Though, degranulation and IL-6 production were only weakly altered. Altogether our data establish the LYN/FcγRIIB/SHIP1 signalosome in the context of FcεRI activation, particularly at supra-optimal Ag concentrations. The fact that SHIP1 tyrosine phosphorylation/activation not only depends on FcγRIIB, highlights the necessity for its tight backup control.Activation of the high-affinity receptor for IgE (FcεRI) follows a bell-shaped dose-response curve. Upon supra-optimal stimulation, mast cell effector responses are down-regulated by inhibitory molecules like the SH2-containing inositol-5′-phosphatase SHIP1 and the SRC-family-kinase LYN. To identify further molecules involved in a negative regulatory signalosome, we screened for proteins showing the same pattern of tyrosine phosphorylation as SHIP1, which is tyrosine-phosphorylated strongest upon supra-optimal antigen (Ag) stimulation. The low-affinity IgG receptor, FcγRIIB, was found to be most strongly phosphorylated under supra-optimal conditions. This phosphorylation is the consequence of passive, Ag/IgE-dependent and progressive co-localization of FcεRI and FcγRIIB, which is not dependent on IgG. Upon supra-optimal FcεRI cross-linking, FcγRIIB phosphorylation is executed by LYN and protected from dephosphorylation by SHIP1. Analysis of FcγRIIB-deficient bone marrow-derived mast cells revealed an ambiguous phenotype upon FcεRI cross-linking. Absence of FcγRIIB significantly diminished the level of SHIP1 phosphorylation and resulted in augmented Ca IgE-bound FcεRIs can be activated via cross-linking by specific multivalent Ag/allergen. Pre-formed mediators like histamine and proteases are immediately released from secretory lysosomes, metabolites of arachidonic acid are generated, and A specific feature of the FcεRI is its unusual bell-shaped dose-response behavior. The cellular response caused by FcεRI cross-linking increases until the use of optimal doses of Ag and descends again at higher  Ag concentrations. Careful studies have demonstrated that the descending part of the dose-response curve is actively controlled by inhibitory molecules and cellular mechanisms .One of these mechanisms is the cytoskeletal rearrangement caused by FcεRI-mediated MC activation. Ag stimulation not only triggers formation of FcεRI complexes, but also their detergent-resistant association to the submembranous cytoskeleton (SMC) . With in3) to PI-3,4-P2  is progressively phosphorylated by LYN and protected by SHIP1 in parallel to increasing Ag concentrations. In line, loss of FcγRIIB significantly decreased SHIP1 phosphorylation upon supra-optimal stimulation. Finally, our observations indicate that FcγRIIB contributes to supra-optimal negative regulation of FcεRI-induced MC activation by serving as a membrane adaptor for SHIP1.To identify further proteins potentially involved in supra-optimal negative regulation, we first addressed if other tyrosine-phosphorylated proteins besides SHIP1 and FcεRI can also interact with the actin cytoskeleton in a detergent-resistant manner upon Ag stimulation. Therefore, we sensitized MCs with the DNP- specific IgE SPE-7 overnight followed by addition of DNP-HSA at an optimal (20 ng/ml) as well as a supra-optimal  concentration. Differentially stimulated BMMCs were lysed first in a mild buffer (L1: 0.5% Triton X-100) and after centrifugation the resulting pellets were further lysed under more stringent conditions , solubilizing proteins still associated to the SMC Figure . After oTherefore, we sought to directly compare the patterns of overall tyrosine phosphorylation in differentially stimulated BMMCs using a stable isotope dimethyl labeling-based quantitative mass spectrometry approach , 16. TheDose-dependent changes in overall tyrosine phosphorylation after differential Ag treatment were quantified. Several proteins were identified that displayed increased phosphorylation at one or more tyrosine residues after supra-optimal compared to optimal stimulation. A selection of these proteins is shown in Figure The strong increase in tyrosine phosphorylation of FcγRIIB at high Ag concentrations was verified by phospho-specific Western blotting Figures . While aVia IgG-containing immune complexes, FcγRIIB is cross-linked to activating Fc receptors on various cell types, as well as the BCR on B-cells , 20. On Additionally, high-affinity binding of free IgE to FcγRIIB under our experimental conditions was excluded. Blocking of FcγRIIB with the CD32/CD16-specific mAb 2.4G2  prior toFurthermore, also the cytokinergic properties of the used IgE (SPE-7)  are not 1κ) used for this experiment is potentially possible, meaning that recruitment of FcγRIIB would not be IgG-independent in the given experimental setup. Nevertheless, strong interaction between anti-IgE and FcγRIIB is unlikely, as the respective antibody was rat-derived, and MCs were of murine origin. Previous studies suggest that compared to murine IgG1, rat IgG1 only weakly interacts with the murine FcγRIIB (IIb antibody (isotype IgG1κ) was used to induce thrombocytopenia in mice, which showed no difference between WT and Fcgr2−/− mice.To also consider the specific multivalent nature of the DNP-HSA that was initially used as an Ag to trigger the observed FcγRIIB phosphorylation, we also tested whether only bivalent cross-linking of the FcεRI could cause this pattern of FcγRIIB phosphorylation. For that purpose, FcεRI cross-linking was either induced with an optimal and a supra-optimal amount of DNP-HSA or optimal and supra-optimal concentrations of monoclonal anti-IgE respectively Figure . Compari FcγRIIB , 28. ParIn conclusion, FcγRIIB tyrosine phosphorylation in IgE-loaded BMMCs in response to stimulation with supra-optimal Ag concentrations occurred in an IgG-independent and thus passive fashion.Lyn−/− BMMCs, we could show that LYN is responsible for the FcγRIIB phosphorylation at Y309 within the ITIM upon supra-optimal FcεRI stimulation -treated WT BMMCs after supra-optimal stimulation. Interestingly, upon phosphatase inhibition FcγRIIB phosphorylation was also observed in response to optimal Ag concentration  for 1 h prior to Ag stimulation rescued ITIM phosphorylation of FcγRIIB in s Figure . Withoutn Figure . This shn Figure . This inFcgr2−/− BMMCs and subsequent phospho-tyrosine-specific immunoblotting revealed that SHIP1 tyrosine phosphorylation was decreased in Fcgr2−/− compared to WT BMMCs in response to both optimal as well as supra-optimal stimulation. BMMCs lacking FcγRIIB were generated from Fcgr2−/− mice. These MCs showed a comparable differentiation process and expressed equal amounts of FcεRI and KIT as the respective WT  in Fcgr2−/−  was performed using IgE- and FcγRIIB-specific antibodies. As shown in Figure Fcgr2−/− instead of WT BMMCs . After supra-optimal stimulation, fluorescence signals were further increased to 2.73-fold compared to the unstimulated control  after Lat. B treatment compared to DMSO controls  of WT and Fcgr2−/− BMMCs. This illustrates that the attenuation of IL-6 release in response to supra-optimal stimulation is less pronounced in Fcgr2−/− compared to WT BMMCs.Regarding production of the pro-inflammatory cytokine IL-6, WT MCs displayed the characteristic bell-shaped dose-response behavior, whereas the pattern for n Figure . After oFcgr2−/− BMMCs  on the cell surface upon Ag-induced fusion of vesicular and plasma membrane by FACS. Interestingly, the comparison of degranulation kinetics by means of the LAMP-1 assay revealed slight but consistent differences. Under optimal conditions, slightly higher amounts of LAMP-1 appeared on the surfaces of WT compared to Fcgr2−/− BMMCs. Contrary after supra-optimal stimulation, LAMP-1 levels were consistently increased in Fcgr2−/− BMMCs, which indicates stronger vesicle fusion to the plasma membrane  of the intracellular Ca2+ concentration, we restored extracellular Ca2+ levels by adding CaCl2 to measure Ca2+ influx through opened store-operated Ca2+ channels. We clearly observed that the differences in Ca2+ mobilization between WT and Fcgr2−/− BMMCs were based on the influx of extracellular rather than the release of intracellular Ca2+ ions. In conclusion, our experiments showed partly enhanced MC activity in Fcgr2−/− compared to WT cells, particularly upon supra-optimal stimulation, which indicated a negative regulatory function of FcγRIIB in response to FcεRI activation.Encouraged by this finding, we analyzed Ag-induced Canulation . Indeed,s Figure . In cont Figures . Besidesn Figure . After tIn this study, we have found that activation of the FcεRI by high, supra-optimal Ag concentrations causes co-aggregation{Formatting Citation} with the inhibitory FcγRIIB, resulting in LYN-dependent, SHIP1-stabilized ITIM phosphorylation of the FcγRIIB. Ag-induced tyrosine phosphorylation of the FcγRIIB was identified in a phospho-proteomic approach analyzing differences in protein tyrosine phosphorylation between optimally and supra-optimally stimulated BMMCs. This was motivated by our previous finding that the dominant negative regulator of MC activation, SHIP1, is phosphorylated strongest in response to high Ag concentrations .FcγRIIB negatively regulates signaling of Fc- and B-cell receptors , 34, to In the present study, we observed passive co-localization of FcγRIIB and FcεRI that is independent of IgG-mediated receptor cross-linking. Instead it is Ag-mediated and progresses with increasing stimulus concentrations. Co-localization is associated with substantial phosphorylation of the FcγRIIB ITIM, which also increases in an Ag-dependent manner. In analogy to IgG-based activation , ITIM phIn our quantitative proteomics analysis especially Y326, but also Y309 within the ITIM of FcγRIIB displayed increased phosphorylation. Phosphorylated Y326 interacts with the SH2-domain of the adaptor protein GRB2, which, via its C-terminal SH3-domain, stabilizes