Puccinia graminis f. sp. tritici (Pgt), is a devastating foliar disease. The Ug99 race group has combined virulence to most stem rust (Sr) resistance genes deployed in wheat and is a threat to global wheat production. Here we identified a coiled-coil, nucleotide-binding leucine-rich repeat protein (NLR) completely linked to the Ug99 resistance gene Sr21 from Triticum monococcum. Loss-of-function mutations and transgenic complementation confirmed that this gene is Sr21. Sr21 transcripts were significantly higher at high temperatures, and this was associated with significant upregulation of pathogenesis related (PR) genes and increased levels of resistance at those temperatures. Introgression of Sr21 into hexaploid wheat resulted in lower levels of resistance than in diploid wheat, but transgenic hexaploid wheat lines with high levels of Sr21 expression showed high levels of resistance. Sr21 can be a valuable component of transgenic cassettes or gene pyramids combining multiple resistance genes against Ug99.Wheat stem rust, caused by  Sr21 that is effective against the Ug99 race group. We developed a diagnostic marker to accelerate its deployment in wheat breeding programs and demonstrated that the introduction of two Sr21 copies in transgenic wheat results in high levels of resistance. An unusual characteristic of Sr21 is its increased resistance to stem rust at high temperatures. We show here that this is associated with the ability of Sr21 to coordinate the upregulation of multiple pathogenesis related genes at high temperatures. These genes slow down the growth of the pathogen and result in the characteristic Sr21 intermediate resistance reaction at high temperatures. A better understanding of this temperature dependent resistance mechanism will be useful for controlling the rust pathogens in our changing environments.Wheat stem rust is a devastating disease that is threatening global wheat production. The emergence of new virulent races of this pathogen in Africa, including the Ug99 race group, has prompted global efforts to find effective resistance genes. We report here the identification of stem rust resistance gene  Puccinia graminis f. sp. tritici (henceforth Pgt), poses an urgent threat to global wheat production since new virulent races have overcome widely deployed resistance genes.More than 700 million tons of wheat are produced every year, but further increases are needed to feed a rapidly growing human population. Reducing yield losses caused by wheat pathogens can contribute to this goal. Wheat stem rust, caused by the fungal pathogen Pgt race was detected in Uganda that was virulent to the widely deployed stem rust resistance genes Sr31 and Sr38 [Pgt races [Sr24, Sr36, Sr9h and SrTmp [In 1998, a new gt races ,3, was vgt races . Since tgt races –8 and acnd SrTmp ), promptSr35 [Sr33 [Sr50 [Sr22 [Sr45 [Sr13 [Pgt resistance genes deployed as gene pyramids or in transgenic cassettes to provide durable resistance [The large and complex nature of the wheat genome has limited the number of cloned Ug99 resistance genes to six, including Sr35 , Sr33 [135 [Sr33 , Sr50 [133 [Sr50 , Sr22 [150 [Sr22 , Sr45 [145 [Sr13 . More resistance ,18.Sr21 was discovered in diploid wheat Triticum monococcum (genome Am), which is closely related to T. urartu, the progenitor of the A genome in polyploid wheat [Sr21, which confers a more effective resistance to the Ug99 race group at high temperatures (20–24°C) than at low temperature (16°C) [T. aestivum) [Sr21 to a 0.19 cM interval on the central region of chromosome arm 2AmL and showed that this region includes a cluster of NBS-LRR resistance genes in the colinear region in the A genome of T. aestivum [The stem rust resistance gene id wheat . Sr21, weratures –24°C thaeratures –24°C thaSr21, which encodes a coiled-coil nucleotide-binding leucine-rich repeat protein (NLR). Six pathogenesis-related (PR) genes showed increased transcript levels in Sr21-resistant genotypes inoculated with Pgt, only when plants were grown at high temperatures. This result provides a tentative explanation for the more effective Pgt resistance conferred by Sr21 at high temperatures. Finally, we identified five different resistance haplotypes of Sr21 and developed a diagnostic molecular marker to accelerate its deployment in breeding programs.Here, we report the identification, validation and characterization of the wheat stem rust resistant gene T. monococcum chromosome arm 2AmL including Sr21 is colinear with an 88-kb region in Brachypodium distachyon chromosome 5 flanked by genes Bradi5g22090 and Bradi5g22200 [B. distachyon genes present in this colinear region  in the proximal group (CJ961291 (RRM2) showed a recombination event with gene CNL1, which was completely linked to the phenotype, completing the proximal side of the physical map. After five cycles of library screening, BAC sequencing and marker development, we identified an additional recombination event between the phenotype and marker 179C18F7R7, which closed the distal end of the physical map.We screened a bacterial artificial chromosome (BAC) library of the resistant ion DV92  using thCNL1, CNL3, CNL5, and CNL6) and three NLR truncated genes with premature stop codons .Sequencing the 405-kb candidate region  that was absent in T. monococcum, whereas the T. monococcum region including cnl2, CNL3, cnl4 and CNL5 was missing in Chinese Spring and Zavitan  and Cscnl1b (Zacnl1b) were similar to T. monococcum CNL1 (~99% identity). Cscnl1a was interrupted by the insertion of a repetitive sequence in CS but not in Zavitan  from diploid accessions G3116  and PI 272557 . Using seven different pairs of gene-specific primers, we amplified CNL3 and CNL5 from DV92 but not from G3116 and PI 272557 , suggesting that this gene was not Sr21. By contrast, CNL1 was PCR-amplified from both resistant accessions (DV92 and G3116) but not from Pgt susceptible accession PI 272557.We sequenced the four NLR genes completely linked to the CNL1 was the best candidate gene for Sr21 among the NLR genes linked to Sr21. This hypothesis was further supported by expression data for these genes in transcriptome databases of DV92 and G3116 [Sr21, we only detected transcripts for CNL1 in both DV92 and G3116 databases  with ethyl methane sulfonate (EMS) and generated 1,151 M1 mutant plants. Among the 1,151 M2 mutant families screened with race BCCBC, we identified seven showing susceptible plants  in the susceptible common wheat variety Fielder. Among the 13 T0 plants, we prioritized five that showed higher transcript levels of CNL1 than Fielder, which has a non-functional copy of CNL1 . Some of the resistant transgenic plants showed even better levels of resistance than the CSSr21 positive control, which carries a Sr21 introgression from T. monococcum . A similar correlation  was observed between the T0 expression levels from all 13 transgenic lines and the copy number estimated by the TaqMan assay  and CSSr21 (hexaploid) and found no significant differences  domain, a central nucleotide-binding (NB) site, and a leucine-rich repeat (LRR) region, typical of many NLR proteins. A nuclear localization signal (NLS) was predicted in Sr21 between amino acids 141 and 152 (cNLS Mapper http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi).We compared the sequences of the database ) with thCNL1 alternative splicing forms, which differ only in their 3’ UTR regions (CNL1-1 to CNL1-5) in the transcriptomes of DV92 and G3116 [CNL1-6 to CNL1-10) in the 3' RACE reactions.Comparing different transcripts and genomic sequences, we identified a 210-bp 5’ untranslated region (UTR) and a 2,159-bp 3’ UTR. The 5’ UTR does not include any introns, whereas the 3’ UTR shows 2 to 4 introns depending on the alternative splice forms . These r regions . We detend G3116  and an aSr21 resistance is modulated by temperature [T. monococcum resistant plants grown at 24°C and 16°C. The plants at each temperature were further divided in mock-inoculated and inoculated with Pgt race BCCBC, and RNA samples were extracted six days after inoculation (80 clones per treatment were sequenced). χ2 tests showed no significant differences (P = 0.81) in the frequencies of alternative splice forms in inoculated vs. mock-inoculated plants (averaged across temperatures) but detected significant differences between temperatures  and CSSr21 (hexaploid). We took samples immediately after moving the plants inoculated with Pgt from a greenhouse (~20°C) to growth chambers at 16°C and 24°C , but detected significantly lower transcript levels in plants grown at 16°C than at 24°C , and in Pgt inoculated plants relative to mock-inoculated plants  (P = 0.02), but no clear trend was observed.For the samples collected at 1, 3 and 6 days post inoculation (dpi), we performed a four-way ANOVA for  0.0001) . A smallSr21 and G3116 described above and primers described in a previous study [PR) genes . For all six PR genes, transcript levels were significantly higher (P < 0.0001) in Pgt-inoculated plants than in mock-inoculated plants  but the interactions between temperature and inoculation were all highly significant in the combined ANOVA  in two-way factorial ANOVAs. In all cases, a strong upregulation of all six PR genes was observed after inoculation with race BCCBC relative to mock inoculation only in the resistant genotype CSSr21  effects for ploidy level and genotype (presence or absence of Sr21), and a very strong interaction between temperature and genotype (P < 0.0001). Plants having the Sr21 resistance gene showed smaller sporulation areas at high temperatures, whereas those without Sr21 showed significantly larger sporulation areas at high temperatures  differences between genotypes and between temperatures. As with the sporulation area, we also detected highly significant interactions between genotype and temperature for both methods  rather than with a rapid