Objective To study the structure of spliced variants of hepatitis B virus (HBV) genomes and elucidate their potential pathogenicity. Methods Amplified the spliced variants of hepatitis B virus genomes by mean of PCR from the serum of the patients with chronic hepatitis B, sequenced and analysis the characteristics of such genomes. Results 10 different types of the spliced variants of hepatitis B virus genomes were obtained with the molecular weight ranging from 765 bp to 2039 bp. There were 6 splicing donor sites and 6 accepter sites, respectively in HBV genomes. All spliced variants showed one or more deletions in the regions coding for core, preS1, preS2 and surface protein, while retained thepathogenic X gene and cis elements which were essential for viral replication and packaging. Conclusions Spliced variants of hepatitis B virus genomes were commonly detected in the serum from chronic Hepatitis B patients, the characteristic structure of such variants implied that they might closely co-related with the pathogenicity of HBV.

OBJECTIVE: To explore the role of N~6-methyladenosine(m~6A) catalytic enzymes(methyltransferases and demethylases) in cadmium-induced oxidative damage in human renal epithelial cells(HK-2 cells), and to analyze the correlation between nuclear factor-erythroid 2-related factor 2(NRF2) and m~6A catalytic enzymes. METHODS: i) HK-2 cells in logarithmic growth phase were randomly divided into control group and 6 cadmium sulfate treatment groups, then treated with 0, 2, 4, 8, 16, 32 and 64 μmol/L cadmium sulfate solution for 24 hours. The cell survival rates were detected by CCK-8 assay, and the appropriate doses of cadmium sulfate were selected for subsequent experiments. ii) HK-2 cells in logarithmic growth phase were randomly divided into control group and low-, medium-, and high-dose groups, and treated with 0, 4, 8, and 16 μmol/L cadmium sulfate solution respectively for 24 hours. Subsequently, the levels of reactive oxygen species(ROS) were detected by fluorescence probe. The mRNA expression of NRF2, the m~6A methyltransferases such as methyltransferase like proteins(METTL) 3, METTL14, METTL16 and the m~6A demethylases such as fat mass and obesity associated protein(FTO), AlkB family of nonheme Fe(Ⅱ)/α-ketoglutarate(α-KG)-dependent dioxygenases 5(ALKBH5) were determined by real-time polymerase chain reaction. RESULTS: i) The survival rate of HK-2 cells was more than 60.00% and lower than that of the control group(P<0.05) after the cells were stimulated with 16 μmol/L of cadmium sulfate. Therefore, 4, 8 and 16 μmol/L of cadmium sulfate were selected as the stimulation concentrations in the follow-up experiments. ii) The relative expression of NRF2, METTL3, METTL14 and METTL16 in HK-2 cells in low-dose group increased(all P<0.05), while the levels of ROS and the relative mRNA expression of NRF2, METTL3, METTL14, METTL16 and FTO in HK-2 cells in medium and high-dose groups increased(all P<0.05) when compared with the control group. There was no significant difference in the expression of ALKBH5 mRNA among these 4 groups(P>0.05). In the correlation analysis, NRF2 mRNA expression was positively correlated with the mRNA expression of METTL3 and METTL16 [Pearson correlation coefficient(r) = 0.61 and 0.66, respectively, all P<0.05]. There was no correlation between NRF2 mRNA expression and METTL14, FTO and ALKBH5(r=0.53, 0.48, and 0.01 respectively, all P>0.05). CONCLUSION: Cadmium sulfate may increase intracellular ROS level, up-regulate NRF2 expression and activate NRF2 signaling pathway as well as enhance the expression of METTL3 and METTL16 in HK-2 cells, thus increasing intracellular oxidative damage and decreasing the cell survival rate.

Objective To assess the effects of hepatitis B virus(HBV) large envelope protein (LHB) on the apoptosis of HepG2 cells and explore the possible mechanism by proteomic approaches.Methods LHB gene was cloned into pShuttle-IRES-hrGFP-1,and the recombinant adenovirus either barboring LHB(Ad-LHB) or empty vector(Ad-GFP) were separately generated.Annexin V-FITC apoptosis detection kit,JC-1 mitochondrial membrane potential assay kit and propidium iodide(PI) staining kit were employed combined with flow cytometry to detect the apoptotic cells infected with Ad-LHB or control of Ad-GFP.The cellular proteins were collected after infection of HepG2 cells by Ad-LHBs or Ad-GFP,and a total of 600 μg proteins were submitted to two-dimensional gel electrophoresis(2-DE) and stained with R350.The gel images were captured by ImageScanner Ⅱ Imaging System,the differentially expressed proteins were identified by ImageMaster 2D Platinum analysis software and picked up by Ettan Spot Picker.After enzyme digestion,the protein samples were analyzed by MALDI-TOF-TOF MS.Results HepG2 cells infected with Ad-LHB were much more prone to apoptosis.There were thirty nine differentially expressed proteins were determined by 2-DE between HepG2 cells infected with Ad-LHB and Ad-GFP,and they were identified ultimately and categorized into thirty three kinds of proteins by MALDI-TOF-TOF MS.Among these proteins,nine were found to be closely related to cell apoptosis,in which CAPN2,eIF3K and PPP2CB were higher expressed in Ad-LHB infected HepG2 cells,and SERPINH1,LASP1,PRDX1,DHRS2,LDHA and PS-MA4 were lower expressed in Ad-LHB infected HepG2 cells.Conclusion LHB could induce apoptosis of HepG2 cells,and several apoptosis-related proteins participated in this process.