the SHIP1-FcγRIIB interaction . This inin vivo , Lyn−/−(129/Sv × C57Bl/6), Lyn−/−Ship1−/− (129/Sv × C57Bl/6), and Fcgr2−/− (C57Bl/6) BMMCs. To each the related WT BMMCs were generated according to the same protocol. Experiments were performed in accordance with German legislation governing animal studies and following the principles of laboratory animal care. Mice are held in the Institute of Laboratory Animal Science, Medical Faculty of RWTH Aachen University. The institute holds a license for husbandry and breeding of laboratory animals from the veterinary office of the Städteregion Aachen (administrative district). The institute follows a quality management system, which is certified according to DIN ISO 9001/2008. Every step in this project involving mice was reviewed by the animal welfare officer. All experiments were approved by the Landesamt für Natur, Umwelt, und Verbraucherschutz NRW (LANUV), Recklinghausen (AZ 84-02.04.2016.A496).Bone marrow-derived mast cells (BMMCs) were generated as described previously . The quo2) were washed in sterile PBS, resuspended in Tyrode's buffer  and adapted to 37°C. Cells were stimulated with different concentrations of Ag  for 30 min at 37°C. The degree of degranulation was determined by measuring β-hexosaminidase release  overnight . Cells were washed in sterile PBS and adjusted to 1.2 × 106/ml in RPMI 1640 containing 0.1% BSA. Cells were stimulated with different concentrations of Ag  for 4 h at 37°C Mouse IL-6 ELISA (BD Pharmingen) was performed according to the manufacturer's instructions. Levels of cytokines varied between experiments due to age of the cells. Qualitative differences or similarities between WT and mutant cells, however, were consistent throughout the study.BMMCs were preloaded with 0.15 μg/ml IgE (SPE-7) overnight  and PE-conjugated anti-CD16/CD32 IgG (BD) for 25 min at 4°C in FACS buffer . Cells were analyzed by flow cytometry using a FACSCanto II (BD).For experiments using Fc-block, MCs were differentially loaded with IgE and incubated with 2.4G2 (BD) as described in the respective experiments. Afterwards cells were stained with anti-IgE IgG (SouthernBiotech) and analyzed by flow cytometry.2) were washed in sterile PBS, resuspended in RPMI 1640 containing 0.1% BSA and adapted to 37°C. MCs were stimulated with the indicated concentrations of DNP-HSA for the indicated periods. MCs were pelleted, washed with FACS buffer and stained with FITC-conjugated anti-LAMP1 immunoglobulins (BioLegend) for 25 min at 4°C in FACS buffer. Cells were then washed twice, resuspended in FACS-buffer, and LAMP-1 surface expression was determined by flow cytometry. Data were analyzed using the FlowJo analysis software (Tree Star).To perform the LAMP-1 assay, BMMCs preloaded with 0.15 μg/ml SPE-7 overnight  and Fura Red (2.75 μM), as well as 5 μl/ml of 20% pluronic F-127 in DMSO  were added, and cells were incubated for 45 min at 37°C. MCs were pelleted, resuspended in RPMI 1640 containing 0.1% BSA, and analyzed in a FACSCalibur (BD Biosciences) with the indicated stimulation procedures. Intracellular calcium release was recorded in the presence of 1 mM EDTA (Sigma). For data analysis the FlowJo analysis software (Tree Star) was used. FACS profiles were converted to line graphs.BMMCs were preloaded with 0.15 μg/ml IgE (SPE-7) overnight, washed with sterile PBS and resuspended at 1 × 102 was edged with a hydrophobic pen (Sigma) to limit the reaction area for PLA reagents. IgE-preloaded BMMCs  were washed in sterile PBS and resuspended in RPMI/0.1% BSA at 3.5 × 106 cells/ml. 1 ml of the cell suspension was added to each cover slip. Cells were incubated at 37°C for 1 h and centrifuged on the cover slips at 1,200 rpm for 5 min at 4°C. Supernatants were aspirated. Temperature was carefully adjusted to 37°C for 20 min with 1 ml of pre-warmed medium. MCs were stimulated with the indicated concentrations of DNP-HSA for 2 min. Cover slips were washed twice with PBS++ . Cells were fixed with 4% paraformaldehyde (PFA) for 10 min in the dark at room temperature (RT) followed by another washing step with PBS++. After quenching with 50 mM NH4Cl for 5 min (RT), cells were washed with PBS++. Samples were further treated according to the instructions provided with the Duolink®;in situ Starter Kit (Sigma) and finally analyzed using a Zeiss LSM 710 . Signal calculations were done with BlobFinder .Cover slips were incubated with 12.5 μg/ml poly-L-lysine for 1 h, washed twice with sterile PBS and dried. An area of approximately 1 cm++ at 3.5 × 106 cells/ml. For each sample 1 ml of the cell suspension was added to a cover slip and centrifuged at 1,200 rpm for 7 min (4°C). Supernatant was aspirated, and cells were fixed with 4% PFA for 10 min in the dark (RT) followed by a washing step with PBS++. After quenching with 50 mM NH4Cl for 5 min at RT, cells were washed with PBS++ and blocked with 5% BSA in PBS++ for 1 h at RT. Antibodies were diluted in 0.2% BSA-PBS++ according to the manufacturer's instructions and incubation with primary/secondary antibodies was conducted for 1.5 h at RT in the dark. If primary and secondary antibodies were used for staining, three washing steps were performed in between with 0.2% BSA-PBS++. After staining cells were washed three times in 0.2% BSA-PBS++. Nuclei were stained with DAPI (Sigma) (1:1000) for 10 min and cells were washed three times as described before. Cover slips were dried and mounted on microscope slides. Samples were analyzed using a Zeiss LSM 710.BMMCs were pretreated as indicated in the respective experiment and concentrated in PBS1K), respectively for indicated periods, pelleted and solubilized in phosphorylation solubilization buffer (PSB)  were washed in sterile PBS and resuspended in RPMI/0.1% BSA. Cells were stimulated with the indicated concentrations of Ag or monoclonal rat anti-mouse IgE antibody   containier (PSB) . Antibod3VO4) was dissolved in distilled water (100 mM). Next pH was adjusted to 10. The solution was boiled up until the yellow color had fully disappeared and pH was readjusted to 10. If the color turned yellow again, the process of boiling and subsequent readjustment of pH was repeated until the solution remained colorless at pH 10. Pervanadate (PV) was prepared immediately before use by mixing 100 mM sodium orthovanadate and hydrogen peroxide in a molar ratio of 2:1 (H2O2:orthovanadate).For the preparation of pervanadate solution, sodium orthovanadate  or a supra-optimal  concentration of DNP-HSA (one batch was left unstimulated as a control), cells were pelleted and resuspended in lysis buffer , 20 μg/ml Aprotinin, 4 μg/ml Leupeptin and 1 mM PMSF). After sonification at 4°C (3 × 1 min per sample), cells were lysed for 30 min on ice. Lysates were cleared by centrifugation  and overall protein amounts were determined and adjusted. Samples were reduced wit 2 mM dithiothreitol (DTT) (Roth) at 56°C for 30 min and alkylated with 4 mM iodoacetamide (IAA) (Sigma) for 25 min at RT in the dark. Next, proteins were digested with the lysyl endopeptidase Lys-C (Wako)  for 4 h (37°C). Samples were then diluted from 8 to 2 M urea with 50 mM ammonium bicarbonate and digested with trypsin (Thermo Fisher)  overnight. For stable isotope dimethyl labeling the on-column method described by Boersema et al. was used . Divergehttps://www.ncbi.nlm.nih.gov/pubmed/27348712) (version 1.5.5.3): peptides corresponding to reverse and contaminant protein entries were removed; a minimum phosphorylation site localization score of 0.75 was set as the minimum; only phosphorylated tyrosine residues were considered. To be included in the result file, a phosphorylation site had to be identified at least once in both technical replicates of one experiment with all ratios. The mean of all normalized ratios  was calculated and is reported in the Supplementary Tables The peptides were trapped on a C18 precolumn  using buffer A (0.1% FA). This was followed by peptide separation on an analytical column  by employing a 260 min or 280 min gradient from 5-40% buffer B  at 230 nl/min. The eluent was sprayed into the mass spectrometer via stainless steel emitters using a spray voltage of 1.9 kV. Mass spectrometry was performed using an Orbitrap Elite (Thermo Scientific). The instrument was operated in data-dependent mode. Full-scan spectra were recorded in the Orbitrap . The top 10 precursors of each full scan were selected for fragmentation using CID in the ion trap . All immunoprecipitations were analyzed in duplicate . The third experiment was carried out using a “label swap.” The resulting raw data files were analyzed by MaxQuant and the built-in Andromeda search engine , 57. Thep-values were calculated by the unpaired two-tailed Student's t-test. For normalized data the one sample t-test including FDR  was used. p-values of *p < 0.05, **p < 0.005, and ***p < 0.0005 were considered statistically significant. Values higher than a p-value of 0.05 were regarded as not significant.All values are expressed as mean ± SD of n observations (n indicated in the respective figure legends). http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (The datasets generated and analyzed for this study are either included in the manuscript and the supplementary files or will be made available by the authors on request to any qualified researcher. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (pository  with theMG carried out experimental design, performed experiments, analyzed data and wrote the manuscript. CP carried out experimental design, performed mass spectrometry experiments, analyzed the respective data and provided experimental and analytical guidance. FN provided fundamental material. MH conceived the study, carried out experimental design, analyzed data and wrote the manuscript. All authors reviewed the manuscript.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.


