Relevance of these phenomena to the human disease is poorly understood. Here, using splenocytes from T1D, autoantibody (Ab+), and non-diabetic (ND) human subjects, we show that a subpopulation of CD5+ B cells that is characterized by expression of FasL (FasLhiCD5+) was significantly elevated in T1D subjects, many of whom had significantly reduced frequency of IL-10posCD5+ B cells compared to Ab+ subjects. The majority of FasLhiCD5+ B cells did not produce cytokines and were more highly resistant to activation-induced cell death than their IL-10posCD5+ counterparts. These results associate expansion of FasL-expressing CD5+ B cells with T1D and lay the groundwork for future mechanistic studies to understand specific role in disease pathogenesis.Fas ligand drives insulitis in the non-obese diabetic mouse model of type 1 diabetes (T1D) and negatively regulates IL-10-producing (IL-10 Type 1 diabetes (T1D) results from the destruction of insulin-producing pancreatic β cells by diabetogenic T cells. Such diabetogenic T cells are held in check by an array of tolerogenic mechanisms that maintain self-tolerance in the steady state , 3. Wher+ Treg cell, the best recognized immunoregulatory cell subtype, plays an essential role in maintaining insulitis in the benign state , which is necessary for insulitis development. This is revealed by the arrest of the disease at a preinsulitis stage in NOD mice bearing homozygous gld mutations of FasL (NOD-gld/gld) or lpr mutations of its Fas receptor (NOD-lpr/lpr). This role of FasL is dominant as FasL-haploinsufficient NOD-gld/+ mice do not develop overt disease and only minor insulitis , 16. On echanism . Very lipression .We began addressing these questions in the present study. Our results associate expansion of FasL-expressing B cells with autoimmune diabetes and implicate them in the deletion of IL-10-producing Breg cells. The results suggest relevance of the long-known role of FasL in regulating susceptibility of NOD mice to autoimmune diabetes to the human disease and lay the groundwork for future mechanistic studies.+), and 10 non-diabetic (ND) subjects. Clinical characteristics of donors are shown in Table Splenocytes used in this study were procured, recovered, and processed by the Network for Pancreatic Organ Donors with Diabetes (nPOD) and delivered in cryovials in liquid nitrogen and then kept at −80°C in our laboratory until used. Samples were from three cohorts: 14 T1D, 8 diabetes-free subjects with T1D-associated autoantibodies . Cells were centrifuged, washed, and counted by using trypan blue, and 1 × 106 cells were used per application, unless stated otherwise. The absence of information regarding the absolute numbers of lymphocytes per spleen  made determination of absolute numbers of each subset per subject just a mere reflection of their frequency among acquired events, and for this reason we presented the data only as frequency of their indicated subsets.To minimize any effect of inter-experimental variation and maintain consistency of gating used to identify positive subpopulations in different diabetic, AbFluorochrome-conjugated monoclonal antibodies (mAbs) specific for human antigens were obtained, unless stated otherwise, from BD Pharmingen , BioLegend , or eBioscience . The following anti-human mAbs (clone) were used: FasL (NOK-1), Fas (DX2), CD19 (H1B19), CD5 (L17F12), CD40 (5C3), CD86 (IT2.2), CD20 (2H7), CD27 (LG.7F9), CD38 (HIT2), CD22 450 (S-HCL-1), CD10 (HI10a), CD1d (51.1), IL-10 (JES3-9D7), active caspase 3 (C92-605), IL-17 (eBio64DEC17), and IFN-γ (B27). Surface expression of human FasL was analyzed by using biotin-conjugated NOK-1 mAb. Previously described , 26 huma+, and ND), and when appropriate, stimulated vs unstimulated samples provided internal controls. For intracellular staining, single cell suspensions were stimulated for 6 h with phorbol myristate acetate (PMA) (50 ng/mL) and ionomycin (500 ng/mL) in the presence of Golgi-Plug (2 mmol/L). Surface staining was followed by cell fixation, permeabilization, and intracellular staining with anti-IL-10, IL-17, TNFα, or IFN-γ mAb as indicated.Single cell suspensions were stained with predetermined optimal concentrations of indicated mAbs. Samples were then washed and acquired using FACS LSRII flow cytometer and analyzed by Flow-JOV10 software. Three types of specificity controls were used. First, compensations were properly set using single-stained samples. Second, Fluorescence-Minus-One unstained samples and those stained with isotype controls were used to specifically gate positive cells and set up quadrants. Third, comparison of positive and negative cells in samples from donors 2 anti-human IgM (Jackson ImmunoResearch) for 24 h at 37°C in the presence or absence of humanized anti-human FasL-neutralizing mAb (RNOK203) or the general caspase inhibitor Z-VAD-FMK (BD Biosciences). During the last 6 h of incubation, cultures were stimulated with PMA and ionomycin in the presence of Golgi-Plug, followed by surface staining and intracellular detection of IL-10 and active caspase 3 using specific mAbs. It is noteworthy that number of cells obtained per sample was limiting and did not allow a more direct and extensive analysis such as multi-color FACS analysis for FasL, Fas, CD5 on B cells without culture with anti-FasL mAb. Such analysis would have directly addressed whether IL-10-producing CD5U test. A two-tailed value of p ≤ 0.05 was considered statistically significant. Statistical analysis and graph preparation were performed by using GraphPad Prism software .Unless otherwise indicated, all data are shown as mean ± SEM. Statistical analysis was performed using the unpaired Mann–Whitney + B cells in the pancreata . B cells are often analyzed as one population, yet they are heterogeneous and contain CD5+ B cells that are viewed as autoreactive and expanded in T1D patients [Figure S2 in Supplementary Material; Ref . On the other hand, the frequency, but not the absolute numbers, of IL-10pos CD5+ B cells was higher in the Ab+ subjects than ND subjects . There were no significant differences, however, in the frequency of IL-10-expressing CD5− B cells (IL-10pos CD5− B cells) among the subjects in the three groups, excluding generalized upregulation of IL-10 in B cells of Ab+ subjects. Consequently and in line with the fact that CD5− B cells comprised the bulk of B cells, the overall frequency of IL-10-expressing cells among total B cells (IL-10pos B cells) was not statistically different among subjects in the three groups . The frequency of IL-10pos T cells, however, was significantly less in T1D as compared to ND, but not Ab+ subjects  was significantly higher in T1D as compared to Ab+ and ND subjects  was generally low and slightly higher in T1D, but the difference among the groups did not reach statistical significance . Taken together, our results show IL-10pos CD5+ B cells are selectively expanded in Ab+ subjects as opposed to selective expansion of FasLhi CD5+ B cells T1D subjects.Next, we determined whether FasL expression is dysregulated in B or T cells in any of the three subject groups. We detected significant differences that, as above, were limited to the CD5s Figure A. The fre Figure B. Likewie Figure B. In addhi and IL-10pos CD5+ cells in Ab+ and T1D indicated that they might represent two separate subpopulations. To directly test this notion, we simultaneously analyzed FasL and IL-10 expression at the single cell level in the CD5+ B cells from T1D, Ab+, and ND subjects by FACS after rapid PMA/ionomycin stimulation to upregulate IL-10. As predicted, we detected two distinct subpopulations that express either IL-10 or FasL in three types of donors  with autoimmune diabetes in humans. Furthermore, our finding that T1D subjects harbor significantly fewer IL-10pos CD5+ B cells than Ab+ subjects .Additional information about procurement procedures, surgical processing, and recovery protocols are detailed in a recent review by Pugliese et al.  and at hAS did the experiments and analyzed the data. AH developed the hypothesis, designed experiments, interpreted the data, and wrote the manuscript. TD contributed to the genesis of the hypothesis and data interpretation and revised the manuscript. HY provided reagents and critically revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Legionella pneumophila, the causative agent of Legionnaires’ Disease, makes use of numerous effector proteins to construct a niche supportive of its replication within phagocytic cells. The L. pneumophila effector SidK was identified in a screen for proteins that reduce the activity of the proton pumping vacuolar-type ATPases (V-ATPases) when expressed in the yeast Saccharomyces cerevisae. SidK is secreted by L. pneumophila in the early stages of infection and by binding to and inhibiting the V-ATPase, SidK reduces phagosomal acidification and promotes survival of the bacterium inside macrophages. We determined crystal structures of the N-terminal region of SidK at 2.3 Å resolution and used single particle electron cryomicroscopy (cryo-EM) to determine structures of V-ATPase:SidK complexes at ~6.8 Å resolution. SidK is a flexible and elongated protein composed of an α-helical region that interacts with subunit A of the V-ATPase and a second region of unknown function that is flexibly-tethered to the first. SidK binds V-ATPase strongly by interacting via two α-helical bundles at its N terminus with subunit A. In vitro activity assays show that SidK does not inhibit the V-ATPase completely, but reduces its activity by ~40%, consistent with the partial V-ATPase deficiency phenotype its expression causes in yeast. The cryo-EM analysis shows that SidK reduces the flexibility of the A-subunit that is in the ‘open’ conformation. Fluorescence experiments indicate that SidK binding decreases the affinity of V-ATPase for a fluorescent analogue of ATP. Together, these results reveal the structural basis for the fine-tuning of V-ATPase activity by SidK.Intracellular pathogenic bacteria evade the immune response by replicating within host cells.  