Histone variants alter the nucleosome structure and play important roles in chromosome segregation, transcription, DNA repair, and sperm compaction. Histone H3 is encoded by many genes in most eukaryotic species and is the histone that contains the largest variety of posttranslational modifications. Compared with the metazoan H3 variants, little is known about the complex evolutionary history of H3 variants proteins in plants. Here, we study the identification, evolutionary, and expression analyses of histone H3 variants from genomes in major branches in the plant tree of life. Firstly we identified all the histone three related (HTR) genes from the examined genomes, then we classified the four groups variants: centromeric H3, H3.1, H3.3 and H3-like, by phylogenetic analysis, intron information, and alignment. We further demonstrated that the H3 variants have evolved under strong purifying selection, indicating the conservation of HTR proteins. Expression analysis revealed that the HTR has a wide expression profile in maize and rice development and plays important roles in development. Histones wrap DNA to form nucleosome particles that compact eukaryotic genomes . Histone31-S87-V89-M90 from H3.3 featuring S31-A87-I89-G90 [In animals, four amino acid substitutions distinguish H3.1 featuring A-I89-G90 , 9, In a-I89-G90 , 9. Phyl-I89-G90 . Arabidopsis thaliana [ Drosophila and mammals [There are 15 HTR genes coding for H3.1, H3.3, and CenH3 proteins inthaliana ; H3.1 anthaliana , which a mammals –14. The  mammals . Arabidopsis and several other plant species [ Capsella rubella might be a new class of H3 variants because of the unique sequence feature. Moreover, the selection and expression analysis suggested the functional conservation of H3 variants.Until now, genome-wide analyses of H3 variants have been conducted in species , 11, 15.http://www.Phytozome.net/) using Arabidopsis H3.1, H3.3, and CenH3 proteins as queries to identify HTRs . The species represented a broad range of the plant lineages from unicellular green algae to multicellular plants (http://www.jgi.doe.gov/). We used a relatively strict criterion to collect HTRs with high-quality sequences. To verify the reliability of our results, each sequence was then searched against the protein conserved domain database (CDD) [http://dx.doi.org/10.1155/2015/341598).We performed BLASTP and TBLASTN searches among sequenced genomes of planta in Phytozome (se (CDD) , SMART dse (CDD) , and these (CDD) , respectThe HTR sequences were then aligned by MAFFT version 7 using the G-INS-i algorithm , followeThe neighbor joining (NJ) phylogeny was performed by MEGA version 6.0 with 1000 replicas; since LG + G is not available in MEGA, we use next available best model JJT + G model, and pairwise deletion. The maximum likelihood (ML) phylogeny was performed by PhyML software , with 10Sequences used for selection analysis are listed in Supplementary Table. H3.1, H3.3, and CenH3 codon alignment was performed using MAFFT version 7 with the G-INS-i algorithm and then loaded into Hyphy  , monocots (Zea mays and Oryza sativa), gymnosperm (Picea abies), lycophyte (Selaginella moellendorffii), bryophyte moss (Physcomitrella patens), and chlorophytes   . Each ma Chlamydomonas reinhardtii, most of them were not previously annotated as HTR genes [After removing incomplete or redundant sequences (nucleic acid sequence redundant) and predicted alternative splice variants, we identified 230 H3 variants (Supplementary Table). The numbers are variable between species . InteresTR genes ; the var Arabidopsis CenH3 AT1G01370 has good statistics supports, enabling us to identify all the CenH3 variants .We first used phylogenetic analysis to define the different H3 groups present in the plant genomes; the phylogenetic tree contains all the H3 variants sequences from (Supplementary Table); the result showed that most of the branches have very low statistics supports (data not shown). However, the clade that containsvariants . All theea abies . One posea abies , 9: the 31Y41H87L90 signature. Members of the H3.1 class are intronless genes and corresponding proteins carry the A31F41S87A90 signature (31Y41(S/Q)87L90 and we defined them as H3.1A [In our analysis, all members of the H3.3  and H3 lysine-36 di- and trimethylation (H3K36me2/me3) are linked with active gene expression; H3 lysine-9 methylation and H3 lysine-27 trimethylation (H3K27me3) are associated with gene repression [We are particularly interested in grass H3 variants and we performed alignments using HTR protein sequences fromand rice . Analysipression , 31. Thepression , 33. Whe Capsella rubella are significantly different from all the other species' HTRs. We performed an alignment compared with Arabidopsis thaliana . The Capsella rubella HTRs were highly conserved within the species. However, even though Capsella rubella is the close relative to Arabidopsis, we did not find the H3.1 or H3.1 signature. Furthermore, we observed multiple deletions, the deletions were found in all the H3-like genes except CenH3, and the corresponding sequences were highly conserved in Arabidopsis. This suggested that CenH3 evolved independently to other H3 variants; the deletion event happened before the duplication of H3-like variants in Capsella rubella.We found that the HTR genes indN and dS is a common way to examine selection pressures on coding regions. Commonly, a dN/dS value of 1 is used to indicate neutral selection and values lesser or greater than 1 to, respectively, indicate purifying and positive selection [dN/dS ratios for each group. The dN/dS values were substantially <1 in all groups, providing a crude indication that the strong purifying selection has been maintained across plants, implying the H3 variants functional conservation. At the individual codon level, most of the residues were under significant negative selection (P < 0.05) . We also observed several sites under relaxed constrain , which may contribute to the functional divergent [Comparing rates of election . To analivergent .To understand the temporal and spatial expression patterns of grass HTR genes, we compared their expression patterns during maize and rice development.Microarray data of 60 different tissues and developmental conditions of maize were used . SeveralWe next analysed the expression profiles of rice HTR genes . The gen Arabidopsis, the H3.1 genes showed high level expression in tissues containing rapidly dividing cells; the H3.3 genes exhibited high level of expression in most of the tissues examined. Arabidopsis H3.1 and H3.3 were proposed as replication dependent and replication independent, respectively [ Arabidopsis [The comparative phylogenetic analysis of plant HTR proteins enables us only to identify the CenH3 variants. Therefore, we performed alignment to classify H3.1, H3.3, and H3-like variants using the established signatures . In Arabectively . Our expbidopsis , indicat Arabidopsis, the H3-like gene At1g19890 is male gamete specific; disruption of this gene might be compensated by other H3 genes [Our analysis identified a big number of H3-like genes. The numbers of H3-like genes are different between species. The H3-like genes' expression patterns are similar with H3.1 or H3.3 or have heterogeneous feature  Figures and 5. IH3 genes . Conside Arabidopsis are known to be crucial for a multitude of physiological and intracellular processes. Here, we report an identification, evolution, and expression of the H3 variants in plants; the comparative genomic analyses of H3 variants establish a framework for understanding the evolutionary mechanisms involved in the origin and expansion of plant HTR genes, and it provides a basis for investigating cellular functions of HTR genes.Histone H3 variants in animals andCapsella rubella HTR proteins. Capsella rubella HTRs have several conserved deletions. The H3.1 or H3.1 signature are absent in Capsella rubella HTR proteins.Supplementary Figure 1: Alignment of Arabidopsis thaliana and P< 12 0.05) and several sites under relaxed constrain(A). The x-axis numbers correspond to the numbering of the amino acids in the Arabidopsis thaliana H3.3 (A) and H3.1(B). The y-axis values are the ratio of dN/dS.Supplementary Figure 2: Analysis of site-specific levels of selection pressure in H3.3 (A) and H3.1(B) proteins. Most of the residues were under significant negative selection (Supplementary Table: Accession numbers of HTR genes used in this study.


Indeed, brain tumors are the leading cause of cancer-related mortality and morbidity in children. Children with DIPG have one-year progression-free survival rates below 25%, and median overall survival of 9 to 10 months with current treatment. Recent genetic studies have revealed that malignant gliomas in children and young adults often show shared missense mutations, which encodes the replication-independent histone 3 variant H3.3. Approximately 30 % of overall GBM and over 70% of DIPG cases harbor the amino-acid substitution from lysine (K) to methionine (M) at the position 27 of H3.3. The H3.3 K27M mutation in DIPG is universally associated with shorter survival compared with patients with non-mutated H3.3. We evaluated whether H3.3-derived peptides that encompass the H3.3 K27M mutation can induce specific cytotoxic T lymphocyte (CTL) responses in human leukocyte antigen (HLA)-A2http://paproc.de/) was used. Four candidate peptides encompassing different amino-acid positions around the H3.3 K27M mutation were synthesized, and peptide-specific CTL lines and clones were generated from peripheral blood mononuclear cells of HLA-A2+ donors by in vitro stimulation with each of the synthetic peptides.For prediction of HLA-A2-binding epitopes, an algorithm integrating peptide binding to HLA (NetMHC 3.4 server) and a proteosomal cleavage site prediction system  induced CTL lines which recognized not only the synthetic peptide loaded on T2 cells but also lysed HLA-A2s Figure . FurtherThese data provide us with a strong basis for developing peptide-based vaccines as well as adoptive transfer therapy using autologous T cells transduced with the TCR.


