehicle kilometres per capita…” (Bastian et al., 2016), they are consistent with their argument that travel trends amongst different socio-economic groups have partially cancelled out.", "Our results are also consistent with the claim that economic recovery and low fuel prices could encourage renewed traffic growth – particularly since we find the income elasticity of car travel to be significantly larger than the price elasticity.", "Our results also suggest that the rebound effect from improved fuel efficiency averaged 26% over this period – which is consistent with the results of other studies.", "Contrary to expectations, we find some evidence that the magnitude of this effect has increased over time, and is now more than twice as large as it was in 1970.", "This contradicts the results of two US studies (Greene, 2012; Small and Van Dender, 2007), but it is important to note that: first, our estimates are based upon the fuel cost per kilometre and hence reflect responses to both fuel efficiency improvements and changes in fuel prices; and second, the variation in fuel cost per kilometre has been much smaller in GB than in the US over this period.", "In any event, this result reinforces the argument that rebound effects should be taken account of when estimating the impact of policies such as fuel efficiency standards.", "Overall, methodological and data limitations severely constrain the range of variables that we can test for with this approach, as well as the level of confidence that we can have in our results.", "For example: our use of aggregate timeseries data severely limits the degrees of freedom available; we do not have data on key variables such as company car ownership; we did not test for possible asymmetric responses to changes in fuel prices (Dargay, 2007); and our use of a lagged dependent variable in the dynamic models potentially introduces bias (Keele and Kelly, 2006).", "Also, technical improvements in fuel efficiency (e.g. improved aerodynamics) may have encouraged a shift towards larger and more powerful cars over this period, but this trend is not captured in our dataset.", "These limitations are common to most econometric studies of travel trends that use aggregate timeseries data and are only partially mitigated by our systematic use of diagnostic tests.", "Hence, to obtain a deeper understanding of both the peak car and rebound phenomena, it is essential to utilise a broader range of data sources, together with a broader range of research methodologies.", "For example, much can be learnt from the analysis of national and regional travel survey data (DfT, 2015e; Headicar, 2013) and consumer expenditure data (Moshiri and Aliyev, 2017), especially when used in combination (Kuhnimhof et al., 2012b) or when compiled for a group of countries (Kuhnimhof et al., 2012a; Schipper and Johansson, 1997).", "Moreover, since the peak car phenomena remains relatively new, the inclusion of more recent data within such studies can improve the level of confidence in the results.", "In sum, there is still much work to do to uncover the drivers of recent travel trends." ]

[ "We find that income, fuel costs and urbanisation are the main drivers of car travel trends in GB.", "Urbanisation may be a proxy for several factors contributing to changes in car use.", "We estimate the direct rebound effect for car travel in Great Britain to be around 26%", "We estimate that the direct rebound effect has increased over time." ]

[ "MULT" ]

[ "Environmental science", "Geography" ]

[ "1000" ]

[ "Land use and land cover change (LULCC) has been suggested to be the most important anthropogenic disturbance to the environment at a local level, causing various microclimatic changes (Mishra and Rai, 2016).", "Anthropogenic influences on the landscape such as alteration in land use through agriculture, forestry, urbanisation and the introduction of invasive alien plant (IAP) species have a profound effect on the functioning of the landscape and ecosystems.", "Further, the present land cover may affect the movement of species as well as determining the availability of land for future use (Singh et al., 2014).", "The modifications generally lead to a degraded environment and thus the importance of maintaining the integrity of ecosystems is fundamental to preserve biodiversity (Singh et al., 2014).", "LULCC has been related to biodiversity loss and thus recent research has arisen to meet land management needs and to assess the role of LULCC in the functioning of the biosphere, through the development of a range of LULCC models (Pérez-Vega et al., 2012).", "LULCC modelling entails the simulation or prediction of the behaviour of the environmental and social systems in the study area over a time period in such a way that it relates to the measured land change (Paegelow et al., 2013).", "In a water scarce country such as South Africa, climate change adaptation is particularly important for catchment management.", "A change in catchment land cover will have a direct effect on the hydrological functioning of a catchment and thus predicting land cover change may help to develop resilience to projected climate changes through, for example, evidence-based water licensing (Palmer et al., 2017).", "The principle drivers of change within the rural areas of southern Africa are linked to five primary drivers, namely commercial afforestation, woody encroachment (both alien and native woody plant invasion), urbanization, increased dryland cultivation and rangeland degradation, and it is now well understood that invasion by alien woody plants is a major driver of grassland transformation and influences the ecosystem services (forage production, water supply, habitat, biodiversity, carbon sequestration and recreation) provided by these rangelands (Münch et al., 2017).", "The storage of carbon in the landscape is driven by biophysical parameters associated with each land cover type and thus changes in land cover proportions across a catchment will impact on the net ecosystem carbon exchange (NEE) of the catchment as a whole.", "Similarly, the ecophysiology of the individual land covers affects the water use of the vegetation within that land cover and changes in land cover proportions within a catchment impact on the hydrology of the catchment as a whole.", "Palmer et al. (2017) determined, through field measurements and satellite imagery, statistics around two biophysical parameters – leaf area index (LAI), and fraction of photosynthetically active vegetation (fPAR) – which are used in NEE and evapotranspiration (ET) modelling.", "This knowledge, combined with predictions of how the land cover will change in the future, precipitates the estimation of future carbon storage and water use within the catchment.", "Research has shown that land use changes will result in changes to the drivers of earth surface conditions that force General Circulation Models (Cao et al., 2015; Pelletier et al., 2015).", "Andrews et al. (2017) stated “A radiative forcing arises in response to changes in land cover (e.g. forest to pasture and crops) predominantly because different surface types have different albedos.", "Forests are generally darker than grasses or croplands and so deforestation tends to increase the Earth's albedo and reflect more solar radiation to space — a negative radiative forcing which causes cooling (e.g. Myhre et al., 2013).", "However the forcing from changes in land-use is further complicated by its impact on hydrology and non-radiative fluxes (e.g. Brovkin et al., 2006; Betts et al., 2007; Davin et al., 2007; Davin and de Noblet-Ducoudré, 2010; de Noblet-Ducoudré et al., 2012) as well the coincidence of land-cover change and snow cover at higher latitudes (e.g. Betts, 2000; Pitman et al., 2011).”", "These changes include variations that are linked to surface albedo; that is the earth's ability to absorb or reflect heat energy.", "For the southern African region, carbon offsets from sequestration may be discounted from the consequences of temperature increases linked to higher albedo.", "In global change science it is vital to consider surface albedo and surface area of a range of different land cover classes, and to recommend policies that will change albedo to further promote the improvements being offered by carbon off-sets.", "Thus for each land cover transition the shift in surface albedo should also be considered.", "Commercial afforestation, IAPs and woody plant encroachment (e.g. Vachelia karroo) all result in an increase in the total above-ground woody standing biomass (O'Connor et al., 2014) in this region.", "In all situations, this is accompanied by an increase in leaf area index (LAI) and possibly a reduction in surface albedo.", "The higher level of green water in these land cover classes is a good absorber of heat, and this may result in further global heating, possibly discounting the positive consequences of carbon sequestration.", "In contrast, rural urbanization (which is different from conventional urbanization as dwellings are more widely spaced, and are interspersed with bare soil) may result in higher albedo.", "Similarly, degraded rangeland, with lower fractional canopy cover, also may have higher albedo (Rotenberg and Yakir, 2010).", "This paper builds on previous work (Palmer et al., 2017; Münch et al., 2017; Gwate et al., 2016; Okoye, 2016) by looking at land cover change trends for two grassland-dominated catchments (S50E and T35B) with different land management systems, in the Eastern Cape Province of South Africa.", "This is a first step in understanding the trend of land cover change on catchment water and carbon fluxes in these catchments.", "Following an integrated multi-factor analysis of drivers of past land cover change within the catchments, the Land Change Modeller (LCM) in IDRISI was used to simulate future land cover scenarios for the year 2030 and postulate preliminary consequences of this change with respect to carbon storage and water use under each land management system.", "The S50E and T35B catchments are located in the Eastern Cape Province of South Africa (Fig. 1).", "In S50E, mixed farming (dualistic farming system) is practiced under communal land tenure arrangements and includes both livestock grazing and crop cultivation (Kakembo, 2001).", "In T35B, the land tenure is predominantly freehold, and land cover comprises extensive dryland cultivation, commercial afforestation and extensive unimproved grassland for livestock production.", "However with an average density of 10% (Kotzé et al., 2010), invasion by woody plants, particularly black wattle (Acacia mearnsii), silver wattle (Acacia dealbata) and poplar (Populus spp.), is a major transformer of grasslands and rangeland production.", "This transformation is aggravated by poor farming practices, including overgrazing, abandonment of cultivation, reduced fire frequency and wood felling that have degraded the vegetation diversity and richness.", "The major fluxes of water and carbon in this socio-ecological system occur through livestock and alien trees.", "Clearing of IAPs in both the catchments is managed by the Department of Environmental Affairs Working for Water (WfW) programme and is premised on increasing water on the landscape in combination with socio-economic development, involving pro-poor interventions (Macdonald, 2004; Oelofse et al., 2016).", "Clearing IAPs that have higher water use relative to indigenous vegetation (Clulow et al., 2011) is expected to increase the proportion of water to maintain other ecosystem services provided by rangelands (Meijninger and Jarmain, 2014; Van Wilgen et al., 2012).", "The S50E and T35B catchments are more fully described in Münch et al. (2017).", "Land cover maps for change analysis were independently produced from Landsat imagery.", "An existing national land cover product for 2000 (Van den Berg et al., 2008) was updated through post-classification editing, while a second land cover data set was derived for 2014 using geographic object-based image analysis (GEOBIA) (Okoye, 2016).", "Theoretical accuracy for land cover change analysis, derived as the product of the accuracies of the independent land cover maps, was lower for T35B (67%) based on lower classification accuracies for 2000 (81%) and 2014 (83%) than for S50E, where accuracies of 83% and 87% respectively for 2000 and 2014 produced a theoretical accuracy of 72% (Münch et al., 2017).", "Conversion labels were assigned as indicators to describe the transition trajectory identified at each intersection of the two land cover maps (Münch et al., 2017; Okoye, 2016; Benini et al., 2010).", "Overall observed land cover change in S50E from 2000 to 2014 amounted to 21%, dominated by increased urbanisation and agricultural intensification.", "However, change could be as high as 42% considering map errors.", "Persistence and intensification of natural or invaded wooded areas possibly IAPs, were identified as a degradation gradient within the landscape, which amounted to almost 10% of S50E.", "In some areas, a return to grassland and bare areas signified abandonment and degradation.", "However, despite a net loss of 5%, grassland still dominates the landscape.", "A smaller overall land cover change (18%) was observed in T35B.", "Plantations increased by 2% (R – Afforestation) through increase in commercial cultivation while grassland was reclaimed (Re) from wooded areas (∼6%), possibly due to eradication of IAPs.", "Urban areas in T35B remained static as out-migration caused a decline in population (Danuta Hodgson, MSc thesis, unpublished data).", "Fig. 1 illustrates the two catchments and the land cover change trajectories identified from the land cover change analysis for 2000 to 2014 (Münch et al., 2017; Okoye, 2016).", "Most land change models follow a data-driven inductive approach, attempting to draw correlations between a multitude of explanatory factors involved using statistical inferences (Overmars et al., 2007), however a deductive model allows the inclusion of relevant driving factors assumed to have causal influence on LULCC, such as political change or climatic disasters.", "Land Change Modeller (LCM), an inductive model integrated into IDRISI Terrset 18.08, provides tools for the assessment and projection of land cover change.", "LCM was developed by Clark Labs in conjunction with Conservation International to provide a suite of tools to address the problems of accelerated land conversion and the analytical needs required in biodiversity conservation (Eastman, 2016).", "In LCM (Fig. 2), land cover is mapped at two time steps (T1 and T2) and the patterns and processes of change are estimated and used for model parameterization/calibration (Mas et al., 2014).", "The approach used in LCM is to analyse changes in land cover between two past time steps (T1 and T2) and use Multi-layer Perceptron (MLP) with explanatory spatial variables to create transition potential maps.", "Markov Chain Analysis assigns the probability of change determined by projecting the historic change to the future, which together with transition potential maps, present a land cover scenario for some future data (T3).", "Concurrently, the individual transition potential maps are aggregated to create a map indicating the propensity of the landscape to experience change.", "Spatial explanatory variables are GIS datasets representing drivers of the observed change (Pérez-Vega et al., 2012) and are typically based on biophysical or socioeconomic criteria.", "Often used datasets include slope, distance to roads and settlements, land tenure and soil types (Mas et al., 2014) and these driver variables are used to model the historical change process (Eastman, 2016).", "The potential explanatory power of a variable can be tested using Cramer's V test where the level of association between GIS datasets representing phenomena thought to be drivers in a particular transition and the land cover in question can be determined.", "Cramer's V is a quantitative measure of association that ranges from 0.0, indicating no correlation (discarded variable), to 1.0, indicating perfect correlation (excellent potential variable) (Megahed et al., 2015) and although these values are not regarded as definitive, they can help in deciding whether to include an explanatory variable in creating a transition potential map for a transition by examining whether the explanatory variable explains the transition for a particular land cover.", "According to Eastman (2016), Cramer's V values of 0.15 or higher are ‘useful’ while those with values of 0.4 or higher are ‘good’.", "Land cover transitions can be grouped into sub-models if the underlying driver of change is assumed to be the same for each transition (Pérez-Vega et al., 2012).", "For example, the processes which affect a land cover being changed from forest to urban may be the same as those which affect grassland being converted to urban.", "This urbanisation transition may be driven by proximity to existing urban areas, proximity to road networks and may have the same topographic driver such as flatter areas are more likely to transition than steep areas.", "In this example, forest to urban and grassland to urban can be grouped in the same transition or sub-model and the explanatory spatial variables would be the same.", "Explanatory spatial variables are assigned to each sub-model on the basis of Cramer's V values and the transition potential of each sub-model is determined through a knowledge based approach to machine learning.", "A neural network consists of a number of interconnected nodes which are simple processing elements that respond to the weighted inputs received from other nodes (Atkinson and Tatnall, 1997).", "The MLP was an advancement in perceptron methods as it is able to separate non-linear data due to it being ‘multi-layer’ and is a popular classification method in remote sensing (Atkinson and Tatnall, 1997).", "MLP is a feedforward neural network in that data flows in one direction from the input layer through the hidden layers which are sets of computational nodes, to the output layer.", "The nodes are linked by a web of connections which are applied as a set of weights and a backpropagation algorithm is used to train the network by iteratively spreading the errors from the output layer to the input (Megahed et al., 2015) by adjusting weights so as to minimise the error between the observed and the predicted outcomes (Pérez-Vega et al., 2012).", "The capability of the model to learn and generalise depends on its architecture (Pérez-Vega et al., 2012) and increasing the number of hidden layers enables the model to learn more complex problems (Atkinson and Tatnall, 1997).", "The training performance is assessed by a precision value expressed in percent and networks that are too small cannot identify the internal structure of the data and produce lower performance accuracies whereas too large networks overfit the training data (Pérez-Vega et al., 2012).", "The aim of the training is to build a model of the data generating process so that network outputs can be predicted from unseen inputs.", "The network output is then compared with the desired output, the error is computed and then back-propagated through the network to adjust weights (Atkinson and Tatnall, 1997).", "Large quantities of data are often required for training (Atkinson and Tatnall, 1997) and thus a small sample of training sites is unlikely to result in an accurate model.", "In the IDRISI MLP, half of the training data are randomly selected for learning and half for validation.", "After the MLP has been trained, validation data are used to calculate a \"skill measure\" (computed as the accuracy of transition prediction minus the accuracy expected by chance) (Mas et al., 2014).", "The MLP thus creates time-specific transition potential maps for each of the sub-models which are expressions of time-specific potential for change (Eastman, 2016).", "However, further steps are required to use this information to predict future land cover classes and also the potential for each cell to either persist or transition between land covers.", "Markov chain analysis is a stochastic modelling approach which has been used extensively for land cover change modelling (Fathizad et al., 2015).", "It assumes that the probability of a system being in a certain state at a certain time can be determined if its state at a prior time is known with the assumption that rates of change observed during the calibration period (T1 to T2), will remain the same during the simulation period (T2 to T3).", "Through cross-tabulation of land cover (Kamusoko et al., 2009) Markov chain analysis determines the amount of land cover change that will occur to the future date (Eastman, 2016).", "In LCM transition probability maps are produced using either logistic regression, MLP trained by backpropagation or a machine learning approach (Mas et al., 2014).", "This provides a probability estimate for each pixel to either be transformed to another land cover or to persist and be calibrated to an annual time step (Kamusoko et al., 2009).", "LCM produces two predictors of future land cover: soft prediction and hard prediction.", "Soft prediction, or potential to transition, is a continuous mapping of vulnerability to change (Eastman, 2016).", "It is calculated by aggregating all the transition potentials and provides an indication of the degree to which the areas have the right conditions to precipitate change.", "The soft predictor thus provides a likelihood of a cell to experience land cover change without providing an indication as to what the new land cover will be.", "The hard prediction procedure used by LCM is based on TerrSet's multi-objective land allocation (MOLA) module.", "MOLA determines a compromise solution by maximizing the suitability of lands for each objective given the assigned weights (Eastman, 2016).", "Land allocation conflicts are resolved by allocating the cell to the objective (land cover class) for which its weighted transition potential is highest based on a minimum distance to ideal point rule using the weighted ranks (Houet and Hubert-Moy, 2006).", "Finally, the transition probability matrix derived from the Markov chain analysis determines how much land is allocated to a class over, T3 – T2, an n-year period.", "In LCM, change is thus modelled through MLP using mathematics and explanatory spatial variables in a trends driven approach (Pérez-Vega et al., 2012).", "Spatial analysis of land cover change using the explanatory variables identifies: 1) maps of the transition potential for each identified land cover transition, 2) a transition potential map indicating the likelihood of each location in the study area to experience change and 3) a scenario land cover map for a selected future date (T3).", "Land cover maps at 30 m pixel resolution (Okoye, 2016), for T1 (2000) and T2 (2014) were used to create 1) transition potential maps for each transition, 2) a projected potential for transition map and 3) a predicted land cover map for 2030 (T3) for S50E and T35B.", "An identical land cover legend consisting of eight land cover classes was used for each time step (Table 1).", "Following Münch et al. (2017), trajectories of land cover change describing both change and persistence were identified and each possible transition of land cover between T1 and T2 was labelled (Table 2).", "Following Pérez-Vega et al. (2012) land cover transitions with common underlying drivers of change were grouped into sub-models.", "Trajectories of land cover change, identifiable at data resolution (30 m) were also labelled (Table 2) as representing (1) intensification - the transition of a lower intensity to a higher intensity usage; (2) afforestation - the planting of commercial trees; (3) deforestation - the clearance of trees; (4) reclamation, degradation and abandonment related to conversion to grassland and bare areas; (5) natural dynamics - seasonal conversions not explained through anthropogenic change; and (6) exceptionality - associated with potential map errors (Münch et al., 2017).", "Of particular importance are areas where another land cover class has potentially been replaced by IAPs (FITB intensification) and where forests (indigenous or alien) and other woody areas have disappeared or been removed (reclamation, deforestation).", "A change from any other land cover class (with the exception of waterbodies and wetlands) were labelled Iu: Urban intensification.", "It was not possible to determine change in the intensity of agricultural activities due to image resolution, but conversion to agricultural practices was identified (agricultural intensification).", "Although persistence (P) — where no land cover change has occurred — can be considered a trajectory, it cannot be considered a transition and thus trajectories representing persistence are ignored by LCM.", "In reality, not all possible transitions occurred between 2000 and 2014 in S50E and T35B.", "Due to the low user's and producer's accuracy for LC classes bare soil (BRS) and wetlands (Wl), Münch et al. (2017) labelled all transitions involving these classes as potential classification error.", "Table 3 displays the labels, transitions and description for each sub-model.", "Small transitions (less than 10 ha) have been removed from the analysis to exclude exceptionalities.", "In choosing explanatory variables, the processes producing land cover change need to be visualised after which a spatial dataset of the particular process must be sourced at appropriate spatial resolution.", "GIS data sets were identified to describe the potential transitions, geo-processing was performed to represent the particular process and abbreviations were assigned to each processed spatial dataset.", "In addition to geographical parameters, Evidence likelihood (EV) which calculates the relative frequency of pixels which belong to the different classes within the areas of change, is recommended where there are low Cramer V values (Eastman, 2016).", "Derived biophysical and anthropogenic datasets were tested for their suitability using Cramer's V where higher values represent stronger relationships between the variable and a particular transition with values higher than 0.4 regarded as good (Megahed et al., 2015).", "Within the context of the communal/traditional farming methods practiced, proximity to parts of the landscape already impacted by people may be considered as potential drivers for degradation.", "For IAP intensification, infestation is more likely to occur in those areas close to existing infestation through the process of seed dispersal.", "Similarly afforestation is more likely to occur in those areas close to existing plantations since infrastructure is already in place to support this.", "Topographic variables can be considered as having a potential constraining or flourishing effect of certain transitions.", "For example, water bodies will not expand into areas with a slope and certain vegetation may not grow higher than a specified altitude.", "Finally, vegetation distribution is influenced by access to water and thus the Euclidean distance from rivers may be used as a proxy for water availability.", "The variables selected for each transition sub-model are shown in Tables 4 and 5 for S50E and T35B respectively.", "Overall Cramer's V values are shown in Table 6 with Cramer's V values for individual land cover classes given in Table 7.", "The skill measure and accuracy rate of each sub-model calculated through MLP, summarized in Fig. 3, are recorded in Table 4 (S50E) and Table 5 (T35B) and shown as maps in Fig. 4.", "The skill measure is based on the 2000 and 2014 land cover maps and compares the number of correct predictions, minus those attributable to random guessing, to that of a hypothetical set of perfect predictions.", "Thus the skill measure is not an evaluation of future performance of the model but rather a gauge of how well the explanatory variables explained change in the past.", "The accuracy and skill measure reveal a wide disparity between the levels of confidence in model predictions for different transitions.", "In S50E, the accuracy varies between 37 and 70 percent, with a correlation of 0.5 between accuracy and number of pixels involved in transition (Table 4).", "Lowest accuracy is associated with Abandonment (A) which also has the lowest skill measure.", "This is followed by Natural dynamic (Dn) with less than 40% accuracy and skill measure of less than 0.3.", "Degradation (De) has a high accuracy due to the large number of pixels involved in this sub-model (persistence of UG), but a low skill measure.", "These anomalies may be explained by the low user's and producer's accuracy for LC classes Wetlands (Wl) (in sub-model Dn) and Bare rock and soil (BRS) (in sub-model De) affecting the MLP (Münch et al., 2017) but may also be an indication that change is not totally controlled by the drivers used in the model.", "Low accuracies amongst those transitions which involve a small number of pixels should be regarded as being of low importance.", "Afforestation (R) and Urban intensification (Iu) in T35B have accuracies higher than 80% and matching high skill measures.", "Natural dynamics (Dn) has the lowest accuracy in T35B similar to S50E.", "Re (reclamation from FITBs) has the lowest skill score of less than 0.1 based on the low class skill ratio for the FITBs to UG transition.", "The probability of a land cover persisting (the diagonal highlighted by *) and of each class transitioning to every other class from the Markov matrix are presented in Table 8.", "Table 9 shows the modelled land cover change based on the proportion of the study area.", "The loss and gain per class is also recorded with the net loss per land cover class indicated.", "In S50E, the probability of UG persisting is approximately 80% with the highest probability of UG being lost are to FITBs (∼4.5%), CLS (∼6.6%) and UrBu (∼8.3%), thus FITBs intensification (If), agricultural intensification (Ia) and urban intensification (Iu) are at the expense of grasslands, constituting ∼12% of the catchment (5,283 ha) as shown in Table 9.", "The probability of 34% FITBs loss to UG (Table 8), possibly due to alien invasive clearing programs, may seem high, but in reality the number of pixels that can in fact transition are limited and the change represents only 4% (1800 ha) of the total area (44,640 ha) in 2030 (Table 9).", "The probability of persistence of FP is low (24%) with a likelihood of transition to FITBs (42%) and UG (34%), which clearly reflects the changes from 2000–2014.", "Classes Wl and BRS also show a very low probability of persisting.", "In T35B, the probability of UG persisting is over 90% with the highest probability of UG being lost are to FITBs (∼3.3%) and FP (3.2%) (Table 8).", "Based on the cross tabulation of land cover classes (Table 9), it was determined that the total change (gain and loss) in the landscape for catchment S50E over all land cover classes was 23% for predicted period 2014 to 2030, compared with 21% for the period between 2000 and 2014 (Münch et al., 2017), assuming a similar map accuracy for the modelled map.", "Since the future scenario model mimics patterns of past measured change, the change intensity, defined as the change per year, remained constant at 1.5% per year for S50E.", "UG, the largest class, also has the largest loss, though this relatively large dormant class, shows a higher change intensity during the modelled period with the loss intensity increasing from 1.27% to 1.34%.", "In contrast to the measured change, a net loss was modelled for FITBs.", "However, the predicted loss falls within the 30% hypothetical error in landscape transition ascribed to error propagation from contributing land cover maps calculated by Münch et al. (2017).", "Net change in FITBs for 2000 to 2014 varied between −0.5% to +1% of total catchment area.", "In T35B, the total change (gain and loss) in the landscape over all land cover classes was only 15.5% for prediction period 2014 to 2030, compared with 18.2% for the period between 2000 and 2014 (Münch et al., 2017).", "The change intensity decreased from 1.3% to less than 1% for T35B.", "FP showed a small net gain.", "Intensification of FITBs were modelled in the upper reaches of the Pot River and Little Pot.", "While FITBs systematically targets UG in transition (If), clearing of FITBs also systematically results in UG (Re), though possibly degraded, with a net loss of FITBs over the period.", "Afforestation (increased FP) is the strongest trajectory in T35B showing a net gain of 1.5% with FP targeting Wl.", "This transition may be the result of the low accuracy of the Wl class in the 2014 input land cover dataset.", "Since the result of this model is a future scenario, typical land cover validation methods cannot be employed since T3 is a future time step.", "Other indicators are thus required to assess the prediction.", "While visual examination reveals spatial patterns, it is subjective and can be misleading.", "Pontius and Millones (2011) suggest that disagreement in land cover maps can be attributed to randomness based on: 1) random distribution of the quantity of each land cover class (quantity disagreement), and 2) random spatial allocation of the land cover classes (allocation disagreement).", "In addition, in this study, the disagreement could also be attributed to errors in the land cover prediction model.", "However, the disagreement statistics can provide an indication of the quality of the future scenario map.", "The disagreement budget between the actual land cover maps 2014 (T2) and 2000 (T1), as well as between modelled land cover classes (T3) and 2014 land cover classes (T2), is provided in Table 10.", "Quantity difference is defined as the amount of difference between the T2 map and a comparison map where the proportions of the classes do not match.", "Allocation disagreement occurs where the quantity per class remains the same but the spatial distribution of the class changes and can be separated into exchange and shift.", "Exchange describes the transition between the misallocated classes.", "Shift refers to the difference remaining after subtracting quantity difference and exchange from the overall difference (Pontius and Santacruz, 2014).", "Even though applied to different land cover maps than proposed (Pontius and Millones, 2011; Pontius and Chen, 2006), the disagreement budget provides a comparison between the measured land cover maps and the future scenario for which there is no validation data.", "Table 10 reveals the classes that account for the largest exchanges and therefore possibly the largest model errors.", "In both measured and modelled transitions, UG had the highest exchange percentage approximately 9% for the 2000–2014 transition and ∼12% for the modelled transition (in bold italics).", "In the measured data (2000–2014) the similarity between categories with similar spectral signatures could cause exchange error, which would be propagated to the predicted LULCC model.", "Fig. 5 shows the overall disagreement budget of the catchment at the two time steps for the two catchments.", "Fig. 5 shows the increase in exchanged pixels in the predicted LULCC model for 2030, with lower quantity disagreement, particularly in T35B.", "The similarity in quantity disagreement between measured and modelled scenarios implies that for S50E the correct number of pixels were allocated to a class.", "The high exchange disagreement for classes UG, FITBs and CLs, as well as FP in T35B suggests that these classes may not be accurately modelled in the 2030 land cover map and that certain transitions were incorrectly predicted.", "This may be expected based on the model accuracies reported for S50E in Table 4 with none of the sub-models having an accuracy level higher than 70%.", "For T35B (Table 5), sub-models for urban intensification (Iu) and afforestation (R) provided accuracies of higher than 80%.", "The disagreement budget for these classes in the two time periods is also similar.", "The sub-models for abandonment (A) and natural dynamics (Dn) presented accuracies lower than 50%, but very few pixels were associated with these transitions.", "Despite these shortcomings, the overall proportion of the land cover within the catchment is likely to be reasonably accurate.", "Aldwaik and Pontius (2012) note that when a large dominant class exists, accounting for a large percentage of the study area, other classes may appear more active by comparison.", "However in this study area, the largest class UG is involved in substantial modelled change, and cannot be excluded.", "In S50E, active transitions occurred from FP (D), FITBs (Re) and CLs (A) to UG with a hypothesized error of 3%, while UG was the target of intensification to FITBs (If) and UrBu (Iu).", "The intensification of FITBs is regarded as a systematically targeting transition as a gain in FITBs targets UG while FITBs also targets the loss of UG.", "The same holds true for urban intensification (Iu).", "This interchange of classes may contribute to the high exchange disagreement.", "In T35B, FITBs intensification (If) and reclamation (Re) systematically targeted UG, implying an exchange of FITBs and UG over the prediction period.", "Land cover change, which is closely linked to rural development initiatives, presents challenges for integrated land and water resources management in the Eastern Cape.", "The aim of this research was to project land cover change trends into the future (2030) to gain an understanding of the implications on biophysical parameters which in turn can guide land management strategies.", "However the complex processes of land cover change are difficult to capture in variables, and model in algorithms, since they are often shaped by dynamic, non-linear human-nature interactions (Camacho Olmedo et al., 2015).", "For this reason, the discussion will firstly focus on potential sources of error in the LCM and then, with these potential limitations in mind, the focus shifts to the implications of the land cover trend projection on biophysical parameters, should no interventions be implemented.", "A land change model must predict both the quantity of each land cover type as well as the location of any change (Pontius et al., 2004).", "The accuracy of an inductive model's output is a function of both the model itself i.e. suitability of algorithms within the model to fulfil the intended purpose, and the accuracy of the input data.", "Thus to anticipate where possible inaccuracies may be entering into modelled output, assumptions within the model can be examined, as can accuracies of input data.", "Fig. 2 shows a flow diagram of the approach taken in LCM which is useful to view in light of this discussion.", "Since the LCM is an inductive approach, past land cover maps are used to empirically model change.", "Errors in the individual input land cover maps will be propagated through the model and produce errors in both future scenario output and the potential to transition map.", "This study is a follow on from the change analysis carried out by the same authors where the land cover maps, their accuracies, and the implications of these accuracies in change analysis are described (Münch et al., 2017).", "The overall accuracies for the land cover maps was reported as 83 and 87% for S50E and 81 and 83% for T35B for T1 and T2 respectively and these levels of accuracy equate to a change accuracy of 67% for T35B and 72% for S50E.", "This may appear rather low however if higher change accuracies are required, for example, change mapped with 75% reliability, the accuracy of input land cover maps at T1 and T2 would need to be about 90% (Fuller et al., 2003), a seldom achievable accuracy level when using mapping land cover from medium resolution satellite imagery.", "Suffice to say, land cover classification is fraught with uncertainties (Münch et al., 2017) and these uncertainties are propagated through to errors in historic change quantification and indeed future scenario mapping too.", "It is therefore important to take cognisance of this limitation and any interpretation of results should be with these accuracies in mind.", "Within the LCM, past land cover spatial distribution is used to estimate both potential to transition and a future land cover scenario as a function of explanatory spatial variables through mathematical modelling (Mas et al., 2014).", "This modelling is based on two assumptions.", "Firstly, in the Markov projection, rates of change are assumed to be constant implying that external forces exerting the change remain constant too.", "In context of increasing human pressure on the land, climate change, and variability in rainfall inter alia, this assumption can be flawed.", "Over short time periods, the impact of the rate of change may not have a significant impact on the projected change, as described by Roy et al. (2014), especially when the scale of the input land cover maps is considered.", "However, climate patterns may be cyclical and if change maps T1 and T2 represent different stages within that cycle (e.g. one in a particularly wet year and the other in a particularly dry year) then rate of change may show much higher than may otherwise be predicted, with the inverse also being true.", "Secondly, in LCM the drivers of change (explanatory spatial variables) are assumed to act identically to create the propensity for change maps.", "Cramer's V is used to test the level of association between a potential explanatory spatial variable and the historic change recorded between T1 and T2.", "The user then decides based on his/her own expert knowledge, the explanatory variables which should be allocated to each transition sub model using Cramer's V as a guide to which variables to include, which in turn will impact on the resulting change propensity map.", "Then even though some explanatory spatial variables may better describe the historic change, once the variable is selected into a sub model, the model does not rank or weight the variable on the basis of its usefulness in describing past change.", "Thus should a variable with a low Cramer's V be selected, it will have as much influence on the change propensity map as a variable with a high Cramer's V. Much research in land change modelling has been based on comparing the outputs between different models (e.g. Mas et al., 2014) however, if a single model can produce different outputs based on the users choice of model parameters, there can be greater variation within the outputs of a single model than between the outputs of different models (Camacho Olmedo et al., 2015).", "Furthermore, the spatial explanatory variables are implemented in the model as stable over time, thus they will have the same influence at T2 all the way through to T3.", "In reality this is unlikely to be the case as some of these variables may also change over time, a topic explored in more detail by Kolb et al. (2013).", "Despite these limitations, in the context of trying to understand appropriate land management interventions for both catchments in light of the trends presented in the results, a qualitative discussion of biophysical parameters that impact on catchment water use, NEE and the surface energy balance and the expected land cover transitions is presented.", "Firstly, from a water resource management perspective, globally, >66% of the total precipitation over land is returned to the atmosphere as ET (Fisher et al., 2005; Mu et al., 2011; Hoff et al., 2010; McMahon et al., 2013; Liou and Kar, 2014) which makes ET very important in catchment water balance.", "During photosynthesis, plants accumulate new biomass as they release water in exchange for atmospheric carbon and ET rates are closely related to the carbon assimilation rates of plants (Franks et al., 2013).", "It is well established that knowledge of land cover can give insight (via ecosystem surface conductance and ET) into the water use of the land surface.", "A transition towards land covers with higher surface conductance will result in higher water use via ET in the catchment.", "In this study the loss of grasslands favouring an increase in anthropogenic land covers (agricultural and FITBs intensification) will result in higher catchment ET for both S50E and T35B with T35B being most impacted.", "Next, from a carbon perspective, fPAR and LAI measured by Palmer et al. (2017) and used in NEE and ET modelling respectively, indicate that both fPAR and LAI are lower for un-improved grasslands than for potential transition classes (Intensification of FITBs and Intensification of CLS and afforestation).", "These transitions will thus represent a gain in both catchment NEE and ET, and a concomitant decrease in run-off.", "From a carbon storage perspective, the transitions will result in more carbon storage, which from a climate change outlook may be seen as a positive change, however in an already water scarce catchment, further water demands by the vegetation will result in a decrease in the availability of water for other land covers.", "Finally when considering the surface energy balance, the changes to surface albedo that will accompany these land cover trajectories are less certain.", "Given that there is a general increase in woody green biomass as a result of both afforestation and continued invasion by IAPs, the findings of Rotenberg and Yakir (2010) make it likely that the decrease in surface albedo from these cover classes will result in an increase in the absorption of energy, with a resultant rise in temperature.", "This decrease in albedo may however be counteracted by an increase in degraded surfaces associated with rural housing and in the unimproved grasslands where continuous grazing by livestock changes species composition and cover.", "Rangeland degradation is commonly associated with the changes in land tenure that are occurring in this catchment (Bennett et al., 2012), and a reduction in the basal cover of herbaceous plants (mainly grasses) is the first noticeable change.", "This results in a surface with higher albedo.", "Since, the land surface reflectance (albedo) affects net surface radiation, dark vegetation with a high LAI will have a lower albedo than open grasslands and it can be postulated (however this must still be measured) that the transitions modelled in the S50E catchment could lead to an overall lowering of albedo in the catchment.", "An increase in net radiation is thought by some to be a driver of global warming, however, Bonan (2008) states that surface warming arising from the low albedo of forests is offset by strong evaporative cooling.", "Thus the impact of a change in albedo in this catchment remains speculative but is a research avenue which should be pursued.", "In this paper, the Land Change Modeller (Idrisi Terrset 18.08) was used, together with land cover mapped for the years 2000 and 2014 (Münch et al., 2017), to model land cover for the grassland dominated S50E and T35B catchments in the Eastern Cape Province for the year 2030.", "It has been postulated that future land cover changes may result in adjustments to biophysical drivers impacting on NEE and catchment water use through ET.", "This work has thus built on previous work (Palmer et al., 2017; Münch et al., 2017; Gwate et al., 2016; Okoye, 2016) as a first step in determining the impact of future land cover change on catchment water and carbon fluxes.", "It was found that in 2014 for S50E (T35B), grasslands represented 57% (80%) of the total catchment area with this figure modeled to decrease to 52% (80%) by 2030 with losses likely to favour a gain in woody plants and cultivated land.", "The results show that the total change (gain and loss) in the landscape over all land cover classes was 21% (18%) for the period between 2000 and 2014 and 23% (16%) from 2014 up to the future scenario for 2030, with the change intensity remaining constant at 1.5% (<1%) per year.", "It was determined that the probability of grasslands persisting is around 80% (>90%) with the highest probability of grasslands being lost to woody encroachment ∼5% (3%) and cultivation ∼7% (<2%).", "Since fPAR and LAI are lower for grasslands than for their transition classes (Palmer et al., 2017), these transitions represent a gain in both catchment NEE and ET, resulting in increased carbon storage, and corresponding increased water use by vegetation.", "It is postulated that these carbon offsets from sequestration may be counterbalanced by temperature increases linked to lower albedo increasing net surface radiation and it is this carbon-water-surface energy flux nexus that requires further research in quantifying impacts.", "The higher LAI will undoubtedly increase catchment-scale ET and reduce run-off.", "The lower albedos will increase surface temperature, and although these may be offset by higher albedo from urbanized and degraded surfaces, the net result from an increase in woody biomass will be a catchment with a lower capacity to provide water to its residents or downstream users.", "The LCM models future scenarios based on trends of historic change and therefore the results represent a future scenario based on no intervention deviating from past interventions.", "The impact of the different land management practices in S50E (dualistic farming system) and T35B (commercial system) can be identified in the historic land cover change trends and in the future scenario.", "It is apparent that under the dualistic farming system, degradation is taking place at a more rapid rate than in T35B where over 90% of current grassland is expected to persist to 2030.", "For those involved in planning in these rural catchments, there should be greater sensitivity amongst policy makers towards the negative effects of further afforestation and uncontrolled invasion of IAPs.", "Finally, the results suggest that rehabilitation and land management initiatives should be targeted in catchments under a dualistic farming system, rather than those which are predominantly commercial systems.", "Lesley Gibson, Zahn Münch: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.", "Anthony Palmer, Sukhmani Mantel: Conceived and designed the experiments; Wrote the paper.", "This work was supported the Water Research Commission [Grant no: K5/2400/4/3].", "The authors declare no conflict of interest.", "No additional information is available for this paper." ]

[]

[ "CHEM", "MATE" ]

[]

[ "1600", "2500" ]

[ "Graphene, and other 2-dimensional (2-d) materials, have shown tremendous potential for applications in the fields of sensors, printed electronics, composites and medicine to name but a few [1].", "Most often, selective chemical functionalization of a 2-d material is necessary to increase compatibility or tailor its properties.", "Many different techniques, both covalent and non-covalent have been investigated [2–5], particularly with respect to graphene [6,7].", "The ability to identify and map chemical groups on the graphene surface is crucial to the development of these functional materials.", "There is often a need for localized functionalization such as on the edge [8] or basal plane [9] of graphene, but there is often little direct evidence that this localization has indeed occurred.", "Traditional bulk analysis, such as combustion elemental analysis, thermal gravimetric analysis and Fourier transform infrared spectroscopy (FTIR) [10,11], are still typically relied upon for characterization of these materials and lack spatial resolution.", "High atomic-resolution quantification has been carried out using solid state nuclear magnetic resonance and x-ray photoelectron spectroscopies [12,13] but both techniques suffer from a relatively high level of surface penetration for 2-d materials and a lack of lateral resolution.", "Raman spectroscopy is now ubiquitous when characterizing graphene materials and can be used to identify mechanical strain, defectiveness and doping of a graphene basal plane [14–16] but again, the spectra have limited spatially resolution, typically ∼1 to 0.5 μm as they are diffraction limited.", "Tip-enhanced Raman spectroscopy (TERS) can increase the spatial resolution to the order of nanometres [17] but is often difficult to set up and can be prone to issues with reproducibility [18].", "Further to this, the polarizability selection rules for a bond to be Raman active often means that, whilst graphene is highly active, many types of ionic functionalization can only be inferred from its effect on doping or disrupting the graphene basal plane [15].", "Due to this, direct identification of functionality on 2-d materials with Raman spectroscopy is not always possible and the interpretation of the Raman spectra is still not standardized for all 2-d materials, particularly with respect to graphene oxide (GO) and reduced graphene oxide (rGO) due to their intrinsically high level of defectiveness [19–21].", "Over the past decade, photothermal induced resonance, or atomic force microscopy-coupled infrared spectroscopy (AFM-IR) has emerged as a promising new technique for high lateral resolution (<30 nm) infrared mapping of materials, including polymers, nanowires and cells [22–24].", "Sub-diffraction limit resolution is achieved during AFM-IR by monitoring the deflection of an atomic force microscopy (AFM) probe in contact with the surface, which probes the rapid transient thermal expansion of the sample accompanying absorbance of the infrared pulse.", "The recorded AFM-IR signal is the amplitude of induced AFM probe oscillation, obtained after fast Fourier transform.", "This has previously been shown to correlate well to infrared absorbance measured using conventional macroscopic FTIR [25].", "Since the IR pulse (10 ns duration), thermal expansion, and damping down of the induced oscillation occur on a shorter timescale than the feedback electronics of the AFM, simultaneous contact-mode topographical measurements and infrared mapping can be performed at a given wavelength [26–29].", "Recently, AFM-IR has been used to map the distribution of sigma and pi-bonded carbon-oxygen groups on GO [30].", "Spectral features which showed spatial variation in this work have been shown to possess a strong thickness dependence in conventional IR measurements [11], and these chemical groups are not the ideal choice to showcase the mapping capability and resolution of this technique due to the local variability observed.", "In this study, we demonstrate the applicability of AFM-IR to identify chemical functionality on monolayer rGO surfaces.", "By functionalized rGO flakes with two different non-covalently bonded pyrene sulfonates, we show that Raman spectroscopy on these materials is not sensitive enough to pick up differences in functionality.", "By employing AFM-IR spectral acquisitions and mapping, even small differences in the spectra are comparable those seen in the bulk FTIR spectra of the pyrene moieties.", "Further to this, we show that the spatial resolution of this technique is similar to that seen in other reports at <30 nm [31].", "This has implications for the in-situ characterization of chemical functionality on graphene where Raman spectroscopy cannot provide enough chemical information and shows that AFM-IR has the potential to advance the ever-growing field of 2-d materials.", "GO was prepared by a modified Hummers method [32] whereby natural flake graphite (30 mesh, 96% C) was oxidized and exfoliated using H2SO4, NaNO3, KMnO4 and H2O2.", "The resulting material was repeatedly washed using a H2SO4 (3% wt.)", "/H2O2 (0.5% wt.)", "mixture and then exfoliated and brought to a pH of 7 using deionized (DI) water.", "To produce the functionalized rGO dispersions, PBS (30 mg) and PCNBS (30 mg) were each added to separate dispersions of GO (10 ml, 1 mg/ml) in water and then bath sonicated (30 min).", "Ascorbic acid (140 mg) was dissolved into each of the samples after which they were heated to 80 °C for 72 h.", "After reduction, the PBS-rGO and PCNBS-rGO dispersions were subjected to bath sonication (15 min) and washing by centrifugation (15,000 rpm, 45 min).", "The supernatant was removed and replaced with fresh DI water.", "This washing process was repeated 5 times.", "The U-rGO dispersion was produced in the same way, with additional high-shear mixing (8000 rpm, 15 min) after washing, to break up the resultant hydrogel and isolating individual rGO flakes.", "To ensure that no excess of pyrene moieties or ascorbic acid was present in the dispersion after the washing process, dialysis in DI water was carried out on all dispersions.", "Dialysis has previously been shown to only remove excess pyrenes in solution and not cause desorption from the graphene plane [33].", "This step was carried out over 12 days, changing the water every 2 days and monitoring the dialysate with UV–Vis spectroscopy to ensure complete separation.", "All dispersions were then spin coated onto 290 nm SiO2|Si for AFM and Raman analysis and 50 nm Au|290 nm SiO2|Si with a 3 nm Cr adhesion layer for SEM and AFM-IR.", "Samples for FTIR were produced by vacuum drying 5 ml of each dispersion.", "SEM micrographs were taken with a Zeiss Ultra Plus microscope with an InLens Detector and 5 keV gun voltage.", "Raman spectra were recorded using an InVia spectrometer (Renishaw) with a 514 nm laser at <1 mW power.", "Bulk FTIR spectra were obtained from 32 co-averages collected in transmission mode using an ATR FTIR spectrometer (Nicolet iS50 spectrometer, Thermo Scientific) with a germanium crystal, operating at 4 cm−1 resolution across a 700–4000 cm−1 range.", "AFM measurements were carried out in tapping mode with a FastScan head (Bruker).", "AFM-IR was performed on a NanoIR2 system (Anasys Instruments) operating with top-down illumination.", "During AFM-IR analysis, specimens were illuminated by a pulsed, tuneable infrared source (optical parametric oscillator, 10 ns pulses at a repetition rate of 1 kHz, approximate beam spot size 30 μm).", "For the present study, AFM-IR images were collected in contact mode at a scan rate of 0.1 Hz using a gold-coated silicon nitride probe (0.07–0.4 N/m spring constant, 13 ± 4 kHz resonant frequency (Anasys Instruments).", "The amplitudes of the infrared-induced oscillations were recorded at a given wavelength using 16 co-averages for 1024 points per 300 scan lines.", "To characterize different functionalities on rGO flakes, three dispersions of rGO were prepared (Fig. 1 a): two functionalized (PBS-rGO and PCNBS-rGO) and one unfunctionalized sample (U-rGO).", "Two substituted pyrenes, sodium 4-(4,5a1-dihydropyren-1-yl)butane-1-sulfonate (PBS, Fig. 1 a i)) and sodium 4-(7-cyano-4,5a1-dihydropyren-1-yl)butane-1-sulfonate (PCNBS, Fig. 1 a ii)) were adsorbed onto the graphene basal plane during the chemical reduction from GO with l-ascorbic acid.", "After deposition of U-rGO, PBS-rGO and PCNBS-rGO on SiO2, the topology of each flake was investigated using AFM.", "Due to the defectiveness of the plane, GO and rGO typically have a flake height of 1 ± 0.2 nm [34] which can be seen in the height profile of U-rGO (Fig. 1 b).", "When compared with this, PBS-rGO and PCNBS-rGO both show an increase in height measurement to 1.7 ± 0.2 nm (Fig. 1 c, d), indicative on a bilayer of pyrene moieties adsorbed onto each face of the rGO plane [35,36].", "To chemically analyse the graphene flakes, Raman spectroscopy was performed to attempt to observe any functionalization on the rGO surface.", "Commonly used to characterize graphene materials, including rGO [37], one of the main advantages of this technique is that it can record spectra at monolayer thickness.", "Unlike pristine graphene, careful deconvolution of the D and G bands in rGO is necessary due to the high level of defectiveness on the basal plane leading to broadening of both primary and secondary absorption bands [20,38].", "After multiple-peak fitting of the broadened D, G and higher-order bands (Fig. 2 a), the Raman spectra of both PBS-rGO and PCNBS-rGO flakes show some evidence of functionalization.", "The G band in graphene, arising from the carbon sp2 E2g breathing mode, is sensitive to doping and shows a narrowing of ∼7 cm−1 (Fig. 2 b) due to charge transfer from the adsorbed pyrene molecules [39].", "The position of the G band can also infer doping of the graphene plane [36] although, surprisingly, there is little measured difference between functionalized and unfunctionalized rGO in this case.", "The ID/IG ratio, the relative intensities of the D and G bands, is often a useful indicator of defectiveness for graphene [40] and shows the same high level of defects for all measured flakes (Fig. 2 b).", "This is to be expected as the graphene basal plane is similar in all samples as they have been subjected to the same processing conditions.", "The U-rGO flakes did undergo additional shear mixing to isolate single flakes but this has been shown not to induce basal plane defects [41] and therefore should not affect this metric.", "While the Raman spectra indicate functionalization by G peak narrowing, PBS-rGO and PCNBS-rGO cannot be further distinguished by this technique.", "Further to Raman spectroscopy, chemical analysis was carried out using FTIR, another commonly employed analytical technique for 2-d materials.", "The FTIR spectra of PBS and PCNBS (Fig. 3 a) show characteristic absorptions for SO3 at 1050 cm−1 (symmetric, νsymSO3) and 1180 cm−1 (asymmetric, νasSO3) [35], with each molecule showing a distinguishable fingerprint for SO3 stretch.", "Both molecules also show pyrene CH out of plane aromatic stretches at 835-880 cm−1 [42,43] PCNBS has an additional peak at 2200 cm−1 corresponding to the CN stretch.", "The aforementioned νSO3 peaks can be seen in the rGO spectra after functionalization (Fig. 3 b) albeit with much reduced intensity and red-shift of 14 cm−1, due to the adsorbed polyaromatic rings being a higher energy of conformation than in the isolated state and therefore shifting the νSO3 vibration [44].", "The polyaromatic pyrene 835 cm−1 band is also visible but the expected CN stretch for PCNBS-rGO is not apparent as it is weakly absorbing and is therefore not strong enough to be observed over the strong rGO background.", "Additional peaks can be seen in all three rGO spectra around 1700-1600 cm−1 (C=O, C=C) and ∼1200 cm−1 (C-O).", "These can be attributed to some residual functionality after the reduction as chemical reduction is yet to be able to fully restore the material to a pristine graphene plane [45].", "rGO, predominantly sp2 hybridized carbon, however generally has a poor FTIR signal due to a low level of permanent dipoles in its structure and a high refractive index making spectral acquisitions difficult.", "While FTIR can identify the presence of PBS and PCNBS within the sample, the bonding between the pyrene molecules and the rGO is entirely noncovalent, therefore their adsorption can only be shown indirectly from the presence of the pyrene and sulfonate stretches at 1036 and 1180 cm−1 in tandem with other techniques.", "It is also worth highlighting that individual monolayer flakes of 2-d materials cannot normally be measured by most conventional ATR FTIR techniques due to its low signal, therefore analysis is normally carried out on bulk powdered samples.", "In order demonstrate the adsorption of pyrene molecules onto an rGO plane, AFM-IR was used to measure the nanoscale infrared response across deposited 2-d flakes.", "The acquisition was carried out by stepping the pulsed incident infrared through the mid-IR region whilst monitoring the amplitude of induced probe oscillations using a photodiode array (Fig. 4 a).", "To illustrate the ability of this technique to produce spectra on single flakes, AFM scans were carried out to identify individual flakes and spectra were acquired on these (Fig. 4 b).", "AFM-IR spectra show a large peak for both functionalized flakes between 1084 and 1036 cm−1 in good agreement with the νsymSO3 bulk FTIR of the pyrene moieties discussed previously.", "It is worth noting that the PBS-rGO spectrum (Fig. 4 b i)) also shows the presence of the 1180 cm−1 (νasSO3) stretch whereas in PCNBS-rGO spectrum it is greatly diminished.", "To further investigate this, and to ensure that we had repeatable and representative spectra of the graphene material, 100 spectra were acquired and averaged.", "Since the sample volume for single isolated flakes of functionalized rGO was small for AFM-IR analysis, and to limit prolonged AFM acquisition times, aggregates of the functionalized rGO were identified and localized infrared spectra were collected at these positions (Fig. 4 d, e, f).", "showing good agreement with the single flake spectra and the bulk FTIR (Fig. 4 c).", "The νasSO3 peak shows dampening and broadening in these average spectra for both materials which we believe is due to the sensitivity of the νasSO3 peak to counter-ions and hydration states [46] are likely to be inconsistent across the materials on the nanoscale.", "The observed strong absorbance can be attributed and directly correlated to the same absorbance peak position and shape for the asymmetric sulfonate stretch, νsymSO3 as seen in the fingerprint region of the bulk FTIR spectra of the functionalizing molecules (Fig. 4 c inset).", "Even with the reduction in asymmetric peak intensity, the distinct νsymSO3 fingerprint of both pyrene moieties can be detected using this technique and show the successful acquisition of nanoscale infrared spectra on rGO.", "Following AFM-IR spectral acquisition, large isolated flakes were selected using SEM (Fig. 5 a) and imaged for photothermal response and topology simultaneously at defined infrared wavelengths of interest, identified from the AFM-IR spectra.", "AFM-IR photothermal response maps were acquired at 1036, 1084 and 1220 cm−1 (Fig. 5 c, d, e, respectively).", "The symmetric sulfonate stretches, νsymSO3, seen at 1084 and 1036 cm−1 were chosen as suitable wavelengths on which to map the localized functionality of the flakes.", "Maps on both PBS and PCNBS-rGO can be seen to give a measurable response down at 1.7 nm flake heights when imaged at both 1036 and 1084 cm−1 in good agreement with the spectral acquisitions.", "The high level of correlation between photothermal expansion and the topological scans clearly shows that the sulfonate groups are present on the rGO flake and evenly distributed across it, in the case of both PBS-rGO and PCNBS-rGO.", "Additionally, mapping at 1220 cm−1 was carried out to ensure that topology was not affecting the IR response.", "The maps acquired at 1220 cm−1 and on the unfunctionalised rGO flake show no signal, ruling out any effect from the rGO plane or folding on the flakes.", "These results go some way to presenting a method for unambiguous infrared mapping on 2-d materials.", "Even with the high anisotropic thermal conductivity of rGO [47,48] limiting thermal expansion out of the plane of the substrate, the response is such that full spectra and resonance mapping can be acquired.", "In addition to mapping the photothermal response, a comparison of the height and IR induced amplitude profiles of the PBS-rGO flake (Fig. 6 a, b) allows the calculation of the lateral resolution of the IR response for graphene materials.", "As the topological scan is carried out simultaneously to the IR response mapping, the two can be superimposed (Fig. 6 c) and used to calculate the lateral resolution [49].", "By examining the gradient of the height profile change when the tip is brought on and off the flake (Fig. 6 c, d), the change in height is seen to be equivalent to the gradient on the same line on the induced amplitude change.", "This metric has been commonly used to examine the different lateral resolutions measured by AFM-IR when analysing different materials [25,30,31,50].", "The height resolution is calculated to be 32 nm when averaged over 20 profiles (each profile in Fig. 6 d is an average of 5 lines).", "The maximum measurement acquired over a 5 line average is 20 nm for a monolayer graphene flake which corresponds to a height of 1.7 nm and at a pixel pitch of 10 nm.", "This directly matches the IR resolution showing that the lateral resolution is limited by the tip in this case.", "The standard deviation in each average, 5 and 8 nm for height and IR amplitude, is less than the resolution of the pixel acquisition (∼10 nm, Methods).", "The lateral resolution is similar to other reports [31,49] but at a 1.7 nm sample thickness.", "By decreasing the tip radius and increasing the number of samples per line acquired when imaging, this has the potential to be further improved, but nevertheless shows a comparable resolution of AFM-IR analysis on 2-d materials to that of state of the art TERS experiments on graphene [51].", "The ability to unambiguously identify and map chemical functional groups on monolayer rGO surfaces has been shown to be achieved using AFM-IR.", "We have demonstrated its ability as a technique which can image functional groups at 1.7 nm thickness and 32 nm spatial resolution, where conventional analysis by Raman and bulk FTIR have limitations in different respects.", "Further to this, it can distinguish between subtle changes in the sulfonate stretches of different functionalizing molecules by analysis of the symmetric vibrational mode even on a high thermal conductivity material.", "We believe that this technique has great potential to be applied to the development of selective functionalization strategies of 2-d materials for their application in the fields of 2-d sensors and devices, particularly where in situ mapping of IR-active surface functionality is not currently possible without destruction of the material.", "C.B., S.M. and A.V. conceived and designed the experiments.", "C.B. carried out samples preparation, AFM, Raman, SEM and FTIR characterisation.", "C.B. and S.M. performed the AFM-IR experiments and analysis.", "K.W.J.H. synthesised the pyrene molecules.", "C.B., S.M. and A.V. co-wrote the manuscript.", "All authors commented on the manuscript." ]

[]

[ "NEUR" ]

[ "Empirical Bayesian beamformer", "Forward model", "Head-cast", "Hidden Markov model", "Inversion", "LORETA", "Magnetoencephalography", "Minimum norm", "Multiple sparse priors", "Resolution", "Resting state" ]

[ "2805", "2808" ]

[ "Magnetoencephalography (MEG) detects electromagnetic fields at sensors outside the head.", "The challenge for the researcher is to infer the neuronal current distribution responsible for the observed data, despite a much higher number of possible sources than sensors.", "The general approach is to restrict the number of potential solutions through a priori assumptions, including the temporal relationship between sources (i.e. source co-variance) and/or the anatomical manifold that gives rise to this function (e.g. the cortical mesh).", "These assumptions are continually being refined and debated (Baillet, 2015; Baillet et al., 2001; Lin et al., 2006; Wipf and Nagarajan, 2009).", "Two recurring issues make it difficult for the community to come to a consensus on optimal source reconstruction methods - the first is the choice of test scenario, the second is the lack of ground truth.", "Firstly, the choice of task, or simulation set-up used to compare source localisations will introduce a selection bias towards a specific temporal pattern predominant in certain cortical areas, which will suit some inversion assumptions but not others.", "Here we set out a framework which utilises diverse spatio-temporal patterns and minimizes selection bias by using a Hidden Markov model (Baker et al., 2014) to parcel endogenous resting-state data into collections of self-similar and quasi-stationary time segments.", "Resting state data have been shown to arise from dynamic spatio-temporal network state fluctuations occurring on the scale of 100–200 ms (Baker et al., 2014; Koenig et al., 2002; Wackermann et al., 1993; Woolrich et al., 2013) that include the rehearsal of the transient dynamic patterns observed during task performance (O'Neill et al., 2017).", "These networks predominate in all M/EEG recordings (even those which are task based) and are key to healthy brain function (Barttfeld et al., 2015; Kaiser et al., 2015; Lewis et al., 2009; Li et al., 2012; Peterson et al., 2014; Philippi et al., 2015; Reineberg et al., 2015; Sheline and Raichle, 2013; Tessitore et al., 2012; Venkataraman et al., 2012; Wu et al., 2014; Wurina et al., 2012).", "We rely on these iterant dynamics, rehearsing multiple task scenarios, to provide a varied and unbiased spatio-temporal repertoire of the source reconstruction problems one might expect from any dataset on which to then test our inversion schemes.", "The second problem then, having identified an appropriate and representative real dataset (as opposed to simulated data), is the lack of access to the ground truth with which to compare recording/inversion techniques.", "Here we leverage new analytic techniques to quantify the sensitivity of MEG source inversion schemes by progressively deforming the anatomical models (Lopez et al., 2013; López et al., 2017; Stevenson et al., 2014).", "Specifically, we quantify how distortions in the MRI-extracted cortical manifold (mesh) affect our ability to predict or model the underlying current distribution (using cross validation error and Free energy).", "The technique assumes that the MEG sensor level data are due to current flow normal to the cortical surface but makes no assumptions about how this current should be distributed.", "The rationale is that the best MEG inversion scheme will be the most sensitive to subtle distortions of the cortical anatomy (as we know that MEG data derives from grey matter structure).", "This spatial distortion metric then provides a principled basis for comparing different a priori inversion assumptions (i.e. different algorithms) and recording techniques.", "We are aiming for a generic method to provide a benchmark to refine inversion (or recording) methods based on human electrophysiological data from multiple labs.", "The paper proceeds as follows: we first parcel resting state datasets into brief epochs using a hidden Markov model (Baker et al., 2014).", "The epochs for the four dominant networks were then amalgamated into four network-specific datasets for each subject and taken forwards for inversion.", "These datasets were then inverted onto a library of subject-specific distorted meshes, for which we had control over the spatial detail available in the forward model.", "For each of these meshes, and for each inversion scheme, we quantified the model fit using cross validation and Free energy metrics.", "As expected, we found that the greater the distortion from the true cortical mesh, the poorer the model fit.", "We then used this spatial quantification to compare different inversion schemes (implemented as different co-variance prior assumptions).", "For these data, we found that the beamformer-based priors (EBB) were the most sensitive to small deviations from the true anatomy.", "In addition to distinguishing between algorithms, here we also tested whether we could use the same methods to distinguish between datasets collected with and without a head-cast (Meyer et al., 2017a; Troebinger et al., 2014a; 2014b), where the accuracy of forward model is more precisely known, and those collected without and found marginal (but not significant) differences.", "Subjects underwent two MRI scans using a Siemens Tim Trio 3 T system (Erlangen, Germany).", "For the head-cast scan, the acquisition time was 3 min 42 s, in addition to 45 s for the localizer sequence.", "The sequence implemented was a radiofrequency (RF) and gradient spoiled T1 weighted 3D fast low angle shot (FLASH) sequence with image resolution 1 mm3 (1 mm slice thickness), field-of view set to 256, 256, and 192 mm along the phase (A–P), read (H–F), and partition (R–L; second 3D phase encoding direction) directions respectively.", "A single shot, high readout bandwidth (425 Hz/pixel) and minimum echo time (2.25 ms) was used.", "This sequence was optimized to preserve head and scalp structure (as opposed to brain structure).", "Repetition time was set to 7.96 ms and excitation flip angle set to 12° to ensure sufficient SNR.", "A partial Fourier (factor 6/8) acquisition was used in each phase-encoded direction to accelerate acquisition.", "For the anatomical scan later used to construct the cortical model, multiple parameter maps (MPM) were acquired to optimise spatial resolution of the brain image (to 0.8 mm).", "The sequence comprised three multi-echo 3D FLASH (fast low angle shot) scans, one RF transmit field map and one static magnetic (B0) field map scan (Weiskopf et al., 2013).", "Scalp surfaces from the head-cast MRI data were extracted using SPM12 (http://www.fil.ion.ucl.ac.uk/spm/) by registering MRI images to a tissue probability map which classified voxels according to tissue makeup (e.g. skull, skin, grey matter etc.).", "The skin tissue probability map was transformed into a surface using the ‘isosurface’ function in MATLAB® and then into standard template library format with the outlines of three fiducial coils digitally placed at conventional sites (left/right pre-auricular and nasion).", "Next, a positive head model was printed using a Zcorp 3D printer (600 × 540 dots per inch resolution) and this model placed inside a replica dewar-helmet with liquid resin poured between the two, resulting in a flexible, subject specific, foam head-cast with fiducial indentations in MRI-defined locations (Meyer et al., 2017a).", "Resting state data was acquired from 12 healthy subjects using head-casts (age: 26.6 ± 3.5 yrs (mean + sd)) and 12 other healthy subjects without head-casts (age: 25.2 ± 6.6 yrs).", "All subjects were right handed, had normal or corrected-to-normal vision, and had no history of neurological or psychiatric disease.", "Informed written consent was given by all subjects and recordings were carried out after obtaining ethical approval from the University College London ethics committee (ref. number 3090/001).", "All subjects underwent a 10 min resting state scan with eyes kept open and instructed to fixate on a central cross on a screen, using a CTF 275 Omega MEG system.", "The head was localised using the three head-cast-embedded fiducials (head-cast subjects) or fiducials placed on the nasion and left and right pre-auricular points (non-head-cast subjects).", "Average range of absolute head movement within the 10 min resting state recording was 0.26 ± 0.06, 0.24 ± 0.05, 1.1 ± 0.54 mm (X,Y,Z directions; ± SEM) for head-cast and 3.2 ± 0.5, 3.0 ± 0.5, 3.3 ± 0.2 (X,Y,Z directions; ± SEM) for non-head-cast data.", "The data were sampled at a rate of 1200 Hz, imported into SPM12 and filtered (4th order butterworth bandpass filter: 1–90 Hz, 4th order butterworth bandstop filter 48–52 Hz) and downsampled to 250 Hz.", "Traditional inverse problem solutions are based on the assumption that the data are stationary during the period of inversion.", "However, resting state data contains rapid dynamics that do not accord well with this assumption (Woolrich et al., 2013).", "Therefore, in order to improve stationarity within a given epoch, we parcellated the data into self-similar periods that capture the resting state network transitions (100–200 ms) using a Hidden Markov Model (HMM) that could identify the rapid formation and dissolution of recurring resting state networks (Baker et al., 2014).", "With this, a ‘statepath’ was estimated for each 10 min resting state block, which tracks the fine spatiotemporal dynamics and allocates each point in time to one of eight dominant network states (Baker et al., 2014).", "For this statepath determination, a copy of each subject's sensor level data was dimensionally reduced using principle component analysis (PCA) to derive 40 components of unit variance and mean (Woolrich et al., 2013).", "With these data, an 8 state Hidden Markov model (HMM; www.fmrib.ox.ac.uk/∼woolrich/HMMtoolbox) was then applied to derive the most probable state at all points in time (the statepath) (Fig. 1A) (Baker et al., 2014).", "The continuous 600s of data was then epoched into 200 ms blocks/epochs (the average scale of individual state transitions (Baker et al., 2014) and each 200 ms data epoch allocated to a new dataset according to the state that was most dominant during that time period.", "This resulted in 8 new datasets each with a collection of epochs that corresponded to a distinct network state as identified by the HMM.", "The four most dominant states for each individual subject (as measured by most time spent in that state) was taken forward for further analysis and spatial estimates averaged across those four inversions for each subject.", "As the HMM was performed on individual, rather than group concatenated data, state numbers did not directly correspond across subjects.", "This however permits superior partitioning within subjects since it allows the model to optimally fit states to the individual subjects, rather than fitting individual data to group states that are (or are not) common across all subjects.", "This resulted in 815 ± 56.9 data segments per partitioned dataset - equivalent to 163s of continuous data.", "Since the HMM selected periods of self-similarity within the resting state, partial correlation maps were examined for all subjects to check that segregation into separate networks had occurred and this segregation did not relate to eye blinks, muscle artefacts or cardiac interference (Fig. 1B).", "We used SPM12 which implements multiple different inversion schemes within a common empirical Bayesian pipeline, whereby all processing steps, besides the choice of prior source co-variance matrix are held constant (Friston et al., 2008; López et al., 2014).", "The choices of prior source covariance matrices used embody four popular inversion algorithms: The traditional unweighted Minimum norm (MMN) in which the source covariance is a diagonal (IID) matrix (Hämäläinen and Ilmoniemi, 1994).", "An (unweighted) LORETA-like solution (Pascual-Marqui, 2002) based on a surface (rather than volumetric) smoothing using Green's function (Harrison et al., 2007) (LOR).", "An Empirical Bayesian Beamformer (EBB) inversion (Belardinelli et al., 2012) in which a direct estimate of prior source co-variance is made based on the sensor-level data (and zero regularization).", "The main difference between the EBB scheme and the classic linearly constrained minimum variance (LCMV) (Van Veen et al., 1997) scheme is that the EBB attempts to fit the measured data (by optimizing model evidence).", "This has the advantage that EBB attempts to explain the measured data and can therefore provide a direct model evidence metric (which can then be compared to other linear inversion algorithms); but the disadvantage is that if there is external noise in the measured data the Bayesian scheme will still attempt to fit it (unless appropriately modelled) whereas the LCMV (spatially filtered) solution will be unaffected.", "The multiple sparse priors algorithm (MSP) (Friston et al., 2008) uses an optimized search to select mixtures of source covariance matrices each describing the activity of separate patches of cortex.", "For each of the different algorithms, the source level estimates were weighted against a diagonal sensor noise covariance matrix (effectively regularizing the solution) within the same empirical Bayes framework.", "For more details on each of these methods and the empirical Bayesian optimization please see (Belardinelli et al., 2012; Friston et al., 2008; López et al., 2014).", "The HMM parcellated datasets originally contained 272 channels and 50 time samples (3 channels were damaged and excluded from the data).", "As part of the cross validation procedure, we excluded 10% of these channels from the inversion stage, leaving 245 channels of data.", "This procedure left 27 unused channels out of the inversion, the data from which were then predicted based on the source estimates and compared to the real sensor data.", "For each of the ten cross-validation folds (each time a random 10% of channels were selected to be left out), the remaining (245 channels of) data were temporally decomposed into 16 orthogonal temporal modes and inverted onto each surface from the library of individualised and distorted cortical meshes (WFS) for each subject.", "Each inversion provided 10 cross-validation scores (the prediction of the hidden channels) which were then converted to a percentage of source data explained (more positive = better model fit) and averaged across the 10 folds and across the four state datasets inverted per subject.", "Both cross validation and Free energy provide metrics which can be used to directly compare different models of the same data, with different independent methods of preventing over-fitting (Bonaiuto et al., 2018; Henson et al., 2009).", "Whilst the Free energy provides a useful relative model fit metric for any given dataset, the absolute value is data dependent and therefore cannot be used to compare between datasets.", "In contrast, the cross validation gives a meaningful quantification of the total amount of data explained and can be used to compare across the different groups (e.g. head-cast versus non head-cast).", "After converting cross validation error to the percentage of total data explained by the model, both the CV % data explained and the Free energy increase with improving model fit.", "Here we performed these inversions using our subject specific libraries of anatomically degraded meshes from the WFS with different levels of distortion and compared them to an inversion performed using the real mesh for each subject.", "We constrained the source estimates to lie on the cortical surface and normal to that surface (Phillips et al., 2002).", "In order to facilitate comparisons of how the anatomical distortion affected the inversions for different subjects with different baseline measures of model fit to their real brain meshes, we normalised the cross validation percentage data explained and Free energy metrics by subtracting the values for the real mesh to derive a relative measure (ΔCV and ΔF).", "As such, a worse fit gives a negative value of ΔCV (and ΔF) and we would predict that as the anatomical complexity of the mesh increases (mesh is less distorted) and approaches that of the real mesh – the quality of the model should improve and the ΔCV and ΔF values should approach zero.", "Across our group of subjects, we determined the level of distortion (harmonic) at which this first becomes statistically distinguishable from the real mesh using a t-test of ΔCV values for each harmonic compared to zero.", "Similarly for ΔF, we use Bayesian model comparison to find out how much distortion is needed to make two meshes distinct.", "For example, using algorithm A we may find that inversions are not much worse (ΔCV and ΔF not significantly different from zero at the group level) using very distorted meshes all the way down to quite a smooth mesh harmonic (e.g. <10) i.e. the inversion algorithm is very insensitive to changes in the mesh shape.", "Alternatively, using algorithm B, we find that even with very subtle distortions of the mesh, that the inversion fit deteriorates substantially, so down at just harmonic 40 (which looks to the naked eye to be very similar to real mesh) the ΔCV and ΔF values are significantly below zero at the group level.", "This point (10 and 40 in this example), labelled the highest distinguishable harmonic (HDH), identifies the minimum amount of mesh distortion that can be reliably distinguished from the real mesh by inversion and can be converted into a conventional spatial metric (mms) by comparing vertex distances.", "The three dimensional Euclidean distance between every vertex from the HDH and the corresponding vertex on the true mesh was therefore calculated to give a distribution of 33,000 distances between the real mesh and the first mesh that can be distinguished from it statistically by inversion.", "We then took the upper, 95th percentile as an estimate of the distance between the two meshes.", "In addition to using the 95th percentile rather than the mean, this method is more conservative than that previously employed (Stevenson et al., 2014), as this directly matches corresponding vertices and therefore reduces the underestimation that could result if, following harmonic distortion, a vertex now lies closer to a non-corresponding other vertex.", "This distance therefore represents an upper bound on the spatial discriminability of both head-cast and non-head-cast resting state data.", "In order to verify our findings, we performed a number of control analyses.", "Firstly, we used the same data but destroyed its correspondence with the MEG sensor locations by randomly shuffling the MEG channels labels and repeated the analysis above 10 times for each subject and averaged over the ΔCV for these different shuffled dataset inversions.", "It is worth noting that sensor shuffling also removes all the (real) spatial correlations between sensors, however since they are not independent, they are not perfectly exchangeable and thus we also performed a secondary control analysis.", "For this, we used the correct sensor labels but instead degraded our data by introducing different amounts of uncorrelated scaled white noise at the sensor level to change the signal-to-noise ratio of the sensor level data (5 dB to - 20 dB) (Troebinger et al., 2014a).", "In both cases (sensor shuffling and noise addition), one would expect the ability to discriminate the true generative model from distorted ones to decrease.", "In order to determine the sensitivity of the inversion to the level of detail in the underlying anatomical mesh model we calculated the ΔCV and ΔF for each subject in the head-cast dataset across their subject-specific library of distorted meshes (Fig. 2).", "This showed increasing cross validation sensor data explained (ΔCV; reduced error) and increasing Free energy (ΔF) for all subjects.", "Statistical group level testing revealed that meshes lower (more deformed) than the 35 harmonic could be distinguished from the real mesh by ΔCV (HDH 35; t11 = −2.49, p = 0.03) and lower than 31 by ΔF (BMC, exceedance p = 0.046; Fig. 2).", "Having confirmed our results using the two metrics – we proceeded with the ΔCV metric which can be compared across subjects and recording methods.", "The effect of source co-variance prior assumptions was then assessed by repeating the process for three other commonly implemented inversion algorithms (MMN, LOR, MSP) on our head-cast dataset.", "These showed lower anatomical mesh discriminability for all alternative algorithms with an HDH for MMN of 25 (t11 = −1.80 -, p = 0.036), an HDH of 25 (t11 = −1.79; p = 0.039) for LOR and an HDH of 17 for MSP (t11 = −2.55; p = 0.027) (Fig. 3A).", "The mean distance between vertices on these meshes and corresponding vertices on the real (MRI-extracted) mesh was then calculated for each subject.", "These were then averaged to give a spatial measure of anatomical discriminability (Fig. 3A), under the different prior covariance assumptions as implemented in the different inversion algorithms.", "This ranged from 3.7 mm for EBB to 6.0 mm for MNN/LOR and 9.4 mm for MSP (Fig. 3B).", "Directly comparing the amount of data explained through cross validation across inversion conditions (using the real mesh) demonstrated a significant difference between EBB and the other 3 algorithms (EBB/MMN; paired t-test – t11 = 7.6,p < 0.001; EBB/LOR; paired t-test – t11 = 7.6,p < 0.001; EBB/MSP; paired t-test – t11 = 14.4,p < 0.001).", "A 2-factor within-subject ANOVA of % cross validation data explained with factors – inversion type (EBB, MMN, MSP) and mesh smoothness (harmonic 1 and harmonic 50) showed a significant interaction between inversion type and mesh harmonic level (F2 = 29.9, p < 0.0001).", "Post hoc examination showed that this was driven by a stronger effect of mesh distortion on the MSP inversion algorithm than EBB or MMN.", "The EBB algorithm was therefore taken forwards for a comparison of head-cast versus non-head-cast datasets (Fig. 4).", "We found that the HDH was higher for the head-cast recorded dataset at 35 (3.7 mm; t11 = −2.49, p = 0.03) than for the non-head-cast related dataset at 29 (5.0 mm, t11 = −2.70, p = 0.021).", "Furthermore, the absolute (non-normalised) amount of data explained through cross validation (CV) was higher in the head-cast (83.2 ± 0.71%) than in the conventional recordings (79.7 ± 1.79%) although this difference was not significant (mixed ANOVA; main effect of hcMEG versus cMEG, F = 3.06; p = 0.09).", "Finally, we checked our analyses by repeating our inversions (Head-cast dataset, EBB algorithm) but after degrading the consistent relationship between our sensor positions and sources by shuffling the sensor labels.", "As expected, this resulted in a breakdown of the previously shown relationship between cross validation and Free energy with mesh distortion.", "Notably we found that lower harmonics (smoother) meshes now showed higher ΔCV (Fig. 5A) (smoother surfaces superior when sensors shuffled).", "Thereafter we degraded our data (without sensor shuffling) by the addition of varying levels of Gaussian white noise (5 to −20 dB) and again repeated our analysis (Fig. 5B).", "This demonstrated that with increasing levels of noise (decreasing SNR), the curve describing the relationship between cross validation and harmonic mesh function flattened and the crossing point (HDH) reduced, indicating that the inversion was no longer able to statistically distinguish the more complex meshes from the real mesh, as would be expected if the data are primarily noise.", "Resting state data is dynamic, emulates task induced network changes, is predictive of behaviour and is simple to acquire in both control and patient populations (Baker et al., 2014; Larson-Prior et al., 2013; Niso et al., 2016; O'Neill et al., 2017; Philippi et al., 2015; Taylor et al., 2017).", "Here we show that it can provide a ready substrate for principled testing of MEG recording methods and inversion assumptions including anatomical forward modelling and functional (co-variance) priors.", "We showed that in moving the cortical surface from a heavily distorted version to the true anatomy, a significant and monotonic improvement occurred in model fit.", "This improvement saturated for some inversion schemes before others, with the beamformer based algorithms (closely followed by Minimum norm) continuing to improve up until the cortical surface deviated by, on average, <4 mm from the ground-truth.", "Critically, rather than compare methods through simulation or a limited task set (with ground truth from another modality), we have presented a method to optimise MEG forward and inverse models whilst minimizing selection bias and based on a plentiful supply of non-invasive human data.", "The HMM constrained the source estimates to select out self-similar and stationary epochs – under the assumption that this would also lead to a clearer picture of the underlying source distributions (essentially segmenting multiple overlapping source distributions into different temporal parcels and therefore simplifying the source reconstruction problem).", "We tested whether this step was justified by comparing the resolution estimates from the HMM parcelled data with arbitrary mixtures of parcels (supplementary Fig. S1).", "We found that the HMM approach demonstrated a significantly higher resolution in each subject compared to the non-HMM dataset (t11 = 3.18, p < 0.009).", "An alternative scheme, avoiding both resting state and the HMM, would be to define stationary periods of time around a common battery of tasks/stimuli to be used (but first agreed upon) universally; however, the concern would be that the ensuing cortical responses might still only occupy a small portion of the possible spatio-temporal solution space.", "We then compared algorithms in two ways: by the model fit (or amount of data predicted), and by the sensitivity of each algorithm to the true anatomy.", "These two tests need not necessarily have been in accord.", "For example, had we used a bunny-shaped blancmange mould instead of a cortical surface, we would still have been able to rank the algorithms based on the amount of data predicted; but we would not have expected any monotonic improvement as features were added to the bunny.", "A related control analysis (Fig. 5) is that when we used the same data but with shuffled lead-fields (destroying the link between the sensors and the anatomy) the amount of data we are able to predict actually decreases as the cortical model approaches the truth.", "It is therefore striking that the models that benefitted most from the true cortical manifold were also those that predicted the most data.", "This adds anatomical validity, confirming that the data being described is indeed generated by pyramidal cell populations normal to the cortical surface.", "Furthermore however, it also allows us to quantify algorithm performance in millimetres without being restricted to or dependent on using a particular processing framework (e.g. SPM) or cost function (e.g Free energy or cross validation) (Stevenson et al., 2014).", "Across anatomy (Fig. 2) and inversion assumptions (Fig. 3), the parametric (Free energy from empirical Bayes) and non-parametric (cross-validation) metrics of model fit were in accordance.", "This helps build confidence in the parametric Free energy metric which is considerably faster, makes use of all the available data, and has a direct probabilistic interpretation (i.e. how much more likely is one model than another?).", "The Bayesian formalism is however predicated on comparing how different models explain the same data; the use of cross-validation, which provides an absolute quantitative measure of data predicted, also allowed us to compare between different datasets (head-cast versus non-head-cast).", "We confirm here earlier reports that within-session head movements are reduced for head-cast versus non-head-cast MEG (Bonaiuto et al., 2017; Liuzzi et al., 2017; Meyer et al., 2017a; 2017b).", "We were surprised that the head-cast and non-head-cast data were statistically indistinguishable in terms of spatial resolution (mixed ANOVA; main effect of hcMEG versus cMEG, F = 3.06; p = 0.09).", "However, we believe that the comparison was particularly conservative in that the non-headcast group showed very little average head movement (3 mm), comparable to the average mesh vertex spacing (1.7 mm).", "Another limitation could be that the models we are using do not sufficiently capture the physics or physiology of the generators of the measured magnetic fields; and that the resolution is constrained by the models and not the recording.", "This could include for example, un-modelled noise sources such as the heartbeat, eye-blinks and other sources of noise.", "Although the HMM states we used were visually inspected to avoid common artefacts such as eye-blinks, it is possible that some of the modelling deficiencies come from failure to explain data that does not arise from the cortex (e.g. heart-beats).", "Finally, the head-cast and non-head-cast cohorts did not overlap and a more sensitive analysis would have been to perform a within-subject comparison.", "Here we used individualised meshes with current flow constrained to be normal to the cortical surface.", "These normal constraints, based on the MRI surface extraction, will themselves have some noise (which will be attenuated by smoothing in the lower harmonic orders) and could be one of the limiting factors in our spatial resolution estimates.", "One further consideration was that our estimates of resolution might have been confounded by absolute levels of signal power.", "We found no evidence for this in any frequency band (supplementary Fig. S2).", "We did, however, find that our subject group was divided into subjects showing a dominant theta/delta peak and those showing a peak in the alpha range (supplementary Fig. S2).", "We found that consistent with previous (MEG within-subject) findings (Stevenson et al., 2014), we achieved a greater sensitivity to spatial distortion in the subjects with a peak in a higher frequency range (p < 0.002).", "This is consistent with invasive observations that higher frequency oscillations tend to have smaller spatial coherence domains (Leopold et al., 2003).", "We should note however that the theta cycle is close to our chosen window length (200 ms) and this could be another reason for the lower spatial resolution estimates in those subjects dominated by lower frequencies.", "We found that the Multiple Sparse Priors algorithm had the least dependence on the true anatomy and also explained the least data.", "We should note however that the MSP-based analyses implemented here were generic and constructed from a limited set of 512 patches (or priors) placed at approximately evenly spaced vertices.", "The MSP algorithm, although perhaps the most elegant and comprehensive method we tested, is also computationally disadvantaged by the need to search over a large space of possible patch/prior combinations and the inherent pitfalls of local extrema in this optimization.", "A more robust way to implement this algorithm would have been to select the best model from many random patch choices (Troebinger et al., 2014b).", "Moreover, the relationship between sparsity assumed by the algorithm, and resting state networks used here for analysis, is beyond the scope of this study.", "We should note that the implementations of all the algorithms we tested were generic and not individually optimized.", "For example, had we used an optimized MNE framework (Gramfort et al., 2014) then no doubt its relative sensitivity would have improved.", "Here we focus on developing the benchmark and showing its application, rather than an exhaustive comparison of algorithms.", "This study was analysed using broadband (1–90 Hz) resting state data.", "Therefore, whether a similar level of spatial discriminability at the mm scale can be demonstrated when more selective data is used (e.g. frequency filtered or spatially restricted) remains to be shown.", "For example, future work might test different frequency bands (eg <30 Hz, >30 Hz) against different anatomy for example infra/supra granular cortical surfaces (Arnal and Giraud, 2012; Bastos et al., 2012; Bonaiuto et al., 2018, 2017).", "Overall, we have here demonstrated and validated a robust and unbiased method for the comparison of inversion and MEG recording methods that can in future be used to evaluate and optimise future techniques." ]

[ "Resting state data provides an unbiased dataset for comparing MEG source estimates.", "We use a hidden Markov model to break the data into stationary segments.", "We reconstruct these data onto a range of distorted cortical surfaces.", "The sensitivity of any algorithm to spatial distortion is quantified in millimetres." ]

[ "MULT" ]

[ "Building energy performance", "Energy efficiency", "Hedonic price", "Mass appraisal", "Real estate market" ]

[ "1000" ]

[ "The dataset features are as follows: 1,042 records, namely single unique properties, and 16 fields, namely property characteristics or other variables.", "Table 1 provides an insight on the measurement scales of the variables, as well as their coding system.", "The measurement scales have been defined according to the theoretical framework postulated by Stevens [7], who defined the following four fundamental scales: nominal (qualitative scale characterized by unordered categories); ordinal (expressing qualitative measures by means of a ranking); interval (expressing quantitative measures by means of successive values); ratio (expressing quantitative measures with a true zero point).", "Within the dataset, coding systems adopted for the variables based upon nominal and ordinal scales does not rely on a preconceived scheme, instead they are driven by the information usually available in residential property advertisements.", "The city of Padua locates in Northern Italy and has been chosen for the survey because of its dynamic real estate market.", "According to cadastral statistics [8], in 2013 the city was accounted for a large building stock with a total of 174,250 real estate units, mainly composed by dwellings (114,702 units corresponding to 65.8% of total), manufacturing buildings (12,654 units; 7.3%), retail stores (9,077 units; 5.2%) and offices (6,486 units; 3.7%).", "The dynamism of the local real estate sector is further witnessed by the amount of transactions recorded in the national observatory on the property market [9].", "Padua has been accounted for 1,618 transactions of residential properties in 2013, namely 1.4% of housing stock.", "Although the ratio was considerably higher just before the 2008 crisis (3,271 dwelling transactions in 2007, 2.9% of housing stock), and in the meanwhile a turning point has been observed in the price trend, the empirical evidence still confirms it may be included among well-performing markets [10–12].", "The maintenance of a real estate market monitor is among the commitments undertaken by the Italian Revenue Agency.", "This monitor provides a full coverage of the national territory, by dividing it into homogeneous zones.", "The zones are identified by means of codes composed by a progressive number following a capital letter.", "In turn, the letters are assigned according to five kinds of locations: B stands for downtown areas; C for inner-ring areas; D for outer-ring areas; E for the outskirts and R for the rural areas.", "Usually, C, D, and E zones encompass both residential and industrial settlements.", "The real estate market monitor divides Padua into 22 zones, as represented in Fig. 1.", "The perimeter of the zones is also provided in the Supplementary materials within this article as a separate kmz file, which can be read with the Google Earth application.", "Owing to the growing interest in building energy efficiency, several regulations have been enacted in Italy since the mid-seventies.", "When the Law 373/1976 entered into force, it constituted the first attempt to impose constraints on the building energy consumptions.", "Subsequently, a major enhancement to deal with the energy saving issue was represented by the Law 10/1991.", "Most recently, trying to meet the increasing consumers׳ demand for market transparency, the Decree-Law 63/2013 – implementing the European Directive 2010/31/EU – has introduced the legal obligation to include energy-related information within real estate advertisements.", "The data which are to be exhibited refer to the global energy performance index and the consequent energy label, as they are reported in the mandatory energy performance certificate [13].", "Nonetheless, during the first time span in which the new regulation was in force, real estate advertisements were allowed to omit the value of global energy performance index.", "This is the reason why the dataset mainly focuses on the energy labels.", "The Italian coding system of energy label relies on eight classes, from A+, which is the best one, to G, the worst.", "Each class correlates to the energy performance index, depending on the climate zone.", "The city of Padua is characterized by a yearly amount of Heating Degree Days (HDD, see European standard EN ISO 15927-6) equal to 2,383.", "Therefore, according to the classification of the national territory (Presidential Decree 412/1993), it falls within the E climatic zone (HDD within the range between 2,100 and 3,000).", "For this zone, Table 2 shows the energy performance index thresholds, for winter heating, corresponding to the aforementioned energy labels.", "During the survey, no cases of dwellings classified as A+ were found, hence they are missing in the dataset.", "The data provided here are useful to investigate the relationship between the energy performance of housing and the expected selling price by owners.", "Such a relationship is predicted to be positive by the literature; nevertheless, its functional form – linear or not – and magnitude are still debated.", "Moreover, georeferenced data allow to analyze the potential spatial patterns of both real estate prices and building energy performances [16].", "The data have been gathered within a research branch devoted to exploring the relationships among building energy performances, the prices in the real estate market, the costs in the construction sector and the provision of affordable dwellings [17,18].", "The survey was conducted during the time span from 2013 April to July.", "A total of 1,042 property advertisements have been accessed, filtering those published on dedicated websites by both private sellers and real estate agents.", "Only residential properties have been considered.", "Main real estate advertisement-posting websites have been inspected daily during the survey period.", "They have been cross-checked to avoid considering the duplicate items.", "When an old advertisement has been found to have been posted again without changes, only initial data have been kept.", "On the contrary, reissued advertisements with changes led to discarding the prior ones, hence oldest data have been replaced by the newest.", "Advertisements are usually concise; moreover, they may contain different kinds of information and make use or not of supporting materials, such as pictures of interiors and exteriors, or floor plans.", "The sample here provided includes only records characterized by complete information with regard to the previously mentioned variables (see Table 1).", "On the contrary, advertisements with missing data have been discarded.", "Based on the location declared in the advertisements, data have been georeferenced but they are characterized by heterogeneous levels of accuracy.", "The sample implements the exact geographical position for dwellings whose advertisement contained the full address.", "The dwellings whose advertisement provided the street name, but not the building number, have been located by referring to the midpoint of the self-same street.", "Otherwise, advertisements providing only succinct information about the location have entailed the need to refer to the midpoint of neighborhoods." ]

[]

[ "ARTS", "NEUR", "PSYC", "SOCI" ]

[ "Eye movements", "Eye tracking", "Face perception", "Social interaction", "Social status" ]

[ "1203", "2805", "3204", "3205", "3310" ]

[ "You look across the card table, into the eyes of your opponent.", "Are you searching their eyes for flickers of information, deciding whether to call their bluff?", "Or by returning their stare are you letting them know that you have nothing to fear?", "In social interactions, people use their eyes to perceive information and also to signal their intentions.", "Yet, the dual functions of gaze have been studied by and large separately.", "While vision psychologists have focused on the information that is present and attended to in a face, social psychologists have focused on how eye contact structures social interaction.", "But, in any one situation of face-to-face contact, or in any one experiment on face perception, we cannot be certain whether gaze serves to encode information or to signal intentions.", "We aim to understand when and how gaze is used for either function by varying the social context in which faces are viewed.", "Research in vision science shows that there is plenty of useful information to perceive in the eyes of another person.", "The eyes are linked to many psychological processes, and so are extensively studied by researchers (Buswell, 1935; Just & Carpenter, 1976).", "They are a remarkably useful source of information during social interaction (Argyle & Cook, 1976; Emery, 2000; Foulsham, Cheng, Tracy, Henrich, & Kingstone, 2010; Kleinke, 1986).", "Following the gaze of another person, for instance, is an important requirement for social learning (Tomasello, Carpenter, Call, Behne, & Moll, 2005).", "Gaze attracts attention from very early ages (Batki, Baron-Cohen, Wheelwright, Connellan, & Ahluwalia, 2000), and learning to interpret what another person thinks and feels by looking at the eyes appears to be crucial to many aspects of social cognition (Charman et al., 2001; Senju & Csibra, 2008).", "Because people are looking at the eyes of each other to seek information, gaze is also a powerful tool for signaling information to onlookers.", "Humans are remarkably sensitive to changes in where others are looking (Gibson & Pick, 1963).", "Indeed, there is evidence that the bright white human sclera has been selected so that group members can perceive each other’s eye movements (Kobayashi & Kohshima, 1997).", "Intentions, desires, obedience and dominance can all be signaled by the eyes.", "For example, gaze can be strategically used to cue an observer’s attention (Kuhn, Tatler, & Cole, 2009), and prolonged eye contact can indicate the intention to deceive others (Mann et al., 2013), social interest (Staas & Willis, 1967), physical attraction (Mason, Tatkow, & Macrae, 2005), and nonverbal dominance (Dovidio & Ellyson, 1982).", "Thus, the eyes can both seek and signal information.", "Previous research has not yet fully dissociated the dual function of social gaze.", "For example, while studies conducted in laboratories found that people tend to look predominantly to targets’ eyes (e.g., Foulsham et al., 2010; Smith & Mital, 2013; Vo, Smith, Mital, & Henderson, 2012), studies conducted in real life situations found that people tend to avoid direct eye contact with targets (e.g., Gallup et al., 2012; Laidlaw, Foulsham, Kuhn, & Kingstone, 2011).", "Indeed, gazing behavior in real life is influenced by the potential for social interactions (Laidlaw et al., 2011), joint attention (Gallup et al., 2012), and social norms (Wu, Bischof, & Kingstone, 2013).", "Such critically relevant social information are often absent when examining eye movements in laboratory settings.", "In fact, it is possible that presenting still images or video clips activates predominantly the observational function of gazing.", "In contrast, in real life interactions gazing likely represents a mixture of both observational and signaling functions.", "Yet, interpreting eye movements from real life observations or comparing between live and prerecorded contexts is difficult.", "Differences in gazing might, at least partially, be explained by the fact that participants actually see different things across these situations.", "In the present research, we developed an experimental paradigm to dissociate the dual function of social gaze.", "We combined the socially relevant information present in real life interactions with the experimental control of the laboratory.", "Unlike previous experiments that contrasted eye movements during real life situations to recorded stimuli (e.g., Laidlaw et al., 2011), in our experiment, participants viewed exactly the same stimuli across conditions.", "We presented the same faces throughout, but varied across blocks participants’ beliefs about the social context.", "We decided to test this paradigm in the domain of social hierarchy, because as with most of the cognitive literature on social gaze, it remains unclear why and when eye movements between people of different social ranks change.", "In many ways, people actively communicate their social rank through dress and demeanor, so that observers can easily perceive it and adjust their behavior accordingly (e.g., Gobel & Kim, 2014; Kraus & Keltner, 2009).", "Indeed, staring into other’s eyes can be used as warning signal (Nichols & Champness, 1971), since prolonged eye contact is perceived as a sign of power (Dovidio & Ellyson, 1982).", "Yet, people also increase attention to higher ranked individuals to monitor their behaviors and learn from them.", "For example, the eyes of higher ranked individuals are looked at more when watching video recordings (Foulsham et al., 2010), and gaze cueing effects are greater for higher than lower ranked faces (Dalmaso, Pavan, Castelli, & Galfano, 2012).", "Therefore, it is possible that eye movements related to social rank could reflect the function of either signaling or perceiving social information, and previous studies have not distinguished these possibilities.", "In the present study, we presented participants with faces of higher and lower ranked targets in different viewing contexts.", "Sometimes participants thought that they were merely observing targets’ faces on the computer screen (one-way viewing), whereas other times participants thought that targets would later watch a video of them looking at targets’ faces (two-way viewing).", "If the primary function of gaze is to perceive information about a target’s social rank, then the viewing condition would not change eye movements, as the same stimuli would display the same visual information across conditions.", "Alternatively, if at least part of the function of gaze is to signal information about one’s own social rank, then viewing condition will interact with the social rank of faces, even when people are looking at faces that are video-recorded.", "We predicted that when looking at higher ranked targets, participants would look longer to their eyes when being unobserved compared to when targets could also see them, as to gain additional information without challenging targets’ superior rank (Emery, 2000).", "In contrast, when looking at lower ranked targets, following literature on rank communication in primates (e.g., De Waal, 1989), we expected that participants would look longer into their eyes when targets could also see them compared to when being unobserved, presumably to signal their own superior rank.", "Sixty students (45 females, Mage = 23.03, SDage = 3.12) took part in this study for €5 pay.", "Participants were French undergraduate and postgraduate students from Parisian universities.", "All participants had normal or corrected-to-normal vision.", "We employed a 2 (target rank: high or low) × 3 (viewing condition: baseline or one-way or two-way) mixed factor design, with the viewing condition as within-subjects factor.", "Participants sat approximately 65 cm in front of a 24″ LCD.", "A SMI 250 remote eye tracker was positioned at the base of the monitor and sampled gaze position 250 times a second, with an accuracy of approximately 0.5 degrees.", "We used eight 5 s long video-clips of Caucasian members of the general public (4 males, 4 females, Mage = 34.50, SDage = 12.12) as they looked directly into the camera with neutral facial expressions (selected from Richardson, Street, Clark, Lotto, & Rice, 2014).", "We overlaid video-clips with white masks so that only the inner parts of the face were visible.", "The video-clip resolution was 640 × 480 pixels, which when presented on screen subtended 34 × 26 degrees of visual angle.", "Before the experiment started, all participants completed a brief questionnaire about their personality and demographic background.", "We told them that we were studying how people form first impressions of each other, and that they would watch a series of video-clips of other participants who had taken part in the experiment previously.", "We informed participants that an eye tracker was measuring their pupil size, and that sometimes we would be video-recording their face.", "Participants watched three blocks of the same eight video-clips (Fig. 1).", "Video-clips were presented in a random order.", "Each trial started with a 1500 ms message announcing that the camera pointing at the participant’s face would soon start recording.", "There was a 3000 ms countdown to the start of the trial.", "Next, a fixation cross appeared for 1000 ms. The video-clip was presented for 5000 ms followed by a message for 1500 ms announcing that the recording was completed.", "The first block was a baseline condition in which participants watched the video-clips without explicit information about the targets.", "The experimenter switched the video camera on and told participants that he needed to make sure that all equipment was operating properly, but that their recordings would not be looked at.", "After the first block, participants were given information about the social rank of the targets.", "The second and third blocks were the one-way and two-way viewing conditions, with the order counterbalanced between participants.", "The only difference between the blocks was that participants received different explanations regarding the video-recording of their faces.", "In the two-way condition, the experimenter told participants that the targets being displayed on screen were scheduled to return to the lab in two weeks’ time, when they would watch the videos that we were filming of the participant.", "For the one-way condition, the experimenter told participants that their recordings would be archived in a drawer, and only watched if he needed to verify the pupil measurement given by the eye tracker.", "We experimentally controlled the social rank of the target faces between subjects.", "After the first block, we informed participants how the past participants had described themselves in the same questionnaire that the participants had filled in at the beginning of the experiment.", "Half of the participants read a description on screen suggesting that all targets occupied higher social ranks, for example, that they obtained postgraduate university degrees and occupied prestigious jobs.", "The other half of the participants read a description on screen suggesting that all targets occupied lower social ranks, for instance, that they failed to obtain postgraduate university degrees and occupied less prestigious jobs.", "After the second block, participants reported their impressions of targets to verify our manipulation.", "Participants rated targets’ social rank characteristics along five 8-point items (anchored: uneducated vs. educated, low status vs. high status, lower class vs. upper class, not prestigious occupation vs. prestigious occupation, inferior social status than the participant vs. superior social status than the participant).", "Our social rank manipulation was successful.", "Since the five manipulation check items were highly correlated (α = .92), we averaged their scores creating a perceived social status scale (M = 5.24, SD = 1.24).", "We submitted this scale to an independent samples t-test.", "Lower ranked targets were perceived as having significantly less status than higher ranked targets (M = 4.26, SD = 0.87, and M = 6.04, SD = 0.86 respectively), t(58) = −7.92, p < .001, d = 2.06.", "Fixation durations were detected by BeGaze 3.3 (SensoMotoric Instruments, Teltow, Germany).", "Overall, fixations to facial features did not differ between baseline (M = 3739 ms, SEM = 142 ms), one-way viewing (M = 3868 ms, SEM = 127 ms), and two-way viewing (M = 3827 ms, SEM = 133 ms) conditions, F(1.81, 106.93) = 2.45, p = .096, ηp2 = .04 (Greenhouse-Geisser corrections applied).", "Therefore we calculated the ratio of total fixation duration to the eye and mouth regions, following one of the most common measures used in the literature (e.g., Klin, Jones, Schultz, Volkmar, & Cohen, 2002).", "These two features are looked at most frequently (Kingstone, 2009; Yarbus, 1965), and differences in these ratios have been observed under different experimental conditions and between different participant populations (e.g., Fox, Mathews, Calder, & Yiend, 2007).", "We found that how participants looked at lower versus higher ranked targets depended on whether participants believed that targets would be looking back at them (Fig. 2).", "A 2 (target rank) × 2 (viewing condition) × 2 (block order) mixed-design ANOVA yielded a significant interaction between target rank and viewing condition, F(1, 56) = 5.21, p = .026, ηp2 = .085.", "Thus, we administered paired samples t-tests to compare gazing between one-way and two-way viewing conditions for each target rank separately.", "Results indicated that when viewing higher ranked targets, participants looked more into targets’ eyes during the one-way viewing (M = .93, SEM = .02) than the two-way viewing condition (M = .88, SEM = 0.02), t(32) = 2.13, p = .041.", "In contrast, when viewing lower ranked targets, participants looked somewhat more into targets’ eyes during the two-way viewing (M = .93, SEM = 0.02) than the one-way viewing condition (M = .90, SEM = 0.02), albeit this difference did not reach levels of statistical significance, t(26) = −1.26, p = .218.", "The effect of block order was marginally significant, F(1, 56) = 3.39, p = .071, ηp2 = .057, but did not interact with any other variables, ps > .28.", "Further analyses took account of any baseline differences in face perception between our participants.", "We computed the differences between one-way viewing and baseline conditions as well as two-way viewing and baseline conditions.", "Analyzing these difference scores also yielded significant results.", "Differences equated to approximately 324 ms increase in looking time on the eyes of lower ranked targets comparing the two-way viewing to baseline condition, and about 367 ms increase in looking time on the eyes of the higher ranked targets comparing the one-way viewing to baseline condition.", "Thus, participants increased direct gaze to the eyes by roughly one or two fixations.", "Finally, we found that neither participants’ gender nor subjective status affected the outcome variables.", "The way viewers look at faces of different rank changes with the belief that they too are being viewed.", "By manipulating this belief alone, we showed that gaze performs a dual function.", "It extracts information from the world and signals information back into the world.", "In our experiment, the social rank of the target face had different consequences for these two functions.", "Our central claim, that social context modulates viewers’ responses to social rank, rests upon the significant interaction between target rank and viewing condition.", "Additionally, we found that when looking at higher ranked targets, participants looked longer to the eyes in the one-way than two-way viewing condition.", "The opposite trend emerged when looking at lower ranked targets, although this difference was not significant.", "Our findings suggest that how social context impacts viewers’ responses to faces differs depending on whether the target holds a higher or lower rank.", "The results are consistent with previous research suggesting that monkeys (Shepherd, Deaner, & Platt, 2006) and humans (Dalmaso et al., 2012) are more attentive and responsive to higher than lower ranked individuals.", "Perhaps because higher ranked targets have a greater potential to influence others, and thus their actions and reactions matter more, participants in our study might have shown greater responsiveness to situational cues, when they were viewing higher ranked rather than lower ranked targets.", "Of course, in other situations, such as direct competition over resources, it might be advantageous to signal superior rank to lower ranked targets.", "Future studies should test how such social contexts impact gazing behavior in relation to social rank.", "Our findings advance a growing literature illustrating that the way we observe the world depends on the beliefs about our ability to interact with it (e.g., Faber & Jonas, 2013; Risko, Laidlaw, Freeth, Foulsham, & Kingstone, 2012).", "For example, people avoid engaging in eye contact when sitting in a waiting room with a live person, but not when viewing that person’s video recording (Laidlaw et al., 2011), and they avoid following the gaze of another person, when that person can see them (Gallup et al., 2012).", "Contrarily, social norms increase gaze contact in other social contexts such as sharing meals, so that when eating salad with another person compared to when eating salad alone, people look more up from their plate (Wu et al., 2013).", "In these previous studies, it cannot be ruled out that the differences in eye movements were, at least to some extent, due to differences in stimuli.", "But in our novel experimental paradigm, visual stimuli were identical across viewing conditions.", "By manipulating beliefs alone, we showed that social context can modulate gazing behavior.", "In order to dissociate the signaling and encoding function of social gaze, we chose to depict all target faces with direct gaze and manipulated participants’ beliefs about the viewing condition.", "In real life, people tend to shift their gaze.", "Indeed, previous research has found that gazing behavior to faces depends on degrees to which stimuli are both social and dynamic (e.g., Speer, Cook, McMahon, & Clark, 2007).", "Future studies therefore could complement our findings by manipulating targets’ rank in more naturalistic videos, where targets move gaze within different viewing contexts.", "We found that eye movements systematically changed when looking at higher and lower ranked targets depending on whether gaze was being used to perceive or to signal.", "In many other situations, however, we still do not know which function is driving gaze.", "Do people with autism spectrum condition look away from the eyes of others (Klin et al., 2002), because they do not seek any information from the eyes, or because they do not wish to signal social engagement?", "Do highly anxious people look more into the eyes of angry faces (Fox et al., 2007), so that they can scan for potentially negative feedback, or because they wish to signal that they are being attentive?", "Our findings highlight that a full understanding of face perception in different contexts needs to consider the dual function of social gaze." ]

[ "We manipulated beliefs about the social context of the viewing situation.", "Change in beliefs alone produced different looking patterns to target faces.", "We were able to dissociate signaling and perceiving functions of social gaze.", "Face perception needs to address the dual function of social gaze." ]

[ "MULT" ]

[ "Economic development", "Economic value", "Free cash flow", "Regional economy", "Regional performance" ]

[ "1000" ]

[ "This article is associated to a Microsoft Excel Worksheet as a supplementary material.", "The data contain time series of free cash flow values on the 85 Russian regions covering the period of 2006–2016.", "No transformation was applied, except for scaling (all in thousands of Russian rubles).", "Missing values (<0.1% of total quantity of levels) were mean-substituted.", "The data file has the “Model description” sheet containing the formalization of data retrieving procedure, the “0-FCF” sheet with the free cash flow indicator values calculated for each of the 85 regions through 2006–2016, sourced by eight factor-values sheets according to the methodology in use.", "Factor data were retrieved from the Federal Statistics Authority of Russia online repository (http://fedstat.ru), formatted by us to the uniform structure, length and sequence of observations (regions).", "Appropriate links to indicators’ webpages are provided on respective data sheets.", "Proper foundations of the free cash flow use and different approaches to its calculation regarding the enterprise level are described in Lehn and Poulsen [4], Richardson [5], and Opler and Titman [6].", "In line with the literature, we denote the free cash flow as the total worth of funds, generated by an economic system over a period, available to be withdrawn by all the types of capital providers “without harming a firm's ability to operate and to produce future cash flows” [7].", "Then, the regional free cash flow is the net worth of an increment to economic potential of enterprises localized within a territory of a given region.", "Economic potential in this case is treated as enterprises' ability to maintain sustainable growth or to increase individuals’ wealth directly (through shareholder value) or indirectly (through the increase of tax revenue or labor demand) [8].", "j stands for a year, j∈[2006; 2016].", "Since we are unable to reliably determine a proportion of cross-regional operations of an enterprise, we assume that all the operations are localized in a region of incorporation.", "Positive free cash flow indicates an increase in value, while the negative means value deterioration, as follows from the cash flow-based methodologies described in Oded and Allen [10].", "Yet, a negative free cash flow value in a specific period does not necessarily mean that a regional property complex is used inefficiently: capital expenditure undermines free cash flow directly but has a potentially positive lagged effect.", "On the other hand, permanently negative free cash flow together with positive operating profit can hardly be an anticipated characteristic of economic development of a region since income reinvestment rate is going to be 100% and above, meaning the increase of debt, thus limiting the possibility to increase quality of life and individuals’ wealth in the long-term perspective.", "The same remark is applicable to permanently positive values of free cash flow, if they are maintained by investment downturn.", "The dataset with supplementary factors – GDP per capita and private sector share in total capital expenditure broken down by regions – allows to verify standard economic growth models from the value point of view.", "Combining the indicator with the private sector share in 2016-total capital expenditure gives a proper look at the data possible use (Fig. 1).", "Thus, the data allow to reclassify regions according to their contribution to value creation and to detect cycles in individual and aggregate dynamics.", "Another possible use of the data is to trace a value growth pattern and to determine the development cycle of a region or of the whole economy (Fig. 2).", "The tracer-chart indicates moderate value creation in 2008–2012 followed by significant volatility afterwards in terms of absolute changes.", "Matching with GDP or GRP dynamics can give an alternative view on growth and development.", "Finally, the data can be decomposed into net operating profit and the net investment to estimate the two broad factors’ contribution to economic performance of a region, in response to Oguz and Knight proposition [11]." ]

[]

[ "BIOC" ]

[ "Glucose", "Islets", "Mitochondria", "Respirometry" ]

[ "1307", "1312" ]

[ "Pancreatic islets rely on mitochondrial respiration to secrete insulin [1], a critical function for maintaining metabolic homeostasis.", "A rise in extracellular glucose levels increases TCA cycle flux, mitochondrial respiration, and ATP synthesis in islet β-cells, which generates molecular signals stimulating and amplifying insulin secretion [2].", "Indeed, even at the level of the whole organism, mitochondrial respiratory function accounts for approximately 90% of total oxygen consumption, 80% of which is coupled to ATP synthesis [3].", "Therefore, measuring oxygen consumption represents the gold standard assessment of metabolic behavior, bioenergetic demand, and mitochondrial function for islet tissue.", "Furthermore, by using different compounds targeting mitochondria, one can determine respiration linked to mitochondrial ATP synthesis and the maximal capacity of cells oxidizing fuels through the electron transport chain [4].", "Islets and β-cells within an islet are known to show remarkable variability in glucose sensitivity [5–10], size [11,12], architecture [8], and cell composition [13] among other factors.", "A small group of “first responder” islets rapidly respond to glucose in vivo by releasing almost their entire load of insulin, while some islets remain dormant and remain unresponsive to glucose [7,14].", "Islet heterogeneity also seems to play a critical role in pathogenesis of metabolic disease, as distinct groups of islets are more susceptible to dysfunction in diabetes [13].", "In addition, one could predict that islet preparations with a low number of “responder islets” and a high number of “dormant islets” would be less likely to efficiently sustain insulin production after their transplantation into subjects with diabetes.", "This could be the case since a high proportion of islets do not survive transplantation, and a low number of responder islets would decrease the probability of providing sufficient insulin in response to glucose.", "Despite the essential role of mitochondrial respiration in insulin secretion [1,15], very little is known about heterogeneity in mitochondrial function among islets from a given population.", "Currently, it has not been possible to address this issue due to a lack of high-throughput methods capable of quantifying mitochondrial function of individual islets within a population.", "The current gold standard for islet respirometry is the XF24 islet capture plate [16].", "Despite being the most sensitive oxygen consumption methodology, the islet capture plate requires 50–80 islets per well, which is almost the entire islet population from one mouse when performed in triplicate.", "Furthermore, only 20 different conditions can be tested simultaneously.", "This experimental platform is not suitable for assessing large numbers of biological samples and lacks the sensitivity to detect respiration of individual islets from heterogeneous populations.", "Other more invasive methodologies can measure oxygen consumption in single islets [17] but require complex microfluidics chips for encapsulating islets after staining with a fluorescent dye.", "Single islet ATP measurements are possible [18] but require viral transduction of biosensors.", "These more invasive manipulations have not been developed for high-throughput measurements, potentially limit the detection of endogenous functional heterogeneity, and could preclude islet use for transplantation.", "Therefore, a rapid, non-invasive, real-time and high-throughput method is needed to quantify islet functional heterogeneity.", "In the present study, we established a non-invasive methodology that enables high-throughput measurement of oxygen consumption in large-sized individual islets from mice and humans.", "We have developed in collaboration with Agilent Technologies a specialized microplate for the XF instrument called the “spheroid plate”.", "This plate contains a perfusion insert in each well that allows stable media flow across islets during mixing, without perturbing islet function.", "The spheroid plate reduces the biological sample size required for mitochondrial respiratory analysis and maintains the functionality for bioenergetics measurements currently used with the XF24 islet capture plate.", "Our new approach allows a populational analysis of metabolic function that revealed heterogeneous glucose sensitivity in large individual islets from both humans and mice.", "We hope our methodology can accelerate islet research and help elucidate mechanisms underlying the metabolic heterogeneity in pancreatic islets.", "Collagenase P, d-glucose, l-leucine, l-glutamine, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and Antimycin A were purchased from Sigma Aldrich (St. Louis, MO).", "Fatty acid-free bovine serum albumin (BSA) was purchased from EMD Millipore (Billerica, MA).", "Fetal bovine serum (FBS) was obtained from Life Technologies (Carlsbad, CA).", "Oligomycin A was obtained from Calbiochem (San Diego, CA).", "Accutase was purchased from Thermo Fisher Scientific (Roskilde, Denmark).", "Seahorse XF96 spheroid microplates, Seahorse XF96 FluxPaks, Seahorse XF Calibrant Solution and Seahorse XF Base Medium Minimal DMEM were acquired from Agilent Technologies (Santa Clara, CA).", "The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.", "All animal care was in accordance with NIH guidelines and the University of California Los Angeles institutional animal care and use committee (Chancellor's Animal Research Committee, UCLA protocol #16-018).", "Mice used for islet isolations were housed at University of California Los Angeles Animal Resources Facility.", "Mice were housed at a maximum of 5 per cage and maintained on a 12 hr/12 hr light/dark cycle at 68–72 °F.", "Mice were provided with water and food ad libitum until the day of islet isolation.", "Human samples were collected from participants in a prospective single center IRB-approved protocol on total pancreatectomy and islet auto-transplant at the University of Minnesota.", "Informed consent was obtained from study participants for general study participation and for pancreatic tissue analyses.", "Islets were isolated from 11 to 16 week old male C57BL/6J mice (Jackson Labs, Bar Harbor, ME) via collagenase P injection into the bile duct, as previously described [16,19].", "Islets were cultured overnight at 37 °C 5% CO2 in islet media (11 mM glucose RPMI 1640 + 10% FBS + 100 U/mL penicillin, and 100 μg/mL streptomycin) prior to experimentation.", "Human islets were obtained from the University of Alberta Diabetes Institute Islet Core (Edmonton, Alberta, Canada) in collaboration with Dr. Patrick MacDonald.", "Islets were isolated from six non-diabetic deceased donors ages 18–71 with 60–95% purity, cultured 7–40 h post-isolation and shipped overnight at 4 °C in CMRL media (Gibco/ThermoFisher, Waltham, MA).", "Upon arrival, islets were further purified from exocrine pancreas and cellular debris by visual inspection and picking.", "Islets were cultured overnight in fresh CMRL media at 37 °C and 5% CO2 before measurement of mitochondrial respiration.", "Human islets were also obtained from the University of Minnesota Schulze Diabetes Institute (Minneapolis, MN, USA) in collaboration with Josh Wilhelm.", "Islets were isolated from living donors with pancreatitis undergoing total pancreatectomy with islet auto-transplantation (Patient 1: 57 years old, non-diabetic female, BMI = 25, HbA1c = 4.8%; Patient 2: 57 years old, non-diabetic male with hyperlipidemia, BMI = 23, HbA1C = 5.8%).", "Islets were shipped overnight (4 °C, with Cryopaks) in Transplant Media (CMRL supplemented with 2.5% human serum albumin, 25 mM HEPES and 20 μg/mL ciprofloxacin).", "Upon arrival, islets were further purified from exocrine pancreas and cellular debris by visual inspection and picking.", "Islets were then cultured for 1–2 h at 37 °C 5% CO2 in CMRL media supplemented with 10 mM niacinamide, 1% (v/v) insulin-transferrin-selenium, 16.7 μM ZnSO4, 5 mM sodium pyruvate, 1% (v/v) Glutamax, 25 mM HEPES, 10% (v/v) FBS, and 1% (v/v) pen/strep.", "Respirometry was conducted on the same day that the islets arrived at UCLA.", "Approximately 1–2% of the final isolated islet product from pancreatitis patients was used for experiments.", "Mouse or human islets (1–32 islets/well) were seeded into wells of a poly-d-lysine-coated (100 μg/mL) XF96 spheroid plate containing 100–175μL/well of warm assay medium (Seahorse XF base medium minimal DMEM, supplemented with 3 mM glucose and 0.1% FBS).", "Islet seeding was done by aspirating islets in a minimal volume of media (∼4–15 μL) and inserting the pipette tip into each well of the spheroid plate, using a Leica S6E microscope to orient the pipette tip directly over the central depression in the well.", "Islets fall out of the pipette tip by gravity and into the central detent of each well.", "Once seeded, islets in the plate were centrifuged at 450 rpm for 7 min with no centrifuge brake, then incubated for 1–2 h at 37 °C in a non-CO2 incubator.", "Mitochondrial respiration was measured using the Seahorse XF96 extracellular flux analyzer equipped with a spheroid plate-compatible thermal tray (Agilent Technologies, Santa Clara, CA).", "Basal respiration was first measured in 3 mM glucose media.", "Islets were then sequentially exposed to glucose (final concentration in well of 20 mM), Oligomycin A (3.5–4.5 μM final concentration), FCCP (1 μM final concentration) and Antimycin A (Ant A, 2.5 μM final concentration).", "To validate individual islet respirometry with the XF96 spheroid plate, individual mouse islets were exposed to either 20 mM glucose or glucose + amino acids (AA, 10 mM each of leucine and glutamine), followed by FCCP (final concentration of 1 μM) and Antimycin A (final concentration of 3 μM).", "For assays testing reproducibility, single islets were seeded in a spheroid plate as described above, and basal respiration in 3 mM glucose media was measured until steady state respiration was achieved.", "Islets were then transferred to different wells in a new spheroid plate and steady state basal respiration was measured again.", "For assessment of islet functional heterogeneity, basal respiration of individual islets was first measured in 3 mM glucose media.", "Islets were then acutely exposed to 20 mM glucose for approximately 60–90 min, followed by Antimycin A.", "Respirometry using the Seahorse XF24 was performed as previously described [16], with pooled islets (50–80 islets/well).", "For direct comparisons between the XF96 spheroid and XF24 islet capture plates, mouse islets from the same islet isolation or human islets from the same donor were used for experiments.", "For calculation of mouse and human islet bioenergetics parameters, average non-electron transport chain OCR values after injection of Antimycin A were subtracted from all OCR measurements.", "All parameters were normalized to basal respiration and reported as % of basal OCR.", "Glucose-stimulated respiration was calculated by dividing the first OCR measurement after injection of 20 mM glucose by the last basal OCR measurement and multiplying by 100.", "Proton leak was calculated by dividing the minimum OCR value after injection of Oligomycin A by the last basal OCR value and multiplying by 100.", "Maximal respiration was determined by dividing the first measurement after FCCP injection by the last basal OCR value and multiplying by 100.", "After oxygen consumption measurement, individual islets in the XF96 spheroid plate were imaged in brightfield (10× objective) with the Operetta high-throughput imaging system (Perkin Elmer, Waltham, MA).", "Islet images were analyzed in ImageJ by manually drawing a line around the perimeter of each islet with the “Polygon selection” drawing feature and selecting the “Measure” function under the “Analyze” tab.", "Human or mouse islets were transferred to a 15 mL conical tube and centrifuged at 1000 rpm for 2 min.", "The supernatant was aspirated, and islets were dissociated by adding accutase (1 mL accutase/1000 islets) and incubating for 10 min at 37 °C.", "After 10 min, islets were pipetted up and down gently to facilitate dissociation.", "Islet cells were transferred to a new tube, and the accutase was quenched with 15 mL of islet media.", "The islet cell suspension was strained with a 40 μm cell strainer (Fisher Scientific, Waltham, MA) and centrifuged at 1000 rpm for 5 min.", "The supernatant was aspirated, and the cell pellet was resuspended in 5 mL of islet media.", "Cells were counted and seeded at 6,000, 8,000 or 10,000 cells per well of a V-bottom 96 well plate (Thermo Fisher Scientific, Roskilde, Denmark).", "Plates were centrifuged at 1000 rpm for 5 min and incubated at 37 °C 5% CO2 for 48–72 h before experiments.", "Data were expressed as an individual islet OCR trace or as means ± standard error of the mean (SEM) or standard deviation of islets derived from 1 to 5 human donors or 1–4 independent mouse islet isolations.", "p-values were calculated by two-way ANOVA with Sidak's multiple comparisons test or linear regression.", "Statistical significance was set at p < 0.05.", "Oxygen consumption can be measured in islets using the XF24 islet capture plate; however, this method requires a significant number of islets per experimental replicate and is not high-throughput [16].", "As a consequence, current respiration measurements average large quantities of islets that may contain individual islets that display unique characteristics.", "The Seahorse XF96 extracellular analyzer has the potential to be a suitable platform to develop a high-throughput methodology for individual islet respiration.", "In contrast to the current XF24 method, the XF96 has sufficient sensitivity to detect respiration in a few thousand cells and has 96 wells instead of 24 wells.", "However, there are a few challenges that need to be addressed.", "The ideal 96 well plate format should: 1) confine the islet within the central region of the well and maintain its proximity to the oxygen sensor, 2) generate a transient small volume measurement chamber to increase signal to noise ratio, 3) protect the islet from oxygen probe-induced damage, and 4) allow free movement of islet cell culture media across the whole islet during media mixing.", "To achieve these goals, two major modifications to the current XF96 plate were introduced, and the new resulting plate was named the “spheroid plate” (Figure 1A).", "On the surface of the well, a small semispherical detent (1 mm in diameter and 0.1 mm depth) was added to confine islets within the central area of the well, aided by poly-d-lysine coating (Figure 1A–B).", "The small detent is located inside a larger circular and flat detent (3 mm in diameter and 0.25 mm depth) (Figure 1B), which holds the new insert (Figure 1A–B).", "The shape of the insert is specifically designed to generate a small volume measurement chamber (approximately 2 μL) below the oxygen probe and facilitates efficient media interchange with the rest of the well through eight narrow vents (Figure 1A).", "The distance between the vents and the well wall is 0.04 mm (Figure 1B), through which media moves laterally and vertically during oxygen probe-mediated mixing (Figure 1B, red arrows).", "The vents are narrow to limit significant oxygen exchange rates between the measurement chamber and the rest of the well when the oxygen probe is in measurement position (Figure 1B).", "Thus, the novel spatial configuration formed by the insert and the large detent is designed to decrease the volume of the chamber during oxygen tension measurements and to maintain the islet in the center of the well during mixing.", "Islets are seeded in each well of a spheroid plate by aspirating the desired number of islets in a low volume (∼4–15 μL) of media with a pipette and carefully placing the islets in the detent at the bottom of the well filled with 100–150 μL of pre-warmed Seahorse assay media.", "A microscope is used to situate the pipette tip directly above the central detent in the middle of the well.", "Once in position, the islets will fall out of the pipette tip and rest in the central detent within the measurement zone.", "Using mouse islets, we validated the capacity of the XF96 spheroid plate to accurately measure islet oxygen consumption (Supplementary Figure 1).", "Figure 1C shows six mouse islets and an individual mouse islet situated in the central detent of a well in the new XF96 spheroid plate after measuring respiration.", "In most cases, the islets remain intact and are not damaged by the oxygen probe during the mixing steps of a Seahorse assay (Figure 1C).", "Unlike the XF24 islet capture plate, the well structure of the spheroid plate obviates the mesh net required to trap islets in the XF24 well measurement chamber.", "This facilitates the islet loading procedure and removes a potential source of measurement variability.", "The XF24 islet capture plate is capable of measuring respiration in human islets [16], but the required large sample size and low throughput of this technique limit its potential as a tool for detecting islet heterogeneity in a population.", "To investigate whether the XF96 spheroid plate may be a suitable high-throughput platform for individual human islet respirometry, we directly compared mitochondrial respiration measurements with either the XF24 or the XF96 methods using the same human islet preparations.", "Using 70 human islets per well, the XF24 yielded stable OCR measurements and showed expected responses to stimulatory glucose, Oligomycin A, FCCP, and Antimycin A (Figure 2A).", "Based on the difference in well size between the XF24 and XF96 instruments, we initially tested 20 humans islets per well in the spheroid plate.", "The XF96 spheroid plate produced stable OCR traces similar to those of the XF24 (Figure 2B), indicating that the XF96 platform can perform human islet respirometry with less than half of the islets required with current XF24 technology.", "When comparing results from five human donors, glucose-stimulated respiration and proton leak were similar between the XF24 and XF96 methods (Figure 2C), but human islets in the spheroid plate showed a significantly higher maximal respiration in the presence of FCCP (Figure 2C) compared to same islets measured in the XF24.", "We then determined whether we could reduce the sample size required for bioenergetics assessment of mouse islets using the spheroid plate.", "To accomplish this, we performed respiratory analyses with 20 mouse islets in each XF96 well and directly compared them to the respiratory traces obtained using 70 mouse islets per well of the XF24 islet capture plate (Figure 2D).", "The XF96 spheroid plate produced very similar OCR traces compared to those generated with the XF24 (Figure 2E), revealing nearly identical glucose-stimulated respiration, proton leak and maximal respiration (Figure 2F).", "We also performed a titration in a wider range of islet number per well and found that the spheroid plate could detect respiration in a single islet (Supplementary Figure 2).", "The spheroid plate drastically reduced the sample size required for islet bioenergetics (Supplementary Figure 2), but characterization of islet heterogeneity requires the sensitivity to measure a population of individual islets.", "In addition, the absence of a clear linear response in OCR vs. islet number at the range between 1 and 6 islets/well suggested that respiration measurements were not possible in every individual islet.", "To this end, we investigated whether the spheroid plate had the sensitivity to consistently profile bioenergetics of individual islets from several different human donors and mouse pancreata, as well as the characteristics of the individual islets in which respiration was successfully detected.", "Because islets vary in size even in the same pancreas [11,12], we measured basal OCR in individual human (Figure 3A) and mouse (Figure 3B) islets of different sizes, with areas <20,000 μm2 (small islets, less than ∼170 μm diameter) to >50,000 μm2 (large islets, greater than ∼290 μm diameter).", "The spheroid plate detected basal respiration in single human islets of all sizes, but many of the islets with an area under 50,000 μm2 showed basal OCR values below background signal (Figure 3A).", "However, the majority of human islets with an area greater than 50,000 μm2 yielded positive basal respiratory values (Figure 3A).", "Similar to human islets, the spheroid plate detected OCR from single mouse islets with an area over 35,000 μm2 (diameter greater than ∼ 210 μm) (Figure 3B).", "Importantly, the detected differences in OCR among different-sized islets were not a consequence of islet displacement from the central measurement area of the well (Supplementary Figure 3).", "To further explore the capability of the spheroid plate to monitor changes in mitochondrial metabolism at the individual islet level, we subjected large-sized single human (Figure 3C) and mouse (Figure 3D) islets to 20 mM glucose, Oligomycin, FCCP, and Antimycin A. Remarkably, individual large-sized human islets yielded OCR traces similar to those produced by the spheroid plate with 20 islets per well in Figure 2.", "Glucose and FCCP stimulated respiration while Oligomycin and Antimycin A inhibited respiration (Figure 3C), enabling calculation of glucose sensitivity, proton leak and maximal respiration in individual human islets (Supplementary Figure 4).", "Importantly, the spheroid plate was able to detect nutrient- and mitochondrial drug-induced changes in respiration of large individual mouse islets (Figure 3D), allowing for population bioenergetics in islets from mice as well (Supplementary Figure 4).", "These results indicate that the XF96 spheroid plate has the sensitivity to consistently assess respiratory function in single mouse islets larger than 35,000 μm2 in area (∼210 μm diameter) and individual human islets larger than 50,000 μm2 area (∼290 μm diameter).", "Islets from the same pancreas show remarkable variability in glucose sensitivity [7,20]; however, little is known regarding the heterogeneity in glucose-stimulated mitochondrial respiration at the individual islet level.", "To assess variability in mitochondrial responses to glucose, we measured glucose-stimulated respiration in large individual human and mouse islets using the spheroid plate.", "Glucose stimulation revealed notable heterogeneity in respiration among both large-sized individual human (Figure 4A) and mouse (Figure 4C) islets.", "Large-sized individual human islets showed variable glucose sensitivity, with an average increase in respiration of roughly 150% of basal levels (Figure 4B).", "However, while some large-sized islets did not respond to glucose, one islet increased respiration over 250% of basal rates (Figure 4B).", "The majority of large-sized individual mouse islets increased oxygen consumption approximately 200% or less compared to basal in the presence of stimulatory glucose (Figure 4D).", "A few islets showed either no response to glucose or a very high glucose response reaching up to 600% of basal respiration (Figure 4D), indicating heterogeneity in glucose-activated mitochondrial activity among large-sized individual human and mouse islets.", "This spectrum of glucose sensitivity was also observed in glucose-stimulated insulin release measured from individual intact mouse islets in the spheroid plate (Supplementary Figure 5).", "Islets vary in size [11,12], cell composition [13], and architecture [8], which are important factors that determine islet function.", "In fact, islets exhibit an appreciable degree of heterogeneity in mitochondrial metabolism [5], an effect that may be due to disparity in islet size and/or cell composition.", "To ensure that the differences in glucose-stimulated respiration were not due to an assay artifact, we reconstituted islets in vitro to form pseudo, or “reaggregated islets.”", "This manipulation simultaneously normalizes the cell composition and controls the size among the formed islets, facilitating further interrogation of the sensitivity of the XF96 platform.", "Islets from the same pancreas were pooled, dispersed into single cells and allowed to reaggregate over the course of 48–72 h (Figure 5A).", "The spheroid plate detected respiration in a single reaggregated islet of 6000 cells (Supplementary Figure 6).", "The statistically significant linear correlation (R2 = 0.999) between the number of plated islet cells and OCR (Supplementary Figure 6) demonstrates that the XF96 spheroid plate can discriminate changes in basal respiration corresponding to an increase of 2000 cells in islet size.", "Notably, dispersion and reaggregation of both human and mouse islets reduced the variability in glucose-stimulated respiration compared to intact islets (Figure 5B–C and Supplementary Figure 6), suggesting that the heterogeneity in glucose-stimulated respiration among individual islets was not an artifact of the spheroid plate.", "Mitochondrial respiration is essential for pancreatic islet insulin release [1], a process that maintains blood glucose levels in response to nutrient consumption.", "The decline or absence of insulin release has dire metabolic consequences, including severe hyperglycemia and diabetes.", "However, these pathophysiological changes in islet secretory function do not occur to all islets uniformly [13].", "Surprisingly, little is known about the mechanisms underlying this functional heterogeneity at the level of an individual islet and whether this heterogeneity extends to mitochondrial respiratory capacity.", "This gap in knowledge may be explained by technical limitations, since it is currently not possible to measure mitochondrial function of single islets from a population in a high-throughput and non-invasive manner.", "In this study, we have developed a novel high-throughput method sensitive enough to detect mitochondrial function at the level of the large individual islet, facilitating populational characterization of islet heterogeneity in a non-invasive manner for the first time.", "Structural modifications made to the spheroid plate increase measurement sensitivity allowing for mitochondrial respiratory analysis of biological samples down to a large-sized intact single islet with an area >35,000 μm2 (diameter ∼210 μm) and a reaggregated islet of 6000 cells.", "This technological advance enabling metabolic profiling at the individual islet level has been utilized to show heterogeneous glucose sensitivity of mitochondrial respiratory function in both human and mouse islets.", "Through populational assessment of islet respiration, we hope our method may be used as a research tool to better understand islet function in health and disease.", "The new design of the XF96 spheroid plate significantly improves the capability of measuring islet respiratory function compared to the XF24 islet capture plate.", "The new spheroid microplate employs a well insert that creates a small enough chamber with adequate media flow to maintain islet function and measure oxygen consumption with the Seahorse XF96 analyzer.", "This insert prevents the requirement of placing a net on top of the well to retain islets within the measurement chamber, a manual and tedious step required for the XF24 method.", "The spheroid plate well modifications simultaneously reduce the number of islets required per well for accurate measurements, increase the throughput of islet bioenergetics and decrease the time required to obtain stable respiratory traces when compared to the XF24.", "Mouse and human islet preparations can now be subjected to more experimental conditions and test compounds to probe mitochondrial function.", "In fact, high-throughput platforms have already been described for drug screening in β-cell spheroids [21], for single cultured cells in sealed sub-nanoliter chambers [22] and in cancer cell spheroids with the spheroid plate [23], but not in islets.", "Ideally, high-throughput islet respirometry should also preserve functionality throughout the course of an experiment and be amenable to downstream analyses.", "The XF96 method improves the kinetics of OCR measurement compared to the XF24 and reduces the number of data points needed to achieve stable glucose-stimulated, Oligomycin-resistant and non-mitochondrial respiration in both human and mouse islets (Figure 2).", "Average assay times for the XF96 spheroid plate were roughly 180 min, compared to approximately 300 min for the XF24 islet capture plate.", "Shorter overall assay times increase the chances for functional islet assays after respirometry, reducing their time in culture.", "Despite this reduction in measurement times, the spheroid plate still shows similar capacity for bioenergetics measurements compared to the current XF24 gold standard method for bulk islet respirometry.", "Thus, we show that the XF96 spheroid plate allows high-throughput measurement of mitochondrial function in intact islets retaining the cellular architecture so critical for their physiological function [11,13].", "Until recently, most islet analyses were performed on larger batches of islets, masking the notable functional heterogeneity that has been uncovered by many groups [5–7,10,12,24].", "For the first time, our spheroid plate facilitates high-throughput mitochondrial respiration profiling of individual human and mouse islets.", "In fact, full mitochondrial stress test profiles can be generated with large single islets, yielding information on glucose sensitivity, ATP production and proton leak and maximal mitochondrial respiratory capacity for an islet population.", "Previous studies have introduced a microfluidic-based technology enabling detection of oxygen levels in encapsulated single islets using an oxygen sensitive dye [17].", "However, our novel spheroid plate-based technology is minimally invasive, does not require any dyes, and detects individual islet mitochondrial respiration in a high-throughput manner.", "Furthermore, mitochondrial stressors can be omitted to solely study glucose-induced respiration and collect the islets after the assay for other purposes (i.e. transplantation).", "Our new methodology revealed considerable heterogeneity in mitochondrial function among large individual islets from the same pancreas.", "Islets from a given pancreas greatly range in size [11] and are known to exhibit unique metabolic characteristics [5,10,25–27].", "Furthermore, even diversity in mitochondrial function within β-cells of a single islet is a well-documented phenomenon that can dictate cellular metabolism [1,27,28].", "However, very little is known about heterogeneity in mitochondrial metabolism at the individual islet level.", "In this study, we observed notable diversity in mitochondrial responses to acute glucose stimulation among large individual human and mouse islets (Figure 4).", "Some islets were very sensitive to a rise in glucose, while other islets showed little or no increase in mitochondrial oxygen consumption, supporting the notion of unique groups of islets with variable glucose sensing capabilities.", "In fact, these differences exist in vivo, as a small group of “first responder” islets rapidly respond to glucose by releasing almost their entire load of insulin, while most islets remain dormant [7].", "It is possible that the observed variability in glucose-stimulated OCR could be due in part to differences in islet cell composition [13], architecture [8], or central islet cell necrosis [29], since these parameters are critical for determining islet capacity for glucose response.", "Importantly, normalization of islet cellular composition and size through dispersion and reaggregation reduced the heterogeneity in glucose sensitivity observed among individual islets, suggesting that the unique glucose responses detected were not an artifact of the spheroid plate.", "However, further work is required to fully understand the factors that contribute to metabolic heterogeneity at the single islet level.", "High-throughput measurement of individual islet oxygen consumption highlighting functional heterogeneity may represent the first step for a critical biomedical breakthrough.", "This type of technology can potentially provide a novel parameter to be tested as a quality control measure of islets used for transplantation.", "Previous studies demonstrated a positive correlation between islet OCR and maintenance of islet function in patients after islet transplantation [30].", "Currently, islet transplantations allow type 1 diabetes patients to produce insulin independently for approximately 1–2 years, with diminishing success 2–5 years after transplantation [31,32].", "A major challenge in islet transplantations is that their success rate is hard to predict [33,34].", "Moreover, insulin secretory capacity measured in donated islets ex vivo does not correlate with maintenance of islet function and viability within the recipient [29,33,35].", "It is possible that islet preparations containing a low number of highly sensitive “responder islets” and a high number of “dormant islets” would be less likely to efficiently sustain insulin production after their transplantation, given that a moderate proportion of islets do not survive transplantation due to issues with engraftment, host immune response, and drugs administered post-transplant [35], among other factors.", "Yet, the current methods for islet quality control analyze pooled islets and would not be able to identify the islet heterogeneity in donor preparations.", "Despite the inherent challenges of islet transplantation, one could expect that transplanting a higher number of functional islets might prolong endogenous insulin production in diabetics.", "Thus, detection of single islet heterogeneity in a high-throughput and non-invasive manner may eventually make the spheroid plate a suitable option for assessing human islets before transplantation, considering the thousands of islets needed for a single successful transplant [33,36].", "To prove its utility for islet transplantation, further studies should be performed to test whether glucose-responsive islets selected by the spheroid plate improve clinical outcomes in rodents and patients after islet transfusions.", "In addition, there are other important caveats that need to be considered if the XF96 method should be adopted as a biomedical tool.", "The XF96 spheroid plate can measure large individual human and mouse islets, but it is not sensitive enough to yield respiratory rates above background levels for a mouse islet with an area <35,000 μm2 or a human islet with an area <50,000 μm2 (Figure 3).", "This presents a challenge, considering smaller islets can be functionally distinct from large islets [12] and are superior in islet transplantations [29].", "However, this challenge may be overcome by pooling the small islets in groups of ∼20 per well.", "Also, our methodology involves loading of islets one well at a time, which reduces the speed and efficiency of workflow.", "This procedural bottleneck may be overcome by automating the islet loading process prior to Seahorse respirometry.", "Finally, once islets with low or high glucose sensitivity are identified, they must be rapidly sorted for down-stream analyses or transplantation.", "While high-throughput islet sorting is currently possible [37], this technology would have to be integrated with the XF96 spheroid plate in order to maximize applicability of single islet respirometry for islet transplantation.", "Overall, the XF96 spheroid plate can be a valuable tool for islet research.", "The new spheroid plate has the sensitivity to detect respiration in larger individual pancreatic islets in a 96 well microplate format, reducing biological sample size and increasing measurement throughput compared to the XF24 islet capture plate [16].", "In addition, the new well insert produces a confined measurement zone which is advantageous not only for islets but for other spheroids in which adherence to the well bottom could pose a significant problem.", "Individual islet respirometry now permits separation of a heterogeneous islet population into glucose-responsive islets and non-responsive islets, facilitating the study of mechanisms underlying glucose sensitivity and enabling the identification of healthy islets for transplantation.", "Aside from glucose, the spheroid plate can also be used to detect heterogeneous islet function in response to other nutrients, small molecule therapeutics and genetic perturbations.", "Thus, our novel populational islet bioenergetics method may be ideal for uncovering mechanisms dictating heterogeneity in islet function and could be further developed into a quality control metric prior to islet transplantation into diabetic patients.", "ML, OSS, LS, and SS conceived this study.", "EPT, SS, ER, ML, and LS designed and performed the experiments.", "KC and AN designed the plate, helped design the experiments and contributed reagents.", "DMW helped with data analysis and built the islet dispersion and reaggregation scheme in Figure 5.", "MA, ZS, JW and PM contributed human islets and reagents.", "MB evaluated all patients undergoing total pancreatectomy with islet auto-transplantation before and after surgery/islet transplants and obtained consent from patients to use their islets for experimentation.", "EPT and ML wrote the manuscript.", "EPT, OS, ER, and ML revised and edited the manuscript." ]

