Altering the biochemical makeup of chromatin by the incorporation of histone variants during development represents a key mechanism in regulating gene expression. The histone variant H2A.B, H2A.B.3 in mice, appeared late in evolution and is most highly expressed in the testis. In the mouse, it is encoded by three different genes. H2A.B expression is spatially and temporally regulated during spermatogenesis being most highly expressed in the haploid round spermatid stage. Active genes gain H2A.B where it directly interacts with polymerase II and RNA processing factors within splicing speckles. However, the importance of H2A.B for gene expression and fertility are unknown.Here, we report the first mouse knockout of this histone variant and its effects on fertility, nuclear organization, and gene expression. In view of the controversy related to the generation of off-target mutations by gene editing approaches, we test the specificity of TALENs by disrupting the H2A.B multi-copy gene family using only one pair of TALENs. We show that TALENs do display a high level of specificity since no off-target mutations are detected by bioinformatics analyses of exome sequences obtained from three consecutive generations of knockout mice and by Sanger DNA sequencing. Male H2A.B.3 knockout mice are subfertile and display an increase in the proportion of abnormal sperm and clogged seminiferous tubules. Significantly, a loss of proper RNA Pol II targeting to distinct transcription–splicing territories and changes to pre-mRNA splicing are observed.We have produced the first H2A.B knockout mouse using the TALEN approach.The online version of this article (10.1186/s13059-019-1633-3) contains supplementary material, which is available to authorized users. Importantly, the structure and the protein interaction surface of a nucleosome can be altered by the substitution of one or more of the major core histones with their variant forms to regulate gene expression and other DNA-dependent processes.Histones and DNA form the core structure of chromatin, the nucleosome core, in which ~ 145 base pairs of DNA are wrapped around a histone octamer comprising of a (H3-H4)Despite being discovered over a decade ago , the in A striking in vitro feature of H2A.B/B.3 is their ability to completely unfold chromatin thus permitting high levels of RNA Pol II transcription [The availability of a H2A.B.3 knockout would therefore be an invaluable tool for investigating the importance of H2A.B in fertility and testing the above hypothesis. However, as the H2A.B.3 protein is expressed from three different genes, this is a technically challenging feat. Our aim was therefore to first devise the most efficient and specific strategy to inhibit the function of all three genes.Previously, transcription activator-like effector nucleases (TALENs) were the choice to perform gene and genome editing , 5, but There are three H2A.B.3-encoding genes  plus a pseudogene (Gm14904) all located on the X chromosome in the mouse (the pseudogene was present in the Ensemble release 57 but subsequently removed)  and by the Cel 1 cleavage assay. The TALEN plasmids were co-transfected into Neuro2a cells in pairs in various combinations .In order to produce H2A.B.3 KO mice, FVB/NJArc mice , hence an extra suffix, JArc) were used. Capped, poly-adenylated, and purified in vitro transcribed mRNA was adjusted to 10 ng/μl for each TALEN pair (101421:101422) and injected into 532 fertilized one-cell embryos and cultured overnight to the two-cell stage. One hundred fifty-three surviving two-cell embryos that displayed normal morphology were implanted in pseudo pregnant females, resulting in the birth of 19 pups. Of these nineteen pups, nine contained TALEN-induced H2A.B.3 mutations  was not amplifiable by PCR. However, when we used mixed primer pairs for PCR amplification , we were able to detect an amplification product. −/x females with wt males (up to four generations), revealed that these additional mutations were lost after G2, but maintained original founder mutations . This can be explained by the fact that TALEN activity can be retained for several cell divisions following their injection into the two-cell stage, thus producing a mosaic genotype, as previously observed [Subsequent Sanger DNA sequencing demonstrated that a fusion between gm14920 and H2Afb2 had occurred (data not shown). Unexpectedly, when the founder L74-5 mouse, which only displayed the deletions and insertions described in Table observed . Collect−/y mice was based on the sequencing of exomes of three related non-mosaic H2A.B.3−/y mice from three consecutive generations (G1-G3). This strategy avoids the problem observed in previous studies where unrelated mice were compared to assess off-target effects [6 and 228 × 106 reads per sample , gm14920 (X∆10Y), and H2Afb2 (X∆64Y) mice. Subsequent crosses between genetically modified H2A.B.3 female mice and wt males produced NM4-G2 and G3 H2Afb3 (X∆5Y), gm14920 (X∆10Y), and H2Afb2 (X∆64Y) mice for exon sequencing  and indels  , gm14920 (10 bp), and a larger deletion of 64 bp in H2Afb2  -deficient mice are subfertile and produce high proportion of defective sperm that appear to resemble the abnormal sperm observed from H2A.B.3TP1 begins to incorporate into chromatin in step 9 elongating spermatids and by step 14, is replaced by the protamines. In wt mice, the incorporation of TP1 occurs as a gradual wave across steps 9–11 spermatid nuclei with an apparent delayed incorporation into pericentric heterochromatin (the intense DAPI-stained regions)  or euchromatin (H3K4me3)  and in the surrounding euchromatin (EU) and calculated their ratio (CC − B)/(EU − B), where B is background autofluorescence. This ratio would decrease if euchromatin contained more H3K9me2 or H3K4me3 than the heterochromatic chromocenter. We also scored two stages of round spermatid differentiation, the early round spermatid (ERS) and late round spermatid (LRS) stage. Indeed, the average (CC − B)/(EU − B) H3K4me3 ratio was smaller in H2A.B.3Given that H2A.B.3 is incorporated into the chromatin of an active gene, we next performed a comprehensive differential gene expression analysis on purified round spermatids using high-throughput 75-bp paired-end sequencing of poly(A)-selected RNA (in triplicate but each mRNAseq library contained mRNA combined from two wt or H2A.B.3 KO mice). One notable initial observation from our analyses was the large variation in the RNA expression profile between different H2A.B.3 KO biological replicates compared to wt replicates, as indicated by a principal component analysis - or protein -based mechanism will ultimately prove to be more specific for genome editing and this will be critical for future medical therapies. In this study, we have successfully applied the TALEN technology, whereby we used one pair of TALENs to specifically and simultaneously disrupt three gene copies of a gene family. Many human diseases are caused by gene copy number variations , and theThe TALEN approach did expose several interesting phenomena. Firstly, the simultaneous knockout of highly homologous H2A.B.3 genes leads to the formation of chimeras between these genes. As every chimera contained a small deletion, it is likely that terminal microhomology, a mechanism of NHEJ, was involved . It is i−/y mice are as fertile as wt mice, whereas other male mice are extremely infertile hints at a possible compensatory mechanism  that caIt is attractive to speculate that the observed increase in H3K4me3 in H2A.B.3 KO round spermatid nuclei may be involved in such a compensatory mechanism, which might explain why our differential gene expression analysis did reveal a major change. On the other hand, the apparent large variation in the gene expression profile between individual H2A.B.3 KO testes suggests that, if H3K4me3 is involved in this compensation mechanism, its ability to compensate is stochastic, which could also explain why some H2A.B.3 KO male mice are fertile and others are less so.It is interesting that TP1 KO mice display defective sperm that appear to resemble the abnormal sperm observed from H2A.B.3 KO mice, i.e., sperm with tails coiled around their heads . This prA major finding is that in the absence of H2A.B.3, RNA Pol II no longer occupies the same territory as splicing speckles becoming more diffuse within round spermatid nuclei. It is therefore attractive to hypothesize that H2A.B.3 might have an important role in the recruitment and/or anchoring of RNA Pol II to active territories of transcription and splicing. This is consistent with previous mass spec data where it was shown that chromatin bound H2A.B.3 directly interacts with RNA Pol II [We have produced the first H2A.B knockout mouse using the TALEN approach. All three H2A.B.3 genes, were successfully genetically modified to inhibit their expression by using only one pair of TALENs. We also describe a robust approach to search for any off-targets and report several interesting TALEN-related phenomena applicable if other multi-copy genes are being considered for genome editing. H2A.B.3 KO male mice were subfertile and reveal several altered phenotypes that could account for this subfertility; an increase in the proportion of abnormal sperm and seminiferous tubules that are clogged. At the molecular level, TP1 chromatin loading and unloading is impaired. Most significantly, a loss of proper RNA Pol II targeting to distinct transcription–splicing territories and changes to pre-mRNA splicing were observed.E. coli competent cells, and single clones were picked and sequence verified.The TALENs were assembled using the method previously described , 22. TheThe Dual-Luciferase Single-Strand Annealing Assay (DLSSA) is based on the Dual-Luciferase Reporter Assay System from Promega . In the DLSSA, the firefly luciferase reporter gene is split into two inactive fragments with overlapping repeated sequences separated by stop codons and one of the three H2A.B.3 genes. Introduction of a double strand break in the H2A.B.3 gene by the TALEN pair initiates recombination between the flanking repeats by the single-strand annealing pathway and produces an active luciferase gene. Neuro2a cells (ATCC #CCL-131) were cultured in Dulbecco’s modified Eagle’s medium  plus 10% FBS and 2 mM L-Glu . One day before transfection, 20,000 cells per well were seeded into a 96-well plate. Cells were transiently transfected using Lipofectamine 2000 (Thermo Fisher Scientific) with four plasmids including a pair of TALENs, the SSA firefly luciferase reporter, and the internal control Renilla luciferase reporter (6.25 ng DNA for each component). The luciferase assay was performed 24 h post-transfection according to the manufacturer’s instructions. TALEN activity was measured as the ratio of the firefly and the Renilla luminescence units.5) were seeded into a 96-well plate the day before transfection. The cells were then transfected with a pair of TALEN DNAs (400 ng of each) using Lipofectamine 2000 (Thermo Fisher Scientific). The genomic DNA was purified 48 h post-transfection. Gene-specific primer pairs were used to amplify each of the H2A gene variants. The primer sequences are H2Aafb3 forward (5′-CAGCAGAAAGCAGCCAAGTGG) and reverse (5′-GCAGGTCAGCCAAGAAGCA); Gm14920 forward (5′-GTACGGTACAAAGGGAG ATG) and reverse (5′-GAGCAGGTCAGCCAAGCAGAG); H2afb2 forward (5′-CAGGTCAGCAGAGAGCAATT) and reverse (5′-CTCCATACTGCTGTAGACCT); and pseudo Lap forward (5′-GTCAGCAGAATGCAGCCAAATAT) and reverse (5′-CAAGCCAGTAGCCAACATCAAG). The PCR products were treated with Surveyor Nuclease (Cel-1) and resolved on PAGE. The nuclease activity of TALENs was measured by quantifying the proportion of the cleaved DNA fragments.The assay has been described in detail previously . BrieflyXbaI and the products were purified . Capped and poly(A)-tailed mRNA transcripts were produced using a mMESSAGE mMACHINE T7 Ultra Transcription Kit (Life Technologies) following the manufacturer’s instructions. mRNA was purified using a MEGAclear kit (Life Technologies) and was eluted in RNase-free water, and single-use aliquots were frozen.Plasmids (in Sangamo backbone) were linearized with Prior to injection, mRNA was diluted to 10 ng/μl in filtered RNase-free microinjection buffer . TALEN mRNA was delivered into the pronucleus of single-cell embryos (FVB), using standard techniques. Injected embryos were cultured overnight to