However, the effects of LRRK2-mediated phosphorylation of these substrates on membrane trafficking remain unknown. Here, using an array of immunofluorescence and pulldown assays, we report that expression of active or phosphodeficient RAB8A variants rescues the G2019S LRRK2–mediated effects on endolysosomal membrane trafficking. Similarly, up-regulation of the RAB11–Rabin8–RAB8A cascade, which activates RAB8A, also reverted these trafficking deficits. Loss of RAB8A mimicked the effects of G2019S LRRK2 on endolysosomal trafficking and decreased RAB7A activity. Expression of pathogenic G2019S LRRK2 or loss of RAB8A interfered with EGFR degradation by causing its accumulation in a RAB4-positive endocytic compartment, which was accompanied by a deficit in EGFR recycling and was rescued upon expression of active RAB7A. Dominant-negative RAB7A expression resulted in similar deficits in EGF degradation, accumulation in a RAB4 compartment, and deficits in EGFR recycling, which were all rescued upon expression of active RAB8A. Taken together, these findings suggest that, by impairing RAB8A function, pathogenic G2019S LRRK2 deregulates endolysosomal transport and endocytic recycling events.Mutations in the gene encoding for leucine-rich repeat kinase 2 (LRRK2) are a common cause of hereditary Parkinson's disease. LRRK2 regulates various intracellular vesicular trafficking pathways, including endolysosomal degradative events such as epidermal growth factor receptor (EGFR) degradation. Recent studies have revealed that a subset of RAB proteins involved in secretory and endocytic recycling are LRRK2 kinase substrates  HoweLRRK2 is widely expressed, suggesting that it may play cellular role(s) common to both neuronal and nonneuronal cell types . LRRK2 r12–in vivo LRRK2 kinase substrates, with RAB8A being one of the most prominent . In addition, and as described previously . In contrast, other RAB proteins tested, including RAB7A, were not phosphorylated to a significant degree . Therefore, the previously reported LRRK2-mediated deficit in endolysosomal trafficking . Upon incubation of cells at 37 °C for either 10 or 30 min to monitor fluorescent EGF degradation, a pronounced delay in the clearance of Alexa555-EGF per cell was observed in the presence of G2019S but not kinase-inactive G2019S-K1906M LRRK2, respectively (C and D). A similar pathogenic LRRK2-mediated deficit in EGF clearance was observed when quantifying intracellular fluorescent EGF-positive puncta per area with no change in the fluorescence intensity of the individual dots (E and F). To further corroborate that the increase in intracellular fluorescent EGF was due to impaired EGFR degradation, biochemical EGFR degradation assays were performed in transfected HEK293T cells expressing G2019S or kinase-inactive G2019S-K1906M LRRK2, respectively. Pathogenic LRRK2 expression did not alter steady-state EGFR expression levels . However, and as described previously (H and I). Therefore, pathogenic LRRK2 interferes with the endolysosomal trafficking and degradation of EGF and the EGFR.Upon binding of EGF, the EGFR is internalized by clathrin-mediated endocytosis and sorted to lysosomes for degradation in a manner dependent on RAB7 . EGFR suFig. 1eviously , cells eFig. S2, A–C). In contrast, GDP-locked inactive RAB8A-T22N was cytosolic and not properly targeted to a tubular recycling compartment . When expressed on their own, neither RAB8A nor RAB8A-Q67L caused alterations in EGF binding or EGFR trafficking, whereas RAB8A-T22N caused a modest decrease in EGF surface binding and a slight delay in EGFR degradation, evident only at t = 30 min (A and B). Importantly, the pathogenic G2019S LRRK2–mediated decrease in EGF binding and the delay in EGFR trafficking were fully rescued when expressing RAB8A-Q67L (C and D). This was not observed with WT RAB8A or with RAB8A-T22N (C and D), although both WT and RAB8A-Q67L were expressed to similar degrees , and none of the RAB8A variants interfered with the coexpression of G2019S LRRK2 . In this cell system, and taking into account a roughly 50% transfection efficiency, overexpression of LRRK2 constructs was about 1–1.5-fold above endogenous levels, and overexpression of RAB8A was around 2.5–4-fold above endogenous levels, respectively .We next wondered how these LRRK2-mediated endolysosomal trafficking deficits may be modulated by RAB8A. In HeLa cells, GFP-tagged WT RAB8A and GTP-locked, constitutively active RAB8A-Q67L were largely localized to a tubular endocytic recycling compartment partially overlapping with the transferrin receptor, with tubular localization more evident in live or in fixed but only briefly permeabilized cells  and had no effect on endolysosomal trafficking when expressed on its own (E and F). However, Rabin8 expression rescued the G2019S LRRK2–mediated deficits in EGF binding and degradation (G and H) without interfering with the expression levels of G2019S LRRK2 , indicating that activating endogenous RAB8A by Rabin8 expression also rescues the LRRK2-mediated endolysosomal trafficking defects.Rabin8 functions as a GEF for RAB8A and activates it by catalyzing GDP release for subsequent GTP loading . Convers34– its own E and F. radation , G and HFig. S2A). Both WT and GTP-locked active RAB11 (RAB11-Q70L) were membrane-associated, whereas GDP-locked inactive RAB11 (RAB11-S25N) was largely cytosolic . When coexpressed with pathogenic LRRK2, both WT and active, but not inactive RAB11, rescued the deficit in EGF binding and degradation (A and B), but none of them had an effect on their own (C and D) even though they were expressed to comparable degrees and did not alter the expression levels of G2019S LRRK2 . As another means to show that the effects reported here were specific, we analyzed the role of RAB18, an endoplasmic reticulum–resident RAB protein  (E and F), and they also displayed no effect on their own (G and H) even though they were expressed to similar degrees and did not affect the levels of coexpressed G2019S LRRK2 . Altogether, these data indicate that the RAB11–Rabin8–RAB8A cascade specifically rescues the endolysosomal trafficking defects associated with pathogenic LRRK2.We next analyzed the effects of RAB11 on the LRRK2-mediated deficits in EGFR trafficking. GFP-tagged RAB11 colocalized with transferrin receptor  . NeitherA and B) , E and Fheir own , G and HFig. S2F). With the exception of RAB8A-T72E, none of these variants significantly altered EGF binding or EGFR degradation when expressed on their own (A and B), with all constructs apart from RAB8A-T22N and RAB8A-T72E expressed to similar degrees . However, the phosphodeficient, but not the phosphomimetic, RAB8A versions fully rescued the effect of pathogenic LRRK2 (C and D) without altering the coexpression levels of G2019S LRRK2 .We reasoned that if LRRK2-mediated phosphorylation of RAB8A causes its inactivation, a phosphomimetic RAB8A variant should be unable to rescue the delay in EGFR degradation. Thus, we expressed WT RAB8A, the phosphodeficient RAB8A-T72A variant, or the phosphomimetic RAB8A-T72D/RAB8A-T72E variants, respectively. Although WT RAB8A was largely localized to a tubular recycling compartment, the phosphomimetic variants displayed a prominently cytosolic localization, and the phosphodeficient RAB8A variant was also partially cytosolic . Strikingly, RAB8A knockdown caused a pronounced deficit in EGF surface binding and EGFR degradation (C and D). Moreover, the deficits induced upon siRNA of RAB8A could be rescued when overexpressing active, GTP-locked RAB7A (RAB7A-Q67L) (E and F). Knockdown of RAB8A was not associated with a change in the steady-state levels of several other RAB proteins, including RAB7A , indicating that the deficits in endolysosomal trafficking are not due to off-target effects on RAB7A protein levels. In addition, an siRNA-resistant version of RAB8A , further indicating that the effects were due to the specific knockdown of RAB8A.Various studies indicate that phosphodeficient and phosphomimetic RAB8A may not be able to properly mimic the dephosphorylated and phosphorylated status of RAB8A, respectively , 39–41. 39–radation , C and D7A-Q67L) , E and FG and H). The fraction of endogenous RAB7 bound to GTP was drastically reduced upon RAB8A knockdown (G and H), similar to what we described previously for G2019S LRRK2 (I). Therefore, loss of RAB8A function effectively phenocopies the pathogenic G2019S LRRK2–mediated defects in endolysosomal trafficking by decreasing RAB7 activity . In contrast, colocalization of fluorescent EGF with RAB8A or RAB11 was minor and was not altered in the presence of G2019S LRRK2 , suggesting that pathogenic LRRK2 did not cause the accumulation of EGF in RAB8A- or RAB11-positive endocytic recycling compartments, respectively.We next wondered whether the delay in EGF degradation in the presence of pathogenic LRRK2 may reflect a redistribution of EGF into a nonendolysosomal vesicular compartment, thereby precluding its efficient degradation. As expected, the active version of endolysosomal RAB7A extensively colocalized with Alexa647-EGF 20 min upon internalization (A and B) and did not alter G2019S LRRK2 expression levels . However, when quantifying the colocalization of Alexa647-EGF with GFP-tagged RAB4 in live mock-transfected versus pathogenic LRRK2-transfected cells, a significant increase was observed in G2019S LRRK2–expressing cells (C and D). Similarly, there was a significant increase in the accumulation of fluorescent EGF in a RAB4-positive compartment upon siRNA of RAB8A as compared with control siRNA (E and F). These data indicate that either G2019S LRRK2 expression or RAB8A inactivation causes alterations in endolysosomal trafficking events, culminating in the accumulation of EGF in a nondegradative, RAB4-positive recycling compartment.Cross-talk between both the recycling and degradative endosomal trafficking pathways has been described to converge on an endocytic RAB4-positive compartment . We therng cells , C and Dol siRNA , E and FA and B), even though both were expressed to similar degrees (C). Shifting cells to 37 °C caused receptor internalization in all cases, but after a 15-min chase, cells showed differential EGFR recycling rates back to the cell surface with an impairment in the presence of G2019S LRRK2 but not the kinase-inactive G2019S-K1906M variant (B). Quantification of the fluorescence intensity of EGFR on the cell surface showed that the receptor did not recycle to the cell surface even upon prolonged chase times (B). These data indicate that pathogenic LRRK2 causes not only a deficit in endolysosomal trafficking but also a defect in endocytic EGFR recycling.The observed accumulation of fluorescent EGF in a RAB4-positive compartment suggests that pathogenic LRRK2 may cause additional alterations in endocytic recycling events. Because the concentration of EGF ligand is known to influence the balance between lysosomal degradation and recycling of the receptor, we next used low ligand concentrations to favor receptor recycling together with an antibody against the extracellular domain of the EGFR in the absence of permeabilization to visualize only surface EGFR . Cells w degrees C. Shifti variant B. Quantise times B. These A–C). Furthermore, siRNA of RAB8A caused a decrease in EGFR surface levels and endocytic recycling deficits of the EGFR, which were rescued upon coexpression of active RAB7A-Q67L but not WT or inactive RAB7A-T22N versions, respectively (D). Similarly, expression of active, but not WT or inactive RAB7A, caused a reversal in the accumulation of fluorescent EGF in a RAB4-positive compartment upon pathogenic G2019S LRRK2 expression with constructs expressed to similar degrees (A–C). In addition, the LRRK2-mediated decrease in EGFR surface levels and the endocytic recycling deficits of the EGFR were rescued by active but not WT or inactive RAB7A versions, respectively (D). Thus, pathogenic LRRK2 expression causes a defect in endolysosomal degradation, which is accompanied by mistargeting of EGF into a RAB4 compartment and by a deficit in endocytic recycling, all phenocopied by knockdown of RAB8A and rescued in the presence of active RAB7A.Because expression of active RAB7A rescued the endolysosomal EGFR trafficking deficits observed upon either siRNA of RAB8A or G2019S LRRK2 expression , we wondectively D. Simila degrees , A–C. Inectively D. Thus, per se may account for the mistargeting of the EGFR into a RAB4A compartment and endocytic recycling deficits as observed upon pathogenic LRRK2 expression or knockdown of RAB8A. Expression of dominant-negative RAB7A (RAB7A-T22N) (A and B). Furthermore, expression of dominant-negative RAB7A, but not WT or catalytically