Hepatitis C virus genotype diversity in Ningxia Hui autonomous region, Northwestern China.

Hepatitis C virus (HCV) genotypes vary greatly in different regions. The aim of this study is to investigate the distribution of HCV genotypes in HCV infected patients, in Ningxia Hui Autonomous Region. Nucleic acid extraction and amplification were performed with test kits on 153 HCV infected patients serum samples. The HCV viral load was measured using reverse transcriptase PCR (RT-PCR) and HCV genotypes were determined. Among the 153 HCV-infected patients, 56 had genotype (GT)1b (36.60%), 45 had GT2a (29.40%), 23 had GT3a (15.00%), 14 had GT3b (9.20%),13 had GT6a (8.50%), 1 had GT1g (0.70%), 1 had GT6xa (0.70%). In GT1b, 21.40% were female and 78.60% were male; in GT2a, 42.20% were female and 57.80% were male;Males were most prevalent in genotypes 1b(39.30%), while female were most prevalent in genotype 2a(46.30%). Rare GT1g and GT6xa were also detected in males. The 41-50 year age group had the highest HCV prevalence of 32.00%. HCV GT1b is the predominant HCV genotype in Ningxia Hui Autonomous Region.

[Construction of TRAF6 ubiquitin site 331 mutant colorectal cancer cell stable line and its effect on biological behavior of colorectal cancer cells].

Objective: To investigate the effect of ubiquitin mutation at position 331 of tumor necrosis factor receptor related factor 6 (TRAF6) on the biological characteristics of colorectal cancer cells and its mechanism. Methods: lentivirus wild type (pCDH-3×FLAG-TRAF6) and mutation (pCDH-3×FLAG-TRAF6-331mut) of TRAF6 gene expression plasmid with green fluorescent protein tag were used to infect colorectal cancer cells SW480 and HCT116, respectively. The infection was observed by fluorescence microscope, and the expressions of TRAF6 and TRAF6-331mut in cells was detected by western blot. Cell counting kit-8 (CCK-8) and plate cloning test were used to detect the proliferation ability of colorectal cancer cells in TRAF6 group and TRAF6-331mut group, cell scratch test to detect cell migration, Transwell chamber test to detect cell migration and invasion, immunoprecipitation to detect the ubiquitination of TRAF6 and TRAF6-331mut with ubiquitinof lysine binding sites K48 and K63. Western blot was used to detect the effects of TRAF6 and TRAF6-331mut over expression on the nuclear factor kappa-B (NF-κB) and mitogen activated protein kinase mitogen-activated protein kinase (MAPK)/activating protein-1(AP-1) signal pathway. Results: The successful infection of colorectal cancer cells was observed under fluorescence microscope. Western blot detection showed that TRAF6 and TRAF6-331mut were successfully expressed in colorectal cancer cells. The results of CCK-8 assay showed that on the fourth day, the absorbance values of HCT116 and SW480 cells in TRAF6-331mut group were 1.89±0.39 and 1.88±0.24 respectively, which were lower than those in TRAF6 group (2.09±0.12 and 2.17±0.45, P=0.036 and P=0.011, respectively). The results of plate colony formation assay showed that the number of clones of HCT116 and SW480 cells in TRAF6-331mut group was 120±14 and 85±14 respectively, which was lower than those in TRAF6 group (190±21 and 125±13, P=0.001 and P=0.002, respectively). The results of cell scratch test showed that after 48 hours, the percentage of wound healing distance of HCT116 and SW480 cells in TRAF6-331mut group was (31±12)% and (33±14)%, respectively, which was lower than those in TRAF6 group [(43±13)% and (43±7)%, P=0.005 and 0.009, respectively]. The results of Transwell migration assay showed that the migration numbers of HCT116 and SW480 cells in TRAF6-331mut group were significantly lower than those in TRAF6 group (P<0.001 and P<0.002, respectively). The results of Transwell invasion assay showed that the number of membrane penetration of HCT116 and SW480 cells in TRAF6-331mut group was significantly lower than those in TRAF6 group (P=0.008 and P=0.009, respectively). The results of immunoprecipitation detection showed that the ubiquitin protein of K48 chain pulled by TRAF6-331mut was lower than that of wild type TRAF6 in 293T cells co-transfected with K48 (0.57±0.19), and the ubiquitin protein of K63 chain pulled down by TRAF6-331mut in 293T cells co-transfected with K63 was lower than that of wild type TRAF6 (0.89±0.08, P<0.001). Western blot assay showed that the protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-HCT116 cells were 0.63±0.08, 0.42±0.08 and 0.60±0.07 respectively, which were lower than those in TRAF6-HCT116 cells (P=0.002, P<0.001 and P<0.001, respectively). The expression level of AP-1 protein in TRAF6-HCT116 cells was 0.89±0.06, compared with that in TRAF6-HCT116 cells. The difference was not statistically significant (P>0.05). The protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-SW480 cells were 0.50±0.06, 0.51±0.04, 0.48±0.02, respectively, which were lower than those in TRAF6-SW480 cells (all P<0.001). There was no significant difference in AP-1 protein expression between TRAF6-331mut-SW480 cells and TRAF6-SW480 cells. Conclusion: The ubiquitin site mutation of TRAF6 gene at 331 may prevent the binding of TRAF6 and ubiquitin lysine sites K48 and K63, and then affect the expressions of proteins related to downstream NF-κB and MAPK/AP-1 signal pathways, and inhibit the proliferation, migration and invasion of colorectal cancer cells.

Further investigation on nuclear transplantation in different orders of teleost: the combination of the nucleus of Tilapia (Oreochromis nilotica) and the cytoplasm of Loach (Paramisgurnus dabryanus).

The nucleus of a blastula cell from Tilapia (Oreochromis nilotica, family Cichlidae, order Perciformes) was transplanted into an enucleated egg of Loach (Paramisgurnus dabryanus, family Cobitidae, order Cypriniformes). From among 3747 nucleo-cytoplasmic hybrid (NCH) eggs two NCH larval fish (0.05%) were obtained; one died on the 6th day and the other died on the 12th day after the operation. Morphological examinations showed that both NCH larval fish had developed normally with an opened mouth except they could not take food after complete utilization of their egg yolk on the 5th day of development. The possible mechanisms for obtaining such inter-order NCH larval fish are discussed. This is the first report indicating that inter-order NCH larval fish can be obtained in spite of their evolutionary divergence.