Objective To determine the causative association between a newly isolated coronavirus and the current epidemic of severe acute respiratory syndrome (SARS) Methods Coronavirus was isolated from the samples of patients with SARS by cell culture Immunofluorescence assay and neutralization test were used to detect the antibodies in serum of SARS patients against newly isolated coronavirus, in order to analyze and determine the association between coronavirus and SARS pathogen Results The antibodies against the novel coronavirus could be detected in 99 of 113 sera from clinically diagnosed SARS patients The results of 10 pairs of double serum detection showed that, antibody titers in the late phase were significantly higher than those in the acute phase, and a highest increase by 128 folds was being found. The neutralization test showed that the antibodies from SARS patients' sera could neutralize the novel coronavirus Conclusion The newly isolated coronavirus was closely associated with and possibly the key pathogen of SARS

Objective:To identify metastasis-associated genes in salivary gland adenoid cystic carcinoma(ACC).Methods:Salivary gland adenoid cytic carcinoma cell line ACC-2 and its highly metastatic ACC-M cells were used to screen the metastasis-related genes in ACC by microarray technology.Two fluorescent cDNA probes labeled with Cy3 and Cy5 dyes,were prepared from the mRNA samples of ACC-2 and ACC-M cells by reverse transcription method.The two color probes were then mixed and hybridized on the cDNA chip constructed by double dots of 1152 human genes,and scanned at two wave lengths.Differentialy expressed genes of the two cell lines were analyzed using computer.Then seven of the differently expressed genes were further validated by RT-PCR technique.Results:Of the 1,152 known genes and expressed sequence tags,26 showed significantly different expression level(minimum 2 fold) between the two cell lines.Among the 26 genes,19 were up regulated(with ratio more than 2) and 7 were down(with ratio less than 1/2).The results of RT-PCR analysis for 7 differently expressed genes were coincident with those of microarray assay.Conclusions:Down regulation of LIFR,LCP1,DPEP1 and ABLIM1,and up regulation of DCC,MMP1 and CNTN2 may be related to the highly metastatic potential of ACC-M cell line.

ObjectiveTo develop an antibody-array system for multiple detection of antibodies against Japanese B encephalitis virus (JEV),Tick-borne encephalitis virus (TBV),Dengue virus ( DENV ),West Nile virus (WNV),Western equine encephalitis virus (WEEV) and East Equine encephalitis virus (EEEV).MethodsRecombined antigens were spotted on array as capture antigens.Specific antibodies were detected by using a sandwich ELISA format.Rabbit antiserum was employed to select and confirm the specificity of antigens and to optimize the conditions of the assay.The detection efficiency of the system was validated by 40 clinical suspected serum samples and compared with the relative ELISA assays.ResultsEleven recombined antigens were selected as diagnostic antigens with high specificity.Better detection could be achieved when scale of antigen concentrations were within 0.125-0.900 mg/ml and the serum dilutions were 1:100-1:1000.When detecting the 26 clinical suspected TBE serum samples,20 were IgG positive (76.9％),and 17 were IgM positive (65.3％) which was 96.1％ and 84.6％ consistent with the relevant ELLSA tests,the 8 clinical suspected JEV serum samples,4 were IgG positive (50.0％),and 5 were IgM positive (62.0％),which was 86.3％ and 90.1％ consistent with the relevant ELLSA tests.As for the 22 DEN serum samples,13 were IgG positive (60％) and 15 were IgM positive (68％) which was 85％ and 93％ consistent with ELISA.The specificity of the assay was 100％ and the sensitivity was higher than the relative ELISAs.ConclusionThe developed antibody-array is highly specific and reliable,which could be used for the detection of antibodies against the 6 arboviruses.