Objective To investigate the relationships between the expression of VEGF,its receptor KDR/flk-1,cNOS mRNA and angiogenesis,cell proliferation,metastasis in human hepatocellular carcinoma(HCC). Methods Immunohistochemical analysis using antibodies against VEGF and its kinase insert domain receptor (KDR) was carried out.cNOS mRNA expression in HCC,liver cirrhosis and normal liver tissue was observed by in situ hybridization.CD34 immunostaining was used to measure the microvascular density(MVD)and proliferative index was evaluated by Ki-67 immunostaining. Results The expressions of VEGF and KDR of HCC were significantly related to MVD,proliferation and metastasis of HCC(P

Objective To investigate the signal transduction pathway of arachidonic acid(AA) and prostaglandin E-2(PGE-2) synthesis in macrophage invaded by Toxoplasma gondii. Methods Synthesis of AA and PGE-2, expression of COX_2 mRNA and protein following stimulation infection by Toxoplasma gondii were evaluated in RAW264^7 cells by ELISA, RT_PCR and Western blotting after treatment with calcium channel blocker verapamil, chelator of extracellular calcium EGTA and inhibitor of CaM trifluoperazine (TFP), selective PKC inhibitor H7. Results Production of AA and PGE-2 induced by tachyzoite was significantly inhibited by EGTA, TFP and BAPTA/AM, and the PGE-2 production was inhibited by H7, with a reduced expression of COX_2 mRNA and protein in a dose_dependent manner. Conclusion The parasite down_regulates macrophage functions by affecting PKC signaling pathways, and triggers a biochemical cascade whose signals ultimately conduct to the secretion of immunosuppressive molecules PGE-2.

ATP-binding cassette protein E (ABCE1) has been annotated as an Rnase L inhibitor in eukaryotes. Previous study showed that the overexpression of ABCE1 was related with the occurrence and clinical stage of lung adenocarcinoma. As an initial investigation into the novel functions of ABCE1, siRNA-expressing vectors targeting sites of the ABCE1 gene were constructed from RNAi-Ready pSIREN-DNR-DsRed-Express vector. Cultured 95-D and NCI-H446 lung carcinoma cells were transfected with the siRNA-expressing vectors using FuGENE 6 and transfection efficiency was determined by using fluorescence microscopy. The expression level of ABCE1 protein was determined by Western blot and immunofluorescence staining. Cell viability was determined by MTT, cell cycle was analysed by flow cytometry.The apoptotic rate was observed by ELISA. Fluorescence microscopy showed a satisfactory transfection efficiency which was about 42.70%. Cell viability and the growth fraction were markedly suppressed, whereas the apoptosis was significantly increased in SiRNA-95-D and SiRNA-NCI-H446 cells than controls(P< 0.05). It can be concluded that the siRNA targeting ABCE1 gene shows a dramatic inhibitory effect on RNA transcription and protein expression and a promoting effect on the apoptosis in 95-D/NCI-H446 cells, which offers a reliable base for the further in vivo experiment.

Objective To obtain protein of the major surface antigen P30 of Toxoplasma gondii by molecularcloning. Methods The gene of P30 (containing the whole P30 gene sequence, without the gene encoding signalpeptide) was obtained by polymerase chain reaction (PCR) using the primer designed according to the DNA sequence ofP30. The recombinant plasmid was constructed using EcoR Ⅰ, Xho Ⅰ and was then transformed into E. coli Top10. Thepositive clones were identified by restriction enzymes and DNA sequence analysis. The fusion protein was induced by IPTGand purified by affinity chromatography using ProBond~(TM) Resin (a kind of nickel-charged sepharose resin) and was identi-fied by SDS-PAGE and Western blotting. Results The products of PCR, cleavage and link reaction were same as ex-pected and the sequence of inserted fragment in the recombinant plasmid was same as reported except one synonymy muta-tion. A 58 kDa fusion protein was induced by IPTG and was purified by chromatography. Conclusion Fusion proteincontaining Toxoplasma gondii P30 was achieved and was provided as experiment material for further research.