hypersensitive reaction.Fluorescent images of e denser , which sT. monococcum and T. urartu accessions with Pgt races MCCFC and BCCBC and confirmed the presence of Sr21 in 44 of them and its absence in 70. We sequenced the complete CNL1 gene from these accessions using three pairs of primers , six as R2 (MG601519), one as R3 (MG601520), six as R4 (MG601521) and three as R5 . S1 is likely a non-functional gene since it carries a frame shift mutation. Surprisingly, polymorphisms between R1 and S1 were concentrated in the CC-NBS domains (87.2% identity) whereas the LRR was more similar to CNL1 (99.2% identity), which suggests a recombination or conversion event. A phylogenetic analysis including the DNA coding regions of the different NLR genes in the CNL1 region, and the closest paralogues in B. distachyon and H. vulgare showed that the CNL1 S1 haplotype is more closely related to cnl7 than to the cluster including the different CNL1 haplotypes (Of the 30 plotypes .T. urartu accessions showed haplotype S2 (MG601524), which was similar to R1 . This haplotype also includes three accessions classified as T. monococcum subsp. aegilopoides in the NSGC. However, analysis of 12 additional genes showed T. urartu haplotypes in these three accessions, suggesting that they are misclassified (we counted them as T. monococcum in the numbers reported above). The S2 haplotype from T. urartu, and the related haplotypes found in wild tetraploid Zavitan and hexaploid cultivars Chinese Spring and Fielder corresponds to the pseudogene Cscnl1b from CNL1 sequence , showed a resistance reaction when inoculated with race MCCFC but were susceptible to BCCBC. Since Sr21 is resistant to both BCCBC and MCCFC, this result suggested that these three accessions carry a Pgt resistance gene different from Sr21.All 40 Sr21 resistant haplotypes introgressed into polyploid wheat, we designed primers based on the C1228W diagnostic polymorphism and the T1500R and H1501S polymorphisms that separate Sr21 resistant and susceptible haplotypes. PCR amplification with primers Sr21TRYF5R5 T. monoco Zavitan . A T. moe 5 Figs  suggestsears ago .Sr21 expression, we detected multiple alternative splicing forms of CNL1, which differed in the intron structure of the 3’ UTR region  and TTKSK (isolate 04KEN156/04) at the USDA-ARS Cereal Disease Laboratory and with race BCCBC (isolate 09CA115-2) at the University of California, Davis (UCD). Assays of response to race TTKSK were performed at 25°C during the day and 22°C during the night with a 16 h photoperiod  and WideSeq at Purdue Genomics Core Facility (https://www.purdue.edu/hla/sites/genomics/wideseq-2/), and were assembled using Galaxy [Sr21 region using the Triticeae Repeat Sequence Database (http://wheat.pw.usda.gov/ITMI/Repeats/blastrepeats3.html) and the genes using BLASTN / BLASTX searches in GenBank (http://www.ncbi.nlm.nih.gov/). These three websites were last accessed February 26, 2018.A Bacterial Artificial Chromosome (BAC) library from the resistant parent DV92  was usedg Galaxy ,47. We iSr21 (Sr21 resistance haplotype R1) with 0.8% ethyl methane sulphonate (EMS). Seeds from 1,151 independent M1 mutants were harvested, and 25 M2 seeds per family were planted and inoculated with Pgt race BCCBC (isolate 09CA115-2) at UCD. Twenty-five M3 seeds from susceptible M2 plants were retested with race MCCFC (isolate 59KS19) at the USDA-ARS Cereal Disease Laboratory.Mutant lines were generated by treating 10,000 seeds from the hexaploid wheat line CSRapid amplification of cDNA ends (RACE) was performed using total RNA extracted from leaves of resistant parent DV92. Both 5' RACE and 3' RACE were performed using the FirstChoice RLM-RACE Kit (Invitrogen) following the manufacturer’s instruction. The PCR products from Nested PCR amplifications were cloned using the TA cloning kit (Invitrogen).Sr21 was amplified from DV92 BAC clone 205J7 using Phusion High-Fidelity DNA Polymerase (New England BioLabs Inc.). This fragment, including the complete Sr21  coding region and introns, 2,772 bp upstream from the start codon, and 2,483 bp downstream from the stop codon, was cloned into the binary vector pLC41Hm. The resulting plasmid pLC41HmSr21 was transformed into Agrobacterium strain EHA105, and was transformed into the Ug99-susceptible wheat variety Fielder (CNL1 haplotype S2) at the UC Davis transformation facility (http://ucdptf.ucdavis.edu/). Primers HptmikiF/R developed from the hygromycin resistance gene and primers S21CNL1F5R5 and Sr21TRYF5R5 developed from CNL1 were used to confirm the presence of transgene . Eighty colonies from every 3' RACE reaction were PCR-amplified and sequenced using the Sanger method.Sequences from the 3' RACE described above and from the transcriptome databases of DV92 and G3116  revealedSr21-resistant diploid T. monococcum ssp. aegilopoides accession G3116 and hexaploid CSSr21 using Spectrum Plant Total RNA Kit (Sigma-Aldrich). First strand cDNA was synthesized from 1 µg of total RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Samples were collected from plants grown under the four treatments resulting from the combination of two temperatures (16°C or 24°C) and two inoculation treatments (Pgt race BCCBC or mock inoculation). Samples were collected immediately after inoculation (0 h) and 1, 3 and 6 days post inoculation (dpi). We quantified Sr21 expression using primers Sr21qRTF1R1 (PR) genes  using primers described before [Total RNA was extracted from leaves of 1qRTF1R1 . The samACTIN levels (the number of molecules in the target / the number of ACTIN molecules) using the 2ΔCT method as described before [We performed the qRT-PCR reactions on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems) using Fast SYBR GREEN Master Mix. Transcript levels were expressed as fold-d before . We calcT. monococcum accessions (28 cultivated and 16 wild type) resistant to races MCCFC and TTKSK and susceptible to races TTTTF, TRTTF and QFCSC, which were previously postulated to carry only Sr21  and BCCBC (isolate 09CA115-2) (at 24°C) for this study. We also confirmed the absence of Sr35 with the diagnostic marker developed from the cloned Sr35 gene [T. monococcum accessions showed heterogeneous resistance reactions to MCCFC  and were listed twice in T. monococcum accessions. The sequenced genomes of diploid wheat T. urartu (http://plants.ensembl.org/Triticum_urartu/Info/Index), tetraploid wheat Zavitan (https://wheat.pw.usda.gov/GG3/wildemmer), and hexaploid wheat Chinese Spring RefSeq v1 (https://urgi.versailles.inra.fr/blast_iwgsc/blast.php) were used to detect the corresponding CNL1 alleles.All r35 gene . Seeds fS1 Fig(A) Proximal chromosome walk group: 22 BACs from the region proximal to Sr21. All BACs were selected from the DV92 BAC library and fingerprinted with restriction enzyme HindIII [CJ961291 and 179C18F7R7 delimit a 405-kb candidate region for Sr21 (blue shaded square). (B) Distal chromosome walk group: 17 T. monococcum BACs from the region distal to Sr21. In both panels, BACs in the minimum tilling path that were sequenced are indicated in red. Colored ovals represent markers used for chromosome walking, whereas colored arrows represent genes identified in the BAC sequences.(PDF)Click here for additional data file.S2 FigCNL1 in 13 transgenic T0 plants based on three technical replicates from a single plant. Numbers below rectangles indicate the copy number of transgenes based on the TaqMan copy number assay. Transcript levels are expressed as fold-ACTIN using the 2ΔCT method. Fielder has a non-functional copy of CNL1.Transcript levels of (PDF)Click here for additional data file.S3 Fig(A) Six T1 plants from family T1Sr21-03 inoculated with Pgt race TTKSK (Ug99). (B) Six T1 plants from family T1Sr21-04 inoculated with TTKSK. The first row of numbers below the figure indicates the average pustule size estimated using the image analysis software ASSESS v.2.0 (n = 3). The second row of numbers is the estimated number of CNL1 copies in the transgenic plants based on a TaqMan copy number assay relative to CSSr21. Pustule size was inversely correlated with the estimated number of CNL1 copies .(PDF)Click here for additional data file.S4 FigCNL1 3’UTR (CNL1-1 to CNL1-10).Yellow rectangles indicate coding regions and dotted blue lines introns. Gray rectangles represent 3’ and 5’ untranslated regions (UTR). The transcriptome databases of DV92 and G3116 and sequencing of multiple clones from 3' RACE reactions revealed ten alternative splice variants of (PDF)Click here for additional data file.S5 FigPathogenesis Related genes PR1, PR2, PR3, PR4, PR5, and PR9 (TaPERO). Diploid T. monococcum (G3116) n = 4 and hexaploid T. aestivum (CSSr21) n = 3. Values were calculated using the 2ΔCT method relative to ACTIN endogenous control . Error bars indicate standard errors of the means. Lack of parallelism between lines in all graphs indicate significant interactions Click here for additional data file.S6 Fig(A-B) Inoculation with Pgt race BCCBC at 16°C (A) and 24°C (B). The hexaploid susceptible line is Chinese Spring  and the resistant line is CSSr21 (“Sr21”). The diploid susceptible line is T. monococcum PI 272557 (“-”) and the resistant line is G3116 (“Sr21”). Similar differences were observed before for Sr21 resistance to TTKSK at 16°C and 20°C [(PDF)Click here for additional data file.S7 Fig(A-D) Race BCCBC growth at 16°C. (A-B) Hexaploid wheat, (C-D) diploid wheat. (E-H) Race BCCBC growth at 24°C. (E-F) Hexaploid wheat, (G-H) diploid wheat. CSSr21 and G3116 are the hexaploid and diploid resistant lines carrying Sr21, respectively. CS and PI 272557 are the hexaploid and diploid susceptible lines, respectively. Pictures were taken five days post inoculation (dpi). Infected leaves were cleared with KOH and stained with WGA-FITC. Interaction graphs are presented in (PDF)Click here for additional data file.S8 FigT. monococcum Ug99 resistant (R) and susceptible (S) accessions. Four bottom lines are CNL1 closest homologs from T. urartu and A-genome of polyploid wheat. Red highlight indicates alleles present in a single haplotype. The first part of haplotype S1 is too divergent and is not presented here.