Legionella pneumophila. The structure and activity of the V-ATPase is altered upon SidK binding, providing insight into the infection strategy used by L. pneumophila and possibly other intravacuolar pathogens.V-ATPase-driven acidification of lysosomes in phagocytic cells activates enzymes important for killing of phagocytized pathogens. Successful pathogens can subvert host defenses by secreting effectors that target V-ATPases to inhibit lysosomal acidification or lysosomal fusion with other cell compartments. This study reveals the structure of the V-ATPase:SidK complex, an assembly formed from the interaction of host and pathogen proteins involved in the infection of phagocytic white blood cells by  Saccharomyces cerevisiae the complex is composed of subunits A3B3CDE3FG3Hac8c′c″def  beamlines at the Canadian Light Source  . Diffrachttp://www.ebi.ac.uk/msd-srv/ssm), and deconSTRUCT  = 0. No significant fluorescence from the protein was observed. The increase in fluorescence due to addition of TNP-ATP was modelled in GNUplot (www.gnuplot.info) with the fit command using the equation  is the total enzyme concentration, [ST] is the total substrate concentration, and m is the increase in fluorescence per unit increase in free substrate. [ES] is the concentration of enzyme bound to substrate given by:Kd is the equilibrium dissociation constant given by Kd = [E][S]/[ES]. The m parameter was estimated from fitting a straight line through the points corresponding to the four highest [TNP-ATP] values in each titration dataset. Each titration dataset was then modeled with fixed m to calculate Ib and Kd. The experimental setup described here is similar to the study by Kubala et al. [1 subcomplex allowed parameters to be estimated from the titration data instead of from separate experiments. This experimental approach is advantageous in situations where protein availability is limited, which was the case in this study. Five replicate experiments were performed for each condition (with and without SidK) using the same batch of freshly purified V1 subcomplex.Where a et al. . However-/pixel/s. Movie frames were aligned using alignframes_lmbfgs and averaged with shiftframes [CTFFIND3 [star_fixmaganiso [TMaCS [alignparts_lmbfgs [correctmaganisotropy_fspace_list [Relion [1 region.3 μL of ~10 mg/mL V-ATPase:SidK sample was applied to nanofabricated holey carbon grids  previousftframes . ContrasCTFFIND3  and corrmaganiso . Candidao [TMaCS  using teo [TMaCS . Candidas_lmbfgs . The aliace_list  and proc [Relion . To miniUCSF Chimera [UCSF Chimera and flexibly fit into the maps with Molecular Dynamics Flexible Fitting (MDFF) [3 map using UCSF Chimera by rigidly docking alpha-helical bundles I and II/III into the map as independent domains. The domains were joined in UCSF Chimera and the geometry of the short connecting loop between the domains was optimized using Modeller [JPred [Maps were visualized and segmented in  Chimera . Atomic g (MDFF) . FlexiblModeller . The secr [JPred .TTEST function in Microsoft Excel 2007) to calculate a p-value. Values less than 0.05 were deemed statistically significant.Where appropriate, results were analyzed using an unpaired, two-tailed Student's t-test  analysis shows co-purification of untagged SidK with intact V-ATPase (left) and the V1 subcomplex (right) via 3×FLAG tags on the V-ATPase A-subunits. B, Micrograph of V-ATPase:SidK3 complexes embedded in vitrified buffer. Examples of V-ATPase:SidK assemblies are indicated by red boxes. Scale bar: 30 nm. C, Trajectories of individual image features from the micrograph in B calculated using alignparts_lmbfgs [D, Representative 2D class averages of V-ATPase:SidK3 complexes from 2D classification. Scale bars: 50 Å.s_lmbfgs . D, Repr(TIF)Click here for additional data file.S2 FigA) and substoichiometric (B) V-ATPase:SidK complexes. Three distinct rotational states of V-ATPase were identified (states 1–3). The numbers in brackets (x:y:z) correspond to the average density of SidK relative to the V-ATPase A-subunit in the ‘tight’ conformation. The average density for SidK was determined by masking the SidK region and calculating the average value of the voxels in that region of the 3D density map. The numbers x, y, and z denote the density of SidK bound to A1, A2, and A3, respectively, where the V-ATPase A-subunit density is 100 and background is 0. Some classes show a mix of complexes with different SidK stoichiometries. C, Further classification allowed separation of substoichiometric V-ATPase:SidK complexes with different SidK binding configurations. Scale bar: 25 Å.Computational processing for images of fully-bound ((TIF)Click here for additional data file.S3 FigA, Cryo-EM maps of V-ATPase:SidK complexes with angular distributions of image orientations. Longer red bars represent a larger number of images while shorter blue bars represent a smaller number of images. Scale bars: 25 Å. B, Fourier shell correlation (FSC) curves for masked maps constructed from gold standard refinement. Based on the 0.143 FSC cutoff, the overall resolutions of the ‘fully-bound’ maps for states 1, 2, and 3 are 6.8, 7.9, and 7.6 Å, respectively, and the ‘substoichiometric’ maps for states 1, 2, and 3 are 6.8, 7.7, and 7.7 Å, respectively. Resolutions were the same for FSCs with and without correction for masking in Relion [n Relion .(TIF)Click here for additional data file.S4 FigA, Local resolution estimates for maps of V-ATPase and V-ATPase:SidK. The poorly-resolved C-terminal region of SidK has a low resolution estimate. Similarly, the C-terminal region of the A-subunit in the ‘open’ conformation (Aopen) is at lower resolution in the V-ATPase map than in the V-ATPase:SidK3 map, indicating flexibility in this region. Scale bars: 25 Å. B, Classification of images with a mask around the V1 region shows that SidK binding increases and decreases the proportion of V-ATPase complexes adopting states 1 and 2, respectively. Three separate datasets were processed independently. Error bars represent one standard deviation. C-E, Overlay of V-ATPase models fitted into density maps of V-ATPase (blue) and V-ATPase:SidK3 (pink). Density of the a-subunit is shown as the transparent gray surface. No significant conformational changes in the V-ATPase were observed. Scale bars: 25 Å. F, protein sequence alignment of S. cerevisiae V-ATPase A-subunit with Homo sapiens V-ATPase A-subunit. Only the regions involved in the interaction with SidK are shown. Identical residues are shown in red.(TIF)Click here for additional data file.S5 FigA, Point mutations F62A and S85E in SidK prevent the protein from co-purifying with detergent-solubilized V-ATPase. B, Point mutations F62A and S85E in SidK prevent the protein from inhibiting detergent-solubilized V-ATPase. *, p<0.01; **, p<0.001.(TIF)Click here for additional data file.S6 Fig3 and the difference map was low-pass filtered to 20 Å with a applied B-factor of 1000 Å2. Slices across the long axis of the structure were averaged for the N-terminal domains of the catalytic region (top row of images) and for the C-terminal domains of the catalytic region (bottom row of images). Residual density is observed in the difference map for the region corresponding to the A-subunit in the 'open' conformation , indicating higher density in this region for the V-ATPase:SidK3 map compared to the V-ATPase map. Scale bars, 25 Å.The map of V-ATPase was subtracted from the map of V-ATPase:SidK(TIF)Click here for additional data file.


In this study, we found that fenfluramine and norfenfluramine disrupted the regulatory association of the sigma 1 receptor (σ1R) with NR1 subunits of glutamate N-methyl-D-aspartate receptors (NMDAR), an effect that was also produced by σ1R antagonists such as S1RA and prevented by σ1R agonists such as PPCC. The antagonists removed σ1R bound to NMDAR NR1 subunits enabling calcium-regulated calmodulin (CaM) to bind to those subunits. As a result, CaM may inhibit calcium permeation through NMDARs. The serotoninergic activity of fenfluramine at 5HT2AR, and likely also at 5HT2CR, collaborated with its activity at σ1Rs to prevent the convulsive syndrome promoted by NMDAR overactivation. Notably, fenfluramine enhanced the inhibitory coupling of G protein-coupled receptors such as 5HT1AR and cannabinoid type 1 receptor with NMDARs, thus allowing the more effective restrain of NMDAR activity. Thus, fenfluramine circumvents the negative side effects of direct NMDAR antagonists and may improve the quality of life of subjects affected by such proconvulsant dysfunctions.Fenfluramine exhibits antiepileptic properties and thus diminishes epileptiform discharges in experimental animal models of Dravet syndrome. Fenfluramine is metabolized into norfenfluramine  Equal loading was verified and adjusted, if necessary, versus actin. In the GPCR immunoprecipitation studies, the middle region was dedicated to the detection of the GPCR, the upper region for co-precipitated NR1C1 subunits and the lower to HINT1. The secondary antibodies were directed to either the heavy or light IgG chains of the primary antibodies, as needed. Thus, the secondary antibodies also reacted primarily with the separated IgG light chains of the accompanying antibodies used to immunoprecipitation of de target GPCR and provided the loading control for the samples in the gel [The primary antibodies to detect immunoprecipitated receptors were: anti-MOR Ct aa 387–398 , anti-CB1R Nt aa 53-66 (GenScript Co), anti-5HT1AR , anti-5HT2AR , anti-NMDAR NR1 , anti-NMDAR NR1 C1 . Other primary antibodies used in this study were: anti-σ1R , anti-CaMKIIpan , anti-CaMKIIα P-T286 , anti-NMDAR NR1 P-S890 , anti-NMDAR NR1 P-S897 , anti-NMDAR NR2A P-Y1325 , anti-NMDAR NR2B P-Y1472 . The anti-HINT1 antibody was raised in rabbits  against the peptide sequence GYRMVVNEGADGGG (aa 93–106). All primary antibodies were detected using the appropriate horseradish peroxidase-conjugated secondary antibodies.p < 0.05. Data were analyzed using one-way ANOVA followed by Dunnett multiple comparisons vs the control group.All graphs and statistical analyses were generated and performed using the Sigmaplot/SigmaStat v.13 package . Significance was defined as


Aedes albopictus, which also inhabits temperate regions, including Europe and the United States of America. In the past, CHIKV has mainly affected developing countries, but has recently caused large outbreaks in the Caribbean and Latin America. No treatment or licensed CHIKV vaccine exists.Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes high fever, rash, and recurrent arthritis in humans. It has efficiently adapted to E. coli and as Fc-fusion proteins generated from HEK293T cells and used for cell-binding assays. Domains A and B bound to all cells tested, independently of their permissiveness to CHIKV infection. Domain C did not bind to cells at all. Furthermore, CHIKV cell entry was promoted by cell-surface glycosaminoglycans (GAGs) and domain B interacted exclusively with GAG-expressing cells. Domain A also bound, although only moderately, to GAG-deficient cells. Soluble GAGs were able to inhibit CHIKV infection up to 90%; however, they enhanced the transduction rate of CHIKV Env pseudotyped vectors in GAG-negative cells.Here, we have identified determinants in the CHIKV cell-attachment protein E2 that facilitate cell binding. The extracellular part of the E2 gene is subdivided into the three domains, A, B, and C. These domains were expressed in These data imply that CHIKV uses at least two mechanisms to enter cells, one GAG-dependent, via initial attachment through domain B, and the other GAG-independent, via attachment of domain A. These data give indications that CHIKV uses multiple mechanisms to enter cells and shows the potential of GAGs as lead structures for developing antiviral drugs. E. coli or as Fc-fusion proteins and the role of glycosaminoglycans (GAGs) on E2 cell binding and CHIKV entry. The two surface-exposed E2 domains, A and B, both bound to cells and domain B bound only to cells expressing GAGs. Domain A bound additionally to GAG-deficient cells and domain C did not bind to cells. CHIKV-pseudotyped lentiviral vector and CHIKV entry were enhanced in cells expressing GAGs. Our results suggest that CHIKV uses at least two entry mechanisms, one GAG-dependent, via attachment through E2 domain B, and the other GAG-independent, via binding of domain A. These data give indications that CHIKV uses multiple mechanisms to enter cells and shows the potential of GAGs as lead structures for developing antiviral drugs. In addition, it shows that domain A and B might constitute good targets for vaccine development.The chikungunya virus (CHIKV) glycoprotein E2 mediates cell attachment and consists of three domains A, B and C. Since the cell entry process of CHIKV is not understood in detail, we analyzed the binding properties of the three E2 domains with proteins expressed in  The mortality rate is very low (0.1%), but the infection rates are high (sometimes 30%) and asymptomatic cases are rare (about 15%). Due to climate change, globalization, and vector switching, the virus will most likely continue to cause new, worldwide outbreaks. Additionally, more temperate regions of the world like Europe or the USA, which have recently reported their first cases, will likely become targets . Th. Th2]. T targets . AlarminCHIKV is a (+) single-stranded RNA virus. Like other alphaviruses, it enters cells by receptor-mediated endocytosis and a subsequent pH-dependent fusion step. CHIKV has two surface proteins that mediate cell entry: the transmembrane glycoproteins E2 and E1. E2 mediates cell attachment and E1 is a class II viral fusion protein , 6]. E2. E26]. Ein vivo replication , , lication , 16], . A. Ain vivlication . Furtherlication . The E2 lication , [8].Since the cell entry process of CHIKV is not understood in detail , we exam2. HEK 293T (ATCC: CRL-1573) cells were incubated in Dulbecco’s modified Eagle’s medium . Jurkat (ATCC: TIB-152) and BHK-21 (CCL-10) cells were grown in Roswell Park Memorial Institute medium . CHO-K1 and pgsA-745 cells were grown in Ham’s F-12 medium  and 2 mM glutamine. Media were supplemented with 10% FBS  and 5% L-glutamine . The glycosaminoglycans chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin and dextran sulfate were purchased from Sigma-Aldrich .All cells used for this study were cultured at 37°C under 5% COThe codon-optimized CHIKV E3-E1 gene (based on isolate “S27”) was synthesized by GeneArt  and cloned into the plasmid pIRES2-eGFP  as described previously .NdeI and BamHI via primers by PCR (template DNA: pIRES2-EGFP-CHIKV E3-E1), cutting the DNA products and the vector, and subsequent ligation. For domain A, two fragments were derived via PCR. One fragment contained domain A itself and the first part of the β-ribbon connector (C-terminus of domain A (aa 1–171)). The other fragment contained the second half of the connector C-terminus of domain B (aa 231–270). The fragments were cloned into the pET-15b vector by a triple ligation (via NdeI and BamHI). The two fragments were linked via a shared SmaI restriction site . By this procedure, E2 domain B was bypassed and replaced with the sequence G4PG5. The constructs also contained an N-terminal poly-histidine-tag for purification.The E2 domain A (including the β-ribbon connector), B (aa 172–231), and C (aa 271–341) genes were cloned into the bacterial expression vector pET-15b. The same was done with the entire extracellular part of the E2 protein (E2ex). Cloning was achieved by adding the restriction sites The primers used for cloning were:Domain E2ex: Dom AI fw 5’-AAAACATATGAGCACCAAGGACAACTTCAAC, Dom C rev 5’-AAAAGGATCCTCACTGGGGCCAGTACTTGTAGG;Domain A: Dom AI fw 5’-AAAACATATGAGCACCAAGGACAACTTCAAC, Dom AI rev 5’-AAAACCCGGGGCCGCCGCCGCCGGGCATGTGCACCTCGATC, Dom AII fw 5’-AAAACCCGGGGGCGGCGGCGGCAACCACAAGAAGTGGCAGTAC, Dom AII rev 5’-AAAAGGATCCTCACTTGGGCACCATGCAGGTC;Domain B: Dom B fw 5’-AAAACATATGCCCGACACCCCCGATAGAA, Dom B rev 5’-AAAAGGATCCTCAGGTCACGGCGGCGTGGC;Domain C: Dom C fw 5’-AAAACATATGGCCCGGAACCCTACCGTG, Dom C rev 5’-AAAAGGATCCTCACTGGGGCCAGTACTTGTAGG).E.coli expression vectors as template: Domain A fw 5’ AAAAGGGCCCAGCACCAAGGACAACTTCAAC, rev 5’AAAAGCTAGCCTTGGGCACCATGCAGGTC; domain B fw 5’ AAAAGGGCCCCCCGACACCCCCGATAGAA, rev 5’AAAAGCTAGCGGTCACGGCGGCGTGGC; domain C fw 5’ AAAAGGGCCCGCCCGGAACCCTACCGTG, rev 5’AAAAGCTAGCCTGGGGCCAGTACTTGTAGG; domain A-ß fw 5’ AAAAGGGCCCAGCACCAAGGACAACTTCAAC, rev 5’AAAAGCTAGCGGGGTCGTGGTGGAAGGG. All mutations were introduced by site directed mutagenesis.The Fc-fusions were constructed by cloning E2 fragments via Apa I, Nhe I sites introduced by PCR, into the vector pCMV2.5-hIgG1Fc-XP . This generated expression vectors that express E2-Fc fusion proteins without any tags. The following primers were used to amplify the E2 fragments from the 600 of 0.5–0.7, and then protein expression was induced by the addition of 1 mM IPTG. After another 2.5 hrs of incubation, cells were harvested and the pellets were frozen at –20°C.Proteins were expressed in BL21-CodonPlus (DE3)-RIPL competent cells  transformed with the pET-15b plasmid containing construct A, B, C, or E2ex. Bacteria were inoculated into 100 ml of LB medium containing ampicillin (0.1 mg/ml) and grown overnight . After 16 hrs, 2 l of LB medium were inoculated with the 100 ml overnight culture. The bacteria were grown to an ODThe recombinant proteins were purified from the bacterial pellets under native  or denaturing  conditions using HisTrap FF Crude columns  and the ÄKTA system  as described by . For A aThe empty Fc control protein and the E2 domain-Fc-fusion proteins were produced in HEK293T cells by transient transfections. Transiently transfected HEK293T cells were grown in DMEM containing 10% FCS. After 48 h, supernatants of the transfected cells were harvested two times at 24 h intervals. The secreted Fc-fusion proteins were purified by affinity chromatography with protein A-agarose, eluted at a pH 2.5, neutralized with 1M Tris, pH 9.0 and dialyzed against PBS pH 7 and stored at -80°C.in vitro-transcribed with T7 RNA polymerase after NotI linearization. The mRNA was transfected into BHK-21 cells using Lipofectamine 2000 . Virus-containing supernatants were harvested 48 hrs later and used to reinfect fresh BHK-21 cells for virus amplification. Infected cells showed a clear red fluorescence. Supernatants were collected and stored at –80°C or used to determine the viral titer.The plasmid pCHIKV-mCherry-490  was in v5 cells). Subsequently, the mCherry-tagged CHIKV (CHIKV-mCherry-490) . V. V24]. VCHIKV cell entry into GAG-deficient cells was reduced; accordingly, the presence of soluble GAGs might inhibit CHIKV entry into GAG-expressing cells or influence entry into GAG-free cells. Therefore, 293T, CHO-K1, and pgsA-745 cells were transduced with CHIKV Env-pseudotyped vectors in the presence of different amounts of soluble GAGs. Dextran sulfate (DX), which consists of long chains of highly sulfated glucose units, was used as a control for the soluble GAGs, in addition to HP. It has a similar charge to HP and the other GAGs, but its structural background is built up of entirely different carbohydrates. The experiment was carried out in a 384-well plate format with vectors encoding firefly luciferase .Transducing 293T and CHO-K1 cells with CHIKV Env-pseudotyped vectors transferring a luciferase gene in the presence of GAGs generally resulted in dose-dependently reduced transduction efficiencies compared to the control without GAGs , top. DXAs shown above, CHIKV replication in GAG-deficient cells was diminished. Accordingly, this raised the question of whether infection of cells is inhibited in the presence of soluble GAGs. 293T cells were infected with CHIKV-mCherry (MOI 1) in the presence of 500 μg/ml of the respective soluble GAGs (but 500 U/ml HP). Six hours later, cells were analyzed by flow cytometry. In conclusion, replication of recombinant CHIKV-mCherry-490 in 293T cells is significantly inhibited by the addition of soluble GAGs. Attachment and/or endocytosis are the critical steps in the viral life cycle where this inhibition occurs.E. coli, and partially purified by Ni2+ affinity chromatography under native (E2 domains B and C) and denaturing (E2 domain A and E2ex) conditions. For domain A and E2ex additional ion-exchange chromatography was performed to remove bacterial protein contaminants. The E2 protein is the cell-binding moiety of CHIKV and has been frequently described to contain determinants recognized by neutralizing antibodies , 8], , . TherefoTheir cell binding was analyzed with CHO-K1, the GAG deficient pgsA-745, 293T and Jurkat cells.293T cells show good transduction efficiencies with CHIKV Env-pseudotyped vectors. In contrast, Jurkat cells have revealed very low transduction efficiencies . The recTo further prove that cell binding of the E2 protein domains is GAG dependent, inhibition of their cell binding by addition of soluble GAGs was analyzed. The recombinant E2 proteins and different cell lines were again incubated at 4°C for binding; however, this time in the presence of 500 μg/ml soluble GAGs. Heparin (HP) was used as a control to ascertain the role of charge in the cell binding. HP is structurally derived from heparan sulfate (HS), but is more heavily sulfated and thus has a higher negative charge density. In summary, binding of E2 domain A to 293T and CHO-K1 cells was not or weakly inhibited by the addition of soluble GAGs. Only the strong negatively charged HP inhibited the interaction of domain A with 293T cells. In contrast, binding of domain B to both cell lines was massively decreased in the presence of HP, HS, CS, and dermatan sulfate (DS). The addition of GAGs to domains A, B, or C had no influence on the binding properties of these proteins towards GAG-deficient pgsA-745 cells.E. coli might not be correctly folded or have incorrect disulfide bond patterns. Therefore the E2 domains were expressed as Fc-fusion proteins in a soluble form in eukaryotic cells by transient transfections of 293T cells. Supernatants containing these proteins were partially purified by protein-A Sepharose chromatography and used for binding assays  on the cell surface. Binding assays revealed that domains A and B bound to CHO-K1, but only domain A bound to pgsA-745 cells, however markedly reduced but still significant. Furthermore binding of domain B to GAG-expressing cells in the presence of soluble GAGs was highly reduced. This suggests that domain B binds GAGs and thereby facilitates CHIKV cell binding. In contrast, domain A binding to GAG-deficient pgsA-745 cells and competition with soluble GAGs only marginally decreased domain A cell binding, indicating GAG-independence.E.coli derived proteins have a native structure. In addition, point mutations in domain A showed the previously observed increase in GAG binding. Deletion of the ß-ribbon connector decreased the GAG binding of domain A, indicating that the ß-ribbon connector is partially responsible for GAG binding. A GAG binding consensus sequence is located in the ß-ribbon connector (-DRKGK- amino acid 251–255) , [lication . These mlication . There alication . Our finlication . The secGAGs on the cell surface are thus not absolutely required for CHIKV cell entry, but they are part of a strategy CHIKV employs in order to enter cells. They might be comparable with T-cell immunoglobulin and mucin (TIM) membrane proteins that promote, although are not absolutely required for, the cell entry of a number of viruses, including CHIKV . The fac


