Serum Exosomal Long Noncoding RNA Growth Arrest-Specific 5 Predicts 3-Month Mortality in Acute-on-Chronic Hepatitis B Liver Failure.

Background: Acute-on-chronic hepatitis B liver failure (ACHBLF) is a clinical syndrome with an extremely high mortality. In this study, we aim to evaluate the potential role of serum exosomal long noncoding RNA (lncRNA) growth arrest-specific 5 (GAS5) in ACHBLF and its predictive value for 3-month mortality. Methods: From December 2017 to June 2022, we enrolled 110 patients with ACHBLF and 42 healthy controls (HCs). Exosomes were isolated from the serum of the participants. Serum exosomal lncRNA GAS5 was detected using quantitative real-time polymerase chain reaction (qRT-PCR). The functional role of lncRNA GAS5 on hepatocyte phenotypes was investigated through loss-of-function and gain-of-function assays. Exosomal labeling and cell uptake assay were used to determine the exosomes-mediated transmission of lncRNA GAS5 in hepatocytes in vitro. Results: The serum exosomal lncRNA GAS5 was identified to be an independent predictor for 3-month mortality of ACHBLF. It yielded an area under the receiver operating characteristic curve (AUC) of 0.88, which was significantly higher than MELD score (AUC 0.73; P < 0.01). Further study found that lncRNA GAS5 could inhibit hepatocytes proliferation and increase hepatocytes apoptosis. Exosomes-mediated lncRNA GAS5 transfer promoted hepatocytes injury. The knocked down of lncRNA GAS5 weakened H2O2-induced hepatocytes injury. Conclusion: We revealed that serum exosomal lncRNA GAS5 might promote hepatocytes injury and showed high predictive value for 3-month mortality in ACHBLF.

Roles of conserved residues in the receptor binding sites of human parainfluenza virus type 3 HN protein.

Human parainfluenza virus type 3 (hPIV-3) entry and intrahost spread through membrane fusion are initiated by two envelope glycoproteins, hemagglutinin-neuraminidase (HN) and fusion (F) protein. Binding of HN protein to the cellular receptor via its receptor-binding sites triggers conformational changes in the F protein leading to virus-cell fusion. However, little is known about the roles of individual amino acids that comprise the receptor-binding sites in the fusion process. Here, residues R192, D216, E409, R424, R502, Y530 and E549 located within the receptor-binding site Ⅰ, and residues N551 and H552 at the putative site Ⅱ were replaced by alanine with site-directed mutagenesis. All mutants except N551A displayed statistically lower hemadsorption activities ranging from 16.4% to 80.2% of the wild-type (wt) level. With standardization of the number of bound erythrocytes, similarly, other than N551A, all mutants showed reduced fusogenic activity at three successive stages: lipid mixing (hemifusion), content mixing (full fusion) and syncytium development. Kinetic measurements of the hemifusion process showed that the initial hemifusion extent for R192A, D216A, E409A, R424A, R502A, Y530A, E549A and H552A was decreased to 69.9%, 80.6%, 71.3%, 67.3%, 50.6%, 87.4%, 84.9% and 25.1%, respectively, relative to the wt, while the initial rate of hemifusion for the E409A, R424A, R502A and H552A mutants was reduced to 69.0%, 35.4%, 62.3%, 37.0%, respectively. In addition, four mutants with reduced initial hemifusion rates also showed decreased percentages of F protein cleavage from 43.4% to 56.3% of the wt. Taken together, Mutants R192A, D216A, E409A, R424A, R502A, Y530A, E549A and H552A may lead to damage on the fusion activity at initial stage of hemifusion, of which decreased extent and rate may be associated with impaired receptor binding activity resulting in the increased activation barrier of F protein and the cleavage of it, respectively.

Demethylated miR-216a Regulates High Mobility Group Box 3 Promoting Growth of Esophageal Cancer Cells Through Wnt/ß-Catenin Pathway.