(PDF)Click here for additional data file.S9 FigCNL1 haplotypes, linked CNL3, CNL5 and cnl7 genes (blue squares), and the closest predicted genes from T. urartu (T.u.), T. dicoccoides (Tdic), Triticum aestivum (Traes), Hordeum vulgare (HORVU), and Brachypodium distachyon (Bradi). Coding DNA sequences were aligned with muscle as implemented in Mega 7, and phylogenetic trees were then generated using the pair-wise deletion method .Neighbor-Joining tree analysis including (PDF)Click here for additional data file.S10 FigSr21 (R1), PI 427971-R (R4) and PI 427796 (R5) carrying the different Sr21 resistant haplotypes. A digested band of 836-bp  was detected in T. monococcum genotypes PI 427971-S and PI 538540 that carry the Sr21 susceptible haplotypes S3 or S4. No amplification product was detected with these primers for susceptible genotypes of T. monococcum PI 272557 (S1), T. urartu PI 428227 (S2), PI 428183 (S2), and the related susceptible haplotypes present in Kronos, Fielder and Chinese Spring (CS) (The undigested 951-bp PCR products (black arrow) are present in genotypes DV92 (R1), G3116 (R3), PI 306540 (R2), CSing (CS) .(PDF)Click here for additional data file.S1 TableT. monococcum BAC library, haplotype analysis, mutant screening, marker-assisted selection (MAS), expression analysis, 3’ and 5’ RACE, cloning and screening for transgenic studies, and copy number assays.Primers for high density genetic map, screening of the (PDF)Click here for additional data file.S2 Table1 plants and a TaqMan copy number assay of the transgenic plants relative to CSSr21 (1 copy).Number of insertions in each transgenic event based on T(PDF)Click here for additional data file.S3 Table2 analysis, the five less frequent alternative splice forms were merged .Alternative splicing forms were identified from multiple 3' RACE reactions . Eighty colonies (using TA-cloning) from every 3' RACE reaction were PCR amplified and sequenced using the Sanger method. Eighty clones were analyzed for each of the four conditions. For the χ(PDF)Click here for additional data file.S4 TablePgt treatments (inoculated and mock-inoculated), and four collection time points after inoculation . (A) Three-way ANOVA at time 0 immediately after inoculation and transfer from greenhouse at 20°C to the chambers at 16°C and 24°C. (B) Four-way ANOVA at 1, 3 and 6 dpi.Transcript levels at two temperatures (16°C and 24°C), two (PDF)Click here for additional data file.S5 TableP values of transcript levels of PR genes in CSSr21 and G3116 plants 6 dpi with Pgt race BCCBC or mock-inoculated at high (24°C) and low (16°C) temperatures. CSSr21 and G3116 indicate P values for the individual two-way ANOVAS. The last row indicates the ratio between the diploid and hexaploid average expression of the PR gene in the Pgt inoculated plants at 24°C.(PDF)Click here for additional data file.S6 TablePgt/host DNA ratio and area of fungal growth detected by fluorescence were determined at 5 dpi. Experiments with BCCBC were performed at 16°C and 24°C and those for TTKSK were performed at 16°C and 20°C and were reported before [Sporulation size was determined at 14 dpi, whereas (PDF)Click here for additional data file.S7 TableTriticum monococcum subsp. aegilopoides, Triticum monococcum subsp. monococcum and Triticum urartu) to five Pgt races TRTTF, TTKSK, TTTTF, QFCSC, and MCCFC. Infection types shown here were based on the previous study [Infection types in diploid accessions (us study .(PDF)Click here for additional data file.S8 TableCNL1 haplotypes among a collection of 74 T. monococcum and 40 T. urartu accessions.(PDF)Click here for additional data file.


Dear Editor,1 Through 2nd Feb, 17 H5N6 cases were documented (http://www.who.int/en/), and H5N6 viruses isolated from both humans and poultry showed marked reassortment.4 We have previously identified three major reassortant H5N6 viruses, including reassortants A, B and C.4 The viruses with these three reassortant forms have caused human infections.In May 2014, China officially reported the first human infection with highly pathogenic H5N6 subtype avian influenza virus (AIV) in Sichuan Province.In November 2016, Hunan and Guangxi provinces reported two additional H5N6 human cases, one from each province. Both human cases were characterized by severe pneumonia and resulted in fatal outcomes. Epidemiologic investigations revealed that both patients had a history of exposure to dead chickens. The Chinese National Influenza Center isolated H5N6 viruses in a biosafety level 3 facility by inoculating 0.2 mL of each original sample into 9- to 11-day-old specific pathogen-free (SPF) embryonated chicken egg allantoic cavities. The isolated viruses were termed A/Hunan/55555/2016  and A/Guangxi/55726/2016 .The eight gene segments of these two viruses were fully sequenced using an Ion Torrent PGM platform . Sequences of HN555 or GX726 viruses have been submitted to the Global Initiative on Sharing Avian Influenza Data (GISAID) with the accession numbers EPI873661–EPI873676.4 All internal genes of the GX726 virus clustered with the A/Environment/Guangxi/44389/2015 (H5N6) virus  genes showed that both HN555 and GX726 viruses belonged to clade 2.3.4.4 II . Neurami6) virus , which rBasic Local Alignment Search Tool (BLAST) results of the viral nucleotide sequences found that most genes of the GX726 virus had the highest similarity with previously isolated H5N6 viruses. However, the PB1 and PA genes of the HN555 virus exhibited the highest nucleotide sequence homology with the A/duck/Guangxi/175D12/2014  and A/duck/Zhejiang/6D7/2013  isolates, respectively . These r5 The amino acid 627E in the PB2 protein suggested a possible low viral pathogenicity in mice.6 No substitutions associated with drug resistance in the NA or M2 proteins were detected, indicating their sensitivity to neuraminidase inhibitors or adamantine.7We next evaluated whether these two novel genotype H5N6 viruses had gained any key mammalian-adapted substitutions. For the HA gene, both viruses had a multiple basic amino acid motif (RERRRKR↓G) at the cleavage sites, indicating their high pathogenicity in poultry. Substitution of Q226L or G228S (H3 numbering) was not detected in either virus, which indicated their preference for avian receptors.In summary, two additional human infections with highly pathogenic influenza A (H5N6) viruses were reported in November 2016 in mainland China. Both homology analysis of nucleotide sequences and phylogenetic analysis of two isolated H5N6 viruses showed that the HN555 virus represented a novel H5N6 genotype and may have been generated by the reassortment of circulating H5N6 viruses with other low-pathogenicity H3 viruses . GX726 r


Historically, the diagnosis of diffuse intrinsic pontine glioma (DIPG) was based on typical imaging findings and clinical characteristics instead of pathology. However, the discovery of mutations in histone H3 variants, and the availability of tumor material for molecular analysis, has led to a paradigm shift in DIPG research and clinical practice. Using data from whole-brain autopsies in a series of nine DIPG patients with known histone mutational status, we here aim to review histopathological characteristics with special focus on intratumoral heterogeneity (ITH) and histone 3 K27 trimethylation (H3 K27me3). All DIPGs showed marked histologic ITH, with 56% even showing focal areas resembling a WHO grade I phenotype. As expected, H3 K27me3 immunoreactivity was lost in the tumors that were H3 K27M-mutated . Strikingly, the H3K27 wildtype tumors  also contained H3 K27me3-immunonegative areas. Our study underscores the importance of the choice of the biopsy site, as ITH in DIPGs could theoretically lead to erroneous histological diagnoses with small biopsies. New in this respect is our finding that a substantial number of otherwise typical DIPGs has areas resembling WHO grade I tumors . Furthermore, our study shows that negative H3 K27me3 immunohistochemistry in a DIPG does not imply a H3 K27-mutant tumor. H3F3A), H3.2 (HIST2H3C), or H3.1 (HIST1H3B and HIST1H3C). Moreover, it was shown that these mutations are spatially conserved and result in both loss of H3 K27 trimethylation (H3 K27me3) and alteration of gene expression profiles, putatively driving gliomagenesis [2016 World Health Organization (WHO) Classification of Tumours of the Central Nervous System introduced “Diffuse midline glioma, H3 K27M-mutant” as a separate WHO grade IV entity, wherein DIPG is a relatively frequent subgroup [Diffuse intrinsic pontine glioma (DIPG), one of the deadliest childhood cancers , shows msubgroup . Consequsubgroup  that wasAll patients fulfilled the diagnostic MRI criteria for DIPG according to Barkovich et al , i.e. a All patients except the youngest one received radiotherapy at time of diagnosis . Per autopsy, an average total of 7 tumor samples from the pons, cerebellar peduncles, and cerebellar hemispheres were obtained. Additionally, an average of 27 samples of histopathologically normal tissue were obtained from adjacent areas cranially and caudally within the brainstem and from more distant cerebral regions, resulting in a total of 306 study samples across all patients.All DIPGs showed marked histologic ITH including areas with WHO grade II–IV histology. Additionally, focal areas were present with a pilocytic astrocytoma- and/or subependymoma-like phenotype , thus resembling WHO grade I lesions Figure . PilocytTable Based on our findings in an autopsy series of nine DIPG patients evaluated in the last eight years at the VU University Medical Center Amsterdam, we underscore that DIPG ITH may lead to misinterpretation