Paramyxoviruses represent a family of RNA viruses causing significant human diseases. These include measles virus, the most infectious virus ever reported, in addition to parainfluenza virus, and other emerging viruses. Paramyxoviruses likely share common replication machinery but their mechanisms of RNA biosynthesis activities and details of their complex polymerase structures are unknown. Mechanistic and functional details of a paramyxovirus polymerase would have sweeping implications for understanding RNA virus replication and for the development of new antiviral medicines. To study paramyxovirus polymerase structure and function, we expressed an active recombinant Nipah virus (NiV) polymerase complex assembled from the multifunctional NiV L protein bound to its phosphoprotein cofactor. NiV is an emerging highly pathogenic virus that causes severe encephalitis and has been declared a global public health concern due to its high mortality rate. Using negative-stain electron microscopy, we demonstrated NiV polymerase forms ring-like particles resembling related RNA polymerases. We identified conserved sequence elements driving recognition of the 3′-terminal genomic promoter by NiV polymerase, and leading to initiation of RNA synthesis, primer extension, and transition to elongation mode. Polyadenylation resulting from NiV polymerase stuttering provides a mechanistic basis for transcription termination. It also suggests a divergent adaptation in promoter recognition between pneumo- and paramyxoviruses. The lack of available antiviral therapy for NiV prompted us to identify the triphosphate forms of R1479 and GS-5734, two clinically relevant nucleotide analogs, as substrates and inhibitors of NiV polymerase activity by delayed chain termination. Overall, these findings provide low-resolution structural details and the mechanism of an RNA polymerase from a previously uncharacterized virus family. This work illustrates important functional differences yet remarkable similarities between the polymerases of nonsegmented negative-strand RNA viruses. RNA viruses replicate and transcribe their genomes using complex enzymatic machines known as RNA-dependent RNA polymerases. The chemical reactions driving nucleotide addition are shared among nucleic acid polymerases but the underlying mechanisms of RNA biosynthesis and the complex polymerase structures are diverse. Of these RNA viruses is the paramyxovirus family, which includes major human pathogens. Paramyxoviruses have common biological and genetic properties but little is known about their replication machinery. Insights into the structure, function, and mechanisms of RNA synthesis of one paramyxovirus polymerase will likely extend to the entire virus family. An emerging, highly pathogenic paramyxovirus is Nipah virus (NiV), which causes encephalitis in humans. We have purified NiV polymerase, probed its enzymatic and biophysical properties and developed it as a model paramyxovirus polymerase. We investigated template strand sequence elements driving RNA biosynthesis for NiV polymerase and obtained a snapshot of NiV polymerase molecular organization using electron microscopy to provide the first structural information on a paramyxovirus polymerase. This work extends previous knowledge by producing the first recombinant paramyxovirus polymerase and using this protein in enzymatic assays to highlight key functional and structural characteristics for the design of new medicines. Paramyxoviruses describe a family of viruses responsible for significant disease, ranging from lower respiratory disease due to parainfluenza virus and measles virus as well as emerging pathogens such as Nipah and Hendra viruses causing severe encephalitis. The replication machinery of these viruses, the polymerase complex, is largely unstudied due to their complexity and size and to date there has been no report of a recombinant paramyxovirus polymerase. The biochemistry and importantly, the potential inhibition of these protein complexes represents a significant opportunity for understanding and preventing disease.Henipavirus genus within the Paramyxoviridae family [Nipah virus (NiV) is an emerging pathogenic paramyxovirus part of the e family ,2. NiV ie family ,4. Due te family . There ae family .Le) and 5′ trailer (Tr) sequences, gene-start (GS), gene-end (GE), RNA-editing, and intergenic sequences in addition to coding regions for six proteins: nucleoprotein (N), large (L) protein, phosphoprotein (P), matrix (M) protein, fusion (F) protein, and glycoprotein (G) [Le region is a bipartite promoter that is highly conserved among Paramyxoviruses [NiV has a nonsegmented negative-strand (NNS) RNA genome of approximatively 18 kilobases . It conttein (G) –10. The tein (G) . The Le oviruses . In NiV,oviruses .Rhabdoviridae family, has recently revealed how the domain organization and enzymatic function are organized in a three-dimensional structure [The L protein of the NNS RNA viruses serves the two main enzymatic functions responsible for RdRp and mRNA cap formation. The L protein ranges in size within NSS viruses but is approximately 250 kDa . Six higtructure . Negativtructure . CRIV antructure . The +doRhabdoviridae) and respiratory syncytial virus . Current mechanistic knowledge of paramyxoviral L proteins has been limited to bioinformatic studies. There has been no report of the purification or biochemical characterization of an active paramyxoviral polymerase, which presents an exciting opportunity to probe the enzymatic properties of the polymerase of an emerging pathogen.Conserved sequences within the RdRp and capping domains suggest that the molecular organization of L proteins from other viruses may be similar. The X-ray crystal structure of the NiV P multimerization domain and the N protein have recently been solved ,21. The de novo and primer-dependent RNA synthesis. We identified sequence requirements for promoter recognition and for polyadenylation by NiV polymerase. We discovered that NiV polymerase is much more prone to stuttering than RSV polymerase, which could explain differences in promoter sequences between pneumo- and paramyxoviruses. Considering NiV’s status as a priority pathogen, we also characterized clinically-relevant nucleotide analogs that inhibit NiV replication by a mechanism of delayed chain termination. The purified enzyme represents a powerful tool for understanding the enzymatic function of the polymerase from a previously uncharacterized family of viruses and has significant implications for the development of novel inhibitors of NiV and other related viruses.Here, we present the first report of the expression, purification and biophysical and biochemical characterization of a paramyxovirus polymerase complex. Visualization of purified NiV polymerase by negative stain-EM shows ring-like particles. Using a combination of enzymatic approaches, we demonstrate that NiV polymerase is active in The paramyxoviral polymerases comprise a large (L) protein and a phosphoprotein (P). We hypothesized that the NiV L protein adopts a structure similar to the VSV L protein given their moderate sequence similarity (35%). Using homology modeling to generate a three-dimensional view of the NiV L protein, our model indicates the RdRp domain adopts a classic right-handed configuration . The seqWe then produced NiV polymerase through the co-expression of the N-terminal FLAG-tag L and P proteins of NiV within insect cells infected with a single recombinant baculovirus clone. The protein was purified by FLAG-tag and size exclusion-chromatography (SEC). Fractions corresponding to a peak near the void volume were pooled, concentrated, and analyzed by SDS-PAGE revealing bands consistent with the expected molecular weights of the L protein (260 kDa) and P protein (80 kDa) Figs  and S1. We probed the molecular architecture of the NiV L(wt)-P complex through visualization by negative-stain transmission EM. The images revealed small, 5–10 nm globular particles and 20–70 nm larger particles that appeared to be clumped or aggregated protein . The sma33P-labeled guanosine triphosphate (GTP) as nucleotide substrate for the reaction. The leader (Le) promoter region at the 3′-end of the NiV genome was chosen to design the 12-mer synthetic RNA template for the RdRp reaction because it contains the authentic sequence recognized by the polymerase during virus replication (33P-GTP incorporation into the nascent complementary RNA strand. In the presence of NiV L(wt)-P, α33P-GTP was not sufficient to promote RNA synthesis without other nucleotides -P was measured in a radiometric assay using αlication . This urleotides , lane 1.leotides , lane 2.e enzyme . The morynthesis , with a ely Figs  and S4.Le bipartite promoter region is highly conserved among Paramyxoviruses [The oviruses . In NiV,Le template. Since the nucleotide sequence at position 5 on the RNA template is not important (33P-GTP to the NiV L(wt)-P RNA complex -P was reduced by ~1,000-fold to 0.015±0.001μM (Elongation of RNA synthesis by NiV L-P was mimicked by using a short 4-nucleotide (nt) primer complementary to the 3′-end of the 12-mer mportant , it was  complex  lane 1.  complex  and S7.  complex . In the  complex , lane 2.±0.001μM . The NiV±0.001μM , lane 3.±0.001μM , lane 4.±0.001μM . GS-5734±0.001μM , lane 5.