active RAB7A (RAB7A-Q67L), caused accumulation of EGF in a RAB4 compartment (C and D), which was rescued upon overexpression of active RAB8A (E and F). Similarly, dominant-negative RAB7A caused a decrease in EGFR surface levels and EGFR recycling, which were rescued upon overexpression of active RAB8A (G). Thus, decreasing RAB7A activity causes the same trafficking deficits as observed upon pathogenic LRRK2 expression or knockdown of RAB8A, and such effects can be rescued by either active RAB7A or RAB8A expression, respectively. Altogether, our data indicate that G2019S LRRK2 expression causes inactivation of RAB8A associated with inactivation of RAB7A and followed by alterations in endolysosomal trafficking events culminating in the accumulation of EGF in a nondegradative, RAB4-positive recycling compartment as well as impaired endocytic recycling.Finally, we wondered whether decreasing active RAB7A levels 7A-T22N)  interfer7A-T22N) , A and Bpartment , C and Dve RAB8A , E and Fve RAB8A G. Thus, in vitro, in contrast to other RAB proteins involved in regulating endocytic recycling (RAB11) or endolysosomal trafficking events (RAB7A). We corroborate Thr-72 as the major LRRK2 phosphorylation site within RAB8A. This residue lies within the switch II domain, a highly conserved region of RAB proteins that regulates the interaction with multiple proteins . All 3XFLAG-tagged RAB8A constructs were generated by Gibson Assembly Master Mix (New England Biolabs). In all cases, the identity of constructs was verified by sequencing the entire coding region.Double myc–tagged WT or pathogenic LRRK2 constructs have been described previously , and tri2. Confluent cells were harvested using 0.05% trypsin and 0.02 mm EDTA in PBS and subcultured at a ratio of 1:4–1:6. Cells were plated onto 6-well plates and the following day, at 70–80% confluence, transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's specifications for 4 h in DMEM followed by replacement with fresh full medium. Double transfections were performed using 4 μg of LRRK2 plasmids and 1 μg of plasmids of interest. Transfected cells were replated the next day at a 1:2 ratio onto coverslips in 24-well plates. Proteins were expressed for 48 h before analysis as described below.HeLa cells were cultured and transfected as described previously . Brieflym siRNA and 1 μg of GFP-RAB7-Q67L or GFP-RAB4 as indicated using 4 μl of jetPRIME transfection reagent  in 200 μl of jetPRIME buffer. The mixture was incubated for 15 min at room temperature and added to 2 ml of full medium per well of a 6-well plate. For knockdown experiments in the presence of WT or siRNA-resistant mRFP-RAB8A variants, cells were transfected with a 50 nm concentration of the indicated siRNA using 4 μl of jetPRIME transfection reagent. Four hours later, medium were replaced with fresh DMEM, and cells were transfected with 1 μg of the indicated RAB8A constructs using Lipofectamine 2000 according to the manufacturer's instructions followed by medium replacement 4 h later. In all cases, cells were passaged 24 h later and processed for Western blotting analysis or fluorescent EGF binding and internalization assays 48 h after transfection. RNAi reagents included Silencer Select Negative Control Number 1 siRNA  and Silencer Select RAB8A . The latter was validated using TaqMan gene expression analysis and found to result in around a 95% reduction of mRNA levels 48 h post-transfection (Thermo Fisher). Knockdown efficacy of this siRNA reagent was confirmed by Western blotting with a sheep polyclonal RAB8A antibody that specifically detects RAB8A .HeLa cells were seeded in 6-well plates at 30–40% confluence 1 day prior to transfection such that they were 70–80% confluent the following day. They were transfected with 25 nHeLa cells were fixed using 4% paraformaldehyde (PFA) in PBS for 20 min at room temperature, permeabilized in 0.5% Triton X-100 in PBS for 3 × 5 min, and incubated in blocking buffer  for 1 h. Coverslips were incubated with primary antibody in blocking buffer for 1 h at room temperature followed by washes in 0.5% Triton X-100 in PBS and incubation with secondary antibodies for 1 h. Coverslips were washed in PBS and mounted in mounting medium with DAPI (Vector Laboratories). For staining with the anti-LAMP1 antibody, 0.5% Triton X-100 was replaced by 0.05% saponin. Primary antibodies included mouse monoclonal anti-LAMP1 , rabbit polyclonal anti-β-coat protein , rabbit polyclonal anti-transferrin receptor , and a mouse monoclonal anti-EGFR antibody against the extracellular domain of the EGFR . Secondary antibodies included Alexa488-conjugated goat anti-mouse , Alexa594-conjugated goat anti-mouse , and Alexa594-conjugated goat anti-rabbit , respectively. To determine the localization of all GFP-tagged RAB protein variants in the absence of antibody staining, cells were fixed, briefly permeabilized in 0.5% Triton X-100 in PBS for 3 min, washed in PBS, and mounted as described above.Images were acquired on a Leica TCS-SP5 confocal microscope using a 63× 1.4 numerical aperture oil UV objective (HCX PLAPO CS). Single excitation for each wavelength separately was used throughout all acquisitions . The same laser settings and exposure times were used for image acquisition of individual experiments to be quantified. Ten to 15 image sections of selected areas were acquired with a step size of 0.4 μm, and z-stack images were analyzed and processed using Fiji.m NaCl, 0.2 m acetic acid, pH 2.5) for 3 min at 4 °C to confirm that labeled EGF was only surface-bound under those conditions. Upon binding, cells were washed twice with ice-cold PBS and transferred to prewarmed serum-free medium to allow uptake of bound Alexa555-EGF/Alexa488-EGF. At the indicated times, cells were fixed with 4% PFA in PBS for 15 min at room temperature and softly permeabilized with 0.5% Triton X-100 in PBS for 3 min before mounting with DAPI.Binding and uptake assays were performed essentially as described . TransfeTo measure the total number of Alexa555-EGF/Alexa488-EGF structures per cell, cells were circled, and a modified NIH Fiji macro (GFP-LC3 macro) was employed. At least 20 and up to 100 independent cells were analyzed for each condition and experiment, and analysis was done by an observer blind to conditions.2 to correct for differences in cell size. Intensity per punctum was then determined by dividing the integrated density of the puncta by the number of puncta  in serum-free medium for 1 h at 37 °C to block novel protein synthesis. Cells were treated with 20 ng/ml nonlabeled EGF for 20 min at 4 °C in serum-free medium in the presence of cycloheximide to allow EGF binding to the receptor. Upon binding, cells were washed twice with ice-cold PBS, then transferred to prewarmed serum-free medium containing cycloheximide, and shifted to 37 °C for 10 min to allow for EGFR internalization (pulse). After the pulse, a mild acidic wash (pH 4.5) was performed on ice followed by two washes with ice-cold PBS. Subsequently, cells were incubated in serum-free medium containing cycloheximide at 37 °C to allow for EGFR recycling back to the plasma membrane, measured at different time points . At the indicated times, cells were fixed with 4% PFA in PBS for 15 min followed by blocking with 10% goat serum in PBS and immunostaining with an EGFR antibody directed against the extracellular domain of the EGFR  followed by staining with an Alexa488-coupled secondary antibody. Permeabilization was omitted from all steps to visualize only surface EGFR. For intensity quantifications, pictures were acquired the same day with the same settings, and analysis was performed with Fiji. Integrated densities of 30–50 cells per condition were determined, background-subtracted, and normalized to 250 μm2 to correct for differences in cell size.Recycling assays were done as described previously . BrieflyFor live-cell fluorescence microscopy to determine colocalization of fluorescent EGF with the various RAB proteins in the absence or presence of pathogenic LRRK2, transfected cells were reseeded onto 35-mm glass-bottom dishes (ibidi) 24 h after transfection. For analysis of colocalization with fluorescent EGF, cells were serum-starved overnight. The next day, medium was replaced by phenol-free, serum-free DMEM (Gibco), and cells were incubated with 100 ng/ml Alexa647-EGF (Invitrogen) for 20 min at 4 °C to allow for fluorescent EGF surface binding. Subsequently, cells were washed twice in ice-cold PBS and incubated for 20 min at 37 °C to allow for internalization of bound EGF before image acquisition.Cells were imaged on a Leica TCS-SP5 confocal microscope using a 63× 1.4 numerical aperture oil UV objective (HCX PLAPO CS) by acquiring individual z-stack images corresponding to the cell center. The JACoP plugin of Fiji was used for the quantification of colocalization of the different GFP-tagged RAB proteins with Alexa647-EGF. After thresholding, the percentage of colocalization was obtained by calculating the Manders' coefficients (M1 for red channel (Alexa647-EGF)), and the percentage of colocalization was obtained by M1 × 100 . A totalm PMSF, 1 mm Na3VO4, and 5 mm NaF). Extracts were sonicated, boiled, and centrifuged at 13,500 rpm for 10 min at 4 °C. Protein concentration of supernatants was estimated using the BCA assay (Pierce), and extracts were immediately resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with primary antibodies overnight at 4 °C. Antibodies used for immunoblotting included a rabbit polyclonal anti-GFP , a mouse monoclonal anti-GAPDH , a mouse monoclonal anti-LRRK2 , a mouse monoclonal anti-RAB8A antibody , a mouse monoclonal anti-tubulin antibody , a sheep polyclonal anti-RAB8A antibody , a rabbit polyclonal anti-RAB7 antibody , a mouse monoclonal anti-RAB11 antibody , and a knockout-validated rabbit monoclonal anti-RAB4 antibody . Membranes were washed and incubated with secondary antibodies  or anti-mouse HRP-conjugated antibody ) for 60 min at room temperature followed by detection using ECL reagents (Roche Diagnostic GmbH). A series of timed exposures were undertaken to ensure that densitometric analyses were performed at exposures within the linear range, films were scanned, and densitometric analysis was performed using Quantity One (Bio-Rad).Cells were collected 48 h after transfection, washed in PBS, and resuspended in cell lysis buffer . Samples were sonicated and centrifuged at 10,000 rpm for 10 min at 4 °C, and supernatants  were analyzed by SDS-PAGE and Western blotting using a mouse monoclonal anti-FLAG antibody , a rabbit monoclonal anti-EGFR antibody , or a mouse monoclonal anti-tubulin antibody  as a loading control.For determination of steady-state levels and EGFR degradation, we employed HEK293T cells because overexpression and transfection efficiencies are higher than in HeLa cells . HEK293Tl-lysine–coated 6-well plates, and degradation assays were performed 48 h after transfection. Cells were serum-starved for 1 h in the presence of 1 μg/ml cycloheximide followed by incubation with 100 ng/ml nonlabeled EGF  for 20 min at 4 °C in serum-free medium containing cycloheximide. After washing in ice-cold PBS, time point 0 was collected by cell scraping. For the remaining time points, cells were incubated in serum-free medium containing cycloheximide at 37 °C for distinct periods of time  followed by cell scraping, and cell extracts were prepared and analyzed by SDS-PAGE and Western blotting as described above.For determination of EGFR degradation, HEK293T cells were transfected and plated onto poly-Escherichia coli strain BL21, and 250 ml of LB was inoculated with a 1-ml overnight culture grown at 37 °C to an OD of 0.6–0.8. Isopropyl 1-thio-β-d-galactopyranoside (EMD Biosciences) (0.5 mm) was added, and bacteria were induced for protein production for 3–4 h at 28 °C. Bacterial cells were pelleted and washed with cold PBS, and cell pellets were frozen at −20 °C. Pellets were resuspended in 5 ml of ice-cold purification buffer , and lysates were sonicated and cleared by centrifugation. Supernatant was diluted with another 5 ml of ice-cold purification buffer, and GST-RILP was purified using 300 μl of a pre-equilibrated 50% slurry of GSH-Sepharose 4B beads  and incubation for 1 h at 4 °C on a rotary wheel. Beads were washed with purification buffer, resuspended to a 50% slurry, and kept at 4 °C. A sample (5 μl) was separated by SDS-PAGE and analyzed by Coomassie Brilliant Blue staining to determine protein purity, and protein concentration was estimated by BCA assay (Pierce). Beads were used with cell lysates within 2 days of preparation. Transfected HEK293T cells (one 10-cm-diameter dish per assay) were collected by centrifugation, washed