This paper reviews the recent literature on numerical modelling of the dynamics of the Greenland ice sheet with the goal of providing an overview of advancements and to highlight important directions of future research. In particular, the review is focused on large-scale modelling of the ice sheet, including future projections, model parameterisations, paleo applications and coupling with models of other components of the Earth system.Data assimilation techniques have been used to improve the reliability of model simulations of the Greenland ice sheet dynamics, including more accurate initial states, more comprehensive use of remote sensing as well as paleo observations and inclusion of additional physical processes.Modellers now leverage the increasing number of high-resolution satellite and air-borne data products to initialise ice sheet models for centennial time-scale simulations, needed for policy relevant sea-level projections. Modelling long-term past and future ice sheet evolution, which requires simplified but adequate representations of the interactions with the other components of the Earth system, has seen a steady improvement. Important developments are underway to include ice sheets in climate models that may lead to routine simulation of the fully coupled Greenland ice sheet–climate system in the coming years. The Greenland ice sheet (GrIS) is the second largest ice body on Earth and is expected to be a major contributor to future sea-level rise . PotentiProjecting future changes and studying the past behaviour of the GrIS and its interactions with other components of the Earth system requires numerical ice sheet models built around a physical description of ice sheet flow Fig. . In its The rate of ice deformation is strongly dependent on ice thickness, surface slope and rheological properties of the ice and the underlying substrate, which change considerably with changing temperature, impurity concentration and water content of the ice. Thermodynamic coupling is needed to model the long-term  evolution as the ice sheet responds to climate changes and might only be neglected for short-term  simulations. Another long-term interaction mechanism of the ice sheet concerns isostatic bedrock adjustment under the varying load of the ice. The millennial to multi-millennial response time scales associated with bedrock changes and thermodynamics have led to the use of spin-up techniques that consist of running ice sheet models with boundary conditions of simulated or reconstructed past climate changes to capture the long-term memory of these processes , 6.However, the present-day configuration of the ice sheet simulated with such long-term reconstructions shows a rather large discrepancy when compared with available satellite and airborne observations , 8. StudGrIS numerical models vary largely in terms of the processes they include, the spatial resolution of their underlying grids and the forcings they apply  dependinAcknowledging considerable overlap between them, we discuss three different approaches to GrIS modelling in this paper: (1) The long-term modelling approach with thermodynamic coupling and isostatic bedrock adjustment, requiring spin-up techniques, (2) data assimilation techniques, typically applied to model initialisation for centennial time-scale projections, making extensive use of high-resolution observations of the present-day ice sheet with inversion techniques, (3) fully coupled ice sheet–climate modelling of different levels of complexity to physically represent feedbacks between the ice sheet, atmosphere and ocean. All three modelling approaches, for example, are represented in the GrIS model initialisation experiments (initMIP-Greenland)  performeDistinguishing between the different approaches is somewhat arbitrary but, in many cases, derives from the specific goals of individual research groups. In fact, closer collaboration across these boundaries could further advance the ice sheet modelling community and should be encouraged. None of the modelling approaches can be completely separated from the others. Aside from appreciating the strengths and limitations of the different approaches for specific model applications, the aim of this review paper is therefore also to stimulate possible directions of future research and cross-fertilisation between different groups. It is in this spirit that we have decided to discuss the different research priorities in one review paper.In this section, we review recent developments concerning specific aspects of large-scale GrIS modelling.Domain discretisation is central to any numerical problem. Models used for long-term simulations nowadays typically apply regular equidistant grids at a horizontal resolution of 5–20 km –17, but As ice is a viscous incompressible fluid, it is best modelled using full-Stokes equations. Due to the intensive computational resources needed to solve these equations and the thin aspect ratio of ice sheets, several approximations based on asymptotic developments of these equations have been derived . These cWhile a Stokes model without any approximation remains the best description of ice sheet flow and has been successfully applied for the GrIS on a century time scale , 32, it Ice sheet thermodynamics are an important element of long-term ice sheet modelling, since ice deformation is non-linearly dependent on ice temperature. In addition, large parts of the GrIS are at the pressure melting point, implying an active role for basal sliding as well. In order to compute both cold and temperate ice properties with a single energy-conserving framework, many models now included an enthalpy framework  and a rhTo aid understanding the evolution of the ice sheet interior, thermodynamic modelling has been used to explain the reconstructed temperature recovered from a Greenland deep ice core . The resAnisotropy is another potential source of discrepancies between observed and modelled ice properties, as rheological laws used in most ice sheet models rely on the isotropic Glen’s flow law . Deep icAn evaluation of the impact of observationally constrained thermal boundary conditions on the modelled thermomechanical state in western Greenland has shown that there is a strong influence of the surface thermal boundary .A study of the geothermal heat flux distribution beneath the GrIS  concludeThe thermal condition at the base of the ice sheet is known to have important consequences for basal meltwater production and hence for the basal friction and sliding velocity . This viAs the GrIS mass loss continues to accelerate , most dyIn addition, the interaction of marine-terminating glaciers with ocean water is recognised as an important forcing mechanism, as oceanic conditions impact both submarine melting and calving. However, modelling such interaction remains a challenging problem, mainly due to the large number of poorly mapped fjord geometries, properties of ocean circulation in the fjords and limited observations of melting along ice fronts. In the absence of detailed physically based methods to estimate interactions between outlet glaciers and the ocean explicitly, parameterisations of the bulk effect of the ice-ocean interaction in Greenland fjords have been developed , 63. TheAn important driver for recent development in the field of GrIS modelling is improving physically based projections of the ice sheet’s contribution to future sea-level change. Many ice sheet modelling studies build further on the legacy of the SeaRISE , 11 and Traditionally, long-term modelling approaches including thermodynamic coupling and isostatic bedrock adjustment were used to provide reconstructions of the present-day ice sheet. Using such a spin-up method, an attempt has been recently made to re-examine the SeaRISE experiments (that were difficult to interpret) with one specific model that is systematically perturbed . ResultsA relatively good match with the observed velocity structure , 73 and In principle, an almost perfect match with the observed geometry can be achieved with nudging techniques , 70, 71 A different approach to improve the match with the observed velocity and geometry consists of allowing for spatially explicit adjustment of model parameters , here stA second category of techniques relies entirely on observations of the ice sheet. Using observations of ice sheet geometry and assuming the ice rheology  is known, basal friction can be treated as the only unknown parameter. In that case, ice sheet initialisation becomes a time-independent data assimilation problem, with the goal of finding optimal model parameters that minimise the misfit with observations, usually surface velocities , 77–79. A new technique to improve basal topography between observations acquired along flight lines was designed, based on the continuity equation and taking advantage of the global coverage in surface velocities . In the The optimisation of the basal topography can also be directly integrated into the data assimilation procedure, either by applying the shallow shelf approximation  or a higInstead of only relying on a modelled ice temperature distribution to define the viscosity, one can complement inversion of the basal drag coefficient with an additional optimisation of the stiffness factor . This fatime-dependent observational data sets for data assimilation [As time series of surface elevation and velocity observations become available, a recent development in centennial scale ice sheet modelling concerns techniques that leverage milation , 82. Themilation , 84, whimilation , where SAnother opportunity to improve estimates of ice sheet configuration lies in the recent radar observation of ice isochrones  that havIn order to validate models and assess their reliability, attempts have been made to compare present-day GrIS simulations to remote sensing observations. Comparison with Gravity Recovery and Climate Experiment (GRACE) data  for the Coupling with the atmosphere in terms of prescribing SMB and temperature boundary conditions in an ice sheet model is common practice and is not further discussed here. Instead, we refer the reader to a review paper on SMB . HoweverWith ISMIP6 , fully cReconstructing the past evolution of the GrIS may have important implications for our understanding of its present and future behaviour, may provide analogues to the present-day climate and is a challenging scientific endeavour in itself. This is usually done with models representing not only the ice flow itself but also other components of the Earth system , often in parameterised or simplified form. While the spin-up of paleo-class ice sheet models to incorporate the long-term thermal memory typically covers the last or several glacial–interglacial cycles , 16, stuSensitivity experiments with different topographic reconstructions were performed to study the conditions during the first inception of the GrIS . The stuAn intercomparison of ice sheet models forced with simulated climate conditions of the mid-Pliocene warm period  indicateSimulations of the GrIS evolution during the LIG were performed with an ice sheet model asynchronously two-way coupled to a Regional Climate Model  to repreThe retreat history of the GrIS during the last deglaciation was reconstructed using an ice sheet model constrained with evidence of past margin positions and relative sea-level indicators , 112. ThGiven the long-term experiments needed in paleo simulations, ice sheet and climate models and their coupling, are typically treated with reduced resolution and/or reduced complexity for synchronously coupled experiments –109, 113We have discussed the literature of recent studies covering overlapping approaches to numerical modelling of the GrIS from paleo applications to future projections. The last few years have seen an important improvement in simulating the present-day dynamic state of the ice sheet, needed as an initial condition for projections of future ice sheet changes. Advances in data assimilation techniques that leverage the growing number of satellite and airborne observations of the ice sheets are at the core of these improvements and have led to modelled ice sheets that reproduce the observed velocity field much better. This is indicative of a growing link between the ice sheet modelling and observational communities, which will also pave the way towards better evaluation and validation mechanisms, which are needed to attach meaningful uncertainty estimates to the projections. However, the relatively short time coverage of satellite data is so far one of the biggest limitations to this effort and further progress is fundamentally dependent on a continuation of high-quality ice sheet observations over time, in order to allow for data assimilation techniques that take