Coronaviruses (CoVs) are generally associated with respiratory and enteric infections and have long been recognized as important pathogens of livestock and companion animals. Mouse hepatitis virus (MHV) is a widely studied model system for Coronavirus replication and pathogenesis. In this study, we created a MHV-A59 temperature sensitive (ts) mutant Wu"-ts18(cd) using the recombinant vaccinia reverse genetics system. Virus replication assay in 17C1-1 cells showed the plaque phenotype and replication characterization of constructed Wu"-ts18(cd) were indistinguishable from the reported ts mutant Wu"-ts 18. Then we cultured the ts mutant Wu"-ts 18(cd) at non-permissive temperature 39.5℃, which "forced" the ts recombinant virus to use second-site mutation to revert from a ts to a non-ts phenotype. Sequence analysis showed most of the revertants had the same single amino acid mutation at Nsp16 position 43. The single amino acid mutation at Nsp16 position 76 or position 130 could also revert the ts mutant Wu"-ts 18 (cd) to non-ts phenotype, an additional independent mutation in Nsp13 position 115 played an important role on plaque size. The results provided us with genetic information on the functional determinants of Nsp16. This allowed us to build up a more reasonable model of CoVs replication-transcription complex.

Objective To evaluate the Flavivirus specific monoclonal antibody(McAb) 2A10 as detective antibody for simultaneously identify tick borne encephalitis virus( TBEV), Japanese encephalitis virus( JEV), dengue ( DEN )-2, DEN-4 and yellow fever virus ( YFV ) by antibody microarray technique.Methods The antibody microarray was developed by spotting TBEV, JEV, DEN-2, DEN-4 and YFV specific McAb on chip as capture antibodies. After incubating with cultured viral supernatants of the above viruses, CY3 labeled detective antibody 2A10 was added to the chips. After reaction, the antibody microarray was scanned and the results were analyzed. By comparing the signal intensities of different spots on chips,the detecting titre and sensitivity of 2A10 for Flavivirus were determined, and the value of 2A10 in detection of Flavivirus was evaluated. Results The hybridization results demonstrated that the titre of 2A10 for Flavi2A10 was specific for Flavivirus and could be used as universal detective antibody for Flavivirus on antibody microarray.

Objective To identify the specific microRNA (miRNA) that can be taken as a molecular marker for human bone marrow-derived mesenchymal stem cells (MSCs). Methods MSCs were isolated and cultured from bone marrow through density centrifugation and then were induced to differentiate into osteoblasts and chondrocytes. Samples of MSCs, osteoblasts and chondrocytes were detected by miRNA microarrays single channel fluorescence chip to determine the expression levels of miRNAs. Significance Analysis of Microarrays ( SAM, version 2.1 ) software was used to analyze the raw data to determine the miRNAs overexpressed in MSCs, which was validated in the same sample using real time reserve transcriptase polymerase chain reaction (RT-PCR). Results MSCs were successfully isolated from bone marrow and induced to differentiate into osteoblasts and chondrocytes in vitro. Microarrays showed that eight miRNAs (has-miR-424, has-miR-34a, has-miR-593, has-miR-10a, has-miR-148a,has-miR-602, mmu-miR-709 and mmu-miR-665) were overexpressed in MSCs but underexpressed in osteoblasts. Three miRNAs including has-miR-424, PREDICTED_MIR189 and mmu-miR-665, were overexpressed in MSCs but underexpressed in chondrocytes. The has-miR-424 expression in MSCs was 6.6times higher than in osteoblasts and 4.4 times higher than in chondrocytes. The results of real time RTPCR showed that the miR-424 was overexpressed in MSCs, 3.6 times higher than that in osteoblasts and 3.1 times higher than that in chondrocytes, which was coincident with the results of microarray. Conclusions The screened MSCs express more miRNAs in comparison with osteoblasts and chondrocytes,play important roles in maintaining self renewal and undifferentiation of MSCs and is a promising specific molecule marker for MSCs.

We constructed a recombinant adenoviral vector expressing human tissue inhibitors of metalloproteinase-1(TIMP-1), and evaluated the inhibition of TIMP-1 secreted by primary fibroblasts after infection with adenovirus-mediated TIMP-1 gene (Ad-TIMP-1) on tumor cell invasion and metastasis in mouse melanoma. It was found that TIMP-1 was detected in the supernatants of cultured mouse primary fibroblasts after infection with Ad-TIMP-1 by indirect enzyme-linked immunosorbent assay (ELISA). The TIMP-1 secreted by Ad-TIMP-1 infected primary fibroblast significantly inhibited B16BL6 cell invasion and metastasis both in vitro and in vivo. We also demonstrated that the primary fibroblasts transfected by Ad-TIMP-1, after being subcutaneously injected into mouse, can secreted TIMP-1 into the blood of mouse and maintained at the therapeutic in vivo levels of TIMP-1. These results suggest that the preparation of Ad-TIMP-1 infected primary fibroblast be an effective method to deliver TIMP-1 gene in vivo, which provides a new strategy of gene therapy and has the potential for clinical applications in the treatment of tumor cell metastasis.
Adenoviridae ; genetics ; metabolism ; Animals ; Female ; Fibroblasts ; metabolism ; Genetic Therapy ; Humans ; Melanoma, Experimental ; pathology ; therapy ; Mice ; Mice, Inbred C57BL ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; genetics ; pharmacology

