MicroRNA miR-212-5p Regulates the MEK/ERK Signaling Pathway by Targeting A-Raf proto-oncogene serine/threonine-protein kinase (ARAF) to Regulate Cowshed PM2.5-Induced NR8383 Apoptosis.

Objective: To investigate the role of miR-212-5p-targeted ARAF during the apoptosis of rat alveolar macrophages induced by cowshed PM2.5. Methods: miRNA and related target genes and pathways were predicted using the KEGG, TargetScan, and other prediction websites. NR8383 macrophages were treated with cowshed PM2.5 to establish an in vitro lung injury model in rats; meanwhile, for the assessment of cell viability, apoptosis, intracellular calcium ions, and mitochondrial membrane potential in NR8383 cells, RT-qPCR was used to detect the expression of miR-212-5p and the target gene ARAF. Results: The bioinformatic analyses showed that miR-212-5p and ARAF were involved in PM2.5-associated cellular damage. Exposure to different concentrations (0 µg/mL, 60 µg/mL, 180 µg/mL, 300 µg/mL) with different durations (0 h, 12 h, 24 h, 48 h) of cowshed PM2.5 resulted in apoptosis, increased intracellular calcium ions, and decreased mitochondrial membrane potential. The miR-212-5p mimic group showed an up-regulation of Bax and cleaved Caspase 3 expression but decreased Bcl2 expression compared to the NC group, and overexpression of ARAF up-regulated the expression of p-MEK1/2 and p-ERK1/2 and simultaneously reversed the above phenomena. Conclusions: miR-212-5p targets ARAF to affect the cowshed PM2.5-induced apoptosis through the MEK/ERK signaling pathway, providing a potential target for relevant farming industry and pathology studies.

Preparation, purification, and biochemical of fat-degrading bacterial enzymes from pig carcass compost and its application.

BACKGROUND: A lot of kitchen waste oil is produced every day worldwide, leading to serious environmental pollution. As one of the environmental protection methods, microorganisms are widely used treating of various wastes. Lipase, as one of the cleaning agents can effectively degrade kitchen waste oil. The composting process of pig carcasses produces many lipase producing microorganisms, rendering compost products an excellent source for isolating lipase producing microorganisms. To our knowledge, there are no reports isolating of lipase producing strains from the high temperature phase of pig carcass compost. METHODOLOGY: Lipase producing strains were isolated using a triglyceride medium and identified by 16S rRNA gene sequencing. The optimal fermentation conditions for maximum lipase yield were gradually optimized by single-factor tests. The extracellular lipase was purified by ammonium sulfate precipitation and Sephadex G-75 gel isolation chromatography. Amino acid sequence analysis, structure prediction, and molecular docking of the purified protein were performed. The pure lipase's enzymatic properties and application potential were evaluated by characterizing its biochemical properties. RESULTS: In this study, a lipase producing strain of Bacillus sp. ZF2 was isolated from pig carcass compost products, the optimal fermentation conditions of lipase: sucrose 3 g/L, ammonium sulfate 7 g/L, Mn2+ 1.0 mmol/L, initial pH 6, inoculum 5%, temperature 25 ℃, and fermentation time 48 h. After purification, the specific activity of the purified lipase reached 317.59 U/mg, a 9.78-fold improvement. Lipase had the highest similarity to the GH family 46 chitosanase and molecular docking showed that lipase binds to fat via two hydrogen bonds at Gln146 (A) and Glu203 (A). Under different conditions (temperature, metal ions, organic solvents, and surfactants), lipase can maintain enzymatic activity. Under different types of kitchen oils, lipase has low activity only for 'chicken oil', in treating other substrates, the enzyme activity can exceed 50%. CONCLUSIONS: This study reveals the potential of lipase for waste oil removal, and future research will be devoted to the application of lipase.

Extraction, Purification, Characterization and Application in Livestock Wastewater of S Sulfur Convertase.