[ "Islets from the same pancreas show remarkable variability in glucose sensitivity.", "Little is known about heterogeneity in mitochondrial respiration in individual islets.", "We developed a novel high-throughput method to measure individual islet function.", "Individual islets within a population show heterogeneous glucose-stimulated respiration.", "Our method may advance islet research by enabling populational metabolic profiling." ]

[ "ENER", "SOCI" ]

[ "Behaviour change", "Behaviour change wheel", "Energy use", "Intervention", "Review", "Workplace" ]

[ "2102", "2103", "2104", "2105", "3301" ]

[ "Non-domestic buildings currently account for around 18% of UK carbon emissions [1] and 20% globally [2]; figures which are set to increase in the future, making workplaces an important focus for energy efficiency and energy saving initiatives around the world.", "Various mechanisms exist to promote energy reductions in non-domestic settings, from voluntary agreements (VAs), adoption of Energy and Environmental Management Systems (EMS), energy audits, use of energy efficient technologies, and occupant engagement [3].", "In this paper we focus on the latter of these options, namely interventions in the workplace which aim to change employee behaviour and by doing so, save energy.", "Literature on this subject is currently sparse and is dispersed across a number of academic disciplines, thus findings need to be integrated to enable key learnings to be identified [3] and to allow appropriate and effective policy and practice to be put in place to promote energy saving in the non-domestic sector [4].", "This paper directly addresses this need by conducting a systematic review of empirical studies worldwide (though restricted to those published in English) to identify the interventions that are most successful at saving energy in the workplace; we also identify and discuss future research needs.", "Every effort has been made to include all studies published by the end of 2015, although we note that the field is expanding rapidly and some literature in this field is difficult to access and often not indexed (e.g. third sector reports).", "As with all reviews, those interested in the rich contextual details contained in the original studies are encouraged to consult those directly.", "Presented here is a high-level and systematic analysis which identifies the trends and gaps in currently available evidence on an international basis.", "Below we provide a brief review of energy conservation in domestic settings and pro-environmental behaviour more broadly, before turning to energy use in the workplace.", "We then describe the framework used to analyse the studies reviewed in this paper.", "Significant headway has been made in understanding energy use in a domestic setting.", "A review of interventions aimed at household energy conservation by social and environmental psychologists reveals that most focus on voluntary behaviour change, rather than changing contextual factors (for example the use of energy efficient equipment) [5].", "Interventions reviewed include the provision of information, feedback and rewards, all of which aim to change individuals’ knowledge and perceptions of energy saving activities.", "The review finds that information produces higher levels of knowledge but not necessarily changes in behaviour; that rewards are generally effective in generating energy savings but that these can be short-lived; and that feedback can also be effective, especially if given frequently.", "Related work suggests that whilst socio-demographic variables like income and household size effect energy use, psychological variables such as attitudes may be more important in determining changes in that use [6].", "A variety of factors are thus important in understanding domestic energy use behaviours, including contextual, socio-demographic and psychological factors; factors that may also be significant in non-domestic settings.", "Domestic and non-domestic settings vary however on a number of counts, creating very different contexts for behaviour change interventions [7].", "The cost of energy use in the workplace is of little relevance to most employees, whilst the sharing of facilities and appliances may create barriers to behaviour change [8].", "Some consider this to represent a ‘tragedy of the commons’ whereby management of a shared resource is delegated to a figure with only limited means of exercising control [9].", "Employees can however be a captive audience and are subject to organisational policies [10], whilst the influence of social and group norms [8] and sense of community [4] may increase motivations to save energy in the workplace [11].", "Common to all workplace settings is the relevance of group dynamics and interactions on energy saving [12], although variation in terms of size and sector also affect the potential for savings [3].", "The field of pro-environmental behaviour has much to offer our understanding of energy use in the workplace.", "Pro-environmental behaviours are behaviours which intentionally seek to reduce the negative impacts of a person’s activities on the natural and built world [13,14], such as reducing one’s use of energy.", "Disciplines including psychology and sociology in particular have sought to understand what drives, limits and sustains pro-environmental behaviour and whilst the aim of this paper is not to deal directly with their theories and models, a very brief and inevitably limited introduction to them is given here as they set the context for most of the empirical investigations that are reviewed.", "Based on assumptions of the ‘information deficit’ model, research in the 1970s suggested that knowledge of how to minimise environmental impacts will translate into pro-environmental behaviour [15].", "This rationalist notion may in some instances be true, but often people do not act in accordance with their values or attitudes, leading to the so-called attitude-behaviour, or value-action, gap [16].", "This gap has been attributed to differences between individuals, contextual influences, temporal discrepancies (i.e. that people’s attitudes change over time), as well as the methodologies used in the measurement of attitudes versus behaviour [17].", "Ajzen’s [18] Theory of Planned Behaviour (TPB) has been particularly influential in research into this gap, suggesting that behaviours are driven by intentions which themselves are driven by a combination of attitudes, subjective norms, and perceived behavioural control [14].", "Another highly influential model in pro-environmental behaviour is Stern’s [13] Value-Belief-Norm (VBN) model which asserts that values relate to an individual’s beliefs which in turn form intentions to act through norms.", "Others have linked pro-environmental behaviour to pro-social behaviour and create models focused on the importance of altruism and empathy [16].", "Psychological understandings of pro-environmental behaviour have frequently been expanded to also include individual, social and institutional constraints.", "Blake [19] for example identifies three barriers to pro-environmental behaviour: individuality (barriers lying within the person), responsibility (barriers from personal feelings that they cannot influence the situation), and practicality (social and institutional constraints).", "Having reviewed many of the major psychological and sociological models of pro-environmental behaviour, Kollmuss and Agyeman [16] conclude that such behaviour represents a complex interplay between multiple factors: demographic factors, external factors (including institutional, economic, and social and cultural factors), and internal factors (including motivation, environmental knowledge, values, attitudes, environmental awareness, environmental involvement, locus of control, and responsibility and priorities).", "Their review highlights that most models fail to deal with the formation of habits and desires for comfort and convenience.", "An increasingly popular approach to researching energy use behaviours pays specific attention to such issues, focusing on everyday social practices and referring not to energy use but rather to ‘practices-that-use-energy’, such as cooking, showering, or using air conditioning [20–22].", "Social practice theory pays attention to the social and collective organisation of practices, conceptualised as broad cultural entities which shape people’s perceptions, their interpretations and the way they act [23].", "Proponents of social practice theory suggest that this provides a more realistic perspective on behaviour change and that it highlights the problems faced in changing behaviours; problems which demand far more than the removal of contextual ‘barriers’ as they involve the organisation of everyday life [23].", "A review of studies in the workplace found that pro-environmental behaviours in this context depend on both individual determinants, e.g. attitudes and organisation-specific influences, e.g. management, however highlights that the interaction between individual and organisational factors is poorly understood [7].", "It is suggested that intervening to promote pro-environmental behaviour in the workplace should focus not only on physical facilitation, but also tailored persuasive communication, and the active engagement of middle management [7].", "A meta-analysis of studies to promote pro-environmental behaviours in the workplace finds that those most impactful interventions are those that involve cognitive dissonance (where differences between actions and pre-existing values or attitudes are highlighted), goal setting, social modelling (providing role models), and prompts (other types of treatment reviewed included ‘making it easy’ for employees, justifying the need for action, providing instructions, giving feedback, offering rewards, and seeking employee commitment) [24].", "With regards to energy use behaviours in the workplace, past research has considered retail, industrial and manufacturing environments [25–27], as well as office-type environments, the latter of which is the context for this paper and will be the focus below.", "Psychological factors found to be important in energy saving are similar to that within pro-environmental behaviour more broadly and include moral norms, pro-environmental attitudes, personal responsibility, and antecedent beliefs [28–32].", "This work additionally notes the importance of engaging employees through all stages of an energy-saving programme, including through participatory interventions which facilitate continual employee involvement [33,34].", "The role of office management and organisational decision-making are also important in creating opportunities to reduce energy use [35–37].", "Managers and the attitudes that they personally hold are central to creating opportunities to reduce energy use, and opportunities exist for human resource management to support the idea that managers are the gatekeepers to environmental performance.", "When it comes to tenanted buildings, building owners can play a role, with an ability to drive sustainability agendas through the requirements that they place on their tenants [38].", "Increased technical monitoring, modelling and measurement of energy within the workplace may also aid energy saving [39–41].", "Engaging building users with energy data can lead to new questions being asked about agency, responsibility and the relations between people and appliances and buildings that use energy, such that creative solutions may be found to reduce demand [42].", "In relation to this, innovative ways of providing feedback on energy use, including in particular visualisation tools have indicated great potential in achieving energy savings [43,44].", "The increasing complexity of both public and private sector workplaces as sites for energy efficiency interventions should not be overlooked, including with regards issues of privacy, trust, responsibility, control, organisational role, productivity, workloads, competing interests, staff relations, budgets, job losses and trade unions [45].", "Academic literature which directly assesses the energy savings of behaviour change interventions in an office-type workplace is relatively sparse.", "Such literature reports on interventions initiated by, or on behalf of, organisations and ranges from the use of computer-based games [46] to comparative feedback [47] holistic programmes involving information, prompts, competitions, peer-education and discussion [48].", "Some programmes combine these tactics with installing new technological features, such as energy efficient lighting and automated control systems [49].", "Trials in the workplace have also been conducted by academic researchers, involving the provision of individualised feedback on desk-based energy use [50] to the distribution of rewards, for example manipulating whether rewards provided are private or public, or monetary or social in nature [51].", "This literature is reviewed in-depth in this paper.", "Policy makers and practitioners are also interested in understanding energy use in the workplace and what can be done to reduce it.", "Bringing together numerous case-studies, a report into low carbon behaviours for the Scottish government found that the most successful behaviour change strategies, relating to energy in the workplace, employed technology and infrastructure upgrades [52].", "Similarly, a report by the UK’s Department of Energy & Climate Change [3] concludes that a combination of technology change, feedback to users, and norm activation tends to be the most successful strategy in delivering lasting changes in energy behaviours in the workplace.", "Such findings have relevance to policy makers working on energy issues worldwide.", "Individually, these pieces of research and reports provide rich and important insights into various aspects of behaviour change interventions to save energy in specific workplace contexts.", "To date however there has been no systematic or comprehensive review to draw together these insights, or to speak more broadly about energy saving interventions in office-type workplaces.", "In this paper, we specifically consider offices in public and private buildings and include universities and schools.", "The scope of the paper is determined by the types of workplace reported upon in the studies we review and the methods employed to engage building users.1", "This paper addresses this gap and adds value to existing studies by identifying the interventions that consistently emerge as successful at saving energy in the workplace.", "It thus synthesises current knowledge, but also extends it, by identifying gaps which remain as challenges for future research.", "This review brings together studies of employer-led behaviour change programmes as well as third sector and academic-led experiments and trials.", "These interventions are based on differing theoretical models and notions of behaviour change; however we do not judge the utility or validity of these, rather our assessment of intervention success is based directly on the energy savings generated i.e. the impact created.", "Whilst the paper is focused on interventions which aim to create changes in behaviour, changes in infrastructure and technology are frequently used in combination with these, thus we end up discussing both voluntary behaviour change and physical and contextual factors.", "In order to systematically analyse the interventions reviewed, we turn to the field of health-related behaviour change, which is arguably far more advanced than environment-related behaviour change.", "We adopt the Behaviour Change Wheel (BCW) as a framework [53,54] for characterising and evaluating the behaviour change interventions reviewed.", "Given the vast array of possible behaviour change interventions, it is important to develop an appropriate framework to assess the specific behaviour under consideration.", "A range of frameworks exist which characterise behaviour change interventions, however many of these are not comprehensive, taking the form of a checklist (e.g. MINDSPACE) [55] or taxonomy (e.g. EPOC’s taxonomy of interventions to change health professional behaviour) [56] rather than being developed and grounded in theory; they are also frequently focused on just one domain (e.g. EPOC) [56].", "One of the most rigorous and coherent frameworks to date has been the BCW.", "The BCW was based on a systematic review of 19 previous frameworks of behaviour change interventions, particularly from health-related research (see Fig. 1).", "It is based on a theoretical model of behaviour that aims to cross cut commonly used models of behaviour (e.g. the Theory of Planned Behaviour) [18] and encompass the important roles of other key conceptual variables highlighted as important within behaviour change literature, e.g. impulsivity, emotional processing [57].", "In this framework, the sources of behaviour are modelled into a ‘COM-B system’ (Capability, Opportunity, and Motivation leading to Behaviour) which theorises that an individual’s Capability, Opportunity and Motivation (described as Sources of Behaviour) interact to generate Behaviour.", "Capability refers to an individual’s psychological and physical capacity to engage in the behaviour concerned; Motivation refers to all those cognitive processes that energize and direct behaviour—not just goals and conscious decision-making but also automatic associations and priming; and Opportunity refers to all the factors that are external to the individual that make the behaviour possible.", "In addition, within Capability, Opportunity and Motivation, there are subdivisions which reflect important distinctions recorded within the literature.", "For example, Opportunity is subdivided into physical and social opportunities; physical Opportunity referring to environmental structures around us and social Opportunity referring to the culture that dictates the way we consider the behaviour (see the centre of the BCW in Fig. 1).", "Around the central hub are nine ‘Intervention Functions’ that describe key features of behavioural interventions aimed at addressing deficits in one or more of the Sources of Behaviour (i.e. Capability, Opportunity or Motivation): Education, Persuasion, Incentivisation, Coercion, Training, Restriction, Environmental Restructuring, Modelling and Enablement; health-related examples of what these involve are given at the end of Table 1.", "Multiple Intervention Functions are associated with each Source of Behaviour.", "These intervention functions have utility in identifying what kinds of behavioural intervention are possible and in classifying behavioural interventions that have already been conducted.", "A further layer of the BCW identifies seven categories of policy that could enable these interventions to occur.", "For a given behaviour in a given context, such as energy saving behaviour in a workplace, the BCW provides a structure to consider strategies that may be useful in promoting changes in behaviour.", "We conducted a systematic review of empirical studies which assess interventions designed to change energy use behaviour in office-type workplaces; note that some focus on a range of pro-environmental behaviours, as well as the use of energy.", "A systematic review aims to collate all empirical evidence that fits eligibility criteria (set in advance) in order to answer a specific research question [58].", "The key characteristics of a systematic review are: a clearly stated set of objectives with pre-defined eligibility criteria for studies; an explicit, reproducible methodology; a systematic search that attempts to identify all studies that would meet the eligibility criteria; an assessment of the validity of the findings of the included studies, for example through the assessment of risk of bias; and a systematic presentation, and synthesis, of the characteristics and findings of the included studies [58].", "We pay attention to each of these characteristics as set out below.", "To be included in the review, papers needed to be a study of a behaviour change intervention in relation to energy use in the workplace and to report on the energy savings generated through the intervention (we use the term ‘workplace’ to refer to an organisational context where employees work alongside each other in an office-type environment; we include offices in private and public building, universities and schools); a study published in or since the year 20002; a study published in English.", "Studies could be of interventions in any country and could be published either in peer-reviewed academic journals or in grey literature (i.e. reports from organisations, governments or the third sector).", "Searches for eligible studies were run on multiple academic online search engines: Scopus, Web of Science (databases: SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, BKCI-S, BKCI-SSH), ABI-INFORM, ASSIA, PsychINFO and Ergonomics Abstract.", "The ECEEE and ACEEE Summer Study Conferences online archives were searched by hand.", "Grey literature was searched by looking at individual company reports from government and non-governmental agencies e.g. DECC, DEFRA, the Carbon Trust and GAP (Global Action Plan), and by using online databases including the Energy Citations Database (DOE), Open EU, and UKOP (UK’s open Publication Platform).", "In order to capture the quite dispersed range of literature in this field, queries used in the search strategy were chosen to cover four main areas; energy-related, workplace, pro-environmental/energy behaviour change, and energy systems.", "Appendix A provides results on the number of papers and reports identified under different combinations of search terms.", "In total, 24 papers reporting on 22 separate interventions were identified which fit the eligibility criteria for inclusion in our review.", "Systematic reviews often include an assessment of the validity of the findings of the studies included [58], and this is sometimes, but not always, a selection criteria [59].", "Given the limited number of papers in existence in this area, we did not set any selection criteria in relation to validity or quality, however we do reflect on these issues for the selected studies overall (see Section 3.3).", "With regards to the systematic presentation and synthesis of findings from the included studies, we use the BCW to characterise and evaluate behaviour change interventions, as outlined above (see Section 1.4).", "Of the 22 interventions identified for review and analysis, 2 were from grey literature.", "Fifteen of the reviewed interventions reported on the results of focused trials or experiments (which often used control groups) conducted by the authors themselves, whilst seven reported on holistic behaviour change programmes initiated in conjunction with employers (see Appendix B for summary details of each study).", "In terms of the interventions reported by the reviewed papers, a variety of workplace settings were examined: 9 were run in commercial companies, 8 in universities, 3 in government buildings and 2 in a school.", "These workplaces were located in the USA (n = 10), UK (n = 5), the Netherlands (n = 3), Canada (n = 2), Sweden (n = 1) and Singapore (n = 1); and differed markedly in size ranging from 22 employees (for individual desk plug-load trials), to 2112 employees (for self-report questionnaires), to energy use behaviour change programmes targeting thousands of employees.", "Interventions lasted from 2 weeks to 7 years.", "Seven of the interventions explored electricity use in general whilst others concentrated more specifically on electricity use in an individual office (n = 1), at the desk (n = 6) or from computers only (n = 3); or electricity for lighting (n = 3), for heating and cooling (n = 2), and for lift usage (n = 1).", "Most of the papers (n = 18) reported changes in energy consumption (electricity or gas) during the course of the intervention, with 4 papers reporting on observing energy-using appliances and equipment, or artefacts of energy use (e.g. the extent to which lights are turned on or off, the coverage of heaters, the use of stairs vs. lifts, the time computers are spent ‘idle’).", "Half of the papers (n = 11) also utilised quantitative questionnaires to survey participants pre- and post-intervention to gather self-reported changes in behaviour due to the intervention, whilst 5 used qualitative methods (interviews, focus groups, participant observation) to understand participant’s experiences during the interventions.", "The remaining papers relied on physical measures of energy use only and did not engage directly with participants.", "Where positive energy savings are reported in terms of measured reductions in energy consumption (rather than observed changes in energy-using appliances or artefacts of energy use), savings range from 5.4% to 50%.", "The 22 studies reviewed in this paper were analysed using the BCW framework.", "These studies were numbered 1–22 for ease of reference, and these numbers are used to identify the studies below and in tables and figures.", "We categorised the methods used in the interventions according to the nine Intervention Functions of the BCW.", "This allowed us to examine whether any of the Intervention Functions were particularly associated with successful energy savings (see Section 3.1), which of the related Sources of Behaviour (i.e. Capability, Opportunity and Motivation) were involved (see Section 3.2), and which had not been previously utilised in this context.", "Table 1 shows how the methods employed in the interventions reviewed correspond to the nine categories of Intervention Functions of the BCW (for full details see Appendix C).", "Some methods fall into multiple categories i.e. constitute more than one Intervention Function.", "Below we work through each Intervention Function in turn, discussing the methods which relate to each category and the energy savings they generate.", "Education in the BCW is described as ‘increasing people’s knowledge or understanding’, an example from the health domain being the provision of information to promote healthy eating [53,p.111].", "All but 1 of the 22 reviewed interventions included methods which constitute Education, including the provision of feedback (regular or one-off), instructions, reminders, checklists, and tips.", "This information concerned generic energy-saving actions, individualised feedback on plug-load energy use, and whole building energy audit results and was provided through a range of media including printed material (brochures, newsletters, posters, signs) and email, or through verbal communication (direct to employees or through peer-education).", "One study, in a school setting, also involved Education through assemblies and lessons which focused on energy efficiency [60] (study 18).", "We highlight that there is some overlap between the categories Education and Environmental Restructuring, the latter of which involves the changing of context – either physical or social – such as the providing of on-screen prompts for GPs in the health domain [53].", "Providing energy-saving prompts by email directly to employees or placing signs by light switches asking employees to turn them off, could be argued to be both educating users as to energy saving actions that can be taken, and to be providing contextual cues to remind people of behaviours that they should already be undertaking (see Section 3.1.7).", "Following Michie et al. [53], we therefore consider reminders, prompts and checklists to also be environmental cues and thus constitute Environmental Restructuring.", "Depending on the language or imagery used, these methods may also constitute Persuasion (see Section 3.1.2).", "Given its ubiquity, it is hard to differentiate the energy saving potential of Education from that of other Intervention Functions which are used in conjunction with it.", "For example study 18 [60] took place in a school and reports on extensive use of Education through assemblies, lessons and assignments focusing on energy-saving measures, as well as through the use of signs and posters in the school and student homes, and through information sent to parents and local officials through newsletters.", "The intervention overall achieved impressive energy savings of 31% but given it also included a prominent Modelling element (Section 3.1.8) through a strong energy champion (the District energy Manager) it is hard to apportion reasons for the savings generated.", "Four of the interventions reviewed specifically focused on the utility of different types of education however.", "Carrico and Riemer [10] (study 1) compared the impact on energy consumption of generic energy advice given through postcards sent directly to individuals, with the impacts of combining this energy advice alongside either emailed feedback on energy use at the building level, or of peer education involving employees acting as points of contact and information for colleagues.", "Feedback and peer-education generated energy savings of 7% and 4% respectively whilst the provision of information alone led to an increase in energy use of 4%.", "Kamilaris et al. [61] (study 19) found that participants considered emails a better means of communicating information than either posters or leaflets, with specific rather than general advice to be most meaningful.", "The study draws attention to the frequency and duration over which feedback information is provided, suggesting emails sent weekly would be effective in providing information but would not annoy employees (as they might do if sent more frequently).", "The study reports sustained energy saving behaviour over a 3 month period post-intervention, indicating the potential for savings over a longer-term.", "Direct information provision (through signs and stickers) and peer education were also compared by Werner et al. [62] (study 8), finding that whilst the former generated little or no impact, the latter generated a reduction in the percentage of classrooms with lights left on after class from 51% to 17%.", "Peer-education provided the opportunity for students to raise questions and concerns and for these to be addressed, thus removing barriers to energy saving behaviour; some students had, for example, expressed a belief that they were not allowed to turn off the lights when leaving the classroom as it was a ‘public space’, a fear which was allayed through the peer-education.", "The third study focusing on the provision of information utilised an interactive poster to persuade employees to use the stairs rather than a lift in their 5 storey building, and thus to save energy [63] (study 14).", "The poster was designed to be persuasive (thus the intervention represents Persuasion too, Section 3.1.2) through images of a tree with leaves growing on it in relation to stair usage.", "Whilst employee pro-environmental attitudes increased over the course of the trial and their use of stairs increased rapidly for a day or two after the poster was installed, there was no statistically significant change in behaviour over the following 2–4 weeks of the study.", "Based on the available evidence, it would appear that targeted information and knowledge passed on by peers is likely to be more effective than general information provided through printed materials such as stickers and posters.", "This is in line with the critique of ‘information-deficit’ models which propose that it is not productive to consider behaviour change simply as requiring a correction in understanding through provision of information [64].", "Indeed users can be considered to have their own expertise and contextualised understanding that can usefully input into developing new policies, support structures and interventions to achieve change [65,66].", "However this is not to say that the provision of information is not sometimes useful and perhaps necessary, particularly when new products and systems are being implemented.", "Important considerations include that information is contextualised and easy to understand, and that information is from a trusted source with sufficient expertise (this can include professionals but also peers) [66].", "Persuasion is defined in the BCW as ‘using communication to induce positive or negative feelings or stimulate action’, for example, the use of imagery to motivate increases in physical activity in the health domain [53,p.111].", "Fourteen of the 22 reviewed interventions reported on methods which constitute Persuasion.", "Methods fall into this category in a number of ways: first, many methods constitute direct persuasion by focusing on the provision of information through the use of graphs, tables, encouraging text, images and interactive displays on either printed or electronic material i.e. visual imagery to induce positive feelings and stimulate action, for example signs and images to make building users feel personally responsible.", "Some other methods use persuasive verbal communication, such as through peer-education discussed above under ‘Education’ (see Section 3.1.1).", "Some further methods are harder to categorise, such as the school motto which was used as a vehicle to promote sustainability, and the articulation of an ‘ethic of sustainability’ [67,69] (study 12a and 12b) and ‘ethos of commitment and can-do attitude’ [48] (study 2).", "These communication tools are aimed at inducing a social environment that will stimulate action, thus constitute Persuasion.", "The school motto and ethic of sustainability referred to above [67,68] (study 12a and 12b) were employed as part of an wide-ranging energy saving programme, encompassing building renovation and new technologies, along with changes in governance and culture, that generated by far the greatest energy savings of any of the reviewed papers, of 50% over 7 years.", "As such it is not possible to distinguish the individual impact of the school motto or ethic of sustainability; rather in this case the programme’s success was put down to its combination of methods and holistic approach.", "Similarly, the example of the ethos of commitment and can-do attitude arises from a wider programme, successful in reducing electricity consumption by 9% [48] (study 2).", "Again, there is clearly some overlap between Intervention Functions.", "Behaviour change initiatives, particularly in the field of pro-environmental behaviour by their very nature have a specific agenda of behaviour change and of persuasion.", "Often therefore when information is presented, it is also presented in a persuasive style or context; Education interventions therefore frequently overlap with Persuasion interventions.", "Not all Persuasion will constitute Education however, as for example the motto, ethic and ethos articulated in the examples provided above aim not to educate i.e. increase knowledge, but explicitly to persuade i.e. induce positive feelings towards the desired behaviour.", "It should be noted that all Persuasion reported in the reviewed papers focuses on promoting positive feelings rather than negative ones.", "The discussion on Coercion (see Section 3.1.4) and Restriction (see Section 3.1.6) may help to understand why this is so.", "Persuasion has the potential to change behaviour, particularly if done by peers and if communicating a deeper message (e.g. ethos of sustainability) rather than superficial information (e.g. to turn off a light).", "It is also noted that in this context, information that it is tailored to and meaningful to the recipient is usually more persuasive, and thus more likely to be acted upon [66].", "Incentivisation in the BCW is described as ‘creating the expectation of reward’, for example through the use of prize draws to induce attempts to stop smoking in the health domain [53,p.111].", "Twelve reviewed interventions reported on methods which constitute Incentivisation, based on two different approaches.", "Incentivisation could involve the provision of rewards in the form of cash, bonuses, food, and other prizes.", "However rewards may also be social in nature i.e. not based on financial or other gains, but rather a sense of achievement, for example the provision of positive descriptive comments in employee reviews.", "Social rewards tend to be given in relation to meeting pre-established targets or goals around performance of energy-saving actions, although goal-setting (with no expectation of reward) is itself also a form of Incentivisation.", "Rewards may be given to employees on an individual basis or based on groups of employees working together.", "Three studies involved Incentivisation in the specific form of on-line games.", "These encouraged competition between (groups of) employees and incentivised them to win by behaving in the most energy-saving way.", "Incentivisation methods reported by 3 of the reviewed interventions were used as part of a holistic behaviour change programme i.e. alongside multiple other methods, thus it is difficult to determine their contribution towards energy savings.", "One study notably relies heavily on Incentivisation [48] (study 2) employing 5 separate methods simultaneously as part of a holistic behaviour change programme (most papers report only 1 or 2) and generates energy savings of 9%.", "Other studies investigating the impact of Incentivisation were part of academic-led trials.", "Handgraaf et al. [51] (study 3) reports that publically given rewards outperformed ones given privately, and that social rewards (grade points with a descriptive comment) outperformed monetary ones (up to €5); in fact public social rewards generated energy savings of 6.4% whilst private monetary rewards led to an increase in energy use.", "One study encouraged competition between employees, although with no tangible rewards beyond social recognition [69] (study 10).", "It involved employees accessing, through an online digital dashboard, the electricity use and carbon dioxide equivalents of their ‘pod’ i.e. clusters of six to eight employees, displayed alongside the same data for all other pods.", "Over a period of 4 weeks, the experiment generated energy savings of 6%.", "Three studies focused on the use of on-line games, one of which, called ‘iChoose’, engaged groups of employees in competition with each other [46] (study 4).", "Although organised through their work, this intervention crossed the domestic/non-domestic divide, as employees were encouraged to record energy saving activities in their own homes, and in doing so gained points for themselves and their team at work (made up of colleagues they worked most closely with).", "There were small monthly cash prizes for individuals in the lead and team prizes at the end of the game.", "Estimated savings of 463 megawatt hours of electricity were made, although notably energy saving activities subsided once the game had finished.", "Drawing on ideas of ‘serious games’, another study developed and trialled an on-line game called ‘Energy Chickens’ in which the health of a pet chicken related to plug-load energy use of the employee [70] (study 15).", "As a result of the game, average energy use reduced by 13% (23% over weekends, and 7% on work days i.e. Monday-Friday) and 69% of employees said the game helped increase their energy awareness, including outside of work.", "This study ranked 4th highest in energy savings amongst those reviewed in this paper, but it should be noted that despite the large reductions in energy use during the game, these savings did not persist and energy use returned to baseline levels.", "Speculating on reasons for the lack of persistence of energy savings, the authors relay how the office IT support stopped people turning off their computers at night and at weekends (as incentivised in the game) so as not to interfere with on-line security and back-up management, highlighting clashes between the policies and goals being presented to employees in that context.", "Another study employed a pervasive game called ‘IdleWars’ to provide feedback on the time a computer was ‘idle’ i.e. had no mouse movements or key strokes for more than 5 min, and to encourage employees to catch or ‘bust’ each other when this happened [71] (study 16).", "Whilst there was no statistically significant change in energy use as a result of the game, the time that computers were left idle reduced by 5.6% over the 2 week trial.", "Whilst the game worked well to engage participants and stimulate discussion around energy use, it inadvertently produced some anti-energy conservation behaviours by being appropriated by employees for fun (e.g. moving a colleague’ mouse whilst they were away from the desk to create another 5 min idle period, which could then be ‘busted’).", "Issues of privacy and productivity were also highlighted by the study, as the game involved touching others’ computers and potentially disruption to work routines.", "It appears therefore that incentives can be effective but must be chosen with care as they do not always work; whilst based on limited evidence, indications are that incentives that are made publically and that are social in nature may be most effective.", "Gaming can also be used to incentivise particular behaviours, but care must be taken in how they might be appropriated and potential impacts on productivity and privacy.", "As has been found in other research however, impacts of incentives may be short-lived and subside when incentivisation finishes [72].", "Interestingly, none of the seven highest performing interventions studied employed Incentivisation.", "Methods which involve Incentivisation are often based on groups of employees working together and invoke a sense of competition and comparison, for example competitions between groups of employees on different floors of an office building (e.g. through public display of energy savings generated by each floor) or comparison with individual colleagues.", "An interesting relationship therefore exists between Incentivisation and Modelling (see Section 3.1.8).", "In the BCW, Coercion is described as ‘creating expectation of punishment or cost’, for example raising the cost of alcohol to reduce excessive alcohol consumption in the health domain [53,p.111].", "No reviewed interventions used methods which fall into this category, although two referred explicitly to a desire not to use negative reinforcement in terms of punishment or restriction.", "The inattention to forms of coercion in behaviour change interventions in the workplace represents a gap in the research, one which also appears to be present in relation to domestic energy use and to other sorts of pro-environmental behaviour.", "This gap may be explained by researchers wishing to avoid the ‘negative connotations’ associated with the punishment or costs inherent to Coercion, or because they fear it may threaten to disrupt the relationship between positive employee attitudes and productivity at work [73].", "It may also be that it is difficult to put Coercion into practice; practically it may be difficult to penalise employees for wasting energy in the workplace and contextual considerations are important here.", "A pay related penalty might be the most obvious penalty.", "Training in the BCW refers to the ‘imparting of skills’, for example advanced driver training to increase safe driving in the health domain [53,p.111].", "Whilst most studies included an element of imparting knowledge and understanding (i.e. Education) none went as far as aiming to impart skills (i.e. Training).", "It should be noted that in the studies reporting on the installation of an energy use feedback device at for example the desk level, there may well have been an element of training in its use; however if this was involved it is not detailed in any of the papers reviewed.", "Interventions tend to focus on either the use of electricity for lighting, computing and other electrical equipment, and/or energy usage for heating or cooling of office spaces.", "The absence of training in these interventions may reflect, at least in part, the prevailing trend of centralising energy management in the workplace, which limits direct employee control of functions such as lighting and local space heating and delegates them to a Facilities Manager [9].", "Accordingly, the inattention to forms of training in behaviour change interventions may exist simply because it is difficult to envisage what training might actually involve.", "Training may be more relevant in more traditional offices with decentralised energy functions where, for example, employees can operate air conditioning interfaces, but often without any knowledge in how to best use them, such as closing windows whilst the air conditioning is active.", "Training may also become more relevant alongside the introduction of new smart energy technologies or systems [8], which return some influence over local conditions to building occupants in order to improve satisfaction levels, which are often low under the centralisation strategy [74].", "In the BCW Restriction is described as ‘using rules to increase the target behaviour by reducing the opportunity to engage in competing behaviours’, for example by prohibiting sales of solvents to people under 18 to reduce abuse of these, in the health domain [53,p.111].", "No reviewed interventions used methods which fall into this category, though in one study [23] (study 11a) they appeared to inadvertently create a similar effect.", "This intervention included the use of ‘Environmental Champions’ (employees who would act as a point of contact and information for colleagues), and found that colleagues were at times unhappy with the idea that these volunteers may be ‘spying’ on their activities, referring to them as the ‘environment-police’; in fact the Champions themselves discussed sometimes feeling uncomfortable in this role.", "The intervention led to a reduction in energy use however, generating 5.4% saving in electricity.", "Based on extensive participant observation, the paper argues that this was little to do with changing employee's attitudes toward, knowledge about, or values regarding the environment, but rather to do with working with the existing ‘officially accredited' rules for office conduct through the introduction and policing of new social expectations and rules involving a pro-environmentalism.", "Again, the inattention currently paid to forms of restriction constitutes a gap in the research.", "It may again be that this gap is, in part, explained by difficulties in envisaging exactly what restriction in relation to energy use in the workplace may involve.", "Informal rules often exist with regards to turning off specific individual equipment after use, and may be introduced alongside environmental restructurings (see below) such as providing recycling bins.", "Policing here tends to be informal through social approval.", "Formal rules are harder to envisage and to enforce, as was discussed regarding Coercion and the potential threat of punishment.", "It may be that there are interesting contrasts with the use of Enablement, which may be considered the reverse of Restriction.", "Further empirical investigations in the area would be useful.", "Environmental Restructuring is referred to in the BCW as the process of ‘changing the physical or social context’ in order to promote a particular behaviour, for example providing on-screen prompts for GPs to ask about smoking behaviour in the health domain [53,p.111].", "Nineteen studies reviewed here (i.e. almost all of them) reported methods which constitute physical Environmental Restructuring, although there are interesting differences amongst these.", "Four different forms of Environmental Restructuring appear within the interventions reviewed: signs and posters (i.e. physical material), electronic feedback devices and ‘dashboards’, retrofit technology, and technology automation.", "Regarding changes to the physical context, 11 of the interventions report on the use of signs, stickers or posters, for example by light switches to provide cues and prompts for employees to turn off the light when not needed.", "Six of the reviewed interventions report on the use of electronic devices and online dashboards to provide real-time energy use feedback (which may implicitly promote awareness and therefore considered use of energy), two use computer screens in gaming interventions, whilst seven use email prompts and checklists.", "Other alterations to physical context focus on technical efficiency; two interventions reviewed here included the use of retrofitting to increase the energy efficiency of office equipment such as lighting or heating whilst six interventions included the use of automation to reduce energy demand, for example by turning off electronic equipment when not used for over a certain time, or the automatic control of lights.", "As noted in Section 3.1.5, these forms of Environmental Restructuring that focus on using technology to improve efficiency arguably reduce the need for employees to alter their behaviour, though there are potential costs to employee satisfaction.", "In terms of the energy saving potential of physical Environmental Restructuring, the use of signs or visual imagery has been discussed above (see Sections 3.1.3 and 3.1.2), suggesting the provision of information alone creates little impact.", "The use of feedback devices, in the form of software tools or physical displays, may offer more potential.", "Murtagh et al. [50] (study 5) report a study involving installation of the ‘MyEcoFootprint’ gadget to provide employees with real-time feedback on desk-based energy use.", "This led to savings in energy (not quantified in the paper) although they diminished over time.", "A study reporting on a piece of pilot research found energy savings of 30% generated by an energy dashboard which provided feedback to employees, as well as the ability to control devices remotely and to set automated on/off timing controls [75] (study 13).", "The full study [76] (study 21) which developed from this pilot reports even higher savings through this combination of online remote (manual) controls (to turn off devices) and automated controls (to schedule turning on and off), with savings of 38% in desk-based electricity use (laptops, monitors, phones, desk light) during the intervention compared to the pre-intervention baseline.", "A further study by Owen et al. [49] (study 6) also examined real time feedback, provided through a dashboard and in relation to energy use for lighting.", "Their trial compared the effect of retrofitted lighting with automatic daylight dimming, to the effect of lighting controlled individually by employees, and a control group with no intervention on lighting (i.e. neither automation nor employee control).", "Employees working under all of these conditions could access the real time feedback provided through the dashboard, but those working with no lighting measures made energy savings of only 2.4%, whilst those with individual switches saved 12.0% and automated lighting saved 12.6%; increasing individual control of lighting and automating lighting controls thus had a similar impact on energy savings.", "Another study [77] (study 22) developed an ‘adaptive proximity controller’ to detect employee activities at their desks and in response to turn computer screens on or off.", "Controlling computer screens in this way generated energy savings of 21% compared to the pre-intervention baseline.", "Other studies which have used automation as part of their intervention strategy have also tended to be successful.", "Metzger et al. [69] (study 10) describe how the use of automated controls to switch off non-essential circuits when a ‘pod’ (i.e. desk area) was empty for 15 min generated energy savings of 20%.", "The highest performing intervention amongst all studies reviewed, generating 50% savings in energy [67] (study 12a), was a holistic behaviour change programme and also included automation to allow shut-off of computer labs, and retrofitting with energy efficient light bulbs.", "The exact contribution of these methods to the overall energy savings is however not known.", "It should be noted that five of the top seven performing interventions involved automation of technology.", "Whilst they may appear separate from behaviour change interventions, technological solutions to workplace energy saving operate both on and in the same physical and social context and so are inextricably part of the story.", "Whilst responsibility for saving energy is in many ways transferred to the new technologies, these necessitate some form of response from employees, either in the form of passive acceptance or more active engagement.", "For example with respect to automated heating and lighting systems, occupants may simply over-ride or subvert the system if they find they do not align with their personal notions of comfort.", "They might for example cover sensors or air vents, or acquire a personal desk lamp or heater.", "Simultaneously, the investment in new technology may have longer-term impacts on the behavioural norms of employees by signalling an organisational commitment to pro-environmental goals.", "In the BCW, Modelling is described as ‘providing an example for people to aspire to or imitate’, for example the use of television series involving safe-sex practices to increase condom use in the health domain [53,p.111].", "In total, 17 of the reviewed interventions report on Modelling, but in a number of different ways.", "Firstly, three interventions report on methods which employ modelling in a very explicit format, through the profiling of particular employees who exemplify good energy conservation behaviour, either individually or through peer-education of others.", "These employees received public recognition for their actions, through official roles and titles, or through recognition programmes that included the giving of prizes.", "All of these modelling interventions are introduced as part of holistic behaviour change programmes, which produced relatively high energy savings: 12% [49] (study 6), 9% [48] (study 2), and 5.4% [23,78] (study 11a and 11b).", "Secondly, 12 studies reported on interventions that involved the comparison or competition between colleagues, either individually or in groups (from small ‘pods’ of 6–8 employees up to competitions between whole floors within a building) or gaming amongst them.", "Comparison may be encouraged through bringing colleagues together to share stories, through ‘opportunities for bragging’, or through public display of the energy savings of other colleagues, groups of colleagues, or an office average.", "Such interventions also often constitute Incentivisation, as discussed above (see Section 3.1.3).", "These interventions tended to occur in conjunction with multiple other interventions, thus the energy saving potential of modelling through comparison and competition is hard to assess.", "One study focused specifically on comparative feedback amongst employees of a US University, with comparison between the 6 separate buildings involved [47] (study 17).", "The year-long energy saving campaign; which involved an online platform of information, a website, email reminders and posters, generated average energy savings per building of 6.5%.", "Pro-environmental attitudes and behavioural intentions amongst participating employees were not found to alter as a result of the campaign, indicating that the energy savings were generated by employee’s motivations to outperform their colleagues in other buildings.", "The study’s authors note however that the energy savings were not sustained after the end of the campaign, although the following year they were higher than in control buildings which also formed part of the study.", "The authors suggest therefore that campaign designers commit to long-term programmes in order to sustain energy savings.", "Another study which explored the role of comparison amongst colleagues reports that employees found this to be the least useful tactic of those employed, which also included the provision of advice, information and team-level energy-use statistics [61] (study 19).", "The study suggests that comparisons on an individual basis may have raised issues of privacy amongst employees, as participants suggested that comparisons between groups of colleagues (in different offices or departments) might have been more useful.", "The scale at which comparison with, or modelling of, colleagues is made is clearly important.", "A further study appealed directly to existing group identity amongst employees as part of an intervention consisting of goal-setting, prompts and feedback [79] (study 20).", "Feedback incorporated group identity through encouraging messages which appealed to the positive traits of the group (as identified by group members prior to the intervention).", "No conclusive results could be drawn from the study in terms of energy savings, but it was found that the intervention changed behaviours and increased feelings of group identity during the experimental period.", "And thirdly, one study reports the widespread recognition of particular teachers, students and the Principal of a school, as playing instrumental roles in achieving energy reductions and providing examples for others to follow [68] (study 12b).", "In this sense, these individuals may be seen as acting as models for others; however this occurred organically as a consequence of the initiative, rather than being its explicit aim.", "This unintentional modelling was part of the highly successful holistic programme which generated energy savings of 50%.", "The other study which reports on interventions within a school also employed Modelling in a similar sense, through the District Energy Manager, described as a strong champion for energy efficiency [60] (study 18).", "It should be noted that all of these examples of Modelling involve colleagues of employees within the same organisation, rather than external individuals, e.g. celebrities, as in the health-related example given above.", "It is hard to draw concrete conclusions about the individual contribution of Modelling to energy savings given its ubiquity (for example as forms of competition and comparison amongst employees) or its presence in holistic programmes alongside multiple other methods, however considering the social environment of most workplaces, it is clearly of interest to those involved in behaviour change interventions in these environments.", "Enablement in the BCW is defined as ‘increasing means/reducing barriers to increase capability or opportunity’,3 with the note that the increase in Capability is specifically beyond what can be achieved with Education and Training, and that the increase in Opportunity is beyond what can be achieved with Environmental Restructuring [53,p.111].", "Examples of Enablement in the health domain include behavioural support given for smoking cessation and surgery to reduce obesity.", "Seven of the reviewed interventions report on methods which constitute Enablement, although in very diverse ways and often as part of holistic behaviour change programmes, again making it difficult to determine their individual utility.", "Two studies report on Enablement through the use of an energy dashboard which enables employees to remotely and automatically control electronic devices at their desks, generating 30–38% energy savings [75,60] (studies 13 and 18).", "Two studies report on Enablement through individualised help and assistance (by a nominated employee) in thinking through the energy use of employees’ desks or workspaces and ways in which this can be reduced; generating fairly high energy savings of 9% [48] (study 2) and 12% [49] (study 6).", "One study reports Enablement in the form of altering after-hours working practices by enabling (and incentivising) employees’ moves to one particular location in the building, so as to reduce energy consumption in other parts [80] (study 9), again achieving fairly high savings of 10%.", "One other intervention reports on Enablement as school students are given control to adjust thermostats [60] (study 18), whilst another reports on four separate methods; providing custodians (i.e. caretakers) and students the opportunity to participate in school governance (including over energy use), providing custodians the ability to alter practices (e.g. when lights are turned on and off at the start and end of the day), allowing students control of spending money saved through energy reductions (which was used to purchase wind energy), and by offering courses in sustainability which the school viewed as important in promoting a wider knowledge of and ethics for sustainability, including around issues of energy [68] (study 12b).", "This study was the highest performing study reviewed, generating energy savings of 50%.", "It should be noted that what sets 7 of the 12 highest performing studies apart from the lower performing studies is their use of Enablement.", "As Enablement was used alongside other forms of intervention it is difficult to attribute success solely to it however its presence within the higher performing and noted absence within the lower performing interventions clearly suggests it may be linked to successful energy savings.", "Enablement seems to be related to a change in levels of employee control and responsibility, for example responsibility for ones’ desk plug load, or control over when lights are routinely turned on and off, or over organisational governance and budgets.", "We suggest that potential forms of enablement be researched further to understand its utility more clearly and identify the most successful forms of its implementation in this field.", "Our review also allows us to draw some conclusions about what makes an effective intervention into energy use behaviours in the workplace.", "We highlight that our conclusions are tentative given the relatively small number of papers reviewed and that many studies use multiple forms of intervention in combination meaning that attributing effects to any one function is impossible.", "Nonetheless, our review provides important descriptive data highlighting intervention functions that have most frequently been associated with success.", "In particular, interventions characterised as involving Enablement, forms of Environmental Restructuring, and those including aspects of social influence, including Modelling, appear to be particularly successful.", "Enablement in particular appears to be a successful intervention function and has been discussed as increasing the means or reducing the barriers to increase a person’s Capability or Opportunity.", "Examples of Enablement include remote control over electronic devices, individualised assistance to employees in relation to their working space, facilitation of late-night workers to a central office area, changes in custodial practices (offering buildings managers more control over energy systems) and participation in governance of the organisation.", "It is an area ripe for further research, particularly in relation to notions of control and responsibility conferred by enabling interventions, which are currently under-researched.", "Indeed with regards to employee engagement in general, interventions which incorporate social influences appear to be generally most successful.", "Modelling, peer-education, public and social incentives, and social persuasion all tend to be associated with successful interventions.", "We discuss further below (see Section 4.1) the particular context of workplaces for such social influences.", "Environmental Restructuring interventions, in particular automation of technologies also hold great potential for energy saving interventions.", "Whilst these technologies do not aim to induce changes in behaviour they appear to create a new context in which energy saving behaviour can take place.", "The negotiation with and acceptance of new technologies by employees has been little researched to date but is worthy of further investigation.", "In the BCW the Intervention Functions (discussed above in Section 3.1) are positioned around a central hub containing three Sources of Behaviour: Capability, Opportunity and Motivation.", "Note that the positioning within the BCW does not reflect associations between the wheel rings but that particular Sources of Behaviour are linked to (and supported through) different Intervention Functions (Table 2).", "Capability, Opportunity and Motivation have already been defined above but it is important to acknowledge that each has two sub components: Capability refers to Physical and Psychological aspects of an individual’s capacity to engage in the behaviour concerned, Motivation refers to Automatic and Reflective brain processes that energize and direct behaviour, and Opportunity refers to external factors that are Physical and Social factors in and which supporting the behaviour [54,p.4].", "Having categorised the methods used in the reviewed studies according to the nine Intervention Functions (Section 3.1), we now go on to relate these to the three Sources of Behaviour (and their sub components) to explore which are associated with the most successful interventions.", "Interestingly, what sets apart the most successful Intervention Functions adopted in workplace energy saving interventions i.e. Enablement, Environmental Restructuring and Modelling, from the others is their correspondence with Opportunity (both Social and Physical), indicating that these may be particularly useful drivers of behaviour to target within interventions.", "None of the less successful Intervention Functions contribute to or provide either Social or Physical Opportunity.", "Interestingly Social Opportunity is related to the concept of ‘social influences’ [53, p.113, Table 2]—already noted as a common feature in successful interventions [2].", "Social influences are interpersonal influences that can have an impact on the way that individuals perceive a situation or behaviour and encompass processes including social pressure, norms, conformity, and comparisons.", "With regards to Physical Opportunity, this Source of Behaviour is related to theoretical ideas of ‘environmental context and resources’ [53, p.113, Table 2].", "As discussed above (see Section 3.1.7), whilst behaviour change programmes aim to alter employee actions in relation to their use of energy, changes in physical infrastructure and technologies are often integral to successful reductions.", "Much remains to be understood about how technologies are accepted and negotiated by employees however, and about their shorter and longer-term impacts on employee behaviour, motivations and willingness to engage in energy savings.", "The communal nature of most workplaces, in that they bring together multiple employees and involve hierarchical management structures, tends to be what sets them apart from domestic settings.", "Social and group norms and dynamics within an organisation have been shown to be important in increasing employee motivations to act [8,10,12,47,79] and this supports findings presented here which also demonstrate how interventions can serve to create new norms and standards regarding energy use in the office.", "Previous research suggests that interventions should target employees based on their pre-existing groupings [8] and that a sense of community is important in motivating energy saving behaviours at work [4].", "The studies reviewed here did not consider – or at least did not detail – all of these things, but they did find that peer-education and modelling of particular employees held potential.", "Personal contact and communications between colleagues with regards to possible energy saving actions can thus alter behaviours.", "Much effort has gone into researching the effectiveness of providing feedback on energy use behaviour, suggesting that in communal settings, such as the workplace, providing feedback on a group basis is more effective than that delivered individually [11,61].", "Findings synthesised here also suggest that whilst the information provided through feedback can be important, the manner in which such information is presented is equally significant.", "Rewards and incentives given publically (rather than privately) are also more likely to be effective, reflecting the importance of social influence.", "Our review is limited by the relatively small number of papers available to review, and our conclusions hinge on their quality.", "Many of the studies made use of self-report questionnaires with participants, typically pre- and post-intervention to gain an understanding of (changes in) behaviour, behavioural intentions, attitudes, values and identities.", "Such self-report questionnaires have been criticised for reporting, for example, what people say they will do rather than what they actually do in practice [50].", "Converged with actual energy saving data, self-report questionnaires are important in order to reveal the underlying drivers for behaviour change.", "We highlight that comparing metrics of success has considerable problems; whilst some studies measure this through digital technologies (i.e. smart meters), others use electricity bills which it is observed makes detecting savings of less than 10% difficult to discern from background variation [50].", "In addition, when assessing energy use, some studies control for changes in the weather whilst others do not.", "The length of interventions, and of research into those, also varies; the majority studies are relatively short term (most examine a few weeks or months), leaving the medium to long-term effects of interventions unknown.", "A number of studies question the wider applicability of their findings as they are based on organisations involved in sustainability or in which employees already demonstrate strong pro-environmental attitudes prior to any behaviour change intervention.", "The studies also represent a fairly homogenous group of workplace environments (individual or open-plan offices, often in Universities, and almost all in the global North) meaning much remains to be understood about the complexity and global diversity represented within the seemingly simple category of non-domestic buildings.", "Details provided on the length of study and numbers of participants vary, with studies of holistic employer-led behaviour change programmes typically containing fewer details than trials led by academics, limiting the understanding gained from the findings.", "The focus and breadth of studies also varies, with academic trials tending to focus on fewer elements of intervention but able to provide greater depth in their assessment of it, whilst employer-led holistic programmes tend to include multiple elements which can be viewed interacting with each other, but with less ability to attribute energy savings to any particular element(s).", "Our review finds that three forms of intervention have not been investigated at all to date; Coercion, Training and Restriction.", "Though we acknowledge there may be important reasons why, in certain contexts, these may not be appropriate or advantageous, a recent meta-analysis of pro-environmental behaviour studies finds that the most successful interventions are often the least studied [24] indicating that these gaps in the literature deserve attention.", "Our analysis has highlighted interventions characterised as involving Enablement, Environmental Restructuring, and those including aspects of social influence as particularly successful.", "It is notable that our analysis highlights the importance of creating physical opportunities to save energy, either through automation or retrofit of technology, or through digital displays and devices to provide information and advice on energy use.", "Whilst some technological solutions may not directly aim to change employee behaviour they certainly create a new context in which behaviour takes place.", "Far greater understanding is needed of the ways in which employees interact with this technology to understand the aspects of these interactions that lead to actual behaviour change.", "We propose that further research is needed in order to drill down to understand which elements of these interventions types are most important in the context of energy savings and when and how these are successful.", "Certain interventions often appear to overlap with each other (e.g. Incentivisation and Modelling) and it is important to understand which aspects are driving behaviour change, though we also highlight that it is possible that certain intervention aspects may work best in interaction with one another.", "There is a clear need for more systematic studies which aim to more conclusively identify successful factors – or combinations of factors – in producing behaviour change.", "Abraham and Michie [81] highlight the importance of assessing the individual and combined effectiveness of techniques used in behaviour change interventions in the health sector and we propose the same is needed within energy saving interventions.", "Our review finds that there is a lack of common definitions of techniques and methods amongst interventions to change energy-use behaviour in the workplace, as well as inconsistencies in the ways in which these are discussed and assessed (cf. the health sector: [81]).", "This problem may indeed be particularly pronounced in this field given the range of disciplines, and indeed organisation types, conducting research in this area.", "We propose that further research in this area provides more depth of description of not only the interventions conducted, but also the sampling and procedures utilised, in order to better allow for replication and the analysis of reasons of failure and success.", "Notably, with regards to sampling, more attention should be paid to all employees within an organisation, with more focus given to those choosing not to participate in interventions to understand their reasons why.", "We propose that interventions conducted should be based on a theoretical framework, rather than just practical considerations, so that the reason for resulting behaviours can be fully understood.", "In particular, it is important to evaluate changes in perceptions and psychological factors alongside changes in energy use so the driving factors for change, or indeed barriers to action, can be better understood.", "Studies should also consider more longitudinal research that explores which interventions create long lasting impacts.", "This provides not only valuable theoretical insight into drivers of change but also some indication as to the useful length of campaigns and frequency with which they should be conducted.", "Much remains to be understood about the influence of the workplace as a context for potential energy saving behaviours.", "Behaviour change interventions operate within the context of competing organisational, institutional and political priorities and it is against this backdrop that the potential for interventions to achieve change must be understood [45].", "Many of the studies reviewed in this paper focused their analysis on the energy savings generated and the efficacy of the intervention tools employed, rather than the wider workplace context, leaving another significant gap for future research.", "Differences between public and private sector workplaces need to be explored, as do those between big and small companies, or those from different parts of the world, or in relation to long-serving or short-term employees.", "Such contexts may influence feelings of responsibility, control, trust and privacy amongst employees, as well as issues of staff relations, productivity, budgets and trade union activity, all of which are important in understanding the potential for energy savings within the workplace [45].", "The influence of local, national and international policies on the potential to save energy in the workplace also deserves attention [3].", "This paper set out to identify what types of behaviour change intervention are most successful at saving energy in an office-type workplace (offices in public and private buildings, universities and schools).", "It did so through an extensive literature review which identified 22 studies of energy saving intervention in offices from around the world, and a synthesis and analysis of these through the Behaviour Change Wheel [53,54].", "We found that interventions which create social and physical opportunities for employees to save energy are the most successful, namely those which constitute Enablement (including direct support and greater control to employees), Environmental Restructuring (particularly automated technologies) and Modelling (various forms of social influence).", "Given the clear importance of governance and culture noted within Modelling interventions, and the potential for process changes noted within Enablement interventions, our findings indicate that energy savings in the workplace depend not only on the individual and collective actions of employees, but importantly also on the attitudes and engagement of management, on wider organisational change, and on investment in energy efficient technology.", "The communal nature of most workplaces demands further scrutiny in order to understand how social influences condition individual and collective actions, including around the negotiation and adoption of new energy technologies.", "We provide recommendations for future research, notably the need to consider forms of intervention not yet researched; Coercion, Restriction, and Training.", "We urge other researchers reporting on energy saving interventions to consider and measure the theoretical reasons behind energy saving behaviour, more systematically report and analyse their findings, and where possible to undertake further longitudinal evaluations; this will enable more accurate identification of those techniques which offer greatest success in saving energy and thus reducing carbon emissions." ]

[ "Studies of international workplace energy saving interventions are reviewed.", "Enablement, Environmental Restructuring and Modelling most promising interventions.", "Coercion, Restriction, and Training intervention types have been little researched.", "Successful interventions create social and physical opportunities for employees.", "Utility of social influence and energy efficient technologies highlighted." ]

[ "MULT" ]

[ "Archaeology" ]

[ "1000" ]

[ "In the Aquitaine basin and the Pyrenees, the Middle Paleolithic (MP) Mousterian culture is followed, in succession, by the Châtelperronian, the Protoaurignacian and the Aurignacian I (a.k.a. Early Aurignacian).", "In Iberia, these initial phases of the Upper Paleolithic (UP) are represented in the Cantabrian strip and in Catalonia but remain unknown to the South of the Ebro basin.", "Based on these observations, the “Ebro Frontier” model hypothesizes that (a) in Valencia, Murcia, Andalusia, Gibraltar, the Mesetan hinterland, and Portugal, the corresponding chronostratigraphic slot is occupied by a late-persisting Mousterian and (b) the pattern is explained by the major biogeographical divide that the Ebro basin would have been at that time (Zilhão, 1993; Zilhão, 2000; Zilhão, 2006a; Zilhão, 2009).", "The paleontological and ancient DNA (aDNA) evidence indicates that, in Europe, extensive admixture occurred at the time of contact between aboriginal Neandertals and in-dispersing groups of modern humans, resulting in the former’s eventual assimilation (Smith et al., 2005; Trinkaus, 2007; Pääbo, 2015).", "The authorship of the Châtelperronian, the Protoaurignacian, and the other so-called “transitional” industries from this time remains debated (Higham et al., 2010; Caron et al., 2011; Hublin et al., 2012; Trinkaus and Zilhão, 2013; Zilhão, 2013; Zilhão et al., 2015; Welker et al., 2016).", "In Western Eurasia, however, the Mousterian is exclusively associated with the Neandertals, while the Aurignacian I and the succeeding Aurignacian II (a.k.a. Evolved Aurignacian), which extend from Asturias in the West to northern Israel in the East, are associated with modern humans only (Verna et al., 2012).", "In this context, the broader paleoanthropological significance of the “Ebro Frontier” model resides in the implication that Neandertals persisted in Southern and Western Iberia longer than everywhere else.", "Within the model, the chronological boundaries of the Middle Paleolithic/Neandertal persistence pattern are given by the difference in age between the earliest archeological cultures (or their phases) that, on each side of the Ebro divide, are unambiguously associated with modern humans: to the North, the Aurignacian I; to the South, the Aurignacian II.", "Given the currently accepted dating of these assemblage types (Higham et al., 2011; Banks et al., 2013a; Banks et al., 2013b), the lag implicated (i.e., the duration of the “Ebro Frontier” pattern) is, at the least, of three millennia, between 40,000 and 37,000 years ago.", "The number of occurrences substantiating that Iberian regions to the South of the Ebro divide were occupied by a late-persisting Mousterian while those to the North were occupied by the Aurignacian I is, however, limited.", "This paucity of occurrences has led to alternative readings of the evidence whereby the late persistence is apparent.", "In such readings, the “Ebro Frontier” pattern would stem from insufficient information on the early Upper Paleolithic, aggravated by (a) Middle Paleolithic-associated radiocarbon dating results that would be inaccurately young, and (b) ambiguity in the definition of the stone tool assemblages implicated (Wood et al., 2013).", "Conversely, it has been argued that no Aurignacian exists in Southern and Western Iberia, their Upper Paleolithic beginning with the Gravettian (de la Peña, 2013).", "Such views imply that (a) the Mousterian persisted even longer (Finlayson et al., 2006; Finlayson et al., 2008), or (b) after a Neandertal extinction event, Southern and Western Iberia remained uninhabited until modern human reoccupation (Bradtmöller et al., 2012; Galván et al., 2014).", "In these scenarios, the role of biogeographical divide played by the Ebro basin under certain climatic and environmental conditions would not have contributed to observed patterns in any significant manner.", "Re-dating and critical examination of old sites and collections (Kehl et al., 2013; Wood et al., 2013) have advanced these debates.", "The scope of the many empirical issues involved, however, requires the excavation of new sites with the potential to settle the key points of contention.", "Here, we report on the progress made in that direction resulting from a decade of fieldwork in Murcia, Southeast Spain.", "When specifically cited, individual radiocarbon results are given as provided by the dating laboratory, i.e., expressed in uncalibrated radiocarbon years Before Present (BP).", "Throughout, however, the discussion is framed in calendar terms, i.e., in years or thousands of years (ka) before the time of measurement for U-series and luminescence dates, and in calibrated years or thousands of years BP for radiocarbon dates.", "We excavated three localities <2 km apart within the Mula basin (Angelucci et al., 2017).", "The Supplementary Information (SI) Appendix provides a succinct geographical description of the area, as well as extensive monographic presentations of the sites’ stratigraphic sequences, dating, human occupation features, and stone tool assemblages.", "The sites are: Cueva Antón (CA; 38°03′51.84″N, 01°29′47.20″W), Finca Doña Martina (FDM; 38°04′43.21″N, 01°29′25.13″W), and Abrigo de La Boja (ADB; 38°04′43.37″N, 1°29′23.17″W) (Fig. 1; Figs. S1.1–S1.2).", "Cueva Antón (SI appendix, chapter 2; Fig. 2) is a cave located in the valley of River Mula (Zilhão et al., 2010a; Angelucci et al., 2013; Zilhão et al., 2016).", "Sandwiched between basal palustrine deposits (complex FP) and well-bedded inundation silts and sands accumulated in recent times during periods of submersion by the reservoir of the La Cierva dam (complex DD), the site contains a thick Upper Pleistocene succession (complex AS).", "The base of this succession (sub-complexes AS2-AS5) is an alluvial fill of MIS (Marine Isotope Stage) 5 age that features discrete anthropogenic lenses recording short-lived occupation episodes — the last of which is layer II-l.", "After an erosional hiatus, broadly coincident with MIS 4, the accumulation of alluvium inside the cave — represented by the basal layers (I-i, I-j, II-a, II-c and II-b; Fig. 2) of the AS1 sub-complex — resumed briefly in MIS 3.", "Layer I-k, an archeologically fertile breccia made-up of wall degradation debris, caps the AS1 deposit, whose surface is erosional.", "Previous work has placed the basal MIS 5 alluvium in the 72–85 ka age range (Burow et al., 2015; Zilhão et al., 2016) and the MIS 3 alluvium and breccia in the 35.1–37.7 ka age range (Table 1; Zilhão et al., 2016).", "Here, the focus lies on layer I-k’s site formation process and stone tool assemblage composition, upon which lie its assignment to the Middle Paleolithic.", "Finca Doña Martina (SI appendix, chapter 3; Fig. 3) and La Boja (SI appendix, chapter 4; Figs. 4 and 5) are rock-shelters located in the Rambla Perea (Zilhão et al., 2010b; Lucena et al., 2012).", "In the regional landscape, this tributary of River Mula likewise communicates the lowlands of the Murcia littoral with the plateaus and mountain ranges extending northward to the Mesetan hinterland.", "Both sites feature stratigraphic successions where a basal Middle Paleolithic is overlain by long Upper Paleolithic sequences.", "The preservation is good for shell but poor-to-nil for bone, and charcoal is abundant — even though, at Finca Doña Martina, chemically weathered (leading to radiocarbon results that are minimum ages only; Tables S3.1-S3.2).", "Layer 8 of Finca Doña Martina yielded a lithic assemblage whose Aurignacian affinities (Figs. S3.31-S3.32) are consistent with the layer’s stratigraphic position between Mousterian layer 9 and Gravettian layers 7b and 6/7.", "At La Boja, the excellent preservation of charcoal and the sub-centimeter precision with which most archeo-stratigraphic units — designated OH (Occupation Horizons) — could be separated provided for a large series of radiocarbon results that, a burrow sample excepted, are in full stratigraphic order (Table 2; Table S4.1; Fig. S4.8).", "The basal Mousterian dates beyond 44 ka and is buried under a thick, multi-ton, roof-collapsed slab.", "The site was re-occupied, in the Aurignacian, once this slab was covered by the accumulation of the sediment forming the IL (Intermediate Level) 4 unit.", "Otherwise archeologically sterile, IL4 includes some post-depositionally intruded material and yielded a date of ca.41 ka.", "This date provides a terminus post quem for the ca.75 cm-thick Aurignacian sequence, which is sealed by another large, roof-collapsed slab.", "Radiocarbon dating places the three basal Aurignacian horizons (OH18-OH20) within the 34.9–38.2 ka interval and the three upper ones (OH15-OH17) within the 33.9–35.6 ka interval.", "Sediment samples from the Mousterian (OH21-OH23) and the Aurignacian (OH17-OH18) of La Boja were also dated by Optically Stimulated Luminescence (OSL) (Table 3; Figs. 6–8 ; Fig. S4.9).", "The multiple-grain dating of the quartz and feldspar minerals places the sequence between 32.6 ± 1.9 ka (C-L3906), for OH17, and 59.9 ± 6.8 ka (C-L3901), for the base of the deposit, below OH23.", "These luminescence ages are in complete agreement with the radiocarbon results for the corresponding Aurignacian and Mousterian horizons.", "The ages of the Late Mousterian in layer I-k of Cueva Antón and of the Evolved Aurignacian in OH18-OH20 of La Boja overlap (Fig. 9).", "As the occupation events recorded at these sites are of short duration, a possible interpretation of this pattern is that the two assemblage types coexisted in the region for an extended period, during which their makers would have made infrequent, alternating incursions into the River Mula and Rambla Perea valleys.", "If so, Middle Paleolithic material ought to exist within the basal Aurignacian of La Boja as (a) discrete, interstratified lenses, or (b) isolated elements mixed in the OH18-OH20 assemblages.", "As neither is the case, the regional contemporaneity between the bearers of the two kinds of stone tool technologies must have been short-lived.", "Therefore, the dating overlap must primarily reflect the statistical uncertainty inherent to radiometric dating.", "Under these priors, CA/I-k and ADB/OH18-OH20 can be treated as two consecutive phases of the regional chrono-stratigraphic sequence.", "Whether the charcoal found in layer I-k of Cueva Antón is anthropogenic, environmentally accumulated, or both, cannot be ascertained.", "However, the basal AS1 alluvium consists of lenses of fine, sandy-silty alluvium deposited in quick succession during low-energy inundation events; such kinds of events are also largely responsible for the matrix of the I-k breccia (Angelucci et al., 2013).", "This record’s resolution implies that any temporal difference that may have existed between human occupation and charcoal deposition must be negligible.", "Nevertheless, to be conservative, the age of the Late Mousterian in layer I-k is best constrained using the terminus post quem represented by the underlying units, layers II-a and II-b.", "That layers II-a and II-b provide indeed a robust maximum age for the human occupation of layer I-k is intimated by the archeological sterility of the basal AS1 alluvium, to which those two layers belong.", "Such sterility precludes interpreting the artefact assemblage in overlying layer I-k as inherited via some sort of local post-depositional process.", "In addition, (a) the stratigraphic integrity of the AS1 package is accredited by the absence of disturbance features across its total thickness and entire excavated extent, and (b) the mode of accumulation of layer I-k implies that its artefact content cannot have been inherited via fluvial transport from an earlier Middle Paleolithic site located elsewhere in the landscape.", "The stone tool refits (Fig. 10; Fig. S2.18), which document on-site production, corroborate the homogeneity, integrity, and in situ nature of both the artefact assemblage and its stratigraphic context.", "There can be no doubt, therefore, that, at Cueva Antón, the human activity recorded in layer I-k post-dates the time of deposition of layers II-a and II-b.", "At La Boja, the age of the successful, hearth-collected sample from OH19 (2014-3348; 33,233 ± 335 BP, VERA-6157_av) is statistically indistinguishable from that obtained for immediately underlying OH20 and represents a direct record of human activity.", "OH19 and OH20 both contain diagnostically Upper Paleolithic, specifically Aurignacian, tool-kits.", "Thus, their dating sets an unambiguous terminus ante quem for the end of the region’s latest Middle Paleolithic.", "Under this reasoning, the earliest possible age of Cueva Antón’s latest Mousterian is 37.1 ka, and the youngest possible age of La Boja’s Aurignacian is 36.5 ka, in calendar years.", "The yellow band in Fig. 9 represents the interval bounded by these dates.", "It was within this interval that, after a coexistence and interaction period of unknown duration, the region’s Neandertal-associated Late Mousterian was replaced by the modern human-associated Evolved Aurignacian.", "Jarama VI, a cave site in the Iberian hinterland once thought to span the MP-UP transition, illustrates well how issues of definition are as much implicated in the Neandertal persistence debate as those of dating accuracy and sample association: upon closer examination, the “Upper Paleolithic” stone tools retrieved in the levels capping the site’s Pleistocene succession turned out to be of Mousterian affinities instead (Kehl et al., 2013).", "Clearly, the robustness of the Mula basin’s chronology also depends on whether the artefact assemblages associated with the dated samples do represent the two sides of the regional transition.", "Figs. 10 and 11 illustrate the key aspects of lithic technology supporting our assignments: method of core reduction, and type of blank that production is designed for.", "In layer I-k of Cueva Antón, the following methods, which are exclusive to the Middle Paleolithic, are found (Figs. S2.17-S2.19): Centripetal, Levallois or Discoid, core reduction, represented by a core, refitted flakes, and debris; Discoid, represented by imported core-trimming, or deliberately overshot, naturally backed flakes bearing notched or denticulated edges; Kombewa, represented by a core discarded in an initial stage of the reduction; and Levallois, represented by an imported laminar flake.", "In La Boja OH18-OH20, only two methods, both unknown in the regional Middle Paleolithic, are found (Figs. S4.39-S4.43): prismatic for the extraction of blades and bladelets, represented by cores, débitage, and refitted sets; and carinated/nosed “scraper” reduction, also including refitted sets and represented by all steps of the sequence (initial large core for long-and-thick blades used as blanks for the extraction of the intended bladelets, the abandoned bladelet cores, the bladelets themselves, and the waste produced as the “scraper” front was reduced, trimmed and reconfigured).", "The Dufour bladelet in Fig. 10 is a typical example of the Roc-de-Combe subtype, an index fossil of the Evolved Aurignacian.", "It comes from OH17, but this and other subtypes of Dufour bladelets occur through the OH15-OH20 sequence (Figs. S4.41-S4.43).", "They are also present, alongside the characteristic carinated/nosed “scrapers”/cores, in layer 8 of Finca Doña Martina (Figs. S3.31-S3.32).", "In OH15 and OH16 of La Boja, backed microliths (Fig. S4.43, nos. 4–5) appear for the first time alongside these characteristic Aurignacian items, suggesting that the emergence of the succeeding Gravettian likely corresponds to a technological transition with no major discontinuity in population, demography, or settlement.", "Well-stratified Portuguese examples show that specialized site occupancy may generate lithic assemblages that, despite their Upper Paleolithic age, lack the period’s diagnostics.", "This evidence questions automatic assignment to the Middle Paleolithic of similar assemblages, the more so if they are small (Wood et al., 2013).", "However, unlike layer I-k of Cueva Antón, those Portuguese assemblages also lack Middle Paleolithic diagnostics: they contain no items (either cores or blanks) indicating that the Discoid, Levallois and Kombewa reduction methods were in use at the time of production.", "A case in point is the assemblage from the EE15 occupation surface of the Lagar Velho rock-shelter (N = 593) (Almeida et al., 2009).", "Here, the idiosyncrasy relates to the situational context (reduction of immediately available quartzite cobbles for the expedient production of cutting edges used in carcass-processing tasks), and is of no wider chrono-stratigraphic consequence.", "The mutually exclusive presence/absence of diagnostic technologies in the Mula basin sites stands despite differences in assemblage size of up to two orders of magnitude, and is consistently seen across time (Table 4).", "In this regard, the Late Mousterian in layer I-k of Cueva Antón is no different from the Middle Paleolithic assemblage of MIS 5 age found in the site’s layer II-l (Tables S2.2–S2.5).", "Likewise, the equivalently small size of the Early Gravettian assemblages in OH13-OH14 of La Boja (Tables S4.22–S4.25) is no impediment for their fully Upper Paleolithic nature to manifest itself through such diagnostics as bladelets extracted from both prismatic and “burin” core-types, the “burins” themselves, and even the technocomplex’s index fossil (a microgravette point).", "Much the same applies to La Boja’s Aurignacian assemblages (Tables S4.10–S4.21).", "At Finca Doña Martina, the lower resolution of the stratigraphic sequence means that each unit samples, and averages out, much longer time intervals.", "Yet, it remains that (a) Levallois and Discoid cores and blanks, sidescrapers, and denticulates are found together in this site’s basal layer 9 (Tables S3.3–S3.5, Figs. S3.29–S3.30) but not in overlying layers 8, 7b and 6/7, while (b) the reverse is true of prismatic, carinated/nosed “scraper” and “burin” core-types, endscrapers, or bladelet tools (Tables S3.7–S3.12; Figs. S3.31–S3.33).", "The variation in the size and composition of these assemblages is primarily due to local factors.", "At Cueva Antón, the patches of dry sediment available for settlement inside the cave during the time of formation of layers II-l and I-k were restricted and surrounded by inundated or boggy riverside terrain (Figs. S2.11, S2.16).", "As shown by the taphonomy of the abundant rabbit bone, the site functioned as an eagle-owl roost throughout, which is inconsistent with frequent or intensive human presence (Sanchis, 2012; Zilhão et al., 2016).", "Likewise, the spatial restrictions to habitation caused by a massive roof collapse explain the small size of the artefact scatter around the hearth in La Boja’s OH13 horizon (Fig. S4.21).", "The spectrum of activities reflected in the use-wear data for layer I-k of Cueva Antón is limited to wood-working (Table S2.5; Fig. S2.19), which is in keeping with the highly transient nature of the occupation(s).", "In the Rambla Perea sites, raw-material economy patterns indicate no significant change in site function across the transition.", "In the residential versus logistical balance of hunter-gatherer settlement-subsistence systems — as gauged by the relative importance of domestic- versus hunting-related stone tools — the scales were somewhat tipped in favor of the latter in the Early Gravettian and the Aurignacian of Finca Doña Martina, but not in the Aurignacian of La Boja (SI appendix, chapters 3–4).", "For the Rambla Perea rock-shelters, lateral variation between two adjacent archeological sites that, in the living past, must have functioned as a single, spatially extensive locus of human activity, suffices to explain the contrasts between coeval lithic assemblages.", "Through time, across the regional MP-UP transition, the use-wear evidence shows that the differences are primarily of a techno-typological nature.", "Hide-working, wood-working, defleshing and the use of projectiles are documented in both the Mousterian and the Aurignacian (Tables S3.6, S4.9; Figs. 12–14 ; Figs. S3.30–S3.32, S4.37–S4.38, S4.41).", "However, (a) hides were processed with sidescrapers in the Mousterian but with endscrapers in the Aurignacian, and (b) projectiles were armed with single, axially-mounted points in the Mousterian but with multiple, laterally-mounted microlithic elements in the Aurignacian.", "In short, synchronic functional variability cannot explain the differences in lithic technology upon which we have assigned the stone tool assemblages of the Mula basin sites to either the Middle or the Upper Paleolithic.", "Ochre is often involved in the processing of hides, as documented by residue on a Mousterian sidescraper from Finca Doña Martina (Fig. S3.30).", "No such residues were found in the lithics from layer I-k of Cueva Antón.", "Thus, the pigment cover of the associated scallop shell (Fig. 15; Fig. S2.20) cannot represent accidental or post-depositional staining by iron oxides brought in for hide-processing tasks or locally produced by diagenetic processes.", "Much the same applies to the ornamental shell assemblage of quite distinct composition found in the Aurignacian of La Boja (Table S4.2; Fig. 15; Figs. S4.32, S4.34).", "This assemblage features ubiquitous red ochre staining even though none was found in the 78 stone tools from OH15-OH20 examined for use-wear (Table S4.9).", "These findings further strengthen the symbolic interpretation previously advanced for Cueva Antón’s ochred scallop (Zilhão et al., 2010a).", "At La Boja, the archeological sequence’s radiocarbon chronology is independently supported by the OSL dating of the basal Mousterian and of the Aurignacian.", "At Cueva Antón, layer I-k could not be OSL-dated for two main reasons: (a) prior to 20th-century burial by silts accumulated during intermittent periods of submersion under the La Cierva reservoir the layer was exposed as a surface for an undetermined amount of time, implying significant uncertainty with regards to environmental radiation parameters; and, (b) coupled with its limited thickness in the cross-sections exposed at the time of sampling, its high stone content (layer I-k is a clast-supported breccia with few fines) made this layer inappropriate for luminescence dating (Burow et al., 2015).", "From within the radiocarbon method itself, the Cueva Antón and La Boja charcoal samples passed all the reliability tests currently available.", "The dates allowing us to bound the Mula basin’s Mousterian-to-Aurignacian transition belong to long series of results that are fully stratigraphically consistent, both internally (within each site) and externally (across sites and with the broader, regional and supra-regional framework).", "At La Boja, the humic fraction was also measured to assess the potential impact of contamination.", "The accuracy of the chronology obtained on the fraction processed with the ABA (Acid-Base-Acid) treatment is supported by (a) the identical results obtained whenever the dating of individual samples was repeated, and (b) the lack of statistical difference between the results obtained for individual samples processed with both ABA and ABOx-SC (Acid-Base-Oxidation with Stepped Combustion) (based on Bird et al., 1999).", "At Cueva Antón, the ABA protocol was found to slightly underestimate the age of the samples, and the success rate of ABOx (26%; five out of 19) was lower than at La Boja (Zilhão et al., 2016).", "However, the Cueva Antón samples surviving the ABOx-SC pretreatment had a high %C, which, following Rebollo et al. (2011), is a good indicator that the material that survived was well preserved.", "In addition, given the aggressiveness of the treatment, the percentage of failed samples is not unexpected; similar rates have been reported when using ABOx-SC for samples derived from contexts dated to broadly the same time interval (Brock and Higham, 2009).", "The dating work carried out at the site of Sima de las Palomas (Fig. 1, no. 2), on the coast of Murcia, ca.60 km to the Southeast of Cueva Antón, provides further support for the late persistence of the Middle Paleolithic in the region — in this case, with diagnostic Neandertal remains found stratigraphically together with the lithics (Walker et al., 2008; Trinkaus and Walker, 2017).", "Correct understanding of the significance of the dates obtained at this key site is hindered by the samples’ provenience notations referring to arbitrary horizontal spits that do not reflect the stratigraphic layout of the sequence — something misunderstood by Wood et al. (2013) and Santamaría and de la Rasilla (2013), although explicitly stated in Walker et al. (2008).", "When the actual stratigraphy is considered, the dating results— obtained by radiocarbon on burnt bone treated with the ABA protocol, U-series on bone using Diffusion/Adsorption (D/A) assumptions, and multi-grain quartz OSL on sediments — are mutually consistent (Fig. 16).", "The U-series dates for Sima de las Palomas show that (a) the accumulation of the lower cemented deposit containing articulated Neandertal skeletons (unit D) took place prior to 46.4 ka, (b) provide a terminus post quem of 53.5 ka for the accumulation of the overlying deposit containing fragmentary Neandertal remains (units A-B and E), and (c) suggest for the base of the latter an age younger than 45.3 ka.", "The OSL result is less precise and, because of the existence of remnants of an older sedimentary fill brecciated against the walls and roof of the cave, could be affected by incomplete bleaching; even so, when its 95.4% probability interval (45.3–64.1 ka) is considered, it agrees with the U-series results.", "Taken together, the OSL and U-series dates are in turn consistent with the two radiocarbon dates from samples retrieved at the same stratigraphic elevation or higher up in units A-B and E.", "The uppermost radiocarbon result (OxA-10666) is from a faunal fragment cemented to a diagnostic Neandertal mandible that was (a) found half-way through the unit A deposit and (b) overlain by ca.50 cm of sediment containing nothing but diagnostic Middle Paleolithic stone tools and diagnostic Neandertal remains.", "As OxA-10666 translates into a calibrated age within the 38.6–42.0 ka interval, the Sima de las Palomas evidence strongly indicates, in line with the Cueva Antón pattern, that the Middle Paleolithic persisted in the region well beyond 40–42 ka.", "In addition, it shows that such a late-persisting Mousterian is indeed a Neandertal-associated technocomplex.", "There is no reason, therefore, to question that the association pertains in those other parts of Iberia where stratigraphy and dating support persistence of the Middle Paleolithic into the same time range: Gibraltar and Portugal.", "At Gorham’s Cave (Gibraltar; Fig. 1, no. 3), an uncalibrated date of 32,280 ± 420 BP (OxA-7857) was obtained for a charcoal sample recovered in stratigraphic association with diagnostic Middle Paleolithic stone tools within Context 24 of the Natural History Museum’s (NHM) 1995–1998 excavations (Pettitt and Bailey, 2000).", "In Middle Paleolithic layer IV of the Gibraltar Museum’s 1999–2005 excavations at the rear of the cave, an uncalibrated date of 32,330 ± 390 BP (OxA-10230) was obtained in the same laboratory, and a separate set of samples yielded uncalibrated dates ranging between 23,780 ± 540 BP (Beta-185345; 2σ) and 32,560 ± 780 BP (Beta-196771; 2σ) (Finlayson et al., 2006; Finlayson et al., 2008).", "In calendar years, these results imply persistence of the Middle Paleolithic in Gibraltar until at least 36.0–37.8 ka (the 95.4% probability interval of the calibration of Beta-196771).", "The Beta samples from Gorham’s all underwent the standard ABA treatment, but the younger ones probably reflect stratigraphic intrusion because, at the rear of the cave, a several millennia-long hiatus makes for direct contact between Mousterian layer IV and Solutrean layer III (Zilhão and Pettitt, 2006).", "The OxA results, in turn, come from samples processed with the gentler RR treatment, which does not include a base wash (Brock et al., 2010; Wood et al., 2013).", "Even though OxA-10230 was a large pine cone scale that, per Bronk Ramsey et al. (2002), made for reliable dating material, Wood et al. (2013) assume that the RR treatment was insufficient to remove all contamination from the Gorham’s OxA samples.", "Based on this assumption, they argue that no confidence can be placed in the notion that the site’s Middle Paleolithic significantly post-dates 40–42 ka.", "However, they did not test the RR results via processing of remaining material in storage, or of new samples, with ABA or ABOx-SC (they report no additional charcoal dating, only failed attempts at extracting collagen from associated animal bone).", "In addition, the RR-treated charcoal samples from the NHM excavations collected lower down in the Gorham’s sequence returned results as old as 51,700 ± 3300 BP (OxA-7790).", "If the latter were to be taken as a byproduct of incomplete decontamination producing a finite result for a sample of infinite radiocarbon age, the unremoved contaminant, if modern (i.e., F14C = 1), could represent no more than 0.16% of the measured carbon.", "For OxA-10230, modeling such a level of contamination shifts the uncalibrated radiocarbon result from 32,330 to 33,069 BP, which is, given the standard deviation, statistically the same thing.", "Against this background, arguing that higher levels of contamination characterized the samples coming from the upper part of Gorham’s Mousterian sequence (but only those…) would be special pleading.", "The more so because the general reliability of the OxA results for the Gibraltar sites’ RR-processed charcoal samples is otherwise implied, in the case of stratigraphic units 53–55 of Vanguard Cave, by their agreement with the luminescence ages obtained for the same deposit: radiocarbon’s RR results were between 41,800 ± 1400 BP (OxA-6998) and 54,000 ± 3300 BP (OxA-6891), OSL’s was 46.32 ± 3.30 ka (OxL-1029) (Pettitt and Bailey, 2000).", "In Portugal, layer 8 of the Gruta da Oliveira cave site (Fig. 1, no. 4) yielded an unquestionably Middle Paleolithic stone tool assemblage (Marks et al., 2001).", "Its radiocarbon dating on burnt bone treated with ABA at Groningen and with RR at Oxford yielded statistically indistinguishable results of, respectively, 31,900 ± 200 BP (GrA-10200) and 32,740 ± 420 BP (OxA-8671) (Angelucci and Zilhão, 2009).", "In calendar terms, these two radiocarbon results, which translate into a 95.4% probability interval comprised between 35.3 and 38.2 ka, are statistically identical to three U-series (D/A) dates on bone from the same layer (Hoffmann et al., 2013).", "The time span indicated by the rich, single-occupation Mousterian open-air site of Foz do Enxarrique, near the Spanish border (Fig. 1, no. 5), is the same (Raposo, 1995).", "Here, the weighted average of the dates obtained by U-series on the tooth enamel of one bovid and two horse samples is 33.6 ± 0.5 ka.", "The accuracy of this chronology is dependent on the uncertain validity of the Early Uptake assumption underpinning the calculation of the ages, while the nature of the association between the dated faunal remains and the stone tools is an open issue.", "Indeed, per Brugal and Raposo (1999), the site’s faunal assemblage is primarily a natural riverside thanatocenosis, with only the cervid component bearing marks indicative of a human activity-related accumulation.", "The two multi-grain, K-feldspar OSL results since obtained at the site for the base of the alluvial sands within which the archeological level is contained (the T5 unit of the local terrace staircase of the Tagus) are, therefore, a better, if less precise estimate of the time of deposition of the stone tool assemblage.", "At 34.8 ± 1.3 and 38.5 ± 1.6 ka (after correction for anomalous fading) (Cunha et al., 2008), the OSL results support an age post-dating 40 ka for the site’s occupation — and, thus, that the Middle Paleolithic persisted in interior Iberia beyond the time of emergence of the Early Aurignacian in the Cantabrian strip and northern Catalonia.", "The persistence of a Neandertal-associated Middle Paleolithic from Iberia’s Mediterranean Southeast to its Atlantic seaboard implies that archeological manifestations of the modern human-associated Aurignacian I not be found across the same territory.", "Such is indeed the case.", "Neither stratigraphic units containing diagnostic assemblages nor isolated index fossils of the Early Aurignacian have been identified in the long cave sequences spanning the MP-UP transition known in those parts of the peninsula: Cova Beneito (Valencia), Cueva Bajondillo (Andalusia), Gorham’s Cave (Gibraltar), and Gruta do Caldeirão (Portugal) (Zilhão, 2006a).", "At these sites, and at others that are either open-air, single-occupation localities, or lack a basal Middle Paleolithic, the earliest Upper Paleolithic is the Aurignacian II (Evolved Aurignacian) or III–IV (a.k.a. Late Aurignacian).", "Technologically, the Aurignacian II is defined by the débitage of carinated and thick-nosed “scrapers”/cores producing characteristically twisted blanks transformed into Dufour bladelets via inverse or alternate retouch, while the Aurignacian III–IV is characterized by the predominance of carinated and other “burin” types of bladelet cores.", "However, as demonstrated at La Boja, the microlithic diagnostics of the Aurignacian II persist to the end of the Aurignacian sequence.", "Therefore, in the absence of reliable dating, or of a technologically representative assemblage of cores and débitage products, the presence of such microliths, even though sufficient to exclude appurtenance to the Aurignacian I, does not exclude assignment to the Aurignacian III–IV.", "When stratigraphic sequences are not resolved to the level of detail seen at La Boja, the possibility that assemblages containing Dufour bladelets correspond to palimpsests that subsume both phases (Aurignacian II and III–IV) cannot be excluded either.", "In Mediterranean Spain, the assemblages from Beneito, the rock-shelter of Ratlla del Bubo (Iturbe and Cortell, 1992), and the cave site of Cova de Mallaetes (Fortea and Jordá, 1976), all in Valencia, and from Bajondillo, are examples of clearly post-Aurignacian I collections that cannot be precisely assigned to one of the succeeding phases of the technocomplex.", "In the Beneito and Ratlla del Bubo assemblages, which remain undated, backed elements are found alongside the characteristic Dufour bladelets.", "This coexistence has led some to question the validity of the industrial diagnosis, or the integrity of the sedimentary contexts (de la Peña and Vega, 2013).", "However, based on the evidence from horizons OH15-OH16 of La Boja, the coexistence suggests instead that the Beneito and Ratlla del Bubo assemblages either are Late Aurignacian or include a component belonging to that phase.", "The Mallaetes context lacks diagnostic stone tools but yielded lozenge bone points in association with a conventional charcoal date of 29,690 ± 560 BP (KN‐I/926).", "The Bajondillo context contains diagnostics suggestive of the Aurignacian II and is dated to 33,690 ± 1195 BP (Ua‐17150) and 32,770 ± 1065 BP (Ua‐18050); however, given the inadequate nature of the samples (of “sediment and charcoal”) and the imprecision of the results, appurtenance to the succeeding Aurignacian III–IV cannot be excluded.", "A related problem exists with the two large, well-studied stone tool assemblages from the open-air Aurignacian sites of the Rio Maior basin, in Portugal: Gato Preto’s is of Aurignacian II affinities but is dated by Thermoluminescence (TL) and therefore with a large 95.4% probability interval, 30.3–45.9 ka; and Vale de Porcos’s, technologically of Aurignacian III–IV affinities, remains undated (Zilhão, 2006b).", "It has been proposed that the diagnostic microlithic tool-type of the Late Aurignacian is an elongated, straight variant of the Dufour bladelet pointed by alternate retouch (Zilhão et al., 2010c).", "This variant is known from layer 2 of the cave of Pego do Diabo, in Portugal, and from the disturbed, surficial deposits capping the Mousterian sequence of Cueva de Zafarraya, in Andalusia.", "At the Portuguese site, the Pleistocene fauna associated with the small assemblage of such Dufours yielded four AMS radiocarbon dates on tooth samples treated with both the Longin and the ultrafiltration protocols.", "Under the stringent criterion of considering reliable only those samples for which both the standard gelatin production and the >30 kDa (thousands of Daltons) ultrafiltered production yielded statistically identical results, the Pego do Diabo deposit accumulated between 29,090 ± 270 BP (VERA-4047) and 30,260 + 330/-320 BP (VERA-4050).", "The earlier result overlaps those for OH15-OH16 of La Boja, but the later one extends the range for another millennium, until ca.33 ka.", "Because the dated fauna is non-anthropogenic, however, it cannot be ascertained whether the “Pego do Diabo points” (a) stand for a “Final” phase, dating beyond 34.0 ka, of the Aurignacian technocomplex in Western Iberia, as the younger result might suggest, or (b) are a component of the ca. 34.0–35.5 ka Late Aurignacian, as indicated by the earlier result.", "If the second hypothesis is retained, the implication would be that the microlithic tool-kit of the Late Aurignacian was more diverse than so far documented in Valencia and Murcia.", "Be it as it may, the Mula basin sites suffice to demonstrate that, by 36.5–37.1 ka, the Aurignacian II was already present in Iberian regions to the South of the Ebro basin.", "This interval is the same during which, based on Bayesian modeling of available dates, Banks et al. (2013b) found that the transition from the Early to the Evolved Aurignacian had occurred to the North.", "This technological transition would therefore seem to have been concomitant with a process of settlement expansion: in Northern Europe, toward the British Isles and equivalent latitudes of Germany and Poland that, during the previous phase, had become devoid of human occupation; in Iberia, toward the lands beyond the Ebro basin, eventually leading to replacement of their late-persisting Mousterian and the assimilation of its Neandertal makers.", "The “Ebro Frontier” model provides a biogeographical and paleoecological framework for the interpretation of these developments in terms of population history.", "In Iberia, the Ebro basin nowadays lies at the interface between two biogeographic regions defined after the distribution of plant communities: Eurosiberian and Mediterranean (Rivas-Martínez, 1987).", "The separation runs along the southern foothills of the Cantabro-Pyrenean mountains but, during the Upper Pleistocene, its very existence and latitudinal placement must have been dependent on the period’s highly variable and frequently oscillating climates.", "During MIS 4, Eurosiberian steppe-tundra environments spilled into and beyond the Ebro basin well into the Iberian core.", "This is shown by the distribution of wooly rhino and mammoth finds: along the Mediterranean coast, down to the Llobregat delta, near Barcelona; in central Iberia, as far West as the Manzanares valley (Madrid) and as far South as the northern flanks of the Sierra Nevada (Granada) (Daura et al., 2013).", "During the Last Glacial Maximum (LGM), Europe’s Upper Pleistocene cold fauna (mammoth, wooly rhino, bison, reindeer) was again present in Catalonia, the Cantabrian strip, and parts of the northern Meseta but absent from Valencia, Murcia, Andalusia, and Portugal.", "These differences in the composition of the large herbivore fauna imply significant environmental gradients within the peninsula during MIS 4 and the LGM, albeit ones that (a) did not follow the present Eurosiberian/Mediterranean divide, and (b) given the shared aspects of stone tool technology and the widespread homogeneity in rock art styles observed through the Gravettian and most of the Solutrean all the way from Portugal, in the West, to the Rhone valley, in the East, did not represent significant barriers to the movement of people, the circulation of objects, or the exchange of ideas.", "We also know that, during periods of extreme aridity such as the episode of iceberg discharge known as Heinrich Stadial (HS) 4, which lasted for a few centuries around ca.40 ka, the kinds of semi-desert environments nowadays confined to northern Almeria and southern Murcia expanded to the Mesetan hinterland and the badlands of the middle and upper Ebro basin (d’Errico and Sánchez-Goñi, 2003; Sepulchre et al., 2007).", "Conversely, during periods of milder, wetter climatic conditions such as Greenland Interstadial (GI) 8 (ca.38.2–36.6 ka), mountain forests and wooded landscapes underwent a very significant expansion below the latitude of 40°N (Fletcher et al., 2010).", "Judging from what happened in the Holocene, during such milder periods human settlement must have retracted to the resource-richer littoral areas, leading to the breaking-up of exchange and communication networks, and favoring the emergence of cultural/biological isolates.", "Based on this evidence, the “Ebro Frontier” model hypothesizes that steppe-tundra environments would have been continuously present in Northern Iberia through the entire MP-UP transition process and that, during this period, the Ebro basin would have functioned as a major physical and biogeographical divide due to: (a) the establishment of semi-desert conditions in the basin itself, the northern flanks of the Iberian Range, and the Mesetan hinterland, in HS4, and (b) the development in adjacent lands to the South and West, both before and after this extreme aridity event, of extensive mountain forests and open woodlands.", "At present, this hypothesis remains difficult to test, because the paleoenvironmental data available are insufficient to reconstruct, with the spatial and temporal resolution required, the impact of these climatic oscillations on the ecosystems of the territory across which the environmental gradient developed.", "However, the divergent cultural-historical trajectories followed either side of the “Ebro Frontier” after ca.45 ka — namely, the failure of the Châtelperronian, the Protoaurignacian and the Aurignacian I to extend southward — do imply the presence of a major, long-lasting barrier to migration, gene flow and diffusion.", "The spread of the Aurignacian II into Southern and Western Iberia signals the disappearance of the conditions underpinning the preceding pattern of cultural divergence, whatever their cause.", "That paleoenvironmental factors must have played a role is nonetheless intimated by the temporal coincidence of the replacement of Iberia’s late-persisting Mousterian (ca.36.5–37.1 ka) with the global climatic transition from GI 8 (the longest and mildest of all MIS 3 insterstadials) to Greenland Stadial (GS) 8 (a “normal” cold phase) (Rasmussen et al., 2014).", "During this transitional period, the Eurosiberian steppe-tundra could and likely did begin to spread into the Iberian core, while the charcoal from sub-complex AS1 of Cueva Antón (12% cryophilous pines, 85% steppic taxa, 3% riverside taxa; Zilhão et al., 2016: Fig. 8, SI Table 2) indicates a descent of the montane pine forest belt from above 1100 m to below 400 m, in agreement with the near disappearance of Mediterranean forest taxa seen at this time in the deep-sea pollen record (Fig. 9).", "The presence of a major biogeographical gradient along the Ebro basin acquires broader paleoanthropological significance because of the period when it happened to be separating modern humans and Neandertals.", "In and of itself, however, the existence at that time of such a gradient, with attendant implications for diffusion and exchange, in no way should be mistaken for something exceptional or unique.", "After the LGM, for instance, the Ebro basin would come to separate moderns (Badegoulian and Early Magdalenian) from other moderns (Upper Solutrean and Solutreo-gravettian) for a comparable duration — three to four millennia (Banks et al., 2009).", "Conversely, prior to 42 ka the Ebro basin had already been separating Neandertals (Châtelperronian) from other Neandertals (Mousterian) — and may well have continued to do so for another couple thousand years if Neandertals were also involved in the manufacture of the Protoaurignacian.", "The Protoaurignacian is well documented along the shores of the Cantabrian Sea, from the Basque sites of Isturitz and Labeko Koba in the East to the Asturian site of La Viña in the West (Zilhão, 2006a).", "Even though no archeologically associated diagnostic human remains have so far been found across the Protoaurignacian’s entire geographical range (Bulgaria to northern Spain) and temporal span (39–42 ka), the genome of the Oase 1 adult male shows that he had had a “pure” Neandertal ancestor only four to six generations back (Fu et al., 2015).", "Combined with the age of the fossil (directly dated by radiocarbon to 37.1–41.4 ka) (Trinkaus et al., 2013), this genomic evidence implies a strong probability of overlap between Neandertals and at least the beginnings of the Protoaurignacian.", "The latter’s industrially “intrusive” characteristics and similarity with the Near Eastern, modern human-associated Early Ahmarian suggest an intrinsic relation to modern human immigration.", "The technological innovations the Protoaurignacian stands for, however, could well have diffused into Neandertal territory well in advance of the arrival of the admixture front.", "Since no evidence exists that an “archeological culture = human type” equation applies to the Protoaurignacian, it remains entirely plausible, therefore, that it was also made by variously mixed Neandertal-modern human, or even “pure” Neandertal populations — and especially so in the West (Trinkaus and Zilhão, 2013; Zilhão et al., 2015).", "If Neandertals were also involved in the making of the Protoaurignacian, then it is only in Aurignacian I times, after 40 ka, that the Ebro basin represented a Neandertal/modern human “frontier.”", "If so, the emergence of such a “frontier” would have been broadly coincidental with the 39.9 ka explosion of the Phlegraean Fields caldera, whose ash fall-out blanketed vast stretches of Italy and Southeastern Europe, severely disrupting food chains for an extended period — the highest trophic levels, including human hunters, being most impacted.", "For the populations of Western Europe, which was not directly affected, the main consequence of the explosion would have been to bring about a release from the constraints of demographic pressure induced across the continental landmass by the previous millennia of population growth and Neandertal assimilation.", "In this scenario, the explosion would have constituted a historically contingent but significant factor contributing to explain why Middle Paleolithic Neandertals persisted for so long in the territories of Europe’s Far West (Zilhão 2009; Fitzsimmons et al., 2013; Marti et al., 2016; Giaccio et al., 2017).", "Whether, at the time of this catastrophic event, the Neandertal/modern admixture front had already reached the Pyrenees and the Cantabrian strip for quite some time or had just arrived there remains an open issue.", "But, whichever the case, the explosion’s impact on the modern human populations of Central and Eastern Europe would have stalled the westward expansion of the front after ca.40 ka.", "If a biogeographical gradient was then extant across the Ebro basin, the demographic crisis caused by the Phlegraean Fields explosion would have enhanced that gradient’s effect.", "And if, with the return to normal stadial conditions, following the end of GI 8, that effect ceased to operate, it would have done so at a time when replenishment of the Central/Eastern European sink created by the explosion would also have reset demographic pressure over the peripheries.", "For Northern Europe, the consequence would have been resettlement.", "For Iberia, it would have been the eventual assimilation of the last of Europe’s Neandertals, as postulated by the “Ebro Frontier” model.", "Both expectations are met by the empirical record.", "The technological and use-wear evidence rejects interpreting layer I-k of Cueva Antón and occupation horizons OH20 and OH19 of La Boja as distinct structural poses of a single, multifaceted system.", "Put another way, the small lithic assemblage in layer I-k of Cueva Antón cannot be interpreted as a functionally specialized, or activity-specific facies of the region’s Evolved Aurignacian.", "Instead, layer I-k of Cueva Antón and occupation horizons OH20 and OH19 of La Boja stand for concrete manifestations of mutually exclusive, long-lasting technologies whose succession, rather than a gradual transition, truly consisted of an abrupt replacement.", "As the efficiency of stone tool production in terms of cutting edge per unit of mass is identical in both technologies (Muller and Clarkson, 2016), the parsimonious reading of this replacement process is that it represents a major break, with demic underpinnings, in regional cultural trajectories.", "The evidence from stone tool technology and the stratigraphic layout of sites is that the pattern derived from the high-precision Mula basin data can be extrapolated to all Iberian regions to the South of the Ebro basin.", "In these regions, artefact assemblages attributable to the earliest phases of Western Europe’s Upper Paleolithic are missing from stratified sites that contain deposits spanning the MP-UP transition, and have never been found as single-component, open-air contexts.", "In addition, no isolated occurrences of their index fossils (e.g., Châtelperronian points/knives, or Aurignacian split-based bone points) have ever been reported among surface, mixed, or post-depositionally disturbed deposits.", "From the basics of Prehistoric Archeology, i.e., from the culture-stratigraphic reasoning providing the framework for all its chronologies, the only inference that one can derive from this pattern is that, southward of the Ebro basin, a late-persisting Mousterian occupies the time slot in which the Aurignacian I is found elsewhere.", "The radiocarbon evidence is entirely consistent with this notion, which available luminescence and U-series independently support, and which no other kinds of radiometric dating results have so far countered.", "A corollary of these findings is that Neandertals persisted until ca.37 ka across Southern and Western Iberia — which carries implications for the authorship of all other aspects of these regions’ archeological record.", "For instance, given their dating and archeological associations, there can be no question that the painted/perforated shells from Cueva Antón and Cueva de los Aviones, as well as the abstract engraving and ornamental use of raptor feathers documented at Gorham’s Cave, stand for manifestations of Neandertal symbolism (Zilhão et al., 2010a; Finlayson et al., 2012; Rodríguez-Vidal et al., 2014).", "Knowing that minimum ages of 40.8 ka for a red disk and 37.3 ka for a hand stencil have been obtained at El Castillo cave (Cantabria) (Pike et al., 2012), and that such motifs exist in Extremaduran and Andalusian sites, it is easy to see how the “Ebro Frontier” pattern may also bear implications for the authorship of cave paintings.", "Recent advances in the field of Genetics increasingly make it clear that, in the Late Pleistocene of Eurasia, the continental extension of rather homogeneous archeological cultures is superimposed on complex ancestry patchworks (Mallick et al., 2016; Pagani et al., 2016).", "This can be explained by a pattern of long-distance diffusion and cultural resilience, which maintained networks over the long-term, combined with extended periods of geographical isolation, which conserved regional genetic variants.", "The “Ebro Frontier” effect makes this mechanism apparent even in the refugia of Southern Europe and especially so at the time of the MP-UP transition.", "This visibility is due to when the frontier formed and for how long it lasted, both allowing the effect to be picked-up with the current resolution of dating techniques.", "Likely, however, similar, broadly coeval but chronometrically less visible Late Pleistocene frontiers must have existed in other parts of Asia and Europe, as well as during the earlier phases of the process of modern human dispersal into these continents.", "The results we report here highlight the need for proper integration of the biological and the archeological evidence when reconstructing Late Pleistocene population histories.", "All lines of evidence are now converging to support replacement-through-admixture, or Assimilation, as the best explanation for the disappearance of the Neandertal and other archaic phenotypes.", "The Iberian evidence suggests this was a time-transgressive evolutionary outcome stemming from dynamic, complex and geographically uneven processes — a punctuated history in which the long-term maintenance of pan-continental networks of gene flow and cultural exchange did not exclude the occurrence of extended periods of significant geographical isolation.", "Excavation proceeded through décapage along observed boundaries, whether natural (e.g., the interface with the underlying geological stratigraphy) or anthropogenic (e.g., the base of distinct occupation floors stacked up within a single natural stratigraphic unit), with subdivisions when necessary.", "Finds were piece-plotted with the help of a laser level, to the nearest centimeter, against grid and site datum.", "Use-wear analysis of stone tools was based on differential interference contrast microscopy, carried out with a BHMJ Olympus model (at × 200 or × 400 magnification), and followed standard recommendations for the cleaning and preparation of the material.", "Large samples of the sediment were floated for the recovery of paleobotanical data; the remainder was entirely dry-sieved using two-sieve stacks (2 and 1 mm mesh-sizes).", "The analysis of pollen, charcoal, mollusk shell and animal bone followed standard protocols.", "Stratigraphic cross-sections were geologically described, drawn and digitally recorded, as were the surfaces exposed at each step of the décapage process.", "At Finca Doña Martina, the DStrectch plug-in for ImageJ was used to highlight color contrasts and produce prints used in the field to help with the décapage of stratigraphic interfaces.", "Photo mosaics were assembled using PT GUI or Microsoft ICE and orthorectified with the University of Venice’s RDF software.", "Elevation maps and 3D models were produced with Surfer.", "Undisturbed soil and bulk sediment samples were collected for micromorphological, phytolith and biomolecular analysis.", "Only securely provenanced, taxonomically classified charcoal samples were submitted for dating.", "All samples were treated with the ABA protocol, and the humic fractions of several samples were also measured (Wild et al., 2008).", "For Finca Doña Martina’s, a milder treatment was used for some, due to poor preservation; in most cases, only the humic fraction could be dated.", "The results for this site are therefore all minimum ages.", "At Cueva Antón, the ABA treatment proved insufficient to remove all contamination, but the chronology of layer I-k reported here is entirely based on results obtained for samples that were processed with the ABOx-SC protocol (Zilhão et al., 2016).", "To check if a similar problem existed at La Boja, some of its samples were also processed with ABOx-SC, in parallel to the standard ABA treatment (Wild et al., 2008) and using a modified version of the procedure given in (Brock et al., 2010), i.e. acid and base treatment at 60 °C.", "In addition, to control for the accuracy of individual measurements, some ABA-treated samples were dated twice.", "The ABOx-SC results and the repeats were in all cases statistically indistinguishable from the original ABA date.", "When more than one result for a single charcoal fragment was obtained, the corresponding average was used.", "Calibration was carried out with the INTCAL13 curve in Calib 7.0 (Stuiver and Reimer, 1993; Reimer et al., 2013).", "The Fig. 9 plot was prepared in OxCal 4.2.4 (Bronk Ramsey, 2009).", "The ADB samples were extracted from macroscopically homogeneous silt-rich deposits (Fig. S4.9).", "Due to the unconsolidated nature of trench walls, it was decided not to drive metal cylinders into the sediment; instead, the samples were extracted with a knife, in complete darkness.", "Coarse grain quartz (100–150 μm) and potassium feldspar (100–200 μm) were extracted using conventional sample preparation techniques (Kehl et al., 2016).", "All measurements were carried out on an automated Risø TL/OSL DA 20 reader equipped with a calibrated 90Sr beta source and an EMI 9235 photomultiplier.", "Multiple-grain quartz samples were measured using the single-aliquot regenerative-dose protocol (SAR) (Murray and Wintle, 2000; Murray and Wintle, 2003), including signal stimulation by blue diodes (470 nm, FWHM = 20) and signal detection through a Hoya U340 filter.", "The initial 0.8 s of the signal minus a background of the last 5 s was used for quartz dating.", "Preheat plateau and dose recovery tests were carried out to check the suitability of the measurement protocol.", "Single-grain quartz dating was not feasible because of low signal intensities.", "Multiple-grain potassium feldspar samples were measured using the post-infrared infrared stimulated luminescence signal measured at 290 °C (pIRIR290) (Thiel et al., 2011).", "Stimulation was carried out with infrared diodes (870 nm, FWHM = 40), and the signals were detected through an interference filter (410 nm).", "The initial 4 s of the signal minus a background of the last 20 s was used in the pIRIR dating.", "Prior IR stimulation temperature tests and dose recovery tests (24 h Hönle Sol2 bleaching) were carried out to check the performance of the measurement protocol.", "Equivalent doses were calculated using the arithmetic mean (AM), except for sample C-L3905, for which we also used the minimum age model (MAM) (Galbraith et al., 1999).", "Additionally, infrared stimulated luminescence measured at 50 °C (IR50) was applied (Wallinga et al., 2000; Preusser, 2003), and the signal was corrected for anomalous fading using the approaches of Auclair et al. (2003) and Huntley and Lamothe (2001).", "Data analysis was carried out using the R luminescence package (Burow, 2017; Kreutzer, 2017; Kreutzer et al., 2017).", "The radionuclide concentrations of the surrounding sediments were measured using high resolution gamma-ray spectrometry.", "The dose rate was calculated using Dose Rate and Age Calculator (DRAC) (Durcan et al., 2015), and included conversion factors (Guérin et al., 2011) and an assumed water content of 5 ± 2%.", "The internal beta dose rate contribution of the feldspar samples was calculated by assuming a potassium content of 12.5 ± 0.5% (Huntley and Baril, 1997).", "The cosmic dose rate was calculated after Prescott and Hutton (1994).", "Dose distributions are displayed as abanico plots (Dietze et al., 2016) (Figs. 6 and 7).", "Equivalent doses calculated with the arithmetic mean and the Central Age Model (CAM) are statistically indistinguishable at 1σ and finally the arithmetic mean was used.", "A typical dose response curve and a decay curve are shown for quartz sample C-L3905 (Fig. 7a).", "Preheat plateau tests (Fig. 7b) indicated that the equivalent dose of the quartz is independent from temperature treatment in the ranges 180–240 °C (C-L3901), 220–280 °C (C-L3904), 180–280 °C (C-L3905), and 240–280 °C (C-L3906).", "Dose recovery tests showed that a laboratory given dose was best recovered using a temperature of 180 °C for samples C-L3901 and C-L3905 and of 260 °C for samples C-L3904 and C-L3906 (Fig. 7c).", "Prior IR stimulation temperature tests carried out for feldspar sample C-L3905 indicated a plateau between 80 °C and 180 °C (Fig. 7d).", "Laboratory doses were recovered with a ratio of the measured to the given dose of 1.07 ± 0.06 (a residual dose of 5 Gy after 24 h of bleaching in the Hönle Sol2 solar simulator was subtracted).", "A representative dose response curve for this feldspar sample is shown in Fig. 7e and the dose distribution in Fig. 7f.", "The laboratory experiments confirmed the suitability of the measurement protocols for both quartz and feldspar minerals.", "Except for sample C-L3903, the quartz OSL age estimates are in stratigraphic order, scatter between 57.7 ± 3.2 ka and 32.6 ± 1.9 ka, and are consistent with the radiocarbon ages obtained for the same units.", "pIRIR290 and IR50 dating was carried out to investigate if the quartz OSL signal was likely to be fully bleached at the time of deposition.", "An internal crosscheck of the two minerals is advisable (Murray et al., 2012) because the pIRIR290 and IR50 signals bleach slower than the quartz OSL signal (Buylaert et al., 2012).", "Comparison of the mean age estimates of all three luminescence signals shows good agreement between the quartz OSL and feldspar IR50 and pIRIR290 ages of sample C-L3901.", "For sample C-L3905, the quartz (35.8 ± 2.8 ka) and IR50 age estimates are younger than the pIRIR290 age (45.4 ± 5.6 ka), which indicates incomplete bleaching of the feldspar pIRIR290 signal at deposition.", "This is supported by the good agreement of the quartz OSL and feldspar IR50 results with the calibrated radiocarbon age (34.9–37.1 ka; VERA-5854) obtained for the same stratigraphic unit, confirming complete bleaching of the OSL and IR50 signals.", "Applying a MAM to the feldspar pIRIR290 dataset results in an age of 37.4 ± 5.3 ka, which demonstrates that the MAM successfully extracts individual equivalent dose values from the distribution that are likely to be fully bleached at deposition.", "For samples C-L3902 and C-L3904, the pIRIR290 age estimates tend to overestimate the quartz and IR50 results.", "It was not possible to extract those individual equivalent doses from the distribution that are likely to have been completely bleached prior to deposition using the MAM.", "Quartz sample C-L3903 appears to be underestimated compared to the underlying samples and we value this result as an outlier.", "João Zilhão: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.", "Diego Angelucci, Valentin Villaverde, Josefina Zapata: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data.", "Daniela Anesin, Thierry Aubry, Ernestina Badal, Dan Cabanes, Martin Kehl, Nicole Klasen, Armando Lucena, Ignacio Martín-Lerma, Susana Martínez, Henrique Matias, Davide Susini, Peter Steier, Eva Maria Wild: Performed the experiments; Analyzed and interpreted the data.", "The authors declare no conflict of interest.", "Archaeological fieldwork and research at Cueva Antón and the Rambla Perea rock-shelters was funded by the Dirección General del Medio Natural de la Región de Murcia, the Municipality of Mula, the University of Murcia, the Fundación Séneca (Murcia), the Ministerio de Ciencia e Innovación (grants HAR2011-24878, HAR2014-52671-P and CGL2012-34717), the Generalitat Valenciana (grant PROMETEOII/2013/016), the Excellence Research Projects Program of the Andalusian Government (grant P11-RNM-7033), and the Leakey Foundation.", "The German Research Foundation’s (DFG) project CRC 806 (“Our Way to Europe.", "Culture-Environment Interaction and Human Mobility in the Late Quaternary”) funded the luminescence dating.", "No additional information is available for this paper.", "Supplementary content related to this article has been published online at http://dx.doi.org/10.1016/j.heliyon.2017.e00435" ]

[]

[ "ENVI", "SOCI" ]

[ "Baltic herring", "Baltic salmon", "Fisheries governance", "Socio-cultural values", "Stakeholder involvement" ]

[ "2308", "3305" ]