in biopsy specimens at two distinct levels when using (immuno) histochemistry only:1. Histologic phenotype. Without exception, all DIPGs showed marked histologic ITH. Remarkably, 56% also showed focal areas with a pilocytic astrocytoma- and/or subependymoma-like phenotype resembling WHO grade I lesions. This finding is of interest, since many consider WHO grade I lesions not part of the histologic spectrum of DIPG, even in case of typical imaging features. Patients with a WHO grade I lesion upon biopsy were therefore often excluded from clinical DIPG trials. With our results we show that this may have potentially been a “false negative” decision. Of note, the survival of the patients with WHO grade I-like tumor regions was as poor as that of the others.2. Immunohistochemical assessment of H3 K27me3/H3 K27 status. As expected, all H3 K27M tumors (78%) lost H3 K27me3 immunoreactivity, irrespective of histological phenotype and grade. However, the two H3 K27 wildtype neoplasms also contained H3 K27me3-immunonegative areas, which were not clearly related to tumor morphology or grade. Using only H3 K27me3 immunohistochemistry may thus lead to a false positive diagnosis of H3 K27-mutant glioma. Focal loss of H3 K27me3 has been reported only once in 2 out of 76 high-grade gliomas [In conclusion, here we demonstrate that histologic phenotype and immunohistochemical staining for H3 K27 status in small DIPG biopsies can be deceptive. From a diagnostic point of view this means that to make the diagnosis DIPG, typical imaging- and clinical signs remain crucial. And, to make the diagnosis of the (overarching) entity diffuse midline glioma, H3 K27M-mutant, as defined in the WHO 2016 classification, next generation sequencing/Sanger sequencing for now seems more reliable than H3 K27M/H3 K27me3 immunohistochemistry. Future studies are needed to assess the correlation between (heterogeneous) focal loss of H3 K27me3 in H3 K27-wildtype DIPGs and the clinical behavior of these tumors. Last but not least, from a therapeutic point of view, the observed spatial heterogeneity in DIPGs suggests that future chemotherapy should be directed against intratumoral polyclonality using multiple drugs, instead of therapies targeting only a single (putative) target. Moreover, combining this with various administration modalities  and (re)irradiation schedules may help to better target the full range of this heterogeneous disease.Whole-brain autopsy was performed in nine DIPG patients diagnosed based on clinical and MRI features  accordinFour-μm-thick sections of formalin-fixed paraffin- embedded material were histochemically stained for Hematoxylin & Eosin (H&E). After heat-induced antigen retrieval in 0.01M citrate buffer (pH6), immunohistochemical staining was performed with antibodies against glial fibrillary acidic protein , neurofilaments 70-200 kDa , oligodendrocyte transcription factor 2 , Ki67 , epithelial membrane antigen , H3.3 K27M  and H3 K27me3 . Immunopositivity was detected with 3,3′-Diaminobenzidine (DAB) as chromogen. Six-μm-thick frozen tissue sections were used for fluorescence immunohistochemistry. Tissue sections were fixed in 4% paraformaldehyde, blocked for 30 minutes in phosphate buffered saline supplemented with 0.1% saponin and 5% normal goat serum, and incubated with antibodies isoform GFAP delta , generating somatic calls for splice sites or coding exons. For Sanger sequencing, the histone 3 sequences of interest were amplified by PCR andsubsequently sequenced by the dideoxy chain-termination method using the ABI PrismTM BigDye Terminator kit , which was run on the ABI Prism Genetic Analyser 3100 automatic DNA autosequencer (Perkin Elmer) and analysed with ABI sequence Alignment Editor software. Primers were designed using Oligo explorer 1.5 software (Genelink – primer sequences available upon request) and blasted against the human genome for specificity (NCBI).All nine tumors were molecularly characterized by next generation sequencing, and histone variants were validated with Sanger sequencing. Genomic DNA was isolated from DIPG tumors and from non-affected counterpart brain regionsusing the QIAamp DNA mini isolation kit . Next generation sequencing was executed by whole-genome paired-end sequencing as performed by Complete Genomics . Primary


DUOX2) impair hydrogen peroxide (H2O2) production and cause dyshormonogenesis. In addition, these mutations have been implicated in autosomal recessive congenital hypothyroidism (CH) with goiter. In this study, we identified DUOX2 mutations that were causative for CH and explored the effects of these mutations on DUOX2 function. Blood samples were collected from 10 infants born with CH and goiter to unrelated parents. We extracted genomic DNA and sequenced all exons by polymerase chain reaction direct sequencing. The effects of DUOX2 mutations were characterized by H2O2 production assays and cycloheximide (CHX) chase experiments. Sequence analysis revealed one novel DUOX2 mutation and one known DUOX2 mutation in unrelated families: c.1060C>T (p.R354W) and c.3616 G>A (p.A1206T). Both mutations impaired H2O2 production. CHX chase experiments demonstrated the DUOX2 mutants had shorter half-lives and degraded more rapidly than wild-type DUOX2. Our study identified two novel DUOX2 mutations in Chinese patients with CH and goiter, which were responsible for the deficit in the organification process.Mutations in the dual oxidase 2 gene ( TG) [TPO) [NIS) [DUOX2) [DUOXA2) [DEHAL1) [Congenital hypothyroidism (CH) is one of the most common neonatal endocrine disorders, presenting with abnormal growth and intellectual impairment. Symptoms are caused by reduced thyroid function. CH occurs in 1 in 3000–4000 births and affects twice as many females as males . WithoutTG) , thyropeG) [TPO) , sodium/O) [NIS) , dual ox [DUOX2) , DUOX ma[DUOXA2) , pendrin[DUOXA2) , and iod[DEHAL1) .TPO) and hydrogen peroxide (H2O2). H2O2 is used as a substrate in the iodination of tyrosine residues and the coupling of iodotyrosine residues to TG [2O2 generation system in the thyroid. Among them, DUOX1 and DUOX2 are essential enzymes in the production of H2O2. DUOXA1 and DUOXA2 enable the DUOX proteins to translocate to the plasma membrane and become fully active [DUOX1 and DUOX2 encode two very similar proteins that are inserted into the apical membrane of the thyroid follicular cell. DUOX2 expression is at least five times higher than DUOX1 [2O2 production causes CH. Defects in DUOX2 cause severe, permanent CH due to complete disruption of thyroid hormone synthesis or milder, transient hypothyroidism caused by insufficient quantities of thyroid hormones [A central part of thyroid hormone synthesis is iodide organification, which requires thyroid peroxidase -dependent generation of H2O2 [ide mRNA , 17. The of H2O2 . The str of H2O2 .DUOX2 mutations in 10 unrelated Chinese children with CH and goiter by standard polymerase chain reaction (PCR)-based sequencing and clarify genotype–phenotype relationships. We also examined the functional effect of the identified mutations on DUOX2 at the molecular level.The aim of this study was to screen DUOX2 from 10 unrelated patients revealed two heterozygous missense mutations in two patients. One was a novel missense mutation (c.1060C>T), which results in an arginine to tryptophan substitution at codon 354 in exon 10  and the perchlorate discharge test was positive (iodine uptake rate >30.51%). Levothyroxine (L-T4) replacement therapy was started immediately at an initial dose of 8.3 μg/kg per day. Replacement therapy was modified during follow-up according to clinical and hormonal evaluations to maintain normal serum TSH and FT4 levels. At 2.5 years of age, TSH, FT4, and FT3 concentrations were normal following a temporary withdrawal of L-T4 therapy for 4 weeks. Therefore, he was diagnosed with transient CH. The patient is now 4.5 years old. His physical and mental development is normal.Patient 1 with a p.R354W mutation was a male infant. He was born after full-term gestation by cesarean delivery from unrelated parents. His birth weight was 2,800 g. There was no family history of thyroid disease. Neonatal screening showed a high level of TSH , so he was recalled at the age of 23 days for further evaluation. At that time, his body weight was 3,600 g and his height/length was 53 cm. Biochemical evaluation showed a serum TSH level of 218 uIU/ml and a FT4 replacement therapy was started immediately at an initial dose of 8.8 ug/kg per day. The dose was adjusted according to clinical and hormonal evaluations to maintain normal serum TSH, FT4, and FT3 levels. When she was 2 years old, concentrations of TSH, FT4, and FT3 were normal following a temporary withdrawal of L-T4 therapy for 4 weeks. The patient is now 6 years old and her physical and mental development is normal, with no evidence of goiter.Patient 2 (P2), was a female subject with a p.A1206T mutation, born by cesarean delivery after full-term gestation to unrelated parents. Her birth weight was 2,650 g. There were no abnormal physical findings until neonatal screening at the age of 6 days, which revealed a high level of TSH (141.5 μIU/ml). The patient was diagnosed with CH according to the neonatal screening program. At the age of 21 days, the patient was recalled to confirm the test results, which showed a high TSH level (186.3 μIU/ml) and a low FT4 level (5.8 pmol/L). At the same time, a 99mTc thyroid scan showed a normally shaped orthotopic but mildly enlarged thyroid gland. L-T2O2 generation was measured after either DUOX2 alone or both DUOX2 and DUOXA2 were expressed in 293T cells , y Figure . One houy Figure .DUOX2 causes hypothyroidism because of insufficient H2O2 production. DUOX proteins require DUOX maturation factors (DUOXA1 or DUOXA2) for the proper translocation of DUOX from the endoplasmic reticulum to the apical plasma membrane, where H2O2 