BACKGROUND: Esophageal cancer (EC) is the eighth most common cause of cancer-associated mortality in humans. Recent studies have revealed the important roles of microRNAs (miRs) in mediating tumor initiation and progression. miR-216a has been found to be involved in the progression of EC, but the underlying mechanisms remain largely unknown. The aim of this study is to explore the mechanism of miR-216a and the downstream molecules in esophageal cancer. MATERIALS AND METHODS: The degree of methylation of miR-216a promoter in EC tissues and cell lines was determined with methylation specific polymerase chain reaction (MSP). The levels of miR-216a and HMGB3 in EC cells were quantified by quantitative PCR (qPCR) and Western blot (WB). EC cell lines were treated with DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AZ), miR-216a mimics, and HMGB3 siRNA to explore the effects of miR-216a and HMGB3 on the proliferation, migration, invasion, and apoptosis of cells. Dual-luciferase reporter assay was employed to verify the binding of miR-216a to the 3'UTR of HMGB2 mRNA. RESULTS: The promoter of MiR-216a was hypermethylated and the expression of miR-216a was down-regulated in EC, while HMGB3 was up-regulated. Dual luciferase reporter assay confirmed the binding of miR-216a to the 3'UTR of HMGB3 mRNA. Demethylated miR-216a and miR-216a mimics elevated miR-216a expression and down-regulated HMGB3, as well as inhibited cell proliferation, migration, and invasion. Inhibiting the expression of HMGB3 played an important role in inducing apoptosis, suppressing cell expansion, and down-regulating the activity of Wnt/ß-catenin pathway. CONCLUSIONS: Hypermethylation in the promoter of miR-216a upregulated HMGB3 and the Wnt/ß-catenin pathway, resulting in enhanced EC progression.

Public Awareness and Mask Usage during the COVID-19 Epidemic: A Survey by China CDC New Media.

An online survey conducted March 18-19, 2020 on the official China CDC WeChat account platform was used to evaluate the effect of public education about masks usage during the new coronavirus disease 2019 (COVID-19) epidemic. Chinese nationals older than 18 were eligible for the survey. The survey collected 5,761 questionnaires from the 31 provinces, municipalities, and autonomous regions of mainland China. 99.7% and 97.2% of the respondents answered correctly that respiratory droplets and direct contact were the main transmission routes. 73.3% of the respondents considered COVID-19 to be 'serious' or 'very serious'. When going to the hospital, 96.9% (2,885/2,976 had gone to a hospital) used a mask during the COVID-19 epidemic, while 41.1% (2,367/5,761) did not use a mask before the epidemic. Among the respondents that used public transportation and went shopping, 99.6% and 99.4%, respectively, wore masks. Among respondents who returned to work, 75.5% wore a mask at the workplace, while 86.3% of those who have not returned to work will choose to use masks when they return to the workplace. The Chinese public is highly likely to use a mask during COVID-19 epidemic, and the mask usage changed greatly since the COVID-19 outbreak. Therefore, public education has played an important role during the COVID-19 epidemic.

Role of N-linked glycosylation of the human parainfluenza virus type 3 hemagglutinin-neuraminidase protein.

