Human infections with avian influenza H7N9 or H10N8 viruses have been reported in China, raising concerns that they might cause human epidemics and pandemics. However, how these viruses adapt to mammalian hosts is unclear. Here we show that besides the commonly recognized viral polymerase subunit PB2 residue 627 K, other residues including 87E, 292 V, 340 K, 588 V, 648 V, and 676 M in PB2 also play critical roles in mammalian adaptation of the H10N8 virus. The avian-origin H10N8, H7N9, and H9N2 viruses harboring PB2-588 V exhibited higher polymerase activity, more efficient replication in mammalian and avian cells, and higher virulence in mice when compared to viruses with PB2-588 A. Analyses of available PB2 sequences showed that the proportion of avian H9N2 or human H7N9 influenza isolates bearing PB2-588 V has increased significantly since 2013. Taken together, our results suggest that the substitution PB2-A588V may be a new strategy for an avian influenza virus to adapt mammalian hosts. Orthomyxoviridae. Antigenic drift (mutation) and antigenic shift (reassortment) are responsible for the rapid evolution of IAV, resulting in influenza outbreaks cannot easily be controlled despite annual vaccination efforts in human and animal populations1575Influenza A virus (IAV) is an important zoonotic pathogen that infects mammals and birds and induces seasonal influenza epidemics and pandemics in humans. IAV is a negative-sense, single-stranded, segmented RNA virus that belongs to the family The first human infection with an H7N9 virus occurred in March 2013, in eastern China121315171819Adaption mutations in viral proteins are important strategies for AIV to cross species barriers and adapt to mammalian hosts23242630323334We previously characterized two H10N8 isolates, a human-origin strain A/Jiangxi-Donghu/346-1/2013 (JX346) isolated from a 73 year-old woman who died from severe pneumonia6 EID50 of each virus. Mice infected with JX346, GH074, or the lethal V virus showed symptoms of illness and weight loss starting from 2 days post-infection (d.p.i). Mice infected with avian-origin G1 or JX102 viruses did not display obvious clinical symptoms and weight loss, although mice infected with JX102 showed a slight weight loss (<5%) around 7 d.p.i. and then recovered  that is lethal in miceecovered , left. Tecovered , right. ecovered , left. Tecovered , right. 6 EID50 of the indicated viruses. The recombinant virus JX102-JX346PB2 containing the JX346-derived PB2 gene was virulent in mice, inducing severe weight loss  in the other 6 gene segmentsght loss  and 100%ght loss . The JX1ght loss . The virght loss . Conversght loss  when comght loss . The JX3ght loss . No virught loss . These rTo identify amino acids in PB2 that contribute to the high virulence of JX346 in mice, we compared the PB2 sequences of JX346 and JX102 and found that there were eleven amino acid differences . The ami22Since JX346-PB2 627E still exhibited a high pathogenicity in mice and in order to demine the influence of the other 10 amino acids besides 627K differing between JX346 and JX102 on polymerase activity, we performed a mini-genome assay using different genotypes of JX346 PB2 containing single substitutions  combined with the K627E mutation in human 293T and avian DF-1 cells. The substituted residues 195D, 292I, 588A, and 598T combined with 627E in PB2 resulted in a significantly lower polymerase activity in HEK293T cells while no significant difference was observed for other substitutions when compared to the PB2 with a single mutant 627E . Both 19Additionally, twelve mutant viruses that possessed the double substitutions in PB2 as described above in the genetic background of the JX346 virus were generated and tested in mice. In contrast to the parental JX346-wt and JX346-PB2 627E viruses, the mutant viruses including JX346-PB2 87D/627E, JX346-PB2 292I/627E, JX346-PB2 340R/627E, JX346-PB2 588A/627E, JX346-PB2 648L/627E, and JX346-PB2 676T/627E were less virulent , whereasWhile 87E, 292V, 340K, 588V, 627K, 648V, and 676M in PB2 play a critical role in the virulence the H10N8 JX346 virus, only the V588A substitution in PB2 resulted in a significantly lower polymerase activity in both human 293T and avian DF-1 cells . Moreove50) and virus replication in mice using mutant viruses. The single substitution A588V in PB2 enhanced the virulence of the avian-origin JX102 virus, evidenced by a reduced the dose of MLD50 and the efficient virus lung replication in infected mice when compared to the parental JX102 virus  or double (A588V/E627K) substitutions in PB2 were generated in the genetic background of the H7N9 avian-origin G1 virus. The results of mini-genome assays in HEK293T cells showed that the polymerase activity was significantly enhanced due to the introduction of PB2 A588V substitution into the G1 PB2 at both 33 °C and 37 °C, as compared with the parental G1-PB2, although it did not result in a similar level of polymerase activity induced by the substitution of E627K or A588V/E627K . In DF1 50 value  (We have shown that the H9N2 V-PB2 627E is not virulent in mice06EID50) . Taken thttp://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) and GISAID (http://platform. gisaid.org) were analyzed. There were four variable amino acids  at this position in PB2 and the Alanine (A) was the most common amino acids . No further human H10N8 infections cases have been reported, most likely because the H10N8 virus has not adapted to mammalian hosts and still prefers to bind avian-like receptors192041The recently emerged H7N9 and H10N8 viruses are able to induce severe respiratory illness and high case fatality rate (35%) in humans, but they do not cause obvious disease and mortality in chickensTo date, it remains unclear why and how avian H7N9 and H10N8 viruses acquire the ability to cross the species barrier to infect humans. A recent study has shown that the internal genes PB2, M, and NP from the endemic H9N2 virus111244Many adaptive mutations in PB2, such as E627K34Neural tropism of the influenza viruses  is an inin vitro results may not represent virus biological features such as virulence in vivo.The conserved structure of the newly defined 627-domain in PB2515253Although many adaptive mutations in PB2 have been identified, the underlying mechanism of influenza host adaptation is unclear. Differing side chains in residues at 588 may affect PB2 structure and its interaction with other viral or host proteins, which is important for influenza virus to adapt to new hosts58i) the effect of the PB2 627K on influenza viruses may be strain-dependent, similar to data previously reported with H5N1ii) the PB2 627K mutation is not the only key factor for mammalian adaption of the novel H7N9 influenza virus and other adaptive strategies to mammalian hosts may be employed for this virus  and H10N8 66.6%) isolates contain the combination of PB2 588V/627K which has been demonstrated to have substantial effects on virus replication capacity and virulence in both H10N8 and H7N9 viruses ,5 and 6,6.6% isolIn summary, PB2-588V identified in our study is important for polymerase activity, virus replication, and virulence in mice, indicating that it may be a new marker of mammalian adaption for the avian influenza virus. Therefore, attention should be given to this new marker during surveillance of avian influenza viruses.MDCK, Human embryonic kidney 293T (HEK 293T) and avian cells DF-1 were grown in Dulbecco’s modified essential medium (DMEM) with 10% fetal bovine serum and antibiotics. The H10N8 A/Jiangxi-Donghu/346-1/2013 (JX346) virus was isolated from a patient with a fatal infection and A/chicken/Jiangxi/102/2013 (H10N8) (JX102) was isolated from healthy chickens in a live poultry market (LPM) that the patient had visited. The phylogenetic and genetic properties of both JX346 and JX102 have been describedEight gene segments from the JX346, JX102, and G1 strains were cloned into a pHW2000 plasmid system and were confirmed using DNA sequencing. Reverse genetic systems for V and V-627E were described previously. The PB2 mutant constructs were produced by site-directed mutagenesis PCR and sequenced. The recombinant and mutant viruses were generated using reverse genetics. HEK293T cells 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 3000 (Invitrogen) according to the manufacturer’s instructions. DNA and transfection reagent were mixed (8 μl p3000 reagent and 7.5 μl Lipofectamine 3000 per well), incubated at room temperature for 5 min, and added to the cells. Six hours later, the mixture was replaced with Opti-MEM (GIBCO) containing 0.2% bovine serum albumin and 1 μg/ml TPCK 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 and sequenced.2 and inoculated intranasally with indicated viruses. Body weight and clinical symptoms were monitored daily for 14 days after infection. The mice were euthanized if they lost more than 25% of their initial body weight. To test virus replication in organs, three mice in each group were euthanized on days 3 and 5 p.i., respectively. Tissue samples including brain, lung, kidney, liver and spleen, from each euthanized mouse were collected for virus titration by an EID50 assay. To determine MLD50 of each virus, three mice per group were anaesthetized with CO2 and inoculated intranasally with 10-fold serially diluted each virus in a 50 μl volume (the dose ranges from 101 to 106 EID50) The MLD50 of each virus was calculated using the method of Reed and Muench.Groups of fourteen 4- to 6-week-old female BALB/c mice, obtained from the Vitalriver Company in Beijing, were anesthetized with COFor polymerase activity assays, RNP complexes composed of PA, PB1, PB2 and NP (200 ng each) were mixed with a luciferase reporter plasmid (200 ng) and a thymidine kinase promoter-Renilla luciferase reporter plasmid (pRL_TK) construct (20 ng), then co-transfected into HEK293T cells in a 12-well plate with Lipofectamine3000 , and incubated at 33 °C or 37 °C for 24 h. Luciferase production was assayed using the dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions. Polymerase activity was normalized to Renilla expression. For the luciferase reporter assays in DF-1 cells, a luciferase reporter containing a chicken RNA polymerase I (Pol I) promoter was constructed as described previously50/cell for 1 h at 37 °C. After 1 h incubation, the cells were washed twice, and then incubated with DMEM containing 0.2% bovine serum albumin (BSA) and TPCK trypsin (1.0 μg/ml) at 37 °C with 5% CO2. Culture supernatants were collected at indicated time points and viral titers were determined by the 50% tissue culture infective dose (TCID50) assay in MDCK cells.Confluent MDCK, HEK293T or DF-1 cells were infected at a multiplicity of infection (MOI) of 0.01 TCIDAll animal experiments involving recombinant H7N9 and H10N8 viruses were reviewed and approved by the Institutional Animal Care and Use Committee at South China Agricultural University (SCAU) and were carried out in accordance with the approved guidelines. All experiments were conducted in biosafety level 3 laboratories and animal facilities at SCAU.Differences between experimental groups were determined by using an unpaired t-test and analysis of variance (ANOVA) in the GraphPad Prism software (GraphPad Software Inc.).How to cite this article: Xiao, C. et al. PB2-588V promotes the mammalian adaptation of H10N8, H7N9 and H9N2 avian influenza viruses. Sci. Rep.6, 19474; doi: 10.1038/srep19474 (2016).