the two-cell stage, then surgically transferred into oviducts pseudopregnant CD1 mice, using standard techniques.Transgenic founders with mutated H2A.B.3 genes were identified among live born mice by PCR , gm14920 (X∆10Y) and H2Afb2 (X∆64Y)). The purified gDNA was subjected to exome enrichment using the Agilent SureSelectXT2 All Exon Kit. The exome capture efficiency was uniformly high with approximately 45–50% of all DNA sequenced being exonic. Sequenced mouse exomes were analyzed using our in-house variant detection pipeline described in detail previously [http://broadinstitute.github.io/picard/), specifically the MarkDuplicate command. SNVs and small indels were called using SAMTools Mpileup [https://sourceforge.net/projects/janda/) were utilized. To reduce the number of total variants called, variants specific to the FVBN mouse strain were downloaded from the Sanger Institute ftp site (ftp://ftp-mouse.sanger.ac.uk/REL-1206-FVBNJ/). As these variants were originally reported relative to reference genome mm9, the UCSC liftOver tool (https://genome.ucsc.edu/cgi-bin/hgLiftOver) was used to map the FVBN variants to mm10. These FVBN-specific variants were annotated and not considered for further off-target analysis.The male mice that were chosen for exome sequencing were genotyped prior to sequencing to confirm that all three generations had the same H2A.B.3 genotype  were removed and subjected to immumohistochemistry and DAPI staining as previously described [The purification was performed exactly as described elsewhere . Briefly6 cells for each replica were used for RNA purification by Trizol. Prior to purification, ERCC RNA spike-in Mix 1 (diluted 1:10) was added to each sample that was already lysed in Trizol. The addition of spike-in control was done at this stage to control for RNA loss during the purification procedure. Purified RNA was resuspended in 50 μl of sterile MQ water. Fifteen microliters of each sample was subjected to Turbo DNase treatment and 3 μl of DNase-treated RNA was used for TruSeq Stranded mRNA LT Illumina library preparation. Samples were sequenced using 75-bp paired-end sequencing on a NextSeq500 platform. Data processing and analysis were as previously described by us [Fblim1 FW GTCTGTGGTTTCTGTCACAAGFblim1 REV GGTAGCAGGGTTCGCACAGSlc37a3 FW GGACTCTGCTAGGCTTCATCSlc37a3 REV CTATACCCAACAAGGGACCCStyx FW CATTCAGCAATCACTCCTGCStyx REV GGTTTTCTTAATGTTAGTAGGAGTraf7 FW GTCTGGAGTATGGACAACATGTraf7 REV CTTCACGGTGCTATCCACAGRound spermatids were purified from six H2A.B.3 KO and six wt males. Three biological replicates for each genotype were obtained by merging cells from two individuals. 4 × 10ed by us . Differeed by us . PrimersAntibodies used in this study were rabbit polyclonal anti-TP1 , rabbit serum anti-H2A.L.2 , rabbit polyclonal anti-Pol II S2 , mouse monoclonal anti-Y12 , rabbit polyclonal anti-H3K4me3 , and mouse monoclonal anti-H3K9me2 .Additional file 1:Table S1. The genomic coordinates of H2A.B.3 coding genes. The degree of homology between the genes is indicated as % Identity. (PDF 49 kb)Additional file 2:Figure S1. The relative expression of H2A.B.3 gene family members in the testis of FVB/NJArc mice. Gene expression was assessed relative to mHPRT using qPCR. (PDF 86 kb)Additional file 3:Table S2. List of TALENs designed to target H2A.B.3 genes. (PDF 51 kb)Additional file 4:Figure S2. TALEN pair specificity tested by the DLLS assay. TALEN pairs designed to target the H2Afb3 gene only (Group 1) or all three H2A.B.3 genes (Group 2). One day before transfection, 20,000 cells per well were seeded into a 96-well plate. Cells were transiently transfected with four plasmids including a pair of TALENs, the SSA firefly luciferase reporter, and the internal control Renilla luciferase reporter. The luciferase assay was performed 24 h post-transfection. The pair 101421:101422 was used to create the H2A.B.3 KO mouse. (PDF 435 kb)Additional file 5:Figure S3. Cel 1 assays. Neuro2a cells were transiently transfected with various TALEN pairs and extracted genomic DNA was amplified by PCR with gene-specific primers and subjected to Cel1 digestion. TALEN activity is represented by the % cleaved  shown below each lane. Cleaved DNA products (arrow heads), uncut DNA (arrow). TALEN pair 101421:101422, highlighted in red, showed the highest activity for all three H2A.B.3 genes. 1,2,3 and P denotes H2Afb3, gm14920, H2Afb2 and pseudo H2A.B.3 genes, respectively. Two biological replicates are shown. Empty GFP-vector was used as a negative control. (PDF 1001 kb)Additional file 6:Figure S4. TALEN-targeted DNA sequences. The alignment of H2A.B.3 genes and the pseudogene. TALENs specific to H2afb3 are in blue and those common for all H2A.B3 genes are in red. (PDF 58 kb)Additional file 7:Figure S5. An example of chimeras formed between gm14920 and H2afb2 in some G1 mice produced by crossing founder L74-5 with a wt. female. (a) genes gm14920 and H2afb2 are not amplified with gene-specific primers in pups #6 and #7, respectively but is amplified in a pup #5. (b) amplification with gm14920-Fw and H2afb2-Rev primers shows the chimeric product in pups #6 and #7. (c) A schematic diagram showing that a chimera was formed between gm14920 and H2afb2 genes with a small deletion between these fused genes suggesting that NHEJ mechanisms were involved. Fw, forward primer; Rev., reverse primer; number; L, DNA ladder. (PDF 198 kb)Additional file 8:Table S3. The exome sequencing coverage. Three consecutive generations of mice from the NM4 H2A.B.3 KO colony were sequenced using paired-end sequencing with a 100 bp read length. (PDF 48 kb)Additional file 9:Figure S6. TALEN-induced deletions of the three H2A.B.3 genes. (PDF 92 kb)Additional file 10:Table S4. Putative SNVs and Indels identified in H2A.B.3 KO mice using the mm10 mouse genome as a reference. (PDF 48 kb)Additional file 11:Table S5. Putative SNVs and Indels identified in H2A.B.3 KO mice after applying the FVB/NJ strain filter. (PDF 48 kb)Additional file 12:Table S6. Predicted putative homozygous and heterozygous polymorphic variants (off-target deletions) in three consecutive generations of H2A.B.3−/y mice. Exome data was referenced to mm10 genome, followed by filtering of FVB/N-specific variants and analyzed for genome polymorphism using Pindel tool. Homozygous polymorphisms: no polymorphic alleles (0/0), both alleles differ from reference (1/1), and both alleles differ from reference and from 1/1 (2/2). Heterozygous polymorphisms: one allele differs from the reference genome (0/1), one allele defers from the reference genome and from 0/1 (0/2), both alleles differ from the reference genome and from each other (1/2). No call, not assigned to any type. (PDF 54 kb)Additional file 13:Figure S7. Computational analysis predicted 19 putative heterozygous deletions shared between all 3 generations of H2A.B.3−/y KO mice. If TALENs introduced off-target mutations in G0 founder mice, then those mutations would be inherited by their progeny as heterozygous. (PDF 83 kb)Additional file 14:Table S7. Nineteen common putative heterozygous polymorphisms identified in all three generations of H2A.B.3−/y mice. (PDF 72 kb)Additional file 15:Figure S8. Exome sequencing reveals all the three expected mutations in H2A.B.3 genes. The screen shots are the output from SVs call by Pindel for all H2A.B.3 genes. The orange border box indicates the deleted region detected for each gene by SAMtools. (PDF 390 kb)Additional file 16:Figure S9. Splicing speckle morphology is not affected by the absence of H2A.B.3. Round spermatids from wt (a) and H2A.B.3−/y (b) were indirectly immunostained with anti-Y12 antibody and counterstained with DAPI. Scale bar is 10 μm. White arrows show accumulation of Y12 signal in DAPI-depleted regions. The immunostaining pattern showed no difference in Y12 localization. (PDF 1236 kb)Additional file 17:Figure S10. H3K4me3 accumulates in euchromatin of H2A.B.3 KO round spermatids. Early round spermatids (ERS) and late round spermatids (LRS) were immunostained with H3K9me2 (panel a—red) and H3K4me3 (panel b—red) and Lectin PNA . DNA was co-stained with DAPI (blue). Scale bar is 10 μm. The quantification of H3K9me2 and H3K4me3 was based on the ratio of (CC − B)/(EU − B). *** ps0.001, ANOVA test. (PDF 1323 kb)Additional file 18:Figure S11. Differentially expressed genes between H2A.B.3KO and wt round spermatids. (a) Principal component analysis of three sequenced H2A.B.3KO and wt mRNAseq libraries (each library combined mRNA from two wt or two H2A.B.3 KO mice). (b) Log2-changes in gene expression are plotted against significance (−log10-FDR). The plot illustrates in red those genes Additional file 20:Figure S12. Loss of H2A.B.3 affects pre-mRNA splicing. The analysis is based on SUPPA2. Illustrated is the distribution of altered splicing events by plotting the difference of percentage-spliced-in  versus transcript abundance of the transcripts associated with the splicing event (TPM) for two different conditions. The light gray dots correspond to  values based on pairwise comparisons of WT versus H2A.B.3 KO replicates  and TPM values averaged across replicates for each condition. The dark gray dots correspond to  values after averaging PSI and TPM values across replicates for each condition. The dark gray dots show the difference in mean (averaged across replicates) PSI values and mean (averaged across replicates and conditions) TPM values. The blue dots represent events based on the replicate-averaged PSI values (the dark gray dots) that are statistically significant at an FDR of 5% Additional file 22:Table S10. Results of the differential splicing analysis showing alternative 5′ splice site usage comparing wt with H2A.B.3 KO round spermatids. (XLSX 31 kb)Additional file 23:Table S11. Results of the differential splicing analysis showing alternative 3′ splice site usage comparing wt with H2A.B.3 KO round spermatids. (XLSX 32 kb)Additional file 24:Table S12. Results of the differential splicing analysis showing alternative first exon usage comparing wt with H2A.B.3 KO round spermatids. (XLSX 90 kb)Additional file 25:Table S13. Results of the differential splicing analysis showing alternative last exon usage comparing wt with H2A.B.3 KO round spermatids. (XLSX 18 kb)Additional file 26:Table S14. Results of the differential splicing analysis showing the use of mutually exclusive exons comparing wt with H2A.B.3 KO round spermatids. (XLSX 15 kb)Additional file 27:Table S15. Results of the differential splicing analysis showing the number of retained introns comparing wt with H2A.B.3 KO round spermatids. (XLSX 15 kb)Additional file 28:Table S16. Results of the differential splicing analysis showing the number of skipped exons comparing wt with H2A.B.3 KO round spermatids. (XLSX 63 kb)Additional file 29:Figure S13. Differential splicing events. The differential splicing events for specific genes between wild type and H2A.B.3 KO identified by SUPPA analysis were visualized in the IGV browser. Representative images are shown for Fblim1 exon 6 , Styx intron 3 , Slc37a3 exon 5  and Traf7 exon 19 . These individual examples of differential splicing events were confirmed by qPCR (c and f). Purified round spermatids from four biological replicas of wild type and H2A.B.3 KO mice were used. β-actin was used as the internal control. Error bars represent standard deviation. Student T-test was used to calculate p values . (PDF 419 kb)Additional file 30:Table S17. PCR primers for genotyping of H2A.B.3 encoding genes in founders and the H2A.B3KO colonies. (PDF 53 kb)Additional file 31:Table S18. List of primers to amplify 19 putative Indels from TALEN mutant mice. Symbols: ∆, deletion. (PDF 72 kb)


