SARS-CoV-2 subgenomic RNA: formation process and rapid molecular diagnostic methods.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which caused coronavirus disease-2019 (COVID-19) is spreading worldwide and posing enormous losses to human health and socio-economic. Due to the limitations of medical and health conditions, it is still a huge challenge to develop appropriate discharge standards for patients with COVID-19 and to use medical resources in a timely and effective manner. Similar to other coronaviruses, SARS-CoV-2 has a very complex discontinuous transcription process to generate subgenomic RNA (sgRNA). Some studies support that sgRNA of SARS-CoV-2 can only exist when the virus is active and is an indicator of virus replication. The results of sgRNA detection in patients can be used to evaluate the condition of hospitalized patients, which is expected to save medical resources, especially personal protective equipment. There have been numerous investigations using different methods, especially molecular methods to detect sgRNA. Here, we introduce the process of SARS-CoV-2 sgRNA formation and the commonly used molecular diagnostic methods to bring a new idea for clinical detection in the future.

LncRNA KCNQ1OT1 ameliorates the liver injury induced by acetaminophen through the regulation of miR-122-5p/CES2 axis.

Long noncoding RNAs (lncRNAs) have been shown to be implicated in acetaminophen (APAP)-induced liver injury (AILI). We applied this study to investigate the role and functional mechanism of KCNQ1 overlapping transcript 1 (KCNQ1OT1) in AILI. The AILI model was established by APAP treatment in mice. The liver injury was preliminarily evaluated by ALT and AST activities via the detection kits. The quantitative real-time polymerase chain reaction (qRT-PCR) was exploited for detecting the expression of KCNQ1OT1, microRNA-122-5p (miR-122-5p), and carboxylesterase 2 (CES2). Protein levels were analyzed via Western blot. 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT) assay, and flow cytometry were separately applied to determine cell proliferation and apoptosis rate. Inflammation was assessed by enzyme-linked immunosorbent assay (ELISA). Dual-luciferase reporter assay was implemented to testify the intergenic combination. The function of KCNQ1OT1 in vivo was explored through KCNQ1OT1 knockdown in mice. APAP triggered the downregulation of KCNQ1OT1 and CES2 in mice serums. KCNQ1OT1 upregulation could relieve the AILI in HepaRG cells, which were abrogated by CES2 downregulation. KCNQ1OT1 served as a sponge of miR-122-5p and miR-122-5p directly targeted CES2. KCNQ1OT1 overexpression abated the AILI through the miR-122-5p/CES2 axis in HepaRG cells in vitro and mice in vivo. The collective results clarified that KCNQ1OT1 weakened the AILI in vitro and in vivo by the miR-122-5p/CES2 axis, providing an explicit molecular mechanism and selectable therapeutic strategy of AILI.

Factors affecting microvascular complications among patients with type 2 diabetes mellitus / 预防医学

Objective @#To identify the factors affecting microvascular complications among patients with type 2 diabetes (T2DM), so as to provide insights into the management of microvascular complications of T2DM.@*Methods@#T2DM patients hospitalized in the Department of Endocrinology of a tertiary hospital in Weifang City, Shandong Province from January 2021 to January 2022 were enrolled, and subjects' basic information, lifestyle and medical history were collected using questionnaire surveys. Fasting insulin, fasting blood glucose and glycated hemoglobin were measured, and factors affecting microvascular complications were identified among T2DM patients using a multivariable logistic regression model and a decision tree model.@*Results@#Totally 1 003 T2DM inpatients were enrolled, including 515 men (51.35%) and 488 women (48.65%), and the prevalence of microvascular complications was 40.18%. Multivariable logistic regression analysis showed that age of 60 years and older (OR=2.510, 95%CI: 1.441-4.374), T2DM duration of 10 years and longer (OR=3.205, 95%CI: 2.242-4.581), fasting insulin of lower than 3.21 μIU/mL (OR=1.749, 95%CI: 1.239-2.469), using of agents or insulin to control blood glucose (OR=1.880, 95%CI: 1.143-3.092), glycated hemoglobin level of 7% and higher (OR=1.751, 95%CI: 1.172-2.615) as factors affecting microvascular complications among T2DM patients. Decision tree analysis identified course of T2DM as a major factor affecting the risk of microvascular complications among T2DM patients, and the prevalence of microvascular complications was 70.22% among T2DM patients with disease course of 10 years and longer and fasting insulin of lower than 3.21 μIU/mL or 16.32 μIU/mL and higher, 44.23% among T2DM patients with disease course of 5 to 10 years and at ages of 60 years and older, and 43.10% among T2DM patients with disease course of less than 5 years and fasting insulin of lower than 3.21 μIU/mL. @*Conclusion@#Advanced age, long course of T2DM, low fasting insulin and high glycated hemoglobin may increase the risk of microvascular complications among T2DM patients.

Expression of cecropin B in Pichia pastoris and its bioactivity in vitro.

Natural cecropin B (CB), consisting of 35 amino acids, is a member of the cecropin family with the highest antibacterial activity. Here, a novel approach for the generation of recombinant CB in the methylotrophic yeast Pichia pastoris was explored. For this purpose, the CB gene was amplified by recursive PCR (rPCR) and cloned into the pPICZαA vector. The SacI-linearized plasmid pPICZαA-CB was transformed into P. pastoris SMD1168 by electroporation. The expression of recombinant CB was induced with 1.0% methanol at pH 5.0 for 60 h at 28°C. Recombinant CB was purified using cationic exchange chromatography; 5.0 mg of pure active CB was obtained from 100 ml of culture broth supernatant. Antimicrobial assays demonstrated that CB has a broad spectrum of antimicrobial activity against both Gram-positive and Gram-negative bacteria. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional CB for both research and industrial purposes.