Objective To study association of hepatitis B virus(HBV) precore (pre c)/basic core promoter(BCP) mutations with the genotype or the progression of liver disease. Methods The serum samples from 148 patients with HBV-relative diseases were collected, including 31 asymptomatic carriers, 32 with chronic hepatitis B (CHB), 40 with liver cirrhosis(LC) and 45 with hepatocellular carcinoma(HCC). The genes covering HBV pre c and BCP were amplified by nested polymerase chain reaction (nPCR). The PCR products were subjected to direct sequencing and the mutations in pre c 1896 and BCP 1762/1764 were determined by sequence analysis. HBV genotypes were also detected in the sera by restriction fragment length polymorphism based on S-gene PCR products. Results Of 148 serum samples of HBV, 128 were successfully genotyped and sequenced. There were 60 genotype B and 68 genotype C. The mutation in pre c (A1896) was significantly higher in genotype B than in genotype C (48.3% vs 29.34%, P

0.05). The sequence of five clones from one serum which was identified precore mutation by mPCR-RFLP were all A1896 mutant strains.Another serum identified mixture infection by mPCR-RFLP , one clone was A1896 mutant strain and four were G1896 wild strains.The results of mPCR-RFLP were verified by cloning.Conclusions Comparison with sequencing, the mPCR-RFLP method is simple,accurate and can be used in large-scale surveys and clinical research.

ObjectiveTo observe the efficacy of L-carnitine in prevention and treatment of toxicity of LFP chemotherapy with gastrointestinal cancer. Methods60 cases of gastrointestinal cancer were divided into 2 groups according to the admission date. The treatment groups received LFP chemotherapy and L-carnitine, while the control group received LFP chemotherapy alone.Both groups received 3 cycles chemotherapy. The gastrointestinal toxicity, neurotoxicity, hematologic toxicity and physical state were compared.ResultsThere were 12 cases of peripheral neurotoxicity in treatment group,the incidence rate was 40.0 ％;but in control group there were 21 patients, and the incidence rate was 70.0 ％. There was a significant difference between the two groups(x2=5.4545,P =0.0195). Anemia in the treatment group was 56.7 ％ (17/30); in the control group the rate was 86.7 ％ (26/30).There was a significant difference between the two groups (x2 =6.698,P =0.0351).After chemotherapy,in the treatment group,there were 4 cases with increasing Karnofsky score ≥ 10,19 patients whose Karnofsky score did not change,and 7 cases with reduced Karnofsky score ≥ 10; in the control group,there was 1 case with increasing Karnofsky score ≥ 10,13 cases with Karnofsky score no changing,and 16 cases with reduced Karnofsky score ≥ 10.The changes in physical state of two groups had a significant difference(x2 =5.711,P =0.0169).The gastrointestinal toxicity,thrombocytopenia,and neutropenia of two groups had no significant difference.ConclusionL-carnitine can reduce peripheral neurotoxicity and hematologic toxicity,improve the patient's physical state in patients received LFP chemotherapy.

OBJECTIVETo investigate the prevalence of transfusion-transmitted virus (TTV) infection and analyze partial nucleotide sequence of TTV isolated from Guizhou area.

METHODSA nested polymerase chain reaction (nPCR) assay with two-set of primers deduced from the first read frame of TTV genome was established, the prevalence of TTV DNA were detected in 395 sera collected from different population in Guizhou area. The partial genes of TTV DNA fragments amplified from two liver disease patients and one hemodialysis patient were analysed and then compared with gene from Japan strain.

RESULTSThe positive rates of TTV DNA in 62 normal adults, 37 blood donors, 50 hemodialysis patients, 107 intravenous drug users (IVDUs), 139 patients with liver diseases were 6.45% (4/62), 8.10%(3/37), 26.00% (13/50), 25.23% (27/107) and 16.54% (23/139), respectively. The homology of TTV DNA sequence was from 99% to 100% within three Guizhou strains. As compared with published sequence, the nucleotide homology is 98% between all three Guizhou strains and Japan strain.

CONCLUSIONSThere are TT virus infection in Guizhou area, the infection rates were higher in hemodialysis patients and IVDUs. The nucleotide homology was high between Guizhou strain and Japan strain.

