Renal homeostasis of oxalate and citrate is controlled by complex mechanisms including epithelial transport proteins such as the oxalate transporter, SLC26A6, and the citrate transporters, the SLC13’s. These transporters interact via the SLC26A6-STAS domain in vitro, however, the role of the Sulfate Transporter and Anti-Sigma factor antagonist (STAS) domain in Ca2+-oxalate stone formation was not investigated in humans. Here, we report two novel human SLC26A6 polymorphisms identified in the STAS domain of SLC26A6 in two heterozygous carriers. Intriguingly, these individuals have low urinary citrate, but different clinical manifestations. Our in vitro experiments indicate that the homolog mutations of SLC26A6(D23H/D673N) and SLC26A6(D673N) alone abolished the expression and function of SLC26A6, and impaired the regulation of SLC13-mediated citrate transport by SLC26A6. On the other hand, the SLC26A6(R621G) variant showed reduced SLC26A6 protein expression and membrane trafficking, retained full transport activity, but impaired the regulation of the citrate transporter. Accordingly, the human SLC26A6(D23H/D673N) carrier showed a dramatic reduction in urinary citrate concentrations which resulted in Ca2+-oxalate stones formation, as opposed to the carrier of SLC26A6(R621G). Our findings indicate that the human SLC26A6-STAS domain mutations differentially impair SLC26A6 expression, function, and regulation of citrate transporters. This interferes with citrate and oxalate homeostasis thus potentially predisposes to Ca2+-oxalate kidney stones.Impaired homeostasis of the carboxylic acids oxalate and citrate, dramatically increases the risk for the formation of Ca Site-directed mutants were generated with the QuikChange Lightning Mutagenesis Kit  and the appropriate primers. The products were verified by Sanger sequencing (Hylabs).versus 435 nm and detected at 530 nm. Cells were incubated with BCECF at room temperature for 5 min and washed with regular solution  for at least 10 min until stabilization of the fluorescent signal. Subsequently, the cells were perfused with regular solution until establishment of a base line. Next, regular solution was replaced with a HCO3− buffered solution  or, with a Cl− free solution (HCO3− buffered solution was papered with gluconate to replace Cl−). All HCO3− buffered solutions were gassed with 5% CO2 and 95% O2.Intracellular pH was measured using a single cell real-time imaging system equipped with anEclipse Ti inverted microscope , PE-4000 LED monochromator , andHamamatsu Flash 4.0LT camera . This system was also utilized foracquisition of the mKate images in +-pyrophosphate, 50 mM NaF, and pH was adjusted to 7.4. 1 mM Na+-orthovanadate, 1% Triton X-100, and a cocktail of protease inhibitors (Roche) were freshly added before each use]. The cells were placed on ice and scraped after addition of lysis buffer. Protein extracts were incubated with Protein G Sepharose beads  and anti-His antibody (1:100)  overnight at 4°C. The beads-protein complexes were incubated for 4 h at 4°C, centrifuged, and washed with lysis buffer four times. Samples were prepared for running on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by heating (37°C for 30 min) in SDS sample buffer. Subsequently, the samples were transferred to nitrocellulose membranes and incubated overnight at 4°C with anti-SLC26A6 (1:500)  and the next day exposed to the appropriate secondary antibody. Signal was developed using enhanced-chemiluminescence (ECL) substrate (CYANAGEN).HEK293T cells were transfected with the indicated plasmids and after 2 days, lysates were prepared [lysis buffer contained phosphate-buffered saline (PBS), 10 mM NaSurface expression was analyzed using biotinylation assay. HEKT cells transfected with the appropriate plasmids were incubated with 0.5 mg/ml EZ-LINK Sulfo-NHS-LC-Biotin  for 30 min on ice. The biotin was quenched using 50 mM glycine and lysates were prepared as previously described. Total protein concentration was assessed by BCA method. The lysates were incubated with Neutravidin agarose resin  for 2 h at 4°C and washed three times using lysis buffer. Protein were isolated by adding 50 μl sample buffer and western blot analysis was performed as previously described. The blots were analyzed after incubation of the membranes overnight either with anti-SLC26A6, anti-tRFP (Evrogen) antibodies, or anti-β-actin  antibody for 1 h at room temperature.+ succinate and 1 mCi 14C succinic acid  per 1.6 mM cold succinate and added to the cells. The cells were then washed twice with incubation solution and 0.5 ml NaOH (1M) was immediately added to lyse the cells. The lysates were then transferred to scintillation vials containing 0.250 ml of HCl (2M). Finally, radioactivity was determined by liquid scintillation counting using a Packard 1900CA TRI-CARB analyzer.Succinate is an established substrate for transport by NaDC-1 and is an accepted surrogate for citrate. HEK293T cells were transfected with the indicated plasmids and washed with PBS . Subsequently, an incubation solution containing 5 mM KCl, 10 mM HEPES, 10 mM glucose, and 140 mM NaCl (pH adjusted to 7.4) was supplemented with 1 mM NaThe putative structure of SLC26A6 (NCBI accession No. NP_075062.2) was predicted using HHPred software  with hig2+-oxalate kidney stone formers and 23 healthy non-stone formers, and identified two SLC26A6 polymorphisms located in the region that encodes for the intracellular STAS domain  or mutants. As shown in As previously reported, SLC26A6 controls oxalate clearance in the intestine and also regulates citrate reabsorption in the kidney proximal tubule . Either The essential function of the SLC26 transporters STAS domain in the interaction and regulation of partner proteins, including NaDC-1, suggests that mutations in this protein region may affect SLC26A6 interaction with and regulation of NaDC-1. To address this, we monitored NaDC-1 function by measuring succinate uptake into HEK293 cells expressing NaDC-1 alone or in the presence of either SLC26A6(WT) or mutants. Our results indicate that SLC26A6 significantly inhibits NaDC-1, yet, both STAS mutations dramatically impair the ability of SLC26A6 to inhibit NaDC-1 , SLC26A6(D674N), or SLC26A6(R621G) and monitored the expression by immunoblot. As presented in As shown in Together, our results, thus far, indicate that D674N (which corresponds to the human polymorphism D673N) dramatically impairs the expression of SLC26A6, and abolishes the trafficking of SLC26A6 to the plasma membrane and, consequently, hampers SLC26A6 transport function. On the other hand, the R621G mutant lowers SLC26A6 total expression and trafficking, however, the residual expression is sufficient to retain full transport activity. Notably, the low surface expression of both mutants relative to WT is insufficient to inhibit NaDC-1, since NaDC-1 inhibition is eliminated by D674N and R621G  abolished SLC26A6 expression, trafficking, transport function, and regulation of the citrate transporter, NaDC-1. Although the patient carries a compound D23H/D673N polymorphism, we focused on the STAS domain missense mutation D673N (we tested the homologous mutant D674N), which impaired protein expression even in the absence of D23H  to EO and by the Israel Science Foundation grants No. 271/16 and 2164/16 to EO. OM was supported by the National Institutes of Health R01DK081423, R01 DK115703, R01 DK091392, and P30 DK079328) and the Charles Pak Foundation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Low pathogenic avian influenza A(H9N2) virus is endemic worldwide and continually recruit internal genes to generate human-infecting H5N1, H5N6, H7N9, and H10N8 influenza variants. Here we show that hemagglutinin cleavage sites (HACS) of H9N2 viruses tended to mutate towards hydrophilic via evolutionary transition, and the tribasic HACS were found at high prevalence in Asia and the Middle East. Our finding suggested that the tribasic H9N2 viruses increased the viral replication, stability, pathogenicity and transmission in chickens and the virulence of mice compared to the monobasic H9N2 viruses. Notably, the enlarged stem-loop structures of HACS in the RNA region were found in the increasing tribasic H9N2 viruses. The enlarged HACS RNA secondary structures of H9N2 viruses did not influence the viral replication but accelerated the frequency of nucleotide insertion in HACS. With the prevailing tendency of the tribasic H9N2 viruses, the tribasic HACS in H9N2 viruses should be paid more attention. Jiahao Zhang, Kaixiong Ma, and Bo Li et al. identify a variant of the H9N2 avian influenza virus within the hemagglutinin cleavage site required for viral infection. They show that this variant leads to higher viral replication in cells and more virulent disease in both chickens and mice. Although frequently updated commercial vaccines were used in areas of endemicity, the influenza A (H9N2) viruses continued to persist in avian and wild birds. The broad prevalence of the influenza A (H9N2) virus in birds naturally increased the risk of transmission to mammals, including dogs, pigs, and humans8. From 2013 to 2018, 27 human cases were laboratory-confirmed in China, Egypt, and Bangladesh as influenza A(H9N2) viruses, while only 13 cases were reported prior to 201212, suggesting an increasing threat to human health. Strikingly, influenza A(H9N2) virus had continuously contributed to some zoonotic spillover events via the reassortment of cross-species variants, including the H5N1 influenza virus in HongKong in 1997, the H7N9 and H10N8 influenza viruses in 2013, and the H5N6 influenza virus in 201517, posing public health concerns.The influenza A (H9N2) viruses were enzootic in birds and undergone continuous evolution in North America, Europe, South Asia and the Middle East. The Eurasian lineage formed three distinct sublineages represented by their prototype strains: A/quail/HongKong/G1/97 (G1-like), A/duck/HongKong/Y280/97 (Y280-like), and A/chicken/HongKong/Y439/97 (Y439-like)18. For infection to occur, the HA0 precursor must be cleaved into the HA1 and HA2 subunits, thereby revealing a fusion peptide for membrane fusion19. Although the infection of H9N2 viruses is usually mild and localized to the respiratory and intestinal tracts due to the restriction of the proteases, the cleavage sites of distinct H9N2 lineages present are with the continuing evolution22. H9N2 viruses varied remarkably in the amino acid at the HACS. The nomenclature of the monobasic, dibasic, and tribasic influenza viruses was designated previously, with basic amino acids occurring once, twice, and three times, respectively. Viruses with the tribasic PAKSKR/G-motif and dibasic PARSSR/G-motif, PAKSSR, and PSRSSR/G-motif viruses in the Y280-sublineages and G1-sublineages had been found at high prevalence in Asia and the Middle East in birds and humans. The monobasic H9N2 viruses bearing one basic amino acid residue at the P1 position were frequently found in the Y439-sublineage and North America-lineage in wild birds; however, there had been no human infections. Thus, the main question is which HACS motifs in distinct lineages confer a replicative advantage to the virus in chickens and mammals and pose an increasing threat to humans.An essential step in infection by influenza A viruses is the proteolytic processing of the trimeric hemagglutinin cleavage site (HACS)24. A previous study