To obtain chemical constituent information of rat plasma after oral administration of Huang-Lian-Jie-Du Decoction (HLJDD), a LC-FT-ICR-MS method has been established, and both positive and negative ions scan modes were include in the analysis. By comparing their retention time, high resolution mass data of HLJDD extracts, blank plasma and dosed plasma, 38 constituents, including 22 prototype compounds and 16 metabolites, were detected in rat plasma after oral administration of HLJDD. In the 22 prototype compounds, 16 constituents were determined unambiguously by comparing with references. In the analysis of metabolites, phase II reactions like glucuronidation and sulfation were the major biotransformation pathways of HLJDD. M11 was observed as the only phase I metabolite in present experiment. The results will be beneficial for the further pharmacokinetics and pharmacological evaluations of HLJDD.
Administration, Oral ; Alkaloids ; blood ; Animals ; Biotransformation ; Chromatography, High Pressure Liquid ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; metabolism ; Flavonoids ; blood ; Iridoids ; blood ; Male ; Plants, Medicinal ; chemistry ; Rats ; Rats, Sprague-Dawley ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry

To obtain chemical constituent information of rat plasma after oral administration of Huang-Lian-Jie-Du Decoction (HLJDD), a LC-FT-ICR-MS method has been established, and both positive and negative ions scan modes were include in the analysis. By comparing their retention time, high resolution mass data of HLJDD extracts, blank plasma and dosed plasma, 38 constituents, including 22 prototype compounds and 16 metabolites, were detected in rat plasma after oral administration of HLJDD. In the 22 prototype compounds, 16 constituents were determined unambiguously by comparing with references. In the analysis of metabolites, phase II reactions like glucuronidation and sulfation were the major biotransformation pathways of HLJDD. M11 was observed as the only phase I metabolite in present experiment. The results will be beneficial for the further pharmacokinetics and pharmacological evaluations of HLJDD.

OBJECTIVETo clone, identify and phylogenetically characterize a clade B-Thai HIV isolate representing the most prevalent virus in Henan province.

METHODSPeripheral blood mononuclear cells (PBMCs) from an HIV-1 infected patient in Henan Province were separated, and co-cultivated with phytohemagglutinin-stimulated healthy donor PBMCs. Proviral DNA was extracted from productively infected PBMCs. The full-length HIV-1 genome was amplified by using the LA Tag long template PCR system. Primers were positioned in conserved regions within the HIV-1 long terminal repeats. Purified PCR products were T-A ligated into a pWSK29-T vector(CNHN 24 clone). Three recombinant clones containing virtually full-length HIV-1 genome were identified by PCR. The full-length genome was sequenced by using the primer-walking approach. Nucleotide sequence similarities were calculated by the local-homology algorithm. Phylogenetic trees of gag, pol and env reading frames were constructed using the Phylip software.

RESULTSHIV-1 C3V4 sequences indicate that the epidemic in this area was B-Thai subtype. V3 loop multiple amino acid sequence alignments showed amino acid alterations at nine positions. The 9,010 bp genomic sequence derived from isolate CNHN 24 contained all known structural and regulatory genes of an HIV-1 genome. No major deletions, insertions, or rearrangements were found. The highest homologies of the gag, pol, vpr, and vif reading frames to the corresponding clade B-Thai RL 42 sequences were 95.42%-97.08%. Phylogenetic trees showed the closest relationship of CNHN 24 and RL 42.

CONCLUSIONThe cloning and characterization of a virtually full-length HIV-1 B-Thai subtype in central China was completed in our laboratory. The data should be helpful to future studies on the genetic diversity of HIV-1.

Amino Acid Sequence ; Base Sequence ; Blood Donors ; China ; Cloning, Molecular ; DNA, Viral ; genetics ; Female ; Genome, Viral ; HIV Infections ; virology ; HIV-1 ; classification ; genetics ; Humans ; Leukocytes, Mononuclear ; virology ; Phylogeny ; Reading Frames ; Sequence Analysis, DNA ; Sequence Homology

Objective To investigate the pathogenic mutations of BRCA1 and BRCA2 in patients with early-onset breast cancer(≤35 years) and explore the relationships between BRCA1/2 mutations and clinical features.Methods Seventy-four patients with early-onset breast cancer were enrolled,who were treated in Hospital 307 between September 2014 and June 2016.High-throughput sequencing was used to test the 49 exon sequences and adjacent sequences of BRCA1 and BRCA2.χ2 test was used to analyze the distribution of BRCA1/2 pathogenic mutations in each group that was set up according to clinical features.Results Fifteen mutations(20.27%) were identified,including 5(6.76%) in BRCA1 and 10(13.51%) in BRCA2.Eleven new pathogenic mutations were discovered,and BRCA1:c.5470_5477delTGCCCAAT was found in one patient.The frequency of BRCA1/2 mutations in the group with a family history of breast cancer or ovarian cancer was higher than in the group without a family history (40.91% vs 11.54%) (χ2=6.534,P=0.011).Conclusion BRCA1/2 pathogenic mutation is significant for early-onset breast cancer,especially for those with a family history of breast or ovarian cancer.The new mutations may be specific to Chinese people.BRCA1:c.5470_5477delTGCCCAAT may be the ancestor mutation among the Chinese.