Adolescent ; Adult ; Aged ; Blood Donors ; China ; epidemiology ; DNA Virus Infections ; epidemiology ; virology ; DNA, Viral ; blood ; Female ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Torque teno virus ; genetics ; isolation & purification

Objective:To provide a potential platform for transferring specific antigen against fish bacterial diseases based on attenuated Lm (EGDe-ΔactA/ΔinlB).Methods: Attenuated Lm (EGDe-ΔactA/ΔinlB) was used to express outer membrane protein K (Ompk),a conserved and effective vaccine candidate in vibrio.The identification of recombinant strains and detection of antigen genes were operated with PCR and RT-PCR,respectively.Results: The results of PCR showed that Lm-Ompk (L-O),Lm-Lmo0576-Ompk (L-L-O) and Lm-P-Ompk (L-P-O) were constructed successfully.The identity of foreign gene was 100％ compared with sequence of NCBI.The analysis of transcription showed that the expressions of Ompk in L-O,L-L-O and L-P-O were significant (P<0.001).Moreover,the expression of Ompk in the condition of antibiotic was higher than that in the BHI without antibiotic (P<0.05).Conclusion: Lm-Ompk (L-O),Lm-Lmo0576-Ompk (L-L-O) and Lm-P-Ompk (L-P-O) were constructed successfully.

Objective To establish a convenient method to detect the genomic population with hepatitis C virus (HCV) at nonstructure 5A (NS5A) region and to determine the correlation between the genomic population complexity at NS5A region and disease stage.Methods The sera from 52 patients with chronic hepatitis C virus infections were analysed using single strand conformation polymorphism (SSCP). In the SSCP, an asymmetric polymerase chain reaction (PCR) was carried out on the 455 bp products of the first round PCR at the NS5A region and the number of band of single strand deoxyribonucleic acid (DNA) which reacted with complemental DNA probe specific for the NS5A region after gel electrophoresis was analyzed.Results In 90% patients with chronic persistent hepatitis, the bands of single strand DNA was limited to one, and in those with chronic active hepatitis or liver cirrhosis, two or more bands of DNA were frequently detected. In about half of patients with hepatocellularcarcinoma, three or more bands were found. The number of bands increased with the progression of liver disease. The multivariate analysis showed that the progression of liver disease was the independent factor of viral diversity (P<0.025) and was not related to the age, sex, the route of infection and the titer of hepatitis C virus ribonucleic acid (HCV RNA).Conclusion These results suggest that the genomic variability of HCV at NS5A region increases with the progression of liver disease, and this may be closely related to the clinical features of type C liver disease.

Objective To determine the concordance in various hepatitis C (HCV) genotyping methods and to investigate the distribution of HCV genotypes in Guizhou area of Southwest China.Methods Serum samples from 206 patients (100 with chronic hepatitis and 106 with hemopathy) were detected for antibody of HCV by second generation enzyme-labelled immunosorbent assay (ELISA). Thirty-five anti-HCV positive samples were detected for HCV RNA by RT-polymerase chain reaction (PCR) and 30 HCV RNA positive samples were determined for their genotypes by three various genotyping methods [PCR with type-specific primers at the core region (primer-set), slot-blot hybridization with type-specific probes at NS5B region (blotting) and the restric fragment length polymorphism analysis of PCR products of 5'NC region (RFLP)]. Ten samples with the known genotype were analysed by the direct sequencing.Results Of 30 samples with positive HCV RNA, the types of 22 could be classified by three methods, and the genotypes determined by various methods had complete concordance. The types of 6 samples could be classified by two methods and 5 had agreement subtypes. The types of two samples could be classified only by RFLP. Overall, 27(90.0%) had subtype 1b infection and 3(10.0%) had subtype 2a infection. The nucleotide sequence of 8 samples with subtype 1b and one with subtype 2a were analysed by the direct sequencing. The subtypes determined by sequence analysis were in complete concordance with those decided by various genotyping methods.Conclusions Subtype 1b is the predominent HCV genotype in Guizhou area, while subtype 2a is less common. There was a good concordance with the genotyping results obtained by various HCV genotyping methods.

OBJECTIVETo assess the association of CYP2C19 gene polymorphisms with the incidence of ischemic stroke among patients receiving clopidogrel therapy following coronary stenting for coronary artery disease.

METHODSClinical data of patients receiving clopidogrel therapy after coronary stenting were retrospectively studied. For a case-control study, 137 patients with acute cerebral infarction and 122 non-stroke patients were selected. Based on the variants of the CYP2C19 gene detected by a DNA microarray assay, the patients were further divided into the wild-type group(CYP2C19*1/*1) and mutant group(defined by the presence of at least one loss-of-function allele, including CYP2C19*1/*2, CYP2C19*1/*3, CYP2C19*2/*2, CYP2C19*2/*3 and CYP2C19*3/*3). The incidences of ischemic stroke in the two groups were compared through a chi-square analysis. The influence of CYP2C19 gene polymorphisms and clopidogrel therapy on the incidence of ischemic stroke was analyzed through multivariable logistic regression.