production takes place. Since Moreno et al. reported the first DUOX2 mutation in 2002 [DUOX2 mutations have been identified, including missense mutations, nonsense mutations, frame shifts, and splice-site mutations [The generation of hydrogen peroxide is a critical step in the synthesis of thyroid hormones. Mutation of utations , 22.DUOXA2 reconstituted system to characterize natural DUOX2 missense variants  at the molecular level and analyze their impacts on H2O2 generation, trafficking, stability, folding and DUOXA2 interactions [2O2-producing activity. The p.R376W [DUOX2 mutations cause completely different CH phenotypes. In vitro experiments have revealed obvious pathogenic functions for some and none for others.Grasberger used a ractions . The p.Q p.R376W  and p.Q3 p.R376W  mutation p.R376W  was founDUOX2. The novel mutation p.R354W is located in the extracellular peroxidase-like domain, and the known p.A1206T mutation is located in the 5th transmembrane helix segments. p.R354 and p.A1206 are highly conserved in other mammalian species and p.R354W and p.A1206T were not identified in 200 alleles from healthy subjects. The H2O2 production assay showed no impairment in H2O2-generating activity by WT DUOX2 protein or mutant proteins. Coexpression of WT proteins with DUOXA2 in HeLa cells significantly reconstituted H2O2 production. Mutant proteins were still unable to produce H2O2, which corresponded with the iodine organification defect observed in patients. We also found that mutant DUOX2 degraded faster than mature WT DUOX2.In our study, we described two missense mutations—p.R354W and p.A1206T in DUOX2 mutations in cases of mild hypothyroidism. Tonacchera identified a novel compound heterozygous mutation p.S911L/p.C1052Y in a child with CH and a eutopic thyroid gland [DUOX2 mutations and suggested the possible involvement of multiple gene defects or epigenetic mechanisms [DUOX2 deletion mutation (p.S965fsX994) in three children with subclinical hypothyroidism and known functional impairment of H2O2 production, confirming that monoallelic mutations of DUOX2 protein can cause different phenotypes [DUOX2 in a patient with permanent CH and PIOD [DUOX2 alleles.Each patient had an increased TSH and decreased T4 serum level after birth. The only clinical sign of hypothyroidism was goiter. Euthyrox treatment was started immediately after the diagnosis of CH, and was continued until 2 years of age. When serum FT3 and FT4 levels had returned to normal, the growth and development were normal. This demonstrated that the phenotypes of the two affected patients were both transient and mild and transient hypothyroidism was diagnosed. We previously identified novel mutation p.A1206T in two unrelated patients with transient hypothyroidism . This isid gland . Hoste [id gland  also repchanisms , 31. De enotypes . Muzza eDUOX2 mutations, genotype–phenotype correlations have become more complex than initially anticipated. The clinical spectrum of patients with DUOX2 mutation ranges from moderate to severe goitrous hypothyroidism. Yoshizawa-Ogasawara described two unrelated Japanese girls with a p.G488R mutation [DUOX2 identified biallelic mutations (p.E327X/p.H678R). This provided a new example of environmental modification of the CH phenotypes by genetic defects, which can potentially distort screening results.With the increasing number of reported mutation . One patmutation . MutatioDUOX2 mutations in Chinese patients with CH and goiter. In addition, we studied the functional significance of these mutants and revealed impaired H2O2 production and faster degradation in mutant DUOX2 proteins. Our findings confirm the genetic heterogeneity of DUOX2 mutations. However, further studies are required to elucidate the underlying mechanisms.In conclusion, we have identified two missense 4 withdrawal. Patients showing persistently high TSH values were defined as having permanent CH. To confirm the diagnosis of transient or permanent CH, all subjects were routinely examined. Blood samples were collected from patients only after obtaining written informed consent from their guardians and the study was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University. All experiments were performed in accordance with the approved guidelines.Ten unrelated CH patients  were recruited through the neonatal screening program in Shandong Province, China, between 2008 to 2013. Blood samples were collected from the heel and TSH levels were measured by enzyme-linked immunosorbent assay according to the same test standard. Subjects with elevated TSH (≥20 μIU/ml) levels were recalled for further evaluation. Serum TSH  and free thyroxin  were determined by electrochemiluminescence assay. CH is diagnosed by high serum TSH levels and low FT4 levels. Ten children were diagnosed with CH and goiter through a 99mTc thyroid scan or thyroid ultrasound examination. They underwent diagnostic reevaluation at 2–4 years of age, based on thyroid hormone testing after 1 month of L-TDUOX1, DUOXA1, DUOX2, TG and TPO mutation screening was negative in all 10 subjects. The human DUOX2 gene is encoded by 33 exons. Gene fragments covering the complete coding sequence, including intron-exon boundaries, of DUOX2 (GenBank NM_014080.4) were amplified using the appropriate primer pairs for exons 1–33 of the DUOX2, as reported previously [Genomic DNA was extracted from peripheral blood leukocytes using standard methods. eviously . IdenticDUOX2 and DUOXA2 open reading frames were amplified using native Pfu polymerase and cloned into pcDNA3.1 (Invitrogen). The primers for DUOX2 were 5′-GGGGTACCCCATGCTCCGTGCAAGACCAGAG-3′ and 5′-GCTCTAGAGCTCAGAAGTTCTCATAGTGGTGC-3′ and the primers for DUOXA2 were 5′-GGGGTACCCCATGACCCTGTGGAACGGCGTA-3′ and 5′-GCTCTAGAGCTCACAGGTTAGTGGTGATACA-3′. The two novel DUOX2 mutations p.R354W and p.A1206T were introduced into the DUOX2 expression vector by site-directed mutagenesis  using the following primers: p.R354W: F 5′-TGCCAGCTGTCATTTCTGGAAGGTCCT-3′ p.R354W:R 5′-AGAAATGACAGCTGGCATTTCTCATGTAG-3′ and p.A1206T: F5′-ATCATGTATGTCTTCACCTCCCACCA-3′ p.A120 6T:R5′-TGAAGACATACATGATGGCCAGGACC-3′. All constructs were confirmed by bidirectional DNA sequencing.cDNA was synthesized with reverse transcriptase M-MLV (Takara) by oligo(dT) priming of total RNA from a normal human thyroid gland. The DUOX2 (containing DUOX2 wild-type (WT) expression vector only), exon 10 (containing R354W DUOX2 mutant expression vector only), exon 28 (containing A1206T DUOX2 mutant expression vector only), DUOX2 and DUOXA2 (containing DUOX2 WT expression vector and DUOXA2 WT expression vector), exon 10 and DUOXA2 (containing R354W DUOX2 mutant expression vector and DUOXA2 WT expression vector), exon 28 and DUOXA2 (containing A1206T DUOX2 mutant expression vector and DUOXA2 WT expression vector), pcDNA3.1(containing an empty expression vector), and non transfected WT cells (no expression vectors). Hela cells and 293T cells were maintained in high-glucose Dulbecco's modified Eagle's medium supplemented with 100 U/ml penicillin, 100 ug/ml streptomycin, and 10% fetal bovine serum under humidified conditions with 5% CO2 at 37°C. The diluted DNA (200 ng DUOX2 WT or DUOX2 mutants with or without 40 ng DUOXA2) and the diluted transfection reagent (with 0.5μl Lipofectamine TM2000) were combined into DNA-Lipofectamine TM2000 complexes at room temperature for 20 minutes. When Hela cells and 293T cells reached 70−80% confluence, the DNA-Lipofectamine™ 2000 complexes were added into each well containing cells and medium and incubated at 37°C in a CO2 incubator for 6 hours, after which culture solution was replaced by complete culture medium.Before transfection, cells were divided into eight groups according to the expression vector to be transfected, including 2O2 production was determined in a reaction with cell-impermeable 10-acetyl-3,7-dihydroxyphenoxazine  in the presence of excess peroxidase, producing fluorescent resorufin [2O2 for 1 hour at 37°C. Relative fluorescence  of the medium was measured and corrected for Amplex Red oxidation in wells containing nontransfected cells. A calibration curve was used to convert changes in fluorescence intensity into absolute nanomoles and the H2O2 concentration was maintained within the linear range of the standard calibration curve throughout. Renilla luciferase activity from co-transfected pRL-Tk plasmid (Promega) was used as internal control [Forty-eight hours after transfection, Hesorufin . Briefly control .Twenty-four hours after transfection, reaction termination was performed by 100 μg/mL cycloheximide . Proteins were collected and extracted at different time intervals as follows: Cell pellets were washed once in PBS and resuspended in three volumes of CelLytic Lysis/Extraction Reagent (Sigma) with 1 mM PMSF. Cell suspension was incubated for 20 minutes on ice and cleared by centrifugation . Total protein concentrations were determined by the Bradford method. Protein extracts were boiled in SDS/β-mercaptoethanol sample buffer. Samples (45 μg) were loaded into each lane of 8% polyacrylamide gels, separated by electrophoresis, and blotted onto polyvinylidene fluoride membranes (Amersham Pharmacia Biotech) by electrophoretic transfer. Western blotting was performed with a rabbit anti-DUOX2 antibody (Abgent) at 1:500 dilution, and bound antibody was detected using horseradish-peroxidase-conjugated goat anti-rabbit secondary IgGs. Detection by chemiluminescence reaction was performed using the ECL kit . Actin was detected as a loading control with a mouse anti-β-actin antibody (Santa Cruz). Three independent experiments were performed.All data are expressed as mean ± standard deviation (SD) and were analyzed by one-way analysis of variance followed by Newman–Keuls multiple comparison test as appropriate (GraphPad Prism version 5 software). A P value of <0.05 was considered statistically significant.


Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses (SIVs) have become predominant in pig populations in China and have recently been reported to have the most potential to raise the next pandemic in humans. The mutation D701N in the PB2 protein, which accounts for 31% of H1N1 SIVs, has previously been shown to contribute to the adaptation of the highly pathogenic H5N1 or H7N7 avian influenza viruses in mammals. However, little is known of the effects of this substitution on the EA H1N1 viruses. Herein, we investigated the contributions of 701N in the PB2 protein to an EA H1N1 SIV (A/Hunan/42443/2015(H1N1), HuN EA-H1N1), which had 701D in the PB2 protein. Our results found that viral polymerase activity, viral replication, and pathogenicity in mice were indeed enhanced due to the introduction of 701N into the PB2 protein, and the increased viral growth was partly mediated by the host factor importin-α7. Thus, substantial attention should be paid to the D701N mutation in pig populations. The EA H1N1 SIVs were first detected in 19792 and have caused several human infections in Europe and Asia since then9. With the increased binding affinity to human-type receptors and the enhanced transmissibility in mammals, EA H1N1 virus has become one of the candidates with the greatest likelihood to raise pandemics11.Influenza A virus infection usually causes substantial mortality and morbidity. As the genetic mixing vessels for avian and human influenza viruses, pigs are the intermediate hosts for the adaption and pathogenicity of avian influenza viruses and, thus, are potentially capable of initiating pandemics in humans15. The D701N mutation in the PB2 protein was originally observed upon the adaption of H3N2, H5N1, and H7N7 viruses in mice18 and was associated with enhanced viral polymerase activity and pathogenicity20. Increased interaction between 701N and cellular importin-α, a component of the nuclear import machinery, was believed to contribute to adaptation, resulting in enhanced nuclear import of vRNPs22.The viral polymerase is a major determinant of interspecies transmission and pathogenesis25. One of the newly emerged reassortants, with two surface genes from the EA H1N1 virus and six internal genes from the 2009pdmH1N1 and classical swine influenza viruses, was reported to have higher pathogenicity in mice compared with the un-reassorted EA H1N1 virus9. These viruses contained amino acid 701D in the PB2 protein. Considering the possibility of EA H1N1 SIVs causing a future human influenza pandemic and the great significance of the 701N mutation in host adaptation, our study focused on the effects of the substitution of the mammalian signature 701N in the PB2 protein on this newly emerged reassortant (A/Hunan/42443/2015(H1N1), HuN EA-H1N1, accession nos. EPI691392-EPI691399 in the GISAID database) isolated from a severely ill child. This virus was a genetic reassortant of the EA H1N1 virus and had a D at position 701 in the PB2 protein9.In public databases, only D is found at position 701 in the PB2 protein of avian H1N1 influenza viruses, while both D and N are observed in swine and human H1N1 influenza viruses, with the percentages of N being 31.175 and 0.126%, respectively (Supplementary Table P < 0.01), with a 3-fold increase  were inoculated into groups of five mice. At the inoculation dose of 101 TCID50, none of the mice died, and the body weight loss was <10% for both viruses , indicating that importin-α7 was required for the efficient replication of both viruses. In wild-type A549 cells, the rgHuN-PB2701N viruses exhibited significantly higher replication levels than the rgHuN-PB2701D viruses at 72 hpi ; in contrast, in importin-α7 knockout cells, growth of these two viruses had no significant difference, despite the lower replication level of the rgHuN-PB2701D viruses  cells, human embryonic kidney (293T) cells and human type II alveolar epithelial (A549) cells were grown and maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , HEPES , penicillin (100 units/ml), and streptomycin .30. In brief, two pairs of small guide RNAs (sgRNAs) targeting exons 5 and 6 of the human importin-α7 gene were designed and cloned into the pX459 vector, and the recombinant plasmids were then co-transfected into A549 cells to perform genome modification. Successful knockout of importin-α7 in A549 cells was confirmed by DNA sequencing and Western blot analysis. For the detection of importin-α7, a rabbit anti-importin-α7 antibody (Abcam) was used, followed by a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Zhongshanjinqiao Biotech). Tubulin (Abcam) was used as a loading control.Importin-α7-KO A549 cells were generated using CRISPR/Cas9 technology as described as previouslyAA CAA GAG; PB2 D701N Reverse, CTC TTG TTT TCT TTG CCC. The presence of the introduced mutations and the absence of additional unwanted mutations were verified by sequencing of the whole cDNA.All eight gene segments of influenza virus A/Hunan/42443/2015(H1N1) were amplified by RT-PCR and cloned into the pHW2000 vector. A plasmid with a single point mutation at residue 701 in the PB2 gene was generated with the following primers: PB2 D701N Forward, GGG CAA AGA A701D and rgHuN-PB2701N) were generated by reverse genetics, as described previously31. In general, the eight gene segments were cloned into the pHW2000 vector and were co-transfected into 293T/MDCK co-cultured monolayers for 24 h. Culture supernatants were propagated in 9–11-day embryonated chicken eggs. Virus stocks were sequenced for verification, and virus titrations were determined on MDCK cells.The wild-type HuN EA-H1N1 virus encoded a 701D in the PB2 protein. The recombinant viruses  to reconstitute vRNPs. Mock transfections were performed with the two reporter constructs only. Luciferase activity was measured after 24 h of incubation according to the Promega Dual-Luciferase Reporter Assay kit protocol.Around 293 T cells were co-transfected with pHW2000-PB2/PB1/PA/NP (0.5 μg each) together with the reporter constructs (0.5 μg each) pFluc  and pRluc . Eight- to ten-week-old female C57BL/6 mice  were anesthetized with 1% pentobarbital sodium and inoculated intranasally with 50 μl of 10-fold serial dilutions of viruses in PBS. Mice receiving PBS were used as controls. Weight loss and survival were observed for 14 days. Mice with weight loss >30% were humanely killed. For detection of virus replication and lung tropism, mice infected with 10The processed lung lobes were embedded in paraffin and cut into 3–4-μm sections. One section was stained with hematoxylin and eosin to check the pathological changes, and another was immunohistochemically stained against influenza virus antigen with mouse anti-NP, kindly provided by Professor Ningshao Xia, the National Institute of Diagnostics and Vaccine Development for Infectious Diseases, Xiamen University. Antigens of influenza viruses were detected using a peroxidase-conjugated anti-mouse/rabbit secondary antibody (Zhongshanjinqiao Biotech).2 and 37 °C. After 1 h of incubation, cells were washed twice with PBS, and infection medium containing TPCK-trypsin was added. At time points 0, 24, 48, 72 and 96 h post-infection (hpi), supernatants were collected, and viral titers were determined on MDCK cells.A549 cells were infected at a MOI of 0.1 with the recombinant viruses for multicycle replication under the condition of 5% COWild-type and importin-α7 knockout A549 cells infected with the recombinant viruses were fixed in 4% paraformaldehyde at 1.5 and 4 hpi. After three washes with PBS, cells were then permeabilized with 0.1% Triton X-100 for 10 min at room temperature. Cells were rinsed once with PBS and then incubated with 2% BSA (blocking reagent) for 30 min at room temperature. Cells were then incubated with mouse anti-NP primary antibody (Abcam) diluted in 2% BSA (1:200) for 60 min at room temperature. After three washes with PBS, cells were incubated with anti-mouse IgG (H+L)/Alexa Fluor 594-conjugated secondary antibody  for 60 min at room temperature. Cells were washed once with PBS and stained with DAPI (Beyotime Biotechnology) for 10 min at room temperature. Cells were then washed with PBS three times and examined for fluorescence using a Zeiss LSM confocal microscope.t test  was calculated using GraphPad Prism software . A P value of <0.05 was considered significant.Student’s Supplementary Table S1. Summary of amino acid at position 701 in the PB2 protein of H1N1 influenza viruses isolated in Asian countries from 2008 to 2017


