Molecular Biology and Biochemistry, Department of

Receive updates for this collection

Implementation of Couples’ Voluntary HIV Counseling and Testing Services in Durban, South Africa

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2015
Abstract: 

Background

Couples’ voluntary HIV counseling and testing (CVCT) is an evidence-based intervention that significantly reduces HIV incidence in couples. Despite the high prevalence of HIV and HIV couple serodiscordance in South Africa, there are few CVCT services.

Methods

From February-June 2013, The Rwanda Zambia HIV Research Group provided support, training, and technical assistance for local counselors and promoters to pilot CVCT services in five hospital-based clinics in Durban, South Africa. Client-level data (age, gender, years cohabiting, pregnancy status, previous testing, antiretroviral treatment (ART) status, neighborhood, and test site) collected as a component of routine CVCT service operation is presented stratified by couple serostatus.

Results

Twenty counselors and 28 promoters completed training. Of 907 couples (1,814 individuals) that underwent CVCT, prevalence of HIV was 41.8 % and prevalence of HIV serodiscordance was 29.5 % (19.3 % M-F+, 10.3 % M + F-). Most participants were 25–34 years of age, and this group had the highest prevalence. Previous individual HIV testing was low (50 % for men, 63 % for women). Only 4 % of couples reported previous CVCT. Most (75 %) HIV+ partners were not on ART, and HIV+ individuals in discordant couples were more likely to be on ART than those in concordant positive couples. Pregnancy among HIV+ women was not associated with previous HIV testing or ART use.

Conclusions

Implementation of standard CVCT services was found to be feasible in Durban. The burden of HIV and couple serodiscordance in Durban was extremely high. CVCT would greatly benefit couples in Durban as an HIV prevention strategy.

Document type: 
Article
File(s): 

Spectrum of Variations in dog-1/FANCJ and mdf-1/MAD1 Defective Caenorhabditis elegans Strains After Long-Term Propagation

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2015
Abstract: 

Background

Whole and partial chromosome losses or gains and structural chromosome changes are hallmarks of human tumors. Guanine-rich DNA, which has a potential to form a G-quadruplex (G4) structure, is particularly vulnerable to changes. In Caenorhabditis elegans, faithful transmission of G-rich DNA is ensured by the DOG-1/FANCJ deadbox helicase.

Results

To identify a spectrum of mutations, after long-term propagation, we combined whole genome sequencing (WGS) and oligonucleotide array Comparative Genomic Hybridization (oaCGH) analysis of a C. elegans strain that was propagated, in the absence of DOG-1 and MDF-1/MAD1, for a total of 470 generations, with samples taken for long term storage (by freezing) in generations 170 and 270. We compared the genomes of F170 and F470 strains and identified 94 substitutions, 17 InDels, 3 duplications, and 139 deletions larger than 20 bp. These homozygous variants were predicted to impact 101 protein-coding genes. Phenotypic analysis of this strain revealed remarkable fitness recovery indicating that mutations, which have accumulated in the strain, are not only tolerated but also cooperate to achieve long-term population survival in the absence of DOG-1 and MDF-1. Furthermore, deletions larger than 20 bp were the only variants that frequently occurred in G-rich DNA. We showed that 126 of the possible 954 predicted monoG/C tracts, larger than 14 bp, were deleted in unc-46 mdf-1 such-4; dog-1 F470 (JNC170).

Conclusions

Here, we identified variants that accumulated in C. elegans’ genome after long-term propagation in the absence of DOG-1 and MDF-1. We showed that DNA sequences, with G4-forming potential, are vulnerable to deletion-formation in this genetic background.

Document type: 
Article
File(s): 

Activation of an Endogenous Retrovirus-Associated Long Non-Coding RNA in Human Adenocarcinoma

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2015
Abstract: 

Background

Long non-coding RNAs (lncRNAs) are emerging as molecules that significantly impact many cellular processes and have been associated with almost every human cancer. Compared to protein-coding genes, lncRNA genes are often associated with transposable elements, particularly with endogenous retroviral elements (ERVs). ERVs can have potentially deleterious effects on genome structure and function, so these elements are typically silenced in normal somatic tissues, albeit with varying efficiency. The aberrant regulation of ERVs associated with lncRNAs (ERV-lncRNAs), coupled with the diverse range of lncRNA functions, creates significant potential for ERV-lncRNAs to impact cancer biology.

Methods

We used RNA-seq analysis to identify and profile the expression of a novel lncRNA in six large cohorts, including over 7,500 samples from The Cancer Genome Atlas (TCGA).