The hit compound N2 had the best antiviral activity against HCV, with a selective index of 32.1. The 5 hit compounds with new scaffolds could potentially serve as NS5B polymerase inhibitors through further optimization and development.The NS5B polymerase is one of the most attractive targets for developing new drugs to block Hepatitis C virus (HCV) infection. We describe the discovery of novel potent HCV NS5B polymerase inhibitors by employing a virtual screening (VS) approach, which is based on random forest (RB-VS), e-pharmacophore (PB-VS), and docking (DB-VS) methods. In the RB-VS stage, after feature selection, a model with 16 descriptors was used. In the PB-VS stage, six energy-based pharmacophore (e-pharmacophore) models from different crystal structures of the NS5B polymerase with ligands binding at the palm I, thumb I and thumb II regions were used. In the DB-VS stage, the Glide SP and XP docking protocols with default parameters were employed. In the virtual screening approach, the RB-VS, PB-VS and DB-VS methods were applied in increasing order of complexity to screen the InterBioScreen database. From the final hits, we selected 5 compounds for further anti-HCV activity and cellular cytotoxicity assay. All 5 compounds were found to inhibit NS5B polymerase with IC Chronic hepatitis C virus (HCV) infection has become a major global health problem because it can result in chronic liver disease, progressing to cirrhosis and hepatocellular carcinoma . There aHCV NS5B polymerase, an RNA-dependent RNA polymerase, is responsible for the replication of positive-strand genomic RNA of HCV . NS5B poIn the past decade, many NS5B polymerase inhibitors have been reported, and they can be classified into non-nucleoside inhibitors (NNIs) and nucleoside or nucleotide inhibitors (NIs) based on their mode of action . NIs actin silico methods have been applied to discover and develop new NS5B polymerase inhibitors [50 values ranging between 46 and 73.3 μM [50 values ranging between 28.8 and 47.3 μM targeting NS5B polymerase [Numerous hibitors –33, inclhibitors . Talele hibitors . A funnehibitors . Musmuca 73.3 μM . Recentllymerase . Computa50 value of 2.01–23.84 μM, and displayed certain inhibitory activities against HCV and no (or weak) cellular cytotoxicity. These compounds can be further optimized and developed into potent and highly active NS5B polymerase inhibitors.In the present study, we discovered a series of novel small molecule NS5B polymerase inhibitor leads using a virtual screening workflow that includes random forest (RB-VS), e-pharmacophore (PB-VS), and molecular docking (DB-VS) methods. The virtual screening workflow is depicted in 50 ≤ 400 nM) and 450 negatives  [50 ≤ 400 nM) and 383 negatives (IC50 ≥ 600 nM) [50 ≤ 400 nM) and 67 negatives (IC50 ≥ 600 nM). The remaining 116 positive (IC50 ≤ 400 nM) compounds , together with the 1000 decoys (the “dl-400” data set) obtained from Schrödinger [50 ≤ 400 nM) with 2190 decoys molecules  , were se 600 nM) –49. The  600 nM) . The tesrödinger , were usoyFinder  for eachData set and chemical database preparation was performed by the LigPrep  with EpirandomForest” package in R [R software were designed for the other parameters.The initial descriptors used in this study were calculated with Dragon 6.0 . The “raage in R  was empl50 or Kd values from 2.4 nM to 140 nM for all three regions of NS5B polymerase. The co-crystal ligand structures and the resolution and affinity values are listed in To improve the potency of identifying active and novel hits from the in silico screening, we used a total of six co-crystal structures of HCV NS5B polymerase  ,60–64 boGlide energy grids were set up for all six prepared protein structures using the Receptor Grid Generation panel in Maestro. The crystal ligands were redocked using the “Refine” option in Glide , and theThe ability to reproduce known inhibitors of the e-pharmacophore hypotheses was evaluated by the three test sets, respectively. Enrichment Factor (EF) was employed for describing the number of known inhibitors recovered when the database is screened. In this study, we focused primarily on EF(1%), which refers to the enrichment in the top 1% of the decoys . The BolThe docking algorithm Glide was used to perform all the molecular docking studies . The sixWe constructed a virtual screening approach by combining the RF-based virtual screening (RB-VS), the e-pharmacophore-based virtual screening (PB-VS) and the docking-based virtual screening (DB-VS) methods. In this investigation, we applied the three virtual screening methods in increasing order of complexity. In the RB-VS stage, a chemical library, including 441,574 compounds from the InterBioScreen database, was screened. The compounds that passed through the RB-VS filter then were processed by a second filtering of PB-VS. In the PB-VS stage, screening molecules were required to match each site in the hypothesis. The distance matching tolerance was designated to 2.0 Å. A fitness score was used to rank the database hits based on their RMSD with the hypothesis involving site matching, vector alignments and volume terms [7 cells/ml. After electroporation, the Huh7.5.1 cells containing virus transcripts were seeded in a 10-cm dish. After the cells were cultured for 4 days, the supernatant was collected and filtered to obtain the stock solution of the hRluc-JFH-1 virus. To obtain the virus titer, the virus stocks were diluted at a gradient of 1:10, and the Huh7.5.1 cells were incubated for 48h at 37°C. Then, the cells were harvested, and the luminescence was detected via the manufacturer’s protocol of the Renilla-Glo™ Luciferase Assay System (Promega).The HCV virus assay was constructed by using the method developed as previously described, albeit with a slight modification –77. Brie4 cells per well at 37°C overnight. All of the synthetic compounds were diluted with DMSO to 10 mM of stock solution. The initial concentration of compounds was 20 μM and then diluted at a gradient of 1:4 to concentrations ranging from 20 μM to 0.02 μM, containing 0.5% DMSO. For the HCVcc system, serial diluted compounds were mixed with a certain titer of HCVcc-hRluc-JFH1 virus, and the final concentration of HCVcc-hRluc-JFH1 virus titer was diluted to the numbers of relative luminescence units (RLU) ranging from 20,000 to 50,000 RLU and then added to the Huh7.5.1 cells. Then, the cells were cultured for 2 days at 37°Cand harvested. The luminescence was detected via the manufacturer’s protocol of the Renilla-Glo™ Luciferase Assay System. EC50 is the concentration of the compound at which the HCV luminescence level in the Huh7.5.1 cells is reduced by 50%. The values of EC50 were plotted by the GraphPad Prism 5 software.Huh7.5.1 cells were seeded in 96-well plates at a density of 2 ×104 cells per well overnight. Cells were incubated with serial diluted compounds for 48 h. The viability of Huh7.5.1 cells was determined in 96-well tissue culture plates using the cell proliferation reagent WST-1 (Roche), and absorbance (OD450 / reference OD630) was measured to detect the cytotoxicity of compounds according to the manufacturer’s protocol for the Cell Proliferation Reagent WST-1. CC50 is the concentration of the compound at which the cell growth was inhibited by 50%. The values of CC50 were plotted by the GraphPad Prism 5 software.Huh7.5.1 cells were seeded in 96-well plates at a density of 2 × 10The SPR experiments were performed using a Biacore T200 optical biosensor . The NS5B protein at 50 μg/ml prepared in buffer of 10 mM sodium acetate (pH 5.5) was immobilized by standard amine coupling to a CM5 biosensor chip. The compounds were injected for 60 s at a flow rate of 30 μL/min using in concentration series between 1 and 250 μM in running buffer (1 × PBS with 5% DMSO). The dissociation was monitored for 300 s. Raw data collected on an SPR biosensor were further processed to eliminate any artifacts such as nonspecific binding and discrepancies in buffer composition. All data processing and analysis was performed using the Biacore T200 Evaluation Software.2, 100 mM ammonium acetate, 1 mM DTT, 500 μM GTP, 250 μM each of CTP, ATP and UTP, 40U of RNasin , 2 μg/ml HCV (-) 3’ TRNA template and 300 ng purified NS5B protein. After 2 h at 30°C, the reaction was stopped by 100 mM EDTA and RNA was purified using the RNaid Kit (Qbiogene). To determine NS5B-catalyzed RNA synthesis, real-time RT-PCR was performed. cDNA was synthesized using AMV-reverse transcriptase  and the primer targeted to NS5B-catalyzed RNA product (5’-caagcaccctatcaggcagt-3’) and then digested with RNAase. PCR was carried out using RNA product–specific primers  and TaqMan RT-PCR Kit according to the instruction. 50% inhibitory concentrations (IC50) were calculated using Statistical SPSS program version 11.5 by probit analysis of regression.The inhibitory effects of the tested reagents on in vitro NS5B activity were determined by NS5B-catalyzed RNA synthesis assay as described previously . PurifieThe training set comprises 772 compounds, including 389 known NS5B polymerase inhibitors and 383 putative noninhibitors. Initially, 4882 molecular descriptors were generated with Dragon 6.0. The initial descriptors were pretreated to discard the “bad” descriptors: (1) descriptors with too many zero values were removed; (2) descriptors with very small standard deviation values (< 0.5%) were removed; and (3) descriptors that are highly correlated with others (correlation coefficients > 95%) were removed. A totalIn the first stage, a full RF model (Model I) was built using all 577 available descriptors. To drop unimportant variables from an RF, the Mean Decrease in Accuracy Decrement importance measure was used. By dropping the less important descriptors (Mean Decrease in Accuracy < 5), a total of 43 descriptors were obtained in the second stage. The 43 descriptors were used to build a second RF model (Model II). In the third stage, the dropping process was repeated until only a few descriptors (with a Mean Decrease in Accuracy > 6) remained. At this point, 16 descriptors were finally chosen to build the third RF model (Model III). The 16 descriptors can be roughly divided into several groups: Walk and path counts (1); Topological indices (1); RDF descriptors (1); GETAWAY descriptors (3); Edge adjacency indices (6); CATS 2D (2); Atom-type E-state indices (1) and 2D autocorrelations (1) . To evaluate and compare the different RF models, the sensitivity (SE), specificity (SP) and overall accuracy (Q) were used as the performance criterions. To evaluate the effect of activity cutoffs on random forest accuracy and performance, we constructed three datasets: (i) Set-150: positives ≤ 150 nM, negatives > 150 nM, (ii) Set-400: positives ≤ 400 nM, negatives ≥ 600 nM, (iii) Set-950: positives ≤ 950 nM, negatives > 950 nM . The resOverfitting is always a weak point limiting the application of machine learning approach . In ordeOf the six NS5B polymerase crystal structures, inhibitors of 3HHK and 3SKA bound to the palm I region, inhibitors of 2BRK and 4DRU bound to the thumb I region, and inhibitors of 2GIR and 3PHE bound to the thumb II region. The six crystal ligands were redocked in the active sites of NS5B to generate e-pharmacophore, respectively. All the redocked structures showed low RMSD values between the docked conformations of ligands and their crystal structures’ binding poses. All the RMSD values were less than 1.0 Å except 3SKA see . Fig 2 sThe e-pharmacophore method incorporating the aspects of structure-based and ligand-based techniques was used to explore the six crystal structures of the NS5B polymerase. This technique not only helps us eliminate the pharmacophore sites that lack significant interactions but also prioritize the sites during virtual screening. Glide XP energetic terms were mapped onto pharmacophore sites to generate pharmacophore hypotheses. These pharmacophore sites were calculated based on the energy and structural information of protein complex. To explore the performance of e-pharmacophore hypotheses, three test sets were employed to evaluate whether the e-pharmacophore models have the ability to differentiate between NS5B polymerase inhibitors and noninhibitors. The test set for the palm I region includes 63 known inhibitors and 1000 decoys, the test set for the thumb I region includes 36 known inhibitors and 1000 decoys, and the test set for the thumb II region includes 17 known inhibitors and 1000 decoys. The enrichment results of all six e-pharmacophore hypotheses were compared for the enrichment factor (EF1%) and BEDROC (α = 160.9 and α = 20.0), based on the recovery rate of inhibitors against the respective ranked test set see . For theThe EF1% and BEDROC values of the six pharmacophore models indicate that the pharmacophore models generated from the six co-crystal structures of the NS5B polymerase could effectively identify the inhibitors in the entire ranked decoy database. Moreover, pharmacophore models developed from the same protein target based on different regions and different ligands are important to identify diverse hits from the database screening. Therefore, the six pharmacophore models were all subjected to the following virtual screening.To determine the docking protocol, the six co-crystal ligands that were retrieved from the palm I (3HHK and 3SKA), thumb I (2BRK and 4DRU) and thumb II (2GIR and 3PHE) regions were docked to their corresponding active sites of the NS5B polymerase. In 2014, Barreca et al  pointed After the validation of the RF model, the e-pharmacophore models and the docking protocols, we constructed a virtual screening process combining the RF-based virtual screening (RB-VS), the e-pharmacophore-based virtual screening (PB-VS) and the docking-based virtual screening (DB-VS) methods. In order to evaluate the performance of the multistage VS approach, we created a validation set that comprises 73 known HCV NS5B polymerase inhibitors and 2190 decoys from PubChem database to assess different VS methods (see The number of hits from each of the 6 e-pharmacophore models and Glide docking (SP and XP) are presented in The final hits found by the virtual screening process combining RB-VS, PB-VS and DB-VS were further inspected for synthesis availabilities (easiness) and expense limitations, and 5 compounds were finally selected . The cytotoxicity testing showed that all compounds displayed no cellular cytotoxicity (CC50 > 100 μM) except the compound N2, which displayed weak cytotoxicity with a CC50 value of 51.3 μM. To further evaluate if the inhibition observed by compound N2 was due to cellular toxicity, we tested the inhibitory activity against HCV of the compound N2 at a concentration of 12.5 μM. The HCV virus titers were suppressed by almost 99%  [To determine the inhibitory activities of the 5 hit compounds, we prepared an HCV cell culture system (HCVcc-hRluc-JFH1) with an HCV genotype 2a JFH-1 virus containing a humanized Rellina luciferase reporter gene   and compCanales)  were theD) for the binding to NS5B were determined for all compounds except N5. N5 might interact with the NS5B, but solubility issues possibly prevented a proper determination of the binding affinity. The D = 4.67–123.1 μM). The ligand-lipophilic efficiency (LLE) calculated from KD and IC50 values has become a popular measure to estimate druglikeness in drug discovery [D = 4.67 μM) and the lowest LLE value of 0.10. Hence, N2 displayed a much worse potential druglikeness and higher logP value than others. 