The knockout mice, hnRNP A1−/−, showed embryonic lethality because of muscle developmental defects. The blood pressure and heart rate of the heterozygous mice were higher than those of the wild-type mice, indicating heart function defects. We performed mouse exon arrays to study the muscle development mechanism. The processes regulated by hnRNP A1 included cell adhesion and muscle contraction. The expression levels of muscle development-related genes in hnRNP A1+/− mice were significantly different from those in wild-type mice, as detected using qRT-PCR. We further confirmed the alternative splicing patterns of muscle development-related genes including mef2c, lrrfip1, usp28 and abcc9. Alternative mRNA isoforms of these genes were increased in hnRNP A1+/− mice compared with wild-type mice. Furthermore, we revealed that the functionally similar hnRNP A2/B1 did not compensate for the expression of hnRNP A1 in organisms. In summary, our study demonstrated that hnRNP A1 plays a critical and irreplaceable role in embryonic muscle development by regulating the expression and alternative splicing of muscle-related genes.Heterogeneous ribonucleoprotein A1 (hnRNP A1) is crucial for regulating alternative splicing. Its integrated function within an organism has not, however, been identified. We generated hnRNP A1 knockout mice to study the role of hnRNP A1  This process plays a crucial regulatory role by altering the function, expression level and localization of gene products. It often occurs in response to the activities of key signalling pathways and is essential for development and differentiation . Given iisorders ,3.cis-regulatory elements located within alternative exons or introns and by trans-acting splicing factors. cis-regulatory elements include enhancers and silencers. Splicing enhancers and silencers are either exonic or intronic, depending on whether they function from an exonic or intronic location. Serine/arginine (SR) proteins and heterogeneous ribonucleoprotein (hnRNP) protein families are well-known trans-acting splicing factors that promote or inhibit splice site recognition. Exonic enhancers bind SR proteins, which promote spliceosome assembly, whereas silencers include proteins of the hnRNP family to interfere with spliceosome construction and exon inclusion [Alternative splicing is modulated by nclusion ,5.in vivo is unclear.The hnRNP A1 protein is one of the most studied hnRNP proteins and is a key player in messenger RNA (mRNA) metabolism and biogenesis. It promotes the regulation of the transcriptional functions of target genes and the alternative splicing of more than 25 identified genes. Other functions regulated by hnRNP A1 include telomere maintenance, mRNA nuclear export, mRNA translation and turnover, and microRNA processing . Howeverin vivo.Muscle was the first tissue studied for tissue-specific alternative splicing. To date, more than 1000 muscle-specific alternative splicing events have been reported . Alternain vivo. We examined the phenotype and physiological functions of these mice and investigated the molecules regulated by hnRNP A1.hnRNP A1 regulates numerous functions in cells. Its integrated function in an organism has not, however, been identified. In this study, we generated hnRNP A1 knockout mice to study the function of hnRNP A1 2.2.1.in vivo, we generated hnRNP A1 knockout mice. The hnRNP A1 gene is disrupted by deleting exons 2–7, a strategy illustrated in figure 1a. Polymerase chain reaction (PCR) was used to confirm that the founder mice were generated successfully (figure 1b). To generate homozygous hnRNP A1 knockout mice, we intercrossed heterozygous hnRNP A1+/− mice. The DNA and protein of hnRNP A1 in the mice were detected using PCR and western blotting (figure 1c). The hnRNP A1 protein was not be detected in the mice when western blotting was used, indicating that hnRNP A1 knockout mice were successfully generated. The number of mice for each genotype was then calculated. Surprisingly, the number of knockout (hnRNP A1−/−) newborn mice  was significantly lower than the expected value  according to Mendelian law, and these newborn mice died within 30 min. However, the number of heterozygous  and wild-type  newborn mice approximated our expectations . These data indicated that some homozygous mice might have been embryonic lethal. To test this hypothesis, we acquired embryo 18.5 (E18.5) fetal mice by using Caesarean section. We obtained 82 fetal mice sourced from more than 10 litters. There were 22 wild-type, 43 heterozygous and 17 homozygous mice. These results were similar to our expectations , supporting our speculation on embryonic lethality in the hnRNP A1 knockout mice. To study the phenotype of hnRNP A1 knockout mice, we used a whole-mount section to perform haematoxylin and eosin (H&E) staining. The body lengths of the knockout mice were shorter than those of the wild-type and heterozygous mice. However, the body lengths of the heterozygous mice were similar to those of the wild-type mice. Although all organs were observed in the knockout mice, the hearts in these mice were round because of dilation of the ventricles (figure 1d).To identify the role of hnRNP A1 2.2.We discovered dilated cardiomyopathy with myofibril hypoplasia in the hearts of the hnRNP A1 knockout mice using histology . The mushnRNP A1 were injected into the embryos of wild-type zebrafish at a concentration of 0.2 mM to specifically knockdown hnRNP A1, we discovered that approximately 77.7% (199 of 256 samples) of zebrafish embryos died prior to 24 h post fertilization (hpf), higher than the percentage of fish with scramble and PBS treatment . When the hnRNP A1 MOs were injected at a concentration of 0.15 mM into the embryos of transgenic line Tg(fli1:EGFP)y1 zebrafish, we discovered that 70.6% (173 of 245 samples) of injected embryos appeared abnormal in the post-dorsal axis at 72 hpf , although the defects were not lethal. Because the entire vascular system of embryos derived from the transgenic line was specifically tagged with a green fluorescent protein reporter, we observed the vasculature in embryos treated with 0.15 mM of hnRNP A1 MO. The development of intersegmental vessels (ISVs) was discovered to be misdirected and appeared to have lateral asymmetry , suggesting that the abnormality in the development of the post-dorsal axis may cause abnormal ISV branching. Additionally, the heart in the hnRNP A1-knockdown embryos showed oedema . Furthermore, the expression levels of hnRNP A1 were significantly decreased in hnRNP A1 knockdown zebrafish . This demonstrated that hnRNP A1 plays a crucial role during embryonic development.To confirm the function of hnRNP A1 in embryonic development, we used zebrafish as a second model animal. When the morpholino (MO) antisense oligonucleotides of 2.3.a), and the blood pressures, especially the systolic pressures, were also higher (b). Electrocardiography (ECG) was performed to examine the electrical activity of the heart. The RR interval, PR interval and P duration of the hnRNP A1 heterozygous mice were less than those of the wild-type mice, although the difference was not statistically significant . These data indicated that hnRNP A1 plays an essential role in cardiac function.The hnRNP A1 knockout mice had heart structure defects and the homozygous mice died within 30 min. Therefore, we investigated the heart function of the heterozygous mice even though they did not possess any obvious phenotype. The heart rates of the heterozygous mice were significantly higher than those of the wild-type mice a, and tho higher b. Electr2.4.hnRNP A1 heterozygous and wild-type mice were different . A total of 101 175 genes were analysed using microarrays. Among them, 6527 genes were upregulated whereas 6163 genes were downregulated in the heterozygous mice compared with the wild-type mice ; 5352 genes were upregulated whereas 6298 genes were downregulated in the knockout mice compared with the heterozygous mice ; and 4355 genes were upregulated whereas 4968 genes were downregulated in the knockout mice compared with the wild-type mice . The common changed genes were analysed using MetaCore software and the data were enriched by process networks. The processes regulated by hnRNP A1 include cell adhesion, muscle contraction, regulation of cytoskeleton rearrangement and development regulation of angiogenesis. The 10 processes affected the most by hnRNP A1 are shown in figure 4a.To understand the underlying mechanism by which hnRNP A1 regulates expression of muscle-related genes, we used exon arrays to determine changes in global gene expression and alternative splicing. The gene expression patterns of the hnRNP A1 knockout and b) that were previously reported to be related to skeletal muscle development [hnRNP A1 heterozygous mice than in wild-type mice (c). These differently expressed genes included mechanistic target of rapamycin (serine/threonine kinase) (mtor), myosin VI (myo6) and notch4. We further studied the protein levels of these molecules using western blotting. The protein expression levels of mtor, myo6 and notch4 were increased in the hnRNP A1-defect mice and consistent with their mRNA expressions . These data indicated that hnRNP A1 modulated the expression level of the muscle development-related genes.Because hnRNP A1 mice had muscle developmental defects, we chose for further study 24 muscle development-related genes b that weelopment . We confype mice c. These 2.5.a. We further confirmed the alternative splicing pattern of muscle development-related genes according to the microarray data. Four genes with alternative splicing patterns were analysed using reverse transcription (RT) PCR: myocyte enhancer factor 2C (mef2c), leucine rich repeat in FLII interacting protein 1 (lrrfip1), ubiquitin specific peptidase 28 (usp28) and ATP-binding cassette, sub-family C, member 9 (abcc9). These genes have been reported to have different splicing forms during muscle