This site is also phosphorylated in cells in a cell cycle dependent manner, consistent with it being a CDK2 substrate. Furthermore overexpression of cyclin E/CDK2 stimulates while the CDK2 inhibitor p27KIP1 represses phosphorylation at threonine 152. This site is located in a bipartite nuclear localization sequence but its phosphorylation was not sufficient to deregulate the subcellular localization of ING5. Although ING5 interacts with the tumor suppressor p53, we could not establish p53-dependent regulation of cell proliferation by ING5 and by phospho-site mutants. Instead we observed that the knockdown of ING5 resulted in a strong reduction of proliferation in different tumor cell lines, irrespective of the p53 status. This inhibition of proliferation was at least in part due to the induction of apoptosis. In summary we identified a phosphorylation site at threonine 152 of ING5 that is cell cycle regulated and we observed that ING5 is necessary for tumor cell proliferation, without any apparent dependency on the tumor suppressor p53.Inhibitor of growth (ING) proteins have multiple functions in the control of cell proliferation, mainly by regulating processes associated with chromatin regulation and gene expression. ING5 has been described to regulate aspects of gene transcription and replication. Moreover deregulation of ING5 is observed in different tumors, potentially functioning as a tumor suppressor. Gene transcription in late G1 and in S phase and replication is regulated by cyclin-dependent kinase 2 (CDK2) in complex with cyclin E or cyclin A. CDK2 complexes phosphorylate and regulate several substrate proteins relevant for overcoming the restriction point and promoting S phase. We have identified ING5 as a novel CDK2 substrate. ING5 is phosphorylated at a single site, threonine 152, by cyclin E/CDK2 and cyclin A/CDK2  The cell cycle of eukaryotic cells is controlled by cyclin-dependent kinases (CDKs) . These eKIP1, which interacts with the kinase complex and negatively controls its activity 8], FoxM1activity ,17. Moreactivity ,19. Cyclactivity ,21,22,23activity ,25,26,27activity ,29,30. Aactivity ,32. Toge+-dependent deacetylase SIRT2 and determined that the enzymatic activity of this protein is negatively regulated by cyclin E/CDK2-dependent phosphorylation both in vitro and in cells. The phosphorylation site is located in an unstructured C-terminal extension of the catalytic domain of SIRT2. Other sirtuins have also potential CDK phosphorylation sites at similar positions relative to the catalytic domain [We have reported on a cyclin E/CDK2 screen that identified 26 proteins as potential substrates . We studc domain . An addic domain . ING1, tc domain . Subsequc domain ,36,37. Mc domain ,39,40.ING5 was identified in a homology search for additional ING family members . SimilarING5 is located is deleted in oral squamous cell carcinoma and odontogenic tumors [Several studies have implicated ING5 in cancer development. For example the region on chromosome 2q37.3 where c tumors ,53,54. Ic tumors . Moreovec tumors . It suggc tumors . In anotc tumors . These fSimilar to other ING proteins, ING5 has been reported to interact with p53 . By enhaHere we describe ING5 as a novel cyclin E/CDK2 substrate. ING5 is phosphorylated at a single site, threonine 152 (T152). We have addressed the functional relevance of T152 phosphorylation by generating non-phosphorylatable and phospho-mimicking mutants and determined their effect on cell proliferation dependent on the tumor suppressor p53. We find that ING5 is required for cell proliferation independently of p53 and that phosphorylation of T152 does not impinge on cell proliferation.6 cells/10 cm dish and serum starved in 0.1% FCS the next day. After 72h the cells were split 1:3 and fed with medium containing 10% FCS, which promotes reentry into the cell cycle [5 cells/6cm plate. Cells were co-transfected, using ExGen500 (MBI Fermentas), with 4.5 μg expression plasmids encoding p27KIP1, ING5, and ING5 mutants or the pEVRF0-HA control vector and 0.5 μg pBABEpuro, a puromycin resistance plasmid. Cells transfected with 5 μg pEVRF0-HA only were used as a negative control. Media was changed six hours after transfection and three hours later cells were selected for sixteen hours with 2 μg/ml puromycin. Ten to twelve days after transfection, plates were washed with PBS and stained in 0.2% (w/v) methylene blue in methanol. Images of each plate were converted to grayscale using Adobe Photoshop 7.0. The numbers of gray pixels were determined and the values of the negative controls were subtracted to determine colony density. For the cyclin E and cyclin A knockdown experiments, ING5 expressing plasmids were transfected first using the Calcium Phosphate method. The next day the cells were split 1:3 and the siRNAs  were reverse transfected using 20 μl HiPerfect/10 cm dish (Qiagen). The following Dharmacon human siGenome SMARTpools were used: cyclin E (M-003213-02), cyclin A (M-003205-02) and control (D-001206-14). Roscovitine and colcemid were purchased from Calbiochem, fulvestrant, tamoxifen and nocodazole from Sigma.HEK293, U2OS, T98G and HeLa cells were cultured in DMEM supplemented with penicillin/streptomycin and 10% fetal calf serum. For MCF7 cells this medium was supplemented with pyruvate and glutamine. HCT116 cells were cultured in McCoy medium supplemented with penicillin/streptomycin and 10% fetal calf serum. Transient transfection assays were performed as described previously [ll cycle . For colAll ING5 constructs were generated by recombining the PCR-amplified ORF into pDonR201 of Invitrogen's Gateway cloning system by virtue of attB1 and attB2-mediated BP-clonase reaction. A subsequent LR-recombination reaction with Gateway-compatible pGex4T2, and pEVRF0-HA resulted in GST- and HA-tagged expression constructs. Site-directed mutagenesis was carried out using the Quick Change protocol (Stratagene) to generate the T152A and T152D mutants. All constructs were verified by DNA sequencing. pCMV-EGFP-tubulin was cloned by standard procedures and used to control transfection efficiency and to identify the transfected cells. For constructs expressing siRNA, the following sequences were cloned into pSUPER :siING5-1: 5’-GGA AGA TAA GAA AGC AGA GAT AGA CAT ACT AGC TGC AGA GTA CAT CTC CA (sense) and 5’-TGG AGA TGT ACT CTG CAG CTA GTA TGT CTA TCT CTG CTT TCT TAT CTT CC (antisense);siING5-2: 5’-GAT CCA GAA CGC CTA CAG CAA ATG TAA AGA ATA CAG TGA CGA CAA AGT G (sense) and 5’-CAC TTT GTC GTC ACT GTA TTC TTT ACA TTT GCT GTA GGC GTT CTG GAT C (antisense);Additional constructs for cyclins and CDKs have been described elsewhere .3VO4, 30 mM NaF, and 30 mM β-glycerophosphate.Expression of human cyclin/CDK complexes occurred in insect cells upon infection with recombinant baculo viruses. The complexes were purified using the GST tag of the cyclins. These procedures and in vitro kinase assays have been described ,66. For 2. The probes were then incubated in the presence of 2.5 U of shrimps alkaline phosphatase (Sigma) at 30°C for 30 min. The reactions were stopped by adding SDS-sample buffer.For phosphatase treatment cells were lysed in RIPA buffer and ING5 immunoprecipitated. Immobilized proteins were resuspended in 50 mM Tris-HCl, pH 8.5, and 1 mM MgClThe rabbit polyclonal anti-ING5 antiserum and the rat monoclonal antibodies 7A11 were raised against a GST-ING5 fusion protein and the rat monoclonal antibodies 3H5 against a peptide phosphorylated at T152.4P2O7, 50 mM NaF, 5 μM ZnCl2, 100 μM Na3VO4, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 units/ml α2-macroglobulin, 2.5 units/ml pepstatin A, 2.5 units/ml leupeptin, 0.15 mM benzamidine), for high stringency lysates in RIPA buffer (as described above).The ING5 pAb 3716 (Abcam), CDK2 pAb H-298 (Santa Cruz), cyclin E pAb M-20 and mAb H-12 (Santa Cruz), cyclin A mAb BF638 (Santa Cruz), cyclin B H20 (Santa Cruz), RB/RB-P Kit #9969 , the actin mAb C4 , ASH2L mAb 4C5 (Activmotif) , and the6/ml cells were harvested and stained with Vybrant DyeCycle violet stain (Molecular Probes). Apoptotic cells were identified by staining with Annexin V-eFluor 450 (eBioscience). Five million cells/ml were diluted in 1 ml binding buffer (eBioscience) and 100 μl of this suspension was incubated with 5 μl Annexin V for 15 min. Subsequently dead cells were stained by diluting the cell suspension with 200 μl binding buffer (eBioscience) and 5 μl PI . Cells were analyzed on a facscanto II and evaluated using the FlowJo software.For life cell FACS analysis, 1x10HeLa cells transiently transfected with constructs expressing HA-tagged ING5 proteins were fixed in PBS containing 3.7% paraformaldehyde for 15 min followed by washing and permeabilization in PBS with 0.1% Triton-X-100 for 5 min. The samples were blocked in PBS with 2% BSA and incubated with the mAb 3F10 as primary antibody. The secondary antibody was conjugated to Alexa555 (Invitrogen). The DNA was stained with Hoechst 33258.32P-γ-ATP [32P-orthophosphate. Immunoprecipitation of ING5 revealed that this protein was phosphorylated in cells  immunoprecipitated overexpressed as well as endogenous ING5 and the mAb 7A11 recognized these proteins on immunoblots . Using tPhosphorylation by CDK2 is determined by the specificity of the kinase and the presence of cyclin binding motifs with the sequence RXL in the substrate . InspectTo address more directly whether T152 is phosphorylated, we generated mAbs against a peptide phosphorylated at T152. We selected mAb 3H5, which detected recombinant ING5 specifically when phosphorylated by cyclin E/CDK2 . TogetheWe were then interested to evaluate T152 phosphorylation in cells. ING5 and ING5-T152A were expressed in HEK293 cells, immunoprecipitated and analyzed for T152 phosphorylation using mAb 3H5. While ING5 was phosphorylated, the mutant protein was not . Upon deKIP1 were co-expressed , including their ability to recognize specific histone modifications and modulating the PTMs of p53 ,73,74,75tion see . Cyclin ING proteins have attracted interest because of their involvement in cancer and because of their effects on the tumor suppressor protein p53 ,36,37. I


PIK3R1, which encodes the regulatory subunit of PI3K, in a metastatic RCC (mRCC), while the mutation was absent in the corresponding primary RCC (pRCC). To identify the function of PIK3R1 in RCC, we examined its expression in normal kidney, pRCC and mRCC by immunohistochemistry and real-time polymerase chain reaction. The expression of PIK3R1 significantly decreased in pRCC and was further reduced in mRCC compared with normal tissue. Besides, its expression levels were negatively correlated with T-category of tumor stage. Additionally, 786-O and A-704 cells with PIK3R1 depletion introduced by CRISPR/Cas9 system displayed enhanced proliferation, migration and epithelial-mesenchymal transition (EMT), and acquired a stem-like phenotype. Moreover, the PIK3R1 depletion promoted the phosphorylation of AKT in the cells. The knockdown of AKT by shRNA reduced p-GSK3β and CTNNB1 expression in the cells, while the depletion of CTNNB1 impaired stem-like phenotype of the cells. Overall, PIK3R1 down-regulation in RCC promotes propagation, migration, EMT and stem-like phenotype in renal cancer cells through the AKT/GSK3β/CTNNB1 pathway, and may contribute to progression and metastasis of RCC.The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway has been identified as an important pathway in renal cell carcinoma (RCC). We have reported a nonsense mutation in  VHL in ubiquitin-mediated proteolysis pathway (UMPP) and alteration of PBRM1 involved in chromatin regulation23Renal cell carcinoma (RCC) is one of the most lethal types of urological cancerPIK3CA, while the regulatory subunit p85α is encoded by PIK3R1In addition to UMPP2PIK3CA and PTEN are frequently altered in RCC6PIK3CA and PTEN, other member of the pathway play a role and be a target of therapy in RCC?The alteration of PI3K/AKT pathway has been found in a broad spectrum of cancersPIK3R1 in human carcinogenesis81213PIK3R1 has been reported as an oncogene in ovarian and colon tumorsPIK3R1 which resulted in decrease of PIK3R1 expression has also been strongly linked with colon cancersPIK3R1 in an mRCC, while the mutation was absent in the corresponding primary renal cell carcinoma (pRCC)Accumulating studies have showed an important role of PIK3R1 in renal cancer cell lines (786-O and A-704 cell lines) using a CRISPR/Cas9 system to achieve haploid knockout of PIK3R1 which significantly decreased the expression of P85α. The mutated renal cancer cells displayed increased abilities of colony formation, tumor formation, migration, epithelial-mesenchymal transition and oncosphere formation. Thus, our current study demonstrates that the downregulation of PIK3R1 contributes to progression and metastasis of RCC.We speculate that ectopic expression of PIK3R1 may be associated with progression and metastasis of RCC. To examine our hypothesis, we firstly analyzed the expression of PIK3R1 in RCC including both pRCC and mRCC by immunohistochemistry (IHC) and real-time polymerase chain reaction (RT-PCR). We discovered that the expression of PIK3R1 in RCC negatively correlated with tumor progression and metastasis. In addition, we induced deletion mutations of P = 0.0014) , pRCC (n = 13) and mRCC (n = 21) was determined by IHC. As shown in 19 0.0014) . Additio 0.0014) . These dPIK3R1 depletion, a CRISPR/Cas9 strategy which was designed according to the nonsense mutation reported previouslyPIK3R1. Firstly, we analyzed the expression levels of PIK3R1 in normal renal cell line (HK2) and RCC cell lines  by using RT-PCR. 786-O and A-704 cell lines showed higher expression of PIK3R1 compared with that of HK2 cell line  sequence of chr5: 67576819C in PIK3R1  and A-704 cells (A-704-mut1 and A-704-mut2) with haploid deletion mutation  (We then applied the CRISPR/Cas9 technologymutation . To detemutation . The pro < 0.01) .PIK3R1 on the growth of renal cancer cells, we carried out colony formation assays for the wild type and mutant cells. 