Development and Application of An Assay for High-throughput Antiviral Compounds Screening against Alphaviruses / 病毒学报

To establish a cell-based rapid luciferase suppression assay for high-throughput screening (HTS) anti-alphaviruses compounds screening, which could cause viral encephalitis, raise the social issues associated directly with public health and huge economic burden to the society. The Gaussia luciferase assay system was used for HTS model for identifying inhibitors of labeled virus XJ160-GLUC. The decreased 50% GLUC activity inhibition ratio was deemed to be the screening positive index. The reaction system in this model was optimized, and the reliability of the model was evaluated. For HTS model's optimization, cells were infected with XJ160-GLUC at an MOI of 0.025 PFU/cell. The supernatant treated with compounds 48h were collected for GLUC expression detection. In the model, Z' factor was up to 0.71, demonstrating that HTS assay for identifying inhibitors that target all aspects of the viral life cycle of XJ160-GLUC was stable and reliable. After screening 8080 compounds (five-in-one), 341 positive samples were selected, and the positive rate was 4.2% with a cutoff at 50% inhibition. Then 1705 compounds were screened subsequently and the positive rate was 1.1% with obtaining 19 positive compounds. These results will lay the foundation for finding the anti-alphaviruses' drug targets.

Relationship between body mass index and long-term prognosis in patients with chronic obstructive pulmonary disease / 中华老年医学杂志

Objective To investigate the relationship between body mass index (BMI) and long-term survival in patients with chronic obstructive pulmonary disease (COPD).Methods A retrospective cohort study was conducted in 1124 patients with completed data among 1528 hospitalized patients with COPD.Vital status was ascertained at death registry and civil affairs department.Clinical characteristics were acquired from medical record.Patients were divided into 4 groups according to the quartile of BMI:BMI＜18.9 kg/m2 (group A),BMI from 18.9 kg/m2to 22.7 kg/m2 (group B),BMI from 22.8 kg/m2 to 26.2 kg/m2 (group C),BMI＞26.2 kg/m2 (group D).The differences in survival curves were compared between groups by using log-rank test.The relationship between all-cause mortality and BMI was evaluated by Cox proportional hazards regression model.Results The median BMI of 1124 patients was 22.7 kg/m2 [(18.9-26.2) kg/m2].270 patients (24.0％) had BMI＜18.5 kg/m2.162 patients (14.4％) died during follow-up.There was significant difference in the survival curves in the four groups (x2 =7.97,P＜0.05).Survival rate was highest in group C and lowest in group A.After adjustment for other factors,BMI was an independent risk factor for predicting long-term survival.The survival rate was respectively decreased by 41 ％ and 35 ％ in group C and D as compared with group A.Conclusions Weight loss is comman in COPD patients.BMI is an independent risk factor for predicting long-term survival.BMI is easily acquired and stable,which is especially suitable to evaluate the prognosis of patients with acute exacerbation.

Association between inflammatory biomarkers and survival in patients with chronic obstructive pulmonary disease: a prospective study / 中华老年医学杂志

Objective To investigate the association between inflammatory biomarkers and survival in patients with stable chronic obstructive pulmonary disease(COPD).Methods A total of 1038 patients with stable COPD from January 2008 to December 2009 were included in a prospective cohort study.Clinical characteristics,pulmonary function tests,6 min walk test and a modified British Medical Research Council dyspnea scale (MMRC) were completed.Fasting blood was obtained to detect inflammatory biomarkers including neutrophils,C-reactive protein(CRP),fibrinogen,TNF-a,IL-6 and IL-8.Participants were evaluated every 3 months,all-cause mortality was used as the end event.Results 120 patients (9.2％) died in the period of follow-up.Kaplan-Meier analysis showed that 1 year,2-year-and 3-year survival rates were 94.4％,88.3％ and 84.0％,respectively.Compared with survivors,those who died had a higher level of inflammatory biomarkers.Cox multivariate regression analysis demonstrated that the independent risk factors for death were neutrophils (HR:1.262,95％CI:1.143-1.512,P=0.035),CRP (HR:1.234,95％CI:1.097-1.624,P=0.029),fibrinogen (HR:1.327,95％CI:1.141-1.619,P=0.026),TNF-α (HR:1.124,95％CI:1.043-1.659,P=0.045),IL-6 (HR:1,429,95％CI:1.237-1.816,P=0.014) and IL-8 (HR:1.188,95％CI:1.024-1.383,P=0.042).C statistical analysis showed that no single biomarker significantly improved the C statistic value on the base of clinical model,but it was further improved by the addition of all biomarkers (C =0.764,P =0.010).Conclusions The level of inflammatory biomarkers in the death with stable COPD is significantly increased.Age.BODE index,neutrophils,CRP,fibrinogen,TNF-α,IL-6 and IL-8 are independent risk factor for the prediction of mortality in patients with COPD.