advantage of the temporal evolution. A limitation of the current generation of data-assimilated ice sheet models used for short-term projections is the missing representation of long-term processes of the bed response, thermodynamics and a prognostic evolution of the basal conditions. This is complemented by process-based models of long-term ice sheet evolution that typically include these physically based descriptions but often at lower spatial resolution and that exhibit considerable mismatch to the observed geometry and velocity at the present day. Recent development from this side of the modelling spectrum is therefore achieved by a continuous increase in model resolution and simulated detail and by an increasing size of model ensembles used for sensitivity studies and exploring various types of uncertainty.Two-way coupling between ice sheet models and AOGCMs has seen significant improvements and is on the way to parity in performance with standalone ice sheet models for the goal of performing future projections on multi-centennial time scales. Aside from the intricate complications that arise from coupling two models with dynamic feedbacks between them, one of the main challenges and limitations has long been to produce realistic enough SMB boundary conditions for the ice sheet. Recent development in dynamic downscaling methods has led to an important step forward in this regard.Paleo studies of GrIS evolution have focused on modelling past warm periods that may help to understand ice sheet behaviour in a warmer than present climate. However, large uncertainties in the climate forcing for the LIG and the mid-Pliocene warm period limit the ability to constrain past ice sheet behaviour and consequently inferences for the long-term future of the ice sheet.Large challenges remain concerning how to combine “the best of both worlds” from long-term spin-up and data assimilation techniques. The goal would be to produce modern ice sheet initial conditions that are close to observations and at the same time include long-term processes like bedrock adjustment and evolving thermodynamics that contain the  memory of past climate changes. This likely requires joint efforts from different corners of the ice sheet modelling community and integration of time series of observations. A big step in this direction would be to define physically based descriptions of basal processes that could replace the inversion for basal drag by deterministic modelling.An emerging development that is expected soon is two-way coupling of GrIS models with regional climate models for future projections. Several groups are currently working on what represents an important step forward to explicitly simulate interactions between evolving ice sheet and climate. Direct coupling between ice sheet models and AOGCMs is underway and should over the next few years become available for multi-centennial future projections, on time-scales where ice-climate feedbacks play an important role.The global ice sheet modelling community has grown significantly in the last several years, leading to new approaches and models with larger user bases. Tighter collaboration between groups across intra-community boundaries should be continued, exemplified by intercomparison exercises like ISMIP6 , MISOMIP


Spilopelia senegalensis) as a biomonitor of environmental contamination with heavy metals from mining practices. Our results revealed the accumulation of heavy metals in the liver, kidneys, and lungs of the laughing dove collected from the mining site. The doves exhibited tissue dysfunction and injury, and decreased antioxidants. These results show the value of the laughing dove as a biomonitor of environmental pollution with heavy metals.Bioindicators and biomonitors are living organisms utilized to appraise the health of the environment or natural ecosystem. Mining, which refers to extraction of valuable materials from the earth, represents a source of heavy metals that can impact the environment, biodiversity, and human health. We investigated the value of the laughing dove s caused by mining activities. S. senegalensis was collected from a gold mining site and a reference site, and metal(loid)s and biochemical parameters were determined. Lead, cadmium, mercury, vanadium, arsenic, copper, zinc, and iron were significantly increased in the liver, kidney, and lung of S. senegalensis from the mining site. Serum transaminases, alkaline phosphatase, creatinine, and urea were significantly elevated in S. senegalensis from the mining site. Lipid peroxidation and nitric oxide were increased, whereas glutathione and antioxidant enzymes were diminished in the liver and kidney of S. senegalensis from the mining site. In addition, multiple histological alterations were observed in the liver, kidney, and lung of S. senegalensis. In conclusion, mining activities provoke the accumulation of metal(loid)s, oxidative stress, and tissue injury in S. senegalensis. Therefore, S. senegalensis is a valuable biomonitor of environmental pollution caused by mining activities and could be utilized in epidemiological avian studies of human health.Environmental pollution with heavy metals (HMs) is of serious ecological and public health concern worldwide. Mining is one of the main sources of HMs and can impact the environment, species diversity, and human health. This study assessed the value of  Bioindicators are living organisms utilized to appraise the health of the environment or natural ecosystem . DiffereMining is the extraction of valuable materials and minerals from the earth. Mining practices create a negative environmental impact; therefore, most of the world’s nations have passed regulations to decrease the impact of pollution from mines ,13,14. EGerbillus nanus in Saudi Arabia  state in the environment and ROS are generated during its transition into the reduced form [S. senegalensis were found to be higher than other HMs. Although it is required for the function of a large number of enzymes and transcription factors within the body, high Zn concentration can be harmful [Fe, Zn, and Cu were also increased in the liver, kidney, and lung of ced form . Fe and ced form  and nephced form , and higced form . Zn conc harmful . TherefoS. senegalensis has not been reported, at least not in Saudi Arabia. The feral pigeons (Columba livia) collected from a ferronickel smelter courtyard in Drenas (Kosovo) exhibited significantly increased concentrations of Pb, Cd, Zn, Cu, and Ni in the liver, kidney, and other tissues when compared with pigeons collected from a control site [The accumulation of HMs in different tissues of pigeons collected from contaminated regions was reported in previous studies; however, the impact of gold mining on rol site . Feral prol site . Hence, rol site . In addirol site . The finrol site .S. senegalensis as a biomonitor of environmental contamination caused by mining activities. The results showed an increase in the concentrations of Pb, Cd, Hg, V, As, Zn, Fe, and Cu, histopathological alterations, increased lipid peroxidation, and decreased antioxidant defenses in different tissues of S. senegalensis collected from the mining site. These data closely reflect the differences in HM concentrations between the mining and control sites and suggest that S. senegalensis provide valuable data for evaluating the impact of environmental pollutants. Moreover, this study might present the scientific basis for employing S. senegalensis in epidemiological avian studies of human health.The current findings introduce information on the value of


The placement of C2 laminar screws is a safe and useful method of axis fixation. The freehand method of screw placement was originally described by Wright et al., and requires detailed knowledge of the C2 posterior element anatomy, relationship to vital neurovascular structures, and technical acumen. The current evidence, surgical anatomy and technical details of screw insertion are investigated and highlighted in this manuscript and surgical video. Fixation of the upper cervical spine has been performed with various techniques ranging from wiring methods to polyaxial screw placement , 2. ScreThe second cervical vertebra represents a specialized segment of the human cervical spine. Its primary purpose is to facilitate rotation of the head by connection to the C1 anterior arch and lateral masses . The intThe bilateral C2 laminae join medially at the base of the spinous process, and extend laterally to the inferior facets. The slope and maximal diameter of the C2 lamina appear to vary significantly with gender on radiographic and cadaveric analysis ,8. The VA cadaveric specimen was acquired and the relevant anatomy and surgical technique pertaining to the freehand method of C2 laminar screw placement was highlighted in a surgical video  or superficial portion. The small ball probe is also used to measure the distance of the tract. A tapered tap is then used along the tract. The small ball probe is once again used to verify that there were no breaches in the created tract. A screw 3.5-4.0mm in diameter is then placed within the lamina. Once this screw is placed, the process is repeated for the contralateral screw. During placement of screws careful consideration is given to the depth at which the screws enter to prevent disruption of the C2-3 joints. Once the screws are placed, rods are cut and contoured to fit into the ipsilateral laminar screw head to join the remainder of the construct. If necessary, a lateral connector may be be used in order to properly connect the rod, although this is avoided to mitigate the potential bio-mechanical disadvantages of lateral connection .The most common fixation methods of the axis involve various techniques of screw fixation, most commonly by using the C2 pars or the C2 pedicle . Use of Wright’s published “crossing” technique utilizes the anatomical advantages of the C2 lamina to ensure proper placement and bio-mechanical stability . Other tThe greatest advantage of laminar screw placement is the reduced rate of neurovascular injury in comparison to the other forms of C2 screw fixation ,8. An adA technical challenge in placing “crossing” laminar screws is the ability to pre-plan the starting points and trajectory to ensure that the screws do not interact to the point of failure after insertion. This can be accomplished by complete and thorough exposure of the C2 posterior elements, including separation of the atlantoaxial membrane from the superior aspect of the lamina in order to gauge the extreme limits of screw placement. Cadaveric studies have indicated that the average spino-laminar diameter and cortical thickness is adequate to receive bilateral screws.The freehand method of C2 laminar screw placement appears to be a safe and effective means of atlas fixation. By demonstrating this skill in a cadaveric model and with the utilization of the latest educational technologies, the technical details of this instrumentation method can be adequately illustrated for instructional and educational purposes.


