Another trackway discovered two years earlier at nearby site A was partially excavated and attributed to a hominin, but curious affinities with bears (ursids) marginalized its importance to the paleoanthropological community, and the location of these footprints fell into obscurity5. In 2019, we located, excavated and cleaned the site A trackway, producing a digital archive using 3D photogrammetry and laser scanning. Here we compare the footprints at this site with those of American black bears, chimpanzees and humans, and we show that they resemble those of hominins more than ursids. In fact, the narrow step width corroborates the original interpretation of a small, cross-stepping bipedal hominin. However, the inferred foot proportions, gait parameters and 3D morphologies of footprints at site A are readily distinguished from those at site G, indicating that a minimum of two hominin taxa with different feet and gaits coexisted at Laetoli.Bipedal trackways discovered in 1978 at Laetoli site G, Tanzania and dated to 3.66 million years ago are widely accepted as the oldest unequivocal evidence of obligate bipedalism in the human lineage Reanalysis of bipedal trackways from Laetoli site A in Tanzania suggest that the footprints were made by a hominin that coexisted with at least one other hominin species. To test the second possibility, Tuttle12 collected data from circus bears trained to walk bipedally and found that their short steps and relatively wide feet were a close match to the site A footprints, although bipedal bears take wider steps. Furthermore, the fifth digit is typically the largest in ursids, solving the ‘cross-stepping problem’, although Tuttle12 noted that humans do occasionally cross-step. He concluded that “until detailed, naturalistic biometric and kinesiological studies are performed on bipedal bears and barefoot humans, we will have to defer choosing among the hominid and ursid hypotheses on Laetoli individual A”4.Tuttle12. White and Suwa8 argued that “reliable identification of these enigmatic prints at Laetoli site A will be impossible until they are more fully cleaned and followed laterally”. Accordingly, we were motivated to relocate and re-excavate site A and conduct a detailed comparative analysis of the prints as well as the locomotion of bears (Ursus americanus), chimpanzees (Pan troglodytes) and humans to test whether the footprints at site A were left by a hominin or an ursid.Complicating matters further, the internal morphology of the site A footprints was never fully cleaned of matrix infill3, we identified the proboscidean trail adjacent to the bipedal footprints. We cleared the surrounding overburden until one of us (K.F.) found the A3 footprint. The area was then brushed clean to expose A1–A5, which have experienced no discernible erosion since their initial discovery  show no evidence of distortion to their perimeters or internal morphologies. Given that track surfaces are likely to represent time scales of hours to days17. If present at all, ursids were rare on the landscape. Although footprint assemblages can include a surprising number of tracks from taxa whose skeletal fossils are rare , there is no clear taphonomic explanation for why ursid tracks would be present but their fossils absent.We recorded 50.9 h of video of wild American black bear behaviour. Unsupported bipedal posture and locomotion occurred only 0.09% of the total observation time, of which 59% was postural and 41% was locomotor  and during bipedalism . We compared these data with human barefoot footprints produced under three conditions: (1) habitually shod (n = 654) walking on a plantar pressure mat19; (2) habitually unshod or minimally shod (n = 41) walking in deformable mud21; and (3) Late Pleistocene tracks (n = 113) from Engare Sero, Tanzania, formed in reworked volcanic ash23  were within 10% of the length of the site A footprints (mean = 161.7 mm). Additionally, we measured the footprints of chimpanzees produced during quadrupedalism  to step length observed at site A fall within the ursid range  and chimpanzee (n = 50) footprints, and the fifth digit to the fourth in bear (n = 5) tracks. The A3 toe impressions match the distinctive proportions of humans and chimpanzees rather than those of bears , resulting in a low corresponding ratio, as expressed in every trackway at Laetoli.By establishing that A3 is a left hominin foot, we can now confirm that cross-stepping occurred. Cross-stepping was never observed in our comparative sample, but humans do it occasionallyThe relative step widths of footprints from sites G and S fall squarely within the modern human distribution . It is thus tempting to assign the site A tracks to A. afarensis; however, this premise requires an examination of foot ontogeny and intraspecific morphological variation that takes into account the mounting fossil evidence of locomotor (and presumably taxonomic) diversity among Pliocene hominins .It is generally accepted that 34), the maker of the site A footprints was smaller in height than other Laetoli trackmakers, which ranged from 111–116 cm (site G1) to 161–168 cm (site S1)35. It is plausible that the site A tracks were made by a juvenile A. afarensis, but this hypothesis is undermined by a distinct footprint morphology from those at sites G or S.Standing between 101 and 104 cm tall  has a slightly more divergent and mobile hallux than its adult counterparts36.The ratio of foot width and length follows a different ontogenetic trajectory in humans and chimpanzees; human feet are consistently narrower than chimpanzee feet Fig. . Footpri37 (38 or midfoot mobility—a characteristic absent from the Laetoli G and S footprints39 (but see ref. 40) and inconsistent with A. afarensis pedal remains41.To explore other instructive traits, we compared proportional toe depth ratios as described by Raichlen and Gordon37 . The mea42). Fig. A. afarensis. We tested this hypothesis by comparing the footprints of humans (n = 10) walking with their preferred gait and then cross-stepping. We found that normal and cross-stepping human footprints differ minimally and do not match in magnitude or direction the differences between the site G and S prints and the site A prints . Bears were present on screen for a total of 50 h 55 min 18 s. For each terrestrial bipedal incident, the length of the event, the approximate age of the bear and the number of steps were recorded. Additionally, steps were evaluated on whether they were completed independently, or the individuals used other environmental objects for balance.U. americanus, P. troglodytes, and Homo sapiens. For bears and chimpanzees, the sample size included all available individuals housed at each location. For the human sample size information see below. Randomization was not relevant to our study as we were interested in measuring footprint characteristics from whole sample populations, as opposed to comparisons within those populations. Blinding was not relevant to the data collected on the non-human comparative species  nor to the data collection on fossilized footprints. The human participants were unaware of the site A tracks at Laetoli and therefore had no knowledge of how the data obtained from their footprints would be used in this study.Comparative kinematic data were collected on three species: U. americanus , whose feet were within 10% of the length (average foot length = 145.7 mm) of the recorded footprints of Laetoli site A (average foot length = 161.7 mm). These orphaned, approximately 20-kg bears were located at the Kilham Bear Center , awaiting reintroduction to the wild. This study examined the bears between the ages of 5–8 months old. Our protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Dartmouth College. The bears were enticed to independently walk bipedally through a constructed mud trackway for either an applesauce or maple syrup reward , the width of the impression for the 1st, 2nd, 4th and 5th digits were measured. The presence or absence of claw impressions was also documented.Data were collected on four juvenile semi-wild n = 46), using a plantar pressure mat at the Ngamba Island Chimpanzee Sanctuary . While this third data set increases sample size and captures intraspecific variation, we recognize that plantar pressure data do not always align perfectly with footprints made in a deformable substrate20.Data for extant chimpanzees were extracted from three sources to collect all the relevant gait metrics. Two published datasets examined the same two subadult individuals housed at Stony Brook University. The third set were recorded on semi-wild individuals  and were supplemented with step length data for the same steps. Three-dimensional kinematic methods and step width calculation have been described previously28. Step length was calculated as the distance between left and right calcaneus markers in the sagittal plane during consecutive hind limb midstance periods. Chimpanzee step lengths are typically asymmetric, so step length was averaged over the two consecutive steps which defined the stride.Chimpanzee stride width data were taken from Thompson et al.42. In brief, chimpanzees traversed a runway, at the centre of which was a pressure mat  and a container of hydrated sediment in which the chimpanzees could produce footprints. This sediment was taken directly from a layer that preserves 1.5 Ma hominin footprints near Ileret, Kenya50. Laterally positioned video cameras were used to record the chimpanzees as they walked along this trackway and produced footprints. Two digitization softwares, MaxTRAQ Lite+ (v. 2.4.0.3) (Innovisions Systems) and ImageJ v.1.4751, were used to quantify various aspects of their gaits, including stride length. Tape measures and digital callipers were used to directly measure the external dimensions of each chimpanzee’s feet. Scaled photographs were taken of the footprints produced in each trial, and these were later measured using ImageJ software.Footprint dimensions and stride length data were recorded on the same two subadult male chimpanzees as above, though at a slightly younger age . The experimental design is described in detail elsewhereData were collected by E.J.M. at the Ngamba Island Chimpanzee sanctuary  managed by the Chimpanzee Sanctuary and Wildlife Conservation Trust (CSWCT) using procedures approved by the Dartmouth College IACUC. The Tekscan plantar pressure mat (PPM) was positioned within a walkway connecting the overnight enclosure to the open forest habitat, near a gate and underneath a solid cross section to help prevent individuals from jumping over the mat using the ceiling bars. This location was determined using the expertise of the sanctuary keepers. The animals were first introduced to a mat shell that was lacking the internal sensors, to habituate them to the novel stimulus. All subsequent data were collected using both the empty and real PPMs, positioned such that they covered the entire width of the walkway to force individuals to walk across one of the two mats. Both mats were covered with thin green sacks to help disguise them from the chimpanzees and facilitate faster removal if necessary. It was determined that the southeast facing direction was the preferred path for the chimpanzees and the sensor-containing PPM was always positioned there from the second collection onwards. Data were collected twice a day; once in the morning (between 06:45 and 08:00) when the chimpanzees were headed to the forest for their first feeding, and once at 18:00, when the chimpanzees were headed into the overnight enclosure to sleep and receive their last feeding. Data were collected on 46 adult chimpanzees . A subset of 54 dynamic pressure records was analysed. Using the associated Tekscan PPM software, Footmat Research (v. 7.10), the pressure recordings were analysed to determine foot length, forefoot width, the width digits 1 and 2, and the linear distance between the centre of digits 1 and 2. The divergence ratio was calculated by dividing the distance between digits 1 and 2 by the individual’s foot length.Data were extracted from previous studies of two modern human populations in order to collect all the relevant foot, footprint and gait metrics.19. Sample size was determined by museum visitor traffic and willingness to participate in a scientific study. In brief, this dataset included 73 children between the ages of 2 and 7 years old  and 581 individuals  between the ages of 8 and 80 