Tumor metastasis is the leading cause of death in patients with advanced gastric cancer (GC). Limited therapeutic regimens are available for this condition, which is associated with a poor prognosis, and the mechanisms underlying tumor metastasis remain unclear. In the present study, increased histone methyltransferase G9A expression in GC tissues correlated with advanced stage and shorter overall survival, and in vitro and in vivo experiments revealed that G9A promoted tumor invasion and metastasis. Moreover, we observed that Reg IV induced G9A via the p-ERK/p-SP1 pathway. SP1 directly binds the G9A promoter and enhances G9A expression, and upregulated G9A then forms a transcriptional activator complex with P300 and GR, thereby promoting ITGB3 expression induced by dexamethasone (DEX) and contributing to GC metastasis. However, the G9A-mediated increase in ITGB3 expression was not dependent on the SET domain and methyltransferase activity of G9A. This study demonstrates that G9A is an independent prognostic marker and promotes metastasis in GC, thus suggesting that it may be a tumor biomarker and potential therapeutic target in GC. Metastasis and invasion are the most important features of GC, and approximately 50% of metastases are peritoneal metastases, which are associated with a poor prognosis2. Because metastatic cells can affect multiple vital organs within the abdomen, the average survival time of patients with peritoneal metastases is <6 months3. Unfortunately, the molecular mechanisms by which advanced GC progress to metastasis remain poorly understood. In view of the detrimental influence of advanced metastasis on survival, exploring the molecular mechanisms of metastasis and developing treatment or prevention strategies is of great significance.Gastric cancer (GC) is a malignant tumor derived from gastric epithelial cells and is associated with high morbidity and mortality4. G9A is involved in autophagy, cell differentiation, embryonic development, cognitive and adaptive behavior, adipogenesis, and other biological processes9. Moreover, recent studies have indicated that G9A expression is elevated in many types of human cancers, including breast cancer10, ovarian cancer11, head and neck squamous cell carcinoma12, lung cancer13 and endometrial cancer14, and G9A has been reported to promote both cell proliferation and metastasis in cancers. For example, G9A-specific small interfering RNAs (siRNAs) result in a DNA damage response and inhibit the proliferation of colorectal cancer cells15. Furthermore, BIX-01294, an inhibitor of G9A methyltransferase activity, induces autophagy-dependent cell death via G9A dysfunction and intracellular reactive oxygen species accumulation in breast and colon cancer cells16. G9A also exhibits methyl-transferase activity and concomitantly represses the downstream effector Ep-CAM, thereby promoting the invasion step of the invasion–metastasis cascade in lung cancer13. Accordingly, the knockdown of G9A expression suppresses the peritoneal metastasis of ovarian cancer11, thus prompting us to study the role of G9A in GC peritoneal metastasis. G9A promotes the methylation of gene promoter region histones or DNA via two different transcription-inhibitory mechanisms18. Alternatively, G9A can act as a scaffold protein promoting the expression of transcription activators, thereby activating gene transcription20.G9A, also known as EHMT2, is a nuclear histone lysine methyl-transferase belonging to the Su(var)3-9 family, which plays a primary role in catalyzing the monomethylation and dimethylation of H3K9 (H3K9me1 and H3K9me2) in euchromatinAccumulating evidence indicates that G9A is associated with tumor development, but the function of G9A in GC remains poorly understood. In the present study, we characterized the expression of G9A and evaluated the correlation between G9A expression and the clinicopathological features of GC. Moreover, the biological functions of G9A were investigated by silencing or restoring G9A expression in GC cells and animal models. Our results indicated that G9A expression strongly correlated with the metastatic properties of GC and may serve as a potential therapeutic target.www.oncomine.org/) and TCGA database (https://cancergenome.nih.gov/). We found that the G9A transcript level was significantly increased in GC tissues compared with matched non-tumor tissues, and the expression of G9A did not differ in LAUREN subtypes of GC  and Tumor, Node, Metastasis (TNM) stage (P = 0.003) but not with other clinicopathologic factors, including tumor differentiation, the extent of invasion, and tumor location  and western blot assays. As shown in Fig. P < 0.01, Fig. P < 0.01, Fig. P < 0.01, Fig. To investigate the effect of G9A on GC cell motility, a wound-healing assay was performed. We observed that G9A knockdown significantly inhibited the wound-healing ability of BGC-823 cells after 48 h . Multiple cell experiments have shown that DNMT1 and G9A together maintain the transfer of methylation information during DNA replication26. In addition, G9A can also increase the levels of DNMT3a and DNMT3b via the ANK domain, thus maintaining DNA methylation at the gene promoter and regulating gene silencing in a manner independent of the methylation enzyme activity of G9A27. Traditionally, G9A has been believed to primarily methylate histones on regular chromosomes to subsequently inhibit the transcription of related genes. However, the latest research shows that G9A can also serve as a scaffold protein that increases the levels of transcriptional activators that activate the transcription of specific genes. Specifically, G9A interacts with multiple transcriptional activators and thereby co-activates gene transcription. Studies have reported that G9A, together with p300, CARM1, and GRIP1, as a coactivator for nuclear receptors in a methyltransferase activity-independent manner29. In human lung cancer cells, G9A recruits CARM1 and p300 to the promoter of the GR target genes ENaCα and CDH16 and promotes the transcription of these genes under the stimulation of glucocorticoids19. In these processes, the role of G9A in the activation of gene transcription does not depend on the methylation enzyme activity but is instead related to the ability of its structural domain to recruit other transcriptional activators. ITGB3, a member of the integrin family and a GR target gene, promotes the peritoneal metastasis of GC by influencing the ability of GC cells to adhere to the peritoneum30. In this study, the knockdown of G9A inhibited the DEX-induced expression of ITGB3, and silencing of ITGB3 attenuated the ability of G9A to promote invasion and adhesion in GC cells. Under DEX stimulation, G9A, P300, and GR formed a transcriptional activator complex, which in turn promoted the expression of ITGB3. ITGB3 was downregulated in G9A-silenced cells, and this effect was reversed after introduction of the G9A-△SET and G9A-mut vectors. This finding further demonstrated that G9A activates gene expression by interacting with nuclear receptors, and this activity is independent of G9A’s methyl transferase activity. Reg IV has been found to be associated with peritoneal metastasis in our previous study, and its overexpression upregulates many adhesion molecules, including ITGB321. In this study, we found that Reg IV affects G9A expression by regulating the ERK/SP1 signaling pathway.In addition, G9A can regulate gene transcription by modifying H3K9 and H3K27 methylation at the promoter region of some genes, in a manner dependent on its histone methyltransferase activity. The single/double methylation of H3K9 (H3K9me1/2) and H3K27 (H3K27me) increases the expression of suppression factors, such as HP1 family members, thus resulting in closer binding between DNA and histone proteins and consequently inhibiting gene transcriptionIn conclusion, G9A does not rely on its methyltransferase activity to concomitantly activate the downstream effector ITGB3. We report the first evidence that the Reg IV-Erk-SP1-G9A complex (with GR and P300)-ITGB3 pathway contributes to GC development. Moreover, high G9A expression correlates with poor survival in patients with GC. These findings suggest that G9A may be an attractive target for suppressing GC metastasis.31, by using antibodies against G9A . The percentage of the positive cancer cells and intensity of the cell staining were scored according to the following rules: Percentage of positive cells was classified as follows: <10% (0), 10–25% (1), >25–50% (2), >50–75% (3), and >75% (4). Intensity of staining was evaluated as negative (0), weak (1), moderate (2), or strong (3). The final scores of the tissue sections were multiplied by the intensity scores and percentage of positive cells scores: 0–2 final scores indicated negative expression, whereas 2–6 final scores represented weak positive expression and 6–12 final scores represented strong positive expression.The tissues used were obtained from the Shanghai Ruijin Hospital at the Shanghai Jiao Tong University School of Medicine. All patients provided written informed consent before enrollment, and the study protocol was approved by the Ethics Committee of Shanghai JiaoTong University School of Medicine of Ruijin Hospital. None of the patients had received chemotherapy or radiotherapy before surgery. All patients had curative intent and underwent radical resection. IHC was performed as previously described2 and saturated humidity atmosphere.Human GC cell lines and human immortalized gastric epithelial cells were purchased from the Shanghai Institutes for Biological Sciences at the Chinese Academy of Sciences. The cells were maintained in 1640 medium  supplemented with 100 IU/ml penicillin, 100 IU/ml streptomycin, and 10% fetal bovine serum. The following inhibitors were used: BIX-01294  against G9A and PD98059 (Selleck) against ERK. All cells were incubated at 37 °C in a 5% COThe G9A shRNAs and overexpression lentivirus were purchased from the Novobio Biotechnology Company in Shanghai, China. The shRNA duplexes were synthesized as follows:5′-CACCGAGAGAGTTCATGGCTCTTTCGAAAAAGAGCCATGAACTCTCTC -3′ (G9a shRNA 1 forward),5′-AAAAGAGAGAGTTCATGGCTCTTTTTCGAAAGAGCCATGAACTCTCTC -3′ (G9a shRNA 1 reverse) and5′-CACCGCATAGATGCCCGTTACTATGCGAACATAGTAACGGGCATCTATGC-3′ (G9a shRNA 2 forward),5′-AAAAGCATAGATGCCCGTTACTATGTTCGCATAGTAACGGGCATCTATGC-3′ (G9a shRNA 2 reverse). G9A was knocked down or overexpressed by lentiviral infection, and 5 μg/ml blasticidin was used to select cells in which G9A was stably knocked down or overexpressed.GC cells were transfected with siRNA or shRNA with Lipofectamine 2000 according to the manufacturer’s protocol. The sense and anti-sense strands of siRNAs and shRNAs are listed in Supplementary Table S31. Primers for qRT-PCR are listed in Supplementary Table STotal RNA was extracted from GC cells and subjected to qRT-PCR as previously described31, with the following primary antibodies: anti-G9A , anti-GAPDH , anti-SP1 (Abcam), anti-p-SP1 (Abcam), anti-Flag , anti-ERK1 , anti-phospho-ERK1/2, anti-H3 , anti-H3K9me2 , anti-ITGB3 (Abcam), anti-GR (Abcam), and anti-P300 (Abcam).A western blot analysis was carried out as previously described31.Wound-healing and Transwell assays were used to study changes in the migration and invasiveness of GC cells according to a previously described protocol5 cells in ultra-low attachment plates. After 48 h of culture, the cells were resuspended in 3--2,5 diphenyl tetrazolium bromide (MTT) and cell absorbance was detected at 570 nm.GC cells undergoing anoikis were identified and quantified with a Cytoselect 24-well Anoikis Assay kit  according to the manufacturer’s instructions. Briefly, anoikis was detected by seeding 1 × 10The adhesion of GC cells to ECM components was evaluated using a CytoSelect 48-Well