Objective To determine Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) sequence types in different geographical areas of China,including Changzhou and Yangzhou cities of Jiangsu province,Wuzhou and Hezhou cities in Guangxi Zhuang Autonomous region,Sanya and Qionghai cities of Hainan province,Jiangmen and Maoming cities of Guangdong province.Methods DNA was extracted using Qiagen DX extraction kits from 88 urine samples which were collected from male patients attending sexually transmitted disease (STD) clinics and positive for nucleic acid amplification test (NAAT) for N.gonorrhoeae.Two rounds of PCR were carried out to amplify the porB and tbpB genes of N.gonorrhoeae followed by gene sequencing.Sequence alignment was performed on the NG-MAST website (http://www.ng-mast.net) to determine the genotype of N.gonorrhoeae.Results The first-round PCR yielded positive results for porB and tbpB in 13.6％ (12/88) and 14.8％ (13/88),respectively,of these urine specimens,and 12 samples were successfully genotyped with the efficiency of genotyping being 13.6％.The amplification efficiency of second-round PCR was enhanced to 71.6％ and 72.7％ for porB and tbpB,respectively,and the efficiency of genotyping increased to 70.5％ (62/88).Compared with the first-round PCR,the second-round PCR showed an increase in amplification efficiency for porB and tbpB by 58.0％ and 57.9％ respectively,as well as in genotyping efficiency by 56.9％.Forty-five genotypes were identified in the 62 samples,including 40 known genotypes and 5 novel genotypes.Of these genotypes,ST1866 was the most abundant (6/62),followed by ST1972 (4/62) and ST3356 (4/62),all of which were from Jiangsu province.The ST532 genotype was identified in 3 samples from Guangdong province,ST2221 genotype in 2 samples from Guangxi Zhuang Autonomous region.Each of the remaining genotypes was identified in only 1 sample and scattered in all of these cities.The 5 novel MAST-genotypes were as follows:porB-892 and tbpB-46 (98％ similarity),porB-130 and tbpB-504 (96％ similarity),porB-2790 and tbpB-32 (99％ similarity),porB-1053 and tbpB-856 (99％ similarity).Conclusions Urine samples can be used for NG-MAST analysis,and two rounds of PCR can enhance the efficiency of genotyping.NG-MAST genotypes appear to be diverse in different geographical areas of China.

OBJECTIVE: To explore the effects of matrine on the proliferation, tumor cell stemness, β-catenin transcriptional activity and AKT/GSK3β/β-catenin signaling pathway in human hepatocellular carcinoma (HCC) HepG2 and Huh7 cells. METHODS: The proliferation and colony formation ability of HepG2 and Huh7 cells treated with 200, 400, and 800 μg/mL matrine were evaluated with MTT assay and colony formation assay, respectively. Real-time quantitative PCR was performed to detect the mRNA expressions of CD90, epithelial cell adhesion molecule (EpCAM) and CD133, and dual-luciferase assay was used to detect the transcriptional activity of β-catenin in the treated cells. The effects of matrine on the expressions of protein kinase B (AKT), P-AKT, GSK-3β, P-GSK-3β, P-β-catenin and β-catenin proteins in the Wnt/β-catenin signaling pathway were assessed using Western blotting. RESULTS: Matrine inhibited the proliferation of the two HCC cell lines in a time- and concentration-dependent manner. The half-inhibitory concentrations of matrine were 2369, 1565 and 909.1 μg/mL at 24, 48 and 72 h in HepG2 cells, respectively, and were 1355, 781.8, and 612.8 μg/mL in Huh7 cells, respectively. Matrine concentrationdependently suppressed colony formation of the HCC cells, producing significant inhibitory effects at 400 μg/mL < 0.01) and 800 μg/mL < 0.001) in HepG2 cells and at 200 μg/mL < 0.05), 400 μg/mL < 0.01), and 800 μg/mL < 0.001) in Huh7 cells. Matrine at 400 and 800 μg/mL significantly inhibited the mRNA expression of CD90, EpCAM and CD133 and the transcriptional level of β-catenin in both HepG2 and Huh7 cells < 0.05 or 0.01). Matrine at 400 and 800 μg/mL also significantly decreased the protein levels of β-catenin, P-AKT and P-GSK-3β and increased the phosphorylation level of β-catenin in both of the cell lines. CONCLUSIONS Matrine inhibits the proliferation, colony formation, and the expressions of tumor stem cell markers CD90, EpCAM and CD133 in both HepG2 and Huh7 cells probably by inhibiting Wnt/β-catenin signaling pathway and the transcriptional activity ofβ-catenin.