786 and A-704 mutated cells displayed enhanced colony formation capability under the condition of separated single cells than that of 786-O and A-704 WT cells , NCAD , VIM  and ZEB1  in 786 and A-704 mutated cells respectively , ECAD (P < 0.05), NCAD (P < 0.01), VIM (P < 0.05), and ZEB1 (P < 0.05) in ACHN-PIK3R1 cells  and RCC cell lines  with RT-PCR. ACHN cell line showed higher expression of NCAD and VIM, compared to HK2 cell line . TherefoR1 cells . In addiR1 cells . These r+, CD133+ and CXCR4+ subset, respectively, compared with 786-O and A-704 WT cells (Cancer stem cells (CSCs) are defined as a subpopulation in tumors which harbors selfrenew, differentiation and serial tumor formation ability2425WT cells .PIK3R1 mutated cells showed that the mutated cells had a much greater ability to initiate tumors and increasing percentage of CSCs compared with the WT cells    There we < 0.05) . Though  < 0.05) .CTNNB1) activation mediated by PIK3R1 depletion, immunoprecipitation with PIK3R1 antibody was carried out by using lysates of 786-O, 786-mut1, 786-mut2, A-704, A-704-mut1, and A-704-mut2 cells. The quantity of p110α immunoprecipitated by PIK3R1 was decreased due to knockout of PIK3R1 in 786 and A-704 mutated cells respectively, compared with 786-O and A-704 WT cells  , compared with 786-O and A-704 WT cells that had the amount of unchanged AKT   and ERK with phosphorylation  in 786 and A-704 mutated cells respectively (In order to determine the molecular mechanism underlying β-catenin ( < 0.05) . Additio = 0.49) . Howeverectively .et al reported that cancer cells acquired cancer stem-like phenotype through alteration of the PI3K/AKT/β-catenin signalingP < 0.01; P < 0.001) , the expression of pAKT , p-GSK3β  and CTNNB1  was decreased in 786-mut1 shAKT and A-704-mut2 shAKT cells respectively, compared with 786-mut1 shCtrl and A-704-mut2 sh Ctrl cells  . To deterl cells . Taken tCTNNB1 in the mutated cells, depletion of CTNNB1 was performed in 786-mut1 and A-704-mut2 cells respectively. The mRNA expression of CTNNB1 was decreased in 786-mut1 shCTNNB1 and A-704-mut2 shCTNNB1 cells, compared to 786-mut1 and A-704-mut2 cells   . After t < 0.01) . By usinrl cells . In orderl cells . These rPIK3CA, AKT and PTEN, are frequently altered, and cause the activation of the pathway in RCC2PIK3R1 expression is reduced in RCC, and that the reduction of PIK3R1 can result in EMT, enhance migration ability, and promote colony formation, tumor formation and cancer stem cell phenotype in renal cancer cells.Over the past 20 years, the functional role of the PI3K/AKT pathway in tumorigenesis has been reported by numerous studiesPIK3R1 may function as a potential cancer suppressor, and that the downregulation of PIK3R1 may promote progression and metastasis of RCC.Our data showed that PIK3R1 expression was reduced in RCC, especially in advanced and metastatic RCC, and the downregulation of PIK3R1 correlated with advanced or metastatic RCC. These results are in consistence with discoveries in hepatocellular and colon cancers16PIK3R1 in renal cancer cell lines. We observed that the haploid deletion mutations of PIK3R1 by CRISPR/Cas9 system led to the haploid knockout and downregulation of PIK3R1, and generated cell lines with the insufficiency of PIK3R1. The binding of p110α to p85α was decreased in 786 and A704 mutated cells with the reduced expression of PIK3R1. We also showed that the downregulation of PIK3R1 functionally activated the PI3K/AKT pathway, as evidenced by the enhanced phosphorylation of AKT in Ser473 residue. The activation of the pathway may be due to the combined effect of several factors. The p85 regulatory subunit of PI3K is necessary to stabilize and recruit p110 catalytic subunit onto cellar membranePIK3R1 has also been shown to have a positive regulatory effect on tumor suppressor PTEN, which negatively regulates the PI3K/AKT pathwayPTEN function that reduces the breakdown of PIP3, and activates the downstream of the PI3K/AKT signaling axisWe then tested the role of Our data show that the reduction of PIK3R1 expression increases the motility and migration capacity of renal cancer cells. A growing body of evidence indicates that the PI3K/AKT pathway is involved in the migratory process of cells, including metastatic cancer cellsNCAD is enriched in cellular protrusions and plays a key role in cell migrationInterestingly, spindle-shaped morphology of mesenchymal cells has been observed in cells with the down-expression of PIK3R1. EMT is conducive to the survival and spread of cancer cellsCTNNB1. Our data are consistent with the study by Zhou et al, who reported that the CSC viability and maintenance of breast cancer stem-like cells required the activation of the PI3K/AKT signaling axisCSCs are defined as cells which are able to produce a new tumorCTNNB1 in renal cancer cells decreased the cancer stem-like phenotype of these cells. Activated AKT can block the kinase activity of GSK3β by phosphorylating GSK3β29Our present study demonstrates that the downregulation of PIK3R1 in RCC enhances stem cells expansion as a result of the activated PI3K/AKT signaling, involving the WNT/CTNNB1 pathway. The PI3K/AKT pathway has always been ascribed to the proliferation and self-renewal of cancer stem-like cellsPIK3R1 in RCC. The important discovery of our study is that PIK3R1 is down expressed in RCC, especially in advanced and metastatic RCC. We observed that the downregulation of PIK3R1 resulted in EMT, increased migration, colony formation, cell sphere, and tumor formation in renal cancer cells, suggesting that the reduction of PIK3R1 expression confers renal cancer cells a selective advantage to translocate, colonize and develop as mRCC, and that PIK3R1 may be involved in the progression and metastasis of RCC through the PI3K/AKT/GSK3β/CTNNB1 pathway. Since the reduction of PIK3R1 expression and activating alterations in PIK3CA can simultaneously be involved in RCC, conformational changes may result from these alterations. These changes would eliminate the repression of PIK3CA caused by PIK3R1To our knowledge, this study is the first report of the functional identification of In summary, our study suggests that PIK3R1 is down-regulated in RCC, especially in advanced and metastatic RCC. The downregulation of PIK3R1 promotes EMT, migration, and cancer stem cell phenotype in renal cancer cells and may contribute to the progression and metastasis of RCC by activating the PI3K/AKT/GSK3β/CTNNB1 pathway. Increasing or restoring expression of PIK3R1 may be a novel treatment strategy for RCC.Antibodies that recognize pAKT (Ser473) , AKT , pERK (Thr202/Tyr204) , ERK , p85α , p110α , β-actin , NCAD , ECAD , VIM (RV202) , ZEB1 (D80D3) , pGSK3β (Ser9) , GSK3β (Y174) , and non-phospho CTNNB1 (Ser33/37/Thr41)  were used in the study. Horseradish peroxidase (HRP) labelled secondary antibody (Beyotime Biotech) was used in western blots. Protein A-Sepharose 4B beads  was used for immunoprecipitation studies.For the protein expression of PIK3R1, the formalin-fixed, paraffin-embedded (FFPE) tissue samples of patient-matched normal renal, pRCC and mRCC tissues from 13 patients who underwent nephrectomy between May 2008 and April 2013 were obtained from the archives of the Zhujiang Hospital of the Southern Medical University . The bio2O2 solution, incubated with 8% bovine serum albumin for 30 min and primary antibody at 4°C overnight, then incubated with corresponding secondary antibody, and subsequently stained with DAB kit (ZSGB Bio). The nucleus was counterstained with hematoxylin. The overall staining for PIK3R1 was measured by the multiplication of staining percentage (0%–100%) and staining intensity on a numerical scale , resulting in an overall product score.Sections (4 μm) were deparaffinized and rehydrated. After antigen retrieval, these sections were treated with 3% HTotal RNA was extracted using RNA isolation kit (Tiangen Biotech) and subjected to cDNA synthesis using PrimeScript RT reagent Kit (Takara Bio). The cDNA was then used for the evaluation of the relative mRNA levels of the indentified gene, running in an ABI 7300 analyzer (Applied Biosystems). SYBR Green I (Tiangen Biotech) was used as the fluorescent probe. Primer sequences for RT-PCR are listed in the 2 in a humidified atmosphere.HK-2 cells were cultured in DMEM/F12 medium (Gibco). Other cells were maintained in RPMI 1640 medium (Gibco). All mediums were supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 U/mL penicillin (Gibco) and 100 mg/mL streptomycin (Gibco), and incubated at 37°C with 5% COPIK3R1 in 786-O and A-704 cell lines was achieved by CRISPR/Case9 system. One 23-base sgRNA (5′-AATGAACGACAGCCTGCACCAGG-3′) was designed to the target site (g.chr5: 67576819) in the PIK3R1 (Gene ID: 5295). The U6-gRNA expression cassette including U6 promoter and +83 bp sgRNA tails was synthesized (Invitrogen) and inserted into pCEP4 vector (Invitrogen) by KpnI and XhoI. The sgRNA was synthesized (Invitrogen) and inserted into two AarI site between U6 promoter and sgRNA tails to form the pCEP4-U6-gRNA expression vector  protocol. Briefly, Cells were transfected at 60% confluency in 12 well with 500 ng of pCEP4-U6-gRNA expression vector, 500 ng of CMV-Cas9 expression vector and 3 μL Lipo-fectamine 2000. After G418 (500 ng/μL) selection for 10 days, G418-resistant colonies were picked and cultured in 24 well plates. Genomic DNA was extracted with E. Z.N.A. Tissue DNA Kit (OMEGA). Targeted cleavage was measured by PCR amplification (forward primer: 5′ CTCATCAGTATTGGCTTACGCTT 3′ and reverse primer: 5′ GGTTGTTTAGACTTTCCACGGTA 3′) and T7 E1 assay. The fragments were determined by Sanger sequencing. To better verify the mutations (insertions and deletions), the PCR product of mutated colonies was then ligated to pMD-18T vector (Takara Bio). Individual mutation and mutational spectra were obtained from Sanger sequence. Cell clones with haploid deletion mutation of Cells were lysed with radioimmunoprecipitation assay buffer (Beyotime Biotech). Protein was determined, separated and transferred to polyvinylidene difluoride membranes . The membranes were blocked, then probed with corresponding primary antibody and HRP-conjugated secondary antibodies (Beyotime Biotech), and detected using BeyoECL PLUS Kit (Beyotime Biotech).For immunoprecipitation assays, cell lysates were incubated with the PIK3R1 antibody overnight and precipitated with protein A-Sepharose 4B beads (Invitrogen) at 4°C for 4 h. The immunoprecipitated proteins were washed, separated on SDS-PAGE, transferred onto a PVDF membrane, and followed by western blot analysis.Briefly, single cells were seeded onto 6-well plates (Corning) at a density of 1000 cells/well and then incubated at 37°C. After two weeks of culture, colonies were stained with 0.5% crystal violet solution for 30 min at room temperature. The colonies from three replicate wells were counted, and pictures were captured digitally.For generation of xenografts, different dilutions of cells were injected with Matrigel (BD Biosciences) subcutaneously into the backs of NOD/SCID mice. The volume of xenografts was serially measured. The mice were sacrificed after 30 days. Animal work was carried out in compliance with the ethical regulations approved by the Animal Care Committee, Southern medical University.5 cells were seeded into a 24 well plate. After overnight incubation, monolayer cells grew to confluency. Artificial wounds were introduced with the P200 pipette tip. The pictures of the wounded area were taken immediately (time 0 h), and at 18 h with an inverted microscope (Olympus Corp). The whole assay was repeated three times.Briefly, 4 × 10Cells were respectively harvested, suspended and added to the upper compartment of transwell inserts . The lower chambers were filled with RPMI-1640 supplemented with 10% FBS. After incubation for 16 hr, cells that had not migrated from the upper chamber were scraped away. The cells that migrated to the lower surface of each membrane were stained and counted under a light microscope in in five fields/well. The whole