years. A pressure-sensitive gait carpet (6.1 m long × 0.89 m wide) with a spatial resolution of 1.27 cm and collecting data at 120 Hz (GAITRite) was used to collect stride length and stride width. For a subset of 33 adults, additional data were collected using a Tekscan PPM and analysed with FootMat Research (v. 7.10) to calculate foot length, the width of digits 1 and 2, and the linear distance between the centre of digits 1 and 2. These measurements were used to calculate a divergence ratio as described above in ‘P. troglodytes’.Data were taken on 654 participants, recruited through the Living Laboratory at the Boston Museum of Science21. Details of the experimental protocol largely mirrored the procedures described above. In brief, subjects generated footprints while walking through a rehydrated sample of the same sediments that preserve 1.5 Ma hominin tracks near Ileret. Video cameras were used to record subjects as they produced footprints, and two digitization software packages (MaxTRAQ Lite+ v. 2.4.0.3 and ImageJ v.1.47) were used to measure stride lengths and other kinematic variables. The external dimensions of subjects’ feet were directly measured with tape measures and digital callipers. Scaled photographs of the footprints produced in each trial were measured using ImageJ.Footprint dimensions and stride length data for 29 Daasanach adults  and 12 children , who live near the town of Ileret, Kenya and grew up either habitually unshod or minimally shod were taken from Hatala et al.52. Sample size was determined by availability and willingness to participate in the study. Five trials were completed with a normal, self-selected walking gait and another five were completed with a cross-stepping gait, as inferred for the Laetoli site A trackmaker. In each trial, a focal footprint was selected, measured in situ, and photographed (25–30 photos per footprint). The lengths and widths of the steps bracketing the track were also measured. Photographs were used to generate 3D models of the tracks using Agisoft Metashape software (v.1.7.3), and average normal and cross-stepping tracks were generated for each subject using DigTrace Pro (v.1.8.1)53. Lengths and widths of these averaged tracks were measured using Geomagic Wrap (v. 2021.0.0) (3D Systems). Regional depths were measured and evaluated using the same methods described below . Within-subject comparisons enabled us to understand how cross-stepping influenced the dimensions of the perimeter and the internal topology of a subject’s footprints.Experiments were carried out by K.G.H. and E.M.W.-H. to investigate whether and how cross-stepping kinematics influence the perimeter dimensions and internal topologies of an individual’s footprints. We could thereby evaluate whether the size and shape of the Laetoli site A tracks could have been generated by a hominin with feet similar to those who left tracks at sites G and S, but while cross-stepping. Detailed methods are provided in Comparative metrics were quantified from a set of modern human footprints from the Late Pleistocene at Engare Sero, Tanzania. These footprints are an important comparison with the Laetoli footprints, as they were generated in a similar circumstance (footprints in volcanic ash) and represent an early population of unshod modern humans.4, were taken from the fossil tracks using the software, ImageJ (v. 1.49), and included foot length, forefoot width, heel width and step width of each footprint at Engare Sero. In some cases, partial footprints were included for measurement, as long as they included the requisite landmarks for those measurements. Overall, data were collected from 151 footprints at the Engare Sero site. Of the 151 footprints, 61 footprints were considered partial footprints and 90 footprints were considered complete footprints. From these, 67 step length and stride width measurements and 105 heel width and ball width measurements were included in our analyses.A scaled 3D orthophoto of the Engare Sero site was created via photogrammetry by B.Z. and C.L.-P. to visualize the distribution of footprint trackways across the entire site using Agisoft Photoscan (now Agisoft Metashape v. 1.4.4). The model was created from hundreds of photos originally taken by the Smithsonian 3D Digitization Program in 2010. Measurements that were defined in Tuttle42. Box and whisker plots and bivariate graphs (using ggplot254) were generated using R (v. 3.6.1), while the table and pie chart were generated using Microsoft Excel (v. 2102).All measurements for Laetoli trackways G and S were obtained from published sources42. In brief, the human comparative sample included 245 footprints produced by 29 adult and 12 juvenile habitually unshod Daasanach individuals traveling at walking speeds. The chimpanzee comparative sample included 45 footprints produced by two individuals walking bipedally. Laetoli samples included only the best-preserved tracks from each site, leaving samples of five footprints from site G that were described by their original excavators as free from taphonomic damage that would obscure track topology , and two from site S (L8-S1-2 and L8-S1-4). For site A, we included tracks A2 and A3, as these were the only two for which we were relatively confident in identifying regions of interest across the entire track. Larger sample sizes would be desirable, but we did not want to sacrifice data quality for quantity by including tracks that were overprinted or that did not appear to represent complete foot anatomy. We did not rely on parametric statistical tests for which larger sample sizes would be a necessity, and instead used an analytical approach that could handle smaller sets of observations (see below).Comparative analyses followed methods similar to resampling analyses published previously42. Using Geomagic Wrap (v. 2021.0.0) (3D Systems), a best-fit plane was fit to the undisturbed substrate surrounding each track, and this was fixed to the xy plane in world coordinate space. In this orientation, depths of the footprint were measured in the regions of the medial and lateral heel, medial and lateral midfoot, and all five metatarsal heads and toes. Raw depth measurements were normalized, within each footprint, to a scale of 0 to 1 in order to compare the topologies of footprints that may vary in depth. However, a Wilcoxon signed-rank test showed that, overall, human and Laetoli track samples did not differ significantly in their depths (P = 0.08). Within-subject means of the 14 normalized depth measurements were calculated, and a between-subject covariance matrix was created using the subject averages for normalized depths at each of the 14 measured regions. An overall ‘human mean footprint’ was also computed by averaging the within-subject mean normalized depths, and this represented a measure of central tendency as described below.For each experimental and fossil footprint, 3D models were constructed using photogrammetry, through a variety of methods described here for Laetoli site A and elsewhere by the authors for other samples55, ggplot254 and reshape256 packages.To represent the range of observed variation in human footprint topography, for 1,000 iterations we randomly sampled a human subject and drew a sample of two of their footprints. We then averaged the normalized depths of those two footprints and computed the Mahalanobis distance (using the between-subject covariance matrix) between this track and the mean of all other subjects’ footprints. Also, for 1,000 iterations we selected a random chimpanzee subject, drew a random sample of two of their footprints and computed the Mahalanobis distance between the average of those tracks and the overall mean human footprint. For the Laetoli tracks, site A and site S samples only included two tracks, so these were simply averaged and the Mahalanobis distance was calculated between each averaged track and the mean human footprint. For Laetoli site G, all possible two-track combinations (ten) were drawn from the sample described above, and the Mahalanobis distance was calculated between the averaged track from each combination and the human mean. In all cases, we calculated multivariate distances using the human between-subject covariance matrix . All analyses described above, and the histogramdisplaying multivariate distances Fig. , were geWhile casts of the site A bipedal footprints existed at one point, all our attempts to locate them (see Acknowledgements for a complete list) were unsuccessful. Prior to our fieldwork at Laetoli in 2019, we modelled the original trackway photogrammetrically using extant photography from the site. Original photography of Laetoli site A was taken by J.R. We obtained his photographs of trackway A through Science Photo Library. All photographs were taken with a Nikon F2 on 35-mm Kodachrome slide film. Digital scans from these slides were used to produce a 3D model of the Laetoli A footprints. Unfortunately, since the images were taken in 1977, they were not recorded with modern photogrammetry processing in mind. Several features of the digitized images limit successful and accurate construction of a 3D model. First, there are only four images of the footprints. One of these images has noticeably different exposure settings that caused significant alignment problems during processing, and thus was excluded. All images were shot at oblique angles, from a relatively narrow range of camera positions. A yellow string defining the site grid lies over one of the footprints, obscuring part of it, and casting a shadow. The images were all taken relatively early in the day, so there are shadows within each footprint that create strong contrasts. The slides were digitized at 4,000 dpi, but they were not scanned with specialized equipment to guarantee geometric accuracy, and this potentially introduced more sources of distortion.12.However, despite the limitations of the images, it was possible to extract 3D data for the Laetoli A footprints. All processing was done in Agisoft Photoscan Pro (v. 1.7.1). The standard processing steps  were run to produce a 3D model, though the process had to be done iteratively to remove noise, ensure accurate alignment of the photos, scale the model appropriately using published measurements, add manual tie points, and refine the model. A DEM  and orthophotograph were exported for further visualization and analysis in ArcGIS (v. 10.6.1). The 3D model was also exported to Autodesk Meshmixer (v. 3.5.474) to create a ‘watertight’ 3D volume that could be 3D printed for further visualization . Photogrammetric reconstruction was validated using published measurements of the footprints. It is important to note however, that there were no published measurements for the depths of the footprints and that the internal anatomy of this reconstruction is potentially misleading because of the incomplete excavation of the footprints51) were taken to create a 3D model of the A trail. This included photo alignment, manual editing of the sparse cloud to remove points with high ‘reprojection uncertainty’, building a dense cloud, building a mesh, refining the mesh, then building a texture. During processing, images were checked for sharpness using the ‘image quality’ tool and any images with significantly lower quality were removed. The model was scaled to the real-world using scale bars placed across the region of interest. Finally, an orthophotograph as well as a DEM  were exported as geotiffs into ArcGIS in an arbitrary local coordinate system for further analysis .A second, more accurate reconstruction was done using photogrammetry from the re-excavated site A bipedal trackway using 57 images taken in June 2019. The images were captured in a systematic manner using a Nikon D7000 camera and Nikon DX AF-S Nikkor 18–105 mm lens. All photos were taken by hand, from an eye level, while walking a series of transects, across the area of interest. Spacing between shots was kept low to ensure a minimum of approximately 65% overlap between adjacent images. All processing was done using Agisoft Photoscan Pro/Metashape Pro (v. 1.7.1). Standard processing steps , Ultimaker Cura software (v.4.8.0) was used to rotate the raw scans and align them with x and y axes. This was a manual process. These rotations were exported to binary-format .stl files. The rotated files were then run through an R script using R version 4.0.3. The R script uses the tidyverse and rgl libraries to load the .stl files into R-friendly dataframes and plot them as contours using ggplot’s geom_contour function. The script is available through GitHub.57  to check model orientation. If necessary, models were reoriented to allow the local ground surface to be level using the "level" tool, and then the files were exported. The correctly oriented model was imported into SAGA GIS using the import stereo lithograph file (STL) tool.  This tool converts the .stl directly to a DEM raster. The rasters were checked in SAGA and a hillshade generated with the analytic hillshading function using the standard sun position setting of 315° azimuth and 45° height. Both the DEM and hillshade were then exported as geotiffs. These geotiffs were imported into ArcGIS for visualization. The DEM was colored using a red-blue colour ramp to indicate relative depth and this was layered onto the hillshade raster using the NAGI fusion methodThree-dimensional surface scans of Laetoli A and plaster casts of bear prints were collected using a Creaform Go!Scan 50.Further information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-021-04187-7.Supplementary InformationAgriotherium) and Supplementary Methods .This file contains Supplementary Discussion (Laetoli A rediscovery and description; Reporting SummaryPeer Review File


