In this study, the prevalence of HIV-1 CRF01_AE intrasubtype recombinants in China is estimated and their contributions to the epidemic are explored.
Available HIV-1 complete genomes of CRF01_AE were retrieved from the HIV database. The two alignments were evaluated with RDP3. Recombinants were defined as cases in which the recombination signal was supported by at least 3 methods with P-values of ≤0.05 after Bonferroni correction for multiple comparisons implemented in RDP3. Phylogenetic analysis was performed to further investigate the role of intrasubtype recombinants in epidemics.
Here, 124 out of the 339 sequences from around the world (36.6 %) showed significant evidence of recombination. Here, 84 of these recombinants were from China, accounting for 54.9 % of local total sequences (84 out of 153). The results indicated non-negligible levels of intrasubtype recombination. Subsequent phylogenetic analysis indicated that a considerable proportion of CRF01_AE strains in China originated from circulating intrasubtype recombinant forms. Three large, well-supported intrasubtype recombinants clusters were identified here. Through a survey of risk factors and sampling cities and provinces, cluster I and cluster II were found to be prevalent primarily among men who have sex with men in major northern cities. Cluster III was prevalent among heterosexuals and intravenous drug users in southern and southwestern provinces.
The current work highlighted the remarkable prevalence of intrasubtype recombination within the CRF01_AE epidemic and emphasized the value of intrasubtype recombinants, which came to circulate in the same manner as intersubtype recombinants.
Electronic supplementary material
The online version of this article (doi:10.1186/s12879-015-1273-5) contains supplementary material, which is available to authorized users.
HIV-1; CRF01_AE; Intrasubtype recombination; Circulating intrasubtype recombinant forms; China
Highly active antiretroviral therapy (HAART) is recommended to control the infection of HIV-1. HIV-1 drug resistance becomes an obstacle to HAART due to the accumulation of specific mutations in the RT coding region. The development of resistance mutations may be more complex than previously thought.
We followed two HIV-1 infectors from a HIV-1 drug resistance surveillance cohort in Henan province and evaluated CD4+ T-cell number and viral load thereafter at ten time-periods and characterized their reverse transcriptase-associated mutation patterns at each time point. Then we constructed the recombinant virus strains with these mutation patterns to mimick the viruses and test the phenotypic resistance caused by the mutation patterns on TZM-b1 cells.
CD4+ T-cell number initially increased and then decreased rapidly, while viral load decreased and then dropped sharply during initial antiretroviral treatment. The number of mutations and the combination patterns of mutations increased over time. According to the phenotypic resistance performed by recombinant virus strains, VirusT215Y/V179E/Y181C/H221Y exhibited high levels of resistance to EFV (5.57-fold), and T215Y/V179E-containing virus increased 20.20-fold in AZT resistance (p < 0.01). VirusT215Y/V179E/Y181C increased markedly in EFV resistance (p < 0.01). The IC50 for VirusT215Y/V179E/H221Y was similar to that for VirusT215Y/V179E/Y181C. VirusT215Y/K103N/Y181C/H221Y induced a dramatic IC50 increase of all the four agents (Efavirenz EFV, Zidovudine AZT, Lamivudine 3TC, and Stavudine d4T) (p < 0.01). As for VirusT215Y/K103N/Y181C, only the IC50 of EFV was significantly increased. T215Y/K103N resulted in a 26.36-fold increase in EFV (p < 0.01). T215Y/K103N/H221Y significantly increased the resistance to AZT and 3TC. The IC50 of EFV with T215Y/V179E was lower than with T215Y/K103N (F = 93.10, P < 0.0001). With T215Y/V179E, Y181C significantly increase in EFV resistance, while the interaction between 181 and 221 in EFV was not statistically significant (F = 1.20, P = 0.3052). With T215Y/K103N, neither H221Y nor Y181C showed a significant increase in EFV resistance, but the interaction between 181 and 221 was statistically significant (F = 38.12, P = 0.0003).
Data in this study suggests that pathways of viral evolution toward drug resistance appear to proceed through distinct steps and at different rates. Phenotypic resistance using recombinant virus strains with different combination of mutation patterns reveals that interactions among mutations may provide information on the impact of these mutations on drug resistance. All the result provides reference to optimize clinical treatment schedule.