assay was repeated three times.3 cells/well in 6-well plates with ultra-low attachment surface (Corning). Cells were maintained in serum-free 1:1 DMEM/F12 (Gibco) supplemented with 20 ng/mL EGF (Invitrogen), 20 ng/mL bFGF (Invitrogen), 2% B27 (Invitrogen), 1% N2 (Invitrogen), and 100 mg/mL streptomycin (Gibco). The number of tumor spheres was counted 14 days after seeding. The whole assay was repeated three times.Single cells were seeded at a density of 5 × 10Cells were harvested and washed twice in ice-cold PBS. Then cancer cells were stained with FITC-conjugated anti-CD133 antibody, PE-conjugated anti-CD44 antibody and FITC-conjugated anti-CXCR4 antibody, respectively, for 30 min on ice. After washing with PBS, the labeled cells were analyzed by flow cytometry (BD FACSAria III).AKT and CTNNB1 and scrambled shRNAs were designed, synthesized and cloned into pSicoR vector: ShCTNNB1: Ccgg CAGATGGTGTCTGCTATTGTACTCGAGTACAATAGCAGACACCATCTGTTTTT g; shCtrl: CcggTTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGA ATTTTTg; shAKT: CcggCCTCAAGAATGATGGCACCCTCGAGGGTGCCATCAT TCTTGAGGTTTTTg; shAKT Ctrl: CcggAGTTCCAGTACGGCTCCAACTCGAGTT GGAGCCGTACTGGAACT TTTTTg. The resulting lentiviral vectors containing the CTNNB1 and AKT shRNA were named shCTNNB1 and shAKT lentivirus. Cells were transfected with the shCTNNB1 and shAKT lentivirus to obtain cell lines stably expressing the CTNNB1 and AKT shRNA.Four shRNA targeted against the different regions of PIK3R1 (NM_181523.2) was amplified by PCR with the following primers (forward: 5'- CGG AAT TCC ATG CAT GGT GAT TAT ACT CTT ACA -3' and reverse: 5'- CGC GGA TCC TCA TCG CCT CTG CTG TGC ATA TAC -3'). The PCR product was purified and cloned into pSin-GFP-vector to obtain Psin-GFP-PIK3R1. ACHN cells were transfected with the pSin-GFP-PIK3R1 lentivirus to obtain a cell line stably expressing the PIK3R1 cDNA.P < 0.05, ** P < 0.01) was regarded statistically significant.Student's t-test was used to comparing the means of two samples. Linear correlation analysis was used to determine relationship between PIK3R1 and NCAD mRNA expression. A value of p less than 0.05 , F.L., D.S., X.Z. and X.W. performed the experiments and analyzed the data. C.L. and S.W. designed the project and wrote the main manuscript text. A.X., P.D., Y.H., D.Z., H.L. (Hongjie Liu), M.Z. and Y.F. edited various parts of the manuscript text. A.X., H.W., Q.X., Y.W. (Yongqiang Wang), Y.Z., Y.W. (Yu Wang) and Z.C. helped with the experimental design. C.L. and S.W. supervised the data analysis and edited text for the manuscript. All authors reviewed the manuscript.Supplementary data


SlAN2 and SlANT1, encoding two R2R3-MYB transcription factors. We show that both can induce ectopic anthocyanin synthesis in transgenic tomato lines, including the fruit. However, only SlAN2 acts as a positive regulator of anthocyanin synthesis in vegetative tissues under high light or low temperature conditions.Anthocyanins are water-soluble polyphenolic compounds with a high nutraceutical value. Despite the fact that cultivated tomato varieties do not accumulate anthocyanins in the fruit, the biosynthetic pathway can be activated in the vegetative organs by several environmental stimuli. Little is known about the molecular mechanisms regulating anthocyanin synthesis in tomato. Here, we carried out a molecular and functional characterization of two genes,  In plants, anthocyanins accumulate as water-soluble polyphenolic metabolites in the vacuole of many (sub)epidermal cell types, where they exert different functions depending on the tissue specificity. In vegetative tissues anthocyanins act as protective compounds after being synthesized in response to different environmental stimuli (e.g. UV irradiation and/or low temperature) and against pathogens, while in reproductive organs they exert an essential role in attracting pollinators and seed dispersers to assure the reproductive success . RecentlPetunia x hybrida) flowers, for example, different members of the R2R3-MYB family, including P. hybrida AN2 (PhAN2)  were used. The anthocyanin-rich phenotype of the T9002 and R9009 transgenic lines was inheritable.The 35S:r pK7WG2   and Photinus pyralis (Pp) luciferase enzymes. The 35S:SlANT1 and 35S:SlAN2 effector constructs were produced by recombining the SlANT1 and SlAN2 entry clones with the vector p2GW7 [SlDFR promoter was amplified from AC genomic DNA using the primers pDFR_GWFW (CACCTTAGTGAAAGACCAACGTG) and pDFR_GWRV (TTTCAGAAATGAAAGGTAAAAAAGAGTC), cloned into pENTR/D-TOPO vector and then recombined with the reporter plasmid pPGWL7 containing the PpLuc gene. A RrLuc-overexpressing vector [Transactivation assay was performed exploiting the or p2GW7 . The SlDg vector  was usedg vector . LuminesSlAN2 and SlANT1, a fragment of the cDNA was amplified using primers designed in order to introduce attB Gateway cloning sites: AN2_attB1: GGGGACAAGTTTGTACAAAAAAGCAGGCTTTGCATTGAAATTGAAGAAG, AN2_attB2: GGGGACCACTTTGTACAAGAAAGCTGGGTTCCATAAGTCAATATCAGTT, ANT1_attB1:GGGGACAAGTTTGTACAAAAAAGCAGGCTAGAAAAATCACCACCATTAAAT, ANT1_attB2:GGGGACCACTTTGTACAAGAAAGCTGGGTTTCCATAAGTCAATTTCAGCA. The fragment was cloned into the pTRV2 using the “Gateway Recombination Cloning Technology” (Life Technologies). Agrobacterium cultures were grown as described in [A. tumefaciens C58C1 strains, containing the pTRV1 and the pTRV2 binary vectors with the silencing fragment (or the empty pTRV2 as control) respectively. Infiltrated seedlings were plenty washed and kept in the dark for at least 12 hours and then grown under stressing conditions to promote anthocyanins accumulation: low temperature (17°C) and limiting soil (e.g. 3 plants in a 80x80x90 mm pot). The RNA was extracted from silenced and non-silenced tissues of three biological replicates and the expression levels of regulatory and target genes were measured as described above.TRV-based T-DNA binary vectors pTRV1, pTRV2 and pTRV2/GATEWAY are from . For botribed in , and celribed in  with a 1Solyc10g086250, Solyc10g086260, Solyc10g086270, and Solyc10g086290, corresponding, respectively, to SlAN2 [N. tabacum), petunia and potato (S. tuberosum) involved in anthocyanin synthesis [Solanaceae family and was clearly separated from anthocyanin MYBs from other dicots, such as A. thaliana or A. majus, and monocots  in transiently transformed protoplasts of A. thaliana. Both SlAN2 and SlANT1 localized into the cell nucleus  was sufficient to activate the anthocyanin biosynthetic pathway. This indicated that the other regulators of the pathway were already expressed where anthocyanin accumulated or that their expression was induced by SlAN2 and SlANT1. To verify these hypotheses we examined by qPCR the expression pattern of the putative regulatory genes in the transgenic lines. Representative plants of single 35S:uit peel . Furtherobserved . On the c plants .A. thaliana protoplasts showed that both SlANT1 and SlAN2 can activate the promoter of SlDFR  a MBW ct parts, ,60SlANT1 and SlAN2 are modulated by environmental factors, we analyzed their expression in vegetative tissues of 4-week-old tomato plants that were exposed for 7 days to high light or low temperature conditions. These factors are known to be major triggers of anthocyanin synthesis and accumulation in plant green parts [SlAN2 following both treatments   could also activate SlANT1 transcription, due to the high number of LREs identified in its promoter.The analysis of a 2 kb nucleotide genomic sequence upstream of y others . Specifis (SlAN2 . Furthers (SlAN2 . The prelements) . Moreovelements) . It is tSlAN2 or SlANT1 was carried out. As shown in SlAN2 caused a strong reduction of anthocyanin accumulation both in leaves and in the stem, as compared to control plants. qPCR analysis showed a strong down-regulation of SlAN2 mRNA in the silenced tissues, confirming the effectiveness of the VIGS, as well as a strong repression of the expression of SlDFR  to promote strong anthocyanin accumulation and Virus Induced Gene Silencing (VIGS) of of SlDFR . Furtherin PhAN4 . On the of SlAN2 , suggestentation  or in chentation . All theAft, produce small amounts of anthocyanins in the fruit skin [atv is instead characterized by high anthocyanin levels in vegetative tissues [Aft and atv results in much higher anthocyanin levels in the fruit [atv locus is presently unknown, while Aft probably encodes a MYB gene, either SlANT1 [SlAN2 [Del, a bHLH-type TF, and Ros1, an R2R3 MYB-type TF, obtaining tomato fruits characterized by an intense purple coloration both in the peel and flesh. Recently, two different tomato R2R3 MYB-type TFs involved in anthocyanin synthesis have been identified and their ectopic expression resulted in increased anthocyanin pigmentation in tomato plants [In recent years, several attempts were made to increase the nutritional value of tomato fruits by inducing the synthesis of anthocyanins. These flavonoids are indeed normally absent in tomato fruits, although some lines, like uit skin . Another tissues . The crohe fruit ,31. The r SlANT1  or SlAN21 [SlAN2 ,35. Atte1 [SlAN2  by expreo plants –36, as cSlANT1 and 35S:SlAN2 plants displayed high expression of SlDFR, encoding a key enzyme of the anthocyanin biosynthetic pathway. This is confirmed by the transactivation of the SlDFR promoter in Arabidopsis protoplasts by the two tomato MYBs  the analysis of Illumina RNA-Seq data  qPSeq data . These of SlANT1 . Overall SlJAF13 . Expressre SlAN2 , implyinSlDFR promoter in transient assays in Arabidopsis protoplasts  [S. lycopersicum and S. pimpinellifolium), other two produce yellow to orange fruits , while the others  produce green mature fruits which in some cases accumulate anthocyanin pigments to various degrees  is conserved (SlANT1 in S. pennellii (SpANT1), since point mutations generate a premature stop codon in the sequence with respect to SlANT1 and Aft-ANT1 [ANT1 during speciation/domestication processes. However only a systematic characterization of these genes in all the wild tomato species would help to elucidate this aspect. Nevertheless, a role for SlANT1 MYB factor in the activation of pigmentation in domesticated tomato under other conditions than those studied here, in different tissues or at different developmental stages of the plant cannot be excluded at this stage.During the evolution of the tomato clade (ersicon)  at leastersicon) . Only tw degrees . Moreoveus paper , nucleotuit peel . Howeverl factor . Furthere on SGN ,40. As i2 allele , that deonserved . To the Aft-ANT1 ,68 Click here for additional data file.S2 FigThe evolutionary history was inferred using the Neighbor-Joining method . The opt(TIF)Click here for additional data file.S3 FigThe evolutionary history was inferred using the Neighbor-Joining method . The opt(TIF)Click here for additional data file.S4 FigSlANT1 , SlJAF13  and SlAN11  in 6 developmental stages and 15 anatomical parts of tomato plants. A large dataset of microarray analyses was selected and queried using Genevestigator.Expression of (TIF)Click here for additional data file.S5 FigSlAN2 promoter (A). Light, abscisic acid, defense and stress and methyl jasmonate responsive elements are highlighted with different colors. Legend of the different responsive elements (B). The analysis was carried out with the PlantCARE Software. Only a sub-set of the cis-acting responsive elements identified was reported.2 kb nucleotide genomic sequence of (TIF)Click here for additional data file.S6 FigSlANT1 promoter (A). Light, abscisic acid, defense and stress, gibberellin, drought and methyl jasmonate responsive elements are highlighted with different colors. Legend of the different responsive elements (B). The analysis was carried out with the PlantCARE Software. Only a sub-set of the cis-acting responsive elements identified was reported.2 kb nucleotide genomic sequence of (TIF)Click here for additional data file.S7 Fighttp://tgrc.ucdavis.edu/index.aspx). A: S. arcanum, accession LA2813 (photo by Scott Peacock); B: S. chilense, accession LA2965 (photo by Scott Peacock); C: S. corneliomulleri, accession LA3157 (photo by Scott Peacock); D: S. pennellii, accession LA1656 ; E: S. peruvianum, accession LA2958 (photo by Scott Peacock); F: S. huaylasense, accession LA1981 ; G: S. chmielewskii, accession LA3663 (photo by Scott Peacock); H: S. habrochaites, accession LA1986 (photo by Scott Peacock); I: S. neorickii, accession. LA2190 .The pictures are available on the TGRC website ((TIF)Click here for additional data file.S8 FigRed shading indicates identical sequences.(TIF)Click here for additional data file.S9 FigSpANT1. Red shading indicates identical sequences.The red box indicates the mutated codon that produce a premature stop in (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableSlAN2, SlANT1, SlAN1, SlAN11, SlJAF13 and SlDFR in different tissues of tomato, analyzed by Illumina RNA-Seq. MG: Mature Green fruit; B: Breaker fruit; B+10: ripe fruit 10 days after breaker stage. Data are the average of two independent biological replicates.Normalized expression (FPKM) of (DOCX)Click here for additional data file.