[ "The way in which fish resources are utilized and managed reflects the socio-cultural values associated with fish and fisheries.", "Different species have different meanings in different contexts and groups (Lam, 2015; Levin et al., 2016).", "Values also affect management decisions, either implicitly or explicitly.", "Thus, governance implies choosing between or reconciling multiple and sometimes conflicting values (De Young et al., 2008; Ives and Kendall, 2014; Kooiman and Jentoft, 2009).", "Furthermore, values also influence the design of institutions and decision-making procedures (Kooiman and Jentoft, 2009).", "However, some values seem to be more actively discussed and promoted in fisheries governance than others.", "Song et al. (2013) found ecosystem conservation, wealth and knowledge to be the most prevalent values in fisheries governance discourse, overall.", "Equality, secure livelihoods, achievement, tradition and influence were discussed to a lesser extent, whereas several other values were rarely acknowledged.", "The less discussed values may nevertheless be important for stakeholders, and ignoring them may reduce the social acceptability of management activities (Ives and Kendall, 2014).", "Therefore, addressing the whole range of values could reinforce the knowledge base of management and enhance the legitimacy of decisions (Linke and Jentoft, 2014; Samhouri et al., 2014).", "In this paper, we explore the multivalued nature of fish resources and discuss its implications for governance.", "We do this by empirically analyzing the values that stakeholders in Finland and Estonia associate with two regional keystone species, Baltic herring and salmon.", "The aims of the paper are: 1) to obtain an overview of the ways in which these fish species are important in different contexts; 2) to discuss the ways in which values are represented in fisheries governance and management at the national level in Finland and Estonia; and 3) to reflect the possibilities to systematically and explicitly engage values in governance.", "We define the values relating to a fish resource as evaluations of the importance of the fish and the fishery to a society (Ignatius and Haapasaari, 2018).", "This definition refers to the socio-cultural dimension of values, and implies a focus on the shared ways in which the fish and the fisheries are important to human communities, and does not view values as something inherent to the environment or to individuals (see also Tadaki et al., 2017).", "We assume that people refer to the shared and context-dependent values when they think about the societal importance of something and communicate it.", "We apply the theory of justification of Boltanski and Thévenot (2006, 1999), which recognizes seven widely shared value bases from which people draw when considering the societal importance of something.", "These value bases are: 1.", "Civic values that reflect solidarity, equality and common welfare, 2.", "Green values that respect nature in its own right and consider conservation important, 3.", "Domestic values that build on traditions and orientate towards preserving the customary past, 4.", "Inspirational values that are founded on an immediate relationship with the environment and the emotions it awakes, 5.", "Fame values that emphasize signs of popularity and visibility, 6.", "Market values that stress the acquirement of money and wealth, and 7.", "Industrial values that highlight the importance of the efficient utilization of resources and the importance of scientific knowledge in the management of the resource (Boltanski and Thévenot, 2006, 1999; Thévenot et al., 2000).", "Although these value bases are widely shared by Western societies, their resonance and interpretation in particular cases may differ depending on context (Ylä-Anttila and Luhtakallio, 2016; Ylä-Anttila and Kukkonen, 2014).", "Therefore, values need to be analyzed in their context.", "We begin with describing the case studies of Baltic salmon and herring fisheries.", "We then present the methodology that we used to analyze the values related to these species.", "The results section presents the value themes discussed by the Finnish and Estonian interviewees.", "After this, we summarize and compare the cases, and discuss the implications for governance.", "Baltic salmon is one of the top fish predators of the Baltic Sea.", "Salmon are born in rivers, migrate to the sea to feed and return to spawn in their natal river.", "During the 20th century, the majority of the Baltic salmon stocks were destroyed or depleted due to the damming of rivers, habitat destruction, pollution and intensive fishing (Romakkanieni et al., 2003).", "The Salmon Action Plan, introduced by the International Baltic Sea Fisheries Commission (IBSFC) in 1997, implied dealing with the stock declines in selected rivers in different Baltic Sea countries through fishing restrictions, stocking and habitat restoration.", "Efforts have also been made to restore salmon stocks in former salmon rivers that were dammed for hydroelectric power production in the middle of the 20th century (Erkinaro et al., 2011).", "Finland currently has two rivers – Tornionjoki and Simojoki – with naturally reproducing wild salmon stock (International Council for the Exploration of the Sea (ICES, 2017).", "The river Tornionjoki is the most important wild salmon river in the whole Baltic Sea.", "Minor natural reproduction has been observed in a few other rives in Finland.", "In Estonia, small-scale wild salmon reproduction takes place in three rivers: Kunda, Keila and Vasalemma (International Council for the Exploration of the Sea (ICES, 2017).", "In addition, in several other rivers reared salmon is released to support natural reproduction.", "Owing to tightened restrictions to protect stocks, commercial salmon fishing has significantly decreased in the last decades (International Council for the Exploration of the Sea (ICES, 2017).", "In 2016, the total allowable catch set by the EU for commercial fishing was 109 034 Baltic salmon, of which Finland’s share was 36 549 salmon (34%) and Estonia’s share 3364 salmon (3%).", "In Finland, commercial salmon fishers operate on the coast and mostly use trap nets.", "The majority of the catch is taken from the Gulf of Bothnia, and a smaller share is caught in the Gulf of Finland.", "In Estonia, salmon is mainly by-catch in the coastal fishery and the majority of salmon is caught by gillnets in the Gulf of Finland (International Council for the Exploration of the Sea (ICES, 2017).", "As commercial fishing has decreased, recreational salmon fishing has increased (International Council for the Exploration of the Sea (ICES, 2017).", "In Finland, the recreational salmon catch was around the same size as the commercial catch in 2016.", "In Estonia, recreational fishers take about one-third of all caught salmon.", "In both countries, angling in rivers is the main form of recreational salmon fishing.", "In Finland, recreational fishers target salmon particularly in the rivers Tornionjoki and Simojoki, which flow into the Gulf of Bothnia, but also in the river Kymijoki that flows into the Gulf of Finland.", "In Estonia, recreational fishing takes place in small rivers flowing into the Gulf of Finland (International Council for the Exploration of the Sea (ICES, 2017).", "Baltic herring is a keystone fish species in the Baltic Sea ecosystem because of its high abundance and its role in the pelagic food web (Casini et al., 2010).", "It is distributed all over the Baltic Sea.", "Herring spawns in coastal waters, and its feeding and spawning migrations are complex.", "(Helsinki Commission (HELCOM, 2013).", "Herring is the most abundant commercial catch species in the Baltic Sea, and stocks are currently considered to mostly be at a biologically sustainable level (International Council for the Exploration of the Sea (ICES, 2018).", "Baltic herring is caught by commercial fishers in all the riparian countries.", "In 2016, around 357 000 tons of Baltic herring (excluding western stocks) was caught, of which Finland’s share was around 137 000 tons (40%) and Estonia’s around 36 000 tons (10%) (International Council for the Exploration of the Sea (ICES, 2018).", "In both countries, Baltic herring is the most important catch species in terms of volume and monetary value.", "The most important herring fishing area for Finland is the Gulf of Bothnia.", "The majority of the Estonian herring catch is fished in the Gulf of Riga and the Gulf of Finland.", "Both countries also catch herring in other parts of the northern and central Baltic Sea (International Council for the Exploration of the Sea (ICES, 2016).", "In Finland, 90–95% of the herring catch is taken by trawlers (Ministry of Agriculture and Forestry of Finland, 2017).", "Estonia trawled around 70% of its herring catch in 2015 (International Council for the Exploration of the Sea (ICES, 2016).", "Coastal fishing with nets and trap nets also takes place during spawning time.", "In Finland, coastal herring fishing takes place along the coast of both the Gulf of Bothnia and the Gulf of Finland and in the archipelagos.", "In Estonia, the Gulf of Riga is the most important area for coastal herring fishery, but herring is also fished in other coastal areas and on the islands (International Council for the Exploration of the Sea (ICES, 2016).", "The use of the catch differs by country and over time, and is largely driven by market conditions (International Council for the Exploration of the Sea (ICES, 2016).", "In Finland, the majority of the catch is currently used as feed for fur animals, but in recent years the use of herring for the production of fish meal and oil has increased.", "In Estonia, most of the catch is exported to eastern European markets to be used in the canning industry.", "The domestic human consumption markets for Baltic herring are small in both countries.", "On average, the Estonians eat more herring than the Finns (BONUS GOHERR, 2016).", "In Finland, the consumption of herring has significantly decreased in recent decades, from about 1.5 kg/person/year in 2000 to 0.3 kg/person/per year in 2016 (Natural Resources Institute Finland, 2017).", "Annual decisions regarding total allowable catches (TAC) for commercial salmon and herring fisheries are taken at the EU level, whereas recreational fisheries are managed at the national level.", "The decision proposals for the TACs are based on scientific stock assessments and related management advice, prepared by the International Council for the Exploration of the Sea (ICES).", "Stakeholders are consulted through the Baltic Sea Advisory Council (BSAC), a regional stakeholder organ representing the commercial and recreational sectors, environmental non-governmental organizations (ENGOs) and a few smaller groups.", "At the national level, each EU member state has its own structures for fisheries management.", "In Finland, fisheries are under the jurisdiction of the Ministry of Agriculture and Forestry.", "In Estonia, the Ministry of the Environment is responsible for stock protection and quotas, whereas the Ministry of Rural Affairs divides the fishing possibilities for commercial fishers and collects fishing data.", "In both countries, commercial and recreational fishers are consulted for management proposals.", "In Finland several ENGOs and associations representing the people living along the salmon rivers also actively engage in fisheries management.", "In both countries, Fisheries Local Action Groups (FLAGs) have been organized to obtain support from the EU to develop the viability of the local communities.", "In recent decades, the management of Baltic Sea fisheries has generally been a contentious issue both at the national and EU level.", "For example, the annual setting of TACs has been disputed (Linke et al., 2011).", "The management of Baltic salmon fisheries in particular has been an issue of conflict between geographical areas, commercial and recreational fishers, and conservationists and resource users (Linke and Jentoft, 2014; Salmi and Salmi, 2010), especially in Finland (Salmi and Salmi, 2010).", "In recent years, Finland’s Ministry of Agriculture and Forestry has tried to ease these disagreements by developing a national salmon strategy in collaboration with stakeholders.", "We analyze the values related to Baltic salmon and herring in Finland and Estonia by applying the justification theory (Boltanski and Thévenot, 2006, 1999).", "This theory represents a communicative approach to values, as it sees them in terms of a common good that is constantly evaluated and negotiated.", "For example, whenever there is a public dispute or a need to provide a good reason for a public decision, people have to emphasize the generality of their arguments.", "In other words, it is not enough for people to consider why something is important for themselves; they must also consider how it might be important for society in general.", "People need to justify their claims by referring to the common good, and other people assess the appropriateness of these claims by using the common good as an evaluative standpoint (Boltanski and Thévenot, 2006, 1999; Ignatius and Haapasaari, 2018).", "The need to justify one’s arguments by referring to the common good is often visible in a decision-making setting, where people with different views and interests meet and are expected to find a common resolution.", "References to the common good may also be made in public discussions, for example, in the media.", "In fact, a person needs to consider the common good and value-based justifications whenever forming a position on the societal importance of something and communicating it (See also Thévenot et al., 2000).", "We analyzed how stakeholders justify the importance of Baltic salmon and herring based on the common good in an interview setting.", "The interview material represents the positions and views of different stakeholders on the importance of these species to society.", "We interviewed 30 stakeholders (17 in Finland and 13 in Estonia) during 2015–2016.", "The interviewees represented commercial and recreational fishers, fishing and rural tourism, ENGOs, fisheries scientists, managers, and local people involved in fisheries.", "The interviewees were selected from organizations that take part in national fisheries governance.", "In regards to commercial and recreational fishing sectors, the emphasis was on regions in which Baltic salmon and herring are important in terms of the number of fishers and the quantity of catches.", "We used a semi-structured interview guide (Bernard, 2002) to ensure that all participants were asked the same questions.", "The interviewees were asked how and why they consider Baltic salmon/herring important for their community/reference group, for other communities and for society in general.", "They were also invited to consider potential counter-arguments and the viewpoints of other groups.", "The seven value bases of the justification theory were used to formulate the thematic questions to further discuss the importance of these species.", "The themes included fairness of management (civic), conservation (green), traditions (domestic), emotional and recreational importance (inspiration), symbolic importance (fame), economic importance (market), and the utilization of the resource and the role of science in management (industrial).", "For example, regarding the importance of traditions, we asked the interviewees whether they were aware of any traditions related to these species and to consider their socio-cultural value.", "The duration of the interviews ranged from 60 to 150 min.", "They were transcribed and grouped into themes according to the seven value bases.", "In the analysis we paid attention to which value-themes were the most discussed and why, and which were not so often referred to and why.", "We also traced similarities and differences between the value-discourses of the two contexts.", "Among the Finnish stakeholders, fairness of management was often a dominant theme in the interviews.", "The interviewees acknowledged that although salmon politics has been conflicted for centuries, stakeholder groups have multiplied and diversified in recent decades.", "The commercial fishers felt that their position had deteriorated due to this development: “Our numbers have decreased, and we are scattered along the coast.", "Therefore, our voice is becoming more and more marginalized, although we are formally consulted.”", "The other groups, including ENGOs, recreational fishers and people living along the salmon rivers, argued that management has become more balanced and just than earlier: “For a long time, commercial fishing at sea was a top priority for the ministry and it didn’t take into account whether some other parties also had right to the natural resource.", "This has now changed, quite totally.”", "These groups considered that the current situation has required and will continue to require active work.", "Fairness was mostly discussed in the context of national fisheries management, but a few commercial fishers and people living along the salmon rivers also highlighted fairness between the Baltic Sea states as an important issue.", "According to them, countries in which salmon rivers are located should have priority rights in salmon fishing, because they have the biggest responsibility for the stocks.", "The Finnish interviewees also frequently discussed conservation values, although conservation was often compromised with utilization values.", "They stressed biologically sustainable stock management.", "The importance of conserving wild salmon stocks was highlighted by the interviewed scientists, representatives of ENGOs, recreational fishers and people living along the salmon rivers.", "They pointed out that emphasis on conservation values has grown in recent years, as commercial quotas have decreased: “In recent years, quotas have safeguarded the stocks quite well, at least in the largest rivers.", "Of course, some of the biological diversity has been lost, but some has also been restored.”", "These groups also stressed the need for more stock-specific fishing and the restoration of river habitats to support the conservation of stocks.", "The commercial fishers generally agreed that safeguarding stocks is important, but also felt that it has been done at their expense.", "They saw open sea fishing, river habitat problems and seals as the main threats to stocks: “Commercial salmon fishing in Finland is not a threat to the stocks because it is coastal trap-net fishing, unlike in the Main Basin, where it is unselective long-line fishery.”", "The majority of the interviewees acknowledged the tradition value of Baltic salmon for the northern salmon river valleys and certain coastal areas.", "Salmon was described as an appreciated seasonal food among the local communities, a cornerstone for the coastal fishing livelihood and part of the local culture.", "In a salmon river area, Baltic salmon was considered an essential part of cultural heritage: “In the old times and even in the 1900s, salmon fishing was a major source of wealth for our area, which heavily relied on fishing, gathering and hunting.", "I think the current importance of salmon stems from this tradition.", "It has been caught here for ages and there is a lot of folklore and documents about it.”", "The commercial salmon fishers emphasized the regional importance of salmon as a seasonal dish, which they help to deliver: “At Midsummer people want to have salmon on their tables, it’s a tradition.”", "Salmon seemed to inspire strong emotions among all Finnish interviewees.", "This inspirational value was linked to the struggle between man and nature and between different groups.", "Salmon was seen as something unique and a source of both joy and sorrow.", "The recreational fishers also viewed salmon as an exciting, impressive sight and a provider of experiences: “Catching a big salmon is like brawling with a man.”", "The people living along the salmon rivers explained that the meaning of salmon is mental or spiritual, and sensory observations confirming that salmon is present were regarded as important: “It is first and foremost an experience to be able to enjoy the power of salmon, something similar to Aurora Borealis.”", "Symbolic values were also attached to salmon, which was considered “the king of fish”, “as valuable as gold”, “a symbol of nature conservation”, “a manifestation of power”, “a pet topic for the media” and “a magnet that attracts different segments of society”.", "Symbolic importance seemed particularly strong in the northern regions.", "The interviewees, depending on their position, called Baltic salmon “salmon of sea area x” or “salmon of river x”.", "In the river areas, salmon was seen as a symbol of hope, and its spawning run was considered an important part of the annual cycle.", "One ENGO representative pointed out that Baltic salmon, as such a charismatic or iconic species, had worked as a flagship for the conservation of other threatened fish species.", "The interviewees also reminded us of the regional economic importance of salmon for coastal fishery and fishing tourism.", "The commercial fishers emphasized that their livelihood depended on salmon, which could bring over half of their yearly revenue.", "The people living along the rivers and the recreational fishers stressed the economic potential of fishing tourism.", "The majority of the interviewees stressed the importance of natural scientific knowledge for the management of stocks.", "However, the commercial fishers claimed that their experiential knowledge should be included in the knowledge base.", "Table 1 sums up the values related to Baltic salmon referred to by the Finnish interviewees.", "In Estonia, the interviewed managers, scientists and recreational fishers often highlighted conservation values, referring both to the sustainable management of the stocks and to the intrinsic value of salmon.", "One manager expressed a humble attitude towards managing the stocks: “Nowadays we can see that nature is the best manager, so what we could do is just help nature recover, so it can manage the natural level.”", "According to one scientist, salmon was an indicator of the wellbeing of the environment: “Salmon is closely connected to the rivers.", "If there are many salmon in the river, it is considered to be in good condition.”", "In relation to this, salmon was seen to symbolize wild nature.", "A recreational fisher regarded salmon as worth preserving for its own sake: “If there is salmon, nature has done its thing and if it’s there, let it be there.", "It is nature.", "Nature has its course, and we’re not allowed to mess it up.”", "Recreational fishers and one manager attached inspirational values to salmon, often combining them with conservation values.", "Seeing the stocks recovering with one’s own eyes was appreciated: “The place was full of big fish, and the view was like ‘wow’.", "I’ll never forget it.”", "Inspirational values were also attached to recreational salmon fishing: “It’s a very interesting fish to catch, because it protects itself very much and is very powerful.", "You must be very smart, you have to know the place and the time, and what equipment to use.”", "The managers and fishers discussed the fairness of management to some extent, but they did not see the representation of different interests in the national policy processes or the allocation of catching opportunities as a highly pressing issue.", "One manager noted: “Salmon has been a small issue; how to divide between recreational and commercial, who has a bigger right to catch salmon during spawning time.", "We’ve come to the decision that almost nobody is allowed to catch.”", "However, another manager raised the issue of fairness in relation to other Baltic Sea countries, claiming that commercial salmon fishing should be a coastal activity: “Then it would be really equal in terms of who really is active, and if you put an effort into the recovery you also see the results.”", "The biological knowledge base of the management of salmon stocks was, overall, regarded as essential and its legitimacy was not questioned.", "The interviewees rarely referred to the tradition value of salmon.", "The economic importance of salmon for commercial fishing was regarded as minor, but the economic potential related to coastal/nature tourism was occasionally mentioned.", "Table 1 summarizes the values related to Baltic salmon referred to by the Estonian interviewees.", "The most emphasized value theme among the Finnish interviewees was the economic importance of Baltic herring for commercial fishing in Finland.", "Economic value was linked to large catch quantities: “Baltic herring is the economic backbone of the Finnish fishing industry due to its volume.”", "The profitability of herring fisheries was an important theme for the commercial fishers.", "All the interviewees saw fish meal and oil industry as an opportunity to increase the economic value of the herring catch.", "One commercial fisher reflected: “Competition for the use of herring catches has emerged only recently, as the Danish fish meal factories have started to take herring.", "(…) Additional competition is always positive in economic terms.”", "All the interviewees considered the use of herring as human food and for the production of fish meal and oil as morally sound.", "They would prioritize the use of herring as food, but because of current very low human consumption, the use of herring as feed in fish farming was seen as the second-best alternative.", "However, the interviewees disagreed on the acceptability of using herring as feed for fur animals.", "A few representatives of the commercial fishing sector saw it as efficient and profitable utilization, but one ENGO representative criticized it from the perspective of global food security: “The majority of fish used for human consumption in Finland is imported.", "We consume mostly Norwegian salmon, which indirectly affects fisheries all over the world, also in those areas where local fish stocks are the most important protein source for local people.", "Then we have this one great, sustainable fish stock just nearby, which is fed to minks.", "The situation is absurd.”", "The commercial fishers acknowledged the importance of herring for food security, but they saw different usages as complementary to each other.", "The interviewees regarded the fishing and use of Baltic herring as an environmentally friendly activity.", "A commercial fisher reflected: “Fishing herring is a very natural source of livelihood.", "The fish stocks are abundant and given by nature itself.", "(…) If we fishers didn’t take care of the fish stocks in the long term too, we’d be mad.”", "Herring fishing was seen as a way to reduce nutrients from the Baltic Sea and herring as a more sustainable food choice than many other animal protein sources.", "The interviewees also believed Finnish herring fisheries to be sustainable.", "One representative of an ENGO commented: “I am proud of our Baltic herring stocks and their management.", "Bothnian Sea herring especially is one of the most sustainably managed fish stocks in the EU and in the world.”", "Fairness of management was an important value theme for the commercial herring fishers.", "They considered it crucial for the sake of justice that the sector would be more involved in the management of the fishery.", "The fishers also saw their involvement as a way of reinforcing the knowledge-base of management and improving the flexibility of management.", "The decision-making framework was seen as rigid and bureaucratic: “Bureaucracy in this sector is horrible.", "They don’t use us active fishers enough as experts.", "It often feels that civil servants act mainly on the basis of their own views.”", "The timeliness of the information used for stock assessments was also considered problematic: “The herring stocks are renewed so quickly that scientific assessments are often outdated by the time they’re published.", "(…) Many of us fishers would like to help, for example, take samples or collect up-to-date information.”", "Other value themes were discussed less in relation to herring.", "Herring was still considered to be a part of coastal culture as traditional food, which also connects people to the sea, for example, through the annual herring markets that have been held in some coastal towns for centuries.", "Symbolically, herring was seen as the ‘people’s fish’, but the image of herring as food was considered poor.", "Inspirational values were sometimes linked to the taste of herring, while the importance of herring for recreational fishers was considered small.", "Table 2 summarizes the values related to Baltic herring referred to by the Finnish interviewees.", "As in Finland, the Estonian interviewees often linked the importance of Baltic herring to its economic value for commercial fishing.", "They also emphasized the importance of herring for the processing industry: “Economically it’s very important for fishermen and for the processing industry that freezes it and sells it.", "Actually, we have vertical integration in the sector, the fishermen’s own processing and freezing and selling of it.", "They’re covering all the chains.", "That makes it more profitable for them.”", "The regional economic importance of the coastal herring fishery was also stressed: “Herring is number one in volume and income.", "In all the numbers.", "(…) It is a kind of a backbone for the fisheries’ community.”", "Although trawl fisheries have been concentrated into large units, the fishers were still worried about profitability.", "The majority of catches is sold to eastern European markets for human consumption, but some fishers pointed out the need to extend these markets to the fish meal and oil industry.", "Still, all the interviewees thought that human consumption should be prioritized over the industrial uses of herring.", "They emphasized that herring should be available for local and domestic consumption.", "Supporting the coastal traditions related to herring fishing and eating was deemed important.", "Herring consumption was also considered an issue of justice as it was seen as nutritious food that low-income people could also afford.", "Consequently, the use of herring in the production of fish meal and oil divided opinions.", "The commercial fishers wanted to see herring used mostly as human food, but found this difficult: “For us, the country that has been focusing mainly on fishing for human consumption, we would very much like to do this in the near future as well, but we see that the world is moving in a different direction.”", "Thus, they saw the fish meal and oil industry as a potential means of increasing the profitability and predictability of commercial fishing.", "Others had reservations: “All that comes from the sea must be mostly used as human food.”", "The environmental values related to Baltic herring were mostly discussed in the context of the sustainable management of stocks.", "The interviewees agreed that biological sustainability is among the top priorities of management, and that most herring stocks are currently at a sustainable level.", "One manager noted: “Baltic herring is caught at a maximum sustainable yield level, and based on this you could give it an environmental certificate.”", "The fisheries scientists expressed more caution, referring to uncertainty in stock assessments and variability in natural conditions.", "A commercial fisher stressed the importance of maintaining a balance in nature: “You have to be a good host.", "You must pay attention to how the stocks are doing.", "Otherwise you lose your entire livelihood.”", "All the interviewees acknowledged the tradition value of herring, particularly for the coastal and island communities.", "The tradition value was related to both herring fishing and herring consumption: “It’s been consumed here since forever, and I couldn’t imagine myself in Estonia or [name of community] without herring or without herring fisheries and herring meals.", "It’s important for cultural heritage, especially on the islands.”", "Some interviewees, including commercial fishers and scientists, referred to historical fishing documents and maps about fishing grounds, while others described the traditional know-how of preparing herring and linked it to inspirational values to a way of creating tasty herring dishes.", "The tradition value was also combined with symbolic values, as many interviewees emphasized the status of herring as the national fish of Estonia and the importance of herring for the identity of certain coastal areas and islands.", "In regard to fairness, the interviewees, including the commercial fishers, seemed relatively content with the management.", "One local fisheries representative noted: “We are in a good position, we can go to our ministries, and I think this is good.”", "Another fishery representative reflected on fairness in terms of allocation of fishing opportunities: “It always depends on who you ask.", "But I find it really hard to name any system that would work better.”", "A few interviewees criticized the knowledge-base of management while others expressed more faith in it: “We want to believe that the specialists who determine how much we are allowed to catch and how to catch are knowledgeable”.", "Table 2 summarizes the values related to Baltic herring, referred to by the Estonian interviewees.", "The interviewed Finnish stakeholders associated a wide variety of values (fairness of management, conservation, traditions, inspiration, symbolic values, knowledge, and economic values) with Baltic salmon whereas in Estonia, diversity was narrower (conservation, inspiration, fairness of management, and knowledge).", "The results suggest that the socio-cultural value of Baltic salmon is more multifaceted and contentious in Finland than in Estonia.", "The frequent references to tradition and symbolic values among the Finnish but not among the Estonian interviewees may reflect the differing cultural histories related to salmon between these two countries, which are also related to geographical differences between the contexts.", "In Finland, Baltic salmon was historically abundant until mid-20th century in many big rivers and an important resource, especially in the north (Erkinaro et al., 2011).", "Salmon also contributed to the development of traditions and local identities in the salmon river areas (Autti and Karjalainen, 2012; Vilkuna, 1975).", "Our study indicates that these values are still discursively maintained.", "Moreover, tradition and symbolic values seem to be important motives for conserving stocks.", "In Estonia, where the rivers and the salmon stocks are smaller (Kesler, 2015), the importance of salmon as a resource and for local traditions and identities has been smaller.", "However, some of the interviewees considered whether people had been alienated from salmon, as the salmon stocks suffered from industrialization (see also Kesler, 2015), and access to fishing was restricted during the Soviet era (see also Vetemaa et al., 2006).", "The finding that the Finns more often emphasized fairness in salmon management than the Estonians suggests that the inclusion of and balancing between different interests has been a long and laborious process in Finland, where the groups involved in salmon policy are more numerous and heterogeneous.", "The Finnish interviewees pointed out that salmon has been a source of contention from times immemorial.", "Representatives of conservation and river fisheries alluded to their former feelings of exclusion from policy-making and saw their current inclusion as being a result of their own hard work.", "They also saw maintaining their representation in policy-making as an issue of justice.", "However, commercial fishers seemed to find this a threat to their livelihood, which in certain areas depends on salmon.", "The Estonian interviewees said that balancing commercial and recreational interests was not easy, but fairness of management did not arouse meandering reflections on social justice.", "This could be because fewer interest groups are involved in policy-making and the economic importance of salmon for commercial fishing is relatively minor.", "The relationship between hydropower and Baltic salmon demonstrates the ways in which different values related to salmon interplay within and between the contexts.", "In both countries, the damming of rivers for electricity production drastically reduced wild salmon stocks during the 20th century.", "The Finnish interviewees saw this as a major environmental, economic, social and cultural injustice, while the Estonian interviewees mostly discussed the environmental damage caused by the dams.", "The Finnish interviewees also talked quite a lot about the legal obligation of the hydropower companies to compensate the damage the dams caused to the fisheries.", "Currently the main compensation method is the annual releases of reared fish into the dammed rivers and their mouths.", "The commercial fishers considered this practice and its continuance crucial for the sake of justice, while the other groups criticized it as insufficient for addressing the environmental, social and cultural aspects of salmon and its fisheries.", "The economic importance of Baltic herring for commercial fishing, as well as the efficient and fair utilization of the catch were emphasized by the stakeholders of both countries.", "The Finnish interviewees also stressed the environmental values related to the fishing and consumption of herring while the Estonian interviewees highlighted the sustainable management of the stocks and the tradition value of herring for the coastal and island communities.", "These results suggest that, overall, the socio-cultural value of Baltic herring is quite similar in these countries, although distinctive nuances also exist.", "The Finnish interviewees saw the use of herring as environmentally friendly behavior, whereas this was not a common view among the Estonians, who valued herring as natural and local food.", "We interpret that the Finns attached environmental values to the use of the catch because of its strong compromise potential for different stakeholders.", "ENGOs, among others, see it as a way to promote sustainable eating habits while at the same time removing nutrients from the sea, whereas the fishing industry also considers it a market opportunity.", "The compromise between environmental and economic values is demonstrated by the effort put into the environmental certification of Finnish herring and sprat fishery (Marine Stewardship Council, 2018).", "The contextual differences between the market areas for herring, and public discussion concerning the environmental impacts of food production and consumption may also explain how the use of herring is framed.", "The importance of herring-related traditions to the Estonian interviewees seems to relate to the fact that in Estonia, coastal herring fishing is still relatively common and is centered in certain regions.", "In 2015, nearly 30% of Estonia’s herring catch was from spawning grounds (International Council for the Exploration of the Sea (ICES, 2016), whereas in Finland the share of coastal fishing from the herring catch was less than 10% (Ministry of Agriculture and Forestry of Finland, 2017).", "The Estonian interviewees also linked the tradition value of herring to the identity of certain areas, of which they seemed to be proud.", "Moreover, coastal herring fishing and dishes were seen as an opportunity to market their region and develop rural tourism.", "In other words, the stakeholders saw the tradition value of herring as being compatible with several other value bases.", "The interviewees in both countries emphasized the importance of prioritizing the use of herring for human consumption.", "At the same time, the domestic consumption of herring has decreased, and the tendency to use the catches for industrial purposes is growing, particularly in Finland (Natural Resources Institute Finland, 2017).", "Including the use of herring in the policy agenda and exploring the possibilities to support the use of herring for human consumption would be in line with socio-cultural values (Pihlajamäki et al., 2018).", "Efforts to promote the use of herring in food production also support the aims of the Common Fisheries Policy of the European Union (Regulation No., 1380/, 2013) to ensure the contribution of fisheries to fish food availability and self-sufficiency.", "Our analysis suggests that in both countries, a combination of several value bases is incorporated in fisheries governance.", "Economic and use values are balanced with conservation values, and expert-steered technocratic decision-making is being reconciled with stakeholder participation (see also Linke and Jentoft, 2016, 2014; Morton et al., 2016; Song et al., 2013).", "The emphasis of the values varied according to the contexts.", "In both countries, the emphasis on conservation values in management has grown in recent decades.", "Ensuring the sustainable harvesting of fish stocks through optimal fishing pressure is increasingly emphasized.", "During the last few decades, the focus of salmon fisheries management has shifted from managing the offshore and coastal fishing of hatchery-reared released salmon towards conserving wild stocks and supporting their natural reproduction.", "In Finland, the former and new approaches exist in parallel, since reared salmon is stocked to compensate for the hydropower-related damage to sea fishery but some activities also aim to restore the salmon populations.", "In Estonia, salmon is stocked only to support natural reproduction, and conservation of the stocks is also implemented by opening salmon migration routes in rivers.", "These actions reflect a stronger emphasis on conservation values.", "In the case of Baltic herring, reconciling the conservation values with economic and use values in governance has been less contentious, because the stocks are in better condition and interest groups less heterogeneous.", "In Finland, a recent governmental development plan for the Blue Economy reflects efforts to combine environmental and economic values with the utilization of the catch, as it searches for new, sustainable and value-adding purposes for the use of herring (Ministry of Agriculture and Forestry of Finland, 2016).", "The Estonian interviewees seemed more satisfied with the knowledge base of fisheries management and the opportunities to participate in governance than the Finns.", "Several Estonian interviewees said that they have possibilities to influence and that policy-makers listen to them.", "The small size of Estonia in terms of both geography and population was often mentioned as a factor facilitating collaboration in governance.", "The differences in satisfaction could also relate to differences in governance practices and the diversity of stakeholder groups as well as attitudes and expectations towards participation and science.", "Overall, understanding contextual differences in satisfaction with management would require an in-depth analysis of both governance practices and management instruments from the stakeholder perspectives.", "Several authors have called for acknowledging the socio-cultural values related to fish resources and incorporating them in governance in a more structured manner (De Young et al., 2008; Lam, 2015; Linke and Jentoft, 2014; Song et al., 2013; Urquhart et al., 2013).", "To examine possible ways to do this, it is beneficial to think of fisheries governance as a process that consists of subsequent stages.", "According to the framework introduced by Linke et al. (2011) fisheries governance process comprises four stages: pre-assessment; appraisal; evaluation and characterization; and management.", "Pre-assessment consists of defining the problem and formulating management objectives.", "Appraisal is about the systematic, knowledge-based assessment of the options for reaching objectives.", "Characterization and evaluation are for determining the most acceptable management options.", "Finally, management is for deciding on and implementing management measures.", "Values are currently incorporated in the stage of characterization and evaluation that aims for a “broadly informed yet basically value-based judgment on the most appropriate management measures” (Linke et al. 2011, p. 140).", "In the EU fisheries governance this is exemplified in the work of stakeholder-led advisory councils that are consulted for management proposals.", "However, Linke and Jentoft (2014) demonstrated that even though stakeholders constantly refer to value issues, these issues are also subordinated to and absorbed into the discussions on natural scientific and technical issues related to data problems and stock assessment methods.", "The authors concluded that to establish environmental and social sustainability in the fisheries sector, value aspects need to be included explicitly and systematically in the instrumental, science-based governance process.", "We agree with these observations and suggest explicit and systematic inclusion of values already in the early stages of governance process, that is, during pre-assessment and appraisal.", "The way in which problems become framed and management objectives formulated is influenced by value judgments regarding the factors to be included, and the scope of the appraisal (De Young et al., 2008; Haapasaari et al., 2012; Linke et al., 2011; Sarewitz, 2004).", "This is particularly the case if governance is viewed as a proactive and strategic endeavor rather than reacting to changes.", "The Common Fisheries Policy of the European Union (Regulation No., 1380/, 2013) highlights the need to move towards long-term planning and objective-setting.", "Since values serve as signposts for what should be, they need to be explicitly discussed in order to define management objectives.", "In practice, the existing and emerging national and/or regional policy forums could be utilized to deliberate on values and draft value-informed strategies for the management and use of fish resources.", "After this, different alternatives for reaching the value-informed goals and implications of management options could be assessed.", "This would require further inclusion of social science in the research institutions that provide science for policy.", "(Linke and Jentoft, 2014.)", "The perspective of the justification theory could be useful in the stages of both pre-assessment and appraisal, since it acknowledges multiple categories of values, but at the same time suggests shifting attention from opposing interests to the common good, which is a requirement for the legitimacy of governance.", "Utilizing justification theory as a tool for identifying socio-cultural values related to fish resources in an interview setting had both advantages and disadvantages.", "The seven value bases helped structure the interviews and their analysis.", "However, because the value bases were broad and enabled the framing of the topic in numerous ways, the interviews were lengthy and their analysis laborious.", "We found interviews useful for identifying the socio-cultural values that stakeholders attach to fish resources.", "The interviews helped us detect points of agreement and disagreement, and to identify less discussed but important values.", "The next step could be to evaluate these findings in a group context to build a shared understanding among stakeholders of the ways in which fish resources are important for society.", "This study demonstrates the multivalued nature of fish resources and highlights the importance of addressing the diversity of socio-cultural values explicitly in governance.", "Although values are currently included in fisheries governance process in the evaluation of management proposals, they also need to be included in the earlier stages of governance process – during pre-assessment and appraisal – to ensure morally reasoned management and use of fish resources.", "Further research is needed concerning potential mechanisms or methods to incorporate values in governance processes.", "An approach capable of accounting for a wide variety of values and orientation towards the common good is required in order to create a meaningful dialogue and deliberation on how to govern common natural resources in a justifiable way." ]

[ "The multivalued nature of fish resources is demonstrated.", "Values related to Baltic salmon and Baltic herring in two countries are compared.", "Emphasis of values represented in fisheries governance depends on context.", "Systematic inclusion of values in governance could enhance the legitimacy of decisions." ]

[ "CHEM", "ENGI", "MATE", "PHYS" ]

[ "FDU-12", "Morphology", "Ordered mesoporous silica", "Stirring" ]

[ "1600", "2211", "2500", "3104" ]

[ "Ordered mesoporous silica materials are applied in catalysis [1,2], molecular adsorption [3,4] and separation [5].", "In addition to their internal textural properties (ordered and adjustable pore size, large surface area and pore volume), the external morphology of these materials also affects their applicability.", "Over the years, many different ordered mesoporous silica materials have been reported, including MCM-41, MCM-48, SBA-15, SBA-16 and FDU-12 [6–10], with different morphological and topological characteristics, which allow for an extensive range of potential applications.", "For instance, mesoporous silica fibres and nanotubes with aligned one-dimensional (1D) pores, such as MCM-41, can be used in nanofluidic applications or to template nanorods.", "However, mesoporous materials with a three-dimensional (3D) pore network system, such as MCM-48, SBA-16, KIT-5, FDU-1 and FDU-12, are advantageous for processes limited by diffusion or prone to pore blockage, for example in catalysis or separation processes, compared to materials with 1D channels [11].", "3D mesoporous materials, with pore networks templated by micellar cubic phases, contain large cavities or cages connected by multidirectional pore entrances of smaller sizes (so-called windows).", "The pore system is templated by micelles formed by a block copolymer.", "The cavity and window size can be controlled by adjusting the synthesis and hydrothermal treatment conditions, such as the temperature, or by adding the right amount of swelling agent [12].", "Different acidic synthesis procedures lead to the formation of fibres, spheres, and mesoporous silica films [8,13–15].", "The optimal morphology depends on the application.", "For example, films are useful for gas sensing [16], while mesoporous spheres are desirable for catalytic and adsorption processes, as this shape facilitates their packing in fixed bed reactors or in liquid chromatography, as the stationary phase [17].", "Tailoring particle morphology is therefore critical to the envisioned application of a mesoporous silica material.", "Several investigations on the effect of the synthesis conditions of FDU-12, such as synthesis temperature, hydrothermal temperature and duration, silica concentration, acid concentration, and salt concentration have been carried out.", "The concentration of HCl and the type of salt used can influence the formation of the silica particles [20–22].", "As discussed, much effort has been devoted to controlling the characteristics of the mesostructure, such as the pore cavity and window sizes of mesoporous silica with cage-like structures, but very little has been reported on controlling the external morphology or macrostructure of FDU-12 silica particles.", "Better understanding of methods to tune the morphology would allow more precise control over properties that are relevant to specific applications.", "Several studies have linked the interaction between hydrolysed silicic species and the micelles formed during the gel phase and hydrothermal treatment, to the growth of a mesoporous silica particle [23,24].", "Chan et al. first proposed a realistic phase separation model for the mechanism of formation of silica mesostructured precipitates [25], which was further developed by Yu et al. for a non-ionic block copolymer templating system [26]; it suggests that mesoporous materials are formed in three stages: cooperative self-assembly of inorganic/organic composites, formation of a liquid crystal-like phase of block copolymer/silica aggregates, and phase separation of the liquid crystal-like phase from the solution with condensation of silica species driving the growth of the solid mesostructures.", "According to this mechanism, the particle shape ultimately results from competition between the free energy of mesostructural self-assembly, the colloidal surface free energy, and energy imparted by other interactions, such as shear.", "A few reports have explored the effect of not stirring (static conditions) [9,26] or stirring for different times [22,27].", "Huang et al. studied the effect of different swelling agents (xylene, toluene, and TMB), time and temperature of synthesis, different silica sources (TEOS versus TMOS), effect of salt (KCl) and the silica/surfactant ratio, but kept the stirring rate constant at 350 rpm or varied within a narrow range between 370 and 450 rpm [28,29].", "Therefore, no reports have mentioned the effect of the stirring rate during synthesis on the formation and morphology of FDU-12 mesoporous silica.", "As will be shown, the stirring rate is not a trivial factor.", "It is one that can govern the structure of these silica materials and influence the uniformity of the macroscopic shape.", "The aim of this study is to demonstrate the effect of the stirring rate on the resultant particle morphology and porous parameters of F127-templated porous silica material.", "Tetraethylorthosilicate (TEOS, reagent grade 98% purity), Pluronic F127 [poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) tri-block copolymer EO106PO70EO106], hydrochloric acid (HCl, analytical grade 37 wt%), trimethylbenzene (TMB, analytical reagent 98% purity), and potassium chloride (KCl, 99 wt%) were purchased from Sigma Aldrich Company Ltd. (Dorset, UK).", "The synthesis procedure was performed based on the report in Ref. [11].", "First, 2.0 g of Pluronic F127 was dissolved in a mixture with 2.0 g of TMB, 5.0 g of KCl and 120 ml of 2 M HCl and stirred for 24 h at 40 °C.", "Then, 8.3 g of TEOS was added to the reaction mixture, and stirred at different rates or left under static conditions for 24 h at 40 °C.", "The different stirring conditions are listed in Table 1.", "Stirring was conducted using an oval magnetic stir bar.", "The mixture was then transferred to a convection oven for hydrothermal treatment at 100 °C for 72 h. Thereafter, the solid product was filtered, washed three times with deionized water, and dried at room temperature overnight.", "The material was calcined at 550 °C for 6 h to obtain the mesoporous silica.", "The resulting mesoporous silica samples were designated FDU-12-X, where X indicates the stirring rate, e.g., FDU-12-100 for X = 100 rpm.", "Scanning electron microscopy was performed with a JEOL JSM-6480LV high-performance SEM (JEOL, Tokyo, Japan).", "The samples for SEM were sputter-coated with gold for 90 s before imaging, to reduce charging.", "Transmission electron microscopy (TEM) was performed with a JEOL 2100 200 kV TEM (Tokyo, Japan) with a Gatan camera (Pennsylvania, USA).", "The samples for TEM were prepared as a suspension in methanol and dropped on a 300-mesh copper grid with a carbon film.", "Powder X-ray diffraction (XRD) patterns were measured with a Stoe Stadi-P copper anode capillary transmission powder X-ray diffractometer in the range of 0.5–3°.", "Nitrogen adsorption and desorption isotherms were obtained with an Autosorb-iQ-MP-XR (Quantachrome, Florida, USA) at −196.15 °C with samples outgassed at 300 °C for 8 h with a pre-treatment at 100 °C for 2 h. Argon adsorption and desorption isotherms for micropore analysis were obtained at −186.15 °C using the same Quantachrome instrument.", "The pore size distribution was calculated from the adsorption branch of an isotherm by the nonlocal density functional theory (NLDFT) analysis method using a spherical model for the pore cavities and the BET surface area was calculated based on relative pressures P/P0 = 0.05–0.3.", "The micropore analysis was obtained from the argon adsorption data.", "SEM images of the samples synthesized using different stirring rates are shown in Fig. 1.", "Mainly agglomerates are observed in these images.", "Particles synthesized in the absence of stirring (FDU-12-0 in Fig. 1(a)), under mild shaking (FDU-12-80′ in Fig. S1(a)) or under mild stirring (FDU-12-100 in Fig. 1(b), FDU-12-200 in Fig. S1(b)) have the best defined polyhedral shapes.", "These silica particles consistently have a hexagonally edged platelet structure, which is 1.6–2.6 μm in size.", "In contrast, when the stirring rate is increased beyond 200 rpm, more and more irregularly shaped particles are formed.", "Samples FDU-12-500 and FDU-12-800 are aggregates composed of poorly defined particles with some composed of the smaller platelets (Fig. 1(c)) or large spheroidal particles (see Fig. S1(e), Supplementary Information).", "It is difficult to attribute a particle size to these samples, because of the agglomeration of the primary particles.", "Monodisperse spheres were not obtained at any stirring rate.", "Literature reports show that SBA-16, which is also templated with F127, can form particles of a uniform spherical shape [27,30–32].", "This spherical shape was transformed to a polyhedral shape by increasing reaction time beyond 60 min (decaoctahedral) and 120 min (rhombdodecahedral) after stirring for 30 min [27] or by varying the surfactant/silica (F127/TEOS) molar ratio, acid concentration and temperature [30].", "Fan et al. [11] also reported FDU-12 silica with a spherical morphology.", "However, this spherical morphology was achieved for particles synthesized at a higher temperature (100 °C).", "Yuan et al. [33] succeeded in forming an FDU-12 material with facets similar to those seen in FDU-12-0 and FDU-12-100 with a surfactant/silica molar ratio of 0.004.", "Although their choice of surfactant was different (F108), the result achieved was similar to that obtained with the F127/TEOS molar ratio of 0.004 for this synthesis.", "Previous work have shown that the morphology of mesoporous silica particles is derived from the shape of the block copolymer micelles, which is influenced by the synthesis recipe and preparation procedure.", "Sayari et al. [9] showed that, to produce short, monodisperse SBA-15 rods, it is essential not to stir the synthesis mixture.", "Zu et al. [34] showed that platelet-like SBA-15 particles are generated when the time of the micelle formation process is short and the stirring rate is high (>800 rpm), while a rod-like morphology is obtained when the micelle formation time is long (several hours), regardless of stirring rate.", "Hwang et al. [27] determined an optimal stirring time of 30 min in order to yield well-defined, microwave-synthesized, cubic SBA-16 particles.", "In our synthesis procedure, samples are stirred for 24 h.", "A long stirring time at high stirring rates may cause the silica particles to overgrow, leading to the undefined shapes observed at higher stirring rates (>200 rpm).", "Mesa et al. [30] explained that, for F127/TEOS systems, a gel-like phase is obtained for the silica particles at temperatures of 40 °C or lower, regardless of the F127/TEOS molar ratio.", "The well-defined shapes seen under static or low stirring rate conditions bears similarities to what is observed when synthesizing rod-shaped SBA-15 particles, which are obtained only under static conditions [9].", "Interestingly, however, the external morphologies do not reflect the internal structure of the samples.", "The porous structure was studied by measuring nitrogen adsorption and desorption isotherms.", "The results are shown in Fig. 2, Figs. S2 and S3.", "The textural properties of all samples are listed in Table 2 and Table S1.", "All samples show a type IV isotherm, characteristic of an ordered mesoporous material.", "The delay in desorption and steepness of the desorption isotherm around the relative pressure P/P0 = 0.45 suggests that the material has a cage-like pore structure.", "The hysteresis loops of all samples occur over the relative pressure range of 0.4–0.75 and are of H2 type, where the steep desorption branch and smooth adsorption branch of this hysteresis loop can be associated to pore-blocking due to pore entrances or windows that are much narrower than the cages to which they lead.", "The results show that the stirring rate has a negligible effect on the textural parameters.", "The majority of the samples have a pore (cavity or cage) diameter of approximately 10.2 ± 0.3 nm.", "The pore size distribution is given in Fig. 3.", "Within statistical errors, Fig. 3 shows a nearly identical, narrow bimodal pore size distribution for all samples with two peaks corresponding to, respectively, the cavities and windows.", "The window size is determined by NLDFT to be ∼2.5 nm.", "The BET surface area of the materials ranges from 542 to 716 m2/g, and the total pore volume from 0.52 to 0.68 cm3/g.", "The micropore volume was determined with argon adsorption to be ∼0.25 cm3/g.", "This demonstrates that the pore structure is uniform and reproducible.", "The results show that there is only a very slight change in the unit cell dimension, indicating that the unit cell remains unchanged, whether stirring or not, and at any stirring rate.", "The lower-indexed, intense Bragg peaks are somewhat broadened due to polydispersity and disorder.", "Crystal defects in a crystalline material could also contribute to peak broadening.", "Assuming that the broadening arises solely from the ordered structure, a domain size is estimated from Scherrer's equation using the full width at half-maximum (FWHM) power of the most intense peak (111) to be 39, 35, 36, and 38 nm for FDU-12-0, -100, -500, -800 samples respectively.", "Here, the domain size represents the lower limit for which stirring affects the macroscopic morphology.", "TEM micrographs were obtained to observe the internal structure and mesophase of all the samples.", "The images generally reveal large domains of well-aligned, ordered pores, which is in agreement with the SAXS results.", "The cubic phase of these structures is clearly seen (Fig. 5), corresponding to the fcc structure resolved using SAXS.", "The FDU-12-800 sample (Fig. 5(D)) shows regions with a more disordered arrangement of pores.", "This is seen in FDU-12-1000 as well (Fig. S6 in the Supplementary Information), indicating that a very high stirring rate (>800 rpm) is detrimental to the formation of a long-distance periodically ordered mesostructure, even though neither the pore diameter nor the local organisation, as illustrated by SAXS, are affected.", "Stirring induces shear, which plays a role in the formation of the particles, and affects, in particular, their shape.", "The results show that the pore diameter of the samples remains essentially unchanged, and the unit cell size a0 for all samples is similar.", "However, there are differences in the particle morphology and also in the mesophase organisation, especially at very high stirring rate.", "This means that fast stirring may influence the surfactant/silica species interactions, albeit only slightly.", "The platelet shape is different from cubic mesoporous silica materials, where the spherical micelles nucleate, grow and aggregate into spheroidal or cubic structures.", "The materials formed under slow stirring conditions have a unique morphology.", "The following hypothesis is proposed to explain the disparity observed in the particle morphology.", "When shaking, stirring slowly, or under static conditions, micelles can nucleate and grow in a more stable environment.", "When stirring rapidly, however, shear on the micelles is pronounced, so that micellar growth is hindered, as well as the assembly with silica precursors to grow large, well defined particles with long-range order of the mesopores.", "Poorly defined morphologies result from rapid reorganisation or reassembly of growing particles.", "With no change in mesostructure observed, it would seem that this material, FDU-12, is more robust under shear than other previously studied materials, such as SBA-15.", "Literature shows that the P123 block copolymer gives a variety of mesostructures, depending on concentration and other synthesis parameters.", "In particular, the cylindrical, elongated P123 micelles of the SBA-15 system are more sensitive to shear during SBA-15 synthesis.", "The F127 block copolymer, on the other hand, primarily yields less extended, spherical micelles.", "This could be the cause for the robustness of the ultimately formed material structures seen in this study and highlights an essential difference with SBA-15 synthesis.", "It was shown that the stirring rate can be employed to tailor the particle morphology of mesoporous silica particles with 3D cubic mesoscopic order.", "Different stirring rates lead to different shear rates that, in turn, influence the aggregation of primary particles, as well as the growth of the silica mesophase.", "Shear, influenced by the stirring rate, can affect the growth of the silica particles and, consequently, the evolution of the structures formed, inside a confined space.", "Well-defined shapes are observed at lower stirring rates, including static conditions, while higher stirring rates lead to indistinct shapes.", "It appears that, for the synthesis of ordered mesoporous silica in the granular state, reaction conditions such as temperature and composition determine the internal structure while the stirring rate has much less effect on the textural parameters, but will affect the final particle morphology.", "This matters, since three-dimensionally connected pore networks, such as those in FDU-12, are beneficial to applications in separations and catalysis, because they ensure accessibility of the internal volume and are less affected by pore blockage." ]

[ "Morphology of FDU-12 mesoporous silica particles is controlled by changing the stirring rate during synthesis.", "When increasing stirring rate, FDU-12 particle morphology changes from well-defined to poorly defined.", "Internal pore structure and three-dimensional pore network topology are unchanged by stirring rate.", "At high stirring rate, the pore network structure changes from regularly ordered to long-range disordered." ]

[ "NEUR" ]

[]

[ "2800" ]

[ "Over one hundred years of research has suggested that sleep and circadian rhythms play an important role in learning and memory.", "Several hypotheses have been put forward to explain how sleep benefits learning, and why disrupted or mistimed sleep and sleep deprivation might affect cognitive function [1,2].", "It has been proposed that sleep facilitates incorporation of memory traces into permanent storage [3], benefits learning through global synaptic down-selection [4], or both [5].", "In recent years substantial progress has been made with respect to understanding the neurobiological mechanisms underlying learning and memory, in particular with regard to the role of glutamate receptors and synaptic plasticity.", "Concomitantly, the molecular and synaptic changes that occur during wake, sleep and sleep deprivation have been investigated in detail.", "Given these recent advances, and given the central importance of glutamate receptors and synaptic plasticity in the relationship between sleep and memory, a view shared by various theories [1,4,6], our aim here is to re-evaluate the relationship between sleep and memory in the light of these recent findings.", "The neural processes associated with sensory processing, motor functions and learning involve intense synaptic and spiking activity, which are computationally and metabolically demanding [7,8].", "Not surprisingly, staying awake for extended periods of time leads to substantial wake-dependent changes in cortical and subcortical network activity [9••,10•,11•], which likely contribute to well-known behavioural deficits after sleep deprivation or disruption [12–14].", "It has therefore been suggested that prolonged wakefulness needs to be off-set by periods of recovery during which metabolic balance and network function must be restored [13,15].", "Such opposing effects of waking and sleep on brain activity and behaviour may be described conceptually within a framework of homeostatic regulation [16].", "Sleep homeostasis is a ubiquitous phenomenon found in many animal species, suggesting that it underscores some fundamental, essential function [16,17].", "According to this framework, sleep pressure (reflected in the dynamics of a hypothetical ‘Process S’) increases as a function of time spent awake and decreases with sleep.", "Like an hourglass, the levels of Process S indicate the time passed awake or asleep and denote how likely we are to be awake or asleep at any given moment.", "The best characterized physiological indicator of sleep-wake history is the level of cortical EEG slow-wave activity (SWA, EEG power between 0.5 and 4.0 Hz) during NREM sleep [18,19], reviewed in Ref. [20].", "In mammals, sleep SWA is high in early sleep and after sleep deprivation, when sleep pressure is increased physiologically, and decreases progressively to reach low levels in late sleep [21–23].", "The dynamics of SWA across the period of sleep have been linked to the levels of synchronisation within cortical networks and neuronal excitability [13,24,25], although the role of subcortical neuromodulation cannot be ruled out [26].", "Importantly, sleep homeostasis is not the only process that is involved in the regulation of waking and sleep across the 24-hour day.", "It is now well established that Process S undergoes a powerful influence from another process—the circadian rhythm (referred to as Process C), which links daily variations in a wide range of physiological variables with the occurrence of waking and sleep [27,28].", "This circadian process is the product of the circadian clock—an intracellular mechanism based upon the rhythmic expression and negative feedback of a number of clock genes and their protein products.", "In mammals, the master circadian pacemaker is located in the hypothalamic suprachiasmatic nuclei (SCN), which regulates peripheral clocks found in cells and tissues throughout the body [29].", "According to our current understanding, the homeostatic process interacts with a circadian drive for arousal, which prevents sleep from occurring at inappropriate times and thus benefits waking functions [16].", "Although several studies have implicated the circadian system in the regulation of synaptic plasticity, learning and memory [30,31•], it is possible that changes in sleep may provide the primary mechanism by which circadian rhythms influence these processes.", "Important insights into the mechanisms underlying sleep regulation have been obtained from a series of molecular studies where the effects of waking and sleep on gene expression in the brain have been investigated.", "These studies showed that several hundred genes were expressed differentially between day and night, or varied between sleep and wake regardless of time of day [32–35].", "Importantly, it was revealed that different functional categories of genes in the brain were selectively associated with wakefulness and sleep.", "Being asleep was associated with differential expression of genes primarily involved in protein synthesis and membrane trafficking.", "Genes up-regulated after waking included molecules involved in energy homeostasis, a number of transcription factors, clock-related genes, heat-shock proteins, genes related to activity-dependent neural plasticity, and glutamatergic neurotransmission-related genes.", "These findings suggested that at least some of the well-known cognitive deficits incurred after sleep loss may arise from changes in synaptic machinery, and specifically changes associated with excitatory glutamatergic signalling and synaptic plasticity.", "Consistent with this notion, synaptic levels of glutamate receptor subunits, including the GluA1 AMPAR subunit (also known as GluR-A or GluR1, encoded by the Gria1 gene), exhibit a pronounced daily variation as a function of sleep-wake history [36••,37].", "For example, it has been shown that if rats were sacrificed after periods of sustained wakefulness or sleep deprivation then GluA1-containing AMPAR levels in synaptoneurosomes in cortex and hippocampus were considerably higher than in a group of animals that were sacrificed after periods of sleep (Figure 1).", "The GluA1 subunit plays an essential role in the trafficking of AMPARs into the post-synaptic membrane and thus supports and maintains synaptic plasticity [38–40], including short-term potentiation (STP) and certain forms of long-term potentiation (LTP) [41–44].", "This is consistent with the proposal that wakefulness is associated with a net increase in synaptic strength, as manifested by increased levels of glutamate receptors in brain areas like the hippocampus and cortex [4].", "In turn, according to this view, sleep is associated with synaptic renormalisation, manifested as a decrease in GluA1-containing AMPAR levels back down to baseline levels [4].", "So what are the behavioural consequences of these diurnal changes in glutamate receptor subunit expression and plasticity at cortical and hippocampal synapses.", "Historically, the relationship between synaptic plasticity and memory has been almost exclusively considered in terms of encoding associative, long-term memories.", "NMDAR-dependent long-term potentiation (LTP) is the dominant cellular model of synaptic plasticity in learning and memory, and the idea that LTP-like processes, in brain areas such as the hippocampus, underlie the acquisition and storage of associative long-term memories has predominated [45].", "Nevertheless, despite this widespread belief and the general acceptance of a hippocampal LTP/associative memory hypothesis, the evidence in support of this theory is equivocal at best [46–48].", "On the other hand, there is increasingly strong evidence that glutamate receptors and synaptic plasticity may underlie a very different kind of memory trace.", "For example, Gria1−/− mice with a global, constitutive knockout of the GluA1 AMPAR subunit, exhibit a robust and selective deficit in a non-associative form of short-term memory that underlies short-term habituation to recently experienced stimuli.", "In marked contrast, these mice are perfectly able to acquire associative, long-term memory tasks, including the Morris watermaze, just as well as their wild-type littermates [49,50••] (see Box 1, Figure 2).", "Habituation describes the decline in the tendency to respond or attend to stimuli that have become familiar as a result of prior exposure.", "Recent studies with Gria1−/− mice demonstrate two forms of habituation: a GluA1-dependent, short-term habituation and a GluA1-independent, long-term habituation [50••].", "Thus, evidence suggests that GluA1-dependent plasticity underlies the short-lasting sense of familiarity for recently experienced stimuli such that the attention paid to these stimuli is reduced [51].", "The relevance of these behavioural findings in Gria1−/− mice for understanding the functional role of sleep is brought into focus by the diurnal wake-sleep dependent rhythm in the synaptic expression of glutamate receptor subunits, including GluA1 [36,37].", "The involvement of GluA1-dependent synaptic plasticity in encoding familiarity for recently experienced stimuli may be reflected in the accumulation of GluA1 subunits in the post-synaptic membranes of hippocampal and cortical neurons during waking experience [40].", "Thus, the increase in synaptic GluA1 after prolonged periods of wakefulness may reflect on-going habituation, leading to a reduction in attention.", "Importantly, this particular kind of GluA1-dependent memory is not permanent, and the sense of familiarity that it supports (and the concomitant reduction in attention) is not lasting.", "Indeed, we suggest that this sense of familiarity may be diminished after restorative sleep [4].", "Thus, an intriguing possibility is that restoration of attentional performance (i.e. dishabituation) is the behavioural endpoint of the network renormalisation that occurs during sleep [52], and glutamatergic plasticity plays an important role in this process.", "It remains to be determined whether sleep merely passively provides an opportunistic process for dishabituation by providing a period of reduced sensory input, allowing the memory traces underlying short-term familiarity to decay, thus resulting in the restoration of attentional capacity, or whether sleep includes an active process (or processes) that leads to the restoration of attention.", "According to this framework, a failure to engage such dishabituation processes could result in persistently low levels of attentional capacity.", "It is important to note that GluA1-dependent habituation is stimulus specific and does not result in a generalised reduction in attention to all stimuli (as noted in Box 1).", "However, according to well-established models of habituation, for example, Wagner’s Sometimes Opponent Process (SOP) model; [53], stimuli are considered to consist of multiple ‘elements.’", "These elements could be considered as the fundamental units that together comprise the various features of a stimulus (i.e. together they represent the visual, tactile, auditory, olfactory and taste components of a given stimulus).", "Different stimuli (particularly in the same stimulus domain) will therefore likely have a proportion of their elements in common.", "In other words, there will be some overlap in terms of the elements that comprise certain stimuli.", "In Wagner’s SOP model, habituation is considered to occur at the level of individual elements (although it also seems likely that habituation may occur at various different levels of stimulus organisation).", "Therefore, over a prolonged period of wakefulness, and with exposure to numerous stimuli, there will be a gradual increase in the habituation to elements of many stimuli.", "Given that there is a degree of overlap in the elements that comprise one stimulus to the next, ongoing stimulus-specific habituation will eventually lead to an overall stimulus-independent, decrease in attentional performance, as the total number of elements that have become habituated gradually increases across waking experience.", "Thus, extended periods of wakefulness will result in reduced attention, whereas sleep provides recovery such that high levels of attention are once again possible during the next waking period.", "A related possibility is that reduced attentional performance after sleep deprivation is associated with the intrusion of sleep-like patterns of neuronal activity into the awake state [54].", "Indeed, several studies have shown that cortical OFF-periods or slow waves are common during waking, especially during quiet states, but also even during active behaviours [9••,12,55–59].", "It seems not unlikely that the occurrence of sleep-like cortical states during waking could lead to attentional deficits, inasmuch as the networks supporting a specific behaviour are experiencing a down-state, although direct evidence supporting this notion is still limited [12,60,61].", "At the same time, the occurrence of neuronal down-states during waking, associated with lowered spiking and synaptic activity, is likely associated with reduced metabolic requirements, or could reflect a shortage of energy substrates [62].", "More generally, the changes in network activity and neuronal excitability across sleep/wake cycles have potentially important implications for energy homeostasis in the brain [4,7].", "It has been shown in humans and animals that cerebral metabolic rates are increased after sleep deprivation or spontaneous wakefulness [15,63].", "Furthermore, after prolonged periods of wakefulness, it may become progressively harder to engage with stimuli when it is required to do so.", "Under normal circumstances, the more often or the longer a stimulus is presented, the less and less attention is paid to that stimulus.", "However, if it then becomes necessary to pay attention to a habituated stimulus (or to a stimulus that contains a high proportion of habituated elements), then the attentional mechanisms or working memory processes will need to overcome or override (and thus effectively work against) that ongoing habituation process.", "Thus it will be cognitively, and potentially metabolically, more demanding, to sustain attention to a habituated stimulus.", "The occurrence of network OFF-periods during sleep deprivation may reflect cortical states, during which the capacity to sustain metabolically-demanding processes, such as paying attention, is reduced.", "On the other hand, the increase in synaptic GluA1 after prolonged periods of wakefulness, leading to habituation, may result in it being progressively harder to pay attention to relevant stimuli when required.", "We posit that network renormalisation during sleep [64•], leading to dishabituation, may reduce the energetic costs of paying attention to stimuli and thus help maintain a viable energy balance in the brain.", "The notion that sleep disturbance or deprivation might lead to deficits in attention is well established [52,65,66].", "For example, numerous studies, both in humans and rodents, have demonstrated that sleep disruption most reliably leads to performance impairments in tasks requiring sustained attention [67–69,70••].", "The consequences of such attentional disruption for cognitive performance following sleep disruption are likely to be many and varied, but may include an impaired ability to acquire new information.", "Consistently, sleep deprivation in mice during the light period, when animals are typically asleep, leads to impaired performance in the novel object recognition task, while sleep deprivation performed in the dark period is not effective [71,72].", "Time of day, arousal state and preceding sleep-wake history may contribute to these effects.", "An inability to attend to the appropriate stimuli at the appropriate times will impede the ability to form new associations and thus to form new longer-term memories, consistent with existing evidence that sleep disturbance or deprivation can disrupt new learning [1].", "Furthermore, reduced attentional capacity may also impact on planned behaviour, particularly in situations in which it is necessary to perform computationally demanding searches for appropriate retrieval cues (either externally or internally) relevant to achieve a future, distant goal [73].", "A further consideration, which may, in part, result from this reduced ability to pay attention (although the two ideas are not wholly dependent on each other), is that sleep disruption may produce a more fundamental change in the nature of behaviour.", "Broadly speaking, behaviour and the sorts of responses that are made can be categorised into two distinct types.", "These are often described as goal-directed versus habit-like (or habitual) behaviours (which are computationally related to model-based versus model-free reinforcement learning respectively; see Box 2).", "It is generally considered that early on when learning a new task, behaviour is predominantly goal-directed, requiring high levels of attention that can be metabolically demanding [74].", "However, if responses and outcomes are predictable and these associations have been acquired, then responding becomes more automatic: it requires less attention and is computationally and possibly therefore metabolically, less demanding.", "Indeed, the switch from goal-directed responding to habit-like behaviour is generally considered as an adaptive response to free up attentional resources for other things once associative learning is at asymptote and the environment is relatively predictable [75,76].", "However, the converse may also be true.", "Namely, that if attentional resources are limited (e.g. as a consequence of sleep disruption/disturbance), then it is much more likely that a model-free/habit-based form of behaviour will predominate over a model-based, goal-directed behaviour which requires high levels of attentional control and higher metabolic resources.", "Indeed, in everyday life there are numerous examples that reduced attention can increase the chances of performing unwanted habits instead of intended actions [77].", "Furthermore, it is increasingly recognised that rather than there existing a simple serial relationship whereby goal-directed, model based behaviours always precede habit-like, model-free responding, organisms are often likely to have access to both forms of behaviour and can rapidly and readily switch between the two, given different environmental circumstances such as changes in stress and arousal [78,79••,80].", "Given the importance of circadian rhythms and sleep deprivation over motivated behaviours (e.g. [81]), it seems plausible that sleep disturbance will fundamentally change the nature of behaviour, making responding more model-free and habitual as a result of reducing attentional capacity.", "Potentially consistent with this account, we have recently demonstrated an important relationship between the nature of behaviour during waking experience and sleep pressure (Figure 3).", "Typically, waking has often been thought of as a homogenous process, which is associated with a continuous, progressive increase in sleep pressure, largely irrespective of the nature of ongoing behaviour or activities.", "However, we recently showed that automatic, stereotypical wheel running behaviour in mice was associated with a marked reduction in neural activity in the motor and somatosensory cortex [9].", "Furthermore, increased engagement with this low attention/low cost automatic, stereotypical behaviour was also associated with a substantially increased capacity to stay awake and an attenuated increase in neuronal excitability across the waking period [9,82].", "Although the psychological processes which determine wheel running in rodents are not clear, this finding might suggest that waking dominated by automatic, model-free behaviours may have a lower ‘cost’ with respect to accumulation of sleep need.", "Thus, while reduced sleep may favour model-free behaviour, conversely engaging in model-free behaviours may reduce sleep requirement.", "The mechanisms underlying the proposed effects of prolonged wakefulness on wake behaviours remain to be determined but may implicate the occurrence of local neuronal OFF-states within relevant cerebral networks.", "Sleep has properties of a local process whereby those networks which have been especially active during waking, or underwent the strongest plastic modifications, may require more intense sleep [4,13,83].", "Interestingly, a recent study showed that merely increasing network spiking activity irrespective of ongoing vigilance state or behaviour does not result in the homeostatic response during subsequent sleep [84].", "By the same token, it is possible that habitual behaviours, which are not associated with any new associative learning and can proceed with reduced levels of attention, may be associated with a reduced accumulation of sleep need.", "Whether the occurrence of OFF periods is merely a marker of energy deficit incurred during intense synaptic activity and plasticity, a consequence of reduced levels of arousal promoting neuromodulators, or an active process contributing to online renormalisation of network function remains to be determined.", "If, as predicted, sleep disruption shifts behaviour to favour more model-free strategies, this may also, in part, explain the significance of sleep disturbance for various neuropsychiatric disorders (Box 2).", "Furthermore, a reduced ability to adopt a model-based approach following sleep disruption could be considered analogous, at least in part, to more recent ideas about the role of sleep in memory consolidation which suggest that its key role is to facilitate the extraction and/or utilisation of the ‘gist’ from a memory, enabling the creation of a schema or world-view [1,5].", "Both scenarios suggest that sleep disturbance would lead to a reduction in model-based engagement with the world.", "This could manifest as either a failure to overcome innate or learned biases or through shallower search through prospective decision trees (e.g. [85]).", "It is important to note, however, that the psychological and neurobiological mechanisms leading to this reduction in model-based behaviours may be quite different according to these two distinct accounts.", "Here we have briefly reviewed evidence which suggests that synaptic homeostasis of GluA1-containing AMPA receptors may provide a mechanism for dishabituation and the restoration of attentional capacity during sleep, maximising the readiness of the animal for behaviour during subsequent periods of wakefulness (Figure 4).", "Attentional deficits following sleep disruption could contribute to many of the learning and memory deficits reported previously in the literature.", "Furthermore, an inability to maintain high levels of attention appropriately (e.g. after sleep deprivation) may also shift cognition and behaviour towards more automatic, model-free, habit-like forms which require low attentional capacity, and away from more goal-directed, model based processing which requires high levels of attention.", "This may also have important implications for understanding the significance of sleep disruption in neuropsychiatric disorders.", "Papers of particular interest, published within the period of review, have been highlighted as:", "• of special interest", "•• of outstanding interest" ]

[ "Synaptic GluA1 subunit levels increase during waking and decrease during sleep.", "GluA1-dependent plasticity is implicated in short-term habituation.", "Sleep may play a role in dishabituation, which restores attentional capacity.", "Sleep disruption favours model-free/habit-based forms of behaviour." ]

[ "MEDI" ]

[ "P300 evoked potential", "Rehabilitation of speech and language disorders", "Speech-Language therapy" ]

[ "2733" ]

[ "Patient evolution at the audiology and speech-language clinic acts as a motivator of the therapeutic process, contributing to patient adherence to treatment and allowing the therapists to review and/or maintain their clinical therapeutic conducts.1", "Electrophysiological measures, in turn, help in the evaluation, understanding and monitoring of human communication disorders, thus facilitating the prognosis definition in each case.2", "The Long-latency auditory evoked potentials (LLAEP) are objective measures used in the electrophysiological hearing assessment, corresponding to the thalamus and cortex activity in response to the auditory stimulus.", "It has an exogenous component, related to auditory sensitivity, and an endogenous component, P300, described in the literature as a cognitive potential.3,4", "P300 is elicited by performing a specific task that usually includes the discrimination between two randomly presented auditory stimuli (a frequent stimulus and a rare one).", "In this sense, the evaluated subject should indicate the stimulus that is considered rare, thus reflecting information about functions such as attention, discrimination, integration and memory.4", "Latency is directly related to the processing of information and the amplitude to the number of information that the stimulus was able to transmit.5", "The development of language, on the other hand, is intrinsically connected to cognitive development.", "It is in the interrelationship of a set of cognitive, linguistic and socio-pragmatic skills that language becomes effective.6", "Therefore, changes in auditory processing, changes in the development of language expression and/or reception, changes in the written language development, phonological disorders and disfluencies may lead to changes in the latency and amplitude of P300.", "Nevertheless, the rehabilitation of these disorders promotes functional and morphological modifications in the central nervous system (CNS) as a consequence of neuroplasticity.7", "Considering the high frequency of language changes, especially in the pediatric population, performing the P300 has gained space in scientific research.8–10", "Thus, the systematic review of this content will provide better planning in future studies, a synthesis of the knowledge gathered so far, in addition to adding new knowledge, subsidizing clinical practice and representing the importance of speech-language audiologists and otorhinolaryngologists’ work.11", "Therefore, the aim of this study is to determine whether speech therapy influences the variation in the latency and amplitude of the P300 auditory evoked potential in patients with speech disorders undergoing speech therapy.", "The review is reported according to the items in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Statement (PRISMA).12", "The strategies aimed at a complete search, including descriptors (DECs and MESH) and Free Terms (TL), based on the four elements of PICO (Patient, Intervention, Comparison, Outcome) present in the title, which consist of: (child or children or preschool) and (event related potential or p300 OR evoked potential) and (language disorders or language therapy or development disorders or rehabilitation of speech or speech therapy).", "The complete strategy is found in the supplementary material (Appendix 1).", "The searches were performed between April and May 2017 and were reviewed in September 2018.", "The following databases were searched: Pubmed, ScienceDirect, SCOPUS, Web of Science, SciELO and LILACS, as well as the gray literature databases: OpenGrey.eu, DissOnline, without language or date restrictions.", "There was no manual search of the included articles to avoid the risk of citation bias.13", "Inclusion criteria were: randomized or non-randomized clinical trials that involved children with language disorders in speech therapy, monitored by P300, compared to children without intervention, as well as the mean values of P300 latency and amplitude in the first and second evaluations, associated with a dispersion measure.", "Exclusion criteria were studies evaluating children with peripheral, cognitive, psychiatric or neurological auditory disorders.", "Repeated articles in different databases were also excluded.", "Titles and abstracts of articles obtained through the search were independently assessed by two investigators who were not blinded to the authors or to the titles of the journals.", "Divergences were resolved by consensus.", "In cases with no consensus, a third author was asked to make the final decision.", "The full texts of potentially eligible articles were acquired and analyzed in full.", "The outcomes sought in the studies were the mean values of latency and amplitude of the P300 components pre- and post-speech therapy associated with a measure of dispersion.", "The data of the published articles were analyzed, and the authors were contacted for additional information.", "In addition to the outcome data, the authors’ names, article title, year of publication, country, age groups, pathology, intervention, number of sessions and studied groups were also extracted.", "A standard form for data storage was created based on the model adopted by Cochrane.14", "Study quality was evaluated according to the recommendations found in the Cochrane Collaboration manual.15", "Two investigators independently assessed the quality of the studies in the following categories: generation of the appropriate sequence; allocation concealment; blinding of the evaluators; and handling of missing data for subsequent final judgment.", "The latency and amplitude variation of the P300 Evoked Potential for both groups (Study Group submitted to therapy and Control Group not submitted to therapy) was compared through a meta-analysis.", "For this purpose, a random effects model was used as a measure of the effect of the mean difference between the groups and as a statistical analysis method.", "An α value of 0.05 was considered statistically significant.", "When it was not possible to obtain adequate data for the analysis, the Cochrane recommendations were followed.", "The statistical heterogeneity between studies was tested using the Cochrane Q Test and inconsistency was tested using the I2 test.", "A p-value < 0.10 was considered statistically significant.", "When necessary, study characteristics considered potential sources of heterogeneity were included in a subgroup analysis.", "Additionally, in case of heterogeneity, the studies were removed one by one to investigate whether that particular study was the source of heterogeneity.", "All analyses were performed using RevMan software (Computer program, Version 5.3.", "Copenhagen: The Nordic Cochrane Center, The Cochrane Collaboration, 2014).", "Of the 1008 titles considered relevant based on the searches in the aforementioned databases, 21 texts were selected for full reading.", "Of these, 18 were excluded16–33 because they did not meet the eligibility criteria (Appendix 2).", "Therefore, three full texts were included in the qualitative and quantitative analysis (Table 1).", "The flow diagram illustrating the search and selection process is shown in Fig. 1 and the mean latencies and amplitudes of the P300 of the articles included in the meta-analysis are shown in Table 2.", "There were no significant differences between the right and left ears for all groups in all included studies.", "Moreover, the Oddball paradigm and the International 10–20 system for electrode placement were used, in addition to a significance level of 5%.", "The other parameters for P300 acquisition can be found in Table 3.", "In the study by Alvarenga,34 which included 20 students with a diagnosis of Developmental Dyslexia, 10 of them were submitted to therapy (GI) and 10 characterized the Control Group (GII).", "Two P300 evaluations were performed in the same interval for both groups.", "After the intervention, GI showed a statistically significant result for P300 latency (p = 0.005).", "The authors concluded that P300 is an efficient tool to monitor the therapeutic evolution of children with Developmental Dyslexia.", "The study by Leite35 evaluated 66 children, 25 of them without phonological disorder (group with typical development) and 41 with phonological disorder (study group), which they divided into two subgroups: 22 comprised the study subgroup A, submitted to 12 speech therapy sessions and re-evaluated by the LLAEP after the intervention, and 19 comprised the study subgroup B, reassessed 3 months after the first evaluation.", "Statistically significant differences were identified between the groups with typical development and study for P300 latencies and amplitudes.", "When comparing the first and the second evaluations, significance was observed for the P300 amplitudes in the study subgroup A (p = 0.039).", "The latency results were not significant for the two subgroups.", "The authors also used a criterion of improvement and non-improvement based on the mean latency and amplitude differences of the LLAEP components of Subgroup B.", "In this evaluation, they reported that after the therapy, improvement was observed in all components of the examination.", "Therefore, they concluded that children with phonological disorders have alterations in P300 and that the audiological/speech-language intervention results in the improvement of results of all the LLAEP components.", "Another study by Leite36 investigated 47 children, using a similar methodology.", "The children were divided into groups with typical development and study groups.", "The group with typical development consisted of 24 children and the study group of 23 children with phonological disorders, with the latter being divided into two subgroups: SG1, consisting of 12 children submitted to 12 speech therapy sessions and who were re-evaluated through LLAEP after the intervention, and SG2, consisting of 11 children who were not submitted to speech therapy and were re-evaluated three months after the initial evaluation.", "They obtained a significant result for P300 latency in the group submitted to speech therapy intervention (p = 0.024).", "The authors did not report the values for amplitude.", "The quality analysis of the included studies is shown in Table 4.", "All included studies were characterized as non-randomized clinical trials.", "Therefore, it is not possible to judge them regarding the categories of random sequence generation and allocation concealment.", "Two of them (Leite,35 2010 and Leite,36 2014) reported the blinding of the evaluators to analyze the latencies and amplitudes of the P300 Evoked Potential, from the inclusion of evaluators blinded to the subjects’ identities and their categories of participation.", "Regarding the handling of missing data, Leite36 2014 reported the abandonment of one member of the therapy group and absence of two members that belonged to the group without intervention in the second evaluation.", "However, he did not report how he treated these data in the statistical analysis.", "It should be noted that regardless of the final judgment found in the table, the three studies show, according to their nature, a high risk of bias due to non-randomization during the selection of their research subjects.", "As the studies are nonrandomized, the groups showed great divergence as early as in the first evaluation.", "Thus, to avoid the phenomenon of regression to the mean, the variations between the final and initial latency and amplitude values would be necessary, as well as the standard deviation associated to these variations.", "Three studies (84 individuals) were evaluated (Fig. 2).", "The mean difference between the latencies of the group submitted to therapy and the Control Group was −20.12 ms with 95% CI of −43.98 to 3.74 ms. The general effect test showed a p = 0.10, revealing that such a difference was not significant.", "For the heterogeneity, I2 = 27% and the value of p = 0.25.", "To avoid the occurrence of reverse causality, as the exposure changes as a result of the disease, a subgroup analysis of the same language disorder (phonological disorder) was performed.", "Thus, the mean difference between the latencies of the group submitted to therapy and the Control Group was −16.59 ms, with 95% CI of −55.11 to 21.9 ms. The test for the overall effect showed a p = 0.40, also revealing that there was no significant difference.", "For the heterogeneity, I2 = 50% and the value of p = 0.16.", "Two studies (61 subjects) were evaluated (Figs. 3 and 4).", "The article by Leite36 did not include the search for amplitude values.", "The mean difference between the amplitudes of the group submitted to therapy and the Control Group was 0.73 uV with 95% CI of −1.77 to 3.23 uV.", "The overall effect test showed a p = 0.57, showing that this difference was not significant.", "For the heterogeneity, I2 = 0% and the value of p = 0.47.", "Three articles met the inclusion criteria of the present meta-analysis, two related to the Phonological Disorder35,36 and one related to Dyslexia.34", "Despite the different language alterations, since the phonological disorder affects orality and dyslexia affects the reading system, both include phonological processing deficits as a basal alteration.", "Moreover, the studies share similarities regarding the subjects’ age and the fact that they include some type of intervention.", "The present review does not aim to find similar or different aspects between the language alterations, nor does it intend to evaluate the therapeutic procedures used.", "Rather it simply aims to determine whether speech therapy influences the variation of P300 latency and amplitude in patients with speech disorders submitted to speech therapy.", "The individual results of the studies that constitute this review state that the stimulation performed by the speech-language intervention is able to reorganize the auditory and cognitive processing abilities, thus observing a reorganization capacity of the brain in the processing of auditory information, based on the brain neuroplasticity capacity.", "They suggest that this effectiveness of the speech-language intervention occurs regardless of variables relevant to the pathology and the intervention, as the results were favorable in different language alterations and in different methodologies applied in therapy.", "Therefore, the effectiveness of the speech-language intervention, found through the P300 analysis, occurs independently of the affected language modality and the strategies or therapeutic resources used by the speech-language therapist.", "The intervention effectiveness is seen through changes in the P300 latency and amplitude in a broad manner, without quantifying the percentage of improvement according to the therapy.", "Therefore, the parameters used in the test acquisition, as well as the methodological characteristics of the studies, are given greater relevance.", "Regarding the test protocols, all the articles followed the recommendations of the International 10/20 System for electrode placement (derivation) and used the Oddball paradigm.", "One of the studies34 did not use the tone burst stimulation for the potential acquisition, using the speech stimulus to obtain specific information regarding auditory discrimination and language processing.", "Although the articles that comprise this review individually indicate that the P300 undergoes changes regarding its amplitude and latency parameters as an effect of the speech-language intervention, the results of the meta-analysis do not show the same thing.", "Regarding the methodological quality, all the studies showed a high risk of bias.", "This statement is based mainly on the impossibility of judging by the random sequence generation and allocation concealment criteria, showing an important selection bias.", "Furthermore, in the study by Leite36 the statistical treatment used in the study was not reported due to the loss of research subjects, which constitutes an attrition bias.", "On the other hand, the concern with the blinding of the evaluators appeared.", "It should be remembered that the article by Leite35 has low risk of bias when considering only its category (non-randomized clinical trial).", "Moreover, one of the studies was excluded for the amplitude comparison due to lack of data.", "This result calls attention to the need for better planning in future research, thus increasing the worth of these investigators’ performance.", "Therefore, the first evaluation already shows a discrepancy in the latency and amplitude mean values due to the several confounding variables in the selection of the groups.", "When discussing this discrepancy, it refers, for instance, to the latencies found in the study by Leite.36", "In it, the group selected for speech therapy intervention had in the first evaluation a mean value of 394.73 ms, whereas the Control Group had 349.55 ms.", "Considering these values, one can observe the distinction between the groups and, therefore, it cannot be affirmed that the values found in the second evaluation strongly consist of the therapy effect or only the phenomenon of regression to the mean.", "Nonetheless, none of the studies attempted to minimize these discrepancies.", "Conversely, to perform the meta-analysis, the Cochrane guidelines were followed14 and the variations of mean latency and amplitude values were calculated, as well as the standard deviation associated with this variation.", "In contrast, the literature points to the success of speech-language intervention in the most diverse disorders.", "Silva and Capellini,37 demonstrated the efficacy of a phonological intervention program in schoolchildren at risk for dyslexia after the application of a specific protocol for assessing cognitive–linguistic abilities pre- and post-therapy.", "Its intervention methodology resembles that proposed by Alvarenga,34 which worked with metaphonological skills and auditory processing, among others.", "Nevertheless, Rosal,38 verified in their study the importance of these same skills for the learning of writing.", "Despite the phonological disorders, different approaches and authors report the good results in the evolution of this patient profile.", "Wiethan and Mota39 gave different contributions of different approaches aimed at treating these alterations.", "Gubiani and Keske-Soares40 also verified the phonological system evolution in patients treated with different therapeutic approaches.", "The divergence found between the individual results of the studies that constitute this review, which affirm that speech therapy influences P300 alterations, and the results of this meta-analysis, which found that speech therapy does not influence the latency and amplitude results of P300, should be interpreted with caution, as they derive from a small number of non-randomized clinical trials.", "The lack of the intervention effect may be much more related to the lack of scientific rigor of the included articles than to the non-evolution of these patients post-therapy.", "The present meta-analysis demonstrates that speech-language therapy does not influence the latency and amplitude results of the P300 Evoked Potential in children with language disorders submitted to audiology and speech-language intervention.", "The authors declare no conflicts of interest." ]

[]

[ "MULT" ]

[]

[ "1000" ]

[ "The data comprise buyer and seller behavior of a total of 384+144 subjects who participated in 8 different treatments of an experiment reported in Krämer et al., 2017: 6 treatments with exogenous benefits (NYOP monopoly, NYOP competition with fixed roles, NYOP competition with a flexible seller; PWYW monopoly, PWYW competition with fixed roles, PWYW competition with a flexible seller) and 2 treatments with endogenous benefits (NYOP, PWYW).", "The data captures for each treatment, round and subject the experimental conditions, subsequent decisions and outcome.", "In the first set of experiments, sessions lasted about two hours and subjects earned on average 18 Euros (about 24 US Dollars at the time of the experiment), including a show-up fee of 4 Euros.", "In the endogenous benefit treatments average earnings amounted to 25 Euros (about 27 US Dollars at the time of the experiment), including a show-up fee of 4 Euros.", "All sessions were conducted at the experimental laboratory of the University of Munich (MELESSA).", "The subject pool consisted mainly of students from a wide range of majors.", "Treatments were implemented using zTree [1] and subjects were recruited using ORSEE [2].", "Table 1 summarizes the data provided with this paper.", "A brief description of the variables is contained in the Excel file variable_description_pwyw_nyop.xlsx.", "In each treatment, subjects are randomly assigned to a role (i.e., buyer or seller) that remains fixed throughout the experiment.", "Each session consists of 24 participants resulting in three markets in the competition treatments (two sellers facing six buyers in each market) and six markets in the monopoly treatments (one seller facing three buyers).", "All treatments are repeated for 20 periods, and subjects are randomly re-matched every period.", "The data comprise eight sessions of the competition treatments and another eight sessions of the monopoly treatments.", "In order to perfectly control the valuations of the buyers and the costs of the sellers we use an induced-value design [5].", "Conditional on using a customer-driven pricing mechanism, sellers may receive a benefit (b) per unit sold.", "In the first set of experiments the benefit is exogenously given.", "In the second set of experiments, benefits are created endogenously in the lab (see below).", "The exogenous benefit is proportional to the number of units sold.", "In order to identify the effect of b, we assigned a strictly positive benefit b randomly to 50% of all markets and the remaining 50% of markets have a benefit of zero.", "Sellers know whether they enjoy a positive benefit from using a customer-driven pricing mechanism while customers know only that these benefits exist in half of all cases.", "At the end of each session we elicit information about risk preferences and social preferences of the participants and their demographic characteristics.", "In treatments PCFlex (PWYW, Competition, Flexible role) and NCFlex (NYOP, Competition, Flexible role) one of the two sellers can choose whether to use posted prices or to use PWYW (NYOP, respectively) while the other (traditional) seller has to use a posted price.", "At the beginning of each period all subjects observe the per-unit cost of the good which is the same for both sellers and drawn from c∈{10, 30, 50}.", "The flexible seller privately learns the per-unit benefit b∈{0, 40} from using a customer-driven pricing mechanism and buyers privately learn their valuations of the good drawn independently from v∈{10, 25, 40, 60, 120, 200}.", "Then each seller decides whether to enter the market.", "Additionally, the flexible seller decides which pricing mechanism to use.", "All buyers and sellers subsequently learn about the market structure.", "Now the posted-price sellers set their prices, and a NYOP seller sets the (secret) threshold price above which s/he is willing to accept all bids.", "Finally buyers decide whether and if so from which seller to buy.", "If they opt for a posted-price seller they have to pay the posted price.", "If they decide to shop with a PWYW seller they get the good with probability 1 and are free to decide how much to pay for it voluntarily (including a price of zero).", "If they go for a NYOP seller they are asked to submit a bid.", "If that bid exceeds the secret threshold price set by the seller in advance, they pay their bid and a transaction occurs.", "In case the bid is below the seller׳s threshold price, they do not receive the good and do not have to pay either.", "Finally, payoffs are realized.", "The competition treatments with fixed roles (treatments PCFix and NCFix) are set up identically to the treatments with flexible roles except for the fact that one of the sellers is constrained to use either PWYW (in treatment PCFix) or NYOP (in treatment NCFix) if s/he decides to enter the market.", "In two monopoly treatments (PM, PWYW Monopoly) and (NM, NYOP Monopoly) there is only one seller who is forced to use PWYW (NYOP, respectively) if s/he enters the market.", "In these treatments, a market consists of only three buyers.", "Costs and benefits are parameterized as in the competition treatments, while buyers’ valuations are drawn from a restricted set, v∈{40, 60, 120, 200}.", "All sellers have the same cost-benefit combination in a given period as in the competition treatments and learn that combination before market entry.", "The exogenous benefit treatments varied our previous designs as follows: Each market consists of two sellers and six buyers.", "The demand is now split into two groups: There are two well-informed buyers, who are fully aware of the market structure, and four follow-up buyers, who have to rely on word of mouth to learn which seller(s) entered the market.", "The purchasing decisions of the well-informed buyers directly affect the market structure for the follow-up buyers, modeling word-of-mouth advertising in a reduced fashion.", "For example, if both well-informed buyers purchase at the PWYW seller, only the PWYW seller is visible to the follow-up buyers.", "However, follow-up buyers may become fully informed about which sellers are in the market if they pay an additional search cost.", "Furthermore, in the new NYOP treatment buyers can still buy from the posted-price seller if their submitted bid was not successful.", "With the beginning of a new period, buyers and sellers are informed about costs (c ∈ {5, 10, 20, 30, 50}).", "In addition, buyers learn their valuations (v∈{10, 25, 40, 60, 120, 200}).", "Then, sellers decide on entry.", "Having observed the sellers’ entry decisions, the two well-informed buyers decide whether and if so with which seller to transact.", "If at least one of the well-informed buyers has opted for the PWYW/NYOP seller, this seller is available for follow-up buyers at no additional cost.", "The same applies for the posted-price sellers.", "If only one seller is available to follow-up buyers, they can invest search costs (csinvest=10) to find out whether the other seller has entered the market.", "If this is the case, they can also purchase from the discovered seller.", "If the market for well-informed buyers is split equally, both sellers are available at no additional search cost.", "After learning about the market structure follow-up buyers make their purchasing decisions.", "In case of submitting an unsuccessful bid to a NYOP seller, the buyer can turn to the posted-price seller if this seller is available.", "Thus, the availability of sellers for follow-up buyers in the NYOP treatment depends on the interaction between the well-informed buyers and the sellers.", "To illustrate this consider the following situation: A well-informed buyer submits a bid to the NYOP seller which is rejected.", "The buyer subsequently purchases the good from the posted-price seller, making both sellers available to the follow-up buyers.", "However, in all cases where both well-informed buyers interact with the same seller only this seller is available to the follow-up buyers." ]

[]

[ "MULT" ]

[ "Food science", "Nutrition" ]

[ "1000" ]

[ "Cereals are major sources of energy for populations around the world.", "Taxonomic classification places them in the Gramineae or Poaceae family which is divided into seven major subfamilies of grasses, Bambusoideae, Oryzoideae, Pooideae, Panicoideae, Arundinoideae, Chloridoideae, and Centothecoideae, 40 tribes and about 600–750 genera [1].", "Wheat, barley, and rye belong to the genus Triticale, oats to Aveneae, rice to Oryzeae, corn and sorghum to Andropogoneae, and millet to Panicease [2].", "Wheat and rice are the most important cereals with regard to human nutrition, and account for up 55% of the total cereal production [3].", "Worldwide, cereals contribute to about half of the human energy requirement and have been grown as staple foods for human consumption and feeds for livestock since the beginning of civilization [4].", "The major cereal grains consumed in descending order are wheat, rice, corn (or maize), barley, oats, rye, millet, and sorghum.", "These grains provide carbohydrates, proteins, B-vitamins, and minerals for a major portion of the world's population [3].", "Cereals are also rich in secondary metabolites (i.e. bioactive compounds) that are important in health promotion.", "The majority of bioactive compounds are located within whole grains, bran or germ rich milling fractions.", "Many studies therefore have been performed to investigate the beneficial effect of whole grains or milling fractions for the general well-being, and for the reduction of oxidative stress and associated disease conditions such as cardiovascular diseases, diabetes, obesity, and cancer [5, 6, 7, 8].", "The beneficial effects of cereals have mostly been attributed to the presence of fibers (e.g. glucans), a group of polysaccharides that are resistant to digestive enzymes in the gastro-intestinal tract, meanwhile their health promoting effects go beyond fibers.", "In women for example, the health effects of whole grains on heart disease remained even after controlling the intake of fibers [9].", "While in men, the bran had a better protection against heart disease than whole grains [10], likely because of both fibres and phytochemicals.", "The benefit of whole grains, therefore, comes from more than one group of compounds that might act in additive or synergistic manners.", "In recent years, peptides and proteins present in foods have gradually been recognized as positive contributors to human health and as such, there has been an increase of studies that focus on the identification and physiological properties of hydrolyzed dietary proteins or purified peptides [11, 12].", "Peptides or their mixtures used in many investigations are generally produced by chemical hydrolysis, enzymatic hydrolysis, or microbial fermentation of protein concentrates or isolates [13].", "In the food industry, the method of choice is enzymatic hydrolysis because of the absence of toxic chemical residues.", "Proteolytic enzymes can come from microbes, plants, or animals and cleave proteins only at certain peptide bonds because of differences in their specificities [14].", "Other factors such as time, temperature, pH, and origin of proteins have an influence on the sequence of resulting peptides and their biological properties.", "After hydrolysis, ultrafiltration and chromatographic procedures are commonly used to obtain completely pure peptides or peptide fractions of different molecular weights, ionisation states or hydrophobicity.", "The majority of bioactive peptides in the literature are short (less than twenty amino acid residues), and their activities are affected by both amino acid compositions and sequences [12, 15].", "Biological functions such as antioxidant, antihypertensive, and antitumor activities have been reported for cereal protein hydrolysates or derived peptides [16, 17].", "This review focuses on those that possess anti-oxidative properties in foods and biological systems.", "Oxidative stress occurs as a result of an imbalance between the amount of oxidants produced and the antioxidant defense mechanism.", "There are endogenous and exogenous oxidants.", "Two groups of oxidants commonly referred to as reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the main contributors.", "However, chlorine, bromine, and sulfide reactive species are contributors as well to the alteration of the redox balance.", "Most oxidants are unstable and short-lived chemical species generated under various conditions in foods and physiological conditions.", "Processes that generate oxidants in food include the length of storage and temperature, heat treatment, the amount of available oxygen, the presence of transition metals, or oxidative enzymes.", "Thermal oxidation of lipids, for example, form unstable primary oxidation products (i.e. hydroperoxides) through initial proton removal and oxygen consumption [12].", "The hydroperoxides are then decomposed into secondary oxidation products such as aldehydes and ketones [18].", "Phytochemicals (i.e. secondary metabolites) such as carotenoids and polyphenols have antioxidant properties, meanwhile, they also possess pro-oxidant function due to the formation of peroxides [12, 19].", "Phenoxyl radicals, often quinones and semiquinones, are formed via auto-oxidation reactions of phenol moieties in the presence of radicals, hydrogen peroxide, and divalent metal ions [19, 20].", "Hydroxyl radicals generated from the reaction of hydrogen peroxide with metals such as ferrous ions are also present in foods [21].", "Light accelerates oxidation, especially in the presence of photosensitizers such as chlorophylls and riboflavin, which become excited upon absorption of energy [22].", "Once excited, chlorophyll or riboflavin reacts with triplet oxygen to produce singlet oxygen by energy transfer and return to their ground singlet state.", "The excited oxygen can diffuse [23] and oxidize electron-rich compounds such as unsaturated lipids, some amino acids and peptides.", "In biological systems, the primary cellular sources of ROS and RNS are the mitochondria where oxidative phosphorylation occurs.", "The mitochondria electron transport chain is composed of four multi-protein complexes I-IV and comprises a series of electron carriers such as ubiquinone, cytochromes, flavoproteins, and iron-sulfur proteins.", "The domains are arranged spatially according to their redox potentials, which vary from −0.320 to +0.380 V [24].", "In complex I, electrons derived from metabolic reducing equivalents of NADH are transferred via multiple redox cofactors to the first mobile electron carrier, oxidized coenzyme Q (CoQ).", "The energy released from the reaction is then captured via ejection of four protons from the mitochondrial matrix into the intermembrane space [25, 26].", "Electrons are also transferred to CoQ from flavoproteins, glycerol 3-phosphate, and succinate dehydrogenase (complex II).", "Reduced CoQ then transfers electrons to complex III for ultimate collection by cytochrome C, which is present in the inter-mitochondrial space.", "The energy liberated is captured via a proton pumping mechanism.", "The last step is the reduction of molecular oxygen to water in four one-electron steps in complex IV.", "It is estimated that 0.2–2% of the total oxygen consumption is reduced to superoxide anion radicals during respiration in mitochondria [27].", "This process leads to the production of other ROS species specifically in complexes I and III (about 90% total production).", "Organelles such as the endoplasmic reticulum and nuclear membranes also contain electron transport chains capable of donating electrons to molecular oxygen, thereby generating superoxide anion radicals and subsequent oxidants [28].", "During their catalytic processes, various enzymes that include xanthine oxidase, cytochrome P450 mono-oxygenase, nitric oxide synthases, lipoxygenases, cyclo-oxygenase, and NADPH oxidase also generate ROS and RNS [26, 28].", "The consumption of oxidized foods and the exposure to radiation of environmental toxins (metals, chlorinated compounds) are in vivo sources of oxidants.", "Common ROS and RNS include free radicals like superoxide anion (O2·−), hydroxyl (HO·), nitric oxide (NO·), nitrogen dioxide (NO2·), and peroxyl (ROO·), as well as non-radical species like singlet oxygen (1O2), hydrogen peroxide (H2O2), ozone (O3), hypochlorous acid (HOCl), nitrous acid (HNO2), peroxynitrite (ONOO−), dinitrogen trioxide (N2O3), and hydroperoxide (ROOH) [5, 29].", "Oxidation of unsaturated fatty acids in foods by 1O2, O2·−, or HO· through successive reactions generate aldehyde compounds that are responsible for off-flavor characteristics of many food products [12, 23].", "Phenoxyl radicals as well as HO· and ROO· can decrease the nutritional value of proteins by reacting with nucleophilic groups, such as the side-chain amine group of lysine or the sulfhydryl group of cysteine in free amino acids, peptides, and proteins [12, 21, 30].", "ROS released by mitochondria are believed to play an important role in conditions associated with oxidative stress such as the aging process, neurodegeneration (e.g. Parkinson's disease), and atherosclerosis [24, 31].", "This is because ROS and RNS induce damage to various biomolecules including DNA strand breaks, base and nucleotide modifications, particularly in sequences containing high guanosine [32, 33].", "The oxidation of amino acid residues can lead to the formation of protein aggregates through cross-linking, loss of enzyme activity, poor metabolic pathways, or cell death [33].", "Carbohydrates can also be oxidized and lead to accumulation of advanced glycation end-products which can further react with other molecules, thereby contributing for example to increase vascular permeability or low-density lipoprotein (LDL) oxidation [34].", "LDL oxidation is a contributor to many diseases such as cardiovascular diseases, arthritis, dementia and metabolic syndrome [35].", "Oxidation of lipids to hydroperoxides can disrupt cell membrane integrity and can also damage proteins [5].", "There is no known beneficial role of oxidants in foods, but they exist in biological systems.", "At low or moderate levels, ROS and RNS play important role in the maturation process of cellular structures and maintenance of the host defense system [29].", "During inflammation and infection, phagocytes (neutrophils, macrophages, monocytes) release free radicals to destroy invading pathogenic microbes as part of the body's defense mechanism [29, 36].", "Although the excess concentration of oxidants is detrimental to organisms, very low levels can be detrimental as well.", "It has been found, for example, that patients with granulomatous disease, a condition that results in defective membrane-bound NADPH oxidase, suffer from multiple and persistent infections because of their inability to produce (O2·ˉ) radicals [37].", "Other beneficial effects of ROS and RNS involve their physiological roles in the function of cellular signaling systems like the sensing of OxyR and SoxR transcription factors which are involved in DNA repair [38, 39].", "Proteins represent about 6–15% of cereals grains.", "In general, there are two main groups of proteins: prolamins and globulins [40].", "In wheat, rye, barley, and corn prolamins constitute 30–50% of total proteins and are called gliadins, secalins, hordeins and zeins, respectively [41, 42].", "In oats and rice, the presence of prolamins is minor and they account for 5–15 % of proteins [40].", "The nutritional quality of cereal proteins is low due to limitations in essential amino acids, mainly lysine.", "This is more pronounced in those cereals with high prolamin contents.", "In addition, these proteins possess limited functionality due to low water solubility and their hydrolysis is viewed as a way to improve not only nutritional values, but also to release bioactive peptides.", "The initial step is the extraction of proteins which can be achieved by solubilisation of cereal flours in solution of salts such as KCl and NaCl, or at alkaline conditions (pH 9–10) [43, 44].", "The alkali method is the most commonly used procedure, meanwhile due to high content sugars in cereals, pre-treatments with polysaccharide degrading enzymes (e.g. cellulase, amylase, viscozyme) have been used to enhance the solubilisation of proteins which are then precipitated at isoelectric points [45, 46].", "The next step involves the cleavage of bonds in the extracted proteins using proteases or by fermentation with microorganisms.", "These steps lead to the production of protein hydrolysates which has been found to possess various activities.", "Fractionation of protein hydrolysates are necessary in many cases to concentrate the activity into specifies fractions or to facilitate the identification of peptides.", "Common methods of fractionation are based on size (membrane filtration, gel chromatography), charge (ion-exchange chromatography), and hydrophobicity (reverse phase chromatography) [47, 48, 49].", "The identification of peptides in the hydrolysates and fractions is often achieved using tandem mass spectrometric and bioinformatic techniques as reviewed in a recent paper [50].", "One of the well known and commonly investigated properties of antioxidant molecules is their ability to scavenge radicals.", "It is, therefore, not surprising that data on the quenching of radicals by hydrolyzed cereal proteins exist in the literature.", "Wheat germ proteins hydrolysed with Proleather FG-F, a protease from Bacillus subtilis, scavenged 81% DPPH (1.6 mg/mL) and 75% O2·− (0.6 mg/mL) radicals [51].", "In other works, alcalase hydrolysed wheat germ proteins quenched DPPH, O2·−, and HO· radicals with EC50 values of 1.3, 0.4 and 0.1 mg/mL, respectively [52].", "In comparison, the EC50 values for wheat proteins fermented with Bacillus Subtilis B1 were 3.2 mg/mL, 6.0 mg/mL and 7.5 mg/mL, respectively [53].", "It appears that alcalase was more effective in releasing O2·− and HO· radical scavenging peptides from wheat germ while Proleather FG-F was better for releasing peptides that quenched DPPH radicals.", "For rice dreg proteins, protamex hydrolysates had better DPPH activity (EC50 8.7 mg/mL) than those from alcalase, neutrase, flavourzyme, and trypsin treatments (EC50 9.9–14.0 mg/mL) [54].", "Meanwhile, rice endosperm proteins hydrolysed with neutrase possessed DPPH (EC50 0.05 mg/mL) and hydroxyl radical (EC50 2.0 mg/mL) scavenging activities, as well as a weak superoxide anion radical scavenging activity [55].", "In a related study, rice bran protein hydrolysate prepared with a mixture of papain and flavourzyme displayed the highest DPPH activity (IC50 6.8 mg/mL) compared to values of hydrolysates prepared with a combination of two proteases comprising papain, flavourzyme, neutrase, protamex, or trypsin [56].", "Wang et al. [57] reported IC50 of 1.57 mg/mL for DPPH radical scavenging activity of a trypsin digest.", "Treatment of rice proteins with neutrase, alcalase, and flavourzyme produced hydrolysates with ABTS· scavenging activities (89–151 μg ascorbic acid (AA) equivalent/mL) relative to treatments with microbial cell extracts (220–280 μg AA eq/mL) [58].", "Based on these studies, rice dreg hydrolysates have much lower scavenging activities than those of brans.", "They are also lower relative to wheat germ or gluten hydrolysates.", "Corn gluten proteins treated with various proteases possess antioxidant activities against HO· (EC50 0.8–7.5 mg/mL), O2·− (EC50 12.5–12.8 mg/mL), and DPPH (EC50 1.0–1.26 mg/mL) radicals [59, 60].", "Hydrolyzed corn proteins and fractions also scavenged ROO· radicals with values of 65.6–191.4 μM TE/g [61].", "Alcalase hydrolyzed Hordein proteins and its fractions had DPPH radical scavenging activities with EC50 values from 0.5 to 3.8 mg/mL or 48–58%, at 0.5 mg/mL, and quenched O2·− by up to 40% at 0.5–1.0 mg/mL [62, 63].", "In a related work O2·− and HO· scavenging activities of barley gluten alcalase hydrolysates were stronger than those of flavourzyme digests, however, DPPH activity was lower [64].", "Hydrolyzed oat bran proteins had ROO· radical scavenging activities of 343–608 μM TE/g and inhibited the formation of O2·− by 20–36% and HO· by 10.2–14.1%, depending on the hydrolysis time and the concentration of pepsin [49].", "Treatment of oat bran proteins with protamex (various concentrations and time) provide similar ROO· (408–712 μM TE/g) activities, but lower HO· (2.4–11.2%) and higher O2·− (24–58%) scavenging activities [65].", "Alcalase and trypsin hydrolysate from whole oat flour ROO· activities were 269 and 434 μM TE/g, respectively while DPPH radicals quenching were 20–35% at 1 mg/mL [43].", "Fractionation of hydrolysed proteins can concentrate activities within certain fractions.", "Wheat pepsin (<3 kDa) and papain (<5 kDa) gluten hydrolysates had higher DPPH, HO·, and O2·− activities relative to fractions with higher molecular weights [66, 67, 68].", "The influence of chemical modifications is illustrated by the increased DPPH and HO· activities after conjugation of wheat gluten hydrolysates (alcalase and flavourzyme) with glucosamine [69], and a decreased ROO· in scavenging activity of pepsin and pancreatin hydrolysates upon deamidation with citric acid [70].", "Polysaccharide degrading enzymes have been used to enhance both extraction yield of proteins and radical scavenging properties of hydrolysates [44].", "Ultrasonic-assisted enzymolysis and pulsed electric field technology increased DPPH or HO· activities of corn protein hydrolysates [71, 72].", "Other strategies to enhance the activity include the choice of protease (however, there appears to be no ideal one because of the influence of concentration), temperature, and duration of hydrolysis.", "In addition, the alkaline pH will affect protein solubility, extraction yield and composition, and the properties of subsequently generated hydrolysates.", "The number of studies performed on pure peptides is limited.", "This is likely because it is labor intensive to fraction and isolate individual peptides using available chromatography and membrane technologies.", "Meanwhile, many peptides have been identified in hydrolysates or fractions with antioxidant activities but only a few have been tested (Table 1).", "Seven peptides derived from oat proteins were evaluated for ROO· radical scavenging activities.", "Amongst them, GLVYI and YHNAP were the most potent with values of 0.67 and 0.61 μM TE/μM peptide, respectively while DVNNNANQLEPR had the least activity, 0.14 μM TE/μM [73].", "Wheat-derived peptide, VLPPQQQY scavenging power against ABTS, DPPH, O2·− and HO· radicals were 371, 3977, 666 and 3629 mmol glutathione/mol peptide, respectively [74].", "Rice bran protein hydrolyzed using trypsin followed by membrane and gel filtration chromatography separations afforded peptide YSK which exhibited high DPPH (IC50 0.15 ± 0.01 mg/mL) [57].", "Four other rice peptides showed DPPH (IC50 0.144–0.161 mg/mL) and ABTS (EC50 0.1070–0.658 mg/mL) scavenging activities [75].", "An antioxidant peptide YFCLT, identified from corn gluten hydrolysate, exhibited excellent ABTS radical scavenging activity with EC50 value of 37.63 μM [76], while peptide CSQAPLA had IC50 values of 0.116 and 0.39 mg/mL in the DPPH and O2·− tests [77].", "There are two main mechanisms by which antioxidant molecules can deactivate free radicals: hemolytic or hydrogen atom transfer (HAT) and single electron transfer (SET).", "Both will produce identical end-products despite the difference in mechanisms [78].", "The two mechanisms may occur in parallel, but one can dominate depending on the structure of the antioxidant peptide and the type of assay that will influence the solubility and partition coefficient.", "Tyrosine containing peptides can act mainly through a HAT mechanism while cysteine, tryptophan and histidine peptides act mainly via SET mechanisms.", "During HAT there is a hemolytic separation of the proton bound to the heteroatom as illustrated in Fig. 1.", "The proton can be directly transferred between the reacting molecules, or can be involved in a solvent assisted proton loss (SAPL) depending on pH and acid-base properties [79].", "Radicals of antioxidant peptides have a significantly longer life than hydroxyl and peroxyl radicals produced in foods and during oxidative stress.", "HAT-based methods like the oxygen radical absorbance capacity (ORAC) assay measure the ability of a molecule (e.g. peptide) to quench peroxyl radical (ROO·) by H-donation.", "It has been found that the reactivity is determined by the bond dissociation energy (BDE) of the H-X group in the antioxidant compound [80, 81].", "Peptides that contain amino groups such as tyrosine, tryptophan, and histidine that can easily donate protons are, therefore, useful in the ORAC assay.", "A study on corn found that peptides HALGA and HAIGA have different activities in the ORAC assay, with the first one being about 1.6-fold more active than the second [82].", "The two peptides have the same properties so one can assume that the replacement of leucine by isoleucine either caused a steric effect or reduced the ability of histidine to donate protons.", "Peptide GLVYIL from oats had higher ROO· scavenging activity than YHNAP.", "It should be noted that the second peptide in addition to tyrosine, contains histidine, which is another proton donating amino acid [73].", "The ROO· activity of the oat peptides shows that the presence of proton donating amino acids and their actual location on the sequence are important.", "In the hydroxyl radical scavenging assay, the presence of cysteine and its location in tripeptides (CQV, QCV, QVC, QCA) derived from rye secalin was critical for their degree of activity.", "The mechanism occurred mainly through the homolytic cleavage of thiol (S—H) bonds as computed using density functional theory [80].", "DPPH and ABTS assays are usually classified as SET reactions, meanwhile, these indicator radicals can be neutralized by reduction via electron transfers but also by quenching via the HAT mechanism [83].", "The reactivity patterns of protein hydrolysates and peptide fractions are difficult to interpret because of the lack of detailed information about the composition and sequences of all peptides.", "The overall mechanism is also affected by the pH because bond dissociation energy and ionization potential of the reactive functional group are important [81, 84].", "Ionization potential values generally decrease with increasing pH as does the electron donating capability.", "The sequential proton-loss electron-transfer (SPLET) mechanism, which involved the deprotonation of the antioxidant molecules followed by an electron transfer, was found to be important for DPPH radical scavenging activities of polyphenols [85, 86] and melatonin, a metabolite of tryptophan [87].", "The method has not been applied to peptides likely because the knowledge of pKa values of an antioxidant molecule is crucial to assess the relative importance of the SPLET mechanism.", "Residues of amino acids that are important for HAT mechanisms are also important for SET assays.", "In the absence of peptide sequences, the amount of some aromatic amino acids in hydrolysates can be used to determine the importance SET-based assays.", "For example, the tyrosine and phenylalanine content of rice bran hydrolysate and ultra-filtered fractions had positive correlations (r > 0.831) with their DPPH and ABTS radical scavenging activities [88].", "In wheat, the presence of tryptophan or tyrosine at the C-terminus of peptides was important for their ABTS radical scavenging activities, however, for DPPH activities the location of these amino acids on sequences was less important [74].", "In a related work, DPPH and ABTS scavenging activities of peptide QQPQPW from corn protein hydrolysate was due to the presence of tryptophan [89].", "Lipid molecules in foods are very prone to oxidation processes which is a major contributor to deterioration during manufacturing, storage, and distribution.", "Various models have been used to study how hydrolysed cereal proteins and peptides can prevent the oxidation of lipids through antioxidant mechanisms.", "Common systems include linoleic acid but also vegetable/animal fats, and food systems.", "Enzymatically generated wheat germ protein hydrolysates showed antioxidant activities in a linoleic system [90].", "In another study, pepsin hydrolysed wheat germ proteins showed better oxidation of linoleic acid compared to hydrolysates generated by other proteases [66].", "The fractionation of wheat gluten based on charge gave fractions that effectively inhibited lipid oxidation during cooking [47].", "In a similar work, alcalase derived protein hydrolysates of rice residue prevented the oxidation of lard more efficiently than those from neutrase, bromelain and papain; the effect was partly attributed the presence of smaller size peptides [91].", "It believed that hydrolyzed cereal proteins could increase meat shelf life since, for example, the incorporation of antioxidantrice peptide fractions into meat products decreased lipid oxidation by 19 and 15% after one and two weeks storage, respectively [92].", "There is also a potential for use of cereal proteins in emulation because hydrolysis can convert them into very good functional and surface active molecules.", "In this regard, hydrolyzed rice proteins were shown to inhibit lipid oxidation in an oil-in-water emulsion [93].", "In addition to hydrolysates, purified peptides have also been shown to prevent the oxidation of lipids.", "An antioxidant peptide isolated from hydrolyzed rice endosperm with the sequence FRDEHKK significantly inhibited the oxidation of linoleic acid [94].", "The tetrapeptide DHHQ, and related DAHK and DHHK isolated from rice albumin showed antioxidant activities by inhibiting the oxidation of copper-induced oxidation of low-density lipoprotein (LDL).", "Oxidation of lipids in food and biological systems can be initiated by radical species but also by the presence of transition metals such as ferrous and copper ions.", "Transition metals are pro-oxidants and they decrease the oxidative stability of lipids through the decomposition of hydroperoxides into free radicals [95].", "Metalloenzymes, especially lipoxygenases, are non-heme iron containing dioxygenases present in plant and mammals and can catalyze the oxidation of polyunsaturated fatty acids [96, 97].", "The rate of initial oxidation is dependent on the reduction potential of the oxidant species which must be greater than that of the lipid.", "Hydrogen peroxide with reduction potential of 320 mV is, therefore, not able to directly initiate oxidation of unsaturated fatty acids (E ∼ 600 mV).", "Hydrogen peroxide does indirectly initiate lipid oxidation because it is the precursor for the formation of HO· radicals, which is a strong initiator (E = 2300 mV) [98].", "Thermodynamically, ROS with E > 1000 mV is capable of oxidizing polyunsaturated fatty acids [99].", "The superoxide anion radical (O2·−, E = 940 mV) is, therefore, not strong enough to attract hydrogen from unsaturated fatty acids [100] while the hydroperoxy radical (HOO·, E = 1060 mV) can.", "Typical oxidative reactions of lipids are displayed in Fig. 2 using linoleic acid as a model.", "Hydrolyzed proteins and peptides like other antioxidant molecules can inhibit the oxidation of lipids in foods or biological systems through three established mechanisms.", "They can work through direct radical scavenging as described above and prevent proton abstraction from the lipid.", "Alternatively, they can donate protons to covert peroxyl radicals like those formed at C-9 and C-13 of linoleic acid (Fig. 2) to form less reactive hydroperoxides.", "These are the so-called chain-breaking antioxidants [101].", "The other main mechanism is via metal chelation.", "The ability of hydrolyzed proteins from rice and corn to inhibit oxidation of lipids in meat, emulsions, and liposomes was believed to be due to their ability to chelate iron or copper ions, as well as their radical scavenging capacity [54, 92, 102].", "Rice peptides FRDEHKK and DHHQ reduced the peroxidation of linoleic acid [94] and Cu2+-induced LDL oxidation mainly through their chelating mechanisms [103].", "Both peptides contain histidine and aspartic acids which certainly contributed to the activity.", "The proposed intermediate states are displayed in Fig. 3.", "Two molecules are required for peptides with one chelating residue but not for those with multiple residues like DHHQ.", "It has been suggested that peptides with a histidine residue at the second or third position had high affinity for Cu2+ [103].", "Peptides can also reduce lipid hydroperoxides to relatively nonreactive lipid hydroxides through non-radical reactions.", "Gluten incubated with linoleic acid hydroperoxides and monolinolein hydroperoxides showed that most sulfhydryl groups were converted to a disulfide and other oxidation products [104].", "The proposed reaction mechanism for the reduction of lipid hydroperoxides to lipid hydroxides can proceed through two-electron transfers from the sulfur of cysteine or methionine to yield sulfenic acid and sulfoxide derivatives (Fig. 4).", "In biological systems, enzymes such as peroxiredoxins or methionine sulfoxide reductases are able to reduce oxidized sulfur.", "Models other than in vitro chemistry and food-based models have been used to assess the antioxidant properties of cereal hydrolyzed proteins and peptides.", "In general, studies on cell and animal models are limited compared to in vitro tests.", "Rat adrenal gland pheochromocytoma (PC12) cells exposed to wheat germ protein alcalase hydrolysates maintained their integrity (i.e. no cytotoxicity) and, at 1 mg/ml, they prevented H2O2-induced oxidative stress by 63.7% [105].", "In another study, wheat peptide fractions (2 mg/mL) prevented oxidative stress induced death in rat jejunal crypt (IEC-6) exposed to indomethacin and increased cell viability by 120% compared to the untreated cells [106].", "Successive separation (<3 kDa) of rice bran hydrolyzed proteins by ion-exchange, size exclusion and RP-HPLC yielded seven antioxidant peptide fractions with anti-proliferative activity on human gastric cancer cells SGC-7901 [56].", "Alcalase hydrolysates of proteins from brewers' spent grain prevented cell death and DNA damage human U937 monocytic blood cells exposed to hydrogen peroxide [107].", "Only a few peptides have been investigated in cell and animal models.", "Peptide RVF from wheat had no cytotoxicity and protected human neuroblastoma cells (SH-SY5Y) from H2O2-induced cell death with an increase viability of 37% [108].", "Peptide FRDEHKK from rice enhanced the viability of t-BOOH induced cytotoxicity in human MRC-5 fetal lung fibroblast cells (74%) and mouse RAW264.7 leukaemic monocyte macrophage (78%) [94].", "Seven peptides derived from oats did not show any cytotoxicity at concentrations of 0–200 μM and four of them (FNDRLRQGQLL, GLVYIL, YHNAPGLVYIL, and DVNNNANQLEPR) protected HepG2 cells from peroxyl radical induced oxidative stress [73].", "In rats treated with non-steroidal anti-inflammatory drugs, oxidative stress often increased and cause tissue damage in the small intestine.", "Daily intragastric administration of a low molecular weight wheat peptide fraction (140–1000 Da) for one month reduced edema and oxidative stress in the small intestine [109].", "In a related work, the same wheat peptide fraction reduced ethanol-induced gastric mucosal damage in mice partly through an antioxidant mechanism [110].", "Microscopic images showed that hepatocyte lesions induced in mice by Calmette-Guerin/lipopolysaccharide were reversed after supplementation of the diet with corn peptide fractions (600 mg/kg bodyweight) [111].", "Low molecular weight (<5 kDa) peptide fraction from alcalase hydrolyzed corn proteins showed hepatoprotective effect against carbon tetrachloride -induced liver injury at 200 mg/kg body weight in mice [112].", "Increased body fat is known to enhance oxidation of fatty acids that can be decreased by the addition of some antioxidant molecules to diets.", "In this regard, supplementation of high fat diets with antioxidant trypsin hydrolyzed oat proteins decreased oxidative stress in mice characterized by higher concentration of free thiol groups in plasma, higher activity of the superoxide dismutase antioxidant enzyme in plasma and liver as well as more vitamins E and C in liver [113].", "The mechanisms of antioxidant molecules at the cellular level can be through radical scavenging, regulation of the activity of redox enzymes or regulation and antioxidant response elements (AREs).", "Most ARES are genes activated by the nuclear factor erythroid 2 related factor 2 (Nrf2) and play a critical role in redox homeostasis, phase II metabolism, and cytoprotection during oxidative stress [114].", "Both reactive oxygen species and reactive nitrogen species are present in vivo.", "Common antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), peroxiredoxins, glutaredoxin (Grx) and glutathione reductase [115].", "Other enzymes such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) have been used to assess the oxidative status of the liver [111].", "In the cytosol and mitochondria, SOD in the presence of copper zinc or manganese converts O2·− radicals into H2O2 which is then converted in the peroxisome to water by CAT.", "In the cytoplasm and extracellular environments, GSHPx converts H2O2 and organic peroxides into water or alcohol derivatives while Prx acts on all peroxides and peroxynitrite (ONOO−) [115].", "Most studied cellular mechanisms of hydrolyzed cereal proteins and peptides include regulation of antioxidant enzymes, available thiol groups, intracellular reactive oxygen species, and reduction of lipid oxidation.", "The hepatoprotective effect of a corn peptide fraction in mice was associated with a decrease in activities of amino acid transferases (AST, ALT) in serum while in the liver there was a decrease lipid oxidation and an increase in SOD activity and glutathione levels [112].", "Wheat peptide fractions reduced ethanol-induced gastric mucosal damage in mice partly through an increased SOD, GPx, total antioxidant capacity level or decreased oxidation of lipid [110, 116].", "The cytoprotective effect of wheat germ protein hydrolysate PC12 cells was explained by an increase in activity of CAT and SOD by 41.13% and 37.74%, respectively while its anti-apoptotic effect was due to the reduction (31.2%) of the fractional DNA content [105].", "A related study explained the protection of stressed IEC-6 cells by small wheat peptides through an up-regulation of 11.1 and 8.6% of SOD and GPx, respectively [106].", "The isolated wheat peptide RVF reduced apoptosis (6–14%) in SH-SY5Y cells and increased Bcl-2/Bax [108] proteins that regulate antioxidant pathway and cell death.", "In the case of brewers' spent grain, the protection was due to the reduction of DNA oxidation (human U937 monocytic cells) and the reduction of interferon-gamma (human Jurkat T cells), a proinflammatory cytokine [107].", "The cytoprotection of HepG2 cells by oat peptides was attributed to several factors including the reduction of intracellular reactive oxygen species, increased cellular glutathione, and increased activities enzymes (SOD, CAT, GPx) [73].", "Cereal peptides and hydrolyzed proteins have been shown to reduce oxidative stress in chemical-based assays, animal models, cell cultures, and food systems.", "Overall, only a small number of pure peptides have been investigated at a cellular level.", "There is, therefore, a need for more studies of diseases or conditions associated with oxidative stress in different types of cells and in animal models.", "There is a lack of human studies, even though peptides from digested food proteins are generally recognised as safe.", "Finally, more chemistry-based studies are also needed to investigate modifications that can occur to peptides due to temperature or high pressure.", "All authors listed have significantly contributed to the development and the writing of this article.", "This work was supported by the National Science and Engineering Research Council of Canada (Grant No: 371908).", "The authors declare no conflict of interest.", "No additional information is available for this paper." ]

[]

[ "ECON", "ENER" ]

[ "Energy Modeling", "Energy Sector", "LEAP", "Mozambique", "Scenarios" ]

[ "2002", "2100" ]