Human parainfluenza virus type 3 (hPIV-3) is a major respiratory tract pathogen that affects infants and young children. The hPIV-3 hemagglutinin-neuraminidase (HN) protein is a multifunctional protein mediating hemadsorption (HAD), neuraminidase (NA), and fusion promotion activities, each of which affects the ability of HN to promote viral fusion and entry. The hPIV-3 HN protein contains four potential sites (N308, N351, N485 and N523) for N-linked glycosylation. Electrophoretic mobility analysis of mutated HN proteins indicated that N308, N351 and N523 sites, but not the N485 site in HN protein, were targeted for the addition of glycans in BHK-21 cells. These functional glycosylation sites were systematically eliminated in various combinations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groups of carbohydrate chains could be analyzed. Removal of individual or multiple N-glycans on the hPIV-3 HN protein had no effects on transport to the cell surface, expression and NA activity. Single glycosylation site mutants (G1, G2 and G4) not only impaired fusion promotion activity but also reduced HAD activity of HN protein, which was even more obvious for all three double mutants (G12, G14 and G24) and the triple mutant (G124). In addition, every mutant protein retained F-interactive capability that was equal to the wild-type protein capability. Interestingly, the F protein that could be co-immunoprecipitated with the G12 mutated protein or immunoprecipitated with anti-F antibody was not efficiently cleaved. For G14, G24 and G124, little cleaved F protein was detected in co-immuoprecipitation F protein assay and its total amounts where in the cell lysates. The mechanism underlying hPIV-3 HN and F protein remained associated before and after receptor engagement and the strength of the HN-receptor interaction modulated the activation of F the protein which could determine the extent of fusion. Finally, we demonstrated that single or multiple N-glycosylation site mutations inhibited fusion at the earliest stages. Taken together, these results indicated that N-glycosylation of hPIV-3 HN is critical to its receptor recognition activity, cleavage of the F protein, and fusion promotion activity, but had no influence on its interaction with the homologous F protein and NA activity.

Complete genome sequencing and analysis of six enterovirus 71 strains with different clinical phenotypes.

BACKGROUND: Hand, foot and mouth diseases (HFMD) caused by enterovirus 71(EV71) presents a broad spectrum of clinical manifestations ranging from mild febrile disease to fatal neurolocal disease. However, the mechanism of virulence is unknown. METHODS: We isolated 6 strains of EV71 from HFMD patients with or without neurological symptoms, and sequenced the whole genomes of the viruses to reveal the virulence factors of EV71. RESULTS: Phylogenetic tree based on VP1 region showed that all six strains clustered into C4a of C4 sub-genotype. In the complete polypeptide, 298 positions were found to be variable in all strains, and three of these positions (Val(P814)/Ile(P814) in VP1, Val(P1148)/Ile(P1148) in 3A and Ala(P1728)/Cys)/Val(P1728) in 3C) were conserved among the strains with neurovirulence, but variable in strains without neurovirulence. In the 5'-UTR region, it showed that the first 10 nucleotides were mostly conserved, however from the 11th nucleotide, nucleotide insertions and deletions were quite common. The secondary structure prediction of 5'-UTR sequences showed that two of three strains without neurovirulence (SDLY11 and SDLY48) were almost the same, and all strains with neurovirulence (SDLY96, SDLY107 and SDLY153) were different from each other. SDLY107 (a fatal strain) was found different from other strains on four positions (C(P241)/T(P241), A(P571)/T(P571), C(P579)/T(P579) in 5'-UTR and T(P7335)/C(P7335) in 3'-UTR). CONCLUSIONS: The three positions (Val(P814)/Ile(P814) in VP1, Val(P1148)/Ile(P1148) in 3A and Ala(P1728)/Cys(P1728)/Val(P1728) in 3C), were different between two phenotypes. These suggested that the three positions might be potential virulent positions. And the three varied positions were also found to be conserved in strains with neurovirulence, and variable in strains without neurovirulence. These might reveal that the conservation of two of the three positions or the three together were specific for the strains with neurovirulence. Varation of secondary structure of 5'-UTR, might be correlated to the changes of viral virulence. SDLY107 (a fatal strain) was found different from other strains on four positions, these positions might be related with death.

'a'-Position-mutated and G4-mutated hemagglutinin-neuraminidase proteins of Newcastle disease virus impair fusion and hemagglutinin-neuraminidase-fusion interaction by different mechanisms.