Allergy to bee and wasp venom can lead to life-threatening systemic reactions. The identification of the culprit species is important for allergen-specific immunotherapy.To determine a panel of recombinant bee and wasp allergens which is suitable for the identification of bee or wasp as culprit allergen sources and to search for molecular surrogates of clinical severity of sting reactions.TM. IgE-reactivity testing was performed using a comprehensive panel of recombinant bee and wasp venom allergens  by ISAC chip technology, ImmunoCAP and ELISA. IgG4 antibodies to rApi m 1 and rVes v 5 were determined by ELISA and IgE/IgG4 ratios were calculated. Results from skin testing, IgE serology and IgE/IgG4 ratios were compared with severity of sting reactions.Sera from eighty-seven patients with a detailed documentation of their severity of sting reaction (Mueller grade) and who had been subjected to titrated skin testing with bee and wasp venom were analyzed for bee and wasp-specific IgE levels by ImmunoCAPA/L of IgE to Api m 1 and/or Ves v 5.The panel of rApi m 1, rApi m 10, rVes v 1 and rVes v 5 allowed identification of the culprit venom in all but two of the 87 patients with good agreement to skin testing. Severities of sting reactions were not associated with results obtained by skin testing, venom-specific IgE levels or molecular diagnosis. Severe sting reactions were observed in patients showing < 1 ISU and < 2kUWe identified a minimal panel of recombinant bee and wasp allergens for molecular diagnosis which may permit identification of bee and/or wasp as culprit insect in venom-sensitized subjects. The severity of sting reactions was not associated with parameters obtained by molecular diagnosis. Apis mellifera) and Vespidae  and the paper wasp are among the most prevalent ones [Systemic allergic reactions to insect venoms affect the skin, the respiratory and gastro-intestinal tract as well as the cardiovascular system and are often life-threatening. According to epidemiologic studies, it is estimated that approximately 1.2–3.5% of the population suffers from severe allergic reactions to insect venom allergens . Venoms ent ones . Hymenopent ones  and showent ones . Accuratent ones , which cent ones . Evidencent ones –13. Howeent ones , 15. Whient ones –17. Anotent ones .Here, we tested a comprehensive panel of recombinant bee and wasp venom allergens for their usefulness to identify the culprit insect in venom allergic patients, which were characterized regarding clinical reactivity by Mueller grading and by skin testing with bee and wasp venom extracts. Furthermore, we investigated if it is possible to establish molecular diagnostics for predicting severities of sting reactions.Sera from eighty-seven patients with bee and/or wasp venom allergy were analyzed in this study Tables  and 2. Fet al [The Slovenian mono-sensitized patients had been tested by skin prick testing with two concentrations of bee and wasp venom extracts  . The skin prick test results were analyzed as described . For theet al . None ofEscherichia coli BL21 (DE3) as C-terminally his-tagged proteins and purified by inclusion body preparation and Ni2+-chelate affinity chromatography [Spodoptera frugiperda (Sf9) insect cells were used as hosts for the baculovirus vector [Synthetic codon-optimized genes coding for Ves v 1 or Ves v 5 were inserted into plasmid pET17b and expressed in tography . Recombis vector . In the s vector . A proteA/L were considered positive and are highlighted in grey in Tables Specific IgE antibodies of serum samples from bee and/or wasp venom allergic patients were quantified by ImmunoCAP tests containing the recombinant (r) allergens rApi m 1 i208), rApi m 3 (i215), rApi m 5 (i216), rApi m 10 (i217) and rVes v 5 (i209) . Specific IgE values ≥ 0.35 kU08, rApi For all sera IgE-reactivities to more than 150 micro-arrayed allergen components were analyzed with a customized allergen-chip based on the ISAC technology  as described . After wE. coli-expressed rVes v 1 was studied by IgE ELISA. Patient`s sera were diluted 1:5 in PBS containing 0.5% wt/vol BSA and 0.05% vol/vol Tween 20 and tested for IgE-reactivity to the allergens and, for control purposes, to human serum albumin (HSA). Bound IgE was detected as described [4 antibody levels specific for rApi m 1 and rVes v 5 were measured in 1:40 diluted serum samples by ELISA as described [A/L)/IgG4(OD) ratios for Ves v 5 were calculated as described [IgE reactivity to purified rApi m 2 expressed in insect cells as folded and non-glycosylated protein and to escribed . IgG4 anescribed . IgE (In a control group (n = 40) of atopic patients without hyperreactivity reactions to insect stings, 28% showed IgE-reactivity to bee venom and 35% to wasp venom in ImmunoCAP measurements (≥0.35kUA/L) .Venom allergic German as well as Slovenian patients were also subjected to skin testing with bee and wasp venom extract. Sixty six % of the German patients, who were thought to be double-sensitized according to allergen extract-based IgE serology, were found to be double-sensitized by skin testing whereas 4% and 30% were identified as mono-sensitized to bee and wasp, respectively . MoleculNone of the sera from the control group, without hyperreactivity reactions to bee and/or wasp venom, showed IgE-reactivity to rApi m 1, but 22.5% and 15% showed IgE reactivity to rVes v 5 by allergen chip and ImmunoCAP measurements, respectively .Skin testing of the Slovenian patients yielded negative results for 18% of the patients  was found for patients with severe reactions  as detected by ImmunoCAP. Using a cutoff of ≥ 0.1 kUA/L for the ImmunoCAP measurements, 57% of the sera showed IgE-reactivity to rApi m 1. There was a significant correlation between IgE levels determined with both methods   .A/L; 88% positive using cutoff ≥ 0.1 kUA/L). Again the levels of Ves v 5-specific IgE determined by both technologies were significantly correlated  (A similar correlation was found for rVes v 5-specific IgE reactivities. We found that 87% of the patients showed IgE-reactivity to rVes v 5 ≥ 0.1 ISU by chip technology and 82% of the sera were positive for rVes v 5 by ImmunoCAP measurements (cutoff ≥ 0.35 kU<0.0001) .Since Api m 1 and Ves v 5 are the marker allergens for bee and wasp sensitization, we investigated if there is an association between skin sensitivity and allergen-specific IgE levels to these allergens . HoweverA/L, <1 ISU) to Api m 1 without IgE reactivity to Api m 3, 4, 5 or 10 showed quite severe sting reactions   who had experienced severe sting reactions  (Similar results were obtained for rVes v 5 . There w4 levels to rApi m 1 and rVes v 5 for patients with identified culprit insect and available Mueller grading (4 levels (OD ≥0.1) were detected only in 5 out 13 sera and for Ves v 5 in 20 out of 30 sera. IgE/IgG4 ratios were therefore calculated only for Ves v 5. For these patients no association between the IgE/IgG4 ratio and the severity of sting reactions was found  [4 ratio specific for Ves v 5 for predicting clinical severity in wasp-sensitized patients but also this parameter was not associated with Mueller grading. Moreover, a considerable percentage of individuals without hyperreactivity reactions to insect venoms showed IgE reactivity to bee and wasp venom extracts and to rVes v 5 indicating that serological IgE test results have no predictive value for clinical severity in bee and wasp sensitization.Due to the fact that for all of the investigated patients detailed data regarding the severity of sting reactions according to Mueller grading and the intensity of skin sensitivity through titrated skin tests were available, we could investigate if surrogate markers can be defined which would allow to predict the severity of sting reactions. This question is very important, because the severity of a reaction varies, ranging from mild local to severe and life-threatening anaphylaxis. Unlike as for food allergy, where it is possible to define threshold levels of allergen-specific IgE and/or IgE reactivity profiles to certain high risk allergen molecules –35, thathnology) . Resultshnology) . In addiImportantly, we found patients who had very low levels of specific IgE against the clinically relevant marker allergens from bee (rApi m 1) and wasp (rVes v 5) but exhibited very severe sting reactions. We consider this result to be of high clinical relevance because it indicates that even subjects with very low IgE levels against these marker allergens can experience severe reactions. Accordingly one may consider informing patients with a positive IgE test results to the non-glycosylated marker allergens  about the potential risk that they may experience a severe sting reaction.S1 FigPie charts showing the percentages of patients with bee and wasp double sensitization (blue), sensitization to bee (yellow), wasp (red) and without positive skin prick test result (grey) according to allergen extract-based serology (left), skin testing (middle) and molecular diagnosis (right) in Slovenian patients with mono-sensitization to (A) bee (n = 11) and (B) wasp (n = 9).(EPS)Click here for additional data file.S2 FigCorrelation of marker allergen-specific sIgE levels to (A) rApi m 1 and (B) rVes v 5 determined by quantitative ImmunoCAP (y-axes: kUA/l) and ISAC allergen-chip measurements (x-axes: ISU) as scatter plots. Pearson`s correlation coefficients for rApi m 1: R = 0.686, p<0.0001and for rVes v 5: R = 0.986, p<0.0001.(EPS)Click here for additional data file.S3 FigA/L) or (B), (D) allergen chip (ISU) (y-axes) were plotted against the lowest concentration of venom giving a positive reaction in intradermal skin testing (x-axes).Specific IgE levels to Api m 1 or Ves v 5 measured by (A), (C) ImmunoCAP (kU(EPS)Click here for additional data file.S4 Fig4 (y-axis) were plotted against the severities of sting reactions (x-axes) for Slovenian and German patients with identified culprit insect.Ratios of Ves v 5 specific IgE/IgG(EPS)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Inhibition of action is involved in stopping a movement, as well as terminating unnecessary movement during performance of a behavior. The inhibition of single actions, known as response inhibition (Inhibition of the urge to respond before or after actions) has been widely investigated using the go/no-go task and stop signal task. However, few studies focused on phase and volition-related inhibition after an action has been initiated. Here, we used functional magnetic resonance imaging (fMRI) to investigate the neural correlates of planning and execution underlying the voluntary inhibition of ongoing action. We collected fMRI data while participants performed a continuous finger-tapping task involving voluntary and involuntary  inhibition, and during the initiation of movement. The results revealed areas of significantly greater activation during the preparation of inhibition of an ongoing action during voluntary inhibition, compared with involuntary inhibition, in the supplementary (SMA) and pre-supplementary motor areas, dorsolateral prefrontal cortex, inferior frontal gyrus (IFG), inferior parietal lobe, bilateral globus pallidus/putamen, bilateral insula and premotor cortex. Focusing on the period of execution of inhibition of ongoing actions, an event-related fMRI analysis revealed significant activation in the SMA, middle cingulate cortex, bilateral insula, right IFG and inferior parietal cortex. Additional comparative analyses suggested that brain activation while participants were planning to inhibit an ongoing action was similar to that during planning to start an action, indicating that the same neural substrates of motor planning may be recruited even when an action is ongoing. The present finding that brain activation associated with inhibiting ongoing actions was compatible with that seen in response inhibition (urge to stop before/after actions) suggests that common inhibitory mechanisms for motor movement are involved in both actual and planned motor action, which makes our behavior keep going seamlessly. Inhibition plays an important role in daily life. For example, inhibition of potentially harmful behaviors such as over-eating and over-drinking, and limiting the time spent playing computer games or browsing the web, are important for maintaining physical and mental health. Inhibition of motor actions has been investigated with the “response inhibition” paradigm using go/no-go tasks and stop signal tasks in which a participant is required to inhibit a planned and/or already initiated action  participated in the present study. The ethics committee of the Hamamatsu University School of Medicine approved the study, and all subjects gave written informed consent. According to the approved procedure, subjects with a current or previous history of neurological or psychiatric disorders and those with metal implants were excluded from the study. Using the Edinburgh handedness inventory . Slice orientation was tilted −20° from the AC-PC line. Two hundred image volumes were acquired in each session, and the first two volumes were discarded to avoid magnetic saturation effects. The total time per session was 8 min and 27 s. To prevent head motion, each subject's head was tightly fixed using cushions. Subjects were instructed to keep their head and body as still as possible during MRI scanning, except their right index finger.MRI scans were conducted with a 3-Tesla scanner . A 15-channel transmitter-receiver coil was used to scan the head. The T1-weighted sequence (Turbo field echo) was used for anatomic referencing of the fMRI recording, coregistration and normalization . For functional scans, T2An MRI-compatible head mounted display  and Presentation software  on a PC  were used to control stimulus presentation. Visual stimuli were presented via the head mounted display . The size of the letters and the plus sign were equal to 2.25° of horizontal and 3° of vertical field of view, respectively. For some subjects, a pair of glasses was attached to the head mounted display to correct their vision. A keypad with four buttons and a computer interface  was used for recording the finger-tapping responses. During the experiment, subjects wore headphones that attenuated the fMRI scanning noise, and remained lying down on the scanner bed with a button pad and an emergency buzzer.A new finger-tapping task was designed to investigate brain activity during behavioral change, and during the planning stages before behavioral