in PBS, and resuspended in pulldown buffer , and lysates were cleared by centrifugation at 13,500 rpm for 10 min at 4 °C. GST-RILP pulldown assays were performed in 1 ml of pulldown buffer containing 300 μg of cell lysate and 60 μl of 50% slurry beads pre-equilibrated in pulldown buffer. Beads were incubated on a rotary wheel overnight at 4 °C and washed twice with ice-cold pulldown buffer, and bound proteins were eluted by adding 40 μl of 1× sample buffer/β-mercaptoethanol and boiling for 4 min at 95 °C prior to separation by SDS-PAGE.GST-RILP pulldown assays were essentially performed as described , 42. Brim GTPγS  for 30 min at 30 °C to activate all available RAB7 prior to immunoprecipitation.Alternatively, active RAB7 levels were determined with a RAB7 activation assay kit (NewEast Biosciences) according to the manufacturer's specifications and as described previously . Briefly2 atmosphere. Cells were transfected with the 3XFLAG-RABs or with WT or G2019S 3XFLAG-LRRK2 plasmid DNAs using polyethylenimine (PEI) with a DNA:PEI ratio of 1:2 (w/w). Cells were harvested 72 h post-transfection for biochemical assays.HEK-293T cells were maintained in DMEM supplemented with 10% fetal bovine serum and 1× penicillin/streptomycin at 37 °C and in a 5% COm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 2.5 mm Na4O7P2, 1 mm β-glycerophosphate, 1 mm NaVO4, 0.5% Tween 20, protease inhibitor mixture, and 1×Complete Mini protease inhibitor mixture (Roche Applied Sciences)). Cleared lysates (1 ml) were incubated with anti-FLAG-M2–agarose beads (Sigma) by rotating overnight at 4 °C. Resin complexes were washed with different buffers , and proteins were eluted in kinase buffer  for 45 min at 4 °C with shaking. Eluted proteins were resolved by SDS-PAGE and stained with Coomassie G-250 to verify protein purity. RAB and LRRK2 protein concentrations were estimated by densitometry against a standard curve of increasing concentrations of bovine serum albumin (BSA) .Cells were lysed in buffer A (20 mm LRRK2 kinase inhibitor PF 06447475 (Tocris Bioscience). Reactions were performed in kinase buffer in the presence of 30 nm LRRK2 and a 300 nm concentration of the respective RAB proteins and with [γ-33P]ATP  and 200 μm cold ATP (Sigma-Aldrich) at 30 °C for 1 h in a final volume of 30 μl. Reactions were terminated with 4× Laemmli buffer and boiling at 95 °C for 10 min. Phosphorylation reaction samples were resolved on precast 4–20% SDS-polyacrylamide gels (Bio-Rad) and transferred onto PVDF membranes. Incorporated radioactivity was detected by a phosphor screen and Cyclone acquisition (PerkinElmer Life Sciences), and the same membranes were probed with HRP-conjugated anti-FLAG antibody to confirm equal protein loading.Phosphorylation reactions were performed either in the presence or absence of 1 μp < 0.05 was considered significant.All data are expressed as means ± S.E. Data were analyzed by one-way analysis of variance with Tukey's post hoc test, and P. R.-R., A. P. T., and S. H. conceptualization; P. R.-R. data curation; P. R.-R. formal analysis; P. R.-R. and S. H. validation; P. R.-R., M. R.-L., J. M.-P., A. B., and E. G. investigation; P. R.-R. visualization; P. R.-R. and S. H. writing-original draft; P. R.-R., M. R.-L., J. M.-P., A. P. T., A. B., E. G., and S. H. writing-review and editing; S. H. resources; S. H. supervision; S. H. funding acquisition; S. H. methodology; S. H. project administration.


In 2019, influenza A(H3N2) viruses carrying an I38T substitution in the polymerase acidic gene, which confers reduced susceptibility to baloxavir, were detected in Japan in an infant without baloxavir exposure and a baloxavir-treated sibling. These viruses’ whole-genome sequences were identical, indicating human-to-human transmission. Influenza virus isolates should be monitored for baloxavir susceptibility. In phase 2 and 3 clinical trials of baloxavir, treatment-emergent amino acid substitutions––I38T or I38F for influenza A(H1N1)pdm09 (pH1N1) virus and I38T or I38M for influenza A(H3N2) virus in the polymerase acidic (PA) protein––were detected  revealed that the whole-genome sequences of these viruses were identical. Both viruses possessed the PA I38T substitution and did not contain wild-type 38I. No amino acid substitutions associated with reduced susceptibility to NA inhibitors were detected.We collected a nasal blow sample from the 10-year-old child 3 days after baloxavir administration and a nasal aspirate from the infant on the first day of oseltamivir administration. Deep sequencing analysis (https://www.medchemexpress.com) and the 4 NA inhibitors approved for use in Japan: oseltamivir carboxylate , peramivir (Sequoia Research Products), zanamivir (Sequoia Research Products), and laninamivir . Because the genomic sequences of A/Kanagawa/IC18141/2019 and A/Kanagawa/IC18144/2019 were identical, we analyzed only A/Kanagawa/IC18141/2019. We determined antiviral susceptibilities by using a focus reduction assay and a fluorescent NA inhibition assay  (50) values using MikroWin 2010 . To interpret the NA inhibitor susceptibility, we applied the World Health Organization criteria of IC50 fold-change values compared with reference IC50 values  to baloxavir compared with the median IC50 value of influenza A(H3N2) viruses isolated in the 2018–19 season in Japan  viruses carrying various types of PA I38 substitutions, 4 of which were isolated from children <12 years of age without prior baloxavir exposure. Almost all mutant viruses isolated from baloxavir-treated patients possessed mixed PA I38T/I, I38M/I, I38R/I, I38T/M/I, I38T/K/I, or I38T/M/R substitutions (>4 after oseltamivir administration (Previous studies reported that oseltamivir-resistant viruses were detected in oseltamivir-treated 1–12-year-old children on day The 8-month-old infant infected with A/Kanagawa/IC18141/2019 in this study had no exposure to baloxavir before specimen collection. The sibling of this infant, infected with A/Kanagawa/IC18144/2019, was treated with baloxavir for a half day before the infant’s symptoms began. A/Kanagawa/IC18141/2019 and A/Kanagawa/IC18144/2019 viruses possessed the same genomic sequences. In Kanagawa, Japan, H3N2 virus activity was highest in February 2019, and an influenza outbreak occurred in the primary school attended by the sibling. Furthermore, during October 2018–February 2019, baloxavir was supplied to medical institutions that together served ≈5.6 million persons in Japan. These observations suggest 2 possibilities: the infant was infected by the sibling who was infected by another host harboring the virus with the PA I38T substitution, or both children were infected by another host harboring the virus with the PA I38T substitution. The median incubation period of influenza A virus is 1.4 days (During our monitoring, 4 of 5 pH1N1 viruses and 26 of 32 H3N2 viruses with the PA I38 substitution were isolated from children <12 years of age. Our results confirm that the frequency of viruses with this mutation is higher in patients <12 years of age than those 12–64 years of age, as previously reported (


Calcium mobilization studies revealed the important role of SCTR on ANGII-mediated calcium mobilization in adrenal gland. The fluo4-AM loaded primary adrenal ZG cells from the C57 mice displayed a dose-dependent increase in intracellular calcium influx ([Ca2+]i) when exposed to ANGII but not from the SCTR-/- ZG cells. Synthetic SCTR transmembrane (TM) peptides STM-II/-IV were able to alter [Ca2+]i in C57 mice, but not the mice with mutated STM-II/-IV (STM-IIm/IVm) peptides. Through enzyme immunoassay (EIA), we measured the aldosterone release from primary ZG cells of both C57 and SCTR-/- mice by exposing them to ANGII (10nM). SCTR-/- ZG cells showed impaired ANGII-induced aldosterone secretion compared to the C57 mice. TM peptide, STM-II hindered the aldosterone secretion in ZG cells of C57 mice. These findings support the involvement of SCTR/AT1R heterodimer complex in aldosterone secretion/release through [Ca2+]i.The involvement of secretin (SCT) and its receptor (SCTR) in angiotensin II (ANGII)-mediated osmoregulation by forming SCTR/ angiotensin II type 1 receptor (AT1R) heteromer is well established. In this study, we demonstrated that SCTR/AT1R complex can mediate ANGII-induced aldosterone secretion/release through potentiating calcium mobilization. Through IHC and cAMP studies, we showed the presence of functional SCTR and AT1R in the primary zona glomerulosa (ZG) cells of C57BL/6N (C57), and functional AT1R and non-functional SCTR in SCTR knockout (SCTR By monitoring in vitro [Ca2+]i changes in a real-time manner upon ANGII stimulation, we investigated the effect of SCTR/AT1aR heteromer formation on calcium influx and consequent aldosterone production.In recent decades, it has been established that secretin (SCT) exerts an important modulatory function in ANGII-mediated osmoregulatory actions. SCTbehavior . With thof AT1aR . Intraceof AT1aR  and atteof AT1aR . All theof AT1aR . Therefo-/-) has been described previously i) as specified by the manufacturer. Briefly, the primary adrenal cortical cells were seeded in 35 mm glass-bottom petri dishes and recovered in F-12K media modified for primary ZG cells (around 12 h). After removing the media, the cells were washed with pre-warmed loading solution  and 0.1% W/V BSA  in HBSS, with 2.5 mM probenecid (Invitrogen)), followed by incubation with loading solution freshly mixed with 1 μM Fluo4-AM and 0.02% Pluronic-127  for 1 h at 37°C with 5% CO2-95% O2. The cells were then washed twice and kept in loading solution. All the loaded cells would be used within 3 h to ensure the viability. During the processing of live cells imaging, the unused cells were kept at 37°C with 5% CO2-95% O2. The Fluo4-AM loaded cells were then examined by the confocal laser scanning microscope . The ZG cells possessed large portion of nuclei, vesicles and round boundaries i in C57 ZG cells   . While, e, STM-I  that is -/- mice. As early as 20 mins after ANGII stimulation, aldosterone levels were significantly elevated in the culture medium of C57 ZG primary cells, and this stimulatory action of ANGII lasted for at least for 4 h. In SCTR-/- ZG cells, the stimulatory action of ANGII was significantly reduced compared to the C57 ZG cells  aldosterone release, we measured cumulative aldosterone release from dispersed ZG cells prepared from C57 and SCTRZG cells , and theCTR axis .2+]i  [2+]i is critical for the subsequent CaMKs-regulated transcription regulators controlling CYP11B2 expression [2+]i changes and aldosterone release upon ANGII stimulation in ZG cells of C57 and SCTR-/- mice, we found that functional SCTR is required for a well-pronounced function of ANGII in Ca2+ mobilization and aldosterone release. Addition of EGTA before and after the treatment of ANGII with ZG cells showed that the extracellular Ca2+ is the major source of [Ca2+]i upon the activation. Therefore, the observed attenuated [Ca2+]i and aldosterone secretion in SCTR-/- is likely to be the result of a lowered activity of the low-voltage-gated Ca2+-channels.ANGII initiates aldosterone production in ZG cells via sustained Ca+i througlum (ER) . This inpression ,29. From-/- mice, ANGII-induced [Ca2+]i is completely inhibited by the presence of candesartan. However, STM-II or STM-IV were only able to partially inhibit the [Ca2+]i, indicating that AT1R alone can mediate the ANGII-induced [Ca2+]i in a heteromer-independent manner. The aldosterone production in SCTR-/- ZG cells in response to ANGII treatment is significantly less than that of the C57 ZG cells, which indicates the disturbed ANGII-aldosterone axis without the functional SCTR that is also consistent with our earlier in vivo results [2+]i, hence reduction in aldosterone secretion. On the other hand, we observed a reduced intrinsic at2R in the SCTR-/- ZG cells. Since, AT2R is commonly known to counteract the effects of AT1R i and aldosterone production in C57 mice, suggesting that even the mutated ones can hinder the formation of the SCTR/AT1aR complex to some extent. However, STM-II and STM-IV exhibited similar inhibitory effects as candesartan, a type 1 receptor antagonist of both AT1aR and AT1bR i in primary ZG cells obtained from C57 and SCTR-/- mice. ANGII (10 nM) was loaded at time 1 min. Fluorescent intensity in C57 mice was rapidly increased and lasted for minutes. While this increase was less pronounced in SCTR-/-. All fluorescent images were acquired under similar settings and were representative of at least five independent experiments. Scale bar represents 10 μm.Shown are the real-time images representing Fluo4-AM labelled free [Ca(TIFF)Click here for additional data file.


