Objective To analyze the drug resistance characteristics,molecular typing,and biological properties of carbapenem-resist-ant Klebsiella pneumoniae(CRKP).Methods A retrospective analysis was conducted on 31 non-repetitive CRKP strains collected clinically from April 2019 to May 2021 at the Third People's Hospital of Nantong.The Vitek 2 Compact microbial analysis system was used for bacterial identification and in vitro drug susceptibility analysis.The broth dilution method was used to determine the minimum inhibitory concentration(MIC)of polymyxin B.The disk diffusion testing was performed to supplement the susceptibility of five com-monly used antibiotics:ertapenem,cefotaxime,cefoxitin,cefoperazone-sulbactam,and tigecycline.The carbapenemase-resistance phenotype of CRKP strains was initially determined by a combined assay of modified carbapenem inactivation method(mCIM)and ED-TA-carbapenem inactivation method(eCIM).Certain carbapenemase resistance genes(blaKPC,blaNDM,blaIMP,blaVIM,and blaOXA-48),AmpC enzyme genes(blaDHA,bla ACC,blaCIT,blaEBC,blaMOX,and blaFOX),extended-spectrum β-lactamases(ESBLs)genes(blaSHv,blaTEM,and blaCTX-M),and nine virulence genes were amplified by PCR and subsequently verified by sequencing.The stringing test was used to screen for hypermucoviscous phenotype strains.The growth curves in vitro and biofilm formation assays,and multilocus se-quence typing(MLST)were performed on 31 isolates.Outer-membrane proteins were extracted and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)to evaluate the expressions of OmpK35 and OmpK36.Results All the 31 isolates were resistant to ampicillin/sulbactam,ampicillin,aztreonam,cefazolin,ceftriaxone,cefotaxime,cefuroxime,ciprofloxacin,pip-eracillin,piperacillin/tazobactam,meropenem,and ertapenem with resistance rate of 100％.The resistance to polymyxin B was ob-served in 32.26％,whereas tigecycline retained 100％susceptibility.In terms of MLST,three sequence types(STs)were identified,with ST15 being the most prevalent,accounting for 61.29％(19/31)of the isolates.All strains produced serine carbapenemase,and only blaKPC-2 was detected among carbapenem resistance genes.The virulence genes fimH and entB were present in all strains(100％,31/31),while the detection rate of mrkD was 80.64％(25/31).Some strains carried virulence genes such as rmpA,rmpA2,and other virulence genes,whereas magA gene was not detected in any isolate.The carriage rates of rmpA2,iutA,and mrkD were higher in ST11 strains than in ST15 strains.The string test was positive in 38.71％of the strains.The growth test showed that there was no significant difference observed in the growth curves among all strains in vitro,and all were able to form biofilms with varying degrees.All ST11 strains exhibited OmpK36 protein alterations,while OmpK35 protein was intact in the 31 strains.Conclusion CRKP strains in this hospital showed high drug-resistance rate,and ST15 was the predominant sequence type.All the isolates carried blaKPC-2 and virulence genes.Enhanced molecular surveillance and strengthened prevention and control measures of CRKP infection are urgently needed.