B7-H3 is a cell surface molecule in the immunoglobulin superfamily that is frequently upregulated in response to autoantigens and pathogens during host T cell immune responses. However, B7-H3's role in the differential regulation of T cell subsets remains largely unknown. Therefore, we constructed a new B7-H3 deficient mouse strain (B7-H3 KO) and evaluated the functions of B7-H3 in the regulation of Th1, Th2, and Th17 subsets in experimental autoimmune encephalomyelitis (EAE), experimental asthma, and collagen-induced arthritis (CIA); these mouse models were used to predict human immune responses in multiple sclerosis, asthma, and rheumatoid arthritis, respectively. Here, we demonstrate that B7-H3 KO mice have significantly less inflammation, decreased pathogenesis, and limited disease progression in both EAE and CIA mouse models when compared with littermates; these results were accompanied by a decrease in IFN-γ and IL-17 production. In sharp contrast, B7-H3 KO mice developed severe ovalbumin (OVA)-induced asthma with characteristic infiltrations of eosinophils in the lung, increased IL-5 and IL-13 in lavage fluid, and elevated IgE anti-OVA antibodies in the blood. Our results suggest B7-H3 has a costimulatory function on Th1/Th17 but a coinhibitory function on Th2 responses. Our studies reveal that B7-H3 could affect different T cell subsets which have important implications for regulating pathogenesis and disease progression in human autoimmune disease. Upon T cell receptor-mediated recognition of MHC antigenic peptides, T cell responses to antigens, including autoreactive antigens, are orchestrated by a group of cell surface signaling molecules. These molecules could be loosely categorized into two distinct types, costimulatory or coinhibitory molecules, based on their functions in regulating T cell responses. Therefore, integrating the functional outcome of costimulatory and coinhibitory interactions determines the fate of a T cell response, which leads to response, unresponsiveness, and death . Variousin vitro T cell response [In 2001, our laboratory initially identified B7-H3 CD276) as a costimulatory molecule that promotes an response  whereas response . Similar as a cosresponse –9. One iresponse –12. To dB7-H3 KO mice were generated in Mayo Clinic and gene targeting mice protocols were approved by the Mayo Clinic Institutional Animal Care and Use Committee. All of the experimental animals used in this study were housed in specific pathogen-free environment and performed under the protocol approved by animal facilities of Johns Hopkins University, Yale University and Sun Yat-Sen University. When mice are paralyzed in EAE induction procedure, they were provided with gel pack and/or moistened food in the cage and mice were monitored daily by both researchers and animal care staff. Animals were euthanized if they were moribund or the experiment was completed. In all cases, mice were humanely sacrificed by using carbon dioxide.5’CGGCTCAGTCACCATCACAGGTAA; B7-H3 reverse, 5’GAGCCGCAATGAGCCTAAGGTCTA; Neo forward, 5’AGACTAGTGAGACGTGCTACTTCCA. The mice were backcrossed to a C57BL/6 or a DBA/1J background for ten generations. The same generations of homozygous or wild type mice were used for experimental analysis.A murine B7-H3 genomic DNA clone was identified by screening a 129/SvJ bacterial artificial chromosome (BAC) library . A 4.6 kb B7-H3 upstream DNA homologous fragment and a 6.5 kb downstream DNA homologous fragment were PCR amplified from a positive BAC clone. The fragments were cloned into a pKO targeting vector that contains a neo resistant gene and a TK gene (Stratagene). The targeting vector with the B7-H3 IgV and IgC domains replaced was transfected into 129/Svj embryonic stem (ES) cells . Correctly targeted ES cells were confirmed by Southern blot analysis and then injected into C57BL/6 mouse blastocytes followed by implantation into foster mothers to generate chimeric mice. B7-H3 mutant mice were identified by PCR analysis of tail genomic DNA. The sequences of the PCR primers are: B7-H3 forward, Mycobacterium Tuberculosis (Difco) and complete Freund’s adjuvant (CFA) (Difco) on day 0; and injected intraperitoneally (i.p) with 400 μg pertussis toxin (Sigma-Aldrich) on the same day and day 2. Mice were scored every other day for signs of EAE on 0–5 scales as follows: 0, no abnormality; 1, complete loss of tail tonicity; 2, flaccid tail and abnormal gaits; 3, hind leg paralysis; 4, hind and foreleg paralysis; 5, death.The wild type (wt) and B7-H3 KO mice  were immunized subcutaneously (s.c.) at both flanks with 200 μl of emulsion comprising 200 μg MOG 33–35 peptide, 400 μg in vitro proliferation and Th cells differentiation experiments). Blood was collected and serum was separated for OVA-specific IgE determination. The trachea was cannulated and the lungs were lavaged 3 times with a total of 1.5 ml of PBS. The bronchoalveolar lavage fluids (BALF) were centrifuged. The supernatants were collected to determine cytokine IL-5 and IL-13 levels and the cell pellets were re-suspended in 1 ml of PBS. Half a milliliter of suspended cells were counted using a Cell Viability Analyzer (Beckman Coulter) to determine the total cell counts in the BALF; the rest was spun onto microscope slides by CytoFuge and stained with Hema3 (Fisher Scientific). BALF differential cell counts were identified by counting 400 cells per slides using a high-magnification microscope. The relative numbers of each cell type were calculated by multiplying the total cell counts by the percentage of each cell type in the BALF cells. For histopathology analysis of lung inflammation, peribronchial and perivascular inflammation was evaluated on a scale of 0–4, for no inflammation (0), few inflammatory cells present (1), a few loci of inflammation (2), multiple loci of inflammation (3), inflammatory cells throughout the lung (4). The scores were presented as mean ± SEM. For mucus-secreting cells in the airways, mucous scores of 0–3 were evaluated for: no positive cells present (0), a few positive cells (1), many positive cells (2), and extensive staining of mucus-secreting cells (3)[Wt and B7-H3 KO mice  were sensitized via i.p. injection of 20 μg OVA protein (Sigma-Aldrich) adsorbed to 4 mg aluminum hydroxide Gel (Sigma-Aldrich) on days 0 and 5. Then, mice were challenged with aerosolized 1% OVA in PBS using a nebulizer (Proneb Ultra II) for 60 minutes on days 12–14. Mice were sacrificed the next day  was dissolved in 10 mM acetic acid at 4 mg/ml and emulsified with an equal volume of CFA (Difco). Wt and B7-H3 KO mice  were immunized s.c. at both flanks with 100 μl of emulsion and a boosted with the same concentration of CII (emulsified in IFA for this immunization) 16 or 18 days after the primary immunization. Animals were evaluated by visual inspection for arthritis incidence; severity was scored individually on a 0–4 scale . Scores 3H-thymidine incorporation during the last 6 hours of the 3 days culture.The brachial and axillary draining-lymph nodes (DLN) cells were isolated from wt or B7-H3 KO mice 10 days post MOG/CFA immunization. Splenocytes were removed from the OVA-immunized wt or B7-H3 KO mice 12 days after their first immunization and from wt or B7-H3 KO CIA mice 20 days after the primary immunization, respectively. The DLN cells or splenocytes were cultured in 96-well culture plates with various doses of MOG peptide, OVA, or CII proteins (heated for 30 minutes at 60°C). The proliferative activity of the T cells was assessed in triplicates by Cytokines in BALF and cultured supernatants were measured using OptEIA kits (BD Pharmingen) for IL-2, IL-12, IL-5, IFNγ and ELISpot Kit (R&D Systems) for IL-13. In the CIA experiments, IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A from the culture supernatants were assessed by CBA mouse Th1/Th2/Th17 Cytokine kit (BD Biosciences) and the data were analyzed using an FCAP Array V2.0.For OVA-specific IgE, ELISA plates were coated with 100 μg/ml of OVA at 4°C overnight. The plate was washed and blocked with 1% BSA for 2 h at room temperature. 