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The presence of SARS-CoV-2 mutants, including the emerging variant B.1.1.7, has raised great concerns in terms of pathogenesis, transmission, and immune escape. Characterizing SARS-CoV-2 mutations, evolution, and effects on infectivity and pathogenicity is crucial to the design of antibody therapies and surveillance strategies. Here we analyzed 454,443 SARS-CoV-2 spike genes/proteins and 14,427 whole-genome sequences. We demonstrated that the early variant B.1.1.7 may not have evolved spontaneously in the United Kingdom or within human populations. Our extensive analyses suggested that Canidae, Mustelidae or Felidae, especially the Canidae family (for example, dog) could be a possible host of the direct progenitor of variant B.1.1.7. An alternative hypothesis is that the variant was simply yet to be sampled. Notably, the SARS-CoV-2 whole genome represents a large number of potential co-mutations with very strong statistical significances (p value
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Objective:To study the protective effect of glucagon-like peptide-1 (GLP-1) on the inflammatory damage induced by high glucose(HG) in placental trophoblasts HTR8/SVneo and its molecular mechanism.Methods:Trophoblasts HTR8/SVneo cells were cultured and divided into low glucose(LG) group treated with low glucose (5 mmol/L), HG group treated with high glucose (25 mmol/L), GLP-1 group treated with high glucose combined with GLP-1, miR-137+ GLP-1 group treated with high glucose combined with GLP-1 after the transfection of miR-137 mimic, miR-137 mimic group transfected with miR-137 mimic, negative control (NC) mimic group transfected with NC mimic, and miR-137 inhibitor group transfected with miR-137 inhibitor, NC inhibitor group transfected with NC inhibitor. Apoptotic rate, expression of miR-137 and IL-6 were measured.Results:The apoptotic rate and the expression levels of miR-137 and IL-6 in HG group were significantly higher than those in LG group. The apoptotic rate and the expression levels of miR-137 and IL-6 in GLP-1 group were significantly lower than those in HG group. The apoptotic rate and the expression levels of miR-137 and IL-6 in miR-137+ GLP-1 group were significantly higher than those in GLP-1 group. The apoptotic rate and the expression level of IL-6 in miR-137 mimic group were significantly higher than those of NC mimic group, the apoptotic rate and the expression level of IL-6 in miR-137 inhibitor group were significantly lower than those in the NC inhibitor group.Conclusion:GLP-1 is able to alleviate the inflammation injury of HTR8/SVneo induced by high glucose through the miR-137/IL-6 pathway.
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Vaccines and antiviral agents are in urgent need to stop the COVID-19 pandemic. To facilitate antiviral screening against SARS-CoV-2 without requirement for high biosafety level facility, we developed a bacterial artificial chromosome (BAC)-vectored replicon of SARS-CoV-2, nCoV-SH01 strain, in which secreted Gaussia luciferase (sGluc) was encoded in viral subgenomic mRNA as a reporter gene. The replicon was devoid of structural genes spike (S), membrane (M), and envelope (E). Upon transfection, the replicon RNA replicated in various cell lines, and was sensitive to interferon alpha (IFN-), remdesivir, but was resistant to hepatitis C virus inhibitors daclatasvir and sofosbuvir. Replication of the replicon was also sensitive overexpression of zinc-finger antiviral protein (ZAP). We also constructed a four-plasmid in-vitro ligation system that is compatible with the BAC system, which makes it easy to introduce desired mutations into the assembly plasmids for in-vitro ligation. This replicon system would be helpful for performing antiviral screening and dissecting virus-host interactions.
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The SARS-CoV-2 infected cases and the caused mortalities have been surging since the COVID-19 pandemic. Viral mutations emerge during the virus circulating in the population, which is shaping the viral infectivity and pathogenicity. Here we extensively analyzed 6698 SARS-CoV-2 whole genome sequences with specific sample collection dates in NCBI database. We found that four mutations, i.e., 5UTR_c-241-t, NSP3_c-3037-t, NSP12_c-14408-t, and S_a-23403-g, became the dominant variants and each of them represented nearly 100% of all virus sequences since the middle May, 2020. Notably, we found that co-occurrence rates of three significant multi-site co-mutational patterns, i.e., (i) S_a-23403-g, NSP12_c-14408-t, 5UTR_c-241-t, NSP3_c-3037-t, and ORF3a_c-25563-t; (ii) ORF8_t-28144-c, NSP4_c-8782-t, NSP14_c-18060-t, NSP13_a-17858-g, and NSP13_c-17747-t; and (iii) N_g-28881-a, N_g-28882-a, and N_g-28883-c, reached 66%, 90%, and nearly 100% of recent sequences, respectively. Moreover, we found significant decrease of CpG dinucleotide at positions 241(c)-242(g) in the 5UTR during the evolution, which was verified as a potential target of human zinc finger antiviral protein (ZAP). The four dominant mutations, three significant multi-site co-mutations, and the potential escape mutation of ZAP-target in 5UTR region contribute to the rapid evolution of SARS-CoV-2 virus in the population, thus shaping the viral infectivity and pathogenicity. This study provides valuable clues and frameworks to dissect the viral replication and virus-host interactions for designing effective therapeutics. One Sentence SummaryFour dominant mutations, three significant multi-site co-mutations, and 5UTR CpG escape contribute to the rapid evolution of SARS-CoV-2 virus.