Objective To analyze the drug resistance characteristics,molecular typing,and biological properties of carbapenem-resist-ant Klebsiella pneumoniae(CRKP).Methods A retrospective analysis was conducted on 31 non-repetitive CRKP strains collected clinically from April 2019 to May 2021 at the Third People's Hospital of Nantong.The Vitek 2 Compact microbial analysis system was used for bacterial identification and in vitro drug susceptibility analysis.The broth dilution method was used to determine the minimum inhibitory concentration(MIC)of polymyxin B.The disk diffusion testing was performed to supplement the susceptibility of five com-monly used antibiotics:ertapenem,cefotaxime,cefoxitin,cefoperazone-sulbactam,and tigecycline.The carbapenemase-resistance phenotype of CRKP strains was initially determined by a combined assay of modified carbapenem inactivation method(mCIM)and ED-TA-carbapenem inactivation method(eCIM).Certain carbapenemase resistance genes(blaKPC,blaNDM,blaIMP,blaVIM,and blaOXA-48),AmpC enzyme genes(blaDHA,bla ACC,blaCIT,blaEBC,blaMOX,and blaFOX),extended-spectrum β-lactamases(ESBLs)genes(blaSHv,blaTEM,and blaCTX-M),and nine virulence genes were amplified by PCR and subsequently verified by sequencing.The stringing test was used to screen for hypermucoviscous phenotype strains.The growth curves in vitro and biofilm formation assays,and multilocus se-quence typing(MLST)were performed on 31 isolates.Outer-membrane proteins were extracted and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)to evaluate the expressions of OmpK35 and OmpK36.Results All the 31 isolates were resistant to ampicillin/sulbactam,ampicillin,aztreonam,cefazolin,ceftriaxone,cefotaxime,cefuroxime,ciprofloxacin,pip-eracillin,piperacillin/tazobactam,meropenem,and ertapenem with resistance rate of 100％.The resistance to polymyxin B was ob-served in 32.26％,whereas tigecycline retained 100％susceptibility.In terms of MLST,three sequence types(STs)were identified,with ST15 being the most prevalent,accounting for 61.29％(19/31)of the isolates.All strains produced serine carbapenemase,and only blaKPC-2 was detected among carbapenem resistance genes.The virulence genes fimH and entB were present in all strains(100％,31/31),while the detection rate of mrkD was 80.64％(25/31).Some strains carried virulence genes such as rmpA,rmpA2,and other virulence genes,whereas magA gene was not detected in any isolate.The carriage rates of rmpA2,iutA,and mrkD were higher in ST11 strains than in ST15 strains.The string test was positive in 38.71％of the strains.The growth test showed that there was no significant difference observed in the growth curves among all strains in vitro,and all were able to form biofilms with varying degrees.All ST11 strains exhibited OmpK36 protein alterations,while OmpK35 protein was intact in the 31 strains.Conclusion CRKP strains in this hospital showed high drug-resistance rate,and ST15 was the predominant sequence type.All the isolates carried blaKPC-2 and virulence genes.Enhanced molecular surveillance and strengthened prevention and control measures of CRKP infection are urgently needed.

Receptor-interacting protein kinase 1 (RIPK1) plays an essential role in regulating the necroptosis and apoptosis in cerebral ischemia-reperfusion (I/R) injury. However, the regulation of RIPK1 kinase activity after cerebral I/R injury remains largely unknown. In this study, we found the downregulation of protein arginine methyltransferase 1 (PRMT1) was induced by cerebral I/R injury, which negatively correlated with the activation of RIPK1. Mechanistically, we proved that PRMT1 directly interacted with RIPK1 and catalyzed its asymmetric dimethylarginine, which then blocked RIPK1 homodimerization and suppressed its kinase activity. Moreover, pharmacological inhibition or genetic ablation of PRMT1 aggravated I/R injury by promoting RIPK1-mediated necroptosis and apoptosis, while PRMT1 overexpression protected against I/R injury by suppressing RIPK1 activation. Our findings revealed the molecular regulation of RIPK1 activation and demonstrated PRMT1 would be a potential therapeutic target for the treatment of ischemic stroke.

As a potential vectored vaccine,Newcastle disease virus(NDV)has been subject to various studies for vaccine development,while relatively little research has outlined the immunomodulatory effect of the virus in antigen presentation.To elucidate the key inhibitory factor in regulating the interaction of infected dendritic cells(DCs)and T cells,DCs were pretreated with the NDV vaccine strain LaSota as an inhibitor and stimulated with lipopolysaccharide(LPS)for further detection by enzyme-linked immunosorbent assay(ELISA),flow cytometry,immunoblotting,and quantitative real-time polymerase chain reaction(qRT-PCR).The results revealed that NDV infection resulted in the inhibition of interleukin(IL)-12p40 in DCs through a p38 mitogen-activated protein kinase(MAPK)-dependent manner,thus inhibiting the synthesis of IL-12p70,leading to the reduction in T cell proliferation and the secretion of interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α),and IL-6 induced by DCs.Consequently,downregulated cytokines accelerated the infection and viral transmission from DCs to T cells.Furthermore,several other strains of NDV also exhibited inhibitory activity.The current study reveals that NDV can modulate the intensity of the innate?adaptive immune cell crosstalk critically toward viral invasion improvement,highlighting a novel mechanism of virus-induced immunosuppression and providing new perspectives on the improvement of NDV-vectored vaccine.