±0.001μM .6 tract and a GAA intergenic region  indicative of polyadenylation  yielded products mainly with the expected size of 5, 11, 12, and 18 nt, respectively (Le promoter sequence containing a poly(U)6 tract. There again, RSV L-P generated products of the expected size, without any strong evidence of polyadenylation c region . The 4-m4 and poly(U)5 templates  tract template to understand the minimum number of uridines required for polyadenylation, while keeping the rest of the sequence unchanged . For allemplates . In addi minimal , lane 1. minimal . This reThis study is the first report of the expression, purification, and biophysical and biochemical characterization of an active recombinant paramyxovirus polymerase. Using an insect cell expression system with an affinity tag on the L protein, we purified the NiV L(wt)-P, NiV L(H1347A)-P, and NiV L(D832A-N833A)-P by FLAG purification followed by SEC. The SEC profile revealed a heterogenous preparation with a significant peak near the void volume for all three L-P complexes . DespiteEscherichia coli cells without L or N proteins, which contrasts with the work presented here [Bioinformatic, biochemical, and biophysical analyses indicate that paramyxoviral P proteins contain disordered and folded regions ,29. It hted here ,22. It hted here ,28,30–32To further probe molecular arrangement and monodispersity of the NiV L-P complex, we visualized the purified NiV L-P complex using negative-stain EM followed by 2D class averaging. Our data revealed two main populations of globular particles, ranging in size from 5–11 nm; four representative class averages are displayed . An addide novo RNA synthesis assay showed that high concentrations of CTP and ATP are needed for the initiation step . Oligonucleotides were synthesized at Dharmacon, Inc . Their sequences with analysis of secondary structures can be found in A homology-based three-dimensional structure of the NiV L protein was generated using RaptorX . To geneThe codon-optimized ORFs for the NiV L and P proteins   were a gift from Michael Lo, cloned separately into pFastBac Dual expression vector. The 3XFLAG tag was added to the N-terminus of the NiV L protein by nested polymerase chain reaction (PCR)  with PrimeSTAR DNA polymerase . The 5′ SacI / HindIII region from the untagged NiV L was then replaced by the 3XFLAG-L PCR fragment. The SacI / XhoI fragment containing NiV P-His was cloned into SacI / XhoI cut pFBD-3xFlag-NiV L to make pFBD-3xFlag NiV L-P-His. The L protein, under the control of the polyhedron promoter, was cloned with an N-terminal 3X FLAG tag. The P protein, under the control of the P10 promoter, was cloned with a C-terminal hexahistidine tag. E. coli DH10Bac was transformed with the pFastBac Dual vector to yield bacmid DNA. A high-titer baculovirus stock was generated after transfection of bacmid DNA into Sf9 cells using Cellfectin. Mutagenesis of the L gene to generate NiV L(H1347A) and NiV L(D832A-N833A) was completed by amplifying the whole template DNA with complementary pairs of mutagenic oligonucleotides (Eton Bioscience) using Kapa HiFi HotStart DNA polymerase , followed by full-length insert sequencing confirmation.Two liters of Sf9 insect cells were infected with the baculovirus stock at a multiplicity of infection of 1 and harvested 72 hours post-infection by centrifugation for 10 minutes at 1000 × g The cells were resuspended in lysis buffer , lysed by microfluidization, and clarified through centrifugation for 30 minutes at 14,000 × g. The clarified lysate was incubated with EZview Red ANTI-FLAG M2 Affinity gel, washed twice with lysis buffer, and the NiV polymerase complex was eluted using 3X FLAG peptide, and concentrated using Amicon Ultra Centrifugal Filters. The sample was further purified by size-exclusion chromatography using a Superdex 200 Increase 10/300 GL at a flow rate of 0.3 mL/min with a running buffer of 50 mM Tris pH 7.4, 150 mM NaCl, 10% glycerol, 0.1% octyl β-D-glucopyranoside, 0.1% n-dodecyl β-D-maltoside, 1 mM DTT. The protein was then concentrated using an Amicon Ultra Centrifugal Filter, subjected to SDS-PAGE, and quantified using a Bradford Assay. The bands associated with the L, P, and HSC70 proteins were digested with trypsin, chymotrypsin, and elastase followed by analysis by nano LC-MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive. The peptides were then loaded on a trapping column and eluted over a 75-μm analytical column at 350 nL/min. Both columns used were packed with Luna C18 resin . The mass spectrometer was operated in data-dependent mode, with MS and MS/MS performed in the Orbitrap at 70,000 full-width at half-maximum (FWHM) resolution and 17,500 FWHM resolution, respectively. The fifteen most abundant ions were selected for MS/MS.2), was applied to a freshly plasma-cleaned grid, blotted to a thin film with filter paper and immediately stained with 1% (wt/v) uranyl formate. Electron microscopy was performed using an FEI Tecnai T12 electron microscope operating at 120keV equipped with an FEI Eagle 4k x 4k CCD camera. Images were collected at nominal magnifications of 110,000× (0.10 nm/pixel) and 67,000× (0.16 nm/pixel) using the automated image acquisition software package Leginon [-/Å2.Samples were prepared on continuous carbon films supported by nitrocellulose-coated 400-mesh copper grids (Ted Pella). A 3 μl drop of NiV L(wt)-P, diluted 60-fold in buffer  were seeded in 96-well plates overnight. The next day, NiV support plasmids consisting of 50, 32, and 50 ng/well of N, P, and L plasmids, respectively, and NiV minigenome (120 ng) prepared in RNase-free TE buffer, were mixed with 0.6 μL/well LT-1 transfection reagent  and 10 μL Opti-MEM/well. Complexes were mixed and incubated for 30 minutes at room temperature before being added to cells. For negative controls, the L plasmid was substituted with an equivalent amount of pcDNA 3.1 plasmid expressing the red fluorescent protein mCherry (Clontech). At 48 hours post-transfection of minigenome and support plasmids, 50 μL of Nanoluciferase assay buffer solution (Promega) was added directly to each well. Well contents were transferred to 96-well opaque white plates, and after 3 minutes, luminescence was read on a plate reader using 0.1 msec integration time .BSRT7/5 cells were a gift from K. Conzelmann , and wer2. Recombinant RSV L-P was produced through the co-expression of RSV L and P proteins in a baculovirus expression system, according to previously described procedures [33P-GTP. Reactions were stopped with the addition of an equal volume of gel loading buffer  denatured at 95°C for 5 minutes, and run on a 22.5% polyacrylamide urea sequencing gel for 2 hours at 80W. The migration products were exposed to a phosphor-screen, scanned on Typhoon phosphorimager  and quantified using ImageQuant .Unless otherwise specified, all NiV and RSV polymerase reactions consisted of 0.2 μM oligonucleotide template derived the NiV leader promoter, 0.2 μM recombinant L-P complex, with a buffer containing 20 mM Tris pH 7.5, 10 mM KCl, 2 mM DTT, 0.5% triton, 10% DMSO, 6mM MgClocedures . In the S1 Fig(a) Representative image at high magnification  used to generate 2D classes  (b) Representative SEC chromatogram of NiV L(wt)-P purification. The sample was run on Superdex 200 Increase 10/300 GL. Fractions associated with the peak at approximately 8mL were collected, pooled, and concentrated.(TIF)Click here for additional data file.S2 FigSDS-PAGE analysis of NiV L(wt)-P, NiV L(H1347A)-P, and NiV L(D832A-N833A)-P.(TIF)Click here for additional data file.S3 Fig(a) Coverage map of NiV L peptides identified after proteolytic digestion and ionization (b) Coverage map of NiV P peptides identified after proteolytic digestion and ionization (c) Coverage map of heat shock cognate 70 protein peptides identified after proteolytic digestion and ionization (d) Example product ion spectrum, for NiV L(wt)-P and (e), an additional, example product ion spectrum for NiV L(wt)-P.(TIF)Click here for additional data file.S4 Fig33P-GTP tracer, a fixed ATP concentration, and CTP concentrations ranging from 0 to 1000μM (lanes 1–6). NiV L(wt)-P and template were incubated in the presence of α33P-GTP tracer, a fixed CTP concentration (1000 μM), and ATP concentrations ranging from 0 to 1000μM (lanes 7–12). (b) The concentrations of ATP and CTP loaded on the gel represented in (a).(a) Product sizes are indicated using a 4-5-6-mer combined RNA size marker (lane M). NiV L(wt)-P and template were incubated in the presence of α(TIF)Click here for additional data file.S5 Fig33P-GTP tracer (lanes 1 and 3), or with α33P-GTP + ATP + CTP (lanes 2 and 4). The total RdRp activity (%) for both templates was quantified and expressed as a bar graph.(a) The 12-mer RNA template from the leader promoter region of the NiV genome and an 11-mer RSV-template derived from the leader promoter region of the RSV genome. (b) Product sizes are indicated using a 4-5-6-mer combined RNA size marker (lane M). NiV L(wt)-P and either NiV or RSV template were incubated in the presence of α(TIF)Click here for additional data file.S6 Fig33P-GTP, ATP, and CTP. (b) Templates loaded in specific wells in gel image presented in (a).(a) Product sizes are indicated using a 4-5-6-mer combined RNA size marker (lane M). NiV L(wt)-P and template described in (b) were combined with α(TIF)Click here for additional data file.S7 Fig33P-GTP tracer (lane 1), α33P-GTP + ATP + CTP (lane 2), α33P-GTP + primer (lane 3), or α33P-GTP + ATP + CTP + primer (lane 4). NiV L(D832A-N833A)-P mutant and template were incubated in the presence of α33P-GTP tracer (lane 5), α33P-GTP + ATP + CTP (lane 5), α33P-GTP + primer (lane 7), or α33P-GTP + ATP + CTP + primer (lane 8). NiV L(H1347A)-P and template were incubated in the presence of α33P-GTP tracer (lane 9), α33P-GTP + ATP + CTP (lane 10), α33P-GTP + primer (lane 11), or α33P-GTP + ATP + CTP + primer (lane 12). (c) Table showing enzyme, NTPs, and primers along with well number.(a) The 12-mer RNA template from the leader promoter region. (b) Product sizes are indicated using a kinase-labeled set of four combined oligonucleotides: 4-, 5-, 6-, and 12-mer primer (lane M). NiV L(wt)-P and template were incubated in the presence of α(TIF)Click here for additional data file.S8 FigKm requirement for ATP. (b) A kinase-labeled primer was used as a marker to estimate size of extension products (lane M). NiV L(wt)-P, template, and primer were incubated in the presence of α33P-GTP tracer (lane 1), and with α33P-GTP combined with variable concentrations of ATP from 1nM to 1μM . (c) Data from multiple experiments was combined to generate a plot of RdRp Activity (%) vs ATP (μM), followed by a calculation of the Michaelis constant (Km).(a) A 12-mer template with a short 4-nucleotide prime was used to analyze the (TIF)Click here for additional data file.S9 Fig33P-GTP tracer (lane 1), α33P-GTP + ATP + CTP (lane 2), α33P-GTP + ATP + R1479-TP (lane 3), α33P-GTP + ATP + 3′dCTP (lane 4), α33P-GTP + CTP + GS-5734-TP (lane 5), and α33P-GTP + 3′dATP (lane 6).(a) A 12-mer template with four bases complementary to a short 4-nucleotide primer was used to analyze primer-extension activity of NiV L(wt)-P. Blue and green arrows indicate incorporation sites for 3′dATP or 3′dCTP at positions +6 or +7, respectively. (b) NiV L(wt)-P, template, and primer were incubated in the presence of α(TIF)Click here for additional data file.S10 Fig33P-GTP tracer (lane 1) or α33P-GTP + ATP + CTP (lane 2). (b) RSV L-P, an 18-mer template, and primer were incubated in the presence of α33P-GTP tracer (lane 1), α33P-GTP + ATP (lane 2), or α33P-GTP + ATP + UTP.(a) RSV L-P, an 18-mer template, were incubated in the presence of α(TIF)Click here for additional data file.S11 Fig(a) The RNA templates used in RNA synthesis reactions with NiV with free energies associated with secondary structure formation . (b) Predicted secondary structures for RNA.(TIF)Click here for additional data file.