Output spike bursts are thought to implement key dendritic computations, such as coincidence detection of bottom-up inputs  and top-down inputs . In this study we used a detailed nonlinear model of L5PC receiving excitatory and inhibitory synaptic inputs to explore the conditions for generating bursts and for modulating their properties. We established the excitatory input conditions on the basal versus the apical tree that favor burst and show that there are two distinct types of bursts. Bursts consisting of 3 or more spikes firing at < 200 Hz, which are generated by stronger excitatory input to the basal versus the apical tree, and bursts of ~2-spikes at ~250 Hz, generated by prominent apical tuft excitation. Localized and well-timed dendritic inhibition on the apical tree differentially modulates Na+, Ca2+, and NMDA spikes and, consequently, finely controls the burst output. Finally, we explored the implications of different burst classes and respective dendritic inhibition for regulating synaptic plasticity.The output of neocortical layer 5 pyramidal cells (L5PCs) is expressed by a train of single spikes with intermittent bursts of multiple spikes at high frequencies. The bursts are the result of nonlinear dendritic properties, including Na The output of most neurons consists of stereotypical electrical pulses, or spikes. Some neurons generate sparse spikes intertwined with groups of spikes in close succession, called bursts. Previous research has implicated bursts in long-range transmission, dendritic computation, and plasticity. However, a detailed account of the spatiotemporal synaptic activation patterns that generate bursts, even in the principal cortical pyramidal neurons, is largely missing. Using experimentally constrained computational modelling we determined the conditions of synaptic activation patterns that induce burst firing in these cells. We found two distinct classes of bursts. They are distinguished by the number of active synapses, on either basal or apical part of the dendritic tree, required for burst initiation, and by the number and frequency of output spikes. We provide an analysis of how location and timing of dendritic inhibition finely edit somatic bursts and show the change in the strength of excitatory synapses during bursts following their dendritic inhibition. Overall, this work offers a deeper understanding of the origin of bursts, including a novel distinction between burst classes and characterization of how inhibition may shape bursts and their correlated synaptic plasticity. Layer 5 pyramidal cells (L5PCs) are considered to be pivotal building blocks of the mammalian neocortex . In rodein vivo tend to fire spikes in a random, Poisson manner such that the timing of each action potential is independent of its predecessors i, values result in “protected” (unchanged) efficacy of the excitatory synapses  to fit experimental findings. Using our model, we tested how L5PC excitatory synapses act under this plasticity rule, and explicitly how inhibition of dendritic Na+, Ca2+ and NMDA spikes (determined by location and Δtinh) controls the manifestation of this plasticity rule.Recent studies associate inhibition of bursts and of dendritic Calearning –44. To sty model ,46  with trunk (1), intermediate tuft (2) or distal (3) inhibition. Note that this maximum value of [Ca2+]i does not fully represent whether an excitatory synapse at the location will be potentiated or depressed, as the time that [Ca2+]i lasts at any given concentration will eventually determine the sign of plasticity. When [Ca2+]i rises from baseline to LTP levels, it goes through LTD levels, so the time above the respective thresholds (together with the learning rate) determines the final synaptic weight (the plasticity sign and magnitude). I.e., if [Ca2+]i crosses θd for a long duration but briefly reaches a maximum over θp, then 2+ hotspot (at location #1) on the apical trunk. The maximum [Ca2+]i at the hotspot is high and nearly invariant to inhibition timing at this inhibitory location  for a magnified effect on synaptic weights. For this proximal inhibition at location #1 the somatic burst is unaffected and therefore so is the Ca+2 spike at the hotspot (blue trace) and the distal NMDA spike (purple trace).ed there C1. Fig 62+ hotspot (at location #2). Well-timed inhibition (|dtinh|< 17 ms) at this location suppressed dendritic spikes, allowing Ca2+ influx only into the distal tuft  do synapses undergo LTP  values for plasticity. In p,d  inhibition of local NMDA spikes controls branch-specific plasticity by transforming LTP to local LTD or to the protected regime, and (3) following the association of bursts, Ca2+ spikes and inhibition with plasticity (see Discussion), we find a correlation between inhibition of bursts and limiting plasticity. The last point is in line with recent experimental studies, showing that limiting plasticity to specific connections relies on inhibition of bursts [We briefly summarize the key results of this section by noting that (1) inhibition located at the hotspot or immediately distal to it suppressed the Caf bursts –44.2+-dependent plasticity rule with whole-tuft excitation as in 2+]i outside the hotspot, meaning minor plasticity effects beyond LTP at the hotspot in the burst-suppressed dendritic tuft .We showed that somatic bursts arise either from a bidirectional stream of excitatory inputs combining basal and tuft synapses, or by generation of a local dendritic voltage plateau due to clustered excitatory input to the apical tuft alone Figs  and S1. ses Figs  and S3. ses Figs , high ex2+-dependent LTP in spines, resembling the classical asymmetric spike-timing dependent plasticity rules (STDP) i (concentrations) during the simulation at different inhibitory locations and times , which creates a more variable and realistic range of phenomena (LTP/D & protected). To generate synaptic weight changes in 2+]i measurements are difficult to control for in these relatively short simulations. Synapses on trees represent predicted weights after learning by this rule.Graphs are created for maximal i measured as in p/d values. The heatmap represents synaptic weight after plastic modification, normalized to initial value and colored on a log2 scale . Modified excitatory synapse location is ordered on x-axis. LTP threshold θp on y-axis. LTD threshold θd is kept at a fixed ratio of 0.6 to θp.[Ca(TIF)Click here for additional data file.S5 Figa. Model neuron as in 2+]i measurement as in 2+ hotspot. b1-3. Maximal [Ca2+]i along the apical dendrite at the three locations (colored electrodes in b) as a function of Δtinh between excitation and inhibition for each inhibition location. Dashed lines mark thresholds, shadings the resulting change–LTP (red), LTD (blue). c1-3. Synaptic weights after repeated execution of the learning rule (Eq (Methods) for 10 input repetitions (60 seconds) and Δtinh = -5 ms at each inhibition distance # . Twenty representative excitatory tuft synapses are color plotted by their respective weights: yellow—LTP (in 2), blue—LTD, and grey—protected.(TIF)Click here for additional data file.


Single-screw extrusion at high screw speeds is established nowadays since it allows for a high mass throughput at a comparatively small extruder size. Compared to conventional extrusion at low screw speeds, a considerable non-linearity in mass throughput appears by exceeding a certain threshold screw speed. In this study, the solid conveying behavior of different plastic granules with varying geometries was investigated in a smooth, a helically and an axially grooved solid conveying zone for screw speeds up to 1350 rpm. These experimental findings are compared to classical analytical predictions in the literature. It is found for the first time that both the shape and size of the plastic granules play a decisive role in determining the threshold screw speed at which a non-linear mass throughput is observed. It is shown that small and spherical granules exhibit a later onset of non-linear throughput compared to larger lenticular and cylindrical shaped granules. Moreover, it is revealed that the mass throughput equalizes for an axially and a helically grooved solid conveying zone at high screw speeds. This is contrary to the low screw speed range where the axially grooved barrel results in a significantly higher throughput than the helically grooved barrel. Thus, the maximum throughput at high screw speeds is limited by the granule stream provided by the hopper opening and is no longer governed by the groove angle. Single-screw extrusion is one of the most important processes in plastics industry. It allows for the mass production of semi-finished products such as foils, plates, tubes and other rather simple profiles . ClassicHigh-speed single-screw extruders are becoming more and more important since the end of the 1990s . The firTo investigate the influence of different screw designs on the decrease in specific throughput at high screw speeds, various screws with different flight pitch and depth were analyzed in , each wi® extrusion system, which consists of a helically grooved feeding and plasticizing zone in combination with a barrier screw, was also already examined for high-speed operation [The suitability of the commercially available Helibarperation ,19. The peration ,21. In tperation ,19.This phenomenon at high speeds is also well known for smooth barrel systems . There aSchneider ,27 invesPeiffer  developedgra to the groove depth hg and to the screw channel depth hs. The first case 1.(a) is equivalent to the case of Grünschloß and assumes a block flow in the groove direction, if the screw channel depth is smaller than two times the mean granule diameter and if the groove depth is smaller than the mean granule diameter. The second case 1.(b) is a mixed case, which assumes slipping between the granule layer in the grooves and the granule layer in the screw channel. This results in a different axial conveying velocity in the grooves and in the screw channel. The case 1.(b) is assumed to take place if the screw channel depth is larger than two times the mean granule diameter and if the groove depth is smaller than mean granule diameter. The third case 2.(a) and the fourth case 2.(b) apply if the screw channel depth as well as the groove depth are larger respectively significantly larger than the mean granule diameter. In contrast to the cases 1.(a) and 1.(b), both cases 2.(a) and 2.