Liver is a unique organ with polyploidy.About 20 to 50 percent of hepatocytes in adult human are polyploid cells,which contain more than two sets of chromosomes.Hepatocytes polyploidization is triggered by the changes in insulin signal during weaning,and regulated by various cell cycle regulator genes to ensure the polyploid cells proportion,ploidy and zonation.The regulation of polyploid hepatocytes is crucial for liver's metabolic and regenerative function,and protecting liver from tumorigenesis.However,in chronic virus hepatitis and nonalcoholic fatty liver disease,the pathological hepatocytes polyploidization can be found in disease progression because of cell cycle checkpoint inhibition and oxidative stress.Distinguishing physiological and pathological hepatocyte polyploidization will be helpful for the understanding the relationship between chronic liver disease and tumorigenesis.

Lateral epicondylitis is a common clinical disease with characteristics of lateral elbow pain, insidious onset and easy recurrence, which can cause forearm pain and decreased wrist strength, seriously affecting patients′ daily life and work. Although there are various treatment methods for lateral epicondylitis with different effects, standard treatments are still lacking nowadays. Platelet-rich plasma (PRP) has good effects on bone and tendon repair, and is now widely used in the treatment of lateral epicondylitis. However, there is a lack of a unified understanding of the technology and specifications of PRP in the treatment of lateral epicondylitis. Therefore, the Sports Medicine Branch of the Chinese Medical Association and Physical Medicine and Rehabilitation Branch of the Chinese Medical Association organized experts in the fields of sports medicine and rehabilitation medicine in China to formulate the "clinical expert consensus on platelet-rich plasma treatment for lateral epicondylitis (2022 version)", and proposed suggestions based on evidence-based medicine mainly from the concept, epidemiology and pathophysiology of lateral epicondylitis, symptoms, signs and imaging manifestations of lateral epicondylitis, PRP concept and application component requirements, quality control of PRP preparation technology, indications and contraindications of PRP in the treatment of lateral epicondylitis, PRP injection in the treatment of lateral epicondylitis, application of PRP in the operation of lateral epicondylitis, related problems after PRP treatment of lateral epicondylitis, evaluation of the results after PRP treatment of lateral epicondylitis, and health and economic evaluation of PRP treatment of lateral epicondylitis, so as to provide guidance for clinical diagnosis and treatment.