In January 2019, two influenza A(H3N2) viruses carrying an I38T substitution in the polymerase acidic subunit (PA), which confers reduced susceptibility to baloxavir, were detected from epidemiologically unrelated hospitalised children in Japan. The viruses exhibited reduced susceptibility to baloxavir but were susceptible to neuraminidase inhibitors. Only one of the two children had been treated with baloxavir. An epidemiological analysis suggests possible transmission of the PA I38T mutant A(H3N2) virus among humans. The cap-dependent endonuclease inhibitor baloxavir marboxil became available in Japan in March 2018 for the treatment of influenza virus infection in patients aged 12 years and older and children younger than 12 years weighing at least 10 kg. Between October 2018 and January 2019, baloxavir was supplied to medical institutions that together serve ca 5.5 million people. In December 2018, we detected influenza AH3N2) viruses exhibiting reduced susceptibility to baloxavir from baloxavir-treated children aged 6 and 7 years [N2 viruseIn January 2019, we isolated two influenza A(H3N2) viruses, A/YOKOHAMA/87/2019 and A/YOKOHAMA/88/2019, from two hospitalised children . Prior twww.fludb.org) including 17,227 PA sequences from A(H3N2) viruses until December 2018 [Deep sequencing analysis of the isolates using MiSeq  revealed that A/YOKOHAMA/87/2019 and A/YOKOHAMA/88/2019 possessed the PA I38T substitution. These PA I38T mutant viruses possessed different PA sequences and therefore originated from different sources of infection. PA I38 is highly conserved in influenza A and B viruses [ber 2018 . Therefo50) values, which were calculated by using MikroWin 2000 software . To interpret the NA inhibitor susceptibility, the World Health Organization (WHO) criteria based on the fold change of IC50 values compared with reference IC50 values were applied [We determined the susceptibilities of the PA I38T mutant viruses to baloxavir and four neuraminidase (NA) inhibitors approved in Japan: oseltamivir, laninamivir, peramivir and zanamivir . Antivir50 values of the viruses to baloxavir and the NA inhibitors are shown in 50 values to baloxavir compared with the median IC50 value of A(H3N2) viruses isolated in the 2018/19 influenza season in Japan. No amino acid substitutions associated with reduced susceptibility to NA inhibitors were detected in either virus. These results indicate that the PA I38T mutant viruses had reduced susceptibility to baloxavir, but remained susceptible to NA inhibitors.The ICIn this study, we detected two PA I38T mutant AH3N2) viruses respectively from two hospitalised children. In addition, during our nationwide monitoring, we detected nine PA I38T or I38M mutant A(H3N2) viruses from baloxavir-treated patients . All of  viruses Of the two children described as infected with a PA I38T mutant virus in this report, one (infected with A/YOKOHAMA/88/2019) had not received baloxavir treatment. Because an influenza outbreak, with possible use of baloxavir, had occurred in this child’s preschool prior to their symptom onset, it could be that the child acquired the mutant virus there. This might indicate that the recently circulating A(H3N2) viruses with the PA I38T substitution have, to some extent, retained replication and possible transmission capability in humans. In this respect, the parents, and a sibling were also diagnosed with influenza 4, 5, or 6 days, respectively, after this child’s symptom onset. Moreover, concerning the second child affected by a mutant virus in this study (i.e. the patient infected with A/YOKOHAMA/87/2019), a sibling of this child was diagnosed with influenza 2 days after the child’s symptom onset. Although we could not obtain specimens from family members of either children, these observations could point to a possible transmission of the PA I38T mutant A(H3N2) viruses among humans.Among the 11 persons infected with PA I38T or I38M mutant AH3N2) viruses in the 2018/19 season in Japan, all but one were children younger than 12 years. In Phase III clinical trials of baloxavir marboxil, the PA I38T and I38M substitutions emerged in 36 9.7%) of 370 A(H3N2) viruses obtained from patients aged 12–64 years and in 18 (23.4%) of 77 A(H3N2) viruses obtained from children aged 6 months to < 12 years [ viruses % of 370 Koszalka et al. reported that influenza viruses circulating in the Asia-Pacific region between 2012 and 2018 were susceptible to baloxavir . In Japa


In cells over-expressing human or various mammalian orthologs of SLC22A24, we showed that steroid conjugates and bile acids were substrates of the transporter. Phylogenetic, genomic, and transcriptomic analyses suggested that SLC22A24 has a specialized role in the kidney and appears to function in the reabsorption of organic anions, and in particular, anionic steroids. Phenome-wide analysis showed that functional variants of SLC22A24 are associated with human disease such as cardiovascular diseases and acne, which have been linked to dysregulated steroid metabolism. Collectively, these functional genomic studies reveal a previously uncharacterized protein involved in steroid homeostasis, opening up new possibilities for SLC22A24 as a pharmacological target for regulating steroid levels.Variation in steroid hormone levels has wide implications for health and disease. The genes encoding the proteins involved in steroid disposition represent key determinants of interindividual variation in steroid levels and ultimately, their effects. Beginning with metabolomic data from genome-wide association studies (GWAS), we observed that genetic variants in the orphan transporter, SLC22A24 were significantly associated with levels of androsterone glucuronide and etiocholanolone glucuronide (sentinel SNPs p-value <1x10 Steroid hormones, ranging from sex steroids such as testosterone to glucocorticoids play key roles in human health and disease. Accordingly, the identification of the genes and proteins involved in their synthesis, disposition and elimination has been the subject of numerous genetic studies. We have been intrigued by recent studies demonstrating that genetic variants in or near a gene encoding SLC22A24 are strongly associated with steroid levels. SLC22A24 is an orphan transporter with no known ligands and no known biological functions. In this study, we use cellular and computational methods to show that SLC22A24 transports steroid conjugates, bile acids, and other dicarboxylic acids. Based on the direction of association of a common stop codon in SLC22A24 with lower levels of steroids, our studies suggest that the transporter functions to reabsorb steroid conjugates in the kidney, a surprising finding, given that conjugation pathways generally function to create polar molecules that are readily eliminated by the kidney. The absence of the transporter gene in many species and its presence in higher order primates both suggest that SLC22A24 plays a specialized role in steroid homeostasis. Overall, our studies indicate that SLC22A24 functions in the reabsorption of conjugated steroids in the kidney. SLC22A24, has puzzled researchers because it has no known role in steroid homeostasis -taurocholic acid (TA) (10–20 fold over empty vector control), [3H]-glycocholic acid (GA) (10–20 fold), [3H]-estrone sulfate (ES) (5–10 fold), [3H]-estradiol-17β-glucuronide (EG) (3–5 fold), and [3H]-androstanediol glucuronide (AG) (5–15 fold)  of 8.6 ± 0.3 μM, 17.5 ± 0.2 μM, 10.5 ± 1.1 μM, and 33.4 ± 2.5 μM, respectively (Cells transiently transfected with 15 fold) . The uptly 5 min . Kineticectively . Similarectively . The kinectively . Using tpara-aminohippuric acid (PAH), uric acid, orotic acid, alpha-ketoglutaric acid and tetradecanedioic acid  and two were monocarboxylic acids  . Howeveroic acid . The preoic acid , whereasoic acid . Furtheroic acid .Collectively, the data indicate that SLC22A24 transports various organic anions, including steroid compounds with anionic moieties  and non-steroid compounds with dicarboxylic acid moieties.3H]-estrone sulfate was not affected by pH between 5.5 to 8.5 and was not dependent on the presence of Na+ ions  20 known inhibitors or substrates of other organic anion transporters; (iii) 17 steroids or bile acids which are used clinically; (iv) 8 dicarboxylic acid metabolites identified in our metabolomic study; and (v) 20 steroid conjugates.The primary goal of the inhibition studies was to confirm that solutes identified in the metabolomic studies were ligands of SLC22A24 and to identify additional ligands of the transporter. The secondary goal was to determine whether known inhibitors of other organic anion transporters in the SLC22 family were inhibitors of SLC22A24. A total of 85 compounds were selected for inhibition studies of SLC22A24-mediated -taurocholic acid uptake by >50%. All 16 bile acids, 5 of the 15 unconjugated steroids, 15 of the 20 steroid conjugates, and 12 of the 22 other anionic non-steroidal compounds inhibited taurocholic acid uptake by >50% , whereas associations were weaker for pregnanediol-3-glucuronide (p = 2x10-7), which was a weaker inhibitor of SLC22A24-mediated taurocholic acid uptake   -taurocholic acid in HBSS) containing either 100 μM estrone sulfate as positive control, 1% DMSO as negative control or 100 μM of the selected test compounds. Each of the 24-well plates contained negative and positive controls. Uptake of [3H]-taurocholic acid was stopped after 10 min by washing twice with ice-cold HBSS. The cells were lysed in 900 μL of lysis buffer (0.1 N NaOH and 0.1% SDS in double distilled water) per well while shaking for up to 90 min. 820 μL of the lysate was then added to 2.5 mL of EcoLite scintillation fluid (MP Bio), and the radioactivity was determined on a LS6500 Scintillation Counter (Beckman Coulter). Values were corrected for protein concentration as determined with a BCA assay kit . All values were determined in triplicate, and final values are expressed as % uptake relative to negative control (1% DMSO). Methods used in the inhibition studies were from previous studies in our laboratory -estrone sulfate (with 1 μM unlabeled estrone sulfate) as a function of time in cells expressing SLC22A24 and SLC22A8. The uptake rates for estrone sulfate and two other steroid glucuronides, estradiol glucuronide and androstanediol glucuronide were evaluated at 1 min. (Middle and right) Uptake of two bile acids, [14C]-glycocholic acid (with 1 μM unlabeled glycocholic acid) and [3H]-taurocholic acid (with 1 μM unlabeled taurocholic acid) as a function of time in cells expressing SLC22A24 and SLC22A8 (only glycocholic acid). The uptake rate for glycocholic acid and taurocholic acid were evaluated at 1 min and 2 min respectively. Each point represents the uptake in SLC22A24- or SLC22A8-expressing cells minus that in empty vector cells. Underlying data are provided in (Left) Uptake of -Estrone sulfate (with 1 μM unlabeled estrone sulfate) was incubated in the Na+ buffer  or Na+ free buffer  for 10 min. Data shown are from the mean ± S.D. for representative experiments of n = 2. (B) [3H]-Estrone sulfate, [3H]-estradiol glucuronide or [3H]-taurocholic acid (with 1 μM unlabeled compound) was incubated in the HBSS buffer adjusted to pH 5.5, 7.4 and 8.5, and the cells were incubated for 10 min. Data shown are from the mean ± S.D. for representative experiments of n = 2. Underlying data are provided in (A) [(TIF)Click here for additional data file.S5 Fighttps://www.uniprot.org/) are shown in parentheses. Red arrow points to the human SLC22A24.The unique and stable entry identifiers from UniProtKB ((TIF)Click here for additional data file.S6 FigGFP fusion constructs were generated for the human, rabbit, horse, and mouse lemur SLC22A24 and the rat Slc22a9/Slc22a24. HEK293 cells were transiently transfected with the GFP fusion constructs. The cells were visualized by confocal microscopy to determine whether the GFP fused protein is expressed on plasma membrane.(TIF)Click here for additional data file.S7 Fig(A) GFP fusion constructs were generated for human SLC22A24 and SLC22A24-322. HEK293T cells were transient transfected with the GFP fusion constructs. The cells were visualized by confocal microscopy to determine whether the GFP fused protein is expressed on plasma membrane. (B) The uptake experiments of various anions were performed for 10 minutes in triplicate wells using trace amount of the radiolabeled compound. Experiments were repeated once. Underlying data are provided in (TIF)Click here for additional data file.S8 Fig(A) Zoom in of the SLC22A24 gene region (reverse direction) between exon 9 and exon 10 along with padding of 10 bases using UCSC browser. The allele “CT” from position 1 and position 2 resulted in formation of two different isoforms (splice variants) of SLC22A24. (B) Zoom in of the SLC22A24 amino acids between exon 9 and exon 10. Arrows show the differences in amino acid positions 533 and 534, which lead to SLC22A24-551 and SLC22A24 proteins.