Vitamin K family molecules—phylloquinone (K1), menaquinone (K2), and menadione (K3)—act as γ-glutamyl carboxylase (GGCX)-exclusive cofactors in their hydroquinone state, activating proteins of main importance for blood coagulation in the liver and for arterial calcification prevention and energy metabolism in extrahepatic tissues. Once GGCX is activated, vitamin K is found in the epoxide state, which is then recycled to quinone and hydroquinone states by vitamin K epoxide reductase (VKORC1). Nevertheless, little information is available concerning vitamin K1, K2, or K3 tissue distribution and preferential interactions towards VKORC1. Here we present a molecular modeling study of vitamin K1, menaquinones 4, 7 , and K3 structural interactions with VKORC1. VKORC1 was shown to tightly bind vitamins K1 and MK4 in the epoxide and quinone states, but not in the hydroquinone state; five VKORC1 residues were identified as crucial for vitamin K stabilization, and two other ones were essential for hydrogen bond formation. However, vitamin MK7 revealed shaky binding towards VKORC1, induced by hydrophobic tail interactions with the membrane. Vitamin K3 exhibited the lowest affinity with VKORC1 because of the absence of a hydrophobic tail, preventing structural stabilization by the enzyme. Enzymatic activity towards vitamins K1, MK4, MK7, and K3 was also evaluated by in vitro assays, validating our in silico predictions: VKORC1 presented equivalent activities towards vitamins K1 and MK4, but much lower activity with respect to vitamin MK7, and no activity towards vitamin K3. Our results revealed VKORC1’s ability to recycle both phylloquinone and some menaquinones, and also highlighted the importance of vitamin K’s hydrophobic tail size and membrane interactions. H), after which vitamin KH is converted to vitamin K epoxide (vitamin KE), showing no biological activity [Vitamin K is of main importance for many physiological processes as the exclusive cofactor of the γ-glutamyl carboxylase (GGCX) enzyme, which catalyzes a post-translational modification: γ-glutamyl carboxylation. This leads to the activation of vitamin K-dependent proteins (VKDPs) involved in bone metabolism ,2, canceactivity .E to vitamin K quinone (vitamin KQ) was evidenced in 1970 [E to vitamin KQ reduction, and then vitamin KQ to vitamin KH reduction [H. VKORC1 presents four strictly conserved cysteine residues among all species: C43, C51, C132, and C135 [E [Biological reduction of vitamin K in 1970 , and the in 1970 ,13. The eduction ,15—no acand C135 ,17. C132and C135 ,19,20. A C135 [E .Vitamin K is a family of molecules composed of phylloquinone (vitamin K1), menaquinones (vitamin K2), and menadione (vitamin K3), and little information about their respective participation in biological processes is available in the literature . VitaminE [Despite the remaining controversy about VKORC1 3D structure—some publications suggested a three-transmembrane helix (3 TM) structure ,34,35,36E , so we uIn silico and in vitro investigations were highly consistent in explaining VKORC1’s ability to support specific vitamin K reduction, and may constitute a great base for the future development of therapies, considering the different forms of vitamin K to use.The present work generated an overall characterization of vitamin K–VKORC1 interactions. We expected to obtain an overview of vitamin K–VKORC1 interactions, giving us structural insights into VKORC1’s ability or inability to reduce some vitamin K forms and to induce VKOR activity into the liver and extrahepatic tissues, with respect to their vitamin K status. www.chemspider.com). The VKORC1 model was used and the molecular dynamics (MD) system was built and prepared as in our previous paper [membed function of GROMACS software. Solvation, energy minimization, 100-ps temperature equilibration, and 1-ns pressure equilibration were carried out using GROMACS routines. 100-ns MD simulations were performed in replica on each vitamin K–VKORC1 complex, and then the two trajectories were concatenated and analyzed as one 200-ns MD simulation.Vitamin K 3D structures were downloaded from ChemSpider website  on vitamin K–VKORC1 or vitamin K–membrane complexes and the VKORC1 residue contributions to BFE were determined using the Molecular Mechanics Poisson–Boltzmann Surface Area (MM-PBSA) method [Distance measurements between the vitamin K carbon-2 atom and C135 α-carbon atom in MD simulations were performed using the ) method  implemen) method . Graphic) method  and 3D f) method .Pichia pastoris as described previously [Q and K3E, respectively. VKOR activity was assessed by performing the same assay adding 1.56 µM warfarin, leading to 40% inhibition of this activity. Vitamin K epoxide and vitamin k quinone solubilities in assay conditions were controlled , which can be likened to the biological affinity between those molecules. The BFE is always a negative value, and corresponds to the energy that one would have to apply to the complex to separate the two biomolecules. The lower the BFE is, the higher the affinity is. BFE calculation was then applied to our complexes  in MD simulations .E, K1Q, and MK4 (regardless of the reduction state) had very close values, from −51.49 to −55.91 kcal/mol, with standard deviations from 3.35 to 4.49 kcal/mol. Nonetheless, the affinity of vitamin K1H towards VKORC1 was lower: the calculated BFE for this complex was −48.48 kcal/mol. Moreover, the high value of standard deviation  revealed instability of vitamin K1H binding to VKORC1 in MD simulations. Vitamin MK7 affinity towards VKORC1 seemed to be higher, as shown by the BFE values that reached −61.67 and −57.42 kcal/mol for vitamins MK7E and MK7H, respectively. However, vitamin MK7Q had a BFE similar to vitamin K1H, with a value of −47.96 kcal/mol. The most striking observation was the high values of standard deviations concerning all states of vitamin MK7  showing great instability of vitamin MK7 binding to VKORC1. Finally, vitamin K3 presented the weakest affinity towards VKORC1, with BFE values from −20.19 to −22.19 kcal/mol.BFE calculations revealed heterogenous values depending on the different forms and states of vitamin K. Complexes formed by VKORC1 and vitamins K1Contribution of VKORC1 amino acids to vitamins K binding was then investigated to carry on this vitamin K–VKORC1 interaction characterization.The calculated BFEs have been decomposed into a single amino acid contribution, allowing us to identify VKORC1 residues of main importance in the overall affinity towards vitamins K . ResidueThe residue F55 was identified as greatest-contributing amino acid to the calculated BFE in each vitamin K–VKORC1 complex. N80, F83, and F87 (carried by TM2) and L112 (located on TM3) residues were highly involved in vitamin K1, MK4, and MK7 binding, whereas TM4 residues A115 and G116 only participated in vitamin MK7 binding. Finally, L22 residue (TM1) contributed in the epoxide state of vitamins K1, MK4, and MK7 binding, whereas G84 (TM2) and Y119 (TM3) residues were involved in their quinone and hydroquinone state binding, respectively. VKORC1 residues appeared as poorly contributing to vitamin K3 binding, which was consistent considering the weak affinity revealed by BFE calculated for vitamin K3–VKORC1 complexes.Structural investigations were then carried out to study crucial amino acids spatial location and to describe their effect on vitamins K binding and orientation towards VKORC1 catalytic site.Amino acids contribution to the previously calculated BFE was shown on the VKORC1 3D structure, leading us to determine their structural relationship towards vitamin K. The presence of hydrogen bonds was also investigated to produce an accurate and overall characterization of vitamin K–VKORC1 interactions.E and K1Q with respect to the C135 residue was conserved in MD simulations, as shown on time = 0 ns and time = 100 ns pictures  and the N80 interaction. Stabilization by F83, F87, G84, L112, and Y119 residues seemed not to be strong enough to keep vitamin K1H close to the catalytic site.Conversely, vitamin K1E and MK4Q were close to C135 residue in MD simulations  and was facing TM2 at the end of MD simulations . Only onFinally, we oriented our latest analysis towards vitamin K–membrane interactions. Indeed, the main difference between vitamin MK7 and vitamins K1/MK4 was the hydrophobic tail, and this tail was likely to interact with the membrane.BFE calculation was thus applied to vitamins K towards membrane, to investigate potential differences between vitamins K affinity regarding the lipid bilayer. The vitamin K–membrane overall BFE was finally compared to the vitamin K–VKORC1 BFE .Distinct BFE profiles were observed depending on the different studied vitamins K, but one point remained constant: vitamins K–membrane and vitamin K–VKORC1 profiles appeared to be symmetrical. When the vitamin K–membrane BFE increased, vitamin K–VKORC1 decreased, and vice versa. This suggested a balance between vitamins K affinity towards the membrane and VKORC1.H) as well as vitamins K1E and K1Q: vitamin K–VKORC1 BFE values remained between −45 and −60 kcal/mol in MD simulations, and the vitamin K–membrane BFE kept constant around −15 kcal/mol. However, the vitamin K1H profile was different with both BFEs, converging to a −35 kcal/mol value at 50 ns. These results agreed with structural observations: vitamin MK4 and vitamins K1E/K1Q remained inside the VKORC1 enzyme and poorly interacted with the membrane, whereas vitamin K1H was sliding away from the catalytic site, allowing contacts between the hydrophobic tail and the membrane  was evaluated using recombinant human VKORC1 expressed in membranes of Pichia pastoris,  preventing determination of kinetic parameters. No VKOR activity was observed in the presence of 100 µM of vitamin K3E. Based on the experimental conditions and LC-MS analysis method used, a detection threshold of 2 pmol of vitamin K3Q produced by 1 mg of VKORC1 per minute is ensured. Thus, this absence of signal indicated a VKOR activity towards vitamin K3E below 2 pmol/min/mg of protein, considered as null.In vitro ability of VKORC1 to reduce vitamins K1astoris, . Human VThe main objective of the present work was the investigation of VKORC1 preferential substrates among vitamins phylloquinone (K1), menaquinones (K2), and menadione (K3). Indeed, VKOR activity is of main importance in all tissues: in the liver to support blood coagulation and in extrahepatic tissues for the activation of osteocalcin and matrix Gla protein (MGP), VKDPs are involved in energy metabolism and calcification processes, respectively. However, vitamin K tissue distribution is heterogenous, with vitamin K1 mainly present in the liver and vitamin K2 found in high levels in extrahepatic tissues . This raE), quinone (Q), and hydroquinone (H) states of each vitamin K, to obtain an overview of their binding to VKORC1. VKORC1 activity towards vitamins K1E, MK4E, MK7E and K3E was finally assessed by in vitro assays.Here we report a molecular modeling characterization of the ability of VKORC1 to bind vitamins K1, K2 (MK4 and MK7), and K3. We also considered epoxide  values compared to vitamin K1E and K1Q. These differences might be explained by the loss of S52 hydrogen bond and N80 residue stabilization, leading to the sliding motion of vitamin KH away from C135  hydrogen bonds  and only L22, F55, and G84 residues contributed to the BFE  which is not possible for the hydroquinone state. The naphthoquinone head is kept close to C135 residue by F55 and N80 residues, whereas the hydrophobic tail is mainly stabilized by F83, F87 and L112 residues.We also evidenced the major role of the vitamin K hydrophobic tail: its absence prevented structural stabilization inside VKORC1 and such VKOR activity (vitamin K3), whereas a long-sized hydrophobic chain induced a membrane effect on vitamins K binding to VKORC1, depending on the vitamins K hydrophobic tail size: Membrane affinity appeared to increase with the hydrophobic tail size, tearing