suggested that the enlarged stem-loop structures of HACS accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids24. Although the H9N2 viruses were LP and did not possess a polybasic cleavage sites, some carry a motif similar to the cleavage sites were found in the HP H5N1 and H7N3 viruses27, indicative of the potential to acquire a polybasic site. In this study, we analyzed the genetic evolution and the structures of HACS among distinct H9N2 lineages, and introduced a scatter plot tool to show the selection and avoidance of certain predominant HACS mutations. Then, we evaluated the viral replication, stability, cleavage efficiency, and virulence in chickens and mice of H9N2 viruses bearing different cleavage site motifs. In addition, the correlation between the frequency of the nucleotide insertion and the size and locations of the stem-loop structures of different HACS among H9N2 viruses was assessed.The H5 and H7 subtype influenza viruses can evolve from low pathogenic (LP) to highly pathogenic (HP) in natural reservoirs, in which virulence can be increased in birds and can then posed threats to humansTo better understand the evolution, prevalence, and molecular features of the cleavage sites of the global influenza A H9N2) viruses, hemagglutinin (HA) sequences were studied from a genetic perspective by performing multiple sequence alignment and phylogenetic analysis. With the flexibility of the P2, P4, and P5 positions in the HACS, the cleavage sites showed variability in different clades as depicted by WebLogo Fig. . The ami viruses,28. Different H9N2 viruses with monobasic, dibasic, and tribasic cleavage sites were generated by site-directed mutagenesis , with an HA-PARSSR/G cleavage motif, was established previouslyHere, we investigated the influence of the HA cleavage mutations on thermal stability. In contrast to the other H9N2 viruses, the rV08-PAKSKR virus displayed a significantly increased infectious titer after incubation at 5 hpi compared with the parental H9N2 viruses Fig. . In cont6 50% egg infection dose (EID50)/200 μl of H9N2 viruses. After 24 h, the ten uninfected chickens were housed with the inoculated groups. All of the H9N2 viruses could be detected in lung samples from the inoculated chickens except with the rV08-PAASDR group  chickens were intranasally inoculated with 10In addition, virus shedding was also detected in tracheal and cloacal swab speciments at 3, 5, 7, 9, and 11 day post-inoculation (dpi). The inoculated chickens in the rV08-PARSSR, rV08-PAKSSR, rV08-PSRSSR, and rV08-PAASDR group shed viruses at 3 and 5 dpi; however, virus shedding in the rV08-PAKSKR group lasted for 7 dpi  in the lungs was significantly higher than the parental rV08-PARSSR virus at 4 dpi, while the virus titers of rV08-PAASDR virus in the lungs was lower than that of the rV08-PARSSR virus (0.0001 < p < 0.001)  of the rV08-PARSSR virus . A random point mutation of a residue in a codon was indicated with a hollow circle overlaying the scatter plots. In our study, we found that the P6 and P1 positions were stable as compared to other residues during the viral evolution. However, substitutions with hydrophobic residues were observed in the P4 and P3  viruses were low pathogenic to birds and had only sporadically infected humans with mild or asymptomatic symptom, their eradication had not been a priority for zoonosis control agencies in many countries. This neglect caused them to continue to share genetic materials with other HP influenza virus of public health significance. Cleavage activation of HA by host protease is a crucial step in the life cycle of influenza virus and ultimately influence the virulence, transmissibility between hosts and adaption to new species32. The minimum sequence requirement of H9N2 viruses is R/K-X-X-R, which consisted of two paired arginine/lysine residues at the P1 and P4 position. However, we found that the HACS of H9N2 virus showed the avoidance of hydrophobic residues at the P2 and P5 positions, and the P2 position seems tolerant of large variations in biochemical properties, especially S337K substitution. Interestingly, our findings showed the mutation pathways that a virus can take to transition between a P2 S and K via a N  were conducted in an animal biosafety level 3 laboratory and animal facilities at South China Agricultural University (SCAU) (CNAS BL0011) in accordance with protocols. All animals involved in the experiments were reviewed and approved by the Institution Animal Care and Use Committee at SCAU and treated in accordance with the guidelines (2017A002).http://www.who.int/en/), the Food and Agriculture Organization of the United Nations (EMPRES-I data sets) (http://empres-i.fao.org/eipws3g/#h=1), and the National Health Commission of the People’s Republic of China (http://en.nhfpc.gov.cn/). We performed a systematic search using PubMed, Internet-based, the Weekly Epidemiological Record (WER) of WHO for serological evidence of human infection. The global distributions of influenza A (H9N2) viruses in birds and humans were available from GenBank (http://ncbi.nlm.nih.gov/genbank/) and GISAID (https://www.gisaid.org) and evaluated using GraphPad Prism 7 (https://www.graphpad.com/) and ArcGIS 10.4 for Desktop (http://www.esri.com/software/arcgis/arcgis-for-desktop/).All of the human-infecting influenza A (H9N2) cases data were obtained from the World Health Organization (WHO) (http://www.gisaid.org/). The H9N2 virus was propagated in the allantoic cavities of 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs at 37 °C for 60 h and then were stored viruses at −80 °C until use. All cells used, including human embryonic kidney (HEK293T) cells, MDCK cells, A549 cells, CEFs cells, Vero cells, and DF-1 cells were provided by the National Avian Influenza Para-Reference Laboratory (Guangzhou) at South China Agricultural University. HEK293T, MDCK, A549, and Vero cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum, 100U/ml penicillin, and 0.1 mg/ml streptomycin and incubated at 37 °C in a 5% CO2 incubator. CEFs and DF-1 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum, 100U/ml penicillin, and 0.1 mg/ml streptomycin and incubated at 39 °C in a 5% CO2 incubator.The virus used, the H9N2 virus A/chicken/Guangdong/V/2008 (rV08-PARSSR)  was isolated from ostensibly healthy chickens in a poultry farm in Guangdong Province of China in 2008. Each segment used in this study is available in the Global Initiative on Sharing All Influenza Data (GISAID) database (http://ncbi.nlm.nih.gov/genbank/) and GISAID (https://www.gisaid.org). The genomic sequence data set  was then created. All sequences were aligned using the MAFFT (version 7.149) program . Maximum likelihood (ML) phylogenies for the codon alignment of the genomic gene segments were estimated using the GTRGAMMA nucleotide substitution model in the RAxML (version 8.2) program. Node support was determined by nonparametric bootstrapping with 1000 replicates. The phylogenetic tree was visualized with the FigTree (version 1.4.3) program (http://tree.bio.ed.ac.uk/software/figtree/). The AAscaterPlot was coded in MATLAB (MathWorks), and the source codes and installation file were provided at http://github.com/WhittakerLab/AAScaterPlot.All of the available genomic sequences with the complete coding regions of influenza A (H9N2) viruses were downloaded from GenBank  were produced by site-directed mutagenesis PCR and then sequenced. The recombinant H9N2 viruses were designated rV08-PARSSR, rV08-PSRSSR, rV08-PAKSSR, rV08-PAKSKR, and rV08-PAASDR, respectively. The five H9N2 viruses were generated using reverse genetics. Briefly, HEK293T cell monolayers in 6-well plates were transfected at 80-90% confluency with 4 μg of the eight plasmids (500 ng of each plasmid) by using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. DNA and transfection reagent were mixed and incubated at room temperature for 5 min, and added to the cells. Four hours later, the mixture was replaced with Opti-MEM (GIBCO) containing 0.2% bovine serum albumin and 1 μg/ml trypsin. After 48 h, the supernatant was harvested and injected into SPF embryonated eggs for virus propagation. Viruses were titrated in embryonated eggs using hemagglutination assays, as recommended by the WHO manual on influenza diagnosis and surveillance. All mutant viruses were confirmed by RT-PCR and sequencing.Eight gene segments from the A/chicken/Guangdong/V/2008 (rV08-PARSSR) strain were cloned into the Hoffmann’s bidirectional transcription vector pHW2000 plasmid system6 EID50/200 μl of the five H9N2 viruses. Chickens were observed for clinical symptoms for 14 days, and serum samples were collected for analysis of virus-specific antibodies. Ten chickens were introduced into the isolators with inoculated chickens 24 h after direct inoculation to assess transmission. Cloacal and throat swab specimens were collected at 3, 5, 7, 9, and 11 dpi from the infected and naïve chickens. Three chickens in each group were dissected to test for virus replication in the organs, including the lung, kidney, duodenum, and brain. Tissue and swab samples were collected for virus titration by an EID50 assay. For the mouse experiments, groups of 4-week-old female BALB/c mice, obtained from the Vital River Company in Beijing, were anesthetized with isoflurane and inoculated intranasally with 106 EID50/50 μl viruses. To test for virus replication in the organs, three mice in each group were euthanized at 4 dpi. Tissue samples including brain and lung tissue samples from each euthanized mouse were collected for virus titration by an EID50 assay.For the chicken experiment, groups of ten 5-week-old SPF chickens  were inoculated intranasally with 1050 assay in MDCK cells. Vero cells grown in 24-well plates were inoculated with five H9N2 viruses containing different HACS motifs at an MOI of 1 for 16 h. Vero cell cultures were then treated with 10 μg/ml of TPCK-trypsin (Sigma) at 37 °C for 15 min. To induce syncytia formation, cells were incubated with fusion buffer with the pH adjusted to 5.0, 5.2, 5.4, 5.6, and 5.8 for 5 min at 37 °C. At 3 h post fusion induction, Vero cells were fixed and stained with Giemsa stain . Images were taken on a cell imaging system .The thermal stability of the HA protein was measured by determining the loss of the virus titer after incubating the viruses at 50 °C temperature for 1–5 h. The titers of heat-treated H9N2 viruses were determined by TCIDCEF and A549 cells were inoculated with the H9N2 viruses at an MOI of 0.01 in the presence of either 1 μg/ml TPCK-trypsin for 24 h in minimal essential medium containing 0.2% bovine serum albumin (BSA) . Proteins from the cell cultures were separated on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and then electrotransferred onto nitrocellulose membranes. Polyclonal rabbit anti-HA antibody from A/Hong Kong/1073/99 H9N2 expressed in baculovirus  was used to detect HA. Goat anti-mouse IgG conjugated with horseradish peroxidase  was used as a secondary antibody, followed by chemiluminescence detection (LI-COR Odyssey). Grayscale analysis of individual bands was determined by using ImageJ, and relative cutting efficiency was calculated according to the equation HA1/(HA0 + HA1).50/cell for 1 h at 37 °C, and CEF cells were infected at an MOI of 0.001 TCID50/cell for 1 h at 37 °C. After 1 h incubation, the cells were washed twice, and then incubated with DMEM containing 