This study was aimed at tracing the molecular typing and drug resistance characteristics of a suspected food poi-soning event caused by Clostridium perfringens in a district of Wuhan City.The FilmArray detection system and multiple fluo-rescence quantitative PCR methods were used to rapidly screen for pathogens in samples from the poisoning event.According to the initial screening results,bacteria were isolated,cultured,and identified by mass spectrometry.Fluorescence PCR was used to detect six virulence genes of the isolated Clostridium perfringens strains.On the basis of whole genome sequencing results,we conducted virulence genes,resistance genes,and whole genome single nucleotide polymorphism genetic evolution(wgSNPs)analyses.Antibiotic sensitivity testing was conducted with the agar dilution method.A total of ten strains of Clos-tridium perfringens were isolated,including eight strains from seven anal swab samples,one strain from fecal samples,and one strain from food samples.Food with suspected contamination had a Clostridium perfringens count of 7.8×106 CFU/g.The PLC(a)toxin gene was detected in all ten gas producing capsule isolation strains,but no other 5 tox-in genes such as CPE were detected,thus confirming that all were type A bacteria producing capsule Clostridium.All strains were 100％resistant to clindamycin and almost completely sensitive to antibiotics such as vancomycin,cefoxitin,and meropenem.Ten strains of Clos-tridium perfringens carried resistance genes such as tetB(P),tetA(P),and mprF,followed by ermQ(70％),ant(6)-Ⅰb(10％),and LnuP(10％).Genetic evolution analysis of wgSNPs indicated that the four outbreak strains clustered together and belonged to an independent subbranch with the suspected food sourcestrains,thus indicating close genetic relationships.In con-clusion,this food poisoning incident might have been be caused by hand torn chickens contaminated with Clostridium perfrin-gens,and the molecular types of the strains revealed high genetic diversity.No multiple drug resistance was observed,but all strains were resistant to clindamycin,an aspect requiring further clinical attention.

Objective To explore the effects of triptolide(TPL)on the proliferation,migration and invasion of gastric cancer cells and its mechanism.Methods Human gastric cancer cell line MKN45 was cultured in vitro and treated with different concentrations of TPL for 48 hours.The cell proliferation inhibition rate was detected by CCK-8 method and the optimal concentration was selected for subsequent experiments.qRT-PCR was used to detect the expression of miR-29b and KDM2A mRNA in cells treated with different concentrations of TPL.MKN45 cells at logarithmic growth phase were randomly divided into the control group(without any treatment),the TPL group(treated with 200 μg/mL TPL),the inhibitor-NC+TPL group(transfected with inhibitor-NC and then treated with 200 μg/mL TPL),and the miR-29b inhibitor+TPL group(transfected with miR-29b inhibitor and then treated with 200 μg/mL TPL).qRT-PCR was used to detect the expression of miR-29b and KDM2A mRNA in each group of cells,and Western blot was used to detect the expression of KDM2A protein.The clone formation ability of each group of cells was detected by plate clone formation assay,and the migration and invasion abilities of each group of cells were detected by Transwell assay.Results TPL at concentrations of 25 μg/mL,50 μg/mL,100 μg/mL,and 200 μg/mL could significantly inhibit the proliferation of MKN45 cells(P<0.05),up-regulate the expression of miR-29b in cells(P<0.05),and down-regulate the expression of KDM2A mRNA(P<0.05).The effect was most obvious at the concentration of 200 μg/mL,so 200 μg/mL TPL was selected for the subsequent experiments.Compared with the control group,the expression of miR-29b in the TPL group increased(P<0.05),the expression of KDM2A mRNA and protein decreased(P<0.05),and the numbers of clone formation,migration and invasion cells reduced(P<0.05).Compared with the inhibitor-NC+TPL group,the expression of miR-29b in the miR-29b inhibitor+TPL group decreased(P<0.05),the expression of KDM2A mRNA and protein increased(P<0.05),and the numbers of clone formation,migration and invasion cells increased(P<0.05).Conclusion TPL can inhibit the proliferation,migration and invasion of gastric cancer cells,and its mechanism is related to the regulation of the miR-29b/KDM2A signaling pathway.