RESULTSA total of 259 patients were enrolled. The case and control groups showed no difference in terms of gender and age. There were 123 cases (47.5%) in the CYP2C19 wild-type group and 136 cases (52.5%) in the mutant group. The incidence of ischemic stroke of mutant group was significantly higher than that of wild-type group (59.9% vs. 44.3%, X2=6.398, P=0.042). Multivariate analysis revealed that loss-of-function polymorphisms of the CYP2C19 gene carried a 1.13 times greater risk for ischemic stroke compared to wild-type genotype (OR=2.13, 95%CI: 1.23-3.71).

CONCLUSIONThe efficacy of clopidogrel for the prevention of ischemic stroke in post-coronary stent patients may be reduced by the insufficiency of the CYP2C19 gene. The dosage of clopidogrel therapy should be adjusted based on its polymorphisms.

Brain Ischemia ; prevention & control ; Cytochrome P-450 CYP2C19 ; genetics ; Genotype ; Humans ; Percutaneous Coronary Intervention ; adverse effects ; Platelet Aggregation Inhibitors ; therapeutic use ; Polymorphism, Genetic ; Stents ; adverse effects ; Stroke ; prevention & control ; Ticlopidine ; analogs & derivatives ; therapeutic use

Objective:To explore the pathogenesis of flunarizine-induced parkinsonism from gut-brain axis perspective.Methods:Thirty male C57BL/6 mice were randomly divided into control group and flunarizine group ( n=15). Mice in the control group were given 0.1 mL 50% polyethylene glycol 400+50% saline by gavage once/d for 2 weeks, while mice in the flunarizine group were given 6 mg/mL flunarizine+50% polyethylene glycol 400+50% saline by gavage at a daily dose of 30 mg/kg for 2 weeks. Body mass was recorded 1, 3, 5, 7, 10 and 14 d after drug administration, and motor function was assessed by rotarod test 14 d after drug administration; 16s RNA sequencing was performed in the feces to observe the intestinal flora; intestinal transit function was detected by Evans blue by gavage; and then, the mice were sacrificed and homogenate or frozen sections (brain and intestinal tissues) were prepared; dopamine-ergic neuron expression was detected by Western blotting; RT-qPCR was applied to detect the expressions of inflammatory factors in the substantia nigra, and immunofluorescent staining was used to detect the expressions of ZO-1 and Claudin-5 in the intestinal epithelial tissues. Results:Compared with the control group, the flunarizine group had lower body mass ratio 1, 3, 5, 7, 10 and 14 d after drug administration (ratio to body mass before drug administration). Compared with the control group, the flunarizine group had significantly shortened residence time in rod rotating and lower rotational speed when falling ( P<0.05). Compared with the control group, the flunarizine group had decreased tyrosine hydroxylase protein in the substantia nigra without significant difference ( P>0.05). Compared with the control group, the flunarizine group had significantly increased interleukin-6 and tumor necrosis factor-α in the substantia nigra (1.00±0.00 vs. 2.79±0.83; 1.00±0.00 vs. 3.39±1.37), significantly lower intestinal Evans blue propulsion rate (80.67%±4.51% vs. 50.67%±6.03%), and statistically decreased ZO-1 and Claudin-5 expressions in the colonic epithelial tissues (27.01±1.41 vs. 16.32±2.83; 37.00±2.80 vs. 24.52±2.12, P<0.05). Totally, 576 microorganisms were noted in both control group and flunarizine group, 744 in the control group alone, and 634 in the flunarizine group alone. The intestinal flora β diversity indices in the 2 groups were significantly different based on weighted Unifrac-principle coordinates analysis (PCoA, PCoA1: 39.88%; PCoA2: 30.69%). Compared with the control group, the microbial colony structure of mice in flunarizine group was dominated by phylum thick-walled bacteria and phylum warty microbacteria, and by families Muribaculaceae, Lachnospiraceae and Akkermansiaceae. Compared with the control group, the flunarizine group had significantly decreased relative abundance of Ackermannia spp. and Lactobacillus spp. in the intestinal flora ( P<0.05). Conclusion:Flunarizine may contribute to the pathogenesis of DIP by causing structural disturbances in the intestinal flora and inducing neuroinflammation based on the gut-brain axis.