HIV-1; Resistance mutations; Recombinant virus; Phenotypic resistance
Transmitted drug resistance (TDR) is an ongoing public health problem in HIV disease treatment. However, little is known about TDR among men who have sex with men (MSM) patients in China. In addition, TDR prevalence among patients with acute HIV infection (AHI) or early HIV infection (EHI) was believed higher than that of patients with chronic HIV infection (CHI), but as AHI is typically either unidentified or crudely defined in large populations, very few direct comparisons have been made. We did a retrospective analysis of TDR in 536 antiretroviral-naive MSM patients from our immunodeficiency clinics at You'an Hospital, Capital Medical University (CMU), in Beijing, China, 2008–2011. The cohort included 266 patients with AHI/EHI and 270 patients with CHI. We analyzed the subtype, estimated the TDR prevalence, and characterized the model of TDR and the predicted drug sensitivity. Additionally, we made a comparison of TDR between the patients with AHI/EHI and patients with CHI.
Our results indicated that among the 536 patients, HIV-1 subtype CRF01_AE accounted for 52.1%, subtype B accounted for 24.8%, CRF07_BC/ CRF08_BC accounted for 21.6% (116/536), and 1.3% were denoted as unique recombinant forms (URFs). A total of 7.8% patients had one or more transmitted HIV-1 drug resistance mutations, representing 6.2% for PI-related mutations, 0.9% for NRTI-related mutations, and 1.7% for NNRTI-related mutations. Although patients with AHI/EHI had a higher TDR prevalence as compared to that of patients with CHI, the difference was not statistically significant. There was no significant difference in TDR model and predicted drug susceptibility between the two groups of patients either.
This study provides important strategic information for public health planning by healthcare officials in China and warrants a comprehensive study with larger patient cohorts from various healthcare centers within China.
Mild cognitive impairment (MCI) is the intermediate stage of the cognitive changes between normal aging and dementia. KLOTH is an age-related gene that may contribute to the risk of MCI. The aim of our study was to explore the association between KLOTHO promoter methylation and MCI in Xinjiang Uygur and Han populations.
DNA methylation assay was performed using the bisulphite pyrosequencing technology among 96 Uygur (48 MCI and 48 controls) and 96 Han (48 MCI and 48 controls) Chinese individuals from Xinjiang province of China.
We found significant association between KLOTHO promoter methylation and MCI in the Han Chinese (CpG1: p = 3.77E-06; CpG2: p = 1.91E-07; CpG3: p = 5.83E-07; CpG4: p = 2.23E-05; CpG5: p = 3.03E-06) but not in the Uygur Chinese. Higher KLOTHO promoter methylation levels were found in Han MCI patients than Uygur MCI patients for all the five CpGs (adjusted p values by age < 0.02).
Our results showed that KLOTHO promoter hypermethylation contributed to the MCI risk in Xinjiang Han Chinese but not in Xinjiang Uygur Chinese. The population difference of KLOTHO methylation in the risk of MCI required further investigation in the future.
Evidence suggests that interaction between key genes mediating signaling and transcriptional networks involving effector T-cell responses may influence an individual’s susceptibility to develop allergic rhinitis(AR).
The aim of this study was todetermine whether specific interactions between key genes involved in effector T-cell pathways are associated with an individual’s susceptibility to develop AR in Han Chinese subjects.
A cohort of 489 patients with AR and 421 healthy controls was enrolled from the Han Chinese population in Beijing, China. AR was established by questionnaire and clinical examination, and peripheral blood was drawn from all subjects for DNA extraction. A total of 96 single nucleotide polymorphisms (SNPs) in 26 reprehensive candidate genes involved in T helper 1 (Th1), Th2, Th17, Th9 and T regulatory cell pathways were selected from the International Haplotype Mappingdatabase for Han Chinese in Beijing (CHB) population, and IlluminaGoldenGate assay was conducted for SNP genotyping. The PLINK software package was used to perform statistical analyses.
Simple SNP-phenotype association analysis using logistic regression showed SNP rs8193036 in IL17A gene, rs2569254 in IL-12 and rs1898413 in RORα weresignificantlyassociatedwith AR.Simple SNP-phenotype association analysis with genetic models demonstrated thatrs2569254 in IL-12, rs1031508 in STAT4, and rs3741809 in IL-26 were likely to be recessive, rs8193036 in IL17A allelic, rs897200in STAT4 genotypic, and rs1898413 in RORα dominant. Epistasis analyses exhibited that 83 SNPs in 23 genes were significantly interactive; of which 59 interactions/SNP pairs demonstrated OR values higher than 2 or lower than 0.5, and 12 interactions/SNP pairs OR values higher than 4 or lower than 0.25. STAT3, RORα and IL-26, involved in Th17 pathway,were the mostfrequentlyinteractive genes.