50 and KD values, which might cause by the fact that only partially occupied NS5B polymerases could achieve maximal anti-HCV activity [In order to explore the binding affinity of hits for HCV NS5B polymerase, SPR was used to evaluate the interaction between the hits and NS5B polymerase. The dissociation constants  are all less than 0.3, which indicated that the chemical structures of Ni are diversified from the Ki compounds.The structures of the 5 hit compounds (N1 –N5) are shown in D value of 123.1 μM. Compound N5 binds to the palm region of NS5B polymerase. From The possible binding modes of N1 –N5 in their respective active sites of the NS5B polymerase are shown in 50 values ranging from 1.61 to 21.28 μM. The cytotoxicity testing showed that no compounds displayed cellular cytotoxicity (CC50 > 100 μM) except compound N2, which displayed weak cytotoxicity with a CC50 value of 51.3 μM. The hit compound N2 has a best antiviral activity against the HCV virus, with a selective index of 32.1. All 5 compounds showed inhibitory potency against NS5B polymerase with IC50 value of 2.01–23.84 μM. These compounds belong to novel and diverse chemotypes and could be further optimized and developed to be potent and highly active NS5B polymerase inhibitors.A virtual screening process including RB-VS, PB-VS and DB-VS was applied to identify the novel NS5B polymerase inhibitors. In this investigation, we applied the three virtual screening methods according to the criterion from simpleness to complexity. RB-VS, chiefly characterized by its rapid and simple computations, was used as the first filter. PB-VS and DB-VS were applied to screen a small subset of compound database after RB-VS because these two methods were time-consuming. The models used for the RB-VS and PB-VS were first established and validated. The RF Model III with 16 descriptors was used in the RB-VS stage. Six e-pharmacophore models from different crystal structures of the NS5B polymerase with ligands binding at the palm I, thumb I and thumb II regions were used in the PB-VS stage. The Glide SP and XP docking protocols with default parameters were used in the DB-VS stage. This multistage approach was then applied to screen a large chemical library including 441,574 compounds from the InterBioscreen database. From the final hits, we selected 5 compounds for further anti-HCV activity and cellular cytotoxicity assay, and all 5 compounds displayed certain inhibition against HCV with ECS1 Fig(TIF)Click here for additional data file.S2 FigThe plot indicates that the OOB error rate tracks the test error rate fairly well once the number of trees is sufficiently large. The plot also illustrates the lack of overfitting once the training error reaches zero.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 File(ZIP)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S6 Table(DOC)Click here for additional data file.S7 Table(DOC)Click here for additional data file.S8 Table(DOC)Click here for additional data file.S9 Table(DOC)Click here for additional data file.S10 Table(DOC)Click here for additional data file.S11 Table(DOC)Click here for additional data file.S12 Table(DOC)Click here for additional data file.S13 Table(DOC)Click here for additional data file.S14 Table(DOC)Click here for additional data file.S15 Table(DOC)Click here for additional data file.S16 Table(DOC)Click here for additional data file.


The T7 residue of CDC26 is critical for its interaction with APC6, a tetratricopeptide repeat-containing subunit of APC/C, and mutation of this residue to Asp (T7D) reduced the interaction of CDC26 with APC6. Replacement of endogenous CDC26 in HeLa cells with exogenous phosphor-mimic T7D-mutated CDC26 increased the elution size of APC/C subunits in a gel filtration assay, implying a change in the APC/C assembly upon phosphorylation of CDC26. Furthermore, T7D-mutated CDC26 promoted the ubiquitination of polo-like kinase 1, a well-known substrate of APC/C. Overall, these results suggest that LATS1/2 are novel kinases involved in APC/C phosphorylation and indicate a direct regulatory link between LATS1/2 and APC/C.In budding yeast, the Mitotic Exit Network (MEN) regulates anaphase promoting complex/cyclosome (APC/C) via the Dbf2-Cdc14 signaling cascade. Dbf2 kinase phosphorylates and activates Cdc14 phosphatase, which removes the inhibitory phosphorylation of the APC/C cofactor Cdh1. Although each component of the MEN was highly conserved during evolution, there is presently no evidence supporting direct phosphorylation of CDC14 by large tumor suppressor kinase 1 (LATS1), the human counterpart of Dbf2; hence, it is unclear how LATS1 regulates APC/C. Here, we demonstrate that LATS1 phosphorylates the Thr7 (T7) residue of the APC/C component CDC26 directly. Nocodazole-induced phosphorylation of T7 was reduced by knockdown of LATS1 and LATS2 in HeLa cells, indicating that both of these kinases contribute to the phosphorylation of CDC26  Cdh1 and the subsequent degradation of mitotic cyclin . The cell lysates were centrifuged at 14,000 g for 20 min at 4°C and the supernatants were incubated for 1 h at 4°C with specific antibodies immobilized on magnetic Dynabeads (Invitrogen), or GST fusion proteins immobilized on glutathione-Sepharose beads . The beads were then separated using a magnetic field or centrifugation and washed with lysis buffer. The bound proteins were eluted by boiling for 5 min in sodium dodecyl sulfate (SDS) sample buffer and then subjected to immunoblotting. For protein dephosphorylation, the cell lysates were treated with 800 units of λPPase (New England BioLabs Inc.) and incubated at 30°C for 30 min. The reaction was terminated by adding SDS sample buffer and boiling for 5 min. For immunoblotting, proteins were separated via SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred electrophoretically to a polyvinylidene difluoride membrane (Millipore). The membranes were blocked with 5% non-fat dried milk in PBS for 30 min at room temperature and then exposed to the primary antibodies for 24 h at 4°C. The following mouse monoclonal antibodies were used: anti-c-Myc (9E10) , anti-FLAG M2 , anti-Cdc27(AF3.1) , anti-β-actin (AC-15) , anti-cyclin A , anti-cyclin B1 (GNS1) , anti-GST , anti-P55 Cdc (H-7) , and anti-Cdh1 (DH01) . The rabbit monoclonal anti-APC6 (D8D8)  antibody was used. The following rabbit polyclonal antibodies were used: anti-APC4 , anti-PLK1 , anti-LATS1 , anti-LATS2 , anti-UbcH10 , anti-Cdc26/Apc12 , anti-Phosho-LATS1  , anti-Claspin , and anti-PTTG-1 . The rat monoclonal anti-HA  and the goat polyclonal anti-APC4 (C-18)  antibodies were used. After incubation with the primary antibodies, the membranes were washed three times with PBS containing 0.1% Tween 20 and then incubated with HRP-conjugated secondary antibodies  for 40 min. After three washes with PBS containing 0.1% Tween 20, the proteins were detected using ECL Plus Western Blotting Detection Reagent  and a chemiluminescence detector . Rabbit antiserum against Mad2 was generated as described previously  ATP , and 1 mM DTT] were incubated at 30°C for 30 min. The reactions were terminated by the addition of 4× SDS sample buffer and then boiled at 95°C for 5 min. The samples were separated by SDS-PAGE (5–20% gradient gels), visualized by Coomassie Blue staining, and subjected to autoradiography. The intensities of the bands were quantified using a BAS-5000 phosphoimager (Fujifilm). For cold in vitro kinase assays, the 20 μl reactions  were incubated at 30°C for 30 min, terminated by the addition of 4× SDS sample buffer, and then boiled at 95°C for 5 min. The samples were separated by SDS-PAGE (5–20% gradient gels) and subjected to immunoblot analyses.GST-LATS1 (amino acids 589–1130) and GST-LATS2 (amino acids 553–1088) were obtained from Carna Biosciences Inc. The 20 μl kinase reactions [20 mM Tris (pH 7.4), 5 mM MgClTwenty-four hours after transfection with plasmids encoding Myc-tagged human ubiquitin or FLAG-tagged PLK1, HEK293T and HeLa cells were incubated with 10 μM MG132 for 12 h before harvest. The cells were then collected, immunoprecipitated using an anti-FLAG antibody, and immunoblotted using an anti-Myc antibody to detect polyubiquitination.The formation of APC/C was examined via gel filtration chromatography using the SMART system and a Superose 6 PC 3.2/30 column . HeLa cells expressing FLAG-tagged wild-type, T7A-mutated, or T7D-mutated Cdc26 were treated with a siRNA against endogenous CDC26 and seeded into 10 cm dishes. The cells were collected, washed with PBS, and lysed in 150 μl of lysis buffer. The lysates were then centrifuged at 15,000 g for 15 min at 4°C, and the supernatants were filtered with a 0.22 μm filter unit (Millipore). The samples were loaded onto a Superose 6 PC 3.2/30 column that had been equilibrated with 0.5% NP-40 lysis buffer. The column was run at a flow rate of 40 μl/min in buffer comprising 150 mM NaCl, 50 mM HEPES-KOH (pH 7.5), and 0.1 mM DTT. Aliquots of the fractions (100 μl) were analyzed by immunoblotting. Protein sizes were estimated using molecular mass size markers .HeLa cells stably expressing GFP-tagged histone H2B were seeded onto a glass-bottomed dish (Iwaki). Time-lapse fluorescence microscopy and differential interference contrast video microscopy were performed using an Olympus LCV110 microscope. Images were acquired using a charge-coupled device camera  equipped with a U Plan Super Apochromatic ×40, 0.95 numerical aperture objective, and then analyzed using MetaMorph software (Molecular Devices).+) were added to the most electronegative areas of the protein. The entire protein was subsequently solvated in a box of TIP3P water molecules [2, respectively. Each system was then heated gradually from 0 to 300 K over a period of 50 ps and maintained at 300 K with a coupling coefficient of 1.0/ps and a force constant of 1.0 kcal/mol-Å2 on the complex. Each system was equilibrated to a free simulation for 1000 ps. Finally, a production run was performed for 300 ns using the NPT ensemble at 300 K with 1.0 atm pressure. During the MD simulation process, coordinates were collected every 1 ps. For each system, free energy calculations were performed for 4000 snapshots extracted from the last 40 ns of the stable MD trajectory using the MM-GSA approach. For each snapshot, the free energy was calculated for each molecular species . The MMGBSA method can be conceptually summarized as follows:The initial coordinates of the CDC6-APC6 complex  were obtolecules  with a molecules , allowinolecules . For equGbind was determined as follows:The ΔGbind) is evaluated a sum of the changes in the molecular mechanical gas-phase binding energy (ΔGMM), the solvation free energy (ΔGsolv), and entropic (-TΔS) contribution:The binding free energy (ΔMM) comprised the electrostatic (Gele) and van der Waals (Gvdw) interactions:The molecular mechanics energy (Gsolv) comprised polar (Gpol) and nonpolar (Gnonpol) contributions:The solvation free energy  and b (a constant) were set to 0.0072 kcal/mol-Å2 and 0, respectively, and SASA was the solvent accessible surface area (Å2) determined using the linear combination of the pairwise overlaps model:The ΔTΔS) was estimated for 50 snapshots taken from the last 40 ns of stable simulation using the mmpbsa_py_nabnmode module implemented in Amber Tools 14. The comparative structural analysis was performed by using a representative snapshot from the clustering of the trajectories of the last 20 ns of stable MD simulation.The conformational entropic contribution Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 FigGST alone, GST-tagged recombinant wild-type (WT) or T7A-mutated CDC26 were incubated with active GST-LATS1 (A) or GAT-LATS2 (B) and cold ATP, and then subjected to immunoblot analyses with the indicated antibodies.(TIF)Click here for additional data file.S2 FigHeLa cells were transfected with a control or CDC26-specific siRNA, treated with nocodazole for 16 h to activate endogenous LATS1, and then subjected to immunoblot analyses using the indicated antibodies.(TIF)Click here for additional data file.S3 FigHeLa cells were transfected with a control or LATS2 expression vector, treated with nocodazole, and then subjected to immunoblot analyses using the indicated antibodies.(TIF)Click here for additional data file.S4 Figt test).(A) The effects of siRNA-mediated knockdown of CDC26 on the proliferation of HeLa cells. The cells were transfected with a control or two different CDC26-specific siRNAs, and cell viability assays were performed on days 0–3 after transfection. (B) The effects of knockdown of CDC26 on cell cycle progression. HeLa cells stably expressing GFP-tagged histone H2B were transfected with a control or CDC26-specific siRNA and synchronized at the G1/S phase by double thymidine block. The time at which nuclear envelope breakdown occurred was set as 0 min and the percentages of cells entering anaphase were counted in three different fields of time-lapse microscopy images. Data are represented as the mean ± standard deviation of these counts. (C) HeLa cells were transfected with a control or LATS1/2 siRNA oligos and cell viability was assessed as in (A). (D) siRNA-mediated knockdown of LATS1 caused the mitotic exit delay. HeLa cells were transfected with a control or two different LATS1-specific siRNAs and treated with nocodazole to arrest at prometaphase. After washing out the nocodazole, percentage of mitotic cells was evaluated by phosphor-Histone H3 and propidium iodide staining. Percentage of cells at mitosis  was counted at the indicated time after release (right). Click here for additional data file.