0.2% BSA  and TPCK trypsin (1 μg/ml) at 37 °C with 5% CO2. Culture supernatants were collected at the indicated time points and stored at −80 °C until use. The MDCK cells infected with rV08-PAKSKR appeared cytopathic effect and began to fall off slightly after 24 hpi. Hence, the mixture of culture supernatants and cell cultures were collected at the indicated time points, and viral titers were determined by the TCID50 assay in MDCK cells.Confluent MDCK cells were infected at a multiplicity of infection (MOI) of 0.001 TCIDRenilla luciferase activities .In brief, we used the modified luciferase reporter plasmid, containing chicken RNA polymerase I promoter, RNA polymerase I terminator, rV08-PARSSR HA segment-derived noncoding region (NCR), and the firefly luciferase gene , respectively.To verify the presence of nucleotide insertions in the different HACS of H9N2 viruses, RNA was extracted from the transfected DF-1 cells with the RNeasy Mini Kit (Qiagen) as directed by the manufacturer. Then, RNA was treated with DNAase (Omega) to eliminate the influence of plasmids. Two-step RT-PCR was conducted with primers . PCR products were purified with a QIAamp Gel extraction kit (Qiagen) and cloned into the http://unafold.rna.albany.edu/?q=mfold), Quickfold program  and RNAfold program from the ViennaRNA Web Services (http://rna.tbi.univie.ac.at/) and the ViennaRNA package were used to predict RNA sequences of the cleavage site regions of the HA genes44. The HACS sequences of low pathogenic H5, H7, and H9 precursors were all avian species  available in the GISAID database (http://www.gisaid.org/). Identical HACS sequences were removed. The representative HA sequences of A/chicken/Bangladesh/29588/2016 (H9N2), A/chicken/Zhejiang/221/2016 (H9N2), A/chicken/Shenzhen/9/1997 (H9N2), A/chicken/Egypt/S4454B/2010 (H9N2), and A/wild waterfowl/Korea/7/2010 (H9N2), A/wild_caterfowl/Dongting/C2032/2011 (H9N2) isolates available in the GISAID’s EpiFlu Database (http://www.gisaid.org/) were used for the structural analysis.The Mfold program (https://swissmodel.expasy.org/), employing the uncleaved H3 monomer (protein Data Bank ID 4BSE) as a template. The corresponding amino acid to a three-dimensional (3D) amino acids structure of the HA protein were mapped using MacPymol (http://www.pymol.org/).We predicted the HA monomer structure using the SWISS-Model website (t test was applied for analysis, and p values less than 0.05 were considered significant and are represented as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For all statistically significant data from at least two biological repeats performed on separate days was used. The exact number of replicates are presented in individual figure legends. Any differences in statistical significance are indicated.Data are presented as the mean ± SD and analyzed using GrapPad Prism 5.0. The independent-samples Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Reporting Summary


Crucially, oral administration of TG protected mice against lethal virus infection and reduced virus titres in the lungs of treated mice. TG-induced ER stress unfolded protein response appears as a key driver responsible for activating a spectrum of host antiviral defences that include an enhanced type I/III interferon response. Our findings suggest that TG is potentially a viable host-centric antiviral for the treatment of influenza A virus infection without the inherent problem of drug resistance.Influenza A virus is a major global pathogen of humans, and there is an unmet need for effective antivirals. Current antivirals against influenza A virus directly target the virus and are vulnerable to mutational resistance. Harnessing an effective host antiviral response is an attractive alternative. We show that brief exposure to low, non-toxic doses of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca There is no entirely satisfactory antiviral treatment for influenza A infections. By directly targeting the highly mutable virus, current and near-market antivirals are inherently susceptible to virus resistance ,2. Thus,2+)-ATPase (SERCA) pump; it impedes the replenishment of Ca2+ in the endoplasmic reticulum (ER) store resulting in net loss of Ca2+ from the ER .18S ribosomal RNA. Human ER stress primers for HSPA5 (FH1_HSPA5 and RH1_HSPA5), HSP90B1 (FH1_HSP90B1 and RH1_HSP90B1) and DDIT3 (FH1_DDIT3 and RH1-DDIT3), human OAS1 primers (FH1_OAS1 and RH1_OAS1), and human IFNB1 (FH1_IFNB1 and RH1_IFNB1) were pre-made designs from Sigma-Aldrich. Primers (synthesised by Sigma-Aldrich) for USSR H1N1 M-gene, human RIG-I (DDX58), IFNL2 and IFNL3 for pig RIG-I and OAS1 were designed using PrimerExpress 3.0.1 (Thermo Fisher Scientific); sequences described in IRF9 and OAS3, and pig ER stress primers for HSPA5, HSP90B1 and DDIT3 were pre-made designs from Sigma-Aldrich.Total RNA was extracted from cells using the RNeasy Plus Minikit . Viral RNA in the spun culture supernatants was extracted with the QIAamp Viral RNA Mini Kit (Qiagen). cDNA was synthesised from 0.5 or 1 µg of total RNA using Superscript III First Strand synthesis kit (Thermo Fisher Scientific). Expression of host genes was performed with a LightCycler−96 instrument , and computation was based on the comparative Ct approach, normalised to Cells were lysed by radioimmunoprecipitation assay (RIPA) buffer  supplemented with 1% phenylmethylsulfonyl fluoride (PMSF) (Santa Cruz), 1% inhibitor cocktail, and 1% sodium orthovanadate, according to manufacturer’s instructions (Santa Cruz). Protein concentration was determined by Bio-RAD protein assay . Primary antibodies were goat antiviral M1 at 1:500 dilution , rabbit antiviral NP at 1:500 dilution  and mouse anti-β-actin at 1:10,000 , and appropriate species-specific secondary antibodies were peroxidase-conjugated for chemiluminescence detection .® 488, Thermo Fisher Scientific) at 2 μg/mL, diluted in 10% serum in PBS, was added for 1 h at room temperature in a humidified chamber. The secondary antibody was removed and the cells thoroughly washed with PBS. After immunostaining for all of the proteins of interest, the cells were washed thoroughly with PBS. ProLong™ Gold Antifade Mountant with DAPI (Invitrogen) was added to each well at a sufficient volume to cover the base of the well and incubated at room temperature for 24 h. The NHBE cells were subsequently stored at 4 °C until imaging with a Leica TCS SP8 confocal microscope using a 63-time oil immersion objective. Integrated density was measured using Image J 1.x 33] and MIn summary, we propose that TG, in orchestrating a spectrum of host antiviral defences, are not readily overcome by virus mutation, and is potentially a viable host-centric antiviral for the treatment of influenza A virus infection without the inherent problem of drug resistance.The use of TG and other structurally-related compounds in antiviral therapy is covered by patent PCT/GB2019/050977.


The first highly pathogenic (HP) influenza A/H7N9 was reported in Guangdong in January 2017. To investigate the emergence and spread of HP A/H7N9 in Guangdong province, we sequenced 297 viruses  A/H7N9, and 220 A/H9N2) during 2016–2017. Our analysis showed that during the fifth wave, three A/H7N9 lineages were co-circulating in Guangdong: the local LP Pearl River Delta (PRD) lineage (13%), the newly imported LP Yangtze River Delta (YRD) lineage (23%), and the HP YRD lineage (64%). Previously circulating YRD-lineage LP during the third wave evolved to the YRD-lineage HP A/H7N9 in Guangdong. All YRD-lineage LP detected during the fifth wave most likely originated from newly imported viruses into Guangdong. Genotype comparison of HP A/H7N9 suggests limited outward spread of HP A/H7N9 to other provinces. The distribution of HP A/H7N9 cleavage site variants on live poultry markets differed from that found in humans, suggesting a V1-type cleavage site may facilitate human infections. Since the first detection of low pathogenic (LP) A/H7N9 avian influenza in the Yangtze River Delta (YRD) region in China in 2013, five epidemic waves of human and poultry transmission took place in China . An impoIn response to the first emergence of HP A/H7N9 in Guangdong province, we used data from our ongoing LPMs environmental surveillance program in the Guangdong province to genetically trace the source of these HP A/H7N9 viruses. In addition, we used genomic data to compare the molecular characteristics of the A/H7N9 viruses in humans, poultry, and the environment. We aim to have a thorough understanding of the origin, spread, and transmission of this novel virus in Guangdong province.Influenza A surveillance was conducted at twenty-eight sentinel hospitals and LPMs in twenty-one prefecture-level cities in Guangdong province during the fifth wave of the A/H7N9 outbreak (November 2016–August 2017). In order to trace the origin of HP A/H7 in Guangdong, we retrospectively screened samples of LPMs collected since January 2016. Environmental samples of LPMs include swab specimens collecting from cages, chopping boards, drinking water, and feather removal machines. Additionally, oropharyngeal and cloacal swabs were taken from apparently healthy chickens, ducks, pigeons, francolins, geese, and quails on LPMs. We collected samples at one retail market and one wholesale market in each of twenty-one prefecture-level cities. All samples were placed in viral transport medium and stored at −80 °C until further processing.Samples were first tested for the presence of influenza A RNA using real-time reverse transcription-PCR (rRT-PCR) assays targeting the matrix protein, followed by rRT-PCR assays specifically targeting the A/H7 and A/H9 genes in twenty-one local Centers for Disease Control and Prevention (CDC) and were further verified by the Guangdong Provincial CDC as described previously . HP A/H7® III One-Step RT-PCR system . Genome sequencing of environmental A/H7 and A/H9 viruses was performed on the next-generation Ion PGM sequencing platform. Sequencing data were analyzed with CLC Genomics Workbench 7.5.1 software. Low-quality reads were trimmed using CLC trimmer with a quality limit set at 0.05. Trimmed reads were then de novo assembled in CLC using default parameters. Contigs with a coverage above 10 were extracted and annotated using a BLAST search against the NCBI non-redundant nucleotide database . Extracted RNA was subjected to reverse transcription and amplification using the SuperScriptdatabase . The seqInformation on live poultry imports from other regions of China to Guangdong province was obtained from the local Ministry of Agriculture. According to the geographic distribution of twennty-nine Chinese provinces and cities, we classified them into six regions. Provinces Hebei, Heilongjiang, Jilin, Liaoning, Inner Mongolia, Shanxi, and Beijing city were grouped as northern regions. Provinces Hubei, Hunan, and Henan were grouped as central regions. Provinces Anhui, Fujian, Jiangsu, Jiangxi, Shandong, Zhejiang, and the city of Shanghai were grouped as eastern region. Hainan is considered the southern region. The north-western region included provinces Gansu, Ningxia, Qinghai, Shaanxi, Xinjiang and the south-western region included provinces of Guangxi, Guizhou, Sichuan, Tibet, Yunnan, and the city of Chongqing.Genome sequences from the present study were combined with all publicly available sequences of influenza A/H7N9 (China) and A/H9N2 (China) virus sequences