change. Subjects were asked to continuously tap a button on a keypad with the index finger of their right hand, under voluntary and involuntary conditions. To clarify the volition of inhibition of ongoing actions, we compared the brain activation between the voluntary and forced inhibition of ongoing actions. In addition, the brain state during the initiation of ongoing actions was also recorded to compare with that of inhibition. There were four conditions in total , presented in short blocks lasting 20 s. Each condition appeared six times per session, in a random order. Three sessions were conducted per subject. In total, each condition was presented 18 times for each subject. In all conditions, a white plus sign was presented as a fixation point.Figure Subjects practiced the tasks for several minutes outside the scanner before MRI scanning, and were instructed to tap a button at a natural speed. However, some participants occasionally failed to produce an action on some trials in the voluntary condition because they were asked to keep tapping or waiting, based on their own judgment. Trials involving an extremely short duration of action (< 1 s), no action, or an extremely long duration of action (>17 s) were excluded from subsequent analysis. Additionally, the performance criterion for each subject included a success rate of more than 90% in all conditions. Three subjects were excluded from the final analysis because of low task performance.To examine each subject's performance, behavioral data from the tasks were analyzed, calculating the average duration of tapping and the average number of taps per second in the period between the presentation of the colored plus sign and the time of execution of behavioral changes. The data from error trials were excluded from this analysis. The timing of execution was defined by the first button-press at the start of the ongoing tapping or the last button-press when the ongoing tapping stopped. Note that the presentation time of the stop or start cue in the forced conditions was not used for determining the timing of execution of behavioral changes to achieve equality between voluntary and forced conditions. These times were used for further fMRI analysis.http://www.fil.ion.ucl.ac.uk/spm/) in MATLAB . Preprocessing of the fMRI was as follows: slice timing correction, realignment, coregistration, spatial normalization to the standard Montreal Neurological Institute (MNI) template, and spatial smoothing using a Gaussian kernel with a full width at half-maximum of 8 mm.fMRI data were analyzed using SPM8 (i) contrast of tapping periods before stopping finger-tapping between voluntary and forced conditions, and (ii) contrast of waiting periods before starting finger-tapping between voluntary and forced conditions. Note that the contrast (i) and (ii) reflect activation of “planning” because subjects were required to decide when to stop or start in the voluntary conditions whereas just waiting in the forced conditions. Especially, the activations related to tapping action itself in both the voluntary and forced conditions were canceled out in the contrast (i). Brain activity related to the execution of voluntary inhibitory action was detected using two contrast values, as above: (i) contrast of execution of inhibition of ongoing actions between the voluntary and forced conditions, and (ii) contrast of initiation of ongoing actions between the voluntary and forced conditions. Because the execution of motor actions must be correlated with a physical movement, the event-related regressors of the timing of an inhibitory or initiatory action were assumed to reflect the neural process of the execution of inhibition of ongoing actions.For each subject, the brain regions were statistically evaluated using a general linear model (GLM) including both explanatory variables of interest and variables of non-interest, and six realignment parameters were used as multiple regressors. To clarify brain activation during the planning and execution phases Figure , the modt-tests for each contrast were conducted to verify the relevant brain regions. For all data, a threshold of uncorrected P < 0.001 for peak-level and a cluster-level family-wise error (FWE) of 0.05 was used for statistical analyses. In addition, two conjunction analyses were performed to confirm the common brain regions related to inhibition of finger-tapping (both voluntary and forced stopping) and planning when to stop or start finger-tapping. A threshold of uncorrected P < 0.001 for peak-level and a cluster-level FWE of 0.05 was also used. Atlases of the human brain , the voluntary stop condition , the forced start condition  and the forced stop condition . Our data showed that the average duration between the sign of the task cue and the start point of motor movements were significantly shorter in the voluntary start condition than in the voluntary stop condition . Conversely, there was no difference between the forced conditions   between the presentation of the colored plus sign and the start of behavioral change were calculated in the voluntary start condition  Figure . These rF = 4.60, P = 0.035], indicating that subjects tapped a button faster in the start conditions than in the stop conditions. In contrast, neither the main effect of active-passive  44), the left insula and the right globus pallidus/putamen Figure . In addiThere was no significant difference in activation during the execution of voluntary stop compared with the execution of forced stop (self-paced > stimulus-triggered). In contrast, brain activation in the occipital regions BA19/37), which include the fusiform gyrus and middle occipital gyrus, were found in the comparison between forced and voluntary stop (stimulus-triggered > self-paced) , the bilateral insular cortex and the right inferior parietal lobe (rIPL) Figure . In contIn the current study, the neural correlates of inhibition of an ongoing behavior were investigated regarding similarities in brain activation in planning and executing to inhibit continuing movements. Behaviorally, in the voluntary conditions, the waiting period before starting the finger-tapping action was shorter than the exercise period before stopping finger-tapping, despite the instruction being the same Figure . The difThe results of the execution phase revealed activation in the MCC, including the pre-SMA, SMA, IPL, IFG, and insula cortex, in the both voluntary and forced conditions Figure . PreviouThe comparison of the execution phase between voluntary and forced conditions revealed brain activation specific to stimulus-triggered movements (stimulus-triggered > self-paced) in occipital regions and motor-related areas Figure . These bThe planning phase was associated with activation in the SMA and pre-SMA, the IPL, the IFG (BA 44), the DLPFC, the insula, the left cerebellum, the primary visual cortex and the globus pallidus/putamen, irrespective of inhibition or initiation condition Figure . InteresBrass and Haggard  discusse5A). This finding suggests that a sequence of simple motor actions generates minimal interference with the process of planning a next action.The conjunction analysis revealed several common regions of activation while planning a motor action, with or without a continuous action being performed Figure , suggestThe current finding of brain regions specific to motor inhibition working commonly during execution and planning may be extrapolated to the pathological symptom named as perseveration seen in disorders of inhibition. The functional breakdown of the brain areas found in the present analysis may be responsible for this perseveration in brain disorders (Gandola et al., In the present study, we used colored visual stimuli to instruct subjects to execute stopping or starting finger-tapping. Especially, the use of visual stimuli such as a stop or start sign in the involuntary conditions compared with the voluntary conditions may be an influencing factor to the results. To clarify this, further study will be required; for example, auditory stimuli instead of visual stimuli would be available to verify the brain regions that related to execution of stopping. In addition, the results showed the laterality of brain activations toward the right hemisphere in both the planning and execution phase Figures , 4. It iTo verify the neuronal processes underlying the inhibition of an ongoing action, we conducted an fMRI study using a finger-tapping task involving self-initiated and stimulus-triggered inhibition of an ongoing action. We attempted to dissociate the inhibition process into planning and execution phases. In the execution phase, inhibition of ongoing action activated the SMA, aMCC, insula cortex, rIPL and rIFG, compatible with the brain activation seen during response inhibition. In addition, activation of the DLPFC, globus pallidus/putamen, premotor cortex, primary visual cortex and cerebellum were observed in the planning phase in both the initiation and inhibition conditions. These results suggest that a common inhibitory mechanism for canceling a motor action is used in both execution and planning of motor action, and that a common planning network of motor action is recruited even when an action is ongoing.KO designed and performed the research, analyzed the data, and wrote the paper. SI and YT performed the research. YO designed the research and wrote the paper.YT was a predoctoral researcher, employed by company Hamamatsu Photonics KK. The remaining 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.


Recently, a Cdk1‐dependent phosphorylation relay within Apc3‐Apc1 subunits has been shown to alleviate Apc1‐mediated auto‐inhibition by which a mitotic APC/C co‐activator Cdc20 binds to and activates the APC/C. However, the underlying mechanism for dephosphorylation of Cdc20 and APC/C remains elusive. Here, we show that a disordered loop domain of Apc1 (Apc1‐loop500) directly binds the B56 regulatory subunit of protein phosphatase 2A (PP2A) and stimulates Cdc20 loading to the APC/C. Using the APC/C reconstitution system in Xenopus egg extracts, we demonstrate that mutations in Apc1‐loop500 that abolish B56 binding decrease Cdc20 loading and APC/C‐dependent ubiquitylation. Conversely, a non‐phosphorylatable mutant Cdc20 can efficiently bind the APC/C even when PP2A‐B56 binding is impeded. Furthermore, PP2A‐B56 preferentially dephosphorylates Cdc20 over the Apc1 inhibitory domain. These results indicate that Apc1‐loop500 plays a role in dephosphorylating Cdc20, promoting APC/C‐Cdc20 complex formation in mitosis.Cell cycle progression and genome stability are regulated by a ubiquitin ligase, the anaphase‐promoting complex/cyclosome (APC/C). Cyclin‐dependent kinase 1 (Cdk1) has long been implicated in APC/C activation; however, the molecular mechanisms of governing this process  The APC/C, an E3 ubiquitin ligase, only becomes active when its co‐activator Cdc20 is associated with it. This study shows that PP2A‐B56 loaded on Apc1 plays a role in mitotic APC/C activation by dephosphorylating Cdc20. The cell cycle is a biological process by which genetic information is accurately duplicated and segregated into daughter cells. At the same time, growth, proliferation and differentiation are controlled accordingly in response to environmental cues, ensuring genome stability. Cyclin‐dependent kinases (Cdks) and the APC/C are some of the most significant cell cycle regulators, controlling the cell cycle through phosphorylation and ubiquitylation, respectively 300, 294–399 in Xenopus). Cdk1‐driven phosphorylation occurs in a coordinated manner between Apc3 and Apc1. Cdk1‐dependent Apc3‐loop phosphorylation acts as a scaffold and recruits a Cdk regulatory subunit p9/Cks protein via its anion binding domain. In turn, this engages a ternary complex Cks‐Cdk1‐cyclin and phosphorylates an auto‐inhibitory domain in a distant subunit, Apc1, in an intramolecular phosphorylation relay 300, acts as a molecular switch, and in unphosphorylated APC/C, it interacts with the C‐box (a short and conserved motif present in the Cdc20/Fizzy family of co‐activators) binding site and sterically hinders engagement of Cdc20. Phosphorylation of Apc1‐loop300 induces a conformational change, resulting in dissociation of Apc1‐loop300 from the C‐box binding site enabling Cdc20 loading for ubiquitylation catalysis Cdk1 and APC/C mutually regulate each other in mitosis, forming a negative feedback loop by which a periodicity of cell division cycle is ensured; there can be no mitotic APC/C activation without Cdk1, and there can be no cyclin destruction and no cell division without APC/C. However, despite the obvious importance of the whole mechanism, the control of APC/C activation and regulation still remains poorly understood. One crucial