S100A8/A9 in patients with HNSCC is associated with increased DNA methylation, less differentiated tumors, and reduced overall survival. Upon ectopic over-expression of S100A8/A9, the cancer phenotype of S100A8/A9-negative carcinoma cells was suppressed in vitro and tumor growth in vivo was significantly decreased. MMP1, INHBA, FST, LAMC2, CCL3, SULF1, and SLC16A1 were significantly upregulated in HNSCC but were downregulated by S100A8/A9 expression. Our findings strongly suggest that downregulation of S100A8/A9 through epigenetic mechanisms may contribute to increased proliferation, malignant transformation, and disease progression in HNSCC.Calprotectin (S100A8/A9), a heterodimeric protein complex of calcium-binding proteins S100A8 and S100A9, plays key roles in cell cycle regulation and inflammation, with potential functions in squamous cell differentiation. While upregulated in many cancers, S100A8/A9 is downregulated in squamous cell carcinomas of the cervix, esophagus, and the head and neck (HNSCC). We previously reported that ectopic S100A8/A9 expression inhibits cell cycle progression in carcinoma cells. Here, we show that declining expression of  Malignant transformation in squamous cell carcinomas (SCC) remains a perplexing process that results in abnormal cellular differentiation, invasivity, and growth regulation, all hallmarks of cancer. In carcinoma cells, calprotectin , a heterodimeric complex of calcium-binding proteins, S100A8 and S100A9, appears to suppress cell cycle progression, growth, and migratory invasion by regulating cell differentiation and adhesion to extracellular matrix (ECM) . EncodedIn different cancers, S100A8/A9 expression varies with cell and tissue of origin, but it is widely reported to be downregulated in cells from head and neck squamous cell carcinoma (HNSCC) -12. The In HNSCC, poor prognosis is highly associated with reduced S100A8/A9 protein levels , 19; loshttp://cancergenome.nih.gov) and other public datasets to decipher molecular and cellular functions associated with expression of S100A8/A9. The interrogation of the TCGA was based on a preliminary genome-wide differential expression profiles from transcriptomic analysis of microarrays developed from healthy human mucosal epithelium and primary HNSCC tissue samples obtained by laser capture microdissection (LCM). Genes regulated by HNSCC were subjected to correlation and pathway analyses to identify, for example, gene networks involved in growth and differentiation in association with S100A8/A9. In separate experiments, genes highly upregulated in HNSCC were further interrogated for their regulation by S100A8/A9 by ectopic overexpression in S100A8/A9-negative carcinoma cells and by knockdown of endogenous S100A8/A9 expression in HNSCC cells using short hairpin RNA (shRNA) interference. To validate that S100A8/A9 regulates gene networks affecting tumorigenesis, we determined whether loss of expression is associated with the development of HNSCC in vivo.To understand S100A8/A9 in the larger context of gene networks, we analyzed the RNA sequencing (RNA-Seq) data from The Cancer Genome Atlas (TCGA) project  with S100A8 and S100A9  with S100A8 and S100A9 expression as indicated by the vertical gray bars , but was not correlated with tumor size (T stage)  (data not shown). Downregulation of S100A8 and S100A9 genes in HNSCC did not correlate with regional lymph node involvement (N stage) . Of these, 425 upregulated and 584 downregulated genes were concordant with the gene sets identified previously with our older microarray data Figure . The lars Figure . The gens Figure . Express) Figure . Express) Figure  and othe) Figure  or dista) Figure , suggestS100A8 and/or S100A9 expression contributes to HNSCC tumorigenesis, we enumerated the number of HNSCC samples (TCGA data) in which S100A8, S100A9, or both were downregulated relative to the mean expression in normal adjacent samples (S100A8 and S100A9 were both reduced in 471 (90.4%) out of 521 HNSCC samples, whereas S100A8 or S100A9 alone was only reduced in 11 (2.1%) and 8 (1.5%) samples, respectively. Based on TCGA RNA-Seq data, patients were then segregated by low (below average) or high (above average) expression of S100A8/A9. The median survival time for the low S100A8 group (n = 275) was 35.5 months and for the high S100A8 group (n = 132) was 64.8 months (n = 0.03) based upon Kaplan-Meier survival plots and log-rank (Mantel-Cox) tests (n = 136) tended towards greater survival than the low S100A9 group , but the difference was not statistically significant p = 0.12) (To show whether reduction of  samples . In TCGAx) tests . The hig = 0.12) .S100A8 and S100A9 expression, TCGA RNA-Seq data was analyzed for DNA methylation and copy number alterations (or CNAs from Genomic Identification of Significant Targets in Cancer (GISTIC)). DNA methylation and S100A8 expression did not correlate; one sample (sample ID# TCGA-CQ-5331) out of 528 cases analyzed showed a K49R missense mutation   of both genes resulted in significant reductions (p ≈ 0.02) in both S100A8 and S100A9 mRNA expression ; overall mRNA expression tended to decrease in association with copy number amplification (gain in multiple copy numbers). When the methylated regions in the proximal promoter and gene body of S100A8 and S100A9 were compared, normal adjacent tissues showed greater overall DNA methylation than in HNSCC samples  but weakly correlated to the levels of S100A8 mRNA expression  Figure , but no n Figure . Interes samples . Promotepression . PromoteS100A8/A9 based on IPA gene network analysis , inhibin beta A (INHBA), follistatin (FST), laminin gamma 2 (LAMC2), chemokine (C-C motif) ligand 3 (CCL3), prostaglandin-endoperoxide synthase 2 (PTGS2), sulfatase 1 (SULF1), and solute carrier family 16 (or monocarboxylate transporter) member 1 (SLC16A1) were not detectable in any of the normal mucosal samples  but were highly expressed  and significantly upregulated in HNSCC Figure . These gs Figure .S100A8/A9 expression contributes to HNSCC, we determined whether S100A8/A9 caused gain or loss of specific predicted functions using the S100A8/A9-negative carcinoma cell line, KB. In this line, we overexpressed S100A8/A9 ectopically [To show that loss of opically   with 107 (of 584) downregulated genes in HNSCC, which were enriched in functions including cellular development and differentiation, cell-to-cell signaling and interaction, and cell morphology as discussed above. Some genes showed known or predicted intergenic signaling relationships with S100A8 and S100A9; these signaling relationships were associated with predicted functions in immunological cell development  than well or moderately differentiated tumors, which were indistinguishable from normal samples based on analysis of microarray data GSE6791 from the GEO database  showed significantly lower migration (p < 0.05) than KB-EGFP cells  , 29. S10S100A8/A9 [S100A8/A9 is likely controlled in HNSCC and how the cancer phenotype is affected.Depending upon cell lineages and tissue types, malignancies differentially regulate and express S100A8/A9. As we and others show, squamous epithelial cells lining the mucosa of the head and neck , esophagus, and cervix constitutively express 100A8/A9 , 33-35. S100A8 and S100A9 mRNA expression profiles. Since the genes encoding S100A8 and S100A9 are located within chromosomal locus 1q21, which contains a cluster of (at least 43) genes known as the epidermal differentiation complex (EDC) [S100A8 and S100A9 was significantly downregulated in HNSCC. Reduction in levels of S100A8- and S100A9-specific mRNAs correlates with the decreases in S100A8/A9 protein levels reported in clinical specimens of HNSCC [www.proteinatlas.org) and in independent studies using HNSCC tumor sections and tissue microarray IHC [Oncomine.org. We do show that S100A12 is weakly correlated to S100A8/S100A9 when compared to other gene transcripts. Since S100A12 did not appear to be broadly associated with HNSCC pathogenesis (TCGA data), we did not further analyze S100A12 although a complimentary role cannot be excluded.To determine whether intracellular S100A8/A9 might regulate cell cycle progression and growth in primary human HNSCCs, we performed correlation analysis of the ex (EDC) , we predex (EDC) . Using Tof HNSCC , 8, 33. rray IHC . In oroprray IHC . WhereasS100A8 and S100A9 dysregulation was similar across all tumor grades (T) and was independent of nodal involvement (N) or distant metastasis (M). Although we did not compare S100A8/A9 expression in subjects based on human papillomavirus (HPV) or smoking status, we recognize that HPV infection has been increasingly associated with tumorigenesis in HNSCC [S100A8 and S100A9 tended to decrease more in HPV-positive than in HPV-negative HNSCC but was not statistically different. In HNSCC, HPV infection can affect gene expression, signal transduction pathways, and tumorigenesis through epigenetic modifications [S100A8 and S100A9 expression is likely an early event in the pathogenesis of HNSCC and is independent of HPV status and copy number gains. As disease progresses, S100A8/A9 levels appear unchanged. Whereas perhaps not contributing to reduced S100A8 and S100A9 expression in HNSCC relative to the normal adjacent tissues, promoter methylation is inversely correlated to the expression levels of S100A8 and S100A9 and appears associated with poor survival of HNSCC patients. Further studies will be needed to confirm this association.Interestingly, in HNSCC , 40, a rications , 42. SinTOP2A, FN1, CENPF, and E2F7 were inversely correlated with S100A8 and S100A9. Whether these genes function upstream or downstream of S100A8/A9 is being addressed in a separate study.The proliferative nature of HNSCC was reflected in the disproportionate upregulation of positive regulators of the cell cycle. Indeed, S100A8 and S100A9, are essential to maintain normal epithelial phenotype; these genes are downregulated in HNSCC as we and others report [S100A8 and S100A9 expression and are enriched in functions associated with cellular development and differentiation, cell-to-cell signaling and interaction, cell morphology, and cellular assembly. S100A8 and S100A9 (and the S100A8/A9 protein complex) appear to be co-regulated and strongly suggested to function in these pathways. S100A8 and S100A9 expression levels show strong concordance across all HNSCC samples. Approximately 90% of the HNSCC samples analyzed show combined loss of S100A8 and S100A9. Only about 2% of the HNSCC samples show single loss of either S100A8 or S100A9 relative to the mean expression in normal samples. In TGCA, the data are insufficient to report HNSCC survival and patient tumor characteristics when only S100A8 or S100A9 was downregulated. Based on our current findings, the combined loss of S100A8 and S100A9, and the resultant reduction in S100A8/A9 protein complex, is strongly suggested to be a key factor contributing to the pathogenesis of HNSCC. Reduced S100A8/A9 expression appears to promote the HNSCC phenotype, since loss of differentiation and cell-to-cell contact growth inhibition can dysregulate cell cycle progression and increase carcinoma growth.Genes located within the EDC on chromosomal locus 1q21, including s report , 44. Loss report -47. As w−/− [−/− mice also fail to express S100A8. Hence, S100A9 alone within cells may be toxic. When we constructed S100A8/A9-positive transfectants in S100A8/A9-negative (no S100A8/A9 protein complex) human carcinoma cells, transfection of S100A9 without S100A8 was unsuccessful and cells died [in vitro studies of either S100A8 or S100A9 expression alone may not be experimentally feasible. Our observation that TGCA data rarely identified HNSCCs with only S100A8 or S100A9 being regulated is consistent with these findings.Note that S100A8 and S100A9 proteins typically form heterodimeric multimers when both complex