Vibrio parahaemolyticus, the main pathogen causing seafood related food poisoning worldwide, has strong biofilm formation ability. ToxR is a membrane binding regulatory protein, which has regulatory effect on biofilm formation of V. parahaemolyticus, but the specific mechanism has not been reported. c-di-GMP is an important second messenger in bacteria and is involved in regulating a variety of bacterial behaviors including biofilm formation. In this study, we investigated the regulation of ToxR on c-di-GMP metabolism in V. parahaemolyticus. Intracellular c-di-GMP in the wild type (WT) and toxR mutant (ΔtoxR) strains were extracted by ultrasonication, and the concentrations of c-di-GMP were then determined by enzyme linked immunosorbent assay (ELISA). Three c-di-GMP metabolism-related genes scrA, scrG and vpa0198 were selected as the target genes. Quantitative real-time PCR (q-PCR) was employed to calculate the transcriptional variation of each target gene between WT and ΔtoxR strains. The regulatory DNA region of each target gene was cloned into the pHR309 plasmid harboring a promoterless lacZ gene. The recombinant plasmid was subsequently transferred into WT and ΔtoxR strains to detect the β-galactosidase activity in the cellular extracts. The recombinant lacZ plasmid containing each of the target gene was also transferred into E. coli 100λpir strain harboring the pBAD33 plasmid or the recombinant pBAD33-toxR to test whether ToxR could regulate the expression of the target gene in a heterologous host. The regulatory DNA region of each target gene was amplified by PCR, and the over-expressed His-ToxR was purified. The electrophoretic mobility shift assay (EMSA) was applied to verify whether His-ToxR directly bound to the target promoter region. ELISA results showed that the intracellular c-di-GMP level significantly enhanced in ΔtoxR strain relative to that in WT strain, suggesting that ToxR inhibited the production of c-di-GMP in V. parahaemolyticus. qPCR results showed that the mRNA levels of scrA, scrG and vpa0198 significantly increased in ΔtoxR strain relative to those in WT strain, suggesting that ToxR repressed the transcription of scrA, scrG and vpa0198. lacZ fusion assay showed that ToxR was able to repress the promoter activities of scrA, scrG and vpa0198 in both V. parahaemolyticus and E. coli 100λpir. EMSA results showed that His-ToxR was able to bind to the regulatory DNA regions of scrA and scrG, but not to the regulatory DNA region of vpa0198. In conclusion, ToxR inhibited the production of c-di-GMP in V. parahaemolyticus via directly regulating the transcription of enzyme genes associated with c-di-GMP metabolism, which would be beneficial for V. parahaemolyticus to precisely control bacterial behaviors including biofilm formation.
Vibrio parahaemolyticus/metabolism* ; Escherichia coli/metabolism* ; Bacterial Proteins/metabolism* ; Transcription Factors/genetics* ; Gene Expression Regulation, Bacterial

Objective:To study the transcriptional regulation of pilABCD by the master quorum sensing (QS) regulator OpaR in Vibrio parahaemolyticus. Methods:Total RNAs were extracted from the wild type (WT) and opaR mutant (Δ opaR) strain. Quantitative real-time PCR (qPCR) was employed to calculate the transcriptional variation of pilA (the first gene of pilABCD operon) between WT and Δ opaR. The regulatory DNA region of pilABCD was cloned into the corresponding restriction endonuclease sites of pHRP309 harboring a promoterless lacZ reporter gene. The recombinant pHRP309 plasmid was then transferred into WT and Δ opaR, respectively, to detect the β-galactosidase activity in cellular extracts using a β-Galactosidase Enzyme Assay System (Promega). The primer extension assay was applied to map the transcription start site of pilABCD using the total RNAs extracted from the WT strain as the template. The regulatory DNA region of pilABCD was amplified by PCR, and the over-expressed His-OpaR was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). Thereafter, the electrophoretic mobility shift assay (EMSA) was applied to analyze the DNA-binding activity of His-OpaR to the target DNA in vitro, and the DNase I footprinting assay was further employed to detect the DNA-binding sites of His-OpaR within the target DNA. Results:The results of qPCR and LacZ fusion assays showed that OpaR activated the transcription of pilABCD, leading to a gradual increase in the expression level of pilA with the extension of culture time. The primer extension assay detected only one transcription start site located at 155 bp upstream of pilA. The results of EMSA and DNase Ⅰ footprinting assays showed that His-OpaR protected two DNA regions located from -246 to -197 bp and -181 to -131 bp upstream of pilA. Conclusions:Vibrio parahaemolyticus OpaR activated the transcription of pilABCD in a direct manner.