available in GISAID and part of A/H9N2 (China) from NCBI. The reference sequence accession numbers are summarized in 8 steps for each alignment and extracted a subset of 2,000 phylogenies from the posterior tree distribution, subsequently used as an empirical tree distribution for phylogeographic analyses. We computed maximum clade credibility trees for each dataset by using TreeAnnotator. We used the discrete phylogeographic method implemented in BEAST to investigate spatial dynamics of H7N9 virus lineages from seven regions in China. The seven regions were central China , southern China (Guangdong), Eastern China , Southwestern China , Northern China , Southeastern China (Fujian), Northwestern China .We estimated molecular clock phylogenies by using the Bayesian Markov chain Monte Carlo approach implemented in BEAST version 1.8. We computed four independent Markov chain Monte Carlo runs of 1.5 × 10Protein sequences of fifty-eight HP A/H7N9 viruses in this study were compared with publicly available HP A/H7N9 sequences using BioEdit. Biologically relevant amino acid substitutions on protein HA, NA, NS1, M2, PA, PB1, and PB2 were compared as described in previous studies .During the study period, 34,106 environmental swabs were tested, ranging from 572 to 3,790 per month during January 2016–August 2017 . SurveilA total of 5.5 billion live birds were imported into Guangdong from different areas of China including twenty-nine provinces from the northwestern, southwestern, northern, central, eastern, and southern regions during the period January 2016–July 2018 . SpecifiWe compared the newly generated sequence data with all Chinese A/H7N9 HP and LP full genomes available in public domains. A total of 57 out of 523 LP A/H7N9 sequences (including 19 genomes from this study) were from Guangdong (11%), 260 (50%), 85 (16%), and 121 (23%) were from eastern, central, and other regions of China, respectively. Of the 125 HP sequences (including 58 genomes from this study), 89 (71%) were collected in Guangdong, 2 (2%) from eastern China, 5 (4%) from central China, and 29 (23%) from other regions.Based on the evolutionary analysis of H7N9 viruses since first outbreak in 2013, the YRD region and the PRD region were identified as the H7N9 outbreak source in China. A/H7N9 viruses are separated in the YRD-lineage and the Pearl River Delta (PRD)-lineage LP A/H7N9 viruses . The YRDThe comparison of genotypes identified a total of 180 genotypes (G1–G180) from the first wave to the fifth epidemic A/H7N9 wave . A totalTo clarify the mechanism behind the genotype diversity of LP and HP A/H7N9 in Guangdong, the internal genes of the viruses within each HA clade were further analyzed. During the fifth wave, three H7N9 lineages were co-circulating in Guangdong: the local LP PRD lineage (13%), the newly imported LP YRD lineage (23%), and HP YRD lineage (64%). Strikingly, in addition to the PRD-lineage LP viruses, a diverse range of YRD-lineage LP A/H7N9 viruses were found during the fifth wave , which sKRTAR↓G (V1), PKGKRTKR↓G (V2), PKGKRTAR↓G (V3), PKGKRIAR↓G (V4), PKRRRTAR↓G (V5), and PKRKRAAR↓G (V6). In the present study, we identified a novel motif ‘PKRKRIAR↓G′ and defined it as V7. Moreover, variants 1, 2, 3, and 5 were also found in the present study. Analysis based on all publicly available HP A/H7N9 and those in the present study showed that most human HP A/H7N9 viruses contain the V1 motif. Variant 1 HP A/H7N9 was found in 93.7 per cent (45/48) of human cases, variants 3, 5, and 6 accounted for 2 per cent (1/48) each. In LPMs’ surveillance samples, variants 1 and 3 accounted for 49.6 per cent (57/115) and 43.4 per cent (50/115) of positive samples, and variants 2, 5, and 7 accounted for 3.5 per cent (4/115), 1.7 per cent (2/115), and 1.7 per cent (2/115), respectively. V1 was widely distributed in the north, east, west and center of Guangdong, while V2–V7 only circulated in the center and east of Guangdong , with an emphasis on variants V1 and V3. Many amino acid substitutions that may be relevant for risk assessment were found both in human and environmental HP A/H7N9, which include G186V on the HA gene, K526R, A588V, and K702R on the PB2 gene, V100A, A402S, and S409N on the PA gene, S31N of M2 gene, N205S on the NS1 gene, and T48A on the NS2 gene . MoreoveThe fifth epidemic wave of avian influenza A/H7N9 started in September 2016 with a sudden increase of human cases in seven provinces . The firSince the first detection of HP A/H7N9 in four cities of Guangdong province, it was detected in nineteen cities in Guangdong and eleven other provinces in China. Analysis based on the HA gene suggested that HP A/H7N9 originated from the Guangdong and eastern China. It seems that HP A/H7N9 viruses spread from Guangdong to other regions . This miSeven different cleavage site variants (V1–V7) were identified in Guangdong in the fifth wave outbreak . CompariOur study was done retrospectively, but showed potential for early warning for vaccine escape variants or circulation of viruses with molecular markers for human infections or antiviral resistance . With thVirus Evolution online.The data used to support the findings of this study are available from the corresponding author upon request.R.B. is a postdoctoral researcher at the Guangdong Provincial Center for Disease Control and Prevention, Guangzhou, China, whose research focuses on the epidemiology, evolution, and transmission of avian influenza viruses in Guangdong province. R.S.S. is a PhD candidate at the Erasmus Medical Centre, Rotterdam, The Netherlands, whose research focuses on the risk-based surveillance of influenza viruses in the market chain.veaa097_Supplementary_DataClick here for additional data file.


To investigate associations between lower-grade glioma (LGG) mRNA-based subtypes (R1-R4) and MR features.n = 145) in The Cancer Imaging Archive (TCIA) who underwent MR imaging. The associations between mRNA-based subtypes and MR features were assessed.mRNA-based subtyping was obtained from the LGG dataset in The Cancer Genome Atlas (TCGA). We identified matching patients (P < 0.05). The time-dependent ROC for the R2 subtype was 0.78 for survival at 12 months, 0.76 for survival at 24 months, and 0.76 for survival at 36 months. In the TCIA-LGG dataset, 41 (23.7%) R1 subtype, 40 (23.1%) R2 subtype, 19 (11.0%) R3 subtype and 45 (26.0%) R4 subtype cases were identified. Multivariate analysis revealed that enhancing margin  and T1 + C/T2 mismatch  were associated with the R1 subtype (AUC: 0.708). The average accuracy of the ten-fold cross validation was 71%. Proportion of contrast-enhanced (CE) tumour  and necrosis/cystic changes  were associated with the R2 subtype (AUC: 0.832). The average accuracy of the ten-fold cross validation was 82%. Haemorrhage  was positively associated with the R3 subtype (AUC: 0.689). The average accuracy of the ten-fold cross validation was 87%. Proportion of CE tumour  was negatively associated with the R4 subtype (AUC: 0.672). The average accuracy of the ten-fold cross validation was 71%. For the prediction of the R2 subtype, the nomogram showed good discrimination and calibration. Decision curve analysis demonstrated that prediction with the R2 model was clinically useful.In the TCGA-LGG dataset, patients with the R2 subtype had the shortest median OS months (Patients with the R2 subtype had the worst prognosis. We demonstrated that MRI features can identify distinct LGG mRNA-based molecular subtypes. Primary brain tumours are one of the top ten causes of cancer-related deaths in the United States . They arA new direction in cancer research has emerged that focuses on the relationship between genomic data and imaging features –12. RadiSurvival analysis revealed that the R2 subtype was significantly correlated with shorter overall survival. Both the R1 subtype and R3 subtype highly expressed an early progenitor-like astrocytoma gene signature. The R4 subtype highly expressed a neuroblastic astrocytoma signature and a neuron-specific signature . This stThe clinical files of LGG patients were obtained from TCGA. MR data were provided by TCIA –20. TCGA3 or < 60 cm3); (L) haemorrhage (negative or positive); (M) multifocal (negative or positive); (N) degree of enhancement (slight or obvious); and (O) necrosis/cystic change (negative or positive). Both neuroradiologists were blinded to LGG mRNA-based subtypes (R1-R4) as well as the clinical data.MR image analysis was performed as previously published by recent studies and our group –23. The P-value of less than 0.05 (two-sided) was considered to indicate statistical significance.We focused on the association of mRNA-based subtypes (R1-R4) and MR features. A colour heat map was drawn to show the correlation patterns between MR features and mRNA expression (R1-R4). Fisher’s exact test, the chi-square test and binary logistic regression analysis were used  for each mRNA-based subtype. We use tenfold cross-validation test. Odds ratios (ORs) as well as their corresponding 95% confidence intervals (CIs) are reported. In the present study, binary logistic regression was repeated for the four LGG mRNA-based subtypes: R1, R2, R3 and R4. The area under the receiver operating characteristic curve (AUC) of each mRNA-based subtype (R1-R4) is reported. Survival analysis was conducted by using Kaplan-Meier analysis and the time-dependent ROC method . A P < 0.05 Fig. In the TCGA-LGG dataset, patients with the R2 subtype had the shortest median OS months  patients  were screened . The demographic and tumour characteristics of all 145 patients  are summarized in Table P < 0.05). Necrosis/cystic change, multifocality, degree of enhancement, proportion of CE tumour, volume, depth, extranodular growth and T1 + C/T2 mismatch were associated with the R2 subtype. Haemorrhage was associated with the R3 subtype (P < 0.05). The degree of enhancement, enhancing margin, proportion of CE tumour and extranodular growth were associated with the R4 subtype .In univariate analysis Tables  and 3, eP = 0.003) and T1 + C/T2 mismatch  were associated with the R1 subtype (AUC: 0.708). The average accuracy of the ten-fold cross validation was 71%. Proportion of contrast-enhanced (CE) tumour  and necrosis/cystic changes  were associated with the R2 subtype  was positively associated with the R3 subtype (AUC: 0.689). The average accuracy of the ten-fold cross validation was 87%. Proportion of CE tumour  was negatively associated with the R4 subtype (AUC: 0.672). The average accuracy of the ten-fold cross validation was 71%.Multivariate analysis Table  revealed3 and absence of necrosis/cystic change were positively associated with the R2 subtype (AUC: 0.832). Haemorrhage was positively associated with the R3 subtype (AUC: 0.689). Proportion of CE tumour > 5% was negatively associated with the R4 subtype (AUC: 0.672).We demonstrated Fig.  that illGlioma is one of the most common primary central nervous system malignant tumours , 25. IntRadiogenomic studies have revealed key imaging differences between certain LGG genetic groups and may aid in the diagnosis of patients with LGG as well as predict survival and guide treatment in patients with LGG , 28–31. The R2 subtype is mostly composed of GIII tumours (77%), tumours mostly of astrocytoma histology (68%), tumours enriched for the methylation subtype M2 (62%) and IDH wild type (67%) tumours. This subtype is correlated with GBM-related