partners are present  in a man−/− , 51; emblls died , 52. CelS100A8/A9 and cell differentiation appears to manifest clinically. Poorly and/or undifferentiated HNSCC primary tumors expressed significantly lower S100A8 and S100A9 mRNA levels than the well and/or moderately differentiated tumors and normal samples. In fact, S100A8 and S100A9 expression levels were similar in normal specimens and well and/or moderately differentiated HNSCC. Thus, reduction in S100A8/A9 expression appears to characterize poorly and undifferentiated tumors associated with more aggressive disease, poor clinical prognosis, and treatment failures. Whether suppression of S100A8 and S100A9 expression is a driver or passenger, dysregulation during tumorigenesis cannot be ascertained from these analyses.The relationship between Candidate marker genes for HNSCC were identified. In HNSCC, MMP1, INHBA, FST, LAMC2, CCL3, SULF1, and SLC16A1 were upregulated using three independent modes of analysis: RNA-Seq data from TCGA, qRT-PCR, and microarrays. These genes were highly expressed and upregulated in HNSCC samples in TCGA and our study and expression was undetectable in normal mucosal tissues by microarray. MMP1, INHBA, and LAMC2 were reported previously as upregulated in HNSCC -55. S100Note, however, that MMP1 expression was relatively low in KB cells, with qRT-PCR cycle threshold (CT) values in upper twenties (data not shown). We previously reported that S100A8/A9 suppressed expression and activity of MMP2 in KB cells while MMP1 mRNA and protein were nearly undetectable [MMP1 and INHBA expression appears to be regulated by S100A8/A9. In TR146 HNSCC cells, knockdown of S100A8/A9 increased expression of both MMP1 and INHBA, which are known to contribute to invasion and tumorigenesis, but other marker genes were unaffected. The discrepancy between TR146 cells and TCGA RNA-Seq data may reflect the heterogeneity in tumor behaviors across individuals and cell lines. Knockdown of S100A8/A9 tended to increase LAMC2 expression, which was implicated in invasion and recurrence of HNSCC . Since sin vitro, ex vivo, and in silico findings, we expected that the malignant and metastatic potential of carcinoma cells would be attenuated when S100A8/A9 is increased in vivo. Indeed, S100A8/A9-producing KB cells showed reduced tumorigenesis in nude mice in comparison to the parental and control KB-EGFP cells. S100A8/A9 expression was also found to suppress tumorigenesis when S100A8/A9-expressing TR146 cells and TR146-shS100A8/A9KD cells were injected in an orthotopic floor of mouth model in athymic nude mice . Since TR146 and transfected KB-S100A8/A9 cells do not release detectable amounts of Mab27E10-reactive S100A8/A9 protein into the medium (data not shown), extracellular signaling through cell surface receptors such as TLR or RAGE by released S100A8/A9 as previously reported [Lastly, the ability of carcinoma cells to de-differentiate into stem cell-like cells, migrate and invade surrounding tissues, metastasize to distant sites and resist apoptosis reflects malignant and metastatic potential , 58. Whereported -32 is lihttp://cancergenome.nih.gov). Briefly, gene expression raw counts representing total transcripts mapped to the hg19 human reference genome using MapSplice alignment and quantitated by RSEM [www.r-project.org). Normalization and differential expression analysis was performed using edgeR Bioconductor package [n = 521) to normal (n = 43) samples. Fold down-regulation with ratio of means less than 1.0 is presented as −1/(ratio of means). Expression correlation analysis was performed using Spearman's rank method, unless stated otherwise, to account for non-linear relationships between gene products, with ρ (rho) ≤ −0.30 or ≥ 0.30 and p < 0.05 as statistical significance criteria.Level 3 RNA sequencing version 2 (RNA-Seq V2) dataset from 528 HNSCC cases that included 43 normal adjacent tissues (NAT) and 521 HNSCC samples was downloaded from The Cancer Genome Atlas (TCGA) website . A total of 528 cases were analyzed for changes in copy number and DNA methylation. The methylated region in proximity and in the gene body of S100A8 and S100A9 was analyzed using Wanderer [Copy number alternation (CNA) and DNA methylation analysis of TCGA HNSCC data was performed using the web-based cBioPortal , 65 TCGAWanderer  and MEXPWanderer .1 to T4 with N0 to N3, and MB to MC. Fifty-four percent of the HNSCC patients were males and 38% females, with ages ranging from 26 to 90 years old (mean and median = 60). No demographic information was available for three patient specimens and ages were unavailable for three others. The tissue samples were handled under strictly maintained temperatures to prevent degradation of RNA. Immediately after collection, the samples were embedded in TissueTek OCT compound and snap frozen at −80°C. Frozen sections were cut to 5 - 8 μm thicknesses followed by a quick hematoxylin and eosin (H&E) staining protocol to identify and differentiate epithelial cells from stromal or infiltrating immune cells in the tissue sections. Briefly, sections were fixed in 70% ethanol for 30 s and stained with H&E followed by three dehydration steps of 30 s each in 70%, 95%, and 100% ethanol. Laser capture microdissection of epithelial cells was performed using a Leica AS LMD  immediately following the staining.Oral mucosal epithelia from healthy volunteers and primary tumor tissues from HNSCC patients were harvested by laser-capture microdissection (LCM) and total RNA was extracted and amplified to generate mRNA for gene expression analysis. Primary HNSCC and normal oral mucosal tissue specimens were obtained from patients undergoing primary malignant resection surgery using a University of Chicago IRB-approved informed consent protocol. Tissues were obtained from 11 non-HNSCC subjects and 50 HNSCC patients with tumors in various TNM stages, ranging from TTo extract RNA, the microdissected cells were directly placed in lysis buffer provided in the RNA purification kit  and homogenized before storing at −80°C. Total RNA was extracted from the captured cells with the RNeasy Micro Kit and treated with DNase to remove genomic DNA. The quantity and quality of purified RNA were determined immediately after purification with the Agilent 2100 BioAnalyzer . The mRNA was labeled and hybridized to Affymetrix Human Genome U133 Plus 2.0 GeneChips.® Expression Analysis Technical Manual . To generate biotin-labeled complementary RNA (cRNA) from the cDNA, double-stranded cDNA was synthesized from total RNA using a Two-Cycle Target Labeling protocol for HNSCC RNA samples or One-Cycle Target Labeling for KB cell RNA extracts, followed by an in vitro transcription (IVT) reaction. Biotin-labeled cRNA was then fragmented and hybridized to Affymetrix Human Genome U133 Plus 2.0 for HNSCC samples or U133A arrays for KB cell samples according to the manufacturer's protocol. The arrays were scanned and the images processed and analyzed using Affymetrix's GeneChip Operating Software (GCOS).Target preparation was performed according to a standard protocol described in the Eukaryotic Target Preparation section of GeneChiphttp://www.broadinstitute.org/cancer/software/GENE-E), followed by Quantile normalization to ensure that gene expression values were comparable across multiple samples (arrays). In addition, gene probes were further filtered by detection calls. When comparing normal and HNSCC samples, HNSCC-specific putative marker genes were selected only when the gene detection call was “Present” and significantly upregulated in at least 90% of HNSCC samples but were “Absent” in all normal samples. To determine differential expression, ANOVA comparisons were performed in Partek Genomics Suite using a false discovery rate (FDR) < 0.05 as significance threshold and fold-change of 2.0-fold as cutoff. Fold-change was determined as the ratio of means of tumor to normal samples. Downregulation fold-change with ratio of means less than 1.0 is presented as −1/(ratio of means). Pathway analysis was performed using Ingenuity Pathways Analysis (IPA)  to identify putative functions associated with regulated genes. These data are not MIAME-compliant since the original CEL data files are not available. By analyzing this database, we formulated questions, which were then used to interrogate TGCA. Comparisons of these primary tissue microarray gene expression data with TGCA showed great consistency as we report.Data mining and statistical analyses were performed using Expressionist Analyst Pro  and Partek Genomics Suite . In brief, expression signals of all probes were first mapped to known gene symbols with median expression value of multiple probes for each gene using GENE-E  or scrambled shRNA control (TR146-shNeg) were cultured with 250 μg/mL of Geneticin to maintain stable knockdown. Cells were plated at 5,000 cells/cm2 in T75 flasks and grown until the epithelial monolayer reached approximately 70% confluency, or 72 h. Cells were then harvested by trypsinization for mRNA and protein expression analysis.KB-EGFP and KB-S100A8/A9 cells were maintained in modified Eagle medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 700 μg of Geneticin (G418 sulfate) per mL in 5% COS100A8 and S100A9 genes along with enhanced green fluorescent protein (EGFP) and neomycin resistant genes for selection (KB-S100A8/A9 cells) and compared to KB transfection control cells containing only the selection genes (KB-EGFP cells) as reported previously [in vitro HNSCC loss of function studies, S100A8 and S100A9 were knocked down in TR146 cells using short hairpin RNA (shRNA)-induced gene silencing as previously described [The S100A8/A9-negative KB carcinoma cell line was transfected to express eviously , 25. Theescribed .® Green QPCR Master Mix  and normalized to GAPDH as a reference gene.The cells were trypsinized, centrifuged and collected as a pellet, and washed once with Dulbecco's phosphate-buffered saline without calcium and magnesium (PBS). Total RNA was extracted and purified using the RNeasy Mini Kit . The final total RNA was eluted in 50 μL RNase-free water. Total RNA samples were quantitated by spectrophotometry for absorbance at 260 nm and adjusted to equal concentrations. Total RNA samples from HNSCC tumor tissue and normal adjacent tissue (NAT) were pooled from three different HNSCC patients as previously described . ReverseFor microscopic examination, KB, KB-EGFP and KB-S100A8/A9 cells were grown under identical conditions on gelatin-coated glass cover slips. Cross-sectional bright field microscopic imaging was performed. Polymerization of actin was visualized by staining the cells with phalloidin conjugated to Alexaflor 594, followed by fluorescence microscopic imaging.TM 48-Well Cell Adhesion Assay Kit based on the manufacturer's protocol . At approximately 70% confluency (∼ 48 h in culture), cells were synchronized by overnight serum starvation. Serum starved cells were then harvested by trypsinization and resuspended in serum free media at 1 × 106 cells/mL. Wells pre-coated with ECM  or BSA were seeded with 1.5 × 105 of serum-starved cells per well, followed by 90 min incubation at 37°C in 5% CO2. BSA-coated wells served as a negative control. The growth medium from each well were then aspirated and each well was gently washed 4 times with 250 μL PBS. After the last wash, the adherent cells were stained with 200 μL of Cell Staining Solution (as provided in the assay kit) for 10 min at room temperature, washed 4 times with deionized water and air dried for 10 min. Stained cells were enumerated by addition of 200 μL per well of Extraction Solution (as provided in the assay kit) and incubated for 10 min on an orbital shaker at room temperature. Extracted samples (150 μL of each) were transferred to a 96-well microtiter plate and optical density (OD) was measured at 570 nm.Cell adhesion assays were performed on extracellular matrix (ECM) protein coated wells using the CytoSelect1/S blockage with 2 μg/mL aphidicolin in MEM + 10% FBS for another 18 h. 1 × 106 synchronized cells in serum-free medium were then seeded in a transwell chamber and allowed to settle for 30 min before MEM supplemented with 20% FBS was added to the outer well to stimulate migration and incubated at 37°C, 5% CO2 for 13 h. After 13 h of incubation, the media in both the transwell chamber and outer well were aspirated and the chamber and outer well were washed once with PBS. Migrated cells adhering to the bottom surface of the transwell membranes were harvested by trypsinization and counted using a hemocytometer.Migration and invasion of carcinoma cells were tested using a transwell system (Costar 3428 with 8 μm pore size) . Cells growing in log phase  were synchronized by serum starvation overnight, followed by Gn = 5 per cell group per experiment) were inoculated subcutaneously with KB, KB-EGFP or KB-S100A8/A9 cells per hind flank at 1 × 106 cells per injection in PBS. At weekly intervals up to 4 weeks post injection, the largest and smallest diameters of each tumor were measured with a caliper. Tumor volume (V) was then calculated from the equation, V = π/6 × larger diameter × 2. Tumor growth was determined as the change in tumor volume (V) over time. Experiments were performed in accordance to all federal guidelines and procedures were reviewed and approved by the University of Minnesota Institutional Animal Care and Use Committee.To determine whether S100A8/A9 expression affected tumor formation in KB carcinoma cells, BALB/c athymic nude mice (