This study suggests that interactions between several SNPs in key genes involved in effector T-cell pathways are likely to influence an individual’s susceptibility to develop AR.
Scavenger receptor class B, member 2 (SCARB2) is essential for endosome biogenesis and reorganization and serves as a receptor for both β-glucocerebrosidase and enterovirus 71. However, little is known about its function in innate immune cells. In this study, we show that, among human peripheral blood cells, SCARB2 is most highly expressed in plasmacytoid dendritic cells (pDCs), and its expression is further upregulated by CpG oligodeoxynucleotide stimulation. Knockdown of SCARB2 in pDC cell line GEN2.2 dramatically reduces CpG-induced type I IFN production. Detailed studies reveal that SCARB2 localizes in late endosome/lysosome of pDCs, and knockdown of SCARB2 does not affect CpG oligodeoxynucleotide uptake but results in the retention of TLR9 in the endoplasmic reticulum and an impaired nuclear translocation of IFN regulatory factor 7. The IFN-I production by TLR7 ligand stimulation is also impaired by SCARB2 knockdown. However, SCARB2 is not essential for influenza virus or HSV-induced IFN-I production. These findings suggest that SCARB2 regulates TLR9-dependent IFN-I production of pDCs by mediating endosomal translocation of TLR9 and nuclear translocation of IFN regulatory factor 7.
The complex epidemic and significant diversity of HIV-1 strains in China pose serious challenges for surveillance and diagnostic assays, vaccine development and clinical management. There is a lack of HIV-1 isolates in current canonical HIV-1 subtype panels that can represent HIV-1 diversity in China; an HIV-1 subtype panel for China is urgently needed.
Blood samples were collected from HIV-1 infected patients participating in the drug-resistance surveillance program in China. The samples were isolated, cultured and stored as neat culture supernatant. The HIV-1 isolates were fully characterized. The panel was used to compare 2 viral load assays and 2 p24 assays as the examples of how this panel could be used.
An HIV-1 subtype panel for China composed of 30 HIV-1 primary strains of four subtypes (B [including Thai-B], CRF01_AE, CRF07_BC and G) was established. The samples were isolated and cultured to a high-titer (106-109 copies/ml)/high-volume (40ml). The HIV-1 isolates were fully characterized by the final viral load, p24 concentration, gag-pol and envC2V3 sequencing, co-receptor prediction, determination of the four amino acids at the tip of the env V3-loop, glycosylation sites in the V3 loop and the drug-resistance mutations. The comparison of two p24 assays and two viral load assays on the isolates illustrated how this panel may be used for the evaluation of diagnostic assay performance. The Pearson value between p24 assays were 0.938. The viral load results showed excellent concordance and agreement for samples of Thai-B, but lower correlations for samples of CRF01_AE.
The current panel of 30 HIV-1 isolates served as a basis for the development of a comprehensive panel of fully characterized viral isolates, which could reflect the current dynamic and complex HIV-1 epidemic in China. This panel will be available to support HIV-1 research, assay evaluation, vaccine and drug development.
We identified a novel HIV-1 circulating recombinant form (designated CRF57_BC) from a total of four patients with no obvious epidemiologic linkage in western Yunnan (Dehong prefecture) in China. Two strains (09CN.YNFL37 and 10CN.DHFL17) were identified in this study. An additional two strains (341 and 1439) were found among strains reported in a previous study. CRF57_BC was composed of subtype B and subtype C, with one subtype B segment inserted into the gag region of the subtype C backbone. Subregion tree analysis showed that the B regions originated from a Thai B lineage and the C regions were from an India C lineage. The emergence of CRF57_BC may reflect the continual generation of various forms of intersubtype recombinants in western Yunnan.
Non-syndromic congenital heart defects (CHDs) develop during embryogenesis as a result of a complex interplay between environmental exposures, genetics and epigenetic causes. Genetic factors associated with CHDs may be attributed to either independent effects of maternal or fetal genes, or the inter-generational interactions between maternal and fetal genes. Detecting gene-by-gene interactions underlying complex diseases is a major challenge in genetic research. Detecting maternal-fetal genotype (MFG) interactions and differentiating them from the maternal/fetal main effects has presented additional statistical challenges due to correlations between maternal and fetal genomes. Traditionally, genetic variants are tested separately for maternal/fetal main effects and MFG interactions on a single-locus basis. We conducted a haplotype-based analysis with a penalized logistic regression framework to dissect the genetic effect associated with the development of non-syndromic conotruncal heart defects (CTD). Our method allows simultaneous model selection and effect estimation, providing a unified framework to differentiate maternal/fetal main effect from the MFG interaction effect. In addition, the method is able to test multiple highly linked SNPs simultaneously with a configuration of haplotypes, which reduces the data dimensionality and the burden of multiple testing. By analyzing a dataset from the National Birth Defects Prevention Study (NBDPS), we identified seven genes (GSTA1, SOD2, MTRR, AHCYL2, GCLC, GSTM3 and RFC1) associated with the development of CTDs. Our findings suggest that MFG interactions between haplotypes in 3 of 7 genes, GCLC, GSTM3 and RFC1, are associated with non-syndromic conotruncal heart defects.