First isolation of Banna virus in northwestern part of Yunnan province / 中华微生物学和免疫学杂志

Objective To identify the Banna viruses isolated in northwestern part of Yunnan prov-ince in order to make the difference clear between the isolates and other Banna viruses isolated in other parts of Yunnan. Methods Three isolates of Banna vires isolated in 2005 and 2006 were identified by morpholo-gy, RNA-PAGE profile and molecular biologic method. Nueleotide and amino acid sequences of segment 12 of the 3 isolates were sequenced and analyzed. Results Three Banna viruses were isolated from mosquitoes collected in northwestern part of Yunnan during 2005 and 2006. Electron microscopy study showed that they are spherical with a diameter of 70 nm, no envelope but two layers of eapsid. It was found that the genome of the 3 isolates composes of 12 segments presenting band profile of 6-6 in RNA-PAGE. Nueleotide acid se-quence analysis about segment 12 showed that the identity was 99% between the 3 new isolates, 98% and 90% between the 3 isolates and the strains isolated in other parts of Yunnan, China and Indonesia, respec-tively. Phylogenetie analysis based on segment 12 gene showed that 3 new isolates clnstered in the same branch with the viruses isolated in other parts of Yunnan. The same difference of amino acids was found between Banna viruses isolated in China and Indonesia strains in the analysis of segment 12. Conclusion Banna virus strains were firstly isolated from mosquitoes collected in northwestern part of Yunnan province. Nueleotide acid sequence analysis of the 3 new isolates showed higher identity with strains isolated in other parts of Yunnan.

Preparation and storage of coltivirus antigen and its application in detection of coltivirus antibodies in serum from patients / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare purified and concentrated coltivirus high titer antigen in order to further detect antibodies against coltivirus in serum sample of patients.</p><p><b>METHODS</b>The coltivirus in C6/36 cells was cultured and harvested at different time, and the titer was titrated. The virus was purified and concentrated by polyethylene glycol (PEG), and stored at -20 degrees and 4 degrees, with and without glycerol, respectively, then the titer of coltivirus antigen was tested by indirect ELISA. By using the antigen, coltivirus antibodies in serum samples from both suspected Japanese encephalitis (JE) and viral encephalitis (VE) patients were detected.</p><p><b>RESULTS</b>The highest titer of coltivirus was found at 3-4 weeks of culturing. The antigen titer could be maintained at least for 6 months, especially antigen with glycerol either at 4 degrees or at -30 degrees even for two years. Totally 1141 serum samples from patients diagnosed clinically as JE and VE were tested. The results showed that 130 samples were coltivirus IgM antibody positive and the average positive rate was 11.4% (130/1141). Among 41 samples of paired-serum from patients in Guangzhou Children's Hospital, 9 samples were positive, the positive rate was 22.0% (9/41) in which 5 samples were diagnosed clinically as VE.</p><p><b>CONCLUSIONS</b>Stable and purified coltivirus antigen was obtained in order to test coltivirus antibodies as well as development of kits. Coltivirus probably can cause summer-autumn encephalitis in China.</p>

Hepatocarcinoma specific IL-1? anti-sense RNA inhibits implanted hepatocarcinoma in mice / 中国肿瘤生物治疗杂志

Objective :To construct hepatocarcinoma specific IL-1? anti-sense RNA expression vector and to explore its effect on the growth of implanted hepatocarcinoma H22 cells in mice and the possible mechanism. Methods:Murine IL-1? anti-sense RNA expression vectors pafpIRES2-antiIL-1?1 and pafpIRES2-antiIL-1?2 under the regulation of minimal alpha-feto protein (AFP) promoter and CMV enhancer were constructed,and further verified by PCR,restriction endonuclease analysis and DNA sequencing. H22 cells transfected with pafpIRES2-antiIL-1? 1 or pafpIRES2-antiIL-1? 2 were divided into 3 groups:H22/mock,H22/antiIL-1?1 and H22/antiIL-1?2 group. Expression of IL-1? was detected by RT-PCR. Transfected H22 cells were subcutaneously injected into mice to establish tumor implanted mouse model. Tumor volume was measured; the cytotocixity of spleen NK against H22 cells was detected by MTT. Results:Hepatocarcinoma specific IL-1? anti-sense RNA expression vectors pafpIRES2-antiIL-1?1 and pafpIRES2-antiIL-1?2 were successfully constructed and were verified by PCR,restriction endonuclease analysis and DNA sequencing. IL-1? expression in H22 cells was down-regulated after transfected with IL-1? anti-sense RNA expression vectors,especially with the pafpIRES2-antiIL-1?2 vector. Hepatocarcinoma cells implanted mouse model was successfully established. Tumor volume and growth of tumor in H22/antiIL-1?2 mice was obviously smaller than that in H22/mock mice,and the cytotocixity of spleen NK against H22 cells in H22/antiIL-1?1 and H22/antiIL-1?2 mice was also greatly enhanced. Conclusion:Hepatocarcinoma specific IL-1? anti-sense RNA expression vector pafpIRES2-antiIL-1? was successfully constructed. It effectively inhibits the growth of implanted hepatocarcinoma in mice probably through specifically blocking expression of IL-1? and increasing cytotocixity of spleen NK.

Construction and identification of a screening vector using enhanced green fluorescent protein(EGFP) as an indicator / 中国海洋药物

Objective To construct and identify the screening vector pUC18-EGFP,using EGFP as an indicator. Methods The EGFP gene was prepared by PCR and cloned into pUC18 resulting the vector pUC18-EGFP. Then DNA fragment was inserted into the MCS of pUC18-EGFP to test its practicability based on green fluorescence. Results The pUC18-EGFP was confirmed correctly by restriction enzyme analyses. The pUC18-EGFP was used to select recombinants. The green strains on the plate were confirmed by restriction enzyme and DNA analyses,which were E.coli harboring recombinants. Conclusion The screening vector PUCl8-EGFP was constructed successfully. Thus,we can select the positive clones on plates based on the green fluorescence of EGFP.