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Pandemic SARS-CoV-2 has infected over 10 million people and caused over 500,000 mortalities. Vaccine development is in urgent need to stop the pandemic. Despite great progresses on SARS-CoV-2 vaccine development, the efficacy of the vaccines remains to be determined. Deciphering the interactions of the viral epitopes with their elicited neutralizing antibodies in the convalescent COVID-19 population inspires the vaccine development. In this study, we devised a peptide array composed of 20-mer overlapped peptides of spike (S), membrane (M) and envelope (E) proteins, and performed a screening with 120 COVID-19 convalescent serums and 24 non-COVID-19 serums. We identified five SARS-CoV-2-specific dominant epitopes that reacted with above 40% COVID-19 convalescent serums. Epitopes in the receptor-binding domain (RBD) of S ill reacted with the convalescent serums. Of note, two peptides non-specifically interacted with most of the non-COVID-19 serums. Neutralization assay indicated that only five serums completely blocked viral infection at the dilution of 1:200. By using a peptide-compete neutralizing assay, we found that three dominant epitopes partially competed the neutralization activity of several convalescent serums, suggesting antibodies elicited by these epitopes played an important role in neutralizing viral infection. The epitopes we identified in this study may serve as vaccine candidates to elicit neutralizing antibodies in most vaccinated people or specific antigens for SARS-CoV-2 diagnosis.
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Objective To investigate the effects of glucagon like peptide 1 (GLP-1) agonist on myocardial hypoxia reoxygenation injury and its molecular mechanism. Methods H9C2 cells were divided into control group, hypoxia reoxygenation(H/ R) group, H/ R+GLP-1 group, and H/ R+GLP-1+phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 group. The cell proliferation activity, apoptosis rate, enzyme contents in the medium and the expressions of apoptosis-related genes were detected. After animal model of myocardial ischemia reperfusion injury (I/ R) was established and was treated with GLP-1 agonist and PI3K inhibitor, serum enzyme contents were detected. Results Hypoxia reoxygenation decreased the myocardial cell proliferation activity and phosphorylated-PI3K(p-PI3K), phosphorylated-protein kinase B (p-Akt), Bcl-2 protein expressions, increased the apoptotic cell number and creatine kinase ( CK), creatine kinase-MB ( CK-MB), lactate dehydrogenase ( LDH) contents in cell culture medium and Bax, caspase-3 protein expressions, which were ameliorated by GLP-1 ( all P < 0. 05). The myocardial cell proliferation activity and Bcl-2 protein expression of H/ R+GLP-1+LY294002 group were significantly lower than those of H/ R+GLP-1 group while the apoptotic cell number and CK, CK-MB, LDH contents in cell culture medium and Bax, Caspase-3 protein expressions were significantly higher (all P<0. 05). Serum CK, CK-MB, and LDH contents in rats of I/ R group were significantly higher than those in control group and I/ R+GLP-1 group. Serum CK, CK-MB, and LDH contents in rats of I/ R+GLP-1+LY294002 group were significantly higher than those in I/ R+GLP-1 group(all P < 0. 05). Conclusion GLP-1 agonist is able to protect the myocardial hypoxia reoxygenation injury via activating PI3K/ Akt signaling pathway.
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Objective:To investigate the extracellular signal-regulated kinase 5 (ERK5) and matrix metallo proteinase-9 (MMP-9) expres-sion levels in osteosarcoma tissues and their clinical significance. Methods:The ERK5 and MMP-9 expression levels in 71 specimens of osteosarcoma tissue and 40 specimens of normal bone tissue were detected by immunohistochemistry. The relationship between ERK5 and MMP-9 expression levels, their clinical characteristics, and prognosis of patients with osteosarcoma were analyzed. Results:The positive expression of ERK5 and MMP-9 in osteosarcoma tissues was 85.9%(61/71) and 74.65%(53/71), respectively, which were significantly higher than those in normal bone tissues at 12.5%(5/40) and 10.0%(4/40) (all P<0.05). The positive expression of ERK5 and MMP-9 was associated with Enneking stage and metastasis (all P<0.05). Kaplan-Meier analysis showed that the survival duration of patients with positive ERK5 and MMP-9 expression levels was shorter than those of the patients in the negative expression groups (all P<0.05). Univariate analysis of COX proportional hazards regression model revealed that tumor size, Enneking stage, metastasis, and positive ERK5 and MMP-9 expression levels are relevant to the overall survival of patients with osteosarcoma (all P<0.05). Multi-variate analysis of COX proportional hazards regression model confirmed that Enneking stage, metastasis, and positive ERK5 and MMP-9 expression levels can act as independent prognostic factors for osteosarcoma patients (all P<0.05). Conclusion:The ERK5 and MMP-9 expression levels are high in osteosarcoma tissues and are related to the clinical characteristics and prognosis of patients with osteo-sarcoma. Thus, ERK5 and MMP-9 expression levels may play important roles in osteosarcoma development and progression.