aspect of APC/C regulation is the engagement of its co‐activators Cdc20 and Cdh1 as apo‐APC/C by itself is not active. Co‐activator engagement is regulated by phosphorylation of both the APC/C and co‐activators. Mitotic co‐activator Cdc20 preferentially binds and activates hyperphosphorylated APC/C promoting mitotic progression, whereas Cdh1, which is inhibited by phosphorylation, functions in telophase and G1 when Cdk activity is low after cyclin B destruction. High‐resolution cryo‐EM has uncovered the detailed structure of the APC/C complex (comprising 14 subunits in vertebrates) providing mechanistic insights Xenopus) inhibits Cdc20 loading to the APC/C, presumably by precluding Cdc20‐Apc6 interaction Phosphorylation of Cdc20 is also an important element that controls the formation of active APC/C‐Cdc20 complex. Cdk1‐dependent phosphorylation of three N‐terminal threonine residues near the C‐box . Using egg extract of Xenopus laevis and reconstitution of apo‐APC/Cs in the MultiBac system, we show here that Apc1‐loop500 has a role in PP2A‐B56 recruitment in mitosis, which in turn dephosphorylates Cdc20 and controls its loading for APC/C activation. Consistently, phosphorylation site mutant Cdc20 can bind sufficiently to the APC/C independently of PP2A‐B56 binding. Furthermore, PP2A‐B56 dephosphorylates Cdc20 more efficiently than the Apc1‐loop300. Our work reveals a mechanism explaining how a mitotic co‐activator Cdc20 can specifically bind to and activate the APC/C in anaphase and therefore initiate sister chromatid separation and mitotic exit.We have identified a B56 binding site within the 300. We therefore hypothesised that these flexible disordered loop domains could also regulate APC/C activity. It has been recently reported that PP2A‐B56 recognises and binds a LxxIxE SLiM on target substrates Xenopus Apc1, hereinafter referred to as Apc1‐loop500) that is located in the N‐terminal WD40 domain of Apc1. This sequence is highly conserved among species including human Apc1  or substitution of three alanines of putative Cdk sites (3A) , compared with MBP‐alone or MBP‐fused Apc1‐loop500‐∆11 (lanes 13 and 15). This result suggests that this flexible Apc1‐loop500 domain can bind B56γ via the B56 binding site. This interaction was not observed in interphase extracts, which have low Cdk activity (lane 10), suggesting that the binding of B56 to Apc1‐loop500 depends on phosphorylation of the loop domain. Consistently, non‐phosphorylatable Apc1‐loop500‐3A failed to bind B56γ even in anaphase extract (lane 16). We also performed Cdk‐dependent in vitro kinase assay and confirmed that WT Apc1‐loop500, but not Apc1‐loop500‐3A, was efficiently phosphorylated by Cdk2‐cyclin A . As was seen for Apc1‐loop500‐∆11, the mutations in the B56 binding site (Apc1‐loop500 L557A/V560A) abolished the ability to bind B56γ , and then tested the ability of mutant apo‐APC/Cs to bind to the co‐activator Cdc20  was observed at almost equal levels in both apo‐APC/Cs. In contrast, in interphase, Cdc20 was able to bind WT APC/C and APC/C1‐L557A/V560A at a similar level . This deletion renders APC/C phosphorylation unnecessary for Cdc20‐dependent activation. Cyclin destruction assays confirmed that unlike WT APC/C, the APC/C1‐∆L300 was able to be activated in interphase by Cdc20  had no effect on Cdc20 binding in interphase extract  and promotes the engagement of Cdc20 with the APC/C for activation Xenopus Cdc20) 300 are exclusively serine  32P‐labelled Apc1‐loop300 fragment incubated in anaphase extracts , which is free from Cdk‐dependent inhibitory phosphorylation 500  was decreased, compared with WT APC/C . In support of this, Cdc20‐5A was able to bind equally to both WT APC/C and APC/C1‐∆11, another B56 binding site mutant APC/C  are similar in both WT and B56 binding site mutant APC/C in anaphase extract  or anti‐pS314/S318 (Apc1‐loop300). Phosphorylation of Cdc20 T79 was significantly reduced upon addition of PP2A‐B56γ, whereas phosphorylation of Apc1‐loop300 remained stable  in the N‐terminal wheel‐like WD40 domain of Apc1  plays an important role in dephosphorylating Cdc20 by directly recruiting PP2A‐B56, thereby allowing productive Cdc20‐APC/C complex formation in mitosis. The Apc3‐loop mediates efficient phosphorylation of Apc3 and Apc1 by directly recruiting Cks‐Cdk1‐cyclin B, whereas the Apc1‐loop300 acts as a phosphorylation‐mediated conformational switch, permitting association with Cdc20. It seems that flexible and disordered loop domains within the APC/C play a central role in the dynamic regulation of the APC/C, in particular through post‐translation modifications (PTMs). This appears to be the case not only for the APC/C but also for other multi‐protein complexes and cellular regulation pathways, e.g. RNA metabolism. Intriguingly, a number of recently identified RNA binding proteins bind to RNAs via disordered loop regions In summary, we find that a disordered loop domain  subunits are gifts from Drs N. Sagata Antibodies used are as follows: anti‐Apc1 , Apc3/Cdc27 , Apc5 , Cdc20 , maltose binding protein  to interphase extracts and incubating for 30–60 min at 23°C Meiotic metaphase II‐arrested (CSF) Xenopus APC/C genes and TEV‐cleavable tandem Strep II‐tag fused to Apc6 at C‐terminus (Apc6‐strep) were generated by MultiBac system . Mutant APC/Cs were generated by PCR‐based mutagenesis, and mutation sites were confirmed by sequencing. To express APC/C complex, High Five insect cells (Invitrogen) at a cell density of 1.5 × 106 were co‐infected with the two recombinant baculoviruses at an MOI (multiplicity of infection) of 1 for each virus and incubated at 27°C for 48 h with shaking (150 rev/min). The cells were harvested, frozen in liquid nitrogen and stored at −80°C. The recombinant APC/Cs were purified with Strep‐Tactin beads and further affinity‐purified by Dynabeads Protein A conjugated to anti‐Apc3 monoclonal antibody (MAb AF3.1). The APC/Cs bound to beads were flash‐frozen and stored at −80°C.Expression and purification of recombinant APC/Cs were performed as described previously 500, spanning amino acid 515–584 of Xenopus tropicalis Apc1, was fused with PreScission protease‐cleavable maltose binding protein (MBP) at the N‐terminus and a TEV‐cleavable 6xHis at the C‐terminus, and subcloned into pET vector. Mutants were generated by PCR‐based mutagenesis and subcloned into pET vector as for WT, and mutation sites were confirmed by sequencing. The Apc1‐Loop300, spanning amino acids 294–399 of X. tropicalis Apc1, was fused with a PreScission protease‐cleavable 3xFlag‐tag at the N‐terminus and a TEV‐cleavable 6xHis at the C‐terminus, and subsequently subcloned into pET vector. For its 7T variant (Apc1‐loop300‐7T), cDNA encoding for the same region but containing threonine substitutions of all the conserved serine Cdk sites  was synthesised  and subcloned into the pET vector as for WT. In both WT and mutant vectors, the Cdk sites (SP or TP) were confirmed by sequencing prior to bacterial expression. Xenopus laevis B56γ was fused with PreScission protease‐cleavable 3xFlag at the N‐terminus and a TEV‐cleavable 6xHis at the C‐terminus, and subcloned into pET vector. After sequencing confirmation, the resultant plasmids were introduced into BL21‐CodonPlus (DE3) and the fusion proteins were expressed at 37°C for 1 h in the presence of 1 mM IPTG. The cells were lysed by 0.3 mg/ml lysozyme and sonicated in lysis buffer . The proteins were purified from clarified lysate using Ni‐NTA agarose beads (Qiagen).The Apc1‐Loop500 fragment (6 μg) was incubated with Cdk2‐cyclin A in the presence of [γ‐32P]‐ATP at 23°C for 10 min, and separated by SDS–PAGE and detected by autoradiography  was incubated with Cdk2‐cyclin A in the presence of [γ‐32P]‐ATP at 30°C for 30 min, and separated by SDS–PAGE and detected by autoradiography  35S]methionine  in a coupled in vitro transcription–translation system . Purified Apc1‐Loop500 fragment proteins were bound to amylose beads (New England Biolabs) by incubating at 4°C for 0.5–1 h. Beads were washed with Tris‐NaCl buffer [20 mM Tris–HCl pH 8.0 and 200 mM NaCl] and XBCSF buffer  and incubated with 35S‐labelled PP2A B subunits in interphase extract in the presence or absence of cyclin BΔ167 at 23°C for 60 min, separated from extract on Micro Bio‐Spin columns (Bio‐Rad), and washed once with XBCSF buffer and then twice with XBCSF buffer containing 0.01% NP‐40. The bound proteins were eluted with SDS sample buffer and analysed by SDS–PAGE and autoradiography. For Cdk phosphorylation‐dependent B56 loading assays, MBP‐fused WT or 3A Apc1‐loop500 fragment (12 μg) was first bound to amylose resin and then incubated at 30°C for 60 min in 20 μl of XBCSF buffer containing 1 mM ATP and 4 mM MgCl2 in the presence or absence of Cdk2‐cyclin A. Beads with MBP peptides (+/− kinase) were washed with XBCSF buffer containing 0.01% NP‐40 and incubated in 20 μl of the same buffer containing purified 3xFlag‐tagged B56γ (4 μg) at 23°C for 30 min. The beads were separated on Micro Bio‐Spin columns (Bio‐Rad), washed once with XBCSF buffer and then twice with XBCSF buffer containing 0.01% NP‐40. The bound proteins were eluted with SDS sample buffer and analysed by SDS–PAGE and immunoblotting.For binding assay with [CSF_HS [XBCSF containing 500 mM KCl and 0.01% NP‐40], eluted with SDS sample buffer and analysed by SDS–PAGE and immunoblotting.The APC/C was immunoprecipitated using Apc3 MAb (AF3.1) immobilised Dynabeads Protein A beads. The bound proteins were washed twice with XB35S]methionine  in a coupled in vitro transcription–translation system , and destruction assays were carried out using Xenopus egg cell‐free extracts (anaphase or interphase extracts). The samples were taken at the indicated time points and analysed by SDS–PAGE and autoradiography. The images were analysed using ImageJ .Destruction assays were performed as described previously Xenopus egg cell‐free extracts in the presence of non‐degradable GST‐fused Xenopus cyclin BΔ167 at 23°C for 60 min and immunoprecipitated using Apc3 MAb (AF3.1)‐immobilised Dynabeads Protein A. The beads were washed twice with XBCSF_HS and once with Ub buffer . The resultant APC/C‐Cdc20 complexes were incubated at 23°C in 20 μl of buffer  containing 0.05 mg/ml E1, 0.025 mg/ml UbcX, 0.75 mg/ml ubiquitin, 1 μM ubiquitin aldehyde, 150 μM MG132 and 2 μl of 35S‐labelled cyclin B (fission yeast Cdc13). The reactions were stopped at the indicated time points with SDS sample buffer and analysed by SDS–PAGE and autoradiography.Ubiquitylation assays were performed as described Three PP2A subunit genes  were PCR‐amplified and cloned into pOENmyc vector with polH promoter and SV40 terminator for Cβ or p10 promoter and HSVtk terminator for Aα and B56γ. PreScission protease‐cleavable GST‐tag or His‐tag was fused to Aα or B56γ at the N‐terminus, respectively. These genes were further cloned into MultiBac vectors creating one pF1 vector‐derivative pFUBB carrying Cβ at MUM1 site and His‐B56γ at MUM2 site. A pU1 vector‐derivative pUUBB carrying Aα at MUM2 site was also created. After sequence confirmation, generation of baculovirus and the expression of PP2A‐B56γ complex were performed as described previously g for 20 min, and the supernatant was centrifuged again for 20 min. The cleared lysate was incubated with GSH beads  at 4°C for 1 h. The beads were washed twice with PP2A wash buffer 1  and once with PP2A wash buffer 2  and suspended into PP2A wash buffer 2 containing PreScission protease and incubated at 4°C for 3.5 h with gently mixing. After a brief centrifugation, the supernatant was incubated with Ni‐NTA at 4°C for 30 min with gentle mixing, and the beads were washed three times with His washing buffer , and the bound proteins were eluted by His elution buffer . All purification steps were performed at 4°C.Cell pellets were thawed on ice and resuspended in PP2A lysis buffer  and lysed by sonication. The lysate was centrifuged at 18,800 CSF_HS without EGTA and once with XBCSF buffer containing 0.01% NP‐40 without EGTA, resuspended into XBCSF buffer containing 0.01% NP‐40 without EGTA and incubated with purified PP2A‐B56γ at 23°C. The reactions were stopped at the indicated time points with SDS sample buffer and analysed by SDS–PAGE and immunoblotting. For time course phosphate removal experiment  and anti‐Cdc20 (B60) antibody‐immobilised Dynabeads Protein A. The beads were washed twice with XBStatistical analyses were performed in GraphPad Prism v6.0. Quantification data are presented as the mean ± SEM from three independent experiments.KF and HY designed research. KF and HY performed research and analysed data. KF and HY wrote the paper. HY acquired funding and supervised research.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Review Process FileClick here for additional data file.