1:50 diluted serum samples were incubated at room temperature for 2h, followed by treatment with HRP-conjugated anti-mouse IgE (BD Pharmingen). After washing, peroxidase substrate was added and the absorbance was read at 450 nm with an automated ELISA reader. For anti-CII IgG, serum was collected from CIA mice 20 days after their primary immunization and the levels of anti-type II collagen Ab (IgG) were assayed using a mouse anti-type II collagen IgG assay kit (Chondrex Inc) according to manufacturer's instructions.DLN cells (EAE model) or splenocytes (asthma model) from wt or B7-H3 KO mice were cultured with MOG peptide (40 μg/ml) or OVA (50 μg/ml) under Th1  or Th2  polarizing conditions for 5 days. 1 μg/ml of anti-CD3 and 1 μl/ml of GolgiPlug was added during the last 6 h. The cells were stained for surface CD4, then fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences) followed by intracellular staining for IFN-γ and IL-4.5’ GACACGGATGCCACCCTACGCTG (forward) and 5’CTGTGATGGTGACTGAGCCGTGAG (reverse). β-actin mRNA levels were determined as a control for each sample. For real-time qRT-PCR, total RNA was extracted using RNeasy (Qiagen) and was reverse transcribed to cDNA using Taqman reverse transcription reagents (Applied Biosystems) according to the manufacturer’s instructions. Primer and probe sets were obtained from Applied Biosystems. Real-time qPCR was performed using the Taqman Universal PCR Master Mix and an Applied Biosystems 7500 Fast Real-Time PCR system. Relative gene expression levels were normalized to the 18S rRNA endogenous control levels.Total RNA was extracted using the RNeasy kit (Qiagen). First-strand cDNAs was synthesized and amplified by RT-PCR using a primer set for mouse B7-H3: 2O2, Block AEC (AbD Serotec), and Avidin Blocking Buffer (Vector Laboratories), respectively. Then, sections were incubated with an anti-mouse biotin-conjugated B7-H3 mAb (5 μg/ml) at 4°C overnight. Subsequently, the Catalyzed Signal Amplification System  was used for staining. The slides were counterstained with hematoxylin. Monoclonal antibody to murine B7-H3 (clone mB7-H3.7H) was generated by immunizing an Armenia hamster with mouse B7-H3Ig recombinant fusion protein, as described previously [Lungs from allergic mice were fixed with Streck Tissue Fixative (Streck). The lung sections were stained with hematoxylin and eosin (H&E) or Periodic Acid-Schiff (PAS) (Sigma-Aldrich) for histological evaluation. Intact spinal cords were flushed out by hydrostatic pressure from mice 20 days after MOG/CFA immunization and fixed in 10% formalin. The sections were stained with H&E to assess inflammatory lesions. The hind paws from normal or CIA mice were collected 40 days after their primary immunization. The skins were removed from their paws and the bones were decalcified using Formical 2000  for 24 hrs followed by fixation with SafeFix II (Fisher Scientific). After deparaffinization and rehydration, the tissue sections were stained with H&E for histological analysis. For immunohistochemistry, endogenous peroxidases, nonspecific binding sites and endogenous avidin/biotin were blocked using 0.3% Ht test. All bars are shown as mean ± standard error of the mean (SEM).All statistics were performed using a two-tailed Student’s The B7-H3 KO mice were generated using homologous recombination in embryonic stem (ES) cells. The targeting vector was constructed to replace exon 2 and exon 3 of the B7-H3 gene with a TK gene and a neomycin resistance gene. Homologous recombination by this targeting vector in ES cells deleted a 2 kb region that encodes for the IgV and IgC domains of B7-H3 . Using Sin vivo function of B7-H3 in autoimmune disease, we first examined the role of B7-H3 on the development of EAE. As shown in To investigate the in vivo findings, we investigated whether there was a difference between wt and B7-H3 KO mouse T cell-specific responses to MOG 33–35 peptide. The brachial and axillary draining-lymph nodes (DLN) were removed from wt or B7-H3 KO mice 10 days after MOG/CFA immunization. Although the DLNs from both the wt and B7-H3 KO mice exhibited swelling, the latter was visibly smaller. The total cell number obtained from DLN in the B7-H3 KO mice was half the number or less of wt mice  were harvested and the total and differential BALF cell counts were determined. The total BALF cell counts in OVA-immunized and challenged B7-H3 KO mice were significantly higher than in wt mice; this increase was mainly observed in eosinophils Fig  and 4B. in vitro. Splenocytes from B7-H3 KO mice exhibited a marked proliferation compared to wt mice  against B7-H3 and B7-H3 KO mice. These studies resulted in inconsistent and conflicting results from different laboratories. In vitro studies using human recombinant B7-H3 showed enhanced T cell growth in the presence of TCR engagement, which led to the hypothesis that B7-H3 is a costimulatory molecule for T cell growth [in vitro systems; these results are inconsistent. For example, infusion of a B7-H3 mAb accelerated the progression of diseases with an enhanced Th1 T cell response in a MOG peptide-induced EAE model [The effect of B7-H3 on the regulation of T cell responses has been evaluated in l growth –18. TherAE model . In addiAE model . While tAE model . FurtherAE model . AlthougAE model . TherefoAE model .et al used GM-CSF with or without IL-12 to prime the lung before using OVA challenge to induce the Th1 or Th2-biased status. In our lung inflammation/asthma model, the B7-H3 KO strain was challenged with OVA in alum adjuvant to bias Th2 responses. Therefore, a significantly enhanced Th2 response was observed in our studies, where indifference was observed in the model produced by Suh and colleagues. In a MOG-peptide induced EAE model, a small increase in the clinical score and accumulation of autoantibodies were observed [Two B7-H3 KO mouse strains were independently generated and characterized in addition to our current study –24. WangIn vitro experiments demonstrated that collagen-specific T cell responses were significantly decreased and IFN-γ, TNF and IL-17 cytokine production was accordingly reduced in B7-H3 KO mice. Our observations suggest that B7-H3 may be an important mediator of pathogenesis and progression in inflammatory arthritis.It is particularly interesting that B7-H3 KO mice were resistant to CIA induction, showed fewer clinical symptoms and a decreased pathogenesis, including diminished infiltration of inflammatory cells to the joints, decreased T cell and antibody responses to collagen, and fewer inflammatory cytokines including those secreted by Th1 and Th17. Although B7-H3 mRNA is widely distributed in various normal tissues, the protein is not typically detectable. In previous mouse studies, B7-H3 was found expressed on osteoblasts which required a late phase of osteoblast differentiation . Using iTaken together, our results support the notion that B7-H3 differentially regulates T cell subsets by costimulating Th1 and Th17 while suppressing Th2 responses. This finding may explain, at least in part, the previously contradictory findings in various model systems. The molecular basis for this differential effect, however, has yet to be characterized. This will largely rely on the discovery of the B7-H3 counter-receptor and, in this regard, a different counter-receptor on these T cell subsets may cause the observed effects. Finally, our findings have important implications for the manipulation of B7-H3 in clinical applications to treat human disease.S1 FigThe paraffin tissue sections were stained with hematoxylin and eosin .(TIF)Click here for additional data file.


When we perform surgical correction for spinal deformity with degenerative changes, nerve root impingement, traction, or nerve root injury could be occurred. Electromyographic (EMG) monitoring provides a real-time measure of impending spinal nerve root injury during instrumented posterior lumbar fusion. However, the utility of intraoperative EMG monitoring is still questionable because of large variance in muscle domination, morphological abnormality and the abnormal EMG signal from a nearby muscle. The aim of this study was identified clinical mapping of lumbar nerve root motor domination under EMG monitoring in the patients who had normal root morphology.We performed corrective surgery for 24 thoracolumbar kyphoscoliosis patients . Average age was 68 y.o. (37-83). All patients underwent the spinal correction by using Ponte osteotomies from L2/3 to L5/S1. They had no severe muscle weakness preoperatively. We exposed bilateral L2-S1 roots and checked root morphology during surgery. Then, we stimulated bilateral L2-S1 roots with 1-10mA by bipolar probe and measured the amplitude of EMG waves in quadriceps femoralis (QF), adductor longus (AL), hamstrings (Ham), tibialis anterior (TA) and gastrocnemius (GC). The first and the second largest EMG amplitudes were investigated in each muscle.There was no abnormality of nerve root morphology. We could detect the largest EMG waves in 202 roots . The second largest wave was detected in 120 roots. The largest EMG wave was observed by L2: QF80%, L3: QF63%, L4: QF45%, L5: TA52% and S1: GC44%. The second largest waves were observed by L2: QF45%, L3: QF48%, L4: QF30%, L5: TA45% and S1: GC41%. The waves of AL were found similar frequency (12-18%) in L2-5, and waves of hamstrings were found similar frequency (10-20%) in L2-S1. The rates of coincidence with right and left roots domination were L2:63%, L3:44%, L4:15%, L5:32%, S1:59%.We have investigated the map of lumbar nerve root domination by EMG monitoring. L2, 3 and 4 root stimulation had reacted most frequently in QF. By L5 and S1 stimulations, the EMG waves were detected in TA and GC. AL and Hamstrings were stimulated with similar frequency by several roots. There was a wide variability in muscle domination of lumbar nerve roots. When we use EMG or other neuro-monitoring during lumbar spinal surgery, we have to pay attention to these variability of distributions of muscle domination.