events such as PTEN mutation, chromosome 10 loss, and EGFR mutation and amplification. Our findings showed that the proportion of CE tumours (> 5%) and the absence of necrosis/cystic changes were positively associated with the R2 subtype (AUC: 0.832). This is the first article to show the connection between the R2 subtype and MR features. For the prediction of the R2 subtype, the nomogram showed good discrimination and calibration. Decision curve analysis demonstrated that prediction with the R2 model was clinically useful.The other RNA subtypes  were populated with IDH-mutant gliomas. R1 lacked 1p/19q codeletion and was comprised of two methylation subtypes, M5 (70%) and M3 (30%), and the vast majority of R1 cases had TP53 and ATRX mutations17. We demonstrated that well-defined margins and the absence of T1 + C/T2 mismatches were positively associated with the R1 subtype (AUC: 0.708). The R3 subtype was entirely composed of IDHmut-codeletion gliomas and was equally distributed across the methylation subtypes M2 and M3. It was also enriched for oligodendrogliomas (85%), mutations in NOTCH1, FUBP1, and CIC, and oligodendrocyte progenitor-specific expression. Our findings showed that haemorrhage was positively associated with the R3 subtype (AUC: 0.689). The R4 subtype highly expressed a neuron-specific signature and a neuroblastic astrocytoma signature. The proportion of CE tumours (<=5%) was positively associated with the R4 subtype (AUC: 0.672). This is the first article to show that MRI features can identify distinct LGG mRNA-based molecular subtypes . Radiogenomics analysis allows researchers to explore the TCGA and TCIA databases for correlations between mRNA-based molecular subtypes and radiological phenotypes.Our study has several limitations. The major limitation of this article was that the sample size of the R3 subtype was only 19 patients (11.0%). The disadvantages of a small sample size might have limited the statistical power to explore additional correlations of the R3 subtype. Our findings should be further investigated and externally validated in larger cohorts of LGG patients. In addition, the MR data are heterogeneous, and in most cases, the images were acquired as part of routine care and not as part of a controlled research study or clinical trial. Our results should be validated using standardized MR imaging.3 and absence of necrosis/cystic changes were positively associated with the R2 subtype. Haemorrhage was positively associated with the R3 subtype. Proportion of CE tumour > 5% was negatively associated with the R4 subtype (AUC: 0.672).Our results revealed connections between LGG mRNA-based subtypes (R1-R4) and MR lesion features. Our findings revealed that ill-defined margins and the absence of T1 + C/T2 mismatches were positively associated with the R1 subtype. The proportion of CE tumour > 5%, volume < 60 cm


The H7N9 virus mutated in 2017, resulting in new cases of highly pathogenic avian influenza (HPAI) H7N9 virus infection. H7N9 was found in a viraemic patient in Guangdong province, China. The present study aimed to clarify the pathogenic characteristics of HPAI H7N9. Virus was isolated from the plasma and sputum of the patient with HPAI H7N9. Liquid phase chip technology was used to detect the plasma cytokines from the infected patient and healthy controls. Mice were infected with strains A/Guangdong/GZ8H002/2017(H7N9) and A/Zhejiang/DTID-ZJU01/2013(H7N9) to observe the virus’s pathogenic characteristics. Serum and brain tissue were collected at 2, 4, and 6 days after infection. The viruses in serum and brain tissue were detected and isolated. The two strains were infected into A549 cells, exosomes were extracted, and virus genes in the exosomes were assessed. Live virus was isolated from the patient’s plasma. An acute cytokine storm was detected during the whole course of the disease. In animal experiments, A/Guangdong/GZ8H002/2017(H7N9) was more pathogenic than A/Zhejiang /DTID-ZJU01/2013(H7N9) and resulted in the death of mice. Live virus was isolated from infected mouse serum. Virus infection was also detected in the brain of mice. Under viral stress, A549 cells secreted exosomes containing the entire viral genome. The viraemic patient was confirmed to have an HPAI H7N9 infection. A/Guangdong/GZ8H002/2017(H7N9) showed significantly enhanced toxicity. Patient deaths might result from cytokine storms and brain infections. Extrapulmonary tissue infection might occur via the exosome pathway. The determined pathogenic characteristics of HPAI H7N9 will contribute to its future treatment. In February 2013, the avian influenza A H7N9 virus was identified as causing human infection, with high mortality and morbidity. To date, five waves of H7N9 infection have caused more than 1500 severe human infections and the mortality remains high, with more than 600 deaths  in ChinaIn a previous study of H5N1 virus infection, viraemia was detected in 82% of the fatal cases, but absent in nonfatal cases. High viral load, and the resulting intense inflammatory responses, are central to influenza H5N1 pathogenesis. Individuals with detectable H5N1 RNA in their blood also had higher pharyngeal viral loads than those without evidence of H5N1 RNA, suggesting that the presence of viral RNA in blood reflects an overall high viral burden, which commonly leads to high mortality . In our Genetic changes to viruses might change their behaviour and pathogenic characteristics . We treaExosomes play an important role in viral infection . StudiesThe work was carried out in accordance with ethical guidelines of the Guangzhou Eighth People’s hospital . Sputum and plasma specimens were collected daily after admission. The patient’s plasma remaining after routine testing was stored in a bio-sample banker. The normal plasma were collected from 10 volunteers . The median age of these volunteers was 26 (interquartile range 23–28 year) year old. These volunteers came to the First Affiliated Hospital of Zhejiang University School of Medicine for regular physical examination. After the physical examination, these volunteers were confirmed healthy. The remaining plasma after routine testing was stored and used in our study.Female specific-pathogen-free (SPF) BALB/c mice, aged 6–8 weeks old, were purchased from the Shanghai Laboratory Animal Center, China. All animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Zhejiang Province and the study was approved by the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University . All experiments with H7N9 virus were performed in a biosafety level 3 laboratory approved by the First Affiliated Hospital, School of Medicine, Zhejiang University (Permit No. 2015-15).The Madin-Darby canine kidney cell line (MDCK) and A549 cell line  were obtained from the ATCC . The cell lines were propagated in growth medium containing Dulbecco’s modified Eagle’s medium  supplemented with 10% foetal bovine serum  at 37°C and 5% CO2. The A/Guangdong/GZ8H002/2017(H7N9) virus used in this study was isolated from a patient in Guangzhou, China, in 2017 (GenBank: MF455313- 455320). We isolated this virus from the patient’s plasma. The A/Zhejiang /DTID-ZJU01/2013(H7N9) virus was isolated from a patient in Zhejiang Province, China, in 2013 . Virus sThe viral RNA of the H7N9 virus was extracted using Trizol  method. The concentration, purity, and integrity of total viral RNA were detected using a NanoDrop 2000 instrument  and an Agilent 2100 Bioanalyzer . The sequencing library was constructed using TruSeq Stranded Total RNA with Ribo-zero Gold , and the process was conducted according to the manufacturer’s protocol. The library was constructed as follows: after digesting ribosomal RNA with Ribo-zero, the RNA was broken into fragments of about 350 nt using Fragment High Mix, and the sequencing library was constructed by synthesizing and purifying cDNA, terminal repair, adding poly-A tail, ligating sequencing connectors, and PCR amplification. After quality checking, the library was sequenced using an Illumina Hiseq Xten sequencer. The sequencing strategy generated 150 bp paired ends (PE150).Clean reads were obtained through data quality control and filtering of raw sequencing data using the NGSQC toolkit . Clean RThe TCID50 (Fifty percent tissue culture infective dose) assay was performed as described previously. The vir2. When the cell density reached 70–80%, the cells were washed three times with PBS to remove the bovine serum. The cells were then infected with the viruses (MOI=0.1). After the adsorption for 2 h, the cells were washed three times with PBS. DMEM supplemented with 1% foetal bovine albumin and 1 µg/ml L-l-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) was added to the culture A549 cells for 48 h when culturing the ZJU01 strain. DMEM supplemented with 1% foetal bovine albumin was added to the culture A549 cells for 48 h while culturing the GZ8H002 strain. The virus culture supernatant was measured by the quantity of RNA and TCID50 titre.A549 cells were cultured in DMEM supplemented with 10% FBS at 37°C and 5% COAfter culture for 48 h, the cells were washed twice with PBS slightly. Cells were then fixed with 4% paraformaldehyde for 20 min. After washed twice with PBS slightly, cells were penetrated with 0.1% Triton X-100 for 20 min. Cells were again washed, blocked for 30 min with 3% bovine serum albumin  and incubated sequentially with 1:400 dilution of mouse anti-influenza A virus nucleoprotein antibody  for 1 h. After washing 5 times with PBS, the Alexa Fluor 488 conjugated goat anti-mouse IgG  with 1:500 dilution was used as the secondary antibody. After washing 5 times with PBS, DAPI  was used as the nuclear counterstain. After washing 5 times with PBS, the cells were observed under the fluorescence microscope.4 TCID50 A/Guangdong/GZ8H002/2017(H7N9) or the A/Zhejiang/ DTID-ZJU01/2013(H7N9) virus. Phosphate-buffered saline  of the same volume was given to the mice in the control group. Mice were observed for illness, weight loss, and death post-infection. Then, the mice were sacrificed, and their serum was collected at 2, 4, and 6 days post-infection (dpi) . The lung and brain were removed. Part of the organ was fixed in 10% buffered formalin, while the other part was used to detect virus levels using quantitative PCR. An H7N9 nucleic acid quantitative detection kit (Cat#Z-RR-0309-02) and Influenza virus A Real time RT-PCR kit (Cat#RR-0051-01) were purchased from Zhijiang biological technology Co., Ltd. Shanghai, China.Mice were inoculated intranasally with 50 µl 10g for 3 h in a Beckman Coulter Optima XPN-80 Ultracentrifuge . The pellet of virus was resuspended by 200 µl of PBS. The viruses were observed using a Tecnai Spirit 120 kv transmission electronic microscope to analyse their structure.The sera collected at 2, 4, and 6 dpi were used to isolate the virus. About 100 µl of serum was injected into the allantoic cavities of 9-day-old SPF embryonated chicken eggs. The embryonated chicken eggs were cultured in an incubator at 37°C for 72 h. The allantoic fluid was harvested and tested using an HA assay. Then 100ul serum from the 6 dpi infected mice was used to detect virus levels using quantitative PCR. Meantime, we collected 1.2 ml of serum from the 6 dpi infected mice and diluted in 40 ml of PBS. The dilution was subjected to ultra-centrifugation at 120000 × 2O2 in methanol. Sections were blocked for 1 h with 