Constructing an efficient and easy-to-operate multigene expression system is currently a crucial part of plant genetic engineering. In this study, a fragment carrying three independent gene expression cassettes and the expression unit of the gene-silencing suppressor protein(RNA silencing suppressor 19 kDa protein, P19) simultaneously was designed and constructed. This fragment was cloned into the commonly used plant expression vector pCAMBIA300, and the plasmid pC1300-TP2-P19 was obtained. Each gene expression cassette consists of different promoters, fusion tags, and terminators. The target gene can be flexibly inserted into the corresponding site through enzymatic digestion and ligation or recombination and fused with different protein tags, which provides great convenience for subsequent detection. The enhanced green fluorescent protein(eGFP) reporter gene was individually constructed into each expression cassette to verify the feasibility of this vector system. The results of tobacco transient expression and laser-confocal microscopy showed that each expression cassette presented independent and normal expression. Meanwhile, the three key enzyme genes in the betanin synthesis pathway, BvCYP76AD, BvDODA1, and DbDOPA5GT, were constructed into the three expression cassettes. The results of tobacco transient expression phenotype, protein immunoblotting(Western blot), and chemical detection of product demonstrated that the three exogenous genes were highly expressed, and the target compound betanin was successfully produced. The above results indicated that the constructed multigene expression system for plants in this study was efficient and reliable and can achieve the co-transformation of multiple plant genes. It can provide a reliable vector platform for the analysis of plant natural product synthesis pathways, functional verification, and plant metabolic engineering.
Nicotiana/metabolism* ; Genetic Vectors/metabolism* ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism* ; Plants, Genetically Modified/metabolism* ; Genetic Engineering/methods* ; Green Fluorescent Proteins/metabolism* ; Gene Expression

Aim To investigate the intestinal protection and possible mechanism of magnolol(MG)in newborn rats with necrotizing enterocolitis(NEC).Methods The rats were randomly divided into control group(Ctrl group),model group(NEC group)and treatment group(MG group).The NEC model was induced by hypoxia,cold stimulation,deep formula milk and LPS intragastric administration in 7-day-old rats for four days.They were killed after five days of treatment with MG(20 mg·kg-1).HE staining was used to observe the intestinal pathological injury.Western blot was used to detect the expressions of IL-1 β,TNF-α,NL-RP3,ASC,caspase-1 and tight junction protein in the distal ileum of rats.Colon contents were collected for 16S rDNA sequencing to understand the gut microbio-ta.Results MG improved the body mass and intesti-nal injury of NEC neonatal rats.The expressions of in-testinal IL-1β,TNF-α,NLRP3,ASC and caspase-1 proteins were down-regulated,and the expressions of Claudin,Occludin and ZO-1 proteins were up-regula-ted.16S rDNA showed that MG increased the diversity of intestinal flora,and at the phylum level,MG in-creased the abundance of firmicutes and bacteroides in NEC model,and decreased the abundance of pro-teobacteria.At the genus level,MG treatment in-creased the abundance of Lactobacillus,unclassified_Muribaculaceae,Racteroides,but decreased the abun-dance of Escherichia_Shigella,Rodentibacter and Fuso-bacterium.Conclusion MG intervention can protect the intestinal tract of NEC rats by potentially improving barrier function,and regulating the intestinal microbiota through the NLRP3/ASC/caspase-1 signaling pathway.

