Comparison and evaluation of laboratory test techniques for severe fever with thrombocytopenia syndrome / 中华实验和临床病毒学杂志

Objective@#Compare the detection result of blood samples of severe fever with thrombocytopenia syndrome (SFTS) patients using different detection techniques, and observe the dynamic characteristics of the virus specific RNA, IgM antibody and IgG antibody, to provide theoretical basis for selection of diagnostic methods of disease.@*Methods@#Acute phase serum of suspected SFTS cases and convalescent serum samples of lab-confirmed cases were collected. Real-time fluorescence quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were used to detect the virus specific RNA, IgM antibody and IgG antibody. The detection results of different methods, the relationship between positive results and the acquisition time, and the dynamic characteristics of viral nucleic acid and antibodies were analyzed.@*Results@#A total of 87 serum samples of the suspected SFTS patients were collected, the positive rate of virus specific RNA, IgM antibody and IgG antibody were 53.41%, 31.03% and 3.41%, respectively. Among 55 confirmed cases of SFTS, the consistent rate of virus specific RNA and IgM antibody detection methods was 36.36%, and the difference between the two methods was significant (χ2=6.82, P=0.009), kappa=-0.257. The sampling intervals of RNA positive samples were all within 12 days, of which the positive detection rate was highest after 7-9 days, and the difference was statistically significant (χ2=10.35, P=0.016). In 34 SFTS convalescent serum samples, all the nucleic acid tests were negative, the positive rate of IgM antibody was 41.18%, which was not significantly different from the acute phase serum samples (P=1.00). The positive rate of IgG antibody was 94.12%, which was significantly higher than that of acute IgG antibody (0%). The dynamic characteristics of IgM and IgG antibody showed that IgM antibody could be detected on the second day after onset, the latest detection time was 74 days after onset, and the highest absorbance value and antibody detection rate occurred in 30-60 days. The earliest detection time of IgG antibody was 12 days after onset, and the last detection time was 100 days.The detection rate of IgG antibody and absorbance value increased rapidly after 30 days, and maintained in a high level. The detection rate of IgG antibody was 100% in 30-60 days.@*Conclusions@#Blood samples taken from SFTS suspected patients within two weeks of onset may be prioritized for detection of viral nucleic acids using Real-time fluorescence PCR or for detection of IgM antibodies by ELISA. Although IgM antibody can be detected 2 days after the onset, the peak appeared much later, so the negative result can’t rule out the diagnosis. IgG antibody has a high seroconversion rate in convalescent samples, and can be used as an auxiliary tool for disease diagnosis.

Elimination of inter-domain interactions increases the cleavage fidelity of the restriction endonuclease DraIII

DraIII is a type IIP restriction endonucleases (REases) that recognizes and creates a double strand break within the gapped palindromic sequence CAC↑NNN↓GTG of double-stranded DNA (↑ indicates nicking on the bottom strand; ↓ indicates nicking on the top strand). However, wild type DraIII shows significant star activity. In this study, it was found that the prominent star site is CAT↑GTT↓GTG, consisting of a star 5' half (CAT) and a canonical 3' half (GTG). DraIII nicks the 3' canonical half site at a faster rate than the 5' star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the DraIII protein was solved. It indicated, as supported by mutagenesis, that DraIII possesses a ββα-metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through site-directed mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases.

Effect of the down regulation of p120-catenin on the proliferation and invasion of human bladder carcinoma cell line T24 in Vitro / 中国综合临床

Objective To investigate the regulating function of p120-catenin (p120ctn) on proliferation and invasion of human bladder cancer cell T24.Methods Special siRNA was used to silence the expression of p120ctn in T24 cells.MTT assay was used to examine the T24 cell growth rate.The invasion of T24 cells and the control cells were measured by transwell chamber assay.Results The silencing of p120ctn could improve the proliferation and the invasiveness of T24 tumor cells.Transwell chamber assay showed that the in vitro invasion function of T24 cells was significant increase after p120ctn-siRNA.Conclusion p120ctn inhibited the bladder cancer proliferation and invasiveness of bladder carcinoma cells.

Differentiation from rat bone marrow stromal stem cells into cardiomyocytes induced by 5-azacitidine in vitro / 中国组织工程研究

BACKGROUND: Bone marrow stromal stem cells (BMSCs) are characterized by rapid amplification and wide differentiation.Thus, to establish in vitro BMSC induction models contributes to the study of tissue engineering.OBJECTIVE: To investigate the possibility of inducing the differentiation of rat BMSCs into cardiomyocytes by 5-azacitidine in vitro.OESING, TIME AND SETTING: The cytological in vitro study was performed at the Central Laboratory of First Affiliated Hospital of Liaoning Medical University from June 2005 to June 2008.MATERIALS: A total of 20 Sprague Dawley rats were supplied by the Experimental Animal Center, Liaoning Medical University.5-azacitidine (Sigma, USA) was used.MHEHODS: Following anesthesia, the rats were used to isolate the femur and tibia. BMSCs were isolated and cultured by the whole bone marrow method + adherent method. When 90% BMSCs were confluence, BMSCs were passaged. BMSCs at the third passage were incubated in a 24-well plate at 2×10~4/well, with 5,10, 15, 20 μmol/L 5-azacitidine. Simultaneously, a blank control group (without inductor) was set. Following 24 hours of induction, BMSCs were incubated in normal medium for 3 weeks.MAIN OUTCOME MEASURES: The following parameters were measured: cell appearance and growth curve, morphological changes following induction, and expression of connexin-43 and a-striated muscle actin.RESULTS: Cultured BMSCs were spindle-shape, with some cell confluence. P3 cells following incubation entered static phase at 1 and 2 days, and entered logarithmic phase at 3 days, reached a peak at 9 days, and then entered platform stage. Cell number became decreased at 12 days. Following induction of 5 μmol/L 5-azacitidine, no significant difference was found in BMSCs.Following induction of 10 μmol/L 5-azacitidine, BMSCs became long and big, extended towards a direction, with the property of myotube formation cells. Following induction of 15 umol/L 5-azacitidine, a few cells survived surrounding the 24-well plate.Following induction of 20 μmol/L 5-azacitidine, cells died. Following induction of 10 μmol/L 5-azacitidine for 3 weeks, expression of connexin-43 and a-striated muscle actin was determined in BMSCs. However, a negative expression was detected in the blank control group.CONCLUSION: BMSCs cannot differentiate into cardiomyocytes by itself. Following in vitro induction, BMSCs can differentiate into cardiomyocytes. Low-dose 5-azacitidine concentration cannot induce the differentiation, but high-dose 5-azacitidine concentration will induce death in a large number of cells. Thus, 10 μmol/L is an optimal concentration.