Objective To evaluate the impact of total parenteral nutrition(TPN)on nutrition status and inflammatory markers in hospitalized fasted patients with inflammatory bowel disease(IBD).Methods A retrospective study was performed and 82 hospitalized fasted IBD patients [male/female=58/24,(39.4±14.5)years] who received TPN entered the study.Among them,38 patients had ulcerative colitis(UC)and 44 patients suffered from Crohn`s disease(CD).Clinical data(gender,age,duration of disease,history of disease,prednisone,immuno-suppressor,and antibiotics)were obtained from medical records.Nutritional parameters,C-creative protein(CRP),and erythrocyte sedimentation rate(ESR)before and after TPN were also obtained.Average caloric supplementation by TPN was(4 437.3±1 199.1)kJ/d and the nitrogen amount was(9.9±1.7)g/d.Median PN length was 15 days(7-54 days).67 IBD patients received a TPN formula with glutamine(≥14 d,25 patients vs.0-14 d,42 patients)and 15 IBD subjects received TPN without glutamine.Malnutrition was diagnosed by body mass index(BMI)and serum albumin level.Results The prevalence of undernutrition was 90.2％(74/82)in the study population.CD patients had a significantly longer history of disease [84(3-288)months vs.24(1-324)months,P<0.001] and a significantly lower BMI [(15.6±1.8)kg/m2 vs.(19.1±3.5)kg/m2,P<0.001] compared with those in UC patients.TPN improved nutritional parameters [serum albumin:(28.7±6.6)g/L before TPN vs.(31.7±5.8)g/L after TPN,P<0.001;pre-albumin:(174.1±85.5)mg/L before TPN vs.(227.2±82.8)mg/L after TPN,P<0.001].Conclusions TPN improves nutritional status in hospitalized fasted IBD patients.However,prospective randomized controlled trials are required to estimate the role of low-to-middle dosage of glutamine in IBD patients.

Objective To investigate the relationship between serum ferritin and nonalcoholic fatty liver diseases in obese children.Methods Obese children aged 6 to 14 years old were enrolled.Duration of obesity, anthropometric parameters (height, body weight, waist circumference, hip circumference), bioelectrical impedance analysis (body fat), serological parameters (liver transaminases, lipid metabolism, fasting blood glucose, fasting insulin, serum ferritin) and liver ultrasonography were recorded.Insulin resistance (IR) index was calculated by homeostasis model assessment (HOMA).All subjects were divided into 3 groups according to liver ultrasound and liver transaminases : simple obese children (SOC) group, obese children with nonalcoholic simple fatty liver (NAFL) group and obese children with nonalcoholic steatohepatitis (NASH) group.Results 86 obese children entered the study, with a mean age of (10.4 ± 1.9) years, including 26 in the SOC group, 28 in the NAFL group and 32 in the NASH group.Waist circumference standard deviation score (SDS or Z-score), waist-to-hip ratio, HOMA-IR index and serum ferritin in the NASH group were obviously higher than those in the NAFL group [2.3 ± 0.3 vs.2.1 ± 0.3, P =0.020;1.0 ± 0.0 vs.0.9 ± 0.1,P=0.014;4.0±1.7 vs.2.9±1.8, P=0.006;(104.1 ±49.6) μg/Lvs.(68.4 ±22.7) μg/L, P=0.004] and the SOC group [2.3 ±0.3 vs.1.9 ±0.3, P=0.000;1.0±0.0vs.0.9 ±0.1, P=0.012;4.0 ±1.7 vs.2.5 ±1.6, P=0.001;(104.1 ±49.6) μg/Lvs.(59.2 ±28.9) μg/L, P=0.001], while there was no significant difference in body mass index Z-score [2.8 ± 0.5 vs.2.7 ± 0.6, P =0.524;2.8 ± 0.5 vs.2.7 ± 0.6, P =0.662].There were no significant differences between the NAFL group and the SOC group in the above indicators [2.1 ±0.3 vs.1.9 ±0.3, P =0.260;0.9 ±0.1 vs.0.9 ±0.1, P =0.952;2.9 ± 1.8vs.2.5±1.6, P=0.283;(68.4±22.7) μg/Lvs.(59.2±28.9) μg/L, P=0.161].Mter controlling age, body mass index, waist circumference, waist-to-hip ratio, triglyceride, and HOMA-IR index, serum ferritin was still positively correlated with the magnitude of nonalcoholic fatty liver diseases in obese children (r =0.335, P =0.002).Conclusion Serum ferritin is probably an independent risk factor for NASH in obese children.