[ "Since recently Mozambique is actively developing its large reserves of coal, natural gas and hydropower.", "Once developed, this could make Mozambique an important player in regional and global energy markets.", "The recent IEA Africa Energy Outlook refers to Mozambique as an emerging large energy producer (together with Tanzania), that soon will join the group of leading energy producers in Africa, including Nigeria, South Africa and Angola (IEA, 2014).", "The International Monetary Fund (IMF) recently predicted that Mozambique could become the world's third largest LNG exporter after Qatar and Australia (IMF, 2016).", "In addition, the country maintains the fourth-largest untapped recoverable coal reserves in the world, of which large-scale exploration started in 2011.", "In terms of planned hydroelectric capacity, Mozambique is in Africa second only to DR Congo.", "Against this background, we present in this paper a new integrated long-run scenario model of the Mozambican energy sector.", "To the best of our knowledge, our model is the first integrated energy modeling and future planning model for Mozambique in the energy studies literature.1", "Our scenario model is based on newly developed and locally collected energy statistics for the recent past as well as information about the latest developments and future plans as regards the production and transformation of energy in Mozambique.", "These data are supplemented with demographic, macro-economic and urbanization trends as well as cross-country based GDP elasticities with respect to biomass consumption, sector structure, vehicle ownership and energy intensity.", "The analysis makes use of LEAP, the Long range Energy Alternatives Planning System – an integrated modeling tool that can be used to track energy consumption, production and resource extraction in all sectors of an economy (Heaps, 2012).", "Hence, we name our model MOZLEAP.", "Our analysis fits in the literature of LEAP-based studies presenting energy planning scenarios at the country level.", "Recent examples include studies on China (Wang et al., 2011), Greece (Roinioti et al., 2012), Japan (Takase and Suzuki, 2011) and Taiwan (Huang et al., 2011; Yophy et al., 2011).", "In addition, and more often, LEAP has been used for sector-level analysis in a country or region, often focusing on the power sector (Bautista, 2012; Dagher and Ruble, 2011; Kale and Pohekar, 2014; McPherson and Karney, 2014), but also on renewable energy planning (Jun et al., 2010).", "Our modelling period starts with historical trends since 2000 and subsequently covers the anticipated surge in natural resources exploration until 2030.", "We model energy demand by households, transport and extractive industries, as well as the sectors agriculture, manufacturing, services, government and other.", "Also we specify electricity demand from neighboring countries in the region, given their essential role in developing the Mozambican electricity market.", "As regards the supply side, we model electricity production on a project by project basis, as well as gas exploration, coal mining, mineral (heavy) sands mining and charcoal production.", "We use the model to explore the potential impact of the expected surge in natural source exploration on aggregate trends in energy supply and demand, the energy infrastructure and economic growth in Mozambique.", "The structure of the paper is as follows.", "In Section 2 we present our methods: the modelling framework, the database that drives our scenario model and the scenarios itself.", "In Section 3 we present and discuss the main elements and results of our modeling exercise.", "Section 4 presents in more detail the future prospects of energy supply and demand.", "Section 5 concludes and discusses key policy implications.", "As mentioned in the introduction, our model makes use of the LEAP framework.", "LEAP is intended as a medium to long-term modeling tool, designed around the concept of long-range scenario analysis (cf. Suganthi and Samuel, 2012).2", "Our model includes a historical period that comprises the period 2000–2010, in which the model is run to test its ability to replicate known statistical data.", "Subsequently, our model generates multiple forward looking scenarios for the period 2011–2030.", "LEAP supports a wide range of different modeling methodologies.", "In essence, the LEAP accounting framework calculates (future) energy demand as the product of activity levels (such as GDP, population, physical production levels) and energy intensity per unit of activity.", "Our energy demand modeling is based on a combination of historical energy and activity level data that we collected and information on demographic and urbanization trends supplied by external sources, locally collected bottom-up information as regards future electricity distribution and cross-country econometric modeling of GDP elasticities with respect to biomass consumption, sector structure and vehicle ownership.", "Fig. 1 and Table 1 summarize, respectively, the structure of the MOZLEAP modelling framework and the MOZLEAP model itself.", "In the next section we describe this approach and its results in more detail.", "On the supply side, we model electricity production, gas exploration, coal mining and mineral sands mining on a project by project basis.", "In addition, we develop and integrate into the LEAP framework a simple biomass model to calculate future paths of charcoal production and biomass consumption in Mozambique.", "On the demand side we adopt a mix of these methodologies to model energy demand by households, transport and extractive industries, as well as the sectors agriculture, manufacturing, services, government and other.", "Also we specify electricity demand from neighboring countries in the region, given their essential role in developing the Mozambican electricity market.", "Most of the energy statistics for Mozambique that we use in our analysis were collected and processed by the Directorate of Studies and Planning (DEP) of the then Ministry of Energy in Mozambique (MoE, 2012).", "The authors of this paper have been actively involved in this process.", "Underlying data were solicited and provided by a range of local institutions, including National Institute of Petroleum (INP), National Company of Hydrocarbons (ENH), Mozambique Petroleum Company (PETROMOC), Cahora Bassa Hydroelectric (HCB), Mozambique power utility (EdM), Mozambique Transmission Company (MOTRACO), National Energy trust-Fund (FUNAE), South African multinational gas and Oil company (SASOL), Matola Gas Company (MGC), Portuguese Petroleum and Gas Company (GALP), VidaGas, National Institute of Statistics (INE), Mozambique Petroleum Import (IMOPETRO) and the Ministry of Planning and Development (MPD).", "Historical data on consumption of traditional biomass have been estimated on the basis of combined information from national survey data published by INE and international data published by the IEA and FAO.", "In addition, we have made use of previously published data on the energy situation in Mozambique (Arthur et al., 2010, 2012; Atanassov et al., 2012; Chambal, 2010; Cuamba et al., 2013; Cuvilas et al., 2010; Batidzirai et al., 2006; Bucuane and Mulder, 2009; Brouwer and Falcão, 2004; Schut et al., 2010).", "Data on existing and future production of mineral resources (coal, natural gas and mineral sands) were compiled on the basis of information gathered from the then Ministry of Mineral Resources (MIREME), KPMG International (2013), United States Geological Survey (Yager, 2012) and the US Energy Information Administration (EIA/DOE).", "In addition, we collected information from press releases by private companies (in Bloomberg, Reuters, Mining Weekly, Mozambique Information Agency-AIM, and other national press), as well as from personal communications with local experts.", "Information on future electricity trade in the region is based on information published in the Integrated Resource Plan by the South African government (SA Department of Energy, 2011) and interviews with local experts.", "Finally, demographic and economic data on Mozambique were obtained from INE, the Ministry of Planning and Development and the Mozambique Central Bank (BM) as well as from the World Bank, the International Monetary Fund (IMF, 2013), the United Nations Department of Economic and Social Affairs (2011 Revision), and the African Development Bank (AfDB).", "All locally collected data, insofar possible, have been checked against data from international sources, including British Petroleum (BP, 2012), International Energy Agency (IEA, 2013a, 2014), United Nations Populations statistics and the World Development Indicators as published by the World Bank.", "Energy scenarios are self-consistent storylines of how an energy system might evolve over time.", "Since this is, to the best of our knowledge, the first integrated energy modeling and future planning study for Mozambique in the energy studies literature, we chose to develop in this paper a limited number of scenarios that are intentionally fairly simple and straightforward.", "Our main goal is to introduce our newly developed scenario model MOZLEAP, and to use it for highlighting major trends in the transformation of the emerging Mozambican energy sector, including the expected consequences for both domestic and international energy markets.", "The development of richer scenarios, including more detail and variation in terms of energy policies, structure of energy demand, energy supply mix options and regional differences, is deliberately left for future work.", "Energy outlooks usually give three basic scenarios – medium, high and low – that are often largely defined by GDP and population growth expectations.", "We follow this approach, but add a fourth scenario that assumes exploitation of Mozambique's natural resources exploration to its fullest potential.", "We label our three basic scenarios as Reference, Reference High and Reference Low.", "Reference is the most likely development path.", "Development of GDP in the Reference scenario is based on baseline projections plus activities of new extractive industry and electricity generation projects that are (almost) sure to be realized, taking into account realistic and somewhat conservative estimates about the output price development in the extractive and aluminum industry.", "Furthermore, it adopts a medium variant of population growth scenarios, a modest decline in household size, a moderate speed of urbanization and somewhat conservative estimates as regards the development of energy intensity improvements across sectors.", "Reference High and Reference Low then refer, respectively, to the optimistic and pessimistic variant of Reference – thus assuming higher (lower) baseline economic growth, lower (higher) population growth, higher (lower) speed of urbanization, faster (slower) decline of household size and higher (lower) output price developments in the extractive and aluminum industry.", "We refer to Table 2 for a brief summary and overview or scenarios.", "Finally, our Extractive scenario describes the expected evolution of the Mozambican energy system if all potential projects of extractive and aluminum industries as well as power generation are realized, including those projects that are yet (very) uncertain.", "In other words, this scenario tells the story of the Mozambican economy and energy sector becoming very much extractive industry driven.", "Because of this focus, we assume all other leading dimensions of the model (population growth, household size, speed of urbanization, energy intensity improvements and output price developments) to be equal to the Reference or Reference High scenario (see Table 2).", "This straightforward set-up, again, is motivated by our aim to show the potential impact of an extractive industry driven development path as caused by the mere expansion of this activity rather than by (optimistic) energy intensity changes or price developments.", "We leave it to future work to analyze the potential impact of price volatility on international natural resource and commodity markets on the Mozambican economy and energy sector, detailing the (future) evolution of international commodity price variation across markets and sectors.", "In this section we present the different parts of our scenario model (see also Fig. 1) in more detail, and show how the expected surge in natural resource exploration relates to aggregate trends in economic growth, population growth, urbanization, biomass consumption, sector structure, vehicle ownership and energy intensity in Mozambique.", "Together with population growth, per capita GDP is a key driving force in our model.", "Evidently, on the one hand energy is an essential production factor that fuels economic growth, while on the other hand increasing standards of living lead to growing demand for energy (GEA, 2012).", "In accordance with this, our model structure assumes that across sectors growing GDP is associated with higher energy use.", "Also, we assume that total biomass consumption and fuel demand for road transport are determined by GDP per capita, either directly (in the case of biomass) or indirectly (in the case of road transport, assuming that vehicle ownership is determined by per capita GDP).", "Finally, we assume that in various sectors of our model the evolution of energy intensity is a function of GDP growth, reflecting the notion of increasing energy efficiency under economic development (Lescaroux, 2011).", "This implies that total GDP growth rate is a weighted composition of baseline and extractive GDP growth rates.", "As regards the evolution of p, our extrapolation methodology assumes a simple, scenario-specific, trend based on expected international market prices of the primary resources involved (LNG, mineral sands, coal, aluminum).", "These prices are partly based on expert judgments for the upcoming years, published in a variety of resources (IEA, 2013b; KPMG, 2013), while for the remaining years price trends are assumed to follow a straightforward but scenario-specific pattern, with annual price fluctuations varying between − 2% and 4%.", "Given the expected large relative size of the extractive industry in the future economy of Mozambique, future price trends for primary resources are deliberately designed to be conservative, in order to avoid an upward bias in future GDP development paths.", "We refer to Table 3 for further details.", "The results of our GDP calculations are summarized in Fig. 2.", "First, this figure clearly shows that Mozambique is extremely poor but at the same time experienced rapid economic growth over the last decade and a half.", "For the period 2000–2015 the average annual growth rate of GDP was about 7.4% (IMF, 2016).3", "In that period, per capita GDP doubled from $250 to just over $500 (the latter equals to about $1100 in PPP terms).", "These levels roughly correspond with 9% of the per capita GDP level in South Africa and 2% of the per capita GDP level in the USA, and imply that still about half of the Mozambican population lives below the local absolute poverty line (Boom, 2011).", "In addition, Fig. 2 shows that our modelling of Baseline GDP (see Eq. (1)) leads to a gradual increase of Baseline GDP per capita to levels of $700–$900 by 2030, depending on the scenario.", "Extractive GDP per capita is expected to increase dramatically over time, from almost zero in 2000 to $70–$90 after 2020 in the Reference scenarios, and up to $225 in the Extractive scenario.", "Together these developments cause total per capita GDP to be in the range of $750–1150 by 2030, depending on the scenario; this equals a 50–125% increase from 2015 levels.", "Third, Fig. 2 shows that GDP growth rates are expected to peak around 2020, especially in the extractive scenario.", "This growth acceleration is largely driven by extractive industry expansion, most notably the assumption that gas processing facilities in the Rovuma Basin, in the far north of the country, will begin production around 2020.", "The International Monetary Fund (IMF) recently predicted that Mozambique's average growth rate during the first half of the next decade could reach over 20% per annum (IMF, 2016).", "Given the political, financial and technical uncertainties in developing these gas fields, we work with more conservative assumptions.", "Nevertheless, we predict growth rates of over 10%, of which one third to almost half will be caused by the extractive sector expansion, depending on the scenario – as can be seen from Fig. 2.", "This resembles the situation in the early 2000s when the extractive sector was an important driving force of GDP growth, due to the opening up of the Mozal aluminum smelter near the capital Maputo.", "Together, our projections imply that over the next decade the extractive sector will comprise about 10–20% of the total economy, depending on the scenario.", "Size and growth of the population helps define critical indicators in our model, such as per capita GDP, the electrification rate, total residential energy consumption, and fuel consumption by passenger cars.", "In addition, these indicators are influenced by the composition of the population in terms of the urban–rural divide and whether or not households have access to electricity.", "Growth of population has been calculated as the product of birth, mortality and net migration statistics, based on information from the National Statistics Institute (INE) that is derived from national censuses 1997 and 2007, supplemented with data obtained from local surveys on, amongst others, infant mortality and HIV prevalence.", "Future projections of these various demographic statistics have been obtained by INE through a combination of extrapolating historical trends, collecting new data from local surveys (after 2007) and the use of demographic modelling software developed by the UN and the US Census Bureau.", "Fig. 3 and Table 3 summarize our key demographic indicators across the various scenarios.", "As regards population growth, all our scenarios for the period 2011–2030 take as their starting point historic data for the year 2010.", "In 2010 Mozambique's population was some 22.4 million in total, of which almost 31% lived in urban areas; average household size was 4.3, average annual population growth was 2.5%, with urban population growing 3.2% per year.", "Subsequently, all scenarios assume population growth to gradually decrease over time.", "In our Reference scenario we expect average annual population growth to decrease to 2.0% in 2030.", "In our Reference Low and High scenarios, we assume this number to be 0.4 percentage point higher and lower, respectively; the Extractive scenario is identical to the Reference High for all demographic indicators (see Table 3).", "As a result, by 2030 total population size is expected to be in the range of 33–37 million people, with 34.8 million people in the Reference Medium scenario (see Fig. 3).", "Growth of urban population is expected to increase to 3.5% per year in 2030 in the Medium scenario; in the Low and High scenarios we assume this percentage to be 50% lower and higher, respectively (see Table 3).", "As a result, by 2030 the percentage of urban population is expected to be in the range of 31–49%, with 39.1% urban people in the Medium scenario (see Fig. 3).", "This implies that in the Medium scenario the number of people living in cities in Mozambique by 2030 is as large as 60% of the entire population in 2010.", "Obviously this will not only reshape the urban landscape in Mozambique over the next 25 years, but also transform (residential) energy demand.", "Finally, in all scenarios we assume the average household size to gradually decrease under influence of income growth and urbanization, from 4.3 persons in 2010 to 4.13–4.25 persons in 2030 (see Table 3 and Fig. 3).", "The extent to which the Mozambican population has access to electricity is expected to change rapidly as a result of intensive (rural) electrification programs and growing income levels.", "In our model the electrification rate is endogenously determined by combining information on electricity network expansion (number of new connections realized) with population growth dynamics as described above.", "In 2010 the national utility EdM realized 100.000 new connections.", "In our Reference scenario we expect this number to increase to 135.000 in 2015, and subsequently decrease to 100.000 in 2030.", "In our Reference Low and High scenarios, we assume that in 2030 respectively 70.000 and 130.000 new connections will be realized (see Table 3).", "Given population growth, this implies that in our model the (household) electrification rate is expected to increase from 15% in 2010 to 34% – 45% in 2030, with 39% in the Reference scenario (see Fig. 3).", "We assume transmission and distribution losses to remain at 5% as from 2011 (see Table 3).", "Biomass intensity per non-electrified household is subsequently derived from total biomass consumption not consumed by electrified households.", "Fig. 4 illustrates the working of our biomass model within the LEAP framework for Mozambique.", "The left-hand side of Fig. 4 shows the evolution of per capita biomass consumption as function of per capita GDP, using actual values for Mozambique.", "Biomass currently is the principal energy source for the majority of Mozambicans, coming from an estimated 30.6 million hectares of forests and representing 80% of the energy consumed by households (Chambal, 2010).", "In our model, total biomass consumption declines with increasing GDP, following an inverted S-shaped patterns as defined by the logistic function of Eq. (4).", "As regards its composition, with rising levels of per capita GDP, consumption of charcoal increases at the expense of fuelwood consumption, under influence of rising income and urbanization – up to some income threshold level, after which it is substituted for modern energy forms such as LPG and electricity.", "The right-hand side of Fig. 4 demonstrates the substitution of fuelwood for charcoal across basic MOZLEAP model runs.", "In our baseline scenario (“Reference”) the percentage share of charcoal in total biomass consumption in Mozambique increases from about 10% in 2000 (historical data) to almost 30% in 2030, thus decreasing the percentage share of fuelwood from about 90% to 70% over the same period.", "In the optimistic (high economic growth) scenario (“Reference:High”) the expected percentage charcoal by 2030 is over 40%, in the pessimistic scenario (“Reference:Low”) it still is expected to double from 10% in 2000 to 20% by 2030.", "Next we calibrate our model by combining this approach with data on the annual evolution of registered vehicles in Mozambique and fuel consumption in the recent past, supplied by, respectively, the Mozambican National Institute of Road Transport (INATTER) and the Ministry of Energy (MoE, 2012).", "Fig. 5 summarizes the results of our methodology for estimating future demand for transport fuels across the various scenarios.", "It shows that the number of vehicles per thousand people is expected to grow from just over 10 in 2010 to about 29–38 in 2030, depending on the scenario.", "With increasing levels of per capita GDP, the implied GDP elasticity of vehicle ownership in our model is decreasing over time, from 2.5% in 2010 to about 1% in 2030.", "In short these numbers mean that the total number of vehicles in Mozambique is expected to increase four to five fold over the period 2010–2030, from just over 370,000 to about 1.6–2.0 million, depending on the scenario; of this total number of vehicles in 2030 in our model 63% consists of cars and 22% of trucks.", "Mozambique has a large potential for the production of biofuel, given its climate and a vast amount of unused arable land.", "At this moment, biofuel production plays only a marginal role in the energy mix.", "However, the country has adopted a National Program for the Development of Biofuels to promote and use agro-energy resources for energy and food security.", "In doing so, the government also aims to encourage socioeconomic development and to reduce the country's dependence on fuel imports (Cuamba et al., 2013; Ecoenergy, 2008; IRENA, 2012).", "The program aims to progressively increase the proportion of biofuel in Mozambique's domestic liquid fuel mix in three phases.", "The pilot phase (2012–2015) is currently being implemented with a fuel blending mandate of 10% for bioethanol and 3% for biodiesel.", "An operational phase (2016 to 2020) will follow, with 15% bioethanol and 7.5% biodiesel blending and conclude with an expansion phase (2021-onwards) of 20% bioethanol and 10% biodiesel blending.", "In our scenarios, we include these phases, but take into account a 5-year delay to reflect the actual situation.", "South Africa's power utility (Eskom) has identified Mozambique as a potentially important supplier of electricity in its Integrated Resource Plan 2011 (SA Department of Energy, 2011) to help addressing its future supply-side challenges.", "Eskom is particularly interested in new hydropower from Mozambique, as the existing electricity generation mix in South Africa is carbon-intensive.", "Already, the Cahora Bassa (HCB) dam represents 40% of Eskom's carbon-free generation.", "One of the scenarios in the IRP is to use 2600 MW of power from Mozambique, including 2135 MW from the new hydro projects.", "Electricity purchases from Natural Gas plants at the Mozambique-RSA border is not looked at in the 2011 Integrated Resource Plan (IRP).", "As of date, South Africa gets 92 MW from Gigawatt plant in Ressano-Garcia, and could get an additional 150 MW from Sasol's plant in the same area.", "According to the IRP, South Africa needs an additional 90 GW of generating capacity by 2030, mostly from renewables.", "Therefore, in our Extractive Scenario, we have modeled 3320 MW of capacity dedicated to Eskom, of which 1900 to 2100 MW would have to be firm.", "As noted before, the LEAP accounting framework calculates (future) energy demand as the product of activity levels (such as GDP, population, physical production levels) and energy intensity per unit of activity.", "Therefore, the final building block of our model is an energy intensity builder that defines for each level of activity the corresponding energy intensity values over time.", "For the period 2000–2010 energy intensity values are calculated based on historical data regarding energy consumption and activity levels on a sector by sector basis.", "Subsequently, future energy intensity values for the period 2011–2030 are calculated on the basis of a variety of simple assumptions, again on a sector by sector basis.", "In this first integrated energy modeling and future planning study for Mozambique we deliberately apply simple and straightforward assumptions that do not vary in itself across scenarios.", "This choice is primarily motivated by our emphasis on exploring the potential impact of the expected unique surge in natural resources exploration in Mozambique on the country's energy supply and demand and economic growth prospects.", "Especially given Mozambique's current status as an extremely poor country with a rapidly expanding energy sector, this made us decide to leave a careful analysis of energy efficiency improvements in end-use sectors – although interesting and important – for future research.", "A summary of our assumptions as regards future energy intensity trends across the various end-use sectors is presented in Table 4.", "We assume that in a poor country like Mozambique electricity consumption per household increases over time under influence of rising GDP, because growing household income leads to increasing demand for electric appliances such as refrigerators and air conditioning.", "As regards LPG, we assume that consumption per household increases over time under influence of rising GDP as well as the degree of urbanization, because growing household income leads to a shift towards modern cooking fuels, while in developing countries LPG is a typical urban fuel for logistic reasons.", "Furthermore, we assume that kerosene consumption per household decreases over time, because of a gradual ‘autonomous’ substitution towards more efficient and cleaner fuels like electricity and LPG.", "Finally, future charcoal and fuelwood intensities are derived from our biomass model.", "In short, we assume that total biomass consumption decreases under influence of increasing per capita GDP, while the share of charcoal in total biomass consumption increases with income and urbanization at the expense of fuelwood.", "For the Agriculture sector we assume that energy intensity increases with about 20% over the course of 20 years, under influence of modernization and mechanization.", "In the Manufacturing sector we assume decreasing energy intensity growth, driven by the opposing forces of modernization and increasing energy efficiency.", "Under influence of increasing domestic natural gas production, we assume that the share of natural gas in this sector increases to 33% by 2030 at the expense of electricity and diesel shares; fuel oil plays a minor role.", "In the sector Commercial Services, we assume that electricity intensity increases with economic growth, while LPG consumption (in hotels and restaurants) again also positively depends on the degree of urbanization – following the same logic as in the residential sector.", "Consequently, we assume a gradual substitution away from biomass consumption.", "Finally, we assume that electricity intensity for Public Lighting increases with economic growth.", "Also, in the sectors Government and Other we assume that electricity intensity will rise under influence of economic growth.", "Energy intensity in the extractive industry is determined by constant values of electricity and diesel consumption per physical unit of production.", "We assume constant energy intensity values and fuel shares for the aluminum smelter MOZAL in the south of Mozambique, based on historical data, because we do not expect changes in its production process.", "Until recently MOZAL, which transforms imported bauxite into aluminum for export, was responsible for about three quarters of total electricity consumption in the country – and thus is a key feature of (future) energy planning and energy policy in Mozambique.6", "Actual values for the mining activities originate from a combination of indicative figures on open-cut coal mining and mineral sands explorations reported in the literature (Bleiwas, 2011; SEE, 2009) and from personal communications with local experts involved in mining activities in Mozambique.", "Finally, fuel efficiency in road transport is assumed to gradually increase over time under influence of economic development, which stimulates increasing import of newer and thus more fuel efficient vehicles.", "In contrast, we assume that fuel intensity for tractors increases because of the expected increasing use of heavy equipment as economic development proceeds.", "As regards the fuel mix, we assume a progressive use of biofuel in the domestic liquid fuel mix, adopting biofuel blending mandates from the government of Mozambique, taking into account a 5-year delay in accordance with the actual situation.", "The energy intensities for the different economy sectors, measured as final energy consumption (in tons of oil equivalence(toe)) by the respective activity level (GDP in million US$), are presented in Table 5, for the Reference and Extractive scenarios.", "The Table shows that according to our model total energy intensity in Mozambique is expected to fall rapidly until 2030.", "This aggregate trend is driven by the combination of a few sector trends.", "First and most important, energy consumption per household is predicted to fall with 20–30% in the period 2010–2030.", "This decrease is mainly caused by the substitution of charcoal and modern energy types for the inefficient source of fuelwood.", "Given that in Mozambique the residential sector is responsible for the major part of energy consumption (see next section), this development obviously has a major impact on the evolution of aggregate energy intensity.", "Second, energy intensity levels are expected to fall relatively rapid in the Manufacturing and, especially, Extractive industry sectors.", "Manufacturing energy intensity changes mainly under influence of an increase in size and value added of manufacturing activities, starting from a very small base.", "The exceptionally high energy intensity level in the Extractive industry sector in 2010 is to be explained by its composition: the MOZAL aluminum smelter and the mineral sand exploration project in northern Mozambique (in Moma) – both of which operate highly energy-intensive processes.", "However, as the Table shows, energy consumption per unit of GDP in the Extractive industry sector is expected to fall rapidly over time because of the expected surge of natural gas and coal exploration, which broadens the production base.", "In contrast, because of our assumptions the other sectors (except Road Transport and Services) show a gradual increase of energy intensity over time.", "Using the modeling framework and assumptions as explained in the previous sections, we present in this section in more detail the future prospects of energy supply and demand in Mozambique, with its implications for the international energy markets.", "As was described in Section 3.1, we constructed extractive industry GDP on the basis of a bottom-up approach, based on information for individual projects in electricity production, gas exploration, coal mining and mineral sands mining.", "Below, we describe these projects in more detail, since they constitute a key element in our scenario paths regarding future energy supply in Mozambique.", "As regards electricity generation, we consider in total 37 projects with a total capacity of almost 11.000 MW.", "Hydro is and remains to be the main source for electricity generation in Mozambique, with the greatest potential lying in the Zambezi River basin at sites such as Cahora Bassa North and Mphanda Nkuwa (Chambal, 2010).", "The existing capacity is around 2200 MW, of which 2075 MW is provided by the Cahora Bassa (HCB) dam.", "In total we consider in our model 15 hydro projects over the entire period 2000–2030, with a total capacity of about 7579 MW.", "In addition, in the period 2011–2030 we consider the construction of 12 natural gas fired power plants with a total capacity of 1114 MW as well as 6 coal fired power plants with a total capacity of 2150 MW.", "Finally, we include 101 MW from diesel generations and 1.2 MW solar power (cf. Cuamba et al., 2013).", "The latter reflects that over the last decade PV solar energy has been gradually adopted in schools and health centers in rural areas, as well as in telecommunications businesses (Chambal, 2010).", "Next to its capacity, for each project we define its transformation efficiency, expected first year of production and merit order.", "As regards the latter, we divide the modeling period into four intervals and attribute merit order by expected first year of production in the basis of 5-years intervals, such that the value one represents existing power plants.", "Furthermore, the Reference scenario includes existing and very likely future power projects whereas the Extractive scenario includes all 37 power projects, including those whose realization is still fairly uncertain.", "We refer to Table A.2 in the Appendix for a detailed overview.", "As regards the exploration of coal, our model includes in total 12 major coal mining projects with, in the Extractive scenario, a maximum total estimated annual production of 108 million tons by 2030.", "In the more moderate Reference scenario we consider 7 mining projects, which together account for 57 million tons per year by 2030.", "We refer to Table A.3 in the Appendix for details.", "For each mining project we define it's expected first year of production, the expansion of capacity over time, and the destination of its production (export versus electricity production).", "As regards Natural Gas production, we of course start by modeling the existing natural gas exploration project by the South African company Sasol in the Pande & Temane gasfields.", "As noted before, the vast majority of natural gas produced from these fields is exported to South Africa through an 865 km pipeline.", "In addition, following the recent gas discoveries in the Rovuma Basin, we include the future construction of 12 so-called on-shore LNG trains plus 4 floating LNG units, each with a capacity of 5 million tons per year.", "Of this total capacity, we include 4 LNG trains (equivalent to 20 million tons per year) in the Reference scenario.", "Total gas production in the Reference scenario is then anticipated to reach just over 23 million tons per year as from 2018, which equals to about 1200 million GJ.", "In the Extractive scenario gas production could reach 65–80 million tons per year in the period 2025–2030, which equals to about 4200 million GJ per year in 2030.", "A detailed list is provided in Table A.4 in the Appendix .", "Finally, on the demand side we model the evolution of several energy-intensive megaprojects, including five mineral sands mining projects and the MOZAL aluminum smelter.", "In the Reference scenario we assume that mineral sands mining only grows marginally from current levels to reach 1.2 million ton per year, with production confined to the existing Moma-Kenmare project.", "In the Extractive Scenario we include four new mining projects, with a total production level of 9.5 million ton by 2030.", "A detailed list is provided in Table A.5 in the Appendix .", "As regards the aluminum company MOZAL, we assume a constant physical production of 547 thousand tons per year in the Reference scenario, and an expansion to 728 thousand tons per year as of 2019 (often referred to as MOZAL-III) in the Extractive scenario.", "In doing so, we implicitly assumed that by 2019, the Center-South “Backbone” transmission line (CESUL) will be accomplished, such that the major power plants from the Zambezi Basin in the North of Mozambique are connected with the dominant economic center of the Maputo area in the South of Mozambique.", "Taken together, the aforementioned assumptions as regards extractive industry evolution in Mozambique, in combination with the previously described future development paths of GDP and population, drive energy production scenarios in our modeling framework.", "In the right-hand side of Fig. 6 we show that in the reference scenario we expect total primary production to increase from almost 14 million tons of oil equivalence (toe) in 2011 to 95 million toe in 2030.", "If Mozambique were to follow the extractive scenario development path, primary production could even increase to a level of over 180 million toe in 2030.", "This equals a 6 to 13-fold increase in primary energy production in less than 20 years.", "Clearly, this means that Mozambique will undergo no less than a revolution at the supply side of its energy sector.", "The left-hand side of Fig. 6 pictures the evolution of aggregate final energy demand across the various scenarios.", "It shows that in the Reference scenario total energy demand is expected to increase to over 12 million toe in 2030.", "This is a 70% increase from the 2011 level of energy demand, and equivalent to an average annual increase of 2.8% as from 2011.", "If Mozambique were to follow the Reference Low development path, total final energy consumption is expected to reach almost 13 million toe in 2030, which equals an average annual increase of energy demand of 3.0% as from 2011.", "In contrast, the lowest level of energy consumption is to be expected if Mozambique were to follow the Extractive Scenario development path – with an estimated total final energy demand of 11.9 million toe in 2030, implying a 2.7% average annual increase over the period 2011–2030.", "The evolution of total final energy demand in the Reference High scenario is very similar to the Extractive scenario, notwithstanding differences in its composition.", "It may appear at first sight somewhat counterintuitive that in the long run the Reference Low scenario yields a considerably higher level of aggregate energy demand than the Extractive or Reference High scenario – surely the latter scenarios include high economic growth and extractive industry expansion.", "Underlying data, however, clearly reveal that this result is to be explained entirely by the evolution of energy demand from the household sector – we illustrate this in Fig. 7.", "Given the relatively small size of the underdeveloped Mozambican economy, the residential sector is and remains responsible for a large part of total energy consumption in Mozambique (over 90% in 2000 and 50–60% in 2030).", "Fig. 7 shows that residential energy demand continues to grow relatively strong over time in the Reference Low scenario, whereas it decreases relatively rapid in the Extractive and Reference scenarios.", "Hence, the diverging energy demand patterns in the right-hand side of Fig. 6 are mainly caused by a straightforward scale effect: over time the number of households becomes much smaller in the Extractive and Reference High scenario than in the Reference Low scenario.", "This feature of our model of course follows from our assumption that population growth is inversely related to GDP growth (see Section 3.2).", "Consequently, it is in the high economic growth scenarios that the weight of the dominant household sector in driving total energy demand decreases most.", "In addition, there is an intensity effect: household energy intensity will decrease relatively rapidly over time in the high economic growth scenarios, given that our model assumes that households substitute away from inefficient biomass consumption towards the use of modern and more efficient energy types like LPG and electricity under influence of increasing per capita GDP (see Section 3).", "As regards the non-residential sectors, Fig. 7 shows that road transport is the second largest sector in terms of energy demand across all scenarios.", "Also, it varies only to a relatively limited extent among the various scenarios, with an annual growth of about 7%.", "As a result, demand for fuel transport is expected to more than quadruple by 2030 as compared to 2010.", "Energy demand growth from the extractive industries is, of course, relatively strong in the Extractive scenario.", "According to this scenario, by 2030 energy demand of extractive industries equals over 1800 thousand toe, which is 80% more than in the Reference scenario.", "As noted before, to a large extent this is driven by the MOZAL aluminum smelter, which dominated the extractive industry sector until 2011.", "By 2030, MOZAL is still responsible for, about 50% of extractive energy demand in the Extractive Scenario; In the Reference scenario this is even 70%.", "in the Reference scenario.", "This is equivalent to 6–8% of total energy demand in the country in 2030, down from 11% in 2010.", "Notwithstanding these high growth rates in demand, the true revolution in the Mozambican energy sector unfolds on the supply side, as noted before.", "To gain more insight in the consequences of the rapid emerging energy production levels in Mozambique, we provide in Fig. 8 a breakdown of total primary energy production by source (top) and destination (bottom).", "As can be seen from the upper part of Fig. 8, natural gas and coal together will make up for 80–90% of these production levels.", "In contrast, at the turn of the century 80–90% of total energy production in Mozambique consisted of wood, with hydro largely making up for the remaining 10–20%.", "Nevertheless, according to our scenarios total energy production from wood and hydro is expected to increase 60–90% over the next 15 years; yet the emergence of natural gas and coal are causing their relative shares in total energy production to decrease to about 3% (hydro) and 10% in 2030.", "The lower part of Fig. 8 shows that 80–90% of total energy production in Mozambique is expected to be destined for export.", "This is especially true for the natural gas and coal production, but to a lesser extent also for hydro, which continues to be exported to South Africa and other neighboring countries in the form of electricity (more on this below).", "The natural destination for coal is India, but Brazil is also expected to be a market for Mozambique's coal export (IEA, 2014).", "Of course, energy production from wood and solar almost exclusively serves the domestic market.", "We developed the first comprehensive long-run scenario model of the emerging energy sector of Mozambique.", "Our analysis made use of the integrated modeling tool LEAP, to track energy consumption, production and resource extraction in all sectors of the Mozambican economy.", "Hence, we name the model MOZLEAP.", "It was our aim to introduce the model, and show its potential as a tool for energy planning and forecasting in the context of the emerging energy sector in Mozambique.", "We have described how the calibration of MOZLEAP is based on recently developed local energy statistics and international data for the recent past, as well as on information about the latest developments and future plans as regards the production and transformation of energy in Mozambique.", "We have shown how future GDP paths were built from a combination of macro trends and bottom-up developments in the extractive industry.", "Moreover, we presented the key mechanisms that drive our model results, including demographic and urbanization trends and cross-country based GDP elasticities with respect to biomass consumption, sector structure and vehicle ownership.", "We have developed four scenarios to evaluate the impact of the anticipated surge in natural resources exploration on energy supply and demand, the energy infrastructure and economic growth in Mozambique.", "Our analysis suggests that until 2030, primary energy production is likely to increase at least six-fold, and probably much more.", "This is roughly 10 times the expected increase in energy demand; most of the increase in energy production is destined for export.", "As a result, Mozambique is rapidly developing into an important player at international energy markets; it may well become one of the leading global producers of natural gas and coal.", "We show that GDP growth rates in Mozambique are expected to accelerate and peak around 2020, which is largely driven by extractive industry expansion, most notably the expected start of large scale gas production in the Rovuma Basin, in the far north of the country.", "The International Monetary Fund recently predicted that as a result Mozambique's average growth rate during the first half of the next decade could reach over 20% per annum (IMF, 2016).", "Given the political, financial and technical uncertainties in developing these gas fields, we worked with more conservative assumptions, but nevertheless predict annual growth rates of over 10% in that period.", "Our projections imply that over the next decade the extractive sector will comprise about 10–20% of the total Mozambican economy, depending on the scenario.", "Consequently, the rise of its energy sector in principle would allow Mozambique to transform the economy and accelerate its much needed development.", "The agriculture sector in Mozambique still is the source of livelihood for more than three-quarters of the population; agricultural productivity is so low that Mozambique remains a net food importer, and malnutrition is persistent.", "The country still ranks among the poorest countries in the world, with around half of the population living below the absolute poverty line and evidence of stagnation in the reduction of poverty rates over the last decade (Ross, 2014).", "Substantial tax revenues from extractive projects may help reduce the structural dependence of the Mozambican government on international aid to finance basic services and much needed investments in, among others, health, education and infrastructure.", "But the recent past as well as our scenario results show that the opportunity to use natural resource wealth for developing an inclusive growth and development strategy in Mozambique poses major for its government.", "This is also demonstrated by the fact that the existing energy strategy of the Mozambican government has thus far been successful to a limited extend only.", "Inter alia, this strategy aims for promotion of new and renewable sources of energy, increased sustainable access to electricity and liquid fuels, diversification of the energy matrix, and institutional coordination and consultation with relevant stakeholders for better development (MoE, 2008).", "Our results show that the energy matrix indeed becomes much more diversified over time, but it also reveals that even with large scale development of the natural gas, coal and hydro potential, total energy production from traditional biomass will increase with at least 60% over the next 15 years.", "This is caused by the sustained dominance of the residential sector in total energy consumption, under influence of the relatively small size of the non-extractive economy in combination with high population growth rates.", "The latter also helps to explain our finding that even under optimistic assumptions the electrification rate is unlikely to exceed 45% by 2030.", "We have shown that PV solar energy has been gradually adopted, but also that it remains very small, percentage wise.", "A similar conclusion holds true for the production and consumption of biofuels.", "According to Chambal (2010), Mozambique has put in place a modern legislative framework for the energy sector in general and the power sector in particular.", "However, he also concludes that implementation and enforcement appear to lag behind considerably and some aspects are still unclear, particularly the contribution that the energy sector is to make to poverty reduction.", "Clearly, the current expansionary strategy for the energy sector in Mozambique makes its economy increasingly vulnerable to a “resource curse”, that may result from (a combination of) resource price volatility on international energy markets, Dutch disease effects, crowding out of productive investments, or discouragement of institutional and capacity building in public administration (Van der Ploeg, 2011).", "Over the last two years GDP growth already slowed down because of a decline in mining sector growth, which was caused by lower international commodity prices and transportation constraints – the delayed development of a new railway line and deep-water port that prevented the scaling up in coal production and exports.", "Large scale development of the country's hydroelectricity potential in the Zambezi river basin has now been delayed for years, due to uncertainty about necessary long-term demand agreements with neighboring countries in combination with interregional network constraints.", "The Mozambican government faces the challenge to balance the need for these and other natural resource related infrastructure with the need for investments in health, education, water and agricultural productivity.", "Moreover, there is a need to weigh the planned expansion of thermal electricity generation capacity on the basis of coal and natural gas, against development of the country's large hydroelectricity potential, also in view of the environmental consequences of both strategies.", "Until recently, megaprojects in the extractive sector contributed little to fiscal revenues, but according to the IMF, by 2020 resource revenues could exceed 40–50% of total revenues and would last for over 35 years (Ross, 2014).", "This would require major improvement of public financial management to ensure that resource wealth is used efficiently and transparently, including the adoption of new fiscal rules that can help safeguard the annual budget from price volatility (Van der Ploeg, 2014).", "In addition, it becomes increasingly clear that there is an enhanced need to manage existing regional imbalances in resource wealth, which is concentrated in the far north of the country whereas other economic development and political power is concentrated in the far south of the country.", "Since recently, Mozambique is facing increasing local institutional and political instability, which has its roots in the country's devastating civil war during the period 1977–1992.", "This includes returning instability in the central region of the country led to attacks by armed insurgents on newly developed mining and transport infrastructures as well as on main roads that connect the northern and southern regions of the country.", "Large scale natural resource exploration is likely to increase these tensions, which underlines the importance of developing an inclusive growth strategy that covers all regions of the country.", "Certainly, this is much more easily said than done, because institutional capacity in Mozambique is still weak in many areas.", "To address the formidable list of challenges described above will therefore require continued improvement of the quality of public administration.", "Hence, the key question, therefore, seems to be whether the expected natural resource boom will encourage or frustrate the required improvements of local institutional capacity." ]

[ "Mozambique is now developing its large reserves of coal, natural gas and hydropower", "We introduce the first comprehensive scenario model of its emerging energy sector", "Until 2030, energy production increases at least six-fold, probably much more", "Mozambique may well become a leading global producer of natural gas and coal", "We discuss the challenges that this resource wealth poses for the country" ]

[ "BIOC", "NEUR" ]

[ "Dopamine", "Imaging mass spectrometry", "Mechanical allodynia", "Nucleus accumbens", "Optogenetics", "Ventral tegmental area" ]

[ "1307", "2804" ]

[ "The dopaminergic pathway, present from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), is important for mediating acute and chronic pain sensations (Ikemoto, 2007; Taylor et al., 2016) and pain-avoidance behavior (Danjo et al., 2014).", "The medial part of the NAc (mNAc) integrates signals from the mesencephalic dopaminergic neurons and other brain areas that process affective information (Britt et al., 2012; Bromberg-Martin et al., 2010).", "Recent studies have shown that one projection of the mNAc, the indirect pathway (Bromberg-Martin et al., 2010), is associated with the affective evaluation of events that shape behavior via interaction of dopamine (DA) with the D2 receptors (Ren et al., 2016).", "This pathway participates in the central representation of pain, and controls the activity of the ascending nociceptive pathways (Ren et al., 2016).", "Accumulating evidence show that input from the VTA neurons, via DA signaling in the mNAc, plays a key role in the indirect pathway.", "We recently reported that activation of dopaminergic neurons in the VTA increases the amount of extracellular DA within the NAc, which blunts pathological allodynia induced by nerve injury or bone cancer (Watanabe et al., 2018a).", "Moreover, manipulating the excitability of mNAc by administering a DA agonist diminishes mechanical allodynia in spared nerve injury models of neuropathic pain (Sarkis et al., 2011).", "In contrast, aversive stimuli inhibit the dopaminergic neurons of the VTA (Danjo et al., 2014; Moriya et al., 2018; Ungless et al., 2004).", "To disentangle the complex sensory and affective components of pain, optogenetic manipulation of sciatic nerve neurons has been used to induce noninvasive excitation of nociceptors in freely moving mice (Iyer et al., 2014).", "Using this method, we observed that exclusive excitation of the sciatic nerves by blue light lowered the pain threshold but did not induce pain sensation, and was accompanied by a mNAc-specific reduction of DA as measured by imaging mass spectrometry (IMS) (Watanabe et al., 2018b).", "IMS measures the biochemical content (Morikawa et al., 2012), for example, the total amount of DA from each pixel in tissue sections (Shariatgorji et al., 2014); therefore, our observation indicated rapid and large fluctuations in the total amount of DA, most likely, manifested by the stored synaptic vesicular pool of DA at mNAc-nerve terminals, which may be associated with the reduced pain threshold.", "However, it remains unclear whether sensitized pain stimulation by optogenetic nerve excitation alters the regional DA content.", "Moreover, the biochemical processes mediating the fluctuation of DA need to be elucidated.", "In this study, we approached this question by visualizing the regional DA content, as well as the metabolic conversion of stable isotope-labeled Tyr (13C15N-Tyr) to DA (13C15N-DA), using IMS.", "The amount of non-labeled DA and de novo synthesized 13C15N-DA from the exogenously administrated 13C15N-Tyr was evaluated within the caudate putamen (CP), mNAc, lateral part of the nucleus accumbens (lNAc), and VTA.", "Using this technique, we demonstrate if mechanical allodynia induced by optogenetic sensory nerve excitation activates medial mNAc-specific dopamine signaling and metabolism.", "Male C57BL/6J mice (8–12 weeks old; Tokyo Laboratory Animals Science Co., Ltd., Tokyo, Japan) were housed (up to six per cage) and maintained in a temperature-controlled room (24 ± 1 °C).", "All mice were maintained under a 12-h light–dark cycle (light was switched on at 8 a.m.), and behavioral tests were performed during the light phase.", "Food and water were provided ad libitum.", "All animal experiments were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, Hoshi University, as adopted by the Committee on Animal Research of Hoshi University.", "After 12–15 h of food deprivation, the mice received oral administrattion of 13C915N1-Tyrosine (13C15N-Tyr; Taiyo Nippon Sanso, Tokyo, Japan; 30 mg/mL in H2O, 10 μL/g body weight).", "After 0.5, 1.0, and 1.5 h, the mice were sacrificed and their brains were harvested and immediately flash frozen in solid CO2 and stored at −80 °C.", "Based on the protocol described by Iyer et al., we used an adeno-associated virus serotype 6 (AAV6) encoding the human synapsin 1 promoter to achieve neuron-specific expression of the blue light–sensitive cation channel, channelrhodopsin-2 (ChR2), (ET/TC) fused to enhanced yellow fluorescent protein (EYFP).", "The AAV6-hSyn-ChR2 (ET/TC)-EYFP or control AAV6-hSyn-EYFP were serotyped with AAV6 coat proteins and packaged at the viral vector core at Nagoya University.", "The final viral concentration was between 1 × 1011 and 3 × 1012 copies/mL.", "Aliquots of the viral solution were stored at −80 °C until use.", "To selectively express ChR2-EYFP or EYFP in the sensory nerves, AAV6-hSyn-ChR2 (ET/TC)-EYFP or control virus was microinjected into the sciatic nerve of C57BL/6J mice.", "Briefly, the mice were anesthetized under isoflurane (3%, inhalation) and a 2-cm incision was made to expose the sciatic nerve.", "The microinjection was performed with an internal cannula (Eicom, Kyoto, Japan) at a rate of 1 μL/min for 8 min using a gas-tight syringe (10 μL; Eicom) and pump (Model ESP-32; Eicom).", "Two weeks after the intrasciatic injection, following 16 h of food deprivation, the mice were orally administered with 13C15N-Tyr.", "Mice received optical stimulation (473 nm, continuous wave [CW], 0.5 h) to the plantar surface of the ipsilateral hind paw, 1.5 h after 13C15N-Tyr administration.", "Thirty minutes after the optical stimulation, the mice were sacrificed the whole brains harvested and flash frozen in solid CO2 and stored at −80 °C.", "To quantify the change in pain sensitivity to tactile stimulus induced by the optical activation of sensory nerves, the paw withdrawal response was measured, in response to a tactile stimulus during blue light stimulation (473 nm, CW) of the plantar surface, using von Frey filaments with a bending force of 0.16 g (AesthesioV R, DanMic Global, LLC, CA, USA).", "Each of the hind paws were tested individually.", "Paw withdrawal in response to tactile stimulus was evaluated by scoring as follows: 0, no response; 1, a slow and slight withdrawal response; 2, a slow and prolonged fiexion withdrawal response (sustained lifting of the paw) to the stimulus; 3, a quick withdrawal response away from the stimulus without flinching or licking, and 4, an intense withdrawal response away from the stimulus with brisk flinching and/or licking.", "Paw movements associated with locomotion or weight shifting were not considered as a response.", "Thin sections (thickness, 8 μm) were cut with a cryomicrotome (CM3050, Leica Microsystems) and thaw-mounted on indium-tin-oxide-coated glass slides (Bruker Daltonics, MA, USA) at −16 °C.", "After sectioning, deuterium-labeled dopamine (D4-DA; 5 μM in 50% MeOH; IsoSciences PA, USA), used as an internal standard, was spray-coated on the sections using a robotic sprayer device (SunCollect system, SunChrom, Friedrichsdorf, Germany).", "To perform on-tissue derivatization of monoamines (Shariatgorji et al., 2014), the sections were manually spray-coated with a solution of 2,4-diphenyl-pyranylium (DPP) tetrafluoroborate salts (Sigma-Aldrich, MO, USA; 1.3 mg/mL in methanol) using an artistic airbrush (Procon Boy FWA Platinum 0.2-mm caliber airbrush, Mr. Hobby, Tokyo, Japan).", "Thereafter, the sections were automatically spray-coated with 2,5-dihydroxybenzoic acid as a matrix (40 mg/mL, dissolved in 50% methanol) using a robotic sprayer.", "Data were acquired with an orbitrap mass spectrometer (QExactive Focus, Thermo Fisher Scientific) coupled with an atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization ion source (AP-SMALDI10, TransMIT GmbH, Giessen, Germany) or a linear ion trap mass spectrometer with another MALDI source (LTQ XL, Thermo Fisher Scientific).", "The raster step size was set at 80 μm (Fig. 1), 120 μm (Fig. 2), or 100 μm (Figs. 3 and 4).", "For the orbitrap mass spectrometer, signals within a mass range between 350 and 400 were acquired with a mass resolving power of 70,000 at m/z 200.", "For the linear ion trap mass spectrometer, signals of DPP-DA (m/z 368 > 232), DPP-D4-DA (m/z 372 > 232), and DPP-13C15N-DA (m/z 377 > 233) were monitored with a precursor ion isolation width of m/z 1.0.", "Thereafter, the spectral data were transformed to image data and analyzed using ImageQuest 1.0.1 (Thermo Fisher Scientific), SCiLS 2019a (Bruker Daltonics) and ImageJ 1.51 (National Institutes of Health, USA) software.", "DA mapping by IMS revealed that in situ DA concentrations were unevenly distributed within the VTA/SN neuronal circuits.", "Tyrosine hydroxylase (TH)-positive neural somas were localized to the VTA/substantia nigra (SN) regions; however, we found that DA was highly concentrated in CP and NAc terminal regions, with concentrations 10-fold higher than that in the SN and VTA (Fig. 1).", "This observation indicated that local de novo synthesis of DA and/or rapid monoamine recycling at the CP and NAc synapses facilitated the accumulation of high levels of DA in the terminal areas.", "Following the observation of regional differences in DA concentration, we sought to assess the mechanism underlying the maintenance of high DA levels in the CP and NAc.", "To answer this, we evaluated the involvement of de novo synthesized DA in these terminal regions.", "Stable isotope-labeled Tyr (13C15N-Tyr) was orally administered to mice, and its conversion to de novo synthesized, labeled DA (13C15N-DA) was visualized in the brain sections comprising the CP and NAc at 0.5, 1.0, and 1.5 h after administration (Fig. 2a–c).", "The results revealed an unexpectedly fast replacement of the total DA pool with the de novo synthesized, labeled DA, within 1.5 h of 13C15N-Tyr administration.", "The newly synthesized DA increasingly accumulated in the terminal area (Fig. 2c and d), and eventually, occupied >10% of the non-labeled DA in the three terminal regions such as: CP, mNAc, and lNAc (Fig. 2e).", "In addition, an uneven rate of DA replacement between the brain regions was observed, with the mNAc exhibiting a lower replacement rate than that in the CP.", "These data indicate that a continuous and large supply of newly synthesized DA accumulates at the terminal regions to produce large storage pools of DA.", "Since DA signaling in the NAc affects the pathological formation of allodynia (Watanabe et al., 2018a), subsequently, we hypothesized that sensitized pain sensation might accompany spatiotemporal DA fluctuation within the VTA/SN circuits.", "To verify this hypothesis, mice expressing ChR2-EYFP or EYFP were fed with 13C15N-Tyr (Fig. 3a) and subjected to von Frey filament test for tactile stimulus during optogenetic stimulation with blue light irradiation.", "The results show a clear elevation in allodynia score in mice expressing EYFP-ChR2 (Fig. 3b).", "Thereafter, their regional DA contents were visually examined in coronal sections at two positions within the CP/NAc and VTA, respectively (Fig. 3c, Supplementary Fig. 1).", "Interestingly, EYFP-ChR2 group exhibiting elevated allodynia score showed significantly higher DA content in the mNAc, than EYFP-group (Fig. 3d and e).", "This spatiotemporal increase in DA accompanied no significant increase in the de novo synthesized 13C15N-DA.", "Moreover, amount of 3-MT, a post-synaptic metabolite of DA degradation, increased over a wide area, including the CP, lNAc, and mNAc (Fig. 3d and e).", "These results suggest that tactile stimulation during blue light irradiation increased the release of DA, followed by its post-synaptic degradation to 3-MT, after which, the mNAc presumably received preferential supply (i.e., de novo synthesis and/or reuptake) of DA.", "Additionally, we found that the VTA showed a higher de novo synthesized DA replacement rate than did the other three regions (Supplementary Fig. 1).", "These data indicate that the VTA has a higher activity of TH-driven DA synthesis and smaller pools of stored DA than that in the terminal regions.", "Optogenetic excitation of the sciatic nerve, without mechanical stimulation, induces aversion behavior in mice expressing ChR2-EYFP (Iyer et al., 2014); thus, we assessed the regional DA dynamics in mice under such condition.", "After the administration of stable isotope-labeled Tyr (13C15N-Tyr), brains were harvested from mice who received and did not receive blue light stimulation for 30 min (Fig. 4a).", "Interestingly, visualization by IMS demonstrated significant reduction of non-labeled DA in the CP and NAc terminal regions; however, no change was detected in the VTA (Fig. 4b and c, Supplementary Fig. 2).", "In contrast, the level of labeled DA remained unaltered in the CP and NAc, whereas slight reduction was observed in the VTA (Fig. 4c, Supplementary Fig. 2).", "Taken together, only those mice which received blue light stimulation without tactile stimulation showed reduced amount of DA in wide areas of the CP and NAc terminal regions, as well as suppressed de novo DA synthesis within the VTA.", "In this study, we demonstrated the presence of a high homeostatic level of DA in the striatum sub-regions, with a continuous supply of de novo synthesized DA.", "The de novo synthesized DA occupied a significant proportion of the non-labeled DA pool in the sub-regions within 1.5 h of 13C15N-Tyr administration.", "Although our results are consistent with that of previous studies demonstrating fast DA turnover (Karoum et al., 1984; Kilts and Anderson, 1987), our technique provided spatially resolved information on the metabolic turnover of DA as high resolution images.", "The DA levels visualized by IMS represent the sum of the intra- and extra-cellular DA concentration at any given pixel.", "Because the concentration of intracellular DA is typically much higher than extracellular DA (Ortiz et al., 2010), the DA dynamics observed in this study predominantly reflect changes in the intracellular DA concentration, most likely present in storage vesicle, localized at the nerve terminals (Kai-Kai, 1984).", "To overcome this technical challenge, we developed a micro-dialysis technique coupled with mass spectrometry (Watanabe et al., 2018b) to enable the comprehensive measurement of extra-cellular metabolites of DA degradation.", "Thus, the evaluation of vesicle-secreted DA and related metabolites, including stable isotope-labeled DA, by the microdialysis-mass spectrometry, is an important complementary approach for further study of DA metabolism.", "Our IMS-based approach revealed elevated levels of DA and 3-MT in the NAc terminals following short-term tactile stimulus, sensitized by optogenetic excitation of the sciatic nerve.", "Thus, it is reasonable to state that DA metabolism, i.e., release and post-synaptic uptake followed by decomposition of DA, was activated during pain sensation, although the reuptake activity was not examined (Fig. 5).", "A recent report supports this suggestion; Mikhailova et al. showed that tail pinch treatment increased extra-cellular DA concentration in the NAc, whereas only touch stimulus did not alter the NAc DA concentration (Mikhailova et al., 2019).", "Other studies reported that pain stimuli, in addition to analgesia, increased the extra-cellular DA concentration in the NAc (Kato et al., 2016; Navratilova et al., 2015), suggesting that activated DA metabolism might be a feedback reaction to sensations of acute pain, and may play a role to attenuate the excess pain stimuli in the NAc.", "On the contrary, in the striatum, optogenetic sensory nerve stimulation or tactile stimulus did not change the level of isotope-labeled DA (Fig. 4c).", "In contrast, a slight but significant decrease in isotope-labeled DA was observed in the VTA by optogenetic sensory nerve stimulation (Supplementary Fig. 2).", "These results suggest that DA production is more stable in the striatum than in the VTA.", "These observations may be congruent to reports which state that acute aversive stimuli induce a robust increase in extracellular DA level in the NAc but inhibits the firing of VTA DA neurons (Holly and Miczek, 2016; Ungless et al., 2004).", "An alternative interpretation of the unaffected de novo DA synthesis in mNAc is that such elevation of DA synthesis might be a slow process, and thus, the present results only show a marginal increase following optogenetic and tactile stimuli, which is statistically insignificant (Fig. 3e).", "In future studies, this hypothesis can be validated by assessing the de novo DA synthesis at a later time point from that of isotope-labeled Tyr administration.", "Nonetheless, large and stable local de novo DA synthesis within the terminal structures of the striatal nerve terminal is suggested to be a major source of DA, as opposed to its transportation from the VTA (Fibiger et al., 1973).", "In support of this suggestion, a previous study showed that restoration of DA synthesis in the striatal sub-regions, by region-specific TH expression alone, is sufficient, at least in part, to recover the affected behaviors in a DA deficient mouse model (Szczypka et al., 2001).", "In summary, we show that the DA neurons in the mNAc increases the biochemical pool of DA in response to the short-term pain sensation.", "However, the detailed molecular mechanism and its pathophysiological significance remains unclear.", "Here, we only examined acute pain stimulation.", "On the contrary, Ren et al. (2016) reported that the basal extracellular DA level is decreased in the spared nerve injury model of chronic neuropathic pain.", "In addition, a single, acute stress promotes long-lasting neuroplastic changes in the VTA dopaminergic neurons (Graziane et al., 2013; Saal et al., 2003).", "Therefore, further study examining the biochemical DA metabolism between acute and chronic pain models should be conducted.", "We believe that our biochemistry-based data offer unique information on DA metabolism in various brain regions linked to the mesolimbic dopaminergic pathway, and on the possible mechanisms by which the changes in the level of this monoamine may shape the sensory and affective components of pain.", "IMS-based visualization of monoamine neurotransmitters represents the sum of intra- and extra-cellular concentrations, most likely, the concentration of vesicular DA stored at the nerve terminals.", "Using this technique, we have revealed high homeostatic level of DA in the mNAc with continuous supply of de novo synthesized DA.", "Optogenetic and tactile stimulation of the sciatic nerve triggers an acute increase in DA level in the mNAc, whereas only optogenetic stimulation caused the opposite, i.e., a reduction in DA level.", "This study demonstrated that rapid and region-specific fluctuations in the concentration of monoamines occur in response to a sensitized, pain stimuli, and might be associated with the formation of allodynia.", "The authors declare no conflicts of interest." ]

[ "Regional DA metabolism in an optogenetic mouse model of mechanical allodynia was examined.", "De novo synthesized DA occupied >10% of total DA pool in the striatum within 1.5 h.", "Pain induced by tactile and optogenetic stimulation increased DA content in the mNAc.", "Optogenetic stimulation without pain reduced DA content broadly in the striatum.", "mNAc-specific DA metabolism might be associated with development of allodynia." ]

[ "EART", "ENVI" ]

[ "Canadian prairies", "Eutrophication", "Hydroclimatology", "Lake Winnipeg", "Nutrients" ]

[ "1901", "2312" ]

[ "Watersheds in the central Canadian prairies exhibit classic cold regions hydroclimatology, with long (4–5 month) cold winters followed by a short (2–5 week) spring characterized by warming temperatures, high surface runoff due to snowmelt over frozen soils, and peak discharge (Glozier et al., 2006; Fang et al., 2010; Cade-Menun et al., 2013).", "In contrast, the remainder of the hydrologic year (summer and fall) is typified by limited discharge because of high evaporation and infiltration rates (Fang et al., 2010).", "The strong seasonality of cold regions hydroclimatology drives the transport and delivery of nutrients and other pollutants from source watersheds to lakes (e.g., Rattan et al., 2017), with the amount of snowpack and the timing and volume of runoff intrinsically linked to seasonal and interannual climate variability (Barnett et al., 2005).", "For example, a three-year study of seasonality of nutrient export from 11 Canadian prairie streams showed that total P (TP) and total N (TN) concentrations and loads both peaked during the snowmelt season (Rattan et al., 2017).", "Similarly, Rankinen et al. (2015) observed that total P and total suspended solids concentrations in selected Finnish catchments tracked changes in seasonal discharge, with highest values in April during snowmelt.", "Because nutrient losses from cold region watersheds are closely tied to hydrology, changes in snowpack depth, the timing and volume of runoff, and the frequency and intensity of rain events could dramatically influence the timing and magnitude of nutrient loss from the land base to proximate streams and downstream waterbodies.", "Analyses of historical data from across Canada have shown significant changes in hydroclimatology over the past 60 years.", "Snow depth and snow cover duration have exhibited decreasing trends since the mid-1970s, with the greatest changes observed during late winter and spring (Derksen and Brown, 2012; DeBeer et al., 2016).", "In particular, annual snow cover duration has decreased by an average of 4 days per decade between 1950 and 2012, equating to a shortening snow cover period of 1 to 2 months in mid-western and western Canada (DeBeer et al., 2016).", "Likewise, analysis of long term trends (1951–2000) in the duration of rainfall events has revealed that the frequency of multi-day rain storms have increased in the mid-western and western region, while the number of single day events has decreased (Vincent et al., 2012; Shook and Pomeroy, 2012).", "These findings of decreases in snow cover (Derksen and Brown, 2012; Dumanski et al., 2015; Vincent et al., 2015), increases in rain precipitation, and persistent rainfall events (Millet et al., 2009; Vincent et al., 2012; Shook and Pomeroy, 2012) over the last five decades have been associated with changes in the hydrology of prairie streams.", "For example, Dumanski et al. (2015) observed dramatic long-term changes in hydrology of the Smith Creek Research Basin in southern Saskatchewan, Canada.", "Results showed that discharge was dominated by snowmelt runoff during the earliest study years (1975–1994), whereas rain on snow contributed to higher runoff peaks in spring during subsequent years (1995–2000).", "Moreover, recent (2011–2014) rain events have produced peaks during snowmelt with secondary discharge peaks also occurring in summer (Dumanski et al., 2015).", "Previous research by Rattan et al. (2017) showed that land-based activities such as fertilizer application, livestock density and sewage were critical factors influencing TN and TP concentrations in Canadian prairie streams whereas physical factors such as water temperature and discharge were the primary determinants of TN and TP loads.", "These findings have implications for water management in the Red River Valley (RRV) of central Canada, influencing the types of beneficial management practices (BMPs) most appropriate for minimizing nutrient loss (tillage method, crop residue management, nutrient management, water retention).", "Yet, to our knowledge, there are no studies that consider the effect of hydroclimatic variability on total, dissolved, and particulate nutrient loss from Canadian Prairie watersheds.", "This study was designed to evaluate the impact of hydroclimatic variability on the timing and quantity of nutrient export from seven agriculturally-dominated watersheds in the RRV of central Canada.", "By examining seasonal changes in dissolved and particulate nutrient export during a snowmelt-dominated hydrological year versus a year with lower snowfall and higher rainfall amounts, we tested the hypothesis that concentrations of particulate forms of N and P are greater in a year when snowmelt is the dominant contributor to runoff.", "However, in a year with a greater influence of rainfall generated runoff, a large share of the P pool and to a lesser extent the N pool will be in the dissolved form because rainfall has a lower erosion potential than snowmelt (Wu et al., 2018).", "This study provides information on the importance of inter-annual variability in climate and hydrology on water quality.", "In addition, our research will support improved decision making, particularly decisions regarding the type and extent of BMPs required to control nutrient export to proximate and downstream receiving water bodies.", "The study was conducted in the RRV of southern Manitoba (Fig. 1).", "Seven sub-watersheds were selected (from 11 sub-watersheds described by Rattan et al. (2017)) to span the range in nutrient producing agricultural activities occurring in this region (four sub-watersheds strongly influenced by seasonal sewage discharge were not included in the present analysis) (Table 1).", "Much of the watershed of the RRV is used for agriculture, predominately cultivation of feed wheat, oats, and grain corn (Manitoba Government, 2018a,b).", "Census and land use data (Manitoba Government, 2018a,b) confirmed that the sub-watersheds were not subjected to significant changes in land use between the two study years, 2013 and 2014.", "For example, human population in southern Manitoba showed no variation from 2013 to 2014 (Manitoba Government, 2018a,b).", "In addition, farm locations, crop cover, and number of large livestock farms showed no significant (p < 0.05) differences between the two study years (Manitoba Government, 2018a,b).", "The well-drained and cropped farmlands in the RRV are situated along a band of very low relief terrain underlain by nearly impermeable glaciolacustrine fine sediments (McCullough et al., 2012).", "This plain is intersected by small wetlands and channelized streams and rivers.", "During snowmelt, stream channels are hydrologically connected whereas during the rest of the year, all streams except mainstem rivers have disconnected reaches as a result of low or no flows caused by high temperatures, limited rain precipitation and increased evaporation (summer and fall) or frozen channels (winter) (Corriveau et al., 2013).", "The region experiences a cold continental climate with the warmest month being July (20 °C) and the coldest January (−14.6 °C) (1981–2010 long term means for Morden, MB; Government of Canada, 2018).", "Annual precipitation averages 426 mm, with 27.1% occurring as snow (1981–2010 records for Morden, MB; Government of Canada, 2018a,b).", "Most snowfall occurs from November to April, with long-term (historical means: 1981–2010) monthly averages of 19–35 mm of precipitation in each of those months.", "Snowmelt typically produces >60% of annual runoff, with soils usually remaining frozen at the time of snowmelt, thereby preventing infiltration of the water rapidly released by the melting snow (Ali et al., 2017).", "Rain precipitation of high intensity can occur in spring, summer or early fall resulting in intermittent flashy discharge (Fang et al., 2010).", "BMPs used in cold climate prairie regions primarily reduce particulate loss.", "These practices include buffer strips and conservation tillage, and have been proven effective in reducing particulate nutrient export (Uusi- Kamppa et al., 2012).", "These practices may not, however, be as effective in controlling the loss of dissolved nutrients entering streams.", "For example, the lack of soil inversion with conservation tillage can result in accumulation of nutrients at the soil surface, resulting in a greater proportion of dissolved nutrients in runoff (Tiessen et al., 2010; Li et al., 2011; Cade-Menun et al., 2013).", "Historical long term means (1980–2010) and contemporary (2013, 2014) air temperature and snow depth data were obtained from the Morden, MB climate station (Government of Canada, 2018a,b), the only station in the region with snow data.", "Snow depth (expressed as centimetres on the ground) was used to identify maximum snow depth, duration of continuous snow cover, and first snow free date.", "The duration of continuous snow cover was defined as the longest length of time within one snow season where snow depth is ≥1 cm.", "The first snow-free date was the first day when snow depth was 0 cm.", "Total precipitation (snow + rain) data were available for only one station (Morden) from Environment and Climate Change Canada (Government of Canada, 2018a,b).", "Rain precipitation data (daily volumes, mm) were obtained from five weather stations (all seven study sites were located within 30 km of a weather station): one station (Morden) from Environment and Climate Change Canada (Government of Canada, 2018a,b) and another four stations (Carman, Altona, Starbuck, and Elm Creek) from Manitoba Agriculture, Food, and Rural Development (Manitoba Government, 2018a,b).", "Multiple-day rainfall events were defined as two or more consecutive days of rainfall where rain precipitation on each day was > 10 mm (Climate and Ocean – Variability, Predictability, and Change, 2009).", "Grab water samples (from a depth of 20 cm) were collected during the open-water seasons of 2013 (April 25th- October 31st) and 2014 (April 7th- October 31st) for determination of total P (TP), total dissolved P (TDP), soluble reactive P (SRP), total N (TN), total dissolved N (TDN), nitrate and nitrite-N (NO3 +NO2), and ammonium-N (NH4-N).", "For all years and sites, samples were collected daily during the rising limb and peak of snowmelt, and weekly during the falling limb.", "Thus, in 2013, samples were collected daily from April 25th to May 13th (19 samples), weekly from May 15th to June 3rd (3 samples) and biweekly thereafter, for a total of 34 samples per site.", "In 2014, samples were collected daily from April 7th to April 27th (21samples), weekly from May 1st to May 31 st (4 samples) and biweekly thereafter, for a total of 42 samples per site.", "Sampling ceased in late October or when current velocity was <0.001 × 103 mm9/s measured over a period of 5 min.", "Samples were not collected during winter as rivers in the RRV (with the exception of the largest) typically freeze to the bottom.", "All water samples were collected in high density polyethylene bottles, stored at 4 °C in a cooler and transported to the Biogeochemical Analytical Services Laboratory (University of Alberta), Edmonton Alberta.", "Samples for SRP and NH4-N were filtered within 24 h of collection and frozen.", "The remaining P and N fractions were stored at 4 °C and analyzed within a week of sampling.", "Samples were analyzed using standard methods (American Water Works Association, 1999).", "Detection limits were 0.001 mg/L for phosphorus (TP, TDP and SRP), 0.007 mg/L for TN, 0.002 mg/L for NO3+NO2, and 0.003 mg/L for NH4-N.", "Particulate P and N were determined by subtracting total dissolved from total concentrations of P and N (Yates and Johnes, 2013).", "Dissolved unreactive phosphorus (DUP) was calculated as the difference between TDP and SRP.", "Dissolved organic nitrogen (DON) was calculated as the difference between TDN and NO3+NO2 and NH4-N (Yates and Johnes, 2013).", "Water levels and temperature were recorded every 30 min at each site with a pressure transducer logger (HOBO, Onset Computer Corporation, MA, USA).", "Daily discharge, the volume of water moving past the study site per day, was estimated by establishing a rating curve based on a power relationship between discharge measured at the nearest Water Survey of Canada site (WSC; Government of Canada, 2018a,b) weighted by the drainage area ratio between the sampling site and the WSC station (Corriveau et al., 2013), and water level measured at the sampling site.", "Significant correlations between the predicted discharge values estimated by the rating curves produced for each sub-watershed and the WSC station confirmed that WSC discharge, corrected for difference in watershed area, was a good predictor of site discharge.", "Stream runoff (i.e., the height (mm) of water that drains from a sub-watershed over a period of time) was calculated as the volume of water flowing out of the sub-watershed over a hydrologic season (snowmelt, spring, summer, and fall) or the sampling year (April-October) divided by sub-watershed area.", "Nutrient loads were calculated as the product of discharge and nutrient concentrations for each hydrologic season (snowmelt, spring, summer, and fall) and the sampling year (April-October).", "For dates with missing nutrient concentrations, values were linearly interpolated between the nearest two sampling dates, as recommended by Schilling et al (2017) when calculating loads using periodic grab samples.", "Comparison of loads calculated using both the linear interpolation method and the Stratified Beale Ratio (an unbiased estimator of nutrient loads; Booty et al., 2014) showed that there were no significant differences (p > 0.05) between the two methods, suggesting both approaches can be used as a useful and reliable tool for estimating watershed nutrient load model validation and calibration.", "All statistical tests (excluding Wavelet analysis) were performed using Minitab ver.", "14 (Minitab ver. 14, 2003).", "Descriptive statistics (mean, standard error, 95% confidence intervals) were calculated, both annually and seasonally, for total precipitation (Morden station), rain precipitation (5 weather stations), surface runoff volume (7 study watersheds) and discharge (7 study watersheds) (Table 2).", "A Mann-Whitney U test was used to compare snow depth between the two sampling years.", "Snow depth data were highly skewed, thus necessitating the use of non-parametric tests (Tiessen et al., 2010).", "Wavelet analysis was used to quantify the degree of variability for each hydrological and climate variable (discharge, runoff volume, snow accumulation, temperature, and rain precipitation).", "This analysis decomposes data on a scale-by-scale basis by applying filters that capture features such as abrupt changes in variability (Blukacz et al., 2009).", "Different filters can be used to decompose variability of the input data and it is critical to select a rigorous filter.", "We used the Daubechies filter (least asymmetric filter, LA8) because it is better at conserving variability at a given scale, than the commonly used Haar filter (Blukacz et al., 2009).", "In addition, wavelet analysis does not have stationarity assumptions (constant mean and variance) like traditional approaches (e.g., spectral analysis) (Gençay et al., 2002, Labat 2005, Dadu and Deka, 2016).", "Recently, wavelet analyses have been used on ecological data sets to quantify spatial patterns in plankton (Blukacz et al., 2009) and in hydrology to quantify temporal patterns in hydrological signals such as precipitation and water levels (Kang and Lin, 2007).", "The relative importance of variability across five temporal scales (ranging from daily to seasonal) was measured as the slope from a log-log plot of wavelet variance as a function of scale, or a wavelet slope.", "A positive wavelet slope indicates that variability increases with scale, whereas the opposite holds for a negative slope (Blukacz et al., 2009).", "All computations were performed using the waveslim package in R Stats (R core team, 2014).", "A nonparametric Mann-Whitney t-test was used to test for significant differences in wavelet slopes between sampling years for each hydrological and climate variable.", "Two–way analysis of variance (ANOVA) was used to identify differences in nutrient concentrations, nutrient ratios (TDP/TP, SRP/TP, PP/TP, TDN/TN, NO3+NO2/TN, NH4-N/TN and PN/TN) and nutrient loads among seasons and between years for the seven sub-watersheds.", "Post hoc Tukey’s test was used to identify significant differences between each level, and interaction plots were used to determine if one factor was dependent on the level of the other factor.", "Also, dissolved (SRP, DUP, NO3+NO2, NH4-N and DON) and particulate (PP and PN) concentrations and loads were expressed as a percentage of TP and TN concentrations and loads.", "In addition, bivariate plots and Pearson’s R correlation analysis were used to quantify seasonal relationships between hydrology and nutrient concentrations (total, dissolved, and particulate) for both study years.", "Prior to analysis, concentration and load data that did not meet normality assumptions (Shapiro-Wilks test) were log-transformed to meet parametric assumptions.", "Total (snow + rain) annual precipitation differed between the two study years.", "In 2013, total precipitation was 433 mm (average 2.14 mm/day) compared to 367 mm for 2014 (average 1.59 mm/day) (Table 2; Morden station data, Government of Canada, 2018a,b).", "Snowfall depth (Fig. 2) showed distinctly different conditions prior and during snowmelt 2013 versus 2014.", "Between November 1st 2012 and March 17th 2013, snow on the ground averaged 16 cm deep.", "The 2012–2013 winter ended on March 18th when snow started melting from a peak depth of 35 cm to depletion on April 19th.", "During the 2013–2014 winter, snow on the ground peaked at 24 cm deep on February 13th and then melted for 45 days, with an average snow depth over the melt of 12 cm.", "In contrast to 2013, snow loss during 2014 occurred in steps, with minimal loss between February 14th and March 4th (and actually small gains in snow depths on certain days) followed by three stepwise losses.", "Comparison of the four weeks prior to the first snow-free day showed that snow depth distribution differed significantly between 2013 and 2014 (U = 9.3, n1 = 32, n2 = 45, p = 0.04; Mann-Whitney test).", "The timing and quantity of rainfall also differed between the two study years.", "In 2013, two isolated storm events delivered a total of 305 mm of rain during spring, summer, and fall combined (averaging 7.62, 1.06, and 0.691 mm/day for spring, summer, and fall, respectively), resulting in discharge peaks in spring but not in summer and fall (Fig. 3).", "In contrast, several rain events occurred between April - October 2014 resulting in a total of 426 mm (averaging 1.82, 3.41, and 1.22 mm/day for spring, summer, and fall, respectively) and; producing discharge peaks during spring, summer, and fall.", "(Fig. 3).", "Results of Mann-Whitney tests showed that the distribution of rainfall differed between 2013 and 2014 for all three seasons (U = 8.4, n1 = 18, n2 = 34, p = 0.03 for spring; U = 10.1, n1 = 92, n2 = 92, p = 0.01 for summer; U = 7.6, n1 = 52 n2 = 33, p 0.03 for fall).", "Comparison of climatologic conditions for the two study years with long-term (1982–2010) normals showed that 2013 was typical of historical conditions.", "In 2013, snow depth at the end of winter was 35 cm (compared to 31 cm for 1981–2010) and snow cover depletion occurred on April 19th (late April for 1981–2010).", "Rain precipitation was 305 mm for April-October 2013 (compared to 409 mm for April-October 1981–2010), 52% of rain volume fell during summer (57% for 1981–2010), and the total number of multi-day rainfall events was two (comparable to the long-term normal).", "In contrast, 2014 was characterized by less snow at end of winter (18 cm), earlier snow cover depletion (March 30th), greater rain precipitation (426 mm April-October), more rainfall occurring during summer, and more frequent multi-day rain events especially during summer.", "During each of the study years, the onset of water flow in the seven study sub-watersheds started several days after snow cover depletion: streams opened and started to run (i.e., current velocity averaged over 5 min > 0.001 × 103 mm9/s) on April 25th in 2013 compared to April 7th in 2014, in both cases when daily maximum air temperatures exceeded 0 °C for three days (Fig. 3).", "Maximum stream discharge occurred during snowmelt for both study years, though greater in 2013 (155 × 103 mm9/s) than 2014 (42 × 103 mm9/s).", "Discharge peaks during summer and fall were relatively smaller than snowmelt peaks and associated with rain events (Fig. 3).", "Greater precipitation (snow + rain) during 2013 resulted in higher mean daily discharge and stream runoff in 2013 (2.30 × 103 mm9/s and 87.9 mm/day, respectively) compared to 2014 (0.912 × 103 mm9/s and 51.4 mm/day, respectively) (Table 2).", "The proportion of the annual stream runoff delivered during snowmelt, spring, summer and fall was 60, 24, 15, and 1% respectively, in 2013 compared to 53, 09, 35, and 3%, respectively, in 2014.", "Application of wavelet analysis to quantify the degree of temporal variability in hydrologic variables revealed significant differences between years (Fig. 4).", "When all five time scales were examined (2–4 days, 4–8 days, 8–16 days, 16–32 days, and 32–64 days), Mann-Whitney tests showed that runoff, discharge and rain precipitation differed (p < 0.05) in variability between 2013 and 2014.", "In 2013, these variables had a negative wavelet slope indicating that variation decreased with time scale and was thus greatest at a smaller time scale (days).", "In contrast, runoff and discharge had a positive wavelet slope in 2014, indicating that variation was greater at a larger time scale (8–16, 16–32 days).", "Wavelet slopes for snow depth did not differ (p > 0.05) between 2013 and 2014.", "Phosphorus concentrations in the seven streams ranged from 0.02 to 3.3 mg/L TP, 0.02–3.1 mg/L TDP, 0.01–2.8 mg/L SRP, and 0.01–3.0 mg/L PP (Table S1).", "One –way ANOVA showed that TP and PP fractions were significantly higher (F(1) = 7.78, p = 0.007 for TP; F = 6.33(1), p =0.011for PP) in 2013 compared to 2014: 0.52 ± 0.38 versus 0.43 ± 0.17 mg/L TP and 0.17 ± 0.10 versus 0.11 ± 0.034 mg/L PP for 2013 and 2014, respectively (mean ± SD).", "There was no significant difference in annual dissolved P concentrations between 2013 and 2014.", "Comparison of nutrient concentrations using one-way ANOVA showed that all P fractions displayed temporal differences (i.e., differences amongst hydrological seasons) within years (p < 0.01).", "Concentrations of TP, TDP and SRP were always greatest during snowmelt and lower during spring, summer and fall (Fig S1).", "In the case of TP and SRP, concentrations were low and not significantly different for spring, summer and fall.", "TDP concentrations were also low during these three seasons but variable in 2014 compared to 2013.", "(Results of pair wise comparisons among hydrological seasons were: F(3) = 9.90, p < 0.001 for TP; F(3) = 8.99, p < 0.001 for TDP, and F(3) = 13.6, p < 0.001, for SRP in 2013 compared to F(3) = 8.91, p < 0.001 for TP, F (3) = 15.6, p < 0.001 for TDP and F(3) = 11.2, p < 0.001 SRP in 2014).", "In contrast, the seasonal pattern of PP differed between years: snowmelt and spring concentrations were greater than summer and fall concentrations in 2013 (F(3) = 9.2, p < 0.001, n = 3) whereas snowmelt, spring and summer concentrations were greater than fall concentrations in 2014 (F(3) = 8.26, p = 0.001) (Fig. S1).", "Pearson’s R correlation was used for analysis of concentration versus discharge plots for snowmelt, spring, and summer hydrologic seasons.", "(Analyses of fall data could not be undertaken because by then, most sites had ceased to flow).", "The results showed that associations between particulate P fractions and discharge differed between hydrologic periods and years (Fig S3).", "The higher TP and PP concentration of 2013 were associated with significant positive correlations, between TP and discharge during snowmelt (R = 0.49, p = 0.001; Fig. 3a) and between PP and discharge during both the snowmelt period and summer (R = 0.62, p < 0.001 for snowmelt; R = 0.48, p < 0.001 for summer; S3 c and i).", "In contrast, PP was not correlated with discharge during any hydrologic period in 2014 whereas TDP was significantly correlated with discharge during the 2014 snowmelt and summer hydrologic periods (R = 0.47, p<0.001 for TDP snowmelt, and R = 0.40, p <0.001 for TDP summer; Fig. S3b and h).", "Two- way ANOVA of P ratios revealed a significant interaction (F (3, 1) = 3.30, p = 0.03) between hydrological season and year for SRP/TP (Table 3).", "Pair-wise comparisons between years showed significant differences (F(3) = 4.21, p = 0.02) for summer and fall with lower SRP/TP for these hydrologic periods in 2013 compared to 2014.", "Within years, pair-wise comparisons showed significant differences in SRP/TP between snowmelt and fall 2013 (F(3) = 3.20, p = 0.03), in contrast to differences between spring and summer (F(3) = 5.22, p = 0.02) and spring and fall (F(3) = 6.31, p = 0.01) 2014 (Fig. 5b).", "Normalizing TP to 100% for all periods showed that in 2013, PP comprised 33% of TP on an annual basis, with the largest share present in fall (43%) and the smallest share in summer (31%) (Fig. 5d).", "In 2014, the PP pool comprised 25% of TP annually, and was proportionately greatest in spring (41%) and smallest in fall (17%).", "Nitrogen concentrations in the seven streams ranged from 0.01 to 12 mg/L TN, 0.01–12 mg/L TDN, 0.01–9.9 mg/L NO3+NO2, 0.01–3.1 mg/L NH4-N, and 0.01–9.6 mg/L PN (Table S1).", "Similar to P, particulate forms of N were greater in 2013.", "However, unlike patterns in P concentrations, annual means of all dissolved N forms were significantly greater in 2014 (F(1) = 7.33, p = 0.03).", "Comparison of seasonal patterns using one-way ANOVA showed that in 2013, snowmelt concentrations of TN, TDN, NO3+NO2, (not shown) and NH4-N were greater and differed significantly from spring, summer and fall means (F(3) = 6.3, p = 0.01 for TN; F(3) = 10.1, p < 0.001 for TDN; F(3) = 7.20, p = 0.001 for NO3+NO2; F(3) = 6.25, p = 0.01 for NH4-N; Fig. S2).", "In 2014, snowmelt concentrations of TN, TDN, NO3+NO2, (not shown) and NH4-N concentrations were also significantly greater compared to spring, summer and fall (F(3) = 7.31, p = 0.02 for TN; F(3) = 24.1, p = <0.001 for TDN; F(3) = 18.5, p < 0.001 for NO3+NO2; F(3) = 8.22, p = 0.011 for NH4-N).", "In contrast, PN concentrations were elevated (F (3) = 8.99, p = 0.002) during both snowmelt and spring 2013 compared to summer and fall, but constant across all four hydrological periods in 2014 (Fig. S2).", "Concentrations of TDN and NH4-N were also greater during snowmelt 2014 than snowmelt 2013 (F(3) = 8.11, p = 0.001 for TDN; F(3) = 10.4, p < 0.001 for NH4-N), whereas PN concentrations were greater in spring 2013 compared to spring 2014 (F(3) = 8.36, p < 0.001).", "Analysis of concentration versus discharge plots showed that associations between N fractions and discharge differed between hydrologic periods and years.", "The higher PN concentrations of 2013 were associated with a significant positive correlation between PN and discharge during snowmelt (R = 0.41, p < 0.001 Fig. S4c, f, and i).", "For both years, TDN summer concentrations showed positive and significant correlations with discharge during the summer (R= 0.33, p = 0.012 for 2013; R = 0.34, p = 0.007 for 2014 (Fig. S4h).", "Two-way ANOVA showed no consistent seasonal or inter-year differences in N ratios (Table 3).", "For NO3+NO2/TN, only seasonal differences were observed such that snowmelt was greater than spring (F(3) = 8.11, p = 0.01) and fall (F(3) = 9.29, p = 0.01), but not summer, for 2013 and snowmelt was greater (F(3) = 8.72, p = 0.01) than all others periods in 2014.", "Analysis of NH4-N/TN identified a significant interaction between season and years (F (3,1) = 3.9, p = 0.01), with pairwise comparisons showing no difference among hydrological periods in 2013, but greater NH4-N/TN ratios during snowmelt (F(3) = 9.26, p < 0.001) compared to other hydrological periods in 2014 (Fig. 6b).", "For PN/TN, only year had a significant effect (F (1) = 3.9, p = 0.03) with ratios, on average, greater in 2013 (Table 3).", "One-way ANOVA on 2013 PN/TN data revealed greater ratios (F(3) = 6.44, p 0.011) during spring compared to other seasons.", "Comparison of dissolved and particulate N fractions as a percentage of TN concentrations showed that on an annual basis, PN concentrations comprised a small portion of TN for both years (15–25%) with little variation (15–20%) among seasons (Fig. 6d).", "On average, TDN represented 76% of TN in 2013 and 80% of TN in 2014.", "Comparison of nutrient loads normalized to 100% showed that in 2013, PP comprised 51% of the TP load on an annual basis, with PP representing the largest share of the TP pool in snowmelt (52%) and the smallest share in fall (27%).", "In 2014, the PP pool comprised 21% of the TP load annually, and was proportionately greatest in spring (38%) and smallest in fall (7%).", "In the case of nitrogen, PN comprised 28% of the annual TN load in 2013 compared to 17% of the annual TN load in 2014.", "A two-way ANOVA examining seasonal and annual variation in nutrient loads showed a significant effect of season (p < 0.01) for all P and N forms (total, particulate, and dissolved), with no interaction between seasons and years (Table 3).", "With the exception of PP in 2014, loads of all P and N forms were greatest during snowmelt (Fig. 7).", "In addition, TP, PP, and PN loads differed between years (Table 4), such that both annual and seasonal total P and particulate P and N loads were greater in 2013 than 2014 (Fig. 7 a, d, h and inset).", "The difference in PP loads between the two years was mirrored in TP loads (i.e., a greater TP load in 2013 than 2014; Table 4) but not TN loads.", "The timing and quantity of nutrient export from seven agriculturally-dominated streams in the Canadian prairies was strongly influenced by differences between two years in weather conditions and, in turn, stream hydrology.", "Differences in maximum snow depth (35 cm in 2013 versus 24 cm in 2014) and timing of snowmelt (occurring three weeks later in 2013 compared to 2014) resulted in 61% more snowmelt runoff and 58% greater snowmelt discharge in 2013.", "These hydroclimatic differences between years were associated with higher concentrations of PP and PN, a greater share of TP concentration as PP, and lower dissolved N concentrations during 2013 when snowmelt-runoff generation predominated.", "The intensity of precipitation events is recognized to influence nutrient delivery, including the quantity and forms of nutrients, in prairie as well as other ecoregions.", "For example, Larson et al. (2013) reported that TP and TN concentrations in two prairie streams (Missouri, USA) increased by 3-fold and 12-fold, respectively, in response to rain events.", "Likewise, Kalkhoff et al. (2016) observed that dissimilarities in nutrient concentrations and loadings between two mid-west USA agricultural catchments were related to differences in rainfall runoff and cropping practices, such that increased concentrations of NO3-N and TP were observed during the months and years with greatest rainfall.", "Research from two sub-watersheds in England (Outram et al., 2016) also identified that the quantity and timing of rain precipitation was directly associated with variation in volume of runoff and discharge and, in turn, the quantity of N or P lost from the catchment.", "Although our analysis spanned only two consecutive years, the results are consistent with other studies showing that runoff and nutrient delivery from agricultural watersheds are related to daily, seasonal or interannual changes in precipitation volume.", "Analysis of hydroclimatic variables showed that 2013 was typical of cold prairie region hydrology whereas 2014 was characterized by less snow, earlier snow cover depletion and more rain precipitation (particularly during summer).", "Wavelet analysis also showed that rainfall patterns differed between the two years, exhibiting greater variability at longer (i.e., weekly) scales in 2014, consistent with occurrence of periodic storm events during summer 2014.", "The differences between our two study years reflect trends observed over the past five decades, notably reductions in snow depth and snow cover duration (Derksen and Brown, 2012; DeBeer et al., 2016), increases in rain precipitation and persistent rain events (Dumanski et al., 2015), and greater delivery of rain during summer (Ehsanzadeh et al., 2012; Dumanski et al., 2015; Vincent et al., 2015).", "Thus our two study years, although sequential, represent two different hydroclimatic scenarios: 2013 was typical of classic Canadian prairie climate and hydrology with a snowy winter, spring rain, and a dry summer whereas 2014 was characterized by lower than normal snow cover, earlier snow depletion, and a wet summer.", "Our finding of classical weather conditions in 2013 compared to wetter, rainier conditions in 2014 was associated with clear differences in stream hydrology.", "In 2013, greater snow depth at the end of winter and less rainfall, with most delivered during spring, resulted in runoff and discharge being greatest during snowmelt (69% of annual runoff) and substantially lower during subsequent seasons.", "In 2014, reduced snow depth followed by a wetter summer still resulted in peak runoff and discharge during snowmelt (53% of annual runoff) but was followed by greater and flashier water delivery in summer.", "This flashier water delivery was evident in the fact that both runoff and discharge showed greatest variability at a weekly time scale in 2014 compared to a daily time scale in 2013.", "Other researchers, examining longer (>60 year) records of prairie hydrology, have previously remarked on the variability of snowmelt discharge and runoff (e.g., Fang and Pomeroy, 2007; Liu et al., 2013).", "For example, Fang and Pomeroy (2007) concluded that spring runoff and stream discharge are inherently unstable in prairie systems, with lower snow depth, higher air temperature, and lower initial soil moisture causing a noticeable reduction in snowmelt runoff and discharge.", "Recent research has linked changes in prairie hydrology to changes in climate.", "For example, in the Red River basin, Ehsanzadeh et al. (2012) noted that abrupt upward shifts in runoff and rain precipitation in the mid-1990s as well as a gradual increase in discharge over a 96-year period were due to climate change and changing farm activities.", "Similarly, analysis of historical long-term normal (˜70 year) records for Smith Creek, a small prairie watershed in Saskatchewan, Canada, revealed an almost 50% decrease in snowmelt-generated runoff and a 27% increase in rainfall-generated runoff.", "This shift in discharge generation from snowmelt to snowmelt + rainfall generated runoff) was interpreted to result from climate change, exacerbated by changing land use and drainage (Dumanski et al., 2015).", "Comparison of nutrient concentrations between 2013 and 2014 revealed that the snowmelt-dominated year (2013) was associated with higher annual TP concentrations as a result of elevated PP concentrations, higher annual PN concentrations and lower annual concentrations of dissolved N.", "While concentrations of all nutrient fractions were greatest during snowmelt (or snowmelt and spring) for both years, the difference in mean annual PP concentrations between years (high in 2013 and low in 2014) and greater dissolved N concentrations in 2014 resulted in a larger share of dissolved forms comprising total P and, to a lesser extent, total N in 2014.", "Previous research in cold climate regions have identified that a large proportion of nutrients in both agricultural runoff and streams are in dissolved form: >75% of TP as TDP in runoff from prairie fields (Hansen et al., 2000; Tiessen et al., 2010), and ˜65% of TP as TDP and >80% of TN as TDN for small prairie streams (Glozier et al., 2006; Corriveau et al., 2013).", "Our results also showed that a large share of the P pool is in the dissolved form during snowmelt but, more importantly, that this fraction differs (p < 0.05) between years (i.e., 64% of TP as TDP for 2013 compared to 75% of TP as TDP for 2014).", "In contrast, the dissolved N share of the TN pool did not differ (p>0.001) between years: TDN represented 78% of TN in 2013 and 80% of TN in 2014.", "Although several prairie studies (e.g., Vanni et al., 2001; Li et al., 2011; Rattan et al., 2017) have quantified nutrient concentrations and fluxes over multiple years, these studies focused on relationships between nutrients and human activity rather than comparing nutrient concentration fractions among years in relation to weather and hydrology.", "Our results showed that dissolved forms comprised a larger share of total nutrient pool in a year when the mechanism for discharge generation was a combination of snowmelt + rainfall generated runoff, versus primarily snowmelt generated runoff.", "The seasonality we observed in nutrient concentrations and nutrient ratios relates to hydrological differences, including antecedent winter conditions.", "During snowmelt, greater concentrations of particulate forms of P and N result from the erosive action associated with melt of the snow cover.", "The deeper snow cover and later melt in 2013 (when air temperatures were warmer and surficial soils had likely begun to thaw) was associated with higher concentrations of PP compared to the shallower snow cover and early melt in 2014.", "In addition, we observed that snowmelt PP and PN concentrations showed strong significant associations with discharge in 2013 but not 2014 (Fig. S3c; S4c).", "These findings strongly suggest that the higher PN and PP concentrations during the 2013 snowmelt were related to the greater (p < 0.01) discharge.", "In similar cold climate regions, Rankinen et al. (2015) and Gonzales-Inca et al. (2018) observed high soil erodibility in partially thawed soil during snowmelt runoff.", "Soil erodibility is high in recently thawed soils, because high water content decreases the cohesive strength of soil aggregates (Van Klaveren and McCool, 1998), therefore snowmelt runoff may be associated with high concentrations of suspended solids and particulate P (Gonzales-Inca et al., 2018).", "Spring was also associated with high concentrations of particulate N and P, likely due to the fact that clay soils dominate the RRV and once wet (i.e., during and following snowmelt), small clay particles stay in suspension almost indefinitely due to Brownian motion (Partheniade, 2009).", "In summer, particulate concentrations showed a sharp decline in 2013 likely due to the limited runoff and presence of vegetation, which protects soil against erosion In contrast, PN showed no decrease in concentration over summer and fall 2014 while PP showed only a small decrease in fall 2014, possibly because summer storms continued to ensure delivery of particulates to proximate streams.", "Comparison of nutrient loads between the two study years showed that particulate loads of both P and N were greater in the snowmelt-dominated year.", "Others have observed higher particulate nutrient export during snowmelt in cold climate regions (Cade-Menun et al., 2013; Liu et al., 2013; Rankinen et al., 2015), though there have been no studies showing that hydrological variation can influence the particulate fractions of N and P between years.", "Our observation of greater particulate loads during the snowmelt-generated year, combined with the fact that the proportion of particulate P differed more between years than the proportion of particulate N, suggests that hydrological variability among years could change stoichiometric N:P ratios in receiving waters (Vanni et al., 2011; Rattan and Chambers, 2017).", "Temporal variation in N:P ratios has substantial implications for aquatic food webs, in particular the productivity and composition of primary producers.", "In Lake Erie, the 2011 record-setting algal bloom was dominated by Microcystis sp. followed by Anabaena sp. (Michalak et al., 2013).", "This species replacement was likely related to shifts in N and P concentrations: Microcystis sp. abundance was made possible by high concentrations of SRP and dissolved inorganic N, whereas N-fixing Anabaena sp. occurred after the Microcystis sp. bloom when dissolved inorganic N was likely depleted (Michalak et al., 2013).", "Our finding that nutrient concentrations, fractionation and export for prairie streams differs between years according to hydrological conditions has implications for water quality, particularly in response to climate change when reduced snowmelt and increased rain events are forecast to occur.", "During snowmelt dominated years, particulate nutrient concentrations and loads are greater and likely to result in increased water turbidity.", "In contrast, during years with reduced snowmelt runoff and greater rainfall, concentrations and loads of particulate N and P are lower in streams dissecting the Red River Valley.", "The difference in nutrient export between snowmelt versus snow + rain dominated years may require changes or adaptations to current BMPs in order to control nutrient losses under a future climate change scenario.", "While measures to reduce nutrient loss such as crop rotation (Liu et al., 2013) and placement and timing of fertilizer application (Flaten, 2011) have been implemented, additional actions may be needed to address the variability in nutrient (particularly P) loss between snowmelt-dominated and snowmelt + rain dominated years.", "In particular, actions may be need to address the difference in seasonality of nutrient loss between snowmelt-dominated years (when snowmelt and spring losses predominate) compared to snowmelt + rain dominated year (when losses occur during snowmelt, spring and summer).", "The authors declare that there are no conflicts of interest associated with this manuscript." ]

[ "Hydroclimatologic changes affected P and N concentrations and fractions.", "Greater fractions of particulate P and N were observed during snowmelt in 2013.", "Greater proportions of dissolved P were observed during snowmelt + rainfall in 2014.", "Effective actions are needed to reduce dissolved P and N export to Lake Winnipeg." ]

[ "CENG", "CHEM", "ENER" ]

[ "Biomass", "Bond Work Index (BWI)", "Coal", "Energy consumption", "Hardgrove Grindability Index (HGI)", "Thermogravimetric analysis" ]

[ "1500", "1605", "2102", "2103" ]

[ "Global coal consumption by power generators is growing annually [1].", "With increasing legislation to reduce emissions from coal fired power stations in Europe [2,3], biomass combustion is playing an increasing role in the UK, Europe and beyond [4].", "In order to minimise costs, biomass is often ground in existing coal mills when used in coal fired power stations, but the fracture mechanics in conventional mills were optimised to exploit the brittle structure of coal which contains pre-existing macro and micro flaws [5].", "This type of breakage does not occur in biomass, which possesses a more orthotropic structure [6].", "Standard grindability tests have been developed for coal, with the Hardgrove Grindability Index (HGI) being the standard test for vertical spindle mills [7,8], and the Bond Work Index (BWI) for tube and ball mills [9,10].", "No standard grindability tests currently exist for biomass, and there have only been limited studies on the use of standard grindability tests for coal on biomass, which have mainly focused on torrefied materials [11–16].", "This paper aims to analyse the applicability of the standard HGI and BWI as a standard test for grindability on a wide variety of commonly used biomasses in the power sector compared to a known coal.", "The Hardgrove Grindability Index (HGI) test is based on Rittinger’s theory that “the work done in grinding is proportional to the new surface produced” [17,18].", "The index varies from 20 to 110, with a lower HGI indicating a coal is harder to grind and more energy will be required to reach the required degree of fineness.", "The test is conducted on a standardised laboratory scale ball-and-race mill and is covered by BS 1016-112:1995 [8].", "A strong, hard coal will often have a high rank and be difficult to reduce in size; a weak, soft coal of lower rank will be easier to grind; but very low rank coals can also be difficult to reduce in size.", "For coal, HGI correlates to compressive and tensile strength measurements which roughly correlate with coal rank, and increasing bulk modulus [5].", "However, Kendall [19] showed that crack propagation becomes impossible via compression once a critical particle size is reached, the length of which is material dependant, and particles below this size are ductile in compression.", "Zuo et al. [20] showed that the relationship between coal size reduction and energy input is a nonlinear curve, so it is difficult to represent coal grindability with a single numerical value.", "Rubiera et al. [21] showed that the HGI of binary coal blends cannot be predicted from the weighted average of the individual coals in the blend, which has important implications in the co-milling and combustion performance of biomass and coal blends, as the actual performance may be quite different to the predicted behaviour for a blend due to the interactions between the blends.", "Vassilev et al. [22] noted that biomass composition and properties varied significantly from coal.", "The observation by Agus and Waters [23] that mills are volumetric devices and that the traditional HGI method favours denser coals with small volumes has led to the HGI test to be modified to use a volume (rather than mass of coal), and this method is commonly used to analyse biomass and coal HGI values experimentally [12,14,24], although industry uses the standardised mass based method [7,8].", "However, these correlations have not been tested on biomass samples commonly used in the power sector.", "An increasing number of legacy coal fired power stations are being converted to burn biomass.", "There is an urgent need to understand the grinding behaviour of a wide range of biomasses in all types of mills, not least because incorrect operation of existing coal mills during biomass milling increases the risk of fires in biomass mill hoppers [37].", "This study aims to investigate the applicability of the HGI and BWI tests for a wide range of commonly used biomasses used in the power generation sector and analyse which biomass characteristics influence the milling behaviour, and the suitability of the test for analysing the grindability of biomass.", "The samples used in this work are either routinely co-fired in coal fired power plants or have been used in biomass co-firing trials, as illustrated in Fig. 1.", "Portuguese wood pellets (mainly pine with a small amount of eucalyptus), Spanish olive cake (a residual waste mix from olive oil production formed of powdered olive pulp (0–850 μm), olive pips (850–3350 μm) and olive pellets/self-formed lumps (3350 μm+) [38]), Russian sunflower husk pellets and Colombian La Loma coal were provided by EDF Energy plc.", "South African eucalyptus pellets, American steam exploded white wood chip pellets, miscanthus pellets, and torrefied white wood chip pellets were provided by E.ON UK plc.", "The particle size range of the biomass particles (prior to densification) was obtained using the British standard BS EN 16126:2012 [39].", "2 litres of boiling deionised water was poured over 300 ± 1 g of each pellet sample and then soaked for 24 h.", "The samples were then dried at 35–60 °C until they reached 5–15% moisture content.", "The samples were then split into two portions; 150 g was used to obtain the moisture content via BS EN 14774-1:2009 [40], and the other 150 g portion was split and sieved according to BS 15149-2:2010 [41] to obtain a particle size distribution.", "Limited information was available on the source and species of the material, for commercial reasons.", "Thermal profiles were produced using TA Instruments Q500 Thermogravimetric Analyser (TGA).", "TGA runs used 10–15 mg of milled sample with a particle size range of 75–300 μm.", "The method used was based on the slow pyrolysis method developed by Lester et al. [42] for analysing the composition of biomass.", "The sample was heated in a furnace at 5 °C/min in 100 ml/min of Nitrogen from atmospheric temperature to 900 °C, after which the gas was switched to air at 100 ml/min.", "The results were processed and analysed in Matlab® 2014a in order to establish the sample composition and peak volatiles release rate on a dry weight basis.", "The composition of the samples is given by moisture, volatile, fixed carbon, and ash contents.", "The peak volatile release rate and corresponding temperature were obtained from the derivative thermogravimetric curves.", "TGA was used to analyse any changes in composition during the BWI test.", "The gross calorific values (H) on a dry weight basis of the samples were found using an IKA C5000 Bomb Calorimeter (Staufen, Germany) in accordance with BS ISO 1928:2009 [43].", "Certified Benzoic Acid tablets were used as a standard, and the sample weight was calibrated to give the same temperature rise as the standard.", "In addition there are the three theories of comminution which describe empirical size reductions, these being Rittinger’s [18], Kick’s [49] and Bond’s [10] theories of comminution which state that:", "The energy required for size reduction is proportional to the new surface area generated [18].", "The equivalent relative reductions in sizes require equal energy [49].", "The net energy required in comminution is proportional to the total length of the new cracks formed [10].", "where P1 is the closing sieve size (μm), G is the grindability (net g/rev).", "The Bond Work Index (Wi) expresses the resistance of the material to grinding to a specified product size, and the higher the value of Wi, the more difficult the material is to grind to the required product size.", "The Work Input W, gives the power required by the mill to grind the product to the required product size.", "In addition, the higher the value, the more power will be required to reduce the material to the required product size for a given mass flow rate.", "The non-linear regression analysis of the BWI and HGI correlations was performed on IBM SPSS Statistics 22.", "where R is cumulative percentage undersize mass (%), d is particle diameter (μm), d′ is the characteristic particle size (μm), defined as the size at which 63.2% (1 − l/e = 0.632) of the particles (by weight) are smaller, and n is the Rosin–Rammler size distribution parameter (dimensionless).", "The Rosin–Rammler parameters were found using the Matlab® GUI Tool developed by Brezáni and Zelenak [54].", "The particle size distributions of percentage retained mass against particle size were plotted on semi-logarithmic plots.", "Geometric mean diameter by mass dgw and geometrical standard deviation Sg was calculated according to BS ISO 9276-2:2014 [55].", "The pre-densified particle size 80% passing particle size (FPP80) was obtained via the particle disintegration test described in Section 2.1.", "The resultant Bond Work Index for FPP80 is defined as WPPi, and the Work Index is WPP.", "Table 2 shows the HGI results for La Loma coal, Spanish olive cake, eucalyptus pellets, wood pellets, steam exploded pellets, and sunflower pellets.", "Miscanthus and torrefied pellets were not tested due to limited quantities of material being available.", "On average, coals used in UK power stations have a HGI around 40–60; the La Loma coal tested in this work falls within this range with a HGI of 46.", "The biomasses tested performed very poorly (HGI of 14–22), indicating a high resistance to grinding.", "Even the steam exploded pellets showed only a nominal improvement (HGI of 29) in comparison to non-treated biomasses.", "The majority of samples showed the same result in the repeat test, or varied by ±1 HGI value.", "Ohliger et al. [12] found very high HGI values for torrefied beech wood, but the crushing ratio (the average particle size before milling divided by the average particle size after milling) for the same samples were lower than that of lignite, indicating that the high HGI values can be misleading when analysed by themselves.", "A HGI equivalent using a Retsch PM100 planetary ball mill was developed by Bridgeman et al. [14], and has been used by Ibrahim et al. [15] to find HGI values as high as 86.4 for torrefied willow.", "These figures have been compared to coals tested as per international HGI testing standards [7,8] by Li et al. [56], and superficially appear to show a vast improvement in grindability, with the potential to be better than some coals.", "However the HGI figures reported by Bridgeman and Ibrahim are not from the same method (and apparatus) and caution is required when comparing HGI values that are not derived from a Hardgrove machine.", "Hardgrove testing machines use compression breakage modes similar to those in a vertical spindle mill, whereas planetary ball mills use high impact breakage modes [57].", "The HGI test was developed for coal fired power plants, and the target 75 μm size is based on what is required for combustion in pulverised fuel coal burners [46].", "However biomass has a target particle size closer to 1000 μm for pulverised fuel burners.", "For a 150 kW pilot burner, the optimal burn conditions for wood feed stocks was 95% of particles (by weight) were smaller than 1000 μm with a moisture content lower than 15% [47].", "Therefore a grindability test which aims to analyse the grindability of biomass to 75 μm is inappropriate, as the target size for optimal combustion of biomass and the setting for the classifier output from full scale mills is an order of magnitude of higher than this.", "In addition, the critical particle size for compressed fracture should be ascertained for biomasses to be milled in coal mills, as below this size the biomass will behave as a ductile material and mills which use compression and impact forces will not be able to further reduce the particle size.", "Coal has a critical particle size of 5 μm, while polystrene’s is 4.48 mm [19], but the critical crack length of biomasses used in PF combustion is unknown.", "Table 1 compares the BWI and HGI tests.", "The HGI test is very constricted in its setup compared to the BWI test.", "The feed sample is already within the target size range (600–1180 μm) and the mass size is small in comparison to the BWI test.", "Even with the modified HGI test the volume is still limited to a small unrepresentative volume (50 cm3) [12,14,15,23,58].", "The BWI test has the advantage of being based on a larger volume (700 ml) and with a variable target size, so the impact of target particle size on the grindability of materials can be investigated.", "The output of the BWI test is also in a more useable form of energy consumption per ton (kW h/t), which allows the method to be compared to other forms of milling such as hammer mills [35] or planetary ball mills [11] which already have modified work indicies based on the theories of comminution.", "Therefore, it can be concluded that HGI is a poor method of testing the grindability of biomass in vertical spindle mills and a new standard grindability test is required to test the grindability of biomasses.", "A larger, more representative, volume of material and a target particle size close to that required for the PF burners and mill classifiers is important.", "It is also important that the grindability test identifies the failure mechanism in use and subsequent impact on particle characteristics.", "Table 2 shows the results of the BWI tests.", "The biomasses all had the same equilibrium sieve size of 1000 μm, while coal was set to 90 μm in order to achieve a particle size close to the 70% passing at 75 μm.", "Miscanthus pellets had the highest Wi at 426 ± 29.5 kW h/t (high variability due to mill choking), with wood pellets (413 ± 3.7 kW h/t) and sunflower pellets (366 ± 0.5 kW h/t) showing similar results.", "Olive cake had a Wi of 136 ± 3.6 kW h/t.", "Surprisingly, eucalyptus pellets showed a much lower Wi at 87 ± 8.7 kW h/t, which was close to that of the steam exploded pellets (64 ± 0.8 kW h/t).", "The lowest Wi for the biomasses was for the torrefied pellets (16 ± 1.1 kW h/t), with the La Loma coal having a similar Wi at 23 ± 0.1 kW h/t with a target size of 90 μm.", "Table 2 also shows the heating value (H) of the samples on a dry basis and what percentage the Work Input (W) represents of this value (W/H).", "As expected, the La Loma coal has the highest calorific value (30,044 J/g).", "The torrefied pellets had a higher heating value than the steam exploded pellets, which was on a par with the other wood pellets.", "The heating values for the samples found in this study corresponded to similar samples tested in literature (14,000–21,000 J/g for biomass [59,60] and 26,000–33,000 J/g for coal [9]).", "The milling energy represents a low percentage of the heating value of the samples, with values ranging from 0.07% for the torrefied pellets, up to 1.86% for miscanthus pellets.", "The work input W and the grindability per revolution G showed a similar order of results.", "Wood pellets had the highest W and lowest G at 102.29 kW h/t and 0.053 g/rev, followed closely by miscanthus and sunflower pellets (95.85 kW h/t, 0.057 g/rev and 93.08 kW h/t, 0.059 g/rev respectively).", "This reversal of order between these samples is due to the difference in feed size (F80) used in the calculations; miscanthus pellets are approximately 6 mm in diameter, while wood and sunflower pellets diameter was approximately 8 mm.", "Miscanthus also had a higher P80 value than wood and sunflower pellets, which means the difference between F80 and P80 is lower for miscanthus than for wood and sunflower pellets.", "The results from the BWI tests show a wide spread of BWI values amongst the biomass samples.", "As expected the wood, miscanthus and sunflower pellets had Wi values an order of magnitude greater than that of the coal, and the olive cake was five times greater than coal.", "However the eucalyptus, which is an untreated biomass, performed almost as well as the steam exploded pellets.", "The torrefied pellets had a significantly higher BWI than the others indicating that the torrefaction process used produced pellets which are more grindable in a tube and ball mill compared to the steam exploded process.", "Fig. 2 shows the mass per size fraction and revolution count for each run of each sample.", "For wood, miscanthus and sunflower pellets the trend is very similar.", "The mass size fraction below the target size of 1 mm stabilises quickly to a constant mass, especially for the wood pellets.", "Even though the revolution count increases from 260 to 2104, the mass produced below 1 mm remains around 100 g after the second run.", "Therefore the increase in revolutions is not producing any more fines, which indicates that the forces within the mill are not sufficient to break down the material.", "This is indicative of mill choking, which is a known issue with biomass in full scale mills, where increasing the revolutions appears to have no impact on the amount of fines produced.", "The decrease in the 3.35 mm+ size fraction indicates that the pellets are breaking down into smaller sizes, so the forces are sufficient to break down the pellets, but the increase in the 1–1.7 mm size in particular shows that the mill is struggling to break down the pellets beyond their pre-densified particle size (FPP80 is 1446 μm for wood, 1311 μm for miscanthus, and 1757 μm for sunflower pellets), and therefore the breakage mechanisms within a tube and ball mill are not suitable for the comminution of ductile materials.", "This test highlights that fundamentally mills that use impact, compression and attrition will struggle to breakdown ductile materials such as biomass to sizes required for PF systems.", "Eucalyptus shows a different behaviour to the other untreated biomasses.", "It has a FPP80 of 1279 μm, which although smaller than the other samples, is still above the target equilibrium size of 1 mm.", "The pattern of mass per size fraction and revolution count for eucalyptus indicates that the forces within the mill are sufficient to break down the material beyond its pre-densified particle size, and as the FPP80 is close to P1, less energy is required to comminute compared to sunflower pellets whose FPP80 is far from P1.", "The percentage of mass produced below 1 mm follows the same pattern as the revolution count, and thus it can be deduced that the revolution count has a direct impact on the amount of fines produced, and that mill choking is not experienced in the same manner as for the other untreated biomasses.", "Olive cake is made of 3 sections: olive pulp (0–850 μm), olive pips (850–3350 μm) and olive pellets/self-formed lumps (3350 μm+) [38], and 43% of the feed sample fell into the sub 1 mm category.", "The graph for olive cake in Fig. 2 does not show a linear or smooth trend compared to the other samples due its heterogeneous nature despite splitting the sample to try to reduce this issue.", "Two distinct patterns emerged; while the revolution count does impact the amount of fines produced, the mass percentage in the 1–1.7 mm size range continually increases as the run count increases.", "This shows that the forces in the mill are sufficient to break down a portion of the olive cake, but not all of it, which could lead to mill choking in a full scale mill over time.", "The La Loma coal showed adequate grindability in the mill for its much finer target size of 90 μm.", "The target for La Loma coal in a tube and ball mill at EDF Energy plc coal fired power station in Cottam, UK, is 70% passing at 75 μm, and from this study the 70% passing from the 90 μm equilibrium size was 68 μm, showing a close approximation to the full scale targets.", "Fig. 2 shows that the mill behaviour for La Loma was similar to that of the olive cake, eucalyptus and steam exploded pellets.", "The two treated biomasses showed very different milling behaviours to the untreated biomasses as well as each other.", "The steam exploded pellets showed a vast improvement on milling performance compared to the untreated pellets and olive cake, but the results were comparable to the eucalyptus pellets.", "The steam exploded had one of the lowest FPP80 at 1286 μm, but also had the finest P80 at 355 μm.", "As with olive cake, eucalyptus pellets and La Loma, the first 100 revolutions led to a high second run revolution count, which quickly reduced as the runs proceeded and the material started to break down.", "For the steam exploded pellets there is virtually no sample in the 1–3.35 mm size range, indicating that the pellets either remain intact or break up into fines during the milling process.", "The torrefied pellets had the highest FPP80 of 1537 μm but interestingly a P80 of 758 μm, which was comparable to the untreated biomasses.", "Fig. 3 shows that the forces within the mill are sufficient to easily break down the pellets, but some remain in the 1–3.35 mm range unlike the steam exploded pellets.", "Fig. 3 shows the combined cumulative distributions for the product of last 3 runs of the BWI test for the biomass samples.", "Apart from olive cake and the steam exploded pellets, the cumulative distributions of the biomass samples were very similar, which is reflected in the P80 values for the samples in Tables 2 and 3 of around 750–800 μm.", "Olive cake had a finer distribution due to the inclusion of 43% of the feed being fines below 1 mm, while the steam exploded produced the finest cumulative distribution of all the biomass samples, which is reflected in its P80 value of 355 μm and in Fig. 3 which shows that the pellets break down into fines rather than larger particles.", "Table 3 shows the Rosin–Rammler data and mean geometric diameter data for the samples.", "There is a good fit for the samples with the Rosin–Rammler distributions (R2 > 0.995), but quite a spread in the Rosin–Rammler distribution parameter n, varying between 0.97 for the steam exploded pellets up to 1.81 for the sunflower pellets.", "Lower n represents a wider distribution, and thus a higher diversity of particle sizes.", "This is also reflected in the higher geometrical standard deviations, Sg, with steam exploded and olive cake having highest values (2.89 and 2.60 respectively).", "This indicates that the product output is very dependent on material type, but for most untreated biomasses the output will be similar in product size and distribution, regardless of the Wi values obtained.", "As expected from other biomass milling studies [52,53] and mathematically, the geometric mean diameter from the ball milling is smaller than the Rosin–Rammler size parameter for all samples.", "Table 4 shows the impact of changing the feed size from pellet diameter (F80) to the 80% passing size for the pre-densified pellet particle size (FPP80) on the Wi.", "By using FPP80, the value of BWI is significantly higher than for the pellet diameter, as the FPP80 creates a lower denominator in the BWI Eq. (6).", "This implies that less energy is required to break the pellets back down to their pre-densified particle size than is involved in breaking the particles into smaller particles.", "The implication is that for biomass pellets there are two stages of milling occurring.", "The first is the breaking down of the pellets into smaller parts or back to the pre-densified particle sizes, and the cohesive forces involved in holding together the pellets are weak and easy to overcome in the mills.", "The second stage of milling is the breaking down of the pre-densified particles into smaller particles.", "This suggests that to reduce energy consumption and potential for mill choking, the pellets should be formed of particles close to the required target size.", "This would reduce the milling to solely the pellet comminution phase and minimise mill choking by eliminating the particle comminution stage.", "Table 5 shows the thermal characterisation of the samples on a dry basis across all the BWI runs.", "There is no appreciable difference in the composition of the samples during the test based on the standard deviations shown for each value.", "The highest fixed carbon for the biomasses was for torrefied pellets at 23.9%, followed by olive cake (18.4%) and the steam exploded pellets at 17.3%.", "Both treated biomasses had higher fixed carbon than the other untreated woody biomasses.", "The percentage component values for the samples in this study corresponded to similar samples tested in literature [59].", "Eucalyptus, torrefied, wood and steam exploded pellets showed a similar peak volatile release rate (0.8–1.1%/°C) and peak volatile release temperature (327–338 °C).", "Olive cake and miscanthus pellets showed a much lower peak volatile release rate (0.5 and 0.6%/°C respectively) and peak volatile release temperature (288 and 285 °C respectively), which is an important consideration in coal mills, which can introduce preheat air between 200 and 300 °C.", "La Loma showed the highest peak volatile release temperature (420 °C), but lowest peak volatile release rate (0.3%/°C).", "However it should be noted that this correlation is based on a very limited number of samples and is only applicable for the test equipment and experimental conditions used to obtain the Wi and HGI values.", "The applicability of two standard grindability methods for coal; Hardgrove Grindability Index and Bond Index test, have been tested on several biomasses and one coal commonly used in the power sector.", "For the BWI test, particle size characterisation, thermal composition and analysis of mill behaviour were used to analyse mill phenomena such as mill choking.", "HGI is a poor indicator of the grindability of biomass in a vertical spindle mill, and can give misleading results when analysed alone.", "Grindability tests which aim to analyse the grindability of biomass to 75 μm are flawed, as the target size for optimal combustion of biomass and the setting for the classifier output from full scale mills is an order of magnitude of higher than this.", "The BWI test can be used to analyse the mill behaviour of biomass in a tube and ball mill.", "Wood, miscanthus and sunflower pellets exhibited mill choking during the BWI test, as the amount of fines produced did not increase with an increasing revolution count.", "Thus the BWI can be used to see if biomass samples are likely to encounter mill choking prior to full scale mill trials.", "The BWI results show that there are two stages of milling occurring in biomass pellets.", "The first is the breaking down of the pellets into smaller parts or back to the pre-densified particle sizes, where the cohesive forces involved in holding together the pellets are weak and easy to overcome in the mills.", "The second stage of milling is the breaking down of the pre-densified particles into smaller particles.", "However, the forces involved in this second stage are much greater than the initial pellet breakage stage.", "Therefore, to optimise milling in a coal mills, biomass pellets should be composed of particles close to the required size so that only the pellet comminution stage occurs.", "Whilst it has been shown that the BWI test is a useful test for analysing and predicting the mill behaviour of biomass in a tube and ball mill, the HGI test is not suitable for predicting the grindability of biomass in vertical spindle mills.", "A new standardised grindability test is therefore required to test the grindability of biomasses in these types of mills." ]

[ "Bond Work Index (BWI) & Hardgrove Grindability Index (HGI) tests for biomass & coal.", "BWI can predict the potential for mill choking of biomass in a tube and ball mill.", "HGI is a poor method of predicting grindability of biomass in vertical spindle mills.", "Pellets should be composed of pre-densified particles close to the target size.", "Approximate correlation between HGI and BWI found for some biomass samples." ]

[ "BIOC", "COMP" ]

[ "Confocal microscopy", "Diffusion", "Fluorescence correlation spectroscopy", "Fluorescence recovery after photobleaching", "GFP", "Live cell", "Nuclear pore complex", "Single particle tracking", "Transport" ]

[ "1303", "1304", "1305", "1311", "1315", "1706" ]

[ "In eukaryotic cells, the cytoplasm and the nucleus are spatially separated by a double membrane, the nuclear envelope (NE).", "Embedded in the NE are the nuclear pore complexes (NPCs), which allow the passage of ions and molecules across the NE and, at the same time, regulate the exchange of larger molecules, such as RNAs, proteins, or ribonucleoprotein (RNP) particles between nucleus and cytoplasm [1].", "The overall shape of the pore is known since pioneering studies, among others, were conducted yeast by electron microscopy (EM) [2] and on Dictyostelium discoideum by cryo-electron tomography (cryo-ET) [3]: the pore is a channel-like structure of about 40–90 nm in length and 40–75 nm in width, showing an asymmetric structure with flexible protein filaments extending out from the pore (approximately 50 nm) into the cytoplasmic environment, and an open basket-like structure extending to about 75 nm into the nucleus.", "More recently, mainly thanks to the straightforward combination of EM and ET with mass spectrometry (MS) analysis, structural modelling, and X-ray crystallography, our knowledge about the finest structural details of the NPC is enormously increasing (for more details see Refs. 3–7).", "Collectively, the achievements brought by structural studies promise to open new perspectives for our understanding of the molecular mechanisms underlying NPC function in normal and altered conditions [8].", "At the molecular level, the whole NPC consists of about 30 different polypeptides designated nucleoporins (Nups), with a very controlled stoichiometry, and a total mass of ~ 125 MDa [9–11].", "Most of the Nups lack a fixed secondary structure but rather contain domains rich in phenylalanine-glycine (FG) repeats [12] which are very flexible.", "These FG-Nups are generally located within the central channel of the NPC, forming a selective barrier that inhibits the efficient translocation of large molecules (> 40 kDa) unless they are chaperoned by transport receptors [13], such as Importin β (Impβ).", "Impβ, one of the major transport receptors, recognizes cargo molecules in the cytoplasm and forms a transport complex either directly or indirectly (i.e. through Importin α, Impα) [14].", "Cargo-receptor complexes are able to interact with Nups at the cytoplasmic filaments or at the peripheries of the central pore [15].", "From here, the cargo-receptor complex is transferred to the FG-repeat domain of nucleoporins in the center of the NPC (e.g. Nup153 [16]), where the FG-Nups offer a functional redundancy of binding sites for Impβ [17,18].", "How nucleoporin-Impβ interaction drives NPC-passage is, however, not a trivial problem, and several models address this issue (for a detailed review refer to [19]).", "In spite of their variety, these models differ mainly in the physical arrangement and mobility (static vs. dynamic) of the FG-domains within the NPC.", "Whatever the organization of the FG-Nups in the NPC, the transport process is terminated by the intervention of Ran guanosine triphosphate (RanGTP), which dissociates Impβ from the FG-Nups at the level of the nuclear basket and causes the release of the cargo molecule into the nucleus.", "The newly formed Impβ-RanGTP complex is selectively transferred into the cytoplasm to initiate a new round of transport.", "Given the highly dynamic nature of the overall process, a variety of time resolved biophysical strategies were applied to nucleocytoplasmic transport of molecules (summarized in Fig. 1).", "They can be roughly divided into three major classes, according to their differential capability to report on the behavior of molecular transport events across the NPC gateway.", "The first comprises perturbation-based approaches, such as Fluorescence Recovery After Photobleaching (FRAP).", "By these methods, the characteristic time of molecular transport across the entire NE can be measured by averaging the behavior of many molecules, across many pores.", "A peculiarity of these methods is that they rely on the change of the optical properties within the sample only, whereas the characteristic dynamics and function of the molecules under study are not altered.", "Originally conceived by Peters in 1974 [20], this technique owes much of its relevance to the discovery and development of auto-fluorescent proteins (FPs) [21], a class of genetically-encoded fluorescent molecules derived from sea organisms such as jellyfish or corals.", "FP-based FRAP was successfully applied to many scientific fields, including biophysics and biomedicine, at cellular and subcellular level [22–25], also taking advantage from the recent development of microscopy setups that allow high-resolution imaging on living cells [26].", "The second class comprises localization-based single particle tracking (SPT) methods.", "By these approaches, the trajectories of single molecules of interest are measured.", "By SPT, the transit times and interactions at the level of isolated NPCs of several transport receptors and model cargo molecules were successfully probed.", "Contrary to other approaches, SPT experiments typically require complex experimental procedures: for instance, the molecule of interest must be purified, properly labelled (without affecting its functionality), and introduced into the cell by microinjection or cell permeabilization.", "Also SPT measurements inherently require bright and isolated particles that must localized and tracked over time many times in order to acquire reasonable statistics.", "Finally, the third class encompasses spatiotemporal fluorescence correlation spectroscopy (FCS)-based approaches, which afford single molecule ‘sensitivity’ in presence of many similarly labelled molecules and in live, unperturbed cells.", "The recently developed spatial extension of FCS, named pair correlation function (pCF) approach, was proven to be particularly suited to study the shuttling of molecules across the NE [27] and other sub-cellular structures (e.g. chromatin territories [28–30]).", "This approach builds on the dual-foci FCS concept [31] and combines FCS and SPT potentialities into a new method in which the time needed for each molecule to be found in a given point in space that is different from the position at time zero can be extracted [32].", "If a barrier to diffusion is present, a longer time will be needed for the same molecule to be found at a position across the barrier, as already demonstrated in the case of molecular transport across the NE [27].", "In the following, the detailed contribution of these three classes of time-resolved methods to our understanding of nucleocytoplasmic transport of molecules will be summarized and discussed.", "A glimpse into the future directions in the field will be also provided.", "Thanks both to the recent advancements in the optical microscopy technologies at our disposal and to the development of new fluorescent labels, several perturbation-based approaches were developed, including photobleaching methods (e.g. FRAP, see below), photoactivation (when nonfluorescent labels become fluorescent after illumination at a selected wavelength, e.g. by using the photoactivable GFP, or PA-GFP [33]), photoswitching (when fluorescence, upon excitation at a certain wavelength, can be switched on or off by light in a reversible manner, e.g. by using the DRONPA [34] or E222Q FP variants [35]) and photophotoconversion (when irradiation of labels at a selected wavelength induces a shift of their fluorescence spectrum toward longer wavelengths, e.g. by using the Dendra [36] and mEos [37] proteins).", "Irrespective of the method, after the photo-perturbation event, what is typically measured is how the fluorescence distribution relaxes toward the steady state.", "Among these strategies, FRAP is by far the most popular one and is widely used as a microscopy protocol suited to study the mobility of molecules and particles [21].", "In a typical FRAP experiment (Fig. 1A), a short and intense light pulse is applied to irreversibly photobleach the fluorescent molecules in a selected region of the sample (e.g. the nucleus, as in Fig. 1A).", "After photobleaching, the “dark” molecules gradually transfer out of the photobleached area, while at the same time unbleached molecules enter it from the surroundings.", "This exchange leads to a fluorescence recovery within the photobleached area (and to the concomitant decrease of fluorescence in the surroundings) that can be monitored by low-intensity excitation (schematic plot in Fig. 1A).", "Analysis of the recovery curves by means of a suitable biophysical model yields insight into the dynamic behavior of the molecule under study.", "Among the dynamical cases amenable to FRAP analysis, a particularly interesting situation occurs when diffusion takes place between two (or more) compartments separated by a permeable membrane, a situation often encountered in biological systems where membrane compartmentalization is at the basis of life.", "Molecular diffusion through the NE falls into this class.", "Concerning the available literature, in a pioneering work by Wei and colleagues [38], real-time imaging and FRAP were used to examine the nucleocytoplasmic shuttling of 27-kDa EGFP molecules in single live cells.", "It was found that EGFP diffuses bi-directionally through the pore, across the NE.", "The ∼ 100-fold slowing down of GFP diffusion at the NE barrier compared to free diffusion within the nucleus or the cytoplasm was interpreted as due to the reduced size of the NPC channel available for diffusion.", "The authors did not report any significant block of EGFP diffusion by depletion of perinuclear Ca2 + stores.", "Also, EGFP bi-directional shuttling showed no variation with the cell cycle.", "These results, obtained on an inert tracer (i.e. GFP), have been considered as a reference for the study of passive diffusion of other molecules through the NPC.", "In fact, GFP is nowadays used as an indispensable benchmark in any attempt to identify the NPC translocation mechanism of endogenous proteins.", "For instance, Sunn and co-workers, based on the GFP reference, were able to show for the first time that the vitamin D receptor B1 (VDRB1) exploits a serum-dependent, active nuclear transport process, while no active nuclear export mechanism was found for the same protein [39].", "In a similar way, FRAP was successfully used to examine the nucleocytoplasmic transport of several other proteins or molecules including virus-derives nuclear localization signals (NLS) such as Tat peptide and the NLS from simian virus 40 (SV40) large tumor antigen (T-ag), members of the Importin family such as Imp13, and transcription factors such as STATs (signal transducers and activators of transcription) cancer regulatory proteins such as the parathyroid hormone related protein (PTHrP) signaling molecule or pRB (retinoblastoma protein) tumor suppressor [40–50].", "Worthy of note, at this point, is the use of reversibly photoswitchable fluorescent proteins (RSFPs) (mentioned above in this section).", "In contrast to standard FRAP or photoactivation strategies, RSFPs enable the reversible optical highlighting of specific pools of molecules and thus the repeated measurements of protein dynamic behavior, definitely increasing the amount of information that can be retrieved from a single measurement.", "In the last 15 years, many of these FP variants have been proposed and new cell imaging applications discovered that exploit their properties [51].", "In the context of nucleocytoplasmic shuttling, it is worth mentioning the pioneering study by Ando and colleagues, conducted on a DRONPA-labelled variant of Erk protein [34].", "Exploiting photoswitching, the authors could identify Erk responses, in terms of translocation in and out of nucleus, in one cell under different, consecutive, stimuli (e.g. with and w/o EGF [34]).", "Concerning photoconvertible FPs, on the other hand, it is interesting to note that they enable to simultaneously detect both non-photoconverted and photoconverted subpopulations of labelled molecules, as showed by Chudakov and colleagues by studying Dendra2 redistribution across the nuclear envelope of living HeLa cells [52].", "The methods described so far, being inherently ensemble-averaging strategies, do not afford information on single molecules but averages over many similarly-labelled molecules.", "This inherent limitation, however, does not prevent perturbation-based methods from providing quantitative information on the molecular (kinetic and thermodynamic) details of the transport process.", "For instance, by combining FRAP with the calibration of intracellular protein (i.e. GFP) concentration, Cardarelli and co-workers were able to quantitatively define the nuclear transport saturability by the estimate of the effective dissociation constant of the NLS-Importin complex in the actual cellular environment [47].", "By this combined approach, the sub-saturation of the transport carriers (importins) by the NLS-cargo molecules was found to be a key factor regulating the overall nuclear import rates in living cells, in contrast to what expected based on the available in vitro data (conducted on purified components of the transport machinery).", "The same approach was then extended to the study of Nuclear Export Signal (NES), its affinity for the export machinery, and the maximum rate of NES-mediated transport at saturation of export carriers [53].", "The measured quantities were found to be remarkably similar to those characteristic of active nuclear import.", "Our results also suggested that active export/import and active export/passive diffusion fluxes must be largely uncoupled, and that a mechanism of differential gating at the NPC level must exist.", "As opposed to perturbation-based techniques, localization-based approaches potentially yield single molecule information, provided that the molecule of interest is properly labelled and introduced into the sample (e.g. a cell, through permeabilization/electroporation) at the desired concentration (steps schematically represented in Fig. 1B).", "In these conditions, localization-based methods can afford valuable information on structural and functional properties of the system under investigation [54].", "A first distinguishing advantage of single molecule tracking experiments is that molecular processes do not need to be synchronized, as opposed to ensemble kinetic measurements, where it is usually challenging to obtain a population of molecules instantaneously triggered to start the process/reaction of interest.", "Also, single-molecule studies yield fluctuations and distributions of dynamical/kinetic parameters that are typically lost in ensemble-averaging experiments.", "Last, connected molecular reactions can be probed with no need to trap intermediates, as in the case of ensemble experiments.", "Of particular interest for biological applications is the detection of single molecules by the use of high-sensitivity CCD camera systems in far-field optical microscopy setups [55].", "The diffraction-limited image of single molecules is typically approximated through a two-dimensional Gaussian function interpolation.", "Although information on the shape of the sub-diffraction particle cannot be usually retrieved, its position can be determined with high precision.", "In particular, the localization accuracy depends on the signal/noise ratio and may reach a few nanometers under optimal experimental conditions [56,57], thus allowing to carefully reconstruct the dynamic behavior (trajectories) of single molecules.", "Historically, single molecules studies have been widely applied to analyze the movement of molecules on membranes (e.g. single receptors and lipid molecules; for a review see [58]), but were concomitantly extended to the study of single-molecule mobility in the 3D interior of cells (for an extensive review see [59]).", "Concerning nucleocytoplasmic shuttling, single-molecule detection in a far-field optical setup was shown to yield significant insight into the molecular details of the transport process.", "For instance, by combining sensitive fluorescence microscopy with microinjection, Babcock and colleagues investigated the transport into the nucleus of influenza genes by real-time, live-cell 3D tracking of single viral ribonucleoproteins (vRNPs) [60].", "The authors show unambiguously that vRNPs are transported within the cytoplasm and nucleus by passive diffusion, while they undergo binding to the NPC, with dissociation rate constants ranging from 1 to 100s.", "Also, the authors demonstrate how the expression of the protein M1 during late infection is able to downregulate the nuclear import of vRNP by directly inhibiting its binding to the NPCs60.", "Concomitantly, in a series of studies conducted both in permeabilized and in microinjected cells, Kubitscheck and colleagues used far-field single-molecule microscopy to measure the distribution of binding sites and the dwell times at the NPC for a series of endogenous transport receptors with and without their respective transport substrates [61,62].", "Based on the obtained results the authors could argue for a molecular transport process with no significant interference between the nuclear import and export processes, in analogy with the FRAP-based results discussed above [53].", "This evidence can be explained, in principle, by assuming that the pores are alternatively involved in export or import processes or that indeed two (structurally?)", "different pore species exist, one deputed to import and another to export.", "An additional scenario, increasingly supported by data, can be that the pore channel embeds independent (i.e. structurally separated) pathways for import and export (see below).", "Interestingly, Yang and colleagues exploited a narrow-field confocal setup to further improve the S/N ratio of standard acquisitions [63,64].", "By this approach, they reached approximately 2 ms temporal resolution and 15-nm spatial resolution for a fixed molecule.", "The spatial resolution is obviously destined to decrease in the case of a mobile molecule, and inherently depends on how fast the molecule is moving.", "In this case, however, the S/N ratio was sufficiently high to directly measure the time a single molecule spends interacting with the NPC.", "The tracking algorithm reveals that molecules spend most of their transport time by randomly moving in the central channel of the NPC with escape from the channel being the major rate-limiting step of the process.", "Worthy of note, the same authors were able to reach a resolution of 9 nm and 400 μs in space and time, respectively, by introducing the SPEED method (Single-Point Edge-Excitation sub-Diffraction microscopy) [65–67].", "With this improved resolution, the transport of several import/export/cargo molecules (e.g. Imp β1, mRNA) was probed in great detail in permeabilized cells.", "Transport pathways were described at molecular resolution and used to build 3D spatial density maps of interactions between the FG-rich central channel and the translocating molecules (for a review see [68]).", "In brief, the authors proved that Importins (with or without their cargoes) and mRNAs use distinct routes from that of the small passive molecules.", "At the same time, they found that active (at least in the case of import) and passive transport are not fully separated in space within the central channel and that the extent of their overlap shall depend on the size of the transiting molecule.", "To tackle the characteristic limitations of SPT-based approaches discussed above (e.g. large labels, complex experimental procedures, high statistic required), fluorescence correlation spectroscopy techniques appear as ideal alternative strategies.", "Among others, the pair correlation function (pCF) method is particularly suited to study nucleocytoplasmic shuttling as it is able to measure the time needed for single molecules to migrate from one point to another within a living cell and in presence of many similarly labelled molecules (schematic representation of the method in Fig. 1C) [32].", "Such a peculiar property of the pCF algorithm makes it suitable to provide a map of molecular transport times among arbitrary points in the cells and to detect the presence of barriers to diffusion with high (millisecond) temporal resolution and diffraction-limited spatial resolution (schematic plot in Fig. 1C).", "Concerning nucleocytoplasmic exchange, the pCF method has been applied to monitor the transport of a model protein substrate (NLS-GFP, as discussed in the previous sections) through NPCs in living cells [27].", "Cardarelli and Gratton demonstrated that the pCF algorithm can easily detect the expected lengthening in the transit time of molecules if two positions across the nuclear barrier are correlated (maximum of correlation in the 100–500 ms range) as compared to two points at the same distance but within the cytoplasmic (or nuclear) compartments.", "On the average, NLS-GFP molecules are slowed down ∼ 40–100 times when they passively diffuse through the pore in the nucleus-to-cytoplasm direction (with respect to free intracellular diffusion), in keeping with several FRAP-based estimates [38,47].", "By contrast, if the same algorithm is applied in the cytoplasm-to-nucleus direction, the role of active, receptor-mediated nuclear import of NLS-GFP becomes evident.", "Accordingly, although passive-diffusion transit times are still measured, the pCF output becomes dominated by shorter transit times (1–40 ms range), characteristic of carrier-mediated transport, as measured by SPT techniques [61–63,69].", "Worthy of note, the NLS-GFP active transit times can be spatially resolved with respect to the distance from the NE.", "In particular, the pCF algorithm detects the fastest cytoplasm-to-nucleus transit times if the starting point is selected close to the NE barrier.", "This result correlates with the localization of endogenous Impα/β carriers, which are typically accumulated at the level of single pores [47].", "Concerning this latter issue, it is worth mentioning that Bianchini et al. recently presented the combination of pCF with STimulated Emission Depletion (STED) to analyze diffusion below the diffraction limit [70].", "The achievable spatial resolution by using overexpressed GFP tagged molecules was found to be around 110 nm in live cells (more than twofold improvement over conventional confocal imaging).", "STED-pCF highlighted how the intracellular environment close to the nuclear barrier affects the mobility of proteins which are actively imported into (or exported from) the nucleus.", "In fact, STED-pCF analysis unveiled the presence of local cytoplasmic and nucleoplasmic constrains to diffusion as well as the presence of slow diffusive component at distances up to approximately 1 μm from either sides of the NE.", "This latter slower component resembles that previously detected for transport complexes between cargo molecules and Importins.", "Remarkably, this level of accurate mapping of diffusion and its regulation is lost in conventional confocal imaging.", "Overall, the broad distribution of transit times (around the maximum of correlation) is a characteristic of the pCF-based analysis of nuceocytoplasmic transport.", "This is due to the fact that, each time that two points are correlated across the NE, the contribution of all the single transport pathways allowed for molecules to travel from one location to the other are averaged together.", "Nonetheless, the single molecule sensitivity afforded by the pCF analysis is precluded to ensemble-averaging measurements, such as FRAP, and is particularly useful if many experimental conditions are to be quantitatively screened with high accuracy.", "As a bright example, Hinde and co-workers exploited pair correlation microscopy to show that polymeric nanoparticles with different shapes but identical surface chemistries moved across the various cellular barriers, including the NPC, at different rates, ultimately defining the site of drug release [71].", "They measured how micelles, vesicles, rods and worms entered the cell and whether they escaped from endosomes and had access to the nucleus via the pore.", "Rods and worms, but not micelles and vesicles, entered the nucleus by passive diffusion.", "Their results demonstrate that drug delivery across the major cellular barrier, the NE, is important for doxorubicin efficiency and can be achieved with appropriately shaped nanoparticles.", "Worthy of mention, recent conjugation of the pCF approach to Number&Brightness analysis opens up new perspectives for the study nucleocytoplasmic exchange in live cells [72].", "In particular, Hinde and co-workers combined the pair correlation approach with molecular brightness analysis into a new method called pCOMB (pair correlation of molecular brightness).", "pCOMB filters the different oligomeric species diffusing within living cells and tracks their mobility based on transit time between two locations.", "Hinde and co-workers successfully used this approach to show the dependence of STAT3 (Signal Transducer and Activator of Transcription 3) intracellular mobility on its oligomeric state.", "They observed that, upon NPC translocation, STAT3 molecules in dimeric state must first bind to DNA to form STAT3 tetramers, which remain bound to DNA but acquire a different mobility.", "Cross-pair correlation (cpCOMB) analysis of the dimer-to-tetramer transition clearly shows that DNA accessibility is a key factor modulating STAT3 tetramer formation.", "Overall, the pCOMB approach was proved well suited for mapping the role of protein oligomerization in the regulation of transcription factor dynamics and function.", "It is worth stressing here that perturbation-, localization-, and fluctuation-based approaches are here presented and discussed not in general, but strictly related to their application to nucleocytoplasmic transport.", "Within such a peculiar biological context each of them shows selected distinguishing benefits or limitations (see Table 1).", "Ensemble averaging methods, for instance, are inherently limited in their ability to report on the molecular details of nucleocytoplasmic shuttling, as they can only provide bulk information on the overall process (averaging the contribution of all the molecules and all the pores in the cell).", "A typical perturbation-based experiment on nucleocytoplasmic transport is also inherently limited in the spatial resolution (S-res in Table 1), that is set by the extent of the photobleached area (and thus typically coincides with the size of the whole nucleus or cytoplasm), and in the temporal resolution (T-res in Table 1), that is typically set by the time needed to capture an image of the whole cell (i.e. from hundreds of milliseconds to seconds, except from line-scan FRAP in which few milliseconds can be technically reached [49]).", "On the other hand, however, perturbation-based measurements do not require complex sample-preparation procedures, can be performed on any standard optical setup and are thus accessible even to non-experts (for both data acquisition and data analysis).", "Localization-based approaches are, in theory, the preferred ones to describe molecular behavior, even in the case of nucleocytoplasmic transport, as they potentially provide information on single molecules (by means of the trajectories) moving across single pores.", "At the same time, however, localization is severely challenged by the movement of the molecule in a 3D environment and this in turn imposes a limit in the temporal resolution accessible (i.e. in this case the time required to collect enough photons for proper localization) and, consequently, in the final spatial resolution at which the trajectories of single molecules are described (please see Ref. 73 for further details).", "In the context of nucleocytoplasmic transport, these intrinsic limitations led to temporal and spatial resolutions in the order of hundreds of microseconds and few nanometers, respectively (e.g. see Ref. 65), which are actually sufficient to describe the dynamic behavior of molecules crossing the NPC.", "It is worth mentioning that, from a technical point of view, localization-based experiments rely on bright and isolated particles to be tracked many times in order to acquire enough statistics.", "Furthermore, most of these experiments require the molecule of interest to be properly purified and labelled (typically by using large particles (gold, quantum dots) that can modify the overall transport dynamics of the protein.", "Also, such experiments often demand for non-standard optical setups (e.g. Ref. 65) and expert users (for both data acquisition and data analysis).", "The spatiotemporal analysis of fluorescence fluctuations combines some of the technical advantages of perturbation-based approaches, such as the use of standard experimental procedures (e.g. use of GFP) and standard optical setups, with the intrinsic single-molecule sensitivity typical of localization-based techniques, but in this case in presence of many similarly labelled molecules.", "In the context of nucleocytoplasmic transport, fluctuation analysis on a standard line-scan acquisition provides a quantitative picture of molecular transit times (averaging the contribution of many single molecules across many pores) with adequate temporal resolution (typically hundreds of microseconds) and spatial resolution limited by diffraction [27].", "Typically, fluctuation-based experiments are accessible even to non-experts, while data analysis requires specific skills.", "Worthy of note, in contrast to the other two classes of techniques, fluctuation spectroscopy offer higher multiplexing capabilities, in terms of its natural compatibility with additional tools, such as multi-channel detection (cross-correlation analysis), super-resolution methods (e.g. STED), additional fluctuation analysis (e.g. Number&Brightness), or feedback-based imaging strategies (see next section).", "As mentioned in the introductory section, intense debate is still ongoing about the molecular details of nucleocytoplasmic transport and the nature of the selective gating imposed by the NPC.", "In particular, the structural and functional spatiotemporal organization of the FG-rich nucleoporins of the central channel (and their role in regulating molecular transport under native conditions) remains largely obscure.", "Available models are mainly based on simplified in vitro attempts to reconstruct the organization of selected components of the pore and are largely contradictory [16,74].", "To get valuable information in vivo, at least three main requisites are desirable: i) high spatial and temporal resolution, in order to properly describe the processes involved in nucleocytoplasmic transport; ii) single molecule sensitivity/resolution; iii) single pore resolution.", "In this regard, as already discussed, each of the presented strategies shows its own limitations.", "Perturbation-based methods, although informative, inherently fail to provide direct single-molecule and single-pore observations.", "SPT-based approaches are valuable as they potentially satisfy all the three requisites.", "Yet, it is not clear whether the experimental conditions required for SPT experiments (e.g. microinjection, permeabilization and use of large, bulky labels) are compatible with unaltered pore function.", "Fluctuation-based analysis, on the other hand, is endowed with single molecule sensitivity and high spatiotemporal resolution under native conditions but are typically conducted across many microns within the cell, thus averaging the contribution of many pores at the same time.", "Worthy of note, high-speed atomic force microscopy (HS-AFM) has been recently proposed as a valuable innovative approach to the study of the spatiotemporal dynamics of the NPC transport barrier [75].", "In fact, Sakiyama and co-workers were able to visualize the nanoscopic spatiotemporal dynamics of FG Nups inside Xenopus laevis oocyte NPCs at a timescale of about 100 ms and conclude that the highly flexible, dynamically fluctuating FG Nups of the central channel rapidly elongate and retract, but do not cohere into a tightly crosslinked meshwork [75].", "Although informative and very promising, the HS-AFM approach still represents an average of pore dynamic behavior on a time scale (hundreds of milliseconds) far from that typical of ucleocytoplasmic transport in vivo through NPCs (milliseconds).", "To tackle this latter relevant timescale, Cardarelli and co-workers proposed to use a feedback-based fluorescence tracking method, previously used to track point-like particles [76], to compensate for local diffusion of the entire NPC in living, unperturbed cells (Fig. 1D) [77,78].", "The measurement is conducted by rapidly orbiting the laser spot around the object of interest (the pore in this case), with a temporal resolution in the millisecond time window and localization precision in the nanometer range.", "Thus, standard analytical tools (e.g. fluctuation analysis) can be potentially brought onto the moving reference system of a single NPC to probe the movement of single molecules with high accuracy [77,78].", "In principle, the correlations in space and time due to single molecules crossing the NPC can be probed by fluctuation analysis along the orbit.", "As a proof of principle, Cardarelli et al. recently exploited feedback-based tracking of the pore position by using GFP-labelled Impβ1 as a dynamic marker of the NPC in live, unperturbed cells [77,78].", "A circular light envelope is formed around the NPC, while the center of mass of the NPC is maintained at the center of the orbit by the feedback routine.", "The combination of this approach with fluctuation analysis allowed monitoring the transport of single molecules across single pores with high spatiotemporal resolution.", "Transport of Importin-β1 was detected as being regulated in such a way as to produce a characteristic distribution of transit times spatially restricted to the pore, and function of the metabolic energy.", "Similarly, the nucleoporin Nup153 (recently implicated in the transport of Impβ) was found to be regulated so as to produce rapid and discrete exchange between two separate positions within the pore.", "Based on these results, it was argued that directed Nup153-mediated Importin motion might represent a key component of the overall selective-gating process within intact NPCs, as envisioned by the polymer-brush model of NPC function [16,79].", "Our data do not exclude alternative patterns of dynamic interactions between the cargo and the NPC; however, they suggest that directed transport rather than passive diffusion may play a relevant role in the regulation of molecular transport across the NPC.", "In summary, the orbital tracking method proved the potentiality to extract information on single-molecule events in a moving, nanoscopic reference system, in presence of many similarly-labelled molecules, and under physiological conditions.", "As such, this approach holds the promise to become a valuable technological platform to address the lingering questions at the level of single pores." ]