Cell Adhesion Assay Kit  according to the manufacturer’s instructions. Matrigel or murine peritoneum was pre-coated in a 24-well plate, and GC cells labeled with carboxyfluorescein succinimidyl ester  were overlaid on the plate bottom. Later, cells were incubated at 37 °C for 60 min and non-adherent cells were removed with three gentle washes with phosphate-buffered saline (PBS). The cells that were adherent to Matrigel or the murine peritoneum were then quantified in MTT assays or counted under a microscope, respectively.GC cells in which G9A was knocked down or overexprssed were resuspended with 0.3% soft agar  in RPMI 1640 containing 10% fetal bovine serum (FBS) and layered on 0.6% solidified agar in RPMI 1640 containing 10% FBS in six-well plates. The plates were incubated for 2 weeks. Colony sizes were photographed under a microscope at 10× magnification.The ChIP assay was performed by using a ChIP kit  according to the manufacturer’s instructions. The resultant precipitated DNA samples were assessed with PCR.32.The luciferase reporter gene and Co-IP assays were performed as previously described6 cells were resuspended in 0.1 ml of PBS and injected into the abdominal cavity. The mice were sacrificed after 80 days, and the number of nodules present in the abdominal cavity and the volume of ascites of each mouse were measured. During this time, we recorded the survival time of nude mice, and survival curves were generated. The organs were excised from nude mice post-mortem for hematoxylin and eosin staining.All animal experiments were conducted according to the Chinese guidelines for animal experimentation and were approved by the Institutional Animal Care Committee of Ruijin Hospital, which is affiliated with the Shanghai Jiao Tong University School of Medicine. Briefly, 4-week-old male BALB/C nude mice were purchased from the Institute of Zoology at the Chinese Academy of Sciences of Shanghai. All experiments were performed in accordance with the official recommendations of the Chinese animal community. To establish the experimental peritoneal metastasis model, 1 × 10t-test, chi-squared test, Fisher’s exact test, Cox proportional hazards regression, and the Kaplan–Meier method. P < 0.05 was considered significant, and P < 0.01 was considered highly significant.The statistical analysis was performed with the SPSS Version 22.0 software . Statistical analyses were performed using a one-way analysis of variance, Student’s Supplementary figure legendsSupplementary tablesFigure S1Figure S2Figure S3Figure S4Figure S5Figure S6Figure S7


H4 avian influenza virus is one of the most prevalent influenza virus subtypes in birds. The evolution and pathogenicity of H4 AIV in domestic birds of China remain largely unclear. In the present study, a total of eight H4 AIV strains isolated in duck farm and live poultry markets (LPM) were characterized. Phylogenetic analysis indicated that these strains are divided into two groups in the Eurasian lineage. Eight genes of MH-2/H4N6 isolated from a duck farm were closely related to three H4N6 viruses from LPM, suggesting a potential AIV link between farms and LPMs. Additionally, the HA, NA, PB2, NP, and NS genes of two other H4N6 viruses isolated in LPM clustered with that of MH-2/H4N6. However, the remaining genes were more closely related to other sublineages, suggesting that MH-2/H4N6-originated viruses reassorted with other viruses in LPM. All H4 viruses replicated in mouse lungs without prior adaptation and all viruses replicated and transmitted among ducks. 29-1/H4N2, MH-2/H4N6, and 420-2/H4N6 viruses caused systemic infection in infected ducks. However, most of the viruses were not adapted in chickens. The present results indicate a potential correlation of AIV between LPMs and farms and suggest that active surveillance of AIV in LPM is warranted in China. Avian influenza (AI), an infectious disease caused by influenza A viruses, is a threat to human and animal health. Wild birds and waterfowl are believed to be the natural hosts and reservoirs of avian influenza viruses (AIVs)12356910Live poultry markets (LPMs) are believed to be the source of AIV in China131414131550) was calculated by using the Reed and Muench methodRespiratory disease was observed in a duck farm in Shanghai, China, in 2009. Subsequently, an H4N6 virus was isolated from oropharyngeal swab samples of the diseased ducks. Since LPM is the major place where live ducks are traded in Shanghai, active surveillance was conducted in the LPM in 2009 to investigate the epidemiology of the H4. Totally, 3,787 oropharyngeal swab samples were collected from ducks and chickens in LPM. Each sample was suspended in antibiotic solution in phosphate-buffered saline (PBS) containing 1,000 U/ml penicillin and 1,000 U/ml streptomycin and centrifuged at 13,800 g for 10 min. The filtered supernatants were inoculated into the allantoic cavity of 9-day-old specific pathogen-free (SPF) embryonated chicken eggs and incubated at 37 ˚C. Allantoic fluid from the incubated eggs was harvested 72 h after inoculation. An HA assay was conducted with 0.5% packed chicken red blood cells and HA-positive samples were subtyped by using an HI assay with anti-sera against AIVs  and RT-PCR using influenza-specific primers as described previously18Viral RNA was extracted and converted to cDNA using reverse transcriptase M-MLV  in a final volume of 20 μl containing: 5 μl RNA, 4 μl 5 × RT buffer, 2 μl dNTPs (10 mM), 2 μl 10 μM specific primer (5′-AGCAAAAGCAGG-3′), 0.5 μl MLV reverse transcriptase, 0.5 μl RNase inhibitor , and 6 μl diethylpyrocarbonate (DEPC)-treated water. The reaction was incubated at 42 ˚C for 1 h followed by incubation at 70 ˚C for 15 min. The eight fragments of each virus were amplified using universal primers, as described previously6 EID50 in 0.1 ml PBS. To assess the transmissibility of H4 virus in ducks or chickens, three naïve ducks and chickens were co-mingled with infected birds. All birds were observed daily for clinical signs until the end of the experiment (14 days). Oropharyngeal and cloacal swabs were collected at 2, 4, and 6 days post infection/contact (dpi/dpc) from the inoculated and contact birds for detection of virus shedding. To elucidate virus replication and distribution in the infected birds, three birds from each group were euthanized at 3 dpi and tissue samples  were collected for virus titration. The remaining birds were euthanized at the end of the experiment. Serum samples were collected at necropsy for seroconversion tests.To assess the pathogenicity of the isolated H4 AIVs in ducks and chickens, groups of 12-week-old influenza virus seronegative shelducks and 6-week-old SPF chickens were used. Six birds were inoculated intranasally with the isolated H4 viruses at 106 EID50 in 50 μl PBS. Eleven other BALB/c mice were inoculated intranasally with 50 μl PBS as mock group. To evaluate viral replication in the respiratory tract and other organs, three mice from each group were euthanized on 3 and 5 dpi. At necropsy, the heart, liver, lung, nasal turbinate, spleen, kidney, and brain tissues were collected and titrated in 10-day-old embryonated chicken eggs. The remaining mice were observed for clinical signs and their body weight was measured until 14 dpi, the serum samples were collected from mice at 14 dpi. For histopathologic examination and immunohistochemistry (IHC), lung samples were fixed in 10% buffered formalin, processed, and stained with hematoxylin and eosin (H&E). An anti-influenza A antibody against the nucleoprotein  was used for IHC staining as described previouslyTo investigate the pathogenicity of the H4 isolates in mice, groups of eleven 6-week-old BALB/c mice  were inoculated intranasally with the viruses at 10All animals were cared for in accordance to the guidelines of the Animal Care and Use Committee of Shanghai Veterinary Research Institute. Animal studies were approved by Chinese Academy of Agricultural Science (Permit number: SHVRI-Po-0120). Data were analyzed using analysis of variance (ANOVA) in GraphPad Prism version 6.0 ; a p-value < 0.05 was considered statistically significant.In 2009, a mild respiratory disease was observed in duck farms in Shanghai. Oropharyngeal swab samples were collected from the diseased ducks for virus isolation. An H4N6 subtype AIV was isolated and designated as A/duck/Shanghai/MH-2/2009 . To trace the origin and distribution of this H4 subtype virus, an active surveillance program was conducted in LPM in Shanghai. A total of 188 AIV viruses were isolated from LPM of Shanghai in 2009, seven out of these isolates are H4 subtype AIV (six H4N6 and one H4N2) with isolation rate of 0.2%. All seven H4 AIVs were isolated from domestic ducks, which were provisionally designated as A/duck/Shanghai/29-1/2009 , A/duck/Shanghai/44-2/2009 , A/duck/Shanghai/46-2/2009 , A/duck/Shanghai/67-2/2009 , A/duck/Shanghai/408-1/2009 , A/duck/Shanghai/420-2/2009 , and A/duck/Shanghai/421-2/2009 . Viruses were amplified in SPF chicken embryonated eggs. The genomes of all eight H4 isolates were sequenced and deposited in GenBank with accession numbers of KX162591 - KX162654.The eight H4 AIV isolates all shared the same amino acid sequence PEKASR/GLF at the HA cleavage site between HA1 and HA2, indicating that all viruses are low pathogenic influenza viruses. All H4 AIVs had 226Q and 228 G (according to H3 numbering) at the receptor binding region, implying that they retained typical avian virus-like receptor specificity . No AIV had a deletion in the NA stalk regions. No mammalian adaptation-associated mutations were observed in the PB2 gene . F26, A/I27, T/V30, N31, E34, and F38 in the M2 protein are amantadine resistance markers. No such substitutions were found, except that four viruses  had a V27I substitution. All of these H4 viruses had a full-length PB1-F2 protein of 90 amino acids except for 29-1/H4N2 (with 58 amino acids). An N66S mutation was found in PB1-F2 of 421-2/H4N6, which could increase the virulence of influenza A virus by inhibiting the early interferon response24The genetic diversity of the H4 AIVs was characterized based on a phylogenetic analysis of eight genes. Standard clade nomenclature system for H5 HA is used for H4 genes classification, which set boundaries of pairwise nucleotide distances >1.5% for between clades and <1.5% for within cladesPhylogenic analysis of internal genes showed that all eight H4 isolates belong to the Eurasian lineage  and 3. T6 EID50. None of the H4 AIVs induced clinical signs of illness in ducks or chickens. All eight viruses replicated in ducks  in LPM enhances the reassortment potential of AIV. Close contact between humans and the LPM provides conditions for cross-species AIV transmission2830Here, we have reported the isolation of a H4 isolate (MH-2/H4N6) from mallards in a farm and traced their genetic and pathogenic characteristics using genetic analyses and animal experiments. Seven other H4 AIVs were isolated from LPMs in Shanghai. The NA genes of all seven H4N6 isolates were located in group 1, though 421-2/H4N6 was distantly related to the other six H4 isolates. The HA genes of the other H4 isolates fell into group 1, which contains the H4 viruses recently isolated in Asia. However, the HA gene of 421-2/H4N6 belonged to group 2, which contains H4 strains isolated in Asia during 2005–2007. Similarly, the 421-2/H4N6 PB2, NP, and NS genes were only distantly related to the other six H4 isolates. Thus, two H4 lineages with different reassortant patterns are co-circulating in LPMs. The MH-2/H4N6 isolated from a diseased duck farm was closely related to five H4N6 viruses from LPM , suggestAquatic birds are the natural reservoir of influenza virus. The virus replicates in bird intestines but does not cause disease32Influenza viruses evolve over time