vitamin MK7 apart from the active site regardless the epoxide, quinone, or hydroquinone state. This last observation is consistent with a previous study showing a decrease in the biological activity of vitamin K as the hydrophobic tail size increases . HoweverE and KQ, but is this mechanism sequential, with vitamin K remaining inside VKORC1? Or does vitamin K need to go outside from the enzyme then come back, while VKORC1 regenerates its reducing power? Does the C51–C135 disulfide bridge constitute the most adequate conformation to bind vitamin KH? The C51–C135 disulfide bridge has been shown to be the primary VKORC1 conformation [As a conclusion, we showed the ability of VKORC1 to reduce vitamins K1 and MK4, but not MK7 and K3. These findings explained VKORC1 ability to support VKPD activation in the liver (mainly containing phylloquinone) and in extrahepatic tissues (in which menaquinones constitute the most of vitamin K stocks). Membrane interactions with the vitamins K hydrophobic tail raised a new questions: Are long-sized hydrophobic tail menaquinones able to act as GGCX cofactors? Are they eliminated (because they are unrecycled by VKORC1) or stocked and then converted to MK (as menadione might be a precursor of menaquinones) ? What woormation , but furNevertheless, this article might constitute a precious starting point for future vitamin K-based therapies, since it gives an overview of the different forms of vitamin K interactions with VKORC1. It also highlighted the importance of membrane consideration into affinity determination, a fact that could also be of great importance for VKORC1 inhibitor development.in vitro activity assays, that revealed VKOR activity towards vitamins K1 and MK4 epoxide, but not regarding vitamins MK7 or K3 epoxide. Structural investigation of vitamins K–VKORC1 complexes indicated seven VKORC1 residues essential for vitamin K binding: S52 and S81 residues were involved in hydrogen bonds, F55 and N80 residues kept the vitamin K naphthoquinone head close to the catalytic site (C135 residue), and F83, F87 and L112 residues stabilized the hydrophobic tail. Finally, this tail size appeared to be crucial, since an absence of hydrophobic tail prevented vitamin K binding (vitamin K3) to VKORC1, whereas a long-sized tail led to a membrane effect pulling vitamin K outside the enzyme (vitamin MK7). Taken together, these results explained VKORC1 ability to support vitamin K reduction in the liver and extrahepatic tissues, depending on their vitamin K status.The molecular modeling study performed here evidenced vitamin K epoxide reductase (VKORC1) ability to bind both vitamin K1 and vitamin MK4 in their epoxide and quinone state, but not in the hydroquinone state. Moreover, vitamins MK7 and K3 were not structurally stabilized inside VKORC1, regardless the epoxide, quinone or hydroquinone state. These results were assessed by


A novel pandemic influenza A(H1N1)pdm09 virus emerged in 2009 globally, and it continues to circulate in humans. The National Influenza Surveillance Network in Taiwan identified five A(H1N1)pdm09-predominant seasons, representing the 2009/2010, 2010/2011, 2012/2013, 2013/2014, and 2015/2016 outbreaks from 2009 to 2016. Independently, a retrospective cohort study (which enrolled 639 infected patients during the five seasons) was conducted at Chang Gung Memorial Hospital to explore the risk factors associated with influenza A(H1N1)pdm09-related complications. A phylogenetic analysis of hemagglutinin (HA) sequences showed that the circulating A(H1N1)pdm09 virus belonged to clades 1, 2, and 8 in 2009/2010; clades 3, 4, 5, and 7 in 2010/2011; clades 7 and 6C in 2012/2013; clades 6B in 2013/2014; and 6B/6B.1/6B.2 in 2015/2016. Compared to individuals infected in non-6B/6B.1/6B.2 seasons , those infected in 6B/6B.1/6B.2 seasons (2013/2014 and 2015/2016) were at higher risk for influenza-related complications , pneumonia , mechanical ventilation , and acute respiratory distress syndrome . For the increased severity of infection during the influenza A(H1N1)pdm09 clade 6B/6B.1/6B.2 seasons, aspects related to the antigenic change of A(H1N1)pdm09 virus, immune response of the host, and environmental factors required further investigation. This novel influenza A(H1N1)pdm09 virus continues to circulate (with antigenic evolution) in humans, and it contributes to normal seasonal epidemics of influenza2.In June 2009, the World Health Organization (WHO) recognized a global human epidemiological event by announcing the detection of the first influenza pandemic of the 21st century that was caused by a novel influenza A(H1N1) virus4. In the 2015/2016 season, several European countries reported a high number of severe cases and outcomes in the groups at risk and healthy young adults; these events were associated with A(H1N1)pdm09 clade 6B.1 infection6. Since 1999, the Taiwan Centers for Disease Control (CDC) has established a nationwide surveillance system requiring contract virologic laboratories to perform continuous virologic surveillance for respiratory viruses, particularly influenza and enteroviruses; this system was established after an epidemic of enterovirus 71 in 19987. The long-term National Influenza Surveillance Network described the epidemiologic pattern of circulating viruses, and it has successfully identified the outbreaks of severe acute respiratory syndrome (SARS)-associated coronavirus and adenovirus9. Moreover, it also identified the novel H7N9 and H6N1 influenza viruses10. Following the large magnitude of A(H1N1)pdm09 activity from July 2009 to January 2010, a second A(H1N1)pdm09 epidemic, evidenced by a sharp increase in the number of hospitalized patients and fatal cases, occurred in the winter of 201011. Subsequently, A(H1N1)pdm09 has persisted at varying levels in the 2015/2016 season, when another A(H1N1)pdm09 epidemic occurred. The 2015/2016 outbreak included a sudden increase in the number of clinical cases with severe complications in adults aged 50–64 years.The 2013/2014 clade 6B-predominant season in the United States represented the first influenza A(H1N1)pdm09 season since the emergence of the virus in 2009, and it was characterized by elevated rates of hospitalization among adults aged 50–64 yearsThus, the present study aimed to describe the A(H1N1)pdm09 epidemiological and virological data obtained from the National Influenza Surveillance Network that reported laboratory-confirmed influenza cases in intensive care units  during a 7-year period extending from the 2009/2010 season to the 2015/2016 season. Independently, we conducted a retrospective cohort study at Chang-Gung Memorial Hospital to analyze the clinical characteristics and complications of patients with A(H1N1)pdm09 virus infection in the five A(H1N1)pdm09-predominant seasons  as identified by the National Influenza Surveillance Network. When these are considered, the results of these two approaches provided a better understanding of the impact of the new influenza A(H1N1)pdm09 variant during predominant seasons on influenza A(H1N1)pdm09 virus-associated complications.Taiwan Influenza Express between July 2009 and May 2016, A(H1N1)pdm09 caused the highest community attack rate  in the 2009/2010 season  .Based on the We identified 639 patients with laboratory-confirmed influenza AH1N1)pdm09 virus infection from the outpatient, inpatient, and emergency departments of Chang Gung Memorial Hosptial (CGMH) in the 2009/2010, 2010/2011, 2012/2013, 2013/2014, and 2015/2016 seasons. This group comprised 248 patients in 2009/2010, 152 patients in 2010/2011, 32 patients in 2012/2013, 81 patients in 2013/2014, and 126 patients in 2015/2016  age of patients in the 6B/6B.1/6B.2 season was older than that in the non 6B/6B.1/6B.2 seasons pdm09-related complications and pneumonia are shown in Table Among the hospitalized patients with laboratory-confirmed influenza A(H1N1)pdm09 infection, male patients and those with underlying conditions were significantly at risk for 30-day mortality  and all-cause in-hospital mortality  as assessed using the multivariable Cox proportional hazard model Table . The sam13, we found that age, underlying disease, and obesity were significant risk factors for A(H1N1)pdm09-related complications. Early antiviral treatment that reduces influenza-related complications, including lower respiratory tract infection, was evaluated14. In the present study, the association between antiviral treatment and the occurrence of complications should be attributed to the antiviral treatment frequently used in patients with influenza-related complications. Furthermore, we observed that the severity of influenza differed significantly, especially in individuals aged ≥6 years, during the 6B/6B.1/6B.2-predominant and 6B/6B.1/6B.2-non-predominant seasons.The present study assessed the clinical characteristics and complications of patients with influenza A(H1N1)pdm09 virus during the five A(H1N1)pdm09-predominant seasons over 7 years, including the 2009/2010, 2010/2011, 2012/2013, 2013/2014, and 2015/2016 seasons. Consistent with previous reports15. In 2013/2014, clade 6B appeared, achieving general dominance over other influenza viruses4. During the 2013/2014 season, an unusually high hospitalization rate in adults aged 50–64 years was observed in the United States and Mexico16. The increased morbidity in middle-aged adults during the 2013/2014 season had been attributed to the low vaccination rate in this age group16. However, that hypothesis cannot explain the unusual number of severe cases because the vaccination rate had already been low during the previous years17. An interesting study has shown that up to 42% of middle-aged adults born between 1965 and 1979, who had been exposed to seasonal H1N1 viruses circulating in 1977, had reduced serologic reactivity with the 2013/2014 A(H1N1)pdm09; notably, the 2013/2014 virus harbors the distinctive K163Q HA antigenic mutation18. In the cohort of individuals born between 1965 and 1979, HA-specific antibodies with activity against A(H1N1)pdm09 must have been produced and shaped by exposure to prior-season H1N1 viruses 19. Nonetheless, the HA-specific antibodies in this cohort failed to recognize the 2013/2014 A(H1N1)pdm09 genetic variant18. This explains the high susceptibility of the members of this middle-aged cohort to the 2013/2014 A(H1N1)pdm09 viruse. Influenza A(H1N1)pdm09 viruses circulating globally in the 2015/2016 season are members of the phylogenetic clade 6B.1/6B.2, which prevailed in Eastern Europe and Western Asia, resulting in widespread influenza activity and severity22. In Russia and Israel, 6B.1/6B.2 caused increases in influenza-associated morbidity and mortality21. The present study showed that individuals in the 6B/6B.1/6B.2 seasons compared with non-clade 6B/6B.1/6B.2 seasons were 1.6-, 1.78-, 2.6-, and 5.5-fold at greater risk for any complications, pneumonia, mechanical ventilation, and the development of ARDS, respectively. Clinically, influenza A(H1N1)pdm09 virus is associated with a greater risk of lower respiratory tract infection24. The 2013/2014 clade 6B and 2015/2016 A(H1N1)pdm09 clade 6B.1/6B.2 variants are more likely to cause severe pneumonia and acute life-threatening respiratory failure in a range of individuals.After the 2009 pandemic, when the 2009 H1N1 virus became a regularly circulating seasonal influenza strain, the WHO recommended the use of the A/California/7/2009 (H1N1)pdm09-like virus as a vaccine strain. In the face of a cumulatively established population immunity, the A(H1N1)pdm09 virus would need to exhibit antigenic changes to avoid suppression by the host immune system. To date, eight genetic virus groups have been identified25. Compared to the A/California/7/2009 vaccine virus, viruses of clade 6B harbor D97N, K163Q, S185T, K283E, and A256T substitutions in HA1. Viruses of subclade 6B.1 harbor further amino acid substitutions S84N, S162N, and I216T and 6B.2 and carry amino acid substitutions V152T and V173I20. More extensive studies