(b) presume that there is no additional mass flow in the grooves [For grooved barrel extruders, Schöppner  distingu grooves ,32. The n, the following linear approach is frequently applied [For predicting the mass throughput  applied :(1)m˙=vaf being the filling degree, which is assumed to be equal one at low screw speeds.With The axial conveying velocity The free channel cross-sectional area of the extruder can be determined according to Equation (3), whereas the first term reduces the free cross-sectional area by subtracting the flight area. The second term is equivalent to the free cross-sectional area of the grooves and thus disappears in smooth barrels.In Equation (3) A and B are fitting parameters without physical meaning.In order to consider the fact that the bulk density of the plastic granule is actually smaller within the extruder compared to the bulk density at high dumping heights, Grünschloß  formulatFurther references regarding the description of the solid conveying zone can be found in a review article of Wilczyński et al. . FurtherIt is known that the classical linear approach in Equation (1) fails to predict the mass throughput if a certain threshold screw speed is exceeded and thus only applies at low screw speeds. This phenomenon was already subject of different studies in the past, which consider the solid conveying at high screw speeds for smooth barrel extruders ,35,36.In contrast to smooth barrel extruders, there is a significant lack of systematic experimental data of the solid conveying behavior in extruders with grooved feed zones, particularly concerning high screw speeds. Thus, the objective of this work is to thoroughly study the solid conveying behavior in a smooth as well as in a helically and an axially grooved feed zone at high screw speeds up to 1350 rpm for the first time. Since the granule geometry is expected to play a decisive role in the solid conveying behavior, three different types of plastics and three different granule shapes are examined. The obtained data will be compared to the classical linear throughput predictions by assuming a nut-screw conveying based on . For attHostalen CRP 100 Black from the company LyondellBasell, Rotterdam, the Netherlands, which is a highly viscous extrusion type that can be used in pipe extrusion processes. The chosen polypropylene (PP) homopolymer is Moplen HP400H from LyondellBasell, Rotterdam, the Netherlands, which is, according to its data sheet, typically used in extrusion blow molded bottles. Besides these two polyolefins, Ultramid B40L from BASF SE, Ludwigshafen, Germany, was selected as a polyamide 6 (PA) which can be used for sheet extrusion. PE-HD and PP were chosen since polyolefins are the most common plastics and successfully processed with grooved barrel extruders. PA was chosen to investigate the effects of a material with distinctly different friction characteristics. Both the virgin PE-HD as well as the virgin PP exhibit a lenticular granule geometry, whereas the virgin PA has an almost spherical shape. Since the PA granule is not perfectly spherical, the diameter was measured at three different positions  is ions see . The thrIn order to obtain an additional grain shape, which is identical for all three materials, a small lab extruder (30/25D) from the Collin Lab & Pilot Solutions GmbH, Maitenbeth, Germany, equipped with a round-hole nozzle with 2.8 mm diameter was used to extrude the respective materials. The extruded strand was cut with a pelletizer unit to obtain cylindrical granules. Due to an imperfect pull-off procedure and gravitational effects the obtained cylindrical granule was not perfectly round, which is schematically shown in Since the geometrical dimensions may affect the solid conveying behavior and the vortex formation in the hopper at high screw speeds, a hundred random grains for each type of granule were measured with regards to its height and its two diameters. The equivalent spherical diameter (ESD) was calculated for classifying the granules based on the four solid conveying cases according to Schöppner, which were previously elucidated. Since doubling the ESD results in values larger than the screw channel depth and since the ESD is larger than the groove depth case 1.(a) can be assumed for five granule types. The only exception is the spherical PA, where case 2.(a) applies, because the ESD is smaller than the groove depth. Furthermore, the average grain mass was measured by weighing a hundred grains for each type see .The bulk density was determined as a function of the dumping height according to the approach of Grünschloß . The meaThe experimental investigations were primarily focused on analyzing the solid conveying behavior in the mere feed zone and thus melting of the plastic should be avoided. Hence, an experimental setup was developed that only includes the solid conveying zone of a common extruder without further zones. The setup shown in This experimental setup enables solid conveying without a back pressure that counteracts on the conveying capacity because the granule can trickle out freely after exiting the feed zone. For all mass throughput measurements, the granule exiting the feed zone was collected for 45 s at a certain screw speed and subsequently weighed. The measurements showed a high reproducibility with a maximum deviation of 5% between independent series of tests. This deviation comes primarily from the human reaction time of putting a vessel below the trickle-out area and removing it immediately after the measurement time.2. The bolt at the left end enabled the progressive tensioning of the spring and the force sensor recorded the applied axial force. The ball bearing ensured that the rotational movement of the screw respectively the cone is neither transferred to the spring nor to the force sensor.For studying the additional effect of a back pressure that usually acts upon the conveyed granule in real extrusion processes, a back pressure element was designed based on previous works  possessiLabVIEW from National Instruments Corporation, Austin, Texas, USA, respectively the calculated pressure oscillated rapidly over time. This was accompanied by a fast horizontal back and forth hammering of the simultaneously rotating conus. The back pressure values given in Chapter 3 are thus mean values with a standard deviation of around ±2 bar. In the following work only the mean values of the back pressure are denoted for simplicity. The maximum spring force of the utilized spring is 8.600 N. This theoretically allows applying up to 150 bar. Nevertheless, due to safety reasons and to avoid possible set-up damage, the maximum applied pressure was set to 80 bar.It was observed that despite intense water cooling both grooved barrels led to a partial clustering of the granule particles. This clustering emerged even in the freely trickling case without the mounted back pressure element in a certain screw speed range, which varied for the PE-HD, PP and PA. Since these solid particle clusters see  lead to In addition, the determined mass throughput of the mere solid conveying zone was compared to an entire extrusion set-up, including melting of the solid granule as well as outflow of the polymer melt through a die. Therefore, the lenticular PE and the lenticular PP were processed using a complete extruder set-up (35/34D). This entire extruder set-up possesses a throttle die, which allows for the adjustment of the flow area within the die and thus for adjusting the die back pressure (see Chapter 3.5). Increasing the die back pressure also leads to an overall pressure increase in the whole barrel. Two kinds of barrel were used: one barrel with a helically grooved solid conveying zone and a subsequent smooth barrel and one barrel which is smooth from end-to-end. In both cases, the geometry of the barrel and of the screw is identical to the geometries of the mere solid conveying zone experiments. For the grooved barrel extruder and for the full-length smooth extruder the solid conveying zone was cooled down to around 60 °C, whereas the residual zones and the die were set to 240 °C. 2 for the smooth barrel, 584.14 mm2 for the helically grooved barrel and 597.83 mm2 for the axially grooved barrel. Obviously, both grooved barrels possess an enlarged free cross-sectional area compared to the smooth barrel. The groove geometries were previously chosen to exhibit nearly the same free cross-sectional area which thus only differs by around 2% between the helically and the axially grooved barrel. First, the mass throughput for each conducted experiment is predicted by using the linear approach of Equation (1). To do so, the free channel cross-sectional area of the respective extrusion barrel is calculated by Equation (3) using the geometry values given in Determining the solid conveying angle to calculate the axial conveying velocity usually requires information about the systems governing friction coefficients. Since the experimental determination of the friction coefficients plastic-plastic, plastic-screw and plastic-barrel is error-prone, a nut-screw conveying according to Grünschloß is assumed for all used materials. This results in equalizing the solid conveying angle with the groove angle with The results of the bulk density determination for different dumping heights are shown in Combining Equations (1)–(3) and using in the respective geometrical values leads to the following equations:f equals one. The equations only depend on the bulk densities of the six different granule types and on the screw speed. It is obvious that the highest mass throughput for a certain type of granule and a certain screw speed is to be expected for the axially grooved barrel. This is due to the significantly higher axial velocity compared to the smooth and the helically grooved barrel. In addition, both grooved barrels exhibit a higher free cross-sectional area in which solid conveying can proceed, compared to the smooth barrel.In these three cases fully filled screw channels are assumed, meaning The results of the mass throughput determination of the lenticular PE-HD are shown in All measured curves in Mounting the back pressure element onto the solid conveying zone with a smooth barrel and applying a back pressure of 60 bar and 80 bar, respectively, results in a small mass throughput decrease in the case of 60 bar and a significant decrease in the case of 80 bar, particularly at low screw speeds. Nevertheless, by exceeding 300 rpm the measured values nearly equalize. Whether this latter observation is caused by real effects or by operational uncertainties of the back pressure element must be evaluated in future work.By examining the mass throughput prediction based on the simple linear law in Equation (1) it becomes obvious that this approach is only reasonable up to around 300 rpm. For the range between 50–300 rpm the mean relative deviation between the calculated mass throughput and the measured mass throughput is approximately 14% for the smooth barrel, 9% for the axially grooved barrel and only 2% for the helically grooved barrel.The results of the cylindrical PE-HD are shown in Quantitative comparison of the cylindrical PE-HD and the lenticular PE-HD always reveals a lower mass throughput for the cylindrical PE-HD for the whole screw speed range. The maximum value at 1350 rpm using lenticular PE-HD is about 400 kg/h, whereas using the cylindrical PE-HD results in a maximum value of around 360 kg/h. This observation also applies for the low screw speed range between 50–300 rpm. Here, the lenticular PE-HD possesses a 5–8% higher mass throughput for the axially grooved barrel, a 7–8% higher throughput for the helically grooved barrel and a 7–13% higher throughput for the smooth barrel, always compared to the cylindrical PE. This can mainly be explained by the bulk density difference at a dumping height of 5.5 mm which is about 7% higher in the lenticular PE-HD case see .Utilizing a smooth barrel and lenticular PE limited the maximum screw speed to 600 rpm since further increase led to an intolerable noise generation due to friction. The applied back pressure of 60 bar and 80 bar led to a considerable reduction of mass throughput for all examined screw speeds.Comparing the calculated throughput with the measured throughput of cylindrical PE reveals a proper prediction accuracy. The relative deviation is around 8% in the case of the smooth barrel, around 8% in the case of the axially grooved barrel and only 1% in the case of the helically grooved barrel, considering the low screw speed range of 50–300 rpm.Again, the calculation of the axially grooved barrel tends to overestimate the throughput. This reveals that the assumed solid conveying angle of 90° is probably somewhat lower in reality. Despite the solid conveying angle of 25° is just a simplified assumption in the case of the smooth barrel, the prediction accuracy is surprisingly well. The very low deviation of the predicted values to the measured values in the helically grooved case reveals that the assumption of equalizing the solid conveying angle with the groove angle is appropriate in this case. To obtain proper