through mutation and reassortment9Although H4 AIVs are low pathogenic AIVs, it is possible that they will eventually gain the ability to transmit efficiently through the accumulation of mutations and reassortments. Therefore, it is important to investigate further the mechanisms of H4 AIVs mutation and reassortment, to prepare for potential pandemics.How to cite this article: Shi, Y. et al. Characterizations of H4 avian influenza viruses isolated from ducks in live poultry markets and farm in Shanghai. Sci. Rep.6, 37843; doi: 10.1038/srep37843 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Toxoplasma gondii is crucial for successful host cell invasion by this obligate intracellular parasite. Despite its tiny body architecture and limited trafficking machinery, T. gondii relies heavily on transport of vesicles containing proteins, lipids and important virulence-like factors that are delivered to these secretory organelles. However, our understanding on how trafficking of vesicles operates in the parasite is still limited. Here, we show that the T. gondii vacuolar protein sorting 9 (TgVps9), has guanine nucleotide exchange factor (GEF) activity towards Rab5a and is crucial for sorting of proteins destined to secretory organelles. Our results illuminate features of TgVps9 protein as a key trafficking facilitator that regulates protein maturation, secretory organelle formation and secretion, thereby ensuring a primary role in host infection by T. gondii.Accurate sorting of proteins to the three types of parasite-specific secretory organelles namely rhoptry, microneme and dense granule in  Toxoplasma gondii is an important food and waterborne pathogen causing toxoplasmosis, a usually mild disease in immunocompetent humans that can turn into a major threat in immunocompromised patients and during primary infection of pregnant woman. T. gondii is a member of the Apicomplexa, a phylum of numerous medically important parasites causing life-threatening diseases in human and animals worldwide. The phylum is typified by specific secretory organelles called rhoptries, micronemes and dense granules that are essential for host cell invasion and host pathway modulation. In Toxoplasma, rhoptries contain two groups, termed rhoptry (ROP) and rhoptry neck (RON), of effector proteins some of which are virulence factors; whereas micronemes secrete MIC proteins that are involved in parasite gliding, host cell attachment and invasion1T. gondii and likely all Apicomplexa have reutilized classical endosomal and endocytic trafficking pathways more typical of higher eukaryotes towards building specialized secretory organelles that release parasite effectors to interplay with host cell signaling pathways as a way to take control over host immunity and ultimately to promote long-term parasitism456767Despite having a single cell architecture, the parasite relies on active and abundant vesicle and protein trafficking. T. gondiiT. gondii Rab5+ and Rab7+ effector complexes likely interact with CORVET and HOPS in a manner similar to mammalian cells to induce membrane fusion within the endolysosome pathway of the parasite14Clearly, regulated vesicular traffic allows different cargoes to correctly reach their specific organelle destinations at the right time4567711TgSORTLRTgSORTLR interacts with clathrin, three components of the AP1 adapter complex, Sec23/24 and three vacuolar protein sorting namely Vps26, Vps35 and Vps9TgSORTLR from TgRab5- to TgRab7-dependent ELC before delivery to Golgi, is also essential for secretory organelle biogenesis and parasite shapeWe have described that transport of MIC and ROP proteins to microneme and rhoptry organelles, respectively, also required an essential sortilin-like receptor named T. gondii counterpart of Vps9 (herein named TgVps9) is a bona fide Rab5 GTP-Exchange Factor (GEF) that is crucial for ROP protein maturation and processing, and its loss leads to a reduced number of rhoptries. Absence of TgVps9 also impairs peripheral microneme biogenesis and disturbs dense granule secretion resulting in an accumulation of novel vesicles present both within and outside the parasite. Together with the rhoptry defect, absence of peripheral microneme formation and dense granule secretion severely affects parasite invasion of host cells. Collectively, these observations support the notion that TgVps9-mediated loading of GTP to TgRab5 is crucial for fine-tuning vesicle sorting to secretory organelles, the latter being essential for T. gondii host cell infection.Here, we report that the TgSORTLRTgVps9 domain operates in vitro as a GEF towards Rab5, by testing activity of a bacterial expressed TgVps9 recombinant protein towards human Rab5, as previously described19849 to aa1134) of TgVps9 was purified from E.coli. Human recombinant Rab5A was purified in its GDP bound form and nucleotide exchange to GppNHp, a non-hydrolysable GTP analogue, was monitored by tryptophan fluorescence measurements. A dose dependent GEF activity of recombinant TgVps9 towards human recombinant Rab5A was detected and compared to human recombinant Rabex5  that stimulate the release of monomeric guanosine diphosphate (GDP)-bound to Rab5, allowing guanosine triphosphate (GTP) to bind and activate Rab5 that, in turn, regulates endosome vesicle trafficking1617t Rabex5 . As expet Rabex5 .TgVps9 possesses GEF activity towards recombinant Rab5, we then sought to determine in T. gondii whether the protein resides in the same subcellular compartment as Rab5. To this end, we chromosomally appended the hemagglutinin (HA) epitope at the 3′-end of the endogenous TgVps9 gene and validated by western blot that the tagged TgVps9 was readily expressed by transgenic tachyzoites .Having established that recombinant hyzoites . HA-taggin vitro GEF activity towards Rab522TgVps9-HA in vivo co-localized with TgRab5A . In addition, VP1 a marker of the plant-like vacuole that is present in close vicinity to ELC co-distributes with TgVps9 (TgVps9 (bottom panel). Taken together, these co-localization studies indicate that TgVps9 is embedded in the endosomal-like compartment (ELC) together with Rab5, a compartment with an established role in the formation of secretory organelles of T. gondii45678Consistent with its TgVps9 function in a clonal homogenous parasite population, we generated conditional anhydrotetracyclin (ATc)-inducible knockout TgVvps9 mutants (named iKOTgVps9) using the strategy depicted in in vitro was verified by PCR using the two specific primers (see nucleotide sequences in Methods) shown in blue arrow  and peripheral micronemes (white arrows) were observed in cytoplasm anterior to the nucleus of ATc-untreated tachyzoites . Elevated numbers of novel vesicles of variable size and morphology were also observed in the PV space delimited by the PVM . We estimated that about 17% of iKOTgVps9 mutants examined by electron microscopy contained these aforementioned 500-nm intra-parasite vesicles while approximately 20% of mutants had novel vesicles of variable size and morphology in their PV space. In addition, we observed a significant reduction of the overall number of rhoptries per mutants using electron microscopy. Specifically, ATc-treated iKOTgVps9 mutants contained less than two thirds the relative number of rhoptries per mutant compared to the parental line. Also, we observed a disorganized ultrastructural morphology with the marked absence of the typical banana-shaped bodies in several TgVps9-depleted mutants , nor the pro-M2AP processing enzyme CPL24252627TgVps9. This suggests that both processing and maturation of MIC proteins likely occurs in the distal sub-compartment of the ELC29TgVps9-independent fashion. It is also tempting to speculate that pre-micronemes and pre-rhoptries may bud from the novel and yet-uncharacterized vesicles that are visible above the nuclei of TgVps9-depleted mutants. As discussed above, TgVps9 displays in vitro GEF towards human Rab5A, comparable to that of Rabex51819202122TgVps9-deficient mutants resemble those previously reported for TgRab5A protein after its encoding gene has been disruptedTgRab5A and Vps11 probably interact through the CORVET-tethering complexesiKOTgVps9 mutants described here.Based on these observations, we propose a model in which hoptries . This moTgSORTLRT. gondii share similarities with those of TgVps9, the latter may participate in regulation of retromer and endosomal lipid content. However, TgVps9 is not associated with the Golgi apparatus like TgSORTLR, suggesting that this parasite Rab5 GEF is likely involved in anterograde transport and secretory organelle formation, rather than protein recycling in T. gondii.Protein trafficking that relies on VPS9 has been described in several other eukaryotic cells such as yeast, which has three Vps9-domain containing proteins, Vps9, Muk1 and Vrl1, all exhibiting GEF activity towards Rab5 paralogsTgVps9 inhibits rhoptry protein processing/maturation, impairs secretory organelle biogenesis and secretion, leading to an inability of TgVps9-deficient mutants to invade host cells and to achieve multiple rounds of invasion, proliferation and reinvasion of host cells. However, there is still a missing link between protein processing, maturation, vesicular traffic and secretory organelle formation. Further investigation of TgVps9 functions that will define how different parasite TgRab proteins are precisely regulated by its GEF activity could provide this missing link.In conclusion, loss of T. gondii tachyzoites of RH strain for CRISPR/Cas9 knockout experiment, RHΔKu80TgVps9 gene (TGME49_230140) and RHΔKu80TATi for inducible knockout (iKO) strain35TgVps9 mutants were cultured in the presence of 1.5 μg/ml anhydrotetracycline (ATc).We used TgVps9 from aa849 to aa1134 was generated using a modified pET19 plasmid that expresses His-tag protein with a TEV cleavage site using the following primers: forward (Recomb-Vps9a.d4_F) CCGGCATATGGCGTCTTCTGCCTCTTTTTCTGCC and reverse (Recomb-Vps9a.d4_R) CCGGGGATCCTTAGCGTTCGCGTTCGCGGTCGTATTC. Human recombinant Rabex5 from aa132 to aa397 and full length Rab5a with a C-terminal CVIL mutation were prepared as previously describedTgVps9 was expressed in BL21 Codon Plus (DE3)-RIPL, and cell pellet was resuspended in buffer A  phosphine (TCEP), 20 mM imidazole pH 8), sonicated, and centrifuged at 20,000 rpm for 1 hour at 4 °C. Cell lysate was incubated with 2 ml agarose Ni-NTA beads, washed with buffer A and subsequently buffer B . Bound protein was eluted using buffer C . His-tags were removed by TEV protease cleavage during overnight dialysis in Buffer A without TCEP and imidazole. In order to remove the His-tagged TEV protease, dialyzed protein solution was incubated with Ni-NTA beads again before the GEF assays were performed.Recombinant protein of the catalytic core of 2, 2 mM dithioerythritol (DTE)), subsequently 200 nM or 400 nM of TgVps9 or 200 nM of Rabex5 were added.GEF activities were analyzed by intrinsic tryptophan fluorescence measurements showing fluorescence changes due to the conformational change from GDP to GTP state19TgVps9-HA knock in parasites. DNA of TgVps9 was cloned in this plasmid using the following primers: forward (F-KI_Vps9) TACTTCCAATCCAATTTAATGCCCCTGCTTGCCCCTCGCCT and the reverse (R-KI_Vps9) TCCTCCACTTCCAATTTTAGCTTTCCTGTCACTATGTTTCGCGTCCG. To obtain iKOTgVps9 mutants, we used pG13-D-T7S4 plasmidTgVps9-5′_F) CCGGCATATGCTTCTAACGGCACCACTTAAGGTGC and reverse (iKOTgVps9-5′_R) CCGGCATA TGTGCGCCTTCTCGTGTCGTCTTG; and another 2-kb DNA containing the coding sequence of TgVps9 gene using the following primers: forward (iKOTgVps9-3′_F) CCGGTGATCAATGTACCCATACGATGTTCCAGATTACGCTCGTCACGGGGAAGAAGACCAGCACGTC and reverse (iKOTgVps9-3′_R) CCGGCCTAGGGGGAGAAGAGGAGACAGAAACATCTCGACTACGACC with the HA-tag sequence underlined in the forward primers inserted at the N-terminus of TgVps9 protein, right after the initiation ATG codon. 