Nuclear medicine molecular imaging has the characteristics of non-invasiveness,high sensitivity,spatiotemporal dynamic visualization,qualitative and quantitative analysis,and by virtue of the advantages of fusion imaging technology,it combines the features of functional metabolism and anatomical structure.Nuclear medicine molecular imaging evaluation is integrated throughout the management of radioactive iodine-refractory differentiated thyroid cancer(RAIR-DTC),including defining RAIR,exploring the lesions,guiding treatment decisions,evaluating efficacy,and assessing prognosis.131I-whole body scan(131I-WBS)is critical for determining RAIR-DTC.Diagnostic 131I-WBS can be used to explore postoperative residual thyroid and suspected iodine-avid metastases before 131I treatment,which is helpful for subsequent 131I treatment decisions.Post-treatment 131I-WBS can further clarify the iodine uptake characteristics of lesions and explore lesions not shown by diagnostic WBS,providing a reference for clarifying the clinical stage of patients and formulating follow-up management plans.The iodine uptake ability of lesions shown by post-treatment 131I-WBS can also predict the therapeutic efficacy of 131I treatment.131I-WBS combined with biochemical changes and other imaging examinations can also be used to evaluate the therapeutic efficacy of 131I treatment.18F-FDG positron emission tomography and computed tomography(PET/CT)is mainly used for high-risk DTC patients with persistently elevated serum thyroglobulin(Tg)or Tg antibody(TgAb)levels and negative 131I-WBS,and can explore and locate lesions.Combining 18F-FDG PET/CT with 131I-WBS provides a thorough evaluation of the overall tumor burden.The uptake of 18F-FDG by DTC metastases indicates poor 131I treatment response and poor prognosis for patients,and is a predictor of rapid disease progression and an increased risk of tumor-specific death.After local or systemic treatment of RAIR-DTC lesions,the early metabolic response to treatment can predict the clinical benefit of patients,allowing for timely adjustment of treatment strategies.In addition,various new radionuclide imaging techniques targeting angiogenesis(such as RGD peptides and prostate specific membrane antigen),fibroblast activation protein and somatostatin receptor can be used as supplementary means when 18F-FDG PET/CT is negative to detect RAIR-DTC lesions.They can also screen patients who qualify for targeted radionuclide therapy based on the uptake ability of imaging agents.These novel theranostics provide new options for progressive RAIR-DTC patients after multiline treatment.

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.

Nuclear medicine molecular imaging has the characteristics of non-invasiveness,high sensitivity,spatiotemporal dynamic visualization,qualitative and quantitative analysis,and by virtue of the advantages of fusion imaging technology,it combines the features of functional metabolism and anatomical structure.Nuclear medicine molecular imaging evaluation is integrated throughout the management of radioactive iodine-refractory differentiated thyroid cancer(RAIR-DTC),including defining RAIR,exploring the lesions,guiding treatment decisions,evaluating efficacy,and assessing prognosis.131I-whole body scan(131I-WBS)is critical for determining RAIR-DTC.Diagnostic 131I-WBS can be used to explore postoperative residual thyroid and suspected iodine-avid metastases before 131I treatment,which is helpful for subsequent 131I treatment decisions.Post-treatment 131I-WBS can further clarify the iodine uptake characteristics of lesions and explore lesions not shown by diagnostic WBS,providing a reference for clarifying the clinical stage of patients and formulating follow-up management plans.The iodine uptake ability of lesions shown by post-treatment 131I-WBS can also predict the therapeutic efficacy of 131I treatment.131I-WBS combined with biochemical changes and other imaging examinations can also be used to evaluate the therapeutic efficacy of 131I treatment.18F-FDG positron emission tomography and computed tomography(PET/CT)is mainly used for high-risk DTC patients with persistently elevated serum thyroglobulin(Tg)or Tg antibody(TgAb)levels and negative 131I-WBS,and can explore and locate lesions.Combining 18F-FDG PET/CT with 131I-WBS provides a thorough evaluation of the overall tumor burden.The uptake of 18F-FDG by DTC metastases indicates poor 131I treatment response and poor prognosis for patients,and is a predictor of rapid disease progression and an increased risk of tumor-specific death.After local or systemic treatment of RAIR-DTC lesions,the early metabolic response to treatment can predict the clinical benefit of patients,allowing for timely adjustment of treatment strategies.In addition,various new radionuclide imaging techniques targeting angiogenesis(such as RGD peptides and prostate specific membrane antigen),fibroblast activation protein and somatostatin receptor can be used as supplementary means when 18F-FDG PET/CT is negative to detect RAIR-DTC lesions.They can also screen patients who qualify for targeted radionuclide therapy based on the uptake ability of imaging agents.These novel theranostics provide new options for progressive RAIR-DTC patients after multiline treatment.