(TIF)Click here for additional data file.S9 Fig(A) Western blot of cells expressing empty vector, SLC22A24, SLC22A24 p.Tyr501Ter, and SLC22A24-322. The latter two were not expressed on the plasma membrane despite detectable mRNA levels. (B) Transcript levels of mRNA measured using Taqman assay in HEK293 Flp-In cells transiently expressing empty vector, SLC22A24, SLC22A24-322 and SLC22A24 p.Tyr501Ter. Underlying data are provided in (TIF)Click here for additional data file.S10 FigThe peptides of interest and their corresponding fragments used to quantify the protein levels of SLC22A24 and SLC22A24-551 are outlined in black. Peptide 1 corresponds to the SLC22A24 and SLC22A24-551 proteins. Peptides 2 and 3 correspond to SLC22A24 and SLC22A24-551, respectively.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 Table(XLSX)Click here for additional data file.S11 Table(DOCX)Click here for additional data file.S12 Table(XLSX)Click here for additional data file.S13 Table(XLSX)Click here for additional data file.S1 AppendixThese sequences in FASTA format is used to create phylogenetic tree in (TXT)Click here for additional data file.S2 AppendixThese sequences in FASTA format is used to create phylogenetic tree in (TXT)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Arabidopsis thaliana, their contribution to antiviral immunity in Nicotiana benthamiana, a model plant for plant-pathogen interaction studies, is not well understood. In this study, we investigated the role of N. benthamiana NbAGO2 gene against TMV (Tomato mosaic virus) infection. Silencing of NbAGO2 by transient expression of an hpRNA construct recovered GFP (Green fluorescent protein) expression in GFP-silenced plant, demonstrating that NbAGO2 participated in RNAi process in N. benthamiana. Expression of NbAGO2 was transcriptionally induced by both MeSA  treatment and TMV infection. Down-regulation of NbAGO2 gene by amiR-NbAGO2 transient expression compromised plant resistance against TMV infection. Inhibition of endogenous miR403a, a predicted regulatory microRNA of NbAGO2, reduced TMV infection. Our study provides evidence for the antiviral role of NbAGO2 against a Tobamovirus family virus TMV in N. benthamiana, and SA  mediates this by induction of NbAGO2 expression upon TMV infection. Our data also highlighted that miR403a was involved in TMV defense by regulation of target NbAGO2 gene in N. Benthamiana.RNAi (RNA interference) is an important defense response against virus infection in plants. The core machinery of the RNAi pathway in plants include DCL (Dicer Like), AGO (Argonaute) and RdRp (RNA dependent RNA polymerase). Although involvement of these RNAi components in virus infection responses was demonstrated in  Caenorhabditis elegans by injection of double-stranded RNA (dsRNA), which was therefore identified as the molecule mediating sequence specific inhibitory effects during RNAi process [Virus diseases threaten crop production and agriculture globally. Plant evolved and developed sophisticated mechanisms to defend against virus infections. RNA interference (RNAi) is an antiviral defense mechanism in plants. The history of our understanding of RNAi for viral defense can be traced back to 1928, when Wingard reported the recovery of virus infected plant from later virus infection . In 1998 process . In 1999 process . RNAi wa process ,7,8. BriArabidopsis thaliana, the functions of these two family proteins were heavily investigated. AGO1 plays key role against viruses like turnip crinckle virus  [Cucumovirus) [Potexvirus) [Potyvirus) [Geminivirus) [Plantago asiatica mosaic virus  [Caulimovirus) [Tobravirus) [Potyvirus) in maize [Tobamovirus) in tobacco [Bromovirus) and rice stripe virus , but not wheat dwarf geminivirus  [Nicotiana benthamiana , several but limited studies were reported on antiviral role of AGO and DCL family proteins. NbDCL4 plays key role against zucchini yellow mosaic virus  in N. benthamiana [Arabidopsis thaliana, but is not functional in N. benthamiana [N. benthamiana [Potyvirus) and the Y satellite of CMV but not TRV or CMV alone [NbAGO2 was broadly investigated in Arabidopsis thaliana, and its antiviral role in N. benthamiana was also reported for TBSV (Tombusvirus), PVX (Potexvirus), TuMV (Potyvirus) and TCV (Tombusvirus) [NbAGO2 in RNAi-mediated antiviral role against infection by TMV, a virus belonging to Tobamoviridae. From its first use in Arabidopsis thaliana in 1996, Artificial microRNA (amiRNA) has been applied in many model and monocot species including Oryza sativa and Brachypodium distachyon [NbAGO2 expression and investigated its role for TMV responses. Our results showed that NbAGO2 played key roles in antiviral responses against TMV in N. benthamiana, and miR403a and SA was implicated in these responses.Although both AGO and Dicer/DCL proteins are involved in RNAi process, their functions are diverged in defense responses against different viruses. Furthermore, there are many AGO and DCL members in plants and they showed specialized roles against different pathogens. In model system usvirus) , cucumbemovirus) , potato exvirus)  and turntyvirus) . AGO2 istyvirus) , TCV, CMtyvirus)  and PVX tyvirus) . AGO4 futyvirus) , CMV [18nivirus)  and Planexvirus) . DCL1 ismovirus) , while Dmovirus)  and TCV movirus) . RDR1 isravirus) , TuMV [2ravirus)  and CMV ravirus) . RDR2 coravirus)  and TuMVravirus) . In othein maize  and agai tobacco . In ricenivirus) . In virothamiana . Althougthamiana . NbRDR6 thamiana . NbRDR6 MV alone . AGO prousvirus) ,33. Therstachyon ,35,36. Vstachyon . We usedN. benthamiana plants were used. Wild type N. benthamiana was provided by Prof. Xiaohan Mo from the Yunnan Tobacco Research Institute  and the GFP16c line was provided by Prof. Feng Li from Huazhong Agricultural University . N. benthamiana seeds were cultured in sterilized nutrient soil and vermiculite (1:1) at growth temperature of 22–25 °C with photoperiod of 14:10 (light:dark) and 70% relative humidity. Three-leaf stage seedlings were used for following experiments. 0.5 mM MeSA  in water solution was sprayed on the leaves and the leaves were collected one day after treatment. Water solution was sprayed as mock treatment. For GFP visualization, TMV-GFP infected plants were illuminated with a hand-held UV lamp at 320 nm or under Nikon SMZ18 microscope .Wild type and 16c GFP (Green fluorescent protein)-transgenic (GFP16c) N. benthamiana leaves infected with TMV and mock solution were collected and frozen in liquid nitrogen after five hours. The collected leaves were ground in liquid nitrogen and soaked with 5 mL 60% ethanol, and the supernatant after centrifugation was used to detect absorbance at 310 nm, which was then converted to SA concentration according to the standard curve [The content of endogenous salicylic acid (SA) was determined by UV Vis Spectrophotometry. A set of standard solutions, containing 5, 10, 20, 30, and 40 μg/mL of SA, were prepared in 60% ethanol solution and one of the standard solution was used for measuring maximum absorption wavelength within a 280–320 nm wavelength range , which was used as a subsequent detection wavelength. The maximum absorption wavelength was 310 nm and the linear regression equation according to the absorbance of five standard solutions was deduced. rd curve .GFP gene, GFP interfering sequences were amplified by Forward primerEcoRV and then was subcloned into binary vector pCB2004B to produce hairpin construct for insert GFP fragment.For silencing of www.benthgenome.com website.For construction of viral pCV (CaLCuV)-based (cabbage leaf-curl virus) amiR vector, following oligonucleotides were designed by amiR design function in amiR1:amiR2:amiR3:Arabidopsis thaliana miR159b as template backbone and the amplicons were cloned into pCVA vector by XbaI/KpnI sites. The same amplicons were cloned into pBI121 vector by XbaI/KpnI sites to produce non-viral binary vector based amiR constructs.The above primers were used to amplify amiR fragment using NbAGO2 gene, open reading frame (ORF) region of NbAGO2 was amplified by the primersN. benthamiana and the amplicon was cloned into pBI121 vector by BamHI/SacI sites.To overexpress GTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAATGGTGCTATCCCCTATCCTGAGCTTgaattc-3′; miR393a, 5′-ctcgagCCAAAGGGCTAATAGCATGATGTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAATCCAAAGGGCTAATAGCATGATgaattc-3′; and miR403, 5′-ctcgagAGTTTGTGCCTAGTGAATCTAAGTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAATAGTTTGTGCCTAGTGAATCTAAgaattc-3′. The restriction endonuclease sites are shown in lower-case letter, and the STTM spacer sequences were underlined. The oligonucleotides and complementary sequences were annealed to produce dsDNA and this dsDNA was then ligated into the pGreen-GUS-Competitor vector by XhoI and EcoRI respectively.For development of STTM (Short tandem target mimic) constructs for miR390a, miR393a and miR403a, the following sequences and their complementary sequences were designed and synthesized. miR390a, 5′-ctcgagGGTGCTATCCCCTATCCTGAGCTTAgrobacterium tumefaciens GV3101 competent cells. The transformants were grown to optical density OD600 = 1 at 28 °C, cells were collected by centrifugation and resuspended in inoculation buffer (10 mM 2-(N-Morpholino)ethanesulfonic acid (MES), 10 mM MgCl2, 200 μM acetosyringone). OD600 values of agrobacterium inoculation solution were all adjusted to 1.0 unless stated otherwise. The inoculation solution was leaf-infiltrated into three-leaf stage N. benthamiana by needleless syringe. For pCV-based silencing experiment, pCVB was simultaneously transformed and co-infiltrated with recombinant pCVA vectors. For virus infection, infectious clone of TMV expressing green fluorescent protein (TMV-GFP), pJL24, was infected as the same way. For pJL24, OD600 value of Agrobacterium inoculation solution was 0.1. In all experiments, inoculation buffer with empty vector was used as mock treatment.The above constructs were transformed into 260/OD280 using NanoDrop2000 Spectrophotometer  and agarose gel electrophoresis. One µg of total RNA was used as template and cDNA was synthesized by random hexamer using GoScript Reverse Transcription System .Leaf tissues were grounded in liquid nitrogen by using mortar and pestle. The powder was homogenized in Trizol reagent  and extracted following manufacturer’s protocols. DNase was used to remove contaminated genomic DNA. Total RNA quality was confirmed by measurement of ODNbAGO2 level by qRT-PCR. The primer sets 5′-ATGTGAAATGGTACGGGCTGAA-3′ and 5′-CAACAAGGTTCCACTGGCATTT-3′ were used for NbAGO2. As an internal control, steady state level of N. benthamiana GAPDH gene was evaluated by primer set 5′-CTGACAAGGACAAGGCTGCT-3′ and 5′-AAGCAGCTCTTCCACCTCTC-3′. The ratio of mRNA level between treatment and mock samples was determined by the 2−ΔΔCT method. At least three biological replicates with two technical replications were carried out, and the differences were evaluated by t-test.The cDNAs were subjected to PCR experiment. The PCR reaction was run on qTOWER 2.2  equipment. Fast SYBR Green Master Mix  was used for evaluation of +, positively charged)  by an electrokinetic capillary apparatus. 