Objective To explore the effects of triptolide(TPL)on the proliferation,migration and invasion of gastric cancer cells and its mechanism.Methods Human gastric cancer cell line MKN45 was cultured in vitro and treated with different concentrations of TPL for 48 hours.The cell proliferation inhibition rate was detected by CCK-8 method and the optimal concentration was selected for subsequent experiments.qRT-PCR was used to detect the expression of miR-29b and KDM2A mRNA in cells treated with different concentrations of TPL.MKN45 cells at logarithmic growth phase were randomly divided into the control group(without any treatment),the TPL group(treated with 200 μg/mL TPL),the inhibitor-NC+TPL group(transfected with inhibitor-NC and then treated with 200 μg/mL TPL),and the miR-29b inhibitor+TPL group(transfected with miR-29b inhibitor and then treated with 200 μg/mL TPL).qRT-PCR was used to detect the expression of miR-29b and KDM2A mRNA in each group of cells,and Western blot was used to detect the expression of KDM2A protein.The clone formation ability of each group of cells was detected by plate clone formation assay,and the migration and invasion abilities of each group of cells were detected by Transwell assay.Results TPL at concentrations of 25 μg/mL,50 μg/mL,100 μg/mL,and 200 μg/mL could significantly inhibit the proliferation of MKN45 cells(P<0.05),up-regulate the expression of miR-29b in cells(P<0.05),and down-regulate the expression of KDM2A mRNA(P<0.05).The effect was most obvious at the concentration of 200 μg/mL,so 200 μg/mL TPL was selected for the subsequent experiments.Compared with the control group,the expression of miR-29b in the TPL group increased(P<0.05),the expression of KDM2A mRNA and protein decreased(P<0.05),and the numbers of clone formation,migration and invasion cells reduced(P<0.05).Compared with the inhibitor-NC+TPL group,the expression of miR-29b in the miR-29b inhibitor+TPL group decreased(P<0.05),the expression of KDM2A mRNA and protein increased(P<0.05),and the numbers of clone formation,migration and invasion cells increased(P<0.05).Conclusion TPL can inhibit the proliferation,migration and invasion of gastric cancer cells,and its mechanism is related to the regulation of the miR-29b/KDM2A signaling pathway.

Epidermal growth factor receptor inhibitor (EGFRI) -related paronychia is a condition clearly related to EGFRI therapy, characterized by periungual erythema, edema, purulent exudates, periungual or subungual granulomatous lesions, and sometimes accompanied by thinning, fragility or even splitting and seperation of nail plates. Inhibition of epidermal function, inflammation and secondary infections, as well as angiogenesis are the core processes in the occurrence and development of EGFRI-related paronychia. This review summarizes epidemiology, pathogenesis, clinical manifestations, prevention and treatment of EGFRI-related paronychia.

This study was aimed at tracing the molecular typing and drug resistance characteristics of a suspected food poi-soning event caused by Clostridium perfringens in a district of Wuhan City.The FilmArray detection system and multiple fluo-rescence quantitative PCR methods were used to rapidly screen for pathogens in samples from the poisoning event.According to the initial screening results,bacteria were isolated,cultured,and identified by mass spectrometry.Fluorescence PCR was used to detect six virulence genes of the isolated Clostridium perfringens strains.On the basis of whole genome sequencing results,we conducted virulence genes,resistance genes,and whole genome single nucleotide polymorphism genetic evolution(wgSNPs)analyses.Antibiotic sensitivity testing was conducted with the agar dilution method.A total of ten strains of Clos-tridium perfringens were isolated,including eight strains from seven anal swab samples,one strain from fecal samples,and one strain from food samples.Food with suspected contamination had a Clostridium perfringens count of 7.8×106 CFU/g.The PLC(a)toxin gene was detected in all ten gas producing capsule isolation strains,but no other 5 tox-in genes such as CPE were detected,thus confirming that all were type A bacteria producing capsule Clostridium.All strains were 100％resistant to clindamycin and almost completely sensitive to antibiotics such as vancomycin,cefoxitin,and meropenem.Ten strains of Clos-tridium perfringens carried resistance genes such as tetB(P),tetA(P),and mprF,followed by ermQ(70％),ant(6)-Ⅰb(10％),and LnuP(10％).Genetic evolution analysis of wgSNPs indicated that the four outbreak strains clustered together and belonged to an independent subbranch with the suspected food sourcestrains,thus indicating close genetic relationships.In con-clusion,this food poisoning incident might have been be caused by hand torn chickens contaminated with Clostridium perfrin-gens,and the molecular types of the strains revealed high genetic diversity.No multiple drug resistance was observed,but all strains were resistant to clindamycin,an aspect requiring further clinical attention.