DPD deficiency is the result of loss-of-function mutations within the dihydropyrimidine dehydrogenase (DPD) gene. The IVS14+1G>A variant is associated with DPD deficiency as a result of a 165-bp deletion in the DPD mRNA. A rare mutation, c.2846A>T, is characterized by a change of the acidic aspartic acid to the aliphatic valine with potential impairment of enzyme activity [In this study, we describe the spectrum of toxicities of fluoropyrimidines in patients carrying the IVS14+1G>A and 2846A>T variants.®) and IVS14+1G>A and 2846T>C DPD variants were screened on a Real-Time Life Sciences® 7900 HT platform. The study was approved by the local Ethics Committee.Data were collected from 550 patients with gastrointestinal, breast, head-neck and pancreatic cancers. They were evaluated for DPD genotype upon development of grade ≥2 non-hematological and ≥3 hematological toxicities (CTCAE v.4) following standard fluoropyrimidine-containing regimens in combination with other cytotoxic agents and/or anti-EGFR and VEGF antibodies. DNA was extracted from blood by the Qiamp DNA Blood Mini Kit , G3/4 mucositis (48%), febrile neutropenia (45%), G3/4 thrombocytopenia (38%), G3/4 anemia (24%), G2/3 hand-foot syndrome (14%), G3 dermatitis (7%) and G2/4 alopecia (7%). The IVS14+1AA patient showed diarrhea G2, mucositis G3, anemia G1, pistrinopenia G3, febrile neutropenia G4, complete alopecia and Staphylococcus aureus sepsis. This patient required 20 days of hospitalization and was managed with antibiotics, platelet transfusion, port removal, G-CSF administration and parenteral nutrition. The patient survived because she was given a reduced 5-FU 250 mg/sqm test dose without folates, while the 2846TT patient deceased after the first cycle of FOLFOX4 treatment because of a diarrhea G3, mucositis G4, febrile neutropenia and piastrinopenia G4.Patients carrying the deleterious IVS14+1G>A and 2846T>C variant alleles display severe toxicities which are fatal in homozygous variant subjects. Although the frequence of DPYD*2A allele is low, the screening for DPD mutation is clinically relevant to avoid the severe toxicities or death in patients treated with fluoropyrimidine-containing regimens. This finding suggests the usefulness of pre-treatment screening of DPD in patients candidates to fluoropyrimidine treatment.


A major determinant in the change of the avian influenza virus host range to humans is the E627K substitution in the PB2 polymerase protein. However, the polymerase activity of avian influenza viruses with a single PB2-E627K mutation is still lower than that of seasonal human influenza viruses, implying that avian viruses require polymerase mutations in addition to PB2-627K for human adaptation. Here, we used a database search of H5N1 clade 2.2.1 virus sequences with the PB2-627K mutation to identify other polymerase adaptation mutations that have been selected in infected patients. Several of the mutations identified acted cooperatively with PB2-627K to increase viral growth in human airway epithelial cells and mouse lungs. These mutations were in multiple domains of the polymerase complex other than the PB2-627 domain, highlighting a complicated avian-to-human adaptation pathway of avian influenza viruses. Thus, H5N1 viruses could rapidly acquire multiple polymerase mutations that function cooperatively with PB2-627K in infected patients for optimal human adaptation. Avian influenza (AI) virus H5N1 subtype strains have been sporadically transmitted to humans with high mortality (>60%), presenting a serious global health threat. In particular, 63% of recent human H5N1 infection cases worldwide have been reported in Egypt, which is now regarded as a hot spot for H5N1 virus evolution. H5N1 clade 2.2.1 viruses are unique to Egypt and probably have the greatest evolutionary potential for adaptation from avian to human hosts. Here, using a comprehensive database approach, we identified various novel polymerase mutations in clade 2.2.1 virus strains, isolated from patients, that enabled enhanced viral replication in both human airway epithelial cells and mouse lungs. Interestingly, the mutations identified acted cooperatively with the PB2-E627K mutation, the most well-known human adaptation mutation, to produce a further increase in viral replication in human hosts. These results provide the first broad-spectrum data on the polymerase characteristics of AI viruses that have been selected in infected patients, and also give new insight into the human adaptation mechanisms of AI viruses. Influenza A viruses replicate and transcribe their genomes in the cell nucleus, catalyzed by a viral polymerase complex composed of PB2, PB1 and PA subunits. Since influenza viruses are segmented single-stranded RNA viruses with negative polarity, each of these genes is encoded in a separate viral RNA (vRNA) segment. The trimeric polymerase complex binds to vRNA and nucleoprotein (NP) to form a ribonucleoprotein (vRNP) for viral replication. The polymerase complex and viral hemagglutinin (HA), the most abundant virion surface antigen, affect the host range of influenza viruses . The avihttp://www.who.int/). H5N1 viruses became endemic years ago in birds in some countries; e.g., China, Vietnam, and Egypt. Continuous circulation has allowed H5N1 viruses to diverge genetically to form phylogenetically and phenotypically distinct clades (designated clades 0 to 9) in different geographic areas. In particular, clade 2.2.1 is unique to Egypt and has striking features. Clade 2.2.1 viruses typically carry mammalian-type PB2-627K [http://www.who.int/). Continuous circulation of clade 2.2.1 viruses has allowed these viruses to diverge genetically to form phylogenetically distinct groups designated clade 2.2.1.1 and clade 2.2.1-B, -C and -D. Among these groups, clade 2.2.1-C has been dominant in Egypt since 2011 and was reclassified as clade 2.2.1.2 [Since its emergence in China in 1996, highly pathogenic AI virus subtype H5N1 has caused 842 confirmed human infections with 53% mortality  database search of the sequences of human and avian H5N1 strains isolated in Egypt during 2006–2011. This yielded 61 human virus PB2, PB1, PA and NP sequences, and 91 PB2, 78 PB1, 78 PA and 81 NP avian virus sequences. At the time of this study (June 2014), the sequences of each gene searched were >70% of the all H5N1 virus sequences for that gene from H5N1 strains isolated in Egypt in the GISAID database: the remaining 30% were partial sequences and, therefore, were eliminated from this study. All sequences were obtained from isolates from clinical swabs or samples that had been passaged no more than three times in eggs and/or cultured cells  for amino acid mutations that presumably were selected during viral replication in H5N1-infected patients by a GISAID  that were either only in human viruses (Category 1) or were more prevalent in human viruses than in avian viruses (Category 2) as candidate human adaptation mutations in clade 2.2.1 viruses . Some muTo investigate the effect(s) of the mutations on viral polymerase activity, we carried out minigenome assays of the polymerases with these mutations in the genetic background of influenza virus A/duck/Egypt/D1Br/2007 (EG/D1) ,30, sincThe minigenome assays showed that several single mutations increased EG/D1 polymerase activity under some conditions. In particular, at 33°C, PB1-P627L and PA-E327K increased polymerase activity 6.3-fold and 2.5-fold, respectively, in vRNA-oriented minigenome assays  and 5.0-P <0.05 by ANOVA with Tukey’s multiple comparison test  of the polymerase mutations on the growth of clade 2.2.1 viruses in human cells using recombinant viruses generated by reverse genetics in the EG/D1 genetic background. For these studies, we selected the 8 inter-segment combinations of mutations with the greatest increase in polymerase activity in minigenome assays  cells, which are anatomically close to the alveoli that are an initial target for H5N1 infection in humans, were infected with one of the mutant viruses or with EG/D1 (wt) at a multiplicity of infection (MOI) of 0.03, incubated at 33 or 37°C, and progeny vRNA production was assayed to 120 h post-infection. The progeny vRNA production of the virus strains in this study are shown in Interestingly, the PB2-D701N human adaptation mutation decreased progeny vRNA production in SAE cells . A similP >0.05 at 37°C and R = 0.11, P >0.05 at 33°C). In contrast, scatter plots (P <0.001) and 33°C . These results suggested that increased viral growth in human airway cells was not only due to an increase in mRNA synthesis (transcription) but also to an increase in both transcription and v/cRNA synthesis (replication) or to a change in the amount of transcription relative to the amount of replication.We next investigated the relationship between polymerase properties and progeny vRNA production of the 29 virus mutant strains able to grow in human airway cells. Scatter plots  showed ter plots  showed tTo clarify whether increased viral growth in human airway cells was due to differences in the efficacy of virus genome replication and/or transcription, we carried out primer extension assays using a polymerase reconstitution system. In this study, 293T cells were co-transfected with plasmids expressing EG/D1 PB2, PB1, PA or NP, each carrying the indicated mutations that had a significant effect on progeny vRNA production in human airway cells, including PB2-E627K, with the PB2-K627E back mutation, and a plasmid expressing cRNA as a template for replication (cRNA to vRNA synthesis). We examined the amounts of vRNA and cRNA. As shown in in vitro results of this study to in vivo infections, BALB/c mice were inoculated intranasally with serial dilutions of 4 virus mutants, selected because they had significant increases of both polymerase activity  of the mutants showed severe weight loss with little or no recovery, and lower survival and earlier death compared to mice infected with EG/D1 (wt) (5 FFU of the mutants or EG/D1 (wt), and lungs were collected for virus assays at 3 and 6 d post-infection. The viral yield in lungs of mice infected with all of the mutants was >130-fold higher at 3 d post-infection and >550-fold higher at 6 d post-infection than in lungs of mice infected with EG/D1 (wt)  . We next/D1 (wt) , indicat/D1 (wt) .in vivo.Lungs of infected mice were examined by histopathology at 3 d post-infection. Mice infected with any of the mutants had much more severe pathological changes in their pulmonary airways and parenchymal tissues than mice infected with EG/D1 (wt)  . Our modInterestingly, the majority of the mutations identified in this study that showed an appreciable effect in increasing progeny vRNA production in human airway cells were clustered around the vRNA and located on two exposed PB-1 domains, the β-ribbon and the β-hairpin. An unusually long β-ribbon, where PB1-D175N, T182I, K198R and K214R were located, contacts the 3’-end of vRNA and the β-hairpin, where PB1-L384S was located, forming a 5’ vRNA binding site together with a PA-arch domain. Thus, the vRNA promoter is sandwiched in a pocket formed by the two domains Figs  and S10.Previous studies to identify human adaptation mutations in influenza viruses have typically relied on comparison of viruses with high and low pathogenicity in mammals. However, a high-throughput screening approach would allow analysis of large numbers of mutations and identification of currently unknown human adaptation mutations. Using a comprehensive database approach in this study enabled us to identify a number of novel putative human adaptation mutations in the polymerase complex of H5N1 clade 2.2.1 viruses. These mutations acted in combination with PB2-627K and increased virus growth in both human airway epithelial cells and mouse lungs. To our knowledge, this is the first report to systematically evaluate the effects of polymerase mutations that have been selected in H5N1-infected patients on AI virus adaptation to infect humans. It should be noted that the present study was limited by the relatively small number of sequences available that were derived from human isolates of clade 2.2.1 viruses, because of the limited number of such sequences in public databases and because emergence of mutations may be stochastic and not necessarily associated with an adaptive advantage. Nonetheless, our database approach led us to identify specific mutations in the polymerase complex that have not been previously reported as human adaptation mutations, indicating the potential of the experimental approach used in this study.H5N1 viruses are endemic in Egypt, which has a substantially higher prevalence of H5N1 viruses than any other country, with collateral human infections . A previPhylogenetic analysis of the PB1, PB2, PA and NP genes in this study –S13 FigsAdaptation of AI virus polymerase in mammals has been studied extensively and the adaptation mutations that have been well-characterized are in the PB2 gene. PB2-627K is recognized as a dominant adaptation mutation in human influenza A viruses and facilitates efficient viral replication in mammalian cells. While several other adaptation mutations  have also been identified in PB2, the majority of these amino acid substitutions have not previously been detected together with PB2-E627K, implying that they had little or no synergistic effect with PB2-627K. In contrast, in this study we found that H5N1 clade 2.2.1 viruses that carry PB2-627K could acquire additional human adaptation polymerase mutations in infected patients, which were advantageous for viral growth in human cells and mice. It was interesting that the mutations identified here were in multiple domains of PB1, PB2, PA and NP that are remote from the PB2-627 domain . The effcis-acting polymerase that forms part of the vRNP, replication is initiated by a trans-acting polymerase complex at the 3’-end of vRNA that is released from the resident polymerase complex [The polymerase structure models in this study indicated that the PB1-D175N, -T182I, -K198R and -K214R mutations, which had appreciable effects on H5N1 human adaptation, structurally and energetically modified the conformation of the PB1 β-ribbon that interacts with the vRNA promoter . These r complex . This in complex .In this study, polymerase activity was not always associated with viral growth in human cells and/or pathogenicity in mice. For example, PA-E327K increased polymerase activity, but had no appreciable effect on progeny vRNA production by EG/D1 (wt) in human cells. In addition, PB2-D701N caused no increase in progeny vRNA production in the EG/D1 genetic background carrying PB2-627K, in spite of the strong effect of PB2-D701N in the minigenome assay. It is possible that mutations such as PA-E327K and PB2-D701N caused the loss of some functional balance in EG/D1 vRNP carrying PB2-627K. For example, both the PB2-E627K and PB2-D701N mutations increased PB2 binding to host importin-α isoforms to indirectly regulate polymerase activity –45, suggSeveral of the human adaptation polymerase mutations identified in this study  have been detected in H5N1 viruses that are circulating in different geographic areas of Asia, with either amino acid E or K at the PB2-627 residue . TherefoIn conclusion, we have identified a number of putative human adaptation polymerase mutations that have not previously been reported. These mutations were widely located throughout the structural domains of the polymerase complex beyond the PB2-627 domain. The adaptation mechanisms of these mutations are probably multifactorial, highlighting the complexity of polymerase functions and of influenza virus polymerase adaptation to new hosts. Our findings also suggested that AI viruses might adapt by multiple pathways to enable their growth in humans. These data provide a novel insight into the functions of the influenza virus polymerase complex and help our understanding of the mechanism of AI virus adaptation to new hosts.All studies using anonymized primary human cells were conducted according to the principles expressed in the Declaration of Helsinki and approved by the Institutional Review Board of Osaka University . All animal experiments were conducted in compliance with Japanese legislation  and guidelines under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, Japan . Animal care, housing, feeding, sampling, observation, and environment enrichment were performed in accordance with the guidelines. The protocols of animal experiments were approved by the Animal Experiment Committee of the Research Institute for Microbial Disease, Osaka University .All experiments with live H5N1 viruses were performed at enhanced biosafety level 3+ (ABSL3+) in the Research Institute for Microbial Diseases, Osaka University, which has been approved for handling H5N1 viruses by the Ministry of Agriculture, Forestry and Fisheries of Japan. All studies with recombinant DNAs were conducted under the applicable laws in Japan and approved by the Biological Safety Committee of the Research Institute for Microbial Diseases, Osaka University  after risk assessments were conducted by the Living Modified Organisms Committee of the Research Institute for Microbial Diseases, Osaka University and, when required, by the Ministry of Education, Culture, Sports, Science and Technology of Japan.in vivo and in vitro experimental work is carried out in class 3 isolators or class 3 biosafety cabinets, which also operate under negative pressure. Air exhausted from the class 3 units is filtered by High Efficiency Particulate Air (HEPA) filters and then leaves the facility via a second set of HEPA filters. Also, users must check pressure gauges when entering and inside the ABSL3+ facility. The ABSL3+ has a dedicated electrical generator in the event of power loss.The ABSL3+ facility of the Research Institute for Microbial Diseases, Osaka University, consists of negative-pressure laboratories in which all Only authorized personal that have received appropriate training can access the ABSL3+ facility. All activities inside the ABSL3+ laboratories are monitored via video cameras. All personnel working at the ABSL3+ facility wear a full Tyvek suit, FFP3 facemasks and multiple pairs of gloves. Furthermore, all personnel conducting this study were vaccinated against seasonal and H5N1 influenza viruses. For animal handling in the facilities, personnel always work in pairs. The facility is secured by procedures recognized as appropriate by the institutional biosafety officers at the Research Institute for Microbial Diseases, Osaka University, and by Japan government inspectors. Antiviral drugs are directly available to further mitigate risks upon incidents.in vivo mouse infection study although this mutant showed reduced progeny vRNA production in human cells compared to EG/D1 (wt) virus.In this study, all the mutations introduced into recombinant EG/D1 virus (clade 2.2.1) were naturally detected as single/multiple intra-segment mutations or inter-segment combinations of the mutations in clade 2.2.1 viruses isolated from patients, except artificial mutation PB2-D701N. Furthermore, a mouse infection study was performed after observing no increase in progeny vRNA production of the selected viruses carrying mutations compared to previously published studies, and the PB2-D701N mutant virus was excluded from the http://platform.gisaid.org). Because there was limited availability of genetic sequences from human isolates of H5N1 viruses since 2012 (only two in 2012 and zero in 2013–2014) at the time of this study (June 2014), we focused on genetic variations of clade 2.2.1 viruses from 2006–2011. These sequences were aligned using the MAFFT program [Published complete sequences of 152 PB2, 139 PB1, 139 PA and 142 NP genes from influenza A virus subtype H5N1 strains isolated in Egypt from 2006–2011 were obtained from the GISAID database ( program . PB2 muthttp://www.brc.riken.jp/lab/cell/english/) and maintained in Dulbecco’s modified Eagle’s medium or Ham’s F-12K medium supplemented with 10% heat-inactivated fetal calf serum (FCS) at 37°C in a humidified atmosphere of 95% air and 5% CO2 as previously described [293T cells, QT-6 cells and Madin-Darby canine kidney (MDCK) cells were obtained from the RIKEN BioResource Center Cell Bank , layered on a 20% sucrose cushion and pelleted by centrifugation at 25,000 rpm for 2 h. Virus pellets were resuspended in PBS and aliquots were stored at −80°C as working stocks. Virus titers were assayed as FFU by focus-forming assays  using MDRenilla luciferase. The total amount of plasmid mixture was 1 μg/well and the PB2:PB1:PA:NP:Pol I:Renilla luciferase ratio was 5:5:5:30:5:1. Cells were transfected with the plasmid mixture using TransIT-LT1 (Mirus) and incubated at 33 or 37°C. At 48 h post-transfection, the cells were lysed and luciferase activity was determined by the dual-luciferase assay system (Promega) according to the manufacturer’s instructions. Luciferase activity values were normalized relative to the Renilla luciferase activity.293T and QT-6 cells (90% confluent in 24-well plates) were transiently transfected with the following plasmid mixture: polymerase II-driven pcXN2 plasmids each expressing the PB2, PB1, PA or NP gene of A/duck/Egypt/D1Br/2007 (EG/D1) , and a hRecombinant viruses were generated with a plasmid-based reverse genetics system in the EG/D1 (wt) virus genetic background as previously described ,49. MutaSAE cells and CEFs were infected in triplicate with the indicated viruses at an MOI of 0.03 and 0.005, respectively. The virus inoculum was removed after 1 h and the cells were washed with PBS, followed by further incubation at 33 or 37°C. At the indicated times post-infection, vRNA titers in the cell culture supernatants were assayed by quantitative real-time RT-PCR as described below.Progeny vRNA was extracted from the culture medium of infected cells with a QIAamp Viral RNA Mini Kit (Qiagen) and assayed by quantitative real-time RT-PCR  using primers targeting the M gene as previously described .Six-well plates with confluent 293T cells were transiently transfected with a plasmid mixture containing 