Congenital heart defects; maternal-fetal interactions; adaptive LASSO; National Birth Defects Prevention Study
To evaluate the association of tumor-derived matrix metalloproteinase 2 (MMP-2) and stromal-derived MMP-2 expression with the prognosis of patients with ovarian cancer, a meta-analysis study was performed, which was aimed to comprehensively review the evidence of MMP-2 as prognostic biomarkers in ovarian cancers.
All relevant studies were searched in PubMed and Web of Science until May 30, 2014. Hazard ratios (HRs) with their 95% confidence intervals (CIs) were used to assess the association between MMP-2 expression (tumor-derived or stromal-derived) and prognosis of patients with ovarian cancer. Pooled odds ratios (ORs) and their 95% CIs were used to assess the correlation of MMP-2 expression with the clinicopathological features of patients with ovarian cancer.
A total of 965 patients in 8 studies were included in this analysis. Among them, tumor-derived and stromal-derived MMP-2 expression was detected in 7 and 5 articles, respectively. The results revealed that ovarian cancer patients with positive tumor-derived MMP-2 expression showed a worse prognosis than did the ones with negative tumor-derived MMP-2 expression (HR, 1.52; 95% CI, 1.06–2.20). However, ovarian cancer patients with positive stromal-derived MMP-2 expression had not. In addition, we also found that tumor-derived MMP-2 expression was associated with distant metastasis (absent vs present; pooled OR, 4.52; 95% CI, 1.56–13.09; P = 0.001).
These results suggested that positive tumor-derived MMP-2 expression could predict a lower overall survival rate and could be an independent dangerous prognostic factor in patients with ovarian cancer.
Tumor-derived; Stromal-derived; MMP-2; Ovarian cancer; Prognosis
We investigated the association between conotruncal heart defects (CTDs) and maternal and fetal single nucleotide polymorphisms (SNPs) in 60 genes in the folate, homocysteine and pathways. We also investigated whether periconceptional maternal folic acid supplementation modified associations between CTDs and SNPs.
Participants were enrolled in the National Birth Defects Prevention Study between 1997 and 2007. DNA samples from 616 case-parental triads affected by CTDs and 1,645 control-parental triads were genotyped using an Illumina® Golden Gate custom SNP panel. A hybrid design analysis, optimizing data from case and control trios, was used to identify maternal and fetal SNPs associated with CTDs.
Among 921 SNPs, 17 maternal and 17 fetal SNPs had a Bayesian false-discovery probability (BFDP) of <0.8. Ten of the 17 maternal SNPs and 2 of the 17 fetal SNPs were found within the glutamate-cysteine ligase, catalytic subunit (GCLC) gene. Fetal SNPs with the lowest BFDP (rs2612101, rs2847607, rs2847326, rs2847324) were found within the thymidylate synthetase (TYMS) gene. Additional analyses indicated that the risk of CTDs associated with candidate SNPs was modified by periconceptional folic acid supplementation. Nineteen maternal and 9 fetal SNPs had BFDP <0.8 for gene-by-environment (GxE) interactions with maternal folic acid supplementation.
These results support previous studies suggesting that maternal and fetal SNPs within folate, homocysteine and transsulfuration pathways are associated with CTD risk. Maternal use of supplements containing folic acid may modify the impact of SNPs on the developing heart.