development [mef2c, lrrfip1, usp28 and abcc9 genes was decreased in the hnRNP A1 heterozygous mice compared with the hnRNP A1+/+ mice (figure 5b). The ratio of full to truncated length of each gene is shown in figure 5c. We also used real-time PCR to quantify each alternative splicing form of these genes (figure 5d). The ratios of the full to truncated length of each gene were decreased, which was consistent with the results of RT-PCR. These data indicated that hnRNP A1 regulated alternative splicing of muscle development-related genes in vivo.Alternative splicing is an essential function of hnRNP A1. We analysed the alternative splicing pattern of genes in the hnRNP A1-deficient mice using the microarray data. A total of 1 188 912 alternative splicing exons were screened in the microarrays. The processes regulated by hnRNP A1 in alternative splicing included neurophysiological and process transmission of nerve impulses, development regulation of angiogenesis, muscle contraction, signal transduction and neuropeptide signalling pathways, and development neurogenesis. The 10 most affected processes are presented in figure 5elopment . The ful2.6.in vivo. We detected the mRNA and protein expression levels of both hnRNP A1 and hnRNP A2/B1 in hnRNP A1 mutant mice. The levels of hnRNP A1 mRNA in various organs of the heterozygous mice were lower than those observed in the wild-type mice . However, the levels of hnRNP A2/B1 mRNA in various organs of the heterozygous mice were comparable with those observed in the wild-type mice (bottom of figure 6a). We further detected levels of hnRNP A1 protein using western blotting. The protein levels in various organs of the hnRNP A1 heterozygous mice were significantly lower than those observed in the wild-type mice (figure 6b). The levels of hnRNP A2/B1 protein in various organs of the heterozygous mice were similar to those observed in the wild-type mice (figure 6c). These data indicated that hnRNP A2/B1 did not compensate for the partial loss of hnRNP A1 expression.hnRNP A2/B1 is a molecular homologue of hnRNP A1. A previous study has shown that hnRNP A2 compensated for the protein expression of hnRNP A1 in HeLa cells with hnRNP A1 deficiency . We examype mice a, top. HhnRNP A1 heterozygous mice had heart function defects. The expression level of muscle development-related genes was different between hnRNP A1-deficient and wild-type mice. The different alternative splicing patterns of muscle development-related genes between hnRNP A1-deficient and wild-type mice were also demonstrated. Furthermore, the in vitro functionally similar hnRNP A2/B1 protein did not compensate for the expression and function of hnRNP A1. These data all indicate the crucial and irreplaceable functions of hnRNP A1 in an organism.In conclusion, the hnRNP A1 knockout mice had muscle defects in the heart, tongue, diaphragm and intercostal muscles, which ultimately caused death. The 3.in vivo is crucial. In this study, we generated hnRNP A1 knockout mice to investigate the role of hnRNP A1 in embryonic development. We discovered that hnRNP A1 knockout mice had muscle differential defects  were used. All animals were housed in the KMU Laboratory Animal Center under a 12 L : 12 D (08.00/20.00) cycle with free access to food and water. The experiments used eight- to 15-week-old 4.2.hnRNP A1 gene was knocked out by deletion of exons 2–7, resulting in frameshift and generating a premature translational stop codon in exon 8. To achieve deletion, a targeting vector was generated using a recombineering method [hnRNP A1 gene was obtained from a bacterial artificial chromosome clone, bMQ-281N24 , derived from 129 strains of mice. The hnRNP A1 gene fragment was inserted between NotI and SpeI of plasmid pL253. This construct was then inserted into intron 2 with loxP from plasmid pL452 and into intron 7 with neo cassette from plasmid pL451. The final targeting construct contained a homologous 5′ long arm of 11.1 kb and 3′ short arm of 6.5 kb. The MC1-thymidine kinase (TK) gene at the end of the hnRNP A1 gene served as a negative selection marker. This vector was confirmed using DNA sequencing. The correct construct was then electroporated into R1 hybrid embryonic stem (ES) cells and selected using both G418 (240 µg ml−1) and ganciclovir (2 µM) for neo and TK, respectively, as described in a previous study [0). These mice were further backcrossed with C57BL/6JNarl mice to produce mice with a pure genetic background. A patent for these mice was issued by the United States Patent and Trademark Office , and they are available from the Rodent Model Resource Center, Taiwan under accession no. RMRC13102.The g method . A 21.9 us study . The gen4.3.hnRNP A1+) were Ex-A1U (EU) 5′-TATAGCGGGATGTGACGTGTTTTG-3′ paired with In-A1 L (IL) 5′-AATGAATCAACACCCCGCAACAAC-3′ and producing products of 451 bp. The primers used for identifying the deleted sequence (hnRNP A1−) were Ex-A1U (EU) paired with Ex-A1 L (EL) 5′-ACTGCACCCACAATGCTTTAAGAG-3′ and producing products of 370 bp. PCR was performed at 95°C for 10 min; 35 cycles of 95°C for 30 s, 58°C for 45 s and 72°C for 1 min; and 72°C for 7 min.Routine genotyping was performed using DNA from mouse tails by PCR. The primers used in PCR to identify the non-mutated sequence  for 12 h. The entire embryos were then treated with 80% ethyl alcohol, trimmed, dehydrated, embedded in paraffin, sectioned at 3 μm thickness, and stained with H&E.4.5.−1. The PR and RR intervals were measured and recorded using a Dual Bio Amp  and PowerLab (ADInstruments). The records were analysed using Chart 5.0 (ADInstruments). Blood pressure and heart rate were measured under general anaesthesia using the BP-2000 Series II Blood Pressure Analysis System . The measurements were repeated 10 times per day.Before ECG was performed, mice were anaesthetized using 1% (v/v) isoflurane in oxygen at a rate of 1 l min4.6.etaCore software . The data discussed in this paper have been deposited in the National Center for Biotechnology Information's Gene Expression Omnibus (GEO) [The hearts of E18.5 mouse embryos were harvested for RNA microarray analysis. The RNA was extracted using TRIzol Reagent . The quality of the RNA was analysed using an RNA 6000 Nano LabChip kit  and a NanoDrop spectrophotometer . Total RNA integrity numbers were between 8 and 10. GeneChip Mouse Exon 1.0 ST arrays  were hybridized according to recommendations of Affymetrix using the Ambion WT Expression Kit protocol  and Affymetrix labelling and hybridization kits. Mouse Exon microarrays were hybridized with 10 µg of labelled DNA. Raw data, transcript data and exon data were controlled using Expression Console Software  at the Institut Curie microarray core facility. The data were analysed using Mus (GEO)  and are 4.7.hnRNP A1, forward 5′-TGG AAG CAA TTT TGG AGG TGG-3′ paired with reverse 5′- GGT TCC GTG GTT TAG CAA AGT-3′; for hnRNP A2/B1, forward 5′-AAG AAA TGC AGG AAG TCC AAA GT-3′ paired with reverse 5′-CTC CTC CAT AAC CAG GGC TAC-3′; and for gapdh, forward 5′-AAG AGG GAT GCT GCC CTT A-3′ paired with reverse 5′-TTG TCT ACG GGA CGA GGA AA-3′. PCR was performed at 95°C for 5 min and 40 cycles of 95°C for 10 s, 60°C for 20 s and 72°C for 5 s. A melting curve was then performed by heating to 95°C, subsequently cooling to 65°C for 60 s, and slowly reheating to 98°C at 0.11°C s−1 with continuous measurement, followed by a final cooling stage at 40°C for 10 s. The alternative splicing forms of muscle-related genes were detected using RT-PCR. The primers for each gene are listed in electronic supplementary material, table S2. PCR was performed at 95°C for 5 min and 30 cycles of 95°C for 30 s, 60°C for 45 s and 72°C for 60 s. The intensity of the signals was measured using LabWorks image analysis software .Total RNA was isolated from tissues using TRIzol Reagent (Invitrogen). A total of 2 µg RNA was reverse-transcribed into cDNA using SuperScript III Reverse Transcriptase (Invitrogen). The quantitative RT-PCR (qRT-PCR) was performed using the Roche LightCycler 480 Real-Time PCR System using the SYBR Green System (Roche). The primers for real-time PCR were as follows: for 4.8.abWorks. The antibodies used in this experiment were anti-hnRNP A1 , anti-hnRNP A2/B1 , anti-Actin , anti-tubulin 4a , anti-notch4 , anti-myo6 , anti-mtor , horse anti-mouse IgG-HRP  and donkey anti-goat IgG-HRP .Proteins were extracted from tissues using RIPA lysis buffer  and separated using SDS-PAGE. After electrophoresis, the proteins were transferred onto nitrocellulose membranes . The membranes were blocked with 5% skim milk solution in TBST  for 1 h. They were further treated with primary antibodies in TBST containing 1% skim milk solution for 1 h. After being washed three times with TBST, the membranes were then incubated with HRP-conjugated secondary antibodies for 1 h. Subsequently, the membranes were immersed in the ECL Plus substrate  for 5 min. The chemiluminescence signals were detected using X-ray film and analysed using L4.9.xcel 2010  and SPSS . A Student's t-test was conducted to evaluate differences between groups. A probability of less than 0.05 was considered statistically significant after normalization.Statistical analysis of the data was performed using Microsoft E