32P or digoxigenin-labelled probes were prepared by the methods described as previously, and hybridization and signal visualization were performed as per the literatures [N. benthamiana NbPR1a gene, a contig sequence with transcript ID of JN247448 from N. benthamiana sequence database (www.benthgenome.com) was used and the primer pairs 5′-GGATGCCCATAACACAGCTC-3′ and 5′-CCTAGCACATCCAACACGAA-3′ were used for amplification of 313bp probe sequence. For N. benthamiana β-actin gene, a partial cDNA sequence (JQ256516.1) was used and the primer pairs 5′-CCCAAAGGCTAATCGTGAAA-3′ and 5′-GCAGCTTCCATTCCAATCAT-3′ were used for amplification of 483bp probe sequence. For TMV detection, nucleotide encoding capsid protein was used and the primer pairs 5′-CAAGCTCGAACTGTCGTTCA-3′ and 5′-GACCAGAGGTCCAAACCAAA-3′ were used for amplification of 352bp probe sequence.Ten µg of total RNA was resolved by agarose gel electrophoresis. RNAs were transferred from the gel to nylon membrane  -based vector pCVA under 35S promoter [NbAGO2 was transformed into Agrobacterium tumefaciens strain GV3101 and agro-infiltrated into N. benthamiana leaves together with pCVB. We used three different regions of NbAGO2 for amiR targeting, and named them amiR-NbAGO2-1, amiR-NbAGO2-2 and amiR-NbAGO2-3 respectively  was used. First, we used pCB2004B-hpGFP to transiently silence GFP expression in GFP16c plant. The result showed that GFP expression was largely silenced by transient overexpression of hpGFP by agroinfiltration, and both GFP fluorescence and GFP mRNA levels decreased by hpGFP expression. One should note that GFP signal in GFP16c plant was mostly localized in vascular tissue. Next, we used this system to investigate the involvement of NbAGO2 in RNA silencing. Down-regulation of NbAGO2 by amiR-NbAGO2 recovered both the GFP fluorescence and GFP mRNA levels in the hpGFP expressed GFP16c plant, while in the control plant GFP was significantly silenced  is reported to be broadly engaged in virus infection and defense response. We also investigated the involvement of SA in the TMV infection process in  by 24 h B. We the 1–4 DPI A. In tob 1–4 DPI B, and Nbthamiana A.NbAGO2 was responsive to TMV infection and MeSA treatment. Involvement of NbAGO2 in TMV infection was further investigated as follows. NbAGO2 was first silenced by pBI121-amiR-NbAGO2 agroinfiltration, and after one day the same leaf region was agroinfiltrated with TMV-GFP, an infectious TMV engineered to express green fluorescent protein, which was retained in the infiltrated area for at least 6 days. The inoculated leaves were monitored daily for the appearance of GFP fluorescence. As shown in NbAGO2 caused stronger accumulation of virus (GFP fluorescence) as compared to control empty vector (pBI121) inoculated leaves. In accordance with this, overexpression of NbAGO2 (Ox-NbAGO2) resulted in weaker virus accumulation, and this effect was abolished by simultaneous amiR-NbAGO2 expression. In addition, virus spread was reduced by treatment with MeSA , TCV, CaMV [OsRDR genes but not OsAGO genes [NbAGO2 was induced by TMV infection in virological model species N. benthamiana. NbAGO2 was shown to be involved in both RNAi and virus defense responses here. NbAGO2 was reported to mediate defense against tomato bushy stunt virus  [NbAGO2 compromise defenses against PVX, TuMV and TCV, but not Tombusvirus and CMV infections [Potexvirus) [AGO2 is a broad spectrum antiviral gene whose activity is achieved by RNAi mechanism. Artificial miRNA was shown to be effective to silence variety of genes in a diversity of plant species including N. benthamiana [Arabidopsis thaliana [N. tabacum [Oryza sativa [Glycine max [Solanum melongena [Medicago truncutula [Triticum aestivum [Vitis vinifera [Solanum tuberosum [Solanum lycopersicum [N. benthamiana AGO family genes via transient expression from both virus vector and binary vector. As infection of virus vector (pCV) hindered subsequent TMV infection, we used pBI121 for expression of amiRNA and its effects on TMV infection. It is interesting to note that AGO2 itself is targeted by miR403a in plants [NbAGO2 by STTM technology influenced TMV infection. As for the miR390a and miR393a, both of them were reported to bind to NbAGO2 protein competitively [NbAGO2 expression was induced by tomato ringspot virus  isolates ToRSV-Rasp1 or ToRSV-GYV, and silencing of NbAGO2 elevated ToRSV accumulation [NbAGO2 was involved in plant responses toward different families of viruses. Induction of RNAi pathway component genes by corresponding pathogens were reported in several manuscripts. In proteins ,53,64. Rnfection . ExpressCV, CaMV  and TCV CV, CaMV ,67. In rGO genes . Here, wusvirus) . CRISPR/fections . Transgeexvirus) . Our datthamiana , Arabidothaliana , N. taba tabacum , Oryza sa sativa , Glycinecine max , Solanumelongena , Medicaguncutula , Triticuaestivum , Vitis vvinifera , Solanumuberosum  and Solapersicum . Here, wn plants , and hertitively , therefomulation . This inNbAGO2 gene against TMV infection was investigated in N. benthamiana. NbAGO2 was demonstrated to be involved in RNAi process in N. benthamiana. NbAGO2 mRNA level was elevated by both TMV infection and MeSA treatment. Suppression of NbAGO2 gene by amiR technology compromised plant resistance against TMV infection. Inhibition of endogenous miR403a, a predicted regulatory microRNA of NbAGO2, reduced TMV infection. Taken all this together, we conclude that SA participates in NbAGO2 mediated TMV response and miR403a is also involved in this response by post-transcriptionally regulating NbAGO2 expression (The role of pression .


In the ODYSSEY CHOICE I trial, alirocumab 300 mg every 4 weeks (Q4W) was assessed in patients with hypercholesterolemia. Alirocumab efficacy and safety were evaluated in a patient subgroup with type 2 diabetes mellitus (T2DM) and who were receiving maximally tolerated statins with or without other lipid-lowering therapies.Participants received either alirocumab 300 mg Q4W  or placebo  for 48 weeks, with alirocumab dose adjustment to 150 mg every 2 weeks at Week (W) 12 if W8 low-density lipoprotein cholesterol (LDL-C) levels were ≥70 mg/dL or ≥ 100 mg/dL, depending on cardiovascular risk, or if LDL-C reduction was <30% from baseline. Efficacy end points included percentage change from baseline to W24 for lipids, and time-averaged LDL-C over W21 to W24.In individuals with T2DM, LDL-C reductions from baseline to W24 and the average of W21 to W24 were significantly greater with alirocumab  vs placebo. At W24, alirocumab significantly reduced levels of non–high-density lipoprotein cholesterol (HDL-C) and other lipids. At W24, 85.9% and 12.5% of individuals in the alirocumab and placebo groups, respectively, reached both non–HDL-C <100 mg/dL and LDL-C <70 mg/dL. At W12, In total, 18% of alirocumab-treated participants received dose adjustment. The most common treatment-emergent adverse events were upper respiratory tract infection and injection-site reaction. No clinically significant changes in fasting plasma glucose and glycated hemoglobin were observed.In individuals with T2DM, alirocumab 300 mg Q4W was generally well tolerated and efficacious in reducing atherogenic lipoproteins. e.g., LDL-C and non–HDL-C) in individuals with T2DM receiving maximally tolerated statin therapy.Alirocumab 300 mg Q4W was generally well tolerated and efficacious for improving lipid levels ( The leading cause of mortality and morbidity among individuals with type 2 diabetes mellitus (T2DM) is atherosclerotic cardiovascular disease –3. Low-dDespite statins and/or ezetimibe, many individuals with T2DM or type 1 diabetes mellitus (T1DM) have elevated LDL-C levels and therefore may be candidates for additional LLT with a proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitor , 8–10. IPresently, the 300 mg every 4 weeks (Q4W) dosing regimen has not been evaluated in individuals with T2DM. This analysis evaluated the efficacy and safety of alirocumab 300 mg Q4W (with possible dose adjustment to 150 mg Q2W) in a study population subgroup with T2DM who received maximally tolerated statins in the ODYSSEY CHOICE I study .Details about the CHOICE I study design and enrolled participants have been reported . BrieflyOnly individuals with a medical history of T2DM and who received maximally tolerated statin therapy with or without other LLTs were included in this subgroup analysis . Study participants were divided into CVD risk categories as previously described  16, 17), 1716, 1Efficacy end points included percentage change from baseline to W24 for calculated LDL-C and time-averaged LDL-C over W21 to W24, as well as percentage change from baseline to W24 for non–HDL-C, apolipoprotein (Apo) B, TGs, HDL-C, lipoprotein (a) [Lp(a)], Apo A1, and TG-rich lipoprotein cholesterol (TRL-C).Additional efficacy end points included the impact of dose adjustment, and the percentage of subjects achieving LDL-C <70 mg/dL, non–HDL-C <100 mg/dL, or both, at W24.β-quantification if TG values were >400 mg/dL. TG and HDL-C levels were determined using Centers for Disease Control and Prevention National Heart Lung Blood Institute Lipid Standardization Program assays . Apo B, Apo A1, and Lp(a) levels were determined using nephelometry. TRL-C level was calculated by subtracting LDL-C from non–HDL-C. Fasting plasma glucose (FPG) levels and glycated hemoglobin (HbA1c) were also assessed.The non–HDL-C level was calculated by subtracting HDL-C from total cholesterol. The LDL-C level was calculated using Friedewald formula and was also determined via Safety was assessed throughout the study. Treatment-emergent adverse events (TEAEs) were defined as any adverse events that developed, worsened, or became serious from the first dose of study drug to the last dose of study drug plus 70 days.All key lipid end points, except for Lp(a), TGs, and goal achievement, were analyzed using a mixed-effects model with repeated measures, with parameters to account for missing data, as previously reported , 18. Lp.The on-treatment population was defined as an all-randomized population who took at least one dose or part of a dose of study drug and had an LDL-C measurement available at baseline and at least one calculated LDL-C value during the efficacy treatment period and within one of the analysis windows up to W24. The on-treatment efficacy analysis included the proportion of individuals achieving predefined lipid goals, and LDL-C levels over time according to treatment status and W12 dose-adjustment status.Safety analyses included all randomized individuals who received at least one dose or part of a study drug. Descriptive statistics were used to analyze safety data.In total, 96 individuals identified as having T2DM and receiving maximally tolerated statins received alirocumab 300 mg Q4W (with possible dose adjustment to 150 mg Q2W at W12), and 50 received placebo. Baseline characteristics were generally similar in the alirocumab and placebo groups . No indiP < 0.0001 vs placebo group; P < 0.0001 vs placebo group; Alirocumab significantly changed LDL-C levels from baseline to W24 by −57.4%  . Mean (SD) FPG baseline levels were 127.0 (39.3) mg/dL for alirocumab and 136.4 (46.9) mg/dL for placebo. Mean (SD) FPG change from baseline at W24 was 9.0 (50.5) mg/dL in alirocumab-treated individuals and 0.8 (42.2) mg/dL in the placebo group (no significant difference between groups).In individuals with T2DM who required additional LDL-C lowering despite maximally tolerated statin (with or without other LLTs), alirocumab 300 mg Q4W (with possible dose adjustment to 150 mg Q2W) significantly reduced levels of LDL-C and non–HDL-C from baseline to W24 . The percentage change in LDL-C from baseline to averaged values from W21 to W24 were −67.9% for alirocumab and +0.9% for placebo. The changes in lipid parameters are consistent with findings from the ODYSSEY DM-INSULIN study  and prevThe variability in LDL-C levels over the 4-week dosing period reflects the pharmacokinetics and pharmacodynamics of alirocumab, with a less pronounced LDL-C lowering effect at 4 weeks after dosing resulting from clearance of alirocumab from the circulation over this period . SimilarAt W12, 18% of alirocumab-treated individuals received dose adjustment, resulting in similar LDL-C reductions at W24 compared with individuals who continued to receive 300 mg Q4W throughout. Similar results were observed in the overall CHOICE I population receiving alirocumab 300 mg Q4W (with possible dose adjustment to 150 mg Q2W) .Several consensus statements and lipid guidelines state it may be reasonable to consider therapy with PCSK9 inhibitors in individuals with atherosclerotic cardiovascular disease and/or T2DM (with target organ damage or with a major cardiovascular risk factor) who are not adequately treated with maximally tolerated statin therapy , 23.Non–HDL-C <100 mg/dL and LDL-C <70 mg/dL have been suggested as a target level in several lipid guidelines and consensus statements for individuals with T2DM and at least one cardiovascular risk factor and/or end organ damage , 10. In In this analysis, alirocumab-treated individuals demonstrated a −33.9% change in Lp(a) from baseline to W24 compared with placebo; similar reductions were observed with alirocumab 150 mg Q2W vs placebo (−25.1%) in pooled data analysis of 10 phase 3 ODYSSEY studies  have been associated with an increased risk of T2DM    . In stud = 0.98) . Alirocu = 0.98) .The results of this analysis suggest that the alirocumab 300 mg Q4W dosing regimen may provide an additional lipid treatment option to the alirocumab 75 or 150 mg Q2W dosing regimens in individuals with T2DM with high to very high CVD risk who are receiving maximally tolerated statin with or without other LLTs.