conotruncal heart defects; single nucleotide polymorphisms; oxidative stress; genetics; folic acid; gene X environment interaction
RghBNG, a gene of unknown function, was cloned from Rehmannia glutinosa by reverse transcription PCR and rapid amplification of cDNA ends. The full-length cDNA of RghBNG was 548 bp with a282-bp open reading frame. It encoded a polypeptide of 93 amino acids with a predicted molecular weight of 10.5 kDa and a theoretical isoelectric point of 9.25. Bioinformatics analysis indicated that RghBNG had no homology to any known plant genes, whereas the RghBNG polypeptide was highly similar to other plant proteins and possessed one conserved B12D protein family functional domain. Phylogenetic analysis revealed that RghBNG encoded for a dicot protein. RghBNG spatial and temporal expression patterns and responses to abiotic stresses and plant growth regulators were investigated by qRT-PCR. RghBNG transcripts were detected in roots, stems, leaves, petals, receptacles, stamens and pistils with the highest and lowest levels respectively observed in petals and leaves of mature plants. Additionally, RghBNG transcripts were detected at three developmental stages of roots, stems and leaves; the highest levels were observed in roots at seedling stage; Transcript levels changed to varying degrees in different tissues and stages; We also studied the effects of abiotic stress and plant growth regulators in roots and leaves. RghBNG expression was significantly increased (p < 0.01) by chromium, gibberellic acid and NaCl, with the highest levels induced by chromium stress; In contrast, 6-benzyladenine reduced expression. These results strongly suggest that RghBNG is involved in R. glutinosa growth, development and response to plant growth regulators and abiotic stresses.
Rehmannia glutinosa; RghBNG gene; Cloning and expression; qRT-PCR; Abiotic stress; Plant growth regulator
Conotruncal heart defects (CTDs) are among the most severe birth defects worldwide. Studies of CTDs indicate both lifestyle behaviors and genetic variation contribute to the risk of CTDs. Based on a hybrid design using data from 616 case-parental and 1645 control-parental triads recruited for the National Birth Defects Prevention Study between 1997 and 2008, we investigated whether the occurrence of CTDs is associated with interactions between 921 maternal and/or fetal single nucleotide polymorphisms (SNPs) and maternal obesity and tobacco use. The maternal genotypes of the variants in the glutamate-cysteine ligase, catalytic subunit (GCLC) gene and the fetal genotypes of the variants in the glutathione S-transferase alpha 3 (GSTA3) gene were associated with an elevated risk of CTDs among obese mothers. The risk of delivering infants with CTDs among obese mothers carrying AC genotype for a variant in the GCLC gene (rs6458939) was 2.00 times the risk among those carrying CC genotype (95% confidence interval: 1.41, 2.38). The maternal genotypes of several variants in the glutathione-S-transferase (GST) family of genes and the fetal genotypes of the variants in the GCLC gene interacted with tobacco exposures to increase the risk of CTDs. Our study suggests that the genetic basis underlying susceptibility of the developing heart to the adverse effects of maternal obesity and tobacco use involve both maternal and embryonic genetic variants. These results may provide insights into the underlying pathophysiology of CTDs, and ultimately lead to novel prevention strategies.
Recombination is one of the major mechanisms underlying the generation of HIV-1 variability. Currently 61 circulating recombinant forms of HIV-1 have been identified. With the development of recombination detection techniques and accumulation of HIV-1 reference stains, more accurate mosaic structures of circulating recombinant forms (CRFs), like CRF04 and CRF06, have undergone repeated analysis and upgrades. Such revisions may also be necessary for other CRFs. Unlike previous studies, whose results are based primarily on a single recombination detection program, the current study was based on multiple recombination analysis, which may have produced more impartial results.
Representative references of 3 categories of intersubtype recombinants were selected, including BC recombinants (CRF07 and CRF08), BG recombinants (CRF23 and CRF24), and BF recombinants (CRF38 and CRF44). They were reanalyzed in detail using both the jumping profile hidden Markov model and RDP3.
The results indicate that revisions and upgrades are very necessary and the entire re-analysis suggested 2 types of revision: (i) length of inserted fragments; and (ii) number of inserted fragments. The reanalysis also indicated that determination of small regions of about 200 bases or fewer should be performed with more caution.
Results indicated that the involvement of multiple recombination detection programs is very necessary. Additionally, results suggested two major challenges, one involving the difficulty of accurately determining the locations of breakpoints and the second involving identification of small regions of about 200 bases or fewer with greater caution. Both indicate the complexity of HIV-1 recombination. The resolution would depend critically on development of a recombination analysis algorithm, accumulation of HIV-1 stains, and a higher sequencing quality. With the changes in recombination pattern, phylogenetic relationships of some CRFs may also change. All these results may be critical to understand the role of recombination in a complex and dynamic HIV evolution.
The aim of this study was to investigate the role of K101Q, Y181C and H221Y emerging in HIV-1 reverse transcriptase with different mutations patterns in phenotypic susceptibility to currently available NNRTIs (nevirapine NVP, efavirenz EFV) and NRTIs (zidovudine AZT, lamivudine 3TC, stavudine d4T) in China.