In this study, we determined the gene expression analysis of IL-17 gene family for early detection of subclinical inflammation among IBD patients.Cytokines have a vital role in the pathogenesis of inflammatory bowel disease (IBD). Interleukin-17 is the signature cytokine of the recently identified T helper 17 (Th17) cell subset. IL-17F is mainly involved in mucosal host defense mechanisms whereas the functions of IL-17B remain largely elusive. In this cross-sectional study, IBD patients divided into two active and inactive groups. Peripheral blood mononuclear cells (PBMCs) from 38 IBD patients which 20 inactive samples and 18 active individuals were collected. Changes of IL-17 F and IL-17B mRNA expression level evaluated by quantitative-real time-PCR.  mRNA expression level of IL-17B and IL-17F in CD, UC, active and inactive groups have been assessed and there were no significant differences (P>0.05). Patients were classified into five different categories as follows: i) 5ASA; ii) 5ASA + Pred; iii) 5ASA + AZA; iv) 5ASA + Pred + AZA; v) 5ASA + Pred + AZA + IFX according to medication usage, expression of IL-17F and IL-17B had no differences (p>0.05).  Evaluation of IL-17B and IL-17F mRNA expression level illustrate no difference among active and inactive patients. Therefore, IL-17B and IL-17F are not biomarkers in an Iranian IBD patients. Inflammatory bowel disease (IBD) is a multifactorial disorder that its symptoms include diarrhea with bleeding, fever and fatigue, Abdominal pain, reduced appetite, unintended weight loss in the gastrointestinal tract , 2. AlthPopulationIn this cross-sectional study, 38 participant subjects  who referred to Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran from 2013 to 2016. A questionnaire including demographic and clinical features filled for all patients. In addition, 6 ml whole blood collected in EDTA tubes and stored at 4˚C until RNA extraction. The consent form was obtained from patients that ethics committee at the Research Center Gastroenterology and Liver Diseases (RCGLD) approved the protocol. RNA Isolation and Real-TimeTotal RNA was extracted from PBMC and YTA RNA Extraction (YEKTA TAJHIZ AZMA) was utilized. Spectrophotometric optical density measurement (260 and 280 nm) was used for evaluation of quality and quantity of RNA. cDNA synthesis used by Revertaid RT Reverse Transcription Kit . For evaluation of IL-17B and IL-17F expression ABI 7500 real-time (2.3 version) PCR system  were employed with Takara SYBR Master Mix instructions . Primers for IL-17B, IL-17F, and Beta-2-Microglobulin (as a housekeeping gene) were designed by using Gene Runner and Primer3 online programs . Statistical AnalysisDetermination of gene expression by Student t-test was apprised. One-way ANOVA and Student t-test were used to examine the IL-17B and IL-17F mRNA expression level between groups. Graph pad prism 5 software  were utilized to draw the graphs. P<0.05 regarded as an indication of statistically significant difference. DemographicsThirty-eight patients have participated in the present study. Accurately, 20 and 18 individuals were in inactive and active phase respectively. To be more specific, 20 patients were in inactive phase, including UC 77.3% and CD 22.7% and 18 patients were in active phase which all of them were UC. In both groups, the rate of the male was 36.8%, the percentage of the female was 63.2%, and there was no significant association between them. The average of patients’ diagnosis age and BMI were 30.68  and 25.7977 respectively .Evaluation of IL-17B and IL-17FmRNA expression level of IL-17B and IL-17F in CD, UC, active and inactive groups have been assessed and there were no significant differences (P>0.05). Additionally, an evaluation of patients who had a history of taking medication was also conducted and there was no association among this groups (P>0.05). Patients were classified into five different categories as follows: i) 5ASA; ii) 5ASA + Pred; iii) 5ASA + AZA; iv) 5ASA + Pred + AZA; v) 5ASA + Pred + AZA + IFX according to medication usage  and I.Revolution of the immune system in IBD patients has been investigated in many studies. It is addressed that impaired mucosal cytokine secretion has a direct effect on pathogenesis of IBD , 12. TheThe present study examined association of IL-17B and IL-17F mRNA expression level illustrate no difference among active and inactive patients. In addition, expression of IL-17F and IL-17B had no differences (p>0.05) in patients who had a history of taking the four types of drug . Thus, these analyses may be used in the prognosis, diagnosis and treatment of IBD.


Osimertinib is a third-generation EGFR-TKI with a high selective potency against T790M-mutant NSCLC patients. Considering that osimertinib can lead to enhanced HER-2 expression on cell surface and HER-2 overexpression is a mechanism of resistance to osimertinib, this study was addressed to investigate the potential of combining osimertinib with trastuzumab emtansine (T-DM1) in order to improve the efficacy of osimertinib and delay or overcome resistance in NSCLC cell lines with EGFR activating mutation and with T790M mutation or HER-2 amplification.The effects of osimertinib combined with T-DM1 on cell proliferation, cell cycle, cell death, antibody-dependent cell-mediated cytotoxicity (ADCC), and acquisition of osimertinib resistance was investigated in PC9, PC9-T790M and H1975 cell lines. The potential of overcoming osimertinib resistance with T-DM1 was tested in a PC9/HER2c1 xenograft model.T-DM1 exerted an additive effect when combined with osimertinib in terms of inhibition of cell proliferation, cell death and ADCC induction in PC9, PC9-T790M and H1975 cell lines. Combining osimertinib and T-DM1 using different schedules in long-term growth experiments revealed that the appearance of osimertinib-resistance was prevented in PC9-T790M and delayed in H1975 cells when the two drugs were given together. By contrast, when osimertinib was followed by T-DM1 an antagonistic effect was observed on cell proliferation, cell death and resistance acquisition. In xenograft models, we demonstrated that HER-2 amplification was associated with osimertinib-resistance and that T-DM1 co-administration is a potential strategy to overcome this resistance.Our data suggest that concomitant treatment with osimertinib and T-DM1 may be a promising therapeutic strategy for EGFR-mutant NSCLC. In patients with advanced Non-Small Cell Lung Cancer (NSCLC) harbouring Epidermal Growth Factor Receptor (EGFR) activating mutations, disease progression occurs after a median of 10–12 months of treatment with EGFR-Tyrosine Kinase Inhibitors (TKIs), such as gefitinib, erlotinib, and afatinib. The acquisition of the EGFR-T790M secondary mutation is the main mechanism of acquired resistance, occurring in 50–60% of the cases  with ImageJ software and the Fold Increase (FI) index was calculated as the ratio between the spheroid volume after 6 days and the volume at T0.Spheroids were generated using LIPIDURE®-COAT PLATE A-U96  as previously described . BrieflyOne million of NSCLC cells were incubated, for one hour at room temperature, with Isotype control Monoclonal Mouse IgG1/R-PE , mouse anti-Human HER-2/PE or mouse anti-Human EGFR/PE  to determine EGFR or HER-2 protein membrane levels as previously described . The anaPC9-T790M and H1975 cells were plated in 96-well plates at a density of 350 cells/well and treated with osimertinib and/or T-DM1 following the indicated schedules in the experiment. Medium with drugs was changed every 7 days. Wells were followed daily over time. Taking into account the different sensitivity to osimertinib, cells were scored as resistant when colonies with at least 20 cells were detected in PC9-T790M cell cultures or when >80% confluence was reached in H1975 cell cultures .Highly purified CD56+ natural killer (NK) cells were obtained by magnetic separation and ADCC was measured with the CytoTox 96 non-radioactive cytotoxicity assay  as previously described .Tyr1068, HER-2 (29D8), p-HER2 Tyr1221/1222 (6B12), AKT, pAKTser473, p-p44/42 MAPK (D13.14.4E), MAPK (137F5), Bim (34C5) and Mcl-1 (RC-13) were from Cell Signaling Technology ; antibody against Actin was from Sigma. HRP-conjugated secondary antibodies were from Pierce  and chemoluminescence system (Immobilion™ Western Chemiluminescent HRP Substrate) was from Millipore . Reagents for electrophoresis and blotting analysis were from BIO-RAD . The chemiluminescent signal was acquired by C-DiGit® Blot Scanner and the spots were quantified by Image Studio™ Software, LI-COR Biotechnology .Procedures for protein extraction, solubilization, and protein analysis by 1-D PAGE are described elsewhere . Antibod6 PC9/HER2c1 cells were subcutaneously injected on the flank of Balb/c-Nude female mice . The animals were housed in a protected unit for immunodeficient animals with 12-h light/dark cycles and provided with sterilized food and water ad libitum. When tumor volume reached an average size of 200 mm3, animals were randomly allocated into four groups (n = 8 tumors per treatment group): control, osimertinib, T-DM1 and osimertinib combined with T-DM1. Osimertinib (10 mg/kg in 1% Tween 80) was given once per day, five times per week, by oral gavage. T-DM1 (15 mg/kg in 0.9% NaCl) was given intraperitoneally twice per week. Tumors were measured as previously described . However, also for this agent, resistance is expected and new therapeutic approaches are needed to prolong its benefit [Osimertinib is an effective treatment in NSCLC patients with T790M EGFR mutation, progressed after first-line therapy with first- or second-generation EGFR-TKIs . MoreoveHER-2 was shown to be overexpressed (IHC 3+) in 2–6% of NSCLC, with gene amplification found in 2–4%, mainly in the adenocarcinoma histotype . In partWe tested T-DM1 in preclinical experiments suggesting its activity against HER-2-driven lung cancer cell lines in vitro . Only reAs previously reported by Scaltriti et al. for lapatinib in breast cancer cells  and by oHere we demonstrated that, differently from trastuzumab, T-DM1 showed anti-proliferative effects in PC9, PC9-T790M and H1975 cells and exerted an additive effect when combined with osimertinib in terms of inhibition of proliferation, cell death and ADCC induction. Because osimertinib is highly effective in mutated NSCLC cell lines, the addition of T-DM1 caused a modest benefit when evaluated in short-term growth assay, by contrast the combination significantly delayed the acquired resistance to osimertinib in PC9-T790M and in the less osimertinib-sensitive model H1975. The combination was effective either when the cells were treated continuously with osimertinib and a simultaneous treatment with TDM1 was cyclically performed  anticipated the appearance of resistance to osimertinib compared to cells continuously treated with osimertinib alone, by contrast in the protocols in which cells were simultaneously exposed to both drugs (osimertinib→osimertinib + T-DM1 or osimertinib + T-DM1→osimertinib), no resistant colonies were detected up to 50 days of treatment. In H1975 cells, all schedules delayed the acquisition of resistance; however, also in this model the less effective was the intermittent schedule (osimertinib→T-DM1) both in terms of percentage of resistant wells and of median time to resistance.Overall, these data may support the use of a combination of osimertinib with T-DM1 using a specific schedule to delay or prevent the acquisition of resistance to the EGFR-TKI, similarly to other combinations of third-generation EGFR-TKIs (with MEK or MET inhibitors) tested in clinical trials currently recruiting patients .In the last part of this study we investigated the role of HER-2 amplification in the responsiveness to osimertinib and the efficacy of T-DM1 in vitro and in vivo to overcome osimertinib resistance. HER-2 amplification, indeed, has been reported as a mechanism of acquired resistance to EGFR-TKIs with a frequency of 12–13% .PC9/HER2c1 cells were exposed to osimertinib in vitro and in vivo in xenotransplanted mice. These cells were significantly more resistant to osimertinib than the parental cells in vitro and tumors grew as the controls indicating intrinsic resistance to osimertinib. T-DM1 significantly reduced tumor growth but the combination of osimertinib with T-DM1 showed a complete suppression of tumor growth, suggesting an important role of T-DM1 in overcoming osimertinib resistance. These data confirm the role of HER-2 amplification as a potential cause of intrinsic resistance to osimertinib, as demonstrated by Ortiz-Cuaran S et al. , and indIn conclusion, the combination of osimertinib with T-DM1 exerted an additive effect on cell growth inhibition when given as a concomitant administration. However, when cells exposed to osimertinib were subsequently treated with T-DM1, an antagonist interaction was observed. The combination of T-DM1 with osimertinib retarded the appearance of resistance in PC9-T790M cells and overcome osimertinib-resistance in cells with HER-2 amplification. Our results may contribute to define new potential future drug-combinations in the treatment of EGFR mutated NSCLC patients.