results, it is also necessary to take into account the bulk density as a function of dumping height.The results of processing lenticular PP and cylindrical PP can be found in Applying a back pressure onto the solid conveying zone led only to a vanishingly small mass throughput reduction for both PP granules with a slightly higher reduction for 80 bar compared to 60 bar, in contrast to the observations of PE-HD. This reveals that not only the granule geometry but also the type of plastic strongly influences the back pressure dependency in the solid conveying zone. In order to properly account for this, the compressibility of the different granules should be considered in future research as a further variable. A description of an experimental and a simulative approach to determine the compressibility can be found in .In the case of lenticular PP, the analytical predictions result in a mean deviation of around 8% for the smooth barrel, around 9% for the axially grooved barrel and only 1% for the helically grooved barrel, considering the low screw speed range from 50–300 rpm. Again, using the helically grooved barrel leads to an extremely well mass throughput prediction, whereas the calculations for the axially grooved barrel overpredict the mass throughput.The cylindrical PP is an exception in the speed range from 50–300 rpm since the measured mass throughput coincides well with the predicted throughput for the axially grooved barrel with a relative deviation of only 3%. Contrary to this, in the helically grooved case the calculation underestimates the measured throughput by around 11%. The exact reason for this quite high deviation in the helically grooved case cannot be given. However, it can be assumed that the measurement of the bulk density for the cylindrical PP is error-prone to a certain extent. Thus, the real bulk density is expected to be higher than the measured bulk density. The granule size distribution of the virgin lenticular PP is notably smaller compared to the broad size distribution of the regranulated cylindrical PP. Hence, it can be assumed that measuring the bulk density for regranulates is more susceptible to error compared to uniform virgin granules. This observation emphasizes the importance of a careful determination of the bulk density as a prerequisite for enabling an accurate mass throughput prediction, especially for granules which possess a broad size distribution.Processing the spherical PA and the cylindrical PA with the helically grooved barrel led to a very high extruder torque which exceeded the permissible motor power. Hence, both series of experiments could not be performed even at low screw speeds. Moreover, a high mechanical wear occurred at the cone of the back pressure element when processing the spherical PA because PA exhibits a significantly higher hardness compared to the previously examined polyolefins. Hence, applying a back pressure was omitted in the case of cylindrical PA to avoid further damage. Due to the previous explanation the maximum back pressure was set to 55 bar for the spherical PA. This resulted in a mean decrease in mass throughput of around 14–18% for both 30 bar and 55 bar compared to experiments without back pressure see .Spherical PA results in a very large mass throughput with around 505 kg/h at 1350 rpm. In the case of an axially grooved barrel a linear behavior is observed until 600 rpm with the specific throughput remaining constant at around 0.52 (kg/h)·min. Using the smooth barrel even results in a linear behavior until 1200 rpm with a specific throughput of around 0.29 (kg/h)·min. The cylindrical PA exhibits an early onset of degressive behavior at 300 rpm for both the smooth and the axially grooved barrel as can be seen When processing the spherical PA in the smooth barrel, the prediction quality is very good with only 3% relative deviation between the calculated and the measured throughput at screw speeds between 50–300 rpm. With regard to the axially grooved barrel, however, the prediction quality is very poor with a relative deviation of about 35%. Nevertheless, even though the calculations for the axially grooved barrel usually exceeded the measured data in the previous cases, this deviation is the highest deviation observed in this work. This can be explained by the fact that applying Equations (5) and (7) assumes a nut-screw conveying, which resembles the solid conveying case 1.(a). However, this assumption is not applicable to the spherical PA since the ESD of 2.68 mm is smaller than the groove depth of 2.8 mm. Hence, to improve the prediction quality the solid conveying case 2.(a) should be used (see Chapter 1.2). This would exclude the additional mass flow in the grooves but still contain a friction increase to the barrel due to the grooves filled with plastic granule and thus a variation of the solid conveying angle.As opposed to this, the results of the smooth solid conveying zone and the full-length smooth extruder differ significantly between 28–48%. The results of the mere solid conveying zone are thus not transferable to entire extrusion processes when a smooth barrel is utilized. In this case, the measured values are always substantially lower for the entire extrusion set-up because of a pronounced counter pressure dependent conveying behavior of smooth barrel systems .The results of melt processing the lenticular PP with the helically grooved entire extruder set-up are depicted in In this study, the solid conveying behavior of different plastic granule geometries was investigated experimentally for the first time using a smooth as well as two grooved barrels up to screw speeds of 1350 rpm. It was found that the shape and size of the plastic granule play a decisive role in determining the start of a degressive mass throughput behavior. A small and spherical granule exhibited an advantageous behavior compared to larger lenticular and cylindrical shaped granules. In the case of the spherical granule, the threshold screw speed is 1200 rpm for a smooth barrel and 600 rpm for an axially grooved barrel. In the case of the lenticular and the cylindrical granules the threshold screw speed is only around 300 rpm for both smooth and grooved barrels.Moreover, it was revealed for the first time that the mass throughput equalizes for an axially and a helically grooved solid conveying zone at screw speeds higher than approximately 800 rpm. This is contrary to the low screw speed range where the axially grooved barrel results in a significantly higher throughput than the helically grooved barrel. Thus, the maximum throughput at high screw speeds is limited by the granule stream provided by the hopper opening and is no longer governed by the groove angle respectively the associated solid conveying angle.Furthermore, it was shown that using a simple linear calculation approach and assuming a nut-screw conveying results in a proper prediction for the helically grooved solid conveying zone at low screw speeds. However, this applies only if the granule possesses a diameter that is larger than the groove depth and if the screw channel depth is smaller than two times the granule diameter (see solid conveying case 1.(a)). Contrary to the accurate prediction in the helically grooved case, using the linear approach always led to an overestimation for the axially grooved solid conveying zone since the actual solid conveying angle in the screw channels is somewhat lower than 90°.A full-length smooth extruder notably differed in the mass throughput compared to the mere smooth solid conveying zone. As opposed to this, the mass throughput for an entire extruder with a grooved feed zone and a subsequently smooth melting and metering zone was in very good accordance with the results of the mere grooved solid conveying zone. This confirms the counter pressure independent behavior of these grooved set-ups. As a next step, a regrind material will be investigated in future work to work out whether using the linear calculation approach, which assumes spherical granules, still holds for plastics with a plate-shaped geometry. Moreover, plastic powder will be examined to investigate whether the late start of degressive throughput behavior is also found when processing spherical materials with a very small particle size. Furthermore, to appropriately incorporate the non-linear behavior at high screw speeds, the obtained experimental results will be used in following work to establish a new analytical calculation model. This new model shall be based solely on physically meaningful parameters such as the average granule mass, the aspect ratio or the pourability of the granule. One possible way could be a coupling of the linear approach with a proper function of limited growth, to account for effects such as a vortex formation in the hopper.Besides this proceeding the solid conveying problem in a grooved barrel can also be addressed via numerical simulation using the discrete element method (DEM) which appears to be another promising way. This DEM approach was previously examined in ,38 for l


Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme. GFAT-1 activity is modulated by UDP-GlcNAc feedback inhibition and via phosphorylation by protein kinase A (PKA). Molecular consequences of GFAT-1 phosphorylation, however, remain poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans. In human GFAT-1, the R203H substitution interferes with UDP-GlcNAc inhibition and with PKA-mediated Ser205 phosphorylation. Our data indicate that phosphorylation affects the interactions of the two GFAT-1 domains to control catalytic activity. Notably, Ser205 phosphorylation has two discernible effects: it lowers baseline GFAT-1 activity and abolishes UDP-GlcNAc feedback inhibition. PKA controls the HP by uncoupling the metabolic feedback loop of GFAT-1.The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5’-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in  The glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting enzyme in the hexosamine pathway producing uridine 5’-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), an essential glycosylation precursor. Here, the authors dissect the mechanisms of GFAT-1 regulation by protein kinase A (PKA)-mediated phosphorylation. N-acetyl-D-glucosamine (UDP-GlcNAc) , acetyl-coenzyme A, and uridine2. Hence, the HP integrates sugar, amino acid, fatty acid, and nucleotide metabolism and is considered an important nutrient sensing pathway. The GlcNAc moiety of UDP-GlcNAc is used as a building block for several macromolecules, including peptidoglycans in bacteria, chitin in fungi and insects, or glycosaminoglycans such as hyaluronic acid in vertebrates8. Moreover, UDP-GlcNAc is an essential precursor for glycosylation reactions in eukaryotes. N-linked glycosylation takes place in the endoplasmic reticulum (ER) and is crucial for proper protein folding and the solubility of proteins9. Mucin-type O-linked glycosylation occurs in the Golgi apparatus and is found on many cell surface and secreted proteins10. In protein O-GlcNAcylation that occurs in cytoplasm and nucleus, UDP-GlcNAc is the donor for the attachment of a single GlcNAc moiety to serine or threonine residues11. This process can fine-tune a protein’s function akin to phosphorylation and often O-GlcNAcylation and phosphorylation compete for modification of the same sites13.The hexosamine pathway (HP) converts fructose-6-phosphate (Frc6P) to uridine 5′-diphospho-Ac) Fig. 1. In all2. Two GFAT paralogs exist that primarily differ in their tissue-specific expression patterns14. GFAT is a modular enzyme composed of a glutaminase domain responsible for hydrolysis of l-Gln into l-glutamate (l-Glu) and an isomerase/transferase domain that catalyzes the isomerization of Frc6P to glucose-6-phosphate (Glc6P) as well as the transfer of ammonia to Glc6P to build glucosamine-6-phosphate (GlcN6P)15. The relative orientation of the two domains with their respective active sites is relevant for catalysis and affected by substrate binding. In the absence of Frc6P, the glutaminase domain is very flexible .6.1.162.gfat-1 mutations cause limb-girdle congenital myasthenic syndrome with tubular aggregates23. This inherited disorder is characterized by defective neuromuscular transmission through impaired neuromuscular junctions, which transmit impulses from motor neurons to skeletal muscle fibers22. Moreover, many studies suggest a role of the HP and GFAT in the