Phenotype testing of currently available NNRTIs (NVP, EFV) and NRTIs (AZT, 3TC, d4T) was performed on TZM-b1 cells using recombined virus strains. P ≤ 0.05 was defined significant considering the change of 50% inhibitory drug concentration (IC50) compared with the reference, while P ≤ 0.01 was considered to be statistically significant considering multiple comparisons.
Triple-mutation K101Q/Y181C/H221Y and double-mutation K101Q/Y181C resulted in significant increase in NVP resistance (1253.9-fold and 986.4-fold), while only K101Q/Y181C/H221Y brought a 5.00-fold significant increase in EFV resistance. Remarkably, K101Q/H221Y was hypersusceptible to EFV (FC = 0.04), but was significantly resistant to the three NRTIs. Then, the interaction analysis suggested the interaction was not significant to NVP (F = 0.77, P = 0.4061) but significant to EFV and other three NRTIs.
Copresence of mutations reported to be associated with NNRTIs confers significant increase to NVP resistance. Interestingly, some may increase the susceptibility to EFV. Certainly, the double mutation (K101Q/H221Y) also changes the susceptibility of viruses to NRTIs. Interaction between two different sites makes resistance more complex.
Nucleoside reverse transcriptase inhibitors (NRTIs); Non-nucleoside reverse transcriptase inhibitors (NNRTIs); HIV-1 resistance mutation; 50% inhibitory drug concentration
Exposure to particulate matter (PM) has been associated with lung cancer risk in epidemiology investigations. Elemental components of PM have been suggested to have critical roles in PM toxicity, but the molecular mechanisms underlying their association with cancer risks remain poorly understood. DNA methylation has emerged as a promising biomarker for environmental-related diseases, including lung cancer. In this study, we evaluated the effects of PM elemental components on methylation of three tandem repeats in a highly-exposed population in Beijing, China. The Beijing Truck Driver Air Pollution Study was conducted shortly before the 2008 Beijing Olympic Games (June 15-July 27, 2008) and included 60 truck drivers and 60 office workers. On two days separated by 1-2 weeks, we measured blood DNA methylation of SATα, NBL2, D4Z4, and personal exposure to eight elemental components in PM2.5, including aluminum (Al), silicon (Si), sulfur (S), potassium (K), calcium (Ca) titanium (Ti), iron (Fe), and zinc (Zn). We estimated the associations of individual elemental component with each tandem repeat methylation in generalized estimating equations (GEE) models adjusted for PM2.5 mass and other covariates. Out of the eight examined elements, NBL2 methylation was positively associated with concentrations of Si (0.121, 95%CI: 0.030; 0.212, FDR=0.047) and Ca (0.065, 95%CI: 0.014; 0.115, FDR=0.047) in truck drivers. In office workers, SATα methylation was positively associated with concentrations of S (0.115, 95%CI: 0.034; 0.196, FDR=0.042). PM-associated differences in blood tandem-repeat methylation may help detect biological effects of the exposure and identify individuals who may eventually experience higher lung cancer risk.
Tandem repeats; DNA methylation; lung cancer
During protoplast regeneration, proteins related to cell morphogenesis, organogenesis and development adjustment were phosphorylated in Physcomitrella patens. These proteins play important roles in regulating postembryonic development in higher plants.
The moss Physcomitrella patens is an ideal model plant to study plant developmental processes. To better understand the mechanism of protoplast regeneration, a phosphoproteome analysis was performed. Protoplasts were prepared from protonemata. By 4 d of protoplast regeneration, the first cell divisions had ensued. Through a highly selective titanium dioxide (TiO2)-based phosphopeptide enrichment method and mass spectrometric technology, more than 300 phosphoproteins were identified as protoplast regeneration responsive. Of these, 108 phosphoproteins were present on day 4 but not in fresh protoplasts or those cultured for 2 d. These proteins are catalogued here. They were involved in cell-wall metabolism, transcription, signal transduction, cell growth/division, and cell structure. These protein functions are related to cell morphogenesis, organogenesis, and development adjustment. This study presents a comprehensive analysis of phosphoproteome involved in protoplast regeneration and indicates that the mechanism of plant protoplast regeneration is similar to that of postembryonic development.
LC-MS/MS; Physcomitrella patens; phosphoproteome; postembryonic development; protoplast regeneration; TiO2 enrichment.