1 × 107 parasites were transfected with 50 μg of linearized plasmid and selected with 2 μM of pyrimethamine. The emerging pyrimethamine-resistant population was cloned by limiting dilution. The clones were checked for plasmid integration by PCR using genomic DNA and two primers: forward (named A or Test_iKOVPS9.F2) ATTACAGCCAGCAGTGGCCAACCGAAT and reverse  GGCGTTGAATCTCTTGCCGACTGTGGAGAGGGAAGTCC.Endogenous gene tagging methodology using pLIC-HA-DHFR plasmidConfocal microscopy was performed as described previously10TgVps9 mutants or parental parasites incubated with ATc or not for 48 h were scrapped and washed twice with PBS. The intracellular parasites were pelleted before suspension by Laemmli buffer  and boiled for SDS-PAGE. 2 × 106 parasites were fractionated on 10% acrylamide gels, which were transferred to nitrocellulose membranes as previously describedIntracellular iKOFor transmission electron microscopy, cells were fixed in 2.5% glutaraldehyde prepared in 0.1 M cacodylate buffer and post-fixed in 1% osmium tetroxide in the same buffer. After acetonitrile dehydration, the pellet was embedded in Epon. Ultrathin sections (90 nm) were cut using a Leica UC7 ultramicrotome and collected on 150 mesh hexagonal barred copper grids. After staining with 2% uranyl acetate prepared in 50% ethanol and incubation with a lead citrate solution, sections were observed on a Hitachi H-600 transmission electron microscope at 75 kV accelerating voltage.Ku80TATi strain and iKOTgVps9 parasites were incubated under ATc condition for 48 h and mechanically lysed by passage through a syringe. 1 × 105 parasites were inoculated to HFF cells and incubated for 1 h at 37 °C. After infection for 1 hour, extracellular parasites were washed out with PBS and used to infect HFF cells before growing for 24 h at 37 °C. These infected cells were fixed by PFA and sequentially stained by GAP45 antibody with DAPI and counted by Axioimager Z1 (Carl Zeiss). Host cell invasion values were calculated using the ratio of intracellular parasite/host nucleus numbers as describedWild type RHΔHow to cite this article: Sakura, T. et al. A Critical Role for Toxoplasma gondii Vacuolar Protein Sorting VPS9 in Secretory Organelle Biogenesis and Host Infection. Sci. Rep.6, 38842; doi: 10.1038/srep38842 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In the 2016–2017 season, the A(H3N2) influenza epidemic presented an unusual early peak pattern compared with past seasons in South Korea. The interim vaccine effectiveness (VE) of influenza vaccination in preventing laboratory-confirmed influenza was estimated using test-negative design through the tertiary hospital-based influenza surveillance system in South Korea. From 1 September, 2016 to 7 January, 2017, adjusted VE of influenza vaccination in preventing laboratory-confirmed A(H3N2) was -52.1% ; -70.0%  in 19–64 years and 4.3%  in the elderly. Circulating A(H3N2) viruses belonged to the three phylogenetic subclades of 3C.2a, differently to A/Hong Kong/4801/2014, the current vaccine strain. Amino acid substitutions in hemagglutinin of circulating viruses seem to contribute to low VE. In conclusion, interim VE analysis presented that the protection of laboratory-confirmed influenza by seasonal influenza vaccination did not show the statistical significance in South Korea in the 2016–2017 influenza season. Influenza has been considered major medically attended acute febrile respiratory illness in humans. Antigenic variation is responsible for influenza being a continuous concern for public health. The 2016–2017 influenza season in South Korea has been characterized by an exceptionally early epidemic of A(H3N2) influenza in December, 2016. A primary clinic-based sentinel surveillance conducted by the Korea Centers for Disease Control and Prevention (KCDC) reported that all the detected influenza viruses up to the week 1  during the 2016–2017 influenza season were identified as A(H3N2) . Logistic regression models were adjusted for sex, age group (19–64 years and ≥65 years), calendar weeks , and comorbidities. Comorbidity was defined as any of following conditions: diabetes mellitus, cardiovascular disease, cerebrovascular disease, neuromuscular disease, chronic pulmonary disease, asthma, chronic kidney disease, chronic liver disease, malignancy, organ transplantation, bone marrow transplantation, immunosuppressant agent use, autoimmune disease, human immunodeficiency virus infection, and pregnancy. Age was adjusted as a continuous variable for age group-stratified analyses on VE. Statistical analyses were performed using SPSS version 20.0 .Nasopharyngeal swabs from twenty-nine A(H3N2) patients collected from week 50 to week 52 (4 to 24 December), 2016, were inoculated into Madin Darby canine kidney cells to isolate influenza viruses. Full length of hemagglutinin (HA) gene of the isolates was sequenced . CorrelaThis study was approved by the Institutional Review Board in Korea University Guro Hospital , Korea University Ansan Hospital (AS11047), Chungbuk National University Hospital (2011-06-044), Hallym University Kangnam Sacred Heart Hospital (2011-06-50), Inha University Hospital (11–1534), The Catholic University St. Vincent's Hospital (VC11ONME0118), Wonju Severance Christian Hospital (CR311025), Chonnam National University Hospital (CNUH-2012-133), Kyungpook National University Hospital (2012-07-030), and Pusan National University Hospital (1208-010-009). Written informed consent was obtained from the participants or their legal representatives.Up to the week 1 of the 2016–2017 influenza season , the peak influenza activity was seen in week 52  . During The median age of the patients was 50 years  and 126 (31.5%) patients were vaccinated against seasonal influenza during the 2016–2017 season. Among 400 patients, 52 (13.0%) were hospitalized. A total of 216 (54.0%) patients were diagnosed with influenza by PCR: A(H3N2), 180; influenza A, subtype not determined, 34; co-detection of A(H3N2) and influenza B, 2 cases. One or more comorbidities were found among 170 patients (42.5%). The demographic characteristics of the study population were shown on There was no difference in the influenza vaccination rate between influenza cases and controls. The adjusted VE of the influenza vaccine was estimated as -35.6%  for influenza A and -52.1%  for A(H3N2) influenza. The age group-specific adjusted VE in preventing A(H3N2) influenza was -70.0%  in 19–64 years age group and 4.3%  in ≥65 years age group .Phylogenetic tree analysis revealed that the isolated A(H3N2) influenza viruses during the 2016–2017 influenza season belonged to the subclades of 3C.2a. However, all the 29 A(H3N2) influenza viruses fell to the another subclades, different from the A/Hong Kong/4801/2014 virus, the current vaccine strain, being designated by group A, B, and C . VirusesA correlation plot using 34 amino acid substitutions in HA from our isolates revealed that the R142K and R142G substitutions were positively and negatively correlated with the D160N substitution, respectively, suggesting that the R142K and R142G were obvious characteristics of viruses in the new subgroup .In the 2016–2017 season, the A(H3N2) influenza epidemic showed an unusual early peak pattern compared with past seasons in South Korea. Also, the peak of influenza epidemic in week 52, 2016, was rather earlier than those of neighboring countries in Asia. Influenza laboratory surveillance information by the Global Influenza Surveillance and Response System showed the peak of number of specimens positive for influenza in week 3, 2017 in Japan and in week 1, 2017 in China .In our study, the interim VE in preventing A(H3N2) influenza during the early 2016–2017 season was as low as -52.1% in adults. The circulating A(H3N2) viruses belonged to the 3C.2a subclades; however, some amino acid substitutions in the HA sequence of the clinical isolates caused them to build distinguishing subclades separately from the current vaccine strain in the phylogenetic tree.Recent studies on the interim analysis of VE showed suboptimal influenza VE in preventing A(H3N2) influenza during the 2016–2017 season. In the US, influenza vaccine was effective to prevent A(H3N2) influenza presenting VE of 43%  in overall aged population, however, in 18–49 years and ≥65 years age group, protection did not reach statistical significance with wide range of confidence interval . In nortPrevious report referred A(H3N2) virus having changes of N171K, I406V, and G484E amino acid substitutions in the HA to as subclade 3C.2a1. Subclade 3C.2a1 viruses are defined with amino acid substitutions of 3C.2a plus N171K in HA1, I77V and G155E in HA2, and often N121K in HA1 as well . HoweverThere are several limitations in this study. First, the sample size is small, as this study is an interim analysis. However, we tried to improve the reliability of our results by including two more weeks after the peak of the early influenza season into the study period. Second, surveillance was performed in tertiary hospitals and the study population is not representative of the population in South Korea. There is a chance that patients with more severe illness could gather to the tertiary hospitals. Therefore, it is possible that the VE estimates are different from those obtained in primary care clinic-based surveillance. However, there is an observation about non-heterogeneity between VE estimates in outpatient settings and inpatient settings . Third, However, this study is noteworthy in estimating the low VE against A(H3N2) influenza in the early period of the 2016–2017 season and in characterizing the genetic features of circulating A(H3N2) viruses in South Korea. Recently, A(H3N2) mismatches between the vaccine strains and circulating strains have raised the global concern about suboptimal VE –21. More


A107) in the efficient replication of EV71. Substitutional mutations of VP1A107 significantly diminish viral growth kinetics without significant effect on viral entry, expression of viral genes and viral production. The results of mechanistic studies reveal that VP1A107 regulates the efficient cleavage of the VP0 precursor during EV71 assembly, which is required, in the next round of infection, for the transformation of the mature virion (160S) into an intermediate or A-particle (135S), a key step of virus uncoating. Furthermore, the results of molecular dynamic simulations and hydrogen-bond networks analysis of VP1A107 suggest that flexibility of the VP1 BC loop or the region surrounding the VP1107 residue directly correlates with viral infectivity. It is possible that sufficient flexibility of the region surrounding the VP1107 residue favors VP0 conformational change that is required for the efficient cleavage of VP0 as well as subsequent viral uncoating and viral replication. Taken together, our data reveal the structural role of the highly conserved VP1A107 in regulating EV71 maturation. Characterization of this novel determinant of EV71 virulence would promote the study on pathogenesis of Enteroviruses.Enterovirus 71 (EV71) is the major causative agent of hand, foot and mouth disease (HFMD) in children, causing severe clinical outcomes and even death. Here, we report an important role of the highly conserved alanine residue at position 107 in the capsid protein VP1 (VP1 A107), which is present in almost all EV71 strains, plays an important role in the replication of EV71, since mutation of VP1A107 significantly impairs viral growth. Detailed mechanistic studies reveal that VP1A107 determines the efficient cleavage of the VP0 precursor during EV71 assembly, and subsequent virion uncoating in the next round of viral infection. We also observed that flexibility of the VP1 BC loop