[]

[ "MATE" ]

[ "Cancer management", "Circulating tumor cell cluster", "Metastasis", "Microfluidic CTC cluster", "Passive detection techniques", "Separation CTC cluster" ]

[ "2501", "2502", "2503", "2504" ]

[ "Metastasis is a complicated, multistep process where cancer cells detach from the primary tumor, migrate to adjacent tissues, invade and travel through the bloodstream or the lymphatic system, survive, proliferate, colonize in distant organs and finally establish a new tumor (Fig. 1a) [1–12].", "These tumor cells that travel through the bloodstream or the lymphatic system are called circulating tumor cells (CTCs).", "After decades of research, our understanding of metastasis is still inconclusive, even though more than a century has been passed since the first report of Thomas Ashworth in 1869 on the presence of circulating tumor cells (CTCs) in the bloodstream [13–15].", "Currently, metastasis is assumed to be responsible for around 90% of cancer-related deceases [16–18].", "Despite decades of research and experiments, cancer therapies have not been sufficient yet, and the mortality rate of cancer metastasis has marginally ameliorated.", "Mechanistic understanding of the metastasis process can lead to the development of anti-metastatic therapies that improve patient mortality [19].", "An increasing number of studies have shown the important role of CTCs in cancer metastases.", "CTCs supply more straightforward and comprehensive information about the tumor [20].", "They can be used for various experimental purposes, e.g., examining the response of cancer cells to chemotherapy, predicting the overall survival, noninvasively monitoring the drug susceptibility, metastatic therapy and as early detection and prognostic biomarkers [21–25].", "Additionally, US food and drug administration (FDA) approved CTCs clinical applications for personalized treatment in metastatic colorectal, prostate, and breast cancers.", "Conventional hypotheses assume that metastasis is established by the invasion and proliferation of individual CTCs into distant organs after the epithelial–mesenchymal transition (EMT), which increases the invasiveness of the CTCs [26].", "However, the discovery of CTC clusters in clinical and animal models [27], the groups of two or more tumor cells with strong cell–cell contacts, has challenged this assumption.", "Individual CTCs might not be the only cause of metastases; rather, multicellular aggregates of CTCs, CTC clusters, may play a significant role [28,29].", "For the first time, in 1954, Watanabe studied metastasis in mouse model and reported the higher potential of CTC clusters in tumor metastases [30].", "In the following decades, the 1970s, experimental studies also demonstrated the higher capacity of CTC clusters in metastases compared to that of single CTCs.", "Fidler et al. found that, if cancer cells were aggregated into clusters before injection, these cells established several-fold more tumors than the equal numbers of individual cancer cells [31].", "Other researches later confirmed this finding [32–37].", "Based on in-vitro quantification methods, it is known that CTC clusters comprise 5–20% of the total CTCs depending on the disease stage in both human and animal models [38–40].", "However, a recent study indicated that the proportion of CTC clusters in the late stage of metastatic cancer is much higher than previously assumed [41].", "Following studies also demonstrated that CTC clusters, despite their rarity, are responsible for seeding ∼50–97% of metastatic tumors in mouse models [42].", "This indicates that CTC clusters have 23 to 100 times higher metastatic potential than individual CTCs [39,42].", "Interestingly, single CTCs with the lower metastatic potential could acquire higher metastatic capability when incorporating with other cells in a cluster [43].", "This justifies the critical role of CTC clusters in cancer metastases.", "Experiments also revealed that the detection of only one CTC cluster in blood at any given time point correlated with significantly lower survival rates in the patients with prostate, colorectal, breast and small-cell lung cancers [28,39,44].", "Altogether, it is quite likely that CTC clusters play a far more significant role in the metastasis process than previously believed.", "CTC clusters are not simply a collection of tumor cells.", "CTC clusters include some other non-tumor cells such as endothelial cells, erythrocytes, stromal cells, leukocytes, platelets, and cancer-associated fibroblasts [39,45–51].", "These non-malignant counterparts were believed to provide advantages for CTC clusters survival.", "Higher metastatic potential of CTC clusters has been reported to be related to several factors.", "These factors include the cooperation of heterogeneous cell phenotypes within the clusters [52], strong cell–cell adhesions, which protect the tumor cells against anoikis [53], and physical shielding against the attacks of the immune cells (Fig. 1b) [54].", "Despite all the research and hypotheses to date, the rarity of CTCs in blood sample (1–100 CTCs per 109 blood cells and even fewer CTC clusters) and deficiencies of the existing separation methods limit our knowledge about CTC clusters.", "Many questions about CTC clusters formation, distribution and properties are still to be answered.", "To address these questions, an efficient separation platform is the first step to capture sufficient viable CTC clusters.", "Such platform makes subsequent molecular, genetic and biological analyses possible.", "Over the past several years, rapid progress in CTCs research has resulted in the development of technology that also can separate CTC clusters.", "However, currently, limited specialized techniques have been developed for the separation of CTC clusters.", "In recent years, great attention has been paid to CTC clusters because of their importance in cancer metastases, and the number of the published articles on CTC clusters has exponentially increased (Fig. 2).", "Despite the recent advances and discoveries, CTC cluster has not yet been reviewed comprehensively.", "Herein, we collate many interesting publications to provide a comprehensive review about CTC clusters from all the related aspects, including separation methods as well as their clinical applications and provide scopes for the future research direction.", "Rarity is a significant challenge for the separation of CTC clusters.", "A 10 ml of a peripheral blood sample from a metastatic cancer patient typically contains 0–100 single CTCs and roughly 0–5 CTC clusters (only about 5–20% of all CTCs) [39] among approximately 50 × 109 RBCs, 80 × 106 WBCs and 3 × 109 platelets [55].", "Another challenge for CTC cluster separation is possible dissociation during the blood sample processing.", "An efficient platform to isolate CTC clusters would have the capacity to separate intact CTC clusters of different shape, size, and composition, autonomously of cell surface markers with minimum manipulation, fast processing time, and vigorous clinical feasibility and validity.", "To date, numerous strategies have been developed for isolating single CTCs from blood sample [56–61] based on the physical (e.g., size, density, deformability, electrophoresis, dielectrophoresis), or biological (e.g., antibody expression) differences of CTCs and non-tumor cells.", "However, only a few platforms have been developed specifically for CTC clusters separation.", "To date, microfluidic devices appear to be the most encouraging platform for separating CTC clusters, as they have several unique features, such as the ability to process whole blood without preprocessing, which results in less cluster dissociation, fast processing time, and collection of live CTC clusters without manipulation.", "Up to now, most studies around clusters have relied on the strategies designed for individual CTCs, which have insufficient efficiency to separate clusters.", "CTC clusters were observed fortuitously, using these platforms, which usually underestimated the number of the CTC clusters due to the limitations of the employed techniques.", "The platforms with the capability of isolating CTC clusters are summarized in Table 2 and are briefly reviewed in this section.", "Recent progress in active separation methods can also aid the development of more advanced CTCs-detecting techniques [62].", "Investigating active detection techniques is out of the scope of current paper that focuses mainly on passive platforms, which are more feasible and have higher potential to be commercialized.", "Antibody-based methods are the most widely used techniques for CTCs separation.", "These methods rely on the expression of cellular surface markers and either isolate cancer cells (positive selection) or remove normal blood cells, thereby enriching cancer cells (negative selection).", "The antibodies mainly pertain to epithelial cell surface markers that are absent from other blood cells [63–66].", "The epithelial cell adhesion molecule (EpCAM) Antibody, cytokeratin antibody (anti-CK) and CD45 are the most common antibodies for distinguishing CTCs and other blood cells.", "However, there are still some limitations in these techniques, such as difficulties in distinguishing between CTCs and non-malignant epithelial cells [67].", "Furthermore, capturing CTCs that have undergone the EMT process cannot be appropriately done using antibody expression techniques.", "One simple technique for detecting and capturing the presence of CTCs in a blood sample is a high-resolution imaging method.", "In this method, blood is first lysed, then the remaining nucleated cells are plated on a surface and stained with antiEpCAM-fluorescent antibodies to discriminate cancerous from other cells.", "However, this technique is incompatible with the applications that require the recovery of viable CTCs because the cells are fixed during processing.", "CytoTrack™ solve this issue by developing a pre-scanner blood sample at high rates (up to 120 million cells/min) and recorded the potential CTCs targets, and operator can select specific cells to be isolated by CytoPicker™ for further analyses and corroboration [68] (Fig. 3a).", "RareCyte also developed a similar platform [69].", "Commercial Epic CTC Platform (Epic Sciences Inc., USA) as another high-speed automated imaging platform uses anti-CK/CD45/DAPI (4′,6-diamidino-2-phenylindole) immunofluorescent staining to detect CTCs.", "The epic platform was reported to be highly efficient for CTC clusters detection [70].", "Ensemble-decision aliquot ranking (eDAR) () [71,72] is another imaging platform that uses multi-color line-confocal to identify and enumerate EpCAM labeled cells.", "In this platform, a switching mechanism steers positive aliquot to slits filtration unit and negative aliquot to waste collection thorough different channels [73] (Fig. 3b).", "CTC clusters with low EpCAM expression were observed in the patient blood samples, utilizing eDAR [73].", "Another technique is CellSearch® [26,74,75] (Veridex, USA), which is a magnetic-activated cell sorting (MACS) method.", "This technique is the first and only clinically validated and an FDA-cleared blood test for CTCs enumeration and separation.", "In this method, a 7.5-ml blood sample is centrifuged to separate solid blood components from plasma.", "Using magnetic nanoparticles coated with antibodies to target EpCAM.", "The cells that have bound to the nanoparticle are pulled to the magnets, and the rest of the cells are removed [76].", "Therefore the CTCs are magnetically separated from other blood cells and subsequently identified with the use of fluorescently labeled antibodies (Fig. 4a) [75].", "In CellSearch method, a CTC cluster is defined as a group, comprising more than two cells expressing EpCAM, cytokeratins (CKs 8, 18, and 19) and DAPI without expression of CD45 [25,53,77–83].", "There are also some techniques that use similar CellSearch principle, labeling CTCs with antigen-specific antibodies linked to magnetic beads like Dynal Magnetic Beads® (Invitrogen, USA), AdnaTest (Adnagen AG) (uses a cocktail of antibodies e.g., EpCAM and MUC-1, and AdnaTest Cancer-type cocktail unlike CellSearch anti-EpCAM antibodies), and EasySep® (negative selection) (Stem Cell Technologies, Canada) [84] that CTC clusters have also been observed using them.", "Beside CellSearch, Vitatex Inc. developed the cell adhesion matrix (CAM) assay [85].", "The CAM assay exploits the invasive characteristic of cancer cells in collagen to isolate metastatic invasive circulating tumor cells (iCTCs).", "When patient blood samples are applied to the CAM-coated tubes (Vita-CapTM) or culture plates (Vita-AssayTM), iCTCs that uptake cell-adhesion matrix preferentially adhere to CAM (Fig. 4b).", "This technique separates CTCs in metastatic prostate and breast cancer [86,87].", "The limited blood sample volumes from cancer patients (5–20 ml) may impose a severe restriction on the separation of rare CTCs.", "CellCollectors® (GILUPI GmbH, Germany) is a European Conformity (CE) approved in-vivo CTCs isolation base on antibody affinity [88].", "The system consists of a needle, which is placed directly in the peripheral arm vein of a patient with up to 1.5 L of blood pass via an indwelling catheter for 30 min.", "The flexible needle is made of stainless steel, a gold coating layer of 2-μm thickness and a hydrogel coating layer with 2–10 μm thickness.", "On the hydrogel layer, antiEpCAM-antibodies are conjugated to identify and isolate the EpCAM-positive CTCs that can be analyzed in downstream analyses (Fig. 4c).", "GILUPI claims that CellCollectors can detect 70% of CTCs in lung, breast, colorectal and prostate cancer patients [89–97].", "Further studies with other tumors are currently is in progress.", "Methods based on biochemical properties also could be combined with and strengthened by microfluidic technologies [98].", "Adams et al. designed a microfluidic device containing a series of the high-aspect-ratio microchannel (35 μm width × 150 μm depth) that were replicated in polymethyl methacrylate (PMMA).", "The microchannel walls were covalently decorated with antibodies directed against cells expressing the EpCAM [99].", "Increasing the throughput of the antibody-based methods, Sequist et al. introduced another microfluidics platform called CTC-chip.", "A standard microscope-slide-sized silicon chip with μm-sized posts array that coated with antiEpCAM-antibodies, to maximize the interaction of the CTCs with the functionalized surface (Fig. 5a) [100].", "The CTC-chip was able to capture CTC clusters in lung cancer [101].", "Geleghorn et al. inspired by CTC-chip described geometrically enhanced differential immunocapturing (GEDI), a theoretical framework for the use of staggered obstacle arrays to create size-dependent particle trajectories that maximize prostate circulating tumor cells (PCTC)-wall interactions while minimizing the interactions of other blood cells [102,103].", "The abundant number and intricate structure of the CTC-chip microposts presented a challenge for clinical research.", "Stott et al. developed a microfluidic device, herringbone HB-chip.", "The HB-chip design utilizes passive mixing of blood cells through the generation of micro vortices created by angled grooves leading to significantly increase the number of interactions between target CTCs and the antibody-coated chip surface (Fig. 5b) [104].", "The device was later optimized geometrically [105].", "HB-chip was one of the first microfluidic platforms that can captures the clusters from metastatic patient blood samples [106].", "Inspired by the HB-chip, Hyun et al. proposed a geometrically activated surface interaction (GASI) to increase the surface interaction between the leukocytes and the anti-CD45 immobilized surfaces for CTCs enrichment through negative selection [107].", "Another group also proposed the same functionalized surface platform for positive selection, modular CTC sinusoidal microsystem [108] (Fig. 5c).", "This microsystem has a higher recovery rate for CTC clusters and has been commercialized by BioFluidica.", "The principle of enhancing antibody–CTCs interaction for CTCs separation has been inspired many similar methods that also have the potential for CTC clusters separation.", "Crammed 100–200 nm pillars were coated with the relevant antibody (anti-EpCAM) [109,110].", "Instead of micropost arrays, some capturing methods inspired by Stott's work use antibody-coated surfaces to increase antibody-CTCs interactions [111,112].", "One of the major limitations in the positive selection of antibody-based methods is its inability to target cancer cells with reduced expression of cancer-associated markers.", "In the EMT process, cells lose their epithelial characteristics and acquire more mesenchymal-like phenotypes.", "Consequently, EpCAM expression significantly decreases, especially in the cells within the clusters [113].", "In addition, EpCAM also can be detected in other diseases such as benign colon disease can be misinterpreted as cancer cells [114].", "Therefore, such positive detection relying on EpCAM expression may disregard some critical subpopulations as the precise number of CTCs may be underrated [115–117].", "One idea to overcome this limitation was proposed to target the actin-bundling protein plastin3, a novel marker that is not downregulated by CTCs during EMT and not expressed in blood cells [118], N-cadherin, O-cadherin, epidermal growth factor receptor (EGFR), the cytoskeletal protein vimentin [119,120], and cancer-specific biomarkers [103,121].", "However, it was reported inexistence vimentin expression among cells within clusters [122].", "In negative selection, leukocytes attachment to CTCs cluster [123] may lead to excluding precious subpopulation of clusters from detection.", "In addition, circulating endothelial cells are CD45−, that can exaggerate the final enumeration of CTC clusters [119].", "Another limitation of antibody-based platforms is the lack of a general marker that could be used for a variety of cancer cells.", "Any marker can distinguish specific tumor cells, but their application is limited by the heterogeneity of tumors and, consequently, the different genetic characteristics of the cell even in the same cancer cells [124].", "Most antibody-based separation strategies have generally been employed towards carcinomas as no specific marker targeting other cancer types (e.g. Sarcomas) exists so far.", "Lately, a new class of CTC-affinitive agents, viz. aptamers, demonstrated a great potential in the detection of CTCs as an alternative to antibodies [125–128] with some advantages such as high affinity, low cost, simple modification, and simple release mechanisms.", "Aptamers are synthetic low-molecular-weight single-stranded DNA/RNA which have been engineered to bind to specific targets, such as cancer cells with high affinity and selectivity.", "Aptamers can bind to cell membrane targets [129,130], and can also be selected against whole cancer cells [131].", "Some research groups developed microfluidics-based cell-affinity devices to capture CTCs using aptamers [132–134].", "Consequently, in antibody-based methods, the prevalence of CTC clusters was rare [64].", "In general, the efficiency of antibody-based methods chiefly depends on two factors: the expression and specificity of the target antigen and the affinity between antigens and antibodies, and the efficiency of labeling process.", "Compared to single CTCs, CTC clusters have smaller surface-to-volume ratios, which reduce the efficiency of antibody-based platforms to detect clusters that can be more obvious in larger CTC clusters [135].", "Although antibody-based methods have been used widely for CTCs separation, there are still some drawbacks, such as high cost and the need precise procedure, which pose challenges for using them pervasively in CTCs detection for clinical applications.", "Differences in physical properties such as cell density, size, and deformability, can be utilized to separate CTCs and CTC clusters.", "For instance, CTCs can be separated by filtration due to their larger size compared to other blood cells (Table 1).", "Most separation platforms based on physical properties use microfluidic technologies.", "Microfluidic platforms not only provide better efficiency in CTCs separation [136] but also facilitate the integration and the automation of high-throughput low-cost sample processing to achieve a real lab-on-chip solution [137].", "Based on the assumption that CTCs especially CTC Clusters are larger than other blood cells (Table 1), microfiltration techniques demonstrate a great potential for attaining high throughput analysis of sample volume.", "ISET® (isolation by size of epithelial tumor cells) (Rarecells diagnostics, France) is developed based on trapping the major epithelial cells (20–30 μm) while passing other cells (6–12 μm) through the pores of predefined size and shape.", "ISET® used a module of filtration (10–12 well) containing polycarbonate track-etch-type membrane, which comprises numerous randomly distributed 8-μm-diameter, cylindrical pores to separate CTCs from blood cells through size and deformability (Fig. 6a) [138].", "RareCells claims that ISET® sensitivity threshold is one CTC in 10 ml of blood.", "ISET® platform also was demonstrated to be able to separate CTC clusters in different metastatic cancer [139–145].", "However, such filtration platforms also retain some larger non-tumor cells, which is why this techniques are considered not very specific.", "CTC clusters from liver and lung cancers captured by ISET® are undetectable by CellSearch, shows more sensitivity of ISET® for CTC clusters separation than antibody-based methods [53,115].", "Another microfiltration platform, called FMSA (flexible micro spring array), enriches CTCs based on their size and deformability.", "FMSA is a 0.5 cm2 filtration membrane with a novel micro spring geometry, which was designed to maximize the throughput and allows for prompt CTC enrichment directly from peripheral blood sample without preprocessing [146].", "Blood sample passed through the FMSA device under accurately controlled pressures.", "Cells with a specific size are trapped in FMSA plate.", "The platform then uses antibodies for immunofluorescent detection.", "CTC clusters were separated from 44% of 7.5-ml whole blood clinical samples of breast, lung, and colorectal cancer in <10 min (Fig. 6b) [146,147].", "CellSieve™ is another filtration platform with ∼160,000 5–9 μm pores, spaced at 20 μm intervals [148,149] (Fig. 6c).", "It was reported detection of CTC clusters in sarcoma patients using CellSieve™ [150].", "CTC cluster separation method based on physical properties can be also combined with and strengthened by microfluidic technology, leading to other microfiltration and microcavity platforms [151], as introduced by Mohamed, Tan, Xu and Zheng and others [152–157] that have potential for CTC clusters separation [158].", "Among shortcomings associated with filtration platforms, clogging is one of the most critical one [159].", "Some groups rectified clogging problem of filtration methods by developing a filter device that could periodically be cleared [160] or by geometrical optimization microcavity [161].", "Filtration platforms allow direct filtering of peripheral blood samples without preprocessing and are more cost-effective compared to antibody-based methods [162].", "However, the intense tension stress and a mechanical lesion at the pore edges of the microfiltration techniques could cause deformation and remodeling [163] that affect the viability and integrity of cells, especially in CTC clusters, thus making the majority of them not suitable for further biological analysis [164].", "Centrifugation is one of the earliest strategies for CTCs separation [165].", "OncoQuick® (Greiner Bio-One, Germany) as a CTCs separation platform is based on density gradient centrifugation [61].", "The kit includes a 50 ml tube that is separated into two sections by a porous barrier.", "The lower section contains the separation medium which prevents blood from mixing with the gradient before centrifugation.", "The upper section accommodates up to 30 ml of the blood sample for processing.", "During centrifugation, the cells are separated according to their densities.", "The denser blood components such as red blood cells and white blood cells migrate through the porous barrier into the lower section.", "Less dense cells, including the CTCs, settle at the interphase layer between the separation medium and the plasma in the upper section [166].", "After washing steps, the captured CTCs can be used for further analyses (Fig. 7a) [167].", "OncoQuick results in higher tumor cell enrichment than in traditional Ficoll density gradient centrifugation [168] but less accurate and less sensitive in CTC enumeration as compared with CellSearch [169].", "Some clinical studies have utilized OncoQuick for CTCs enrichment [170–173], CTC clusters were observed in some of their results.", "A more advanced density gradient centrifugation technique is the RosetteSep® (Stem Cell Technology, Canada), which is based on the negative selection.", "RosetteSep® is a physical-biochemical-based method where antibodies crosslink a variety of unwanted cells, specifically leucocytes and RBCs, forming aggregates termed ‘rosettes’.", "The ‘rosettes’ sediment in the erythrocyte layer during the centrifugation step using a gelatin density gradient.", "The CTCs are negatively enriched in the mononuclear layer (Fig. 7b) [174].", "Centrifugal spiral microfluidic devices utilize inertia and the Dean flow [175–178] for CTCs separation.", "These microchannels have been designed with various cross-section geometries such as the rectangular [179,180], trapezoidal [181,182] and the stair-like [183] configurations.", "Two contrary inertial force (FL) and shear gradient force (FW) are dominant on particles with size ratio a/h > 0.1 (where a is particle diameter and h is channel height), while the secondary flow (Dean vortex) in curvilinear channels controls the movement of smaller particles.", "Inertial lift forces confine CTCs to a specific region of the channel cross-section, while smaller blood cells continue to be entrained along the Dean vortices.", "Using this method, CTCs and blood cells are focused to distinct streams within the microchannel and can be collected through two separate outlets.", "The throughput of these devices is shown to be reasonably high (as 7.5 ml of blood per 8 min) with high separation efficiency [182].", "These devices are also used for cell retention in perfusion culture flask [184], cell fractionation, and filtration [185].", "Hou et al. developed a spiral microchannel with intrinsic dean drag and inertial lift forces for size-based separation of CTCs from the blood sample.", "Dean flow fractionation (DFF) platform facilitates simple coupling with downstream biological assays of cancer cells (Fig. 8) [179].", "A year later Warkiani et al. upgraded the DFF platform with a trapezoidal cross-section (ClearCell® FX) [182] for ultra-fast label-free CTCs separation from peripheral blood samples using the Dean drag force coupled with the inertial lift force.", "This technique utilizes the intrinsic Dean vortex present in a curvilinear microchannel, along with inertial lift forces that focus large cells like CTCs against the inner wall to separate cancer cells based on size.", "The trapezoidal cross-section, averse to the common rectangular cross-section, can alter the core position of the Dean vortex, to achieve more effective separation (Fig. 8).", "With upgraded DFF, single CTCs and clusters successfully were isolated.", "More than 80% of the spiked cancer cells were recovered from 7.5 ml of blood within 8 min [182].", "This method is particularly attractive because of its simplicity and the high processing rates, approximately 0.5–1 ml/min for RBC-lysed blood samples.", "The high throughput makes DFF beneficial for applications that require the isolation of CTCs from large volumes of blood, such as early detection.", "Recently, using this device, CTC clusters were observed in the head and neck cancer [186].", "Clusters are on average larger than individual CTCs and healthy blood cells.", "However, strategies that rely solely on size-based separation may have limitations when applied to CTC clusters.", "The majority of clusters consist of 2–4 individual CTC.", "Individual CTC size varies dramatically, ranging from 12 to 30 μm even within the same patient [187,188].", "This overlap size range of large single CTCs and leukocytes (∼6–20 μm) with clusters (Table 1) [189] can lead to reducing the size disparities of most clusters with large singles and leukocytes.", "On the other hand, clusters often assume alignments that mask their most extended axes during size-based separation [55].", "Some technique discussed so far have been designed and developed specifically for separation single CTCs.", "CTC clusters were incidentally observed in many of these single CTC isolation platforms.", "Addressing the drawbacks of these platforms, Sarioglu et al. fabricated an exclusive platform for separation of intact and viable CTC clusters [190].", "The team developed the Cluster-Chip, to capture CTC clusters independently of tumor-specific markers from the unprocessed blood.", "CTC clusters are isolated through bifurcating triangular pillars as traps under low–shear stress conditions that preserve their integrity.", "The Cluster-Chip captures CTC clusters by relying on their cell–cell junction (Fig. 9a).", "This platform is able to capture CTC clusters in 30–40% of patients with metastatic breast, prostate, and melanoma cancer at a blood sample flow rate of 2.5 ml/h [190].", "The recovery of clusters immobilized on micropillar arrays is challenging due to the requirement of an operation temperature of 4 °C and flow with shear stress greater than physiological one for releasing the CTC clusters [55].", "Recently Inspired by Cluster chip Gao et al. amended an earlier size-based CTC separation platform [191], which captured CTC clusters and single CTCs separately [192].", "To address this limitation, Au et al. proposed a two-stage continuous microfluidic chip that separates intact CTC clusters from blood samples [193].", "This platform designed to utilize deterministic lateral displacement [194] to sort clusters based on geometric properties such as size and asymmetry.", "The first stage separates larger clusters based solely on their large size; using standard cylindrical DLD micropillar arrays to deflect particles with shortest axial diameters of 30 μm or more.", "The second stage was designed with asymmetric hybrids of elliptic cylinders and “I”-shaped pillars with the 30 μm ceiling.", "The second stage imposes the clusters that failed to be captured in the first stage to align their longitudinal axes “flat” in the flow direction (XY plane) (Fig. 9b).", "Therefore, the second stage sorts CTCs by discriminating asymmetric clusters from symmetric single cells.", "This strategy isolates 99% of clusters containing 9 or more cells and 66% of smaller clusters from whole blood.", "In a DLD-Chip, CTC clusters experience physiological or even lower shear stress and have short residence times.", "This platform separates clusters with over 87% viability and unhindered proliferation abilities.", "However, this strategy is limited by its relatively slow blood flow rate of 1 ml/h.", "Another microfluidic device deliberately designed to isolate CTC clusters is “antibody-functionalized 3D scaffold gelatin-microchip”, which can efficiently separate clusters by combining antibody recognition and physical barricade effect of the scaffold structure [195,196].", "Improving capture efficiency of marker-dependent strategies by CTCs-antibody interaction increment idea [197], Cheng et al. coated the 3D PDMS scaffold with multiple thermosensitive gelatin layers and functionalized it with anti-EpCAM antibodies.", "This scaffold with porous structure generates uncontrolled migration of cells that leads to increasing cell–structure interaction.", "After pumping blood sample into the scaffold chip at a flow rate of 50 μl/min to capture CTCs, gelatin hydrogel dissolves at physiological temperature (37 °C) and washing with PBS (Phosphate-buffered saline), allowing the cell-friendly release of CTCs for further analysis (Fig. 9c).", "Using this microchip, free individual and cluster CTCs were successfully obtained from the blood sample of cancer patients.", "This platform captured more than 88% of MCF-7 single CTCs with 60–70% recovery ratio and 82%–100% of two-to over nine-cell cluster with 50–100% recovery ratio respectively, with the high viability of more than 90% [195].", "Besides all the platforms mentioned above, some additional methods have been developed to detect or separate CTC clusters.", "Ge et al. proposed a novel strategy integrating subtraction enrichment and immunostaining-FISH (SE-iFISH®) (immuno-fluorescence in situ hybridization) [198].", "The integrated platform enables effective depletion of WBCs RBCs by immunomagnetic and centrifugation, to establish a high-throughput detection of CTCs irrespective chemical markers and physical properties.", "The SE-iFISH platform was able to efficiently detect CTC clusters from prostate cancer [199].", "A photoacoustic technique exploits strong optical absorption of melanin to image and sense melanoma CTCs in vivo.", "This platform utilized linear-array-based photoacoustic tomography (LA-PAT) technique for label-free high-throughput in-vivo CTC cluster detection.", "In addition, LA-PAT can quantify the number of cells in the CTC clusters and study their kinetics in the blood circulation by analyzing the contrast-to-noise ratios of the photoacoustic signals [200].", "Jiang et al. utilized both physical and biological properties of CTC clusters to introduce a new isolation technique treats platelets as a marker for the separation of platelet-cloaked CTC clusters.", "In this method, CTCs were targeted by capturing platelet-covered cells.", "This platform incorporated a two-step microfluidic strategy.", "The first step depletes free platelets by size, using deterministic lateral displacement (DLD).", "The second step isolates platelet-covered clusters, using the herringbone CTC chip (HB-Chip), which as mentioned induces micro vortices to enhance cell-capture surface interactions.", "This platform enabled the separation of CTCs from ∼60% of epithelial lung and breast cancer, and also 83% of mesenchymal melanoma cancer [201].", "Ozkumur et al. developed a three-step strategy that combines microfluidics and magnetic-based in which small CTC clusters were observed [202].", "After the magnetic labeling of cells in whole blood, DLD was used to deplete RBCs, platelets, and other small blood cellular debris from the sample.", "Next, inertial focusing was utilized to align nucleated cells within a microfluidic channel by introducing asymmetrically curved channels.", "These Channels help to extenuate the cellular collisions and ensure cellular displacement only as a function of magnetic force in the next step.", "At the last step, CTCs (positive selection) or WBCs (negative selection) immunomagnetically deflect into the collection channels.", "CTCs can be used for various clinical purposes, e.g., examine the cancer cells respond to therapeutic regimes, predict overall survival, noninvasively drug susceptibility monitoring, metastatic therapy and as early cancer detection and diagnostic biomarkers.", "Due to the higher metastatic potential, CTC clusters serve as a noninvasive method with high potential for diagnosis, prognosis, and treatment in many academicals studies and clinical trials.", "In the following sections, we discuss experimental attempts of using CTCs in a clinical context (Table 3).", "According to ClinicalTrials.gov there have been more than 400 recruiting clinical trials that utilize CTCs up to January 2019.", "The extreme effort is required on CTC clusters applications.", "Conventional tumor biopsy posses disadvantages such as sampling bias, sampling difficulty, and harm to patients.", "Since CTCs are present in the peripheral blood of carcinogenesis cancer patients even in the early stage [203], detection them from blood sample, especially CTC clusters due to their higher metastatic potential, as liquid biopsy could be a great alternative to conventional tumor biopsy [204] for cancers prognosis and diagnosis.", "Recent experimental studies have revealed a direct and robust association between the presence of CTC clusters recovered from venous patient blood and the significantly reduced survival rate as well as lousy prognosis in some types of cancer [28,38,205–210].", "Researchers also demonstrated the correlation of CTC clusters number present in a blood sample with worse progression-free survival (PFS) and overall survival (OS) [39,152,209,211–213], but any correlation between the number of CTC clusters and tumor type or stage [38,63,214,215].", "However, in a recent study, the presence of CTC cluster in a blood sample of the patient was correlated with resistance to therapy in epithelial ovarian cancer (EOC) [216].", "The molecular analysis of CTCs facilitates the identification of the molecular drivers of cancer in the patient body [217].", "In a recent work, molecular profiling of epidermal growth factor receptor variant type III (EGFRVIII) was shown to be a good indicator of squamous cell carcinoma of head and neck [41].", "After the immunohistochemical analysis, EGFRVIII expression observed in CTCs of a patient, also was detected in the primary and the metastatic tumors of the same patient [41].", "In a study of Gasch et al. [218], the mutations of PIK3CA, KRAS, and BRAF genes were analyzed using Sanger sequencing for predicting the resistance against anti-EGFR therapy in five patients.", "The PIK3CA gene displayed two different mutations in two separate CTCs in a patient, which indicates the CTC analysis capability to inform us about the tumor mutational heterogeneity.", "Using sensitive deep-sequencing genomic analyses of CTCs in patients with prostate and colorectal cancer demonstrated that mutations in CTCs resemble mutations in both the primary tumor and metastases [119,219–221].", "Zhang et al. recently utilized CTCs-derived organoids in genetic analyzing of lung adenocarcinoma CTCs to detect ALK instability and rearrangement [222].", "Therefore, relinquish these mutations in therapeutic regimes can affect the efficacy of drugs against mutinied targets [223,224].", "Recent studies have specified that CTCs could be used in frequent genetic profiling to monitor the evolving mutational outlook and drug sensitivity patterns for individual patients [222,225–227].", "Giesing et al. showed antioxidant genes of CTC clusters analyzing as a novel method with superb prognostic and predictive properties for monitoring treatment regime [228].", "The authors suggested that the antioxidant genes helps CTC clusters as a survival and defence mechanism in confronting with immune surveillance.", "Recently, CTC-derived organoid cultures have emerged as a novel technique in medical research and precision medicine as they preserve tumor tissue heterogeneity and drug-resistance responses, and thus are suitable for high-throughput drug screening [229,230].", "CTC-derived organoids could help to identify mutations in CTCs and epigenetic information about tumors, and screen treatment regimes in real time [63,227].", "Some groups recently have developed microfluidic platforms to study tumor cell–drug interactions were assessed.", "Subsequently, the pharmacological efficacy of chemotherapeutic drugs [231,232], tumor cells for drug responsiveness [233] and resistance [234] were monitored.", "Molnar et al. demonstrated that the CTC clusters number in blood sample reflects the chemotherapeutic sensitivity in colorectal cancer [235].", "Recently, Khoo et al. developed a platform to evaluate drug response on CTC clusters using patient-derived CTCs cultures.", "The team designed tapered microwells on microfluidics platform to allow CTC clusters formation without pre-enrichment and subsequent drug screening in situ [236].", "In addition, the elevation of CTCs number in peripheral blood is associated with macroscopic progression of tumor.", "Another study [237,238], demonstrated that CTCs number (≥5 per 7.5 ml blood) with and without CTC cluster [239] after the first chemotherapy could be a biomarker of disease progression and monitoring of treatment strategy.", "The authors proposed that the patients with unchanged blood levels of CTCs represent cancer cell resistance to the adopted therapy, so they should shift to other treatment regimes.", "The improvement of long-term survival is still disappointing.", "For most of the approved new cancer drug regimens, the survival time is only 1–2 months [240,241].", "A major reason for these modest gains is that these drugs were not developed to directly target agents responsible for metastasis [63], especially CTC clusters [242].", "A novel strategy for combating metastasis could be to dissociate CTC clusters into less potent individual CTCs in the circulation (e.g., by weakening the adhesion energies between cancer cells within clusters).", "Choi et al. tried such a strategy practically using urokinase [123,243].", "The authors claimed that urokinase could lyse fibrin to dissociate CTC clusters and as a result reduced the prevalence of metastasis in animal models.", "In addition, they demonstrated a reduction in the number of CTC clusters that incubated with urokinase in vitro.", "In-vivo urokinase utilizing in the blood of treated mice showed a decreased number of CTC clusters compared to the control.", "Therefore, the results suggest that urokinase disintegrates CTC clusters into individual CTCs [123].", "However, some researchers do not agree with disaggregation of CTC clusters in the bloodstream as a metastasis treatment.", "They caution that urokinase treatment may also include the risk of increasing invasiveness of tumor cells and metastatic spreading, resulting in the opposite effect of that, as reported by Choi et al. [244].", "In the field of cancer drug development, Gao et al. used CTCs-derived organoids for testing the new version of androgen receptor antagonist (enzalutamide) and PI3K-kinase pathway inhibitors (Everolimus and BKM-120) [245].", "Overall, despite of all experimental studies in CTC cluster, currently, the clinical importance of CTC clusters remains elusive.", "Further study is requisite to exploit the full potential of CTC clusters in real-world clinical applications.", "CTC cluster analysis as a noninvasive liquid biopsy is a new expanding field that can introduce unprecedented horizon in early cancer diagnosis and therapy assessment in clinical trials.", "Nevertheless, due to inefficient separation platforms and heterogeneous biology, there are still many fundamental unsolved issues about CTC clusters.", "As such, to date, it is not clear the metastatic potential of included tumor cells in a cluster compared to single CTCs and the effect of CTC cluster size and cell number on its metastatic potential.", "Whether dissociating CTC clusters into single CTCs can effectively reduce their metastatic risk.", "How the associated non-tumor cells included in CTC clusters increase their survival and more efficient distant colonization, as well as CTC cluster collective migration are among the outstanding questions in CTC cluster biology.", "Despite the significant progress in separation methods, substantial work still needs to be done to achieve a platform to efficiently identify, enumerate, and isolate intact CTC clusters in a reasonable time with minimal manual intervention.", "Subsequent developments in CTC cluster separation technologies will enhance our knowledge about these multicellular aggregates and their contribution to metastasis progression and can translate laboratory-based concepts to clinical applications in real-world settings.", "Complementary studies should be undertaken to characterize CTC clusters and to utilize their clinical value.", "Monitoring treatment regime is a great potential field of interest toward individual treatment.", "Therefore, the next step after developing an efficient separating platform for CTC cluster is ex-vivo patient-derived CTCs culturing.", "However, to date, no techniques have been presented for CTC clusters culturing.", "The future research should focus on developing strategies for long-term culture of patient-derived CTC clusters.", "Due to their higher metastatic potential, CTC clusters are expected to be utilized broadly in cancer and metastasis clinical trials in the coming years.", "We envision that liquid biopsy and qualitative and quantitative monitoring of CTCs, especially CTC clusters, will allow the clinician to establish more effective personalized treatments.", "The authors declare no conflicts of interest." ]

[]

[ "BIOC", "IMMU", "MEDI", "VETE" ]

[ "Booster", "China", "Eradication", "Inactivated poliovirus vaccine", "Oral poliovirus vaccine", "Poliomyelitis", "Poliovirus", "Vaccine" ]

[ "1313", "2400", "2725", "2739", "3400" ]

[ "Oral poliovirus vaccine (OPV) has been the mainstay of poliomyelitis control in many countries since the 1950s.", "Nonetheless, there are several disadvantages in continuing vaccination with OPV in countries where wild-type poliovirus has been eradicated.", "Despite its otherwise remarkable safety profile, OPV may rarely cause vaccine associated paralytic poliomyelitis (VAPP) due to reverse mutations in the RNA genome of the attenuated vaccine strains resulting in neurovirulence [1].", "An estimated two to four VAPP cases per 1000,000 birth cohort are expected to occur each year in countries using OPV only [1].", "In addition, outbreaks of poliomyelitis caused by circulating vaccine-derived strains have been documented and remain a potential threat in countries where OPV continues to be used [2,3].", "Inactivated poliovirus vaccines (IPV) have been available since the 1950s and enhanced IPV formulations with improved immunogenicity were introduced in the 1980s.", "The inactivation of IPV ensures that reverse mutations and neurovirulence are unable to occur.", "IPV is highly immunogenic administered as IPV alone or in mixed IPV-OPV schedules [4].", "IPV manufactured by GSK Vaccines has been used alone or in combination with diphtheria, tetanus and pertussis vaccine antigens since 1996 [5].", "The standalone IPV Poliorix™ (hereafter referred to as IPV; GSK Vaccines, Belgium) containing the three poliovirus types is currently licensed in more than 20 countries, and more than 12 million commercial doses have been distributed.", "Routine use of IPV and IPV combination vaccines has confirmed their positive benefit-risk profile in developed countries [4,6].", "In 2015, wild-type poliovirus 1 remains endemic in Pakistan and Afghanistan [7].", "For the first time, no wild-type polio cases have been recorded in Africa for more than 12 months [7].", "Poliomyelitis due to wild-type poliovirus type 2 has not been documented since 1999 and poliovirus type 3 since 2012.", "In anticipation of the planned withdrawal of poliovirus type 2 from OPV, the World Health Organisation (WHO) recommends that all children receive at least one IPV dose in order to maintain immunity to poliovirus type 2 [1].", "In addition, the WHO recommends that an all-IPV schedule be considered in countries with high vaccine coverage and a low risk of importing wild virus [1].", "In China, the last case of domestic wild-type poliomyelitis was reported in 1994 and the country was certified as polio-free by WHO in 2000.", "Since then, imported wild-type poliovirus outbreaks have been infrequently reported [8].", "However, several outbreaks of vaccine-derived poliovirus infections have been reported during the last decade [2,3].", "These outbreaks and the continuing risk of VAPP in vaccinees highlight the need to consider the risks associated with continued OPV use in the national poliomyelitis immunisation policy.", "The Chinese poliomyelitis immunisation schedule comprises 3 doses of OPV at 2, 3 and 4 months of age, with one booster dose at 4 years of age.", "We conducted four clinical trials (two pilot safety studies: A and B, and a large randomised controlled study: C and D (extension of study C) to assess the immunogenicity, reactogenicity and safety of IPV when administered for primary vaccination according to the Chinese immunisation schedule, and as a booster in the second year of life.", "The study protocols and associated documents were reviewed and approved by the Guangxi Institutional Review Board.", "The studies were conducted in accordance with the Declaration of Helsinki, Good Clinical Practice principles and all applicable regulatory requirements.", "Written informed consent was obtained from each subject's parent/legally acceptable representative prior to enrolment.", "Study A was conducted at the Cangwu Centre for Disease Control and Prevention, Longxu town, Cangwu County, Wuzhou City, Guangxi Province.", "Study B was conducted at the Wuzhou Centre for Disease Control and Prevention, Wuzhou, Guangxi.", "Study C and D were conducted in two centres: the Cangwu Centre for Disease Control and Prevention in Wuzhou City and the Mengshan Centre for Disease Control and Prevention, Mengshan Town, Mengshan County.", "Studies A, B and C (NCT00937404, NCT01021293 and NCT00920439, respectively) were conducted between 04 August 2009 and 05 July 2010.", "Study D (NCT01323647) was conducted between 23 April 2011 and 19 September 2011.", "Two open, single group, pilot safety studies were conducted to assess the safety and reactogenicity of IPV when administered as a 3-dose primary vaccination in infants (Study A) and as a booster dose in toddlers primed with three doses of OPV (Study B) (Table 1).", "The sample sizes were chosen to provide at least 20 evaluable subjects, as required by the Chinese Regulatory authority guidelines [9].", "Study C was a randomised, controlled trial to assess the immunogenicity, safety and reactogenicity of IPV when administered in a 3-dose primary vaccination schedule.", "The primary study objective was to demonstrate non-inferiority of IPV as compared to OPV in terms of the immune response to poliovirus types 1, 2 and 3 one month after the third vaccine dose (Table 1).", "A randomisation list was generated at GSK Vaccines, Belgium and was used to number the vaccines.", "Treatment allocation at the investigator site was performed using a central, web-based randomisation system.", "A blocking scheme ensured that balance between treatments (1:1 ratio) was maintained.", "The randomisation algorithm used a minimisation procedure accounting for centre [10].", "Subjects vaccinated in Study C were invited to return at 18–24 months of age to participate in Study D in order to investigate antibody persistence after primary vaccination with IPV or OPV (Control group).", "Study D also assessed the immunogenicity, safety and reactogenicity of a booster dose of IPV administered to children who had received three priming IPV doses in Study C.", "Participants in Study A and C were healthy infants between 60 and 90 days of age and born with a gestational age of 36 to 42 weeks.", "Participants in Study B and D were healthy toddlers 18 to 24 months of age.", "Toddlers participating in Study B had received three priming doses of OPV in the first year of life as per Chinese recommendations.", "Toddlers participating in Study D had received primary vaccination in Study C.", "Infants and toddlers were excluded from participation if they had evidence of previous or intercurrent poliomyelitis disease or vaccination (other than the doses specified in the protocol of the booster studies).", "Children were excluded if they had a history of seizures or progressive neurological disease, any immunosuppressive condition, a history of allergic reactions likely to be exacerbated by any vaccine component, or major congenital defects or serious chronic illness.", "Administration of a vaccine not foreseen by the study protocol was not permitted within 30 days prior to vaccination, nor was its planned administration during the study period; with exception of combined diphtheria-tetanus-pertussis (DTP), Haemophilus influenzae type b (Hib) conjugate vaccine and hepatitis B vaccines.", "Children were excluded if they had received ≥14 days of immunosuppressants or other immune-modifying drugs since birth, immunoglobulins and/or any blood products since birth or their planned administration during the study period.", "Each dose (0.5 ml) of IPV contained 40 Dalton (D) antigen units of inactivated poliovirus type 1, 8 D antigen units of inactivated poliovirus type 2 and 32 D antigen units of inactivated poliovirus type 3, with 2-phenoxyethanol as preservative.", "One IPV vaccine lot (AIPVB021B) was used for study A and B and a second lot (AIPVB023C) was used for Studies C and D. IPV was administered intramuscularly into the thigh using a 25 gauge needle of at least 1 inch (2.54 cm) length.", "The Chinese OPV vaccine (lot 20090401, administered to the Control group in Study C) was cultured on Monkey Kidney Cells.", "Each dose (2 drops, 0.1 ml) contained a total amount of live virus of ≥106.15 Median Cell Culture Infective Dose (CCID50), with ≥106.0 CCID50 poliovirus type 1, ≥105.0 CCID50 poliovirus type 2 and ≥105.5 CCID50 poliovirus type 3.", "Primary vaccination with IPV or OPV was administered at 2, 3 and 4 months of age (Studies A and C).", "Booster vaccination with OPV is not recommended under the Chinese vaccination schedule until 4 years of age.", "Booster vaccination with IPV was administered at 18–24 months of age in Studies B and D. Children in study D also received combined diphtheria-tetanus-acellular pertussis and Hib vaccine (DTPa/Hib, Infanrix™Hib, GSK Vaccines, Belgium), co-administered at separate sites to IPV in the IPV group, and administered alone to the Control group.", "The occurrence of redness, swelling, pain at the injection site, drowsiness, fever (axillary temperature >37.0 °C), irritability/fussiness and loss of appetite that occurred within four days (day 0–3) after each dose were recorded on diary cards.", "Symptoms were graded on a 3-point scale where grade 3 (severe) was defined as redness or swelling >30 mm, fever >39.0 °C, ‘cried when limb was moved/spontaneously painful’ for pain, drowsiness that ‘prevented normal activity’, ‘crying that could not be comforted/prevented normal activity’ for irritability/fussiness, and ‘did not eat at all’ for loss of appetite.", "The occurrence of gastrointestinal symptoms was additionally solicited in Study C. Grade 3 gastrointestinal symptoms (nausea, vomiting, diarrhoea and/or abdominal pain) were defined as ‘preventing normal activity’.", "All other adverse events including events that required medical attention (defined as hospitalisation or an unscheduled visit to/from medical personnel, including emergency room visits) were recorded for each participant for 30 days after each vaccination (31-day follow up).", "Serious adverse events (SAEs) were captured from the first vaccination until one month after the last study vaccine dose.", "Blood samples were collected from a subset of children (the first 316 vaccinated in each group) before the first dose and one month post-dose 3 in Study C, from all children prior to booster vaccination in Study D, and from all children in the IPV group in Study D one month after the booster dose of IPV.", "Serological assays were performed at the Chinese National Institute for Food and Drug Control laboratory in Beijing.", "Anti-poliovirus type 1, 2 and 3 antibodies were measured using a virus micro-neutralisation test adapted from the WHO Guidelines for WHO/EPI Collaborative Studies on Poliomyelitis [11].", "The lowest dilution tested was 1:8.", "Titres were expressed in terms of the reverse of the 50% inhibitory dose.", "An antibody titre ≥8 was considered seroprotective.", "Statistical analyses were performed using SAS® version 9.1 or later on Windows XP Professional, and StatXact-7.0 or later procedure on SAS.", "The analyses of safety were performed on the Total vaccinated cohorts, which comprised all children who had received at least one dose of the study vaccine.", "The analysis of immunogenicity was conducted on the According-to-protocol (ATP) immunogenicity cohort who comprised all eligible children who complied with protocol-defined procedures and for whom data concerning the immunogenicity endpoint measures were available.", "The ATP persistence cohort in Study D included all children who had completed their full 3-dose primary vaccination course in Study C, who had not received non-study polio vaccination and for whom serological results were available at the persistence time point.", "Seroprotection rates with exact 95% confidence intervals (CI) and geometric mean titres (GMTs) with 95% CIs were calculated for each of the three poliovirus antigens at each time point.", "Antibody titres below the cut-off of the assay were given an arbitrary value of half the cut-off for the purpose of GMT calculation.", "One month after dose 3 in Study C, the standardised asymptotic 95% CIs for the group difference in seroprotection rates (Control group minus IPV group) were computed.", "As agreed with the Chinese Regulatory Agency, non-inferiority was concluded if the upper limit of the 95% CI on the difference (Control group minus IPV group) in the percentage of seroprotected subjects was ≤10%.", "In exploratory analyses, the 95% CIs for the GMT ratio between groups (Control group over IPV group) for each of the three poliovirus antigens were computed.", "In Study C the analysis used an ANCOVA model including the vaccine group as fixed effect and the log-transformed pre-vaccination titre as the co-variable.", "The ANCOVA model was selected because infants received polio vaccination for the first time, and the ANCOVA method allowed adjustment for potentially variable pre-vaccination titres (‘adjusted GMTs’).", "In Study D, an ANOVA model on the logarithm10 transformation of the titres prior to the booster dose was used as all subjects had receive primary vaccination against poliovirus.", "Potential differences were highlighted if the 95% CI for the GMT ratio between groups did not contain the value ‘1′.", "Potential differences should be interpreted with caution as no adjustment for multiplicity for these comparisons was accounted for in the planning of the exploratory analyses.", "To assess the impact of missing data due to children lost to follow-up on the results of Study D, a sensitivity analysis was performed using a general linear mixed model.", "In order to meet Chinese Regulatory guideline requirements for 20 subjects, 25 infants and toddlers were enrolled in the pilot studies (Study A and B).", "With 284 children in the ATP immunogenicity cohort of each group in Study C, the study had 91% power to reach the primary objective assuming 90% seroprotection for each poliovirus type in the Control group, with alpha equal to 2.5%.", "Assuming that approximately 80% of these children participated in the extension study and that 10% would be non-evaluable, the expected 95% CI around a post-booster seroprotection rate of 97.0% in 395 children would be (94.8; 98.4).", "25 children received at least one primary vaccination dose of IPV in Study A, and 25 received a dose of IPV at 18–24 months of age in Study B, after OPV priming according to the Chinese recommended schedule (Fig. 1).", "Pain at the injection site was the most frequently reported solicited local symptom after primary vaccination (Study A), reported in 12.0% of children (Fig. 2).", "Irritability/fussiness was the most frequently reported solicited general symptom (56% of children).", "After the booster dose (Study B), the most frequently reported solicited local and general symptoms were redness (20.0% of children) and fever (24.0% of children).", "No grade 3 local or general symptoms were reported in either study.", "At least one unsolicited symptom during the 31-day (day 0–30) follow-up period after vaccination was reported in 60% of children in Study A and 40% of children in Study B. No grade 3 unsolicited symptoms and no SAEs were reported in either study.", "There were 1100 children who received primary vaccination with IPV or OPV in Study C.", "In Study D, a booster dose of IPV was administered to 470 children primed with IPV in Study C (Fig. 1).", "There were a similar number of males and females in the IPV and Control groups (Table 2).", "Pain at injection site was the most frequently reported solicited local symptom after primary vaccination with IPV, reported in 20.5% of children in Study C (Fig. 2).", "Grade 3 pain was reported in 0.5% of children and grade 3 redness and swelling in 0.2% of children.", "Irritability/fussiness was the most frequently reported solicited general symptom in both groups (44.4% in the IPV group and 39.3% in the Control group) (Fig. 2).", "The most frequently reported grade 3 symptom in both groups in Study C was irritability, reported in 1.8% and 1.6% of children in the IPV and Control group, respectively.", "Grade 3 fever was uncommon (0.4% of children in the IPV group and 0.5% in the Control group).", "At least one unsolicited symptom during the 31-day (day 0–30) follow-up period after vaccination was reported in 28.2% (155/550) of children in the IPV group and 29.5% (162/550) in the Control group.", "Grade 3 symptoms were recorded in 0.7% (4/550) of children in the IPV group (bronchitis, nasopharyngitis, upper respiratory tract infection) and 0.5% (3/550) in the Control group (bronchitis, upper respiratory tract infection).", "None of the grade 3 symptoms were considered by the investigator to be causally related to vaccination.", "At least one symptom (solicited or unsolicited) that required medical attention was reported in 8.5% (47/550) of children in the IPV group and 8.0% (44/550) of children in the Control group.", "12 SAEs were recorded in nine children in Study C: three children in the IPV group (diarrhoea, herpes zoster, hydrocephalus) and six children in the Control group (bronchopneumonia, epilepsy, upper respiratory tract infection, bronchitis, enteritis and in one child, bronchopneumonia with abdominal distension, cardiac failure and respiratory failure).", "There were no fatal events in any study.", "None of the SAEs were considered by the investigator as causally related to vaccination, and all had resolved by the end of the study.", "After the booster IPV dose, the most frequently reported solicited local symptom was pain (10.5%) with a maximum intensity of Grade 1 for 33 (7.1%) subjects (Fig. 2).", "With the exception of fever, solicited general symptoms appeared to be reported less frequently after the booster dose than after primary vaccination (Fig. 2).", "The most frequently reported solicited general symptom after the IPV booster was fever (33.4% of children).", "Grade 3 fever was reported for eight children (1.7%).", "At least one unsolicited symptom during the 31-day (day 0–30) follow-up period after vaccination was reported in 4.7% (22/470) of children in the IPV group.", "One grade 3 symptom (rash), reported in one child (0.2%), was considered by the investigator to be causally related to vaccination.", "At least one symptom (solicited or unsolicited) requiring medical attention was recorded in 6.4% (30–470) of children.", "No SAEs were reported after booster vaccination with IPV in Study D.", "One SAE (fever), reported for a child in the Control group, was considered by the investigator as causally related to DTPa/Hib vaccination.", "Non-inferiority of the immune response elicited by IPV versus Chinese OPV vaccine was demonstrated according to the pre-specified statistical criteria: the upper limit of the standardised asymptotic 95% CI on the group difference for the percentage of seroprotected subjects was <10% for all poliovirus types (Table 3).", "One month after the third dose, 100% of children in the IPV group and at least 98.3% in the Control group had seroprotective antibody titres for each poliovirus type (Table 4).", "The anti-poliovirus GMTs were 30 to 300 times higher than the seroprotection cut-off in the two groups.", "Exploratory analyses suggested that the anti-poliovirus type 1 and 2 GMTs were higher in the Control group than the IPV group, and were higher in the IPV group for poliovirus type 3 (Table 3).", "At 18–24 months of age the percentage of children with seroprotective antibody titres was at least 94.7% for each poliovirus type in the IPV group, and at least 96.1% in the Control group (Table 4).", "Exploratory analyses continued to indicate that persisting anti-poliovirus type 1 and 2 GMTs were higher in the Control group than the IPV group, and were higher in the IPV group for poliovirus type 3.", "Results of the sensitivity analysis correcting for subjects lost between the primary study and the persistence time point showed that GMT ratios calculated using the model were similar to the observed GMT ratios for all three polioviruses (data not shown), indicating that subjects lost between the primary and the booster study did not affect the conclusions of Study D.", "One month after the booster vaccination all children in the IPV group had seroprotective antibodies for each poliovirus.", "Antibody GMT increased by at least 22-fold for each poliovirus, and post-booster GMTs were higher than those observed one month post-primary dose 3.", "Three primary doses of IPV were immunogenic in children vaccinated at 2, 3 and 4 months of age, with responses that were non-inferior to Chinese OPV in terms of seroprotection.", "The majority of children continued to have seroprotective antibodies for all three poliovirus types at 18–24 months of age, suggesting that IPV induced durable immunity.", "A booster dose of IPV elicited seroprotection in all subjects, as well as marked increases in GMTs consistent with an immune memory response.", "We observed lower GMTs, but not seroprotection rates, for poliovirus types 1 and 2 in children who received primary vaccination with IPV compared to OPV.", "Lower GMTs were also observed following IPV-containing than OPV-primary schedules in a recent study in Chinese infants [12].", "Similar trends were observed in studies conducted using other IPV vaccines (alone or in combination) and schedules in other countries including the United States [13].", "This difference is likely to be of little clinical importance in view of the high percentages of children who achieved the internationally-accepted seroprotective threshold [14], the large increases in titres observed after the booster dose and because the GMTs are 30 to 300 times the assay cut-off.", "IPV was well tolerated with few grade 3 local or general symptoms reported after vaccination.", "In the controlled Study C, the occurrence of solicited general symptoms appeared to be similar in the IPV and OPV groups.", "The incidence of fever was higher after the booster dose of IPV than after primary vaccination, but grade 3 fever (>39 °C) was uncommon.", "Study C and D provide IPV immunogenicity and safety data in a large cohort of children compared with the recommended Chinese OPV vaccination schedule.", "A potential limitation of the studies is that safety of an IPV booster after OPV priming was only assessed in a small cohort in pilot Study B, and immunogenicity of an IPV booster after OPV priming was not evaluated.", "However, the immunogenicity and safety of IPV after OPV is well established, and at least one dose of IPV after OPV priming is recommended by WHO [1].", "The current Chinese schedule recommends a booster dose of OPV at 4 years of age.", "In view of the somewhat lower titres achieved after primary vaccination with IPV compared to OPV, as well as the robust booster responses observed in our study after the 18–24 month IPV booster dose, administration of the IPV booster dose in the second year of life will help to ensure durable immunity against poliovirus in an all-IPV schedule.", "IPV has successfully controlled poliomyelitis in countries where its continuous and exclusive use has occurred; such as Iceland, Sweden, Finland and the Netherlands.", "In the Netherlands and Sweden, importation of wild-virus and occurrence of wild type polio in unvaccinated religious groups has been successfully contained, demonstrating herd effects of IPV [15,16].", "Importation of poliovirus type 1 to Israel (a sub-tropical country that has used IPV exclusively since 2005) in 2013 resulted in no cases of poliomyelitis, but evidence of transmission with virus detected from environmental and stool samples [17].", "The long-term potential for continued poliovirus transmission in settings of high faecal-oral transmission or in sub-tropical and tropical settings where IPV is implemented exclusively is not known.", "The role of IPV in poliovirus control will continue to increase as the world moves towards eradication [18].", "This is reflected in WHO guidelines that now recommend at least one IPV dose be administered to all children, and by the growing number of countries transitioning from OPV to an all-IPV schedule [1].", "As yet, success of IPV in preventing poliovirus disease and transmission in developing countries and tropical settings has not been demonstrated.", "The results of four studies in infants and toddlers suggest that IPV is immunogenic with a clinically acceptable safety profile when administered at 2, 3, 4 and 18–24 months of age IPV could feasibly be incorporated into Chinese vaccination schedule with the advantage of eliminating the risk of VAPP and vaccine-derived poliomyelitis outbreaks.", "Poliorix and Infanrix are trademarks of the GSK group of companies", "This study was sponsored and funded by GlaxoSmithKline Biologicals S.A. GlaxoSmithKline Biologicals S.A. was involved in all stages of the study conduct and analysis and also took charge of all costs associated with the development and the publishing of the manuscript.", "MH is a freelancer contracted through Chiltern International, a for-profit company, by GSK to undertake this research.", "SK, OVM, KH and SR-G are employees of GSK group of companies and OVM and KH declare having GSK stocks.", "CGL reports having received a grant from GSK for undertaking serum testing as part of this study.", "Y Li, Y Liu, R–CL, HZ and XC have no conflicts to declare." ]

[ "IPV safety and immunogenicity were assessed in the Chinese vaccination schedule.", "IPV was non-inferior to Chinese OPV in terms of seroprotection rates.", "IPV had a clinically acceptable safety profile in four studies.", "IPV could feasibly be incorporated into Chinese vaccination schedule." ]

[ "ENGI", "MATE" ]

[ "Drug delivery system", "Electrohydrodynamic", "Mathematical modelling", "Progesterone" ]

[ "2210", "2211", "2500" ]

[ "In the development of oral formulations, the aqueous solubility of the drug plays a key role in the extent of drug absorption [1].", "For any therapeutics to be successfully absorbed in the body, it needs to be present in the form of an aqueous solution at the desired site of action [1,2].", "The solubility is the limiting factor for a drug to attain the desired concentration in systemic circulation in order to achieve pharmacological response for an orally administered drug [3].", "However, many drug molecules under development are poorly soluble: that critically limits their absorption, resulting in poor bioavailability and pharmacokinetics in vivo [2,3].", "Progesterone is an endogenous steroidal hormone that is involved in all aspects of reproduction and used to control reproductive function.", "It is being used for hormone replacement therapy (HRT) in menopausal women, who failed to ovulate and stop producing progesterone from their ovaries [4], this would drop down the hormone levels in the body, hence results in various effects such as hot flashes and night sweats.", "In addition, recent studies have reported the applicability of progesterone in preventing preterm birth.", "This is defined by World Health Organization as birth before 37 weeks of pregnancy are completed [5], which is the largest cause of neonatal morbidity and mortality in infants [6,7].", "Progesterone has an essential role in maintaining pregnancy [8,9], and it is involved in establishing uterine quiescence [10].", "The various administration routes of progesterone include oral, parental (intramuscular and subcutaneous) and topical (as a cream of vaginal gel), of which peroral delivery is the most preferred route of administration.", "However, progesterone suffers from a short half-life and low water solubility; these lead to inconsistent bioavailability and high first pass metabolism [11].", "Biodegradable polymers are promising candidates for oral drug delivery with their ability to shield the drug from external harsh chemical and enzymatic degradation of the gastrointestinal tract (GIT) as well as bypass the first pass metabolism through their unique uptake mechanism [12,13].", "A previous study reveals that the dissolution rate of poorly water-soluble drug can be enhanced by incorporating it into a polymeric matrix thereby inducing its amorphous state [14], again making this a preferable oral delivery technique.", "In addition to this, long term sustained drug delivery systems can also be obtained using biodegradable particles, in which progesterone can be administered via depot injection [15] and released for a prolonged period of time.", "In a depot injection, the medication is deposited in a localized mass via intramuscularly, subcutaneously or intradermal injection.", "This offers advantages over long-term daily injection which can compromise patient compliance.", "Biodegradable microparticles in the field of controlled drug release, where the drug is continuously released from the particles within a period of time, have generated immense interest.", "They are able to deliver a wide range of drugs through various administration routes and enhance drug bioavailability and protect the drug from degradation [16].", "The drug release behavior can be controlled by altering polymer compositions including molecular weight, monomers ratio and the nature of terminal groups for copolymers [17].", "It has been reported that other than the physicochemical properties of polymers, factors such as particle size [18,19], morphology and porosity [20], as well as drug loading [21] also play important roles in deciding the drug release kinetics.", "Appropriate selection of the biodegradable polymeric materials is essential to develop a successful particulate delivery system.", "Among the variety of materials available to fabricate these solid particles, copolymers have received much attention in recent years as different physicochemical properties can be achieved by altering their polymer composition.", "Poly (d, l‑lactic‑co‑glycolic acid) (PLGA) is one of the most frequently used biodegradable polymers in drug delivery applications [22,23].", "PLGA is approved by the FDA as a drug delivery vehicle and it preferred for this purpose because of its favorable biodegradability, biocompatibility and tunable physicochemical properties [24,25].", "The copolymers with various molecular weights and copolymer compositions are commercially available.", "The ability to alter lactide: glycolide ratio, molecular weight and the nature of terminal groups enables precise control over drug release profile [25].", "In general, therapeutics can be orally released in a controlled manner via transport through the polymer or be triggered in response to environmental stimuli or released during polymer dissolution [26].", "In addition, the bioavailability of hydrophobic drugs can be improved due to the hydrophilic property of PLGA, thus enhancing oral bioavailability [27,28].", "Polymeric micro- and nanoparticles can be administered as oral, injectable and inhalable drug delivery systems, and can be fabricated using various methods including solvent evaporation [29], emulsion [30,31], spray-drying [32,33], porous glass membrane emulsification [34] and coacervation [35].", "However, most of these conventional approaches suffer from some limitations such as broad particle-size distribution, low drug encapsulation efficiency [36], particle agglomeration and difficulties for incorporation of hydrophilic drugs [30,37].", "Moreover, non-degradable surfactants are employed in some of the approaches which might cause unwanted effects in pharmaceutical applications.", "In addition, for highly sensitive and active pharmaceutical ingredients, inactivation or degradation is possible owing to high temperature, organic solvent exposure and high shear stress during the particle fabrication process [31,38].", "Electrohydrodynamic atomization (EHDA), also known as the electrospray technique, is believed to overcome the drawbacks associated with conventional techniques and has been deliberately chosen as a method for the fabrication of bioactive molecule loaded micro and nanostructured materials in the application of drug delivery [39–41].", "In this method, drug molecules are firstly incorporated into the polymer solution and infused through a capillary nozzle.", "A high voltage is applied to atomize the solution and the electric charge generated on the droplet, competing with the surface tension and resulting in near-monodisperse microdroplets in the cone-jet mode and further solidify to form microspheres in a single-step process.", "EHDA also has the potential to avoid particle aggregation due to self-dispersing of the droplets owing to Coulombic repulsion [42].", "Moreover, electrospraying has the advantage of high drug encapsulation efficiency [43] and can enable encapsulation of both hydrophobic and hydrophilic drugs [28,39].", "In this work, electrospraying was utilized to produce progesterone loaded PLGA particles.", "Many studies have investigated the formulations of PLGA particles for controlled release of different pharmaceuticals [16,18,25,43,44] and encapsulated hydrophobic drugs within different polymer matrices [11,27,28].", "Progesterone-loaded PLGA microparticles have been prepared by several researchers by use of different techniques, including microfluidic [26], hydrogel template [45] and solvent evaporation-based methods [46].", "However, there is a lack of systematic reports on applying single step electrospraying in producing progesterone-loaded particles, specifically using PLGA copolymer with different monomers ratios and concentrations which can provide different drug release profile in order to meet various requirements of drug delivery.", "Given the hydrophobicity of progesterone and that oral delivery is the most preferred route of administration, a particular challenge and objective was to enhance its dissolution by incorporation of the drug into the PLGA matrix.", "The overall objective of this study was to carry out a systematic investigation on the effects of the electrospraying technique, polymer concentration and use of two different copolymer compositions (PLA/PGA ratio of 50:50 and 75:25) on the resulting particle properties, and release behavior (in vitro) using progesterone.", "PLGAs with different copolymer ratios (1.", "PURASORB PDLG 5002A, 50:50 DL-lactide/glycolide copolymer, molecular weight of 17,000 Da, inherent viscosity 0.2 dl g−1; 2.", "PURASORB PDLG 7502A, 75:25 DL-lactide/glycolide copolymer, molecular weight of 17,000 Da, inherent viscosity 0.2 dl g−1) was obtained from Corbion (Amsterdam, The Netherlands).", "Progesterone (molecular weight of 314.46 g mol−1) and N, N‑Dimethylacetamide (DMAc, anhydrous, 99.8%) were obtained from Sigma-Aldrich (Poole, UK).", "Progesterone-loaded PLGA particles were produced using electrospraying (Fig. 1).", "PLGA solutions with different concentrations (2 and 4 wt%) and copolymer formulations (50:50 and 75:25) were prepared by dissolving the polymer in DMAc at ambient temperature (20°C).", "Progesterone (2 mg/ml) was added to polymer solution at each concentration to make polymer/drug ratios of 20:1 and 10:1.", "PLGA solutions with no drug (2 and 4 wt%; 50:50 and 75:25) were prepared as a control study.", "The polymer solutions were loaded into a 10 ml plastic syringe and fed into a stainless-steel needle with internal diameter of 0.84 mm at a constant flow rate implemented by a syringe pump (Harvard PHD 4400, Edenbridge, UK).", "A high positive electrical potential varying from 12.0 to 16.0 kV relative to the ground was supplied using a high voltage power supply (Glassman Europe Ltd., UK).", "Once the droplets were ejected from the stable cone-jet, particles were collected at a working distance of 200 mm below the needle exit, either on a glass microscope slide, or a grounded collector.", "The cone-jet formed at the tip of the needle during particle production was monitored using a Leica DMS300 camera.", "All experiments were conducted at ambient temperature (20 °C) and relative humidity of 40–55%.", "Each experiment was repeated 3 times.", "The density (ρ) was measured using a standard density bottle (DIN ISO 3507-Gray-Lussac).", "Surface tensions (γ) of all the solutions were determined using Wilhelmy's plate method using a Kruss tensionmeter (Tensiometer K9, Hamburg, Germany).", "The viscosity (μ) was estimated using a U-tube viscometer (VWR, size E).", "The electrical conductivity (κ) was measured using a conductivity probe (Jenway 3540 pH/conductivity meter).", "The characterization of all solutions was undertaken at ambient temperature.", "The mean value and standard deviation of ten readings is reported.", "In order to investigate how PLGA copolymer ratio and concentration influence the size, morphology and eventually the release profile of the particles, four solutions with various PLGA concentrations (2 and 4 wt%), different copolymer ratios (50:50 and 75:25) were prepared.", "Progesterone was added to each solution to make four more solutions incorporating the drug.", "The measured values for these parameters are summarized in Table 1.", "Particles were collected on glass slides and initially examined using an optical microscope (Nikon Eclipse ME 600) fitted with a camera (Micropublisher 3.3 RTV, 3.3 megapixel CCD Color-Bayer Mosaic, Real Time Viewing camera; Media Cybernetics, Marlow, UK).", "Particles were further observed using a field emission scanning electron microscope (SEM; Hitachi) for particle size and morphology analysis.", "The micrographs were taken from different areas for each sample and 300 particles were randomly chosen to measure both width and length using ImageJ (Brocken Symmetry Software).", "To measure the zeta potential of particles, a dilute suspension of particles was prepared in distilled water.", "The zeta potential was estimated with ZetaPlus™ zeta potential analyzer (Brookhaven Instruments, Holtsville, NY) at room temperature on the basis of electrophoretic mobility under an electric field.", "For each formulation the mean value of 5 measurements were taken.", "XRPD patterns of samples were analyzed.", "The X-rays are generated by a cathode ray tube filtered to produce monochromatic radiation directed towards the sample.", "Cu Kα radiation was used with wavelength 1.5418 Å and graphite monochromatic filtering wave at a tube voltage of 40 mV and tube current of 15 mA.", "The interaction of the incident rays with the sample produces constructive interference (and diffracted rays).", "The diffracted intensity was recorded in the 2θ angle range from 5 to 40° at a scanning speed of 0.02°/min.", "The infrared spectra of two PLGA formulations, progesterone and drug-loaded particles, were recorded using a Fourier Transform Infrared spectrophotometer.", "A small quantity of each sample was placed onto the sample holder and scanned in the frequency range of 400–4000 cm−1.", "To investigate the release profile of progesterone-loaded particles, in vitro dissolution studies were conducted in phosphate-buffered saline (PBS) with pH 7.4.", "One compressed phosphate-buffer saline tablet (PBS, yields 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4 at 25 °C, Sigma-Aldrich, UK) was dissolved in 200 ml of deionized water.", "22 mg of progesterone-loaded particles were encapsulated in a dissolvable polymer capsule.", "The solubility of 2 mg of pure progesterone was investigated as a control group under the same release conditions in order to investigate how solubility of poorly water-soluble progesterone can be enhanced after electrospraying in the PLGA matrix.", "The polymer capsule was then put in a stainless-steel sinker basket.", "The sinker basket was subsequently submerged into 100 ml PBS and the temperature of the solution was kept constant at 37 °C.", "At predetermined time intervals, samples of 3 ml were withdrawn from each solution and immediately replaced with fresh PBS already stored at 37 °C.", "Dissolution studies were also conducted on pure progesterone under the same protocol as the control group.", "The samples were filtered through a membrane (0.22 μm) to filter any impurities and avoid any interference in absorbance.", "Three replicates of each sample were analyzed for drug concentration using UV–visible spectrophotometer at 224 nm to calculate the drug percentage.", "A calibration equation to calculate the progesterone concentrations of unknown samples was produced by measuring the absorbance of different concentrations of progesterone from 1 to 20 μg/ml.", "The release studies were conducted over 7 days and repeated three times for each set of PLGA particles and the results of cumulative amount released was plotted as a function of time.", "The release of drugs from PLGA particles is known to be a complicated process, involving both diffusion of the drug out of the particles and inward diffusion of water leading to modification of the PLGA.", "Detailed mathematical models of these processes have been set up [47,48], and although they can be applied quite generally they have most often been used with perfect sink boundary conditions, that is, the drug concentration at the surface has been set to zero.", "In the experiments reported here the drug solubility is low, and the solution in the container is saturated after about 120 min and remains approximately constant.", "In the saturation regime the rate of release of the drug will be determined by the intervals between sampling the solution and replenishing it rather than by the rate at which the drug can escape from the particles.", "Four different formulations of drug-loaded particles and PLGA particles were successfully prepared using electrospraying under different operating conditions (Table 2).", "The model drug, progesterone, was encapsulated in PLGA polymers by co-dissolving in DMAc before electropraying into fine droplets.", "The effects of PLGA concentration and copolymer composition were evaluated by particle characterization and drug-release behavior in this study.", "Other important parameters such as applied voltage and solution flow rate were optimized to achieve a stable cone-jet and uniform size distribution.", "Particles were produced both with and without progesterone to investigate the effects of polymer/drug ratio and copolymer formulation of PLGA on particle size and morphology.", "Initially, drug free particles were produced as the control system using PLGA with two copolymer ratios (50:50 and 75:25), and for each copolymer formulation, particles were prepared using PLGA concentrations of 2 and 4 wt% (F5, F6, F7 and F8, respectively).", "Drug free particles were generated at constant flow rate of 3 μl/min and the voltage required to form a stable cone jet was between 13.0 and 16.0 kV.", "The particles size and morphology were studied using SEM, and the representative SEM images of each formulation are shown in Fig. 2.", "In general, all particles were produced with a smooth outer surface.", "The results showed that both concentration and copolymer ratio of the polymer influenced the diameter of progesterone-free and progesterone-loaded particles.", "Larger particles were produced with higher concentrations of PLGA while the outer surface of the particles stayed smooth, which can be attributed to the higher viscosity of the 4 wt% polymer solutions (values are shown in Table 1).", "The copolymer ratio and addition of drug were found to have no significant influence on particle size.", "SEM images (Fig. 2) showed that the solution from PLGA copolymer ratio of 50:50 for both polymer concentrations resulted in formation of spherical particles, while solutions from PLA: PGA copolymer ratio of 75:25 produced elongated or irregular particles.", "In Eqs. (5), (6), and (7) [50] d is the particle diameter (m), c is a constant, Q the liquid flow rate (m3 s−1), ρ the liquid density, I the current, ε0 the permittivity of vacuum, γ the liquid surface tension in ambient air, and κ the liquid electrical conductivity.", "In order to investigate the effect of copolymer formulation on progesterone release profile, particles obtained from the same polymer concentration are required to have similar dimensions.", "Thus, flow rate was kept constant at 2 μl/min in order to achieve comparable particle size distribution.", "The diameter of the particles obtained from all formulations are in the range 0.72 and 1.66 μm and presented in Table 2, however note well that some are oblong in shape.", "Zeta potential is an important characteristic of polymeric particles, it is a measurement of surface charge of the particles in a suspension.", "In a sense, zeta potential can influence particle stability, as the absolute value of zeta potential increases the amount of charge on the particle surface.", "This results in larger electrostatic repulsive interaction between particles which prevents the aggregation of the particles and leads to the formation of more stable particles.", "A physically stable particle suspension should have a minimum zeta potential of ±30 mV [51].", "The zeta potentials of all the formulations ranged between about −30 mV and −50 mV.", "It showed slight increases in negativity with an increase in polymer concentration and addition of drug.", "PLGA composition was found to have no significant influence on zeta potential.", "The physical form of the drug, polymer and drug-loaded particles were tested using XRPD and the patterns are shown in Fig. 3.", "The diffractogram of progesterone shows strong characteristic peaks at 2θ values of 10.48, 12.64 and 16.9 degrees, which indicate that it is crystalline in its pure form.", "Both PLGA diffractograms have the characteristics of amorphous material.", "Electrosprayed particles in all formulations were tested, and no crystalline peaks were observed.", "A possible explanation for this is that progesterone is molecularly dispersed within the PLGA polymeric matrix and almost complete amorphization was achieved.", "FTIR spectra were used to detect whether there was any interaction between PLGA and progesterone during the electrospray process.", "FTIR spectra of progesterone, PLGA polymers (75:25 and 50:50) and progesterone-loaded particles with different polymer/drug ratio are shown in Fig. 4.", "The pure progesterone sample exhibited characteristic peaks including bound OH (2800–3000 cm−1) and CO stretching (1600–1750 cm−1).", "PLGA polymers showed vibration CO stretching at 1752 cm−1 and CO stretching at 1000–1250 cm−1.", "Additionally, weak peaks for CO stretching were observed between 1600 and 1750 cm−1 in four different progesterone-loaded formulations, suggesting the presence of intact progesterone.", "Thereby, the FTIR results indicate that there were no chemical interactions between polymer and progesterone.", "Copolymer ratio is one of the key factors affecting the behavior of drug release.", "By manipulating the copolymer ratio, the release kinetics of the drug can be controlled.", "Fig. 5 shows cumulative percentages of released progesterone against time plots of electrosprayed microparticles from four formulations.", "Narrow error bars indicate good reproducibility.", "Progesterone release from all four formulations showed a biphasic release pattern.", "Phase I in this biphasic release profile is usually described as initial burst release, which can be due to three reasons: first, drugs that are freely and weakly bound on the particle surface, second, drug release from the pores and cracks of particles with various morphology [52] and third, drug diffuses out from the polymeric matrix.", "Later in the modelling analysis, phase I can also be described as diffusion dominated release with a reasonable value for diffusion constant.", "Phase II is predominately governed by drug diffusion, either through water-filled pores or polymer.", "The release study showed increase in PLA:PGA ratio from 50:50 to 75:25 decreased the rate of progesterone release.", "Drug-loaded PLGA particles of lower PLA:PGA ratio for both polymer concentrations (50:50 2 and 4 wt%) cumulatively released 862 and 792 μg in 2 h, respectively.", "On the other hand, higher copolymer ratio (75:25 2 and 4 wt%) PLGA particles showed only 741 and 654 μg cumulative release in 2 h, respectively.", "For diffusion-controlled drug release, water-uptake and swelling ability of polymer will potentially affect the diffusion rates within the polymer matrix.", "Increase in release rate on decreasing PLA:PGA ratio can be attributed to subsequent increase in hydrophilicity.", "Water-uptake and polymer hydration occurs immediately upon immersion in water or administration in vivo [53].", "Lower PLA:PGA ratio (PLGA 50:50 vs. 75:25) is considered to be more hydrophilic and thus water absorption rate might increase to a greater extent [54].", "This action has been found to be a pore-forming process and leads to an increase in drug diffusion [55,56].", "Although, these pores are not big enough for drug transport in the early stage of drug release, a porous network will be established as more and larger pores formed [55,56].", "For small hydrophobic drugs, transport through the polymer phase may occur [54,57].", "Progesterone is a small hydrophobic drug with average molecular weight of 314.46 g mol−1 and low water solubility of 8.81 mg l−1 (25 °C) [58].", "Since drug must enter the water phase before it can be released, the more hydrophilic the polymer, the faster its water absorption.", "Hence, PLGA particles with less lactide content, which is therefore more hydrophilic, would provide a better environment for drug to be released [52].", "In case of PLGA particles of higher polymer concentration, about 70% of progesterone was released after 2 h, which is before the onset of polymer dissolution.", "Some of the drug could have been released due to being loosely attached to the surface, but it is unlikely that only few of the drug molecules would have been properly encapsulated in particles.", "Therefore, it is logical to assume that the release of progesterone was dominated by diffusion through the polymer.", "In case of low drug loading (4 wt% PLGA +5 wt% progesterone), the total amount of drug released after 2 h was less (792 μg) compared to 2 wt% PLGA drug-loaded particles (862 μg).", "For the other copolymer composition (75:25) also, particles with relatively higher polymer concentration showed less cumulative drug release (654 μg) as compared to the lower polymer concentration (741 μg).", "Besides, an increase in the surface area: volume ratio results in higher release rate of hydrophobic drugs [18,59].", "It was noticed that, with the increase in polymer concentration, particle size increases resulting in decreased surface area: volume ratio.", "Therefore, less buffer penetration and hence reduced drug release rate occurs.", "Also, the increase in particle size may also result in increased drug diffusion distance, which may be another contributing factor.", "It was noticed that, the main difference was on the initial burst release.", "The release profile after 48 h in the same copolymer composition seems to have similar patterns.", "This indicates that after 48 h, PLGA copolymer ratio is playing a dominating role.", "It is observed that progesterone is not completely released from most of the formulations during the 168 h of measurement, where only 43, 50, 83 and 94% of progesterone was released from F1, F2, F3 and F4, respectively, indicating that there was still drug entrapped within the polymer network.", "PLGA erosion typically does not occur within 7 days and this explains the rest of drug within the polymer matrix.", "As shown in Fig. 5 different progesterone release profiles have been achieved using different PLGA copolymer ratios and concentrations.", "Administration of progesterone by the oral delivery offers high compliance for patient and is the most preferred route [60].", "However, progesterone suffers from low bioavailability and destruction in the digestive system which take place faster than its absorption [61].", "As observed, unlike encapsulated progesterone, pure progesterone molecules suffer from extremely low water solubility.", "As shown in Fig. 5, only 380 μg (24%) progesterone has been dissolved, which is less than half of that from all the other formulations.", "The dramatic increase in dissolution rate for progesterone loaded PLGA formulations was partially explained by the physical form change of progesterone (Fig. 3) after being electrosprayed with PLGA into particles, which indicates that progesterone is molecularly dispersed within the polymer matrix.", "As observed, about 85 and 76% of progesterone has been released from formulation F3 and F4 in 24 h, respectively, which can be desirable when rapid treatment is required and make it a preferable oral delivery system.", "A woman who has her ovaries surgically removed will experience a sudden drop in progesterone level.", "Such loss of hormone can lead to health problems unless adequate hormone replacement is provided.", "In such a scenario, rapid oral delivery of progesterone is preferred.", "In formulations F1 and F2 only 43 and 37% of progesterone was released after 2 h, respectively, and about 50% was released after 7 days of measurement.", "This release profile of progesterone is beneficial for sustained release such as by depot injection.", "As mentioned previously, progesterone has a role in maintaining pregnancy and preventing preterm birth in women considered to be at high risk [6].", "To date, the most common routes of administration of progesterone are intramuscular injection and vaginal suppository [62].", "In reference [62] the suppository was made and characterized by a new process [63–65].", "Progesterone delivered by regular injection may lead to inflammation at the injection site, resulting pain, redness and sterile abscess.", "Yet, the suppository material for vaginal delivery also has its limitations such as escaping from the vagina causing inconvenience and uncertainty as to the drug dose absorbed.", "For women considered to be at high risk of extremely preterm (<28 weeks) and very preterm (28–32 weeks) delivery, long-term administration of progesterone is required in order to maintain its level.", "Through depot injection [15], the drug is slowly released from the deposits in a localized mass.", "We believe that such an approach holds several potential benefits including that it ensures sustained release of progesterone at low doses over prolonged periods and prevention of injection site problems by reducing the frequency of dosing.", "A depot injection is favorable for patients who have difficulty swallowing medication or remembering to take medication regularly.", "In general, our data indicates that the electrosprayed polymeric particles are beneficial for formulation of poorly water-soluble drugs for oral administration since they have great potential to enhance the dissolution rate of the drug.", "The electrospraying process effects the poorly aqueous-soluble molecules at the particulate level through nanonization and amorphization [66].", "Under the impact of electrical forces, drugs are dispersed into small droplets ranging from micro- to nanosize, which allows solvent to evaporate easily and, as a result, immediate solidification takes place.", "By virtue of rapid solvent evaporation amorphous state of drug in the system is generated which attributed to randomly “frozen” drug molecules in the solid polymer matrix forming solid dispersion.", "Not only a predictable and controlled drug release can be designed by manipulating the polymer concentration and copolymer ratio but also a protection of encapsulated active pharmaceutical ingredients from degradation and loss of bioactivity.", "We have fitted Q(t) in Eq. (4) to the experimental release rates up to 105 min, and the results are shown in Fig. 6.", "Using the particle sizes in Table 2 we can extract diffusion coefficients for progesterone in PLGA, and the results are shown in Table 3.", "It appears that the diffusion coefficient in the 50:50 formulation is about ten times greater than in the 75:25 formulation.", "As a check of accuracy of these values, we have performed a similar analysis of previously published data [45], which gave a graph showing the release profiles of progesterone from 50 μm diameter 50 μm high microsylinders of 50:50 PLGA.", "Fitting those results gave a diffusion coefficient of 2.1 × 10−17 m2 s−1, which is in reasonable agreement with our results from much smaller particles.", "In this work, the release kinetics of hydrophobic drug (progesterone) from drug/PLGA particles with different copolymer ratios and polymer/drug ratios were measured in vitro.", "It was found that the copolymer composition and polymer/drug ratio influence drug release kinetics at 37 °C.", "Overall, drug release rate was increased with increasing polymer concentration and with an increase from the PLGA with lower PLA: PGA copolymer ratio.", "In addition, this study also suggests that hydrophobic drugs can be successfully encapsulated into PLGA particles using electrospraying and the dissolution property of hydrophobic progesterone had been significantly improved after electrospray which can result in higher bioavailability.", "In conclusion, the drug release can be controlled significantly by varying the polymer concentration and copolymer composition for various applications." ]

[ "Single step electrospraying was applied with high processing yield to prepare drug delivery systems.", "Solutions of PLGA with different monomer and drug ratios were used for producing progesterone-loaded microparticles.", "Encapsulation efficiency of 80–90% was achieved in selected formulations.", "The release kinetics were studied in vitro and the progesterone release rate was controlled.", "The dissolution rate of hydrophobic progesterone in the prepared formulations was significantly improved." ]

[ "ENVI" ]

[ "Airport emissions", "Particle number size distributions", "Photonucleation", "Positive Matrix Factorization", "Traffic emissions", "Ultrafine particles" ]

[ "2300" ]

[ "It has been widely reported that atmospheric particulate matter (PM) has a negative impact upon human health, with 7 million deaths per year attributed to the exposure to air pollution (WHO, 2018).", "Disentangling the impact on public health of the different sources contributing to PM would allow targeted policies to reduce emissions.", "Sources of mass concentrations of PM ≤10 µm and ≤2.5 µm in aerodynamic diameter (PM10 and PM2.5, respectively) have been identified and quantified in different cities and regions around the world (e.g. Amato et al., 2016; Pancras et al., 2013; Viana et al., 2008; Wang and Shooter, 2005), but less is known about the sources and their contribution to ultrafine particle (UFP, particles ≤0.1 µm) number concentrations (PNC) (Vu et al., 2015).", "Previous studies suggest that sources dominating PNC differ from those dominating particle mass concentrations as detailed in Table S1 for the four European cities studied in this work.", "The quantification of the sources affecting UFP is very important as epidemiological studies suggest that negative health effects may be enhanced with decreasing particle size (Meng et al., 2013; Sioutas et al., 2005), although the associations between UFP and mortality or hospital admissions have been inconsistent in the literature (Kettunen et al., 2007; Lanzinger et al., 2016; Ohlwein et al., 2019; Samoli et al., 2016; Stafoggia et al., 2017; Tobías et al., 2018).", "In urban areas, road traffic constitute the main source of UFP (Kumar et al., 2014; Morawska et al., 2008; Shi et al., 2001) but few attempts have been made to quantify its contribution to PNC (Beddows et al., 2015; Friend et al., 2012; Kasumba et al., 2009; Kim et al., 2004; Liu et al., 2014; Ogulei et al., 2006; Pey et al., 2009; Sowlat et al., 2016; Squizzato et al., 2019; Zhou et al., 2004, 2005).", "Emissions from vehicles are dependent upon the vehicle technology and the properties of fuels and lubricant oils, as well as the driving conditions (Jones et al., 2012; Kittelson et al., 2002; Lähde et al., 2009; Maricq et al., 2002; Rönkkö et al., 2017, 2014; Sgro et al., 2008).", "Primary particles in the vehicle exhaust include soot particles with mean particle around 30–100 nm (Kittelson, 1998; Maricq et al., 2002) and solid core particles in the nucleation mode, usually below 10–15 nm (Rönkkö et al., 2017, 2013; Sgro et al., 2008; Yao et al., 2005).", "Undiluted vehicle exhaust emissions, which are at very high temperatures, contain also a variety of different gaseous components, mainly volatile organic compounds (VOCs) and sulphuric acid.", "Since the saturation ratio of these gaseous compounds rises as the gas cools, these compounds either condense or nucleate to the particle phase immediately after the exhaust discharge to the atmosphere (Casati et al., 2007; Kittelson, 1998; Shi and Harrison, 1999).", "Which process predominates, condensation or nucleation, depends on the availability of pre-existing particle surface area (condensation sink; McMurry and Friedlander, 1979) along with the dilution and cooling rate (Morawska et al., 2008).", "Those nucleated particles have been named delayed primary aerosols by Rönkkö et al. (2017) since they are typically present in the particle phase in normal ambient air temperatures.", "Secondary particles are also generated from gaseous precursors from vehicle exhaust emissions when fully diluted within the ambient air and, driven by photochemistry, are oxidised by reactive species.", "This oxidation causes VOCs to turn into less volatile species, enhancing secondary aerosol formation by condensation and new particle formation (Gentner et al., 2012; Robinson et al., 2007; Volkamer et al., 2006).", "Besides traffic, other sources are known to contribute to UFP.", "Ports and shipping emissions have been identified as source of UFP.", "Shipping emissions are characterised by high concentrations of VOCs and sulphur dioxide that result in the formation of secondary particles by nucleation and condensation processes (Kasper et al., 2007).", "Particle number size distributions (PNSD) from shipping emissions are characterised by an ultrafine mode with mode diameters ranging between 20 and 50 nm (Healy et al., 2009; Kasper et al., 2007).", "Sulphur content on shipping fuels has started to be controlled, with two areas in northern Europe in which sulphur emissions are tightly limited, including the Baltic Sea.", "Gas- (Brewer et al., 2016; Yu et al., 2018) and, specially, coal-fired power plants (Wang et al., 2011a) and airports (Cheung et al., 2011) are other relevant sources of ultrafine particles that may affect PNC in urban environment.", "Ultrafine PNSD are complex.", "In contrast to the mass concentration, which is predominantly conservative, particles undergo several processes that modify their PNC and size such as new particle formation (nucleation), evaporation, condensation, deposition, and coagulation (Harrison et al., 2018).", "Therefore, freshly emitted particles may have a PNSD that may be transformed as these particles move away from the source (Zhu et al., 2002).", "Accordingly, primary particles would mainly influence the air quality near the emission source, while secondary particles would become more relevant as they travel away from the source to the urban background (UB; Morawska et al., 2008; Yao et al., 2005).", "Therefore, there is a need for identification, characterisation, and quantification of the contribution of different sources contributing to PNC.", "Long-term measurements in different stations provide essential information for understanding the intricate relationship between local emission sources, particle atmospheric transformations, and meteorological processes.", "Numerous studies have investigated the effect on PNC and PNSD of specific sources in urban areas (Brines et al., 2015; Dall’Osto et al., 2013; Keuken et al., 2015; Zhu et al., 2002).", "However, long-term PNSD measurements are still scarce (Hofman et al., 2016; Pey et al., 2008; Reche et al., 2011b; Squizzato et al., 2019; Sun et al., 2019; Von Bismarck-Osten et al., 2013; Wang et al., 2011b) since UFPs are not a regulated pollutant.", "In addition, many different approaches are used to carry out source contribution analysis and direct comparison of results among studies is difficult.", "However, a number of source apportionment studies with Positive Matrix Factorization (PMF) applied to PNSD have been done before, mainly in the United States (Kim et al., 2004; Ogulei et al., 2007; Squizzato et al., 2019; Zhou et al., 2004) but also in China (Liu et al., 2014), Australia (Friend et al., 2012), and Europe (Czech Republic: Leoni et al., 2018; Germany: Yue et al., 2008; UK: Beddows et al., 2015).", "The aim of this work is to identify sources and processes and quantify their contributions to urban ambient concentrations of PNC, by using the same methodological approach applied to long-term measurements of PNSD in four European cities with varying climatic and emission patterns: Barcelona, Helsinki, London, and Zurich.", "This study is based on data from four UB stations located in four European cities (Fig. 1; Table 1) with different climatic conditions, emission sources, and urban morphology.", "Two street canyon sites (one in Helsinki and one in London) were also evaluated to compare with the sources observed at the UB sites.", "Barcelona is located in the Northeast coast of Spain, in the western Mediterranean Basin.", "With 1.6 million inhabitants (3.6 million in its metropolitan area) in 2016 (Eurostat, 2018).", "Barcelona has one of the highest cars densities in Europe (5,500 registered cars km−2; DGT, 2018), far above the traffic densities commonly observed in other cities across Europe (1,000–1,500 cars km−2).", "The traffic fleet is characterised by a high proportion of diesel cars (40%) and buses (89% in 2015; DGT, 2018).", "Moreover, Barcelona holds one of the main harbours in the Mediterranean Basin, which may be a significant source of air pollutants that can be often carried by the sea breeze towards the city.", "The airport is located in a near city (about 10 km away from the monitoring station), with around 300,000 operations per year (AENA, 2018).", "The measurements were carried out at the Palau Reial UB monitoring site (BCN, Table 1) located in southwest Barcelona that is influenced by vehicular emissions from one of the main traffic avenues of the city (at an approximate distance of 300 m) with an average traffic density of 70,000 vehicles/working day.", "Helsinki is a coastal city situated in southern Finland, at the shore of the Gulf of Finland.", "It is the largest city in Finland with 0.6 million inhabitants (1.1 million if considering the metropolitan area) in 2016 (Eurostat, 2018).", "Helsinki’s car density is around 1,400 cars km−2 (Statistics Finland, 2018).", "On average, Finland had a share of diesel vehicles of 26.8% in 2016 (http://www.aut.fi/en/statistics/long-term_statistics/share_of_diesel_cars).", "Helsinki’s port was the busiest passenger port in Europe in 2017 with 12.3 million passengers (Port of Helsinki, 2018).", "Ships crossing the Baltic Sea must run on fuel with low sulphur content (International Maritime Organization MARPOL regulation).", "The airport has around 85,000 landings per year (FINAVIA, 2018) and it is about 13 km N from the UB station (HSK).", "The SMEAR III (Station for Measuring Ecosystem–Atmosphere Relationships) UB station in Kumpula, Helsinki (HSK, Table 1), is located in an area with different urban land uses varying from allotment gardens to office areas (e.g. University of Helsinki) and single family house areas with relatively low traffic loads.", "The station is close (about 200 m) to one of the main roads to the city centre with an average traffic density of 50,000 vehicles/day (Järvi et al., 2009).", "The traffic-monitoring site in Helsinki is adjacent to Mäkelänkatu and can be classified as a street canyon (HSK_SC, Table 1) with a traffic density of 28,000 vehicles/working day.", "In its 42 m of width, Mäkelänkatu has six lanes with two tramlines and rows of trees in the middle.", "The road is flanked by four- and five-storey buildings (height-to-width ratio of 0.4; Rönkkö et al., 2017).", "HSK_SC is located about 870 m southwest of the HSK site.", "London is in southeastern England (UK).", "Greater London has 8.7 million inhabitants, making it the largest city in the European Union (Eurostat, 2018).", "Car density in London is around 1,700 cars km−2.", "In 2015, the proportion of diesel cars in the urban areas of England was of 46% in vehicles-kilometres (number of vehicles on a traffic network multiplied by the average length of their trips measured in kilometres, as measure of traffic flow) and the proportion of diesel buses in London was 83% vehicle-kilometres (UK NAEI, 2014).", "London is surrounded by several airports, some of them over 30 km away.", "The busiest is Heathrow with around 480,000 movements/year in 2018 and is located approximately 18 km from the UB station at North Kensington.", "The North Kensington UB monitoring station (LND, Table 1) is placed in the grounds of Sion Manning School in St Charles Square and is mainly a residential area.", "The London traffic monitoring station (LND_SC, Table 1) is sited in the kerbside of Marylebone Road, one of the most heavily trafficked of the city with over 80,000 vehicles/working day.", "It is a street canyon with six lanes (height-to-width ratio of 0.8) that experiences frequent congestion.", "The LND_SC station is about 4 km to the east of the LND station.", "Both stations belong to the London Air Quality Network.", "Meteorological parameters for both stations were obtained at Heathrow Airport, including solar radiation (UK Met Office, 2006).", "Zurich is located in northeastern Switzerland and is the smallest of the cities in this study.", "The city of Zurich has 0.4 million inhabitants (0.6 million including the metropolitan area) and a car density of 2,000 vehicles km−2.", "In Switzerland, and presumably also in Zurich, the vehicle fleet consists of 27.2% diesel vehicles, 71.2% gasoline vehicles and 1.6% hybrid and electrical powered vehicles (BFS, 2016).", "The measurements were carried out at the Zurich-Kaserne UB station (ZRC, Table 1) that is part of the Swiss National Air Pollution Monitoring Network (NABEL).", "It is located in a public courtyard in the city centre.", "The roads surrounding the station have low traffic intensity and the area is not affected by major emissions from industries but it is close to a district with high density of restaurants (west).", "The biggest train station in Switzerland is located about 300 m away northeast.", "Zurich airport, with around 270,000 movements per year, is located 10 km north.", "There is a military base (converted to a civil airport with joint military use in 2014) with very little volume of air traffic.", "It is located 9 km NE from the monitoring station.", "The instrumentation used for measuring aerosol and gaseous pollutants at the different stations is described in Table 2.", "For the present study, the data were averaged to hourly values.", "The periods under study varied depending on the data availability for each site (from 2007 to 2017).", "Different instruments and measuring configurations were used for PNSD measurements at the different sites, and, thus, the measured size ranges varied: in BCN from 11 to 478 nm (Scanning Mobility Particle Sizer Spectrometer, SMPS TSI 3936), in HSK from 6 to 700 nm (Differential Mobility Particle Sizer, DMPS), in HSK_SC from 6 to 800 nm (DMPS), in LND and LND_SC from 17 to 604 nm (SMPS TSI 3080), and in ZRC from 10 to 487 nm (SMPS TSI 3034).", "Most of the instruments for measuring PNSD were fitted with a dryer (except the one in Barcelona and Zurich) as recommended by the EUSAAR protocol (Wiedensohler et al., 2012) and were corrected for diffusional losses (except in Zurich).", "These differences, particularly in the lower size cut, complicate the comparison of the number concentration of smallest particle and total PNC.", "The SMPS and DMPS underwent several checks for quality control and assurance.", "On a daily basis, all instruments were checked to ensure they were turned on and working correctly.", "The impactors and inlets were cleaned on a weekly or biweekly basis.", "Flow rates were measured at least once a month (twice a month at most of the stations) to ensure the flow was within ±10%.", "Once per year, the instruments were either sent for complete maintenance (and the high-voltage supply of the DMA was checked) or participated in a calibration workshop with other SMPS or DMPS (e.g. Gómez-Moreno et al., 2015).", "Black Carbon (BC), PM mass (except HSK), and gaseous pollutant (NO2, NO, SO2, CO, O3) concentrations were also monitored (Table 2).", "The instruments for BC and PM monitoring were also checked frequently: the impactors were cleaned at least once per month and the concentrations were compared to gravimetric measures for PM and elemental carbon determinations for BC.", "Flow is very stable for both types of instruments and were checked at least twice per year in all cities.", "Both PM and gaseous monitoring are performed according to the European Union standards (Directive 2008/50/EC).", "PMF is a descriptive model and there are no objective criteria with which to choose the best solution.", "PMF was performed with Multilinear Engine 2 (ME-2, Paatero, 1999), to identify and quantify the sources of PNSD.", "ME-2 was used instead of the USEPA PMF 5.0 because the latter software accepts a limited number of observations.", "The hourly averaged PNSD data were combined with the hourly concentrations of gaseous pollutants (NO2, NO, SO2, CO, O3).", "Adding additional species (other than PNSD) can help to separate and identify the sources.", "It can also decrease the rotational ambiguity because of increased numbers of edge points (Emami and Hopke, 2017; Li et al., 2019).", "BC was not included in the PMF analyses because the data coverage was low for some of the stations.", "However, we performed a sensitivity analysis (data not shown) to test the influence of including BC in the factor profiles and contributions and we obtained very similar results.", "PM mass concentrations were also excluded for three reasons: (1) because they were not available for HSK, (2) ultrafine particles contribute very little to PM mass concentrations and are generally uncorrelated with them, and (3) several different sources may be affecting PM mass concentrations and, thus, the mass values may not add useful source information to PMF as PM composition data would.", "Although CO and SO2 were not available for HSK_SC, these species were used for the other sites because UB stations are the focus of this study and the HSK_SC dataset only covered a short period.", "Only when missing values corresponded to less than 25% of the number of size bins and gaseous pollutants within a timestamp (that is, at least 75% of the variables had data available), we imputed the concentrations.", "If no data were available for any PNSD size bin during a specific timestamp, it was excluded from the analysis.", "The missing cells within included observations were imputed using the ‘mice’ (Multivariate Imputation by Chained Equations) R package (Van Buuren and Groothuis-Oudshoorn, 2011).", "The corresponding uncertainties for imputed data were set to twice the imputed value.", "The PNSD source profiles and contributions obtained from the ME-2 analyses were scaled to the measured concentration by regressing the measured total PNSD against the ME-2 contribution matrix.", "We performed the analyses to all-year dataset.", "Previous literature suggest performing the analyses by season to account for the substantial season-to-season variability in temperature and solar radiation (Ogulei et al., 2007).", "We compared all-year results with the results obtained when performing PMF separately by season: summer (June, July, and August), winter (December, January, and February) and transitional period (March, April, May, September, October, and November).", "We obtained the same number of sources and most of them showed a correlation coefficient > 0.98.", "However, some differences were observed for specific sources, as shown by some goodness-of-fit measures (Table S3).", "These differences were mainly due to transfer of contributions between two sources (often of the same nature such as two traffic sources) for one of the seasons during the year.", "For some stations, carrying out a PMF for each season led to inexplicable strong jumps in the source contributions from the last day of a season to the first of the following season).", "Thus, we selected to present the results of the all-year dataset as it captured a smoothed season-to-season variability and ensured continuity of the sources throughout the year.", "Data management, descriptive statistics, and plots were performed with the R statistical software (v 3.5.1.,", "R Core Team, 2018) and the package openair (Carslaw and Ropkins, 2012).", "Summary statistics for the different pollutants and meteorological data are presented in Table 3 for all monitoring stations.", "PM10, PM2.5 and NO2 average concentrations were below the European limit values of 40, 25, and 40 µg m−3 (annual average), respectively, in UB stations from all cities.", "PNC and PNSD are difficult to compare between stations, as the measurement protocol and, specially, the measured particle size differs considerably.", "To quantify the effect of the differences in the size ranges, we calculated the total PNC for a reduced common range of approximately 17–480 nm (Table S4) and resulted in a reduction of 45% of the full PNSD range for HSK_SC, 27% for HSK, 26% for ZRC, 17% for BCN with almost the same concentration for the London stations.", "Thus, when interpreting the source contributions, we should take into account these differences and that these differences will affect mainly the sources in the nucleation mode.", "Concentrations of BC and the gaseous pollutants that are tracers of combustion processes were highest at the London stations (and particularly the traffic station, LND_SC), followed by HSK_SC, BCN, ZRC, and finally, with the lowest concentrations, the UB station of HSK.", "The average temperature over the measurement period were lowest in Helsinki, followed by similar temperatures in London and Zurich, and the highest being in Barcelona.", "As expected, solar radiation increased from north to south, with the lowest average being observed in Helsinki and the maximum in Barcelona.", "The datasets for each site were independently analysed by PMF.", "Those PMF solutions that had the most physically meaningful profile and temporal behaviour were selected after comparing solutions with different number of factors (Ogulei et al., 2007; Vu et al., 2015).", "The factors that were common in the different stations, although with slightly different profiles, were Nucleation, two traffic factors (Fresh traffic and Urban, the latter dominated by traffic emissions but influenced by other urban sources including biomass burning in some cities during the cold periods), and Secondary aerosol.", "In addition, in Helsinki Biogenic contributions were identified as a separated factor (further details in the sources below).", "For all stations PMF predictions of PNSD correlated very well with the observed values (Fig. S1).", "For BCN, new particle formation through photonucleation processes had such a high impact that Photonucleation was separated by PMF as a differentiated factor from nucleation particles emitted by road traffic (and nucleation particles from traffic exhaust were included in the Fresh traffic factor, which was specifically labelled as Traffic (nucleation + fresh) for the BCN site).", "At the other stations, however, the low correlation between traffic tracers such as BC and NOx and the Nucleation factor indicated that Nucleation also included particles generated by photonucleation processes, as there were high concentrations of the Nucleation source under low NOx concentration conditions (Fig. S2).", "Four factors were identified for BCN site.", "The profiles of the factors are presented in Fig. 2, both for the PNSD and gaseous data.", "The sources identified from the profiles were Photonucleation (size mode diameter: 13 nm), fresh traffic with a size mode of 31 nm but also including nucleation particles (labelled as Traffic(nucleation + fresh)), a second traffic source mixed with urban background emissions with a size mode of 76 nm (Urban, mostly traffic emissions but slightly influenced by other urban emissions), and Secondary particles with the coarsest particle size (mode: 175 nm; Table 4).", "The Secondary source showed a bimodal distribution, with a minor peak at 18 nm.", "A bimodal distribution was also observed by Pey et al. (2009) in BCN by using a completely different approach.", "Pey et al. (2009) observed a minor peak in the 23–30 nm and a major one at 310–800 nm, with a much higher contribution of the accumulation mode than the nucleation.", "The bimodal distribution of secondary aerosols has also been observed in other locations (e.g. Gu et al., 2011; Kasumba et al., 2009; Squizzato et al., 2019).", "Pey et al. (2009) attributed the secondary source to consist mainly of accumulation mode ammonium-sulphate and ammonium nitrate particles.", "The profiles for the gaseous pollutants also support the source identifications from the size distribution (Fig. 2): Photonucleation and Secondary particles were explained by high contributions from O3, while the traffic sources were associated with high NO2.", "Although photonucleation particles were successfully separated from traffic nucleation particles by PMF in Barcelona, the minor peak at midday and the correlation between NOx and Traffic (nucleation + fresh) (Fig. S3) suggested that the Traffic nucleation factor still included particles generated by photonucleation processes.", "Following the same methodology detailed above for the splitting of the Nucleation source in the other stations, the difference between the Traffic (nucleation + fresh) and the NOx* factor was assigned to be photonucleation particles.", "These residual photonucleation particles were added to the Photonucleation source and subtracted from Traffic (nucleation + fresh).", "With the exception of the source profile, all results presented in this work include the correction for the Photonucleation and Traffic (nucleation + fresh) sources.", "The daily, weekly, and monthly patterns for each source are shown in Fig. 3 and were inspected to determine the correspondence with the source identifications.", "Traffic (nucleation + fresh) and Urban showed a daily pattern coinciding with the typical traffic peaks (Table 1).", "Traffic (nucleation + fresh) had an average annual contribution of 5207 ± 5468 pt cm−3 (Table 5) and showed the lowest concentrations during the summer months (summer average: 3556 ± 2977 pt cm−3) and the highest during late autumn (autumn average: 6223 ± 6330 pt cm−3).", "The daily patterns by season are presented in Fig. S4.", "Similarly, Urban showed the lowest concentrations during August (2352 ± 1186 pt cm−3), although the concentrations were more constant throughout the year (2835 ± 2103 pt cm−3) than for Traffic (nucleation + fresh) due to the contribution of other urban background sources.", "Both sources related to traffic were greatest during weekdays and showed the least concentrations on Sunday.", "Moreover, Traffic (nucleation + fresh) and Urban showed high correlation with BC (r = 0.76 and r = 0.83, respectively; Table S5).", "Photonucleation showed a peak around midday, coinciding with the greatest levels of solar radiation (r = 0.51, Table S5), with negligible contributions during nighttime.", "Photonucleation also showed a small peak at 07:00 of misclassified particles that probably correspond to traffic emissions.", "Photonucleation did not show a consistent weekly pattern, with similar concentrations throughout the week.", "Contributions were minimal during the winter months (1506 ± 2690 pt cm−3, average for 12–14 solar h: 3049 ± 2989 pt cm−3) with significant contributions from June to August (3743 ± 5536 pt cm−3, average for 12–14 solar h: 9153 ± 8554 pt cm−3).", "The Secondary source (675 ± 572 pt cm−3) seems to be influenced by traffic emissions since the morning peak and the greatest contributions on Fridays could be seen in the daily and weekly pattern.", "The correlation coefficient with BC is moderate (r = 0.58).", "The contributions from the Secondary from our study is much lower than the 2000–6000 pt cm−3 reported by Pey et al. (2009) for BCN in 2004 determined by means of Principal Component Analysis, however, sulphate levels were reduced by a large proportion since 2009 to the study period of this work (Pandolfi et al., 2016).", "Five factors were identified for Helsinki: Nucleation (mode = 11 nm), Fresh traffic (22 nm), Urban (50 nm), Biogenic (100 nm), and Secondary (224 nm).", "The same factors were identified in the street canyon site, HSK_SC.", "However, at the HSK_SC the size modes were shifted towards smaller particles due to the proximity to the source for Nucleation (7 nm), Fresh traffic (13 nm), and Urban (45 nm).", "Although the Biogenic was characterised by the same size mode (100 nm) in both stations, the Secondary showed slightly larger particles (256 nm) at HSK_SC than at HSK (Table 4).", "Similar to BCN, the Secondary source also showed a bimodal distribution at both Helsinki stations with the minor peak being at around 33 nm in HSK and 30 nm in HSK_SC (Fig. 2).", "As indicated previously, the Nucleation source was split into Photonucleation and Traffic Nucleation (Fig. S2).", "The gaseous profiles indicate high contributions of NO2 to the traffic sources (including the mixed source of Photonucleation and Traffic Nucleation) in both stations (Fig. 2).", "O3 was mainly associated with the Biogenic source.", "The daily and weekly concentration patterns corroborate the source identifications from the PNSD profiles (Fig. 3).", "Wood combustion contributed to Urban and also to Fresh traffic during winter, which, besides boundary layer height evolution over the year, would partly explain the higher contributions during the cold periods (Fig. S4; Ripamonti et al., 2013).", "Nevertheless, the main source of UFP number concentrations would still be traffic in HSK and especially in HSK_SC.", "In fact, in HSK_SC, BC attributed to biomass burning was 14% of total BC concentrations (Helin et al., 2018).", "The average annual concentrations were 1391 ± 1482 pt cm−3, 1871 ± 2546 pt cm−3, and 2219 ± 2269 pt cm−3 in HSK and 2811 ± 4050 pt cm−3, 5636 ± 7056 pt cm−3, and 4156 ± 3884 pt cm−3 in HSK_SC for Traffic Nucleation, Fresh traffic, and Urban, respectively (Table 5; refer to Table S6 for average contributions over the same period at HSK and HSK_SC).", "Regarding the Biogenic and Secondary sources, they did not present a specific daily or weekly pattern, with similar levels throughout the day and the week (Fig. 3).", "Annual averages were 1001 ± 923 pt cm−3 for Biogenic and 198 ± 219 pt cm−3 for Secondary in HSK and 1734 ± 1439 pt cm−3 and 226 ± 217 pt cm−3 in HSK_SC, respectively.", "However, the annual pattern showed important differences between these two sources.", "Biogenic had its peak during the warmer months (Fig. 3) when biogenic emissions (e.g. monoterpenes, isoprene) were at their maximum (Rantala et al., 2016).", "Biogenic VOC emissions are important precursors for new particle formation (Kirkby et al., 2016; Yan et al., 2018), which may reach the urban environment after they have grown up to around 100 nm.", "Although biogenic VOC emissions peak in summer, the frequency of new particle formation events is more common during spring and autumn due to the inhibition of new particle formation under high isoprene concentrations (Kiendler-Scharr et al., 2009) and because in cooler temperatures the particle-phase might be favoured by supersaturation.", "Since we observed the highest concentrations during summer, the Biogenic source may also have the input of particles from polluted air masses from long range transport, which is particularly intense in the summer months due to controlled burning and forest fires from other regions in Eastern Europe (Niemi et al., 2009).", "It may also be mixed with secondary aerosols from anthropogenic origin, as we would expect the levels of Biogenic source to be lower of what we found during the winter when temperatures are often negative.", "The influence of anthropogenic secondary aerosols on Biogenic is also strengthened by the comparison of the contribution of the sources for the simultaneous periods in HSK and HSK_SC (Table S6), when the Biogenic source is higher in HSK_SC than HSK (we would expect similar levels if there were no influence of anthropogenic and traffic emissions).", "On the other hand, although concentrations of Secondary were low all year round, they showed a trend with its minimum during the summer months to some extend explained by the evaporation of ammonium nitrate due to the higher temperatures during summer (Pakkanen et al., 2001).", "The Secondary source may account mainly for anthropogenic emissions, with the total secondary contribution being the addition of the Secondary and Biogenic sources.", "Photonucleation in HSK (annual average = 313 ± 1044 pt cm−3) was higher during the spring (509 ± 1494 pt cm−3) and autumn (310 ± 969 pt cm−3), which coincides with the periods of higher new particle formation in Finland reported in the literature (Dal Maso et al., 2005; Laakso et al., 2003).", "By looking at the annual pattern of both HSK and HSK_SC (Fig. 3), we suspect that part of the subsequent growth of nucleated particles may be apportioned to the Urban source since this source showed maxima in spring and autumn (Laakso et al., 2003).", "Although the methodologies are not directly comparable, our results are in agreement with the cluster analysis by Hussein et al. (2014) for HSK.", "Four factors were selected as the solution for PMF in both LND and LND_SC.", "The sources were identified as Nucleation (mode = 21 nm in LND), Fresh traffic (37 nm), Urban (81 nm), and Secondary (294 nm).", "Although we were expecting a shift towards smaller particles in LND_SC due to proximity to the traffic source, we obtained the same or very similar mode diameters for Nucleation (21 nm) and the sources from traffic emissions (34 and 81 nm for Fresh traffic and Urban, respectively).", "We obtained a much smaller mode for the Secondary aerosols at LND_SC (93 nm; Table 4) but the profile shows a high contribution of particles from around 80 to 300 nm (Fig. 2).", "The different shape of the profile for the Secondary factor in Marylebone may consist of a mix including also traffic non-exhaust emissions as well as cooking, as the latter was identified to be much larger in LND_SC than LND (Ots et al., 2016).", "PMF was previously applied to PNSD data from 2011 to 2012 from LND (Beddows et al., 2015) and four factors were also identified with very similar profiles to ours.", "Fig. 3 shows the diel patterns for Traffic nucleation, Fresh traffic, and Urban.", "These sources followed the typical pattern of BC and NOx (common traffic tracers) previously described in literature for LND (North Kensington) and LND_SC (Marylebone; Reche et al., 2011a).", "In LND, Urban was strongly correlated with BC (r = 0.84; Table S5) while Fresh traffic showed a moderate correlation (r = 0.53) and Traffic nucleation was surprisingly, and unlike what happened at the other sites, not correlated with BC (r = 0.09).", "For LND_SC, correlation coefficients for BC were stronger (0.52 for Traffic nucleation, 0.72 for Fresh traffic, and 0.84 for Urban) as expected for a street canyon station.", "Compared to the UB stations in the other cities, LND concentrations of traffic sources were relatively high during the night with a midday local minimum.", "Moreover, compared to the other cities both LND and LND_SC had stable concentrations of the traffic sources all year round and did not show a minimum over the summer months (Fig. 3).", "Annual average concentrations (Table 5) were much lower at LND (527 ± 625 pt cm−3 for Traffic Nucleation, 2948 ± 2149 pt cm−3 for Fresh traffic, 1770 ± 1821 pt cm−3 for Urban) than at LND_SC (1451 ± 1242 pt cm−3, 5087 ± 2903 pt cm−3, 5037 ± 2903 pt cm−3, respectively), especially for the source with the smallest particle size which may be associated with fresher emissions (Table S6 shows the annual average for the same periods on both stations).", "The Urban source may also be affected by emissions from biomass burning, which may contribute particularly to the evening peak during the winter months (Fuller et al., 2014; Young et al., 2015).", "Urban showed a higher and maintained evening peak during autumn and winter, which was not present during spring and summer (Fig. S4).", "In LND, Photonucleation was higher during the summer months (398 ± 932 pt cm−3) than the rest of the year (annual average: 203 ± 619 pt cm−3), with very little contribution during the winter (Fig. S4).", "The same pattern for Photonucleation was found at LND_SC but with lower contributions (annual average: 165 ± 506 pt cm−3; summer: 302 ± 723 pt cm−3; winter: 47 ± 195 pt cm−3) as photonucleation processes are usually more important under clean atmospheres (Spracklen et al., 2006).", "The high PNC in LND_SC may have prevented new particle formation; this point is explored more fully by Bousiotis et al. (2019).", "Besides the fact that London sites had the largest lower size cut-off, the high PNC in London may also explain the lower Photonucleation contribution in comparison with BCN and HSK.", "Barcelona has considerable pollution levels, however, it experiences very high solar radiation levels and a frequent midday clean up by sea breeze also transporting SO2 from shipping.", "On the other hand, Helsinki has lower solar radiation but a much cleaner atmosphere (Table 3).", "For both London sites, contributions of the Secondary aerosol were stable during the day with the minimum found in the early afternoon in LND (Fig. 3), probably associated with the thermal instability of ammonium nitrate and semi-volatile organic aerosols and with better dispersion conditions of higher wind speeds and mixed layer heights.", "The annual trend followed the one described for nitrate in London (Revuelta et al., 2012), with lower concentrations during summer (also related to nitrate evaporation) and with higher concentrations from February to April.", "In LND the weekly pattern of the Secondary source showed lower contributions during the weekend (clearly observable when plotting normalised concentrations), which may indicate the influence of local traffic emissions on the secondary particles (also, the correlation with BC reached r = 0.50).", "Average annual contributions were 184 ± 269 pt cm−3 for LND and 589 ± 551 pt cm−3 for LND_SC.", "The coarser nature of the Secondary source made it to be the most correlated with PM mass concentrations (r = 0.81 and r = 0.80 for PM2.5 in LND and LND_SC, respectively).", "Similar to previous sites, four factors were identified as contributors to PNSD in ZRC.", "With the exception of the Secondary factor, the profiles for ZRC were quite similar to those in LND although the mode diameter for Nucleation (15 nm for ZRC) was shifted towards smaller sizes due to smaller lower cut-off of the instrumentation used in ZRC (Fig. 2).", "The gaseous profile for the Nucleation factor was mainly explained by NO2 and O3 suggesting the influence of traffic emissions and the presence of photonucleation particles.", "Fresh traffic (33 nm) had a similar mode diameter to BCN while for Urban (67 nm) the mode diameter was between the ones found in HSK and BCN (Table 4).", "The Secondary factor showed a bimodal distribution with the main peak in the coarser mode (246 nm).", "The average contributions of Traffic nucleation, Fresh traffic, and Urban were 1621 ± 1309 pt cm−3, 4906 ± 4626 pt cm−3, and 2426 ± 1854 pt cm−3, respectively.", "Traffic nucleation and Fresh traffic clearly showed the morning traffic peak, while the afternoon peak was followed by Fresh traffic and Urban (Fig. 3).", "The diel patterns by season indicate that the relative difference between the morning and afternoon peak was much higher during summer and autumn (Fig. S4).", "This suggests that both Fresh traffic and Urban, were possibly affected by other combustion sources, such as solid fuel burning for heating during the cold period or for recreational activities.", "The daily pattern by day of the week (Fig. S5) allows identification of the drivers of high concentrations in the afternoon peak as these occurred on Friday and Saturday evenings during the warmer months.", "There is a recreational area with barbequing near the monitoring station.", "PMF was not able to separate the charcoal or wood combustion used for the barbeques from the traffic sources.", "Traffic emissions are the main contributors to Fresh traffic and Traffic nucleation (and to a lesser extent to Urban) since the concentrations diminish considerably during the weekends.", "The Secondary source is very stable over the day (annual average: 316 ± 275 pt cm−3), with a slightly lower concentration in the early afternoon.", "Similar to other sites, the greatest concentrations of Secondary aerosols were observed during the colder months, especially February and March, suggesting that ammonium nitrate may be an important component of the Secondary source (since it evaporates during the warmer periods).", "Minguillón et al. (2012) reported that during wintertime, ammonium nitrate contributed 63.7% of total PM1 mass concentrations (8.8 µg m−3), which diminished to only 1.5% (0.1 µg m−3) during the summertime.", "This result suggests that the Secondary chemical composition may change over the year since there was not as large a reduction in the PNSD sources.", "Photonucleation contributions showed the expected pattern, with highest levels at midday/early afternoon and during the summer months (summer average: 390 ± 926 pt cm−3; annual average: 316 ± 275 pt cm−3).", "Fig. 4 shows the relative contribution of the sources identified by PMF at the different sites.", "Traffic emissions were by far the highest contributor in all stations, with contributions ranging at maximum (as some traffic sources were mixed with other sources such as biomass burning) from 71 to 91% of the total PNSD.", "The variability in the absolute average contribution of the combination of traffic sources among the UB sites (ranging from 5245 ± 3480 pt cm−3 in LND to 8952 ± 6281 pt cm−3 in ZRC; Table 5) partly corresponds to the differences on the lower size cut of the measurement instrument.", "Generally, the lower the size cut, the greater the number of traffic-emitted particles included in the analysis.", "This explains the lowest traffic contribution (in absolute terms) in London, where we would expect the contrary (LND has the highest BC and NO2 concentrations among the UB sites; Table 3).", "In addition, the relative contribution of the different traffic sources may also be explained by the influence of other sources to these factors as well as the distance to the road of the monitoring site and meteorology, as these variables would affect the amount of time from emission until measurement and the type of physical processes that the particles undergo.", "For instance, condensation and coagulation implies a growth of the particles (and a number reduction in the case of coagulation), while evaporation is associated with shrinkage (Backman et al., 2012; Yao et al., 2010).", "The high contribution of traffic emissions to total PNSD has been consistently reported in literature for urban environments around the globe, including the cities in this study (Beddows et al., 2015; Brines et al., 2015; Dall’Osto et al., 2012; Friend et al., 2012; Liu et al., 2014; Pey et al., 2009; Posner and Pandis, 2015; Squizzato et al., 2019; Vu et al., 2015; Wang et al., 2013).", "Photonucleation is by far more important in the city with the highest insolation, which is consistent with previous studies (Brines et al., 2015).", "In BCN, Photonucleation represented on average the 23% of the total PNSD, reaching 35% during the period with higher solar radiation (summer) but being significant during the whole year.", "In the rest of the UB sites, Photonucleation contributed on average around 3–4% (Fig. 4), reaching a maximum of 7% in LND (summer) and HSK (spring), and a 5% in ZRC (summer).", "The contribution of Photonucleation in BCN (2586 ± 4310 pt cm−3) was approximately an order of magnitude higher than at the other sites: 313 ± 1044 pt cm−3 in HSK, 203 ± 619 pt cm−3 in LND, and 282 ± 795 pt cm−3 in ZRC.", "Note that HSK and ZRC lower size cuts (6 and 10 nm, respectively) are lower than in BCN (11 nm) while in LND, the lower size is 16 nm.", "Generally, Secondary aerosols were the source contributing the least to PNSD (although they would be a large fraction of the mass) due to their regional origin: 3% in LND, HSK, and ZURICH and 6% in BCN.", "Its relative contribution was generally constant over the year.", "In HSK, the combination of Biogenic and Secondary added to a total relative contribution of 17%.", "Finally, to provide a consistent comparison, Table S7 shows the annual average of the contributions at the UB stations for the same period at the four cities.", "The relative contributions for the simultaneous period were very similar to the ones observed for the full period of analysis.", "Fig. 5 shows the polar plots for the different sources and BC, PM10, and SO2 (if available) in the UB stations, indicating the wind speeds and directions that were associated with higher concentrations of each specific source.", "The polar plots for the street canyons are presented in Fig. S6.", "Traffic sources in BCN were mainly of a local origin since they were predominant at low wind speeds.", "BCN is influenced by a nearby important avenue with high traffic intensity.", "BC followed a similar polar distribution than Traffic (nucleation + fresh) and Urban.", "Although Secondary particles were also associated with low wind speeds, high contributions of this source were also observed with winds from the E blowing both at low and high wind speeds.", "The behaviour of Secondary particles is broadly mimicking that of PM10, corroborating the secondary and regional origin of the Secondary source.", "SO2 concentrations were highest for winds blowing from 100 to 180° (especially 160–180°) that is the location of the harbour.", "Barcelona is registering very low SO2 levels compared other European cities (Henschel et al., 2013), especially since 2008 when power generation was restricted to only natural gas in the metropolitan area.", "Thus, currently the main source of SO2 in the city is shipping.", "Photonucleation particles were associated with light breezes blowing from the S (180–190°).", "The airport is located about 10 km SSW (185 – 205°) from BCN.", "Although Photonucleation is associated with wind directions that may come from the harbour and airport, we cannot link their emissions with Photonucleation due to the low wind speeds and low frequency of winds, especially from the direction of the airport (Fig. S7).", "Traffic sources in HSK were associated with N and S wind components (Fig. 5).", "Traffic nucleation was particularly associated with N winds.", "There are several major roads located N that may have some influence on Traffic nucleation, but we would expect a similar or even higher effect (due to particle growth by condensation or coagulation) on Fresh traffic, which is not the case.", "In this direction there is also located the airport, and air traffic is a well know emitter of huge amount of ultrafine particles in the nucleation mode (Hu et al., 2009; Keuken et al., 2015; Masiol and Harrison, 2014; Mazaheri et al., 2009) that can be detected few kilometres away (Cheung et al., 2011; Hudda et al., 2016).", "The association with high wind speeds may indicate that nucleation particles have to travel fast in order to be detected in such a small size in our receptor station in the UB.", "Therefore, there might potentially be a contribution from airport emissions to the Traffic nucleation source.", "However, the nucleation mode particles in Helsinki might also originate from several other sources, such as wood combustion in fireplaces and sauna stoves, oil-combustion in boilers, and regional nucleation events.", "Both Secondary and Biogenic were associated with winds from the E and, particularly for Secondary, covering the angle range to the S - SE.", "At HSK_SC the local wind direction results from the orientation of the street canyon relative to the prevailing wind.", "Thus, prevailing winds are associated with NW and SE (Fig. S7).", "The Biogenic source is associated with N winds (Fig. S6), which, according to the street canyon recirculation dynamics, may actually indicate regional S winds as it did in HSK.", "With the exception of Nucleation particles (both Traffic & Photonucleation) in LND, all sources were mainly associated with calm episodes and E winds.", "This is in agreement with LND being located west of central London and mainland Europe.", "Urban showed a very local contribution (similar to BC) associated with low wind speeds.", "On the other hand, Traffic nucleation and Photonucleation were clearly associated with winds blowing from the W – SW sector.", "Again, although it is difficult to evaluate and quantify, this is pointing towards emission contributions from Heathrow airport (one of the busiest in the world).", "Moreover, W – SW winds were also associated with clean air masses that favour new particle formation and, therefore, it is difficult to rule out the possible contribution from the airport.", "Harrison et al. (2019) have also shown an influence of aircraft emissions upon PNSDs at London stations and further analysis of new particle formation events in London is provided by Bousiotis et al. (2019).", "At LND_SC, the highest concentrations of traffic sources were observed with E or W winds (same direction as the street canyon) and for winds blowing from the S sector.", "The street canyon recirculation explains the influence of S winds as the station is located in the southern side of the road and, therefore, receives the local pollution of the road when winds blow from the S (Harrison et al., 2019).", "In ZRC, Fresh traffic and Urban sources seem to be mostly associated with SE winds while Traffic nucleation was mainly associated with winds from the N sector.", "Secondary particles were mainly associated with local contributions and NE winds.", "NNE winds are often associated with cold weather forcing the semi-volatile components into the particle phase (particularly ammonium nitrate).", "The same results were obtained with the conditional probability function plots (Fig. S8) that indicate the probability of concentrations above the 90th occurring by wind direction and are useful for identify source-areas.", "The polar plots in Fig. 6 were used to identify possible airport influences on the Nucleation particles of the different UB stations.", "Fig. 6 shows the polar plot for the Nucleation factor (before the splitting into Traffic and Photonucleation) in the first column, the Traffic nucleation source in the second, the Photonucleation source in the third column, and the Photonucleation source for those periods in which there were relatively high Photonucleation concentrations even when the environmental conditions were not favourable for the photonucleation process (i.e. low solar radiation – below 80 W m−2 – during the winter months).", "These periods were selected because if photonucleation was low, then airport contributions might be easier to identify.", "We focused on only the UB stations since the high contribution of traffic emissions in the street canyons might obscure the presence of nucleation particles from the airport.", "In Fig. 6, the two dashed lines indicate the angular range of influence of airport emissions: winds blowing from 185 to 205° (SSW) may carry airport emissions to BCN, from 345 to 10° (N) to HSK, from 246 to 260° (WSW) to LND, and 0–22 (NNE) ° to ZRC (the military airport has not been considered due to the low air traffic).", "In all cases, Nucleation particle were observed as being directionally dependent and coincident with the location of city airports.", "At all the UB stations, Nucleation particles were associated with those wind directions that were from the corresponding airport.", "ZRC has a more complex topography that would induce funnelling and, therefore, the highest concentration were a bit shifted toward NNW instead of NNE.", "In BCN, the low wind speed may indicate a local origin of the Nucleation particles (either Traffic or Photonucleation) instead of the airport.", "Alternatively, both HSK and LND showed the coincidence of the airport wind directions with the greatest contributions for Traffic Nucleation and Photonucleation (under low radiation conditions).", "However, we note that in HSK there is also several main roads and residential areas with high residential wood combustion emissions at the same direction with the airport.", "It is difficult to isolate airport emissions contributions without additional tracers; however combining the evidence across the four cities is indicative of airport emissions influencing urban background Nucleation concentrations.", "Aircraft emissions are characterised by a PNSD peaking in the nucleation mode (Hu et al., 2009; Keuken et al., 2015; Masiol and Harrison, 2014; Mazaheri et al., 2009).", "So far, few studies have reported observing an impact of airport emissions in PNSD within urban environments located few kms away (Cheung et al., 2011; Hudda et al., 2016; Keuken et al., 2015).", "BC highest concentrations were not associated with wind directions from the airport (Fig. 5) and this may indicate that traffic or wood burning emissions are not the main source of Nucleation particles when the wind is blowing from the airport.", "On the other hand, not identifying high BC concentration from airport direction would be in accordance to aircraft emissions, as aircraft plumes are characterised by very high PNC (especially in the nucleation range) but not BC (Keuken et al., 2015).", "Further studies are needed to confirm the potential impact of aircraft emissions and isolate them from other sources.", "Positive Matrix Factorization (PMF) was used for receptor modelling the source contributions to long time series of hourly size segregated number particle concentrations at six monitoring stations (four urban background and two traffic stations) in four European cities that were affected by different meteorology and emission patterns: Barcelona, Helsinki, London, and Zurich.", "We identified common sources across all cities: Photonucleation, traffic emissions (3 sources, from fresh to aged emissions: Traffic nucleation, Fresh traffic – mode diameter between 13 and 37 nm-, and Urban – mode diameter between 44 and 81 nm), and secondary particles.", "PMF was able to separate the Photonucleation factor only for Barcelona, while a manual split of the Nucleation factor (into Photonucleation and Traffic nucleation) was performed for all the other stations using NOx concentrations as a proxy for traffic emissions.", "Traffic emissions were the main contributor in all stations, with a potential maximum contribution ranging from 71 to 94% of PNSD.", "For London and Zurich stations, no significant variability among seasons was observed.", "On the other hand, the high levels of solar radiation in Barcelona led to an important contribution of Photonucleation particles (ranging from 14% during the winter period to 35% during summer).", "Moreover, a source identified as Biogenic emissions was only present in Helsinki importantly affecting both the UB and street canyon stations, particularly during summer (23%) but also during spring (14%) and autumn (12%).", "When looking at wind directions that were favouring Nucleation particles, we observed that in most cases the highest concentrations took place when the wind was blowing from the airport location.", "Particularly, we could easily identify periods when the urban background station in London was affected by emissions from Heathrow.", "Although less clear, we found that airport emissions might be as well affecting background PNSD concentrations in Helsinki, Zurich, and Barcelona.", "This work provided a detailed characterisation of the sources affecting ultrafine particles in four urban environments in Europe, confirming the great contributions from traffic emissions but also highlighting some differences such as the role of photonucleation in high insolation cities.", "The contribution of the different sources to PNSD will be used in future studies to evaluate the impact of these specific sources on human health.", "Ioar Rivas: Conceptualization, Methodology, Software, Formal analysis, Writing - original draft, Funding acquisition.", "David C.S. Beddows: Methodology, Software.", "Fulvio Amato: Conceptualization, Methodology, Resources.", "David C. Green: Conceptualization, Methodology.", "Leena Järvi: Investigation, Data curation.", "Christoph Hueglin: Conceptualization, Investigation.", "Cristina Reche: Investigation, Data curation.", "Hilkka Timonen: Investigation, Data curation.", "Gary W. Fuller: Conceptualization, Methodology.", "Jarkko Niemi: Investigation, Data curation.", "Noemí Pérez: Investigation, Data curation.", "Minna Aurela: Investigation, Data curation.", "Philip K. Hopke: Methodology.", "Andrés Alastuey: Conceptualization, Resources.", "Markku Kulmala: Conceptualization, Resources.", "Roy M. Harrison: Conceptualization, Methodology, Supervision.", "Xavier Querol: Conceptualization, Methodology, Supervision, Resources.", "Frank J. Kelly: Conceptualization, Supervision, Funding acquisition.", "We developed a methodology (inspired by the one proposed by Rodríguez and Cuevas, 2007) to split the Nucleation factor into two sources: Photonucleation and Traffic nucleation.", "To this end, we used NOx as a proxy for traffic emissions within the Nucleation factor.", "Considering that at the morning peak hour most of the Nucleation particles would be from traffic, we multiplied NOx concentration by a scaling factor (the ratio between Nucleation and NOx) so it matched Nucleation concentrations at morning peak hour (08:00 h local time, except for HSK_SC that was at 07:00 h).", "The scaling factor was different for each day (day-specific) to account for the possible variations in the NOx to Nucleation ratio due to: (1) long-term changes in the fleet (differences in the fuel proportion and the emission control technologies incorporated in the vehicles; Park et al., 2017; Rönkkö et al., 2013); (2) driving conditions (Wang et al., 2010); and (3) the dependency on meteorological and dilution conditions of particle formation (affecting to nucleation particles) in diluting engine exhaust (Charron and Harrison, 2003; Gidhagen et al., 2005; Rönkkö et al., 2006).", "For daytime hours (from 08:00 to 21:00 h local time), if solar radiation was over 10 W/m2, the product of hourly NOx concentrations by the day-specific factor was assigned to the Traffic nucleation source, while the rest was assumed to be Photonucleation (Photonucleation = Nucleation – Traffic nucleation; Fig. S2).", "During the night (22 h – 07:00 h) all Nucleation particles were assigned to Traffic nucleation as no photonucleation would be expected.", "In this work, we use the term 'factor' when referring to the direct results from PMF (e.g. for the profile description) and the term 'source' when discussing about the final sources after the splitting of the Nucleation factor.", "Regarding the different traffic factors (including the one labelled as Urban) identified at each station, the profiles obtained from this work (Fig. 2) are within those reported at the existing literature.", "As stated in the reviews by Morawska et al. (2008) and Vu et al. (2015), diesel exhaust emissions are within the size range of 10–130 nm while gasoline emissions are within 15–60 nm.", "Exhaust plumes are characterised by the emission of primary particles (a carbonaceous mode) and secondary nucleation particles (Morawska et al., 2008).", "Exhaust primary particles are within the range of 30–500 nm (Casati et al., 2007; Vu et al., 2015) and are mainly composed of agglomerates of solid carbonaceous material (soot: graphitic carbon and lesser quantities of metallic ash with condensed or adsorbed hydrocarbons and sulphur compounds).", "The carbon core diameter has been reported to be down to 2.5 nm in cars (Sgro et al., 2008) and 10 nm in heavy-duty vehicles (Rönkkö et al., 2013).", "Heavy-duty vehicles may also emit coarser primary particles within the accumulation mode (100–1000 nm; Morawska et al., 1998).", "Nucleation particles form from the hot exhaust gases while they cool down and condense to produce large numbers of particles in the nucleation range (<30 nm) (Morawska et al., 2008).", "Binary nucleation of H2SO4-H20 or ternary nucleation of H2SO4-NH3-H2O are the main mechanisms of particle formation (Meyer and Ristovski, 2007; Shi and Harrison, 1999).", "The small sulphuric acid core starts growing by condensation of hydrocarbons (Tobias et al., 2001).", "In this study, the mode diameters of the profiles of the different traffic factors fall within the abovementioned size ranges (Table 4).", "The Nucleation factors showed a size range of 7–21 nm and, thus, traffic nucleation particles may be the principal contributor.", "The lower size range is especially dependent on the lower size-cut of the instrument used for PNSD measurements but in all cities a source with a mode diameter below 25 nm was resolved (Table 4).", "The size range of mode diameters for Fresh traffic (14–37 nm) may correspond to fresher traffic emissions of core carbon particles with organic compounds condensed and absorbed, including smaller carbon agglomerates.", "Urban encompassed larger carbon agglomerates (as supported by the correlation of BC and Urban always being higher than for Fresh traffic and Traffic nucleation; Table S5) as well as aged traffic particles that had grown enough to be in this size-range.", "The latter was corroborated by the daily patterns of Urban concentrations that usually showed a longer and delayed peak in comparison with Fresh traffic (Fig. 3)." ]