must be conducted to identify the potential antigenic differences between clade 6B and subclade 6B.1/6B.2; such studies are expected to improve our understanding of how A(H1N1)pdm09 evolved (and continues to evolve) and how it affects and interacts with the human immune system. In the 2017/2018 season, the WHO has selected a new vaccine virus, which is the A/Michigan/45/2015 (H1N1)pdm09-like virus (a member of the 6B.1 subclade), as the influenza vaccine virus component for the northern hemisphere.Research has shown that clade-6B A(H1N1)pdm09 viruses, including those in subclades 6B.1 and 6B.2, were antigenically distinguishable from the A/California/7/2009 vaccine virus when tested with the human post-vaccination sera11.The National Influenza Surveillance Network coordinated by the Taiwan CDC was established more than 10 years ago. Policies favoring government funding for vaccines and antiviral agents have been consistent during the subsequent intervals. Between 2009 and 2015, government-funded vaccines have been administered primarily to those aged 6 months to 12 years, elderly individuals aged ≥65 years, healthcare workers, and individuals with underlying diseases. Individuals aged 13–64 years were not included in the government-funded vaccination program. Elementary school children aged 7–12 years had the highest influenza vaccination rate, with coverage reaching 60–70% annually26. The vaccine coverage rate in non-elderly adults and elderly individuals would have been low during each of the outbreaks, particularly during the first wave, given that no A(H1N1)pdm09 vaccine was available in 2009/2010. Thus, the increased severity of influenza during the 2013/2014 and 2015/2016 seasons is unlikely to reflect a decreased rate of vaccination. The study has shown that Taiwan experienced the greatest burden of influenza-related complications due to A(H1N1)pdm09 clades 6B/6B.1/6B.2 in the sixth year of its circulation. The reasons for the increased impact of influenza-related complications remain uncertain. Aspects related to the antigenic change of A(H1N1)pdm09 virus, immune response of the host, and environmental factors required further investigation. This report shows the importance of influenza disease surveillance and requires that the influenza A(H1N1)pdm09 virus should always be considered.The present study is limited by its observational nature and the incorporation of a retrospective investigation. A potential bias may exist due to the exclusion of all cases with A(H1N1)pdm09 infection for 7 years. Nonetheless, no change was observed in terms of admission or management procedures during these outbreaks. The surveillance and reporting system in Taiwan has long been established. Taken together, the increased frequency of complications in 2013/2014 and 2015/2016 is unlikely due to detection bias. In addition, the major drawback of this study was the lack of documentation about the history of influenza vaccination in the records used to generate this study. However, a study by Taiwan CDC has reported that 95% of patients with complications in the 2015/2016 season had not received the influenza vaccineTaiwan Influenza Express, a weekly online influenza surveillance report, has been published by the Taiwan CDC from July to May of each year since 2005 (http://www.cdc.gov.tw/english/submenu.aspx?treeid = 00ED75D6C887BB27&nowtreeid = 8F1E239D0FD8877A)27. This report includes the total number of respiratory specimens; isolate number of influenza A(H1N1), influenza A(H3N2), and influenza B; and case number of laboratory-confirmed influenza cases in intensive care units (ICUs), a class of events that is considered a category 4 nationally notifiable disease. However, data on weeks 19–37 are not available annually. A confirmed case involved a patient who had acute influenza-like illness (temperature ≥ 38 °C with either cough or sore throat) and nasopharyngeal/throat or bronchoalveolar lavage samples harboring influenza A(H1N1)pdm09 virus as detected using real-time (RT) reverse-transcription polymerase chain reaction (PCR) assay or via viral culture28. For the purposes of the present study, each season was defined as extending from July of the same year to May of the following year. The annual population figures provided by the Department of Household Registration Affairs of the Interior Ministry were used for the calculation of the incidence of laboratory-confirmed influenza A(H1N1)pdm09 cases in the ICU.The network consists of eight regional commissioned laboratories located in the northern (n = 3), central (n = 2), southern (n = 2), and eastern (n = 1) parts of Taiwan. These laboratories have steadily collected more than 8000 respiratory specimens for surveillance per year, including more than 1000 influenza virus specimens annually, all of which are sent to the Taiwan CDC for the monitoring of influenza viral activity. The 2 in patients aged between 2 and 18 years; and 3) BMI > 28 kg/m2 (Chinese criteria) in patients > 18 years29. Medical conditions associated with a high risk for influenza complications were defined based on those listed by the United States Advisory Committee on Immunization Practices30. Patients with confirmed pneumonia on radiography, acute respiratory distress syndrome (ARDS), acute onset of cardiovascular, neurologic condition, respiratory failure with mechanical ventilation; those who were admitted in the ICU; and those who died were considered to have influenza-related complications. Pneumonia on radiography was diagnosed based on the presence of a consolidation, infiltrate, or opacity31. ARDS was defined according to the standard criteria32. The primary study outcome was the occurrence of (any) influenza-related complications. The secondary study outcomes were pneumonia, mechanical ventilation, ARDS, 30-day mortality, and in-hospital mortality.Based on the data obtained from the National Influenza Surveillance Network, 2009/2010, 2010/2011, 2012/2013, 2013/2014, and 2015/2016 were considered as A(H1N1)pdm09-predominant seasons. Therefore, we conducted a retrospective observational cohort study for the 2009/2010, 2010/2011, 2012/2013, 2013/2014, and 2015/2016 seasons. This study was performed at the Chang Gung Memorial Hospital (CGMH), a 4000-bed, university-affiliated teaching hospital that is located in northern Taiwan and provides both primary and tertiary care. In addition, CGMH is one of the 8 regional commissioned laboratories of the Taiwan CDC. Patients who had acute influenza-like illness (temperature ≥ 38 °C with either cough or sore throat) and had influenza A(H1N1)pdm09 virus as detected using RT-PCR assay or via viral culture using respiratory specimens were included in the study. Patients whose data are not available were excluded. The institutional review board of CGMHT approved the study, and it was carried out in accordance with the relevant guidelines and regulations. Informed consent was waived due to the study’s retrospective nature. All medical records of the enrolled patients were reviewed. Demographic characteristics, underlying medical conditions, clinical course, antiviral treatment (oseltamivir or zanamivir), mechanical ventilation, admission to an ICU, and death were recorded using a structured questionnaire. Body mass index (BMI), a measure of obesity, was calculated for patients whose height and weight data were available. Obesity was defined as follows: 1) body weight ≥ 95th percentile in children < 2 years of age; 2) BMI ≥ 25 kg/m33. Sanger sequencing of the viral HA and NA genes was performed to establish clade designation and to detect differences in amino acid33. The obtained amplicons were assembled into a full-length 1,701-bp span for HA and 1410-bp for NA using DNASTAR Lasergene . Newly reported sequences in this study were deposited at the GenBank database under the accession numbers shown in Fig. 34. The percentages of replicate trees  are shown next to the branches in which the associated taxa clustered together in the bootstrap test. Phylogenetic analysis in this study was conducted using MEGA735.A total of 82 isolated influenza A(H1N1)pdm09 virus were randomly selected for the analysis of viral hemagglutinin (HA) and neuraminidase (NA) genes across the five seasons. The RNA was extracted using the QIAamp Viral RNA mini kit  according to the manufacturer’s instructions. RT-PCR and primer pairs used for sequencing HA and NA genes were performed, as previously describedP value < 0.1 in the univariate analysis were included in the multivariate model. The Hosmer–Lemeshow goodness-of-fit test was performed to assess the overall fit of the model. All statistical operations were two-tailed. P values ≤ 0.05 were considered statistically significant.Continuous variables were presented as medians and interquartile ranges (IQRs); categorical variables were presented as numbers and percentages. All analyses were performed using the Statistical Package for the Social Sciences software package version 22.0 . The incidence rate ratio (IRR) was generated using Poisson regression with 95% confidence intervals to compare the rates of laboratory-confirmed influenza A(H1N1)pdm09 cases in the ICU per 100,000 populations across different seasons; 95% confidence intervals for which the upper and lower bounds did not include 1 were considered as statistically significant. Differences in categorical variables were compared using the chi-square test or a Fisher’s exact test. Continuous variables were compared using the Kruskal–Wallis one-way analysis of variance test. Multivariate logistic regression analysis and multivariate Cox proportional hazards model were used for outcome analysis. The variables included sex, season, age group, onset to presentation, underlying condition, obesity, smoking, alcoholism, and antiviral therapy. Variables with a Supplementary Dataset 1


Propylea japonica is one of the most common pests of many crop systems throughout East Asia, and has often been used in the testing of non-target impacts during environmental risk assessments of genetically engineered plants. The present study assessed the suitability of nine frequently used reference genes for comparisons of P. japonica gene expression. Expression stability was compared across developmental stages, sex, a range of tissues, and following exposure to different temperatures. Data were analyzed using RefFinder, which integrated the results obtained using NormFinder, geNorm, BestKeeper, and the ΔCt method. This led to the identification of unique sets of reference genes for each experimental condition: ribosomal protein S18 (RPS18) and elongation factor 1 α (EF1A) for developmental stage comparisons, RPS18 and EF1A for sex comparisons, EF1A and ribosomal protein L4 for tissue comparisons, and RPS18 and EF1A for analyses of temperature-mediated effects. These reference genes will help to enhance the accuracy of RT-qPCR analyses of P. japonica gene expression. This work represents an initial move towards building a standardized system for RT-qPCR analysis of P. japonica, providing a basis for the ecological risk assessment of RNAi-based insect control products.Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique commonly used in molecular biology to analyze RNA expression. The selection of suitable reference genes for data normalization is a precondition for credible measurements of gene expression levels using RT-qPCR.  Cq) values obtained using this method  - 1) × 100.The RT-qPCR reactions were conducted in accordance with our previous study . The melgeNorm [NormFinder [BestKeeper [ΔCt method [RefFinder (http://150.216.56.64/referencegene.php), providing a stability ranking of the candidates. The optimal number of reference genes for target gene normalization was determined by pairwise variation (Vn/Vn+1) using V-values calculated by geNorm [n/Vn+1 cutoff value of ≤ 0.15 signified that the additional n + 1 reference gene was unnecessary; this indicated the appropriate number of reference genes for RT-qPCR data normalization;The stabilities of the nine reference genes were assessed using the following four approaches: geNorm , NormFinrmFinder , BestKeestKeeper , and thet method . Finallyy geNorm . A Vn/VnP. japonica and each was visualized as a single amplicon (All reference genes were expressed in amplicon . The speamplicon .2) for each standard curve. The standard curve for each gene is also shown ; 2) glyceraldehyde-3-phosphate dehydrogenase (GAPDH); 3) elongation factor 1 α (EF1A); 4) α-tubulin (TUBA); 5) ribosomal protein L4 (RPL4); 6) ribosomal protein S18 (RPS18); 7) heat shock protein 90 (HSP90); 8) arginine kinase (ArgK); 9) vacuolar-type H+-ATPase subunit A (V-ATPase A). M, DL100 DNA marker.PCR reaction templates: 1) (TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


