Copper is a trace element essential for the maintenance of normal physiological functions in cardiovascular system,and its transport and metabolisms are regulated by various copper proteins such as copper-based enzymes,copper chaperones and copper transporters.The disturbance of copper level or abnormal expression of copper proteins are closely associated with the development of cardiovascular diseases such as atherosclerosis,hypertension,ischemic heart disease,myocardial hypertrophy and heart failure.Thus,intervention of copper ion signaling pathways is expected to be an effective measure for treating cardiovascular diseases.Some copper complexes,such as trientine,copper-aspirinate complex and copper(Ⅱ)diethyldithiocarbamate,have been found to play a role in the prevention and treatment of cardiovascular diseases and possess potential prospects.Exploring the role of copper in maintaining normal cardiovascular status and the potential application of copper complexes in the treatment of cardiovascular diseases may lay a foundation for finding new targets for prevention and treatment of various cardiovascular diseases,and provide new ideas for clinical treatment of cardiovascular diseases.

Objective:To explore the effect of significant variation in non-structural protein 1 (NSP1) of 2019 novel coronavirus (2019-nCoV) on binding to 5′UTR, and to provide clues for the development of antiviral drugs and vaccines.Methods:The bioinformatics analysis of 2019-nCoV genome database was conducted to select the amino acid variation sites (T12A, R124L, N128I, K141A, GHVMV82-86DEL and KSF141-143DEL) that may affect the structure of NSP1 and the ability binding to 5′UTR. PSIPRED online tool was used to analyze the secondary structure of the variants, mCSM-NA was used to predict their binding ability to RNA, and DynaMut webserver was used to analyze the influence of variants on protein stability. The variant plasmids were constructed and transfected into HEK-293T cells. The dual luciferase reporter gene assay and RNA binding protein immunoprecipitation (RIP) assay were used to detect the binding ability of the NSP1 variant for viral 5′UTR.Results:Bioinformatics analysis predicted that except for R124L mutation, the other five variants could change the secondary structure of protein, and the mutations of T12A, R124L, N128I and K141A could reduce the binding ability of RNA, while the mutations of T12A, R124L and N128I reduced the stability of protein. The experimental results showed that R124L, N128I, GHVMV82-86DEL and KSF141-143DEL significantly weakened the binding ability of NSP1 to 5′UTR.Conclusions:Some mutations or deletion of NSP1 amino acids could change the secondary structure and significantly weaken the binding ability of NSP1 to the 5′UTR, suggesting that the pathogenicity of the virus may be changed, which could provide a theoretical basis for the development of antiviral drugs and vaccines.

Objective:To investigate the expression and clinical significance of serum miR-193-5p and miR-196-5p in children with IgA nephropathy (IgAN).Methods:The 95 children with IgAN (IgAN group), 80 children with non IgAN nephritis (non IgAN group) and 50 normal subjects (control group) in Sanya Women and Children's Hospital of Shanghai Children's Medical Center and Sanya People's Hospital were selected to be included in this study. According to Oxford classification score (MEST), children with IgAN were divided into 35 cases with MEST≥3 points and 60 cases with MEST<3 points. The serum levels of miR-193-5p, miR-196-5p, IgA/C3 and galactose deficient IgA1 molecule (Gd-IgA1) in each group were compared. The receiver operating characteristic (ROC) curve was drawn to analyze the value of miR-193-5p, miR-196-5p, IgA/C3 and Gd-IgA1 in the diagnosis of IgAN. Pearson correlation analysis was used to analyze the correlation between the expression levels of miR-193-5p, miR-196-5p and IgA/C3 and Gd-IgA1.Results:The serum levels of miR-193-5p, miR-196-5p, IgA/C3 and Gd-IgA1 in IgAN group were significantly higher than those in non IgAN group and control group (all P<0.001). The levels of miR-193-5p, miR-196-5p, IgA/C3 and Gd-IgA1 in MEST≥3 group were significantly higher than those in MEST<3 group (all P<0.001). ROC curve analysis showed that the area under the curve for the combined diagnosis of IgAN by miR-193-5p, miR-196-5p, IgA/C3 and Gd-IgA1 was 0.958 (95% CI: 0.902-0.998), with sensitivity of 98.6% and specificity of 86.4%. Pearson correlation analysis showed that the expression levels of serum miR-193-5p and miR-196-5p in IgAN children were positively correlated with IgA/C3 and Gd-IgA1 (all P<0.001). Conclusions:The expression levels of serum miR-193-5p and miR-196-5p were significantly increased in children with IgAN, and the combined detection of IgA/C3 and Gd-IgA1 has high value for the diagnosis of IgAN in children.