3% bovine serum albumin  in PBS and incubated sequentially overnight at 4°C with 1:200 dilution of polyclonal rabbit anti-H7N9 antibodies. Antibody binding was detected using EnVision System reagents . All slides were counterstained with haematoxylin.The amount of virus was evaluated using quantitative PCR. Organ tissue was prepared for haematoxylin eosin (HE) staining. Immunohistochemical (IHC) assays were also conducted. Paraffin sections of organs were de-waxed and then subjected to heat treatment in citrate buffer, and endogenous peroxidase activity was quenched using 0.3% HThe plasma of the patient was collected from the patient with HPAI H7N9 infection. Ten normal plasma samples were collected from the healthy volunteers. The cytokines and chemokines in human plasma was measured using the Bio-Plex Pro™ Human Cytokine Screening Panel, 48-Plex . Serum samples from the mice were detected using Bio-Plex Pro™ Mouse Cytokine Grp I Panel 23-Plex . The detection was based on the Bio-Plex 200 system (Bio-Rad) and performed according to the manufacturer instructions. g to remove the cells. Then 40 ml virus culture supernatant was obtained. The supernatant was then subjected to ultra-centrifugation at 120000 × g for 3 h in Beckman Coulter Optima XPN-80 Ultracentrifuge. The pelleted exosomes were resuspended in 200 µl of PBS. The exosomes were then observed using a Tecnai Spirit 120 kv transmission electronic microscope.The culture of the two strains of viruses in A549 cell model was described in the previous part. After 48 h of culture, the culture supernatant was collected by centrifugation at 500 ×To separate the virus from the exosomes, exosomes were purified using an Exosome-human CD63 Isolation kit . The exosomes were bound to the magnetic beads marked by CD63. The viruses were separated by washing with solution buffer. Then, 40 µl of magnetic beads were washed with 200 µl of isolation buffer (PBS with 0.1% BSA). The pre-enriched exosome solution was titrated with isolation buffer for the magnetic beads and mixed well. The beads were incubated overnight for 22 h at 4°C with mixing. The bead-bound exosomes were washed by adding 400 µl of isolation buffer and mixed gently by pipetting. The tube was placed on a magnet for 1 min and the supernatant was discarded. These steps were repeated three times. The purification of exosome was verified, with no H7N9 detected in supernatant (Cq > 38 was considered negative). The last supernatant was collected and the bead-bound exosomes were resuspended by 300 µl of isolation buffer. The identification of H7N9 genes in the exosome was performed using an H7N9 nucleic acid quantitative detection kit. Then, 100 µl of bead-bound exosomes solution was injected to the allantoic cavities of 9-day-old SPF embryonated chicken eggs and cultured at 37°C for 72 h. The allantoic fluid was harvested and tested using an HA assay.P-values < 0.05 were considered statistically significant.Statistical analyses of data for weight and survival rate were performed using Graphpad Prism 5 . Statistical analyses of the data for cytokines and chemokines were conducted using the Mann–Whitney test. Statistical analyses of the data for H7N9 gene expression in A549, embryonated chicken egg and mouse serum were conducted using a one-way analysis of variance (ANOVA) test with Tukey’s multiple comparison test. Statistical analyse of the data for H7N9 gene expression in exosome-enclosed viral particle was conducted using paired t test. Statistical analyses of the data for H7N9 gene expression in the brain and lung leachates were conducted using a two-way ANOVA test with Bonferroni post-tests. The one-way analysis of variance (ANOVA) test with Tukey’s multiple comparison test was used to compare the TICD50 titre of H7N9 virus isolated from the exosomes. The values are presented as means ± SD for the indicated sample sizes. The patient suffered a cytokine storm in the hospital. Forty-eight cytokines and chemokines were tested. Among them, the levels of 32 cytokines and chemokines including IL-1ra, IL-6, IP-10, IL-10, IFN-γ, MCP-3, IL-18, HGF, MCP-1, MIG, IL-2Rα, IL-12(p40), IL-8, MIP-1α, PDGF-BB, G-CSF, CTACK, MIF, SCF, HGF, IL-16, SCGF-β, RANTES, TNF-α, FGF basic, IL-2, IL-17, MIF, MIP-1β, SCF, SDF-1α, and TRAIL were elevated in the patient. The levels of ten cytokines and chemokines including MIG, IL-1ra, IL-6, IP-10, IL-10, IFN-γ, MCP-3, IL-18, HGF, MCP-1 were dramatically higher in this patient  comparedThe HPAI virus could be isolated from both the sputum and the plasma. Deep sequencing of the HPAI virus isolated from the sputum and the plasma were performed. The genome of ZJU01 strain was used as the reference genome. A circos diagram was constructed to present the mutations in the different genes . In the circos diagram, the innermost circle represents the virus isolated from the plasma. The other circles represent the virus isolated from the sputum collected at different times. The HPAI virus isolated from the sputum (three sputum samples from the same patient) and the plasma were highly similar. There were only rare mutations between them. However, the genes of the HPAI virus had many mutation in the eight viral genes compared with ZJU01 strain A.FigureWe further compared the replication ability of these two virus. When cultured in embryonated chicken eggs, the replication ability of A/Guangdong/GZ8H002/2017(H7N9) virus was better than that of A/Zhejiang /DTID-ZJU01/2013(H7N9). The quantity of RNA and the TCID50 titre of A/Guangdong/GZ8H002/2017(H7N9) was higher than those of A/Zhejiang /DTID-ZJU01/2013(H7N9) B,C. WhenTo determine whether the A/Guangdong/GZ8H002/2017(H7N9) strain had acquired any key molecular substitutions associated with increased virulence and transmissibility in mammals, or with antiviral drug resistance, we performed deep sequencing of the HPAI H7N9 virus. As shown in We observed illness, weight loss, and death in the mice after H7N9 challenge. Various amounts of weight change were noted in all three groups A. The miWe isolated the H7N9 virus from the mouse serum. In the A/Guangdong/GZ8H002/2017(H7N9) infection mice, the live virus was detected persistently in the serum until death . The serWe collected the serum of mice infected with A/Zhejiang/DTID-ZJU01/2013(H7N9) and A/Guangdong/GZ8H002/2017(H7N9) in early (2 dpi) and late stages (6 dpi) of infection and detected the serum cytokine levels. In the early stage of infection, IL-6, G-CSF, and IL-13 levels were high in the A/Zhejiang/DTID-ZJU01/2013(H7N9) and A/Guangdong/GZ8H002/2017(H7N9) infection groups E-G. In tThe histopathology of organ tissues was evaluated using HE staining. The extent and characteristics of the lesions varied among the groups. The mice infected with A/Zhejiang/DTID-ZJU01/2013(H7N9) had multifocal interstitial inflammatory hyperaemia and exudative pathological changes in the lung, with larger lesions in the lung tissue and fusion of multiple patchy lesions. These histopathological changes were severe at 2 dpi. In the following days, the pathological changes of the lung tissue persisted, but gradually improved. At 6 dpi, the pathological change became moderate. This was consistent with the improvement in the mice’s general health A,B. The High levels of the H7 virus gene were detected persistently in the lungs of the H7N9-infected mice C. At 2 dIn both infection groups, HE staining of brains presented inflammatory cell infiltration B, E. In We also detected the expression of the H7 gene in brain leachate C. The H7The TCID50 of brain leachate was only detected at 6 dpi in the A/Guangdong/GZ8H002/2017(H7N9)-infected mice D. In theThe exosomes were enriched using ultra-centrifugation. The morphology of the exosome was viewed using TEM. The typical shape of exosomes was observed in the different groups. The shape and size of the exosome varied from 50 to 200 nm. The culture supernatant from A549 cell model without virus infection was observed using transmission electronic microscope. The exosomes were showed by blue arrow A. The cuAfter purification, the H7N9 virus genes in the exosomes were detected using quantitative PCR. The exosome-enclosed viral particle is a very small part compared to the total viral particle. The H7 gene content of total viral particle and exosome-enclosed viral particle were presented in HPAI H7N9 virus infection starts in the respiratory tract. After infection, HPAI H7N9 virus replicates in the lung. Meanwhile, the patient suffered from persistent viraemia and a cytokine storm was observed his plasma. Live H7N9 virus was detected the patient’s blood. These phenomena were confirmed using animal experiments. How is the virus transmitted from the lung to the blood? One possible explanation is that inflammation leads to the disruption of the lung-blood barrier. Another possibility is that the virus is released into the blood through via exosomes. When the H7N9 virus exists in the blood, the virus might cause extrapulmonary infection. We speculated that the virus could infect the brain, after observing this phenomenon in the mouse model . HoweverIn the present study, HPAI H7N9 infection of a patient proved to be deadly. This patient presented with viraemia and died 3 days after admission. Whether there is live virus in the blood and whether it could lead to extrapulmonary infection have been two important questions. Previous studies detected viral RNA from LPAI H7N9, H5N1, and H1N1 infections ,8. H5N1 In the previous studies of H5N1 and pandemic 2009 H1N1 virus infection, viraemia was commonly existed in the fatal cases, but was absent in nonfatal cases . In prevIn our previous study, we observed that the levels of 34 of the 48 cytokines and chemokines tested were significantly elevated in plasma samples from patients infected with LPAI H7N9. We further found that the levels of MIF, SCF, MCP-1, HGF, and SCGF-β were highly positively correlated to disease severity. In addition, the profile of mediators MIF, SCF, MCP-1, HGF, SCGF-β, IP-10, IL-18, and IFN-γ is an independent outcome predictor . Cytokinin vitro study, avian H7N9 virus could effectively infect human brain cells. Meanwhile, the virus could be transcribed, replicate its viral genome, and generate infectious progeny virus [Influenza virus infection of the respiratory tract is associated with a range of neurological complications. The incidence of severe pandemic H1N1 influenza-associated neurological complications was estimated to be 1.2 per 1,00,000 persons among children and Asian Pacific patients ,33. The ny virus . In the However, we must say that we haven’t fully revealed the potential mechanism behind this feature of the 2017 H7N9 strain. But we observed the GZ8H002 strain might have acquired the possible key molecular substitutions associated with increased virulence and transmissibility in mammals. As we know, the presence of KRTA insertion in the cleavage sites enhanced the extrapulmonary infection ability and virulence of HPAI virus ,39. ThisExosomes are cellular secreted vesicles that are free to enter cells in a receptor-independent manner. Exosomes have been found to be closely associated with viral infection , and canIn summary, the viraemic patient was confirmed to be infected with HPAI H7N9. Cytokine storms and potential brain infections might be the cause of patient death. Exosomes might be the pathway that leads to infection in extrapulmonary tissues.