Objective To evaluate the associations between polymorphisms of LEPR Gln223Arg,LEPR Pro 1019Pro and the risk on obesity.Methods A computerized search on literature was carried out in Wanfang,CNKI,VIP databases and CBM,PubMed,EMBASE databases to collect articles published between 1979 and 2010 concerning the associations between polymorphisms of LEPR Gln223Arg and/or LEPR Pro 1019Pro and risk of obesity in the Chinese population.Odds ratios (ORs) were used to assess the strength of the association,and 95％ confidence intervals (CIs) to present the precision of the estimates.Meta-analysis was performed using the STATA statistical software.Results Fifteen literature were collected for Meta-analysis by the uniform inclusion and exclusion criteria.There were 1096 obese patients and 949 controls for polymorphisms of LEPR Gln223Arg in 9 papers,together with 961 obese patients and 818 controls for polymorphisms of LEPR Prol019Pro in 8 papers.Overall,there were significant associations between decreased risk of obesity and LEPR Gln223Arg polymorphisms (-668 A→G) (G versus A,OR=0.66,95％CI:0.49-0.89; AG and GG versus AA,OR=0.50,95％CI:0.32-0.77; respectively).There were significant associations between increased risk of obesity and LEPR Prol019Pro polymorphisms (-3057 G→A) (A versus G,OR=1.61,95％CI:1.15-2.26; AG and AA versus GG,OR=1.50,95％CI:1.08-2.08; respectively).Conclusion Variant alleles at both LEPR-668 and LEPR-3057 were associated with obesity in the Chinese Han-dominated population.

Objective:To investigate the subtype distribution of HIV-1 strains prevalent in four areas of China,and to study the characteristics of gag gene variation and changes in antigen epitopes under the host immune pressures. Methods:The plasma of HIV-1 infected people from Henan, Guangdong, Sichuan and Beijing in China were collected. Virion RNA was extracted directly from plasma after the virion was condensed. The gag gene was amplified by RT-PCR and nested-PCR.Sequences were subtyped by Genotyping Tool software, and phylogenetic analysis of gag gene were performed using the MEGA 4.1 software.The gene distances intra each subtype were calculated by Distance program. The Ks/Ka ratios were calculated using SNAP program. The variation analysis of CTL antigen epitopes restricted by main HLA-Ⅰ specificities in China was performed.Results:Six subtypes or circulating recombinant forms(CRFs)of HIV-1,including B',CRF07_BC,CRF01_AE,B,CRF08_BC and CRF02_AG,were identified in four areas of China.The gene distances intra each subtype were CRF01_AE>B>CRF08_BC> CRF07_BC>B' listed in order of size, meanwhile the order of Ks/Ka ratios was CRF01_AE>B>CRF08_BC>B'>CRF07_BC. Far more diversity of antigen epitopes in P17 region was observed than that in P24.Epitope mutations intra subtypes were CRF01_AE>B>B'>CRF07_BC listed in order of size. Conclusion:Itseems that CRF01_AE is under the strongest immune pressures,and displays the most diversity of gene and variation of epitopes intra subtypes prevalent in China, followed by subtype B, B' and CRF07_BC. The discrepancy of epitope mutations intra the subtypes is significant.

OBJECTIVETo investigate the factors of the human immunodeficiency virus (HIV) associated with sexual-transmission in central China.

METHODS(1) Cross-sectional study: couples that one was HIV positive were selected in Henan and Hebei province of China. The couples must be 20 - 50 years old with normal function on sexual intercourse. Cordant couples that subsequently infected partners were at risk of infection solely through sexual contact with the HIV-seropositive partner and the discordant couples that the seronegative partners were at risk of infection solely through sexual contact with the HIV-seropositive partner, were selected. Plasma viral load, CD4 cell count were tested. (2) Case-control study was used to compare 7 sexual transmitted cases and 56 nontransmitted controls with respect to the frequency of sexual intercourse, plasma viral load and CD4 cell count.

RESULTS(1) A total of 87 couples that at least one partner was HIV positive were recruited include 56 discordant couples and 7 cordant couples with whom sexual transmission had happened. The rate of sexual transmission was 11.1% among those at the risk of sexual transmission. (2) Of the discordant couples, male positive rate 25%, female positive was 75%. (3) The risk for transmission was higher in those couples with the frequency of unprotected vaginal sexual intercourse (> or = 4 times per month) than the reference group (< 4 times per month) (Fisher's exact test, P = 0.047, OR = 8.0). Median plasma viral load was significantly higher in the antecedent infected partners of cordant couples than the positive partner of discordant couples (378,285.71 vs 136,578.57 copies/ml, t = 3.591, P < 0.01). The odds ratio was 22.0 for plasma viral load > or = 100,000 copies/ml compared with the reference group of < 100 000 copies/ml (Fisher's exact test, P = 0.016). The CD4 cell count and CD4/CD8 of the transmitted group were significantly lower than that of the nontransmitted (t = 2.767, P < 0.05; t = 6.06, P < 0.05).