OBJECTIVES: To determine the effects of heptad repeat regions (HRs) and N-linked carbohydrate sites of the Newcastle disease virus hemagglutinin-neuraminidase (HN) protein on fusion of HN and fusion (F) proteins and HN-F interaction. METHODS: We mutated six 'a' residues in the HRs and four asparagines in N-linked carbohydrate sites to alanine in the HN protein. A vaccinia-T7 RNA polymerase expression system was used to express HN cDNAs in BHK-21 cells to determine the HN functions. Deglycosylation was treated with PGNase F digestion. The formation of HN-F protein complexes was determined by the coimmunoprecipitation assay. RESULTS: Each HR-mutated protein interfered with fusion and the HN-F interaction. The G4-mutated protein not only impaired fusion and HN-F interaction but also decreased activities in cell fusion promotion, hemadsorption and neuraminidase. CONCLUSIONS: It is assumed that two different mechanisms for mutations in these two regions are responsible for the decreased fusion promotion activity and the reduced ability of interaction with F protein. Mutations in the HRs impair fusion and HN-F interaction by altering the transmission of a signal from the globular domain to the F-specific region in the stalk, but the G4 mutation modulates fusion and HN-F interaction by the misfolding of some important structures.

[Study on functions of N-carbohydrate chains in human parainfluenza virus type 3 hemagglutinin-neuraminidase protein].

To determine the functions of N-carbohydrate chains in human parainfluenza virus type 3 hemagglutinin-neuraminidase(HN) protein, a PCR-based site-directed mutagenesis method was used to obtain N-glycan mutants. Protein electrophoresis rate, cell surface expression,receptor binding activity, neuraminidase activity and cell fusion promotion activity were determined. The HN proteins of single mutants (G1, G2, and G4) and multiple mutants (G12, G14, G24 and G124) migrated faster than the wild-type (wt) HN protein on polyacrylamide gels, while G3-mutated protein and wt HN protein migrated at the same position. There was no statistic difference in cell surface expression and neuraminidase activity between wt and each mutant HN protein (P>0.05), but receptor binding activity and cell fusion promotion activity of each mutant protein was reduced to significant extent (P<0.05). G1, G2 and G4 mutants exhibited re duced receptor binding activity, which was 83.94%, 76.45% and 55.32% of the wt level, respectively. G1, G2 and G4-mutated proteins also showed reductions in fusion promotion activity, which was 80.84%, 77.83% and 64.16%, respectively. Multiple mutants with G12-, G14-, G24- and G124- substitutions could further reduce receptor binding activities, 33.07%, 20.67%, 19.96% and 15.11% of the wt HN level, respectively. G12, G14, G24 and G124 mutants exhibited levels of fusion promotion activity that were only 46.360, 12.04%, 13.43% and 4.05% of the wt amount, respectively. As N-glycans of hPIV3 HN protein play an important role in receptor binding activity and cell fusion promotion activity of HN protein. We propose that the loss of N-glycans change the conformation or orientation of globular domain that is responsible for receptor binding and lower receptor binding activity and cell fusion promotion activi ty.

[Expression and bioassay of rubella virus E1-374 glycoprotein in yeast cells].

OBJECTIVE: To express the rubella virus E1-374 glycoprotein in Pichia pastoris and study the immunogenecity of the recombinant protein. METHODS: The cDNA of protein E1-374 was cloned into the expression vector pGAPZalphaA and transformed into Pichia pastoris GS115 cells by electrotransfection. The expressed protein was confirmed by indirect immunofluorescence and demonstrated immunoreactivity by Western Blot. Rubella virus IgG antibody was assayed with ELISA after mice were inmmunized by E1-374 glycoprotein. RESULTS: SDS-PAGE analysis and Western Blot analysis of E1-374 protein revealed this protein to be 46.89 x 10(3). Antiserum (1:100) and E1-374 (5.5 microg/ml) was chosen for ELISA optimization. The intra-assay coefficient of variation for the ELISA was 0.36%-12.45%. CONCLUSION: Protein E1-374 was highly expressed in Pichia pastoris cells, and it was a good choice to prepare rubella virus recombinant protein vaccines.