or the VP1A107 region directly associates with viral infectivity. Collectively, our data suggest a structural role of the highly conserved VP1A107 residue in regulating viral maturation, which is instrumental for the viral uncoating and viral replication. Discovery of this novel determinant of EV71 virulence would promote the study on pathogenesis of Enteroviruses.Enterovirus 71 (EV71) causes hand, foot and mouth disease in children. In this work, we have discovered, for the first time, that the alanine residue at position 107 of the capsid protein VP1 (VP1 Enterovirus 71 (EV71) is a small, non-enveloped, positive-stranded RNA virus, a member of the human enterovirus species A in the Enterovirus genus, the Picornaviridae family –4. In reThe EV71 genome is protected by an icosahedrally symmetric capsid that is made of 60 copies of viral capsid proteins VP1 and VP3 and an average of 58 to 59 copies of capsid proteins VP2 and VP4, in addition to 1 to 2 copies of their capsid precursor protein VP0. During virus assembly, the viral capsid precursor P1 is firstly cleaved to yield VP0 (36 kDa), VP1 (32 kDa) and VP3 (27 kDa), forming a heterotrimer protomer. Five protomers then join together to form the pentameric assembly subunit, twelve of which self-assemble into an empty particle named as procapsid . ProcapsStructural studies have revealed the processes that occur during enterovirus infection of cells. Cellular receptors first attach to the virus capsids, often binding to a canyon-like cavity surrounding the five-fold axis , 16. Thi−3 substitutions per site year-1 [EV71 is genetically diverse. For example, the VP1 gene undergoes an estimated mutation rate of 4.2–4.5×10e year-1 . In addie year-1 , 23. In in vitro transcribed full-length viral RNA, both strains were able to induce cytopathic effect (CPE) 24 hours after transfection. However, a significant delay was observed for EV71-CCA to cause CPE compared with EV71-HP. For example, CPE was observed in 100% of Vero cells 3 days after transfection with the EV71-HP RNA  that underline the distinct replication kinetics of the EV71-HP and EV71-CCA viruses, we swapped the genomic sequences between these two viruses and generated a panel of chimeric viruses . Replica50/ml, P<0.01). Conversely, changing the T672 to A672 in EV71-CCA increased viral titer from the original value of 2.00 –lgTCID50/ml to 6.00 –lgTCID50/ml (P<0.01), a level observed for the EV71-HP virus (A107) has undermined the replication of EV71-CCA.We next aligned the sequences of the P1 protein region between EV71-HP and EV71-CCA, and found three non-conservative amino acid mutations, including leucine (HP)/proline (CCA) at position 329 in capsid protein VP3, alanine (HP)/threonine (CCA) at positions 672 and 854 in capsid protein VP1 . In ordeHP virus . In contHP virus . These rWe next asked which stage of EV71 replication is affected by the A107T mutation in VP1. To this end, equal amounts of full-length RNA of EV71-HP and EV71-CCA were transfected into Vero cells. The neutralizing antibody was then added into cell culture in order to block a second round of viral infection and thus stop viral spread. Viral replication was monitored by measuring the capsid protein VP1 in Western blot . SimilarIn order to identify the defect(s) that have led to the impaired infectivity of the EV71-CCA particles, we first quantified viral genomic RNA and the amount of capsid protein in the EV71-HP and EV71-CCA virions . InteresVP1 capsid protein plays a central role in the attachment of EV71 virions to host receptors , 24. We Next, we further investigated whether the EV71-HP and EV71-CCA viruses were internalized by cells at different rates. Vero cells were inoculated with equal amounts of these two viruses. Following synchronized attachment to cells at 4°C and cell internalization at 37°C, virions that did not enter the cells were removed by digestion with subtilisin. VP1 protein of the internalized virions was analyzed by Western blot . The resEV71 virion uncoating after its entry is a prerequisite for productive infection, enabling the release of viral RNA genome into the cytoplasm. EV71 uncoating involves conformational changes in capsid, generating 3 types of virus particles: the mature virions sedimentated at 160S, the A-particles sedimentated at 135S and the empty viral particles sedimentated at 80S . The firWe noted a considerably higher amount of VP0 protein in the mature virions of the EV71-CCA strain compared to the EV71-HP strain that were sedimentated to fractions 12 to 14 (160S) . The pre107 compared to their parental strains  and HP (A672T), both viruses have shown remarkable change in viral infectivity by a single amino acid substitution at VP1 strains . These t strains . The qua strains . This coEV71 virion uncoating is a prerequisite for enabling the release of viral RNA genome into the cytoplasm. Due to the insufficient VP0 cleavage, the inefficient uncoating of the EV71-CCA strain shall result in delayed genomic RNA release and consequently RNA replication. In agreement with the hypothesis, we found that RNA replication in EV71-CCA was delayed in the early stage of the infection compared with that of EV71-HP . This prA107 is located on the βC of VP1 . VP1-107A is found in virtually all the available sequences of VP1 βC. This high conservation of VP1A107 indicates its important role in EV71 replication.Recent crystal structures  of the eC of VP1 , one of C of VP1 . To deteA107 residue, we substituted this amino acid for other residues with different electrostatic potential and the side chain in both EV71-HP and EV71-CCA strain and examined the replication of these viral mutants. The results in To further investigate the function of the VP1We next investigated the structural mechanism underlying the replication ability of the above VP1 mutated viruses through MD simulations of EV71-HP (A107) and the A107T, A107R, A107D and A107S mutants. In the analysis, the root mean square deviation (RMSD) values of the backbone Cα atoms were calculated and plotted in 50/ml exhibited rapid growth phenotype, including A, S, E and D at VP1107 in both the EV71-HP and EV71-CCA strains. However, substitution of VP1107 for T, K and R resulted in a slow growth phenotype, and these mutants produced lower than 3 of -lgTCID50/ml. These data indicate that viral replication ability is well correlated with viral growth phenotype. Substitution of the VP1107 residue generated significant variations in viral infectious titers and viral growth phenotypes, which provides an opportunity to explore the structural basis for the replication ability of these various mutants. However, the RMSF analysis used above did not reveal much difference for most of substitutional mutants except for A107. As the hydrogen-bond interaction plays a crucial role for the structure and function of protein, we further investigated the hydrogen-bond networks associated with the VP1107 residue for each amino acid substitution. The results in 107 residue, and the data are summarized in 107 residue is required for efficient replication of EV71.In addition to viral infectious titers, we also examined the growth phenotypes of mutant viruses in cultured cells by monitoring the induction of CPE. As shown in Interestingly, D94 is involved in hydrogen-bonding only in the two mutants that exhibited slow growth phenotype. For A107T, the side-chain hydroxyl group of T107 forms strong H-bond with the main chain oxygen atom of D94 with the percentage occupancy of 71% , left. F107 residue is required for efficient VP0 cleavage and virion maturation of EV71.Finally, we examined the VP0 cleavage of these mutants tested above. As shown in A107, regardless of different EV71 lineages, indicates the important function of this residue in EV71 replication.A number of virulence determinants for EV71 have been reported, which may influence virus assembly, attachment, replication, or cell tropism. In this study, we have identified a single amino acid A107 in VP1 as the genetic determinant of viral virulence that functions through regulating viral maturation. This A107 in VP1 is conserved in the 3777 EV71 strains that are available in the GenBank database. This high conservation of VP1A107 impairs the cleavage of capsid precursor protein VP0, which occurs at the final stage of enterovirus assembly, involving an inward radial collapse of the provirion to yield the mature virus particle. Infectivity analysis showed that a single amino acid substitution (A to T) at VP1107 caused slow viral growth and dramatically reduced virus titer (3.50 –lgTCID50/ml), concurrent with impaired VP0 cleavage. This correlation between diminished viral infectivity and impaired cleavage of VP0 among the substitutions of the VP1107 residue further strengthens the important role of VP0 cleavage in viral infectivity [Our mechanistic studies showed that mutation of VP1ectivity .195 result in the assembly of a highly unstable 150S virus particle resembling mature virions except carrying the uncleaved VP0 [107 mutated virus. The defective uncoating consequently prevents release of genomic RNA, resulting in reduction in viral replication. In agreement with this, we found that RNA replication in EV71-CCA was delayed in the early stage of the infection compared with that of EV71-HP  in the conserved, positively charged 5-fold region of the VP1 capsid protein were identified as important sites in EV71 for mouse adaptation and neuropathogenesis in cynomolgus monkey –34. Furt107A is critical for EV71 replication, and is highly conserved among all viral genogroups. The results of mechanistic study suggest that the VP1107 residue modulates the flexibility of the VP1 BC loop, regulates VP0 cleavage and subsequent viral uncoating. Characterization of this novel determinant of EV71 virulence may provide new opportunities for vaccine development and drug discovery.In summary, we have demonstrated that VP1in vitro. Both strains were plaque-purified in Vero cells. Isolated plaques were passaged on RD cells to increase the titer for use in subsequent assays.African green monkey kidney (Vero) cells (ATCC CCL-81) and human rhabdomyosarcoma (RD) cells (ATCC CCL-136) were maintained in Dulbecco’s modified Eagle’s medium. EV71 strain HP (EV71-HP) was isolated from the patient with the nervous system diseases in Fuyang outbreak during 2008. EV71 strain CCA (EV71-CCA) was a cell adapted virus that were generated by series passage Viral RNA was extracted from the culture medium of infected cells with Trizol LS reagent according to the manufacturer’s instructions. cDNA synthesis was performed by reverse transcription PCR as described previously .The full-length cDNA clones of EV71-HP (pEV71-HP) and EV71-CCA (pEV71-CCA) were constructed as described previously . The comcDNA clones of various point mutations were prepared by site-directed mutagenesis via PCR with specific pairs of primers using the infectious clone of pEV71-HP or pEV71-CCA as the template. The sequences of the primers used for site directed mutagenesis are as follows.FY-672(S)+: CTAACCCAAATGGTTATTCCAACTGGGACATAGAC;FY-672(S)-: GTCTATGTCCCAGTTGGAATAACCATTTGGGTTAG;FY-672(E)+: CTAACCCAAATGGTTATGAGAACTGGGACATAGACATA;FY-672(E)-: TATGTCTATGTCCCAGTTCTCATAACCATTTGGGTTAG;FY-672(D)+: CTAACCCAAATGGTTATGACAACTGGGACATAGACAT;FY-672(D)-: ATGTCTATGTCCCAGTTGTCATAACCATTTGGGTTAG;FY-672(T)+: CTAACCCAAATGGTTATACCAACTGGGACATAGAC;FY-672(T)-: GTCTATGTCCCAGTTGGTATAACCATTTGGGTTAG;FY-672(K)+: CTAACCCAAATGGTTATAAGAACTGGGACATAGACATA;FY-672(K)-: TATGTCTATGTCCCAGTTCTTATAACCATTTGGGTTAG;FY-672(R)+: CTAACCCAAATGGTTATAGGAACTGGGACATAGACATA;FY-672(R)-: TATGTCTATGTCCCAGTTCCTATAACCATTTGGGTTAG;ZD-672(A)+: CTAACCCAAATGGTTATGCCAACTGGGACATAGAT;ZD-672(A)-: ATCTATGTCCCAGTTGGCATAACCATTTGGGTTAG;ZD-672(S)+: CTAACCCAAATGGTTATTCCAACTGGGACATAGAT;ZD-672(S)-: ATCTATGTCCCAGTTGGAATAACCATTTGGGTTAG;ZD-672(E)+: CTAACCCAAATGGTTATGAGAACTGGGACATAGATATA;ZD-672(E)-: TATATCTATGTCCCAGTTCTCATAACCATTTGGGTTAG;ZD-672(D)+: CTAACCCAAATGGTTATGACAACTGGGACATAGATAT;ZD-672(D)-: ATATCTATGTCCCAGTTGTCATAACCATTTGGGTTAG;ZD-672(K)+: CTAACCCAAATGGTTATAAGAACTGGGACATAGATATA;ZD-672(K)-: TATATCTATGTCCCAGTTCTTATAACCATTTGGGTTAG;ZD-672(R)+: CTAACCCAAATGGTTATCGCAACTGGGACATAGATA;ZD-672(R)-: TATCTATGTCCCAGTTGCGATAACCATTTGGGTTAG.HindIII to produce linear DNA template, purified by phenol/chloroform extraction, ethanol precipitation and then dissolved in RNase-free water. For in vitro RNA transcription reaction, 1 μg of linear DNA template was transcribed by MEGA script high yield transcription T7 kit in total volume of 20 μl. The in vitro transcripts were then purified by phenol/chloroform and dissolved in RNase-free water. 