We investigated potential patient-to-patient transmission of hepatitis C virus (HCV) in two hemodialysis centers in Beijing, China. Approximately 8.25% (32/388) hemodialysis patients were HCV antibody positive, and 4.90% (19/388) were HCV RNA-positive, which consisted of 2a genotype (1/19) and 1b genotypes (18/19). Using next generation sequencing (NGS) approach, MiSeq platform, we sequenced HCV, targeting hypervariable region 1 (263 base-pairs) of genotype 1b specimens and obtained 18 to 243 unique HCV variants. Analysis of phylogenetic tree, viral epidemiology signature pattern (VESP) and Shannon entropy indicated no obvious HCV similarity for most HCV infections but limited HCV variants from Patient 31 (P31) were closer with respect to evolutionary relationship with Patient 24 (P24). However, it was unlikely that HCV was transmitted directly from P24 to P31 in the hemodialysis center. Otherwise, their genetic distance (3.92%-8.92%), would have been much less. Moreover, P31 was infected less than two years before specimen collection, and other external high risk factors existed for these two patients. Thus, our data indicated no evidence of patient-to-patient transmission of HCV in the two hemodialysis centers, suggesting that current HCV infection control measures are effective. As reppolicies , 4. In 2policies  and 2120Because other external high risk factors existed for these subjects, such as blood transfusion and surgery, concluding that hemodialysis was the infection source was difficult. To investigate the potential for patient-to-patient transmission we studied patient data to identify potential risks during hemodialysis at both centers and to propose measurements to prevent new HCV infections.Molecular evolutionary analyses have been used to assess genetic similarities and to investigate transmission of clinical pathogens –9. HowevThus, we developed an NGS approach (MiSeq platform) for tracing HCV infection, and investigated potential HCV patient-to-patient infections at two hemodialysis centers in Beijing, China. Because other patients also attended the same centers regularly, we could compare subjects to determine whether HCV infections were due to patient-to-patient transmission within centers and reveal potential deficits in infection control measurements.All hemodialysis patients  were enrolled from July to November 2011. Serological testing indicated that 15 patients (7.69%) in Center A (P1-P15) and 17 patients (8.81%) in Center B (P16-P32) were anti-HCV positive. HCV RNA qualitative testing confirmed HCV RNA-positive rates for patients in Center A and Center B were of 4.10% (8/195) and 5.99% (11/193), respectively. All hemodialysis patients were anti-HIV negative and 3 patients  were infected with HBV prior to hemodialysis. Given that no epidemiological relatedness was found between patients from the two centers, patients from one center was a control for phylogenetic analysis for patients from the other center. Information for 19 patients, including possible risk factors, medical records, and patient demographics were collected. Ethical approval to conduct this study was granted by the Institutional Review Board of the National Center of AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention. Written informed consent was obtained from all participants for their clinical records to be used in this study.H1-TGG CTT GGG ATA TGA TGA TGA ACT (1293–1316) and H2- GCA GTC CTG TTG ATG TGC CA (1598–1617); internal primers, H3-GGA TAT GAT GAT G CTG GT (1301–1320) and H4-ATG TGC CAR CTG CCR TTG GTG T (1584–1605).To identify viral genotypes, the HCV NS5B region (H77 positions: 8276–8615) and core/E1 region (869–1292) were amplified with nested PCR and then sequenced directly using Sanger’s method. Primers and PCR conditions have been previously published , 15. Thehttp://hcv.lanl.gov/content/sequence/VIRALIGN/viralign.html). MAFFT [http://www.hcv.lanl.gov/) and worked as external controls. Genetic distance and a neighbor-joining (NJ) tree were calculated with a Kimura 2-parameter model (provided by MEGA6.06). Statistical differences of inter-personal distances were tested with SPSS 19.0 software using a Mann-Whitney U test. P < 0.05 was viewed as significant. Viral epidemiology signature pattern (VESP) analysis and Shannon entropy-two analysis were calculated with online tools available at the HCV database.PCR for the first round was: 30 s at 98°C; followed by 30 cycles for 10 s at 94°C, 30 s at 67°C, and 2 min at 72°C. The second round was identical to the first round, except the annealing temperature was reduced to 60°C. Paired-end sequencing of 250 base-pairs was performed on a MiSeq platform. Sequencing libraries were built using Nextera XT DNA Sample Preparation Kit . A data cleaning strategy was performed according to published methods . We adde). MAFFT . Forty rAll HCV RNA-positive specimens were amplified successfully using nested PCR and subsequently genotyped. Genotypes of HCV from P26 was 2a and the other 18 were 1b. The transmission possibility between P26 and other patients could therefore be excluded. As a result, only 18 specimens with HCV genotype 1b were subsequently analyzed by NGS. We summarized the primary characteristics of 19 patients who were HCV RNA-positive in An average of 94 unique HCV variants (ranged 18–243) from each specimen was determined by NGS (GenBank accession numbers: KR909903-KR911598). The genetic distance between any two patients in Center A ranged from 7.53% (between P2 and P13) to 27.48% (between P3 and P8). The genetic distance between patients in Center B ranged from 6.51% (between P24 and P31) to 27.27% (between P16 and P18). The closest distance between any patients in Center A and another patient in Center B was 8.90%. The phylogenetic tree indicated a relatively close evolutionary relationship of two patients, P24 and P31, who received hemodialysis at Center B . Other sVESP analysis confirmed that 11 sequence sites on P24’s signature pattern were significantly different from that of the 6 variants of P31. However, there were 15 significantly different sites between the signature pattern of P24 and P31’s remaining variants. The number of different sites between P24 and any controls was from 40 to 57. Similarities could be observed between P31 and controls, which ranged from 41 to 57. Shannon Entropy-Two analysis of P24 and the 6 variants of P31 suggested that 7 out of 263 sequence sites have stronger variable characteristic, with 4 in P24 and 3 in P31. A less conserved trend (23/263) was observed between P24 and P31’s remaining variants, with 13 variable sites in P24 and 10 in P31.We investigated HCV status and genotypes among patients receiving hemodialysis at two separate centers (A and B) from July to November 2011. Anti-HCV positive rates for patients in both centers were consistent with published data , which mWe obtained more unique variants with an NGS approach compared with conventional methods , 20 and


Full-genome analysis was conducted on the first isolate of a highly pathogenic avian influenza A(H5N1) virus from a human in North America. The virus has a hemagglutinin gene of clade 2.3.2.1c and is a reassortant with an H9N2 subtype lineage polymerase basic 2 gene. No mutations conferring resistance to adamantanes or neuraminidase inhibitors were found. In December 2013, an HPAI A(H5N1) infection was reported in a Canadian resident who recently returned from China. No human or poultry HPAI A(H5N1) infections had been previously reported in North America.Since the 1997 emergence of highly pathogenic avian influenza (HPAI) A(H5N1) virus in Hong Kong, China, 648 HPAI A(H5N1) infections and 384 associated deaths in humans have been reported. During 2013, Cambodia reported the most human infections, followed by Egypt, Indonesia, China, and Vietnam  samples, bronchoalveolar lavage (BAL), and cerebrospinal fluid (CSF) samples tested positive for influenza A(H5N1) virus by various molecular testing methods, including sequencing, at the Provincial Laboratory for Public Health and the National Microbiology Laboratory, Public Health Agency of Canada  included 1,378 bp of the hemagglutinin (HA) gene , 1,350 bp of the neuraminidase gene (BAL), 810 bp of the matrix gene (NP), and 687 bp of the polymerase basic 2 (PB2) gene (NP). These sequences were identical to corresponding sequences obtained from the isolate, suggesting the absence of cell culture–induced changes.http://blast.ncbi.nlm.nih.gov/Blast.cgi) analysis of each gene of A/Alberta/01/2014 showed that 7 of 8 genes shared ≥99% identity at the nucleotide and protein levels with HPAI A(H5N1) viruses of avian origin. However, the PB2 gene showed 98% nt similarity and 99% aa identity to avian influenza A(H9N2) viruses collected in China. Phylogenetic analysis of each gene , may improve virus replication in the upper respiratory tract of ferrets  identified only 2 aa substitutions in the HA1 region, R189K and G221R. Although position 189 was within the putative antigenic site B, the overall conservation of sequence suggests that A/Alberta/01/2014 is a close antigenic match to the candidate vaccine virus.In agreement with Xu et al.  virus provides valuable insight into the presence of mutations that may reflect adaptive changes, altered virulence, and/or transmission phenotype. Because of the unique manifestation of neurologic symptoms and encephalitis reported in this patient, additional studies are needed to understand the broader aspects of virus heterogeneity and its role in this fatal case.Case details and methods for a full-genome analysis of highly pathogenic avian influenza A(H5N1) virus isolated from clinical samples of an infected human, North America, 2013.Neighbor-joining phylogenetic trees of the polymerase basic 1, polymerase, nucleoprotein, neuraminidase, matrix, and nonstructural protein genes of highly pathogenic avian influenza A(H5N1) viruses with A/Alberta/01/2014.


E. coli flagellar types H1 and H12 show a high serological cross-reactivity and molecular serotyping appears an advantageous method to establish a clear discrimination between these flagellar types. Analysis of fliCH1 and fliCH12 gene sequences showed that they were 97.5% identical at the nucleotide level. Because of this high degree of homology we developed a two-step real-time PCR detection procedure for reliable discrimination of H1 and H12 flagellar types in E. coli. In the first step, a real-time PCR assay for common detection of both fliCH1 and fliCH12 genes is used, followed in a second step by real-time PCR assays for specific detection of fliCH1 and fliCH12, respectively. The real-time PCR for common detection of fliCH1 and fliCH12 demonstrated 100% sensitivity and specificity as it reacted with all tested E. coli H1 and H12 strains and not with any of the reference strains encoding all the other 51 flagellar antigens. The fliCH1 and fliCH12 gene specific assays detected all E. coli H1 and all E. coli H12 strains, respectively (100% sensitivity). However, both assays showed cross-reactions with some flagellar type reference strains different from H1 and H12. The real-time PCR assays developed in this study can be used in combination for the detection and identification of E. coli H1 and H12 strains isolated from different sources.The  Escherichia coli are ubiquitous as commensals in the gut of humans and warm-blooded animals. Apart from their role as beneficial microbes, some E. coli strains are known to behave as human and animal pathogens, causing a wide spectrum of extraintestinal and enteric diseases, with urinary tract infection and diarrhea as most frequent    and non-motile (NM) E. coli strains have to be examined  genes encoding H19, H25, and H28 flagella we have observed a high genetic variability among fliCH19, flicH25, and fliCH28 alleles, respectively. To some part, this sequence alterations were associated with some O-groups of strains which allowed the development of real-time PCR protocols for specific typing of flagellar variants encoded by enterohemorrhagic E. coli O145:H25, O145:H28, and O121:H19 strains  express H1 or H12 flagella  strains carrying capsular polysaccharides  that cause cystitis, pyelonephritis and urosepsis  O83:H1 strains were associated with Crohn's disease in human patients  at the Federal Institute for Risk Assessment (BfR) in Berlin, Germany and from the French Agency for Food, Environmental and Occupational Health and Safety (Anses) in Maisons-Alfort, France. E. coli strains used for specificity study included in particular the E. coli reference strains belonging to serogroups O1-O181 and H-types H1-H56  in 25- μl reaction volumes, a LightCycler Nano  in 10 μl reaction volumes or with a LightCycler 1536  in 1.5-μl reaction volumes according to the recommendations of the suppliers. Primers and TaqMan probes were used at 300 nM final concentrations. The following thermal profile was applied to all instruments: enzyme activation at 95°C for 1–10 min as recommended followed by 40 cycles of denaturation at 95°C and annealing at 60°C.fliC gene variants to their respective H-types was performed as previously described  and primers for specific detection of all genetic variants of thirteen fliCH1 and eight fliCH12 genes (this work). Real-time PCR probes and primers used in this work were designed with the software Primer Express V3.0 (Applied Biosystems) and are described in Table Mapping of fliC homologs were determined and have been submitted to European Nucleotide Archive (ENA). The GenBank Accession numbers are listed in Table The nucleotide sequence of the PCR products were determined as described  was found in 16 (42.1%) of the H12 strains and associated with five different O-groups. Fourteen (45.2%) of the E. coli H1 strains produced Stx, however most of these were from pigs with edema disease  and harbored the stx2e gene. O:H types known to be associated with E. coli causing extraintestinal infections of humans  were detected among the investigated H1 strains. Interestingly, strains belonging to these serotypes originated not only from humans but also from animals and food. Certain strains belonged to serotypes which have not been previously associated with clinical disease and their role of pathogens for humans and animals is not yet known.The es Table . The thiE. coli flagellar antigens H1  and H12   at the nucleotide level and 98.98% identity and 99.16% similarity at the amino acid level (7 amino acids (aa) exchanges). Additional fliC nucleotide sequences from six E. coli H1 and five E. coli H12 strains were obtained in this work  , 340 (Asn/Lys), 361 (Gly/Asp), 391 (Thr/Lys), 396 (Asn/Asp), and 430 (Asn/Lys). The six flagellar type specific aa sequence differences were all located in the variable region of the fliCH1 and fliCH12 genes.An alignment of the amino acid sequences of thirteen y) Table , generats Figure . The aa E. coli fliCH1 and fliCH12 translation products explains the serological cross-reactivity which was previously described for H1 and H12 antigens   were found between target-sequences and fliCH1 and fliCH12, primers and probes , H49 (3/3), H31 (1/2), H34 (2/7), H41 (1/2), H45 (3/4) as well as with one O6:H4 strain. Weak cross-reactions were also observed with one O2:H25 strain and one O153:H25 strain. Cross-reactions with the fliCH12 real-time PCR-assay were observed with H7 (9/9), H28 (6/6), H31 (1/2), H34 (2/7), H41 (1/2), as well as one O20:H9, one O55:H19 and one O153:H14 strains. In contrast to the respective reference strains, cross-reactions were not observed with either real-time PCR-assay with two other H15 and one H52 strain tested , the fliCH1 and fliCH12 gene specific assays were found 100% specific. Thus, 100% of H1 and H12 strains would be accurately typed with this system.Overall, molecular typing of E. coli strains belonging to 26 different O-serogroups, O-untypable and O-rough strains. With one exception (O79:H1 and O79:H12), H1 and H12 strains did not share common O-serogroups which would indicate that flagellar types H1 and H12 have separated from each other not very recently in evolution. They may have evolved independently following rearrangements in the O-group loci of ancestor strains carrying the closely related H1/H12 flagellar types and do not directly derive from a common O-group ancestor.The genetically and serologically closely related flagellar antigens H1 and H12 were found in heterogeneous types of fliC genes from thirteen H1 and eight H12 strains we identified six H-type specific aa changes at positions 302, 340, 361, 391, 396, and 430. All these are located in the variable part of flagellin determining antigen specificity  and fliCH12 sequences  (97.5% similarity) is less than that found between different alleles of fliCH28 (92.0% similarity)   or MGB-replacements (XS-probe) are needed to ensure specificity between closely similar DNA-targets as previously shown for fliCH19 allelic discrimination  were detected in this work. Interestingly, these were not only from humans but also found in animals and food. It was previously described that animals, food and water can be a source of pandemic ExPEC strains .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.