PDL1 mRNA was measured using RNA in situ hybridization (RISH). The overall prevalence of PD-L1 protein expression was 25.2% in tumor cells and PDL1 mRNA expression was 11.9%. There was a strong positive correlation between PD-L1 IHC and RISH results . In adenocarcinoma, PD-L1 protein and mRNA expressions significantly correlated with poorly differentiated histologic subtype . PD-L1 expression was also associated with genetic alteration in adenocarcinoma. High PD-L1 expression level was associated with EGFR-naïve and KRAS-mutant subgroup . With a 1% cut-off value, PD-L1 protein expression showed a short overall survival duration in early stage adenocarcinoma with marginal significance . Our study revealed that PD-L1 expression varied with histologic subtype and genomic alteration status in lung adenocarcinoma, and activation of the PD-L1 pathway may be a poor prognostic factor especially in early stage lung adenocarcinoma. In addition, PDL1 RISH showed promising results in predicting PD-L1 protein expression in NSCLC.In this study, we present the clinicopathological features associated with PD-L1 protein and mRNA expression in a large Asian cohort of patients with non-small cell lung cancer (NSCLC) and assessed the prognostic implications of PD-L1 expression, particularly in early stage NSCLC. We retrospectively analyzed 687 NSCLC specimens (476 adenocarcinoma and 211 squamous cell carcinoma) using tissue microarray. PD-L1 immunohistochemistry (IHC) was performed using Dako 22C3 pharmDx assay and  Researchers have recently become interested in developing immunotherapies for the treatment of non-small cell lung cancer (NSCLC), particularly monoclonal antibodies targeting the programmed cell death-1 (PD-1) receptor and its ligand (PD-L1) . InteracBesides acting as a predictive biomarker, PD-L1 shows inconsistent results among various studies as a prognostic biomarker. Studies investigating the prognostic role of PD-L1 and its association with clinicopathological features and driver mutations in NSCLC have yielded quite different results –13. ThisPDL1 mRNA exhibited a positive nonlinear relationship with PD-L1 protein using this assay, suggesting the potential applications of the RNAscope assay in future clinical studies [The variety of PD-L1 immunohistochemical (IHC) assays, involving the use of different antibodies and interpretation criteria, may also contribute to the lack of consistent results . Given t studies ,16.In this study, we evaluated the clinicopathological features associated with PD-L1 protein and mRNA expression in a large Asian cohort of patients with NSCLC and investigated the prognostic implications of PD-L1 expression, particularly in early stage NSCLC.th edition of the American Joint Committee on Cancer staging manual [Our cohort consisted of 687 patients with NSCLC, including 476 with adenocarcinoma (ADC) and 211 with squamous cell carcinoma (SqCC) who underwent surgical resection between May 2003 and December 2012 at Seoul National University Bundang Hospital. None received pre-operative chemotherapy or radiation therapy. Clinicopathological information was obtained from clinical records and pathology reports. The pathologic staging was based on the 7g manual . The stuAll resected tumor specimens were fixed with formalin and then stained with hematoxylin and eosin (H&E). All H&E slides were carefully reviewed by two of the authors (H. Kim and J.H. Chung) to confirm the original diagnosis and classify the histological subtype. ADC in situ and minimally invasive ADC samples were excluded from the study. All other invasive ADC samples were categorized as lepidic, papillary, acinar, micropapillary, solid, or invasive mucinous according to the 2015 World Health Organization Classification of Lung Tumors . These hThe slides were independently reviewed by two pathologists (H. Kim and J.H. Chung) to select the most representative sections. The most representative tumor area was carefully marked on the H&E-stained slide of each sample tissue. A TMA was constructed using 2-mm-diameter cores derived from the representative tumor areas selected at random of the FFPE tissue blocks from each case by SuperBioChips Laboratories .TMAs were sectioned at a thickness of 4-μm and stained using the Dako pharmDx assay. Briefly, the slides were stained with anti-PD-L1 22C3 mouse monoclonal primary antibodies with the EnVision FLEX visualization system on a Dako Autostainer Link 48 instrument , along with negative control reagents and cell line run controls, as per the manufacturer’s instructions. The IHC slides were scored independently by two pathologists (H.J. Kwon and H. Kim). PD-L1 was considered positive in tumor cells only in cases of at least 100 viable tumor cells, if membranous staining alone or membranous and cytoplasmic staining together was present. Membranous staining in tumor cells directly adjacent to immune cells was not considered positive if the surface touching immune cells was the only stained part. The percentage of stained cells in the overall area of the tumor (Tumor Proportion Score) was scored regardless of intensity . Cases wPDL1 mRNAs were measured using RNAscope assays  following the manufacturer’s instructions [ructions . BrieflyPD-L1 mRNA signals were in the tumor compartment or mesenchyme, as visualized by red dotted or clustered patterns. No standard scoring criteria for PD-L1 mRNA expression in NSCLC had been determined; therefore, we adopted the RNAscope system scoring guidelines (“RNA scope score”): 0 (no staining or < 1 dot per 10 cells); 1 (1–3 dots per cell); 2 (4–9 dots per cell); 3 (10–15 dots per cell); and 4 (> 15 dots per cell and > 10% dots in clusters) [PD-L1 RNA scope and proportion scores showed a linear correlation , cases showing either an RNA scope score of 1 or more or an RNA proportion score of 1 or more were designated as PD-L1 mRNA positive.lusters) . We alsoEGFR mutations in exons 18–21 and KRAS mutations at codons 12, 13, and 61, as described previously [ALK gene was assessed using fluorescence in-situ hybridization with an ALK probe  and a 15% cut-off value, as described previously [Polymerase chain reaction and DNA sequencing with FFPE tissue samples were used to analyze eviously . Rearraneviously .p values of less than 0.05.Statistical analysis was carried out using Stata Statistical Software version 14  and R program . Spearman’s test and logistic regression were performed to compare assays and determine appropriate cut-off values. Cohen’s ĸ coefficient of agreement was obtained to cross-check the results. A Kaplan-Meier analysis was performed to construct survival curves, and statistical significance was assessed using log-rank tests. A multivariate analysis was performed by Cox proportional hazards regression modeling. All statistical tests were two sided, and statistical significance was accepted for Clinicopathological characteristics are summarized in p < 0.05). PD-L1 mRNA expression was detected in 11.9% (82/687) of patients. In subgroup analysis, SqCC showed higher mRNA positivity than ADC . The overall prevalence of PD-L1 protein expression in tumor cells was 25.2% 173/687; . With a PD-L1 mRNA expression . As the TPS of PD-L1 protein expression increased, PDL1 mRNA expression was observed frequently  pairwise between assays at two PD-L1 IHC cut-off values (1% and 50%). OPA with 1% and 50% IHC cut-off values were 80.1% and 91.1%, respectively . Positivp < 0.001 and p = 0.002, respectively; EGFR-mutated group than in the EGFR-negative group , PD-L1 protein expression was higher in the KRAS-mutated group than in the KRAS-negative group (p = 0.017). Smoking history, pathological stage, and ALK status were not associated with PD-L1 status. In SqCC, only tumor size was associated with PD-L1 protein expression with marginal significance .Next, we investigated the associations between PD-L1 status and clinicopathological parameters in ADC and SqCC. In ADC, both PD-L1 protein and mRNA expression were correlated with histologic subtype (p = 0.02)  in multivariate analysis after adjusting for the conventional clinicopathological covariates .We performed survival analysis to investigate the prognostic role of PD-L1 protein and mRNA expression in ADC and SqCC. In ADC, lymphovascular/perineural invasion and pathologic TNM stage were independent poor prognostic factors for disease-free survival (DFS) and overall survival OS; . PD-L1 e = 0.02)  and tendvariates . For PD-PD-L1 mRNA expression and compared PD-L1 protein and mRNA expression; the results showed that PD-L1 mRNA was a potential surrogate marker, with a positive correlation with protein expression.In this study, we demonstrated that PD-L1 expression was significantly associated with histologic grade and genetic alteration status in lung ADC. We also found that PD-L1 protein expression was an adverse prognostic marker for OS in patients with early stage lung ADC. We also assessed Our study showed that PD-L1 protein and mRNA expression levels were significantly associated with high histologic grade and solid subtype of ADC. Our results are in line with the results of several studies which reported the correlation between PD-L1 expression in tumor cells and poor differentiation and solid histology –12. ThisFrom biological point of view, elevated expression of PD-L1 poorly differentiated lung ADCs compared with well-differentiated ADCs might account the inactivation of effector-immune cells through PD-1 receptor signaling which could ultimately enhance tumor progression. This relationship supports the results of our study that ADCs with high expression of PD-L1 are associated with poorly differentiated histology and poor prognosis.EFGR mutation status and PD-L1 expression in NSCLC is still unclear. Although preclinical studies have suggested that EGFR-driven NSCLC inhibits antitumor immunity through activation of the PD-1/PD-L1 pathway in an intrinsic manner, epidemiological studies have suggested that EGFR-mutant NSCLC is more likely to exhibit decreased PD-L1 expression. Two recent pooled analyses have provided further support for this inverse relationship. In one study, patients harboring EGFR mutations were more likely to have decreased PD-L1 expression  [EGFR wild-type status  [EGFR mutations and PD-L1 expression could be the variability, including the assessment of biomarkers from a single lesion site at a single time point, which often provides poor insights into spatiotemporal dynamics. For example, PD-L1 expression has been shown to fluctuate during EGFR tyrosine kinase inhibitor (TKI) and post-progression [EGFR TKI treatment, which could exclude the temporal heterogeneity of PD-L1 status. PD-L1 protein and mRNA expression were lower in the EGFR-mutated group than in the EGFR-negative group. Low PD-L1 expression in EGFR-mutated ADC may be related to the lower prevalence of PD-L1 in the Asian population than in Western populations. In our cohort, PD-L1 expression was observed in 16.2% of patients with a 1% cut-off and in only 6.1% of patients with a 50% cut-off. Several reports have shown low PD-L1 prevalence in lung ADC of Asian patients [EGFR mutation prevalence may be one of the causes of ethnic differences in PD-L1 expression.The relationship between 10–2.93) , and in  = 0.01) . One reagression . In our patients . Thus, tThe prognostic impact on PD-L1 expression in NSCLC is still controversial ,27, but In this study, PD-L1 expression was found as frequently in stages I and II  as in stages III and IV , indicating that aberrant expression of this ligand may be an early event. Patients with early stage NSCLC may have a more intact immune system and the potential for long-lasting immune priming against micrometastases . TherefoPDL1 mRNA expression. Further investigation of the mechanism of PD-L1 protein expression bypassing mRNA expression, and minimizing the analytic variables of RISH assay is necessary to reduce discrepancy between the two assay.Finally, we assessed PD-L1 expression using IHC and RISH. Many studies have attempted to assess PD-L1 expression using different techniques, including RISH ,16. The The lack of response data to anti-PD-1/PD-L1 drugs is the major limitation of our study. The issue on utilizing PD-L1 mRNA assay should be made based on data from clinical trials of the drug under consideration and further studies are needed with the therapeutic responses.EGFR-naïve status in lung ADC. In addition, PD-L1 expression may be a prognostic marker in patients with early stage lung cancer.In conclusion, elevated PD-L1 expression was associated with poorly differentiated histology and S1 Table(XLSX)Click here for additional data file.