AIM: To delineate the specific mutation responsible for retinitis pigmentosa(RP)in a Yi pedigree, and to analyze the correlation of RHO gene mutation with clinical phenotype.METHODS:A comprehensive clinical evaluation was conducted on the proband diagnosed with RP and other familial members, complemented by a thorough ophthalmic examination. Peripheral blood samples were obtained from the proband and familial members, from which genomic DNA was extracte. Subsequent whole exome sequencing(WES)was employed to identify the variant genes in the proband. The identified variant gene was validated through Sanger sequencing, then an in-depth analysis of the mutation genes was carried out using genetic databases to ascertain the pathogenic mutation sites. Furthermore, an exhaustive analysis was performed to delineate the genotype and phenotype characteristics.RESULTS:The RP pedigree encompasses 5 generations with 42 members, including 19 males and 23 females. A total of 13 cases of RP were identified, consisting of 4 males and 9 females, which conforms to the autosomal dominant inheritance pattern. The clinical features of this family include an early onset age, rapid progression, and a more severe condition. The patients were found to have night blindness around 6 years old, representing the earliest reported case of night blindness in RP families. The retina was manifested by progressive osteocytoid pigmentation of the fundus, a reduced visual field, and significantly decreased or even vanished a and b amplitudes of ERG. The combined results of WES and Sanger sequencing indicated that the proband had a heterozygous missense mutation of the RHO gene c.1040C>T:p.P347L, where the 1 040 base C of cDNA was replaced by T, causing codon 347 to encode leucine instead of proline. Interestingly, this mutation has not been reported in the Chinese population.CONCLUSION:This study confirmed that the mutant gene of RP in a Yi nationality pedigree was RHO(c.1040C>T). This variant leads to the change of codon 347 from encoding proline to encoding leucine, resulting in a severe clinical phenotype among family members. This study provides a certain molecular, clinical, and genetic basis for genetic counseling and gene diagnosis of RHO.

AIM: To delineate the specific mutation responsible for retinitis pigmentosa(RP)in a Yi pedigree, and to analyze the correlation of RHO gene mutation with clinical phenotype.METHODS:A comprehensive clinical evaluation was conducted on the proband diagnosed with RP and other familial members, complemented by a thorough ophthalmic examination. Peripheral blood samples were obtained from the proband and familial members, from which genomic DNA was extracte. Subsequent whole exome sequencing(WES)was employed to identify the variant genes in the proband. The identified variant gene was validated through Sanger sequencing, then an in-depth analysis of the mutation genes was carried out using genetic databases to ascertain the pathogenic mutation sites. Furthermore, an exhaustive analysis was performed to delineate the genotype and phenotype characteristics.RESULTS:The RP pedigree encompasses 5 generations with 42 members, including 19 males and 23 females. A total of 13 cases of RP were identified, consisting of 4 males and 9 females, which conforms to the autosomal dominant inheritance pattern. The clinical features of this family include an early onset age, rapid progression, and a more severe condition. The patients were found to have night blindness around 6 years old, representing the earliest reported case of night blindness in RP families. The retina was manifested by progressive osteocytoid pigmentation of the fundus, a reduced visual field, and significantly decreased or even vanished a and b amplitudes of ERG. The combined results of WES and Sanger sequencing indicated that the proband had a heterozygous missense mutation of the RHO gene c.1040C>T:p.P347L, where the 1 040 base C of cDNA was replaced by T, causing codon 347 to encode leucine instead of proline. Interestingly, this mutation has not been reported in the Chinese population.CONCLUSION:This study confirmed that the mutant gene of RP in a Yi nationality pedigree was RHO(c.1040C>T). This variant leads to the change of codon 347 from encoding proline to encoding leucine, resulting in a severe clinical phenotype among family members. This study provides a certain molecular, clinical, and genetic basis for genetic counseling and gene diagnosis of RHO.

During the preparation of functional gradient materials (FGM) with plasma immersion ion implantation-ion beam enhanced deposition (PIII-IBED), the combination strength between the coating and the substrate would be greatly affected by the implantation dose and the distribution of implanted ions in substrate. According to the requirements of FGM, an idea of multi grades energy implantation had been suggested, with which the Gauss peak could be moved toward the surface, and the concentration of implanted ions could be maximized at the surface. In this study, the distribution of carbon ions implanted into titanic alloy substrate have been simulated theoretically, and the tentative idea have been confirmed experimentally.
Alloys ; Carbon ; Coated Materials, Biocompatible ; chemical synthesis ; Computer Simulation ; Heart Valve Prosthesis ; Titanium