Objective To construct short hairpin RNA (shRNA) expression plasmids against the inhibitor of differentiation 2 (Id2) gene and establish a suitable cell model for the role of Id2 in proliferation and differentiation of human Ewing sarcoma cell.Methods Three shRNA sequences targeting Id2 gene were designed and inserted into the pGPU6/GFP/Neo (-shRNA-Id2) expression vectors.The recombinant pGPU6/GFP/Neo-shRNA-Id2 plasmids were introduced into Ewing sarcoma RD-ES cells by liposome-mediated transfection.The knock-down efficiency of Id2 in infected RD-ES cells was verified by Western blot assay.The cell growth and cell cycle changes were evaluated by cell counting and flow cytometry between transfected cells and control cells.Results The Id2 expression decreased 54 ％ and 57 ％,respectively,in RD-ES cell line which were transfected with the shRNA-Id2-543 and shRNA-Id2-593 plasmids compared with the control group cells by Western blot analysis.The cell growth assay demonstrated that the cell number in transfected cells was significantly decreased during 6-7 d compared with the control group (P ＜ 0.05).The cells at the S-phase of cell cycle were increased [(36.60±1.53) ％ and (44.89E2.46) ％ vs (29.73±2.03) ％,P ＜ 0.05],and no significant changes at the G2 phase or even reduction in the transfected cells.Conclusions Id2 stable knock-down cell lines are successfully established.The reduced expression of Id2 is related with decreased cell growth and cell cycle arrest in the S-phase.Id2 maybe plays an important role in proliferation of Ewing sarcoma cell.

Early recognition and treatment for early warning electrocardiogram (ECG) of sudden death are very important to prevent and treat malignant arrhythmia and sudden death. Previous studies have found that R-on-T and T wave alternation, and QT interval prolongation are closely related to malignant arrhythmia or sudden death, which are included in the critical value of ECG.By analyzing the ECG characteristics of 4 patients with sudden death, we found that although the causes of the patients were different, there were transient prolongation of QT interval after premature contraction in 12 lead ECG, followed by malignant arrhythmia or sudden death. Thus, we thought that the transient prolongation of QT interval after premature contraction had a high value for warning malignant arrhythmia or sudden death. This phenomenon should be paid enough attention to reduce the risk of sudden death.
Arrhythmias, Cardiac/diagnosis* ; Death, Sudden ; Death, Sudden, Cardiac ; Electrocardiography ; Humans ; Long QT Syndrome/diagnosis*

Objective To investigate the expression level of neutrophil extracellular traps (NET) in the peripheral blood and liver tissue of primary biliary cholangitis (PBC) patients and its correlation with clinical biochemical parameters. Methods A total of 24 PBC patients who were admitted to Renji Hospital, Shanghai Jiao Tong University School of Medicine, from August 2016 to August 2020 were enrolled, as well as 8 patients with primary sclerosing cholangitis (PSC) and 19 patients with autoimmune hepatitis (AIH) matched for age, and 19 healthy individuals were enrolled as healthy control group (HC group). The serum level of myeloperoxidase (MPO) was measured, and its correlation with clinical indices were analyzed. Immunofluorescence assay was used to measure the expression of NET in the liver of PBC patients, and an in vitro experiment was to compare the ability of peripheral blood neutrophils to produce NET between PBC patients and healthy controls. Normally distributed continuous data were expressed as mean±standard deviation, and the independent samples t -test was used for comparison between two groups; for the non-normally distributed continuous data expressed as M ( P 25 - P 75 ), the Kruskal-Wallis H test was used for comparison between multiple groups, and the Mann-Whitney U test was used for comparison between two groups. A correlation analysis was performed for MPO level and liver-related laboratory markers, and the Spearman's correlation coefficient was calculated. Results The serum level of MPO in the PBC group was increased to 811.21 (450.67-1 216.20) ng/mL, which was significantly higher than that in the AIH group [468.58 (142.63-812.43) ng/mL] and the HC group [357.54 (203.52-811.21) ng/mL] ( P < 0.05), suggesting that there was a significant increase in the production of NET in peripheral blood of PBC patients. The PSC patients had a serum MPO level of 763.56 (489.59-1 633.14) ng/mL, which was significantly higher than that in the HC group ( P < 0.05). MPO level was positively correlated with alkaline phosphatase ( r =0.500, P < 0.05), gamma-glutamyl transpeptidase ( r =0.426, P < 0.05), alanine aminotransferase ( r =0.521, P < 0.05), and aspartate aminotransferase ( r =0.547, P < 0.01). Confocal immunofluorescence showed colocalization of H3Cit and MPO in the liver of PBC patients. In vitro experiment showed that compared with the HC group, the PBC group had an increase in NET produced by peripheral blood neutrophils after in vitro stimulation and an increase in spontaneous production of NET. Conclusion There is an increase in NET in peripheral blood and liver of PBC patients, and the content of peripheral blood NET is positively correlated with biochemical parameters of liver function. NET may become a novel biomarker for assessing the severity of PBC.