2 μg transcribed RNA was transfected into 2×105 Vero cells using lipofectamine 2000. Clone-derived viruses were then passaged in RD cells to increase virus titers and stored at -80°C for subsequent experiments.All EV71 cDNA constructs were digested with 50 assay by the Reed and Muench method.Virus supernatants were harvested when above 75% infected cells showed cytopathic effect. Cell debris was removed by centrifugation at 5,000g for 10 minutes, and then the supernatants were filtered through a 0.45 μm filter. The titer of virus stocks was determined using a TCID5 Vero cells per well were seeded in a 6-well plate 24 hours in advance. A series of 1:10 dilutions were made by mixing 25μl of virus sample with 225 μl of DMEM medium. 200 μl of dilutions of viral supernatant were inoculated to individual well of 6-well plate. The plates were incubated at 37°C with 5% CO2 for 1 hour with being shaken every 15 minutes, and then the virus inoculum were replaced with 1 ml of DMEM medium containing 1% agarose. After 7 days of incubation at 37°C with 5% CO2, the agarose layer was covered with 1 ml of 1% agarose containing 0.01% neutral red to stain live cells at 37°C for4 hours in the absence of light. After staining, the number and the diameter of plaques were measured.Virus plaque morphologies were determined by plaque assay as described previously . BrieflyMAB979, a mouse monoclonal antibody that specifically recognizes EV71 VP0/VP2 capsid proteins (Millipore), MAB1255-M08, a mouse monoclonal antibody that specifically recognizes EV71 VP1 capsid protein (Abnova), and anti-β-actin antibody (Sigma-Aldrich) were used as primary antibodies. Infected cells or concentrated virus samples were lysed in loading buffer and resolved by electrophoresis in a 12% SDS-PAGE. Proteins were transferred onto a PVDF membrane, blocked with 5% skim milk, and probed with primary antibodies, including anti-EV71 VP0/VP2 antibody (diluted 1:1000), anti-EV71 VP1 antibody (diluted 1:1000), or anti-β-actin antibody (diluted 1:5000) at 4°C overnight. After incubation, the 1:5000 diluted HRP-conjugated secondary antibody was added, followed by incubation for 2 hours at room temperature. After four wash steps with PBS plus 0.1% Tween 20 (PBST), signals were detected using Western Lighting chemiluminescence reagent (Millipore) and the densitometry of specific blots was calculated using the ImageJ software.in vitro transcribed viral genomic RNAs were transfected into cells using lipofectamine 2000. 2 hours post transfection, the antibody MAB1255-M08  was added into the medium, followed by 48 hours incubation.Vero cells were seeded in 6-well plates 1 day prior to the assay. 2 μg 4 cells per well1 day prior to virus infection. 100 μl of virus dilutions were added to the cells. After 1 hour of incubation at 4°C for simultaneous adsorption, the unbound viruses were removed by three wash steps with PBS. The cells were then fixed in the wells with 4% paraformaldehyde for 20 minutes at room temperature, followed by blocking with 5% skim milk-PBS. The antibody solution  prepared in PBS containing 5% skim milk was added into each test well and incubated for 1 hour at 37°C. After four times washing with PBST, HRP-conjugated anti-mouse IgG secondary antibody (1:5000 dilution) was added into each well and incubated for 40 minutes at room temperature. TMB peroxidase substrate was added and allowed to react for 30 minutes at room temperature. The reactions were stopped by adding 0.5M H2SO4. The absorbance was recorded at OD 450 nm using a microplate reader.The virus binding assay was performed using cell-ELISA. In brief, Vero cells were seeded in 96-well plates at a density of 106 suspended Vero cells in PBS were incubated with 300 μl virus supernatant for attachment at 4°C for 1 hour. After fixation by 1% paraformaldehyde, the cells were washed twice by PBS and re-suspended in 100 μl anti-EV71 VP1 antibody solution . After incubation at 4°C for 1 hour, cells were washed four times by cold PBS containing 2% bovine serum, and then stained with FITC-conjugated anti-mouse IgG secondary antibody diluted at 1:100 and incubated on ice for 1 hour. After three washes with PBS, the stained cells were re-suspended in PBS, and the signals were detected using a Guava easyCyte flow cytometer and analyzed using the Guava ExpressPlus Assay.The ability of EV71 binding to the cells was also determined by flow cytometric analysis. A total of 2×10For viral RNA quantification, 250 μl of the purified virus sample was used for RNA extraction by TRIzol LS (Invitrogen) according to the manufacturer’s instruction. cDNA was synthesized with reverse transcriptase of Moloney murine leukemia virus (Takara), and its level was quantified by real time PCR  using the primers EV71-RT-F (5’-gcagcccaaaagaacttcac-3’) and EV71-RT-R (5’-atttcagcagcttggagtgc-3’) that are located in the capsid protein VP1 gene. The copy number of the viral genome RNA was calculated by generating a standard curve using serial dilutions of plasmid containing DNA sequence for the VP1 gene.EV71 supernatants were purified by ultracentrifugation at 37,000 rpm for 2.5 hours at 4°C in a Beckman SW41Ti rotor through a 15% sucrose cushion. Protein concentrations of the samples were measured using the Bradford assay. Viral genomic RNA copies were determined by real time RT-PCR as described above. Viral genome copy numbers were expressed as copies/ml for each sample, and were then divided by the protein concentration (μg/ml), the final values represent the genome/capsid ratios.GAPDH was used as the internal control to normalize the total RNA of the sample. The negative strand RNA amount were expressed as fold increase relative to that of sample collected at 0.5 hour post infection.Vero cells were seeded in 6-well plates 1 day prior to the assay. The cells were inoculated with equal amount of HP strain or CCA strain, followed by incubation at 4°C for 1 hour. After synchronized viral adsorption, the cells were washed twice with PBS to remove the unbound virions. Then the cells were incubated at 37°C in the medium of DMEM. The virus infected cells were collected by Trizol reagent at indicated time points post infection. For detection of replication of negative strand genomic RNA, the total RNAs of the infected cell samples were extracted and subjected to RT-PCR quantification as described previously. In brief, cDNA was synthesized with reverse transcriptase of Moloney murine leukemia virus (Takara) using the primer 1F (5’-ttaaaacagcctgtgggttg-3’) specific to the negative strand genomic RNA. Then the cDNA of negative strand genomic RNA was quantified by real time PCR  using the primers EV71-RT-F (5’-gcagcccaaaagaacttcac-3’) and EV71-RT-R (5’-atttcagcagcttggagtgc-3’) that are located in the capsid protein VP1 gene. The gene of Vero cells were seeded in 6-well plates 1 day prior to the assay. The cells were inoculated with equal amount of HP strain or CCA strain, followed by incubation at 4°C for 1 hour. After synchronized viral adsorption, the cells were washed twice with PBS to remove the unbound virions. Virus internalization was initiated by incubation at 37°C for 30 minutes in the medium of DMEM containing 2% FBS. 100 μg/ml final concentration of subtilisin A was then added to the medium and incubated at 37°C for 15 minutes to remove the un-internalized virions. After digestion, PMSF was added to a final concentration of 5 mM to inactivate subtilisin A. The cells were lysed and subjected to western blotting analysis for detection of the VP1 protein.EV71 culture supernatant was harvested by three freezing-thawing cycles. The cell debris was removed by passing through a 0.45 μm filter, and then the supernatant was concentrated by centrifugation at 37,000 rpm for 2.5 hours at 4°C in a Beckman SW41 Ti rotor through a 15% sucrose cushion. The concentrated virions were further purified by a 15%-35% discontinuous sucrose gradient ultracentrifugation at 33,000 rpm for 70 minutes. After centrifugation, gradients were fractionated from the top in 0.5 ml aliquots. One half of each fraction was analyzed by RT-qPCR for quantification of viral genomic RNA and the rest was subjected to western blotting to detect viral proteins.van der Waals interactions was set to 10.0 Å, along with a time step of 2.0 fs. Long-range electrostatic interactions are treated with the Particle Mesh Ewald (PME) method. The SHAKE algorithm was applied to constrain all bonds involving hydrogen atoms. All of the MD results were analyzed using the ptraj module of the Amber 11 software package.VP1 protein configuration was extracted from the empty EV71 particle (PDB ID 3VBU). Residues 211–217 were missing in the PDB file. We added this loop before MD simulation. Four mutants  were generated using the Maestro software. A total of 10ns MD simulations were carried out for EV71-HP and mutants of VP1. MD simulations of EV71-HP and all these mutants of VP1 were performed using the Amber11 software package. The amber ff99SB force field was applied for the protein. TIP3P water molecules were utilized to solvate the complex, extending at least 10 Å from the protein. The counter ions (Cl-) were added for charge neutralization. To remove the bad contacts, the system was subjected to energy minimization. Firstly, the water molecules and ions were refined through 2,500 steps of steepest descent followed by 2,500 steps of conjugate gradient, keeping the protein fixed. Secondly, the whole system was relaxed by 10, 000 cycles of minimization procedure with 5,000 cycles of steepest descent and 5,000 cycles of conjugate gradient minimization. After that, the system was heated from 0 to 300 K by 500ps position restraint simulation. 2ns MD simulations without any restraints were sequentially performed to equilibrate the system. Finally, a length of 10 ns trajectory was computed at 300K under constant pressure. During the simulation, the cutoff distance for The purified virus samples were lysed in loading buffer and resolved by electrophoresis in a 12% SDS-PAGE. After electrophoresis, the gel was fixed with 100 ml of the fixing solution (adding 50 ml of ethanol and 10 ml of acetic acid to 40 ml of ultrapure water) in a clean tray for 20 minutes or longer time. Then the gel was washed by 30% ethanol solution and ultrapure water in sequence. After that, the gel was stained with the ProteoSilver Plus Silver Stain Kit (Sigma) according to the manufacturer’s instructions.P value of < 0.05 was considered statistically significant. In all figures, error bars indicate the standard deviations for 3 independent samples. Three asterisks indicate a P value lower than 0.01.Unpaired two-tailed Student’s tests or one-way analysis of variance with the Tukey posttest were conducted to compare results obtained from the different experimental groups. S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigThe protein bands of VP0, VP1, VP2 and VP3 were indicated respectively.(TIF)Click here for additional data file.