During May 20–October 13, 2018,The U.S. influenza surveillance system§§ for all reporting jurisdictions were minimal and, although a small number of jurisdictions have reported the geographic spread of influenza activity¶¶ as local, approximately 60% of all reporting jurisdictions reported sporadic activity. Data from CDC’s National Center for Health Statistics Mortality Surveillance System indicated that the percentage of deaths attributed to pneumonia and influenza remained below the epidemic thresholdDuring May 20–October 13, data obtained from the U.S. Outpatient Influenza-Like Illness Surveillance Network (ILINet) indicated that the weekly percentage of outpatient visits to health care providers for influenza-like illness (ILI)CDC serves as the WHO Collaborating Center for Surveillance, Epidemiology, and Control of Influenza, one of six WHO Collaborating Centers for Influenza in the WHO Global Influenza Surveillance and Response System (GISRS).https://www.ncbi.nlm.nih.gov/genbank or EpiFlu: https://www.gisaid.org/). CDC antigenically or genetically characterized 680 influenza viruses collected and submitted by U.S. laboratories and laboratories outside the United States during May 20–October 13, including 351 influenza A(H1N1)pdm09 viruses, 185 influenza A(H3N2) viruses, and 144 influenza B viruses.The components for the Northern Hemisphere 2018–19 influenza vaccines were selected in February 2018, during one of the twice-yearly WHO-sponsored vaccine consultation meetings. The recommended Northern Hemisphere 2018–19 trivalent influenza vaccine composition included an A/Michigan/45/2015 (H1N1)pdm09-like virus, an A/Singapore/INFIMH-16–0019/2016 (H3N2)-like virus, and a B/Colorado/06/2017-like virus (B/Victoria lineage), with an additional influenza B virus (B/Phuket/3073/2013-like [B/Yamagata lineage]) recommended for quadrivalent vaccines.Phylogenetic analysis of the A(H1N1)pdm09 hemagglutinin (HA) genes of viruses collected globally since May 20, 2018, identified viruses belonging to HA genetic subgroup 6B.1 . All of Among 185 influenza A(H3N2) viruses collected and sequenced since May 20, 2018, phylogenetic analyses indicated cocirculation of multiple clades and subgroups of HA genes. The HA genes of the viruses belonged to genetic groups 3C.2a, 3C.2a1, and 3C.3a, with 3C.2a predominating . The majThirty-six influenza B/Victoria-lineage viruses were phylogenetically analyzed. All HA genes belonged to genetic group V1A and 31 (86.1%), belonged to subgroup V1A.1, represented by B/Colorado/06/2017, the reference virus representing the B/Victoria lineage component of Northern Hemisphere 2018–19 influenza vaccines. The V1A.1 subgroup is characterized by a two amino acid deletion in the HA at residues 162–163. One virus belonging to the genetic group V1A-3DEL was identified. This virus had a three amino acid deletion (amino acid residues 162–164) in the HA; similar viruses were identified sporadically in several countries in recent months. Eighteen of 19 antigenically characterized B/Victoria lineage viruses (10 in and nine outside the United States) were well inhibited by ferret antisera raised against cell-propagated B/Colorado/06/2017-like viruses.Phylogenetic analysis of the influenza B/Yamagata lineage viruses sequenced showed that all HA genes belonged to genetic group Y3 . Among 6The WHO Collaborating Center for Surveillance, Epidemiology, and Control of Influenza at CDC tested 347 influenza virus specimens collected during May 20–October 13 from the United States and worldwide for resistance to oseltamivir, peramivir, and zanamivir, the influenza virus neuraminidase inhibitor antiviral medications currently approved for use against seasonal influenza. Among 134 influenza A(H1N1)pdm09 viruses (63 in and 71 outside the United States), 132 influenza A(H3N2) viruses (57 in and 75 outside the United States), and 81 influenza B viruses (44 in and 37 outside the United States) tested, all were susceptible to all three medications. High levels of resistance to the adamantanes (amantadine and rimantadine) persisted among influenza A(H1N1)pdm09 and influenza A(H3N2) viruses, which is consistent with the current recommendation to avoid use of these medications against influenza at this time.The WHO recommendations for influenza vaccine composition for the Southern Hemisphere 2019 season were made at the WHO Consultation and Information Meeting on the Composition of Influenza Virus Vaccines held September 24–27, 2018, in Atlanta, Georgia. The recommended components for the 2019 Southern Hemisphere egg-based influenza trivalent vaccines are an A/Michigan/45/2015 (H1N1)pdm09-like virus, an A/Switzerland/8060/2017 (H3N2)-like virus, and a B/Colorado/06/2017-like virus (B/Victoria lineage). For egg-based quadrivalent vaccines, an additional component, B/Phuket/3073/2013-like virus (B/Yamagata lineage), is recommended. It was recommended that the A(H3N2) component of non–egg-based vaccines be an A/Singapore/INFIMH-16–0019/2016-like virus. Compared with the composition of the 2018 Southern Hemisphere influenza vaccine formulation, these recommendations reflect an update to the influenza A(H3N2) component for egg-based vaccines, a change in the influenza B lineage included in the trivalent vaccine and a change in the influenza B/Victoria component. Compared with the composition of the Northern Hemisphere 2018–19 influenza vaccines, these recommendations reflect only one change, an update to the A(H3N2) component used in egg-based manufacturing. Fourteen human infections with novel influenza A viruses were reported in the United States during May 20–October 13. Influenza viruses that normally circulate in swine and not humans are called “variant” viruses when detected in humans and designated with the letter v after the subtype. One infection was associated with an influenza A(H3N2)v virus, and 13 were associated with influenza A(H1N2)v viruses. All but one infection occurred among persons aged <18 years. The A(H3N2)v virus infection was reported from Indiana in a patient who reported swine contact at an agricultural fair in the week before symptom onset. All A(H1N2)v virus infections were reported in August from three states: California (six cases), Ohio (four), and Michigan (three). Eleven of the 13 patients reported contact with swine at agricultural fairs, one reported attendance at an agricultural fair but no contact with swine, and one reported neither contact with swine nor attendance at an agricultural fair. Limited human-to-human transmission might have taken place with this last A(H1N2)v infection; however, no ongoing or sustained human-to-human transmission associated with any of these infections was identified. None of the novel influenza A virus infections resulted in hospitalization, and all patients recovered.The genome of the one A(H3N2)v virus(A/Indiana/27/2018) was closely related to A(H3N2)v viruses detected during 2017 and viruses known to circulate in the U.S. swine population. Antigenic testing showed reduced inhibition by ferret antisera raised to the nearest A(H3N2)v candidate vaccine virus (CVV), but postvaccination antisera from adults vaccinated with the 2017–18 influenza vaccine reacted with the virus at titers that were within fourfold of those against the homologous reference virus, A/Michigan/15/2014, representing the A(H3N2) component of the 2017–18 seasonal influenza vaccines. Postvaccination sera collected from children, however, had lower titers to this virus than to the A/Michigan/15/2014 homologous virus titer. These studies indicate that vaccination with the 2017–18 seasonal influenza vaccine might offer less protection against this A(H3N2)v virus for children than adults.All of the A(H1N2)v viruses detected had HA gene segments from the delta 2 sublineage of the swine influenza virus H1 HA lineage. The HA and neuraminidase gene segments of these viruses were closely related to 2017 and 2018 A(H1N2) influenza viruses circulating in the U.S. swine population, including swine identified at the agricultural fairs attended by infected persons and viruses sporadically detected in previous A(H1N2)v zoonotic infections. Antigenic testing demonstrated that all of the 2018 A(H1N2)v viruses were well inhibited by ferret antisera raised to the nearest CVV. HI reactivity of pooled, child and adult postvaccination antisera from persons vaccinated with the 2017–18 vaccine was below the limit of detection for all viruses tested. These studies indicate that vaccine viruses specially developed to prevent A(H1N2)v virus infections would be protective; however, vaccination with the seasonal vaccine would not offer any protection.In the United States, ILI activity remained below baseline levels during May 20–October 13, 2018; low levels of laboratory-confirmed influenza were reported as a result of a mix of influenza A and B. In the Southern Hemisphere, low levels of influenza activity were observed with a predominance of A(H1N1)pdm09 viruses. Analysis of available viruses suggests that minimal drift of viruses has occurred. https://www.cdc.gov/flu/about/disease/burden.htm). Influenza vaccination prevents millions of medical visits, tens of thousands of hospitalizations, and thousands of deaths each year, even with vaccine effectiveness estimates in the range of 40%–60%. Health care providers should urge their patients to get vaccinated by the end of October, if they have not already been vaccinated. Vaccination efforts should continue throughout the influenza season.Vaccination before the onset of influenza activity is the primary strategy to prevent influenza-associated illness and its potentially serious complications. A recent report indicated the high prevalence of influenza illnesses resulted in approximately 79,000 deaths and 960,000 hospitalizations during the 2017–18 influenza season  viruses since the selection of viruses for the 2018–19 Northern Hemisphere vaccines was made in February. However, the influenza A(H3N2) component for egg-based vaccines was updated to address genetic and antigenic changes that occur when A(H3N2) vaccine viruses are propagated in eggs. The A(H3N2) component was updated because sera against egg-propagated A/Switzerland/8060/2017 (H3N2) virus showed better reactivity with an increasing number of circulating A(H3N2) viruses than sera generated against egg-propagated A/Singapore/INFIMH-16-0019/2016. No changes were recommended for the A(H3N2) component of cell-manufactured or recombinant vaccines. It is difficult to predict which influenza virus will predominate or what the season will be like, but there will likely be cocirculation of influenza A(H1N1), A(H3N2), and B influenza viruses.https://www.cdc.gov/flu/spotlights/vaccine-reduces-risk-severe-illness.htm). These benefits are especially important for persons at high risk for serious influenza complications, including persons aged ≥65 years, children aged <5 years, pregnant women, and persons with certain underlying long-term medical conditions, including heart and lung disease, or diabetes.Annual influenza vaccination is the best method for preventing influenza infection and its potentially serious complications. In the United States, annual influenza vaccine is recommended for all persons aged ≥6 months who do not have a contraindication pdm09 viruses predominating in most regions. In the United States, influenza activity was low in all regions, typical for this time of year. Fourteen influenza variant virus infections were reported in the United States, and most were associated with exposure to swine. Annual influenza vaccination is recommended for all persons aged ≥6 months who do not have a contraindication. Providers should encourage influenza vaccination now prior to the increase of influenza activity.


