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 investigate the therapeutic methods and curative effect of combined interven-tional therapy on hepatic metastasis from colorectal cancer. Methods Hepatic artery angiography and port-calheter-system( PCS) were undertaken in 35 cases of liver metastasis from colorectal cancers. Therapeutic measare were divided into transcatheter arterial infusion(TAl) in 10 cases and transcatheter arterial emboliza-tion(TAE) in 25 cases according to their angiographic features. Drugs included oxalic acid platinum, CPT11 , MMC and 5-Fu + CF. Laser ablation under the CT guidance was taken after 2-3 courses of treatment, including 9 cases of ethanol ablation besides laser ablation. The curative effects were determined after laser ablation by checking with CT and CEA one month later. Results The lesions shrank in 17 cases, while 13 remained the same and enlarged in other 5 after 2-3 courses of TAE or TAI. CEA reduced in 6 cases, with no change in other 5 and increas in another 3 among the 14 positive CEA cases. CT scan showed spotty gasification in 29 cases and whole gasification in 6 after additional ablation treatment. Among them, the foci dwindled in 8 cases treated by solely laser ablation, with no change in 5 and increase in 7. The foci dwindled in 5 cases, 2 remainal the same and increase in 2 of 9 cases treated by the combination of laser ablation and percutaneous ethanol injection (PEI). Among 8 unchanged or increased CEA cases after TAE or TAI, showed decrease in 3 cases, other 4 remained the same and another increased after ablation. Conclusions 1. The blood supply of hepatic metastastic foci from coloreclal cancer is mainly fed by hepatic artery and subselective transcatheter angiography showed tumor stain clearly. 2. By means of PCS and TAI or TAE, using oxalic acid platinum, CPT11 and 5-Fu regular chemical therapy provided a better curative effect for metastasis. But 3. TAI or TAE can not eliminate all foci laser combining with ethanol ablation would be the effective complement.

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 .

Objective To construct 1.3-fold-overlength infectious clone of hepatitis B virus isolated from Chinese patients , observe the expression of plasmid in Huh7 of liver cancer cells and establish genome of HBV in vitro. Methods HBV DNA in serum was extracted from HBV patient. SOE-PCR was performed to produce a 1.3-fold-overlength genome of HBV. The plasmid was named pHBV1.3 (C). After that,pHBV1.3 (C) was transfected into Huh7 cells, HBV related viral antigens and DNA were detected by ELISA,Western Blot and Fluorescence quantitative PCR. Furthermore, adefovir dipivoxil, a clinic anti-viral drug, was utilized to test the sensitivity of the new infectious clone. Results An infectious clone of pHBV1.3 (C) was successfully constructed. HBV gene carried in pHBV1.3 (C) could be efficiently replicated and expressed in Huh7 cells. Adefovir could inhibit HBV replication in this HBV cell model. Conclusions A recombinant plasmid containing 1.3-fold-overlength of HBV genotype C was successfully constructed. This construct is competent to support viral transcription and replication in vitro , suggesting that infectious cells are expected to be a new model of HBV infection in vitro.

Objective To analyze the epidemiological characteristics of pathogens of children with hand , foot and mouth disease (HFMD) in Nantong area, in order to provide the basis for the treatment and prevention of HFMD. Methods Multiplex RT-PCR (fluorescence PCR method) was used to detect EV71, CoxA16 and non-infectious cases of EV71, CoxA16 of children with HFMD in Nantong. VP1 gene of strains isolated from 12 cases of EV71 was amplified and underwent phylogenetic tree analysis. 208 cases clinical data of children with HFMD were analyzed and summarized. Results In 208 cases of children with HFMD swab samples, EV71 of 64 cases were positive (30.8%); CoxA16 of 28 cases were positive (13.5%); 40 cases of other enterovirus were positive (19.2%). Phylogenetic tree analysis showed that EV71 epidemic strains are C4 subtypes in Nantong region. The clinical manifestations of enterovirus infections in EV71 , CoxA16 and other enterovirus are caused by rash, fever and other symptoms, the difference of which was not statistically significant (P > 0.05). In laboratory examination, EV71 infection made white blood cell count higher than CoxA16 and other enterovirus, but the difference was not statistically significant (P>0.05). Conclusions EV71, CoxA16 and other enterovirus intestinal virus are main pathogens of HFMD in Nantong. EV71 C4 subtype is the main popular subtype of EV71 in Nantong. The elevation of white blood cell count of HFMD is the main clinical manifestations and characteristics , but can not serve as the identification of children infected with EV71, CoxA16 and other enterovirus.

Objective To investigate the effects of Ginaton on acute lung injury (ALI) induced by lipopolysaccaride (LPS).Methods The acute lung injury rat model was induced by receiving 5 mg/kg LPS injection in tail vein.A total of 63 Wistar rats were divide into 3 groups without caring sex.Saline control group,LPS treated group and Ginaton treated group.The samples of different groups were collected 2 h,6 h,and 10 h after tail vein administration.Serum soluble cell ashesion molecule-1 (sICAM-1) was measured by ELISA,immunohistochemical staining and Western blotting of NF-kB were performed on sections of lung specimens.Results The acute lung injury rat model was successfully induced by receiving LPS injection in tail vein.The sICAM-1 levels increased more in Ginaton treated group than those in Saline control group (P＜0.01),and the increase in LPS treated group were the highest.By immunohistochemical staining,the positive NF-kB cells in Ginaton treated group were much less than those in LPS treated group (P＜0.01),and in Saline control group were least (P＜0.01).The results of the Western-blot method were consistent with immunohistochemical method.Conclusion ALI could be induced by LPS,Ginaton showed a protective effect through probably inhibiting activation of NF-kB on LPS-induced-ALI in this animal model.

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 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.

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