E. coli and S. cerevisiae, ClpB, and Hsp104, cooperate with the Hsp70 chaperone system in the solubilization of aggregated proteins. Aggregate solubilization relies on a substrate threading activity of ClpB/Hsp104 fueled by ATP hydrolysis in both ATPase rings . ClpB/Hsp104 ATPase activity is controlled by the M-domains, which associate to the AAA-1 ring to downregulate ATP hydrolysis. Keeping M-domains displaced from the AAA-1 ring by association with Hsp70 increases ATPase activity due to enhanced communication between protomers. This communication involves conserved arginine fingers. The control of ClpB/Hsp104 activity is crucial, as hyperactive mutants with permanently dissociated M-domains exhibit cellular toxicity. Here, we analyzed AAA-1 inter-ring communication in relation to the M-domain mediated ATPase regulation, by subjecting a conserved residue of the AAA-1 domain subunit interface of ClpB (A328) to mutational analysis. While all A328X mutants have reduced disaggregation activities, their ATPase activities strongly differed. ClpB-A328I/L mutants have reduced ATPase activity and when combined with the hyperactive ClpB-K476C M-domain mutation, suppress cellular toxicity. This underlines that ClpB ATPase activation by M-domain dissociation relies on increased subunit communication. The ClpB-A328V mutant in contrast has very high ATPase activity and exhibits cellular toxicity on its own, qualifying it as novel hyperactive ClpB mutant. ClpB-A328V hyperactivity is however, different from that of M-domain mutants as M-domains stay associated with the AAA-1 ring. The high ATPase activity of ClpB-A328V primarily relies on the AAA-2 ring and correlates with distinct conformational changes in the AAA-2 catalytic site. These findings characterize the subunit interface residue A328 as crucial regulatory element to control ATP hydrolysis in both AAA rings.The members of the hexameric AAA+ disaggregase of  AAA+ proteins constitute a protein superfamily sharing the ability to convert the chemical energy derived from ATP hydrolysis into mechanical work. AAA+ proteins share the AAA domain, which is defined by a region of ~230 amino acids in length, comprising conserved Walker A and Walker B motifs for nucleotide binding and hydrolysis. The AAA domain also drives protein oligomerization, frequently into hexameric ring-like structures with a central pore. The catalytic active site is located at the subunit interface of AAA domains involving conserved elements from both subunits  harbor two AAA domains  and solubilize aggregated proteins in concert with a cognate Hsp70 chaperone system  and R756 (AAA-2). Arginine fingers are essential for ClpB/Hsp104 disaggregation activity  and size exclusion chromatography  following standard protocols. Purifications of DnaK, DnaJ, GrpE, Luciferase, and Casein-YFP were performed as described previously  containing an ATP Regenerating System . Luciferase activities were determined with a Lumat LB 9,507 (Berthold Technologies) MDH disaggregation was monitored by turbidity measurement at an excitation and emission wavelength of 600 nm (PerkinElmer LS50B spectrofluorimeter).ClpB disaggregation activities were determined by following the disaggregation of heat-aggregated Malate Dehydrogenase  and 0.05 μM urea-denatured firefly Luciferase at 25°C as described  are caused by analysis of different protein purification batches.ATPase activities of ClpB (0.5 μM) was determined in Reaction buffer in absence or presence of substrate (10 μM casein) using a NADH-coupled colorimetric assay (Sigma) by measuring the decrease of NADH absorption at 340 nm in a BMG Labtech FLUOstar Omega plate reader. Minor differences in ATPase activities determined for ClpB wt and mutants (To determine the affinity of ClpB (wt and derivatives) for the fluorescent nucleotide analog mantADP equilibrium titrations of 1,25 μM mantADP with different ClpB concentrations were performed at 30°C using a FP 6500 JASCO Spectrometer (Excitation: 360 nm/Emission: 400–500 nm). The affinity for mantADP can be determined using the following equation:0: fluorescence of fluorophor (mantADP); Fmax: maximum fluorescence observed; [E]0: total concentration of ClpB (μM); [L]0: total concentration of fluorophor (μM); Kd: dissociation constant of the complex (μM).with F: observed fluorescence; FTo determine the affinity of ClpB wt and derivatives for ADP and ATPγS competition titrations were performed. 1,25 μM mantADP were initially mixed with 1,25 μM ClpB and pre incubated for 5 min at 30°C. Subsequently this complex was titrated with solutions of ADP and ATPγS and mantADP fluorescence as determined as described above. The affinities for the unlabeled nucleotides were determined using the following equations:0: starting fluorescence without nucleotide; F1: maximum fluorescence observed; [ATP]: concentration of ADP/ATPγS (μM); Ki,app: apparent dissociation constant.with F: observed fluorescence; Fi,app: apparent dissociation constant; Ki: dissociation constant; [mantADP]: mantADP concentration (μM); Kd (mantADP): dissociation constant of mantADP.with KUnfolding and degradation of Casein-YFP (0.25 μM) was determined in Reaction buffer in presence of 4 μM BAP (wt or derivatives) and 6 μM ClpP and an ATP regenerating system. YFP fluorescence was followed on a Perkin-Elmer LS50B spectrofluorimeter .2, 2 mM DTT). One micromolar ClpB was incubated at 25°C in presence of ATPγS for 5 min. Crosslinking reactions were started by addition of 0.1% glutaraldehyde. Reactions were stopped after 2 and 10 min by addition of 1 M Tris pH 7.5 and crosslinking products were analyzed by SDS-PAGE (4–15%) followed by Sypro Ruby Staining (ThermoFisher).All tested ClpB variants were dialyzed against Reaction Buffer B  in presence of 2 mM ATPγS. Next ClpB was diluted 20-fold into respective D2O-based low salt MDH buffer to initiate amide hydrogen exchange. The exchange reaction was quenched by the addition of 1 volume of ice-cold quench buffer (0.4 M potassium phosphate pH 2.2) and injected into an UPLC (Waters) setup, following online peptic digestion. ClpB peptides were analyzed on an electrospray ionization quadrupole time-of-flight mass spectrometer  as described  and mi (m/z) using the Bruker Compass software :HX-MS experiments were performed as described earlier . A fully deuterated ClpB sample was generated by incubating Dt is the observed average mass of the peptide at time point t, mass0% is the observed average mass of the undeuterated peptide and mass100% is the observed average mass of the fully deuterated peptide.where massE. coli cells harboring plasmid-encoded clpB alleles were grown in the absence of IPTG overnight at 30°C. Serial dilutions were prepared, spotted on LB-plates containing different IPTG concentrations and incubated for 24 h at indicated temperatures.E. coli ΔclpB cells harboring IPTG-inducible YFP-tagged clpB alleles were grown to mid-exponential growth phase in the presence of 100 μM IPTG at 30°C. Cells were subjected to heat stress (20 min at 43°C) followed by a recovery period (120 min) at 30°C. One milliliter cell cultures were taken before and after heat stress and during recovery and centrifuged. For snapshot imaging, cells pellets were resuspended in 100 μl icecold PBS buffer and immobilized on 1% (w/v) agarose pads (in 1x PBS). Agarose pads were sealed with Apiezon grease and covered with cover slips. Imaging was performed using the xcellence IX81 wide field system (Olympus) with a Plan Apochromat x100/1.45 numerical aperture oil objective, a Hamamatsu OrcaR2 camera and the according filter settings (YFP). For image analysis ImageJ was used and for statistical analysis at least 100 cells were counted to determine the % of cells without and with foci pre, after heat shock and during the recovery phase.Arabidopsis thaliana, analyzing the plant homolog of ClpB, Hsp101. The authors isolated the Hsp101-A499T mutant, which harbors a point mutation in M-domain helix3 and exhibits cellular toxicity at increased temperatures (38°C), a phenotype not observed for hsp101 null mutants , or strongly reduced toxicity (A328I). Toxicity upon expression of A328I/K476C was only observed at 42°C in presence of 100 μM IPTG  and urea-denatured Luciferase as model substrates. We focused our analysis on A328L, A328I, and A328V variants as those mutants either suppressed K476C toxicity  or exhibited toxicity on its own (A328V) Figure . DisaggrA similar trend was observed when using aggregates of urea-denatured Luciferase as alternative substrate Figures . The disclpB-A328V expression in E. coli might therefore be different from ClpB-K476C.Taken together, the disaggregation activities provide a rationale for suppression of ClpB-K476C toxicity by A328L and A328I mutations, as they strongly reduce disaggregation activities and abrogate the high disaggregation activity of K476C. While ClpB-A328V represented the most potent A328X mutant in protein disaggregation, its activity was reduced and clearly different from hyperactive ClpB-K476C. The molecular basis for cellular toxicity noticed upon The hyperactive state of ClpB and Hsp104 M-domain mutants is linked to very high ATP hydrolysis rates in presence of substrate  very high ATPase activity in presence of substrate . These low expression levels do not affect growth of E. coli cells. Protein aggregates forming in E. coli cells during heat stress are deposited at the cell poles  or AAA-2 (E678A) allowing for ATP binding but abolishing ATP turnover in the respective AAA+ ring. Additionally, we combined the A328V and K476C mutations with the Walker A mutation K611Q, causing deficiency in ATP binding at the AAA-2 ring. We did not include a respective Walker A mutant of AAA-1 as it shows oligomerization defects  mass spectrometry (MS). HX-MS determines the solvent accessibility and structural flexibility of the peptide backbone. Amide hydrogens are protected from HX if engaged through hydrogen bonds in secondary and tertiary structures. We compared the HX-MS patterns of peptic peptides from ClpB wt, ClpB-A328V, and ClpB-K476C in the presence of ATPγS Figure . The HX-In the presented work we analyzed the role of intersubunit communication in controlling ClpB ATPase and disaggregation activity. We selected the conserved A328 residue for analysis as it is located in a strategic position at the AAA-1 subunit interfaces close to the essential arginine fingers R331 and R332. Furthermore, the identical residue was identified as intragenic suppressor of a toxic, gain-of-function Hsp101 M-domain mutant : high ATPase and unfolding activities and temperature-dependent cellular toxicity. ClpB-A328V, however, differs from M-domain mutants and constitutes a novel class of hyperactive ClpB mutants. HX-MS analysis revealed that M-domain motif2 of ClpB-A328V does not show increased deprotection as compared to ClpB wt, indicating that M-domain motif2 is not displaced from the AAA-1 ring in contrast to ClpB-K476C. This shows that the initial molecular event resulting in high ATPase rates of ClpB-A328V and ClpB-K476C is different. A328V might, however, activate an allosteric network, which is also involved in ATPase activation upon M-domain dissociation. The latter event must include additional activation steps as both classes of hyperactive ClpB mutants (A328V and K476C) differ in disaggregation activities and ATPase regulation. Accordingly, coupling A328V or K476C to Walker B mutants that prevent ATP hydrolysis at the AAA-1 or AAA-2 ring, had distinct consequences on ATPase activities. Preventing nucleotide binding or hydrolysis at AAA-2 entirely abrogated ATPase activity of ClpB-A328V, in contrast to ClpB-K476C  ClpB-E279A has lower ATPase activity  to BB and AM.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.