ARG1 locus (chr6:131894344-131905472) and their functional effects on type 2 diabetes mellitus (T2DM) have not been thoroughly elucidated to date. The present study aimed to investigate an association between variant alleles at ARG1 locus and T2DM in patients. Two ARG1 single nucleotide polymorphisms (SNPs) were characterized in a representative sample of 500 patients with T2DM and 500 healthy volunteers. Serum lipid profile was studied by spectrophotometric analysis, while serum arginase-1 concentrations were determined by an enzyme-linked immunosorbent assay. The regions, encompassing target SNPs (rs2781665 and rs2781666), were amplified by polymerase chain reaction and genotypes were assigned by restriction digestions. A statistically significant increase was observed in the serum hs-CRP and arginase-1 levels in the subjects with T2DM than in controls . The variant genotypes of rs2781666 and rs2781665 were significantly associated with T2DM when compared with controls (P< 0.0001). Moreover, type 2 diabetic patients showed higher frequencies of T allele at rs2781666 and rs2781665 compared to the controls . Haplotype T-T (chr6: 131893247-131893559) mapped at rs2781665-A/T and rs2781666-G/T displays higher frequency in the subjects when compared to the healthy ethnically-matched control samples (P <0.0001). We wish to propose, the first ever observation to our knowledge that concluding high levels of arginase-1 and the ARG1 polymorphisms are possible causes to confer/augment the risk of T2DM in subjects originates in Pakistan.Genetic polymorphisms mapped in the  ARG1 gene and their association with T2DM have not been explained until recently.Type 2 diabetes mellitus (T2DM) is a chronic disease affecting 451 million public around the world . T2DM iARG1 (HGNC: 663) gene may modulate individual's susceptibility to diabetic complications  and HapMap database has been investigated to determine the association with cardiovascular disease phenotypes in European populations . Here we show an association between ARG1 SNPs, mapped in the upstream promoter region and T2DM in patients.Arginase-1 (EC 3.5.3.1) originally detected in hepatocytes, is also expressed in various cell types including endothelial cells, vascular smooth muscle cells and macrophages . Howeveal., 2007). Ethnical., 2007). The prThe research study presented here includes clinical, biochemical and genetic analyses of samples collected from patients with T2DM and their ethnicity-matched controls. The protocol was designed by following the Helsinki Declaration (2002), and was approved by the ethics review board (ERB) of COMSATS University Islamabad, Pakistan and Institutional Review and Ethics Board (IREB) of the Postgraduate Medical Institute, Peshawar, Pakistan. An informed consent was obtained from all participants of the study focused on the genetic, biochemical and, demographic data collection and analyses. One thousand samples, including 500 (mean age 44.3 ± 3.5 years) documented subjects with T2DM and 500 (mean age 44.9 ± 7.8 years) healthy ethnicity-matched controls were ascertained in this study. The standard inclusion criterion was applied based on negative blood relation between the groups. The diagnosis of T2DM was done by considering the fasting plasma glucose at ≥126 mg/dL or 2-hours plasma glucose at ≥200 mg/dL . All assays were performed according to the manufacturer's instructions using standard protocols. HbA1c was measured using an immunoturbidimetric method . Arginase-1 levels in the serum samples were determined by using Reddot Biotech (Canada) immunoassay kit. Arginase-1 standard was run on micro test plate and the antigen concentration, in ng/mL, was determined from the standard curve, measured by AMP Diagnostics ELISA reader (Austria). High-sensitivity C-reactive protein (hs-CRP) was estimated by Tina-quant C-reactive protein (latex) high sensitive assay using a Roche/Hitachi-904 chemistry analyzer, Roche Diagnostics; Indianapolis, USA  kits for the determination of fasting blood glucose levels, total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) from serum samples using SPECORDARG1 was investigated by polymerase chain reaction (PCR) using forward primer 5'-CGG AAG GAT CTT TAA GGT GCC-3' and reverse primer 5' -CCA TGT GTC CGA TGC AGT TCT G-3'. For the genotyping of rs2781665 polymorphism, forward primer 5' AAT ATC TAG GCA ATA TGA GGA ATA CC-3' and reverse primer 5'- CCT ATT GGT GGG AAA GAA C-3' were used. The PCR reaction mixture contained 3 µL (Approximately 150 µg) of genomic DNA, 2.5 µL (125 µg) of each forward and reverse primer, 5 µL of 10X PCR buffer (0.2 M of (NH4)2SO4, 0.8M Tris-HCl (pH 8.8) and 0.2 % w/v of Tween-20, 4 µL of 25 mM MgCl2 , 1 µL of the 20 mM dNTPs , and 0.5 µL (2.5 U) of Taq DNA polymerase (5 U/ µL)  in 31.5 µL of deionized water. PCR reactions were performed in ProFlex PCR System . PCR was carried out at: an initial denaturation step at 95 °C for 10 min, followed by 40 cycles at 95 °C for 30 s, 60 °C for 1 min, and 72 °C for 2 min, and a final extension at 72 °C for 10 min. PCR products were resolved in 2 % agarose gel through electrophoresis. TaiI  restriction enzyme was used for the digestion of 294 bp fragment harbouring rs2781666 SNP mapped in upstream promoter of ARG1. RFLP was performed in 0.2 mL tubes  in a total volume of 20 µL containing 12 µL of amplified products, 2 µL of 10X G buffer , 0.3 µL of TaiI enzyme, and 5.7 µL of deionized water. MaeIII restriction enzyme was used to investigate the 264 bp fragment harbouring rs2781665. The digested products were resolved in 3 % agarose gel through electrophoresis and visually inspected under ultraviolet light to assign genotypes, segregating in subject with T2DM, and healthy controls.Polymorphism (rs2781666) mapped in http://osse.bii.a-star.edu.sg/calculation2.php). P value < 0.05 was considered as statistically significant.Basic and clinical parameters of the study samples are mentioned as mean ± SD from the mean. Statistical analysis was performed with GraphPad Instat 3.05 . Comparison of basic and clinical variables was carried by Chi-square test and independent samples t-test. Hardy-Weinberg equilibrium was calculated by using Arlequin V3.0. Chi-square and Fisher's exact test were used to calculate the genotype and allele frequencies between the groups. Odd ratio and 95 % CI in the binary-logistic model were calculated for the assessment of association by using MedCalc Software . Statistical power calculation was done by using OSSE online tool . There was a significant difference in the distribution of genotypes for rs2781666 and rs2781665 between cases and controls , and ARG1 rs2781665-A/T polymorphism (P< 0.0001) were independently associated with T2DM. However, age and BMI were not associated with the T2DM in this study  is a predominant isoform, which is responsible for the oxidation of l-arginine to l-citrulline and nitric oxide (NO) in the vascular system . HoweveARG1 locus with T2DM remained significant in multivariate model when confounding variables were taken in consideration. The conclusion of this study is supported by prior observation in an experimental study showing that arginase-1 expression is up-regulated in the coronary artery of diabetic subjects . Subseqal., 2001) after tal., 2001; Wang etal., 2001) emergedARG1 locus and their associations to T2DM have not been studied to date. Therefore, it was of great interest to investigate the association of rs2781665 and rs2781666 at ARG1 locus to T2DM. Interestingly, data from the current investigation revealed a significant association of aforesaid SNPs to T2DM. The variant genotypes, at both loci, have shown a significant link to T2DM. The risk of the variant alleles at rs2781665 and rs2781666 were 1.9 and 1.7-fold respectively, higher in the subjects of T2DM. We observed that T-T haplotype rs2781665/rs2781666 (chr6: 131893247-131893559) at ARG1 locus confer the risk of T2DM. Being first ever attempt to study the genetic variants at ARG1 locus and their associations to T2DM is incomparable and invites the scientific community to reproduce/exclude our findings by designing similar studies in different populations and /or sub-populations. However, Sediri and colleagues . Studieues (2010). LikewiARG1 polymorphism with arginase-1 levels and hs-CRP in patients. The variant genotype at rs2781666 and rs2718665 are associated with significantly higher levels of arginase-1 and hs-CRP in diabetic cases. The functional correlations between ARG1 polymorphisms and the disease phenotype are still unknown. However, in another case-control study, the variant genotype of rs2781666 at ARG1 locus was significantly linked to the increased arginase activity in patients diagnosed with essential hypertension (EH). High arginase was also significantly associated to the reduced concentrations of nitrite and nitrate in subjects of EH . The fial., 2018). Positial., 2014). Data fARG1 in Pakistani samples. The second, we could not determine the nitric oxide catabolites in this study; therefore, we intend to extend our findings to investigate the relationship between arginase and nitric oxide in diabetic patients. Therefore, replicate studies are invited to achieve a better and accurate conclusion on the baseline results provided in our study. Our study has some limitation: the study is restricted to two tag SNPs of ARG1 polymorphisms at rs2781666 and rs2781665 to T2DM in subjects originated in Pakistan. The variant genotype/minor allele T at rs2781666 and rs2781665 are contributory factors in escalating the level of arginase-1. Therefore, we wish to propose single-nucleotide polymorphisms at ARG1 locus are likely to be associated in generation of T2DM phenotypes in Pakistani samples.In conclusion, we have demonstrated the association of arginase-1 levels and Authors are thankful to the Higher Education Commission, Islamabad, Pakistan, for funding under the project NO.21-523/SRGP/ R&D/HEC/201. We wish to thank all individuals for their volunteer participation in the study. The authors are thankful to the Humayoon Shafique Satti for their help in sample collection. The authors are thankful to Maahil Arshad, Victoria Park Collegiate Institute, Toronto, Canada, for proofreading the final draft of manuscript.No conflict of interest has been disclosed by the study investigators.


