The xylose reductase of Pichia stipitis is one of the most important enzymes. It can be used to build up recombinant Saccharomyces cerevisiae strain for utilizing xylose and producing ethanol. Intercellular redox imbalance caused by NADPH preference over NADH for Pichia stipitis xylose reductase (PsXR) has been considered to be one of the main factors for poor ethanol productivity. Some key amino acids of PsXR, which affect the activity or coenzyme preference, were investigated in our previous study. In this study, Lys21 were rational designed for site-directed mutagenesis to alter coenzyme specificity of PsXR from NADPH and NADH into single NADH. The wild gene and mutagenesis genes were ligated into pET28b, and were transferred into E.coli BL21(DE3). After induced by IPTG, the xylose reductases were purified. Purified mutants K21A (Lys21-->Ala), K21R(Lys21-->Arg) were characterized by steady-state kinetic analysis. The results showed that the coenzyme dependence of K21A was completely reversed to NADH.
Aldehyde Reductase ; metabolism ; Amino Acid Substitution ; genetics ; Coenzymes ; pharmacology ; Escherichia coli ; genetics ; metabolism ; Ethanol ; pharmacology ; Lysine ; genetics ; Mutagenesis, Site-Directed ; NAD ; metabolism ; NADP ; metabolism ; Pichia ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Recombination, Genetic ; Xylose ; pharmacology

To analyze the prevalence, genomic characteristics and clinical relevance of carbapenem-resistant bacteria in untreated hospital wastewater, and to provide a reference basis for in-hospital assessment of public health situation and prevention of cross-infection. In March 2023, untreated wastewater in the wastewater treatment station of the Second Affiliated Hospital of Soochow University and wastewater in the U-shaped wastewater pipes of the hand-washing sinks in 26 wards were collected, centrifuged and diluted, and the drug-resistant bacteria were isolated by using LB solid plates containing meropenem (2 μg/ml) for species identification, drug sensitivity analysis, carbapenenase gene PCR detection and whole genome sequencing. The genome sequence was identified for drug resistance genes. Retrospective research was used, combining multilocus sequence typing (MLST) and single nucleotide polymorphism (SNP) analysis, to compare their homology with clinical isolates of the same quarter. The results showed that 56 carbapenem-resistant gram-negative bacteria were isolated from hospital wastewater, originating from 13 genera, of which 17 were isolated from the total hospital wastewater, with Aeromonas spp. as the most dominant genus (35.3%, 6/17), and 39 were isolated from the wastewater of 17 wards, with Pseudomonas spp. as the most dominant genus (30.8%, 12/39). All common wastewater isolates from our hospital were multidrug-resistant bacteria, with up to 100% resistant to some second-and third-generation cephalosporins. A total of 8 carbapenemase genes originated from wastewater isolates, including blaKPC, blaNDM, blaIMP, blaVIM, blaIND, blaOXA-58-like, blaOXA-48-like, and blaOXA-427-like. 39 wastewater isolates carried the carbapenemase genes, and the total wastewater of the hospital carried the highest isolation rate of blaKPC-2 bacteria (35.3%, 6/17) and the highest isolation rate of blaIMP-8 bacteria (31.8%, 7/22) were found in the wastewater from 26 wards. 14 wastewater isolates were found to carry both carbapenemase genes, with a total of 6 combinations. A new blaIMP-101 isoform was also identified for the first time. 4 wastewater isolates and 11 clinical isolates were screened for inclusion in the SNP analysis, in which only 15 SNPs differed between the two strains of ST11 Klebsiella pneumoniae of clinical and wastewater origin, which was highly homologous. In conclusion, the presence of multiple multi-drug resistant conditionally pathogenic bacteria in untreated hospital wastewater has the potential risk of spreading drug-resistant genes in the environment. The highly homologous Klebsiella pneumoniae isolated from hospital wastewater and clinics indicates the close association between hospital wastewater and clinical infections. Hospitals need to strengthen the monitoring of drug-resistant bacteria and drug-resistant genes in the wastewater environment, to prevent the widespread dissemination of drug-resistant bacteria and drug-resistant genes in hospital wastewater and to prevent nosocomial infections caused by drug-resistant bacteria in wastewater.

Mechanically-induced hemolysis is an important index for evaluating the blood compatibility of medical devices.It mainly evaluates the risk of mechanical hemolysis under simulated clinical use conditions of medical devices.The existing hemolysis test standards mainly evaluate the risk of material-induced hemolysis and cannot evaluate the risk of mechanically-induced hemolysis.Although some institutions have conducted research on mechanically-induced hemolysis caused by medical devices,systematic evaluation standards have not yet been established.This study mainly summarizes the evaluation process,selection of evaluation models,and steps for assessing mechanically-induced hemolysis,providing a reference for relevant evaluations and standard establishment.

To analyze the prevalence, genomic characteristics and clinical relevance of carbapenem-resistant bacteria in untreated hospital wastewater, and to provide a reference basis for in-hospital assessment of public health situation and prevention of cross-infection. In March 2023, untreated wastewater in the wastewater treatment station of the Second Affiliated Hospital of Soochow University and wastewater in the U-shaped wastewater pipes of the hand-washing sinks in 26 wards were collected, centrifuged and diluted, and the drug-resistant bacteria were isolated by using LB solid plates containing meropenem (2 μg/ml) for species identification, drug sensitivity analysis, carbapenenase gene PCR detection and whole genome sequencing. The genome sequence was identified for drug resistance genes. Retrospective research was used, combining multilocus sequence typing (MLST) and single nucleotide polymorphism (SNP) analysis, to compare their homology with clinical isolates of the same quarter. The results showed that 56 carbapenem-resistant gram-negative bacteria were isolated from hospital wastewater, originating from 13 genera, of which 17 were isolated from the total hospital wastewater, with Aeromonas spp. as the most dominant genus (35.3%, 6/17), and 39 were isolated from the wastewater of 17 wards, with Pseudomonas spp. as the most dominant genus (30.8%, 12/39). All common wastewater isolates from our hospital were multidrug-resistant bacteria, with up to 100% resistant to some second-and third-generation cephalosporins. A total of 8 carbapenemase genes originated from wastewater isolates, including blaKPC, blaNDM, blaIMP, blaVIM, blaIND, blaOXA-58-like, blaOXA-48-like, and blaOXA-427-like. 39 wastewater isolates carried the carbapenemase genes, and the total wastewater of the hospital carried the highest isolation rate of blaKPC-2 bacteria (35.3%, 6/17) and the highest isolation rate of blaIMP-8 bacteria (31.8%, 7/22) were found in the wastewater from 26 wards. 14 wastewater isolates were found to carry both carbapenemase genes, with a total of 6 combinations. A new blaIMP-101 isoform was also identified for the first time. 4 wastewater isolates and 11 clinical isolates were screened for inclusion in the SNP analysis, in which only 15 SNPs differed between the two strains of ST11 Klebsiella pneumoniae of clinical and wastewater origin, which was highly homologous. In conclusion, the presence of multiple multi-drug resistant conditionally pathogenic bacteria in untreated hospital wastewater has the potential risk of spreading drug-resistant genes in the environment. The highly homologous Klebsiella pneumoniae isolated from hospital wastewater and clinics indicates the close association between hospital wastewater and clinical infections. Hospitals need to strengthen the monitoring of drug-resistant bacteria and drug-resistant genes in the wastewater environment, to prevent the widespread dissemination of drug-resistant bacteria and drug-resistant genes in hospital wastewater and to prevent nosocomial infections caused by drug-resistant bacteria in wastewater.