Furthermore, virus-specific CD8 T cells were more activated in the absence of NCR1, resulting in exacerbated immunopathology, documented by weight loss, and superior virus control early during chronic LCMV infection. Transfer experiments of virus-specific CD8 T cells into NCR1 deficient hosts revealed a direct cross talk between NK and CD8 T cells. Studies on the splenic microarchitecture revealed pronounced disorganization of T cells in infected NCR1gfp/gfp mice, resulting in enhanced immunopathology and disruption of the T cell niche upon chronic LCMV infection. Our data show a novel pathway employed by NK cells to regulate antiviral CD8 T cell responses, namely direct recognition and elimination of activated CD8 T cells via NCR1 early during infection to protect the host from an overshooting T cell response.Besides their function in recognizing cancerous and virally infected cells, natural killer (NK) cells have the potential to shape adaptive immune responses. However, the mechanisms employed by NK cells to negatively regulate virus-specific CD8 T cell responses remain to be fully defined. Using activating receptor natural cytotoxicity receptor (NCR) 1 deficient (NCR1 Arenaviridae family, is a well-established mouse model for acute and chronic virus infections, and it has allowed the identification of many immunological principles that were subsequently confirmed in human infections, such as CTL escape or CD8 T cell exhaustion. NK cells belong to the first line defense, being activated early following infection or exposure to malignant cells, and mediate their antiviral or anti-tumoral effect by direct cytotoxicity and inflammatory cytokine secretion. While NK cells are dispensable for control of LCMV, NK cells have the potential to shape adaptive immunity by regulating T cell responses. The absence of NK cells leads to increased T cell immunity and thereby, to faster eradication of the virus. However, the detailed mechanisms of how NK cells control antiviral T cell responses is still poorly defined. Here, we identified the activating NK cell receptor NCR1 to be involved in the regulation of CD8 T cell responses during acute and chronic LCMV infection. The absence of NCR1 led to a more robust CD4 and CD8 T cell response and to superior viral control in acute and chronic LCMV infections. However, the increased CD8 T cell responses led to severe immunopathology in the setting of chronic infection. Hence, NK cells curtail CD8 T cell responses to protect the host from immunopathological damage in an NCR1 dependent manner.LCMV, which is part of the  Candida glabrata, leading to systemic clearance of the fungus [Natural killer (NK) cells play an essential role in eliminating cancerous and virus infected cells, they co-express Eomesodermin (EOMES) and Tbx21; T-box expressed in T cells (T-bet) and are able to produce cytotoxic mediators such as perforin/ granzyme B and Interferon (IFN)γ . In conte fungus . In a mue fungus .Besides recognizing and eliminating altered-self and virally infected cells, there is a growing body of evidence that NK cells can influence adaptive immune responses, resulting in either positive or negative effects on the emerging T cell response . NK cellgfp/gfp) mice, we found that the absence of NCR1 led to elevated virus-specific CD8 T cell numbers and accelerated control of the infection. During chronic LCMV infection, the frequency of virus-specific CD8 T cells was increased in absence of NCR1 and NCR1-deficient mice exhibited more pronounced immunopathology compared to wild type (WT) hosts. Moreover, pronounced splenic disorganization in absence of NCR1 compromised the T cell niche, likely provoking the reduction of total CD8 T cell numbers in LCMV infected NCR1-deficient mice. Transfer experiments revealed a direct NK-T cell cross talk with elimination of activated CD8 T cells in an NCR1-dependent manner. Our study unravels a mechanism whereby NK cells directly eliminate virus-specific CD8 T cells via NCR1. The absence of this regulation results in an overshooting CD8 T cell response that leads to better virus control, but also to aggravated immunopathology.Activated T cells evolved mechanisms to protect themselves against NK cell mediated killing. Sensing type I IFN rescues CD4 and CD8 T cells, as T cells lacking the receptor for type I IFN (IFNAR) were directly eliminated by NK cells , 26 via gfp/gfp mice at steady-state [icre mice, which were reported to be hyper-responsive [gfp/gfp mice. In naïve mice and in mice infected with LCMV WE for 2 days, NK cell numbers were comparable between C57BL/6 and NCR1gfp/gfp mice  was slightly higher in absence of NCR1, the absolute number of mature NK cells was comparable 2 days after LCMV WE infection  into C57BL/6 (Ly5.2+) and NCR1gfp/gfp (Ly5.2+) mice and analyzed CD8 T cell responses in various organs 7 dpi. Numbers of CD8 T cells were comparable in naive NCR1gfp/gfp and C57BL/6 mice  during infection (+ CD62L-) were subjected to NCR1 mediated regulation by NK cells, because naïve CD8 T cells (CD44- CD62L+) were comparable in NCR1gfp/gfp and WT mice  that has been previously described [in vivo activation and were transferred into LCMV infected NCR1gfp/gfp or WT hosts. Survival of transferred P14 T cells was assessed 4 hours later  at the same time  were increased in frequency and numbers upon chronic LCMV infection (gfp/gfp and NCR1 blocked WT mice compared to untreated C57BL/6 mice (gfp/+ mice and isotype (αIgG1 κ) treated WT mice did not show an increased CD8 T cell response upon chronic LCMV infection  in NCR1gfp/gfp mice upon LCMV infection, suggesting a faster maturation process [hi CD27lo NK cells in the spleen were comparable between NCR1gfp/gfp and C57BL/6 mice. Further analysis of NK cells in WT and NCR1gfp/gfp mice revealed no alterations in quantity and quality upon LCMV infection  were more abundant in NCR1gfp/gfp compared to WT mice, explaining the increase of total numbers of LCMV-specific CD8 T cells despite comparable frequencies in NCR1gfp/gfp, αNCR1 ab treated WT and untreated WT mice. In contrast, during chronic infection, the frequency of virus-specific CD4 and CD8 T cells was highly increased in NCR1gfp/gfp mice, but numbers of virus-specific CD8 T cells and transferred P14 T cells were comparable to WT mice due to a general decrease of total CD8 T cell numbers in NCR1gfp/gfp mice in chronically infected NCR1ent mice . Hence, Even though NK cells were shown to kill APCs and thereby limit T cell responses in LCMV and MCMV infection , 42, we In vivo cytotoxicity assays, based on transfer of activated polyclonal as well as virus-specific CD8 T cells into LCMV infected recipients with WT or NCR1 deficient NK cells revealed that activated CD8 T cells were eliminated in an NCR1-dependent manner, demonstrating a direct NCR1-mediated regulation by NK cells  of the Swiss Federal Government. The protocol was approved by Cantonal Veterinary Office of the canton Zurich, Switzerland (Permit number 147/2014 and 115/2017).+) mice expressing a TCR specific for LCMV peptide gp33-41 described previously [+) mice expressing a TCR specific for LCMV peptide gp61-80 were described earlier [gfp/gfp mice, the endogenous Ncr1 gene is replaced by GFP [The mice were housed and bred in specific pathogen-free facilities. The following strains were used: C57BL/6J mice , purchased from Janvier Elevage . P14 transgenic  and np396-404 (FQPQNGQFI) were purchased from NeoMPS . Blocking NCR1 was performed by i.p. injections of 200μg αNCR1   one day before and after infection, and afterwards twice a week. Isotype control was applied using 200μg of Ultra-LEAFTM Purified Mouse IgG1 κ  one day before and after infection, and afterwards twice a week.The LCMV isolates WE, the mutant strain LCMVWE 8.7 (LCMV8.7) , LCMV do+ T cells were isolated from naïve P14 mice using positive selection with CD8+ beads  according to manufacturer’s protocol and adoptively transferred into recipient mice 1 day prior to virus infection. CD4+ T cell were isolated from naïve Smarta mice using positive selection with CD4+ beads  according to manufacturer’s protocol and adoptively transferred into recipient mice 1 day prior to virus infection.CD8Lymphocytes were harvested from spleen, lung and lymph nodes as previously described . For DCsDetermination of virus-titers was done as described before .+ magnetic beads . 20’000 YAC-1 cells were co-cultured with DX5+ NK cells in triplicates in a ratio of 1:1, 1:10 and 1:25. After 4 h incubation at 37°C, surface stainings were performed in PBS and the samples were washed. The apoptotic markers, Annexin V-PB (BioLegend UK Ltd) and 7AAD (BD Biosciences) were diluted in Annexin V Buffer (BD Biosciences) and incubated for 30 min at 4°C followed by transfer on ice and direct acquisition of the samples.Activated NK cells were harvested from donor mice 2 days after LCMV WE infection and isolated using DX5gfp/gfp mice and CD8 T cells were positively MACS sorted. To distinguish the donor CD8 T cells from the host, donor CD8 T cells were CTV (CPDeF450)  labeled before transfer. For this, CD8 T cells were washed in PBS and resuspended in 10 μM CTV and incubated for 10 min at 37°C followed by addition of FCS to quench the labeling. Cells were washed twice in PBS containing 5% of FCS. 8x106 labeled CTV+ CD8+ T cells were adoptively transferred into day 2 1x106 ffu LCMV WE infected recipient mice. After 4 h, lymphocytes of spleen and lung were isolated and analyzed by flow cytometry.Activated CD8 T cell were obtained from day 7 LCMV WE infected NCR16 naïve P14 T cells into NCR1gfp/gfp mice following co-infection with LCMV8.7 and VVG2. After 5 days, splenic CD8 T cells were positively MACS sorted and transferred into day 3 LCMV WE infected recipient hosts, following a 4 h incubation. Spleens were harvested and the recovered number of P14 T cells was assessed. Prior to transfer, the amount of P14 T cells among all CD8 T cells from donor mice was analyzed by flow cytometry.Activated virus-specific CD8 T cells (P14 T cells) were obtained by transfer of 1x1010 μm thin sections were prepared from frozen spleens embedded in Optimal Cutting Temperature (O.C.T.) compound (Sakura). The sections were air-dried and fixed in ice-cold acetone for 10 min at RT followed by a washing step with PBS. The samples were blocked using 10% FCS in PBS for 1 h at RT. After washing with PBS, antibody dilutions were performed in PBS containing 1% FCS  and CD45.1-PB(A20) (BioLegend)). After incubation for 1 h in the dark, the sections were washed with PBS and the slides were mounted with Mowiol (Sigma-Alrich Chemie GmbH). The next day, images were acquired on the Visitron Confocal System inverse confocal microscope, Visitron Systems GmbH) with 10x magnification  at RT. Acquisition of the pictures was performed using Evolve 512 EMCCD cameras (Photometrics). Data was analyzed using Volocity software .t test, unpaired Mann-Whitney U-test and multiple time points were analyzed applying two-way ANOVA followed by Sidak’s multiple comparisons test using GraphPad . Statistical significance was determined with *p< 0.05, **p< 0.01, ***p< 0.001 and ****p< 0.0001.Statistical significance was determined by two-tailed unpaired S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.