A total of 507 ESCC cases and 1,496 controls were enrolled. We found that LEPR rs6588147 AA genotype was associated with ESCC risk . In the stratified analyses, LEPR rs6588147 G>A polymorphism increased the risk of ESCC . However, LEP rs7799039 A>G decreased the risk of ESCC . In addition, LEPR rs1137101 G>A polymorphism decreased ESCC risk in some subgroups . Our findings suggest that LEPR rs6588147 G>A polymorphism is associated with the increased risk of ESCC; however, LEP rs7799039 A>G and LEPR rs1137101 G>A polymorphisms may be protective factors for ESCC.Single nucleotide polymorphisms (SNPs) in energy metabolism related gene may be key agents in the development of human malignancies. In this study, we aimed to examine the association of  In China, esophageal cancer (EC) is the fourth most commonly diagnosed cancer in males and the fifth in females, with an estimated 477,900 new patients and 375,000 related deaths occurring in 2015 [transcription factor 7-like 2 (TCF7L2) gene maps to the long arm of chromosome 10q25.3. TCF7L2 belongs to the high mobility group-box (HMGB) family [et al. reported that TCF7L2 expression was associated with a poor prognosis of ESCC [TCF7L2 rs7903146 locus might exert its enhancer function by interacting with HMGB1 [TCF7L2 single-nucleotide polymorphisms (SNPs) are proposed susceptibility factors for the development of cancer. Previous studies indicated that TCF7L2 rs7903146 (C/T) polymorphism might influence the risk of breast cancer [TCF7L2 rs290481 T>C polymorphism located on near the 3’ end of this gene. Ling et al. reported that this SNP was associated with hepatocellular carcinoma susceptibility with marginal significance [TCF7L2 SNPs and ESCC risk was not explored.The ) family  and is a) family , 7. Ishi of ESCC . A previt cancer . TCF7L2 ificance . HoweverLeptin (LEP) gene maps to chromosome 7q31.3. LEP is secreted by white adipose tissue and has been identified to be involved in endocrinologic metabolism [LEP gene might influence the risk of cancer [LEP rs2167270 G>A was associated with the risk of esophageal adenocarcinoma [LEP polymorphisms. It is suggested that rs7799039 A>G polymorphism in the upstream region of LEP gene can affect leptin expression, possibly at the transcriptional level, thereby altering adipose secretion levels of the hormone [LEP rs2167270 G>A is a 5’-utr SNP and may play regulatory roles in translation and stability of mRNA. LEPR rs1137100 G>A, rs1137101 G>A polymorphisms are missense SNPs and may alter the structure and the function of LEPR protein. Doecke et al. found LEPR rs1137100 G>A, rs1137101 G>A polymorphisms influence the risk of esophageal adenocarcinoma in Caucasians [LEPR rs6588147 G>A polymorphism locates on the intron region of LEPR gene. Slattery et al. found that LEPR rs6588147 G>A polymorphism affected risk of colon cancer among men [LEPR rs1137100 G>A, rs1137101 G>A and rs6588147 G>A polymorphisms and ESCC risk remains unknown in Asians.The tabolism . It is ttabolism , type 2 tabolism  and pathtabolism , 17. LEPtabolism . LEP comtabolism –22. Thusf cancer –25. In aarcinoma . There aucasians . LEPR rsmong men . HoweverTCF7L2, LEP and LEPR polymorphisms with the risk of ESCC in Eastern Chinese Han populations. The TCF7L2 rs7903146 C>T, rs290481 T>C, LEP rs7799039 A>G, rs2167270 G>A and LEPR rs1137100 G>A, rs1137101 G>A and rs6588147 G>A polymorphisms were genotyped by SNPscan genotyping assays in 507 ESCC cases and 1,496 non-cancer controls.In this case-control study, we aimed to examine the potential association of P < 0.05). However, the mean ± SD of height was not significant (P > 0.05). The proportion of smoking and drinking was significantly higher in ESCC patients compared with controls (P < 0.05). Locus information of TCF7L2, LEP and LEPR polymorphisms is listed in Table TCF7L2 rs7903146C>T, rs290481 T>C, LEP rs7799039 A>G, rs2167270 G>A and LEPR rs1137100 G>A, rs1137101 G>A and rs6588147 G>A SNPs were 99.50%,99.45%, 99.50%, 99.40%, 99.50%, 99.50% and 99.50%, respectively. Minor allele frequency (MAF) in controls is listed in Table TCF7L2 rs7903146C>T, rs290481 T>C, LEP rs7799039 A>G, rs2167270 G>A and LEPR rs1137100 G>A, rs1137101 G>A and rs6588147 G>A genotypes in controls conform to Hardy-Weinberg equilibrium (HWE).There were 2,003 participants in the present case-control study including 507 ESCC patients  and 1,496 non-cancer controls . The age and sex were well matched in two groups  = 1.87, 95% confidence interval (CI) = 1.02–3.43, P = 0.042 and AA vs. GG/GA: crude OR = 1.93, 95% CI = 1.06–3.53, P = 0.031 .In addition, after a logistic regression analysis, we found that TCF7L2, LEP and LEPR) polymorphisms and focused on their susceptibility to ESCC. The association between LEPR rs6588147 G>A polymorphism and the increased risk of overall ESCC was identified. We also found that LEPR rs6588147 G>A polymorphism increased the risk of ESCC in <63 years and male subgroups. LEP rs7799039 A>G was associated with the risk of ESCC in ≥63 years and BMI ≥ 24 kg/m2 subgroups. In addition, LEPR rs1137101 G>A polymorphism decreased the risk of ESCC in ever smoking and ever drinking subgroups.The pathogenesis of ESCC was very complex. Multiple factors  may contribute to the etiology of ESCC. Understanding of the individual’s heredity background may be helpful for the prevention and treatment of ESCC. In this study, we selected energy metabolism and insulin-sensibility relative gene . When each subject was interviewed, a questionnaire was used to obtain demographic variables and risk factors. And weight and height were also measured. In this study, a BMI ≥ 24 was considered as the criteria for obesity and overweight .TCF7L2 rs7903146C>T, rs290481 T>C, LEP rs7799039 A>G, rs2167270 G>A and LEPR rs1137100 G>A, rs1137101 G>A and rs6588147 G>A genotypes were assessed by the SNPscan™ kit , which is a double ligation and multiplex fluorescence PCR [TCF7L2, LEP and LEPR polymorphisms were confirmed.Genomic DNA was carefully isolated from EDTA-anticoagulated blood of recipients by using a Promega DNA blood mini kit . ence PCR . For quaTCF7L2, LEP and LEPR genotypes, sex, age, BMI, smoking and drinking status) were compared with Chi-square test (χ2). Deviations from the HWE for TCF7L2, LEP and LEPR genotypes distribution in controls were evaluated by an internet-based calculator (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl) [TCF7L2 rs7903146C>T, rs290481 T>C, LEP rs7799039 A>G, rs2167270 G>A and LEPR rs1137100 G>A, rs1137101 G>A and rs6588147 G>A polymorphisms with ESCC susceptibility were evaluated by crude ORs and 95% CIs. Multivariate linear regression adjusted for age, sex, BMI, alcohol use and smoking status was used to determine the relationships between TCF7L2 rs7903146C>T, rs290481 T>C, LEP rs7799039 A>G, rs2167270 G>A and LEPR rs1137100 G>A, rs1137101 G>A and rs6588147 G>A polymorphisms and ESCC risk with quantitative traits. Data analysis was conducted with SAS software for windows . A P < 0.05 (two-tailed) was accepted as the criterion of statistical significance.Continuous variables  are expressed as mean ±standard deviation (SD). Comparisons between ESCC patients and controls were carried out with Student’s t-test. The categorical variables (e.g. hwa1.pl) –49. The


More than 70% of the patients in each disease group had total vitamin D < 20 ng/mL (P < 0.001). In CRVD patients, CD3 and CD19 correlated (P < 0.05) with 25(OH)D3. In CAD patients, CD8, CD4, CD19 and CD4/CD8 correlated (P < 0.05) with 25(OH)D2, and CD8 correlated (P < 0.05) with 25(OH)D3. In T2DM and hypertension patients, CD8, CD3, CD19 and CD4/CD8 correlated with 25(OH)D3. Progressive trends (P < 0.05) towards increased CD8 and CD4/CD8 were observed in vitamin-D-deficient T2DM and hypertension patients. Significant differences (P < 0.05) in CD8 were observed in vitamin-D-deficient CAD patients, whereas significant differences (P < 0.05) in CD8 and CD19 were observed in CRVD patients. Higher CD8 and CD4/CD8 in 25(OH)D-deficient T2DM and hypertension patients suggested a Th1 lymphocyte profile induction. Increases in CD8-positive lymphocytes suggested a similar, less pronounced effect in vitamin-D-deficient CRVD and CAD patients.Hypovitaminosis D is associated with age-related illnesses, including hypertension, cardiovascular disease (CRVD), cerebrovascular disease (CAD) and type 2 diabetes mellitus (T2DM). In our retrospective observational study, blood samples of elderly healthy controls (n = 461) and patients with age-related diseases  were subjected to flow-cytometry in order to determine correlations between age-related diseases and cluster of differentiation 4 (CD4), CD8, CD3, and CD19 lymphocyte markers, as well as serum levels of 25-hydroxyvitamin D Vitamin D is known to function in a wide range of homeostatic processes including calcium absorption and bone remodeling2. The major vitamin D metabolites in serum are 25-hydroxyvitamin D2 (25(OH)D2) and 25-hydroxyvitamin D3 (25(OH)D3), with the serum level of 25(OH)D3 most often much higher than the level of 25(OH)D24. The dermal 25(OH)D3 conversion after exposure to solar radiation serves as the major source of vitamin D5, whereas 25(OH)D2 is present in relatively few foods including egg yolk, fish and mushrooms and is used to fortify mainly milk, orange juice and cereals. Recent research has revealed alternative vitamin 3 pathways leading to various (OH)D3 products other than 25(OH)D3 in skin, placenta and porcine adrenal gland8.Vitamin D refers to a group of lipid-soluble compounds that act as precursors for the production of the biologically active secosteroid, 1,25-dihydroxyvitamin D, which is primarily produced in the kidney9 and observational studies based on 25(OH)D concentrations revealed beneficial effects for patients with a variety of illnesses11 including metabolic syndrome (MetS)13, obesity14, certain cancers16, a number of autoimmune disorders17, hypertension19, cardiovascular disease (CVD)20, and T2DM22. However, whether vitamin D deficiency contributes to the pathology of these diseases remains largely unclear.Vitamin D deficiency is a global pandemic24, with women often exhibiting vitamin D deficiency at younger ages than men25. Serum markers of both innate and adaptive immunity are associated with the vitamin D status27. It has been demonstrated that 1,25(OH)2D3, the most biological active metabolite of vitamin D, acts on T and B lymphocytes to modulate both the cytotoxic and antibody-producing functions of lymphocytes29. Previous studies have shown that 1,25(OH)2D3 synthesis could activate innate immune responses, while suppressing adaptive immune responses31. A decreased CD4+/CD8+ ratio, resulting from reduced CD4+, was reported in patients with acquired immune deficiency syndrome (AIDS), suggesting CD4+/CD8+ reversion was an important indicator of immunodeficiency diseases and viral infection32. Similarly, T cell variations have been correlated with cancer outcomes34 and T2DM35.Investigations worldwide have shown that vitamin D levels wane in the elderly39, a set of conditions associated with chronic diseases that are highly prevalent in the elderly, including CVD, neurodegenerative diseases and glucose metabolism disorders41. Animal model and cell culture studies have demonstrated beneficial immunomodulatory effects of vitamin D, which is thought to induce an immunological transition from a Th1/Th17 response to a Th2/Treg response based on the cytokine profiles of immune cell types measured following vitamin D treatment42.Vitamin D deficient elderly persons frequently exhibit a progressive increase in serum levels of acute-phase reaction proteins and proinflammatory cytokines37. Blockade of the IL-6 receptor with tocilizumab has been shown to suppress Th1 and Th17 signaling and stimulate beneficial Th2 responses in patients with early-stage rheumatoid arthritis43, and suppression of Th17 signaling with the vitamin D receptor agonist, elocalcitol, was shown to reduce autoimmunity in non-obese diabetic mice44. Studies of immune cells have also shown that treatment with 1,25(OH)2D3 reduced the expression of IL-6 and various other proinflammatory factors45. Given the high prevalence of elevated serum IL-6 in vitamin D deficient elderly persons, we retrospectively analyzed the records of a large population of elderly Chinese people to investigate whether the immune marker profiles of lymphocyte subsets correlated with vitamin D levels in patients with illnesses more common diagnosed in the elderly such as T2DM, cerebrovascular disease (CRVD), coronary artery disease (CAD), hypertension and cancer46.Observational studies of elderly people in Italy and Ireland found that the serum level of IL-6 was inversely associated with the vitamin D statusWe analyzed the records of patients’ ≥40 years of age who were treated for various conditions at the Hospital of Shanghai Zhongshan-Xuhui between May 2012 and August 2016. Patients with hepatic failure, serum creatinine >120 µmol/L, hyperthyroidism, hypothyroidism, immune disorders or infection were excluded from our study. Patients receiving hormone medication were also excluded from our analysis. A total of 461 healthy volunteers (control subjects) were also enrolled in our study. Informed written consent was obtained from all of the patients and control subjects prior to their participation in our study. The study was approved by the Institutional Review Board of ZhongShan-Xuhui Hospital, and was carried out in accordance with the principles of the Declaration of Helsinki for ethical research involving human subjects.2  and deuterated 25(OH)D3   as internal standards for 25(OH)D2 and 25(OH)D3, respectively. The analysis was performed using an API 4000 LC-MS/MS