Osteosarcopenia is a geriatric syndrome referring to the co-existence of osteoporosis and sarcopenia.Its pathogenesis involves factors such as genetics, mechanics of the musculoskeletal system, endocrine regulatory mechanisms and molecular signaling pathways.In clinical practice, aside from comprehensive assessment of risk factors, screening of bone density, muscle strength, muscle mass and the overall body function must also be undertaken.Intervention measures primarily include therapeutic exercise, nutritional support and drugs.

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 rapidly construct hepatitis B virus(HBV)adefovir(ADV)‐resistant strain(RT A181T)infectious clone by using SOE‐PCR ,to observe the expression of recombinant plasmids in Huh7 cells and to establish the in vitro cell model of HBV ADV‐resistant strain(RT A181T) .Methods The conservative primers were designed ,the full‐length HBV genome was amplified from serum in the patients with ADV‐resistant chronic hepatitis B .Then ,the 1 .3‐fold genome‐length infectious clone pHBV1 .3 was constructed by using the SOE‐PCR technique ,which was transfected into human hepatoma cells Huh7 cell line .ELISA ,Western Blot and Real‐time PCR were adopted to detect infectious cloning replication and expression ,meanwhile the antiviral drugs lamivu‐dine(LAM ) and ADV were used to verify the infectious clone copy and inhibition of expression .Results The HBV ADV‐resistant infectious cloning plasmids pHBV1 .3 was successfully constructed .This plasmid could be effectively replicated ,transcripted and ex‐pressed in Huh7 cell lines .LAM could effectively inhibit the replication and expression of the infectious clone ,while ADV could not inhibit the replication and expression of the infectious clone .Conclusion The constructed infectious clone plasmids pHBV1 .3(RT A181T ) of HBV ADV‐resistant strain can efficiently replicate and express protein in vitro ,its transfected cells can be used in the study of HBV replication mechanism and antiviral study .

Objective To evaluate the safety and feasibility of totally laparoscopic cholecystolithotomy.Methods Patient baseline characteristics of all 34 totally laparoscopic cholecystolithotomy (TLC) were collected in a database.This group was compared with 34 matched patients who underwent the laparoscopic cholecystectomy (LC) in the same period.Retrospectively,intraoperative and postoperative data were added.Results Operatingtime was significantly longer in the TLC group(124.56 min vs 78.50 min,P ＜0.01).The mean hospitalization expenses of operation was significantly higher in the TLC group(10 970.85 yuan vs 8 666.72 yuan,P ＜0.01).Although not significant less patients have the symptoms of postoperative dyspepsia or diarrhea were seen in the TLC group compared with the LC group (2 vs 6,P =0.26).Intraoperative details and postoperative results such as,blood loss,hospital stay,exhaust time,abdominal bleeding,bile leakage,incision infection have no significant difference.One case of gallstone recurrence was detected in TLC group.No stone recurrence was reported in common bile duct in LC group.Conclusions TLC is effective and feasible for chronic calcular cholecystitis and is particularly favorable for thepatients with medical insurance.However,this approach is technically demanding and should be performed by experienced surgon.

Objective To analyze enterovirus 71 (EV71) molecular epidemiology characteristics in Nantong region of Jiangsu , 2014 .Methods The pharyngeal swab samples were selected from 52 children with hand ,foot and mouth disease .After extracting the virus nucleic acid ,the EV71 VP1 gene of the virus were amplified by RT‐PCR amplification .The phylogenetic tree was con‐structed between isolated and EV71 reference strains from other parts of the country .Results 12 strains EV71 VP1 gene were iso‐lated from 52 cases of clinical specimens .The nucleotide and amino acid homology of 12 strains EV71 VP1 gene was 93 .4% -99 .1% ,the phylogenetic tree analysis showed that 12 strains of VP1 gene all belong to C4 genotype C4a subgenotypes .Conclusion The isolated strains in Nantong in 2014 are all the EV71 C4a subgenotypes of C4 genotype ,the same with the most of isolates in re‐cent years .