Proinsulin (Pins) is the precursor of insulin. The expression of proinsulin in Escherichia coli forms inclusion body, so that the recombinant protein should be processed with multiple steps to form active insulin. With the development in biotechnology, cell-free protein synthesis (CFPS) system is becoming a valuable tool in protein expression by decoupling the cell growth with protein production, which allows it to express proteins that would interfere with cell physiology. In this study, we synthesized soluble proinsulin in CFPS system in order to establish a new approach for both insulin expression and delivery. The soluble proinsulin was successfully expressed in CFPS system by fusing proinsulin with two types of fluorescent protein. The expression of Pins-mCherry was confirmed by Western blotting analysis, and the Pins-eGFP titer was (12.28±3.45) μg/mL in CFPS system. These results implicated that the proinsulin was expressed partially in soluble form. Here, for the first time, we successfully expressed soluble proinsulin in CFPS system by fluorescent protein fusion. These results provide useful information in developing new insulin expression and delivery method.

Objective:To evaluate myocardial microcirculation perfusion with myocardial contrast echocardiography (MCE) in patients with acute myocardial infarction after percutaneous coronary intervention (PCI), and to explore the prognostic value of different types of myocardial microcirculation perfusion.Methods:This is a prospective cohort study. Patients with acute myocardial infarction who underwent successful PCI in Nanfang Hospital of Southern Medical University and Kanghua Hospital of Dongguan City from October 2019 to June 2021 were selected. All the enrolled patients completed MCE examination within 72 hours after PCI. According to the examination results, the patients were divided into normal microcirculation perfusion group, delayed microcirculation perfusion group, and blocked microcirculation perfusion group. Adverse cardiovascular events including all-cause death, cardiovascular death, and angina re-hospitalization were followed up, and left ventricular ejection fraction (LVEF) review results were collected at six months to one year after surgery. Kaplan-Meier survival curve was used to investigate the difference in the incidence of adverse cardiovascular events in different myocardial perfusion groups, and Cox regression analysis was used to evaluate the effect of myocardial perfusion on adverse cardiovascular events.Results:A total of 113 patients with acute myocardial infarction were included, aged (56.3±11.5) years, with 88(78%) males. There were 31 cases in the normal microcirculation perfusion group, 43 cases in the delayed microcirculation perfusion group and 39 cases in the blocked microcirculation perfusion group. LVEF was reviewed in 49 patients, and LVEF in the delayed microcirculation perfusion group was significantly improved compared with baseline at follow-up ((63.3±1.2) % vs. (58.6±1.8) %, P=0.043), and there was no statistically significant difference between the other two groups (all P>0.05). The median follow-up time was 473 days, during follow-up period 30 adverse cardiovascular events occurred. Kaplan-Meier survival curve analysis showed that there was a statistically significant difference in the incidence of adverse cardiovascular events among the three groups ( Plog-rank=0.029). Cox regression analysis showed that abnormal microcirculation perfusion (defined as delayed and blocked microcirculation perfusion) was an independent predictor of adverse cardiovascular events in patients with acute myocardial infarction after PCI ( HR=1.90, 95% CI1.16-3.12, P=0.011). Conclusions:Microcirculatory perfusion decrease or lost is common in patients with acute myocardial infarction after PCI. Timely restoration of blood flow reconstruction can save heart function when microcirculatory perfusion decreases. Microcirculatory perfusion is a predictor of adverse cardiovascular events in patients with acute myocardial infarction, and patients with poor myocardial perfusion are more likely to experience adverse cardiovascular events.

Objective: To discuss the taxonomy and molecular characteristics of one virus strain (SZC30) isolated from Culicoides in Yunnan. Methods: Culicoides were collected with light trap method in the Wulong Village of Shizong County of Yunnan Province in July, 2013. BHK-21 and C6/36 cells were used for virus isolation. The positive isolates were inoculated into brain of one-day suckling mice. Alphavirus and Getah virus specific primers were used to amplify the genome of the virus isolation by RT-PCR. The products of RT-PCR were sequenced. Clustal X1.83, DNAStar, Mega5.1 were used for bioinformatics analysis. Results: Totally 3 500 culicoides were collected and divided into 41 batches for virus isolation. One isolate (SZC30) produced cytopathic effect (CPE) on BHK-21 and C6/36 cells; the result of RT-PCR with Alphavirus and Getah virus NS1 specific primer were positive; the sequence analysis of NS1 gene suggested that SZC30 and two Getah virus strains (YN0540, SC1210) from China were in the same evolutionary branching, the nucleotide homology were 97%-100%, and the amino acid was 97%-100%. Conclusions SZC30 isolated in Yunnan province was identified as Getah virus.

As a ligand-dependent transcription factor,retinoid-associated orphan receptor γt(RORyt)that controls T helper(Th)17 cell differentiation and interleukin(IL)-17 expression plays a critical role in the pro-gression of several inflammatory and autoimmune conditions.An emerging novel approach to the therapy of these diseases thus involves controlling the transcriptional capacity of RORyt to decrease Th17 cell development and IL-17 production.Several RORyt inhibitors including both antagonists and inverse agonists have been discovered to regulate the transcriptional activity of RORyt by binding to orthosteric-or allosteric-binding sites in the ligand-binding domain.Some of small-molecule inhibitors have entered clinical evaluations.Therefore,in current review,the role of RORyt in Th17 regulation and Th17-related inflammatory and autoimmune diseases was highlighted.Notably,the recently developed RORyt inhibitors were summarized,with an emphasis on their optimization from lead compounds,ef-ficacy,toxicity,mechanisms of action,and clinical trials.The limitations of current development in this area were also discussed to facilitate future research.

Objective: To investigate the positive rate for 2019-nCoV tests and co-infections in Wuhan district. Methods: A total of 8 274 cases in Wuhan were enrolled in this cross-sectional study during January 20 to February 9, 2020, and were tested for 2019-nCoV using fluorescence quantitative PCR. Both respiratory tract samples (nasopharynx, oropharynx, sputum and alveolar lavage fluid) and non-respiratory tract samples (urine, feces, anal swabs, blood and conjunctival sac swabs) were collected. If both orf1ab and N genes are positive, they are classified as nucleic acid test positive group; if both orf1ab and N genes are negative, they are classified as negative group; if single gene target is positive, they are classified as suspicious group. Individuals were divided into male group and female group according to sex. At the same time, 316 patients were tested for 13 respiratory pathogens by multiplex PCR. Results: Among the 8 274 subjects, 2 745 (33.2%) were 2019-nCoV infected; 5 277 (63.8%) subjects showed negative results in the 2019-nCoV nucleic acid test; and 252 cases (3.05%) was not definitive (inconclusive result). The age of cases with COVID-19 patients and inconclusive cases was significantly higher than that of cases without 2019-nCoV infection (40 vs 56, t=27.569, P<0.001; 52 vs 56, t=6.774, P<0.001). The positive rate of 13 respiratory pathogens multiple tests was significantly lower in 104 subjects who were positive for 2019-nCoV compared with those in subjects who were negative for 2019-nCoV test (5.77% vs 18.39%, χ²=24.105, P=0.003). Four types of respiratory tract samples and five types of non-respiratory tract samples were found to be positive for 2019-nCoV nucleic acid test. Conclusion The 2019-nCoV nucleic acid positive rate in male is higher than in female. Co-infections should be pay close attention in COVID-19 patients. 2019-nCoV nucleic acid can be detected in non-respiratory tract samples.