The rapid spreading of HIV drug resistance is threatening the overall success of free HAART in China. Much work has been done on drug-resistant mutations, however, most of which were based on subtype B. Due to different genetic background, subtypes difference would have an effect on the development of drug-resistant mutations, which has already been proved by more and more studies. In China, the main epidemic subtypes are CRF07_BC, CRF08_BC, Thai B and CRF01_AE. The depiction of drug resistance mutations in those subtypes will be helpful for the selection of regimens for Chinese. In this study, the distributions difference of amino acids at sites related to HIV drug resistance were compared among subtype B, CRF01_AE, CRF07_BC and CRF08_BC strains prevalent in China. The amino acid composition of sequences belonging to different subtypes, which were obtained from untreated and treated individuals separately, were also compared. The amino acids proportions of 19 sites in RT among subtype B, CRF01_AE and CRF08_BC have significant difference in drug resistance groups (chi-square test, p<0.05). Genetic barriers analysis revealed that sites 69, 138, 181, 215 and 238 were significantly different among subtypes (Kruskal Wallis test, p<0.05). All subtypes shared three highest prevalent drug resistance sites 103, 181 and 184 in common. Many drug resistant sites in protease show surprising high proportions in almost all subtypes in drug-naïve patients. This is the first comprehensive study in China on different development of drug resistance among different subtypes. The detailed data will lay a foundation for HIV treatment regimens design and improve HIV therapy in China.
A rapid increase in the number of HIV cases in the men who have sex with men (MSM) population has been observed in China; however, little information is available on the genetic characterization of HIV prevalent in this population. In this study, 95 HIV-1-seropositive drug-naive patients from the Beijing MSM population were enrolled. The genetic characterization and transmission of drug resistance of HIV-1 were examined based on full-length gag, pol, and partial env gene sequences. Three subtypes, including CRF01_AE (56.0%), B (30.8%), and CRF07_BC (12.6%), were identified. Close phylogenetic relationships were found among these strains with isolates from other populations in Beijing and MSM isolates from Hebei province, which suggested that the Beijing MSM population might act as a bridge for HIV transmission between MSM and other high-risk populations. Drug-resistant mutations were identified in 5.3% of sampled individuals. Our results provided detailed genetic data and would be helpful for understanding the transmitting pattern of HIV strains between MSM and other populations.
Alpha-2-adrenergic receptor (ADRA2A) is involved in the sympathetic nervous system and plays a role in the regulation of insulin secretion and lipolysis. Recent studies have indicated that the ADRA2A polymorphisms are associated with type 2 diabetes (T2DM) in Caucasians and African Americans. The present study aimed to evaluate the association between the ADRA2A polymorphisms and T2DM in a Chinese Han population. Two single-nucleotide polymorphisms (SNPs) rs521674 and rs553668 in the ADRA2A gene were genotyped in 2094 Chinese subjects (1042 T2DM patients and 1052 nondiabetic controls) by using the TaqMan allelic discrimination technique. A single-locus analysis indicated that SNP rs553668 was associated with T2DM (p=0.04). Further analysis indicated that the association of SNP rs553668 was found in T2DM patients with body mass index (BMI)<25 kg/m2 (p=0.03), but not in the patients with BMI≥25 kg/m2 (p=0.56). This association was still significant in a recessive model (p=0.01, odds ratio=0.68, 95% confidence interval=0.51–0.92). In conclusion, the present study provides evidence that the ADRA2A polymorphism, rs553668, is associated with lean T2DM patients in a Chinese Han population. Further investigation to explore the role of ADRA2A in the regulation of body weight has been taken into our consideration.
Yunnan, Guangxi and Henan are the provinces with the most severe HIV epidemic in China, which were also among the first group of areas providing free ART in 2004. However, little comprehensive data are available on prevalence of HIV subtype and baseline drug resistance in drug-naïve populations. In this study, 1746 treatment-naïve HIV-positive individuals were randomly selected from new-reported cases in Henan, Guangxi and Yunnan. Among of them, subtypes and drug resistance of 1159 strains were determined by amplifying and sequencing full-length pol genes. Significantly different distributions of HIV subtypes prevalent in three provinces were identified (P<0.01). CRF08_BC was found dominant in Yunnan (59.8%), while CRF01_AE was dominant in Guangxi (77.3%) and subtype B was dominant in Henan province (93.9%). The total prevalence of drug resistance was 7.1%. The highest prevalence of HIV drug resistance was found in Henan (12.2%), followed by Yunnan (5.6%) and Guangxi (3.3%). The results of this study suggest that genetic drug-resistance should be tested before initiation of ART in China, especially in Henan province. Furthermore, the prevalence of HIV drug resistant strains should be considered separately in different areas in China before the change of different free ART regimens.