H3 subtype influenza A virus is one of the main subtypes that threats both public and animal health. However, the evolution and pathogenicity of H3 avian influenza virus (AIV) circulating in domestic birds in China remain largely unclear. In this study, seven H3 AIVs (four H3N2 and three H3N8) were isolated from poultry in live poultry market (LPM) in China. Phylogenetic analyses of full genomes showed that all viruses were clustered into Eurasian lineage, except N8 genes of two H3N8 isolates fell into North American lineage. Intriguingly, the N8 gene of one H3N8 and PB2, PB1, NP and NS of two H3N2 isolates have close relationship with those of the highly pathogenic H5N8 viruses circulating in Korea and United States, suggesting that the H3-like AIV may contribute internal genes to the highly pathogenic H5N8 viruses. Phylogenetic tree of HA gene and antigenic cross-reactivity results indicated that two antigenically different H3 viruses are circulating in LPM in China. Most of the H3 viruses replicated in mice lung and nasal turbinate without prior adaptation, and the representative H3 viruses infected chickens without causing clinical signs. The reassortment of H3 subtype influenza viruses warrants continuous surveillance in LPM in China. Orthomyxovirus, is classified into subtypes according to the serological reactivity of their surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA)2368Influenza A virus (IAV), a member of the genus H3 subtype influenza viruses, known as having wide host range from birds to various mammalian species including humans, pigs, dogs, and horses121316151822In China, live poultry markets are also served as slaughterhouses in which chickens, ducks, quails, guineafowl, and pigeons etc. are kept and mixed for days. Comingling of different poultry species makes live poultry market a major source of AIV dissemination, reassortment and interspecies transmission2728Oropharyngeal swabs were collected from fowls at live poultry markets in Shanghai, Jiangsu, Guangzhou, Zhejiang, Shandong and Hebei provinces during 2009 to 2011. Virus isolation was performed by inoculating of 9-day-old specific-pathogen-free (SPF) embryonated chicken eggs with filtered swab supernatant. Embryos died within 24 h after inoculation were discarded. The allantoic fluid was collected from embryonated chicken eggs 72 hours after inoculation, of which hemagglutination assays (HA) were run with 0.5% packed chicken red blood cells. Hemagglutination inhibition (HI) assays were used to determine the HA subtype by using a panel of positive serum against H1 to H16 subtypes. HI tests were performed in accordance to WHO guidelines. All HI assays were performed in duplicates. For further verification, subtype specific real-time RT-PCR was performed with HA-specific primers targeting H3 genes as previously reportedMadin-Darby Canine Kidney Cells (MDCK), Human lung adenocarcinoma epithelial cells (A549) and DF-1 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 5% bovine calf serum (BCS), 1× L-glutamine and 1× antibiotics. Cells were inoculated with the respective viruses in MEM infecting medium containing 0.3% BSA (Sigma), 1 mg/mL TPCK-treated trypsin (Sigma) and 1× antibiotics. To analyze virus growth kinetics, confluent MDCK, A549 and DF-1 cells were infected with each virus at a multiplicity of infection (MOI) of 0.01. The supernatants from infected cells were collected at different time points  and viral titers were determined in embryonated chicken eggs.A QIAGEN Viral RNA Isolation Kit was used to extract vRNA from 140 μL allantoic fluid from infected SPF embryonated eggs. The isolated RNA was eluted into 30 μL diethylpyrocarbonate-treated water. Two-step RT-PCR was employed to amplify each viral gene segment. The first-strand cDNA was transcripted by using reverse transcriptase M-MLV  with universal primer (5′-AGCAAAAGCAGG-3′) for influenza A virus in a final volume of 20 μL per manufacturer’s protocol. Eight fragments  of each virus were amplified respectively by using the Hoffman universal primers for influenza A virus as described previouslyMultiple sequence alignment and the phylogenetic and evolutionary analyses were conducted by using MEGA6.0. Briefly, phylogenetic trees of all eight full length gene segments  of seven H3 isolates were generated by applying the neighbor-joining method with Kimura’s two-parameter distance model and 1000 bootstrap replicatesHI assays were used to determine the antigenic cross-reactivity among H3 viruses from different groups in phylogenetic trees of HA. Four representative isolates  from group 1 and group 2 were chosen for antigenic cross-reactivity tests based on the phylogenetic trees of HA gene. Antisera against 74-1/H3N2, 120-1/H3N8, 854-2/H3N8 or B1646-2/H3N2 respectively were generated by immunizing SPF chickens twice with UV inactivated viruses. HI tests were performed in accordance to WHO guidelines.50) by the Reed and Muench method336 EID50 of each virus in 50 μL phosphate-buffered saline (PBS). The mock group was inoculated intranasally with 50 μL of PBS. Body weights, clinical signs were monitored daily, and three mice from each group were euthanatized at 3 and 5 days post infection (dpi) respectively. Lung, spleen, nasal turbinate, heart, and brain tissue were collected aseptically. Then, the tissues were homogenized and the homogenates were titrated in 10-day-old embryonated eggs. For histopathological examination, tissue samples of lungs were fixed in 10% buffered formalin, and processed and stained with hematoxylin and eosin (H&E). The lung sections were scored by a pathologist in a blinded fashion. The scores assigned from 0 to 3 reflecting the severity of microscopic lung lesions were based on damage of epithelial layer, damage of alveolar septal and alveolar, and lymphocyte infiltration. Those statistical variables were subjected to comparisons for all pairs by using the Tukey–Kramer test. Pair-wise mean comparisons between inoculated and control groups were made using the Student’s t-test ; a P-value < 0.05 was considered statistically significant.Seven H3 subtype influenza viruses were purified and propagated by inoculating 9-day-old SPF chicken embryonated eggs. Allantoic fluid was harvested and titrated in 10-day-old SPF embryonated eggs to determine the 50% embryo infectious dose  were collected for viral titration. The remaining birds were euthanized at the end of experiment. Serum samples were collected for serology tests.To assess the pathogenicity of the isolated H3 AIVs in chickens, three representative H3 viruses  from group 1 and group 2 in phylogenetic trees of HA were chosen for chicken study. Groups of seven 12-week-old SPF chickens were used, inoculated intranasally with 10All animal studies in this study were conducted in accordance to the guidelines of the Animal Care and Use Committee of Shanghai Veterinary Research Institute, and all animal studies protocols are approved by Chinese Academy of Agricultural Science (Permit number: SHVRI-Po-0120).87 out of 1000 oropharyngeal swabs collected from fowls during 2009 to 2011 were identified as influenza virus positive according to the hemagglutination assay and RT-PCR amplification of M segment. Seven viruses were determined as H3 subtype AIV based on the HI results. Further sequencing of the HA and NA gene revealed that four H3N2 and three H3N8 viruses were isolated. Consequently, the seven isolates were named as A/duck/Shanghai/120-1/2009 , A/duck/Nanjing/A1591-1/2010 , A/chicken/Nanjing/B854-2/2011 , A/duck/Hebei/B1645-2/2011/, A/duck/Hebei/B1646-2/2011 , A/duck/Hebei/B1647-1/2011  and A/duck/Shanghai/74-1/2009 . B854-2/H3N8 virus was isolated from chicken and all the others were from ducks. No obvious clinical signs were observed in either of the fowl. Full genomes of all seven H3 isolates were sequenced and deposited in GenBank, as accession numbers listed in Growth kinetic results showed that all H3 viruses replicated efficiently in MDCK cells, and replicated to different titers in A549 and DF-1 cell . NotablySeven H3 isolates shared the same amino acid sequence (PEKQTR/GLF) at the cleavage site between HA1 and HA2, indicating that they are low pathogenic strains. 98Y, 134G, 135K, 136S, 137G, 138A, 153W, 155V, 183H, 190E, 194L, 195Y, 224R, 225G, 226Q, 227S, 228G, and 229R were observed in hemagglutinin at the receptor-binding pocket area of all seven isolates. None of these residues have been reported to be involved in recognition of human-like receptors, suggesting that all the isolates prefer avian-like receptors. E/D at position 627/701 of the polymerase PB2 protein of the seven H3 isolates suggested that these isolates were short of efficient adaptation to mammals. According to sequences of NA, all isolates presented susceptibility to neuraminidase inhibitors 36Based on phylogenetic trees, HA segments of seven H3 isolates are grouped into Eurasian lineage. Three H3N2 isolates from Hebei  were clustered in group 1 including H3 subtype influenza viruses from Netherlands and Korea . The remPhylogenic analysis showed that internal genes of all seven H3 isolates distributed in group 1 and group 2 within the Eurasian lineage. Notably, in group 1, the PB1, NP and NS genes of three H3N2 isolated from Hebei  had close relationship with those of highly pathogenic H5N8 virus emerged in Korea and United States  and 4. SPA gene of A/duck/Shanghai/120-1/2009 (H3N8) also has close relationship with the highly pathogenic H5N8 virus . PA geneTo test the antigenic cross-reactivity among H3 viruses isolated in this study, based on the phylogenetic trees of HA gene, four representative H3 isolates B1646-2/H3N2 from group 1 and 74-1/H3N2, 120-1/H3N8, and 854-2/H3N8 from group 2 were chosen for antigenic analysis. The results showed that B1646-2/H3N2 from group 1 showed low cross-reactivity with sera against viruses  from group 2. Similarly, antiserum against B1646-2/H3N2 had low reactivity with the viruses of 74-1/H3N2, 120-1/H3N8 and 854-2/H3N8 . The pre6 EID50 viruses intranasally. No clinical sign was observed in either group, and all H3 isolates caused only slightly weight loss in mice (data not shown). The virus titration results showed that all the H3 isolates replicated in lung and nasal turbinate without prior adaptation  were chosen for the chicken infection study. The results showed that no clinical sign was observed in the infected chickens, and all three viruses replicated inefficiently in chickens. B854-2/H3N8 and 120-1/H3N8 were only detected in pancreas. B1646-2/H3N2 was detected in trachea, lung, pancreas and intestine with low titers . These tMultiple subtype influenza viruses  have circulated and evolved in live poultry markets which was considered to be a prolific source of AIVs in China523394041424345The highly pathogenic avian influenza (HPAI) subtype H5N8 virus was introduced into Korea and North America, and Europe in 2014Phylogenetic analysis of HA genes and antigenic cross-reactivity results indicated that two antigenically different lineages H3 viruses are co-circulating in LPM in China. The H3N2 influenza viruses from group 1 had close relationship with H3 viruses circulating in Korea. The chicken study indicated that the three representative viruses  from group 1 and 2 replicated and shed inefficiently in domestic chickens, despite the B854-2/H3N8 was isolated in chicken. It suggested that duck-originated viruses have not adapted chicken.Mammalian adaptation associated residues  were absent in all the seven H3 viruses, although all these H3 viruses replicated efficiently in MDCK cells. Further mice study suggested that most of the isolates replicated efficiently in mice without prior adaptation. Viral titers in lungs of certain H3N2 viruses (i.e. 1646-H3N2) are comparable to those of H5N2 and H5N8 in infected mice reported recentlyTaken together, the seven H3 viruses isolated from LPM in China reassorted with different influenza viruses, indicating that intense reassortment is occurring among different subtype influenza viruses from different areas. However whether the reassortment happened in LPM or before the fowl introduced into it remain unclearly. Nevertheless co-circulation of these viruses in LPM will enhance the reassortment. Hence, continuous surveillance in LPM is warranted.How to cite this article: Cui, H. et al. Phylogenetic analysis and pathogenicity of H3 subtype avian influenza viruses isolated from live poultry markets in China. Sci. Rep.6, 27360; doi: 10.1038/srep27360 (2016).