Objective:To establish a rapid, sensitive and specific detection method for important imported viruses based on the recombinase aided amplification (RAA) method and clustered regularly interspaced short palindromic repeats-associated protein 13a (CRISPR-Cas13a) system.Methods:In this study, we selected Japanese encephalitis virus (JEV), Yellow fever virus (YEV), West Nile virus (WNV), Middle East respiratory syndrome coronavirus (MERS-CoV)、Ebola virus (EBOV), Dengue virus (DENV), Rift Valley fever virus (RVFV), Zika virus (ZIKV) as subjects, and designed specific RAA primers and CRISPR RNA(crRNA). The sensitivity and specificity of the method were evaluated. We detected suspected clinical samples of dengue fever and compared with the fluorescent reverse transcriptase-polymerase chain reaction (RT-PCR) technology. Clinical simulation samples of the remaining seven viruses were also detected.Results:The RAA method combined with CRISPR-Cas13a can detect eight pathogens within 40-52 min at 39 ℃. The sensitivity was 1-10 copies/μl. There was no cross-reaction among eight viruses and all clinical samples could be detected by this method.Conclusions:The established RAA combined with CRISPR-Cas13a detection method can sensitively, specifically and quickly detect eight imported infectious disease pathogens.

COVID-19 ; Coronavirus Nucleocapsid Proteins ; Humans ; Nucleocapsid Proteins ; Phosphoproteins ; SARS-CoV-2

Objective:To establish a rapid method for detecting the activity of 2019-nCoV2.Methods:The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection system was screened after propidium monoazide (PMA) treatment and exposure to heat-inactivated 2019-nCoV samples, and the PMA pretreatment conditions were optimized to establish the 2019-nCoV PMA-RT-qPCR detection method . The established method was used to detect virus inactivated by different temperatures and chlorine-containing disinfectant, to evaluate its effect in detecting virus activity.Results:For the PMA-RT-qPCR assay, 200 μmol/L of PMA, 10 min of incubation time, 15 min of exposure time, and the CDC ORF1ab detection system were selected; there was no significant difference in the result of PMA-RT-qPCR and direct RT-qPCR for the active virus; the Ct values of PMA-RT-qPCR for virus inactivated by 95 ℃ and chlorine-containing disinfectant were higher than that of control groups at different dilutions; only partial dilutions of 70 ℃ and 56 ℃ heat-inactivated virus had higher Ct values than control groups. Conclusions:The established PMA-RT-qPCR for 2019-nCoV activity detection method has a good detection effect on the virus inactivated by 95 ℃ heat and chlorine disinfectant, and provides an auxiliary means for judging the infectivity of the virus in the sample.

Objective:The entire genome sequences of 25 novel coronaviruses in Jiangsu province were analyzed and their evolutionary characteristics were studied.Methods:High-throughput sequencing was used to sequence the throat swab samples from confirmed cases. Single nucleotide polymorphisms were analyzed using CLC Genomics Workbench 12.0 software. Evolution characteristics were analyzed by MEGA 5.1.Results:A total of 52 single-base substitution mutations were detected in 25 strains. Phylogenetic analysis showed 25 stains were clustered into two clades. Viruses in clade 1 contain 8 682 and 28 144 CT SNP links. While viruses in clade 2 contain mutations in those two bases, i. e., 8 682 (ORF1ab: C8 517T, synonymous mutation) and 28 144 (ORF8: T251C, L84S). Among clade 2, five stains subclustered into one group based on SNP links in 24 034 (S: C2 472T, synonymous mutation), 26 729 (M: T207C, synonymous mutation), and 28 077 (ORF8: G184C, V62 L). There were no significant differences in the distribution of different clades/subclusters in the population and the disease types.Conclusions:We have found some SNPs occurred in new coronaviruses. The effects of different SNPs on virus transmission and pathogenicity need to be further studied.

