Simultaneous detection of viable Salmonella spp., Escherichia coli, and Staphylococcus aureus in bird's nest, donkey-hide gelatin, and wolfberry using PMA with multiplex real-time quantitative PCR.

Salmonella spp., Escherichia coli, and Staphylococcus aureus are common microbial contaminants within the homology of medicine and food that can cause serious food poisoning. This study describes a highly efficient, sensitive, specific, and simple multiplex real-time quantitative PCR (mRT-qPCR) method for the simultaneous detection of viable Salmonella spp., E. coli, and S. aureus. Primers and probes were designed for the amplification of the target genes invA, uidA, and nuc. Dead bacterial genetic material was excluded by propidium monoazide (PMA) treatment, facilitating the detection of only viable bacteria. This method was capable of detecting Salmonella spp., E. coli, and S. aureus at 102, 102, and 101 CFU/ml, respectively, in pure culture. PMA combined with mRT-qPCR can reliably distinguish between dead and viable bacteria with recovery rates from 95.7% to 105.6%. This PMA-mRT-qPCR technique is a highly sensitive and specific method for the simultaneous detection of three pathogens within the homology of medicine and food.

Efficacy and safety of umbilical cord-derived mesenchymal stem cells in Chinese adults with type 2 diabetes: a single-center, double-blinded, randomized, placebo-controlled phase II trial.

BACKGROUND: To determine the efficacy and safety of umbilical cord-derived mesenchymal stem cells (UC-MSCs) in Chinese adults with type 2 diabetes mellitus (T2DM). METHODS: In this single-center, double-blinded, randomized, placebo-controlled phase II trial, 91 patients were randomly assigned to receive intravenous infusion of UC-MSCs (n = 45) or placebo (n = 46) three times with 4-week intervals and followed up for 48 weeks from October 2015 to December 2018. The primary endpoint was the percentage of patients with glycated hemoglobin (HbA1c) levels of < 7.0% and daily insulin reduction of ≥ 50% at 48 weeks. Additional endpoints were changes of metabolic control, islet ß-cell function, insulin resistance, and safety. RESULTS: At 48 weeks, 20% of the patients in the UC-MSCs group and 4.55% in the placebo group reached the primary endpoint (p < 0.05, 95% confidence interval (CI) 2.25-28.66%). The percentage of insulin reduction of the UC-MSCs group was significantly higher than that of the placebo group (27.78% versus 15.62%, p < 0.05). The levels of HbA1c decreased 1.31% (9.02 ± 1.27% to 7.52 ± 1.07%, p < 0.01) in the UC-MSCs group, and only 0.63% in the placebo group (8.89 ± 1.11% to 8.19 ± 1.02%, p˃0.05; p = 0.0081 between both groups). The glucose infusion rate (GIR) increased significantly in the UC-MSCs group (from 3.12 to 4.76 mg/min/kg, p < 0.01), whereas no significant change was observed in the placebo group (from 3.26 to 3.60 mg/min/kg, p ˃ 0.05; p < 0.01 between both groups). There was no improvement in islet ß-cell function in both groups. No major UC-MSCs transplantation-related adverse events occurred. CONCLUSIONS: UC-MSCs transplantation could be a potential therapeutic approach for Chinese adults with T2DM. Trial registration This study was registered on ClinicalTrials.gov (identifier: NCT02302599).

[Preparation of monoclonal antibody against enterovirus 71 capsid protein VP1].