development and progression of diabetes or cancer26. Both disorders are characterized by an aberrant glucose metabolism, and the HP links altered metabolism with aberrant glycosylation. Especially elevated O-GlcNAcylation contributes to the pathogenesis of diabetes and cancer. Altered O-GlcNAcylation was also reported to play a critical role in neurodegenerative diseases, heart disease, and inflammation28.A disturbed function of GFAT-1 due to 31 and through phosphorylation33. In human GFAT-1, Ser205 and Ser235 are phosphorylated by cAMP-dependent protein kinase A (PKA)35. Ser205 phosphorylation has reported effects on GFAT-1 activity, although the published data are contradictory: On the one hand, phosphorylation of Ser205, and the corresponding Ser202 in human GFAT-2, were reported to increase activity35. On the other hand, it was described that Ser205 phosphorylation leads to decreased GFAT-1 activity34. Phosphorylation of the second PKA site Ser235 in GFAT-1, which is not conserved in all eukaryotes and is absent in GFAT-2, seems not to affect the enzymatic activity36. Ser243 was shown to be phosphorylated by adenosine monophosphate (AMP)-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II, but the effect on GFAT activity is not clear: depending on the study, phosphorylation at this site is reported to be either activating or inhibiting38.Given that GFAT drives the metabolic flux of the HP, its regulation is of great physiological importance and in eukaryotes it occurs through UDP-GlcNAc feedback inhibitionCaenorhabditis elegans forward genetic mutagenesis screen we identified gain-of-function mutations in gfat-1 that suppress tunicamycin-induced proteotoxic stress39. These mutations result in single amino acid substitutions in GFAT-1 and increase UDP-GlcNAc concentrations in the worms that also show improved protein quality control as well as a significant lifespan extension39. Recently, we found that loss of regulation by UDP-GlcNAc feedback inhibition is one gain-of-function mechanism and identified a critical role of the interdomain linker in UDP-GlcNAc inhibition40. Although UDP-GlcNAc binds to the isomerase domain of GFAT-1, this interaction inhibits the glutaminase function and consequently the combined GlcN6P production41. We proposed that UDP-GlcNAc disturbs the tight coupling of the active sites by interacting with the interdomain linker and by interfering with the relative orientation of the two domains40  allele due to the GFAT-1 R203H gain-of-function substitution.Previously, we performed a forward genetic screen in nce Fig. . To ident-1 Fig. . To testase Fig. , the enznts Fig. . Given tele Fig. . We concC. elegans to humans   Fig. . SubsequµM) Fig. . While AµM) Fig. . In the µM) Fig. . Togethe34  for both active sites  compared to wild type GFAT-1 (kcat/Km: 21.3 mM−1 sec−1)  Table . These d6-tag  and the UDP-GlcNAc inhibition remained unaffected by the C-tail lock mutation  R203H mutant points to the predominant role of the defective feedback inhibition as the relevant gain-of-function mechanism.We describe two independent gain-of-function mechanisms of GFAT-1 R203H. First, when UDP-GlcNAc levels are low, the reduced Ser205 phosphorylation in GFAT-1 R203H results in higher GFAT-1 activity compared to wild type GFAT-1 that is inhibited by Ser205 phosphorylation. Second, when UDP-GlcNAc levels are high, wild type GFAT-1 is under stronger feedback inhibition than GFAT-1 R203H, resulting in elevated cellular UDP-GlcNAc levels in the mutant. The observation of high UDP-GlcNAc levels in whole worm lysates in the 40. In general, domain movements are common in the family of glutamine amidotransferases and the function of GFAT depends on the relative orientation of the glutaminase and isomerase domains44. Mouilleron et al. reported a structure of the E. coli GFAT active site mutant C1A (PDB 3OOJ) with a drastically changed orientation of the glutaminase domain relative to the isomerase domain, where one monomer adopts an inactive orientation without forming the ammonia channel45. This structure underpins the high flexibility of the two domains relative to each other. In wild type E. coli GFAT, the glutaminase domain adopts a specific position relative to the isomerase domain after Frc6P binding16. The presence of Frc6P activates the glutaminase function of GFAT more than 100-fold in E. coli and 70-fold in C. albicans and it is very likely that this substrate-induced activation occurs in the human GFAT as well41. Also, l-Gln binding induces a rotation of the glutaminase domain relative to the isomerase domain in E. coli GFAT19. These domain movements upon substrate binding are required to enable specific interactions between the isomerase and glutaminase domains that are necessary for catalysis. It is well-established that phosphorylation can introduce conformational changes within loops or domains through altered electrostatic interactions46. A well-studied example for a phosphorylation-induced change in domain orientation is rabbit muscle glycogen phosphorylase, whose domains are shifted by ~50° relative to each other after phosphorylation48. The phosphorylation of GFAT-1 at Ser205 would introduce, at physiological pH, a negatively charged phosphate group, potentially affecting ionic interactions in close proximity. Arg202 of the glutaminase domain and Glu425 of the isomerase domain likely form a salt-bridge between the two domains and this interaction might be affected upon Ser205 phosphorylation  to mediate signals of neurotransmitters and hormones, such as glucagon or adrenaline55. The downstream protein targets of the cAMP-PKA signaling pathway regulate glucose homeostasis by inhibition of glycolysis and glycogen synthesis, as well as by triggering glucose release through stimulation of glycogenolysis and gluconeogenesis55. Our previous data indicated that GFAT-1 is under a constant UDP-GlcNAc inhibition in vivo40. Presumably, uncoupling of the feedback inhibition maintains GFAT-1 activity and ensures steady UDP-GlcNAc production when PKA activity is high. Given that UDP-GlcNAc is an essential building block and precursor for all glycosylation reactions in mammals, this mechanism is optimally positioned to ensure a constant UDP-GlcNAc supply. In all, our findings illuminate how the different means of GFAT-1 regulation, kinase signaling and feedback inhibition, are coordinated at the molecular level to fine-tune metabolic flux in the HP.Very few enzymes are known in which phosphorylation interferes with feedback inhibitionC. elegans were maintained at 20 °C on nematode growth medium (NGM) agar plates with E. coli OP5056. The mutant was outcrossed to the wild type N2 strain. Strains used in this study: N2 (Bristol), AA2559 [gfat-1(dh783)], MSD544 [gfat-1(syb3246)] with the S202D substitution. The homozygous syb3246 allele is lethal and mutants were maintained as a balanced heterozygous strain (gfat-1(syb3246)/mIn1 using: [θ] = θ/(10 n c l) in deg cm2 dmol−1 .GFAT-1 was dialyzed in 10 mM potassium phosphate buffer pH 7.5, 0.5 mM TCEP, 0.5 mM Na®-4 (HORIBA) spectral fluorimeter at a concentration of 1.5 mg/ml in SEC buffer without Frc6P at 20 °C. Excitation wavelength was set to 295 nm and the spectra were recorded in a range of 305 to 450 nm in a quartz cuvette with 1.5 mm path length. The bandwidth for the excitation monochromator was 4 nm and for the emission monochromator 2 nm.Fluorescence emission spectra were measured with a FluoroMax2 on treated polystyrene culture dishes (Corning) in DMEM media with high glucose  with pyruvate  supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco).HEK293 cells (ATCC) were cultured at 37 °C, 5% CO6-tag between Gly299 and Asp300 (pcDNA3.1-hGFAT1-His299). Internally tagged GFAT-1 was subcloned from pFL-hGFAT1-His299 using NheI and HindIII restriction sites. For each variant, one well of a 6 well plate was transfected with 2 µg of plasmid DNA with Lipofectamine® 2000 (LifeTechnologies™) according to the manufacturer’s protocol. Selection was performed with 500 µg/ml G418 for several weeks. For Western blot analysis, cells were washed, and proteins were extracted with RIPA-buffer  NP40, 0.5% (w/v) deoxycholate, 0.1% (w/v) SDS; pH 7.5). Cell debris was removed by centrifugation, protein concentration of the supernatants were determined using Pierce™ bicinchoninic acid (BCA) protein assay kit (Thermo Scientific) according to the manufacturer’s protocol. Two micrograms of cell lysate was separated by reducing SDS-PAGE and transferred to PVDF membranes. Primary antibodies against human GFAT1 and α-TUBULIN were used. Chemiluminescence of the appropriate secondary rabbit or mouse HRP-conjugated antibodies after incubation with ECL HRP substrate  was detected using a ChemiDoc™ Quantity One® system (Bio-Rad).Cells stably overexpressing GFAT-1 variants were generated by transfection of HEK293 cells with pcDNA3.1 plasmids with human GFAT-1 containing an internal HisThe following antibodies were used: GFAT1 , α-TUBULIN , rabbit IgG , and mouse IgG .Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data1Reporting Summary


A convenient, environment-friendly, and cost-effective method to keep anti-icing for a long time was highly desirable. Slippery lubricant layers were regarded to be effective and promising for anti-icing on different surfaces, but the drought-out of lubricants and the possible detriments to the environment were inevitable. By combining super-high molecular weight sodium polyacrylate (H-PAAS) with polyolefin through a one-pot method, a self-sustainable lubricating layer with extremely low ice adhesion of un-freezable water hydrogel was achieved at subzero conditions. The lubricant hydrogel layer could auto-spread and cover the surface of polyolefin after encountering supercooled water, frost, or ice. Due to the reduction of storage modulus in the interface, the ice adhesion of the specimen surfaces was far below 20 kPa, varying from 5.13 kPa to 18.95 kPa. Furthermore, the surfaces could preserve the fairly low adhesion after icing/de-icing cycles for over 15 times and thus exhibited sustainable durability. More importantly, this method could be introducing to various polymers and is of great promise for practical applications. Due to precipitation and cold conditions, ice froze and accumulated on various horizontal and vertical surfaces, such as power lines , airplanThe traditional passive anti-icing surfaces mainly included nanostructured superhydrophobic surfaces (SHS) ,10,11 an2CH(CH3)]n) granules were obtained from Macklin Biochemical Co., Ltd. . Super-high molecular weight sodium polyacrylate powder ]n) with Mn of 30,000,000 was purchased from Heowns Biochemical Company .Polypropylene , and then, the mixture was poured into a double screw extruder  to make PP-H-PAAS granules after mixing for 30 min at 180 °C. Finally, circular specimens (PP-H-PAAS) with a diameter of 7 cm and a thickness of 3 ± 0.2 mm were produced by an injection molding machine  under a pressure of 0.7 MPa for 30 s. The H-PAAS mass fractions in the specimens were 0%, 5%, 10%, 15%, 20%, which were labeled as 0, 1, 2, 3, and 4, respectively.As was shown in The air-conditioning system was composed of a thermostatic bath, temperature control platform, and a T-type micro temperature probe. The surface temperature of the specimen was accurately controlled from 25 to −15 °C.di) were determined when transparent hemispherical shape changing to a dark opaque triangular-like peak.According to our previous research , in situThe CAH also measured the specially modified optical angle meter as shown in A was the ice covering area, F was the vertical tension. Given that As shown in ce layer , a plastThe icing/de-icing cycle test was carried out on the Specimen 3 by repeating the same procedures of ice adhesion measurement for 15 times. Each time, after the drawing machine was used to divorce the spindle and the ice layer from the center of the specimen surface, another three globes were inserted to control the gap height at 2 mm. Afterward, the deionized water was injected to form an ice layer again for next icing/de-icing cycle. After completing the de-icing process, the ice layer still adhered to the bottom of the spindle as shown in The surface morphology and elemental analysis of the specimens were recorded by the scanning electron microscope  equipped with an energy diffraction spectrum (EDS) at 5.0 kV. Besides, the roughness of the specimen surfaces was