Mycobacterium tuberculosis (M. tb) that are difficult to treat. A current therapeutic strategy attempting to circumvent this issue aims to enhance drug delivery to reduce the duration of the antibiotic regimen or dosage of first-line antibiotics. One such agent that may help is cyclic peptide [R4W4], as it has been shown to have antibacterial properties (in combination with tetracycline) against methicillin-resistant Staphylococcus aureus (MRSA) in the past. The objective of this study is to test cyclic peptide [R4W4] both alone and in combination with current first-line antibiotics (either isoniazid or pyrazinamide) to study the effects of inhibition of M. tb inside in vitro human granulomas. Results from our studies indicate that [R4W4] is efficacious in controlling M. tb infection in the granulomas and has enhanced inhibitory effects in the presence of first-line antibiotics.Tuberculosis (TB) is currently one of the leading causes of global mortality. Medical non-compliance due to the length of the treatment and antibiotic side effects has led to the emergence of multidrug-resistant (MDR) strains of  Mycobacterium tuberculosis (M. tb) is the etiological agent that is responsible for causing tuberculosis (TB). According to the World Health Organization (WHO), it is estimated that around 9 million people are suffering from active TB disease with an approximate global mortality of 1.43 million people annually  are one such alternative therapeutics against antibiotic-resistant pathogens since they may act as effectors and regulators of the immune system as well as inhibitors of bacterial cell growth . Additioresidues , enablin4W4] causes a significant decrease in the viability of M. tb and works effectively with first-line antibiotics such as isoniazid (INH) and pyrazinamide (PZA) by modulating the levels of cytokine release, oxidative stress, and autophagy.Our study findings indicate that  (4 μg/ml), [R4W4] (8 μg/ml), INH and [R4W4] (4 μg/ml), INH and [R4W4] (8 μg/ml), PZA and [R4W4] (4 μg/ml), and PZA and [R4W4] (8 μg/ml). All treatments that included INH and PZA used minimum inhibitory concentrations . Treated infected PBMCs were cultured for 8 days at 37°C in the presence of 5% CO2 for granuloma formation. At the conclusion of the 8-day incubation, each well was terminated. Supernatants of each well were collected and aliquoted into sterile Eppendorf tubes by treatment categories. Sterile cold 1 × PBS was added into each experimental well without cover glasses to lyse the in vitro granulomas. To further dislodge the in vitro granulomas, each well was scraped using a sterile micropipette. Lysates from each category were then collected and aliquoted into sterile Eppendorf tubes by treatment category. Wells with cover glasses were treated with 4% paraformaldehyde (PFA) for 1 h at room temperature to affix in vitro granulomas to their respective cover glasses for imaging analyses. PFA-treated wells were also washed three times with PBS to remove any cellular debris that could impede imaging analyses.PBMCs  supplemented with albumin-dextrose-catalase (ADC)  were inoculated with previously collected supernatants and lysates. Plates were then incubated at 37°C for a minimum of 4 weeks. Plates were then read, and colony-forming units (CFUs) were counted.In order to quantify the intracellular survival of Cytokine analyses were conducted to quantify the produced levels of TNF-α, IFN-γ, and IL-10 in collected supernatant samples. The sandwich enzyme-linked immunosorbent assay (ELISA) was used. Assays and analyses were conducted per the assay manufacturer's protocol .in vitro granulomas were permeabilized with Triton-X for 2 min and stained overnight with FITC-conjugated CellROX and with antibodies against LC3B conjugated with PE. Cover glasses were washed with PBS and mounted on clean glass slides with mounting media containing 4′,6-diamidino-2-phenylindole (DAPI). Slides were observed under the fluorescent microscope. Fluorescent images were captured, and the fluorescent intensity was quantified using the ImageJ software.Cover glasses with fixed p < 0.05 was considered statistically significant.All data analyses for this study were conducted using GraphPad Prism 8.0. . A one-way ANOVA  with Tukey corrections was performed for datasets greater than two groups. Reported values are in means with each respective category. Data represent ±SE from experiments performed in eight different individuals. Analyses with a 4W4], led us to test its efficacy against M. tb., which has a thick peptidoglycan layer along with other lipid layers on the cell wall. [R4W4] has already been shown to have potent antibacterial activity against pathogenic gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) with an inhibitory concentration (MIC) of 2.67 μg/ml (1.95 μM). In addition, a follow-up 24-h study showed bactericidal activity against MRSA with the combination of two times the MIC of [R4W4] and four times the MIC of tetracycline (0.5 μg/ml) (4W4] have been reported using MTS proliferation assay against two cancer cell lines (human ovarian adenocarcinoma SK-OV-3 and human leukemia CCRF-CEM) and one normal human embryonic kidney HEK 293T cell line at 24-h incubation. The cyclic peptide [R4W4] demonstrated more than 84% cell viability at a concentration of 20.5 μg/ml in both cancer and normal cell lines  against MRSA  with lone INH and PZA at MIC did not result in complete clearance of M. tb.  and the growth of M. tb was monitored for 8 days  in 7H9 was significantly reduced by 2.2-fold in the group treated with [R4W4] at 4 μg/ml concentration (p = 0.0223)   . There w 0.1709) .M. tb inside the in vitro granulomas treated with [R4W4] at 4 and 8 μg/ml. The sham control group of human M. tb-infected granulomas was compared against in vitro granulomas treated with [R4W4] at 4 and 8 μg/ml, respectively. Compared to the control group, intracellular M. tb survival was significantly reduced in both groups treated with [R4W4] at 4 μg/ml (p = 0.0348) and 8 μg/ml concentrations (p = 0.0423)  (in vitro granulomas with R4W4 caused a reduction in the M. tb burden (We then determined the viability of  0.0423) . In comp 0.2719) . These fb burden .4W4] in altering the underlying immune effector mechanisms against M. tb infection, we first measured the levels of several related cytokines that cause the activation (TNF-α and IFN-γ) and inhibition (IL-10) of immune effector mechanisms. TNF-α expression was significantly downregulated by [R4W4] at both 4 μg/ml (p = 0.036) and 8 μg/ml concentrations (p = 0.0003) compared to sham-treated control categories  compared to sham control . These findings indicate that [R4W4] supplementation augments immune responses against M. tb infection by downregulating the levels of IL-10  leading to oxidative stress. It is important to point out that the survival of M. tb is dependent on the extent of ROS produced by the host immune cells. Exacerbation in the levels of ROS can damage the immune cells causing diminishment in the effector responses against M. tb infection  in reducing the burden of M. tb in the in vitro granulomas. Infected PBMCs were treated with [R4W4] at 4 and 8 μg/ml concentrations and cultured in the presence and absence of INH or PZA at their respective MIC. Consistent with our previous observations, there was a significant and 18-fold reduction in viability of M. tb in INH-treated granulomas (p = 0.0314). Treatment with INH in conjunction with [R4W4] at either 4 μg/ml (p = 0.0287) or 8 μg/ml (p = 0.0292) concentration resulted in a further reduction in the viability of M. tb when compared with the control category  and 8 μg/ml (p = 0.3803) concentrations  along with a notable decrease in the levels of IL-10 (p = 0.1336) in the granuloma supernatants from INH + [R4W4] treatment categories when compared to untreated and INH-alone treated groups . A further decrease in the viability of M. tb was observed with PZA + [R4W4] treatments. The fold reduction in the viability of M. tb was 511 and 136 in granulomas treated with PZA + [R4W4] at 4 μg/ml and PZA + [R4W4] 8 μg/ml, respectively, when compared to untreated granuloma. The levels of TNF-α, IL-10, and IFN-γ were measured in the granuloma supernatants from control, PZA-treated, and PZA + [R4W4]-treated groups  treatment resulted in an increase in the levels of IFN-γ (4W4] showed a significant increase in the intensity of CellROX staining compared to the PZA-alone group (4W4] treatment caused a significant increase in the intensity of LC3B staining compared to the control and PZA-alone group. In comparison to the untreated granulomas, the fold increase in the intensity of LC3B staining was 2 and 2.4 in granulomas treated with PZA + [R4W4] at 4 μg/ml and PZA + [R4W4] at 8 μg/ml, respectively (4W4] caused increased production of IFN-γ, decreased levels of IL-10, increased autophagy, and improved control of M. tb infection. These findings further support the previous findings that decreased IL-10 along with increased IFN-γ will favor effective control of M. tb infection (Although not significant, PZA + [Rof IFN-γ . PZA trene group . There wectively . Our resnfection .4W4] elicits direct antimycobacterial ability against virulent Erdman strain of M. tb at 4 μg/ml. Treatment of in vitro granulomas with [R4W4] both alone and in combination with first-line antibiotics such as INH and PZA at MIC concentration resulted in a further decrease in the viability of M. tb. The addition of [R4W4] to granulomas both in the presence and absence of PZA led to a reduction in the levels of TNF-α and IL-10, and elevation in the levels of autophagy. Our study findings, therefore, indicate that [R4W4] causes a significant decrease in the viability of M. tb and works in conjunction with first-line antibiotics by modulating the levels of cytokine release and autophagy. In conclusion, we believe that [R4W4], along with anti-TB treatment, would not only attenuate the side effects of antibiotics but can also enhance the immune responses to eliminate the active M. tb infection.Our study findings demonstrate that [RThe datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Institutional Review Board of Western University of Health Sciences. The patients/participants provided their written informed consent to participate in this study.AA synthesized cyclic peptide. RK and VV conceived the study, developed study design, analyzed the data, and prepared the manuscript. JH, DA, and RC conducted the studies and drafted the manuscript. AY, KT, RA, and TN provided technical assistance. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Seeds have greatly contributed to the successful colonization of land by plants. Compared to spores, seeds carry nutrients, rely less on water for germination, provide a higher degree of protection against biotic and abiotic stresses, and can disperse in different ways. Such advantages are, to a great extent, provided by the seed coat. The evolution of a multi-function seed-coat is inheritably linked to the evolution of tissue polarity, which allows the development of morphologically and functionally distinct domains. Here, we show that the endothelium, the innermost cell layer of the seed coat, displays distinct morphological features along the proximal-distal axis. Furthermore, we identified a TRANSPARENT TESTA transcriptional module that contributes to establishing endothelium polarity and responsiveness to fertilization. Finally, we characterized its downstream gene pathway by whole-genome transcriptional analyses. We speculate that such a regulatory module might have been responsible for the evolution of morphological diversity in seed shape, micropylar pore formation, and cuticle deposition. The seed coat consists of cell layers that surround, nourish, protect, and facilitate the dispersal of the fertilization product(s) . As a whIn Arabidopsis, the seed coat comprises an outer (oi) and an inner (ii) integument . Both iitransparent testa (tt). TT loci are involved in different aspects of tannin deposition: biosynthesis, transport, and regulation  or Wassilewskija (Ws-2) were used as wild type controls as appropriate. The tt16-1 mutant was isolated in the Ws-2 accession and then backcrossed to the Col-0 accession more than three times  and subsequently transferred to soil for further characterization.The p method . TransfoGAPDH, AT4G12590, AT4G02080, and AT3G25800) , including RNase-Free DNase Set (Qiagen) treatment during washing, according to the manufacturer’s instructions, and subsequently stored at −80°C. The Superscript Reverse Transcriptase II kit (Invitrogen) was used to generate cDNA from 1 µg of total RNA. Each cDNA sample was diluted 1:125 in water. Quantitative PCRs were performed with the SYBR Green kit (Bio-Rad) on a Bio-Rad CFX real-time PCR machine. For each reaction, 4.4 µL of diluted cDNA were added to 5 µl of SYBR Green and to 0.3 µl of each primer (10 µM) (3G25800) , and sub3kbProTT1:gTT1 was performed from Arabidopsis (Col-0) genomic DNA (https://www.ncbi.nlm.nih.gov/gene/840386 ) using the gene-specific primers (5′- GGGGACAAGTTTGTACAAAAAAGCAGGCTCTAACCATTTGCTTGTGTCAACA -3′) and (5′- GGGGACCACTTTGTACAAGAAAGCTGGGTCAAAAAACAAAGTCTCGGAGACAG -3′) carrying the attB1 and attB2 Gateway recombination sites. 3kbProTT1:gTT1 was recombined into the pDONR207 vector (Gateway recombination). Compared to what annotated on the TAIR website (www.arabidopsis.org), the 3kbProTT1:gTT1 DNA fragment misses an adenine in position −1250 of the promoter and the CATATATATATATATATATATATATATATATATA sequence in position 605–638 of the intron. PCR amplification of gTT1 was performed using the gene-specific primers (5′- GGGGACAAGTTTGTACAAAAAAGCAGGCTCAATGGAGTCACCACCACTATACGAGA -3′) and (5′- GGGGACCACTTTGTACAAGAAAGCTGGGTCAAAAAACAAAGTCTCGGAGACAGAT -3′) carrying the attB1 and attB2 Gateway recombination sites. For GR-inducible analyses, the gTT1 sequence was recombined (Gateway recombination) into the pR1R2ΔGR binary vector  were harvested.−1), sucrose (7%) solution, as previously described  analyses were conducted as previously described . GFP andescribed . mPS-PI escribed . Transmiescribed .Modified pseudo-Schiff propidium iodide (mPS-PI) or calcofluor stained and GFP or citrine fluorescent samples were analyzed with a Leica TCS-SP5 or Leica TCS-SP8 spectral confocal laser scanning microscope (Leica Microsystems). For TEM, samples were examined with Hitachi HT7700 electron microscope operated at 80 kV (Elexience—France), and images were acquired with a charge-coupled device camera (AMT). For confocal microscopy, pictures showing mid-plane longitudinal sections of seeds were captured when possible. Otherwise, three dimensional z-stacks were acquired, and the mid-plane longitudinal sections were obtained with the Volume Viewer plugin of the Image J software .P and L represent perimeter and periclinal length of the cell, respectively. In the case of a perfect rectangle, GP represents the ratio between anticlinal and periclinal lengths.Quantitative analyses of cell area and roundness were conducted as previously described . Ratio bPro35S:gTT1-GR main inflorescences at stage 6.50 . The function SqueezeVar of the Limma library was used to smooth the specific variances by computing empirical Bayes posterior means. The library kerfdr was used to calculate the adjusted p-values.For each array, the raw data comprised the logarithm of median feature pixel intensity at wavelengths 635 nm (red) and 532 nm (green). For each array, a global intensity-dependent normalization using the loess procedure  was perfwww.arabidopsis.org). GO enrichment analyses were conducted using the gene ontology enrichment analysis and visualization tool , TT1 (AT1G34790), SHP1 (AT3G58780), SHP2 (AT2G42830), STK (AT4G09960), ML1 (AT4G21750), PDF2 (AT4G04890), CR4 (AT3G59420), TT2 (AT5G35550), TT8 (AT4G09820), TTG1 (AT5G24520), TTG2 (AT2G37260), FIE (AT3G20740), and MSI1 (AT5G58230). Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: Methods). The chalazal region of the endothelium was characterized by smaller and rounder cells, when compared to the other regions . Whereas the periclinal cell length was on average greater than the anticlinal cell length all along the wild type endothelium, tt1 seeds exhibited anticlinally elongated cells in a sub-domain of the micropylar region . RNA in situ hybridization experiments showed TT1 expression in the endothelium but high background signal levels did not allow to conclusively address TT1 expression in other integument cell layers . The promoter region of CmWIP1, TT1 orthologue in melon, contains retrotransposons whose methylation regulates CmWIP1 expression  upstream of TT1 genomic sequence translationally fused to GFP (3kbProTT1:gTT1-GFP). Seven independent 3kbProTT1:gTT1-GFP lines showed fluorescence in the nuclei of endothelium cells from stage 2-IV of ovule development  and seeds (2 DAF). TT1 expression was equally reduced before and after fertilization in the tt16 mutant when compared to the wild type  (PROTODERMAL FACTOR 2 (PDF2), and CRINKLY4 (CR4) epidermal cell fate genes , and TTG2 encode for transcription factors that regulate the flavonoid late metabolism in developing siliques. TT2 and TT8 were down-regulated whereas TTG1 up-regulated in tt1 seeds at 4 DAF, when compared to the wild type (The henotype  and has henotype . To furt35S promoter (Pro35S:TT1-GR). Dexamethasone (DEX) treatment releases the GR transcription factor chimeric protein from a cytoplasmic HEAT SHOCK PROTEIN 90 complex that prevents its nuclear translocation and therefore its functionality . Addition of DEX to Pro35S:TT1-GR plants caused leaf elongation and narrowing similar to plants that overexpress TT1 (Sagasser 2002), indicating that the TT1-GR chimeric protein retains function. To minimize sample handling, we analyzed entire inflorescences bearing ovules and seeds up to the early torpedo embryo stage. To prevent indirect transcriptional effects of the inducible TT1-GR protein, we infiltrated Pro35S:TT1-GR inflorescences with cycloheximide (CHX), an inhibitor of protein synthesis. We then treated the samples with DEX or a mock solution for 1 h. To discount for the unspecific effect of DEX, we conducted an identical experiment with wild type inflorescences. Transcript levels across the entire genome were measured by hybridization to CATMA microarrays  under the control of the constitutive cauliflower mosaic virus roarrays . To incrPRX36 co-expression network  , the DIR network , three a network , a mucil network  PECTIN M network , and the network   genes  genes , which aP) genes .CmWIP1, TT1 orthologue in melon, has been shown to repress the expression of the melon 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE 7 (ACS7), an ethylene biosynthetic enzyme that regulates sex determination. In line with this study, we revealed that TT1-GR represses ACS4, an orthologue of ACS7, and regulates the expression of four ethylene responsive factor genes (ARABIDOPSIS TAPETUM 1 (ATA1), the orthologue of the TASSLE SEED 2 gene that controls sex determination in maize  , which regulates directional cell growth and cortical microtubule organization  and MULTICOPY SUPPRESSOR OF IRA1 (MSI1) Polycomb Group (PcG) proteins, which prevent the fertilization-independent expansion of the integuments . A fractlization . Howevery spaces , as obseTT1 in the ii1’ cell layer than previously described (TT1 expression solely in newly developed ii1’ cells, right after endothelium periclinal cell division, and in the most proximal ii1’ cells. Compared to previous analyses, we tested a longer promoter region containing transposon sequences, which have been shown to regulate TT1 orthologue expression in melon (TT1 genomic sequence. These data suggest that Arabidopsis TT1 expression in the ii1’ might be negatively regulated by transposons or TT1 intronic sequence. In an alternative non-exclusive scenario, TT1 protein might be rapidly degraded in the ii1’ cell layer. TT1 protein pattern across fertilization, constantly present in the endothelium while only transiently in most ii1’ cells, might explain for the ii1’ unique responsiveness to fertilization.The ii1’ cell layer differs from the other cell layers for its sub-epidermal position and origin by periclinal cell divisions. It has been previously shown that sub-epidermal outer integument cell stripes, developing in a fraction of wild type seeds, respond to the FIE and MSI1 repressive mechanism as epidermal integument cell layers . This stescribed . We detein melon , and TT1TT1 expression in the ii1’ is activated by TT16. It has been shown that TT16 regulates ii1’ cell expansion and patterning along the proximal-distal axis (tt1 and tt16 seeds display ii1’ cells with an anticlinally over-elongated shape when compared to the wild-type (tt16 seeds do not display empty spaces in between ii1’ cells, thus suggesting that TT1 and TT16 also play independent functions as observed in the endothelium micropylar region.Our data show that tal axis . In lineild-type . NeverthThe datasets generated for this study can be found in the Microarray data from this article were deposited in the international repository GEO, Gene Expression Omnibus , accessiOC performed the research, analyzed the data, and helped to write the article. JL, WX, and DG helped to perform morphological analyses, SP performed the transcriptomic analysis and CP performed the transmission electron microscopy analysis. LL helped to analyze the data and write the article. EM designed the research and wrote the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.