system  equipped with a Shimadzu liquid chromatograph . The accuracy of low, medium, and high concentration controls was 85–115%, and the precision (CV) was <15%. The inter- and intra-assay coefficients of variation were <10%. Analyst 1.5 software  was used for data collection and quantitative analysis.Serum samples collected from patients and healthy volunteers were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) using deuterated 25(OH)D47. Cells from whole blood samples were subjected to flow cytometry analysis to determine the proportions of lymphocyte subsets in peripheral venous blood within 2 h of collection, based on binding of the following monoclonal antibodies (mAbs): Anti-CD4, anti-CD8, anti-CD3, anti-CD56 and anti-CD19 , The stained cells were sorted using a FACS Aria flow cytometer (BD Biosciences) using appropriate isotype controls for gating, and the data were analyzed using FlowJo software .CD3 represents the total T cell number (thymus dependent lymphocyte) and CD19 is a cell surface receptor complex component that regulates B lymphocyte responsesP < 0.05 indicating a statistically significant difference between the two groups.All statistical analyses were performed using SPSS, version 18.0 . Continuous variables are presented as the median ± interquartile range (IQR). Differences between patients with different diseases were compared using variance analysis. Categorical variables were summarized as a percentage of the whole and a chi-squared test was used to detect differences. Pearson correlation analysis was used to evaluate the correlations between indicators in two different groups, with Demographic and clinical data for the patients and control subjects are presented in Table 48, which is consistent with the findings of previous studies of the prevalence of vitamin D deficiency in the elderly50. The 25(OH)D3 and 25(OH)D2 serum concentrations of healthy control subjects differed significantly compared to some patients , the latter of which was likely due to the regular use of vitamin D dietary supplements. Total serum vitamin D in the healthy control group was slightly below the reference range lower limitts Table .2 and 25(OH)D3 in the various disease groups using correlation analysis, and the results of the analysis are presented in Table 3 and positive correlations between CD19 and 25(OH)D3 and CD56 and 25(OH)D2. Given that 25(OH)D3 is the greater contributor to vitamin D status, these results suggest that vitamin D deficiency might correlate with a shift from humoral immunity to cell mediated immunity in CRVD patients. However, the lack of significant correlations for CD4, CD8, and CD4/CD8 in CRVD patients as well as the missing significance of total 25(OH)D clearly limit such an interpretation.We investigated the relationships between lymphocyte immune markers and the serum levels of 25(OH)D2 and significant negative correlations between CD8 and 25(OH)D3, CD4 and 25(OH)D2, CD19 and 25(OH)D2 and CD4/CD8 and 25(OH)D2. However, reduced 25(OH)D2 significantly correlating with increased CD4, CD19, and CD4/CD8 as well as missing significance of total 25(OH)D, limits the interpretation of these results in CRVD patients.In patients with CAD, we observed a significant positive correlation between CD8 and 25(OH)D3 negatively correlated with CD8 and CD3, and positively correlated with CD19 and CD4/CD8, which also was visible for total 25(OH)D. These results suggest that vitamin D deficiency correlates with the induction of Th1 immunity in T2DM patients, and that patients with T2DM might benefit from 25(OH)D3 dietary supplements.In T2DM patients, the level of 25(OH)D3 and total 25(OH)D negatively correlated with CD8 and CD3 and positively correlated with CD19 and CD4/CD8, a finding which suggests that patients with hypertension might also benefit from 25(OH)D3 dietary supplementation.In patients with hypertension, 25(OH)D2 and 25(OH)D3 with lymphocyte subsets, the results for kidney disease, digestive disease and fracture/osteoporosis groups were insufficient to draw any reliable inference of the effects of serum levels of vitamin D metabolites on these diseases.Though there were some correlations between 25(OH)D2 and 25(OH)D3 levels in the CRVD, CAD, T2DM and hypertension groups, the results of which are presented in Table 3 levels , and into 3 categories based on the serum level of 25(OH)D2 . Significant differences were observed in the number of patients in the various 25(OH)D3 and 25(OH)D2 categories for all of the disease groups (Table 3 and <1 ng/mL 25(OH)D2 categories for all of the CRVD, CAD, T2DM and hypertension groups. Overall, these results are consistent with the findings of previous studies regarding the high prevalence of vitamin D deficiency in elderly persons50. These results suggest that either vitamin D deficiency contributes to the pathology of CRVD, CAD, T2DM and hypertension or alternatively that these diseases modify the maintenance of serum vitamin D levels in a similar manner.Based on our findings regarding correlations between the levels of vitamin D metabolites and lymphocyte subsets, we performed a stratified analysis of the relationship between immune cell markers and vitamin D in patients with the most prevalent age-related diseases represented in our study. To this end, we first performed a stratified analysis of 25(OH)D2 and 25(OH)D3 concentrations established in the previous section.To complete our study, we performed an analysis of immune cell markers and serum vitamin D levels in CRVD, CAD, T2DM and hypertension patients, that were stratified according to the categories of 25(OH)D3 concentrations, whereas only the distribution of CD4-positive cells was significantly different across the various categories of serum 25(OH)D2 levels. The higher proportion of CD8-positive cells in 25(OH)D-deficient CRVD patients and the higher proportion of CD19-positive cells in CRVD patients with 25(OH)D3 levels >20 ng/mL suggests that low serum vitamin D promotes a Th1 immune marker profile in CRVD patients.As shown in Table 2 levels Dls Table . In T2DMes Table , with th3 concentrations (Table 3-deficient patients (<20 ng/mL), and a greater proportion of CD4-positive cells observed in patients with serum 25(OH)D3 >20 ng/mL. This finding is supported by the trends in CD3 and CD19, which suggest a transition from B-cell-mediated immunity to T-cell-mediated immunity, with progressively lower serum levels of 25(OH)D3. The percentage of CD3-positive and CD56-positive cells also differed significantly across the 25(OH)D2 concentration categories, but the lack of clear trends in CD3 and CD56 with regard to serum 25(OH)D2 concentration confounded our estimation of the effects of serum 25(OH)D2 levels on lymphocyte immune markers in patients with hypertension. Overall, these data suggested that patients with hypertension might benefit from dietary supplementation with 25(OH)D3.In patients with hypertension, the percentage of cells with CD4, CD8, CD3 and CD19 as well as the CD4/CD8 ratio differed significantly across the various categories of serum 25(OH)D49, especially elderly women52. Previous studies have shown that a vitamin D deficiency is a risk factor for a number of immunity-related pathologies including autoimmune diseases53 and graft rejection55. Cell culture studies have shown that 1,25(OH)2D3 suppressed T cell proliferation, causing a shift in the T cell phenotype from Th1 to Th256. Immunological studies in mice with experimentally-induced colitis showed that treatment with 1,25(OH)2D3 suppressed Th1 and Th17 cells and induced the differentiation of Th2 and Treg cells58. Studies on experimental autoimmune encephalomyelitis in mice showed that 1,25(OH)2D3 induced Treg cells61 and modulated the induction of Th17 T-cells by inhibiting the actions of IL-1761.Hypovitaminosis D is a global pandemic and is highly prevalent in older persons53, end-stage liver disease62, T2DM63, cancer53, obesity9 and CVD65, all of which are more prevalent in the elderly. However, whether vitamin D deficiency contributes to the pathology or progression of these diseases remains largely unclear11. Treatment with 1,25(OH)2D3 preferentially inhibits Th1 lymphocytes and induces Th2 cells68, as well as Treg lymphocytes in the presence of IL-269. Studies of pathogen-induced immunity suggest that the immune status of leukocytes in vivo is primarily driven by the local activation of vitamin D70. Therefore, the findings of in vitro studies using 1,25(OH)2D3 may not provide a completely accurate representation of the role of vitamin D in T-cell activation in vivo.Hypovitaminosis D is also associated with a number of unrelated diseases including osteoporosis71, and the primary effect of vitamin D treatment involves changes in gene expression70. Age has been shown to negatively impact T-cell clonal expansion and the repertoire diversity that can impair T-cell-mediated immune responses73. Studies of soluble proinflammatory factors and serum markers of oxidative stress have implicated low-level inflammation in the pathology of MetS, CVD74, T2DM75, obesity76, and hypertension77. Like hypovitaminosis D, these diseases are highly prevalent in the elderly46. Two recent studies of elderly persons in Europe with37 and without hypertension78 found that vitamin D deficiency was associated with an elevated serum level of the proinflammatory cytokine IL-6. However, the link between age-related changes in T-cell physiology and coinciding changes in the serum cytokine profiles of elderly people is unclear, and comprehensive studies of lymphocyte immune markers in patients with age-related diseases are scarce.Among human leukocytes, CD8-positive lymphocytes have a relatively high density of vitamin D receptors80, serum levels of 25(OH)D3 and 25(OH)D2 were significantly correlated with three or more immune markers in CRVD, CAD, T2DM and hypertension patients (Table 3 and 25(OH)D2 that were ≤20 ng/mL and ≤2 ng/mL, respectively D3 and 25(OH)D2.There are, however, certain limitations to our findings that should be considered in the interpretation of our results. Although the sample sizes of the CRVD, CAD, T2DM and hypertension groups were relatively large, our healthy control group was substantially smaller, and our observational analysis was limited to outpatient data recorded at a single hospital. In addition, we did not examine serum cytokine profiles in our patients, which could have provided further information regarding the Th2 to Th1 transition represented by the trends in CD8-positive cells and the CD4/CD8 ratio. Furthermore, we did not exclude patients receiving vitamin D dietary supplements or those whose lifestyle or dietary practices might have influenced their vitamin D status, all of which might have partially masked trends in the number of vitamin D-deficient patients in each group and the percentages of lymphocyte immune markers in the stratified analyses. In addition seasonal 25(OH)DIn summary, we found a number of correlations between lymphocyte immune markers and vitamin D deficiency in middle-aged and elderly CRVD, CAD, T2DM and hypertension patients Fig. , but fut


Escherichia coli bacteriophage UFV-AREG1. This phage was isolated from cowshed wastewater and showed specificity for enterohemorrhagic E. coli O157:H7 (ATCC 43895), E. coli 0111 (CDC O11ab) and E. coli (ATCC 23229).Here, we present the genome sequence of the  In general, bacteriophages show great diversity, morphology, biochemistry, and genetic content . The stuEscherichia coli O157:H7 (CDC EDL-933), E. coli 0111 (CDC O11ab), and E. coli (ATCC 23229). Bacteriophage UFV-AREG1 can reach concentrations of over 1012 PFU/ml in laboratory conditions when the host is E. coli O157:H7. Transmission electron microscopy showed a phage with an icosahedral head of 114 by 86 nm and a contractile tail of 117 by 23 nm. These parameters allow the classification of UFV-AREG1 as a member of the Myoviridae family (The bacteriophage UFV-AREG1 was isolated from cowshed wastewater showing specificity to Escherichia phage HY01 (KF925357) as the reference. Genes were predicted from the genome using Prodigal version 2.50 (http://blast.ncbi.nlm.nih.gov) against the GenBank and UniProt databases.The genome of UFV-AREG1 was sequenced using Illumina HiSeq by Macrogen , yielding a total of 10,319,880 paired-end reads 100 bp in length. The sequenced reads were trimmed for quality (minimum Q30 score), and the genome sequence was assembled with a coverage of 5,600× using Geneious version 9.1.2 (Biomatters) with the genome of Escherichia phages HY01 and PEC4 (KR233165), sharing 91.8% and 91.5% nucleotide sequence identities, respectively. The 274 predicted proteins show high similarity to previously described enteric phages and other T4-like phages. As a T4-like phage, the genome of UFV-AREG1 was also opened to the rIIa gene. The BLASTx searches showed that the genome of UFV-AREG1 differs from the Enterobacteria T4 (NC_000866) phage in 30 ORFs. Most of the proteins encoded by these ORFs do not have putative conserved domains. Among these proteins, four annotated ones in UFV-AREG1 related to the T4 phage were found: one receptor recognition protein (ORF 244), one transferase protein (ORF 42), and two structural proteins (ORFs 29 and 121).The UFV-AREG1 genome comprises 170,788 bp of a linear dsDNA with a density of 1.60 gene/Kb and a G+C content of 35.3%, which is within the range observed in T4-like phages (35.0% to 43.0%). UFV-AREG1 is more similar to E. coli phages and their genetic diversity.The best understanding of the gene and proteins encoded by the UFV-AREG1 bacteriophage can provide new information about KX009778.The genome sequence of UFV-AREG1 is available in GenBank under the accession number