[ "Ultrafine particle sources were identified and quantified in four European cities.", "Common sources were Photonucleation, different Traffic sources, and Secondary.", "Maximum contribution of traffic sources ranged 71–94% of total particle number.", "Airport emissions contributed to nucleation particles in urban background areas." ]

[ "CENG", "ENGI" ]

[ "Enhanced heat transfer", "Heat exchanger tube", "Swirl", "Twisted tape", "Twisted tube" ]

[ "1507", "2201" ]

[ "Applying twisted tubes and twisted tapes has long been a method enhancing passive heat transfer in shell and tube heat exchangers.", "Numerous applications are found in chemical processing plants, power plants and solar air/water heaters, and air conditioning equipment, among many others.", "Twisted-tubes are used to improve the heat transfer coefficient of the tube side of shell and tube heat exchangers, which plays an important role in reducing heat exchanger size [1–7].", "According to its geometry, a twisted tube produces a swirl/secondary flow that can increase the mean velocity, effective axial Reynolds number and temperature gradient of a working fluid [1,2,8–13].", "Tan et al. [14] examined the enhanced heat transfer and pressure loss behavior in a twisted oval tube with various axis ratios and twist ratios.", "Compared to a circular tube, the twisted oval tubes enabled higher heat transfer and had a greater pressure drop than a smooth circular tube.", "Their results also showed that both heat transfer and pressure loss increase with the axis ratio, and both decreased with an increasing twist ratio.", "Tan et al. [15] studied the heat transfer enhancement and friction factor behavior of a twisted oval tube fitted with a twisted tri-lobed tube.", "Their results indicated that the twisted tri-lobed tube gave a higher thermal enhancement than a twisted oval tube.", "Sun et al. [16] performed experiments to determine the thermal enhancement in a built-in twisted belt on the exterior of thread tubes using several nanofluids as the test fluid at various mass fractions.", "Their results indicated that the optimum heat transfer rate and friction factor were using an enhanced tube through which a Cu/water nanofluid at a 0.05% mass fraction flowed.", "Pozrikidis [17] studied the effect of s twisted tube installed in square cross-section channels on the flow behaviors.", "Their results showed the secondary flow that developed over the tube cross-section affected the axial flow.", "Recently, tape inserts were applied in twisted tubes for enhanced heat transfer.", "Eiamsa-ard et al. [18] coupled a three-start spirally twisted tube in which a triple-channel tape was mounted.", "The effect of tape width ratio and tape arrangement was also studied.", "The experimental results showed that heat transfer increased with the tape width ratio while a twisted tube in which a tape in belly-to-neck arrangement was mounted gave a better thermal performance factor than another arrangement.", "Omidi et al. [19] reported the influence in a heat exchanger with a tri-lobed cross-section fitted with a Y-tape on the thermal behavior.", "They observed that the twisted tube in which a Y-tape was mounted gave a higher thermal performance than a twisted tube alone.", "Cheng et al. [20] carried out the heat transfer and fluid flow behavior in a twisted tube with various cross section shapes in laminar flow region.", "Their result showed that the cross-section shape of twisted tube caused insignificant effect on the heat transfer rate.", "The maximum thermal performance factor of 2.69 was found by using of the twisted pentagon tube with twist pitch ratio of 0.17.", "Soleimani et al. [21] reported the heat transfer and pressure loss in a heat exchanger using natural gas as the testing fluid.", "Their results showed that the application of twisted tube helps in decreasing the gas coil length up to 25% as compared to the straight tube.", "Li et al. [22] studied the air side heat transfer rate of twisted oval tube bundles with various twist pitches, longitudinal tube pitches, outer major axis to outer minor axis ratios (A/B), transverse tube pitches, and longitudinal tube row numbers.", "They observed that the twisted oval tube bundles gave high heat transfer rate than those the circular tube bundle.", "Makarov et al. [23] investigated the heat transfer in twisted tubes with square cross sections using helium-xenon mixture as the working fluid.", "Effect of the twisted tube cross-section shape and twist step on the heat transfer rate and hydraulic resistance was reported.", "According to the results of previous works, combinations of twisted tubes and tape inserts are more effective in enhancing heat transfer due to the combined actions of tubes and tapes since stronger turbulence and flow fluctuation are generated.", "This research examines the use of compound devices consisting of a twisted tube consisting of trapezoidal shaped channel and classic twisted tapes.", "Twisted tube surfaces act as secondary flow and turbulence generators near the tube wall, while twisted-tapes function as swirl flow promoters.", "The objectives of this study are: (a) to examine the thermohydraulic performance of a twisted tube, and (b) to evaluate the compound effects of a twisted tube and twisted tapes on heat transfer and friction characteristics.", "Twisted tapes with four different twist ratios, defined as a ratio of twist length to tape width; y/w = 2.0, 3.0, 4.0 and 5.0, were tested to find the optimal conditions.", "All tests were performed at similar Reynolds numbers, 4500 ≤ Re ≤ 16,000, based on the tube's hydraulic diameter.", "The details and images of the twisted tube used in the current study are shown in Fig. 1.", "The copper twisted tube had a length of 800 mm with a trapezoidal cross-section.", "The characteristic geometries of twisted tubes are (1) tube outer diameter (D), (2) tube outer to base (d), (3) fin apex angle (R), (4) thickness (t), and (5) loop length (l).", "The twisted tape was made of straight aluminum tape with a thickness of 1.0 mm (δ), a width (w) of 15 mm.", "Twisted tapes with various twist ratios (y/w) of 2.0, 3.0, 4.0 and 5.0 were used.", "In each run, a twisted tape swirl generator was mounted inside the twisted tube as shown in the figure.", "The experimental setup is shown in Fig. 1.", "The test/heating tube was made up of a twisted tube under uniform wall heat flux conditions.", "The experimental setup components and their functions are as follows: T-type thermocouples to measure the local surface/wall temperatures (Tw), resistance temperature detectors (RTDs) to measure the entrance and exit air temperatures (one RTD at the entrance and three RTDs at the exit), electric heater to supply heat to the fluid flowing through the copper tube, a data logger, a multimeter, two digital pressure gauges, rotameter, a three horsepower electric motor, and a personal computer for storing and displaying the collected data.", "A calm section with a length of 1.5 m was located upstream of the test section.", "The surface wall temperatures (Tw) were measured at 15 stations around a perimeter of the tube.", "Each T-type thermocouple was installed in a 0.5 mm deep groove on the outer tube surface.", "All experimental data were recorded under steady flow/state conditions.", "Heat transfer (Nu) can be written in term of the Nusselt number as:Nu = hD/k", "The pressure drop (ΔP) across the twisted tube length (L) was monitored using a digital pressure gauge mounted across the test section.", "The pressure loss is expressed in terms of the friction factor (f) as:f = (2ΔP D / L ρU2)", "The Reynolds number is calculated based on the hydraulic diameter of the twisted tubes as:Re = ρUD/μ", "Practically, thermal performance should be higher than 1.0, which reflects an overall energy savings by the heat transfer augmentation.", "Details of the data uncertainties were also quantified [26,27].", "The data uncertainties of Nu and f are written as:", "Figures Fig. 2(a and b) depict the variation of the Reynolds number with the Nusselt number (Nutt) and Nusselt number ratio (Nutt/Nup) for a twisted-tube mounted with twisted tape at various twist ratios (y/w = 2.0, 3.0, 4.0 and 5.0).", "It can be seen that the Nusselt number increases with the Reynolds number (Re).", "At a given operating Re, the Nusselt number of the twisted tube mounted with twisted tapes (Nutt) is consistently higher than that of the twisted tube alone.", "The numerical velocity contours (Fig. 3) show that the twisted-tube installed with twisted tape creates double swirl flows both in the core and near wall regions, leading the vigorous fluid mixing, while the twisted-tube alone generates only near-wall swirl flow.", "Additionally, these double swirl flows also increase the effective flow length and the fin effect of the tape, which further enhance thermal performance of the heat exchanger.", "The temperature contours also show that the twisted tube with twisted tape has a larger contact surface area, stronger swirl flow and a thinner thermal boundary than the twisted tube alone.", "Consequently, the combined device yields higher Nusselt numbers than the twisted tube alone.", "The results shown in Fig. 2(a and b) also indicate that heat transfer is increased as twisted-tape ratio decreases since a tape with smaller twist ratio gives stronger swirl and thus better fluid mixing between the core-flow and twisted wall regions.", "The heat transfer rate of the twisted tube installed with twisted-tape having y/w values of 2.0, 3.0, 4.0 and 5.0, are 70.4%, 59.9%, 50.7% and 41.8% higher than those of the twisted tube alone, and 172%, 155%, 140% and 126% higher than those of the smooth tube, for Re between 4500 and 16,000.", "Furthermore, the twisted-tape with the lowest twist ratio (y/w = 2.0) offers better heat transfer than the ones with larger twist ratios of y/w = 3.0, 4.0 and 5.0 by around 6.6%, 13.1% and 20.2%, respectively.", "The twisted tube offers 59.4% higher heat transfer than that of the smooth tube alone.", "The effect of using a twisted-tube equipped with twisted tapes on the flow friction is shown in Fig. 4(a and b).", "It can be seen that the use of the twisted-tube mounted with twisted tapes results in a 6–12 times higher friction factor (f) than a twisted-tube alone.", "The higher friction loss is primarily caused by 1) dissipation of dynamic pressure of the fluid caused by the combined effects of the flow obstruction of twisted tube and twisted tape with the double swirling flows induced by the enhanced devices, and 2) the enhancement of fluid contact area.", "The friction factor slightly increases with the twist ratio (y/w).", "The twisted tape with the lowest twist ratio (y/w = 2.0) yields a higher friction factor than with twist ratios, y/w, of 3.0, 4.0 and 5.0 by around 14.7%, 30.2% and 48.6%, respectively.", "Additionally, the twisted tube combined with twisted-tapes with y/w values of 2.0, 3.0, 4.0 and 5.0 causes, respectively, friction loss up to about 276%, 228%, 189% and 153% of the twisted tube alone and up to 12, 10.4, 9.2 and 8 times higher than a smooth circular tube.", "The twisted tube friction losses were up to 3.2 times higher than the smooth circular tube.", "The influence of a twisted tube combined with twisted-tapes on the thermal performance is shown in Fig. 5(a and b).", "A twisted tube combined with twisted-tapes results in higher thermal performance than a twisted tube alone.", "The increase in thermal performance with decreasing Re is due to the lower friction factor ratio at low flow rates.", "It was found that using a twisted tube combined with twisted-tapes is more feasible in terms of energy savings at lower Re.", "Additionally, the thermal performance increases with reduced twist ratios (y/w).", "The thermal performance of the twisted tubes compounded with twisted-tapes having y/w = 2.0, 3.0, 4.0 and 5.0 are in the ranges of 1.11 and 1.28, 1.09 and 1.25, 1.08 and 1.23, and 1.06 and 1.21.", "These are higher than those of a twisted tube alone by around 9.6%, 7.6%, 5.8% and 4.1%, respectively.", "The twisted-tape with the lowest twist ratio (y/w = 2.0) yields higher thermal performance than the ones with twist ratios, y/w, of 3.0, 4.0 and 5.0 by 1.9%, 3.6% and 5.3%, respectively.", "The thermal performance of the twisted tube alone is in the range of 1.02–1.17.", "In the present work, the heat transfer enhancement and pressure loss in a twisted tube installed with the twisted tape were experimental studied.", "The thermohydraulic performance results under constant pumping power criteria were also reported.", "The major conclusions from the present work can be summarized as follows:", "In the experiments, the twisted tube surface generates secondary flow and promotes turbulence near the tube wall, while twisted-tape placed in the core tube induces swirl flow along the core tube.", "Utilizing twisted tubes combined with twisted-tape increases the heat transfer, pressure loss and thermal performance compared to a tube without twisted tape.", "These results are attributed the stronger turbulence and better fluid mixing caused by the combined effects of swirl flows induced by both the tube and tapes.", "Heat transfer increases as the twisted-tape ratio (y/w) decreases, attributed to the stronger swirling intensity that helps in promoting destruction of thermal boundary along the tube wall.", "The twisted-tape with the lowest twist ratio, y/w = 2.0, causes higher heat transfer than the ones with twist ratios (y/w) of 3.0, 4.0 and 5.0 by up to 6.6%, 13.1% and 20.2%, respectively.", "The thermal performance factor increases with reduced twist ratios (y/w) and Re.", "The highest thermal performance factor is obtained using the twisted tube combined with twisted-tapes with twist ratios (y/w) of 2.0, 3.0, 4.0 and 5.0 are found to be 1.28, 1.25, 1.23, and 1.21, respectively.", "The twisted-tape with the lowest twist ratio (y/w) of 2.0 yields higher thermal performance than the ones with twist ratios of 3.0, 4.0 and 5.0 by 1.9%, 3.6% and 5.3%, respectively.", "The use of a twisted tube compounded with twisted-tapes is more feasible in terms of energy saving at lower Reynolds numbers.", "The result is applicable for design the compact heat exchanger design in order to minimize pumping power and optimize the energy consumption in the thermal system.", "The authors declare there are no conflicts of interest." ]

[]

[ "NEUR", "PSYC" ]

[ "Alzheimer's disease", "Dementia", "Frontotemporal", "Nonverbal sound", "Physiology", "Progressive aphasia", "Pupillometry", "Semantic" ]

[ "2805", "3205", "3206" ]

[ "Disambiguation of potentially relevant, ‘salient’ stimuli from the busy multisensory background is accomplished efficiently and largely automatically by the healthy brain.", "However, successful processing of sensory salience depends on a number of subprocesses: these include accurate parsing of the sensory environment, representation of particular sensory objects, assignment of emotional and reward value, and linkage to physiological and motor effector mechanisms that govern an appropriate behavioural response (Beissner, Meissner, Bar, & Napadow, 2013; Critchley, Corfield, Chandler, Mathias, & Dolan, 2000; Kirsch, Boucsein, & Baltissen, 1995; Zhou & Seeley, 2014).", "Each of these subprocesses entails complex neural computations that are likely a priori to be vulnerable to the effects of neurodegenerative pathologies.", "The canonical syndromes of frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD) are associated with altered emotional, physiological and behavioural responses to salient sensory signals (Fletcher, Downey, et al., 2015a, in press; Fletcher, Nicholas, et al., 2015c, 2015d; Hoefer et al., 2008; Zhou & Seeley, 2014).", "These are most strikingly exemplified by the phenotypes of disrupted hedonic valuation and aberrant reward processing that characterise FTLD (Fletcher, Downey, et al., 2015a; Perry et al., 2014), though AD may also produce abnormalities of sensory salience coding (Fletcher, Downey, et al., 2015a, in press; Fletcher, Nicholas, et al., 2015c, 2015d).", "Such abnormalities further suggest a physiological substrate for the higher order disturbances of emotional and social cognition that frequently accompany these diseases (Downey et al., 2015; Kumfor & Piguet, 2012; Omar et al., 2011; Warren, Rohrer, & Rossor, 2013; Woolley et al., 2015), with implications for biomarker development and management strategies.", "The salience of a sensory signal generally depends on attribution of its meaning and this is well illustrated in the often ambiguous realm of sounds.", "Perceptually similar sound sources can have very different biological implications (compare, for example, the rumble of thunder and the growl of a large predator).", "Auditory salience cues such as loudness, movement (looming) and affective valence are coded physiologically in pupillary and other autonomic responses (Fletcher, Nicholas, et al., 2015c, 2015d; Neuhoff, 2001) mediated by distributed cortico-subcortical brain networks (Beissner et al., 2013; Critchley et al., 2000; Mueller-Pfeiffer et al., 2014).", "In addition to these well recognised examples, auditory semantic ambiguity is also a candidate salience cue: there is a biological imperative to resolve the identity of potentially meaningful sounds, and the ability to do this efficiently and accurately is likely to have conferred survival and reproductive advantages during human evolution.", "In this context, ‘potentially meaningful’ sounds would include naturally occurring, spectrotemporally complex sounds sharing perceptual characteristics with animal (including conspecific) vocalisations.", "It might be predicted that the processing of such sounds would engage brain mechanisms for coding salience, particularly under adverse listening conditions where the identity of the sound source is difficult to determine.", "Coding such ambiguous sounds for salience would direct attentional and behavioural resources to the sound source so that its identity can be determined rapidly with an appropriate behavioural response.", "From a clinical perspective, diseases of the auditory pathways tend to render sounds more difficult to identify and ‘adverse listening conditions’ might also be produced by brain diseases that degrade central mechanisms of auditory semantic analysis: in this situation, the perceptual features of sounds will be coded more or less accurately but sounds will be ambiguous (and therefore, potentially salient) because the attribution of meaning to auditory percepts is impaired.", "However, it has not been established whether semantically significant or semantically ambiguous sounds have physiological salience correlates.", "In particular, the interaction of semantic and salience mechanisms has not been explored in neurodegenerative diseases that that might disrupt these mechanisms differentially.", "Here we investigated physiological and neuroanatomical correlates of this putative ‘semantic salience’ response in a cohort of patients representing canonical dementia syndromes (semantic dementia – SD; behavioural variant frontotemporal dementia – bvFTD; progressive nonfluent aphasia – PNFA; typical amnestic AD) relative to healthy older controls.", "We studied patients representing a range of dementia syndromes in order to assess the extent to which putative salience responses might differentiate or transcend syndromic categories.", "Semantic deficits are not restricted to a particular syndrome: while SD is the paradigmatic disorder of the human semantic system (Lambon Ralph, Sage, Jones, & Mayberry, 2010), less severe or less consistent auditory and other semantic deficits have been documented in each of the other neurodegenerative syndromes included here (Golden et al., 2015; Goll, Crutch, Loo, et al., 2010, Goll et al., 2011; Hardy et al., 2015; Hsieh, Hornberger, Piguet, & Hodges, 2011).", "Moreover, these diseases have been shown to have distinct profiles of pupil reactivity to salient sounds (Fletcher, Nicholas, et al., 2015c, 2015d).", "We measured pupil responses to sounds that varied in semantic content, constituting two sound conditions: real nonverbal sounds with prior semantic associations and synthetic sounds that lacked any such associations.", "Pupil responses in these two sound conditions were compared and assessed in relation to nonverbal auditory semantic function in each group.", "Neuroanatomical correlates were determined using voxel-based morphometry (VBM) of patients' brain MR images.", "We hypothesised that healthy older individuals would show larger pupil responses to real than synthetic sounds and that the magnitude of this difference would vary inversely with nonverbal auditory semantic function across the patient cohort.", "In particular, we hypothesised an exaggerated pupil response to real sounds in the SD group, as severely degraded sound identification in these patients would preclude disambiguation of these potentially salient sound sources.", "We further hypothesised an anatomical correlate of this semantic salience response in anterior temporal cortex previously implicated in auditory semantic analysis (Golden et al., 2015; Goll, Ridgway, Crutch, Theunissen, & Warren, 2012; Hsieh et al., 2011) and in the central autonomic control network previously implicated in programming physiological salience responses (Critchley et al., 2000; Wang, Boehnke, Itti, & Munoz, 2014; Wang, Boehnke, White, & Munoz, 2012; Zhou & Seeley, 2014).", "Forty consecutive patients fulfilling consensus criteria for dementia syndromes (bvFTD, n = 13; SD, n = 11; PNFA, n = 6; typical AD, n = 10) and 20 healthy older individuals with no history of neurological or psychiatric illness participated.", "No participants had a clinical history of hearing loss or pupillary disease.", "All patients with AD (but no other patients) were receiving treatment with acetylcholinesterase inhibitors; four patients were receiving antidepressant medication (two SD, one PNFA, one AD).", "In order to assess any effect from peripheral hearing function on experimental performance, screening pure tone audiometry was conducted in each group using a previously described procedure (Golden et al., 2015).", "A general neuropsychological assessment corroborated the clinical diagnosis for each of the disease groups; all patients had a consistent profile of regional brain atrophy on MRI and none had radiological evidence of significant or strategic vascular damage.", "Cerebrospinal fluid total tau and beta-amyloid1-42 assays (available for 18 patients: seven AD, five bvFTD, four SD, two PNFA) and Florbetapir PET brain imaging (available for six patients: four SD, two PNFA) further supported the clinical diagnoses (AD, CSF total tau: beta-amyloid1-42 ratio >1; other groups, CSF total tau: beta-amyloid1-42 ratio <1 and Florbetapir-PET negative for amyloid deposition).", "Genetic screening of the cohort revealed nine patients with a pathogenic mutation (five C9orf72; four MAPT).", "Demographic, clinical and general neuropsychological data are summarised in Table 1.", "The study was approved by the local institutional ethics committee and all participants gave informed consent in accord with the principles of the Declaration of Helsinki.", "Nonverbal sounds were derived from publically-available sound libraries (www.freesound.org, www.freesfx.co.uk) and sampled a range of human, animal, environmental and mechanical sounds.", "In a pilot experiment, 20 healthy younger individuals (median age 28 years (range 23–37), six male) were asked to identify an initial set of 180 sounds: those that were misidentified by two or more (>10%) of the healthy younger pilot control group were excluded in order to ensure all sounds in the main experiment were intrinsically highly familiar and identifiable (final stimulus sets listed in Tables S1 and S2).", "Audio samples were converted to digital wavefiles sampled at 44.1 kHz, and edited so that all sound stimuli were two seconds in duration (brief sounds such as hiccoughs that tend to be naturally periodic were repeated within this interval) and mean overall intensity (rms value) was fixed across stimuli.", "In addition, as loudness modulates pupillary dilatation in healthy individuals (Steiner & Barry, 2011) the peak volume of each sound as played through the experimental sound delivery system was measured using a sound level meter and incorporated as a nuisance covariate in subsequent analyses.", "Ten nonverbal sounds rated as highly identifiable by the pilot healthy control group were selected and spectrally inverted in Matlab® to produce synthetic (‘meaningless’, M−) versions of the sounds that were similarly acoustically complex but lacking the semantic associations of the real (‘meaningful’, M+) sounds.", "The M+ and M− sounds had matched mean overall intensity; M− sounds had higher measured mean peak loudness than M+ sounds.", "All sound stimuli were presented via Audio-technica ATH-M50 headphones from a notebook computer at a constant, comfortable listening level (at least 70 dB) in a quiet room.", "Autonomic responses to sounds were assessed using pupillometry.", "Sounds in the M+ and M− conditions were all presented twice in randomised order, yielding a combined playlist of 40 trials (Table S1).", "Four additional sounds (two M+, two M−) were presented prior to the playlist proper as familiarisation trials but were excluded from the final analysis.", "During pupillometry, participants were seated before a computer monitor in a dimly but uniformly illuminated room.", "The experimental trial design is schematised in Fig. 1.", "Pupil area was measured from the right pupil using an infra-red camera (Eyelink II; SR Research, Canada) mounted on a headset just below the line of sight while the participant fixated a 1 cm white circle in the centre of the monitor screen.", "All experiments were run using Eyelink II software.", "Each experimental trial was triggered once adequate visual fixation was achieved and pupil area was measured (sampling rate 250 Hz) over the entire trial duration.", "During each trial there was an initial brief silent interval (two seconds), followed by the sound stimulus (two seconds) and a final silent equilibration interval (seven seconds).", "On completion of the recording period, the participant was asked to rate how pleasant and how alerting they found the sound on modified Likert scales using a wireless mouse cursor, to provide indices of sound affective valence and arousal (no time limit was imposed).", "In off-line analysis, baseline pupil size and pupil responses were calculated and artefacts were identified and removed using a customised algorithm in Stata12®.", "This algorithm calculated maximal pupil response as change from baseline pupil area for each trial; baseline values were calculated as the mean value over the initial two second silent interval of the trial.", "Artefacts were chiefly blinks, easily detected due to their characteristic temporal trajectory; pupil data were discarded for the interval 50 msec prior and 750 msec following an artefact, to allow for completion of an ensuing light reflex (as determined from data collected in the healthy young control pilot group).", "In a simple regression model, the total proportions of data points removed due to artefacts did not differ significantly (p > .05) between sounds or between experimental groups.", "Maximum pupil area during a given trial was positively correlated with pupil area during the brief silent interval.", "In order to avoid this potentially confounding influence, the log ratio of maximal pupil area to baseline pupil area was used as the metric of pupil response for each trial (pupilmax).", "We assessed auditory semantic cognition in the patients and healthy control participants using a novel nonverbal auditory semantic task that did not rely on cross-modal labelling of sounds.", "Pairs of real (M+) sounds (n = 60, incorporating the 10 M+ sounds presented in the pupillometry experiment; see Table S2) were created in which the constituent sounds were associated either with the same sound source (e.g., a goose honking, a goose's wings flapping; 21 trials) or with different sources (e.g., a goose honking, a child yawning; 39 trials).", "Sound pairs in the ‘same’ and ‘different’ conditions did not differ systematically in the acoustic similarity of their component sounds; sound pair classification therefore relied on a semantic decision based on recognition of the sounds and could not be based on perceptual criteria.", "The auditory semantic test was administered separately from the pupillometry session.", "Sound pairs were presented in randomised order; the sounds comprising each pair were presented serially with a .5 sec inter-sound delay.", "The task on each trial was to decide whether the paired sounds came from the same source or different sources (‘Are the sounds made by the same kind of thing or different kinds of things?’),", "and the participant could respond verbally or by pointing to the corresponding written word on a prompt sheet.", "Participants were given practice trials before commencing the test, to ensure they understood the task and the response criteria; during the test, no feedback about performance was given and no time limits on responses were imposed.", "All data were analysed in STATA12.1®.", "Before calculating mean pupilmax responses for analysis, individual pupilmax responses to each sound were adjusted for any potential confounding effects of sound loudness, pleasantness and arousal (based on control participant rating data), as these factors are known to drive pupillary reactions (Goldwater, 1972).", "As individual participants had different numbers of missing data points, the relative effect of these potential confounds might further differ between participants: average effects of loudness and affective factors on pupilmax were therefore estimated separately using a linear mixed effects model with crossed random effects for participant and sound and this constant value for each sound was added or subtracted from the crude pupil responses.", "Mean pupilmax responses to different sound conditions and performance on the sound pair semantic classification task were then compared between participant groups (after adjusting for any demographic differences between groups) and correlations with peripheral hearing function, medication use (whether or not the participant was taking relevant, acetylcholinesterase inhibitor or antidepressant medication), and disease severity metrics (symptom duration and a nonverbal executive measure, reverse visual spatial span) were assessed using linear regression models.", "The difference in mean pupilmax responses to the M+ and M− sound conditions was assessed within each group with paired t-tests.", "In order to assess how well the M+ sounds presented in the pupillometry experiment indexed patients' general auditory semantic competence, we performed a separate subanalysis of the semantic classification task to assess just those trials that contained one of the M+ sounds presented in the pupillometry experiment (a subset of 14 semantic classification trials).", "In all analyses, a threshold p < .05 was accepted as the criterion for statistical significance.", "For 26 patients (12 bvFTD, 10 SD, four PNFA), a sagittal 3-D magnetization-prepared rapid-gradient-echo T1-weighted volumetric brain MR sequence (echo time/repetition time/inversion time 2.9/2200/900 msec, dimensions 256 × 256 × 208, voxel size 1.1 × 1.1 × 1.1 mm) was acquired on a Siemens Trio 3T MRI scanner using a 32-channel phased-array head-coil.", "Pre-processing of brain images was performed using the New Segment (Weiskopf et al., 2011) and DARTEL (Ashburner, 2007) toolboxes of SPM8 (www.fil.ion.ucl.ac.uk/spm) under Matlab 7.0® and following an optimised protocol (Ridgway et al., 2008).", "Normalisation, segmentation and modulation of grey and white matter images were performed using default parameter settings and grey matter images were smoothed using a 6 mm full width-at-half-maximum Gaussian kernel.", "A study-specific template mean brain image was created by warping all bias-corrected native space brain images to the final DARTEL template and calculating the average of the warped brain images.", "In order to adjust for individual differences in global grey matter volumes during subsequent analysis, total intracranial volume (TIV) was calculated for each patient by summing grey matter, white matter and cerebrospinal fluid volumes following segmentation of all three tissue classes.", "In the VBM analysis, separate voxel-wise linear regression models were used to assess associations in the combined patient cohort between regional grey matter volume (indexed as voxel intensity) and parameters of interest.", "Contrasts assessed positive and negative (inverse) correlations between regional grey matter volume and both overall pupil reactivity (individual overall mean pupilmax across the sound stimulus set) and differential pupil reactivity to sound semantic content (individual difference in mean pupilmax between the meaningful and meaningless sound conditions: M+ > M−).", "Age, TIV and syndromic group membership were included as covariates of no interest in each model.", "To help protect against voxel drop-out because of potentially marked local regional atrophy in particular scans, we applied a customised explicit brain mask based on a specified ‘consensus’ voxel threshold intensity criterion (Ridgway et al., 2009), whereby a voxel was included in the analysis if grey matter intensity at that voxel was >.1 in >70% of participants (rather than in all participants, as with the default SPM8 mask).", "Statistical parametric maps of regional grey matter volume correlating with pupil response parameters of interest were examined at threshold p < .05 after family-wise error (FWE) correction for multiple voxel-wise comparisons both at whole brain level and separately within three pre-specified regional volumes of interest.", "These regional volumes of interest were based on previous neuroanatomical work and encompassed the temporal lobes anterior to Heschl's gyrus (previously implicated in semantic analysis and signalling the behavioural value of sounds and other sensory objects: Goll et al., 2012; Golden et al., 2015; Hseih et al., 2011), insular cortex (implicated as an ‘autonomic hub’ in salience processing: Critchley et al., 2000; Zhou & Seeley, 2014) and dorsal brainstem including superior colliculi (previously identified as a key integrative site of autonomic effector response: Wang et al., 2012; Wang et al., 2014).", "Regional volumes were created by manually tracing from the template mean brain image using MRICron®.", "The participant groups did not differ in mean age and patient groups did not differ in mean symptom duration; gender distribution and years of education varied between participant groups and were therefore included as nuisance covariates in subsequent analyses.", "Participant groups did not differ in peripheral hearing performance nor did peripheral hearing performance correlate with pupil response or auditory semantic measures, after exclusion of one outlier patient with bvFTD (performance >2 standard deviations beyond rest of group).", "Baseline pupil size did not differ significantly between groups; the PNFA group but not the other groups showed a significant change (increase) in baseline pupil size over the course of the experiment (p < .05).", "Mean pupilmax values over the entire sound stimulus set and for the M+ and M− conditions separately did not differ between experimental groups.", "Over the course of the experiment, healthy individuals showed significant attenuation (p < .01) of pupilmax responses to M− but preserved responses to M+ sounds; the AD group showed significant (p = .03) attenuation of pupilmax responses to M+ sounds, while other patient groups showed no significant attenuation of pupil responses over time.", "M− sounds were rated as less pleasant than M+ sounds by the healthy control group (p < .01), but not the patient groups, while M+ and M− sounds did not differ significantly in mean arousal ratings (see Table S1).", "After adjusting for these factors and for measured peak loudness, the healthy control group showed a non-significant trend (p = .09) toward increased mean pupilmax responses to M+ compared with M− sounds (see Fig. 2).", "All patient groups showed significantly greater mean pupilmax responses to M+ than to M− sounds (SD, AD, p < .01; bvFTD, PNFA, p < .05); a post hoc analysis of the small subgroup of patients with pathogenic mutations suggested some heterogeneity within the bvFTD group [significantly greater mean M+ responses than M− responses in MAPT mutation cases (p < .01), a trend to a significant difference in C9orf72 mutation cases (p = .1) but no significant difference in sporadic bvFTD cases (p = .51)].", "The increased pupil response to M+ compared M− sounds was significantly larger (p < .05) in the SD group and the AD group (though not in other patient groups) than in healthy controls; the magnitude of this differential pupil response did not differ significantly between patient groups.", "There were no correlations between overall pupil reactivity or the magnitude of the differential pupilmax response to M+ versus M− sounds and overall disease severity (as indexed by symptom duration and reverse visual spatial span) or medication use.", "Mean performance for each of the participant groups on the nonverbal auditory semantic classification task is summarised in Table 1.", "The healthy older control group overall achieved high sub-ceiling scores on this test.", "Relative to the healthy control group, the PNFA group showed no auditory semantic deficit while each of the other syndromic groups showed impaired performance (all p < .01); comparing patient groups, the bvFTD, SD and AD groups performed worse (p < .01) than the PNFA group.", "Auditory semantic performance profiles for the patient groups relative to the healthy control group were similar for the subset of sounds also represented in the M+ condition of the pupillometry experiment.", "Patients' auditory semantic performance did not correlate with mean overall pupil reactivity or with mean pupil responses to M+ or M− sounds separately.", "However, auditory semantic performance did show a significant inverse correlation with the magnitude of the difference in mean pupil responses to M+ versus M− sounds, in the combined patient cohort (r2 = .2; p < .01; see Fig. 2) and in each of the patient groups showing an auditory semantic deficit (bvFTD r2 = .8, p < .01; SD r2 = .6, p < .05; AD r2 = .9, p < .01).", "Grey matter associations of pupil response parameters for the combined patient cohort are summarised in Table 2 and statistical parametric maps are presented in Fig. 3.", "After FWE correction for multiple voxel-wise comparisons, grey matter associations were not identified at whole brain level but were identified within the pre-specified regional anatomical volumes of interest.", "Overall pupil reactivity to sound (mean pupilmax across the stimulus set) was significantly (pFWE < .05) positively correlated with grey matter in the region of the superior colliculus; no grey matter areas showing a significant inverse correlation with overall pupil reactivity were identified.", "The magnitude of the difference in pupil responses to M+ versus M− sounds was significantly (pFWE < .05) inversely correlated with grey matter in left anterior superior temporal cortex; no grey matter areas showing a significant positive correlation with this pupil response difference measure were identified.", "Here we have demonstrated that dementia syndromes have different profiles of autonomic responses to real and synthetic nonverbal sounds, after controlling for elementary acoustic and affective factors.", "This differential autonomic response was present in all patient groups but was largest (and significantly greater than the healthy control response) in patients with SD and AD.", "Moreover, the magnitude of the differential response was inversely related to auditory nonverbal semantic competence across dementia syndromes but was not related to overall autonomic reactivity, more general disease severity or medication effects.", "Considered together, these findings suggest that impaired semantic processing of nonverbal sounds confers an enhanced physiological salience signal in these dementia syndromes that is separable from other salience cues such as emotional value and arousal potential.", "Little information is available concerning the physiological coding of semantic salience.", "However, semantic load and in particular, semantic ambiguity along behaviourally relevant dimensions (such as threat) have been shown to modulate cerebral and autonomic responses to both visual and auditory stimuli in the normal brain (Farrow et al., 2012; Werner & Noppeney, 2010).", "In neurodegenerative diseases, ‘primitive’, behaviourally relevant cues to moving (approaching vs withdrawing) sound sources have been shown to modulate autonomic responses and this modulation was enhanced in SD relative to other dementia syndromes (Fletcher, Nicholas, et al., 2015c).", "Though the evidence remains limited, these previous findings in the healthy brain and in neurodegenerative disease are in line with the present data.", "If the damaged semantic system cannot identify potentially meaningful sounds, then this unresolved ambiguity may render such sounds behaviourally salient and engage physiological effector mechanisms for salience coding.", "This autonomic response to auditory semantic salience, though amplified in dementia syndromes, was also evident in attenuated form in the healthy control group here: healthy controls showed a trend toward enhanced pupil responses to real compared with synthetic sounds but (unlike the patient groups) showed habituation of pupil responses to synthetic sounds over the course of the experiment.", "These data suggest that both highly familiar real sounds and synthetic sounds of the kind presented in the pupillometry experiment are rapidly disambiguated by the normal semantic system (either as meaningful auditory objects or as meaningless sound events).", "In this formulation, SD as the paradigmatic disorder of the human semantic system is an important test case: it is noteworthy that here as in previous work (Fletcher, Nicholas, et al., 2015c) patients with SD showed enhanced sensitivity to auditory salience.", "Compared with other canonical dementia syndromes, patients with SD would be predicted to have the most marked and selective difficulty with disambiguation of meaningful sound sources while retaining relatively intact mechanisms for perceptual coding of sound features and programming autonomic responses (Beissner et al., 2013; Bozeat, LambonRalph, Patterson, Garrard, & Hodges, 2000; Goll, Crutch, Loo, et al., 2010).", "We do not argue that autonomic responses per se are normal in SD: while the present study did not address these processes directly, other work suggests that the coupling of cognitive to autonomic effector processing of sound stimuli is altered in SD as well as in bvFTD and AD (Fletcher, Nicholas, et al., 2015c, 2015d).", "However, the processing of complex sounds such as those presented here engages hierarchical and distributed mechanisms both in the healthy brain and in SD (Goll, Crutch, & Warren, 2010; Goll et al., 2012), providing candidate neural substrates for relatively intact physiological signalling of auditory salience in patients with dementia.", "The complex spectrotemporal structure of the stimuli used here would allow the damaged semantic system to encode perceptual features characteristic of real sounds but lacking in the synthetic (spectrally inverted) sounds; the presence of such spectrotemporal features could label natural sounds for further semantic analysis even if identification were not achieved.", "A VBM analysis of our combined patient cohort identified neuroanatomical correlates of pupillary responses to sound in a distributed cortico-subcortical network.", "Midbrain grey matter in the region of the superior colliculus was associated with overall pupillary reactivity to sound.", "The superior colliculus is involved in orienting responses (Krauzlis, Lovejoy, & Zenon, 2013; Kustov & Robinson, 1996; Wang et al., 2014, 2012) and in processing potential threat in ambiguous stimuli (Farrow et al., 2012).", "This region mediates output to the eye, head and neck and arm and shoulder via the thalamus from cortical areas including the frontal eye fields.", "Stimulation of the superior colliculus results in coordinated head and eye gaze shifts (Freedman, Stanford, & Sparks, 1996) and more recently, it has been shown that stimulation at thresholds below those necessary to evoke saccadic eye movements results in pupillary dilatation in both monkeys and owls (Netser, Ohayon, & Gutfreund, 2010; Wang et al., 2012), suggesting a role for this region in coding salience responses.", "The present evidence suggests a need for some caution in interpreting the role of superior colliculus in salience coding in neurodegenerative disease.", "While we did not identify an overall significant impairment of general pupil reactivity across the patient cohort, this autonomic parameter varied between patient groups (for example, the AD group but not the other groups showed significant attenuation of pupilmax responses to real sounds over time), suggesting that larger patient cohorts may be required to more fully elucidate the role of this midbrain effector region in salience processing in dementia syndromes.", "Enhanced differential pupillary response to auditory semantic salience (real vs synthetic sounds) in this patient cohort was associated with atrophy of left anterior temporal cortex.", "This neuroanatomical association is in line with the inverse association between pupillary salience responses and auditory semantic impairment in the present study and corroborates previous work implicating an anterior temporal lobe network in semantic processing of sounds and other sensory objects (Lambon Ralph et al., 2010; Visser & Lambon Ralph, 2011).", "The present evidence further suggests that this cortical region is involved in mediating autonomic responses to sound meaning.", "Although the links between semantic memory and autonomic networks remain poorly defined, current formulations increasingly emphasise the role of distributed cortico-subcortical networks including the anterior temporal lobe in ‘appraising’ the behavioural significance of sensory stimuli and programming appropriate physiological responses (Kafkas & Montaldi, 2012; Zhou & Seeley, 2014).", "This may be particularly relevant under conditions of perceptual or semantic ambiguity (Farrow et al., 2012; Zekveld, Heslenfeld, Johnsrude, Versfeld, & Kramer, 2014): it is therefore plausible that this linkage might be upregulated in the setting of a damaged semantic system.", "This study has several limitations that could be addressed in future work.", "Larger patient cohorts would improve power to detect effects on semantic salience processing and potentially, further stratify dementia syndromes based on both cross-sectional and longitudinal profiles of autonomic reactivity.", "It would be of particular interest to assess genetic mutation cohorts with defined molecular substrates that are potentially associated with specific auditory salience signatures (Fletcher, Downey, et al., 2015a, in press): inclusion of mutation carriers would also allow assessment of the biomarker potential of autonomic indices from earliest clinical disease stages, and might be achieved via multi-centre collaborative studies (Rohrer et al., 2015).", "Our work leaves open the possibility that other autonomic modalities (such as skin conductance) might show differential sensitivity to semantic impairment, and ideally these modalities would be compared directly.", "The effects of medications that affect autonomic function should be assessed directly, both in order to calibrate for any confounding impact on endogenous autonomic responses and to determine modulatory effects with therapeutic potential.", "The linkage between semantic and autonomic processing could be more directly explored using functional neuroimaging; this is particularly pertinent as the interaction of these mechanisms might differ between diseases (Fletcher, Nicholas, et al., 2015d).", "More fundamentally, the present study suggests a hypothesis concerning the role of auditory semantic ambiguity in triggering physiological salience responses that should be assessed in the healthy brain by manipulating sound stimulus ambiguity and semantic associations systematically.", "Taking the above caveats into account, our findings suggest that autonomic responses index semantic impairment across dementia syndromes.", "The present study should be regarded as preliminary: the sensitivity, specificity and translatability of physiological metrics require further systematic substantiation.", "Nevertheless, such ‘physiological phenotyping’ of dementia syndromes might be developed as a useful tool in these diseases.", "The dementias collectively present substantial problems of nosology, diagnosis and disease tracking; these issues are particularly pressing for diseases in the FTLD spectrum for which syndromic boundaries are often difficult to define but robust biomarkers that can be applied across syndromes are lacking.", "Capturing disease effects near the time of clinical conversion or in the later stages of disease is challenging, as existing neuroanatomical and neuropsychological biomarkers are relatively insensitive; yet accurate disease diagnosis and tracking will be essential to assess the impact of therapeutic interventions in future clinical trials.", "Studies such as this one could in future be used to guide revised diagnostic criteria for neurodegenerative syndromes, informed by pathophysiological data.", "Moreover, there is considerable interest in identifying new biomarkers with wider applicability across diseases and disease stages.", "Autonomic salience signalling warrants further evaluation both as a window on disease neurobiology and as a candidate novel physiological biomarker that could potentially complement or extend the range of conventional cognitive instruments." ]

[]

[ "BIOC" ]

[]

[ "1303", "1304", "1307", "1311", "1312", "1315" ]

[ "The biosynthesis of cytochrome c is achieved by different systems in different organisms [1].", "The heme group is covalently attached to apocytochrome in a critical and poorly understood step in this post-translational modification process.", "A characteristic and highly conserved CXXCH motif occurs in the cytochrome amino acid sequence; the two cysteine side chains are covalently linked to the vinyl groups of the heme to form holocytochrome c.", "The histidine in the motif usually acts as a ligand to the iron in the heme.", "In the mitochondria of yeast and animals a single protein attaches heme to cytochrome c; the protein is called holocytochrome c synthase (HCCS) and is also referred to as cytochrome c biogenesis System III or heme lyase.", "HCCS functions in the mitochondrial intermembrane space, upon import of the apocytochrome from the cytosol [2].", "The import of apocytochrome c, coordinated by HCCS, has a close relationship with the heme attachment.", "There is an absence of signal sequence cleavage from the protein in the process of import, except the N-terminal methionine [3].", "Although apocytochrome c is able to reversibly enter and exit through the mitochondrial outer membrane [4], several studies show that there is an interaction between the apocytochrome c and HCCS to form a complex [5,6].", "The mechanism whereby the mitochondrial HCCS attaches heme to apocytochrome remained largely uninvestigated for many years, save for a finding that it would not attach heme to structurally related bacterial c-type cytochromes and that one or more CP motifs (a cysteine-proline sequence has often been considered as a heme binding motif in several proteins) in the HCCS sequence were found to be important for HCCS function [7].", "The situation has changed in the past three years because the importance of the CP motifs has been questioned [8] and several studies have shown that the N-terminal region of the mitochondrial cytochrome c sequence is critical for attachment of heme [9–12].", "Arguably surprisingly, a conserved phenylalanine at position 15 in the yeast protein and 11 in horse heart, but in each case separated by only three residues from the CXXCH motif, and for many years thought only to be essential for cytochrome c structural integrity, has been shown to be essential for heme attachment but not necessary for properly folded protein.", "This has left open questions including whether phenylalanine in this position is absolutely essential in the sense of both the nature of the residue and its exact position relative to the CXXCH motif.", "It was especially notable that the cytochrome c of Paracoccus denitrificans, which is not processed by HCCS, has a phenylalanine residue spaced only one residue differently than in mitochondrial cytochromes c [13].", "Some previous studies have focused on the importance of the complete CXXCH motif which is near to the N-terminus in mitochondrial cytochromes c.", "The two cysteine residues and the histidine residue in the motif are generally highly conserved with a few exceptions.", "There have been reports of both success [14,15] and failure [16] of heme attachment to SXXCH and CXXSH variants of yeast and human cytochrome c.", "A CXXCR variant of yeast cytochrome c was also reported to be able to be expressed by plasmid-based expression systems, but not by the native cytochrome biosynthetic system of yeast itself [17–19].", "In the prokaryotic world, various heme-containing proteins have motifs different from CXXCH motif, such as the CXXCK motif of NrfA, a bacterial nitrite reductase.", "Analysis of the CXXCK, CXXCM and CXXCR variants of a double-cysteine variant of cytochrome b562 had been carried out with the cytochrome c maturation (Ccm) system that is found in many Gram-negative bacteria including Escherichia coli; all the variants failed to mature [20].", "There are also studies reporting the outcome of varying the number of residues between the two cysteines or mutating these residues [11,21], with mixed results.", "There is less information about the tolerance of HCCS to similar changes in its substrate.", "Here we utilise well-established systems with either one-plasmid or two-plasmid methods of HCCS-cytochrome c co-expression in E. coli [22,23] for the investigation of tolerance of absence of the cysteine and histidine residues in the CXXCH motif and of variations in the N-terminal sequence.", "In this type of approach a failure to observe heme attachment to a variant cytochrome could result from either failure of catalysis by HCCS, severe instability of the apo proteins or attachment of heme followed by rapid degradation of unstable protein.", "We and others [11,12] introduced the control of seeking to mature the same cytochrome variants in the E. coli periplasm using the Ccm system, or System I, which has much broader substrate specificity than System III; it is able to carry out heme attachment under most circumstances when a CXXCH motif is present [24] irrespective of neighbouring residues.", "By applying this strategy to cytochrome c variants, we thus have a way to distinguish HCCS substrate recognition properties from the features responsible for structural integrity of the cytochrome c protein.", "In addition, there are examples of heme attachment by mitochondrial extracts [25] to cytochrome c fragments [9].", "In this context, we also investigated several truncations of Saccharomyces cerevisiae cytochrome c to test for heme attachment to these fragments by HCCS.", "The present paper describes experiments that address these issues, some of which have also been addressed in complementary ways in other recent papers to which we return in the Discussion.", "There is also some ambiguity as to which residues in the CXXCH motif are essential for processing, with reports that variation at the H is possible and that either of the cysteine residues can be changed.", "These issues are also considered in the present paper.", "A system with HCCS and cytochrome c on two different plasmids (two-plasmid system), from which iso-1 cytochrome c (CYC1) and HCCS from S. cerevisiae are expressed from separate plasmids, as well as a system with both genes on one plasmid (single-plasmid system) in which the horse cytochrome c and S. cerevisiae HCCS are encoded on the same plasmid, were used [13,23].", "The single-plasmid system has been optimised for high levels of production of horse holocytochrome c, facilitating detection and analysis of the product.", "The two-plasmid system has the advantage that both genes are from the same organism which might simplify interpretation of interactions between the two proteins.", "Nonetheless, yeast HCCS matures both yeast and horse cytochromes c.", "The QuikChange® Site-Directed Mutagenesis Kit with KOD polymerase (Stratagene) was used with the wild-type plasmids (see Table 1) as templates to generate the variants.", "The constructs were all sequenced (Geneservice, Oxford) and then transformed into E. coli strain BL21(DE3).", "A list of the constructs and strains is shown in Table 1.", "Horse heart cytochrome c or Strep-tagged yeast cytochrome c were expressed in E. coli strain BL21(DE3) with yeast HCCS.", "Periplasmically targeted Strep-tagged yeast cytochrome was constructed by the addition of the Pseudomonas aeruginosa cytochrome c551/552 pre-sequence to pOScyc1.", "The resulting plasmid was then co-expressed with the E. coli cytochrome c maturation system genes on plasmid pEC86 in BL21(DE3) [26].", "Isopropyl β-d-1-thiogalactopyranoside (IPTG) was used as the induction agent.", "Cells were cultured in LB (Luria–Bertani) media or 2-YT media supplemented with 100 μg/ml ampicillin (and 33 μg/ml chloramphenicol in the case of the two-plasmid system) for 20 h in a 30 °C incubator with 200 rpm shaking.", "125 ml cultures in 250 ml flask were used in most experiments.", "500 ml cultures in 2 l flasks were used when a larger quantity of cell extract was required for analysis.", "The periplasm was separated to produce spheroplasts using a procedure described previously [27].", "Cytoplasmic extraction was performed by sonication of the spheroplasts and 35 min centrifugation at 16 000×g.", "Cell extracts were analysed by SDS–PAGE gel electrophoresis (pre-cast 10% Bis-Tris gels, NuPage) followed by heme-staining as described [28].", "UV–vis spectroscopy was performed on extracts using a Perkin Elmer UV–visible Lambda 2 spectrometer.", "A 19% (vol/vol) pyridine solution in 0.15 M NaOH was used to determine pyridine hemochrome spectra [29].", "The position of the α-band in the visible spectra of denatured hemoproteins in a pyridine solution is indicative of the saturation of the heme vinyl groups and therefore whether no, one or two heme-protein thioether bonds are present.", "Western blots were used to determine the level of expression of cytochrome c in cell extracts.", "A conjugated Strep alkaline phosphatase antibody (IBA) was used for detection.", "SIGMAFAST BCIP/NBT tablets were used for development.", "The cysteine residues and the histidine residue in the CXXCH of the cytochrome c of Equus caballus were substituted with alanine, lysine, methionine or arginine producing the variants C15A, C18A, H19K, H19M and H19R.", "These were co-expressed with S. cerevisiae HCCS and analysed by heme-stained SDS–PAGE gels, as shown in Fig. 1 (A).", "The C15A variant (AXXCH) alone was matured, as seen by a heme-staining band at the expected molecular mass, and all the other variants did not undergo heme attachment.", "The gels were overloaded to maximise detection.", "The level of holocytochrome c for the C15A variant is lower than wild-type according to the intensity of the absorption spectra normalised for cell mass.", "According to UV–vis spectra, as shown in Fig. 1 (B), the wavelengths of the absorption maxima in the pyridine hemochrome spectra were shifted, i.e. the α peak shifted from 550 nm to 553 nm, as would be expected for a cytochrome c with a single bond between the heme and the protein.", "A stop codon was introduced in different positions in the gene of S. cerevisiae iso-1 cytochrome c (CYC1) to remove different lengths of the C-terminal region, producing three variants: G29X, H45X and K60X.", "The X indicates the position of the inserted stop codon, as indicated on the sequence alignments in Fig. 2 (A).", "The truncated variants and the wild type protein were co-expressed with S. cerevisiae HCCS in E. coli; Fig. 2 (B) shows the results of heme-stained SDS–PAGE analysis of cytoplasmic extracts.", "Heme-staining bands were observed with predicted molecular masses (∼6.5 kDa and ∼8 kDa) for H45X and K60X variants, but no reproducible product for the shortest variant, G29X, was detected.", "The low levels of heme-containing truncations can be ascribed to relative instability of these molecules.", "We explored further what, if any, other residues, could substitute for the crucial F15, identified by us and others as key for recognition by HCCS.", "Four additional replacements (Fig. 3) of the phenylalanine in the N-terminal region of S. cerevisiae cytochrome c were made (F15Y, F15W, F15I and F15E).", "A F15A variant (which was reported in previous work [12]) was also included in this analysis for comparison with the new variants.", "The results of SDS–PAGE analysis and UV–vis absorption spectra in Fig. 4 (A) and (C) show that the F15A and F15E replacements abolished holocytochrome c production completely, whereas the maturation of cytochrome c is attenuated, but not abolished, to different extents in both the F15W and F15I variants.", "The F15Y variant retains a high level of heme attachment.", "This implies the aromatic R-group of residue F15 is important in recognition by HCCS, as replacement of phenylalanine by both tryptophan and tyrosine had smaller impacts on the level of heme attachment than replacement with other residues including the hydrophobic isoleucine.", "This might indicate the importance of involvement of an aromatic residue in a π–π or π-cation interaction.", "The wavelengths of the peaks in the α-band region of the pyridine hemochrome UV–vis absorption spectra of the variants were at 550 nm as for the wild type, as shown in Fig. 4 (C), indicating that the two heme-to-protein thioether bonds had formed in these cases.", "However, the Western blot result in Fig. 4 (B) shows that while F15A and F15E variants produce no holocytochrome c, there was a significant level of apocytochrome c produced in the cytoplasmic extracts for both variants.", "On the contrary, the overall cytochrome c (apo and holo) levels of F15W and F15I variants are lower than the others.", "One possibility for the abolition of the holocytochrome c production for F15E and F15A could be due to intrinsic structural disturbance introduced by the replacements, instead of the failure of recognition by HCCS.", "The observation of readily detectable levels of apocytochrome of the F15A and F15E variants in cytoplasmic extracts suggests that the lack of heme attachment to these two variants is caused by loss of recognition by HCCS, rather than structural instability of the proteins in either the apo or holo forms.", "In order to further demonstrate the importance of the F15 residue in the context of recognition but not structural significance, a periplasmically targeted yeast cytochrome c was constructed and tested by co-expressing with the Ccm system from the plasmid pEC86.", "The 23-amino-acid long periplasmic targeting sequence MKPYALLSLLATGTLLAQGAWAED from P. aeruginosa cytochrome c551/552 was inserted at the beginning of the sequence of Strep-tagged S. cerevisiae cytochrome c. Periplasmically targeted variants F15A, F15Y, F15W, F15I and F15E were made from this construct.", "Despite efforts to optimise the procedure, periplasmic extraction of the wild-type and variants were incomplete as large amounts of periplasmically targeted cytochrome remained as contaminants in the cytoplasmic portion after the extraction.", "All periplasmically targeted variants, except F15E, expressed readily detectable quantities of holocytochrome c. Fig. 4 (D) shows SDS–PAGE heme-staining analysis of whole-cell extracts for the five variants and wild-type cytochrome c.", "The periplasmically targeted F15I variant was matured to a similar level as the F15Y and F15W variants.", "The larger size of F15Aperi and F15Eperi is attributed to incomplete cleavage of the targeting signal sequence as observed previously [12].", "Both of them also show a much lower level of production of holocytochrome c than wild-type.", "The key point here is that the Ccm apparatus of E. coli that functions in the periplasm essentially recognises only the CXXCH motif and thus variant mitochondrial holocytochrome c with F15A and F15E can be formed, whereas the HCCS system is unable to process these variants.", "Taken together with the detection of apo F15A and F15E proteins, this is strong evidence that the requirement for a particular residue at position 15 is a consequence of the demands of HCCS and not the demands for structural integrity of holocytochrome c.", "Amino acid R18 in S. cerevisiae cytochrome c is a highly conserved residue, and is directly adjacent to the CXXCH motif in the amino acid sequence on the N-terminal side.", "Its significance in HCCS substrate recognition was analysed in previous work.", "The equivalent residue to this arginine in the N-terminal region in P. denitrificans c550 is a lysine which has an adjacent asparagine residue; however, in S. cerevisiae cytochrome c there are three residues (KTR) between the phenylalanine F15 and CXXCH motif but in P. denitrificans just two residues (NK).", "The sequence alignment of two deletion variants with the wild type S. cerevisiae cytochrome c and the P. denitrificans c550 is shown in Fig. 3.", "To test the importance of this spacing difference and its possible implication in the substrate recognition by HCCS, two variants del16K and del17T in yeast cytochrome c were made by deleting either lysine or threonine.", "Fig. 5 shows the heme-stained SDS–PAGE, UV–vis absorption spectra and Western Blot analysis of the cytoplasmic extracts of cells expressing the deletion variants co-expressed with yeast HCCS.", "Neither of them had heme attachment completely abolished; however, the levels were significantly diminished.", "The total cytochrome c levels of the two deletion variants are also significantly lower than wild-type, according to the Western Blot in Fig. 5 (B).", "The pyridine hemochrome UV–vis spectra in Fig. 5 (C, inset) shows while the levels of the two deletion variants are significantly lower than that of wild-type, the wavelengths of the absorption maxima of the variants are shifted from 550 nm (wild-type) to 552.5 nm (α peak).", "As it was necessary to examine the possible role of intrinsic instability in the variants, the same deletion variants were also made in the periplasmically targeted S. cerevisiae cytochrome c.", "These were co-expressed with the Ccm system from pEC86.", "In Fig. 5 (D), the wavelengths of the peaks in the α-band region of the pyridine hemochrome UV–vis absorption spectra of the variants are same as the wild type value at 550 nm.", "The heme-stained SDS–PAGE analysis of cytoplasmic extracts results shows that both of the variants are produced in considerable amounts by the Ccm system as shown in Fig. 5 (E).", "The Ccm apparatus is able to mature the deletion variants of yeast cytochrome c without a change in the nature of the heme binding.", "Thus cytochrome c seems capable of folding to a stable structure once heme is added to the deletion proteins by the Ccm system.", "Attempts at the reciprocal experiment, addition of an alanine residue to the P. denitrificans cytochrome c550 to increase the spacing between F and CXXCH to three, did not result in the production of holocytochrome c in detectable amount (not shown).", "In summary, HCCS appears to have misattached heme to the two deletion mutants but when the same two proteins were expressed in the periplasm, the Ccm system processed them ‘normally’ as judged by both a pyridine hemochrome spectra and relative intensities on a heme-stained gel.", "Thus the cytochrome c itself does not appear to be compromised by the deletions but rather it is the action of HCCS that is affected.", "The misattachment is most likely the formation of a single thioether bond rather than the usual two in the HCCS-processed proteins.", "This work confirms conclusions from several previous studies which showed that HCCS can attach heme to an AXXCH motif and supports the majority of studies [14,15,30] but very little or no heme attachment to CXXAH motif.", "The visible absorption spectra of the AXXCH variant are consistent with a single thioether bond and this variant is comparable with the wild type c-type cytochromes from trypanosomes.", "The absence of the second thioether bond in the latter type of protein has been shown to cause little perturbation to the overall structure of the cytochrome [31].", "However, although HCCS can in principle form the ‘trypanosome’ type cytochrome c, with one thioether bond those organisms lack the HCCS system but contain a putative novel System V for cytochrome c maturation.", "Interestingly, a recent study has shown that the cytochrome c maturation system in those organisms cannot produce a correct version of a CXXCH-containing cytochrome [32], thus adding to the evidence that a system distinct from HCCS is functional in that group of organisms and that HCCS needs the entire CXXCH motif for optimal performance.", "The failure of heme to attach to the H19M, H19K and H19R variants further demonstrates the importance of the histidine residue and thus it seems that the HCCS system cannot process CXXCR.", "No cytochrome c biogenesis specific system, except specific variants of Systems I or II will recognise anything except histidine.", "Detectable amount of two truncated forms of S. cerevisiae cytochrome c were observed.", "The absence of reproducible heme attachment of the shortest fragment truncated, G28X, is probably due to degradation.", "The holocytochrome levels for the two longer fragments are significantly lower than the wild type, probably due to the reduced stability, and thus degradation of these N-terminal fragments of cytochrome c.", "The heme attachment to the truncated protein is consistent with the recent demonstration that heme was attached to an N-terminal fragment fused to a PDZ domain [33].", "The truncation experiments also add to the evidence that the methionine residue M83, an axial ligand to the heme iron in holo cytochrome c, is not required for HCCS activity; it has been reported earlier that the M83A protein is readily produced by HCCS [34], certainly to a greater extent than the truncations.", "Within the N-terminus of mitochondrial cytochrome c, the importance of the phenylalanine residue F15 (yeast numbering) for maturation has been firmly established.", "The formation of stable and folded F15A (or F11A for the horse heart and human proteins) cytochrome c through periplasmic maturation using the Ccm system has shown that, contrary to a long-held assumption, the conserved F at position 11 is not crucial for the fold of the protein but rather for the action of HCCS.", "The consequences of substitution of F15 led to mixed results – F15Y variant retained a similar (80–90%) level, as judged by pyridine hemochrome analysis of holocytochrome production as the wild-type, whereas F15W and F15I variants had levels around 40% and 20% of that in the wild-type.", "One possible explanation for these observations is the aromatic nature of the R-groups of tyrosine (substantially) and tryptophan (moderately so) are recognised by HCCS and are compatible with a stable folded holo-cytochrome c.", "The bulky isoleucine is less successful in imitating phenylalanine in tertiary structure.", "Nevertheless, F is the preferred residue, on the basis of experiments described in the present paper, and that can explain its conservation.", "Glutamate and alanine, with very different R-group characteristics, are not able to replace the aromatic phenylalanine, hence holocytochrome production was abolished.", "However, as the presence of high levels of apocytochromes F15E and F15A variants demonstrates, these apo forms were available as potential substrates for HCCS and thus the absence of cytochrome maturation in these variants may be explained by lack of substrate recognition by HCCS.", "Our finding that the Y15F variant is well tolerated by HCCS from yeast is of interest in respect of the recent report of a similar finding for human HCCS [30].", "In the latter case it was speculated that this might be an adaptation in the human enzyme to allow it to recognise both cytochrome c and cytochrome c1, the latter having Y at the corresponding position.", "However, our work shows that even though yeast has a dedicated enzyme for cytochrome c1 assembly the HCCS can still tolerate Y in that position.", "The importance of an F residue at position 15 (yeast numbering) is now well established but it has been noted that apocytochromes c2 of both P. denitrificans and Rhodobacter capsulatus each contain an F residue, but at the equivalent of position 16; these apo proteins are not matured by HCCS [30,35].", "We found that deleting a K or a T between the F and the CXXCH motif in the mitochondrial cytochrome, and thus making the spacing similar to the bacterial proteins, attenuated the production of holo cytochrome c by HCCS but far from abolished it.", "Other had we found that inserting an alanine in the P. denitrificans sequence between the F and the CXXCH did not confer recognition by HCCS (unpublished results), which agrees with the results of a similar experiment [30] using human HCCS and R. capsulatus cytochrome c2.", "However, in two recent related studies that appeared after completion of the present work, recognition of a bacterial (R. capsulatus) cytochrome c by HCCS could be achieved either by a K11 insertion between in combination with an E9L substitution [35] or insertion of an A together with mutations of two glutamate residues at the N-terminus (E8K_E10I_Ala) in R. capsulatus cytochrome c2 [30].", "In both the latter cases the spacing between F and CXXCH was increased to three residues but, there are clearly additional factors at work than just the spacing difference in different cytochrome amino acid sequences.", "Thus, it could be suggested that the phenylalanine residue and spacing in the N-terminal region of cytochrome c are both essential for optimal heme attachment performance by HCCS, although some heme attachment can occur with a spacing of two.", "The shift in wavelength of the alpha peak of the spectra of the deletion variants of S. cerevisiae indicates a change of nature in heme binding to the CXXCH motif locally, due to the change in spacing between residues F15 and C19." ]

[ "Holocytochrome c synthase (HCCS) does not attach heme to cytochromes lacking the histidine in the CXXCH motif.", "HCCS can recognise C-terminally truncated cytochromes c.", "The aromatic nature of, or possibly shape complementarity to, F15 in cytochrome c is important for recognition by HCCS.", "The spacing of the phenylalanine relative to the CXXCH is a recognition feature." ]

[ "BIOC", "CHEM" ]

[ "Glutathione", "Hydrogen sulfide", "Oxidative stress", "Persulfides", "Reactive species interactome", "Redox status", "Thiol-maleimide michael addition" ]

[ "1308", "1605" ]

[ "Many biological processes that have previously been associated with an overproduction of reactive oxygen species (ROS) and/or an impaired antioxidant and free radical scavenging capacity were thought to culminate in ‘oxidative stress’, cell death and tissue damage.", "More recently, such conditions are interpreted to reflect situations in which a shift in redox poise has occurred, affecting both global and regional redox status and cysteine-based redox signaling [1–3].", "Importantly, unfavorable outcomes may not be inevitable if intervened early on.", "Consequently, there is an increased interest in analytical methods that provide a more refined mapping of the associated metabolic changes and enable further study of the underlying mechanisms.", "In parallel with those developments, there has been a resurgence of interest in the biological effects of hydrogen sulfide (H2S)2 and related oxidation products such as persulfides and polysulfides [4–6].", "No specific biological targets have yet been identified for those compounds, and many of their effects appear to be mediated by interaction with metals, heme proteins or redox interaction with other sulfur species.", "Moreover, sulfide is involved in post-translational protein modification (persulfidation, S-sulfhydration) whereby reactive cysteine groups involved in redox signaling are modified, resulting in altered chemical reactivity and protein function [7].", "Interestingly, ROS were shown to chemically and functionally interact with nitric oxide (NO) and other reactive nitrogen species (RNS), as well as with sulfide-related reactive sulfur species (RSS) such as thiyl radicals, persulfides and polysulfides.", "Our group recently proposed that the interaction of ROS, RNS and RSS with each other and with their thiol targets constitute a ‘reactive species interactome’ (RSI) that regulates fundamental cellular processes involved in stress signaling [3].", "The RSI enables single cells and whole organisms to sense and adapt to alterations in nutritional, metabolic and environmental conditions [3].", "A corollary of this concept is that assessment of only a few constituents of this RSI as “markers of oxidative stress” (e.g. oxidized lipids) will not be sufficient to provide useful insight as they cannot adequately capture the complexity of the chemical interactions within the RSI.", "This may explain why the redox community has been struggling in successfully identifying the key regulatory nodes and operating principles of this interaction network, and to e.g. understand how specific thiols and redox switches like the nuclear factor erythroid 2 [NF-E2]-related factor 2 (Nrf2) contribute to redox homeostasis in health and disease.", "Thus, the renewed interest in sulfide related phenomena, antioxidant processes and redox signaling demands quantification of pertinent redox-regulated events in much higher resolution than hitherto accomplished.", "Given that glutathione is the major intracellular low-molecular-weight (LMW) aminothiol, much of the earlier ‘oxidative stress’ literature revolved around pathways of formation and degradation of this ubiquitous and (according to Helmut Sies) “inevitable” antioxidant [8], besides the measurement of protein, lipid and DNA oxidation products as biomarkers of an increased oxidative burden.", "As a result, many different methods have been developed to quantify glutathione and related metabolites in blood and tissues [9–12], and the ratio of reduced over oxidized glutathione (GSH/GSSG) has been used for decades as a sensitive oxidative stress biomarker [11,13,14].", "In many of these methods the sulfhydryl (-SH) group of the aminothiol is derivatized with an electrophile such as monobromobimane (mBB), iodoacetic acid (IAA), iodoacetamide (IAM) or N-ethylmaleimide (NEM).", "This reaction step serves two purposes: i) it prevents ambient air mediated oxidation of the thiols to their corresponding disulfides [10,15] and ii) it conveniently forms a fluorescent product [16], often improving sensitivity limits for detection.", "More recently, the cysteine/cystine (Cys/CysSS) redox couple emerged, along with GSH/GSSG, as a powerful predictor of cardiovascular mortality [17].", "Circulating total homocysteine has been used as an integrative biomarker of folate and methionine metabolism, cobalamine deficiency and cardiometabolic risk for some time [18], while the mixture of “free thiols” and “protein-bound thiols” (including glutathione, cysteine, and homocysteine; please refer to Box 1 for definitions of terms in italics) was proposed to reflect the plasma redox thiol status more than 2 decades ago [19,20].", "Most of the methods used to determine these species employ either high pressure liquid chromatography (HPLC) or a similar chromatographic separation technique coupled to a ultraviolet, fluorescence or electrochemical detector.", "In more recent applications these detectors have been replaced by mass spectrometers, potentially offering more specific and sensitive measurements [21,22].", "There is accumulating evidence that these mesurements may be applied in clinical settings to predict morbidity and mortality from redox diseases [3].", "Interestingly, even the rather simple spectrophotometric measurement of total free thiol availability using a non-selective free sulfhydryl probe (Ellman's reagent) in plasma or serum has been demonstrated to have an astonishing power to predict graft failure and mortality in renal transplant patients and cardiovascular mortality in a heart failure cohort [23,24].", "This suggests that the single free SH group of circulating serum albumin may be another integrative biomarker of redox-sensitive events in vivo [3].", "S-cysteinylated and S-glutathionylated albumin have also been proposed to represent useful biomarkers of oxidative stress [25,26], and albumin itself may be an important transporter of low-molecular weight thiols by allowing the formation of reversible mixed disulfides.", "The complexity of the methionine recycling, transsulfuration and glutathione metabolic pathways suggests that no single biomarker will adequately capture overall metabolic and redox status of all of these pathways at a global level.", "Many different assays for the quantification of sulfide in seawater or simple aqueous buffer systems exist, but not all are suited for the detection of sulfide in biological material [27,28].", "Modifications of a century-old colorimetric technique, the methylene blue assay [29,30], have been used by many research groups to detect sulfide in blood, and reports from several groups hinted at associations between blood pressure or metabolic status and methylene blue reactive material (interpreted to reflect sulfide levels).", "While the overall specificity of the methylene blue assay for sulfur and its low running costs continue to contribute to its popularity, doubts have been raised as to its reliability for the quantification of sulfide in complex biological media [31].", "In the last couple of years a large number of fluorimetric probes have been developed to detect sulfide [32], but many retain some cross-reactivity with other RSS.", "Moreover, an HPLC-based method with fluorimetric detection that employs the reaction of sulfide with mBB has been developed [33–35] and is now widely used for sulfide quantification in biological material.", "Most recently, the same reaction principle was exploited to develop a more specific detection technique for sulfide using liquid chromatography hyphenated to tandem mass spectrometry [36].", "In spite of all these developments, still relatively little is known about specific pathways of sulfide metabolism (other than its oxidation to thiosulfate and sulfate) in humans, and/or how changes in circulating sulfide concentrations relate to plasma thiol redox status.", "Some of the electrophilic compounds used to derivatise aminothiols before chromatographic separation, such as mBB [16] and NEM [37,38], are known to also react with sulfide.", "Surprisingly, this potential does not yet seem to have been exploited in more recent approaches using mass spectrometry to measure aminothiol concentrations together with sulfide.", "The aim of the present study was to describe the development of an analytical platform that allows simultaneous determination of the thiol redox metabolome, including total and free thiols, and their corresponding disulfides as well as sulfide in complex biological matrices such as human blood, saliva and urine.", "We here present first proof-of-concept results of our own method development using NEM as alkylating agent for thiols and sulfide to achieve this objective, and discuss advantages, pitfalls and limitations.", "Unless otherwise stated all reagents and materials were of the highest purity available and purchased from Sigma-Aldrich (Gillingham, UK or Munich, Germany).", "Dual stable isotope (13C, 15N) labelled reduced (GSH) and oxidized glutathione (GSSG) were from Cambridge Isotopes and obtained from CK Isotopes Ltd (Newtown Unthank, UK), N-ethyl-d5-maleimide (d5-NEM) was from Sigma-Aldrich.", "Sodium persulfide (Na2S2) was from Dojindo Europe (Neuss, Germany).", "Hanks´ balanced salt solution + Ca2+ + Mg2+ (HBSS+) was obtained from Invitrogen, bovine serum albumin (BSA) from Carl Roth (Karlsruhe, Germany) and HPLC-grade solvents from either VWR (Darmstadt, Germany) or Fisher Scientific (UK).", "Argon gas (> 99.99%) was from BOC Group (Guildford, UK).", "Ultrapure N2 gas was produced by a nitrogen generator (Parker Balston, UK).", "Blood was taken from healthy volunteers of either gender (20–58 years old) with informed consent to participate before enrollment.", "Procedures were approved by the ethics committees of the Heinrich Heine University of Dusseldorf (ClinicalTrials.gov Identifier: NCT02272530) and the University of Southhampton (ERGO 30507/31426) and conducted in accordance with the Declaration of Helsinki.", "The chromatography system used in most of the studies described was a Waters Aquity ultrahigh performance liquid chromatography (UPLC) system with a thermostatted autosampler (kept at 5 °C) and an ultrahigh performance binary pump, coupled to a triple-quadrupole mass spectrometer (Xevo TQ-S, Waters) equipped with a heated electrospray ionization source (ESI).", "Chromatographic separation of the target analytes was achieved using a 1.6 µm Modus 100 × 2.1 mm Aqua UPLC column (Chromatography Direct, Runcorn, UK) kept at a temperature of 30 °C; mobile phase A was H2O with 0.15% formic acid and 5 mM ammonium formate and mobile phase B was 95% acetonitrile with 5% H2O, 0.15% formic acid and 5 mM ammonium formate.", "The chromatographic gradient starts at 99% A, decreasing to 60% A over 4.5 min, before dropping to 0% A over 0.5 min and being held at that level for 1.5 min.", "The column is brought back up to 99% mobile phase A over 0.5 min and held at 99% for a further 1 min to equilibrate.", "A flow rate of 0.2 ml/min was used throughout, and total run time including equilibration was 8 min.", "An injection volume of 5 µl was used, with a wash step every ten injections consisting of 100% mobile phase B for 5 min, followed by a blank using the regular gradient to control for potential carry-over and column equilibration.", "We found that carry-over was negligible with two needle rinse steps (300 µl methanol, followed by 600 µl H2O /acetonitrile 90/10%, v/v) between injections.", "Our initial separation attempts used an Aquity UPLC CSH C18 (1.7 µm) 2.1 × 100 mm column (Waters) kept at 30 °C; mobile phase A was H2O with 5 mM ammonium formate and mobile phase B was 95% acetonitrile with 5% H2O and 5 mM ammonium formate.", "The chromatographic gradient started at 95% A, decreasing to 40% A over 5 min before returning to 95% A over 1 min and being held at that level for a further 1 min, resulting in a total run time of 7 min at a constant flow rate of 0.2 ml/min.", "Experimental results depicted in Fig. 3 and some panels of Fig. 4 were obtained using these separation conditions, as indicated in the figure legend.", "Mass spectrometry settings were as follows: capillary voltage 2.80 kV, source offset 6 V, desolvation gas flow 800 L/h, cone gas flow 150 L/h, nebulizer pressure 7.0 bar, collision gas (argon) flow 0.14 ml/min, desolvation temperature 250 °C.", "All analytes were detected using positive ionization in multiple reaction monitoring (MRM) mode with specific precursor/product ion combinations identified and cone and collision energies optimized for each individual compound during direct infusion of authentic standards.", "For this, compounds were dissolved in NEM-containing buffer at a concentration of approx. 1 µM and diluted in water containing 5 mM ammonium formate.", "In positive ionization mode the precursor (parent) compound is usually the protonated form of the starting material, and the fragmentation profile is dependent on the cone and collision energies applied; optimal detection parameters for these were established separately for each compound during direct infusion of authentic standards by varying those parameters and selecting the most suitable product (daughter) ion by intensity and specificity.", "In order to maximise sensitivity, accuracy and selectivity specific MRM time windows were used to minimize the number of concurrent MRM transitions being monitored.", "For quantification purposes the MRM transitions were therefore only monitored during the time window where the relevant compounds elute.", "For specifics including the chemical structures of analytes and m/z values of precursor>product couples monitored see Table 1.", "Signals were captured and data processed using MassLynx v.4.0 and Quanlynx v.4.0 software (Waters).", "Quantification of the compounds of interest was accomplished by comparison of peak areas to external standards with variations in ionization efficiency (and potential loss during ultrafiltration) adjusted using stable isotope labelled internal standards.", "Each class of compound, i.e. oxidized thiol, reduced thiol and sulfide-related species, has a representative internal standard.", "For the oxidized thiols, 13C415N2-GSSG was used; for the reduced thiols 13C215N-GSH was derivatized with an excess of NEM and used as internal standard.", "For sulfide, a deuterated stable-isotope labelled standard (32S-(d5-NEM)2) was prepared by reacting an exact amount of d5-NEM with a two-fold molar excess of Na2S in ammonium phosphate buffer pH 7.4 for 30 min at RT, and removing excess sulfide by bubbling for 60 min with high-purity argon.", "A mixed internal standard working solution was prepared by combining all three stable-isotope labelled compounds in ammonium phosphate buffer pH 7.4.", "Specific MRM conditions were established to monitor each of the internal standards (Table 1), and the ratio between the signal intensity of each analyte and its corresponding internal standard was used for quantification.", "Stock solutions of standards containing each analyte of interest were prepared fresh for each analysis in pH 7.4 ammonium phosphate buffer (except for cystine (CysSS) and homocysteine (HCys), which were prepared in 0.1 N HCl, while sulfide, persufide and methanethiol were first dissolved in water) and serially diluted in buffer to construct a concentration response curve for calibration; each concentration of standard was spiked with the same final concentration of stable isotope standards.", "Preliminary LC-MS/MS experiments carried out with mixed thiol standards at room temperature (RT) and pH 7.4 revealed that the reaction with NEM was complete for all analytes in under 10 min.", "Those results were confirmed by spectrophotometric monitoring of NEM consumption under the same conditions (302 nm; Cary 60 UV/vis spectrophotometer), demonstrating rapid kinetics for the reactions of NEM with GSH, cysteine (Cys) and Hcys (at a 1:1 molar ratio, reactions were complete within 12–15 s) whereas the reaction between NEM with sulfide (2:1 molar ratio) required 10 min to run to completion.", "Optimal pH was assessed by comparing reactions of NEM with either aqueous standards of select thiols (GSH, Cys, HCys, sulfide and stable-isotope labelled GSH) or by addition of NEM stocks prepared at different pH to human plasma.", "For those experiments, 100 mM NEM stock solutions were prepared in different ammonium phosphate buffers adjusted to pH 6.0, 7.4, 8.0 and 9.0 and used within 5 min of preparation.", "Freshly prepared aqueous solutions of thiols were individually reacted with NEM at these different pH values at a final concentration of 10 mM NEM, and after 5 min waiting aliquots of these NEM-thiol incubates were combined and analysed immediately.", "Endogenous thiols contained in human plasma (obtained by centrifugation of EDTA blood using the rapid centrifugation protocol as detailed in Section 2.8) were derivatized using freshly prepared NEM stock solutions of different pH at a 1:10 v:v ratio (10 mM NEM final concentration).", "Careful assay validation for linearity, over a range of biologically relevant concentrations, and a preliminary assessment of precision, reproducibility and limits of detection were carried out using authentic standards, according to standard procedures, and are shown in Table 1 and Fig. 1.", "Full validation awaits further assay refinement with inclusion of additional analytes of interest.", "Venous blood was collected from healthy human volunteers using a 21-gauge (0.80mmx19mm) butterfly needle and BD Vacutainer™ tubes.", "Following gentle mixing by inversion, blood was processed immediately as described below.", "Saliva was collected using oral swabs (Salivette™, Salimetrics, Carlsbad, CA, USA) followed by centrifugation; a mid-stream urine sample was collected using a sterile urine container.", "Sample aliquots were measured either fresh or transferred into cryovials (Nunc), snap frozen in liquid nitrogen and stored at − 80 °C until analysis.", "For human plasma analysis, whole blood was collected directly into EDTA BD vacutainer™ tubes and gently mixed by repeated inversion; 1 volume of NEM (100 mM stock solution in PBS) was added to 9 volumes of blood within 1 min, resulting in a final conc of 10 mM NEM (1:10 dilution), again followed by gentle mixing.", "Stabilized, anticoagulated blood was then centrifuged for 1 min at 3000 × g to separate plasma from cellular components, snap frozen in liquid nitrogen and kept at − 80 °C until analysis.", "Before analysis, NEM-derivatized plasma samples were supplemented with the internal standard (dual stable-isotope labelled GSSG and NEM-derivatized GSH as well as d5-NEM-derivatized sulfide in ammonium phosphate buffer, 1:1 v/v; target concentrations 100 nM GSH, 200 nM GSSG, 100 nM sulfide), and afterwards samples were cleared by ultrafiltration using spin columns with a 10 kD cut-off (Millipore).", "Each step of the sample preparation was carefully optimized and validated.", "To compare detection of thiols and sulfide in plasma with that in serum, whole blood was also collected into serum BD vacutainers, incubated for 60 min at RT to allow clotting, and then centrifuged at 3000 × g according to the manufacturer's protocol.", "To test the effects of other anticoagulants, whole blood was collected into BD vacutainers containing EDTA, heparin or citrate, diluted 9:1 with NEM (final NEM concentration 10 mM), centrifuged at 800 ×g for 10 min at 4 °C (see Fig. 3).", "Centrifugation speed was optimized by comparing GSH levels in plasma obtained by standard/low-speed centrifugation of whole blood (800 × g, 10 min, 4 °C) with short/high-speed centrifugation (1 min 3000 ×g or 3 min 3000 ×g at 4 °C).", "Hemolysis was excluded by assessing plasma free hemoglobin concentration by UV–visible spectroscopy [39].", "Sample stability and stabilization conditions were determined by focussing primarily on the levels of GSH, GSSG and sulfide (although other thiols were assessed in parallel) when comparing the addition of different concentrations of NEM (1, 5, 10, 20 mM final concentration) to whole blood, or when NEM addition was delayed by specified periods of time (30 s; 5, 10, 15 and 20 min).", "To test the effects of time of addition of NEM in whole blood, and also the effects of freeze/thawing on stabilized samples, the same blood sample was divided into identical aliquots, which all underwent the indicated procedures (addition of NEM in whole blood or plasma, repeated freeze/thawing, etc,).", "To determine the concentration of total thiols, free thiols and bound-thiols (see Box 1), NEM stabilized plasma was divided into two aliquots: (a) to assess total thiols (= free thiols + bound thiols) one aliquot was treated with 50 mM dithiothreitol (DTT) for 30 min, which reduces LMW disulfides and mixed disulfides with proteins; (b) to determine free thiols one aliquot was left untreated.", "In both aliquots thiol concentrations were assessed following NEM derivatization by LC-MS/MS analysis, as indicated above.", "Bound thiols were calculated by subtracting free thiols from total thiols.", "The same procedure was carried out with the blood cell pellet (comprising red blood cells (RBCs), white blood cells, and platelets), urine and saliva samples, except that incubation time with DTT for urine and saliva was only 10 min.", "Optimal reaction conditions for the reduction of disulfides by treatment with DTT were established for each biological matrix.", "To this end, samples were reacted with 10, 25, 50 and 100 mM DTT for 10, 20, 30 and 60 min at RT before addition of NEM; appropriate volumes of a 100 mM NEM stock solution in ammonium phosphate buffer were then added to neutralize all SH equivalents of DTT and yield an additional 10 mM excess of NEM to ensure full capture of additionally released thiols.", "An alternative incubation sequence was tested in which DTT was added to NEM-treated human plasma to achieve (following neutralization of the excess NEM) a similar final DTT concentration.", "Results obtained with either sequence were virtually identical.", "To test for the presence of acid-labile sulfide in blood cells, urine and saliva, 50 µl of NEM-stabilized sample was placed in a septum-sealed screw-cap vial and 200 µl 0.1 N HCl was added through the septum using a gas-tight syringe.", "After 10 min of incubation at RT the pH was neutralized by addition of 20 µl 1 N NaOH, followed by vortexing and addition of 20 µl 100 mM NEM through the septum.", "Internal standards were added after a further 10 min of incubation, and samples were then subjected to ultrafiltration followed by LC-MS/MS analysis as described above.", "It is important to note that acid treatment will liberate sulfide from both LMW and protein-bound sulfane sulfur (and possibly other sulfur species).", "25 µl of a solution consisting of 100 mM NEM with 20 mM EDTA in PBS (final [NEM] = 10 mM; final [EDTA] = 2 mM) were added to 225 µl of whole blood (9:1 (v/v) dilution).", "Blood cells and plasma were separated via centrifugation of whole blood at 3000 g for 3 min at 4 °C.", "Plasma was collected, and proteins were precipitated by mixing plasma 1:1 (v/v) with 150 µl of an ice-cold solution containing 5% SSA and 10 mM NEM in Mili-Q water.", "Additionally, 15 µl of the internal standard (either 2 mM glutathione ethyl ester in Milli-Q water or the same dual-labelled stable isotope GSH and GSSG standard mix as indicated above) solution was added (1:10 relative to plasma volume).", "The sample was mixed by vortexing, sonicated for 20 s and centrifuged at 10,000 ×g for 10 min at 4 °C.", "The supernatant was collected and the pellet extracted once more with the same volume of ice-cold 5% SSA containing NEM (10 mM) solution as before.", "After centrifugation at 10,000 ×g for 10 min at 4 °C, supernatants were combined and measured with UPLC-quadrupole time of flight (QToF) mass spectrometry using an injection volume of 10 µl.", "UPLC-QToF analysis was performed on an Agilent 1290 Infinity UPLC System with a binary pump and autosampler coupled to an Agilent 6550 iFunnel QToF system.", "For analyte separation a Zorbax Eclipse Plus C18 RRHD 2.1 × 50 mm 1.8 µm column from Agilent was used.", "Mobile phase A was 0.1% formic acid in Milli-Q water, and mobile phase B was acetonitrile.", "Separation was accomplished using the following conditions: 0–2 min: 99% A; 2–7 min: 99 − 1% linear gradient.", "An isocratic flow at 1% A was then run for 5 min (7–12 min) to wash the column.", "After 12 min in total, the proportion of A was increased again to 99% and kept constant for 4 min.", "The method takes a total of 16 min at a flow rate of 0.6 ml/min.", "Temperature was held constant at 20 °C.", "Analytes were ionized in positive mode (ESI) using the following conditions: gas temperature 220 °C, drying gas 12 L/min, nebulizer: 35 psig, sheath gas temperature: 330 °C, sheath gas flow: 11 L/min, capillary voltage: 2.5 kV, nozzle voltage: 1 kV and fragmentor voltage: 30 V. Analytes were determined using extracted ion chromatograms of the total ion count.", "Analyte levels were typically assessed in technical duplicates or triplicates for at least two independent biological samples, as indicated in figure legends.", "Data were analysed with GraphPad Prism 7.0c for Mac.", "Outlier were identified by applying the ROUT method [40].", "Data were tested for normal distribution by using Shapiro-Wilk normality testing.", "Data are reported as mean ± SD (for technical replicates) or SEM (for biological replicates), as indicated in the figure and table legends.", "Group differences were tested either using 1-way or 2-way ANOVA as required by the experimental setting, followed by an appropriate multiple comparison post-hoc test (Dunnet's) or t-test (when just two groups were being compared) as indicated in the figure legend.", "Significance was assumed when p < 0.05.", "Method development started by selecting key compounds of interest according to the scheme summarized in Fig. 1A, including HCys, homocystine (HCysSS), Cys, CysSS, GSH, GSSG, cysteinylglycine (CysGly), glutamylcysteine (GluCys), and sulfide.", "This was followed by establishing appropriate conditions for the detection of these analytes by LC-MS/MS.", "To this end, authentic standards of reduced thiols were reacted with excess NEM in buffered solution at pH 7.4 and mixed with disulfide standards to find suitable separation conditions by UPLC (see reaction scheme for reduced thiols and sulfide with NEM depicted in Fig. 1B).", "During the initial phase of development a standard C18 reversed phase column with a simple gradient elution profile was used for analyte separation, allowing us to make a number of general observations regarding their elution profile.", "We found that symmetric and mixed disulfides (which do not react with NEM as they lack a free thiol group) show limited retention on reversed phase material, clustering together close to the solvent front; this was followed by the group of maleimide-adducts of aminothiols, with S-(NEM)2 eluting last.", "Thus, derivatizing reduced thiols with NEM clearly prolongs their retention time on the column, particularly when the derivatization product involves two succinimide groups, as with sulfide.", "For some compounds more than others double peaks were observed, a phenomenon previously described for Cys and GSH [41,42].", "This has been attributed to the formation of two diasteromers that differ in position of the sulfur atom in relation to the maleimide nitrogen, depending on where the reduced thiol adds to the double bond of the pyrrole ring.", "Modifying elution conditions afforded reasonably good separation of NEM-containing adducts, but remained problematic for disulfides.", "We therefore opted for a multimodal resin in order to improve retention of the highly polar analytes.", "The mixed mode Aqua UHPLC column was found to offer suitable properties, combining polar separation characteristics with the stability and non-polar separation of a traditional C18 column.", "This allowed for better separation of the more polar thiols such as GSSG and HCysSS; however, CysSS was still not retained well by the column and eluted with a poor peak shape close to the solvent front.", "The order of elution of other derivatives remained largely the same (Fig. 1C).", "Therefore, further optimization experiments were carried out on the Aqua UHPLC column according to the separation procedure described in Section 2.3 of the Materials and Methods section.", "Table 1 lists the structures of analytes of interest along with the specific cone/collision energies used for their detection and typical retention times observed using these separation conditions.", "Since GSH is a major intracellular antioxidant and the GSH/GSSG ratio in plasma and tissues widely regarded as the ‘gold standard’ for the assessment of systemic or cellular redox status, we chose stable-isotope labelled GSH and GSSG to account for variations in ionization efficiency and possible loss of analyte during sample preparation.", "Consistent with the known dynamics of glutathione in blood [9] on spiking fresh or previously frozen plasma we observed a rapid loss of internal standard, unless plasma was treated with NEM before addition.", "Since this was observed with both reduced (fast) and oxidized (slower) internal standard, we opted to alkylate the stable-isotope labelled GSH standard and only added internal standards to the biological sample pre-incubated with NEM for at least 10 min.", "This measure effectively prevented any loss of internal standard even after sample deproteinization (see Section 3.9 below).", "The disappearance of both reduced and oxidized thiols was also observed on spiking blood plasma with Cys and HCys.", "Considering the unique chemistry of sulfide and its reaction with two instead of one molecule(s) of NEM (Fig. 1B), we opted to include an additional stable-isotope labelled internal standard for this analyte.", "Preliminary studies with 34S-(NEM)2 showed that the degree of ionization quenching of the sulfide adduct in a variety of biological matrices indeed differed from that of the dual-labelled GSH adduct on occasion, justifying a separate internal standard for sulfide.", "However, those earlier experiments also revealed that this particular compound was not ideal as an internal standard; besides only differing by two m/z units (34S versus 32S) the natural abundance of the 34S isotope (4.2%) means that high concentrations of endogenous sulfide can lead to interference, necessitating the introduction of cumbersome mathematical correction factors.", "This problem was overcome by the use of the pentadeuterated NEM analogue, N-ethyl-d5-maleimide (d5-NEM) in preparing the internal standard, which coelutes with the regular NEM adduct of sulfide without showing any overlap due to the large difference of 10 m/z units.", "Therefore, a defined mixture of 13C415N2-GSSG, 13C215N-GS-NEM and 32S-(d5-NEM)2 was prepared in ammonium phosphate buffer pH 7.4 and added to the mixture of internal standards of all analytes reacted with NEM, and compounds were detected using the specific MRM conditions previously established by direct infusion of individual compounds as listed in Table 1.", "The ratio between the signal intensity of each analyte and its corresponding internal standard was used for quantification in the biological matrix (Fig. 1D).", "A standard calibration set was prepared to determine linearity, precision, limits of detection (LOD), applicability and reproducibility of the method, whilst also allowing quantification of the analytes in different biological matrices.", "Accurately weighed amounts of analytical-grade reduced thiols were dissolved in the appropriate volume of NEM-supplemented ammonium phosphate buffer and allowed to react for 5 min at a 10-fold molar excess of NEM over thiol.", "Accurately weighed sulfide was first dissolved in water before addition of a defined volume of this concentrated stock to NEM-containing buffer in a septum-sealed reaction vial under otherwise identical reaction conditions.", "NEM-derivatized thiol and sulfide solutions were then combined with roughly equimolar concentrations of the oxidized thiols to yield a stock solution of all analytes to be tested.", "This mixed standard was serially diluted with buffer to yield targeted final concentrations of 10 μM, 5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 10 nM and 5 nM when added to the biological sample (2 × more concentrated in the mixed standard).", "For most compounds this is well within the physiological range, except for cellular/tissue GSH levels (being in the low mM range), which may therefore necessitate appropriate dilution of samples before analysis.", "To each dilution of the standard calibration set and the biological samples the same volume of internal standard was added yielding final concentrations of 100 nM GSH, 200 nM GSSG, and 100 nM sulfide.", "This allowed adjustment of peak areas by taking into account the ratios between analyte and internal standard.", "All analytes showed excellent linearity over a wide range of concentrations (> 3 orders of magnitude), with overall r2 values of between 0.994 and 0.999 (Fig. 1F).", "Using an injection volume of 5 µl achieved sensitivity limits were all in the nanomolar range, differing by class of compound and structure, with a general order of disulfide>thiol adduct with one NEM>sulfide (two NEM/thiol).", "Limits of detection and quantification, defined as signal-to-noise ratio of > 3 and > 10, are listed in Table 1.", "Assay precision will be determined at a later stage of method development.", "All analytes of interest could be readily detected in fresh human plasma and quantified without problems in each sample measured.", "Between-run reproducibility was tested for a pooled plasma sample over several days and was better than 20% overall.", "Fig. 1 shows a typical chromatogram of standards (panel C) and a representative plasma sample (panel E).", "Our next efforts focussed on exploring what effects variations in reaction conditions such as pH and sample preparation (blood collection, cell separation, and stabilization) may have on analyte concentrations detected.", "Reaction conditions were optimized to perform well under biologically relevant conditions.", "At neutral pH, NEM rapidly reacts with thiols [43], and this was experimentally confirmed to extend to physiological pH using a thiol standard mixture.", "Since the reaction was found to be complete in under 10 min no additional work was undertaken to further optimize reaction temperature or time as this was considered well within the timeframe required for the processing of biological samples, their placement into the sample compartment of the UPLC, and initiation of the first chromatographic run.", "Reaction pH is known to be critical for thiol alkylation by NEM for a number of reasons (see Discussion for details), and pH values in the range of 6.5–7.5 have been used in most previous studies.", "Human blood plasma deviates very little from its normal physiological pH of 7.40 under most conditions and is naturally buffered to maintain its pH. We therefore sought to experimentally test these assumptions by carrying out a series of experiments with select analytes (GSH, Cys, HCys and sulfide), varying the pH from 6 to 9 in an aqueous buffer system; those experiment were complemented by additional incubations of NEM with human plasma at the same pH values.", "The results of these studies are displayed in Fig. 2.", "The results reveal clear differences between the reaction of NEM with structurally distinct thiols.", "Whereas little difference was found for the calibration curves for sulfide between pH 6 and 9, small differences were observed for GSH; these differences became increasingly prominent with HCys and Cys.", "With Cys, a 10-fold difference in product yield was observed between pH 7.4 and pH 9.", "When fresh human plasma was added to NEM dissolved in buffer of different pH, the changes in peak areas were not as dramatic as with the mixture of reduced thiols in aqueous solution.", "Yet, a few notable differences were observed: peak areas decreased slightly with alkalinization for GSH and Cys and increased rather more markedly for sulfide; a small increase compare to physiological pH was also observed upon acidification with Cys and sulfide.", "Taken together, this data suggests that adjustment of the reaction pH from 7.4 to either more acidic or more alkaline conditions affects product yield and/or stability of the reaction product to a different extent with different thiols, with the most dramatic variation observed with Cys.", "If the calibration curves of the authentic standards at the respective pH would be used, this would result in an overestimation by a factor of 10 in the case of Cys.", "For sulfide, the same changes in pH appear to result in increased sulfide amounts to become available for reaction with NEM; whether this was a result of decomposition of metastable per/polysulfides or due to enhanced release of sulfide from non-covalent binding sites remains unclear at this stage.", "All further experiments were therefore carried out using NEM-containing stock solutions kept at pH 7.4.", "Since the physiological pH of plasma, saliva and cellular samples is almost always near this pH there is in fact no need to add a stabilizing buffer for these compartments; however, the pH of urine may vary from slightly acidic to alkaline, depending on nutritional habits and metabolic status; thus, checking pH after addition of NEM-containing buffer would seem to be advisable.", "Having demonstrated that in principle our platform technology is robust and allows sensitive, specific and simultaneous measurement of multiple thiols, disulfides and sulfide in plasma our next efforts focussed on demonstrating its versatility.", "To this end, we tested how cumbersome it would be to include an additional analyte into the existing method.", "Herein we show examples for three very different compounds: methanethiol (methyl mercaptan, CH3SH), N-acetylcysteine (NAC), and coenzyme A (CoA).", "Establishing specific MRMs, optimizing cone/collision energies and subsequently testing where the compound elutes on the column takes approximately one day per analyte using UPLC-MS/MS.", "NAC and CoA did not differ much from other thiols in terms of fragmentation properties.", "CH3SH was found to be extremely sensitive to in-source fragmentation, requiring a very low cone voltage for detection; it was also very well retained on the column, eluting between the other thiols and sulfide (see Fig. 1C).", "NAC fell within the same time window where other aminothiols elute, whereas CoA showed an unusually strong interaction with the mixed mode column.", "This resulted in extreme peak tailing, preventing inclusion of CoA in the current method.", "Ongoing efforts focus on developing a short method for discrete measurement of this particular analyte using a C18 column.", "Thus, in principle, many new analytes can be incorporated into the method within one day, although some compounds (here exemplified by CoA) may require additional development effort and/or alternative chromatography/elution conditions.", "To allow this method to be used in translational studies [3], we focussed on the optimization of analysis of thiols and sulfide primarly in biological matrices.", "Since blood is in contact with all tissues, it is likely to represent not only a convenient biomarker matrix but also to play a central role in systemic thiol and sulfide metabolism.", "A further advantage is that blood is relatively easily accessible.", "Therefore, in the present study we focused on optimizing sample preparation for blood (although these procedures can be easily adapted to tissues and cells).", "To avoid changes in thiol speciation (in particular sulfide) while minimizing interference with chromatographic separation and analyte detection, we opted to remove proteins by simple ultrafiltration over a 10 kDa membrane.", "The advantages of this approach to sample clean-up over protein precipitation methods include the avoidance of analyte dilution, pH changes and addition of organic solvents, which may interfere with the separation (please see Discussion for further comments).", "The choice of anticoagulant used to collect blood may have a significant impact on the concentrations of thiols/disulfides and sulfide measured, and previous work on glutathione emphasized the importance of metal chelators to minimize artificial oxidation [44].", "Our first efforts to identify optimal conditions for combined thiol/sulfide analysis in whole blood therefore focussed on the choice of anticoagulant (Fig. 3A).", "For reasons of practicality we limited our comparison to using commercial vacutainer tubes.", "In all cases, NEM was added immediately after filling and before centrifugation at 800 ×g for 10 min (the standard protocol used in many of our earlier studies).", "A final sample concentration of 10 mM NEM was chosen in all the pilot work.", "This had been successfully used by our group and others in the context of NO related research to prevent changes in concentrations of nitrite, nitrate and nitroso species in blood and tissues [45].", "Blood was collected from three different individuals into EDTA, heparin, citrate or serum vacutainers and tested in direct comparison (Fig. 3A).", "Citrated samples had a greatly reduced sulfide concentration compared to other anticoagulants, likely due to the acidic nature of this anticoagulant perhaps favoring volatilization of sulfide.", "Blood collected in heparin tubes showed slightly higher levels of GSH and sulfide compared to EDTA, but lower levels of Cys and CysGly.", "Serum tubes were unsatisfactory for measurement of physiological levels of thiols and sulfide as they showed greatly reduced concentrations of the majority of thiols measured (except GluCys) and sulfide.", "Whether this was due to excessive protein thiolation as a result of the coagulation process remains speculative at this juncture and was not further investigated.", "In any case, these results seem clearly to rule out serum or citrate tubes for thiol redox measurements.", "Since the concentrations of most analytes measured were rather similar using either EDTA or heparin anticoagulated blood and the former may have an advantage inasmuch as it minimizes thiol oxidation by metal chelation (obviating the need to add another chelating agent) all further validation experiments were carried out using EDTA as anticoagulant.", "All thiols are susceptible to oxidation when exposed to ambient air, and sulfide is in addition volatile; it would thus seem advisable to add alkylating agents such as NEM as soon as possible to a biological sample in order to avoid artificial shifts in the ratio of reduced to oxidized thiols.", "For blood, we reasoned that it would be advantageous to add NEM immediately after collection.", "As expected, delaying the addition of NEM to whole blood resulted in lower levels of GSH being detected in plasma (Fig. 3B).", "In contrast, sulfide showed an apparent increase in concentration the longer the blood was left without derivatization.", "Irrespective of specific mechanisms involved it would seem advisable to add the alkylating agent as soon as feasible to the biological sample of interest.", "Our next efforts revolved around finding the optimal concentration of NEM to achieve complete alkylation (Fig. 3C).", "The most abundant thiol in human plasma is the single free sulfhydryl group of Cys-34 of serum albumin [46], which is not analysed in this method but will still react with (and thus consume) NEM.", "It has a plasma concentration in the order of 0.3–0.4 mM in healthy human individuals; to some extent, NEM can also react with amine residues consuming additional equivalents.", "Thus, to ensure that all of the low-molecular weight thiols and sulfide are fully derivatized a range of NEM concentrations (1–20 mM final concentrations) were tested.", "There was no significant change in concentration between 1 mM and 5 mM NEM for the majority of analytes.", "However, the concentration of NEM used for derivatisation of whole blood had a dramatic effect on the apparent plasma levels of GSH and sulfide recorded (Fig. 3C).", "Higher NEM concentrations significantly increased the levels of GSH and sulfide measured, while those of other thiols were either not affected or their concentrations were slightly reduced.", "Sulfide levels showed the largest variation with different NEM concentrations, tripling between 1 mM and 10 mM NEM and plateauing between 10 mM and 20 mM (Fig. 3C).", "A striking observation was that, at the two highest concentrations of NEM tested (10 and 20 mM), measured plasma GSH concentration was considerably higher than the range typically reported in the literature [44].", "By contrast, GSH and sulfide concentrations increased to a much lesser extent when NEM was added to plasma after its separation from RBCs (and other blood cells) by centrifugation (compare Fig. 3E and C).", "This data suggests that the observed elevated plasma concentrations of these two analytes, when higher NEM concentrations were added to whole blood, were an artefact resulting from leakage and/or transport of material from blood cells.", "This phenomenon was not specific to this particular thiol alkylating agent since even higher levels of sulfide were found when mBB instead of NEM was used as thiol alkylating agent and added to whole blood under otherwise identical conditions.", "A direct comparison of mBB, NEM and IAM (Fig. 3D) revealed that the latter resulted in the lowest sulfide levels detected, demonstrating that the nature of the alkylating agent applied can have a marked influence on the levels of sulfide detected.", "As centrifugation time itself contributes to the delay between collection of blood and prevention of thiol oxidation by addition of NEM and consequent loss of GSH (as observed in Fig. 3B) and other thiols the centrifugation protocol was also optimized for centrifugation speed (800–3000 ×g), centrifugation time (1–10 min) and hemolysis of RBCs (Fig. 4A,B).", "The latter is of critical importance due to the very high concentration of GSH and sulfide within RBCs, such that small numbers of ruptured cells can lead to dramatic increases in the apparent plasma concentration of these analytes.", "None of the centrifugation protocols tested induced hemolysis of RBCs as assesed by UV–vis spectrophotometry of the plasma obtained after centrifugation, a simple procedure that can detect concentrations of free hemoglobin as low as 10 nM (a low-grade hemolysis corresponds to absorbance values of > 0.2 [39]).", "Nevertheless, a consistent finding was that the longer/low-speed centrifugation (800 ×g, 10 min) resulted in considerable leakage of GSH from the cells (Fig. 4A – GSH, compare pink vs. blue bar).", "Therefore, the optimal protocol chosen to preserve the natural speciation of thiols and sulfide in blood was to add NEM to a final concentration of 10 mM as quickly as possible upon collection of blood, combined with a short/rapid centrifugation procedure (1 min at 3000 ×g).", "For the most part, samples will have to be collected over longer periods of time (and in case of multicenter studies even at different locations) requiring storage in the frozen state before analysis.", "We therefore felt it was important to ensure that signal intensities of key analytes did not suffer a dramatic loss upon freeze/thaw.", "As shown in Fig. 4C, instead of any significant decrease, some concentrations notably increased after freeze/thawing, especially CysGly whereas others such as sulfide did not change at all.", "The only exception was NAC, which dropped by 30–50% after a single freeze/thaw compared to immediate analysis.", "However, another obvious finding from these studies was that the extent of concentration changes observed varied dramatically from individual to individual and between analytes, ranging from a few percent to > 3-fold.", "Further stability tests with NEM-treated samples showed no significant changes in the levels of the analytes when kept for extended time in the refrigerated autosampler compartment or subjected to an additional freeze/thaw (Fig. 4D).", "Therefore, human plasma samples stabilized with NEM can be frozen without apparent loss in concentration of analytes; however, absolute concentrations measured differ from those apparent on immediate sample analysis.", "We sought to demonstrate the accuracy of our platform technique by comparing the results obtained by an independent laboratory.", "Instead of ultrafiltration, this laboratory used a protein removal technique accepted and optimized for GSH/GSSG mesurements, i.e. by precipitation with sulfosalicylic acid (SSA), along with a different chromatographic method (regular C18 instead of mixed mode material) and mass spectrometry principle (ToF high resolution instead of triple quadrupole; see Methods for details).", "Two other thiols (Cys, HCys) and one disulfide (HCysSS) were also included in the comparison in addition to GSH/GSSG.", "The concentrations of these analytes were compared between both methods in plasma from 10 healthy human volunteers (Fig. 5).", "On comparison of Method A (ultrafiltration and triple quadrupole) with Method B (SSA deproteinisation and QToF) we found that deproteinization of samples with SSA leads to a statistically significant underestimation of GSH and GSSG, while Cys, HCys, HCySS yielded consistently higher concentrations (Fig. 5A).", "These differences are evident on Bland-Altman comparison of the methods (Fig. 5B).", "Total amounts of a specific thiol in a biological sample can be determined by reduction of all disulfides by DTT before NEM addition and analysis, while the corresponding free thiol is assesed in DTT untreated samples.", "The bound thiol levels are obtained by calculating the difference of total thiols-free thiols.", "First, to ensure complete reduction of all disulfides (including sulfide) we determined the minimally effective concentration of DTT and the incubation time required to achieve the highest final concentration of LMW thiols and sulfide in the reaction solution.", "Complete reduction was achieved by treatment of plasma with 50 mM DTT for 10 min at RT (sample workflow is shown in Fig. 6A).", "Similar results were obtained by analyzing other biofluids, including saliva and urine, whereas human blood cells required 100 mM DTT and 30 min incubation for complete reduction (see below).", "There were no significant differences in the final concentration of thiols in plasma samples reacted with NEM before reduction compared with samples reduced with excess DTT and then reacted with NEM.", "As a proof-of-concept study, we determined the steady-state concentrations of free reduced and oxidized thiols as well as the total thiol content in plasma of 10 healthy human volunteers (Table 2).", "The concentrations listed correspond broadly to values previously reported in the literature [46].", "However, the distribution of circulating concentrations among healthy human volunteers highlights a number of aspects that are usually less obvious by looking at averages.", "First, there are considerable differences in concentration of free thiols between individuals, varying from 2 to 3 fold in some cases to as much as 10-fold for other analytes.", "Second, the ratios of reduced over oxidized thiols varies markedly between glutathione, cysteine and homocysteine.", "Third, amounts of bound thiols are generally higher than circulating concentrations of free reduced thiols; this is particularly striking for GluCys and sulfide for example, where bound sulfide exceeds free sulfide by a factor of 100.", "Persulfide and methanethiol were not detected in any plasma sample.", "However, solutions of NEM-derivatized standards of persulfide (formally, the disulfide of H2S in its anionic form) were found not to be stable over time, representing a methodological limitation that is not easily overcome without switching to another alkylating agent.", "Overall, these data show that our method can also be applied for high-throughput determination of free and protein-bound thiols in human plasma.", "In addition to the possibility of measuring free thiols and subjecting aliquots of the same biological specimen to additional reduction with excess DTT (before or even after addition of NEM) to liberate bound thiols, another sample aliquot can be pretreated with acid, neutralized and then reacted with NEM to measure acid-labile sulfur (and other thiol-containing species that may be released upon this treatment; see Fig. 6A for workflow).", "Fig. 6B-D shows exemplary results using this sample workflow for blood cells and two other biofluids, urine and saliva.", "It is immediately apparent from this comparison that not only absolute concentrations differ dramatically between different biospecimen, but also the distribution of thiols (i.e. the proportion of free versus bound, and acid-labile).", "Similar to plasma (Table 2), bound cysteine exceeded free levels by more than an order of magnitude in urine and saliva; however, bound glutathione was only found in saliva, not urine or blood cells.", "Somewhat lower levels of cysteinylation but essentially no glutathionylation were found in blood cells.", "Significant amounts of acid-labile sulfur are present in saliva, urine and blood cells.", "Unexpectedly, not only sulfide concentrations were increased following sample acidification, but some thiols also showed clearly higher levels compared to untreated controls.", "This was particularly evident for methanethiol, concentrations of which increased approximately 50-fold upon acidification in urine; methanethiol concentrations in blood cells and saliva remained below the quantifiable limit.", "It is important to point out that under acidic conditions the NEM-sulfide adduct is likely to originate not only from reaction with free HS- but also with both protein-bound and LMW polysulfides due to the susceptibility of these compounds to acid hydrolysis.", "In summary, an additional straightforward sample pre-treatment allows further expansion of the measurement opportunities to include acid-labile sulfur species in biological samples.", "While there seems to be some overlap between DTT-reducible and acid-labile material distinct differences remain, suggesting different chemical characteristics of the biological material susceptible to reduction and acid-treatment.", "We here outline a novel specific, highly sensitive and robust mass spectrometry-based approach for the measurement of the thiol redox metabolome capable of quantifying total and free thiols, and their corresponding disulfides as well and sulfide in complex biological matrices such as blood, saliva and urine.", "Specifically, we describe our validation work focusing on i) the development of the analytical procedure, i.e. suitable detection and separation conditions, quantification strategy (external and internal standards, linearity, and versatility of the method); ii) optimization of sample preparation, using blood as starting material (choice of anticoagulant, sample stabilization, centrifugation and storage); iii) versatility of the method with examples of how further compounds of interest can be included for a more comprehensive coverage of the thiol redox metabolome in the future; iv) determination of the accuracy for detection of glutathione redox status as compared to an optimized, well established method; v) analysis of the thiol metabolome (including total and free thiols and their corresponding disulfides, as well as hydrogen sulfide) in plasma samples of 10 healthy human volunteers, as well as in blood cells, urine and saliva.", "Taken together, this data indicates that the approach can be used to characterize the redox metabolome in patient cohorts and animal models of disease.", "It can therefore be applied in clinical and translational studies in search for novel prognostic and diagnostic strategies for patient stratification; moreover, it may assist in identifying novel interventional approaches for the treatment of redox diseases [3].", "There is no shortage of analytical methods for the determination of biological thiols (for recent reviews see [47,48]), and many more assays appeared in just the last couple of years.", "Several of those more recent additions to our analytical armamentarium use a combination of some form of chromatographic separation (HPLC, UPLC or capillary electrophoresis) and mass-spectrometry for detection.", "In most (albeit not all) cases, the latter offers better selectivity and higher sensitivity compared to earlier spectrophotometric, fluorimetric, chemiluminescence or electrochemical detection approaches.", "While there are plenty of choices for the quantification of reduced thiols, much fewer techniques exist for the measurement of oxidized thiols, in particular at their low natural abundance; however, this is of utmost importance for an accurate assessment of thiol redox equilibria.", "Given the significance of glutathione as cellular antioxidant the majority of analytical developments revolved around GSH/GSSG.", "Since the pioneering analytical work of Reed et al. [15] most investigators are aware of the need to prevent thiol oxidation in order to avoid an artificial elevation of disulfide levels.", "However, not only GSH/GSSG but also the extracellular cysteine/cystine ratio is an important read-out of the whole body redox poise (and apparently uncoupled from the glutathione redox) [1], and the ratio of cystine and glutathione has recently been proposed as a new biomarker for oxidative stress in the cardiovascular disease setting [17].", "Yet, an even simpler measurement of total serum free thiols by means of Ellman's reagent has been shown to predict mortality in patient cohorts [23,24].", "How the latter relates to glutathione and cysteine redox state is currently unknown.", "While the complexity of changes in extracellular thiol levels has been recognised and the dynamic interaction between free reduced and oxidized as well as protein-bound thiols been defined as “plasma redox thiol status” more than two decades ago [49] the main factors and regulatory element that define these interactions in whole blood remain ill defined.", "This uncertainty is accompanied by new developments that appear to highlight the involvement of sulfide and associated metabolites such as inorganic and organic persulfides and polysulfides [5,6,26].", "This interaction gives rise to oxidized thiols in which the sulfur of the SH group of Cys, GSH or sulfide (and likely other thiols) is bound to one or more sulfur atoms; the properties of the resulting thiol derivatives differ fundamentally, both chemically and biologically, from the originating thiols [50,51].", "Moreover, even seemingly well characterized compounds such as glutathione seem to have additional actions beyond those of their classical antioxidant function, which would seem to warrant further exploration [52].", "Yet, many pitfalls surrounding its analysis in cells and biofluids (some of which have been known for decades) often appear to have been neglected and remain to be better characterized to avoid the enormous variations in GSH/GSSH ratios reported in the literature [53].", "Even sophisticated, validated and robust methods for the simultaneous determination of glutathione and cysteine redox state [54] would seem to leave room for improvement inasmuch as pre-analytical sample handling is somewhat cumbersome and time-consuming, involving multiple steps, and total assay time is rather long by today's standards.", "More importantly, however, the technique used in this particular case (which is based on the technique established by Reed et al. [15]) is unsuitable for the analysis of thiols that do not also contain an amino group such as sulfide or methanethiol.", "Hence, novel analytical methods with improved sensitivity and specificity, a simplified workflow and higher speed of analysis without compromising robustness and versatility are required to allow future study of the complex interactions between different elements of the thiol redox metabolome in larger numbers.", "Herein, we provide one example for how such an approach might be put into action.", "Here we describe the necessary steps needed to establish an analytical platform for high-throughput characterisation of the thiol metabolome in biological matrices.", "As soon as the analytical instrumentation is available, the first fundamental step to this end is to define the relevant target analytes as well as the internal standards for their quantification; from there on, one can proceed with optimization of detection and separation conditions as well as a critical assessment of linearity, precision, LOD and reproducibility of the method.", "These steps are clearly dependent on the type of instrumentation available, but in part also on the different chemistry of the analytes intended to be covered.", "However, the main objective of the present method development was not to identify ideal conditions for analysis, but rather to find conditions that are fit-for-purpose for high-throughput redox profiling of large numbers of samples under everyday conditions.", "To this end, we found that it was not the instrumentation available but rather the sample preparation that represented the most important factor which defines the qualitative and quantitative results.", "We chose human blood as suitable starting material for our development.", "The reason for this choice was severalfold: not only is it a readily accessible and relatively abundant starting material, but it also represents a sort of systemic equilibration medium that is in contact with all other compartments of the body [3].", "The redox buffering capacity of the blood (and in particular of RBCs) is notoriously high, and it appears to be kept constant by multiple enzymatic and non-enzymatic mechanisms [3,55].", "For the same reasons the determination of ‘redox reserve’ (total thiols) and ‘redox status’ (ratio of reduced/oxidized thiols) in blood may be relevant to understand the status of biological fitness and resilience of an individual (or animal) under study.", "The first critical steps during development were therefore to find a way to stabilize the sample, to separate cellular components from the plasma without damaging RBCs (which contain millimolar concentrations of GSH [55]), in a quick and straightforward way, compatible with rapid and reproducible collection of clinical specimens in a critical care setting, for example.", "We found that one of the easiest ways to collect blood in this context was to choose commercial EDTA vacutainers since this anticoagulant is also a metal chelator, which may help avoiding secondary metal-dependent oxidative reactions (such as the Fenton reaction).", "Reduced thiols in EDTA anticoagulated blood can then be stabilized by treatment with an excess of alkylating agent, added within the first 1–2 min after blood withdrawal.", "In the present study we used NEM for this purpose, whilst others have used different alkylating agents such as IAM or mBB.", "We opted for NEM due to its superior speed of reaction with SH groups at physiological pH (see below).", "Regardless of the nature of alkylating agent employed in this context, we observed that addition of these chemicals to whole blood leads to an artifactual increase in GSH and sulfide concentrations in plasma if centrifugation is carried out at low speed for too long.", "This problem was circumvented by use of a high-speed/short-centrifugation protocol for cell separation, indicating that this phenomenon was an artefact possibly due to an alkylation-induced increase in RBC membrane permeability.", "Indeed, NEM is well known to affect RBC membrane properties, inducing a loss of membrane asymmetry and exposure of phophatidylserine as well as to modifying critical thiols in ion channels and transporters, thereby affecting ion exchange processes, decreasing cell deformability and increasing membrane leakage [55].", "Another important factor that requires careful consideration in methodological developments is the choice of protein removal method.", "Biological fluids such as plasma are complex matrices, the constituents of which can potentially cause interference with chromatographic separation and analyte detection by e.g. lowering ionization efficiency or ion suppression.", "In the present study, biological samples were cleared before analysis, and the majority of proteins were removed by ultrafiltration over a 10 kDa cut-off membrane using centrifugal columns.", "The classical methods described for determination of aminothiols, specifically GSH, using either chromatographic techniques or enzymatic assay, also normally include a protein removal step.", "Many standard deproteinization procedures are based on addition of acids (trichloroacetic acid, SSA) or metals like zinc sulfate/NaOH.", "Clearly, either of these methods would cause drastic changes in pH, which may affect detection of sulfur species and sulfide [27].", "In fact, sulfur species are well known to be particularly labile under acidic conditions; on the other hand, sulfur can be released from thiols under alkaline conditions [27], leading to an artificial elevation of sulfide.", "In addition, zinc sulfate is known to trap free sulfide and form a water-insoluble ZnS precipitate.", "Alternative protein removal processes include addition of polar organic solvents such as methanol, acetonitrile or acetone, or solid phase extraction.", "We observed that using organic solvents for protein precipitation decreases the recovery of GSSG; moreover, the high concentration of organic solvent in the sample sometimes interferes with the chromatography (which often requires an almost complete aqueous phase to start with).", "Therefore, in order to quantify aminothiols and sulfide using an identical sample processing procedure, clearing samples by ultrafiltration, which does neither change the pH nor add any acid or solvent, to us appears to be best practice, at least for matrices such as plasma.", "A further important step was to determine the stability of the samples after freeze/thaw.", "We found that except for NAC (levels of which dropped by 30–50% after freeze/thaw compared to immediate analysis) concentrations of all other thiols did not drop but rather increased in concentrations after freeze/thaw.", "The reason for this behavior is not immediately obvious, but clearly there is no problem with the stability of the thiol-NEM adduct.", "We suspect that this observation may be related to the stability of thiol derivatives that previously escaped detection (perhaps a metastable compound that decomposed during the freeze/thaw process such as a persulfide and/or polysulfide).", "However, levels of sulfide did not change under the same conditions, suggesting that the product of decomposition of these per/polysulfides might be a higher oxidized form not captured by our assay.", "Interestingly these effects of freeze/thaw were not observed when proteins were removed by addition of SSA.", "This suggests an alternative explanation that is even more intriguing: conceivably, aminothiols may exist not only in free and protein-bound form (whereby binding is via a mixed disulfide and therefore DTT-reducible), but a fraction of them also binds to proteins in a non-covalent fashion.", "When protein conformation is changed by precipitation with acid/organic solvents or as a consequence of a freeze/thaw process this non-covalent association is perturbed, making more aminothiols available for trapping by the excess NEM present.", "This scenario might also explain the apparent differences in absolute concentration of analytes (which were independent of the detection method) when plasma was processed by ultrafiltration versus acid precipitation; moreover, it might offer an explanation for our observation that not only sulfide, but also the concentration of some aminothiols increases upon acid treatment.", "The notion of non-covalent protein binding of thiols is also consistent with the observed concentration-dependence of NEM in plasma - why should levels of certain aminothiols (and sulfide) otherwise further increase with higher NEM concentrations unless they are removed from an equilibrium?", "This concentration-dependent phenomenon was particularly prominent with sulfide (see Fig. 3E), and consistent with a recent report that H2S non-covalently binds to albumin and hemoglobin [36].", "These aspects of the thiol redox metabolome would seem to warrant further investigation as it might offer yet another, hitherto unrecognized level of redox regulation by modulation of protein association.", "Many different thiol alkylating agents and strategies have been developed over the years, and compounds used not only differ in chemical structure and reactivity but also in the mechanism by which they interact with thiols.", "Therefore, selection of the most appropriate compound for thiol derivatization will be determined by the purpose of its use [56,57], for example whether the goal includes either labelling or blockage of reactive thiol groups in proteins, thiol quantification in biological samples, or the prevention of autoxidation.", "NEM is well known for its usefulness in characterizing sulfur metabolism due to its propensity to rapidly react with SH groups [58].", "As a result of its reaction with protein thiols and SH-containing cofactors it can inhibit intermediary metabolism, block vesicular transport, activate specific ion channels and interfere with cell replication, for example.", "Half a century ago, it was also employed for the spectrophotometric measurement of SH groups [59].", "NEM and related maleimides have since been widely used for the analysis of thiols, including GSH [60,61], for the prevention of GSH autoxidation [61] to stabilize human blood for later quantification of thiols/disulfides [62]; even in a similar workflow for sulfur pathway analysis using a different chromatographic separation [63].", "It has also been known for some time to react with sulfide [38,43].", "However, to the best of our knowledge this property has not been used for development of a combined method for thiol and sulfide detection using mass spectrometry.", "Of note, the compound's specificity for thiols is not absolute.", "At high concentrations, NEM can also react with amino groups of peptides/proteins and aminothiols [64].", "In fact, its specificity for SH groups strongly depends on reaction conditions; the side-reaction with amines does not pose a major problem as long as the pH of the reaction solution is kept near neutral.", "However, reagent concentrations in excess of those found optimal for the majority of analytes in a specific biological matrix (determined to be 10 mM NEM for plasma in the present study) may lead to lower apparent concentrations of select analytes.", "Cys is a case in point and has been shown to undergo intramolecular transamidation to form a cyclic reaction product; this reaction can run to completion under alkaline conditions [64], and this may well account for the progressive descrease in apparent Cys levels in Fig. 3C (when increasing NEM concentrations were tested) as well as the drop in reaction yield when pH was changed from 7.4 to 9 (Fig. 2B).", "Yet, apart from these examples and in spite of its widespread use as a pharmacological and analytical tool for decades, it is somewhat surprising that the reaction mechanism of NEM with thiols has not been investigated in much more detail (this could change in the near future as the underlying chemistry also forms the basis for numerous applications in materials chemistry including the surface modification of porous polymeric monoliths used in chromatography columns [65,66]).", "In order to appreciate NEM's uniqueness among thiol-reactive compounds and explain some of our experimental findings with different aminothiols and sulfide (as well as polysulfides) it may be informative to briefly review key mechanistic aspects of the chemical reaction.", "This is of importance since reaction conditions not only affect the stability of NEM and the final reaction product (both of which are subject to alkaline hydrolysis [43,67,68], but also speed and nature of the reaction itself as well as its specificity for SH groups over reaction with amines.", "The pH plays a central role not only for reagent and product stability, but also because it affects, along with the pka of the SH group, the amount of thiolate available.", "It is a fortunate coincidence for analytical chemistry that the reaction of NEM with thiols proceeds optimally around the physiological pH, i.e. in the range of 7–7.5.", "NEM and related maleimides are electron-deficient compounds (electrophiles) that participate in chemical reactions by accepting an electron pair of a nucleophilic compound such as a thiol.", "As a maleic acid derivative it also has an activated double bond with which thiols can react.", "The pH-dependence of NEM's reaction with GSH and Cys [43,69] indicated that only the thiolate (RS-) and not the undissociated thiol (RSH) reacts with NEM.", "The mechanism involves a nucleophilic addition of the thiolate (a strong nucleophile) onto the olefinic (C˭C) bond of the maleimide ring, forming an intermediate carbon-centered anion; as a strong base this immediately abstracts a proton from the conjugate acid (RSH) or from water, yielding the thioether (see Scheme 1).", "Reactions of a nucleophile with an α,β-unsaturated carbonyl (or another electron-withdrawing group) are known as ‘Michael addition reactions’ of which the thiol-NEM reaction is a special case.", "Since the sulfur can add to either side of the double bond it gives rise to different diastereomeres (see Fig. 1B), sometimes complicating the chromatographic separation of thiols [41] by leading to double peaks.", "Formation of the maleimide-thiol adduct was long thought to be irreversible (unless subjected to electrolysis).", "More recent studies, however, have documented retro reactions to occur even under physiological conditions, explaining e.g. the faster-than-expected cleavage of certain anti-cancer immunoconjugates and PEGylated products in circulation [70].", "For analytical purposes, this is not a concern as such reactions typically proceed over many hours/days and require elevated temperatures.", "The reaction of NEM with sulfide deserves special attention not only because two molecules of NEM bind to either side of the sulfide-sulfur (Fig. 1B and Table 1), but also because the same reaction can occur with polysulfides (as demonstrated here for hydrogen persulfide; see Section 3.10).", "The NEM adduct of S22- (Table 1) was found to be unstable and, over time, decomposed to yield additional S-(NEM)2.", "The reason for the instability of this thiol adduct is not known, but may be related to the ease with which sulfide chains can break and/or how favorable e.g. the NEM-S˙ radical is as a leaving group.", "Whatever the reason, the instability of S2(NEM)2 is in line with recent findings by Akaike's group demonstrating that NEM is unsuitable for the determination of cysteinepersulfide/polysulfide concentrations in biological tissues (Suppl Fig. 2 in [71]).", "Extrapolating those results to hydropolysulfides (Sx2- where x can be 2–7) one may expect that part of the sulfide detected by NEM in plasma does not originate from free HS- but be derived from organic and inorganic per- and polysulfides.", "Our comparison with mBB (the current “gold-standard” for sulfide measurement) suggests that this reagent may suffer from the same limitations.", "Thus, measured sulfide concentrations with these reagents may represent the sum of free HS- and per/polysulfides, and this warrants further investigation.", "If in addition free sulfide is also in equilibrium with protein associated forms it may be difficult, if not impossible, to determine the concentration of free sulfide in a given biological system – however, the sum of free and non-covalently bound sulfide and polysulfide (or simply “NEM-sensitive sulfide”) may still be a biological meaningful measure.", "NEM has a number of advantages over other thiol alkylating compounds.", "This includes foremost the speed of reaction with thiols, which is considerably faster than that of mBB or IAA/IAM.", "This makes it ideally suited for the prevention of autoxidation of thiols to their disulfides during sample preparation.", "Another advantage is that as an uncharged molecule, it easily penetrates cell membranes – a property it shares with mBB and IAM.", "While this can be an advantage for the analysis of tissue and intracellular biothiols, we here show that the same property can also lead to an overestimation of true plasma levels unless certain sample handling conditions are met.", "An advantage that is particularly relevant for mass spectrometric detection is the fact that reaction with NEM improves the ionization efficiency of thiols [72], which explains the excellent sensitivities achieved using ESI-MS/MS, for example.", "Not only can thiols be oxidized (in vivo and artificially during sample handling) to form symmetric disulfides (e.g. from GSH, GSSG and from Cys, CysSS), they also engage in the formation of mixed disulfides leading to formation of both LMW and protein disulfides.", "It was proposed that these protein-bound asymmetric disulfides may either represent a post-translational modification or be involved in inter-organ thiol transport [3].", "It is important to note that formation of protein-bound asymmetric disulfides may mask detection of a particular thiol, if only free thiol concentrations are assessed.", "Therefore, if determination of the total amount of a specific thiol (like HCys) is required for diagnostic or prognostic purposes, total (bound+free) thiol concentrations are determined following reduction of the covalent bond between the protein thiols and the LMW thiol.", "Indeed, determination of total HCys was demonstrated to be more predictive of certain pathophysiologies as compared to free HCys [73].", "For this purpose we subjected the biological samples tested (plasma, blood cells, urine, and saliva) to reduction by DTT.", "To ensure complete reduction of all bound thiols (incl sulfide) we determined that the minimally effective concentration of DTT and incubation time required to achieve the highest maximal concentration of LMW thiols and sulfide in the final reaction solution was 50 mM DTT and 10 min incubation at RT for all biological matrices tested except blood cells.", "The latter matrix required 100 mM DTT and 30 min incubation for complete reduction, emphasizing the importance to validate each step of the analytical procedure for the biological matrix intended to be investigated.", "For plasma analysis DTT was added to NEM-stabilized plasma.", "However, no significant difference in final concentrations of total thiols were found between samples that had been reacted with NEM before reduction and those that were reduced with excess DTT and then reacted with NEM; this demonstrates that, in contrast to alkalinization which leads to hydrolysis and ring-opening of the thioether [67], the thiol-maleimide adducts formed are stable under acidic conditions.", "Acid-labile sulfur is found in bacteria, mammalian and plant cells/tissues, and various analytical methods are available for its quantification [74,75].", "However, origin and biological role of this “pool of sulfide” remains enigmatic.", "While its presence in cells and tissues has been ascribed to metal-sulfide complexes such as iron-sulfur proteins (e.g. aconitase involved in intermediary metabolism) and Fe-S clusters in complexes of the mitochondrial respiratory chain, sources in biofluids are believed to be mainly organic or inorganic per- and polysulfides.", "Thus, the additional sulfide demonstrated here to be captured as NEM-adduct following sample treatment with acid may originate from a variety of sources including protein-bound and LMW polysulfides.", "Some but not all of the DTT-reducible thiols may be acid-labile, although these two classes have been shown earlier to be chemically distinct [76].", "Herein we show that this method may easily be adapted to measure additional very different compounds, including CH3SH, NAC and CoA.", "CH3SH is a bacterial metabolic product that also arises as a result of methylation of sulfide and methionine transamination in mammalian cells [77–79], and a human methanethiol oxidase has been identified, which metabolizes CH3SH to H2O2, formaldehyde and sulfide [80].", "We found CH3SH to be very well retained on the column, eluting between the other thiols and sulfide but to be extremely sensitive to in-source fragmentation.", "NAC assumes particular importance in bioavailability monitoring following its oral application as antioxidant or as a mucolytic drug.", "Determination of CoA in plasma and other body fluids may also assume a particular importance for redox-related studies, because it is a quantitatively important thiol involved in intermediary metabolism that engages in S-thiolation reactions in much the same manner as glutathione [81,82].", "We had some technical difficulties with this particular analyte as the NEM adduct of CoA was retained too strongly by the column due to multiple mechanisms of retention, making it impossible to measure circulating plasma concentrations of this important intermediary metabolite along with other thiols and sulfide in the same run.", "Nevertheless, we are confident this can be resolved using either a different separation column or modified chromatographic conditions, and this is a focus of our ongoing efforts.", "This method may be also implemented to assess further key intermediates of the methionine recycling and transsulfuration pathway by the addition of further related compounds such as methionine, S-adenosyl-methionine, S-adenosyl-homocysteine and cystathionine to the existing method, eventually enabling to capture the sulfur metabolome in its entirety.", "It is important to note that sulfide levels assessed here by NEM derivatisation are higher compared to methods using other protocols employing mBB [33,35].", "In our hands, absolute “free” sulfide levels are strictly dependent on the alkylating agent used, and may depend on the chemistry of the reaction of the alkylating agent; very different concentrations in apparent sulfide concentrations were found with aliquots of the same sample using NEM, IAM or mBB.", "The reason for these differences warrants further elucidation.", "Of note, other reports demonstrated that e.g. dBB can extract sulfur from other sources [83], and that NEM reacts differently with polysulfides as compared to IAM or IAA [71].", "The platform technology we present here for the measurement of reduced and oxidized thiols in combination with sulfide related metabolites appears to be robust, sensitive and versatile.", "Its strengths include the possibility to measure the thiol redox metabolome including total and free thiols and their corresponding disulfides as well as sulfide in complex biological matrices; the technique can be easily combined with other compatible analytical procedures for detection of NO metabolites and nitrosospecies, as established by us before [45,84], using NEM for stabilization of thiols.", "This may provide a deeper understanding on the systemic thiol redox state within the RSI.", "We made an effort to simplify workflows for sample preparation and storage, realizing there will be a need to translate critical steps into easy-to-follow standard operating procedures, in particular when measurements have to be carried out in large numbers, human studies are run in a multi-center fashion and/or biobanked samples are used.", "Lack of reproducibility and robustness or a very particular sample handling requiring specialist training could easily make a particular method wrongly appear unsuitable for use in clinical studies, or worse perhaps, lead clinicians towards incorrect treatment decisions.", "Despite careful optimization, some technical limitations remain.", "In the current configuration these relate to the relatively poor retention of CysSS on the column, which together with an unknown and variable interference from plasma constituents causes ionization quenching, leading to an underestimation of the true concentration of this disulfide in plasma.", "This appears less of a problem (or no problem at all) in other biological matrices such as RBCs and murine tissues and may be partially overcome by using the standard addition method to determine absolute concentrations.", "Moreover, as discussed above, sulfide levels appear higher than with other methods, calling for a systematic comparison of the leading methods described for sulfide assessement [27,31].", "Furthermore, there may be a need for additional methods to cover the per- and polysulfides as important compounds bridging aminothiol and sulfide chemistry.", "As discussed above, a major limitation of the application of NEM is the fact that it cannot be used for detection of per- and polysulfides, for which the use of β-(4-hydroxyphenyl)ethyl iodoacetamide was carefully optimized and successfully applied in cells and tissues by the group of Akaike and co-workers [71].", "The inclusion of these compounds for assessment of the redox metabolome would seem to be a sensible complementary approach to the analytical platform we here introduced.", "We have outlined a novel specific, highly sensitive and robust mass spectrometry-based approach for quantification of the thiol redox metabolome.", "During the development of this analytical approach, we realized that the majority of variability that can be appreciated in the literature assessing aminothiols (and even more in the literature describing the methods to measure sulfide) originates from sample processing procedures rather than the actual analytical method itself.", "The use of authentic standards and stable isotope labelled internal standards (which are readily available for most aminothiols and sulfide) allow optimization of these methods with limited effort.", "The choice of analytes to be measured and the availability of the standards should drive the optimization procedures.", "However, procedures of sample stabilization, for cellular separation and/or isolation (including centrifugation and eventually tissue digestion with detergents), conservation and freeze/thaw cycles all profoundly affect the results produced and therefore also require careful optimization for each biological matrix of interest.", "Although the analytical approach with NEM presented here is not suited to cover the per- and polysulfide interface, the principles described here may be applied to find an approach allowing detection of an even broader spectrum of metabolites belonging to the thiol redox metabolome.", "In conclusion, these principles can be applied to assess the thiol redox metabolome in patient cohorts and animal models of disease, thus allowing the identification of the regulatory nodes of redox signaling, while enabling diagnosis, risk assesment, patient stratification and prognosis of redox diseases." ]