Objective: To investigate the pyroptosis induced by different enteroviruses in human neuroblastoma cells SH-SY5Y and the differences among them. Methods: SH-SY5Y cells were infected with nine strains of enterovirus respectively, including enterovirus A71 (EV-A71), Coxsackievirus A (CA), Coxsackievirus B (CB), Echovirus (Echo). The cellular morphology of infected and control groups were observed and activity of Caspase-1 of infected and control groups were detected by flow cytometry at 48 h post infection. Results: The activity of Caspase-1 induced by EV-A71 was higher than control (P<0.001), and the activity of Caspase-1 induced by EV-A71 isolated from severe case was significantly higher than that induced by EV-A71 isolated from common case (P<0.001). The activity of Caspase-1 induced by CA was at a lower level. The activity of Caspase-1 induced by CB and Echo were both significantly higher than that induced by EV-A71 and control (P<0.001). Cytopathic effects (CPE) were found to be related with the activity of Caspase-1. Conclusions EV-A71, CB and Echo all could induce pyroptosis mediated by Caspase-1 in SH-SY5Y cells, but the ability of CA to induce pyroptosis was at a lower level, which may provide evidences for further study on mechanism of neuropathy caused by enterovirus.

Objective: To analyze the clinical manifestations and results of etiological examinations of 17 elderly patients with influenza A (H1N1) viral pneumonia, and to understand the clinical features of pneumonia and molecular characteristics of influenza A (H1N1) virus infection in the elderly. Methods: The elderly patients with pneumonia who were hospitalized in the Department of Respiratory Diseases of Nanjing First Hospital from January 2018 to March were enrolled. The cases were confirmed by nucleic acid examination for influenza virus and the clinical data were collected. After the amplification of the whole genome of influenza virus, the high throughput sequencing and bioinformatics analysis were performed. Results: The mean age of the 17 enrolled patients was 73.8±10.8. All of them had at least 1 underlying disease, and 7 cases had co-infection. Respiratory symptoms and fever were the most prominent clinical manifestations. Lesions in both lungs were found in 76.5% of the patients. The result of high throughput sequencing showed that all the viruses were highly homologous to the vaccine strain, and the HA gene belonged to the 6B.1 subgroup. Furthermore, three variations of antigenic locus (H138Y, S74R and S164T in HA) and a drug-resistant variation (H275Y in NA) were detected in the circulating strains. Conclusions Elderly patients with influenza A (H1N1) virus pneumonia often have underlying diseases and are prone to have co-infection. The molecular characteristics of the virus and the variation of key amino acid loci should be closely monitored in order to provide evidence for epidemic prevention and clinical antiviral treatment.

Objective: To establish a rapid and sensitive isothermal amplification assay for the detection of human Adenovirus. Methods: Primers and probe used for recombinase polymerase amplification(RPA)were designed based on the conserved region of the adenoviruses hexon gene. After optimizing the reaction temperature and times, the products of RPA were detected by capillary electrophoresis and lateral flow dipstick(LFD). Sensitivity and specicity of the assay were evaluated. The diagnostic value of the RPA-LFD assay was verified using clinical samples which were simultaneously tested by real time PCR assay. Results: The analytical sensitivity of RPA-LFD assay was 2 copies DNA molecules per reaction and no cross reaction with other pathogens was observed. Compared with real-time PCR assay, the sensitivity, and specificity of the present assay were all 100%. Conclusions The RPA-LFD assay developed in this study has the characteristics of high specificity, sensitivity, rapid and no requirement of expensive equipment which provided a new tool for rapid detection of human adenovirus.