These protein partners interact witha common area on the S100B protein surface, validating the method of using theconsensus sequence for S100B target search. In addition, each S100B targetprotein distinguishes itself by additional contacts with S100B. This perspectivesuggests that the combination of sequence homology search and structuralanalysis promises to identify newer S100B-binding partners beyond the use of theconsensus sequence alone as the given example in the XPB subunit of the TFIIHgeneral transcription factor. XPB is a helicase required for both transcriptionand DNA repair. Inherited xpb mutations are associated with human diseaseXeroderma Pigmentasum, Cockayne syndrome, and trichothiodystrophy. S100B proteinis likely associated with much more biological pathways and processes. Webelieve that S100B will attract more and more attentions in the scientificcommunity and S100B related studies will have important implications in humanhealth and medicine.The S100B protein is an intra- and extracellular signaling protein thatplays a role in a multitude of cellular processes and abnormal S100B isassociated with various neurological diseases and cancers. S100B recognizes andbinds effector proteins in a calcium-dependent manner. S100B has been shown tointeract with the actin capping protein CapZ, protein kinase C, Hdm2 and 4, RAGEreceptor, and p S100proteins exist exclusively in vertebrates, with 24 members observed in humans -[L/I]-x-W-x-x-I-L). Pioneered by Ivanenkov et al. , the seql target .Specifieraction . SubsequROS-GC1 ,22, micrROS-GC1 ,19–29.2+−boundstate [2+−dependent manner [2+− bound S100B with short peptides derived from itsknown targets have been evaluated by NMR and X-ray crystallography, includingp53, the NDR kinase, the RAGE receptor, RSK1 and CapZ [The NMR structure solution of the bovine S100B apo-form yielded the firststructural view of a S100B protein .Since tndstate . Other tt manner . Severaland CapZ ,29,43–4853 was first proposed by Baudieret al. based on the similarity of the p53 PKC-phosphorylation site tothat of the myristoylated alanine-rich C kinase substrate (MARCKS) protein[53capable of interacting with S100B was identified in the p53oligomerization domain [53 oligomerization [53 C- terminus[53 carboxy-terminal regulatory domain (CTD)could be phosphorylated by PKC, and this activity could be inhibited by S100B[53 transcriptional activity, preventing itsstimulation of cell cycle arrest and apoptosis [The S100B interaction with p protein. MARCKS  protein. In addin domain . S100B trization , as wellterminus. It was by S100B. Such inpoptosis .53activity, small molecule screening studies have revealed several inhibitors ofthe S100B-p53 interaction including pentamidine (Pnt) in the form ofpentamidine isethionate, an antiprotozoal drug currently approved for treatmentof Pneumocystis cariini pneumonia [53 expression, and decreased cell migration [www.clinicaltrials.gov, identifierNCT00729807). Structure solution of the S100B-Pnt complex showed two Pntmolecules bound per monomer, occupying two sites adjacent to the p53binding surface [53 binding site [To counteract the deleterious effects of S100B on pneumonia ,52. Pnt  surface . Furthering site . These fing site –57.Theying site . With th53-CTDpeptide (amino acids 367–388) revealed an induced folding of the peptide,normally unstructured, into an α-helical structure has the smallest ΔG  in the p53peptide-S100B complex.To quantitatively compare the interaction energies of the S100B- peptidecomplexes, we analyzed the structure models using the Proteins, Interfaces,Structures and Assemblies (PISA) server ;the resin silico andin vitro quantitative data suggests that known structures canbe used as the guidance to identify new S100B targets of high-affinity. As the poolof available S100B-target peptide complex structures grows, it has become clear thatthe S100B-interaction consensus sequence is largely limiting in that the specificresidues involved in interaction with S100B vary greatly between peptide sequences[This apparent correlation between the equences. To reflThe Xeroderma pigmentosum complementation group B (XPB) helicase is thelargest subunit of the general transcription factor II (TFIIH) complex. XPBplays vital roles in both transcription and nucleotide excision repair . Being a53-CTD, and so we propose that XPB should interact with S100B ina similar fashion to that of the p53-S100B complex [53 C-terminal interaction[53 in a competitive manner. A similar mode ofregulation was proposed for the S100B-RSK1 complex by Gogl et al. Based on theirstructural modeling, S100B binding could block ERK2 binding to RSK1, and thusinhibit the phosphorylation of the RSK1 activation loop [Several structural studies have provided a framework for how XPBfunctions as a molecular wrench to melt dsDNA both alone ,65 and i complex . Intereseraction. XPB couion loop . This inion loop . In a siion loop . Alterna53 extremeC-termini share low sequence homology (~23% using BLAST), most of theconserved residues in p53 form direct contacts with S100B in theS100B-p53 peptide complex (PDB entry 1DT7)  . Specifi 494–782 . During  494–782 .Furthereraction .In this review, we analyzed the structural basis of the S100B-peptideinteractions on available data and literature information. Besides the commonbinding area shared by all S100B interacting proteins, the additional contactsprovided by each target warrant the limitation of sequence only search inidentifying new S100B target proteins. We believe that the combinatorialsequence/structure-homology approach for S100B target identification will expand newS100B targets beyond the use of sequence-based searches alone. The identification ofthe XPB protein as a new S100B target likely reveals a new means of regulatingtranscription and/or DNA repair through S100B. With the diversity of targets alreadyidentified, the processes involving S100B, and other S100 proteins, are likely farmore extensive than what we currently know and could have important implications inhuman health and medicine.


Due to the importance of mTORC1 pathway in DR, we investigated the effects of Sch9, a yeast homolog of mammalian S6K1 and a major substrate of mTORC1 on H2S production in yeast Saccharomyces cerevisiae. We found that inhibition of the mTORC1-Sch9 pathway by SCH9 deletion, rapamycin or myriocin treatment resulted in a dramatic decrease in H2S production. Although deficiency of SCH9 did not alter the intracellular level of methionine, the intracellular level of cysteine increased in Δsch9 cells. The expression of CYS3 and CYS4, two transsulfuration pathway genes encoding cystathionine gamma-lyase (CGL) and cystathionine beta-synthase (CBS), were also decreased under mTORC1-Sch9 inhibition. Overexpression of CYS3 or CYS4 in Δsch9 cells or WT cells treated with rapamycin rescued the deficiency of H2S production. Finally, we also observed a reduction in H2S production and lowering of both mRNA and protein levels of CGL and CBS in cultured human cells treated with rapamycin to reduce mTORC1 pathway activity. Thus, our findings reveal a probably conserved mechanism in which H2S production by the transsulfuration pathway is regulated by mTORC1-Sch9 signaling.Endogenous hydrogen sulfide mediates anti-aging benefits of dietary restriction (DR). However, it is unclear how H Physiologically, H2S plays an important role in protecting the nervous system and the cardiovascular system of animals [The roles of Hydrogen sulfide , cystathionine beta-synthase (CBS), cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulfurtransferase (3MST). The production of H2S can be controlled by the expression of these enzymes, the bioavailability of their substrates, and enzyme activity modulating factors [2S production is complicated and more studies are required to clarify how it is controlled under physiological or pathological conditions.Endogenous production of H2S production due to sulfide amino acids restriction is essential for anti-aging benefits of dietary restriction (DR) [2S production as well [SCH9, an homologue of mammalian S6K1 in Saccharomyces cerevisiae and one of direct substrates of mTORC1, mimics DR and provides anti-aging benefits [2S production even though it mediates at least some effects of DR. Since the mTORC1-Sch9 pathway in yeasts responds to DR [2S production via sulfide amino acids metabolism.Recently it was suggested that endogenous Hion (DR) . Similar as well . Mechani as well , 10. Inhbenefits –13. Howeds to DR , 15 and ds to DR –18, we sSCH9 extends yeast lifespan through mechanisms shared with lifespan extension by calorie restriction (CR) [2S mediates the benefits of CR, we first compared H2S production in Δsch9 mutant cells to WT cells. While WT cells released measurable amounts of H2S, Δsch9 cells produce barely detectable amounts of H2S  , 16. Sins of H2S  and 1B. nt cells  and 1B, 2S production by Δsch9 cells was also observed by measuring the reduction of methylene blue in different yeast strain backgrounds (BY4741 and BY4742) (2S in these two yeast strains by using WSP-1 fluorescent showed the same trends (2S production in yeast [2S production in WT cell under CR (2S production was still significantly impaired in Δsch9 cell even under CR (Decreased H BY4742) . Measuree trends . Consistunder CR . Howeverunder CR .2S production, two inhibitors, Myriocin and Rapamycin, that indirectly lower Sch9 activity were used to inhibit the phosphorylation of Sch9 at the activation loop and the hydrophobic motif, respectively [2S production.To investigate if phosphorylation of Sch9, which correlates with its kinase activity and is required for Hectively  . The supplementation of Cys/PLP decreased the cell viability .The S. cerevisiae strains and plasmids used in this study are listed in For protein extraction, trichloroacetic acid yeast cell extracts were prepared according to a method described previously , 27. HeLTotal RNA was extracted from yeast cell or HeLa cell with RNAiso Plus  or Beyozol reagent (Beyotime) respectively. For yeast cell, cells (OD600nm of 5) were pretreated with 50 U lyticase at 30 °C for 30 min to increase extraction efficiency. Reverse transcription reactions were conducted using a PrimeScript RT reagent kit with gDNA eraser . The primers for real-time quantitative PCR (RT-qPCR) are indicated in 2S were pasted on the top of inner wall of 125 ml culture flasks containing 25 ml media. 1 L culture flasks containing 250 ml media were used for TB50a cells which do not intensively produce H2S. H2S reacts with the lead acetate on the strip, creating a darkened band. The length of the darkened band is proportional to the amount of H2S produced during fermentation [Lead acetate strips for measuring Hentation , 29. The2S detection was also performed in centrifuge tubes [2S dissolved in medium. Methylene blue decolorization by H2S were monitored at 663 nm and normalized to biomass.The methylene blue assay described previously for Hge tubes . 36ml ce2S levels were also determined using the H2S fluorescent probe WSP-1  [2PO4, 10 mM Na2HPO4 and 136.75 mM NaCl, pH 7.4), the pellet was resuspended in 1 ml PBS. 10 μl of cells was added to a microscope slide, and WSP-1 fluorescence was monitored using CEWEI LWD200-37FT fluorescence microscope . Alternatively, the fluorescence was measured at 465/515 nm excitation/ emission using an f4500 fluorescence spectrometer . The fluorescence intensity was normalized according to the OD value.Intracellular free Han, USA) . 1 OD of2S production, growth media was supplemented with or without 10 mM Cys and 10 mM pyridoxal-5′-phosphate (PLP) and a lead acetate strip was placed above the media in a 25 ml cell culture flask incubated in a CO2 incubator at 37 °C for indicated time.293T or HeLa cell was grown in DMEM (Invitrogen) supplemented with 10% FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin with or without 250 nM rapamycin. To measure HThe extraction and estimation of cysteine content in yeast cells were done as described previously . Cells gAll data from at least three independent experiments. Error bars are presented as averages ± SD. Statistical analysis and comparisons were performed using two-tailed, unpaired Student t tests.Supplementary Figures