Sulfide is a toxic pollutant in the farming environment. Microbial removal of sulfide always faces various biochemical challenges, and the application of enzymes for agricultural environmental remediation has promising prospects. In this study, a strain of Cellulosimicrobium sp. was isolated: numbered strain L1. Strain L1 can transform S2-, extracellular enzymes play a major role in this process. Next, the extracellular enzyme was purified, and the molecular weight of the purified sulfur convertase was about 70 kDa. The sulfur convertase is an oxidase with thermal and storage stability, and the inhibitor and organic solvent have little effect on its activity. In livestock wastewater, the sulfur convertase can completely remove S2-. In summary, this study developed a sulfur convertase and provides a basis for the application in environmental remediation.

Development of a microbial protease for composting swine carcasses, optimization of its production and elucidation of its catalytic hydrolysis mechanism.

BACKGROUND: Dead swine carcass composting is an excellent method for harmless treatment and resource utilization of swine carcass. However, poor biodegradation ability of traditional composting results in poor harmless treatment effect. Researches report that the biodegradation ability of composting can be improved by inoculation with enzyme-producing microorganisms or by inoculation with enzyme preparations. At present, the researches on improving the efficiency of dead swine carcass composting by inoculating enzyme-producing microorganisms have been reported. However, no work has been reported on the development of enzyme preparations for dead swine carcass composting. METHODOLOGY: The protease-producing strain was isolated by casein medium, and was identified by 16 S rRNA gene sequencing. The optimal fermentation conditions for maximum protease production were gradually optimized by single factor test. The extracellular protease was purified by ammonium sulfate precipitation and Sephadex G-75 gel exclusion chromatography. The potential for composting applications of the purified protease was evaluated by characterization of its biochemical properties. And based on amino acid sequence analysis, molecular docking and inhibition test, the catalytic hydrolysis mechanism of the purified protease was elucidated. RESULTS: In this study, a microbial protease was developed for swine carcass composting. A protease-producing strain DB1 was isolated from swine carcass compositing and identified as Serratia marcescen. Optimum fermentation conditions for maximum protease production were 5 g/L glucose, 5 g/L urea, 1.5 mmol/L Mg2+, initial pH-value 8, inoculation amount 5%, incubation temperature 30 °C and 60 h of fermentation time. The specific activity of purified protease reached 1982.77 U/mg, and molecular weight of the purified protease was 110 kDa. Optimum pH and temperature of the purified protease were 8 and 50 °C, respectively, and it had good stability at high temperature and in alkaline environments. The purified protease was a Ser/Glu/Asp triad serine protease which catalyzed substrate hydrolysis by Glu, Arg, Ser, Asp and Tyr active residues. CONCLUSIONS: In general, the microbial protease developed in this study was suitable for industrial production and has the potential to enhance composting at thermophilic stage. Moreover, the catalytic hydrolysis mechanism of the protease was further analyzed in this study.

In Vitro Activity of Various Antibiotics in Combination with Tigecycline Against Acinetobacter baumannii: A Systematic Review and Meta-Analysis.

Given that tigecycline-based combination therapy is recognized as a valuable option for the treatment of tigecycline-resistant Acinetobacter baumannii, we conducted this systematic review and meta-analysis to assess the overall evidence of its effectiveness. The synergy rate was defined as the primary outcome that was calculated separately for time-kill, Etest, and checkerboard microdilution methods. The secondary outcomes were bactericidal activity and the efficacy of combination treatment on the development of resistance. In total, 37 published papers and 16 conference proceedings were included. Nine classes consisting of 22 antibiotic types in combination with tigecycline against 1,159 A. baumannii strains were reported in the analysis. For the time-kill studies, combination therapy showed a synergy rate of 37.9% (95% confidence interval [CI], 30.7-46.5); the highest synergy rate was 67.4% (95% CI, 27.3-91.9) for tigecycline in combination with colistin. Moreover, combination with amikacin or colistin could efficiently inhibit the development of tigecycline resistance. Compared with checkerboard microdilution and Etest methods, time-kill studies always showed higher synergy rates. Altogether, these results suggest that the in vitro tigecycline-based combinations resulted in moderate synergy rates and that several combinations could suppress the resistance of A. baumannii to tigecycline, which should be further confirmed in animal models and clinical trials.