tested by the atomic force microscope . The scan size was 20 µm × 20 µm, the scan rate was 60 µm/s and sample lines were 256. The chemical composition was further investigated by X-ray photoelectron spectroscopy . The number of scans was 5, the source gun type was Al K Alpha, the spot size was 400 µm, and the energy step size was 0.100 eV.S) was defined asThe water swelling ratio  formed in the interface of specimen and water. The emergence of the lubricant hydrogel layer lowered the DCA, so it took more than one hour for the whole CA equilibrium process, as shown in According to Equation (1), the water swelling ratio was about 300. To determine the dynamic mechanical properties of the hydrogel, the hydrogel on the surface of specimens was collected in the cup of the DMA instrument. As shown in di of specimens. The Tdi at −10 °C increased in the H-PAAS mass fraction. For the SHS, the higher and bigger the contact angle was, the longer the delay time of ice formation [− of H-PAAS exposed to the external surface quickly to capture water under the side-chain extension motion and extended an extra thin lubricant hydrogel layer between the water phase and substrate material phase as seen in + of polyelectrolyte proliferated in the lubricant hydrogel layer, the lower the freezing point due to the hydration [+ and -COO− having entered the hydrogel layer and these ions could reduce the freezing point to delay icing. On the other hand, the main chains of H-PAAS were long enough to take nearly 2 h for these main chains to stretch completely when encountering water, which led to the continuous decrease in the DCA value as shown in + and -COO− of side chains of H-PAAS kept entering the hydrogel layer. The decrease in the DCA value also meant the decrease of the contact line, which resulted in the consequence that the interface area between the water phase and specimen phase kept increasing. This could damage the formed ice crystallization and delayed icing. Therefore, the super cooler water near the lubricant hydrogel layer was difficult to freeze and the icing time was delayed. Therefore, Specimen 4 attained a super long unfrozen level of 3048 s, which was 126-fold of PP.ormation . Quite dydration ,38,39. N+ and -COO− combined with water molecules began to form an H-PAAS-LHL and spread to further sites which drove the H-PAAS main chains to move, contributing to the auto-spread of H-PAAS-LHL. In consequence of this, both H-PAAS islands and PP sea were covered by a smooth H-PAAS-LHL as seen in + iron on the topmost H-PAAS-LHL layer lowered the freezing point and formed an ultra-thin un-freezable water layer. The slippery layer beneath ice decreased the ice adhesive strength of the surface of compound materials.As shown in + ions which was of less weight and could move randomly in the water phase at a higher speed. However, the -COO− anions were fixed on the H-PAAS main chains by the C-C binding, so the displacement of -COO− anions was limited. That is why the migration speed of Na was more rapid than that of O.To intensively investigate the detailed chemical composition changes before and after water absorption, XPS tests were performed. Receding contact angle was another important factor in close relation with the ice adhesion strength. The ice adhesion strength of the specimens was shown in 1.56 MPa , the τicDurability was another essential aspect considered for an icephobic material . To evalA novel icephobic engineering raw material was introduced by blending polypropylene with super-high molecular weight sodium polyacrylate through a one-pot method. Objects with icephobic properties were produced by simple molding processing to form a lubricant hydrogel on the surfaces even in a cold environment. The designed surfaces performed greatly low ice adhesion properties. Compound materials with 20% H-PAAS attained a super long unfrozen level of 3048 s, which was 126 fold of the pure PP. The adhesion strength on the sample surfaces was all below 20 kPa. The minimal value (5.14 kPa) was obtained with 15% H-PAAS being added. Furthermore, the surfaces could maintain this low adhesion level during icing/de-icing cycles for over 15 times. Therefore, a convenient, environment-friendly, self-sustainable, and cost-effective method on different materials provided more opportunities for producing polymeric materials with durable and anti-icing properties.


Anemona nemorosa and Muscari sp. have not yet been described for herbaceous species that of Jasminum nudiflorum corroborates findings for woody species. Flowers of Galanthus nivalis and Scilla forbesii were ice tolerant. For all herbs, it became clear that the soil acts as a thermal insulator for frost susceptible below ground organs and as a thermal barrier against the spread of ice between individual flowers and leaves. Both ice barrier types presumably promote that the reproductive organs can remain supercooled, and can at least for a certain time-period escape from effects of ice formation. Both effects of ice barriers appear significant in the habitat of the tested species, where episodic freezing events potentially curtail the reproductive success.In the temperate zone of Europe, plants flowering in early spring or at high elevation risk that their reproductive organs are harmed by episodic frosts. Focusing on flowers of two mountain and three early-flowering colline to montane distributed species, vulnerability to ice formation and ice management strategies using infrared video thermography were investigated. Three species had ice susceptible flowers and structural ice barriers, between the vegetative and reproductive organs, that prevent ice entrance from the frozen stems. Structural ice barriers as found in  As reproductive organs of plants are most sensitive to freezing , and espRanunculus glacialis L. could be proven to tolerate ice formation without freezing injury . Cross-G. nivalis). For each experiment, ice nucleation temperatures of vegetative vs. reproductive plant parts was compared, regressing plant parts as fixed and individual as random factors on the ice nucleation temperature. For the experiment with J. nudiflorum, in which only one twig was used and for the second experiment with Muscari sp., in which only two flower stalks were used, no statistical comparison was possible. For the first experiment with G. nivalis, where the assignment of leaves to particular individuals was impossible  implemented in the function “lmer” in the package “lmerTest” . LME allpossible  an ANOVAp < 0.05 .


Danio rerio) and antityrosinase activity of the ethanolic extract and its phytochemicals from Ceylon olive (Elaeocarpus serratus Linn.) leaves were investigated in this study. Among the leaf extract and four soluble fractions, the ethyl acetate soluble fraction exhibits the best antityrosinase and antimelanogenesis activities. One phenolic acid, gallic acid, and two flavonoids, myricetin and mearnsetin, are isolated from the active subfractions through the bioassay-guided isolation; their structures are elucidated based on the 1D and 2D NMR, FTIR, UV, and MS spectroscopic analyses. These compounds have significant antityrosinase activity whether using l-tyrosine or l-DOPA as the substrate; mearnsetin shows the optimal activity. In the enzyme kinetic investigation, both gallic acid and mearnsetin are the competitive-type inhibitors against mushroom tyrosinase, and myricetin acts as a mixed-type tyrosinase inhibitor. Leaf extract and an ethyl acetate soluble fraction show effective performance in the inhibition of melanin formation in zebrafish embryos. Mearnsetin also possesses a promising antimelanogenesis effect, which is superior to the positive control, arbutin. Results reveal that the Ceylon olive leaf extract and its phytochemicals, especially mearnsetin, have the potential to be used as antimelanogenesis and skin-whitening ingredients.The melanogenesis inhibition effect in zebrafish ( Melanin plays an essential role in many biochemical functions; it provides protection for the skin from ultraviolet (UV) damage, the elimination of reactive oxygen species (ROS), and other biochemical reactions ,2,3. TheArctostaphylos uva-ursi), the extract of green tea, alpha hydroxyl acid (AHA), ascorbic acid, and so on  × 100. Then, the half-maximal inhibitory concentration (IC50) of the specimen was calculated from the concentration response curve.In vitro antityrosinase assay was a spectrophotometric analysis based on the methods previously described ,34,35. ll-tyrosine/l-DOPA) concentration was varied at 0.5, 0.75, 1.0, 1.25, and 1.5 mM. The reaction was similar to antityrosinase assay as described above; 40 µL of specimen solution and 70 µL of potassium phosphate buffer  were mixed, which was followed by adding 50 µL of 200 unit/mL tyrosinase, after incubation at 25 °C for 10 min. Then, we added 40 µL of substrate into the well and mixed thoroughly, and kinetic measurements of the solution were measured immediately for a period of 3 min at the detected wavelength (475 nm). Both kinetic parameters, Michaelis–Menten constant (Km) and maximum velocity (Vmax), were calculated from the Lineweaver–Burk linear equation +, in agreement with the molecular formula C7H6O5. Myricetin (ES2): yellow needle; mp 358 °C; UV (MeOH) λmax (log ε) 252.5 (4.43) and 374.0 (4.50) nm; IR (KBr) νmax 3421, 1663, 1596, 1520, 1229, 1202, and 1171 cm−1; EI-MS m/z 319.36 [M+H]+, molecular formula C15H10O8. Mearnsetin : yellow powder; mp 184 °C; UV (MeOH) λmax (log ε) 259.5 (4.21) and 364.5 (4.26) nm; IR (KBr) νmax 3409, 2960, 2927, 2853, 1661, 1599, 1507, 1208, and 1163 cm−1; EI-MS m/z 333.0 [M+H]+, molecular formula C16H12O8. The NMR data of these compounds are summarized in Three compounds . Similar results were observed in the l-DOPA used as the substrate; the efficacy of three phytochemicals was superior to that of arbutin.Antityrosinase activity of these compounds from active subfractions were represented in m and Vmax, are determined by the enzyme initial rate at the different substrate concentrations in the Lineweaver–Burk plot; the intersect at the y-axis is equivalent to 1/Vmax, and the intersect at the x-axis is −1/Km. In the enzyme kinetic study of terpenoid compounds from the citrus peel essential oil against tyrosinase, citral acted as a noncompetitive inhibitor, and myrcene was a competitive inhibitor [A true enzyme inhibitor contains four modes of inhibition, including competitive, uncompetitive, mixed type (both competitive and uncompetitive), and noncompetitive . Kineticnhibitor . The inhnhibitor .l-tyrosine and l-Dopa, the linear regression line of different concentrations of mearnsetin had the same intercept on the y-axis and the increasing slope. The kinetic parameters of gallic acid, myricetin, and mearnsetin are summarized in m and a constant in Vmax were observed, indicating that both gallic acid and mearnsetin were the competitive inhibitor of tyrosinase. It indicated that gallic acid and mearnsetin could bind to free tyrosinase with high affinity and prevent substrate (l-tyrosine or l-DOPA) binding to the active site of tyrosinase. Myricetin was found to be a mixed-type inhibitor, containing competitive and uncompetitive inhibition, since Km was increased and Vmax was decreased. Uncompetitive inhibition demonstrated that compounds bind to the tyrosinase–substrate complex but not to the free tyrosinase.The inhibition type on tyrosinase of three phytochemicals was elucidated by an in vitro enzyme kinetic study. Danio rerio) is a novel and valid model organism for melanogenesis research in recent studies [Zebrafish  were evaluated in this study. Among leaf extract and its four fractions, ethyl acetate soluble fraction had the best antityrosinase activity. Phytochemicals, including gallic acid, myricetin and mearnsetin, were isolated and identified from the active subfractions by bioassay-guided chromatography and spectral analyses. The order of antityrosinase activity of these compounds was mearnsetin > myricetin > gallic acid. The in vitro enzyme kinetic study reveals that gallic acid and mearnsetin were competitive inhibitors of tyrosinase, and myricetin was a mixed-type inhibitor. Results from the zebrafish in vivo assay revealed that an ethyl acetate soluble fraction and three phytochemicals possessed significant melanogenesis inhibition effects. The antimelanogenesis effect of mearnsetin was superior to that of the positive control, arbutin, in zebrafish.