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To study the effect of glucagon-likepeptide 1(GLP-1)receptor agonist on insulin resistance and hepatic oxidative stress in rats with diabetes mellitus combined with nonalcoholic fatty liver disease. 36 male SD rats were served as the experimental animal and randomly divided into control group, model group, and GLP-1 group. The rats of control group were given routine diet with intraperitoneal injection of normal saline, those in model group were given high fat diet and intraperitoneal injection of normal saline, while GLP-1 group rats were fed with high fat diet and intraperitoneal injection of liraglutide. After 4 weeks of treatment, insulin resistance, lipid metabolism, liver injury and oxidative stress were all assessed. Serum fasting blood glucose, fasting insulin, total cholesterol, triglyceride, alanine transaminase(ALT), aspartate transaminase(AST)levels and total cholesterol, triglyceride contents in liver tissue, and as well as homeostasis model assessment for insulin resistance(HOMA-IR)levels of model group were significantly higher than those of control group, complex insulin sensitivity index(ISIcomp)level was significantly lower than that of control group; serum fasting blood glucose, fasting insulin, total cholesterol, triglyceride, ALT, AST contents and HOMA-IR levels of GLP-1 group were significantly lower than those of model group, ISIcomp level was significantly higher than that of model group; superoxide dismutase(SOD), glutathione peroxidase(GSH-Px), catalase(CAT)contents in liver tissue of model group were significantly lower, while malondialdehyde content and SOD, GSH-Px, CAT, NF-E2 related factor-2(Nrf-2), antioxidant response element(ARE), heme oxygenase-1(HO-1), quinone oxidoreductase-1(NQO-1), glutathione thiol transferase(GST)mRNA expression were significantly higher than control group; SOD, GSH-Px, CAT contents and SOD, GSH-Px, CAT, Nrf-2, ARE, HO-1, NQO-1, GST mRNA expression in the liver tissue of GLP-1 group were significantly higher, while malondialdehyde content was significantly lower than that of model group. GLP-1 receptor agonist reduces insulin resistance and liver oxidative stress injury in diabetic rats with nonalcoholic liver disease.
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Wnt/β-catenin signaling pathway participates in cancer cell proliferation,invasion and metastasis and effectively induces drug resistance.It is also the key signal to mediate cancer carcinogenesis.Recent studies in vitro indicate that disturbance of Wnt/β-catenin signaling pathway can increase the sensitivity of the cancer cells to chemotherapeutic drugs.In-depth researches and analysis of tumor drug resistance induced by Wnt/β-catenin will provide potential targets and possible therapeutic means for the treatment of tumors.
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Objective: To investigate the effects of receptor interacting protein kinase 4 (RIPK 4) gene-silencing on epithelial-mesenchymal transition (EMT) of osteosarcoma U-2OS cells. Methods: The specific siRNA targeting RIPK 4 gene was transfected into U-2OS cells by liposome to establish RIPK 4 gene-silencing U-2OS cell line. Then the change of RIPK4 expression level was detected by Western blotting. The migration and invasion abilities of U-2OS cells after RIPK 4 gene-silencing were detected by Transwell chamber assay. The morphological changes of U-2OS cells were observed under an inverted optical microscope. Finally, the expression levels of EMT makers E-cadherin and vimentin in U-2OS cells were detected by Western blotting. Results: After transfection with RIPK4-siRNA, the expression level of RIPK4 protein was significantly decreased in U-2OS cells (P < 0.05). The migration and invasion abilities of U-2OS cells in RIPK4-siRNA transfection group were significantly reduced (both P<0.05). The morphology of U-2OS cells conversed from mesenchymal phenotype to epithelial phenotype. The expression level of E-cadherin was significantly up-regulated in RIPK4-siRNA transfection group (P < 0.05), whereas the expression level of vimentin was significantly down-regulated (P < 0.05). Conclusion: RIPK 4 gene-silencing can inhibit the occurrence of EMT in osteosarcoma U-2OS cells, and reduce the migration and invasion abilities of osteosarcoma cells.
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The 3' untranslated region (UTR) of the hepatitis C virus (HCV) is believed to function in the initiation and regulation of viral RNA replication and protein translation by interacting with the viral and host components. To examine host proteins interacting with the HCV 3'UTR, biotinylated 3'(+)UTR, and its reverse complementary 5'(-)UTR were used in RNA pull-down assay. Cellular proteins from Huh7 cells pulled down by biotinylated RNAs were identified by 2DE/MALDI-TOF MS and 1DE/LC/MS methods. Totally, 10 proteins could be identified from both methods, among which six bound specifically to the 3'(+)UTR, three proteins to the 5'(-)UTR only, and one protein bound to both. Three identified proteins (PCBP2, G3BP1, and DDX1) were selected for further investigation into their possible roles on the HCV replication. Differently regulating effects on HCV replication by siRNA-mediated silencing of these proteins were observed, indicating a complex role of 3'UTR binding proteins on HCV replication.