500 ng pcXN2 plasmid expressing EG/D1 PB2, PB1 and PA, 3 μg pcXN2 plasmid expressing EG/D1 NP and 500 ng pPol plasmid expressing EG/D1 segment 6 RNA. At 72 h post-transfection, cells were collected in Trizol reagent (Invitrogen) and RNA was purified according to the manufacturer’s protocol. Primer extension analysis was carried out using specific DIG-labeled primers for segment 6 vRNA and cRNA, with cell 5S ribosomal RNA (5S rRNA) as an internal control. Primer extension was carried out with SuperScript III reverse transcriptase (Invitrogen). After heating at 65°C for 5 min and quickly cooling on ice, transcription products were analyzed on 8% polyacrylamide gels containing 7 M urea in TBE buffer, transferred onto positively charged Nylon Membranes (Roche) and baked at 80°C for 4 h. For chemiluminescence detection, anti-DIG antibody linked to alkaline phosphatase, Fab and CDP-Star (Roche) were used according to the manufacturer’s instructions. The band intensities were quantified by ImageJ software and vRNA expression was calculated.293T cells were transfected with equal amounts of expression plasmids (250 ng PB2 and 500 ng PB1). At 16 h post-transfection, the cells were harvested with sample buffer and heated for 20 min at 100°C. The cell lysates were then separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore). Immunoblot analysis was performed using anti-influenza PB2 or PB1 antibody (GeneTex) and HRP-conjugated secondary antibody. The antibodies were visualized with Pierce Western Blotting Substrate Plus (Thermo Scientific) and exposed on Amersham Hyperfilm ECL . The band intensities were quantified by ImageJ software and total protein expression was calculated.5 FFU virus and virus titers were assayed as FFU in MDCK cells. For histopathology analysis, mouse lungs were collected 3 d post-inoculation with 2.5×105 FFU virus, fixed in 4% buffered paraformaldehyde, embedded in paraffin, cut into 5 μm sections, stained with hematoxylin and eosin, and examined by light microscopy. Immunohistochemical staining for the H5 antigen was performed [Five-week-old female BALB/C mice (Japan SLC), under mixed anesthesia (medetomidine-butorphanol-midazolam), were inoculated intranasally with 75 μl samples of serial 10-fold dilutions of virus in PBS. The mice were observed daily for 14 d for weight loss and mortality. Mouse lungs were collected 3 and 6 d post-inoculation with 2.5×10erformed  on deparThe sequences of 139 PB1 genes from influenza A virus subtype H5N1 strains isolated in Egypt from 2006–2011 were obtained from the GISAID database. Phylogenetic analysis was performed using MEGA4 software  for the http://www.chemcom.com). Residues 195–198 are missing in 4WSB and, therefore, the structure was built by MOE structure preparation tools (the parameter was default). Consensus vRNA promoter sequences (PDBID code 4WSB) were fit into the mutant models. After homology modeling, molecular optimization was carried out between the polymerase complexes and the vRNA promoter, and the interaction energy (IntE) and distance between each polymerase residue and the vRNA promoter residues in the complexes were calculated. The plausibility of this model was supported by the observation that the configuration of the EG/D1 structure modeled from the influenza A virus crystal structure (Protein Data Bank ID code 4WSB) corresponded to the polymerase complex crystal structure of influenza B virus (Protein Data Bank ID code 4WSA) including the complete PB1 β-ribbon [The crystal structure of the heterotrimeric polymerase complex of influenza virus A/little yellow-shouldered bat/Guatemala/060/2010 (H17N10) bound to the vRNA promoter (Protein Data Bank ID code 4WSB)  was usedβ-ribbon , within Statistical analysis was carried out using GraphPad Prism Version 6 software (GraphPad Software Inc.).S1 Fig(A) In the vRNA-oriented minigenome assay, human polymerase I transcribes vRNA-like luciferase RNA, which is subsequently transcribed by the influenza virus polymerase complex and nucleoprotein. Thus, reporter mRNA can be transcribed directly from the vRNA-like molecule. (B) In the cRNA-oriented minigenome assay, human polymerase I transcribes cRNA-like luciferase RNA, which must be replicated to a vRNA-like molecule before reporter mRNA transcription. Thus, reporter gene expression is dependent on influenza virus polymerase-mediated replication in the cRNA-oriented minigenome assay. Thick arrows indicate RNA synthesis steps that should be efficient in each minigenome assay system.(PDF)Click here for additional data file.S2 FigRenilla luciferase-expressing plasmid as an internal control. For comparison, cells were also transfected with plasmids expressing WSN (H1N1) PB2, PB1, PA and NP, and with the other plasmids listed above. After 48 h at 37°C, luciferase activities were measured and normalized to the internal Renilla luciferase activity. Each data point is the mean ± SD of three independent experiments. The asterisks indicate a P value <0.01 (ANOVA with Tukey’s multiple comparison test).293T cells were transfected with plasmids expressing EG/D1 PB1, PA, NP, and EG/D1 PB2 (wt) or the PB2-K627E mutant, and with a human polymerase I-driven plasmid expressing a vRNA-luciferase reporter gene and a (PDF)Click here for additional data file.S3 FigRenilla luciferase-expressing plasmid as an internal control. Luciferase activities were assayed at 48 h post-transfection and normalized to the internal Renilla luciferase activity. The data were expressed relative to the results for EG/D1 (wt) polymerase. (A) Comparison of luciferase activity at 37 and 33°C in vRNA-oriented minigenome assays in 293T cells. (B) Comparison of luciferase activity at 37 and 33°C in cRNA-oriented minigenome assays in 293T cells. (C) Comparison of polymerase activity in 293T and QT-6 cells in vRNA-oriented minigenome assays at 37°C. (D) Comparison of polymerase activity in 293T cells and QT-6 cells in cRNA-oriented minigenome assays at 37°C. Each data point is the mean ± SD of three independent experiments. Bars with light and dark colors indicate single/intra-segment multiple mutations and inter-segment combinations of mutations, respectively.293T and QT-6 cells were transfected with plasmids expressing an inter-segment combination of mutations in EG/D1 PB2, PB1, PA and NP as indicated, a human or chicken polymerase I-driven plasmid expressing a vRNA-oriented or cRNA-oriented luciferase reporter gene, and a (PDF)Click here for additional data file.S4 FigP value <0.05 and <0.01, respectively, when compared with the EG/D1 (wt) virus titer (Student’s t test).SAE cells were infected with EG/D1 (wt) or a virus strain with the indicated polymerase mutation(s) at an MOI of 0.03 and incubated at 37°C (A) or 33°C (B). The culture supernatants were harvested at the indicated times post-infection and assayed by quantitative real-time RT-PCR to determine the amount of progeny vRNA. Each data point is the mean ± SD of the log10 number of vRNA copies/ml from three separate experiments. Single and double asterisks indicate a (PDF)Click here for additional data file.S5 FigCEFs were infected with EG/D1 (wt) or a virus strain with the indicated polymerase mutation(s) at an MOI of 0.005 and incubated at 37°C. The culture supernatants were harvested at the indicated times post-infection and assayed by quantitative real-time RT-PCR to determine the amount of progeny virus RNA. Each data point is the mean ± SD of the log10 number of viral RNA copies/ml from three separate experiments.(PDF)Click here for additional data file.S6 FigP value <0.01 (ANOVA with Tukey’s multiple comparison test). Representative results of primer extension assays for EG/D1 and the indicated mutants are shown.Primers specific for virus segment 6 were used in this assay. 5S rRNA levels served as an internal loading control. After quantitation of the band intensities using ImageJ software, the amount of expression for each vRNA (upper panel) was calculated relative to that for EG/D1 (wt). The asterisks indicate a (PDF)Click here for additional data file.S7 Fig293T cells were transfected with mutant PB2 or PB1 expression plasmids. At 16 h post-transfection, cells were harvested and protein expression levels were analyzed by Western blotting using anti-PB1 or PB2 antibodies. After quantitation of the band intensities using ImageJ software, the amount of expression for each PB1  or PB2  was calculated relative to that for EG/D1 (wt). Representative results of western blots for EG/D1 and the indicated PB1 mutants  and PB2 mutants  are shown. NS indicates statistically not significant (ANOVA).(PDF)Click here for additional data file.S8 FigFive-week-old BALB/c mice (5 mice per group) were inoculated intranasally with serial 10-fold dilutions of the indicated viruses. The body weight of infected mice was monitored for 14 d post-infection. The mean ± SD of the percent body weight change for each group of mice is shown. The numbers in the graphs show the numbers of surviving animals.(PDF)Click here for additional data file.S9 FigThis phylogenetic tree was reconstructed from sequence data for the PB1 genes of the 139 H5N1 clade 2.2.1 viruses available in the GISAID database. Subgroups of clade 2.2.1 viruses and the amino acid mutations that are conserved in each of the branches or specific to human virus strains are shown on the right. Green is used to highlight human virus strains.(PDF)Click here for additional data file.S10 Fig(A) A plausible structural model of the EG/D1 PB1 β-ribbon and β-hairpin with the vRNA promoter. The locations of vRNA contact sites and the mutations identified in this study are shown in blue and red, respectively. Other structures are omitted for clarity. (B−D) Mutant polymerase structures with the mutations identified in this study superimposed onto EG/D1 (wt) PB1, showing the flexibility of the long β-ribbon. (B) Close-up of residue 198 in the EG/D1 PB1 β-ribbon with vRNA, with the K198R mutation superimposed. (C) Close-up of vRNA contact residue 188 in the EG/D1 PB1 β-ribbon with vRNA , with the mutant polymerase structures carrying K198R/D175N (red) and K198R/K214R (blue) superimposed. (D) Close-up of vRNA contact residue 203 in the EG/D1 PB1 β-ribbon with vRNA , with the mutant polymerase structures carrying T182I/K214R (pink) and K198R/D175N (cyan) superimposed. (E) Close-up of residue 384 in the EG/D1 PB1 β-hairpin with vRNA, with the L384S mutation superimposed. Potential interactions between the vRNA contact residues and neighboring vRNA are represented by broken lines and distances (Å) are indicated with the same colors as the mutant polymerase structures.(PDF)Click here for additional data file.S11 FigThis phylogenetic tree was reconstructed from sequence data for the PB2 genes of the 152 H5N1 clade 2.2.1 viruses available in the GISAID database. Subgroups of clade 2.2.1 viruses and the amino acid mutations that are conserved in each of the branches or specific to human virus strains are shown on the right. Green is used to highlight human virus strains.(PDF)Click here for additional data file.S12 FigThis phylogenetic tree was reconstructed from sequence data for the PA genes of the 139 H5N1 clade 2.2.1 viruses available in the GISAID database. Subgroups of clade 2.2.1 viruses and the amino acid mutations that are conserved in each of the branches or specific to human virus strains are shown on the right. Green is used to highlight human virus strains.(PDF)Click here for additional data file.S13 FigThis phylogenetic tree was reconstructed from sequence data for the NP genes of the 142 H5N1 clade 2.2.1 viruses available in the GISAID database. Subgroups of clade 2.2.1 viruses and the amino acid mutations that are conserved in each of the branches or specific to human virus strains are shown on the right. Green is used to highlight human virus strains.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.