Antarctica’s largest contributor to sea level has sped up further as a major section of its floating ice shelf collapsed. Speedup of Pine Island Glacier over the past several decades has made it Antarctica’s largest contributor to sea-level rise. The past speedup is largely due to grounding-line retreat in response to ocean-induced thinning that reduced ice-shelf buttressing. While speeds remained fairly steady from 2009 to late 2017, our Copernicus Sentinel 1A/B–derived velocity data show a >12% speedup over the past 3 years, coincident with a 19-km retreat of the ice shelf. We use an ice-flow model to simulate this loss, finding that accelerated calving can explain the recent speedup, independent of the grounding-line, melt-driven processes responsible for past speedups. If the ice shelf’s rapid retreat continues, it could further destabilize the glacier far sooner than would be expected due to surface- or ocean-melting processes. In the decades since it and neighboring Thwaites Glacier were called “the weak underbelly of the West Antarctic Ice Sheet,”  has spedBeginning in 2015, the Copernicus Sentinel 1A/B synthetic aperture radar (SAR) satellites began collecting data every 12  and 6 days  over PIG and much of its catchment. Here, we use these data to investigate changes in PIG’s flow speed as its ice shelf retreated by nearly 20 km since late 2017. While most of the past speedup was due to processes concentrated at the grounding line, earlier work has shown that partial loss of the outer shelf also can cause speedup of the grounded ice  shows a stepped retreat of the central shelf by ~26 km from 2015 to 2020  due to a2, equivalent to ~20% of the modeled shelf’s initial area.Although past PIG speedups have been associated with thinning and related grounding-line retreat, the timing of the recent events suggests that the loss of buttressing from the outer shelf is responsible for the recent speed increase. While smaller calving events have not produced substantial variation in speed  as a sinWith or without the shelf loss, when the prognostic model starts, there are transients associated with model-data mismatch (fig. S1), which tend to dissipate within a few years. There are additional transients caused by the melt function, which is different than the unknown melt history under which the observed thickness and velocity used to initialize the model evolved. To isolate the response due to the shelf loss, As just noted, the speedup shown in Both the correspondence of shelf loss with the speedup and the agreement between the modeled and observed responses suggest that the recent speedup can be largely attributed to the loss of a large (20%) section of the shelf from 2017 to 2020. This loss of area reduced the ice shelf’s ability to buttress the ice stream by reducing its connection to the embayment’s sidewalls, thus reducing lateral resistive stresses on the shelf. While not at the scale of a complete collapse of the ice shelf  that weakens the ice-shelf margins (through our inversion for the flow-law coefficient), but this damage does not evolve with time. Given the model-data agreement, post-2017 damage need not be invoked to explain the bulk of the speedup . MoreoveThe likelihood of further retreat of PIG’s ice shelf in the coming years to decades remains unclear. Although surface melt events occasionally occur, there is no evidence of melt ponds causing hydrofractures that would have contributed to the recent calving (movie S1). Furthermore, climate models indicate that these conditions are not likely to occur in this century for PIG or many other Antarctica glaciers , there iIrrespective of the exact cause, recently initiated rifting processes appear to have driven much of the ice-front retreat via calving at a nonsteady rate  (We use an open-source, shallow-shelf, ice-sheet model, icepack, to simulate the response of PIG to a partial loss of the ice shelf (B (We use an inverse solver with Tikhonov regularization to determine basal shear stress for a regularized Coulomb friction model constrained by the 2017 observations used in an earlier study  is repreTo simulate calving, we created a new mesh with a shelf front based nominally on March 2020 conditions . We thenThe model was constrained with the average velocity field from 2017 Sentinel 1A/B data, with coverage gaps in the upper catchment filled with data from the MEaSUREs Version 2 velocity map of Antarctica (From January 2015 to March 2020, the central part of PIG’s ice-shelf front retreated by 26.3 km . IgnorinMooring observations in front of PIG started early 2009 , thereby


Cotinus coggygria Scop. was evaluated in vitro. The results showed that fisetin exhibited strong inhibitory activity with an IC50 value of 4.099 × 10−4 mM. Enzyme kinetic analysis revealed that fisetin is a non-competitive inhibitor of α-glucosidase, with an inhibition constant value of 0.01065 ± 0.003255 mM. Moreover, fluorescence spectrometric measurements indicated the presence of only one binding site between fisetin and α-glucosidase, with a binding constant (lgKa) of 5.896 L·mol−1. Further molecular docking studies were performed to evaluate the interaction of fisetin with several residues close to the inactive site of α-glucosidase. These studies showed that the structure of the complex was maintained by Pi-Sigma and Pi-Pi stacked interactions. These findings illustrate that fisetin extracted from Cotinus coggygria Scop. is a promising therapeutic agent for the treatment of T2DM.The inhibition of α-glucosidase is a clinical strategy for the treatment of type 2 diabetes mellitus (T2DM), and many natural plant ingredients have been reported to be effective in alleviating hyperglycemia by inhibiting α-glucosidase. In this study, the α-glucosidase inhibitory activity of fisetin extracted from  Diabetes mellitus (DM), associated with carbohydrate metabolism disorders and characterized by high blood glucose levels, has become a severe global non-communicable disease . With 46Cotinus coggygria Scop., which is distributed in Asia and North America  . According to Equation (3), qK was calculated to be 6.0501 × 1012 L·mol−1·s−1 Fol−1·s−1 B, which aK) and binding site (n) were determined according to the double logarithmic regression curve equation [The binding constant (equation :(4)lg with the lowest discrete optimized protein energy (DOPE) score as the template ,40. A RaThe theoretical binding mode between fisetin and α-glucosidase is shown in The amino acids in the 4 Å surrounding fisetin were analyzed in detail. As shown in Yue et al.  conducte50 value of 4.099 × 10−4 mM and Ki value of 0.01065 ± 0.003255 mM. The fluorescence quenching of α-glucosidase by fisetin was a static procedure. A single fisetin binding site was identified on α-glucosidase with a binding constant  of 5.896. Comparison of the kinetic parameters between the IC50 and Ki slope showed that the inhibition tended to be non-competitive. Molecular docking was used to simulate binding interactions. The Pi-Sigma and Pi-Pi stacked interactions played a significant role in the inhibition ofα-glucosidase by fisetin. This study provides the foundation for the potential use of fisetin as an α-glucosidase inhibitor. However, we still need further in vivo studies on α-glucosidase to confirm our in vitro results.In the present study, the inhibitory effect of fisetin on α-glucosidase activity was investigated using enzyme inhibition, fluorescence quenching coupled with kinetic analysis, and molecular simulation. We found that fisetin has the potency to non-competitively inhibit α-glucosidase, with an IC


The lateral habenula (LHb) is a key brain region implicated in the pathology of major depressive disorder (MDD). Specifically, excitatory LHb neurons are known to be hyperactive in MDD, thus resulting in a greater excitatory output mainly to downstream inhibitory neurons in the rostromedial tegmental nucleus. This likely results in suppression of downstream dopaminergic ventral tegmental area neurons, therefore, resulting in an overall reduction in reward signalling. In line with this, increasing evidence implicates aberrant inhibitory signalling onto LHb neurons as a co-causative factor in MDD, likely as a result of disinhibition of excitatory neurons. Consistently, growing evidence now suggests that normalising inhibitory signalling within the LHb may be a potential therapeutic strategy for MDD. Despite these recent advances, however, the exact pharmacological and neural circuit mechanisms which control inhibitory signalling within the LHb are still incompletely understood. Thus, in this review article, we aim to provide an up-to-date summary of the current state of knowledge of the mechanisms by which inhibitory signalling is processed within the LHb, with a view of exploring how this may be targeted as a future therapy for MDD. The lateral habenula (LHb) is a brain structure within the epithalamus which is now well established to have a causative role in the pathogenesis of major depressive disorder  in rodents, is one of the primary afferent structures to the LHb. This structure is thought to be primarily GABAergic in nature  of the hypothalamus also contains distinct populations of excitatory and inhibitory neurons which provide convergent input to individual LHb neurons , and project to the midbrain  has recently been shown to modulate both excitatory and inhibitory synaptic transmission within the LHb , a technique whereby electrodes are implanted within the brain to modulate neuronal activity  in mice triggers internalisation of both GABAB receptors and G protein-gated inwardly rectifying potassium (GIRK) channels, along with a concurrent increase in protein phosphatase 2A activity, which is known to regulate the expression of these channels. Consistently, pharmacological inhibition of protein phosphatase 2A normalises GABABand GIRK expression and alleviates depressive symptoms (Lecca et al., Modulating the electrical firing pattern and synaptic activity of LHb neurons In summary, as the mechanisms by which inhibitory signalling within the LHb are processed become clearer, so too do the potential strategies by which this can be exploited as a therapeutic strategy in MDD. The consistency between pre- and postsynaptic modulation of LHb neurons in relation to depressive behaviour is striking- inhibitory afferents promote reward-like states, while excitatory afferents promote aversion, and consistently activation of postsynaptic inhibitory GABA and glycine receptors appears to have an antidepressant effect, as does blockade of excitatory AMPA and NMDA receptors. As such, novel therapies for MDD may attempt to capitalise on this knowledge by aiming to selectively modulate such pathways.All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.