[ "The thiol redox metabolome comprises free and bound thiols, disulfides, and sulfide.", "We developed a LC-MS/MS platform for detection of the thiol redox metabolome in biological samples.", "Thiols in blood, urine and saliva are stabilized by derivatization with N-ethylmaleimide, also preventing oxidation.", "Total and acid-labile thiols are determined following addition of DTT and acid.", "Accuracy was investigated by comparison with an established method to assess GSH and GSSG.", "Applicability was shown for plasma of 10 healthy volunteers, blood cells, urine and saliva." ]

[ "MULT" ]

[ "Gasification kinetic", "Particle size", "Pyrolysis", "Syngas", "Triplochiton scleroxylon" ]

[ "1000" ]

[ "Constant increase of population leads to a move up in energy requirements.", "Because of the anxiety to global climate change, environmental pollution and limited resources of fossil energies, governments are constrained to migrate progressively from fossil energies to clean energies.", "That explains why valorization of renewable energies is nowadays preconized and encouraged.", "This lead to a firm surge of biomass with a least carbon source of energy, liquid fuels as gaseous fuels and biofuels [1,2].", "Biomass is the fourth largest source of renewable energy in the world and which produces zero carbon emission [3,4].", "Thermochemical conversion is a bio-refinery platform where biomass is transformed into high sub-value products.", "Gasification is one of the best among thermochemical conversion processes.", "It converts biomass into syngas, which is a mixture of carbon monoxide and hydrogen.", "That source of energy has a wide range of applications.", "Hydrogen and carbon monoxide for example are synthetized to produce variety of chemicals and transport fuel.", "These products are classified into three large groups: chemical feedstock (ammonia), energy feedstock (carbon dioxide) and transport fuels (biogas) [5,2].", "Cameroon has the second largest forest in the sub-Saharan Africa and the East region of Cameroon produces about 1.5-million m3 of forest residues annually [6].", "Only 20,000 m3 are recovered to produce 600 tons of charcoal per year, much of the scraps are burned in forest side and others abandoned in sawmills [7].", "This huge amount of biomass can be converted into precious volume of renewable energy rather than burning in the agricultural fields.", "This will not only solve the issue of dumping agriculture and forest wastes, but it would overcome the ever increasing energy crisis and environmental pollution problems in the country.", "Teodora et al. [8] noticed that sawdust resulting from wood exploitation and processing is mostly stored in un-controlled manner and can contribute to environmental pollution.", "In the same vein sawdust which is a source of biomass is used for the production of solid fuels for generating heat in residential use (for cooking) and in boilers.", "Different researchers observed that thermochemical conversion process converts all component of biomass into bio-energies [5,2], meanwhile biochemical conversion process requires mainly chemical pretreatment and uses enzymes which are expensive.", "Majority of conversion processes are limited with narrow tolerance range of proximate analysis of the converted biomass: particle size and moisture content.", "For instance, the greater the particle size than the accepted range will produce more syngas in the gasification process, but will reduce the speed of gas diffusion and at the same time reduce the quality of the syngas produced in the reactor [9].", "Pyrolysis kinetic models of biomass are classified into three reactions: one-stage global single reaction, one stage multiple reactions and two stage semi-global reaction [10].", "Firstly, one-stage global single reaction uses experimental data obtained from mass loss rates.", "Secondly, one-stage, multiple reactions where biomass is decomposed into two parallel reactions to produce char and different gases.", "The distribution of products is determined by one-stage, multiple reactions.", "At industrial scale, modelling work from experimental study has been viewed on determining the biomass evaporation time for particles sizes.", "That parameter is to be considered for the design of the reactor.", "These expressions can be reviewed for temperature variation and shape of particle [11,12].", "They involve several free parameters that have to be fitted to existing experimental data and, therefore, can vary significantly depending upon the feedstock and operating conditions.", "Rapagna et al., [11] observed experimentally that the particle size affects the efficiency of gasification in terms of production of gas and amount of tar formed.", "Majority of gasifiers are designed with wood fuels but little work is carried out to use agro residues as fuels.", "Triplochiton scleroxylon has been valorized for cooking and heating especially in developing countries, few authors have used Triplochiton scleroxylon for the production of biofuel especially for syngas production.", "On the regard of the preceding literature review, the present study focuses firstly on the production of syngas using sawdust of Ayous as feedstock, and secondly on the modelling of kinetic of the gasification process.", "The biomass samples were the residues of Triplochiton scleroxylon (Fig. 1) which were collected from a furniture factory in Ngaoundere (Cameroon).", "These biomass samples were shredded into particle by different laboratory sieves.", "Characterization of these biomass samples was carried out using the proximate and chemical analysis.", "The dry matter content of sawdust was determined by thermo gravimetric method, which is the reference method for determining the water content or total solids in foods described by AOAC [13].", "The ash content was determined by AOAC method 923.03.", "The difference between the dry matter and the mass of ash corresponds to the mass of organic matter.", "Moisture content of biomass was noticed by heating material at 105 °C for 24 h in an oven.", "The weight difference between initial and later stage was considered as moisture content of the respective biomass.", "Volatile matter of the biomass was determined by AOAC method 923.03.", "The difference between volatile matter free biomass and biomass ash was calculated as fixed carbon.", "Chemical analysis of Triplochiton scleroxylon was carried out.", "The cellulose, hemicellulose content was done according to AOAC Standards 973.18 and 2002.04 [14,15] and lignin content was determined according to the standard insoluble Klason lignins [16].", "Fig. 2 describes the experimental device in which syngas was produced.", "Sawdust of Ayous with specific particle size (d < 500 µm, 500 µm < d < 1 mm and 1 mm < d < 1.9 mm)) is introduced in a cylindrical batch reactor (25.6 cm3) set on a hot plate (600 °C).", "Both elements are then placed on electronic balance in order to follow the mass loss over time during gasification process.", "Temperature sensor is to control the temperature inside the reactor during the process.", "The produced syngas is collected through a flexible pipe and stored in an inverted glass tube (syngas collector).", "The flammability test is carried out by igniting syngas at the outlet valve.", "The aim consists of heating the sample in an environment poor in air so that to avoid combustion process.", "Before introducing the sample in the batch reactor, this one is first pretreated.", "In fact, each sample of sawdust spends 24 h inside an oven of 105 °C in order to better dry the sample and to eliminate the moisture content.", "That pretreatment enabled to minimize the steam of water produced during the drying phase which begins as soon as the reactor is placed on the hot plate.", "Cool water in the syngas collector is to decrease temperature of the mixture so that to condensate the vapour water.", "Experiment of each sample has been conducted trice by operating in the same experimental conditions.", "Term k is a rate constant given by the Arrhenius law.", "With the Arrhenius law constants available, either from experiment or from the literature, and assuming a value for the carbon conversion, according to the integration form, a plot of its left side versus time yields a straight line of which the slope is the value of rate constant k. Term m0 is the initial mass of the biomass (g).", "The terms mc and mb are the mass of char remaining after complete conversion (g) and the decomposition of the mass (g) respectively.", "The proximate and chemical analysis results for Triplochiton scleroxylon are shown in Table 1.", "This proximate analysis which was done for the characterization of Triplochiton scleroxylon showed that the biomass with good quantity of volatile matter and fixed carbon is favourable for the gasification of the organic material.", "The material with excessive moisture content required excessive energy to dry at initial stage, but its yield depends upon fixed carbon and volatile matter [18,19].", "Kim et al. [20] carried out proximate analysis and noticed that when the ash content increases the combustion volume decrease which impact the heat transfer, surface of biomass and also the quantity of oxygen into the biomass during combustion.", "During pyrolysis there is an endothermic reaction in the reactor so as to evaporate moisture in the biomass; the greater the moisture content of the biomass the longer the pyrolysis process will last in the reactor.", "Biomass having high moisture content impacts the quality and nature of syngas and bio-oil.", "The recommended value of moisture content in biomass intended for the bio-oil production is less than 10 wt% [21].", "Phase separation and quality of bio-oil in terms of density and viscosity are observed in bio-oil due to higher moisture content [22].", "The present work is not in accordance with other authors because the obtained moisture content is equal to 18.67 ± 0.58 wt%; this may be due to climatic conditions during the sampling of raw material.", "In order to bring out the repeatability of results, each experiment has been conducted trice and only the average data have been represented.", "Table 2 shows the average relative error of each experiment and on which it can be observed that the maximal relative error is less than 10%.", "That is a proof that results of various repetitions of each experiment are approximately identical.", "So, during gasification of samples, some parameters such as the mass loss of feedstock and temperature inside the reactor were noticed.", "Figs. 3–5 represent the mass loss and temperature profiles of sawdust with particle size less than <500 µm, using 9 g, 7 g and 5 g of sample, respectively.", "It is noticed in these figures that, temperature increases progressively over the time till becoming constant while the mass of sawdust in the reactor decreases until becoming constant.", "The simultaneous stabilization of both parameters marks the end of the gasification process in the reactor.", "The three masses have been converted into syngas after 14 min, 12 min and 10 min, respectively.", "The average temperature inside the reactor of these gasification tests was around 326 °C.", "Figs. 6–8 represent the mass loss and temperature profiles of sawdust with particle size 500 µm < d < 1 mm, using 9 g, 7 g and 5 g of sample, respectively.", "It is also noticed in these figures that temperature increases progressively over the time until becoming constant while the mass of sawdust in the reactor decreases progressively until becoming constant.", "Times for both parameters to reach the stabilization point were worth 20 min, 17 min and 12 min, respectively.", "These three gasification experiments performed with an average temperature inside the reactor of 340 °C.", "Figs. 9–11 represent the mass loss and temperature profiles of sawdust with particle size 1 mm < d < 1.9 mm, using 9 g, 7 g and 5 g of sample, respectively.", "It can also be noticed in these figures that temperature increases progressively over the time until becoming constant while the mass of sawdust in the reactor decreases progressively until becoming constant.", "Times for the mass and temperature to reach the steady state were worth 30 min, 24 min and 18 min, respectively.", "These three gasification experiments performed with an average temperature inside the reactor of 373 °C.", "It can be observed in all these above figures that the mass of sample reduces progressively till becoming constant; this implies that one part of sawdust has been converted into syngas associated with other volatile products.", "The remaining mass in the batch reactor represents the char formed during gasification process.", "It can be noticed in Table 3 that the formed char during each test represents in average 32% of the mass of sample initially introduced in the reactor.", "Therefore, the mass loss rate, which represents the gasification speed of feedstock, has been deducted from the slope of the linear regression of the mass loss curve.", "For the three particles sizes used in the present paper (d < 500 µm, 500 µm < d < 1 mm and 1 mm < d <1.9 mm), the gasification speeds were equal to 0.430 g/min, 0.359 g/min and 0.228 g/min, respectively.", "According to these results it can be observed that the reduction of the particles size of sawdust decreases its residence time in the reactor.", "According to results presented in Table 3, there is no significant variation on bio-fuel production when the particles size of sawdust is reduced (Fig. 12).", "Whatever the particles size, the production in syngas and volatile products does not change and remains around an average percentage of 67.96%.", "However a significant difference is observed in the residence time of feedstock in the reactor.", "As illustrated on Fig. 13, the less the particles size is the less is the residence time of sawdust in the reactor to be transformed into syngas and other chemical products.", "Due to the fact that the heat source is under the reactor, heat will progressively diffuse from the bottom to the top wall of the reactor.", "The gasification process will thus evolve following the diffusion of heat.", "Since the sensor has been set at the centre of the reactor, the increasing profile of temperature (Figs. 3–11) represents the duration for the gasification process to reach the centre of the reactor.", "So, the less the particle size is the more heat diffuses rapidly in the feedstock inside the reactor during gasification process.", "In order to verify whether the produced gas is effectively the syngas; a flame test was carried out at the end of each experiment.", "The syngas produced ignited producing a blue flame (Fig. 14).", "The fact that the produced flame was completely blue testifies the purity and the dominance of hydrogen among other gases.", "The present study reveals that Triplochiton scleroxylon, commonly called Ayous (in Cameroon) is a good substrate for syngas production; and that the particle size has an influence on syngas production, especially on residence time in the reactor.", "It was also noticed that the char produced is mostly equal to 32% of the amount of biomass introduced initially in the reactor.", "Future works will be focused on the quantification of the real syngas produced.", "A. Ali designed the project and made suggestions towards the improvement of the manuscript; P. Onguene Mvogo designed and prepared the experimental device; B. Tchouanti Nzali conducted experiments and wrote the manuscript; and R. Mouangue made critical review and also proposed suggestions towards its improvement.", "All the authors read and approved the final manuscript." ]

[]

[ "SOCI" ]

[ "Community-based system dynamics", "Diet", "Group model building", "Latin America", "Transport" ]

[ "3306", "3312", "3319" ]

[ "Over half of the world's population, more than 4.1 billion people, lives in cities (World Bank, 2015).", "In general, many health outcomes are better among urban populations than rural populations (Bai et al., 2012), although much heterogeneity exists both between and within cities.", "Many factors associated with urbanization can be good for health (e.g. greater employment opportunities, higher income levels, and access to more robust educational and health care systems), but others can be detrimental (e.g. sedentary lifestyles, consumption of processed foods, crowding, exposure to air pollution, and social inequalities) (Bai et al., 2012).", "Important questions remain regarding the dynamic processes that drive the health of urban residents (Diez Roux, 2015; Glouberman et al., 2006), as well as the policies and interventions that may be most effective in promoting the health of urban residents and the environmental sustainability of cities (Devi, 2015).", "Latin America is among the world's most urbanized regions, with over 80% of people living in cities (World Bank, 2015).", "Many cities in Latin America have implemented innovative public health (e.g., sweetened beverage taxes, food labeling) and urban planning policies (e.g., bus rapid transit, cycling infrastructure) to improve health, sustainability, and quality of life (Colchero et al., 2016, 2017; Corvalán et al., 2013; Heinrichs and Bernet, 2014; Ramos et al., 2017).", "The unique policy environments within these cities can serve as a model for identifying approaches to improve the health and sustainability of cities across the globe, particularly those in low- and middle-income countries.", "Two influential mechanisms through which city living impacts health are via factors related to food and transport systems.", "Despite living further from the places where food is produced, urban dwellers typically have access to a wide variety of healthy and unhealthy foods in restaurants, supermarkets, small corner stores, and other retailers (Fleischhacker et al., 2011; Richardson et al., 2012).", "Higher incomes coupled with greater access contribute to increased consumption of animal products, sugar, and processed foods among city dwellers in countries undergoing urbanization, which has fueled increases in obesity and chronic disease (Drewnowski and Popkin, 1997; Popkin, 1999).", "Other pathways through which urban environments likely affect diet include via city policies (e.g., food labeling, retailer restrictions), increased exposure to food marketing, and diet-related social norms, knowledge, and attitudes (Popkin, 1999, 2006).", "Pathways through which transport impacts the health of city residents include effects on active transport, sedentary behaviors and physical activity, commuting times, air pollution exposures and traffic related injuries (Frank and Engelke, 2001; Mueller et al., 2017).", "Urban planning decisions and urban transportation policies can impact health through their effects on automobile use, congestion, public transportation quality and availability, land use mix and pedestrian or cyclist– oriented development (Giles-Corti et al., 2016).", "The multiple, interacting pathways through which cities impact health can be thought of as a complex adaptive system (Bai et al., 2012; Diez Roux, 2015).", "Among the hallmarks of a complex adaptive system are factors at multiple levels of influence (e.g., individual-, neighborhood-, and policy-level factors) and feedback loops.", "For example, consider the variables that impact city residents' choice of commute mode.", "The decision to commute via private vehicle or public transit may be influenced by city-level factors such as congestion, adequacy of the road network, and access, quality, and affordability of public transit (Cervero, 2002).", "The decision may also be influenced by interpersonal factors –active transportation methods like walking and cycling may be seen as more normative among individuals with friends and colleagues who commute via these modes (Bopp et al., 2013; Campbell and Bopp, 2013).", "Personal factors, including an individual's socio-demographic characteristics (e.g., income, car ownership) and commuting context (e.g., distance) may also influence their choice (De Witte et al., 2013).", "Many of the factors that influence population health are likely to be interrelated and to influence each other via feedback loops, which make these systems adaptable.", "For example, consider a city with a transit network that includes roads, a bikeway, public transportation and transit oriented development.", "Under the initial conditions of the city, commuters will choose a transit mode based on context (e.g., relative prices), their commute characteristics (e.g., time by commute mode, distance, elevation change, neighborhood walkability and mixed land use), and preferences (e.g., attitudes towards bicycling).", "As the city's population grows, additional commuters may use the road network and urban sprawl may lengthen commuting distance.", "As a result, the city's road network will become more congested and driving times will increase.", "This change in driving times may cause some commuters who drive to re-evaluate their mode choice and switch to an alternative mode that is not as affected by congestion, like public transportation or cycling.", "As more and more people switch, the system will evolve a new state with a new distribution of drivers, cyclists, and public transportation users.", "This is an example of a balancing feedback loop, a type of stabilizing feedback structure that helps to regulate the effects of changes (i.e., population growth that adds new commuters) imposed on a system (Meadows, 2008).", "These complex dynamics of feedback and delays result in structures that may resist policy intervention or that may respond to policies in counterintuitive or counterproductive ways (Sterman, 2006).", "Identifying the feedback loops that impact people's behaviors can help us better understand why a complex system acts the way it does and can help build support for policy interventions to achieve healthier outcomes.", "Few studies have explicitly employed systems approaches to inform our understanding of drivers of health in cities.", "However, there are a range of methods available to help describe and explore the structure and function of complex adaptive systems that include both formal simulation modeling and qualitative approaches.", "Formal models can help us understand how urban policies can be used to improve health, particularly when the pathways through which the policy achieves its effects are fairly well understood (Beran et al., 2019; Rutter et al., 2017).", "For example, promotion of walking and cycling for transport can reduce vehicle emissions and congestion in cities, as well as promote active lifestyles among city dwellers.", "However, these benefits must be weighed against the costs of increased exposure to air pollution among pedestrians and cyclists (Giles and Koehle, 2014; Giles-Corti et al., 2016).", "An alternative approach to reducing congestion and emissions might be to improve public transportation infrastructure and implement options like bus rapid transit in combination with transit oriented development together with transit oriented development (Cervero and Dai, 2014).", "Formal models can help to quantify the costs and benefits that might be expected under these alternative approaches and serve as a policy decision support tool.", "Group model building is an approach to understanding how stakeholders perceive the variables, relationships, and feedback loops that comprise a complex adaptive system to co-create qualitative causal maps and system dynamics simulation models (Hovmand, 2014; Hovmand et al., 2015b).", "Community based system dynamics modeling (CBSD) prioritizes building stakeholders’ capabilities in the use of tools from the field of system dynamics (P Hovmand, 2014).", "Group model building and CBSD have been shown to improve understanding of a problem, promote systems thinking, and lead to consensus for action (Andersen et al., 1997; Hovmand, 2014; Richardson and Andersen, 1995; Vennix, 1996).", "The methods have been used across diverse disciplines (e.g., business, public health) in the public and private sector, and are well-suited to generate insights about the complex systems that drive health in cities (Brennan et al., 2015; Hovmand, 2014; Rouwette et al., 2002).", "SALURBAL (Salud Urbana en América Latina) is a multi-country, interdisciplinary research team spanning 14 institutions and 8 countries across Latin America and the U.S. that is studying how urban environments and policies impact health, health equity, and environmental sustainability in Latin American cities (Diez Roux et al., 2018).", "One of the aims of SALURBAL is to use the tools of complex systems to gain insights into the complex and interrelated drivers of health in Latin American cities, and to identify effective policy levers.", "To address this aim, SALURBAL is using complex systems simulations (i.e., agent-based models) combined with a series of CBSD workshops with a diverse group of regional stakeholders.", "These workshops were broadly focused on understanding the key variables and feedback loops via which food behaviors and transport impact health and environmental sustainability in Latin American cities, and on identifying food and transport policies that could be used to improve health and the environment.", "We chose to concentrate on the food and transport systems based on the large role that diet and physical activity play in driving the burden of chronic disease in Latin American cities (Gakidou et al., 2017), and because they represent policy areas of particular interest to the region (LAC-Urban Health and SALURBAL, n.d., November LAC-Urban Health and SALURBAL, n.d.).", "In this study, we describe the objectives, design, and implementation of the study, present key evidence regarding achievement of each objective, and reflect on insights generated throughout the process.", "The design process for the three CBSD workshops started with a 2.5-day training and planning session that brought together substantive experts from the research team with methodological experts on the use of CBSD.", "The session used a combination of lectures and hands-on experiential exercises to introduce key principles in system dynamics modeling, as well as the use of structured workshops and scripted activities (Hovmand et al., 2015b).", "As an example, a common script used in workshops is “Initiating and Elaborating a Causal Loop Diagram,” in which participants work together to draw causal maps that illustrate feedback loops that explain a dynamic behavior of interest.", "In the training session, participants elaborated two separate causal maps conveying their perspectives on traffic injuries and active transport.", "These activities were used to generate initial insights about these issues from a feedback perspective and motivate a discussion around how to adapt these scripts for use in workshops with stakeholders who had experience and deep knowledge of food and transportation systems in Latin American cities.", "At the end of the training session, the team developed a list of explicit (i.e., communicated to participants prior to workshops) and implicit (i.e., values and principles that influenced the workshop approach) objectives to inform the design of the workshops (see Table 1), identified team members to serve key roles on the core modeling teams (CMTs), and laid out a framework for the design process.", "The CMTs consisted of a representative from the country hosting the workshop, at least one modeler, someone with knowledge of the participants, someone familiar with the objectives of the study, and at least one expert in CBSD methods.", "The CMTs for each workshop were responsible for convening over the two months preceding each workshop to select and adapt scripts, develop a facilitation manual for each workshop, work with team members from relevant countries to identify participants, and communicate on an ongoing basis with the larger SALURBAL study team and systems working group.", "In order to build capacity within our team (Objective E2), facilitation was led by members of the research team with substantive expertise in food and transport systems, and team members with methodological expertise in CBSD served as process coaches (please see Scriptapedia for descriptions of roles) (Hovmand et al., 2015a).", "Between November 2017 and May 2018, the SALURBAL team conducted one CBSD workshop in each of three cities: Lima, Peru; São Paulo, Brazil; and Antigua Guatemala, Guatemala.", "In general, participants were purposively identified by SALURBAL team members from countries in the same region where each workshop was to be held (e.g., the Lima workshop included participants from Peru, Argentina, Chile, and Brazil).", "Participants were also targeted for recruitment based on their expertise in either food or transport systems.", "Within these content areas, three types of participants were targeted: elected and administrative policymakers, members of civil society (e.g., nonprofits), and academics.", "Additionally, two private sector stakeholders with relevant domain expertise were identified by Brazilian team members and recruited to participate in the São Paulo workshop.", "No private sector stakeholders were identified for recruitment in the Lima or Antigua Guatemala workshop.", "For each workshop, an effort was made to include 8–12 participants from each of the food and transport systems content areas (i.e., a total of 16–24 participants per workshop), and to include participants from diverse countries (with the exception of the São Paulo workshop, due to language constraints).", "Each participant targeted for recruitment was emailed an invitation letter describing the SALURBAL study and purpose of the CBSD workshops; follow-up emails were sent by team members in the country hosting each workshop.", "Each of the three workshops took place either during a full-day session (Lima) or a 1.5-day session (Antigua Guatemala and São Paulo).", "Workshops were conducted in the predominant language of the host country (i.e., Spanish or Portuguese).", "The structure of each workshop was guided by the facilitation guide produced by the workshop's CMT (please see the supplement for a sample facilitation guide).", "The project employed an iterative approach whereby the structure of each workshop was slightly altered based on context and lessons learned from preceding workshops, as described in the next section.", "In general, workshops were composed of an introductory session with a general presentation about SALURBAL and an overview of complex systems thinking, followed by a series of scripted activities that were led by a facilitation team (Hovmand et al., 2015b).", "Each facilitation team included a convener, two community facilitators (i.e., one with domain expertise in food and one in transport), two modeler facilitators, at least four recorders, and 1–2 process coaches.", "Generally, the convener introduced the project at the beginning of the workshop and helped close the workshop.", "Scripted exercises were typically led by the modeler facilitator in collaboration with the community facilitator.", "The modeler facilitators also developed causal loop diagrams (CLDs) that aggregated and combined variables and feedback loops across multiple CLDs produced by sub-groups of participants (see Supplemental Figure S5).", "Other roles on the facilitation team generally provided support (e.g., the recorders took notes throughout each workshop that were later used to inform analytic decisions, as described below).", "Further details describing each of these roles can be found on Scriptapedia (Hovmand et al., 2015a).", "The agenda for each workshop included a series of scripted activities (see Table 2).", "While the scripts provided a structure for the critical components of each activity, they also allowed enough flexibility for the facilitation team to make adaptations both in the planning phases immediately before, and on-the-fly during the course of any given activity.", "This was done to ensure that each script achieved its intended objectives.", "A Graphs Over Time script encouraged participants to think dynamically and to define the boundary of the problem under investigation (e.g., a prompt was to “please think of a factor that influences healthy eating in cities”).", "In the Causal Loop Diagramming script, the modeler facilitator asked sub-groups of 3–4 participants to build a causal loop diagram that explains the feedback structure that generates the dynamic behavior of one to two factors in the food system or transportation system, and how that factor can influence a healthy urban environment.", "During the Action Ideas script, participants were asked how they would intervene on the systems depicted by the CLDs, the size of the potential impact and feasibility of the proposed interventions, and whether there might be unintended or secondary consequences.", "Each participant completed every scripted activity, either as part of the full group or in parallel among sub-groups of participants.", "As shown in Table 2, many of the scripted activities produced artifacts.", "We primarily report on artifacts produced during the Causal Loop Diagramming script (i.e., CLDs) and the Action Ideas script (i.e., a list of action ideas).", "Although we do not describe artifacts produced in the Hopes and Fears or Graphs Over Time scripts, it is important to note that these scripts and the artifacts produced were critical to the workshops and supported subsequent scripts.", "For example, the Graphs Over Time script was used to help participants think about food and transport as dynamic variables that change over time.", "The variables identified during this script informed those that emerged in the CLDs developed in the subsequent script.", "The CMTs made subtle changes to the activities and structure of the second and third workshops in response to lessons learned and participant feedback in the first workshop.", "The Lima workshop took place during a single day, but the workshops in São Paulo and Antigua Guatemala were extended to 1.5 days to allow participants more opportunities to work across sectors and refine their thinking together, as well as a night for participants to reflect on the knowledge gained during the first day.", "A key difference in the structure of the workshops is that in the Lima workshop there was a single causal loop diagram (CLD) workshop completed by small groups of food or transport domain experts.", "The São Paulo and Antigua Guatemala workshops included an additional CLD activity that was completed by small “mixed” groups of participants with domain expertise in food and participants with expertise in transport.", "The purpose of this combined CLD activity was to promote transdisciplinary thinking between the groups and to understand how stakeholders conceptualize connections between the systems driving food behaviors and transport.", "The São Paulo and Antigua Guatemala workshops also included additional time for the facilitation team to present the synthesized penultimate CLDs to participants, to receive feedback, and to modify the feedback structures to more accurately represent participants’ perceptions of the underlying dynamics.", "Another important difference between workshops was that we slightly modified the Action Ideas script based on lessons learned from the preceding workshops, in order to facilitate further integration between the CLDs and the intervention ideas.", "In Lima, participants wrote their action ideas onto a piece of printer paper, but did not map the action ideas directly onto the CLDs.", "In São Paulo, participants had printouts of the CLDs that they could use individually to either visualize or trace out how an action idea would work within the context of the variables and relationships in the CLD.", "In the final workshop in Antigua Guatemala, participants worked with the modeler facilitator to add their action ideas directly onto the synthesis CLD produced by the group.", "This final iteration facilitated critical thinking regarding how interventions work within the context of a system, as well as group discussion because action ideas were “mapped onto” the synthesis CLD in real time while being projected for participants' review and feedback.", "These iterations in design were purposeful to facilitate and encourage more explicit consideration of how policies could work within the system of variables and feedback loops depicted in the CLDs.", "For example, consideration of the CLDs can help participants to identify leverage points for intervention in the system (i.e., places where a small change can produce a large shift in system behavior), as well as sources of resistance that may impact the intervention's effectiveness (Meadows, 2008).", "Three team members reviewed the CLDs created during all three workshops and conducted a content analysis to identify common themes in the variables in the CLDs.", "All three team members were from Latin America.", "One had deep methodological expertise in system dynamics modeling and domain expertise in transport.", "The other two had exposure to complex adaptive systems, primarily through participation in the 2.5-day training and planning session at the beginning of the project; one had domain expertise in transport, while the other had expertise in food.", "The purpose was to develop a qualitative understanding of the themes that emerged in each of the workshops, as well as to provide some general insight into the frequency with which variables with similar themes were present across workshops.", "Each team member used Qualtrics software to group all of the variables included in the final CLDs from the regional workshops into thematic piles.", "A separate team member then “labeled” the theme that emerged from each pile, compared the piles generated by each team member, and developed a list of the themes that emerged and their corresponding variables.", "The themes, labels, and variable lists were then presented back to the SALURBAL systems working group and feedback was solicited.", "No conflicts in labeling or theme identification were identified by the working group.", "After all workshops were completed, we conducted a multi-stage synthesis.", "The purpose was to produce a single “synthesis” CLD that, in the simplest way possible, captures the major variables and feedback loops that drive change over time in food behaviors and transport in Latin American cities, as identified by participants in the three workshops.", "Inputs to the synthesis included the final CLDs produced in each of the workshops, as well as reports and notes from each workshop.", "In total, there were four input CLDs: one transport CLD and one food system CLD from Lima, and one cross-domain CLD each, from São Paulo and Antigua Guatemala.", "Each CLD was produced during a series of iterative synthesis scripts that used CLDs produced by sub-groups of participants (i.e., during the Causal Loop Diagramming script).", "Please see Supplemental Figure S5 for a flow diagram showing how CLDs were combined in this study.", "The aim of the first stage of the synthesis was to develop a list of the feedback loops in each input CLD, and to identify those feedback loops that were common across multiple CLDs.", "Three team members (one with content expertise in food and methodological expertise in complex systems; two others who were recorders during at least one of the workshops) began by identifying each of the major feedback loops and variables featured in the CLD produced during the São Paulo workshop; we started with this CLD because it had the most feedback loops.", "We then did the same for the other CLDs.", "As we identified each feedback loop, we qualitatively assessed whether there was a high degree of overlap between it and those previously identified (e.g., whether the dynamics described by a feedback loop from Antigua Guatemala were very similar to those identified in São Paulo).", "Key considerations in this assessment were text descriptions of each CLD created by workshop participants, as well as recorders’ notes from the workshops.", "In the second step of the synthesis, the same three team members developed an “aggregate” CLD that included all unique feedback loops identified across the three workshops.", "In cases where feedback loops from multiple input CLDs described the same fundamental dynamics (as determined in the previous step of the analysis), the principle guiding the analysis was to reduce the variables and relationships in the CLD into the most parsimonious structure possible that captured the underlying dynamics.", "During this aggregation, we generally retained variable labels developed by participants.", "Notably, there were no disagreements about the directionality (i.e., + or -) of the relationships between common variables described by participants.", "Lastly, in a third stage, three team members (one who participated in the previous round of synthesis, one with content expertise in transport, and one with methodological expertise in CBSD) conducted multiple stages of refinement in order to produce a “synthesis” CLD.", "The purpose of the synthesis was to develop a concise CLD with a reduced number of feedbacks that depicted fundamental dynamics described by participants during the workshops.", "At each stage, we compared the synthesis CLD with supporting information from the workshops (e.g., workshop notes, reports, and original artifacts) to ensure integrity and correspondence to the original intentions of workshop participants.", "Concurrent with each stage of synthesis, we presented the initial versions of both the aggregate CLD and synthesis CLD to team members involved in the planning and implementation of the workshops; team members provided verbal and written feedback that we then used to guide revisions.", "The study protocol was reviewed by the Drexel University Institutional Review Board, which determined that the study did not consist of human subjects research.", "Investigators in Brazil received formal approval for the Brazil workshop in accordance with the requirements of their local Institutional Review Board.", "A total of 62 stakeholders participated in the three workshops, including 32 with expertise in the food systems domain and 30 in the transport systems domain (see Table 3).", "Latin American countries were broadly represented, with participants representing ten different countries.", "There was a fairly even mix of stakeholders from academia, civil society, and policymaking across all three regions.", "In Brazil, two stakeholders from the private sector also participated in the workshop.", "Following each workshop, participants received a report and the facilitation guide that was used to implement the workshop in Spanish or Portuguese.", "The purpose was to provide a tangible deliverable via which participants could see some of the results of the time they committed to the project, to serve as a vehicle for continued engagement with SALURBAL, to facilitate collaboration between participants, and to facilitate continued systems thinking and use of these types of methods in participants’ own work.", "The report was prepared by the CMT and included background about the study, the explicit and implicit objectives guiding the workshops, an overview of participant information (i.e., number of participants by country and affiliation), the summary agenda, the CLD/s produced during the workshop, feedback loops and descriptions provided by participants during the workshop, action ideas, and reflections and insights that emerged from the workshop.", "Both participants and members of our team gained experience in CBSD and use of causal loop diagrams to understand urban health.", "Fifteen team members participated in the two-day training in CBSD methods.", "Furthermore, 25 team members participated in the CMT for at least one of the three workshops.", "This included two methodological experts with experience in CBSD, who helped guide the process of designing the workshops.", "Thirty-nine team members participated in the facilitation team for at least one of the three workshops, meaning that they contributed to the implementation and execution of the workshops themselves.", "Roles played by team members during the workshop include conveners, facilitators, reflectors, process coaches, recorders, and observers, as well as five team members from the country hubs that provided logistical support.", "No formal evaluation was conducted to assess pre-versus post-workshop changes in knowledge or understanding dof systems approaches, use of CLDs, or impacts on problem understanding.", "In Supplemental Figures S1-S4, we present the four CLDs produced across the three workshops.", "This includes one food and one transport CLD produced in Lima, and one CLD that includes both systems produced in each of São Paulo and Antigua Guatemala.", "There were 98 variables across the four CLDs, including several that were common across more than one CLD.", "For example, all three workshops included consumption of ultra-processed foods (UPF) as an important individual-level behavior within the food system, and car use and congestion as key system-level behaviors within the transport system.", "A key step in understanding how stakeholders view the systems that drive healthy eating and transport is identifying themes in the variables included in the CLDs.", "The research team identified eight themes via an inductive pile-sorting exercise, to which we assigned the following names: 1) health outcomes, 2) individual behaviors, 3) system behaviors and outputs, 4) time use, 5) built environment and access, 6) knowledge and attitudes, 7) policy and policymaking, and 8) social equity.", "In Table 4, we present these themes and variables.", "In Fig. 1, we present the synthesis CLD that includes the major feedback loops from CLDs from all three workshops.", "The synthesis CLD includes four balancing and six reinforcing feedback loops.", "In Table 5, we present a description of each feedback loop.", "The descriptions were developed by participants and were translated and lightly edited for clarity by the research team; the research team used these descriptions to create brief, informal names for each feedback loop.", "There were also important differences in the variables and feedback loops described across workshops.", "For example, participants in Antigua Guatemala (please see Appendix Figure S4) described an important feedback structure linking gender equity in the labor force, gender roles, and time use.", "Specifically, participants posited that an increase in women in the labor force promotes gender equity, which can affect the distribution of household responsibilities between men and women.", "This reduces the pressure on women to be the sole preparer of food in the household and, in turn, contributes to more women entering the workforce.", "Conversely, in societies with a highly gendered distribution of household responsibilities, the constraints on women's time will prevent many from entering the workforce.", "An example of a feedback structure unique to the São Paulo workshop was a reinforcing loop related to urban agriculture and pollution (please see Appendix Figure S3).", "As urban and peri-urban agriculture increase, air pollution will eventually decrease as carbon dioxide is removed from the air.", "Because ozone pollution causes oxidative damage to plants that reduces crop productivity (Lobell and Gourdji, 2012; Wilkinson et al., 2012), the decreased air pollution leads to more and more productive urban and peri-urban agriculture.", "Collectively, stakeholders identified clear connections between the food and transport systems, respectively, and health.", "As shown in Table 4, participants in all three workshops included at least one health outcome as a variable in their CLD.", "Specific variables within the health domain included general health and health problems, chronic disease, obesity, and health care costs.", "While no variables explicitly mentioned health equity, stakeholders did describe feedback loops pertaining to social equity.", "One example is the feedback loop related to gender equity described above.", "A second example described by participants in the Lima workshop related to commuting patterns and residential segregation.", "Specifically, many of the poorest residents of Latin American cities live in peri-urban slums on the outskirts of cities.", "This increases travel times between these individuals’ homes and workplaces, which limits the number of hours they can work and, as a result, their income.", "As cities grow, these individuals are often pushed even further outside of the city center, which lengthens their commutes even further.", "Participants identified 52 action ideas to improve health through intervention in the food and transport systems (please see Table S1 in the Appendix for the list of action ideas).", "In general, about half of these action ideas consisted of policies or interventions within the domain of the built environment and access.", "For example, participants in the Lima and Antigua Guatemala workshops identified taxes on ultra-processed foods as a high-impact strategy to improve health.", "Participants in all three workshops also identified urban planning strategies via which cities can be redesigned to facilitate healthy choices, including by improving walkability and safety, conserving vital public spaces, increasing housing density, and promoting mixed-use planning so that people can access food sources, recreation facilities, and other resources within their own communities without using cars.", "Participants in the Lima and São Paulo workshops identified several action ideas related to policy and the policymaking process.", "Several policies were intended to address specific barriers or challenges within the food and transport systems.", "Within the food system, this included legislation to explicitly recognize that vacant areas may be used for urban gardening, as well as creation of operating regulations for urban farms.", "Within the transport system, this included development of improved fuel standards.", "Action ideas also reflect stakeholders’ recognition of the importance of the policymaking process itself – one example is an idea to incorporate advocacy into public policy agendas for urban redesign.", "Participants in the Antigua Guatemala and Lima workshops identified several action ideas to improve nutrition knowledge and attitudes.", "These included policies related to comprehensible nutrition labeling, social marketing and nutrition education, bans on food marketing directed at children, and development of dietary guidelines.", "The study successfully achieved several of its objectives, particularly those related to stakeholder engagement, capacity building, and working with stakeholders to develop explicit representations (e.g., CLDs) of their mental models.", "In reflecting upon the planning and implementation of the workshops, our team identified several insights regarding both the process and our understanding of stakeholders’ perceptions of the systems through which food and transport systems impact health.", "A major emphasis of the SALURBAL study is to help develop the research infrastructure, partnerships, and expertise necessary to advance public health and health policy research within the Latin American context.", "The process of planning and implementing the CBSD workshops was a clear opportunity for capacity building, because most research team members, including those from both Latin American and U.S. institutions, had little practical experience with these methods but high interest in using them.", "The team-based approach to planning and implementing the workshops was a purposeful design decision on the part of the team members that were experienced in these methods.", "This design decision is an important aspect of the study, and likely impacted both the structure of the workshops and artifacts (e.g., causal loop diagrams, action ideas) produced.", "The approach allowed for a high number of team members to learn about and gain practical experience with the methods.", "Key contributors to team members' learning included the two-day training that took place at the study's outset, as well as the guided planning and implementation process in which at least one methodological expert worked with the core modeling and facilitation teams.", "Given that use of complex systems methods in public health is in its early stages, we believe that this type of facilitated learning model can be quite useful for diffusion of these methods.", "Several members of the research team have leveraged the experience gained through the development and implementation of the workshops to utilize CBSD methods in other aspects of their work.", "One example is that team members from Colombia have used CBSD methods to investigate the potential effects of a new cable car project (i.e., TransMiCable) that connects a low-income, self-built neighborhood on steep hillsides in Bogotá to the main station.", "These investigators used a similar process and workshop design to understand how the TransMiCable implementation will affect a range of environmental and social determinants of health, healthy behaviors, and health outcomes.", "A second insight related to the process of implementing the workshops is that our team was struck by participants’ ability to describe the structure and function of the food and transport systems in incredible depth with only minimal training related to complex system structures and CLD notation.", "As described earlier, participants were able to identify a broad range of feedback loops, as well as to articulate examples of how these loops function.", "A third insight is that stakeholders viewed their participation in the workshops quite favorably and felt that the tools (e.g., CLDs, common language) and experiences to which they were introduced helped them to see the issues with which they work from a different perspective.", "For example, one of the stakeholders worked for a government agency and initially questioned why he was asked to participate in an academic workshop.", "By the end of the workshop, this participant mentioned the utility of the complex systems approach and told a facilitator that he planned to seek external funding to continue to use CBSD and systems thinking within the context of his agency's work.", "Participants also expressed their appreciation for the materials they received after the workshop.", "Finally, the workshops were not devoid of challenges or resistance.", "In follow-up interviews, multiple participants reflected that the workshops allowed for disagreements (e.g., in the relationships between variables) and fostered productive conversations to find common ground and resolve these disagreements.", "For example, some participants created CLDs that had feedback loop structures that mirrored those included in the introductory presentation, others felt limited to only including variables for which data had been collected.", "Some participants struggled with how to represent differences between groups (e.g., gender) in the CLD, or with drawing graphs of variables over time, instead drawing graphs of correlations between variables.", "Another source of friction was related to disagreements between participants regarding key variables and relationships to including in CLDs.", "These types of challenges are common in CBSD because it is an interpersonal process that encourages participants to make their mental models explicit and negotiate a revised view of a problem that integrates new and different perspectives.", "The design and facilitation of workshops emphasized the creation of CLDs as boundary objects, which are tangible representations of dependencies and interactions that cross disciplinary, organizational or social lines (Black and Andersen, 2012).", "Use of CLDs and other visual representations as boundary objects can be used to turn disagreement and conflict into collaborative problem-solving discussions (Black and Andersen, 2012).", "We found that, as is common in many CBSD workshops, the work of developing models helped participants to embrace conflict and generate new insight and meaning.", "Some of the challenges and sources of resistance we encountered in the workshops were anticipated and discussed by the core modeling team and facilitation team prior to the first workshop, while others emerged during the first and second workshop and discussed in planning and training for the remaining workshops.", "In reflecting upon the workshops and findings, one of the insights we found interesting is that stakeholders were more readily able than we had anticipated to identify connections between the systems driving food and transport behaviors.", "For example, CLDs in each of the workshops included time use variables in both the food and transport systems.", "Participants in Antigua Guatemala further described how the size of cities is interrelated to both food and transport.", "An increase in car ownership can contribute to urban sprawl and, as cities grow, the distance between food producers and consumers becomes greater.", "This is just one example of the complex and dynamic interrelationships between the pathways through which living in cities impacts the health of urban dwellers.", "A second insight is that some of the feedback loops described by stakeholders may help explain secular trends in food behaviors and transport, as well as policy responses.", "An important secular trend in many countries, including several in Latin America, is increased UPF consumption over the last several decades (Monteiro et al., 2010; Popkin and Hawkes, 2016; Rivera et al., 2004).", "The synthesis CLD suggests several dynamic mechanisms that may have contributed to these increases.", "For example, as UPF consumption rises, the food industry can fund lobbying activities to stifle policy interventions and can purchase food advertising that promotes and normalizes UPF consumption (i.e., feedback loops R4 and R5) (Kassahara and Sarti, 2018; Rovirosa et al., 2017; Théodore et al., 2014).", "In contrast, enactment of government policies to promote healthy eating occurs over a longer time horizon and occurs only after serious health effects emerge (e.g., the obesity epidemic).", "These feedback loops may help to explain why expansive policies to improve healthy eating have recently passed in Peru (i.e., front-of-pack warning labels), Chile (i.e., front-of-pack warning labels and marketing regulations), and Mexico (i.e., sugar-sweetened beverages and junk food tax).", "These countries are fairly far along in the nutrition transition (Drewnowski and Popkin, 1997; Popkin, 1999, 2006), but evidence from Colombia and elsewhere suggests that the process of passing health-protecting legislation like excise taxes on sweetened beverages and tobacco taxes can be lengthy and require civil society engagement in advocacy, acquisition of evidence that includes monitoring of health consequences, estimation of relationships between variables, and policy evaluation (Moodie et al., 2013; Villar Uribe, 2017).", "This evidence can be used to inform public opinion, counter the effects of industry lobbying, and support policy decisions (Villar Uribe, 2017).", "A third insight is that feedback loops may vary in their strength and implications in different contexts and starting conditions.", "For example, the synthesis CLD highlights two different mechanisms through which cities may respond to the problem of congestion.", "One of the feedback loops (R1) suggests that cities can attempt to ease congestion by investing in more and bigger infrastructure for cars (e.g., more and bigger roads and freeways).", "This type of investment, however, can contribute to urban sprawl.", "In contrast, cities can also respond to congestion by investing in active and public transit infrastructure with the goal of facilitating a shift in commuters’ transit mode choices and promoting healthy behaviors like physical activity (B1).", "Cities that are diffuse, that have previously invested in road infrastructure, or that have very high rates of car ownership may be less likely to invest in active and public transit infrastructure than cities that are more dense, have made these investments in the past, and that have less of a “car culture.”", "The importance of context and starting conditions can help explain why some cities (e.g., Guatemala City) invest predominantly in road infrastructure while others spend more on active and public transit infrastructure (e.g., Curitiba, Bogotá).", "In our view, it is premature to say whether we achieved some of the study's objectives.", "For example, the third explicit objective was to use insights from the workshops to identify and prioritize research questions and practice implications that our team will address using complex systems modeling in the future.", "Our team has begun development of two agent-based models: one to explore policies to improve active transit, the other to identify policy approaches to reduce consumption of ultra-processed foods.", "The CLDs produced in the workshops were among several inputs to the design of these models; however, the range of variables and relationships included in the CLDs was typically much broader than would fall within the boundary of any agent-based model that we could reasonably develop.", "That said, outputs from the workshops informed the outcomes and policy levers to be explored in each of the models.", "As the larger SALURBAL study continues to develop, an important step will be to further consider how insights from the workshops, including the CLDs, can be built upon through the use of other modeling efforts (e.g., formal system dynamics models).", "The final implicit objective of the study was to test the waters for a potential simulation model, and to assess the potential for dissemination of complex systems approaches beyond academia.", "The workshops were extremely useful for demonstrating the high potential of participatory complex systems methods like CBSD modeling, but we did little to assess participants’ receptivity to simulation models.", "As the CMTs planned each of the workshops, it become clear that introducing stakeholders to simulation models would take substantial time out of the agenda that could be better used with scripted activities to introduce more general principles in systems thinking.", "Introducing stakeholders to simulation-based complex systems methods may take place in follow-up activities as SALURBAL evolves.", "This may, for example, include facilitated table-top exercises in which policymakers can use the agent-based models to explore policy counterfactuals.", "In considering our synthesis CLD and other findings, it is important to note that the purpose of the CBSD workshops was not to develop an authoritative depiction of the underlying systems that drive food behaviors and transport across Latin American cities or even within the specific cities in which the workshops were held.", "Some of the differences between variables and relationships between variables that we observed across workshops may be driven by the interests of the specific stakeholders present or chance and don't necessarily reflect regional differences in the real systems being depicted.", "Developing a representative depiction of the systems that drive health in a given Latin American city (or region), for example, might involve a series of workshops conducted serially with multiple cohorts of participants.", "If conducted across multiple cities (or countries), such an approach would allow for a more nuanced examination of commonalities and differences in the variables, feedback loops, and policies identified across different contexts.", "In contrast, our approach was designed to support our research team and stakeholder partners to view the systems with which we work from a broader perspective, and to generate insights to refine and prioritize future collaborative work.", "The artifacts (e.g., CLDs, action ideas) produced during the workshops reflect the unique knowledge and experiences of stakeholders and may be of high relevance in some urban contexts in Latin America and less relevant in others.", "Stakeholders were purposively recruited by SALURBAL team members based on team members' professional networks and knowledge of regional stakeholders with relevant domain expertise.", "As such, our findings may not be generalizable to other stakeholders or regions in Latin America.", "Similarly, it is worth considering that important variables and feedback loops that link the food and transport systems to health and the environment may not be included in the CLDs or other artifacts produced during the workshops.", "For example, stakeholders in the three workshops did not focus on mechanisms via which food and transport behaviors contribute to and are influenced by global climate change.", "It is difficult to assess the extent to which the omission of these mechanisms reflect the structure of the underlying systems being explored, stakeholders’ unique perspectives regarding these systems, workshop design (e.g., structure of scripted activities, prompts, time constraints), or the model boundary (e.g., we used the Graphs Over Time script to explicitly set the boundary to include only factors that occur within cities).", "We used an unscripted approach to develop the synthesis CLD (Fig. 1).", "The unscripted approach relied on artifacts and notes from each workshop to identify the main narratives variables and feedback loops.", "Facilitation team members from each workshop were also involved in developing and reviewing multiple iterations of the synthesis model.", "We acknowledge, however, that this process does create space for bias in filtering the variables and feedback loops included in the synthesis model.", "The only bias that we knowingly used was one that favored the inclusion of feedback narratives, as that is the basis of the SD approach.", "Due to time and budgetary constraints associated with facilitating workshops across multiple cities, we did not validate the synthesis model by systematically soliciting participants’ feedback.", "In future research, it is worth considering a study design that would employ series of workshops that would enable participants to review, consider, and refine a synthesis model in an iterative fashion and help to elucidate common and divergent system structures that emerge across varying urban contexts.", "Finally, the action ideas developed by participants were an important set of artifacts produced during the workshops.", "As mentioned in the methods section, we adapted the “Action Ideas” script prior to the final workshop in Antigua Guatemala to ask participants to explicitly draw how each intervention would affect the variables and relationships included on the CLD produced by the group.", "In contrast, during the previous two workshops we asked participants to consider how each intervention would impact the system but did not ask them to explicitly draw these impacts on the diagram.", "As a result, we are unable to assess how each action idea may (or may not) influence the systems of variables and feedback loops elucidated by participants, or how the workshops and CLDs may have impacted participants’ thinking with respect to intervention approaches.", "The CBSD workshops were a method for meaningful, early engagement of policy and practice stakeholders.", "The guided process of planning and implementing the workshops was a unique approach that helped build our diverse team members’ capacity for CBSD methods and deepen our capacity for systems thinking.", "The workshops were an important vehicle for introducing stakeholders to complex systems thinking, and generated artifacts and insights that are useful for understanding the mechanisms through which the food and transport systems impact health.", "We hope that this study can serve as a blueprint for other multi-disciplinary, stakeholder-engaged studies that use the tools of complex systems to examine drivers of urban health.", "The Salud Urbana en América Latina (SALURBAL)/Urban Health in Latin America project is funded by the Wellcome Trust [205177/Z/16/Z].", "More information about the project can be found at www.lacurbanhealth.org.", "JDM received funding from the joint grant of Research Offices at the Universidad de Los Andes and the Universidad de Ibagué (project 19-528-ESP)." ]

[ "CBSD methods can promote systems thinking and help identify policy approaches.", "Stakeholders identified 98 variables driving food behaviors and transport in cities.", "Balancing and reinforcing feedbacks drive change in food behavior and transport.", "Feedback loops can also explain policy responses and lack of policy responses.", "Stakeholders identified 52 policies that leverage variables and feedbacks." ]

[ "CENG", "MATE" ]

[ "AE", "Acoustic emission", "Composites", "Mechanical properties of adhesives", "Metals", "Shear-lag model", "Stress analysis" ]

[ "1500", "2502", "2507" ]

[ "The use of adhesively-bonded double-lap joints (DLJs) is preferred over conventional joining techniques such as bolting, riveting and welding.", "They offer many advantages including, for instance, the ability to join dissimilar materials such as steel and fiber reinforced composites, weight savings, improved stress distribution along the bond-line and enhancement of the corrosion and fatigue resistance characteristics [1–3].", "Such characteristics are essential, especially in maritime applications.", "However, the dominant damage mechanisms in these joints may vary based on various factors including: loading conditions, environmental conditions, mechanical properties of adhesive and adherends, adhesive thickness and also the adhesion quality between the adhesive and adherends.", "These damage mechanisms include, but are not limited to: interfacial failure between the adherends and adhesive layer (i.e., adhesive failure), cohesive failure within the adhesive layer and failure within the adherends [4,5].", "Therefore, a deep and thorough understanding of the mechanics of the bi-material adhesively-bonded joints is essential to fully utilize their capabilities in industrial applications.", "Experimentally, various techniques have been employed to study the stresses and damage evolution in adhesively bonded joints including, but not limited to, Digital Image Correlation (DIC) [6–8] and Acoustic Emission (AE) [9–11].", "Thäsler et al. [6] proposed a method to measure and track the crack growth in fatigue-loaded adhesively bonded CFRP joints using DIC.", "They applied their technique to single and double-lap shear adhesively-bonded joints.", "Moreover, Saeedifar et al. [7] characterized the damage in bi-material steel-to-composite double-lap adhesively-bonded joints using the combination of AE and supervised clustering techniques.", "In addition, they successfully validated the classified damage mechanisms against DIC, Fiber Optic Sensor (FOS) and camera results.", "Beside experimental techniques, to understand the mechanics and stress state in DLJ, analytical approaches can represent an alternative to numerical modelling providing closed-form solutions with an acceptable level of accuracy and robustness.", "Comprehensive literature reviews [12–14] are available detailing the performance of various analytical models in predicting the stress distribution for both single and double-lap joints for similar and dissimilar adherends.", "Da Silva et al. [12] concluded that almost all the available analytical models are two-dimensional, which is sufficient as the stresses in the width direction are generally lower than the loading direction.", "In addition, most of the models are limited to the linear-elastic regime for both the adherends and the adhesive as non-linearity introduction leads to very complex solutions which might require to be solved numerically at the end.", "They also discussed the limitations of the classical analytical models which include: i) neglecting the adherends’ shear deformation, ii) assuming only linear adhesive response and iii) overestimating the stress at the end of the overlap by violating the stress-free condition.", "This overestimation results in a conservative failure load prediction when designing DLJs.", "As an attempt to overcome these limitations, the improved shear-lag model, by Tsai et al. [15] for double-lap adhesively-bonded joints, was proposed to take into account the shear deformation in the adherends.", "Moreover, Liu et al. [16] introduced a closed-form solution to the stress/strain distribution in bi-material bonded joints using ductile adhesive.", "In their model, they accounted for the adhesive plasticity.", "Again based on a shear-lag formulation which was validated against experimental results, the model was able to analyze an arbitrary thermal and mechanical loading condition.", "Moreover, Shishesaz et al. [17] investigated the effect of adhesive thickness and viscoelasticity on the shear stress distribution in DLJs using analytical models.", "Results reported a drop in the peak shear stress to almost 1/3 of its initial value by increasing the thickness from 0.01 to 0.1 mm.", "In addition, a defined ratio of the viscous effect to the shear modulus of the adhesive is found to have an adverse effect on the peak shear stress in the joint.", "Although this study investigated the adhesive thickness effect, still the maximum thickness investigated (~0.1 mm) is considered very thin for applications such as maritime applications, which is the focus of the current study.", "To the authors’ knowledge, there is almost no study in the literature investigating the potential of using shear-lag analytical approaches for relatively thick (~8 mm) adhesively-bonded bi-material DLJs.", "Thus, the objective, of this study, is to determine to what extent analytical models “such as the improved Tsai model” can be reliable in predicting and understanding the stress state in bi-material thick adhesively-bonded joints in the light of using DIC and Finite Element (FE) models.", "Two different structural adhesives, representing a ductile (Methacrylate-based) and brittle (Epoxy-based) types, are used to bond CFRP skins to a steel core.", "The fabricated joints are loaded in tension while damage evolution is monitored by DIC and AE.", "Results from the DIC displacement/strain map are used to validate the distributions obtained from the analytical Tsai-improved model and a linear-elastic FE model.", "Besides, the damage and failure analysis is studied by correlating the AE cumulative energy with the macroscopic load-displacement of the DLJ and the DIC displacement distributions at specific locations.", "Finally, a summary of the concluding remarks of this research study is provided.", "The steel used in this study is high strength structural shipbuilding steel AH36 with Young's modulus of ~ 200 GPa, yield strength of 350 MPa, ultimate strength of (400–550) MPa and Poisson's ratio of 0.26.", "The CFRP laminates are produced using vacuum infusion.", "In order to avoid galvanic corrosion, a thin layer of chopped-glass fiber fabric was used on both sides.", "The lay-up was quasi-isotropic, with the 0°-direction in the length direction of the final specimen geometry.", "The thickness of the steel plate and the CFRP laminate used is 8 mm and 2.5 mm respectively.", "The first adhesive used to bond the steel and CFRP adherends is a two-component toughened epoxy adhesive with a mixing ratio of 2:1 by volume.", "The adhesive tensile modulus is ~ 2 GPa with a tensile strength of ~ 35 MPa and strain to failure of 10% as per the manufacturer datasheet.", "The second adhesive used is a two-component Methacrylate adhesive with a mixing ratio of 10:1 by volume.", "The adhesive tensile modulus is ~200 MPa with a tensile strength of ~ 15 MPa and strain to failure of 40–60% as per the manufacturer datasheet.", "The DLJ specimen consists of two steel cores separated by a gap “Teflon insert” and bonded with thick adhesive layers (~8 mm) to CFRP skins as depicted in Fig. 1.", "The shop primer on the steel was removed by grit blasting up to SA2.5.", "Then, the adherends’ surfaces were cleaned and degreased with isopropanol prior to bonding.", "The test setup schematic depicted in Fig. 2 consists of four main data acquisition systems.", "The Universal Testing Machine (UTM) records the load and the cross-head displacement.", "The corresponding displacement and the strain contour measurements are acquired by processing in-situ images captured by a 3D DIC system from one side.", "A camera continuously monitors the cross-section of the specimen during the loading process from the opposite side.", "And one Acoustic Emission (AE) sensor is placed on the lower steel adherend to capture the AE signals generated during the test.", "Tensile testing was conducted as per the guidelines of ASTM D3528 standard for “Strength Properties of Double-lap Shear Adhesive Joints by Tension Loading” [18].", "Universal Testing Machine (Zwick Roell) with 250 kN load cell is used to apply a displacement controlled tension with a rate of 1.27 mm/min according to the standard.", "Three specimens are tested for each adhesive type.", "One AE sensor was mounted on the steel, and the AE signals recorded were used for all the analysis.", "The AE sensor was a broadband, resonant-type, and single-crystal piezoelectric transducer from Vallen Systeme GmbH, AE1045S-VS900 M, with external 34 dB preamplifier and an operating frequency range of [100–900 kHz].", "To eliminate the surrounding noises, the threshold was set to 50 dB.", "An AMSY-6 Vallen, 8-channel acoustic emission system with the sampling rate of 2 MHz, was used to record the AE signals.", "Ultrasound gel was applied between the surfaces of the sensor and the specimen to ensure good acoustical coupling.", "A standard pencil lead break procedure [19] was used to check the connection between the specimen and the AE sensor surface prior to the mechanical test.", "To further investigate the mechanics of the DLJs, a 2D linear elastic FE model is implemented using ABAQUS Standard.", "Dimensions of the actual tested specimen, detailed in section 2.2, are used as an input for the model geometry.", "Due to two planes of symmetry, only a quarter of the joint is modeled (see Fig. 4).", "Roller boundary conditions replacing the planes of symmetry are defined while the gap edge is defined as a free surface.", "The load is applied as a constant displacement boundary condition along the steel “inner adherend” edge.", "A zoomed-in picture is highlighted to show the mesh density used in this study.", "In addition, a plane strain condition was used for the simulation while specifying the width in the elements definition.", "In this case, the three-dimensional free-edge effect [22] is neglected.", "The element type used was CPE4R, a linear quadrilateral reduced integration element.", "The total number of elements was 1840 elements.", "Besides, The materials’ mechanical properties are detailed in Table 1.", "The load-displacement curves of the DLJ specimens are depicted in Fig. 5.", "The load-displacement response of the Epoxy-based specimens is almost linear up to the final failure, which can be described as a brittle fracture.", "On the contrary, the load-displacement response for the MMA-based specimens has a nonlinear behavior from the beginning of the test up to the final failure with a significant plasticity and damage progression leading to a ductile fracture.", "It is clear from the load-displacement data that there is a trade-off between the strength and the ductility “strain to failure” of the adhesives.", "In the case of the MMA adhesive, the displacement to failure is approximately 4 times higher than the Epoxy-based; while the strength of the Epoxy is almost 1.6 times higher than the MMA-based counterpart.", "Using the FEM model, the shear stress distribution through the thickness of the double-lap joint is determined for both adhesive types as depicted in Fig. 6.", "The shear stress distribution across the thickness confirms the assumptions made earlier for the derivation of the analytical model.", "The shear stress is linear in the outer adherend “CFRP” and the inner adherend “steel”, with the mid-plane experiencing zero shear stress.", "In addition, the stress is almost constant in both adhesives, especially the MMA based.", "This confirms the assumption made to derive the equations for the analytical model.", "The displacement contour map, obtained from the DIC analysis during the experimental testing, is compared against the FEM predicted displacement map in Fig. 7.", "For both adhesives, there is a good agreement between the DIC and FEM displacement distribution across the thickness of the joints.", "Moreover, the gradient of displacement in the MMA-based adhesive is found to be much larger than the Epoxy-based case.", "Due to the ductile nature of the MMA-based adhesive, it experiences larger deformation (see Fig. 7).", "This directly results in higher shear strain imposed/transferred via the MMA-based adhesive for the same applied deformation “displacement”.", "The AE signals captured during the tensile testing of the DLJs, for both adhesive types, are correlated with the DIC displacement maps and the camera images to confirm the different damage events.", "Fig. 9 demonstrates the load-displacement curve for the Epoxy-based DLJ as well as the cumulative AE energy.", "The cumulative AE energy is calculated as the summation of the energies of the individual AE signals captured during the test.", "As expected from the brittle nature of the adhesive, the stiffness of the joint is almost linear with a sudden drop of the load (see point 1 in Fig. 9b) at approximately 1.25 mm then linear again up till failure (see point 2 in Fig. 9b).", "The same pattern is confirmed from the cumulative AE energy curve as point 1 shows a sudden increase in the cumulative energy indicating strain energy released due to a damage event.", "From the DIC displacement/strain map and the line plot of the displacement in the center of the joint at the gap “Teflon insert” position, a clear discontinuity in the displacement is observed (see Fig. 9c).", "This discontinuity corresponds to the crack propagation in the thick adhesive layer leading to the strain concentration in the DIC map (see Fig. 9a).", "The second sudden increase in the Cumulative AE energy curve occurs instantaneously at the final failure.", "This increase is significantly larger than the first one as it corresponds to the rupture of the joint at multiple locations.", "The DIC and camera images clearly indicate the multiple fractures in the adhesive layer as well as debonding at the steel/adhesive interface “adhesive failure” and delamination in the CFRP skin.", "Unlike the Epoxy-based adhesive, the MMA-based DLJ demonstrates a non-linear load-displacement response with cumulative AE energy increase at different displacements, as shown in Fig. 10a.", "The exact same approach used to understand the source of these increases, combining the input from the DIC strain/displacement maps and the camera images, is utilized.", "Four main points on the load-displacement, based on the cumulative AE energy curves, are identified.", "The initiation of the crack in the adhesive layer (at point 1 in Fig. 10a) results in energy dissipation captured by the AE system and a discontinuity in the DIC displacement map (see line plot in Fig. 10b and DIC map at point 1).", "Then, at higher loading levels (Points 2 and 3), the crack in the middle of the joint continues growing (see Fig. 10b) in addition to steel/adhesive debonding “adhesive failure” occurring at the lower edge of the DLJ (see Fig. 10c).", "Finally towards the end of the test, the debonding at the steel/adhesive interface from the center of the DLJ and the lower edge join (see point 4 in Fig. 10a) leading to the largest increase in the cumulative AE energy at point 4.", "These observations are supported by the DIC strain map in Fig. 10a–c.", "Fig. 11 demonstrates the post mortem fractured DLJs for the two types of specimens investigated in this study.", "From the visual inspection, it is clear that different damage mechanisms can be spotted for the two adhesive types.", "In the case of the Epoxy-based adhesive joint (see Fig. 11a), a transverse crack extending from the gap leads to a cohesive failure in the adhesive under mode I. On the other side of the joint, a complete adhesive failure occurs at the steel/adhesive interface.", "Moreover, interlaminar delamination in the CFRP skin is obvious.", "For the MMA-based adhesive joint (see Fig. 11b), again crack propagation “cohesive failure” from the gap under mode I is observed.", "At the crack tip singularity, local adhesive failure occurs at the CFRP/adhesive interface.", "In addition, some adhesive failure at the steel/adhesive interface can be spotted in the center of the specimen at the vicinity of the gap.", "Larger adhesive failure at the steel/adhesive interface occurs at the free edges of the joint.", "However, there are no signs of any interlaminar delamination in the CFRP skin in this case.", "The mechanics and stresses of double-lap Steel-to-CFRP adhesively-bonded joints, with thick adhesive layer, loaded in tension were investigated experimentally using Digital Image Correlation (DIC) and Acoustic Emission (AE), analytically using a one-dimensional closed-form solution and numerically with Finite Element analysis.", "The double-lap bi-material joints were fabricated of a steel core adhesively bonded to two CFRP skins with adhesive thickness of ~ 8 mm, using an Epoxy-based and MMA-based adhesives.", "From the analysis performed, the following conclusions can be drawn:", "The improved Tsai analytical model, used for thick adhesive layers and assuming a constant shear stress through the thickness, proved to yield an acceptable level of accuracy to predict the stress state in thick-adhesive bi-material DLJs.", "Unlike similar adherends DLJs, it was observed that the stress distribution symmetry along the bond-line vanishes for dissimilar adherends.", "Moreover, the peel stresses distribution predicted by the FEM suggested that highest peel stress occurs at the vicinity of the gap in the center of the DLJ", "The gradient of displacement in the MMA-based adhesive, through the joint thickness obtained from FEM and DIC, is found to be larger than the Epoxy-based case.", "This is due to the ductile nature of the adhesive as opposed to the Epoxy-based counterpart.", "AE cumulative energy and DIC displacement maps successfully identified the damage evolution for DLJs during testing." ]

[]

[ "AGRI", "ARTS", "EART", "ENVI", "SOCI" ]

[ "Biomes", "Eurasia", "Late Quaternary", "Pollen", "Vegetation" ]

[ "1105", "1204", "1907", "2306", "3302" ]

[ "Northern Eurasia (north of ca. 40°N and from 10°W to 180°E) is a large landmass with distinct gradients in climate.", "The last deglaciation led to dramatic changes to the Earth system, and in the North polar amplification likely enhanced regional responses to climate forcing (Serreze and Barry, 2011).", "The geography of Eurasia altered dramatically as the extensive shallow coastal shelves bordering the Arctic Ocean and the shallow Bering Strait region, which were subaerial for much of the last glacial-interglacial cycle, were flooded by eustatic sea-level rise.", "The biotic system saw major shifts in vegetation structure and composition (Andreev and Tarasov, 2013; Lozhkin and Anderson, 2013; Willerslev et al., 2014), continental-scale ecotones between forest and steppe and forest and tundra being particularly sensitive recorders of climate change (e.g., Pielke and Vidale, 1995; Thompson et al., 2004; Williams et al., 2011).", "Changes in the size of mammalian populations and local and global extinctions occurred (Sher, 1997; Stuart et al., 2004; Barnosky et al., 2004).", "There were changes in the terrestrial carbon sink, such as the development of peatlands (Smith et al., 2004), in patterns of thermokarst and permafrost degradation (Zimov et al., 2006; Walter et al., 2007) and in other nutrient cycles (McLauchlan et al., 2013).", "To date, the late-Quaternary changes in Eurasian vegetation have not been comprehensively summarized and mapped using palaeodata.", "How well key climate changes are depicted in palaeoclimate simulations can be examined via comparison with spatio-temporal vegetation changes on a large scale.", "Recent climate simulations have used contiguous time-steps over long periods (i.e., independent simulations for a set of time-slices; Singarayer and Valdes, 2010), or transient runs (i.e., continuous simulation over a given time span; e.g., Liu et al., 2009).", "Several comparisons have been made for the last glacial maximum (LGM), 6 kyr cal BP and present (e.g., Kaplan et al., 2003; Wohlfahrt et al., 2008; Bartlein et al., 2011), and Hoogakker et al. (2016) examined snapshots of compiled environmental data through the last glacial cycle with simulated data.", "To be effective, this type of exercise requires suitable comparator datasets.", "Pollen data remain by far the most abundant and widespread source of information about late-Quaternary vegetation change.", "Compared with other regions, the Siberian sector is described by relatively sparse palaeodata, but more pollen data are becoming available and can be compared with information from other sources, e.g. plant macrofossils (Binney et al., 2009) or ancient DNA (Willerslev et al., 2014).", "Here we build on the efforts of the PAIN project (e.g., circumpolar vegetation reconstructions for 6ka and 21 ka: the Pan-Arctic INitative; Bigelow et al., 2003) plus new data synthesized in an international workshop to develop a series of pollen-based biome reconstructions from 21 to 0 kyr cal BP for Eurasia north of 40°N.", "We used dated pollen records from geo-archives to map vegetation biomes using the biomization algorithm of Prentice et al. (1996) modified by Bigelow et al. (2003).", "We present time-slice maps of biomes and describe the most prominent changes, assess uncertainty in the biome assignments, and discuss the key emergent patterns in relation to changing Quaternary climates, distribution changes of key plant taxa and the sustainability of the Eurasian megafauna.", "We also summarize the dynamics of major plant functional types (PFTs) as they relate to key land-cover properties.", "The study region extends from the Iberian Peninsula to easternmost Siberia north of 40°N (Fig. 1).", "In our results and discussion we distinguish eastern and western sectors delineated by the 40° E meridian.", "West of ca. 40°E lies largely mountainous Europe; to the east are the extensive Russian and western Siberian plains.", "A series of broad lowlands characterize the region east of the Taimyr Peninsula into northeast Siberia.", "These lowlands are bordered by a series of north-south mountain complexes.", "The southern fringe of the study region is marked by the Mediterranean Sea in the west and central Asian mountain ranges and basins further east.", "Potential current natural vegetation includes temperate and mixed forests, boreal forest, temperate grassland (steppe) and alpine and arctic tundra (Fig. 2).", "West of 40°E in particular, extensive forested regions have been transformed by agriculture.", "Considerable climatic variation is reflected in the range of modern biomes that characterize the continent (Fig. 2).", "A latitudinal maritime-continental gradient is evident: steppe and desert regions occupy the arid continental interior south of ∼50°N, while the presence of Atlantic and Pacific Oceans in the west and east, respectively, is linked to temperate conditions and deciduous or mixed-deciduous forest cover.", "In Europe, the southern margins of the study region feature a Mediterranean climate and evergreen sclerophyll vegetation.", "Mean annual precipitation across the boreal forest zone (north of ∼50°N) ranges from over 1000 mm in mountainous Scandinavia to ∼200 mm in northeast Siberia; evergreen coniferous forest in the western part of the continent grades to deciduous coniferous forest in the east.", "Annual temperature ranges in the eastern boreal region can be > 60 °C.", "The northern edge of the landmass and higher elevations support tundra, with cool growing season means below 10 °C and cold winters.", "During the cold, dry glacial maximum centred on 21 kyr cal BP glaciation was relatively restricted compared with earlier phases of the last glacial cycle.", "Ice covered Scandinavia and adjacent areas but barely affected the western and central Siberian lowlands (Svendsen et al., 2004).", "In northeast Siberia (here defined as lands lying east of the Verkhoyansk Range), glaciation was largely restricted to the mountains (Barr and Clark, 2012; Velichko and Faustova, 2009).", "Thus much of the study region remained unglaciated between the LGM and present.", "Palaeoecological records of full-glacial age from across the region from the Atlantic to the Pacific have long been interpreted as representing treeless vegetation that supported a diverse grazing megafauna (Guthrie, 1990) and small bands of human hunter-gatherers (Pitulko et al., 2004).", "However, growing evidence points to persistence of boreal and temperate tree taxa across the region in a variety of refugia (Stewart and Lister, 2001; Birks and Willis, 2008; Binney et al., 2009; Huntley et al., 2013).", "Subsequent vegetation changes are likely linked with the demise of much of the grazing megafauna (with the direction of causality still contested; Zimov et al., 1995; Stuart et al., 2004; Zimov, 2005; Campos et al., 2010).", "Deglaciation was marked by climate oscillations, most notably the Younger Dryas (12.9–11.7 ka; Rasmussen et al., 2006 and Lowe et al., 2008), although this feature is not recorded across the whole study region.", "With a shift to warmer and moister interglacial climates, treeless vegetation was replaced by boreal or temperate forest over much of the region, and wetland extent increased (Lozhkin et al., 2011; Andreev and Tarasov, 2013).", "Recent millennia have seen human transformation of vegetation cover, particularly in Europe (e.g., Gaillard et al., 2008; Kaplan et al., 2009; Trondman et al., 2015; Fyfe et al., 2015).", "Biomization of pollen data is the process whereby inferred vegetation can be represented by a relatively small number of biomes that describe major structural, functional and floristic differences.", "Key to the process is preparation of a high-quality database, which requires considerable checking of data and metadata and decisions about site chronologies, taxonomy and biomization rules.", "The Supplementary On-line Material (SOM) contains details of methodological approaches.", "Below are brief descriptions of the main features of the sites, samples used, and key decisions made in database construction and biomization.", "The pollen database used in this study was constructed by the authors from several sources, including extant public databases and previously unpublished data.", "Because of geographic differences in site densities, we present results both across the total study area and separately for Europe and adjacent Asia.", "Table S1 in the Supplementary Online Material (SOM) provides basic metadata for the biomized samples and accompanying text describes quality control decisions.", "The total number of sites is 5858, the majority of which are modern surface/core-top samples (Fig. 3).", "Data from more than one discrete section from the same (terrestrial) exposure were amalgamated to form one site.", "All sites had their own radiocarbon chronologies (i.e., no sites were dated by correlation).", "We used previously generated, calibrated chronologies when available.", "For new, uncalibrated chronologies or chronologies that used conventional radiocarbon ages, we used CLAM v. 1.2 (Blaauw, 2010) to calibrate dates and construct new age models.", "For extant age models in 14C years, we converted ages using the “quick calibration curve” (NEOTOMA, Eric Grimm, http://www.neotomadb.org/, based upon the INTCAL04 calibration curve of Reimer et al., 2004).", "Fig. 3 shows the temporal distribution of samples, which were classified in to age slices.", "We used pollen values of the sample closest to the mid-point of the 1000-yr calibrated age slice.", "To avoid samples falling into more than one age slice, samples were omitted when the reported error on the age calibration/age model was >500 yr, except for 21 kyr cal BP, for which we experimented with both a 500-yr and a 1000-yr limit.", "All ages are given in calibrated years before present, depicted by kyr BP in the following sections.", "Great care was taken to produce a dataset with a consistent taxonomy.", "The checked, redundancy-free taxonomy includes 2874 taxa.", "We incorporated a diversity of minor herb types attributable to open-land biomes.", "Potential natural vegetation is used to estimate the effectiveness of modern biomization patterns in the northern extra-tropics because tree pollen dominates modern spectra, even in predominantly agricultural areas, and previous evaluations of modern vegetation-pollen patterns have shown a relatively good match (Prentice and Webb, 1998).", "While several maps of Eurasian potential natural vegetation exist, none has the appropriate resolution of vegetation categories for comparison with our biomized data.", "We therefore created a map specifically to assess the distribution of pollen-based biomes against modern vegetation (see SOM for more details).", "We excluded the Mediterranean sclerophyll biome.", "While occurring today within the study area boundaries, it was more restricted in the past, and only a few sites are affected by its removal.", "The map of potential natural vegetation displays polygons that all relate to the biomes used in the biomization (Fig. 2, Table 1).", "We used a restricted distribution of desert, as desert sites typically classify as steppe pollen biomes.", "We collapsed the cool-temperate evergreen needle-leaved forest biome into the cool mixed forest because the groups of constituent taxa for both biomes in northern Eurasia are almost identical.", "Inclusion of both introduced unnecessary noise into biome assignments while adding little extra information on land-cover change.", "The BIOME 6000 approach to synthesizing pollen data into vegetation biomes (Prentice and Webb, 1998) focuses on the contribution of plant-functional types (PFTs) to the total pollen sum, using fuzzy logic to calculate the most probable dominant biome.", "It is therefore not species-dependent.", "The known biases of pollen representation are moderated using a simple square-root transformation.", "Binney et al. (2011) showed that this simple function performs moderately well in balancing the relative over-production of most boreal tree/shrub taxa against the under-representation of most forbs.", "The pollen biomes used are a subset of those simulated by BIOME 4 (Kaplan et al., 2002).", "This means our dataset has the potential for direct comparison with palaeovegetation simulations.", "We follow the biomization approach used by Bigelow et al. (2003), except we added desert (DESE; see Table 1) and collapsed two mixed forest biomes (see Section 3.2 above).", "We defined 30 PFTs (see Table 2).", "A total of 752 taxa were used in biomizations; the remainder were omitted because they only occurred once or twice, or because they were taxonomically or ecologically ambiguous.", "The taxon-PFT table (Table S2) and the PFT-Biome (Table S3) are presented in the online supporting material.", "We used a biomization program written in Matlab and described in Binney et al. (2011).", "The biomes are primarily presented as biome “dotmaps”.", "Each site with a sample in the age slice of interest is mapped according to a colour-coded key.", "Where dots overlap, we chose to map the biomes giving preference to open-land biomes, which resulted in a visual bias towards this biome type.", "Following Bigelow et al. (2003), the Larix pollen values were multiplied by a factor of ×15 prior to biomization, because of the importance of the taxon across Siberia and its exceedingly low level of representation in pollen spectra.", "For NE Siberia (E of the Verkhoyansk Ranges, ca. >", "155° E) we made the assumption that Pinus pollen (sub-genus Haploxylon) derived from P. pumila, the only taxon in the region today.", "Percentages for all taxa were calculated using the total terrestrial pollen sum, and a square-root transformation of the pollen percentages was applied prior to biomization (Prentice and Webb, 1998; Binney et al., 2011).", "A slightly different biomization scheme has been applied to the northern high latitudes in previous work by Tarasov et al. (1998, 2000, 2013).", "Tundra remains one biome, rather than being sub-divided, and Larix is given a multiplier of ×20.", "A visual comparison of biome results suggests the latter is an inconsequential difference.", "However, because there is no dry tundra category (DRYT) in the Tarasov et al. studies, the constituent taxa for steppe (STEP) differ significantly between the two systems.", "For the LGM, particularly in the northern portion of the study area, our biomization procedure typically registers DRYT where Tarasov et al. (2000) register STEP.", "The nature of the zonal vegetation of the LGM (dry, cool conditions) was addressed by Kaplan et al. (2003), who described a bioclimatic envelope drier than most contemporary tundra and cooler than steppe.", "In much palaeoecological literature, this vegetation has been described as “steppe-tundra” or “tundra-steppe” (see Anderson et al., 2004; Andreev and Tarasov, 2013; Lozhkin and Anderson, 2013).", "Samples from site records unavailable to us but biomized by Tarasov et al. (2000) are plotted for 21,000 yr BP (see section 4.3 below).", "Summaries of structural-functional changes in land cover were created by defining “super PFTs” (unique groupings of PFTs in broad types, such as woody deciduous and graminoids) and calculating sums for each time slice for the eastern and western sectors of the study area.", "No taxon occurred in more than one super PFT.", "Using the biomization taxon-PFT look-up table, we summed scores for the following groups: grasses, sedges, forbs (excluding Artemisia), Artemisia, Ericales, Sphagnum, woody angiosperms and gymnosperms.", "Artemisia is in its own PFT because it represents shrub as well as forb taxa, particularly in STEP, and it has high pollen representation.", "The super-PFT values were summed for each age slice and a super-PFT sum created, which was used to calculate a percentage value for each super-PFT.", "Additionally, summed percent values of i) woody biomes and ii) non-woody biomes were calculated separately for areas to the west and east of 40°E for 500-yr time-slices before, during and after the Younger Dryas stade (YD, i.e., 13,500 to 10,500 kyr cal BP).", "This separation allows temporal patterns across the majority of the northern Eurasian land mass to be assessed without being masked by the large number of European sites (see Fig. 4a and b).", "In certain situations, pollen values do not clearly portray important aspects of the vegetation.", "For example, Larix poses a problem to all pollen-based reconstructions because of the extremely low pollen representation (see above).", "Small (possibly refugial) populations of various tree species may also be virtually undetectable in the pollen record (Stewart and Lister, 2001; Birks and Willis, 2008).", "Records for key woody taxa from the New Eurasian Macrofossil Database (NEMDB; Binney et al., 2009; http://oxlel.zoo.ox.ac.uk/resources/) enhance the relatively sparse pollen data coverage during the last glacial maximum (time slices 21–18 kyr cal BP) and help delimit northern treeline.", "The database contains numerous records of woody taxa at (and beyond) modern treeline, including Larix, Pinus, and Picea.", "Pollen sites are point data while mapped vegetation is in the form of raster data, and point-to-pixel comparisons are not the most effective way to assess how well mapped vegetation and pollen-based biomes match.", "Pollen sites integrate vegetation on a range of spatial scales (Bigelow et al., 2003; Sugita, 2007a,b) and thus the relevant size of a ‘point’ varies from site to site.", "Furthermore, vegetation representation by pollen is affected by other scaling factors.", "For example, where there is relatively flat topography (and relatively homogenous vegetation) pollen-based data usually match regional vegetation well.", "In mountainous regions there is a complex vegetation mosaic, the pollen signal is mixed, and the probability that at any one pollen site/grid cell will match the vegetation is lower.", "Northern Eurasia offers the opportunity to evaluate this effect as it has both extensive plains and mountain complexes, a factor which we incorporated into our assessment of the effectiveness of modern pollen biomization.", "We compared modern biome identifications with mapped vegetation using an error matrix approach.", "To test the influence of topography on biomization effectiveness, we created a ruggedness index for all 100 × 100-km cells over Eurasia, using an elevation model at 1-km resolution.", "The ruggedness index reflects the sum of the slopes of every 1-km cell within each 100 × 100 km cell.", "The larger the slope sum, the more rugged is the terrain.", "Then we computed, for each pollen site, whether the vegetation map grid cell identity and the biomization result matched (0 = error, 1 = match).", "All positive matches and all errors were grouped separately and tested for difference in ruggedness using a t-test for two samples of unequal variance (the latter tested with an F-test).", "A qualitative visual inspection of the biomized modern sample dataset overlain on potential natural vegetation shows good matches between pollen biomes and vegetation across Siberia, central Asia and northern Europe (Fig. 4a).", "Evergreen boreal forest (TAIG), cold deciduous forest (CLDE), cool mixed forest (COMX) and steppe (STEP) are delineated by a dominance of matching biome points but also include a scatter of other biome types.", "The four tundra biomes that occur along the Arctic coast and on high-arctic islands are moderately well reproduced by the pollen (Fig. 4a).", "Dry tundra (DRYT) is virtually absent in modern tundra vegetation in this region but is mapped in high-elevation areas of central Asia.", "In western and central Europe (Fig. 4b), the boreal forest zone is clearly demarcated by the pollen.", "Mountainous regions are dominated by a mixture of temperate deciduous (TEDE) and cool mixed forest (COMX) biomes, with the latter predominant at the boreal-nemoral ecotone.", "Cool conifer forest (COCO) is not mapped as an extant vegetation biome (Fig. 2), but some pollen sites are classified as COCO.", "Steppe (STEP) is identified at sites east of the Black Sea.", "It is notable that the open-land biome dry tundra (DRYT) is common across the northern European lowlands, the British Isles and the northern Iberian Peninsula, where the potential natural vegetation would be forest but the majority of land is currently used for agricultural production.", "While any vegetation map on this broad scale inevitably simplifies ecotonal boundaries and within-biome heterogeneity, the mapped locations of major biomes and their ecotones are well matched by the pollen: steppe-forest in southern Siberia and central Asia, the boreal-deciduous forest in Europe and western Asia, and the longitudinal ecotone between the two coniferous biomes in Siberia.", "A numerical table of the results of the error analysis (Table S4, Table 3) quantifies i) the success with which a given pollen biome predicted its mapped biome and ii) for a given mapped biome, the rate at which it was successfully predicted by pollen.", "The critical patterns are discernible in the mapped data upon close inspection.", "For example, the mapped distribution of cold evergreen needle-leaved forest (TAIG) is well identified by pollen (84% of mapped points in this biome are TAIG).", "However, pollen records classified as TAIG are often in error, having a success rate of only 34% (i.e. many TAIG points fall into other biome polygons).", "Cool mixed forest (COMX) and temperate deciduous forest (TEDE) biomized points predicted the location of the mapped biomes with moderate success (43 and 41%, respectively).", "Here, much of the error can be explained by a reciprocal relationship (i.e., TEDE polygons are frequently characterized by COMX pollen biome points, and COMX polygons by TEDE points).", "These two biomes are physiognomically similar in their constituent taxa.", "Non-forest biomes are not well defined quantitatively because non-forest regions receive tree pollen from forested regions, which can cause pollen spectra to be biomized as forest biomes (see section 3.3.", "above).", "Furthermore, extremely poor relationships may be partly a function of few pollen points and/or small vegetation areas.", "The relationship of landscape ruggedness with biomization success was strongly significant (p = 4.86 e−13).", "A logistic regression on the binary data (1, Match, 0, error), showed that the variable ruggedness (slope sum) is a predictor of biomization success (p = 7.20 e−12).", "As radiocarbon errors tend to increase with age of sediment (Fig. 3) and older sites tend to be sampled at low temporal resolution, several possible samples were excluded from older time-slices.", "This, together with the fact that old sites are rare, means that prior to 14 kyr cal BP, site coverage is sparse, particularly in Siberia.", "The 21-ka time slice, however, features a larger dataset as 21 kyr cal BP is a critical time slice for comparison with simulations derived from climate modelling.", "For this time slice we relaxed the error limit, which added several more sites (for all sites see Fig. S1, 21 kyr cal BP).", "Maps illustrate both pollen biome assignments and macrofossil occurrences through time.", "(Fig. 5; see Fig. S1 for the complete set of pollen-biome maps).", "At 21 kyr cal BP, treeless biomes dominate Eurasia.", "Most sites record either steppe (STEP; as defined either by Tarasov et al., 2000 or Bigelow et al., 2003) or dry tundra (DRYT, Bigelow et al., 2003, this biomization).", "Sites in northeast Siberia, along the north coast of Siberia and in Europe are dominated by forms of tundra, with DRYT the most common.", "Sites further south in central Asia are also STEP or DRYT.", "The Siberian lowlands have few data points, but, given the results of the topographic test, it is likely that tundra biomes would characterize much of this region.", "A few sites, however, record evergreen or deciduous coniferous forest (TAIG, CLDE), notably in the Caucasus and across mountainous central Asia to the Pacific Coast and Japan.", "In southern and central Europe, a few sites record forest biomes intermittently between 21 and 17 kyr BP.", "Subsequently, there is an increase in TAIG in southern and central Europe, and TAIG sites appear further east across the eastern European plain 16-15 ka.", "Eastward, tundra still dominates, but CLDE sites occur in both northeast and central Siberia from 15 kyr cal BP.", "A major change is seen around 14 kyr cal BP.", "At this time, the number of palaeorecords increases markedly (Fig. 3).", "While DRYT is still common, in the far eastern region it tends to be replaced by low-high shrub tundra (SHRU).", "There is a continuing increase of TAIG, and temperate deciduous forest (TEDE) appears at some sites in southern and central Europe.", "The central Asian region remains steppe or DRYT.", "Some sites show CLDE in central and eastern Siberia.", "By 13 kyr cal BP, a mixture of DRYT and woody biomes characterizes the whole region, with TAIG and TEDE in the western region and TAIG and CLDE in the east.", "However, far eastern Siberia features a mixture of SHRU and DRYT.", "Treeless biomes are rare across Eurasia by 9 kyr cal BP.", "Of the forest biomes, TEDE dominates in Europe and TAIG and CLDE across Siberia.", "The Near East and central Asia still indicate STEP/DRYT along the southern margins of the study area.", "By 8 kyr cal BP, the arrangement of biomes approaches the modern pattern.", "The 7 kyr cal BP map includes data from the BIOME 6000 database, and these extra sites allow good demarcation of the steppe region across western and central Asia.", "Holocene treeline changes along the northern forest-tundra ecotone in central and eastern Siberia, where Larix forms the treeline, are clarified by the combination of the macrofossil record with the pollen biomes.", "In particular, a southward retreat from the Arctic Ocean coast after 5 kyr BP is discernible.", "From 2 to 0 kyr cal BP, DRYT increases noticeably in Europe.", "The percent summed PFT abundances, as recorded by the taxon scores (which are subject to square-root transformation) change with time (Fig. 6a and b).", "The observed patterns may partly reflect biased spatial sampling of the vegetation through time.", "For example, prior to 14 kyr BP, more southerly sites are an important geographic component, and the 21 kyr BP (eastern) value of 20% conifers reflects sites in China and Japan recording coniferous forest.", "While the fine details of changes are probably not significant, a few major patterns are worth considering.", "Across the whole region the main trend is that non-woody PFTs (grasses, sedges, forbs, including Artemisia) are dominant until 15 kyr cal BP.", "The square-root transformation favours the forb PFT, as nearly all forbs are underrepresented and have low pollen values.", "This feature highlights two effects: i) proportions of biomized forbs and graminoids, which constitute the herbaceous component of the vegetation, are approximately equal and ii) the proportion of herbaceous PFTs continuing into the Holocene is far greater than is typically interpreted from pollen diagrams.", "Between 15 and 8 kyr cal BP the non-woody PFTs are reduced and the deciduous woody PFT expands.", "The deciduous woody PFT includes Betula, Alnus and Salix, which can represent tundra shrubs as well as trees.", "Thus this increase may be interpreted as an expansion of forest growth forms or of tundra shrubs, depending upon region.", "In the Holocene, this PFT expands in the west considerably more, reflecting the greater importance of deciduous trees in western Eurasian forests, while the coniferous woody PFT expands in the east, reflecting the dominance of coniferous forest.", "Although Ericales and Sphagnum form a small proportion of all values, these PFTs, which are indicative of mesic and wetland conditions, increase beginning about 15 kyr cal BP and continue to increase until the present in the western region.", "The plots of the percent tree and non-tree pollen biomes for time slices before, during and after the Younger Dryas stade (∼12.9–11.7 kyr cal BP) are shown in Fig. 7.", "A drop in the abundance of woody biomes is clear at 12 ka.", "However, most of the signal is derived from the western sector (Fig. 7b); there is no clear pattern in the east.", "When mapped in large numbers, the modern pollen data delineate major biomes and their ecotones relatively well (see Table 3).", "Error patterns are explainable by six factors that are not mutually exclusive: geographic proximity to other vegetation biomes; floristic similarity between pollen biomes; over-representation of woody vs non-woody taxa; the reliability of modern vegetation mapping; site type; and site density.", "The degree of geographic proximity refers to the location of the pollen site relative to areas of vegetation transition.", "For example, there is overlap of boreal pollen biomes to tundra vegetation.", "This is because the generally abundant pollen types Pinus (pine) and Picea (spruce) are key constituents of the pollen biome TAIG and tend also to occur in pollen spectra derived from adjacent shrub tundra.", "In the European mountains, TEDE and COMX pollen biomes are intermingled, probably because the pollen biomes differ by only a few key taxa and the vegetation mosaic has a relatively fine spatial scale.", "The degree of floristic similarity of pollen-based biomes determines how robust the assignments are in relation to small changes in pollen percentage values.", "This is particularly seen in the interchangeability of desert (DESE), STEP and DRYT, which share a large sub-set of taxa and are defined by forb or xeric shrub taxa that mainly have low pollen productivity.", "In fact, it is not possible to distinguish steppe from desert in our modern pollen data.", "The tendency for woody taxa to produce relatively large quantities of pollen creates a bias that is not fully corrected by the square-root transformation, so typically forest biomes map on to non-forest areas such as tundra and steppe (see above).", "Where several biomes form a hierarchy of floristic subsets reflecting net primary productivity levels (e.g. the set of tundra biomes, or STEP and DESE), errors typically run in the direction of a more productive pollen biome being assigned to a less productive vegetation type, due to the pollen representation bias of woody taxa over most herbaceous taxa (in the case of tundra) or non-local pollen over virtually no local pollen (in the case of deserts).", "The exception is CLDE, which is characterized by Larix, a taxon with extremely low pollen representation.", "The evergreen coniferous pollen signal (TAIG) within the deciduous (Larix-dominated) conifer biome of eastern Siberia is likely due to the presence of Pinus sibirica, which occurs as far east as the Lena River and Lake Baikal and which has high pollen productivity.", "On the other hand, Pinus pumila is mostly restricted to the north-easternmost part of Asia and thus only contributes to the CLDE biome; here the CLDE is better represented.", "In the southeast coastal region, P. koreana likely contributes pollen to the forest biomes.", "Mapping the modern land cover as potential natural vegetation may be an unrealistic approach in Europe and western Asia, where across large areas forest biomes have been replaced by agricultural land.", "Here there is a striking over-abundance of DRYT assignments, and, to a lesser extent, STEP.", "It is difficult to define an agricultural biome through pollen, as the indicators of cultivation are a suite of taxa with poor pollen representation (but see Fyfe et al., 2015; Trondman et al., 2015).", "DRYT and STEP are both defined by the presence of grass (Poaceae), which is also a major component of agroecosystems, and it is not surprising that they appear across cultivated areas in these regions.", "The examination of modern patterns has implications for interpreting the palaeorecords.", "Perhaps the clearest of these is site density.", "For older time slices, when sites are sparse, the modern analysis suggests that more reliance might be placed on extrapolating records across uniform topography (e.g., the Eurasian plains) than when interpreting palaeovegetation for topographically complex regions (i.e., mountain regions).", "A similar caution relates to ecotones, where biome assignment may be more ambiguous than for sites located more centrally within a biome.", "Furthermore, the occurrence of pollen of woody taxa in the record, assuming relative pollen productivity does not change over time, may bias results towards reconstructing a biome with more vegetation biomass than was actually present.", "Ideally, site type should be considered when evaluating the regional applicability of an assigned biome.", "Bigelow et al. (2003) showed that lacustrine records reflect a more regional pollen signal and moss polsters a local signal.", "Siberian records derived from yedoma (icy silt deposits), thus basically from palaeosols, will tend to give a local signal.", "The dataset has incomplete metadata, meaning only a subset of sites can be typed.", "Of 5920 total site entities (including localities with multiple sections), 810 are lakes and 1717 are located in non-lake situations (e.g, moss polster, peat, yedoma); the remainder are undefined (Fig. 8).", "Biome maps and PFT trends clarify large-scale changes in biome distributions since the LGM, shifts in PFT dominance in space and time, and the temporal trends in establishment of forested biomes during deglaciation (Figs. 5–7).", "The widespread dominance of DRYT, plus occurrences of steppe and other tundra types, indicates that the full-glacial landscape of Eurasia was largely treeless (see also Tarasov et al., 2000; Bigelow et al., 2003).", "Deglaciation and the accompanying major rearrangement of the climate system is reflected by dramatic changes in the dominant biomes, particularly the expansion of forest biomes and woody plant cover in general.", "The spread of forest biomes appears underway by 15 kyr cal BP in Europe and complete across the region by 13 kyr cal BP.", "Thus, the two millennia centred on 14 ka mark the critical transition from predominantly glacial to predominantly interglacial climate.", "The marked increase in sites recording data between 15 and 13 kyr cal BP (centred on 14 kyr cal BP) is not an artefact of sample number and dating errors (see above).", "Both lakes and peatlands require enough moisture to exist, and the increase of sites at this time most likely represents a region-wide increase in effective moisture, probably largely driven by increased precipitation.", "Notably, the abundance of Sphagnum begins to increase at this time (there is also a later increase, in the early Holocene, particularly west of 40° E).", "As site coverage increases from 14 kyr cal BP, the northern edge of the steppe region of central Asia and the northern treeline become clear in the maps.", "A moderate drop in woody PFTs occurs in Europe (W of 40° E) during the 12.5 kyr cal BP time slice (Fig. 7).", "This may reflect some retreat/thinning of forest cover during the Younger Dryas (YD) stade, which is expressed strongly in NW Europe but far more weakly with distance eastward from the Atlantic (Kokorowski et al., 2008).", "At 12 kyr cal BP and in subsequent time slices in central and eastern Siberia, CLDE sites are scattered across the region, suggesting an expansion of cold deciduous (Larix-dominated) forest at about this time.", "While the low number of sites in this region limits conclusions, there is no clear YD signal.", "From 11 to 9 kyr cal BP TAIG in Europe retreats northwards and COMX expands.", "This likely reflects the appearance of deciduous taxa at higher elevations and latitudes than before.", "The well-documented northern expansion of TEDE (Huntley, 1988; Prentice et al., 2000) 8–4 kyr cal BP is seen in Scandinavia.", "The northern treeline, represented by TAIG in the west and CLDE in the east, can be seen extending to the Arctic Ocean across much of Eurasia by 9000 kyr cal BP.", "There is a suggestion of treeline retreat in eastern and central Siberia between 5 and 3 kyr cal BP (see also Binney et al., 2009).", "From 3 kyr cal BP to present, DRYT - interpreted as a signal of open agricultural land - increases across Europe (see below).", "A major concern about current warming of the northern high latitudes is the extent to which woody vegetation (forest or shrub tundra) will expand at the expense of herbaceous tundra.", "So-called “arctic greening” has been widely reported (e.g., Sturm et al., 2001; Goetz et al., 2005; Forbes et al., 2010), and its consequences include modification of the land-surface feedback to climate (e.g., Chae et al., 2015).", "A similar change has been described as forest and shrub-tundra biomes replaced herbaceous biomes across northern Eurasia during deglaciation (Velichko, 2009).", "How well past woody vs herbaceous cover can be simulated based on palaeoclimate model output therefore has utility as regards future climate change and its potential impact on arctic vegetation.", "The mapped fossil records can be compared with the vegetation modelling results of Allen et al. (2010), who used a DVGM (LPJ-guess) and a version of the general circulation model HadCM3 (Singarayer and Valdes, 2010) to estimate annual net primary productivity (aNPP) of different PFTs for Eurasia during the late Quaternary.", "From 21 kyr cal BP to 16 kyr cal BP, pollen biomes and PFTs are dominated by herbaceous taxa, while a few sites record TAIG or CLDE in Pacific Russia, Japan and Southern Europe.", "This mirrors the simulated pattern of high aNPP for mesophilous herbs from Eastern Europe to Chukotka (Allen et al., 2010).", "However, in Europe, boreal and summer green trees are simulated to have highest productivity.", "This pattern is not borne out by the pollen, which indicates DRYT and STEP dominated to the Atlantic coast at 21 kyr cal BP, suggesting the climate of Europe is simulated as warmer, or more likely moister, than it probably was.", "After 21 kyr cal BP and until 16 kyr cal BP some pollen sites record forest biomes, particularly in coastal Asia and southern Europe, and, by 16 kyr cal BP, in the eastern European lowlands beyond the ice sheet.", "TEDE, COMX, and TAIG occur in Pacific coastal China and Russia and in Japan, TAIG and TEDE in Europe.", "At 14 kyr cal BP, the simulated boreal evergreen aNPP suggests conditions warm and moist enough to support tree growth in at least some locations, namely mountainous regions in southern Europe and in the north European plain; the pollen data support this.", "However, pollen data from mountainous central Asia indicate STEP and tundra biomes, whereas the simulation suggests boreal evergreen dominance.", "In this region of complex topography, the pollen data should be considered less reliable; however, the error is in the opposite direction from what might be expected (see section 3.3 above), which implies a mismatch between data and simulation.", "Scattered localities record CLDE in Siberia.", "The simulated data also suggest some tree growth may have been possible across Siberia, but the highest aNPP is still seen in mesophilous herbs.", "Thus both simulated and observed PFTs suggest largely open landscapes across the northeast at 14 kyr cal BP, but with trees present.", "Finally, climate simulations for 7 kyr cal BP tend to show central Asia as dry, a feature related to strong evaporative land-surface feedback in models (Wohlfahrt et al., 2008).", "While the biome maps represent only the northern part of central Asia, there is little evidence of an expansion of dry-land biomes, which might be expected with heightened aridity.", "The abundances of the super-PFTs (Fig. 6) provide rough indications of trends in vegetation properties that reflect disturbance and/or biogeochemical processes.", "For example, while tundra and steppe do burn, boreal coniferous forest will have a greater effect on the occurrence of fires, associated changes in surface albedo and release of gaseous and particulate burn products to the atmosphere, as the fuel availability and burn severity in this biome is far greater than in the other biomes.", "In the eastern region, the conifer super PFT minimum values of <10% at 18-17 kyr cal BP gradually increase during deglaciation to reach ∼30% by 9–8 ka, from which time the Siberian boreal forest likely contributed significantly to total global fires.", "The increases in Sphagnum and Ericales, beginning about 14 kyr cal BP and accelerating at the beginning of the Holocene, are an approximation of wetland initiation.", "Thus, they broadly mirror the importance of wetlands as a component of land cover across Eurasia and the potential for increased carbon sequestration and methanogenesis from the onset of the Holocene.", "The biomized PFT values indicate that traditional pollen-based interpretations probably under-estimate the contribution of forbs to the vegetation at 21 kyr cal BP and in subsequent millennia.", "For example, other forbs are equal in abundance to Artemisia (Fig. 6), and forb PFT values are similar to those of graminoids.", "This finding matches that of Willerslev et al. (2014), who proposed based on sedimentary DNA evidence, that forbs had an equal, if not dominant, role in full-glacial vegetation along with graminoids.", "Macrofossils (e.g., Kienast et al., 2005) also point to an important role for forb taxa in the full-glacial vegetation.", "The significance of this is yet to be fully investigated.", "While grasses were an important element of forage, relatively high diversity and biomass of forbs provides a variety of forage types and plant chemistry, broad options for self-medication (Forbey et al., 2009), and seasonal variation in availability of forage.", "This, along with actual primary productivity levels (Allen et al., 2010), may have contributed significantly to the carrying capacity of the vegetation that supported the megafauna.", "The biome data show that from ∼16 kyr cal BP, Europe supported a mosaic of woody and herbaceous vegetation.", "This persisted through the YD, with some fluctuations in proportional cover, until forest biomes became dominant at 10 kyr cal BP.", "In contrast, central and eastern regions featured predominantly treeless vegetation until 14-13 kyr cal BP.", "Thus, as suggested by Lister and Stuart (2008), different mammalian species were confronted with rather different vegetation configurations in the western and eastern regions, which in turn would have affected adaptation and survival of their populations through deglacial changes.", "The change to forest and shrub-tundra biomes from herbaceous biomes across northern Eurasia during deglaciation has been linked with population reduction and eventual extinction of grazing herbivores (e.g., Stuart et al., 2004; Guthrie, 2006; MacDonald et al., 2012).", "Between 14 and 10 kyr cal BP, woody biomes, either SHRU or CLDE, gradually replaced DRYT in central and eastern Siberia, except along the northern coast, where DRYT persisted.", "This pattern supports the observation from radiocarbon-dated fossil data that populations of grazing mammals such as the woolly mammoth (Mammuthus primigenius) gradually retreated northwards during deglaciation so that by the onset of the Holocene, ca 11 kyr cal BP, populations were highly restricted in far-northern areas.", "Notably, DRYT is recorded on Wrangel Island through the Holocene, the locality that supported the last Holocene Eurasian population of mammoths (Vartanyan et al., 1993; Lister and Stuart, 2008).", "The biome data summarize a large number of spatially distributed pollen sites and clarify that forest biomes occurred, at least locally, in unglaciated northern regions from ∼17 kyr cal BP onwards.", "Finds of macrofossils of Picea and Larix across Eurasia (Binney et al., 2009; Fig. 5) also support this conclusion.", "Whether these populations were present through the LGM is less clear.", "The vast northern Eurasian region is topographically complex enough to have featured a wide range of local climates, and it is likely that refugial populations persisted, even under the generally cool, dry conditions of the full glacial.", "Using simulations, Huntley et al. (2013) demonstrated that short-lived interstades that punctuated glacial conditions allowed for temporary expansion of tree populations.", "The temporal resolution of our data is such that the chances of pollen sites recording such localized (in time and space) tree population expansions are low.", "The reverse, a temporary contraction of tree cover, is shown in the Younger Dryas chronozone, as this period is within the temporal resolution of the data (Fig. 7).", "However, it should not be firmly concluded that putative refugial populations (observed or simulated) necessarily contributed extensively to local modern populations, as the spread of new individuals from adjacent regions and widespread gene exchange would have occurred over several millennia as climates rapidly changed (Semerikov et al., 2013; Edwards et al., 2014).", "Further study on the genetic impact of northern refugia on the development of modern forest populations is required.", "The biome DRYT is characterized by grass and a range of plant families (Asteraceae, Brassicaceae, Caryophyllaceae, etc.) that are typical of temperate meadow communities as well as tundra.", "Thus DRYT appears as a common biome in Europe in the past two millennia, particularly at 1 kyr cal BP and in the modern dataset.", "Its distribution demarcates areas of Europe where deforestation was already widespread in medieval times and which are now intensive agricultural regions.", "The absence of the DRYT (or similar) option explains why in the northern continents, previous biomizations did not identify agricultural landscapes (e.g., Prentice et al., 2000).", "The issues of pollen representation that lead to the underestimation of open land in traditional pollen studies are summarized by Trondman et al. (2015), who apply a new algorithm (Sugita, 2007a) to pollen data in NW Europe and demonstrate that the proportion of open land at 3 kyr cal BP and within the most recent millennium are considerably greater than previously thought, which is in accordance with our biomization results.", "Similarly, Fyfe et al. (2015) combine PFTs into land-use groups, and by numerically emphasizing key agricultural indicator taxa, argue that open land is detectable as early as 6 ka.", "All these approaches, including the relatively straightforward biomization methodology used here, operate by modifying pollen values to reduce bias due to differential pollen productivity.", "All studies emphasize that the degree of deforestation in Europe has been greater than previously thought, particularly over the past 1000–2000 years.", "This database analysis reflects both the strengths and weaknesses of large pollen datasets.", "Large-scale patterns in vegetation cover are usefully approximated by pollen data, and, at the continental scale, the resultant patterns can be used to assess critical changes in surface properties or compare with simulated vegetation data.", "However, error rates in assigning biomes, particularly in regions of complex topography, need to be borne in mind when interpreting past vegetation.", "For Eurasia, as far as incomplete metadata show, the majority of sites are terrestrial and thus are biased towards a local record.", "These limitations argue against creating land-cover estimates by interpolation of pollen data points alone.", "However, the large number of sites (more than mapped), which have been quality-checked, the associated error information, and the high level of floristic detail may make this database useful for a range of further analyses." ]

[ "Pollen biomes for 1000-yr time slices from 21 to 0 ka BP are mapped for Eurasia.", "Tundra and steppe biomes dominated 21–14 ka BP, but forest biomes were also present.", "From 14 ka BP woody biomes expanded and wetland indicators became more prominent.", "Extensive taxon list underlines importance of forbs in glacial-age non-woody biomes.", "The grass-forb tundra biome maps European agricultural land in recent millennia." ]

[ "BIOC" ]

[]

[ "1307", "1309" ]