The distribution of HIV-1 subtypes and genetic characterization of CRF01_AE in Guangxi, southern China were identified. The distribution of HIV-1 genotypes based on gag, pol, and partial env sequences (n=349) was as follows: CRF01_AE (66.5%), CRF08_BC (19.2%), CRF07_BC (7.2%), URF (4.6%), subtype B (1.7%), and subtype B′ (0.9%). CRF01_AE predominated in all geographic regions and risk populations and there were multiple introductions of CRF01_AE strains in Guangxi. We found a peculiar CRF01_AE monophyletic lineage distinct from other CRF01_AE viruses, and we designated it “CRF01_AE-v” for convenience. CRF01_AE-v circulating in both heterosexuals and injecting drug users (IDUs) had accounted for 39.7% of CRF01_AE. It showed a selective advantage in the Guangxi population and formed its own characteristic compared with all the CRF01_AE references. Our results suggested that CRF01_AE-v was a new variant of CRF01_AE and it might lead to a new epidemic in Guangxi.
Thirty HIV-1 URF_01AE/ B′ complete or nearly full-length genome sequences sampled within Southeast Asia were obtained from the Los Alamos HIV Sequence Database. Phylogenetic and recombinant analyses revealed that three sequences indeed displayed the identical recombinant structure. Of note, the three subjects, harboring novel CRF01_AE/B recombinants, did not have apparent epidemiological linkage. They fulfilled the criteria for the designation of a new circulating recombinant form (CRF) and constituted the 52nd CRF identified in the worldwide HIV-1 pandemic. In this chimera, two short subtype B segments were inserted into a backbone of CRF_01AE. The breakpoints corresponded to HXB2 nucleotide positions 2930, 3251, 8521, and 9004 approximately. This CRF is the first one identified by neatening and analyzing the sequences already presented in the Los Alamos HIV Sequence Database. This indicates that we should pay attention not only to explicit subtype sequences but also to those classified as a unique recombinant form (URF) so far.
Zhengzhou is the capital of Henan province, where severe HIV prevalence was found in former paid plasma donors. In recent years, the HIV epidemic in men who have sex with men (MSM) increased rapidly in the city. To explore the subtype distribution and genetic characterization of HIV in MSM in Zhengzhou city, phylogenetic analysis was fulfilled based on the full-length gag, pol, and partial env gene. A total of 31 HIV-1-seropositive MSM individuals were enrolled. The full length gag, pol, and partial env gene were amplified and sequenced. Multiple subtypes, including CRF01_AE (45.2%), subtype B (38.7%), and CRF07_BC (16.1%), were identified. Close phylogenetic relationships among our strains with strains from the Henan local area, Hebei MSM population, Beijing area, and Liaoning area were found, suggesting a multiple introduction of HIV into the population. The results will provide clues for prevention and for changes in behavior in the Henan MSM population and also detailed sequence data for vaccine design.
Assessing the prevalence of HIV-1 drug-resistance and the mutation patterns associated with resistance in the geographical regions implementing free antiretroviral therapy (ART) in China is necessary for preventing the spread of resistant strains and designing the regimens for the subsequent therapies with limited resources.
Plasma samples in different cities/prefectures were collected at Yunnan Provincial Hospital of Infectious Disease from January 2010 to December 2011. Genotyping of drug-resistant individuals was conducted using an in-house assay on plasma samples. Viral load, CD4 T cell counts and demographic data were obtained from medical records and an administered questionnaire.
A total of 609 pol sequences (515 ART-failure and 94 therapy-naïve individuals) derived from 664 samples were obtained. The prevalence of drug-resistance was 45.1% in the ART-failure individuals. Of these, 26.8% harbored HIV strains dually resistant to nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors, and 14.8% harbored HIV strains resistant to only one drug category. Mutations such as M184V/I, K103N, V106A, Y181C and G190A were common among the ART-failure individuals, and the frequencies of M184V/I, K103N and V106A were 28.2%, 19.2%, and 22.1%, respectively. The percentages of individuals exhibiting intermediate or high-level resistance to 3TC, FTC, EFV and NVP drugs were 28.4%, 28.2%, 37.3%, and 37.5%, respectively. Factors such as ethnicity, transmission route, CD4 counts, viral load and the duration of ART were significantly correlated with development of drug resistance in the ART-failure individuals.
The high prevalence of HIV drug-resistance observed among the ART-failure individuals from 2010 to 2011 in Yunnan province should be of increasing concern in regions where the implementation of ART is widespread. Education about the risk factors associated with HIV drug resistance is important for preventing and controlling the spread of HIV drug-resistant strains.