Objective To prepare murine monoclonal antibody (mAb) against enterovirus 71 (EV71) capsid protein VP1. Methods VP1 protein was expressed and purified. BALB/c mice were immunized with inactivated and purified EV71 virus, and mAb specific to EV71 VP1 was generated by hybridoma technique. Indirect ELISA was used to test antibody titer and antibody subclass identification. The expression of VP1 protein was detected by Western blot in EV71-infected RD cells. The expression and distribution of VP1 protein was detected by immunofluorescence cytochemistry in EV71-infected RD cells. Results Six antibody strains were obtained, among which three were IgG2a and three were IgG2b, all of which could be used for ELISA, Western blot and immunofluorescence cytochemical staining. 2D7 exhibited neutralization capacity with 50% inhibitory concentrations(IC50) of 9.892 µg/mL. Conclusion Six strains of monoclonal antibodies with excellent reactivity were obtained, which laid a foundation for the further studies on the identification and diagnosis of EV71 as well as the functional of VP1 protein.

Chronic intermittent hypoxia induces the pyroptosis of renal tubular epithelial cells by activating the NLRP3 inflammasome.

Obstructive sleep apnea syndrome (OSAS) is a respiratory disorder and chronic intermittent hypoxia (CIH) is an important pathological characteristic of OSAS. Injuries on renal tubular epithelial cells were observed under the condition of CIH. Pyroptosis is a programmed mode of cell death following cell apoptosis and cell necrosis, which is mediated by NLRP3 signaling. The present study aims to investigate the effects of CIH on the pyroptosis of renal tubular epithelial cells and the underlying mechanism. Firstly, CIH was induced in two renal tubular epithelial cell lines, HK-2 cells and TCMK-1 cells. As the aggravation of hypoxia, an increasing trend of elevated apoptotic rate was observed in HK-2 cells and TCMK-1 cells, accompanied by the excessive release of ROS and LDH, and upregulation of NLRP3. Subsequently, the CIH model was established on rats. The pathological analysis results indicated that in CIH rats, the glomerular bottom membrane and mesangium were slightly thickened and edema was observed in the renal tubule epithelium. More serious injury was observed in the moderate intermittent hypoxia group. The expression level of IL-1ß and IL-18 was promoted as the aggravation of hypoxia, accompanied by the elevated production of LDH and ROS. The expression level of cleaved Caspase-1, Caspase-1, GSDMD, TLR4, MyD88, NF-κB, p-NF-κB, and NLRP3 was found significantly upregulated as the aggravation of hypoxia. Lastly, the pathological changes in rats induced by CIH were dramatically abolished by MCC950, a specific inhibitor of NLRP3. Collectively, CIH triggered the pyroptosis of renal tubular epithelial cells by activating the NLRP3 inflammasome.

Resveratrol affects the migration and apoptosis of monocytes by blocking HMGB1/NF-κB pathway.

BACKGROUND: Chronic inflammation is now recognized as a causal factor of aging. Resveratrol is a non-flavonoid compound that widely exists in plant species, exerting anti-inflammatory effects in vitro and in animal models. The chemotaxis of inflammatory cells and secretion of cytokines are key characters in inflammation response. METHODS: The effects of lipopolysaccharide (LPS) and high mobility group box-1 (HMGB1) chromosomal on the migration, inflammatory response, and apoptosis of monocytes were detected. THP-1 cells were used to study the effects of resveratrol treatment on LPS- and HMGB-induced monocytes. We aimed to investigate the effect of Resveratrol on monocyte migration and the expression of a special cytokine named HMGB1 in THP-1 cells. RESULTS: Resveratrol obviously inhibited THP-1 migration induced by LPS. LPS increased the expression of HMGB1 and its release in THP-1 cells, which were both decreased by resveratrol. Resveratrol inhibited the activity of NF-κB-p65 and the translocation of NF-κB-p65 from nucleus to cytoplasm induced by LPS. In addition, Resveratrol increased LPS and HMGB1-inhibited monocyte apoptosis. Resveratrol inhibited the LPS-induced HMGB1 secretion and its activation through NF-κB pathway. The THP-1 migration induced by LPS was inhibited by resveratrol. CONCLUSIONS: Resveratrol may inhibit monocyte migration and induce apoptosis by blocking downstream HMGB1/NF-κB/MCP-1 signaling pathways, thereby reducing systemic inflammation.