Since the spring of 2013, human infections with H7N9 viruses have been detected in China. Some of these viruses have become highly pathogenic. Highly and low pathogenic avian influenza H7N9 viruses are currently co-circulating with the seasonal influenza A viruses H3N2 and H1N1pdm09. Prompt identification and isolation of H7N9 patients is one measure to prevent the spread of H7N9 virus and help prevent a pandemic. The majority of commercially available point-of-care rapid influenza diagnostic kits can differentiate between influenza A and B viruses, but cannot distinguish between H7N9 viruses and seasonal influenza A viruses. Accordingly, we have developed a rapid diagnostic kit specific for the H7 subtype that is accessible, easy to use. Although the detection limit of this H7 kit is one-tenth lower than that of a commercially available rapid influenza A and B diagnostic kit of similar design, except for the specificity of the monoclonal antibodies used, this kit is highly specific, detecting only H7-subtype influenza viruses, including the recent highly pathogenic H7N9 viruses from humans, and does not show any non-specific reactions with other HA subtypes. This H7 kit will be of value for the early detection of H7N9-infected patients. Human infections with low pathogenic avian influenza (LPAI) H7N9 virus were first reported in the spring of 2013 in China . As of 1In the 2016–2017 influenza season, the fifth and largest wave of LPAI H7N9 occurred in southern China . In addiPrompt identification and isolation of H7N9 patients is one means to prevent the spread of H7N9 virus. However, we cannot distinguish influenza virus subtypes based on the symptoms of patients. Although the severity of H7N9 virus infection is generally higher than that of seasonal H3N2 and pdmH1N1 virus, but lower than that of H5N1 virus, the initial symptoms of human infection with avian H7N9 virus are similar to those caused by other subtypes . In addiThe research protocol for experiments with mice was approved by and is in accordance with the TAUNS Laboratories, Inc., Shizuoka, Japan . We used swabs from two healthy volunteers under a research protocol approved by the Research Ethics Review Committee of the Institute of Medical Science, University of Tokyo . Written informed consents were obtained from both subjects. All experiments with H7N9 viruses were performed in biosafety level 3 (BSL3) laboratories at the University of Tokyo  and at the Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences .EPI439504; PB1, EPI439508; PA, EPI439503; HA, EPI439507; NP, EPI439505; NA, EPI439509; M, EPI439506; NS, EPI439510)]. The resulting products were cloned into pHH21 plasmids. All constructs were subsequently sequenced to verify the absence of unwanted mutations. Then, Anhui/1 was generated by use of plasmid-based reverse genetics. Transfectant virus was collected and amplified in Madin–Darby canine kidney (MDCK) cells, cultured in Eagle’s minimal essential medium (MEM) containing 0.3% bovine serum albumin (BSA) and 0.75 μg/ml tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-trypsin. Amplified Anhui/1 virus was purified through a 10–50% sucrose density gradient and resuspended in phosphate-buffered saline (PBS). To inactivate Anhui/1, β-propiolactone  was added to the viruses, and incubated at 4°C for 16–24 h. Inactivation of Anhui/1 was confirmed by passaging the viruses twice in 10-day-old specific pathogen-free embryonated chicken eggs and then performing hemagglutination assays.A/Anhui/1/2013(H7N9) (Anhui/1) virus was engineered by using reverse genetics as described previously . BrieflyTo produce monoclonal antibodies (MAbs), 7-week-old female BALB/c mice  were immunized with inactivated Anhui/1 plus an adjuvant (unpublished data) four times at 2-week intervals and boosted at 5 or 6 days after the final immunization. Two or three days after boosting, hybridoma cell lines were established with the spleen cells of the immunized mice and myeloma cells (Sp2/O-Ag14) by using methods previously described . The reaTable 1): A/Chicken/Egypt/119S-NLQP/2011(H5N1) was kindly provided by Dr. A.-S. Arafa, Animal Health Research Institute, Egypt; A/duck/Gunma/466/2011(H7N9) was kindly provided by Dr. T. Saito, National Institute of Animal Health, Japan; A/Anhui/1/2013(H7N9) and A/Shanghai/1/2013(H7N9) were kindly provided by Dr. M. Tashiro, National Institute of Infectious Diseases, Japan; A/Guangdong/17SF003/2016(H7N9) and A/Taiwan/1/2017(H7N9) were kindly provided by Dr. T. Odagiri, National Institute of Infectious Diseases, Japan; A/duck/Hong Kong/301/78(H7N2) and A/seal/Massachusetts/1/80(H7N7) were kindly provided by Dr. H. Kida, Hokkaido University, Japan; all other viruses were isolated in our laboratory) were propagated in MDCK cells with MEM containing 0.3% BSA and 0.75 μg/ml TPCK-trypsin. The titers of these viruses were determined by using 50% tissue culture infectious dose (TCID50) in MDCK cells (Table 2) were propagated in the allantoic cavity of 10-day-old embryonated chicken eggs. The titers of these viruses were determined by using 50% egg infectious dose (EID50) in embryonated chicken eggs. The EID50 was calculated by using the method of Influenza viruses of various subtypes (CK cells . Recent We tested six frozen swabs (four from chickens and two from the surrounding environment) that were collected between 2014 and 2015 in China as part of our surveillance activities. H7N9 viruses were isolated from all six frozen swabs (data not shown). Six other swabs, collected in 2013 in Indonesia, were used to demonstrate the lack of reactivity with the sample matrix: two from chickens, two from ducks, and two from quails. No viruses were isolated from these six swabs (data not shown). We also tested clinical swabs from two healthy volunteers as negative controls.1–106 TCID50/100 μl or 101–106 EID50/100 μl) or undiluted virus solution were tested. One hundred microliters of sample was mixed with 700 μl of the extraction buffer . Three drops (80–120 μl) of the mixed solution were then dropped on to the sample placement region of the kit. Results were visualized after 3–8 min of incubation at room temperature. The minimum viral titers required for a positive reaction were determined twice or three times independently. The average titers of these two or three experiments were then considered the detection limit of the kit.The sensitivity and specificity of the H7 kit was compared with that of the commercially available influenza rapid diagnostic kit ImunoAce Flu , which can detect both influenza A and B viruses. Tenfold serially diluted (with MEM containing 0.3% BSA or PBS) viruses . To develop the kit, two MAbs, BL-101 and BL-102, produced from inactivated Anhui/1-immunized mice were selected on the basis of the result from the immunochromatographic assay. MAb BL-101 was conjugated with platinum-gold-colloid (2.5 μg of the antibody and platinum-gold-colloid complex/kit) to make a complex with the H7 antigens in the samples at the reagent pad region of the kit. MAb BL-102 was immobilized to the nitrocellulose membrane to capture the antibody at the result region (0.2 μg of antibody/kit) .The detection mechanism of the H7 kit is based on an immunochromatographic method that is almost identical to that of the commercially available rapid diagnostic kit ImunoAce Flu . ImunoAce Flu uses colloidal-platinum-gold beads, which are platinum-coated colloidal-gold beads; the detection sensitivity of these beads is more than twice that of colloidal-gold beads . BrieflyTable 1). All of the viruses reacted with ImunoAce Flu, which can detect type A and B viruses, whereas the H7 kit detected only H7 subtype influenza viruses and did not show any non-specific reactions with any other HA subtypes (Table 1). The detection sensitivity of the H7 kit was significantly lower than that of ImunoAce Flu. ImunoAce Flu gave positive results with 102–104.7 TCID50 of the viruses, whereas the H7 kit required 105.7–107.3 TCID50 of viruses (Table 1).The specificity and sensitivity of the H7 kit were tested against 15 strains of various subtypes .We analyzed the detection sensitivity of the H7 kit for currently circulating H7N9 viruses in China. Both ImunoAce Flu and the H7 kit gave positive results with all of the viruses tested. The detection limit of ImunoAce Flu ranged from 10Table 3). Both kits gave negative results with six frozen swabs from birds  from which we could not isolate influenza viruses (data not shown). In addition, both kits gave negative results with fresh clinical swabs from two healthy volunteers (data not shown). To examine whether materials in the clinical swabs interfered with the test results, we tested the detection limits of the ImunoAce Flu and H7 kits against A/Guangdong/17SF003/2016 (H7N9) with a fresh swab from a healthy volunteer. We found that the detection limits of both kits were identical to those for the virus alone as described in Table 1 (data not shown). This result indicates that materials in clinical swabs do not affect the test results.We also performed a retrospective analysis on frozen swabs from chickens and the surrounding environment that were collected between 2014 and 2015 in China. We selected six frozen swabs from which H7N9 viruses had been previously isolated, and which were positive with ImunoAce Flu. The H7 kit gave positive results with tow out of these swabs . Although we did not confirm how many live viruses there were in each frozen swab, the detection sensitivity of the H7 kit for viruses in frozen swabs was almost the same as that for isolated viruses (Tables 3).To determine the relative quantity of virus in each frozen swab, we performed real-time RT-PCR. Due to the limited amounts of samples available, we were able to perform real-time PCR only once. The cycle threshold (Ct) values of three H7N9 isolates, A/pigeon/Shanghai/S1069/2013, A/chicken/Guangdong/S4021/2014, and A/duck/Shanghai/SD016/2015, were used to generate a standard curve  and almost the same as that of their control kit, Capilia® Flu A+B (103.1–104.4 EID50/test) (50/test) . We prev50/test) . This in50/test) . Here weTable 3). Although the sensitivity of the H7 kit was 10 times lower (>105.59 EID50/100 μl) than that of ImunoAce Flu (>104.31 EID50/100 μl) (Table 3), these results demonstrate that the H7 kit could be used to detect H7 virus in swabs from patients. In addition, this H7 kit can detect viruses of different H7 subtypes (Table 1). This H7 kit will therefore be useful not only for analyzing human samples in the clinical setting but also in the field of veterinary medicine for earlier detection of HPAI H7 viruses. We tested only one H7N2 and one H7N7 virus and did not test other H7 viruses. Therefore, although this H7 kit has potential as a diagnostic kit for H7 viruses in general, its ability to detect H7 viruses other than H7N9 viruses needs to be tested in the future. In addition, identification of the epitopes of the MAbs used in our kit will help us to understand the potential changes in the sensitivity of the kit to H7 HA as the virus continues to change.In our retrospective analysis of six H7N9-positive frozen swabs, the H7 kit detected virus in two of these six swab samples that were positive with ImunoAce Flu (Since 2016, human infections with HPAI H7N9 viruses have been detected in China . To dateKI-H, JS, XW, YS-T, MI, KM, KN, SY, SW, HC, and YK conceived the experiments. KI-H, JS, XW, YS-T, MI, KM, TSL, KN, SY, and SW conducted the experiments. KI-H and YK wrote the manuscript. All authors reviewed the manuscript.YK has received speaker’s honoraria from Toyama Chemical Co., Ltd. and Astellas Pharma Inc., has received grant support from Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Company, Limited, Toyama Chemical Co., Ltd., TAUNS Laboratories, Inc., Otsuka Pharmaceutical Co., Ltd., and Denka Seiken Co., Ltd., and is a co-founder of FluGen Inc. KM, KN, and SW are employed by TAUNS Laboratories, Inc. TAUNS Laboratories, Inc. has patents “Japan patent JP3886000B” and “United States patent US7713328.” The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.