Aim To investigate the intestinal protection and possible mechanism of magnolol(MG)in newborn rats with necrotizing enterocolitis(NEC).Methods The rats were randomly divided into control group(Ctrl group),model group(NEC group)and treatment group(MG group).The NEC model was induced by hypoxia,cold stimulation,deep formula milk and LPS intragastric administration in 7-day-old rats for four days.They were killed after five days of treatment with MG(20 mg·kg-1).HE staining was used to observe the intestinal pathological injury.Western blot was used to detect the expressions of IL-1 β,TNF-α,NL-RP3,ASC,caspase-1 and tight junction protein in the distal ileum of rats.Colon contents were collected for 16S rDNA sequencing to understand the gut microbio-ta.Results MG improved the body mass and intesti-nal injury of NEC neonatal rats.The expressions of in-testinal IL-1β,TNF-α,NLRP3,ASC and caspase-1 proteins were down-regulated,and the expressions of Claudin,Occludin and ZO-1 proteins were up-regula-ted.16S rDNA showed that MG increased the diversity of intestinal flora,and at the phylum level,MG in-creased the abundance of firmicutes and bacteroides in NEC model,and decreased the abundance of pro-teobacteria.At the genus level,MG treatment in-creased the abundance of Lactobacillus,unclassified_Muribaculaceae,Racteroides,but decreased the abun-dance of Escherichia_Shigella,Rodentibacter and Fuso-bacterium.Conclusion MG intervention can protect the intestinal tract of NEC rats by potentially improving barrier function,and regulating the intestinal microbiota through the NLRP3/ASC/caspase-1 signaling pathway.

Epidermal growth factor receptor inhibitor (EGFRI) -related paronychia is a condition clearly related to EGFRI therapy, characterized by periungual erythema, edema, purulent exudates, periungual or subungual granulomatous lesions, and sometimes accompanied by thinning, fragility or even splitting and seperation of nail plates. Inhibition of epidermal function, inflammation and secondary infections, as well as angiogenesis are the core processes in the occurrence and development of EGFRI-related paronychia. This review summarizes epidemiology, pathogenesis, clinical manifestations, prevention and treatment of EGFRI-related paronychia.

To perform a comprehensive analysis of the pathogenic causes of a food poisoning case in a district of Wuhan Cit-y,we investigated the molecular epidemiological relationships among pathogenic bacteria,to aid in traceability analysis of food-borne disease outbreaks,as well as clinical diagnosis and treatment.The pathogenic bacteria in this food poisoning case were i-solated and identified according to GB789.4-2016.The isolated strains were subjected to genotyping with pulsed field gel elec-trophoresis(PFGE).Drug resistance gene analysis,multi-locus sequence typing(MLST),and genome-wide single-nucleotide polymorphism analysis(wgSNP)were conducted via whole genome sequencing(WGS).The evolutionary tree for cluster analy-sis was constructed in fasttree software.Drug susceptibility testing was conducted with the broth microdilution method.A total of 12 strains of Salmonella were detected in seven anal swab samples and two fecal samples from the case,as well as three anal swab samples from unaffected individuals.The serotype of the strains was Salmonella typhimurium.The strain exhibited severe multiple drug resistance,including resistance to amikacin,ampi-cillin,cefazolin,gentamicin,piperacillin,and tetracycline,but susceptibility to other antibiotics.The coincidence rate between drug resistance genes and drug resistance phenotypes was high.PFGE revealed that nine strains from this food poisoning case were highly homologous.WGS revealed that the MLST type was ST19,and varying numbers of SNPs(1-6)were present a-mong strains.The phylogenetic tree revealed nine isolated strains forming a distinct cluster,differing from other Salmonella strains in the database and belonging to a novel clonal branch.The single nucleotide site in the strains was highly homologous to that of GCF in Jiangxi_020221795.1.The food poisoning case was caused by Salmonella typhimurium ST19,and all nine iso-lated strains originated from the same source.The chef is closely connected to this food poisoning case.This strain of Salmo-nella typhimurium belongs to a new clonal branch and exhibits multiple drug resistance.