Context: Bergenin, isolated from the herb of Bergenia purpurascens (Hook. f. et Thoms.) Engl., has anti-inflammatory, antitussive, and wound healing activities. However, whether bergenin affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear.Materials and methods: In this study, the inhibitory effects of bergenin (100 μM) on the eight human liver CYP isoforms  were investigated, enzyme kinetics and time-dependent inhibition studies were also performed in vitro using human liver microsomes (HLMs).:Results The results showed that bergenin inhibited the activity of CYP3A4, 2E1 and 2C9, with IC50 values of 14.39, 22.83 and 15.11 μM, respectively, but other CYP isoforms were not affected. Enzyme kinetic studies showed that bergenin was not only a non-competitive inhibitor of CYP3A4, but also a competitive inhibitor of CYP2E1 and 2C9, with iK values of 7.71, 11.39 and 8.89 μM, respectively. In addition, bergenin is a time-dependent inhibitor for CYP3A4 with Kinact/IK value of 0.025/3.50 μM−1 min−1.Discussion and conclusions: The in vitro studies of bergenin with CYP isoforms indicate that bergenin has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP3A4, 2E1 and 2C9. Further clinical studies are needed to evaluate the significance of this interaction. Bergenia purpurascens (Hook. f. et Thoms.) Engl., a traditional Chinese medicine, possesses anti-inflammation and antidiarrhoeal abilities and is used clinically for the treatment of diarrhoea, dysentery and other gut-associated diseases  enzymes are important phase I enzymes in the biotransformation of xenobiotics, and CYP450 enzymes can be inhibited or induced by a variety of drugs and chemicals that can give rise to toxicity or treatment failure , testosterone (CYP3A4), coumarin (CYP2A6), chlorzoxazone (CYP2E1), dextromethorphan (CYP2D6), diclofenac (CYP2C9), S-mephenytoin (CYP2C19) and paclitaxel (CYP2C8) were used as probe substrates to determine the effects of bergenin on eight CYP enzymes. In addition, enzyme kinetic studies were conducted to determine the inhibition mode of bergenin on CYP enzymes.The purpose of this study was to investigate the effects of bergenin on eight major CYP isoforms in human liver microsomes (HLMs). d-Glucose-6-phosphate, glucose-6-phosphate dehydrogenase, corticosterone (≥98%), NADP+, phenacetin (≥98%), acetaminophen (≥98%), 4-hydroxymephenytoin (≥98%), 7-hydroxycoumarin (≥98%), 4′-hydroxydiclofenac (≥98%), sulphaphenazole (≥98%), quinidine (≥98%), tranylcypromine (≥98%), chlorzoxazone (≥98%), 6-hydroxychlorzoxazone (≥98%), paclitaxel (≥98%), 6β-hydroxytestosterone (≥98%), clomethiazole (≥98%) and furafylline (≥98%) were obtained from Sigma Chemical Co. . Montelukast (≥98%) was obtained from Beijing Aleznova Pharmaceutical . Coumarin (≥98%), diclofenac (≥98%), dextromethorphan (≥98%) and ketoconazole (≥98%) were purchased from ICN Biomedicals . Pooled HLMs were purchased from BD Biosciences Discovery Labware . All other reagents and solvents were of analytical reagent grade.Bergenin (≥98%) and testosterone (≥98%) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products . O-deethylation for CYP1A2, testosterone 6β-hydroxylation for CYP3A4, coumarin 7-hydroxylation for CYP2A6, chlorzoxazone 6-hydroxylation for CYP2E1, dextromethorphan O-demethylation for CYP2D6, diclofenac 4′-hydroxylation for CYP2C9, S-mephenytoin 4-hydroxylation for CYP2C19 and paclitaxel 6α-hydroxylation for CYP2C8. All incubations were performed in triplicate, and the mean values were utilized. The typical incubation systems contained 100 mM potassium phosphate buffer (pH 7.4), NADPH generating system , the appropriate concentration of HLMs, a corresponding probe substrate and bergenin (or positive inhibitor for different probe reactions) in a final volume of 200 μL.As shown in v/v), and 1% neat methanol was added to the incubations without inhibitor. The final microsomal protein concentration and incubation times for the different probe reactions are shown in The concentration of bergenin was 100 μM, and the positive inhibitor concentrations were as follows: 10 μM furafylline for CYP1A2, 1 μM ketoconazole for CYP3A4, 10 μM tranylcypromine for CYP2A6, 50 μM clomethiazole for CYP2E1, 10 μM quinidine for CYP2D6, 10 μM sulphaphenazole for CYP2C9, 50 μM tranylcypromine for CYP2C19, 5 μM montelukast for CYP2C8. Probe substrates, positive inhibitors (except for dextromethorphan and quinidine which were dissolved in water) and bergenin were dissolved in methanol, with a final concentration of 1% (50). iK values were obtained by incubating various concentrations of different probe substrates  in the presence of 0–50 μM bergenin.A 100 μM bergenin was used to initially screen for its direct inhibitory effects towards different human CYP isoforms. For the CYP isoforms whose activities were strongly inhibited, secondary studies were performed to obtain the half inhibition concentration , the reactions were terminated by adding a 100 μL acetonitrile internal standard mix and then placed on ice; the corresponding metabolites were determined by HPLC.To determine whether bergenin could inhibit the activity of CYP3A4, 2E1 and 2C9 in a time-dependent manner, bergenin (20 μM) was pre-incubated with HLMs (1 mg/mL) in the presence of an NADPH-generating system for 30 min at 37 °C. After incubation, an aliquot (20 μL) was transferred to another incubation tube  containing an NADPH-generating system and probe substrates whose final concentrations were approximate to IK and Kinact values for the inactivation of CYP3A4, the incubations were conducted using higher probe substrate concentrations  and various concentrations of bergenin (0–50 μM) after different pre-incubation times (0–30 min), with a two-step incubation scheme, as described above.To determine the Vmax and mK. Inhibition data from the experiments that were conducted using multiple compound concentrations were represented by Dixon plots, and inhibition constant (iK) values were calculated using non-linear regression according to the following equation:I is the concentration of the compound, iK is the inhibition constant, S is the concentration of the substrate and mK is the substrate concentration at half the maximum velocity (Vmax) of the reaction. The mechanism of the inhibition was inspected using the Lineweaver–Burk plots and the enzyme inhibition models. The data comparison was performed using the Student’s t-test and performed using IBM SPSS statistics 20 .The enzyme kinetic parameters for the probe reaction were estimated from the best fit line using least-squares linear regression of the inverse substrate concentration versus the inverse velocity (Lineweaver–Burk plots), and the mean values were used to calculate To investigate whether the bergenin affects the catalytic activity of CYP enzymes, the probe reaction assays were conducted with varying concentrations of bergenin . Specifi50 values of 14.39, 22.83 and 15.11 μM, respectively.The enzyme-inhibition study showed that inhibition of CYP3A4, 2E1 and 2C9 by bergenin was concentration-dependent, with ICiK values of bergenin on CYP3A4  and clinical (in vivo) interaction studies in the drug development.In clinical practice, many patients undergo multiple-drug therapy. Multiple-drug therapy possesses several advantages, such as simultaneously treatment of diseases, or multi-drug therapy for the treatment of complex chronical disorders, resulting in a better treatment outcome compared with monotherapy. However, many herb–drug interactions resulting from the concurrent use of herbal drugs with prescription and over-the-counter drugs may cause adverse reactions such as toxicity and treatment failure  over the in vivo concentration of the inhibitor  were less than 300 ng/mL (0.9 μM). Therefore, the rat plasma concentrations of bergenin were much lower than the iK and IC50 values of bergenin determined in this study, and severe drug–drug interaction might not occur if bergenin were co-administered with the substrates of the CYP3A4, 2E1 and 2C9. However, due to the pharmacokinetic differences of human and rats, further in vivo system studies are needed to identify the interactions of bergenin with CYP isoform in humans.The results of this study indicate that bergenin may influence the n et al.  have repIn conclusion, the effects of bergenin on the activity of CYP enzymes were systematically investigated. The results showed that bergenin could inhibit the activity of CYP3A4, 2E1 and 2C9, while the activities of other CYP enzymes were not affected. Therefore, to avoid adverse drug interactions, it is recommended that bergenin should not be used with other drugs metabolized by CYP3A4, 2E1 and 2C9.