CONCLUSIONSThe frequencies of heterosexual-transmission in central China were relatively low. The risk of heterosexual transmission was related to the frequency of sexual intercourse. Higher plasma viral load and lower CD4 count was strongly correlated with high risk of heterosexual transmission.

Adult ; Blotting, Western ; CD4 Lymphocyte Count ; China ; Coitus ; Cross-Sectional Studies ; Disease Transmission, Infectious ; Female ; HIV ; immunology ; HIV Infections ; transmission ; Heterosexuality ; Humans ; Male ; Middle Aged ; Risk Factors ; Spouses ; Viral Load

BACKGROUNDVirus with nucleoside reverse transcriptase inhibitors (NRTIs) or nonnucleoside reverse transcriptase inhibitors (NNRTIs) resistant mutations show different evolution tendencies when the anti-viral therapies are interrupted. Understanding the replication fitness of drug-resistant virus is important for the study of the prevalence of drug-resistance. For this purpose, we characterized the replication capacity of HIV-1 virus carrying lamivudine (3TC) or nevirapine (NVP) resistant mutations.

METHODS3TC and NVP resistant variants were induced in vitro by selecting wild type virus in the presence of drugs. For the competitive replication assay, drug-resistant variants were cocultured with wild-type virus in the presence or absence of drugs. The ratios of the viral species were determined over time by using a real-time RT-PCR-based assay.

RESULTS3TC-resistant (M184I mutation) and NVP-resistant (Y181I mutation) virus should be selected in vitro in two different ways. The competitive replication assay showed that the ratio of virus carrying a M184I mutation increased from 98.8%, while the wild type virus decreased to 1.2% after 4 passages in the presence of 3TC; the percentage of virus carrying the Y181I mutation increased to 90.5%, while wild type virus decreased to 9.5% in the presence of NVP. In the absence of drugs, the ratio of virus carrying the M184I mutation decreased to 5.3%, while wild type virus increased to 94.7%; the ratio of virus carrying Y181I increased to 75%, while wild type virus decreased to 25% after 4 passages.

CONCLUSIONSThe NVP-resistant virus is fitter than wild type virus even in the absence of NVP that may be the reason that NNRTIs-resistant virus is spreading quickly.

Cell Line ; Drug Resistance, Viral ; genetics ; HIV-1 ; drug effects ; genetics ; growth & development ; physiology ; Humans ; Lamivudine ; pharmacology ; Mutation ; Nevirapine ; pharmacology ; Reverse Transcriptase Inhibitors ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Virus Replication ; genetics ; physiology

Acquired Immunodeficiency Syndrome ; drug therapy ; metabolism ; virology ; Adult ; Anti-HIV Agents ; pharmacology ; therapeutic use ; Drug Resistance, Viral ; Female ; HIV-1 ; drug effects ; physiology ; Humans ; Male

OBJECTIVETo establish a cohort of human immunodeficiency virus (HIV) discordant couples for follow-up studies and to collect data on frequency of HIV heterosexual transmission and related factors.

METHODSA total of 52 HIV discordant couples were identified by face to face interview and serological testing, in which the HIV negative individuals had no HIV infection behaviors including injecting drug use, blood transfusion or having sexual partners other than his/her own wife/husband. Three times of follows-up studies were carried out in 0.5 year, 1 year and 2.5 years to collect information on their sexual practices and condom use through face to face interview together with 20 ml whole blood collected to test HIV antibody, CD4+ T cell count and viral load.

RESULTS(1) In the period of 2.5 years follow-up, no HIV seroconversion and HIV transmission was found. (2) The frequencies of sexual intercourse between once per month to once per week were 65.4%, 72.9%, 71.7% and 80.0% at the time of cohort setup: 0.5 year, 1 year and 2.5 years of follow-up respectively. The rates of "occasional use" to "never use" condoms were 76.9%, 66.6%, 69.1% and 60.0% at the time of cohort setup as: 0.5 year, 1 year and 2.5 years of follow-up, respectively. No significant difference between different times of follow-up for sexual intercourse or condom use. (3) 85.4%, 66.6% and 60.0% of the HIV positive individuals kept their CD4+ T cell count stabilized or raised during the 0.5 year, 1 year and 2.5 years follow-up period, respectively. However, 66.7% of them showed stable or declined viral load in the period of 2.5 years follow-up. It appeared that stable or raised CD4+ T cell and the stable/declined viral load happened simultaneously.

CONCLUSIONNo transmission was identified in this study. The stabilized CD4+ T cell count and viral load might be account for the reason of no transmission while the biological factors from host and virus related with transmission need to be further studied.

CD4 Lymphocyte Count ; China ; epidemiology ; Cohort Studies ; Coitus ; Condoms ; Contraception Behavior ; Female ; HIV ; physiology ; HIV Infections ; epidemiology ; immunology ; transmission ; virology ; Humans ; Male ; Rural Health ; Spouses ; Viral Load