[ "Hyaline cartilage forms the load-bearing surface of articular joints and is required for friction-free movement.", "The tissue is avascular and aneural and is composed primarily of an extracellular matrix rich in type II collagen and proteoglycans.", "It is maintained by a single cell type-the chondrocyte, which occupy <3% of the tissue volume.", "Articular cartilage is mechanically important in the joint, but it is vulnerable to damage though acute injury, or during joint disease.", "Partly due to its avascular nature, it has poor intrinsic capacity for repair, which predisposes the joint to developing osteoarthritis (OA).", "An important clinical aim is thus to repair focal defects and eventually larger lesions caused by the degeneration of the cartilage during OA.", "This requires strategies to replace damaged areas with new cartilage and the most promising of these strategies is cell-based treatments with donor cells.", "Autologous chondrocyte implantation (ACI) was developed to treat focal cartilage defects (Brittberg et al., 1994; Filardo et al., 2012), but complications such as chondrocyte hypertrophy resulting in vascular invasion and calcification have impeded progress (Hettrich et al., 2008; Pelttari et al., 2006).", "Moreover, the need for 2 operations and invasive harvesting of intact cartilage, together with de-differentiation of cultured chondrocytes during monolayer expansion, has hindered wider application (Kang et al., 2007).", "There is thus limited evidence that this approach can provide a permanent or large-scale solution.", "Stem cells, which can respond to developmental signals to create chondrocytes, are an alternative source of cells.", "Mesenchymal stem cells (MSCs), such as from human bone marrow, can be induced to form chondrocytes (Pittenger et al., 1999) and have been used for cartilage repair (Wakitani et al., 2011), but they have limited capacity for expansion as a bulk supply of cells (Stolzing et al., 2008).", "Human pluripotent stem cells (hPSC) in contrast can undergo unlimited expansion and can differentiate into any cell type in the body (pluripotency).", "This offers the potential to generate chondrocytes for the treatment of cartilage repair (Cheng et al., 2014a).", "Thus, hPSCs offer an alternative source of cells for allogeneic cell-based cartilage repair.", "Induced pluripotent stem cells (iPSCs) derived from adult somatic cells provide the opportunity to generate joint disease models and, in due course, may also form a source of therapeutic cells.", "Our lab previously developed a directed differentiation protocol for hESCs, which exploits normal developmental signals to generate chondrogenic cells with high efficiency and purity (Cheng et al., 2014a; Oldershaw et al., 2010).", "To activate lateral plate and chondrogenic mesodermal induction we used BMP4, a growth factor shown through mouse knockout studies (Winnier et al., 1995) to be essential for murine mesoderm formation and implicated in mesenchymal condensation and the generation of skeletal elements in limb bud development (Bandyopadhyay et al., 2006; Tsumaki et al., 2002).", "Using our protocol we showed that implanted hESC-derived chondroprogenitors could repair an osteochondral defect in immunocompromised Nude rats (Cheng et al., 2014b).", "Although these chondroprogenitor cells formed cartilage in vivo they responded poorly to the in vitro conditions under which adult bone marrow MSCs formed cartilage matrix in 3D cell cultures.", "From these results we concluded that this protocol does not proceed through an adult MSC-like stage, but generates cells resembling embryonic limb bud chondrogenic mesenchyme (Griffiths, Ronshaugen and Kimber unpublished).", "Hence, we refer to these cells as chondroprogenitors.", "We therefore set out to investigate ways to direct these cells to a more mature chondrocyte phenotype with enhanced production of extracellular matrix.", "BMP2, BMP4, and BMP7, as well as GDF5, are all expressed in the joint and thought to play roles in its development and the formation of cartilage (Francis-West et al., 1999; Gaissmaier et al., 2008; Hatakeyama et al., 2004; Zou et al., 1997).", "BMP4 has been used extensively to drive diverse mesoderm specification from hESCs (Laflamme et al., 2007; Xu et al., 2002; Zhang et al., 2008) and is used routinely in chondrogenic differentiation (Diekman et al., 2012; Lee et al., 2013; Craft et al., 2015).", "However, BMP2, also a known chondrogenic factor (Bandyopadhyay et al., 2006), has been employed in some studies in the differentiation of hESCs to chondrocytes with promising results (Yamashita et al., 2015) as have other TGFβ-family growth factors (Nakagawa et al., 2009).", "We therefore investigated the effects of substituting, or adding other TGFβ-family growth factors, which are expressed during cartilage development in vivo and have reported pro-chondrogenic properties.", "We proposed that these alternative growth factors might enhance the chondrogenic phenotype and boost expression of extracellular matrix genes.", "HUES1 and MAN7 human embryonic stem cell lines were cultured as previously described (Cheng et al., 2014b; Oldershaw et al., 2010).", "Briefly, hESCs were cultured on mouse embryonic fibroblasts (iMEFs, mitomycin C inactivated), at 6 × 104/cm2 on 0.1% gelatin (Sigma) coated plates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% (v/v) knockout serum replacement, 2 mM l-glutamine, 1% (v/v) nonessential amino acids (NEAA), 0.1 mM β-mercaptoethanol, 1% (v/v) penicillin/streptomycin (all Invitrogen) and 10 ng/ml FGF2 (Autogen Bioclear, Wiltshire, UK).", "Cells were passaged using TrypLE™ (Invitrogen).", "For differentiation cells were transferred for at least 7 days to chemically defined feeder-free culture (Baxter et al., 2009).", "Cells were lifted from the iMEF layers with TrypLE™, and plated onto fibronectin-coated (Millipore) tissue culture flasks with feeder-free medium: 50:50 F12:DMEM (Lonza) supplemented with l-glutamine, 1% NEAA, 0.1 mM β-mercaptoethanol, 1% penicillin/streptomycin, 0.1% bovine serum albumin (BSA) (Sigma), 1% (vol/vol) N2 supplement 2% (vol/vol), B27 supplement (both Invitrogen), 10 ng/ml activin A, 4 ng/ml neurotrophin 4 (Peprotech, London, UK) and 20 ng/ml FGF2 (Baxter et al., 2009).", "Alternatively, they were cultured feeder free, on mTESR medium (Stem Cell Technologies) on Vitronectin substrate (Thermo).", "The hESCs were differentiated in a basal medium (DMEM:F12, 2 mM l-glutamine, 1% (vol/vol) ITS, 1% (vol/vol) nonessential amino acids, 2% (vol/vol) B27, 90 μM β-mercaptoethanol) supplemented with appropriate sequential addition of growth factors as indicated in Fig. 1, as previously described (Oldershaw et al., 2010); Stage 1 (day 1–3): Wnt3a (25 ng/ml R&D Systems), Activin-A (reducing from 50 to 10 ng/ml, Peprotech) and BMP4 (40 ng/ml; Peprotech), followed by Stage 2 (day 4–8): BMP4 (40 ng/ml), Follistatin (100 ng/ml) and GDF5 (20 ng/ml) (all Peprotech) and finally Stage 3 (day 9–14): GDF5 (20 rising to 40 ng/ml), FGF2 (20 ng/ml) and NT4 (2 ng/ml) (all Peprotech) (Fig. 1).", "In some experiments BMP2 (40 ng/ml days 3–8, 20 ng/ml days 9–10) was substituted for the same concentration of BMP4 (both Peprotech), or at the concentration shown in Fig. 1.", "Cells were passaged on day 5 and day 8 with a change of substrate from fibronectin to fibronectin: gelatin 50:50 at day 8.", "For some experiments from day 11 to day 14 of the protocol, GDF5 was either replaced by BMP7 (100 or 300 ng/ml; Peprotech) together with either TGFβ1 (10 ng/ml; Peprotech) or TGFβ3 (10 ng/ml; Peprotech), or BMP7 (300 ng/ml) and TGFβ1 or TGFβ3 were added in addition to GDF5.", "Gene expression was analysed on days 4, 8 and 14 of the protocol.", "The proliferation and apoptosis of chondrogenic cells was assessed in triplicate samples using Celltiter-Glo™ reagent and Caspase-Glo 3/7 and quantifying luminescence signal on a Glomax multidetection system (Promega).", "The numbers of aggregates were counted manually in 3 wells of 6 well plates (HUES1) or 17 wells of 24 well plates (MAN7).", "The largest 20 aggregates for each growth factor were identified by eye and the size was measured using Image J. For gene expression analysis the aggregates were isolated manually, and RNA was collected from the aggregate cells and non-aggregate cells.", "cDNA was analysed by QRT-PCR.", "Total RNA was extracted using mirVana™ miRNA Isolation Kit (Life Technologies), reverse transcribed using M-MLV reverse transcriptase (Promega) and candidate genes expression (normalised to GAPDH) assessed using SYBR Green PCR Master Mix (Applied Biosystems) with an ABI PRISM 7500 Real Time System (Applied Biosystems).", "At least three separate biological repeats analysed in triplicate were performed.", "RIPA buffer cell extracts were resolved on 10% SDS gels and transferred to nitrocellulose membranes.", "After blocking, an appropriate antibody anti-SOX9 antibody (Abcam, 1 μg/ml), anti-GAPDH antibody (Cell Signalling, 1:2000), anti-type II collagen antibody (Abcam, 1 μg/ml or Santa Cruz), anti-SMAD1/5/9 (Abcam, 1 μg/ml) or anti-p-SMAD1/5/9 (Cell Signalling, 1 μg/ml) was added overnight followed by appropriate LI-COR secondary antibody and membranes exposed using Odyssey CLx Imager (LI-COR).", "Cells cultured on fibronectin coated plastic were fixed using 4% paraformaldehyde fixed and stained with primary antibody (SOX9, Millipore, 5 μg/ml) or IgG control followed by one hour in appropriate secondary antibody in blocking buffer.", "Samples were collected from pluripotent hESCs and at different times after the start of differentiation for analysis of COL2A isoforms, COL2A1-IIa and COL2A1-IIb.", "hESCs were cultured either with BMP4 or BMP2 from day 3–10, RNA was isolated, reverse transcribed and subject to PCR.", "Reaction conditions were denaturation 45 s, 94C; Annealing 45 s, 60C; elongation 45 s, 72C for 35 cycles and expression was analysed using 1.5% agarose gel electrophoresis.", "Primers were forward CTGCTCGTCCCGCTGTCCTT and reverse AAGGGTCCCAGGTTCTCCATC (SIGMA).", "Single-cell suspensions were fixed in ice-cold methanol (10 min at −20 °C) and permeabilized before incubation with primary antibody (mouse anti-OCT4 [Cell Signalling], goat anti-human SOX9 [Millipore]).", "Flow cytometry was conducted using a BD Biosciences Fortessa and the software Diva gating was used for analysis.", "SW1353 cells were seeded at a density of 2.1 × 105 cells in each well of 24 well plates, then treated with different concentrations of BMP2 and BMP4 for three days.", "Total protein was collected, and alkaline phosphatase activity was measured using fluorescence substrate 2′-[2-benzothiazoyl]-6′-hydroxybenzothiazole phosphate on a Promega Glomax multi-detection system.", "Animal work had local ethical approval and was carried out under Home Office licence as required for animal husbandry and experimentation in the UK.", "Osteochondral defects (2 mm) were made in the trochlea groove of the femur in athymic RNU rats (Charles River, Germany) as previously described (Cheng et al., 2014b).", "Fibrin constructs were formed by resuspending 3 × 106 cells in 50 μl fibrinogen followed by adding 50 μl thrombin and implants (containing 2 × 105 cells) were cut to fit the defects using a biopsy punch (Cheng et al., 2014a).", "Defects without implant or with cell-free fibrin gel served as controls.", "Joints were fixed in 10% neutral buffered formalin, decalcified and embedded in paraffin for histology.", "Histological scoring for cartilage repair by 3 independent observers was by Pineda's method (Pineda et al., 1992; Orth et al., 2012).", "Data are shown as the mean ± SEM.", "An unpaired t-test for data from two groups, or one-way ANOVA for data from groups of three, or more was used to compare data with post hoc Tukey's test for gene expression comparison and Bonferroni's test for histology analysis.", "P values < 0.05 were considered to be significant.", "With the aim of generating more mature chondrogenic cells with a greater capacity to produce matrix in vitro, we looked at the effect of BMP7, TGFβ-1 and TGFβ-3 as alternatives to, or in addition to, GDF5, in stage 3 of our protocol (day 9–14).", "Cells cultured with GDF5 showed typical chondrogenic cell morphology and produced cell aggregates, but cells cultured without GDF5, but with BMP7 together with either TGFβ-1 or TGFβ-3 showed pronounced cell death.", "In addition, QRT-PCR showed that the removal, or replacement of GDF5 in the protocol caused a dramatic decrease in COL2A1 expression, without affecting the expression of SOX9 (Supplementary Fig. S1).", "When we added BMP7, together with either TGFβ-1, or TGFβ-3 (GDF5 still present) there was less cell death, but there was a similar reduction in COL2A1 with no change in SOX9 or ACAN expression (Supplementary Fig. S1).", "Although BMP4 has been shown to be most active in early mesodermal patterning in mice, BMP2 has been shown in different chondrogenic systems to have strong pro-chondrogenic effects (Toh et al., 2005; Duprez et al., 1996).", "We therefore tested the substitution of BMP4, used from day 3 to 10 to induce mesoderm and later chondrogenic mesoderm, with BMP2 (Figs. 2 and 3).", "Results using 2 hESC lines of different origin (HUES1 and MAN7), both showed that with BMP2 there was earlier appearance of cell aggregates during stage 2, which typified chondrogenic differentiation, whereas they were delayed until stage 3 in the protocol with BMP4.", "Moreover, with BMP2 the aggregates were significantly larger (Fig. 2c), although there was no significant change in overall cell expansion, or apoptosis (Fig. 3).", "In the cultures under the conditions tested, there was an approximately 8-fold increase in cell number by day 10 in the presence of either BMP2 or 4, which was as reported previously (Oldershaw et al., 2010).", "However, during the protocol there was a small but significant increase in cell number with BMP2 compared to BMP4 between day 5 and day 9 (Fig. 3), suggesting an early increase in proliferation.", "At the end of the protocol the cells in aggregates were manually separated from those in between aggregates and they were assessed separately for chondrogenic gene expression.", "This confirmed that the cells in aggregates were enriched in the chondrogenic transcripts COL2A1, SOX9 and ACAN, while cells outside aggregates had higher expression of collagen COL1A1, which is more associated with non-chondrogenic fibroblastic cells (Fig. 2d).", "With the aim of generating more mature chondrogenic cells with a greater capacity to produce matrix in vitro, we looked at the effect of BMP7, TGFβ-1 and TGFβ-3 as alternatives to, or in addition to, GDF5, in stage 3 of our protocol (day 9–14).", "Cells cultured with GDF5 showed typical chondrogenic cell morphology and produced cell aggregates, but cells cultured without GDF5, but with BMP7 together with either TGFβ-1 or TGFβ-3 showed pronounced cell death.", "In addition, QRT-PCR showed that the removal, or replacement of GDF5 in the protocol caused a dramatic decrease in COL2A1 expression, without affecting the expression of SOX9 (Supplementary Fig. S1).", "When we added BMP7, together with either TGFβ-1, or TGFβ-3 (GDF5 still present) there was less cell death, but there was a similar reduction in COL2A1 with no change in SOX9 or ACAN expression (Supplementary Fig. S1).", "Although BMP4 has been shown to be most active in early mesodermal patterning in mice, BMP2 has been shown in different chondrogenic systems to have strong pro-chondrogenic effects (Toh et al., 2005; Duprez et al., 1996).", "We therefore tested the substitution of BMP4, used from day 3 to 10 to induce mesoderm and later chondrogenic mesoderm, with BMP2 (Figs. 2 and 3).", "Results using 2 hESC lines of different origin (HUES1 and MAN7), both showed that with BMP2 there was earlier appearance of cell aggregates during stage 2, which typified chondrogenic differentiation, whereas they were delayed until stage 3 in the protocol with BMP4.", "Moreover, with BMP2 the aggregates were significantly larger (Fig. 2c), although there was no significant change in overall cell expansion, or apoptosis (Fig. 3).", "In the cultures under the conditions tested, there was an approximately 8-fold increase in cell number by day 10 in the presence of either BMP2 or 4, which was as reported previously (Oldershaw et al., 2010).", "However, during the protocol there was a small but significant increase in cell number with BMP2 compared to BMP4 between day 5 and day 9 (Fig. 3), suggesting an early increase in proliferation.", "At the end of the protocol the cells in aggregates were manually separated from those in between aggregates and they were assessed separately for chondrogenic gene expression.", "This confirmed that the cells in aggregates were enriched in the chondrogenic transcripts COL2A1, SOX9 and ACAN, while cells outside aggregates had higher expression of collagen COL1A1, which is more associated with non-chondrogenic fibroblastic cells (Fig. 2d).", "Comparing gene expression between BMP2 and 4 induced cells (Fig. 3) revealed that the pluripotency-associated markers, OCT4 and NANOG, decreased early in both cultures.", "They also both showed similar SOX9 expression, but the SOX9 cofactor SOX5 was significantly increased at day 14 in BMP2 cultures.", "The strong chondrogenic drive of BMP2 was revealed most markedly by major increases in the expression of both COL2A1 and ACAN, which were increased by 4 and 8.5-fold respectively in the presence of BMP2.", "This chondrogenic enhancement was also supported by a 2-fold decrease in the expression of COL1A1.", "There was also no change in COL10 expression, which was very low in both BMP2 and BMP4 cultures.", "The effects of BMP2 were similar in both hES cell lines tested.", "Since BMP2 increased the expression of genes associated with chondrogenesis we validated the effect by measuring proteins expression in hESC chondrogenic cells at the end of stage 3.", "Collagen produced by the cells is constitutively secreted into the extracellular matrix, we therefore used a protein transport inhibitor cocktail (Brefeldin A and Monensin) for 6 h prior to collecting cell lysate in order to compare cellular Collagen in the 2 cultures at day 14.", "In both BMP4 and BMP2 cell lysates a strong type II collagen protein band was detected (Fig. 4a).", "The BMP2 cultured cells showed a stronger band for SOX9 protein compared to BMP4 cells at the end of stage 3 (Fig. 4b).", "Parallel flow cytometry showed >95% of cells were SOX9 positive at the end of stage 3 in both BMP4 and BMP2 cultures (Supplementary Fig. S2).", "Since BMP2 increased the expression of genes associated with chondrogenesis we validated the effect by measuring proteins expression in hESC chondrogenic cells at the end of stage 3.", "Collagen produced by the cells is constitutively secreted into the extracellular matrix, we therefore used a protein transport inhibitor cocktail (Brefeldin A and Monensin) for 6 h prior to collecting cell lysate in order to compare cellular Collagen in the 2 cultures at day 14.", "In both BMP4 and BMP2 cell lysates a strong type II collagen protein band was detected (Fig. 4a).", "The BMP2 cultured cells showed a stronger band for SOX9 protein compared to BMP4 cells at the end of stage 3 (Fig. 4b).", "Parallel flow cytometry showed >95% of cells were SOX9 positive at the end of stage 3 in both BMP4 and BMP2 cultures (Supplementary Fig. S2).", "Since mature chondrocytes produce an alternatively spliced transcript, COL2A1 IIB, in which exon 2 is omitted, while in prechondrogenic mesoderm the prevalent form is IIA, in which exon 2 is retained (Ryan and Sandell, 1990; McAlinden, 2014) we measured transcript expression to detect if this transition occurred in our cultures.", "The analysis of splice-form specific transcripts showed that in both BMP2 and 4 cultures the predominant isoform at day 14 was IIA, but a weak band for IIB was also detected (Fig. 4c).", "Interestingly in this analysis the expression of both isoforms of COL2A1 but notably IIb occurred earlier in the BMP2 cultured cells than in BMP4 cultures.", "BMP2 and BMP4 are often used interchangeably in research and bind to the same receptors.", "The above results suggest that BMP2 generates more chondrogenic signals in the hESC cultures, than BMP4.", "This may result from BMP2 synergising with additional co-factors creating higher BMP2 potency.", "To assay activity independently, we measured the induction of alkaline phosphatase in the chondrosarcoma line SW1353 (Fig. 5a).", "The results suggested that intrinsically BMP4 was more potent than BMP2 at 10-20 ng/ml in SW 1353 chondrosarcoma cells.", "Although this may not hold for developing chondrogenic cells this result suggests that the greater effect of BMP2 on hESCs was unlikely to be due to a simple difference in its potency.", "We next examined if signalling from the two BMPs was similar (Fig. 5d).", "BMP binding to its heterodimeric receptor activates phosphorylation of SMAD1, 5 and 9 which further interact with co-SMADs and after translocation to the nucleus drive changes in gene transcription.", "Flow cytometry analysis of day-14 cells, showed no significant difference between BMP4 and BMP2 treatments in the number of cells positive for SMAD1/5/9 and p-SMAD1/5/9.", "This suggested that receptor signal transduction through the SMAD pathway from BMP2 and 4 was similar in day 14 hESC chondroprogenitors.", "Confirming this, when the ratio of p-SMAD1/5/9 to total SMAD1/5/9 was normalised to GAPDH in extracts from cells examined on day 10, there was no difference between BMP2 and BMP4 (both at 20 ng/ml, Fig. 5e).", "To test whether fibrin gels containing BMP2 induced progenitors promote improved cartilage repair in vivo we embedded hESC-chondrogenic cells from day 14 in fibrin matrix and implanted them in 2 mm diameter osteochondral defects in the trochlea groove of the femur of Nude rats as previously described (Cheng et al., 2014b).", "Control defects were left empty or repaired with fibrin gel alone, and repair was analysed at 6 and 12 weeks (Fig. 6).", "Two animals were removed from the study because in each case dislocation of the patella had occurred during joint surgery.", "The empty control defects retained a marked cavity and showed no evidence of organised repair by 12 weeks.", "When fibrin alone was used, repair with a more uniform surface was generated, but with little type II collagen immunostaining and only a few isolated areas of Safranin O staining.", "In contrast, in the defects repaired with cells embedded in fibrin, there was strong type II collagen immunostaining and Safranin O positive areas and there was also good integration with the surrounding tissue.", "Cartilage repair was assessed in sections using the system developed by Pineda (Pineda et al., 1992).", "The repair associated with the fibrin gels containing cells induced with BMP2 appeared better than those with BMP4 cells and when the sections were scored blind by 3 observers a significant improvement was detected with BMP2 cells.", "TGFβ-family growth factors play roles during mesenchymal condensation and cartilage formation, (Cheng et al., 2014b; Steinert et al., 2003).", "Therefore, we examined the effect of TGFβ-1, TGFβ-3 and BMP7 on the differentiation protocol in enhancing the hESC-chondrogenic phenotype.", "We found that replacing GDF5 with BMP7 together with TGFβ-1 or -β3 from day 11 produced no improvement in SOX9 expression and reduced COL2A1 expression.", "This is in distinction to Nakagawa et al. (2009) who reported chondrocyte formation from hESCs in the presence of BMP7 and TGFβ-1, albeit under a less defined culture regimen including serum.", "In the absence of GDF5, which in other tissues can function as a survival factor (Jaumotte and Zigmond, 2014; Sullivan et al., 1998), hESCs showed pronounced apoptosis.", "This suggests that GDF5 has a survival effect on human chondrogenic cells, especially since adding GDF5 in the presence of BMP7 and TGFβ-1 suppressed cell death.", "However, in the BMP7/TGFβ-1/GDF5 protocol, these growth factors did not enhance chondrogenesis, as there was a decrease in expression of COL2A1 and no significant increases in ACAN, or SOX9.", "The specific role of GDF5 in maturation of the chondrocytes and establishment of the joint hyaline cartilage is difficult to assess, because of the loss of cells in its absence.", "However, as well as a role in enhancing cartilage formation (Hatakeyama et al., 2004; Thomas et al., 1996; Thomas et al., 2006; Roelofs et al., 2017; Shwartz et al., 2016), GDF5 is suggested to play a role in modulation of BMP action during the formation of the joint and the interzone and at a time when permanent cartilage is well established (Tsumaki et al., 2002; Thomas et al., 2006).", "In contrast, substituting BMP2 for BMP4 from day 3 led to enhanced expression of chondrogenic transcription factors and importantly increased COL2A1 and ACAN expression at the end of the protocol in both hESC lines examined.", "We confirmed that cell aggregation reflected an enriched chondrogenic cell population with increased COL2A1, ACAN and SOX5 expression and the size of the condensations formed was increased with BMP2, suggesting a larger proportion of chondrogenic cells.", "Although initial proliferation was slightly greater in the presence of BMP2, confirming the published effect on proliferation (Shu et al., 2011), final culture expansion in the protocol remained the same for both BMPs and there was no evidence of differing apoptosis.", "Although COL2A1 and ACAN expression were substantially increased in BMP2 cultures, there was no significant change in the expression of SOX9, but there was increased expression of SOX5, which acts co-ordinately with SOX9 in driving matrix gene expression and may have been a factor limiting the transcription of the COL2A1 gene.", "BMP2 and 4 have often been used interchangeably in experiments in cell and developmental systems.", "Therefore, it was surprising that using BMP2 at the same concentration as BMP4 showed such a pronounced effect on matrix gene expression.", "The two growth factors have high structural homology, they are the most closely related of the BMP-subfamily and interact with the same receptors (BMPR1a, b [ALK3, ALK6], BMPR2 [ALK4]).", "However, although Flow cytometry and immunoblotting showed little difference in SMAD signalling between BMP2 and BMP4 derived cells, additional signal pathways may be triggered by BMP2 resulting from effects on receptor lateral mobility and clustering (Guzman et al., 2012).", "Analysis showed that by the end of the protocol SOX9 protein was increased in BMP2 cultured cells compared to BMP4.", "This may be a consequence of additional signals from BMP2 resulting in increased cell rounding and cytoskeletal relaxation inherent in cell aggregate formation, which is known to promote chondrocyte differentiation (Woods et al., 2005) and has been reported to increase the half-life of SOX9 transcripts in chondrocytes (Tew and Hardingham, 2006).", "In our cultures there was no large increase in SOX9 transcript levels in BMP2 cultures, but it is possible that there are additional direct effects on protein translation through the cell shape and cytoskeletal changes accompanying aggregate formation.", "The differential effects of BMP2 and BMP4 may therefore be modulated by their different interactions with extracellular matrix components that regulate their association and clustering with receptors at the cell surface (Sieber et al., 2009).", "This is likely to be governed by the rapid changes in matrix components during differentiation.", "Although BMP2 is established as involved in osteogenic differentiation (Shu et al., 2011), we saw no evidence of COLX expression during differentiation and it is clear that during early development BMP2 has roles in limb development (Duprez et al., 1996) initiation of chondrogenesis, chondrocyte proliferation (Bandyopadhyay et al., 2006) and stimulates synthesis of cartilage collagen II and proteoglycans including aggrecan (Blaney Davidson et al., 2007).", "In order to assess any benefit of BMP2 in the differentiation protocol we tested the repair potential of BMP2 and BMP4 hESCs in vivo.", "The results showed that implanting fibrin gels containing BMP2 and BMP4 induced-cells produced effective tissue repair significantly better than with cell-free fibrin implants.", "The histological scoring showed that BMP2 induced cells produced significantly better quality of repair at 12 weeks compared to BMP4 induced cells.", "In previous work (Cheng et al 2014b) we have shown that hESC-derived chondrocytes can be found in the implant area at 8 or 12 weeks after implantation suggesting that they contribute to the repair process.", "This current work therefore suggests that BMP2 generated chondroprogenitors support better cartilage matrix repair in vivo compared to those generated by BMP4.", "Our protocol provides an experimental in vitro system permitting detailed analysis of the mechanisms driving human chondrogenesis in development and in tissue repair.", "For the latter we are now applying our refined protocol to clinical grade hESCs, which, would be suitable for repairing human cartilage defects.", "The procedure we developed also provides a platform for studying early development and joint disease mechanisms and by using iPSCs from patients it enables the study of genetic diseases affecting joint cartilage.", "The following are the supplementary data related to this article.", "Effects of different growth factors in stage 3 of chondrogenic protocol in HUES1.", "Chondrogenic protocol was carried out as described before until day 10, from day 11 to day 13 cells were treated with different combination of growth factors and on day 14 cell morphology was observed and photographed under phase contrast (a).", "RNA was collected on day 14 for gene expression analysis (b).", "FACS blot for SOX9 labelled cells differentiated with either BMP2 (dark grey) or BMP4 (light grey) in the chondroprogenitor differentiation protocol.", "Supplementary data to this article can be found online at https://doi.org/10.1016/j.scr.2019.101497.", "The data that support the findings of this study are available from the corresponding author upon reasonable request." ]

[ "BMP2 stimulates increased chondrogenic gene expression from hESC chondroprogenitors compared to BMP4.", "Other TGFΒ members were not effective.", "BMP2 promoted COL2a1 positive aggregate formation.", "with no effect on culture expansion or apoptosis." ]

[ "NEUR" ]

[]

[ "2805", "2808" ]

[ "The dopamine receptor D4 (DRD4) gene locates on chromosome 11p15.5.", "As summarized previously (Swanson et al., 2000), this gene has a polymorphism in a coding region—a variable number of tandem repeats (VNTR) of a 48-base pair sequence in exon 3 (Lichter et al., 1993) that codes for a variation in the third intracellular loop of the D4 receptor.", "This gene is expressed in a wide range of brain areas (Mrzljak et al., 1996; Van Tol et al., 1992).", "A specific allele, the 7-repeat allele, of this polymorphism has been robustly associated with the risk of attention deficit hyperactivity disorder (ADHD) via multiple meta-analyses (Li et al., 2006).", "However, in Asian populations, the 7-repeat allele is scarce or absent (Li et al., 2006) and perhaps partly because of this, the prevalence of ADHD is quite low (Faraone and Biederman, 1998).", "On the other hand, another allele, the 5-repeat allele, of this polymorphism has also been shown to be associated with an increased risk of ADHD via meta-analysis, and is relatively more frequently observed in Asian samples than the 7-repeat allele (Li et al., 2006).", "It has been shown that the DRD4 gene's polymorphisms lead to the difference in how well the receptors bind with dopamine and similar compounds (Van Tol et al., 1992), and it has been assumed this basic difference leads to the difference of the phenotype.", "The initial study (Van Tol et al., 1992) has just shown the inefficiency of receptors from 7-repeat alleles to bind compared with those of receptors from 2-repeat alleles and 4-repeat alleles.", "However, the 5-repeat allele has shown the same phenotype as the 7-repeat allele with regard to the effects on ADHD; it may therefore be assumed that the pharmacological aspects and effects of the 5-repeat allele on various characters can be parallel to those of the 7-repeat allele.", "Previous genetic studies have shown the effects of the 7-repeat allele in several behavioral variables (Szekely et al., 2011) and regional gray matter structures (Durston et al., 2005; Monuteaux et al., 2008; Shaw et al., 2007).", "In particular, using a large sample size, Shaw et al. (2007) have shown that the 7-repeat allele carriers have a thinner right orbitofrontal/inferior prefrontal and posterior parietal cortex.", "Like other polymorphisms findings, some of the initial results regarding DRD4 polymorphism with less statistical power cannot be replicated (Kluger et al., 2002).", "A recent study showed a robust association between the 7-repeat allele and a slow processing speed (Szekely et al., 2011).", "Another interesting recent finding was the association between the 7-repeat risk allele and decreased performance of the creativity measured by a divergent thinking test in an Israeli sample (Mayseless et al., 2013).", "It should be noted that unlike Asian samples, the Israeli sample is known to have a high prevalence of ADHD (Cohen et al., 2013) as well as a high prevalence of the risk allele of ADHD (Mayseless et al., 2013), which is supposed to partly contribute to the cultural difference in the prevalence of ADHD (Faraone and Biederman, 1998).", "In the Israeli sample, this association between the 7-repeat risk allele and decreased performance of creativity measured by a divergent thinking task is despite the previously known robust association between impaired selective attention and increased divergent thinking (for the full discussion of this matter, see Takeuchi et al., 2011b).", "However, to date, the following points have not been investigated.", "(a) The association between the polymorphism of DRD4 and brain microstructural properties measured by diffusion tensor imaging (DTI), such as fractional anisotropy and mean diffusivity (MD); (b) the association between the polymorphism of DRD4 and neural activity during externally directed cognitive tasks using a sample size with a statistical power that allows the analysis of this type of polymorphism (N > a few hundred at least, for estimates, see Supplemental Discussion); and (c) the neural and psychological effects of the 5-repeat allele of DRD4 in a sample with a relatively low risk of ADHD.", "We investigated these issues using DTI, an n-back working memory functional magnetic resonance imaging (fMRI) paradigm, and psychological measures, in several hundred Asian samples.", "The fractional anisotropy (FA) measure of DTI has proved useful in revealing the neural basis of ADHD and provided insights into the etiology of the psychiatric diseases (Konrad and Eickhoff, 2010).", "It also correlated with cognitive functions, including ones that were shown to be associated with the DRD4 gene in normal adults (Jung et al., 2010a; Takeuchi et al., 2013b).", "On the other hand, the MD measure of DTI shows a unique property compared with MD, and reduced MD is likely to reflect the increase in tissue density due to the shape of the neuron or glias, synapse, or enhancement of tissue organization (strengthening of axonal or dendritic backbones and surrounding tissue) (Assaf and Pasternak, 2008; Sagi et al., 2012).", "Consistently, patients with neuronal degeneration have shown an increase in MD (Andreone et al., 2007; Kantarci et al., 2001; Nusbaum et al., 2001).", "In addition, further patients with ADHD show an increase in MD (Konrad and Eickhoff, 2010).", "Furthermore, a recent investigation elucidated a unique property of MD in the gray matter.", "This is partly due to its sensitivity toward neural plasticity (Abe et al., 2014; Sagi et al., 2012).", "More pertinent to the present study's subject is its association with dopamine.", "For example, MD in the dopaminergic system's areas (MDDS) has been shown to be more sensitive to the pathology of neurodegeneration of dopaminergic systems than other MRI measures and PET measures of dopamine receptor binding (Péran et al., 2010; Seppi et al., 2004).", "It can sensitively detect the neural plasticity involving dopamine function change (Razek et al., 2011; Takeuchi et al., 2014c).", "It also shows robust correlation with the motivational state, which is highly relevant to dopamine as well as traits crucially related to dopamine (Bjørnebekk et al., 2012; Laricchiuta et al., 2013; Picerni et al., 2013; Takeuchi and Kawashima, 2013).", "Thus, DRD polymorphism may well be associated with these DTI measures, and its investigation can reveal new insights into the effects of DRD4 polymorphisms.", "Corresponding to the three aforementioned uninvestigated issues, we hypothesized that the effect of the 5-repeat allele of DRD4 is reflected in microstructural properties in a wide range of areas where the DRD4 gene is expressed.", "We also hypothesized that it is reflected in reduced task-induced deactivation (TID) in the areas that are deactivated during attention-demanding tasks (default mode network; DMN).", "This is because TID in the DMN is thought to reflect inefficient attention reallocation (Takeuchi et al., 2011b) and reduced TID has been associated with increased inattentional tendencies such as increased mind wandering in nonclinical groups (Brewer et al., 2011).", "On the other hand, in clinical ADHD groups, the patient group tends to show a decrease in activities during attention-demanding tasks in areas of the lateral prefrontal and certain areas in the parietal cortex that are activated during the tasks, although some studies have also shown reduced TID in the DMN (Brown et al., 2012; Brown et al., 2011).", "Furthermore, it has been suggested that one of the reasons why the risk alleles of psychiatric diseases are widely prevalent is that these alleles have adaptive effects on several processes, such as creativity, when they are not accumulated in the individual's genome (Horrobin, 2002).", "Therefore, considering these and the previously reported robust association between impaired selective attention and increased creativity measured by divergent thinking, we hypothesized that the risk allele of ADHD in the present sample leads to increased divergent thinking performance.", "The present study revealed the neural and psychological effects of the DRD4 5-repeat allele in a healthy Asian sample, which is known to have a low prevalence of ADHD, as well as those of the robust risk allele (7-repeat allele) for ADHD.", "The 5-repeat allele was associated with worse processing speed performance, higher self-reported impulsivity, and less tendency to maintain attentional focus to the external world in this sample.", "These results are consistent with the previous finding of the association of slow processing speed with another risk allele (7-repeat allele) of the DRD4 gene (Szekely et al., 2011), and the association of the 5-repeat risk allele with the risk of ADHD (Szekely et al., 2011).", "We revealed that 5-repeat allele was significantly associated with increased originality in creative performance.", "Furthermore, the 5-repeat allele was significantly associated with increased MD in the widespread gray and white matter areas of the cerebral cortex, and with subcortical areas located mainly in the globus pallidus, amygdala, midbrain areas, brain stem, and others.", "This is largely consistent with our hypothesis, as discussed below.", "Finally, partly congruent with our hypothesis, the 5-repeat allele was associated with reduced TID in the precuneus areas in both the attention-demanding task and simple sensorimotor task; similar patterns were observed in the posterior cingulate cortex and in areas located around the midbrain and hippocampus.", "The extent of the area with significant association between MD and the DRD4 5-repeat allele was consistent with the previously reported distribution of DRD4.", "5-Repeat allele carriers showed increased MD in the widespread gray and white matter areas of the cerebral cortex, globus pallidus, amygdala, midbrain areas, and others.", "As described in the Introduction section, an increasing number of studies stress the importance of MD, and it is not surprising that physiological correlates are found in the gray matter areas.", "The present widespread results of diffusivity measures are congruent with the findings of the studies of polymorphisms, studies of normal development, studies of a certain disease that showed variables that were investigated in these studies affect brain mechanisms in a wide range of white matter areas (Taki et al., 2013; Tost et al., 2012; Whitwell et al., 2011) in analyses limited to white matter.", "Previously, a positron emission tomography (PET) study in humans showed that DRD4 is notably expressed to a greater extent in the neocortex than in the striatum (Boy et al., 1998).", "Furthermore, molecular studies revealed that DRD4 is expressed highly in the neocortex, midbrain, amygdala, and globus pallidus, and lowly expressed in the striatum and hippocampus (Mrzljak et al., 1996; Van Tol et al., 1992).", "Therefore, these previous findings of the distribution of the expression of DRD4 and the present extent of the association between the DRD4 5-repeat allele and MD were very concordant, suggesting the validity of the present findings.", "Perhaps the neural mechanisms in the areas in which DRD4 is expressed are globally affected via the DRD4 5-repeat allele.", "There are several possible mechanisms via which the 5-repeat allele of DRD4 can affect MD.", "MD is often believed to be a measure of overall water content (Moseley et al., 2002).", "Possible obstacles, such as the presence of fewer or smaller cellular structures (e.g., capillaries, synapses, and macromolecular proteins), may prevent the free diffusion of water molecules and may also be expected to cause a decrease in the MD value (Ni et al., 2010).", "Among normal samples, cognitive learning affects MD values after only 2 h of training in the relevant tissues and together with the results of animal experiments, an increase in the number of synaptic vesicles and swelling of astrocytes because of the increased activity has been suggested to underlie these MD changes (Johansen-Berg et al., 2012; Sagi et al., 2012).", "In the long run, changes in other physiological mechanisms, such as dendritic sprouting and angiogenesis, may affect MD (Johansen-Berg et al., 2012).", "On the other hand, the risk alleles of the DRD4 gene are less sensitive to dopamine stimulation (Asghari et al., 1995).", "A regular decrease in the related neural activity in the carriers of risk alleles of DRD4 may lead to an increase in MD through the decrease in the swelling of astrocytes, number of synaptic vesicles, dendritic sprouting, etc.", "The areas that exhibited significant differences in MD in the present study, as well as areas in which the DRD4 gene was highly expressed, included areas of the mesocortical dopaminergic system and mesolimbic dopaminergic system.", "Neurons in the mesolimbic system project to the nucleus accumbens, hippocampus, etc. from the ventral tegmental area; this system plays a key role in the reward mechanism of the brain (Carlson, 2001).", "Finally, neurons in the mesocortical system project to the prefrontal cortex from the tegmental ventral area, and are involved in cognitive control and in other higher-order cognitive functions (Carlson, 2001).", "Blunted response in these networks may lead to the disruption of cognitive, behavioral deficits, and TID changes, suggesting the insufficient attentional reallocation (as discussed below) observed in the 5-repeat allele carriers.", "In addition, these physiological mechanisms can mostly impact gray and white matter and are congruent with the findings of MD found in both gray and white matters.", "As discussed in our previous study (Takeuchi et al., 2011b), the reduced TID observed in the precuneus areas, posterior cingulate cortex, hippocampus, and midbrain areas in the 5-repeat allele carriers may suggest their impaired ability to suppress irrelevant cognitive activity during a cognitive task.", "These areas are the regions that are deactivated during cognitive tasks (Takeuchi et al., 2011b).", "The precuneus areas, posterior cingulate cortex, and hippocampus are supposed to be engaged in cognitive activities such as self-related mental representations and episodic memory retrieval during rest (Cavanna and Trimble, 2006).", "It has been argued that the magnitude of TID in these areas during cognitive tasks reflects the reallocation of attention from the semantic processes that occur during rest to the cognitive processes involved in performing the task (McKiernan et al., 2003).", "Therefore, the reduced TID observed in these areas in 5-repeat allele carriers may indicate that they are not able to inhibit or suppress cognitive activity irrelevant to the task performance (task-unrelated thought) during cognitive tasks.", "These neural characteristics may lead to (or reflect) an increased risk of attentional and behavioral deficits in 5-repeat allele carriers.", "This type of inattentiveness that cannot suppress or filter out irrelevant information (thought) may be important for creativity measured by divergent thinking.", "As discussed previously (Takeuchi et al., 2011b), one speculation about the implication of the fact that individuals who are unable to suppress DMN activity is that such an inability to suppress seemingly unnecessary cognitive activity may actually help them associate two seemingly distant ideas represented in different networks.", "This idea of creativity has been long stated, and a vast amount of studies showed that inattentive subjects who cannot filter irrelevant information are creative (for summary, see Takeuchi et al., 2011b).", "The inability to suppress an irrelevant network when one network is recruited may lead to the intrusion of thoughts from the irrelevant network and may allow the combination of two isolated ideas.", "Consistent with this notion, a reduced TID in the areas of the DMN in subjects with higher creativity measured by divergent thinking as well as subjects at a risk of schizophrenia, which is known to be robustly associated with higher creativity measured by divergent thinking (Takeuchi et al., 2011b; Whitfield-Gabrieli et al., 2009).", "In addition, some recent previous functional imaging studies of creative cognition have reported that regions of the DMN could be critically involved in creativity (Fink et al., 2010; Fink et al., 2012).", "Furthermore, other studies of resting state paradigms have revealed that resting state brain activity involving the DMN is important for creativity measured by divergent thinking (Takeuchi et al., 2012a; Wei et al., 2013).", "Some structural studies have indicated that the brain structure of the key nodes of the DMN is associated with creativity (Fink et al., 2013).", "In addition, DMN is thought to be critically involved in self-referential or introspective cognitive activities, such as those involving autobiographical memory, and social cognition, such as the theory of mind (Buckner et al., 2008; Fair et al., 2008; Schilbach et al., 2008).", "Perhaps related to this, we previously summarized the overlap or associations between self-reflection or access to self- and social cognitive functions and showed associations between social cognitive functions and creativity measured by divergent thinking (Takeuchi et al., 2014b).", "Based on this neuroimaging evidence, it has been pointed out the DMN is important for creative cognition (Jung et al., 2013) and the present findings may implicate the importance of the DMN itself and functions related to DMN, such as self-related cognition and social cognition (not only that of attention).", "We propose possible speculative mechanisms of the association between the 5-repeat allele and originality.", "While subscales (dimensions) of divergent thinking tend to be highly correlated, originality shows rather distinct properties, and some previous studies have demonstrated specific changes in originality (Kharkhurin, 2008).", "One speculative reason why originality was specifically affected in 5-repeat allele carriers is that while the originality of the idea is increased by the aforementioned mechanisms, decreased processing speed associated with the 5-repeat allele may hinder other dimensions of divergent thinking because of the association between divergent thinking and processing speed (Rindermann and Neubauer, 2004).", "To support the former association (DMN activity during the task and originality), we investigated the association between originality/fluency and brain activity during the task.", "The results indicate that the originality dimension may be specifically (after adjusting for the number of responses) associated with reduced TID in the area of the posterior cingulate cortex during the sensorimotor task, similar to the DRD4 polymorphism, but the overlap of the two is not statistically definitive (see Supplemental Methods, Supplemental Results, and Supplemental Fig. 1).", "However, brain lesions of certain areas irrelevant to attention processes have been shown to increase originality (Shamay-Tsoory et al., 2011).", "Originality is rated by how infrequent the idea is, and schizotypy, which is associated with atypical thinking patterns or experiences (Claridge et al., 1996), is known to be uniquely associated with originality of ideas (Green and Williams, 1999).", "Thus, the odd or atypical thinking pattern caused by certain disruptions of the brain networks may lead to originality (odd ideas).", "At present, these possibilities are pure speculation, and future studies are required to explore this issue more thoroughly.", "We propose possible speculative mechanisms of the association between the 5-repeat allele and originality.", "While subscales (dimensions) of divergent thinking tend to be highly correlated, originality shows rather distinct properties, and some previous studies have demonstrated specific changes in originality (Kharkhurin, 2008).", "One speculative reason why originality was specifically affected in 5-repeat allele carriers is that while the originality of the idea is increased by the aforementioned mechanisms, decreased processing speed associated with the 5-repeat allele may hinder other dimensions of divergent thinking because of the association between divergent thinking and processing speed (Rindermann and Neubauer, 2004).", "To support the former association (DMN activity during the task and originality), we investigated the association between originality/fluency and brain activity during the task.", "The results indicate that the originality dimension may be specifically (after adjusting for the number of responses) associated with reduced TID in the area of the posterior cingulate cortex during the sensorimotor task, similar to the DRD4 polymorphism, but the overlap of the two is not statistically definitive (see Supplemental Methods, Supplemental Results, and Supplemental Fig. 1).", "However, brain lesions of certain areas irrelevant to attention processes have been shown to increase originality (Shamay-Tsoory et al., 2011).", "Originality is rated by how infrequent the idea is, and schizotypy, which is associated with atypical thinking patterns or experiences (Claridge et al., 1996), is known to be uniquely associated with originality of ideas (Green and Williams, 1999).", "Thus, the odd or atypical thinking pattern caused by certain disruptions of the brain networks may lead to originality (odd ideas).", "At present, these possibilities are pure speculation, and future studies are required to explore this issue more thoroughly.", "There may be some incongruence between these previous findings of the association between attention-related cognition and TID in the DMN and the previous findings of brain activity of subjects with ADHD as discussed below.", "Although some studies have revealed a reduced TID in the DMN in subjects with ADHD during attention-demanding tasks (Brown et al., 2012), most findings have revealed reduced activation in the areas that are activated during attention demanding tasks, particularly the lateral prefrontal areas (Burgess et al., 2010).", "Although we found a tendency in 5-repeat allele carriers toward reduced activation in the left lateral prefrontal cortex, due to the nature of whole-brain analysis (insignificance doesn't mean the lack of associations at all), it is not possible to say 5-repeat allele carriers are or are not associated with the reduction in brain activity in the lateral prefrontal cortex; therefore, the implications of lack of significance are statistically unclear.", "However, importantly, it has been shown that reduced activity in the lateral prefrontal cortex of ADHD subjects is mediated by their reduced working memory capacity (Burgess et al., 2010; Valera et al., 2010).", "To the best of our knowledge, no studies have reported an association between 5-repeat allele carriers (or 7-repeat allele carriers) and reduced working memory capacity.", "The incongruence of the lateral prefrontal cortex activity may come from this difference.", "In addition, because the increased cognitive load caused by the task difficulty would be expected to result in increased TID, the fact that subjects of ADHD may feel the task is difficult (and may have more cognitive load during the task) may lead to a less frequent occurrence of reduced TID in the DMN in subjects with ADHD.", "However, these are speculations and future studies are required to reveal this issue by, for example, focusing on subjects with ADHD with intact working memory capacity.", "We failed to observe significant effects or tendency of the polymorphism of DRD4 on working memory performance as well as on general intelligence performance measured by non-verbal reasoning.", "This may be odd considering the implication (efficiency of attentional reallocation and suppression of task-irrelevant thoughts) of reduced TID in DMN, which was shown to be related to this polymorphism in the present study, as discussed above, as well as associations among attention, ADHD, and working memory performance (and general intelligence related to working memory capacity) (Engle et al., 1999; Martinussen et al., 2005).", "In studies of common polymorphisms, effects on cognitive functions tend to be weak.", "Thus, the lack of significance should not be treated as evidence of the lack of effects.", "However, we suggest one possible explanation for this result, as described above, as follows: it seems that working memory deficit in patients with ADHD is associated with alterations in the activity of the lateral prefrontal cortex and not that of DMN (Burgess et al., 2010; Valera et al., 2010).", "From a neuroimaging perspective, there are various aspects of and systems in the attention system (Corbetta et al., 2008), and the suppression of task-irrelevant cognitive activity may not be so strongly associated with the performance of working memory.", "For example, one study reported that the correlation coefficient r of the frequency of the intrusion of task-irrelevant thoughts and working memory performance was about − 0.23, explaining 5% of variance in working memory performance (Brewin and Smart, 2005).", "Thus, such a potentially weak indirect association between the polymorphism of the DRD4 gene and working memory performance might have resulted in the negative findings in this study.", "We failed to observe significant effects of the polymorphism of DRD4 on FA.", "Particularly, in the whole-brain analyses, the lack of significant effects was not evidence of the lack of effects.", "However, the lack of significance may be incongruent with some of the previous findings, in which significant effects of polymorphisms of dopamine-related genes, such as those of catechol-O-methyltransferase (COMT) (Papenberg et al., 2014; Thomason et al., 2010), were found with a smaller sample size.", "Furthermore, FA is thought to reflect, in part, the degree of myelination (for details of this discussion, see Takeuchi et al., 2013b), and previous physiological studies have pointed out a profound association between the myelination process and dopamine (Káradóttir and Attwell, 2007; Lindholm and Jazin, 2007).", "Moreover, these previous physiological studies have suggested that the dopamine receptors D2 and D3 play key roles in mediating these associations.", "Thus, the lack of significance may, in part, be ascribed to the type of dopamine receptor we investigated.", "There is at least a few potential pitfall or limitation of this study.", "This study investigated the effect of the 5-repeat allele in an Asian sample.", "The Asian population has a unique characteristic regarding the polymorphism of DRD4 and ADHD, as described in the “Introduction.”", "Whether this finding is generalizable to other samples remains to be investigated.", "Furthermore, in this study, although we excluded subjects with psychiatric disorders on the basis of voluntary self-reports, we did not diagnose ADHD officially.", "The symptoms of disorders such as ADHD are known to have continuity with the normal sample (Harrington, 2001); thus, the potential inclusion of the actual ADHD is not thought to substantially alter the nature of the study.", "In addition, the low prevalence of ADHD in Asian and Japanese samples is well known and focusing on a high education sample plus the presented exclusion criteria may further lower the risk of ADHD (Faraone and Biederman, 2005).", "Consistent with this notion, the basic evaluation of another sample, which was recruited in the same manner, suggested that the sample that was recruited in the same manner as that in the present study contained few ADHD subjects (see Supplemental Methods and Supplemental Results).", "Nonetheless, the exact prevalence of ADHD in the present sample was not known; if available, this information may have provided some additional information.", "Another limitation was common to our previous studies and other studies that use college cohorts (Jung et al., 2010b; Song et al., 2008; Takeuchi et al., 2011c; Takeuchi et al., 2010a, b; Wei et al., 2013).", "As previously described (Takeuchi et al., 2012a), we used young healthy subjects with high educational backgrounds.", "Limited sampling of the full range of intellectual abilities is a common hazard when sampling from college cohorts (Jung et al., 2010b).", "Many possibly unique sample characteristics of college cohorts may bias some findings.", "Higher educational background is also known to be associated with a lower ADHD prevalence (Faraone and Biederman, 2005), which should have further lowered the risk of ADHD in the population.", "Whether our findings would also hold across the full range of population samples and a normal distribution must be determined using larger and more representative samples (Takeuchi et al., 2012a).", "Focusing on highly intellectual subjects with a low risk of ADHD was certainly warranted for the purpose of this study, given the association between higher intelligence and higher creativity measured by divergent thinking among subjects with normal and inferior intelligence (Sternberg, 2005) and the assumption of this study that the risk allele of ADHD should lead to higher performance of creativity measures as described in the Introduction section.", "Finally, as can be predicted from the significance level of some of the significant results, when the present sample was split into two groups randomly, the present results of the effects of the polymorphism will not become significant in both split samples, even at an uncorrected level in psychological results.", "The relatively large sample is a strength of this study, and the results were congruent with the theoretical background as well as the previous meta-analyses of the clinical findings involving this polymorphism.", "Nonetheless, the general effects of the common polymorphism are still weak, and these results and the significance level suggest that attempts should be made to replicate the results.", "This study revealed the psychological and neural effects of the 5-repeat allele of DRD4, which is robustly associated with the risk of ADHD.", "The present study newly suggested that the 5-repeat allele led to the widespread disruption of the neural networks and inefficient attentional reallocation, which was reflected in a reduced TID in the areas deactivated during cognitive tasks.", "These phenomena may lead to an increased risk of ADHD.", "Furthermore, the increased originality of creative thinking observed in the 5-repeat allele carriers may support the notion of the side of adaptivity of the widespread risk allele of psychiatric diseases (Horrobin, 2002).", "The following are the supplementary data related to this article.", "Positive correlation between originality/fluency and brain activity during the sensorimotor task.", "(a) Significant positive correlation between the originality dimension score/fluency dimension score of the S-A creativity task and brain activity during the 0-back sensorimotor task.", "Significant results are superimposed on the anatomical rendering.", "Results are shown with a threshold of P < 0.05, corrected for multiple comparisons at the cluster level with the voxel-level cluster-determining threshold of P < 0.05, corrected for voxel-level FDR.", "(b) The result of the positive correlation between the originality dimension score/fluency dimension score of the S-A creativity task and brain activity during the 0-back sensorimotor task.", "Results are shown with a threshold of P < 0.05, corrected for voxel-level FDR (but without the extent threshold).", "Findings were overlaid on a single-subject T1 SPM5 image.", "The red color represents the T-score.", "Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.neuroimage.2015.01.053.", "Seven hundred and seventy-eight healthy, right-handed individuals (433 men and 344 women; 20.7 ± 1.9 years) participated in this study as part of an ongoing project investigating associations among brain imaging, cognitive functions, aging, genetics, and daily habits.", "Data derived from the subjects in this study are to be used in other studies irrelevant to the theme of this study.", "Some of the subjects who participated in this study also became subjects of intervention studies (psychological and imaging data recorded before the intervention were used in this study) (Takeuchi et al., 2013a).", "Psychological tests and MRI scans not described in this study were performed together with those described in this study.", "All subjects were university, college, or postgraduate students or subjects who had graduated from these institutions within 1 year before the experiment, and had normal vision.", "They were recruited using advertisements on bulletin boards at Tohoku University or via email introducing the study.", "These advertisements and emails specified the unacceptable conditions in individuals with regard to participation in the study such as handedness (left-hand), the existence of metal in and around the body, claustrophobia, the use of certain drugs, history of head trauma, psychiatric and neurological diseases, and previous participation in related experiments.", "We provided a routine laboratory questionnaire to all potential experimental subjects for the assessment of psychiatric illnesses and recent drug use history.", "In this questionnaire, each subject answered questions related to their current or previous experiences of any list of illnesses and listed drugs that they had recently taken.", "Handedness was evaluated using the Edinburgh Handedness Inventory (Oldfield, 1971).", "These assessments were performed when the (potential) subjects first came to the laboratory, and if they did not meet the aforementioned criteria, they were not included in the study.", "The family history of psychiatric disorders was not assessed because of its sensitive nature.", "Written informed consent was obtained from each subject in accordance with the Declaration of Helsinki (1991).", "This study was approved by the Ethics Committee of Tohoku University.", "Genotyping of the DRD4 gene was performed as described in previous studies (Pérez-Edgar et al., 2014).", "Genomic DNA was prepared from saliva samples that were collected in the laboratory using the Oragene DNA kit (DNA Genotek, Ottawa, Ontario, Canada).", "For details of following procedures, see Supplemental Methods.", "The genotype distributions for the full samples of 765 Asian subjects who were successfully genotyped were: 2-repeat allele/2-repeat allele (N = 12, 1.7%), 2-repeat allele/3-repeat allele (N = 2, 0.3%), 2-repeat allele/4-repeat allele (N = 128, 16.7%), 2-repeat allele/5-repeat allele (N = 6, 0.8%), 3-repeat allele/4-repeat allele (N = 5, 0.7%), 4-repeat allele/4-repeat allele (N = 542, 70.8%), 4-repeat allele/5-repeat allele (N = 66, 8.6%), 4-repeat allele/6-repeat allele (N = 1, 0.1%), and 5-repeat allele/5-repeat allele (N = 3, 0.4%).", "This distribution was in line with the Hardy–Weinberg equilibrium (P > 0.05).", "Among all the subjects who participated in this experiment, 13 subjects failed to come for saliva sample collection, declined to offer a sample, or their sample could not be correctly processed.", "In the final sample, participants were classified as 5-repeat carriers (5R +; n = 75; 72 heterozygotes, three homozygotes, 34 females, aged 20.9 ± 1.8 years) or noncarriers (5R −; n = 690, 306 females, aged 20.7 ± 1.8 years).", "The DRD4 genotype groups did not differ in age or sex (P > 0.10).", "The lack or scarcity of 7-repeat alleles is common in Asian or Japanese populations (Aoki et al., 2013; Li et al., 2006).", "Subsequently, neuropsychological tests of basic cognitive performance and questionnaires for psychological variables relevant to ADHD or the DRD4 gene were administered.", "[A] Raven's advanced progressive matrix test (Raven, 1998), a non-verbal reasoning task. [", "B] A (computerized) digit span task, which is a working memory task (for details, see Takeuchi et al., 2011b). [", "C] Tanaka B-type intelligence test (Tanaka et al., 2003), a non-verbal mass intelligence test used for third-year junior high school and older examinees, does not include story problems but uses figures, single numbers, and letters as stimuli.", "In all subtests, the subjects have to solve as many problems as possible before a certain time (a few minutes).", "For the details of these subtests, see Takeuchi et al. (2013a). [", "D] The Stroop task (Hakoda's version) (Hakoda and Sasaki, 1990; Takeuchi et al., 2012b), which measures response inhibition and impulsivity.", "Hakoda's version is a matching-type Stroop task requiring subjects to check whether their chosen answers are correct, unlike the traditional oral naming Stroop task.", "The test consists of two control tasks (Word–Color task and Color–Word task), a Stroop task, and a reverse-Stroop task.", "In this study, we used the Word–Color and Color–Word tasks as measures of simple PS and the Stroop and reverse-Stroop tasks as measures of inhibition.", "In each task, the subjects have to complete as many of the exercises as possible in 1 min.", "For the details of these subtests, see Takeuchi et al. (2014b). [", "E] Simple arithmetic tasks, similar to the ones constructed by Grabner et al. (2007), measured multiplication performance consisting of two forms of one-digit times one-digit multiplication problems (a simple arithmetic task with numbers between 2 and 9).", "The two forms of each task are the same, but the numbers used in the problems are ordered differently.", "Each form of the simple tasks have to be completed in 30 s. Average performance was used as a performance. [", "F] The SA creativity test (Society_For_Creative_Minds, 1969), which measures creativity through divergent thinking, involves three types of tasks (generate unique ways of using typical objects, imagine desirable functions for ordinary objects, and imagine the consequences of unimaginable things happening).", "The SA test scores the four 4 dimensions of the creative process (fluency, originality, elaboration, and flexibility) (Takeuchi et al., 2010a).", "(a) Fluency—Fluency is measured by the number of relevant responses to questions and is related to the ability to produce and consider many alternatives.", "Fluency scores are determined by the total number of questions answered after excluding inappropriate responses or responses that are difficult to understand.", "(b) Flexibility—Flexibility is the ability to produce responses from a wide perspective.", "Flexibility scores are determined by the sum of the (total) number of category types that responses are assigned based on a criteria table or an almost equivalent judgment.", "(c) Originality—Originality is the ability to produce ideas that differ from those of others.", "Originality scoring is based on the sum of idea categories that are weighted based on a criteria table or an almost equivalent judgment.", "(d) Elaboration—Elaboration is the ability to produce detailed ideas (Society_For_Creative_Minds, 1969).", "Elaboration scores are determined by the sum of responses that are weighted based on a criteria table or an almost equivalent judgment.", "Please refer to our previous studies (Takeuchi et al., 2010a, b) for more extensive details, including those on the psychometric properties of this test, sample answers to the questionnaire, and the manner in which the tests were scored. [", "G] The cognitive reflectivity–impulsiveness questionnaire (Takigiku and Sakamoto, 1991) was used to assess individual differences in reflectivity and impulsivity.", "This is a self-reported questionnaire and has been previously used for measuring individual reflectivity and impulsivity (Sasaki and Kanachi, 2005). [", "H] The External-Preoccupation Scale (Sakamoto, 1998) was used to measure the maintenance of external focus on a specific object.", "Data for this scale were collected only from a subset of the subjects (678 successfully genotyped subjects). [", "I] The Novelty Seeking Scale, Japanese version (Kijima et al., 1996), of the Temperament Character Inventory (Cloninger et al., 1993) was used to measure novelty seeking tendency.", "fMRI was used to map brain activity during typical cognitive tasks.", "The n-back task, a typical task for fMRI studies with conditions of 0-back (simple cognitive processes) and 2-back (working memory).", "The n-back task was performed during fMRI scanning, as described in our previous study (Takeuchi et al., 2011a,b, 2014a).", "We have provided the same information for the readers' sake.", "Participants received instructions and practiced the tasks before entering the scanner.", "During scanning, they viewed stimuli on a screen via a mirror mounted on a head coil.", "Visual stimuli were presented using Presentation software (Neurobehavioral Systems, Inc., Albany, CA, USA).", "A fiber-optic, light-sensitive key press interface with a button box was used to record participants' behavior.", "We used a simple block design and the n-back WM task (Callicott et al., 1999) to tap brain activities during the WM task.", "There were two conditions (0- and 2-back).", "Each condition had six blocks, and all n-back tasks were performed in one session.", "The subjects were instructed to recall stimuli [visually presented four types of Japanese letters of vowels] seen “n” times previously (e.g., two letters shown previously for the 2-back task and the currently presented letter for the 0-back task).", "Two buttons were used during the 0-back task, and the subjects were asked to push the first button when the target stimuli were presented and the second button when the other stimuli were presented.", "During the 2-back task, the subjects were asked to push the first button when the currently presented stimuli and the stimuli presented two letters previously were the same, and to push the second button when the currently presented stimuli and the stimuli presented two letters previously were different.", "Our version of the n-back task was designed to require individuals to push buttons continuously during the task period.", "The task level of the memory load was found to be above the stimuli for 2 s before the task started (cue phase) and remained unchanged during the task period.", "Each letter was presented for 0.5 s, and a fixation cross was presented for 1.5 s between each item.", "Each block consisted of 10 stimuli.", "Thus, each block lasted for 20 s.", "A baseline fixation cross was presented for 13 s between the task and the presentation of the next condition's task level (2 s).", "Thus, the rest period lasted for 15 s.", "There were six blocks for each 2- and 0-back condition.", "MRI data acquisition was conducted using a 3 T Philips Achieva scanner.", "Forty-two transaxial gradient-echo images (echo time = 30 ms, flip angle = 90°, slice thickness = 3 mm, FOV = 192 mm, matrix = 64 × 64) covering the entire brain were acquired at a repetition time of 2.5 s using an echo-planar sequence.", "For the n-back session, 174 functional volumes were obtained.", "Diffusion-weighted data were acquired using a spin-echo EPI sequence (TR = 10,293 ms, TE = 55 ms, FOV = 22.4 cm, 2 × 2 × 2 mm3 voxels, 60 slices, SENSE reduction factor = 2, number of acquisitions = 1).", "The diffusion weighting was isotropically distributed along 32 directions (b value = 1,000 s/mm2).", "Additionally, using a spin-echo EPI sequence (TR = 10,293 ms, TE = 55 ms, FOV = 22.4 cm, 2 × 2 × 2 mm3 voxels, 60 slices), 3 images with no diffusion weighting (b value = 0 s/mm2) (b = 0 images) were acquired.", "From the collected images, FA and MD maps were calculated (Takeuchi et al., 2011c).", "Preprocessing and analysis of functional activation data were performed using SPM8 implemented in Matlab.", "Here we describe the summary versions; however, the full details and methodological considerations have been provided in Supplemental Methods.", "Before analysis, BOLD images were re-aligned and re-sliced to the mean image of the BOLD images, which was then realigned to mean images of b = 0 images, as described previously (Takeuchi et al., 2011b).", "Because the mean b = 0 image was aligned with the FA image and MD map, the BOLD image, b = 0 image, FA image, and MD map were all aligned.", "Subsequently, using a previously validated two-step new segmentation algorithm of diffusion images and the previously validated diffeomorphic anatomical registration through exponentiated lie algebra (DARTEL)-based registration process (Takeuchi et al., 2013c), all images, including gray matter segment [regional gray matter density (rGMD) map], white matter segment [regional white matter density (rWMD) map], cerebrospinal fluid (CSF) segments [regional CSF density (rCSFD) map] of diffusion images, were normalized.", "The voxel size of normalized FA images and MD images and segmented images, was 1.5 × 1.5 × 1.5 mm3.", "The voxel size of normalized BOLD images was 3 × 3 × 3 mm3.", "Normalized rGMD maps were smoothed (3 mm full-width half-maximum) and taken to the second-level analyses of functional activities.", "Next, we created average images of normalized rGMD and rWMD images from the normalized rGMD and rWMD images from the subset of the entire sample (63 subjects) (Takeuchi et al., 2013c).", "Subsequently, for the analyses of MD images from the normalized images of the (a) MD, (b) rGMD, and (c) rCSFD maps, we created images where areas that were not strongly likely to be gray or white matter in our averaged normalized rGMD and rWMD images (defined by “gray matter tissue probability + white matter tissue probability < 0.99”) were removed (to exclude the strong effects of CSF on MD throughout analyses).", "These images were then smoothed (6 mm full-width half-maximum) and carried through to the second-level analyses of MD.", "Next, from the average image of normalized WM segmentation images from the 63 subjects mentioned above and from the created mask image consisting of voxels with a WM signal intensity > 0.99.", "We then applied this mask image to the normalized FA image; therefore, we retained only areas that are highly likely to be white matter from the normalized FA images.", "These images were smoothed (3 mm full-width half-maximum) and carried through to the second-level analyses of FA.", "Individual-level statistical analyses were performed using a general linear model (GLM).", "A design matrix was fitted to each participant with one regressor in each task condition (0- or 2-back in the n-back task) using a standard hemodynamic response function (HRF).", "The cue phases of the n-back task were modeled in the same manner, but were not analyzed further.", "Six parameters obtained by rigid body correction of head motion were regressed out by putting these variances into the regressor.", "The design matrix weighted each raw image according to its overall variability to reduce the impact of movement artifacts (Diedrichsen and Shadmehr, 2005).", "We removed low-frequency fluctuations with a high-pass filter, using a cut-off value of 128 s.", "After estimation, beta images were smoothed (3 mm full-width half-maximum) and taken to the second level analyses.", "For the reasons why we used lower smoothing values in functional activation analyses and used 2-back > rest and 0-back > rest contrasts instead of 2-back > 0-back contrast in this study, see Supplemental Methods.", "Behavioral data were analyzed using SPSS 18.0 (SPSS Inc., Chicago, IL).", "Differences in the scores for the cognitive measures between the carriers and non-carriers of the 5-repeat allele of the DRD4 gene were analyzed using analysis of covariance (ANCOVA).", "Additional covariates for each analysis were age and sex.", "In psychological analyses, results with a threshold of P < 0.05, corrected for false discovery rate (FDR) using the graphically sharpened method (Benjamini and Hochberg, 2000), were considered statistically significant.", "The correction for multiple comparisons using this method was applied to the results of abovementioned 16 ANCOVAs.", "The 16 dependent variables are psychological measures that were described in the Psychological measures subsection and are listed in Table 1 (namely RAPM, Digit span, Tanaka-B type intelligence test, Word–Color task, Reverse Stroop task, Color–Word task, Stroop task, Simple arithmetic, Total SA creativity test score, SA creativity test–fluency score, SA creativity test–flexibility, SA creativity test–originality, SA creativity test–elaboration, cognitive reflectivity–impulsivity, External–Preoccupation, and Novelty seeking).", "They are all hypothesized on the basis of the study hypothesis, previous studies of polymorphisms of the DRD4 gene in the Introduction section (Durston et al., 2005; Szekely et al., 2011), and clinical conditions of ADHD (Barkley, 1997).", "In particular, carriers of the risk allele of ADHD were thought to have higher divergent thinking measures (the study hypothesis in the Introduction section); lower performance in intelligence, processing speed, and working memory; and a higher tendency for attention diversion toward external stimuli, disinhibition, and novelty seeking.", "FDR-based methods have been shown to be more powerful and sensitive than other available approaches to multiple statistical testing (See Benjamini and Hochberg, 1995 for a full discussion; Genovese et al., 2002).", "Comparing carriers and noncarriers of risk alleles and assuming that the risk is inferred even in heterozygotes of the risk alleles is correct for this polymorphism.", "This is because the 5-repeat allele is not so frequent in any population (0%–8%) (Li et al., 2006); thus, in any studies, homozygotes of 5-repeat alleles are rare (Comings et al., 1999; Qian et al., 2003).", "However, through the accumulation of these studies with the lack of homozygotes of 5-repeat alleles, meta-analysis had provided convincing evidence that 5-repeat alleles increase the risk of ADHD (Li et al., 2006).", "Further the mechanisms of how DRD4 gene polymorphisms work in a different manner are shown to be through the relative efficiency of how efficiently agonists bind with the translated receptors (Asghari et al., 1995).", "In this context, the possible mechanism that two risk alleles are required to express phenotypes is ruled out because dopamine combines with the receptors of risk alleles and even a single risk allele's receptor can still function but the receptors of each allele compete with the receptors of other alleles.", "In group-level imaging analyses, we tested for group-wise differences in functional activity during the attention-demanding and simple sensorimotor tasks, MD and FA, across the brain.", "We performed voxel-wise ANCOVAs to test group differences in functional activity analyses and MD analyses.", "Biological Parametrical Mapping (Casanova et al., 2007) implemented in SPM5 allowed to use these voxel-wise ANCOVAs by including images representing regional values as covariates.", "The analyses were performed using this software.", "In the analyses of MD, the dependent variables were as follows: the existence of the 5-repeat allele, the signal intensities of preprocessed (masked) rGMD and rCSFD images at each voxel, age, sex, and total intracranial volume.", "The analyses were limited to the gray + white matter mask that was created above.", "rGMD and rCSFD images were included as covariates to exclude the effects of the extent of these tissues on MD and remove the possibility that the extent of tissue instead of MD itself impacted the obtained results; however, we did not include white matter density as covariates because tissues were gray matter, white matter, or CSF in the areas relevant to the analysis.", "In the analyses of functional activity, the dependent variables were as follows: the existence of the 5-repeat allele, signal intensities of preprocessed (nonmasked) rGMD images at each voxel, accuracy and RT of each task (accuracy and RT of 0-back task for 0-back task's analyses, and accuracy and RT of 2-back task for 2-back analyses), volume-level mean frame-wise displacement during the scan for the n-back task (Power et al., 2012), and total intracranial volume.", "For the analyses of FA, we performed normal whole-brain ANCOVA with correction for the effects of age, sex, and total intracranial volume.", "Analyses were limited to the white matter mask that was created above.", "There were no significant group (based on the existence of the 5-repeat allele) differences in these variables, which were used as covariates (P > 0.1, ANOVA).", "The total intracranial volume was included as a covariate, considering its effects on DTI measures (Takao et al., 2011).", "Multiple comparison correction was performed using the false discovery rate (FDR) approach (Genovese et al., 2002), and areas that surpassed the extent threshold (Friston et al., 1996) based on this cluster-determining threshold were reported.", "Among the subjects who were successfully genotyped for the DRD4 gene, diffusion-weighting data were successfully obtained from 756 subjects and functional activation data (including behavioral parameters) were successfully obtained from 738 subjects using the above-mentioned parameters, and were analyzed in the group-level analyses.", "Analyses of covariance corrected for the effects of age and sex revealed that 5-repeat allele carriers had a significantly lower performance in the Word–Color and simple arithmetic tasks, lower score of cognitive reflectivity (vs. impulsivity), lower score of external preoccupation, and higher score on the originality dimension of the S-A creativity task.", "For all results and statistical values, see Table 1.", "Interestingly, 5-repeat allele carriers had significantly higher scores on the originality test; this was the only superior result for this group among performance-type cognitive tests.", "Visual inspection of the histogram of this originality score shows a slight deviation from the normal distribution, and with this sample size, the Kolmogorov–Smirnov test for non-normality of distribution showed significant results (P < 0.05).", "However, the nonparametric test of the difference in originality scores between the two groups showed a similar statistical value (P = 0.024); thus, the non-normality of the test fails to explain the difference.", "To reveal the nature of this result, we tested the association between the score on the originality dimension of the SA creativity task and other measures after correcting for the effects of age and sex through multiple regression analyses.", "Results revealed that the originality dimension of the SA creativity task showed a significant positive correlation with the total score and scores on other dimensions of the SA creativity task and other cognitive tests requiring speed, e.g., Tanaka-B type intelligence test, Word–Color task, Reverse Stroop task, and simple arithmetic task (P < 0.05), but not with others.", "However, these significant correlations are all positive correlations and cannot explain the specific positive association between the 5-repeat allele and the originality dimension, given that the 5-repeat allele carriers tended to show inferior performance in these tasks.", "In addition, to reveal the specific association of the originality dimension with other cognitive variables, we calculated the originality dimension score/fluency dimension score (originality of answers after the number of responses is adjusted), which has sometimes been used (Eisenman, 1969), and after correcting for the effects of age and sex, we investigated its association with other cognitive variables through multiple regression analyses.", "However, none of the cognitive variables used in this study showed significant associations with this value (P > 0.05).", "A whole-brain ANCOVA corrected for the effects of age, sex, and total intracranial volume revealed an absence of significant effects of the DRD4 5-repeat allele on FA.", "Analyses of covariance corrected for the effects of age, sex, and total intracranial volume revealed that 5-repeat allele carriers had a significantly higher MD in: (a) the extensive cluster that spread across gray and white matter areas located mainly in and close to the frontal, parietal, and cingulate cortices; (b) anatomical cluster that spread across gray and white matter areas located mainly in and close to the amygdala, ventral tegmental area, globus pallidus, and thalamus; (c) areas located in the brainstem; and (d) anatomical cluster that spread mainly around the fusiform gyrus (Fig. 1).", "For the statistical values, see Table 2.", "The subjects performed the task properly in terms of accuracy because the accuracies in both groups were higher than 99% in both the 0-back and 2-back tasks (Table 3).", "This means for subjects in both the groups, 2-back task conditions were not over the capacity of working memory and we did not have to take into account the nonlinear brain activation changes, which appear as the cognitive load becomes higher and after the working memory capacity is exceeded (Callicott et al., 1999).", "The reaction time and movements were also comparable between two groups (Table 3).", "Because these variables were not for validated measures to measure abilities, we did not include them in analyses to evaluate the effects of the DRD4–5-repeat allele, these variables did not exert effects on the DRD4–5-repeat allele after correction for age and sex (P > 0.1, ANCOVA).", "These results suggest that fMRI activation results are not explained by these variables.", "ANCOVAs corrected for confounding factors revealed that 5-repeat allele carriers had a significantly higher activity during the 0-back sensorimotor task in the areas of the precuneus and in the posterior cingulate cortex and anatomical cluster that spread across the thalamus, ventral tegmental area, and hippocampus (Fig. 2a, Table 4).", "Furthermore, ANCOVAs corrected for confounding factors revealed that 5-repeat allele carriers had a significantly higher activity (beta estimates) during the 2-back working memory task in the area of the precuneus (Fig. 2c, Table 4).", "Similar tendencies were observed in the posterior cingulate cortex and areas located around the hippocampus and midbrain, where significant differences were observed in the analysis of the 0-back task.", "The extensive regions located around these areas showed deactivation during the wide range of externally directed attention-demanding cognitive tasks (Takeuchi et al., 2011c, See also Figs. 2b, d).", "This suggests that “higher activity” (beta estimates) in the extensive regions located around the precuneus in 5-repeat allele carriers actually corresponds to the reduction of TID or reduction of deactivated areas around these areas during the 0-back and 2-back tasks in 5-repeat allele carriers.", "Furthermore, ANCOVAs revealed that 5-repeat allele carriers had weak tendencies of lower activity during the 2-back working memory task (but not during the 0-back task) in the areas of the caudal and rostral parts of the left dorsolateral prefrontal cortex (regions of a priori hypothesis; x, y, z = − 21, 24, 60, respectively, t = 3.33, P < 0.001, uncorrected; x, y, z = − 30, 39, 6, respectively, t = 3.27, P < 0.001)." ]

[ "Effects of 5-repeat (5R) allele of dopamine D4 receptor (DRD4) gene were examined.", "5R carrier was associated with higher originality in the creativity task.", "5R carrier was associated with lower mean diffusivity in the wide-spread areas.", "5R carrier was associated with lower task-induced deactivation in the precuneus." ]

[ "AGRI" ]

[ "Antibacterial activity", "Antioxidants", "In vitro regeneration", "Organic elicitors", "Phenolic compounds" ]

[ "1110" ]

[ "Coleonema pulchellum Williams (Rutaceae) is an evergreen, erect and dense shrub, which occurs from the western to the eastern Cape in South Africa.", "It is an ideal aromatic garden plant which remains beautiful with pink flowers throughout the year.", "The plant contains phenylpropenes, phenylpropanoids and terpenoids which exhibit antimicrobial properties (Brader et al., 1997).", "In vitro plant cell cultures have been carried out in the recent past for biosynthesis of compounds with pharmaceutical properties (Palacio et al., 2008).", "Plant tissue culture techniques offer a viable tool for in vitro regeneration in the conservation and commercial propagation of medicinal plants (Pattnaik and Chand, 1996).", "The capacity for plant cell, tissue and organ cultures to produce and accumulate many of the same valuable chemical compounds as the parent plant in nature has been recognized since the inception of in vitro technology.", "At the present time, many valuable compounds are produced by in vitro plant cell cultures (Baskaran et al., 2012).", "However, in other cases production requires more differentiated organ cultures such as shoots and roots (Karuppusamy, 2009).", "No studies on in vitro propagation of C. pulchellum have been reported.", "The aim of this study was to investigate the potential of C. pulchellum in vitro cultures to synthesize phenolic compounds and to evaluate the changes in phenolic content due to plant growth regulators and organic elicitors used in the propagation process.", "The antioxidant and antibacterial activities were also evaluated in in vitro regenerated plants in comparison with parental (wild) plants.", "Folin & Ciocalteu phenol reagent, gallic acid (3,4,5-trihydroxibenzoic acid), N6-benzyladenine (BA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), thidiazuron (TDZ), casein hydrolysate (CH), glutamine (GM), mebendazole (MBZ), haemoglobin (HB), catechin hydrate, 2,2-diphenyl-1-picryl hydrazyl (DPPH), Mueller-Hinton (MH) broth, neomycin, and p-iodonitrotetrazolium chloride (INT) were obtained from Sigma-Aldrich Co. (Steinheim, Germany).", "Ferric ammonium sulphate, sodium nitrite, sodium hydroxide, aluminium chloride and sodium hydrogen carbonate (BDH Chemicals Ltd., Poole, England); agar (bacteriological) (Oxoid Ltd., Basingstoke, England); dimethyl sulfoxide (DMSO), acetone and methanol (Merck KGaA, Darmstadt, Germany) were used.", "All chemicals and standards used in the assays were of analytical grade.", "Young shoot-tip explants (approximately 2 cm) of mature C. pulchellum plants were collected from the Botanical Garden, University of KwaZulu-Natal, Pietermaritzburg, South Africa.", "The explants were washed under running tap water for 10 min to remove loose dirt, disinfected with 4% (v/v) previcur for 2 min and then decontaminated in a 3.5% sodium hypochlorite solution for 15 min.", "The explants were then washed five times with sterile distilled water.", "Aseptic shoot-tip explants (10 mm in length) were cultured on MS medium (Murashige and Skoog, 1962) supplemented with 30 g l− 1 sucrose, 8 g l− 1 agar and different plant growth regulators (PGRs) and organic elicitors (OEs) alone or in combination for high-frequency rapid shoot multiplication.", "The concentration and combinations of PGRs are indicated in Tables 1 and 2.", "The pH of the medium was adjusted to 5.8 with 0.1 N NaOH and/or 0.1 N HCl before gelling with 8 g l− 1 agar and autoclaved at 121 °C for 20 min.", "The cultures were maintained at 25 ± 2 °C under a 16 h photoperiod and PPF of 40 μmol m− 2 s− 1 provided by cool white fluorescent light (Osram L 58W/640, Germany).", "Proliferating shoots (at least 3.0 cm in length) were obtained from shoot-tip explants on MS medium containing 4.4 μM BA, and transferred to half- and full-strength MS media supplemented with different concentrations of auxins (IAA, IBA and NAA) and 8 g l− 1 agar.", "In addition, half-strength MS medium supplemented with 5.4 μM NAA, 8 g l− 1 agar and different concentrations (10–40 g l− 1) of sucrose was also tested.", "The optimized root induction medium (half-strength MS basal medium plus 5.4 μM NAA, 30 g l− 1 sucrose and 8 g l− 1 agar) was used in all treated shoots for root induction.", "Media devoid of PGRs were used as controls.", "The pH of the media was adjusted to 5.8 with 0.1 N NaOH and/or 0.1 N HCl prior to addition of agar and autoclaving at 121 °C for 20 min.", "All cultures were maintained at 25 ± 2 °C under cool-white fluorescent light at an intensity of 40 μmol m− 2 s− 1 with a 16 h photoperiod.", "All rooted shoots were removed gently from the rooting medium after 8 weeks, and transferred to terracotta pots (95 × 120 mm, 500 ml) containing a 1:1 (v/v) vermiculite:sand mixture and irrigated with tap water every second day.", "These plantlets were maintained in the greenhouse (25 ± 2 °C under natural photoperiod conditions and a midday PPF of 950 ± 50 μmol m− 2 s− 1) for acclimatization.", "Plant survival (%) was recorded after 10 weeks.", "Ten-week-old in vitro-grown shoots, one-year-old ex vitro-grown plants (grown on MS + 4.5 μM TDZ) and parental plant tissues (leaves, stem and roots) of C. pulchellum were oven dried at 50 °C to constant weight.", "The dried materials were ground into fine powders using a grinder (IKA®, USA).", "The finely ground samples (0.1 g) were extracted with 10 ml 50% methanol in a sonication bath (Branson model 5210, Branson Ultrasonics B.V., Soest, Netherlands) for 20 min on ice.", "The methanolic extracts were then filtered under vacuum through Whatman No. 1 filter paper and the filtrates were immediately used for the determination of total phenolics and flavonoids.", "Dried and finely ground plant extracts for antibacterial and antioxidant assays were extracted in a sonication bath on ice for 1 h (1.0 g extract in 40 ml of 70% acetone), and concentrated in a rotary vacuum evaporator (Büchi, Switzerland) at 30 °C.", "The concentrated extracts were then dried over a stream of cold air at room temperature, re-dissolved in 50% DMSO to known concentrations and immediately used for the determination of antibacterial and DPPH radical scavenging antioxidant activities using appropriate solvent controls.", "The Folin & Ciocalteu assay (Singleton and Rossi, 1965) was used for the determination of total phenolic compounds.", "As control a blank that contained 50% aqueous methanol was used instead of sample extracts.", "A standard curve of gallic acid was used to quantify the total phenolic content in the extracts.", "Total phenolic compounds were expressed in mg gallic acid equivalents (GAE) per g dry weight (DW).", "The aluminium chloride (AlCl3) method was used to quantify total flavonoid content (Zhishen et al., 1999).", "Suitable aliquots of catechin were used to derive a standard calibration curve.", "Total flavonoid content was expressed in mg catechin equivalents (CE) per g DW.", "The minimum inhibition concentration (MIC) of the extracts was evaluated using the serial micro-dilution assay (Eloff, 1998).", "Four bacterial strains Gram-positive (Enterococcus faecalis ATCC 19433 and Staphylococcus aureus ATCC 12600) and Gram-negative (Escherichia coli ATCC 11775 and Pseudomonas aeruginosa ATCC 10145) were used.", "Overnight bacterial cultures were diluted with sterile Mueller-Hinton (MH) broth to a final inoculum concentration of 106 colony forming units (CFU)/ml.", "Plant extracts were re-suspended in 50% DMSO to a concentration of 25 mg/ml.", "A positive control, neomycin (100 μg/ml) was used against each bacterial strain.", "Sterile distilled water, 50% DMSO, bacteria-free MH broth, and each bacterial strain, with no extract added completed the set of controls.", "The assay was done in triplicate.", "INT (0.2 mg/ml) dissolved in distilled water was used as the indicator of bacterial growth after incubation at 37 °C for 1 h.", "Data were collected after 10 weeks of culture for shoot multiplication and 8 weeks for rooting experiments.", "All PTC experiments were repeated three times with 50 explants per treatment.", "The data were subjected to one-way analysis of variance (ANOVA) using SPSS version 18.0 for Windows (Chicago, USA).", "Significantly different means were separated using Duncan's multiple range test (p = 0.05).", "Shoot-tip explants of C. pulchellum were cultured on MS medium with different cytokinins [benzyladenine (BA), meta-topolin (mT) and thidiazuron (TDZ)] at various concentrations (4.2–22.7 μM) to evaluate their potency on shoot multiplication.", "Different morphogenic characteristics were noticed in these in vitro cultures (Table 1).", "The shoot regeneration rate varied significantly between the control and PGR-containing treatments after 10 weeks of culture (Table 1).", "TDZ (13.6 μM) produced significantly higher shoot numbers (37.0 shoots per explant) after 10 weeks of culture (Table 1; Fig. 1A).", "However, these shoots exhibited shoot-tip necrosis symptoms (Table 1).", "On the other hand, BA, mT and lower concentrations of TDZ (4.5 μM) produced normal shoots (Table 1; Fig. 1B).", "The present study indicates that shoot-tip necrosis, a physiological disorder commonly observed in in vitro cultures affecting a wide range of plants, depends on the type and concentration of cytokinin in the culture medium.", "It is a phenomenon whereby the apical shoot initially becomes brown and later dies (Lakshmi and Raghava, 1993; Bairu et al., 2009).", "Benzyladenine was successfully used to control shoot-tip necrosis in chestnut cultures (Piagnani et al., 1996).", "Excessive yellowing of leaves and necrosis was observed at higher concentrations of 2-isopentenyladenine (2iP), kinetin or TDZ in Mexican redbud plants (Mackay et al., 1995).", "In the present study, various combinations and concentrations of PGRs were used to develop high-frequency rapid normal shoot multiplication from shoot-tip explants.", "A combination of TDZ (13.6 μM) and IAA (2.9 μM) significantly increased normal shoot production compared to other treatments (Table 1; Fig. 1C), indicating cytokinin–auxin synergistic effects in shoot proliferation.", "These cultures induced different types of basal callus after 10 weeks of culture (Table 1).", "A low auxin concentration in combination with high cytokinin concentration promoted shoot proliferation in other species of Rutaceae (Bohidar et al., 2008; Vennel Raj and Basavaraju, 2012).", "The importance of organic elicitors (OEs: casein hydrolysate, haemoglobin, glutamine and mebendazole) alone and in combination with cytokinin (TDZ and BA) was examined for rapid shoot proliferation (Table 2).", "Organic elicitors showed significant rapid shoot proliferation in C. pulchellum.", "The promotional effects of elicitors on shoot regeneration have been reported in other plant species (Baskaran and Jayabalan, 2005; Baskaran and Van Staden, 2011).", "In this study, GM and HB treatments increased the number of shoots after 10 weeks of culture (Table 2; Fig. 1D).", "The HB treatment produced normal shoots as well as green compact basal callus (GCBC) after 10 weeks of culture.", "Shoots produced in the CH treatment exhibited shoot-tip necrosis symptoms, root growth and green friable basal callus (Table 2; Fig. 1E).", "A combination of TDZ and GM significantly increased shoot number (Table 2), but the shoots were also necrotic after 10 weeks of culture (Table 2; Fig. 1F).", "Combinations of CH, MBZ or HB with TDZ produced hyperhydric shoots as well as green compact basal callus (Table 2; Fig. 1G).", "Hyperhydricity is characterized by a glassy or swollen appearance to the tissue, usually resulting in reduced multiplication rates, poor quality shoots and tissue necrosis (Ziv, 1991).", "Their organs are translucent, in some cases less green and easily breakable (Ziv, 1991).", "In this study, combinations of HB or GM and BA produced normal shoots with a significantly higher shoot length (Table 2; Fig. 1H).", "These organic elicitors can be considered to mimic PGRs in the culture medium.", "Hyperhydricity has been reported to be influenced by cytokinin type and concentration (Kadota and Niimi, 2003), which is in agreement with our present findings.", "The current investigation showed that OE and PGR combinations in the MS medium enhanced adventitious shoot multiplication.", "Excised shoots from MS medium containing 4.4 μM BA were rooted using both half- and full-strength MS media with different types of auxins (Fig. 1I).", "Both half- and full-strength MS media containing 5.4 μM NAA induced higher numbers of roots after 8 weeks of culture (Table 3).", "Root length was significantly higher in full-strength MS medium containing 5.4 μM NAA after 8 weeks of culture (Table 3).", "These cultures produced normal shoots and roots as well as white friable basal callus (Table 3).", "In this study, concentrations of sucrose (10–40 g l− 1) were also tested in half-strength MS medium with 5.4 μM NAA.", "Different rates of root growth were observed after 8 weeks of culture (Table 3).", "The medium containing 30 g l− 1 sucrose was more effective in induction of root growth compared to other sucrose concentrations (Table 3).", "The present study revealed the importance of sucrose concentration on root induction in C. pulchellum.", "A similar trend was also observed in Persian walnut (Vahdati et al., 2004).", "Half-strength MS medium supplemented with 5.4 μM NAA was selected as the standard growth medium for root induction in subsequent experiments.", "All the PGR and OE-treated shoots produced roots with different morphogenic characteristics (Tables 1 and 2).", "Root induction rate varied significantly between the shooting media after 8 weeks of culture (Tables 1 and 2).", "GM-produced shoots were the most amenable to root induction, and produced significantly higher root numbers (12.0 roots per shoot) and root lengths (13.24 cm) after 8 weeks of culture (Table 2; Fig. 1J).", "These cultures induced green roots and white friable basal callus (Table 2).", "The present study indicates that beneficial root growth depends on type as well as concentration of PGRs and OEs in the media (Tables 1 and 2).", "The in vitro-rooted shoots were removed from the culture media after 8 weeks and successfully acclimatized (100%) in a vermiculite:soil mixture (1:1 v/v) in the greenhouse (25 ± 2 °C) under natural photoperiod conditions with a midday PPF of 950 ± 50 μmol m− 2 s− 1 (Fig. 1K).", "The results on the effect of cytokinins and OEs are presented in Table 4.", "Total phenolic and flavonoid contents were higher in the leaves and stems compared to the roots for both acclimatized and natural plants.", "Shoot cultures provide a viable option for the production of secondary metabolites, especially where the compounds are synthesized and/or stored in the aerial plant parts (Amoo et al., 2012).", "Cytokinin type and OEs had a significant effect on total phenolic and flavonoid contents.", "In particular, casein hydrolysate significantly upregulated the levels of total phenolic compounds and flavonoids (Table 4).", "Total phenolic and flavonoid contents were also upregulated in in vitro C. pulchellum cultures treated with BA.", "Different types and concentrations of PGRs have been reported to regulate developmental processes and modify the concentration of secondary metabolites in plants differently (Palacio et al., 2008; Baskaran et al., 2012).", "A combination of GM and BA significantly increased the in vitro accumulation of phenolics and flavonoids, compared to when the compounds were used singly.", "The results show that the concentration of secondary metabolites in in vitro cultures is influenced by the composition of the medium (Abbasi et al., 2010; Baskaran et al., 2012).", "Natural phenolic compounds and flavonoids are widely known for a range of biological activities such as antimicrobial, anti-cancer and cardiovascular regulatory effects (Brader et al., 1997; Palacio et al., 2008; Moyo et al., 2010).", "All the treatments exhibited a dose-dependent increase in DPPH radical scavenging activity (Table 4).", "In vitro treatment using cytokinins and organic elicitors had a significant effect on DPPH radical scavenging activity, in particular BA, TDZ and CH as well as their respective combinations.", "Natural antioxidants play a crucial role in ameliorating the detrimental effects of oxidative stress caused by reactive oxygen species as well as being food additives that inhibit the oxidation of nutrients in foodstuffs.", "There is a strong correlation between disease pathogenesis and oxidative stress levels, hence the use of natural antioxidant compounds is considered an effective therapeutic approach against several diseases (Moyo et al., 2010).", "The potential use of in vitro technology for the synthesis of antioxidant compounds from medicinal plants has been widely reported (Matkowski, 2008; Abbasi et al., 2010; Amoo et al., 2012; Khateeb et al., 2012).", "The antibacterial activity of C. pulchellum extracts is presented in Table 5.", "The tested extracts had better activity against Gram-positive Enterococcus faecalis.", "In vitro extracts derived from mT and CH-treated plants exhibited high antibacterial activity against E. faecalis with MIC values of 0.781 and 1.041 mg/ml, respectively.", "Furthermore, CH-treated C. pulchellum plants had a low MIC (1.56 mg/ml) against the Gram-negative bacteria E. coli.", "Isolated phenylpropenes from C. pulchellum had good antimicrobial activity (Brader et al., 1997).", "The authors attributed the observed antimicrobial activity to the reactivity of the phenolic hydroxyl group (Brader et al., 1997).", "In a similar study, Khateeb et al. (2012) reported the antimicrobial activity of in vitro-derived secondary metabolites of Cichorium pumilum.", "The present study shows that C. pulchellum contains biologically potent therapeutic phytochemicals with high antibacterial and antioxidant activities.", "The present study demonstrated that C. pulchellum shoot-tip explants are a good starting material for in vitro culture establishment.", "A combination of TDZ and IAA produced a significant number of normal shoots while lower TDZ concentrations favourably affected the induction of normal shoots.", "The best rooting was achieved with half-strength MS medium containing 30 g l− 1 sucrose and NAA.", "In vitro and ex vitro-grown C. pulchellum treated with combinations of OEs and PGRs exhibited high antioxidant and antibacterial activities.", "This study showed that in vitro regenerated C. pulchellum plant tissues and organs contain the active phytochemical constituents, which have medicinal properties.", "The present micropropagation system can be recommended for the mass propagation of C. pulchellum plants.", "The acclimatized in vitro-derived plants can be used as an alternative to natural populations of C. pulchellum." ]

[ "In vitro propagation for Coleonema pulchellum is reported for the first time.", "More normal and healthy shoots were obtained with 4.5 μM TDZ.", "In vitro and ex vitro-grown C. pulchellum exhibited high bioactivities.", "The protocol can be applied for mass propagation and plant transformation studies." ]

[ "AGRI", "EART" ]

[ "Alaska", "Autumn phenology", "Boreal forest", "Radiative transfer analysis", "Satellite SOS and EOS", "Time-lapse camera", "Tundra", "Understory vegetation" ]

[ "1111", "1903", "1907" ]

[ "In the Arctic and sub-Arctic regions, including Alaska, warming trends have been accelerating and the increased trend in surface temperature in the region over the past decade is twofold higher than that in the whole northern hemisphere (Bekryaev, Polyakov, & Alexeev, 2010; Hinzman et al., 2013; IPCC, 2013).", "It is of particular interest whether the carbon uptake by terrestrial vegetation increases or decreases due to the change in phenology under climate change (Barichivich et al., 2013; Forkel et al., 2016; Goetz, Bunn, Fiske, & Houghton, 2005; Oechel, Laskowski, Burba, Gioli, & Kalhori, 2014) because the impact of earlier spring onset could be moderated by enhanced ecosystem respiration in the prolonged autumn period (Piao et al., 2008; Ueyama, Iwata, & Harazono, 2014).", "Observation and modeling of start of growing season (SOS), end of growing season (EOS), and growing season length provide essential information on how terrestrial vegetation responds to climate changes (Buermann et al., 2014; Keenan et al., 2014; Nagai et al., 2013b; Piao et al., 2011; Richardson et al., 2013b; Schwartz, Ault, & Betancourt, 2013; Verbyla, 2008; Xu et al., 2013).", "In the Arctic and sub-Arctic regions, the earlier SOS trends were found in previous studies (Buermann et al., 2014; Delbart et al., 2008; Hogda, Tommervik, & Karlsen, 2013; Myneni, Keeling, Tucker, Asrar, & Nemani, 1997; Piao et al., 2011).", "On the other hand, a relatively limited amount of information is available on large-scale EOS variations and its long-term trends (Jeong & Medvigy, 2014).", "Recent studies have found a trend for later EOS in many zones in Europe (Garonna et al., 2014), North America (Zhu et al., 2012), and for temperate vegetation over the northern hemisphere (Jeong, Ho, Gim, & Brown, 2011), suggesting that rising temperature could affect the phenological events.", "However, the environmental factors (e.g. photoperiods and temperature changes) that control the changes in EOS are not clearly understood (Delpierre et al., 2009; Jeong & Medvigy, 2014; Richardson et al., 2013a).", "The estimation of SOS and EOS utilizes the seasonal patterns in satellite vegetation indices (VIs).", "In the Arctic and sub-Arctic regions, an increase in the normalized difference vegetation index (NDVI) (green-up) in spring is partially affected by the timing of snowmelt (Dye & Tucker, 2003; Jonsson, Eklundh, Hellstrom, Barring, & Jonsson, 2010; Kobayashi, Delbart, Suzuki, & Kushida, 2010; Kobayashi, Suzuki, & Kobayashi, 2007; Suzuki, Kobayashi, Delbart, Asanuma, & Hiyama, 2011).", "A systematic bias has also been found at EOS, and snow is thought to affect the satellite-based EOS estimation (Zhu et al., 2012); however, it has not been investigated quantitatively.", "Because boreal forests in Alaska are sparser than forests in lower latitudes (Canada and the U.S.), the seasonality of understory plants has a substantial impact on satellite signals (Pisek & Chen, 2009; Rautiainen & Heiskanen, 2013; Yang, Kobayashi, Suzuki, & Nasahara, 2014).", "In interior Alaska, black and white spruce are the dominant species.", "The spectral reflectance of these species with evergreen needles is relatively unchanged throughout the growing season (Nagai et al., 2012), while it is likely that satellite phenology metrics should be greatly influenced by understory plant phenology.", "However, how the satellite-based phenology metrics are influenced by the forest overstory status, understory plant phenology and other factors such as snow and observation conditions remains less investigated in Alaska.", "Thus, comparisons with ground-based datasets are essential.", "Time-lapse camera images provide the seasonal information on surface conditions and have been used widely for the detection of phenological events at ground level (Richardson et al., 2007; Woebbecke, Meyer, Vonbargen, & Mortensen, 1995).", "In this study, we quantified the latitudinal gradient of SOS, EOS, and the timing of snow cover in Alaska using ground-based time-lapse digital camera images, and then compared these events with SOS and EOS determined using the satellite data.", "In the ground-based analysis, we used firsthand time-lapse camera images obtained at 17 sites (six tundra sites and eleven boreal evergreen forest sites) along a latitudinal transect through the state of Alaska, USA (61°N to 71°N), which covers the boreal forest and tundra ecosystems (Fig. 1).", "We evaluated SOS and EOS by the three phenology algorithms using two satellite datasets (Terra-moderate resolution imaging spectroradiometer (MODIS) and SPOT-VEGETATION).", "Through the comparison, we investigated the discrepancies in satellite phenology metrics and the influence of snow and understory phenology in tundra and boreal forests.", "To quantitatively evaluate those impacts, we performed a detailed radiative transfer simulation.", "The study sites are distributed on a north–south transect across Alaska between a latitudinal range of 61°N to 71°N (Fig. 1).", "All ground observation sites are within a longitudinal band between 144°W to 157°W.", "These sites contain two distinct ecosystems: evergreen needleleaf forest and tundra.", "Evergreen needleleaf forests are found in the south of the Brooks Range (68°N), while the tundra ecosystem is typical of the north.", "The dominant overstory tree species in interior Alaska is black spruce (Picea mariana).", "Most of the understory layer is covered with rusty peat moss (Sphagnum fuscum) and splendid feather moss (Hylocomium splendens).", "The colors of rusty peat moss and splendid feather moss vary spatially and seasonally (brown to green).", "The understory layer is also partly covered with tussocks formed by herbaceous perennial cotton-grass (Eriophorum vaginatum) (Kim, Kodama, Shim, & Kushida, 2014; Nakai et al., 2013).", "The dominant vascular plants of the understory are low shrubs and herbs such as Labrador tea (Ledum groenlandicum), bog bilberry (Vaccinium uliginosum), dwarf birch (Betula nana), and cloudberry (Rubus chamaemorus) (Kim et al., 2014; Nakai et al., 2013).", "These vascular plants are deciduous.", "The camera observation sites in the tundra are located in heath tundra and moist acidic tundra areas.", "The moist acidic tussock tundra is dominated by tussock sedge (Eriophorum vaginatum), and dwarf shrubs (Betura nana, Carex bigelowii, Vaccinium vitis-idaea, and Ledum palustre) (Euskirchen, Bret-Harte, Scott, Edgar, & Shaver, 2012; Kim et al., 2014; Oechel et al., 2014).", "The dry heath tundra is dominated by Dryas spp., lichen, and dwarf shrubs (Environmental Data Center Team, 2014; Euskirchen et al., 2012).", "We used time-lapse camera images obtained from 17 sites across Alaska (Table 1 & Fig. 1).", "The 6 northern sites (denoted as T-1 to T-6) are located in tundra and the other 11 sites are in boreal forests (denoted as F-1 to F-11).", "At these sites, there are three different time-lapse camera systems: GardenWatchCams (Brinno Inc., Taiwan), webcams, and a fish-eye camera (Nikon Coolpix 4500 with an FC-E8 fisheye lens) (Table 1).", "These camera systems took the images of tundra and forest understory vegetation at a nadir or horizontal view.", "The sampling interval was from 15 min to 6 h depending on the camera setting.", "Images that were bright enough to obtain color information and in which there were no distinct sunlit or shade variations were selected.", "Areas within images showing tundra or understory vegetation were extracted for analysis.", "T-4, T-5, and T-6 were located near Toolik Lake (Environmental Data Center Team, 2014).", "The camera system in T-6 (Atigun Gorge) was mounted on a 10 m tower near the Atigun River bridge crossing of the Dalton highway.", "The two other cameras (T-4 and T-5) were mounted on towers attached to buildings at the Toolik Field Station.", "The T-5 cameras faced toward Toolik Lake and surrounding shrubs, and the T-4 cameras faced south toward the Brooks Range.", "The camera images taken at around noon are available publicly (http://toolik.alaska.edu/edc/abiotic_monitoring/image_library.php).", "The other three tundra sites were located to the north of Toolik Lake.", "The T-2 and T-3 sites were wet sedge and tussock tundra, respectively.", "At these sites, GardenWatchCams (Brinno Inc., Taiwan) were installed roughly 1 m above the ground (Kim, 2014).", "For the T-1 (Barrow) site, images were taken from a web camera installed near the eddy covariance tower in moist acidic tundra (Kwon, Oechel, Zulueta, & Hastings, 2006; Zona et al., 2009).", "Images were recorded every 15 min from April to September 2006.", "Data around noontime were used in this study.", "The sites F-1 to F-11 were located in black spruce forests in interior Alaska (Iwata et al., 2011; Iwata, Ueyama, Iwama, & Harazono, 2013; Kim, 2014; Nagai et al., 2013a; Sugiura, Nagai, Nakai, & Suzuki, 2013; Ueyama et al., 2014).", "The F-6 and F-7 sites are close to each other; however, the forest conditions are different: F-6 is a matured black spruce forest and F-7 is a pre-matured black spruce forest after the fire in 2004.", "Except for F-6, the time-lapse camera images were installed at the forest understory.", "These time-lapse cameras monitored understory conditions from a nearly horizontal view and any changes in the land surface, such as the snow cover and understory vegetation changes, were easily identified.", "Images were recorded every 1–4 h throughout the season from 2010 to 2012.", "However, because these cameras were installed in remote sites under severe climate conditions, there were several large gaps in the data due to technical problems, such as disorientation of the camera by gusts of wind and damaged batteries in cold weather.", "We selected the years and sites (Table 1) for which understory images provided adequate seasonal coverage.", "The camera at the F-6 site was a fish-eye camera (Nikon Coolpix 4500 with an FC-E8 fisheye lens) installed at a height of 17 m on the eddy covariance tower of the Ameriflux site (US-Prr) (Ikawa et al., 2015; Nagai et al., 2013a).", "The camera faced down and captured an image of the understory surface every hour.", "The exposure and white balance were set to automatic over the growing season (Nagai et al., 2013a).", "Monthly 0.5° air temperature data from the Climate Research Unit (Harris, Jones, Osborn, & Lister, 2014) were used to evaluate the latitudinal gradient of air temperature.", "We extracted the temperature datasets close to the study sites.", "To compare with phenological events, we used the monthly temperature in May for SOS and an average of August and September in EOS.", "SOS and EOS were determined using time-lapse camera data and satellite datasets.", "For time-lapse camera data, we first extracted solar noontime images because they exhibited the best brightness condition.", "Then we defined the region of interest (ROI) from the images to extract the red, green, and blue digital numbers.", "The image areas with spruce tree branches and sky were excluded.", "The true SOS and EOS values were determined by a visual interpretation (DSOS_V and DEOS_V).", "This was performed for the selected sites (F-1, F-4, F-5, F-7, F-9, F-10, and F-11 in Table 1), where the cameras were installed close to the herbaceous plants and deciduous dwarf shrubs.", "The dates of DSOS_V and DEOS_V were recorded when the leaf emergence and senescence were recognized clearly in the ROI.", "This visual interpretation was performed for herbaceous plants and deciduous dwarf shrubs within ROI to exclude seasonal color changes in moss and lichen.", "In autumn, we determined EOS when all leaves in the ROI were senesced and turned brown.", "For time-series satellite datasets, we applied three algorithms to determine SOS: the local NDVI threshold method (White, Thornton, & Running, 1997), the NDII method (Delbart, Kergoat, Le Toan, Lhermitte, & Picard, 2005), and the method of Zhang et al. (2003).", "In the local NDVI threshold method, SOS was determined when normalized NDVI exceeded 0.5.", "In the NDII method, SOS was determined as the point when the NDII in spring reached a minimum value and then increased above a spring threshold.", "This threshold was determined by the method used in Delbart et al. (2005), which is 20% of the seasonal NDII amplitude.", "The method of Zhang et al. (2003) was applied to NDVI datasets.", "Green-up (D1) and maturity (D2) were estimated by the fitting method described by Eq. (3).", "The satellite-based SOS estimate was determined as the midpoint of D1 and D2 following a study of reflectance and NDVI seasonality in Siberia (Kobayashi et al., 2007).", "Fig. 3 shows examples of the seasonal variations in NDVI, NDII and GEI.", "For autumn timings, the local NDVI threshold method was used to determine EOS.", "The method of Zhang et al. (2003) should be theoretically applicable in the same manner as for SOS.", "However, in our study area, the temporal patterns of NDVI in autumn were complex due to multiple occurrences of snowfall and snowmelt until the ground was covered with lingering snow.", "Thus the NDVI fitting using the logistic function frequently failed.", "The estimated SOS and EOS from satellites can be affected by clouds (Kobayashi & Dye, 2005).", "The effects of clouds during ± 2 compositing periods (a total of five consecutive samples: ± 16 days for MODIS and ± 20 days for SPOT-VEGETATION) around SOS and EOS were evaluated based on the cloud flag information.", "For MODIS, when the two cloud-related bits in the State Flag data were both zero, we considered the data to have no cloud contamination; otherwise, the observation was considered to be partially or fully affected by clouds.", "Similarly, for SPOT-VEGETATION data, when the two cloud-related bits in the Status Map (SM) data were both zero, we considered the data to be cloud-free; otherwise, the observation was considered to be partially or fully affected by clouds.", "Cloud fractions (%) for each location were quantified as the proportion of the number of cloudy samplings in ± 2 compositing periods during SOS and EOS events.", "The last and first dates of lingering snowpack were evaluated by the time-lapse camera images.", "We followed the criteria provided by Sugiura et al. (2013), which is: (1) when the length of lingering snowpack cover exceeds 30 consecutive days, the period from the first day to the last day is the period of lingering snowpack and (2) “the existence of snow cover” is the condition, in which more than half of the surface is covered by snow.", "Based on their criteria, we determined the last (snowmelt) and first (snowfall) dates of lingering snowpack from time-series images.", "A three-dimensional Monte Carlo ray-tracing radiative transfer simulation was performed to examine the cause of seasonality in satellite spectral reflectance over sparse black spruce forest sites.", "In this study, a spatially explicit three-dimensional model, the Forest Light Environmental Simulator (FLiES), was employed to simulate bidirectional reflectance seasonality in red and NIR (Kobayashi & Iwabuchi, 2008; Kobayashi et al., 2012).", "The performance and reliability of FLiES for simulating light transmittance through a canopy and bidirectional reflectance factors have been investigated in the previous works (Widlowski et al., 2011, 2013).", "Required inputs for the model include simulated forest landscape data including individual tree position and size.", "The shape of the tree crown was abstracted as a spheroid and the crown objects were divided into two domains: the outer domains were filled with leaves and inner domains were filled with woody materials.", "Forest landscape data were constructed based on the field census measurements at the F-6 site (PFA), which located ~ 32 km from Fairbanks, interior Alaska.", "Individual tree positions, canopy heights, and crown widths were based on the data of Kobayashi, Suzuki, Nagai, Nakai, and Kim (2014).", "The tree density, canopy cover, and maximum overstory leaf area index (LAI) were 3967 trees ha− 1, 15% and 0.73, respectively.", "We also constructed a denser forest landscape simulation based on the abovementioned data to understand how the characteristics of sparse tree stands in a black spruce affect the determination of SOS and EOS.", "In this landscape data, we maintained an identical tree density as the original data and doubled the size of crown heights and crown widths.", "The canopy cover and maximum overstory LAI of this denser landscape were 45% and 2.4, respectively.", "There are minor, but non-negligible seasonal changes of overstory LAI even in boreal evergreen forests (Chen, 1996).", "To consider this seasonal changes and the potential contamination of minor deciduous trees in evergreen spruce forests, we performed the simulation for three different seasonal overstory LAI conditions: (1) constant overstory LAI throughout the season, (2) overstory LAI changes seasonally by 20%, and (3) overstory LAI changes seasonally by 50% (Table 2).", "Seasonal changes in understory optical data are summarized in Table 2 based on measurements reported in Kobayashi et al. (2014).", "Other optical variables—such as needle reflectance and transmittance, and woody reflectance—were 0.0741, 0.0232, and 0.0509 in red and 0.466, 0.387, and 0.0906 in NIR, respectively.", "These variables were constant throughout the season.", "We simulated five days from spring to autumn.", "In spring (DOY 140) and autumn (DOY 274) (Table 2), two understory conditions were simulated: with and without snow.", "The reflectance of snow value used was measured by Suzuki et al. (2011).", "The solar zenith angles of the simulated conditions were near the satellite overpass time (LST 10:30 am).", "The satellite-based SOS and EOS, the ground-based SOS and EOS, and the last (spring) and first (autumn) lingering snowpack dates estimated for each site and year are summarized in Fig. 4.", "The growing season lengths (the black bar between SOS and EOS) from the ground-based time-lapse images were quantified for 21 out of 31 cases (site × year) (see Supplemental Fig. 1).", "For the other 10 cases, due to bad quality or missing images, the image data from spring to autumn were not fully available.", "For those sites, we only estimated one of two phenology timings (SOS or EOS) depending on the data availability.", "The date of ground-based SOS and EOS were determined by the analysis shown in the following section (Section 3.1).", "The satellite-based SOS and EOS in Fig. 4 are the ensemble of all methods estimated from two satellite datasets (SPOT-VEGETATION and Terra-MODIS).", "Overall, correlation coefficients (R) of ground-based and satellite-based events were 0.64 for SOS and 0.49 for EOS, respectively.", "The error estimates for the methods used are described in Section 3.2 and Fig. 5.", "The satellite-based SOS and EOS, the ground-based SOS and EOS, and the last (spring) and first (autumn) lingering snowpack dates estimated for each site and year are summarized in Fig. 4.", "The growing season lengths (the black bar between SOS and EOS) from the ground-based time-lapse images were quantified for 21 out of 31 cases (site × year) (see Supplemental Fig. 1).", "For the other 10 cases, due to bad quality or missing images, the image data from spring to autumn were not fully available.", "For those sites, we only estimated one of two phenology timings (SOS or EOS) depending on the data availability.", "The date of ground-based SOS and EOS were determined by the analysis shown in the following section (Section 3.1).", "The satellite-based SOS and EOS in Fig. 4 are the ensemble of all methods estimated from two satellite datasets (SPOT-VEGETATION and Terra-MODIS).", "Overall, correlation coefficients (R) of ground-based and satellite-based events were 0.64 for SOS and 0.49 for EOS, respectively.", "The error estimates for the methods used are described in Section 3.2 and Fig. 5.", "In the spring period, D1 was 10.3 days earlier than DSOS_V and D2 was 46.0 days later than DSOS_V on average.", "The DSOS_V was thus consequently represented by D1_75% + D2_25% (the weighted average of D1 [75% weight] and D2 [25% weight]).", "The mean bias of D1_75% + D2_25% was 1.2 days (Table 3).", "In the autumn period, both D3 and D4 were earlier than DEOS_V (54.6 days for D3 and 5.2 days for D4).", "The ground-based EOS was represented by D4 – 5.2 days.", "The biases of the estimated SOS and EOS were diverse among the methods (Fig. 5).", "Among three satellite-based methods for determining SOS and EOS, no particular method was most consistent with the ground-based measurement.", "In spring, the satellite-based SOS was between 2.9 and 12.7 days earlier (positive biases) than the ground-based SOS in boreal forest sites, excepting the NDII method.", "In tundra sites, direction of the biases varied by the methods employed.", "Three methods (NDVIs, NDIIS, NDIIM) calculated SOS later than the ground-based SOS (bias = − 6.9 days [SOS (NDVIs)], − 16.4 days [SOS (NDIIS)], and − 13.3 days [SOS (NDIIM)]) and three methods (NDVIM, LogisticS, LogisticM) calculated an earlier SOS than the ground-based SOS (bias = 10.2 days [SOS (NDVIM)], 3.4 days [SOS (LogisticS)], and 10.0 days [SOS (LogisticM)]).", "The NDII method showed the smallest biases (0.26 days for SOS (NDIIS) and − 0.58 days for SOS (NDIIM)) and RMSEs (9.4 and 10.0 days) for forest sites.", "However, in tundra sites, both the biases and RMSE for the NDII method were largest among the methods (− 16.4 days for SOS (NDIIS) and − 13.3 days for SOS (NDIIM)) and RMSEs (30.3 and 20.4 days).", "The surface in most tundra sites is inundated just after snowmelt and NDII is sensitive to not only the amount of vegetation but also the surface wetness.", "Therefore, the NDII did not drop rapidly after snowmelt, but rather gradually decreased as a combined effect of wetness and green vegetation development from spring to summer.", "Consequently, the date of the minimum NDII, which is considered as SOS, was delayed until around the early summer.", "For autumn EOS, the large differences between satellite-based and ground-based EOSs were found for both boreal forest (mean bias − 25.7 days and RMSE 30.2 days for SPOT-VEGETATION and mean bias − 26.0 days and RMSE 30.7 days for MODIS) and tundra sites (mean bias − 16.0 days and RMSE 18.4 days for SPOT-VEGETATION and mean bias − 20.3 days and RMSE 22.3 days for MODIS).", "The differences for boreal forest sites were 5.7 days (MODIS) to 9.7 days (SPOT-VEGETATION) days greater than those of tundra.", "The possible causes of these EOS differences are analyzed in Section 3.4.", "Persistent cloud cover potentially introduces errors to the estimation of the increase in SOS and EOS.", "In our study sites and periods, 78.5% of SOSs and EOSs were estimated under low cloud atmospheric conditions (CF < 20%) (Fig. 6).", "As the SPOT-VEGETATION S10 has longer compositing periods, the cloud fraction of SPOT-VEGETATION was less than that of MODIS.", "For both SPOT-VEGETATION and MODIS, the cloud fractions during EOS were larger than during SOS.", "The increase in cloud fraction is prone to cause later SOS or earlier EOS estimates, which is the opposite of the bias direction in our results (Fig. 5).", "Thus, our result implies that, while the effects of cloud were marginal at our study sites, the effects of the cloud cover potentially underestimated the bias in SOS and EOS evaluated in Fig. 5.", "The timing of snow cover and phenological events in 2011 and 2012 showed consistent latitudinal gradients across the forest-tundra ecotone in the study area (Fig. 7).", "The relative timing of snow (snowmelt in spring and snowfall in autumn) versus ground-based and satellite based phenology differed between spring and autumn periods.", "In spring, the snowmelt preceded satellite-based and ground-based SOS by 16 to 19 days.", "In autumn, the ground-based EOS preceded satellite-based EOS by 19 to 26 days and the satellite-based EOS almost coincided with the snow events.", "For both years, the latitudinal gradient of phenological events (slopes shown in Fig. 7) were steeper in spring than those in autumn, except for the case of spring snowmelt in 2011.", "The latitudinal gradients of the ground-based SOS and EOS were 3.4 days degree− 1 and − 2.7 days degree− 1 for 2011 and 4.9 days degree− 1 and − 3.7 days degree− 1 for 2012, respectively.", "The latitudinal gradients of air temperature in spring (May) and autumn (an average of August and September) were − 1.4 °C degree− 1 and − 0.89 °C degree− 1 in 2011 and − 1.3 °C degree− 1 and − 0.81 °C degree− 1 in 2012, respectively.", "Thus, the changes in SOS with regard to air temperature (dSOS/dTa) were − 2.5 days °C− 1 in 2011 and − 3.9 days °C− 1 in 2012.", "The changes in EOS with regard to air temperature (dEOS/dTa) were 3.0 days °C− 1 and 4.6 days °C− 1 in 2011 and in 2012, respectively.", "The correlation between EOS and the timing of snow cover onset was weaker than that between SOS and the timing of snowmelt (Fig. 8).", "The correlation coefficients of satellite-based and ground-based SOSs against the latest continuous snow date were 0.79 (p < 0.01) and 0.58 (p < 0.01), respectively.", "On the other hand, the correlation coefficients of satellite-based and ground-based EOSs against the earliest continuous snow date were 0.63 (p = 0.017) and 0.19 (p = 0.88), respectively.", "The simulated NDVI displayed seasonal patterns that were typical of those observed in the satellite NDVI data (Fig. 9).", "Although NDVI depends on satellite view angles, those geometries (three view zenith and azimuthal angle cases in Fig. 9) did not result in substantial differences in NDVI (coefficient of variation < 3.5%) except for the snow periods (up to 28%).", "The understory reflectivity accounts for 86–99% in red and 65–83% in NIR (canopy cover = 15%) (Fig. 9d–f) and 48–97% in red and 25–66% in NIR (canopy cover = 45%) (view zenith angle and azimuth angle = 0°) (Fig. 9a–c).", "After the snowmelt, NDVI increased during the summer (summer-2) because of the decrease in understory red reflectance and increase in the NIR reflectance (Table 2).", "However, this NDVI increase (spring-1 to summer-2) accounted for only 8.6–17% of total NDVI seasonal amplitude (Fig. 9a–f).", "Seasonal variations in NDVI were caused by the contribution of understory observed from the satellite (Fig. 9).", "The results slightly depended on the zenith and azimuth angles, but the seasonal changes were similar among the satellite view angles.", "The reflectance contribution of the understory gradually decreased from spring-1 to autumn-2, indicating that satellites view more of the canopy overstory (greener-needles) rather than brownish understory.", "Moreover, this seasonal change of overstory and understory contributions was larger in the red domain than that in the NIR domain.", "Consequently, the NDVI in late autumn was greatly influenced by the greener overstory needles.", "In addition, the NDVI in late autumn (autumn-2) increased again under both canopy cover of 15% and 45% conditions due to the very high solar zenith angle.", "The seasonality in overstory LAI caused the changes in the NDVI seasonality.", "For example, the constant overstory LAI case (Fig. 9a) exhibits almost constant NDVI throughout the growing season (from spring-2 to autumn-1).", "In contrast, the overstory LAI with 50% seasonality exhibits the bell-shaped NDVI seasonal patterns (Fig. 9c).", "Changes of up to 50% in overstory LAI caused 6.0–9.0% differences in the NDVI for a canopy cover of 45% (Fig 9a-c), and 3.3–4.9% differences for a canopy cover of 15% (Fig. 9d–f).", "As the LAI seasonality increased, the reflectance was more influenced by the understory in the spring and autumn rather than greener overstory, and the seasonal amplitude of NDVI (NDVImax − NDVImin) was enhanced from 0.55 to 0.66 (with snow season) and 0.078 to 0.106 (snow free period) for a canopy cover of 45% and 0.73 to 0.78 (with snow season) and 0.096 to 0.125 (snow free period).", "In spring phenology, satellite-based SOS metrics are known to be partially affected by the snowmelt in the Arctic and sub-Arctic regions (Dye & Tucker, 2003).", "Despite this issue, the satellite-based SOS may be related to leaf emergence in spring because leaf emergence happens immediately after the snowmelt.", "We estimated SOSs using multiple methods and data in Alaska to avoid the bias due to the method employed (White et al., 2009).", "Based on the comparison of six satellite-based SOSs (Fig. 5), we recommend using an ensemble average SOS because individual methods showed relatively high errors and their tendencies varied with the ecosystem type (forest or tundra).", "In fact, the ensemble SOS was the best performing of the methods employed (Fig. 5).", "NDVIs showed a relatively good performance in our comparison (Fig. 5); however, a single method and satellite data potentially leads to erroneous results due to atmospheric influence (e.g. residual clouds).", "Indeed, our comparison revealed that the NDII based methods (NDIIS and NDIIM) had the largest biases (− 13.3 to − 16.4 days) while Delbart and Picard (2007) found that the bias between satellite-based SOS calibrated by NDII and the ground SOS measurements near the Toolik lake (close to the T-4 & T-5 sites) was less than our results (− 3.25 days).", "This was potentially caused by the periods compared, the difference in the satellite data used, atmospheric conditions, and the differences of the ground phenology observation methods.", "The satellite-based SOS was slightly earlier than the ground-based SOS, but its latitudinal gradient was consistent with the leaf appearance of the forest understory and tundra vegetation (Fig. 7).", "Thus the satellite-based SOS can be useful as a measure of spring tundra and forest understory leaf emergences.", "The consistency between the ground-based and satellite-based EOSs was weaker than the relationship of ground-based and satellite-based SOS for boreal forest and tundra sites because of large biases between those two EOSs (19 to 26 days, Fig. 7).", "The satellite-based EOS was more relevant to the first date of lingering snowpack.", "On the other hand, the ground-based EOS was independent of snow events (Fig. 8).", "At tundra sites, the EOS biases were smaller than those of boreal forest sites.", "This is because, unlike the black spruce forests, the deciduous plants are directly exposed on the canopy surface.", "The leaf color change can be directly viewed from satellites.", "However, due to the abrupt decrease in NDVI caused by snowfall, the NDVI threshold (defined as 50% of the NDVI seasonal amplitude) did not correspond with EOS events.", "In addition to the lingering snowpack, there may be other issues on the uncertainty of the EOS estimation.", "Nagai et al. (2014) found that the autumn phenology in a cool-temperate deciduous broad-leaved forest occurred continuously because of different phenological timings among species and their spatial heterogeneities.", "This causes the additional uncertainty in determining the EOS.", "Beck et al. (2007) proposed a different threshold for spring and autumn phenology events.", "Using their approach the EOS is determined by a higher threshold value.", "If the variation in reflectance in tundra plants is similar to the understory reflectance in boreal forests (Table 2), the EOS in tundra sites may be estimated to be 84% of the NDVI seasonal amplitude.", "In boreal forest sites, however, it may not be straightforward to calibrate the biases between satellite-based and ground-based EOS in boreal forests by simply adjusting the threshold.", "The plant canopy radiative transfer simulation suggests that the appropriate NDVI threshold can vary with the canopy cover, overstory LAI seasonality, and solar zenith angles (Fig. 9).", "Assuming that the crown cover of 15% in Fig. 9 is more common in Alaska, the EOS in forest sites may be estimated to be 86% of the NDVI seasonal amplitude when overstory LAI is assumed to be constant throughout the growing season.", "For the determination of the appropriate threshold, a reliable forest cover map is required.", "Also, evergreen boreal forests show a little LAI seasonality (Chen, 1996).", "This weak seasonality does not significantly affect the NDVI threshold in sparse forest; however, as the canopy cover increases or minor deciduous trees contaminate the spectral signals, the NDVI threshold is likely to be affected by those overstory seasonal changes.", "The time-lapse cameras provide precise in-situ phenology measurements that can be used for the validation of satellite phenology estimates.", "When applying this technique to boreal forest understory, there are several issues to be carefully considered due to the limitations of image quality and the region of interest in camera images.", "The species composition within the camera images influences the timing of phenological events.", "At our study sites, the regions of interest of the understory vegetation within the camera images were defined to be as large as possible to avoid an irregular phenological pattern of particular plants and to obtain the average signature of different plants.", "Although most evergreen forest understory is covered with herbaceous plants, deciduous dwarfs, moss, and lichen as described in Section 2.1, the species composition in camera images was not identical.", "This could cause uncertainty in obtaining the average understory phenology in each forest site.", "Likewise, we focused on the phenology of herbaceous plants and dwarf trees.", "While Moss and lichen also show seasonally, it was not taken into consideration in this study.", "However, even if moss and lichen are present, because they grow under the herbaceous plants and dwarf trees, their phenological changes may have less impact on the satellite signal.", "The effect of moss and lichen should be investigated further through much finer-resolution images, with information regarding the spatial fraction of each element.", "The timings of SOS and EOS along 61°N to 71°N derived from satellite and time-lapse camera images revealed the latitudinal gradients from both measurements were almost consistent.", "Zhu et al. (2012) estimated the latitudinal gradients of SOS and EOS across the North American continent from 35°N to 70°N.", "The exact comparison may not be appropriate because of the difference in satellite data used (NOAA-AVHRR GIMMS), study periods (1982 to 2006) and area (North America).", "However, it is interesting to compare the consistency of both results.", "The rate of change calculated by Zhu et al. (2012) was 2.6 days degree− 1 for SOS and − 2.8 days degree− 1 for EOS for 35°N to 70°N.", "When evaluated from 60°N to 70°N (from Fig. 2 in Zhu et al. (2012)), both SOS and EOS were approximately 2.5 days degree− 1 and − 2.5 days degree− 1.", "This study showed 3.5 to 5.7 days degree− 1 for SOSs and − 2.3 to − 2.7 days degree− 1 for EOSs, respectively.", "Zhu et al. (2012) reported latitudinal symmetry for SOS and EOS, but we found a higher latitudinal gradient in SOS than in EOS, indicating that the SOS was less sensitive to changes in air temperature (dSOS/dTa of − 2.5 to − 3.9 days °C− 1) than EOS (dEOS/dTa of 3.0 to 4.6 days °C− 1).", "The average air temperature in the Arctic region rose up to 1.36 °C in the first decade of the 21st century (Bekryaev et al., 2010; Hinzman et al., 2013).", "If the timings of SOS and EOS were only regulated by air temperature, it would be expected that the temperature increase would have an impact on the timing of phenological events in the Arctic.", "Assuming dSOS/dTa and dEOS/dTa evaluated from the latitudinal gradient of phenology and air temperature (Fig. 7) are used for decadal scale phenology change, in the first decade of the 21st century SOS may be 4.4 days earlier and EOS 5.2 days later following a 1.36 °C warming across the state of Alaska.", "This assumption derived from the temperature dependency of phenology should be validated by a more detailed analysis based on long-term ground-based observations.", "The satellite-retrieved SOS and EOS have been extensively used for large-scale phenological studies.", "Despite their numerous spatial and trend analyses in SOS and EOS, there is still little information on how the latitudinal gradient of satellite-based SOS and EOS are related to changes in seasonal surface condition (e.g., snow), plant phenology, and satellite observation conditions in the Arctic and sub-Arctic regions.", "Using time-lapse camera images collected at 17 sites across Alaska, we quantified the phenological events along the latitudinal range from 61°N to 71°N.", "In SOS events, we found that the satellite-based SOS can be a measure of spring understory and tundra plants green-up when the SOS values estimated by multiple methods are averaged.", "A large difference was revealed between ground-based and satellite-based EOS.", "The satellite-based EOS was much more relevant to snowfall events than the senescence of understory and tundra plants in Alaska.", "While there are several limitations in using satellite- and ground-based phenology estimates, quantification of latitudinal gradient of phenology can be useful information in understanding the influence of warming to plant phenological timings.", "As understory plants in black spruce forests have a significant impact on the carbon exchanges, the reliable information of understory plants phenology is important.", "Our study demonstrates the possible impact of phenology in spruce forest understory and tundra ecosystems in response to climate change in the warming Artic and sub-Arctic regions.", "The following is the supplementary data related to this article.", "The time-lapse camera images for the important timings, SOS − 10 days, SOS, SOS + 10 days, DOY ~ 210 (summer), EOS − 10 days, EOS, and EOS + 10 days.", "Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.rse.2016.02.020." ]

[ "Forest understory and tundra phenology is estimated across Alaska from 61°N to 71°N.", "Satellite estimates of spring onset were consistent with ground-based data.", "From 84 to 86% of the NDVI seasonal amplitude can be used as a threshold for EOS.", "The latitudinal gradients of SOS and EOS were 3.5 to 5.7 and − 2.3 to − 2.7 day/°N." ]