Objective To identify the infection and the replication of Tick-borne encephalitis virus(TBEV) in human neuroblastoma cells.Methods After being inffected with TBEV,the cell culture supernatant of human neuroblastoma cell line SK-N-SH was collected and assayed at different time points.Byusing real-time RT-PCR and plaque assay to measure the titer of virus in the supernatant,the replication andproliferation of TBEV in human neuroblastoma cell was identified.Meanwhile,the morphological change of SK-N-SH after TBEV infection was also visualized by observation under microscope and immunmquorescenceassay.Results Real-time RT-PCR and plaque assay both demonstrated that TBEV could replicate effectively in SK-N-SH cells,the peak titer could reach 2.92× 107 PFU/ml on 3 days post-inoculation.And significant morphological change occured on infected SK-N-SH cells after 2 days post inoculation.By immunofluorescence assay,the virus particles could be detected and visualized.Conclusion TBEV can replicate andproliferate effcctively and cause significant cell morphological changes in human neuroblastoma cell SK-N-SH,which demonstrated that SK-N-SH could be a suitable cell model for TBEV culture.

OBJECTIVETo identify the clinical predictors of atrial fibrillation (AF) after coronary artery bypass grafting (CABG).

METHODS322 consecutive patients who had undergone isolated CABG were reviewed. Preoperative, intraoperative and postoperative data were collected. Patients were grouped according to whether AF appeared postoperatively.

RESULTSAF occurred in 75 patients (23.3%). Most cases of AF (85.6%) appeared on or before the third postoperative day. The mean age for patients with AF was 62.5 years compared with 56.7 years for patients without AF (P < 0.05). The mean aortic crossclamp time for patients with AF was 67 min compared with 60.3 min for patients without AF (P < 0.05). The mean duration of cardiopulmonary bypass for patients with AF was 109.6 min compared with 97.3 min for patients without AF (P < 0.05). The mean duration of mechanical ventilation for patients with AF was 19.1 h compared with 15.7 h for patients without AF (P < 0.05). Multivariate logistic regression analysis was used to identify the following independent predictors of postoperative AF (P < 0.05): age > or = 65 years (OR 2.7; 95% CI 1.5 to 5.1), lesions in the right coronary artery (OR 2.5; 95% CI 1.4 to 4.5), and early postoperative withdrawal of beta blocker (OR 3.9; 95% CI 2.1 to 7.7).

CONCLUSIONSAF remains the most common complication after CABG. Age and lesions in the right coronary artery can influence the incidence of AF, and beta blocker and magnesium may be the most economical and effective prevention for AF early after CABG.

Adult ; Age Factors ; Aged ; Atrial Fibrillation ; diagnosis ; etiology ; Coronary Artery Bypass ; adverse effects ; Coronary Vessels ; pathology ; surgery ; Female ; Humans ; Male ; Middle Aged ; Multivariate Analysis ; Postoperative Complications ; Prognosis

Objective:To deeply explore the supportive care needs of patients with inflammatory bowel disease, in order to provide reference for the development of supportive care strategies.Methods:A qualitative description was conducted based on the supportive care needs framework. Objective sampling method was used to select 17 patients with inflammatory bowel disease from the First Affiliated Hospital with Nanjing Medical University from August to November 2021 for semi-structured in-depth interview and directed content analysis was used to analyze the interview data.Results:The final analysis yielded a total of 5 categories. These were physiological need, information need, practical (daily life) need, emotional and social support need, psychological and spiritual needs. They were all within the framework of supporting care needs.Conclusions:In the future, targeted education and diversified social support should be implemented based on the needs of patients with inflammatory bowel disease and from the perspectives of hospital, family members and peers.