Results

We identified the tumor-specific expression of a novel lncRNA that we have named Endogenous retroViral-associated ADenocarcinoma RNA or ‘EVADR’, by analyzing RNA-seq data derived from colorectal tumors and matched normal control tissues. Subsequent analysis of TCGA RNA-seq data revealed the striking association of EVADR with adenocarcinomas, which are tumors of glandular origin. Moderate to high levels of EVADR were detected in 25 to 53% of colon, rectal, lung, pancreas and stomach adenocarcinomas (mean = 30 to 144 FPKM), and EVADR expression correlated with decreased patient survival (Cox regression; hazard ratio = 1.47, 95% confidence interval = 1.06 to 2.04, P = 0.02). In tumor sites of non-glandular origin, EVADR expression was detectable at only very low levels and in less than 10% of patients. For EVADR, a MER48 ERV element provides an active promoter to drive its transcription. Genome-wide, MER48 insertions are associated with nine lncRNAs, but none of the MER48-associated lncRNAs other than EVADR were consistently expressed in adenocarcinomas, demonstrating the specific activation of EVADR. The sequence and structure of the EVADR locus is highly conserved among Old World monkeys and apes but not New World monkeys or prosimians, where the MER48 insertion is absent. Conservation of the EVADR locus suggests a functional role for this novel lncRNA in humans and our closest primate relatives.

Conclusions

Our results describe the specific activation of a highly conserved ERV-lncRNA in numerous cancers of glandular origin, a finding with diagnostic, prognostic and therapeutic implications.

Document type: 
Article
File(s): 

Single-Molecule Real-Time Transcript Sequencing Facilitates Common Wheat Genome Annotation and Grain Transcriptome Research

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2015
Abstract: 

Background

The large and complex hexaploid genome has greatly hindered genomics studies of common wheat (Triticum aestivum, AABBDD). Here, we investigated transcripts in common wheat developing caryopses using the emerging single-molecule real-time (SMRT) sequencing technology PacBio RSII, and assessed the resultant data for improving common wheat genome annotation and grain transcriptome research.

Results

We obtained 197,709 full-length non-chimeric (FLNC) reads, 74.6 % of which were estimated to carry complete open reading frame. A total of 91,881 high-quality FLNC reads were identified and mapped to 16,188 chromosomal loci, corresponding to 13,162 known genes and 3026 new genes not annotated previously. Although some FLNC reads could not be unambiguously mapped to the current draft genome sequence, many of them are likely useful for studying highly similar homoeologous or paralogous loci or for improving chromosomal contig assembly in further research. The 91,881 high-quality FLNC reads represented 22,768 unique transcripts, 9591 of which were newly discovered. We found 180 transcripts each spanning two or three previously annotated adjacent loci, suggesting that they should be merged to form correct gene models. Finally, our data facilitated the identification of 6030 genes differentially regulated during caryopsis development, and full-length transcripts for 72 transcribed gluten gene members that are important for the end-use quality control of common wheat.

Conclusions

Our work demonstrated the value of PacBio transcript sequencing for improving common wheat genome annotation through uncovering the loci and full-length transcripts not discovered previously. The resource obtained may aid further structural genomics and grain transcriptome studies of common wheat.

Document type: 
Article
File(s): 

Knowledge of HIV Serodiscordance, Transmission, and Prevention among Couples in Durban, South Africa

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2015
Abstract: 

Objective

Couples’ voluntary HIV counseling and testing (CVCT) significantly decreases HIV transmission within couples, the largest risk group in sub-Saharan Africa, but it is not currently offered in most HIV testing facilities. To roll out such an intervention, understanding locale-specific knowledge barriers is critical. In this study, we measured knowledge of HIV serodiscordance, transmission, and prevention before and after receipt of CVCT services in Durban.

Design

Pre- and post-CVCT knowledge surveys were administered to a selection of individuals seeking CVCT services.

Methods

Changes in knowledge scores were assessed with McNemar Chi-square tests for balanced data and generalized estimating equation methods for unbalanced data.

Results

The survey included 317 heterosexual black couples (634 individuals) who were primarily Zulu (87%), unemployed (47%), and had at least a secondary level education (78%). 28% of couples proved to be discordant. Only 30% of individuals thought serodiscordance between couples was possible pre‐CVCT compared to 95% post-CVCT. One-third thought there was at least one benefit of CVCT pre‐CVCT, increasing to 96% post‐CVCT. Overall, there were positive changes in knowledge about HIV transmission and prevention. However, many respondents thought all HIV positive mothers give birth to babies with AIDS (64% pre-CVCT, 59% post-CVCT) and that male circumcision does not protect negative men against HIV (70% pre-CVCT, 67% post-CVCT).

Conclusions

CVCT was well received and was followed by improvements in understanding of discordance, the benefits of joint testing, and HIV transmission. Country-level health messaging would benefit from targeting gaps in knowledge about serodiscordance, vertical transmission, and male circumcision.

Document type: 
Article
File(s): 

Conserved Genetic Interactions between Ciliopathy Complexes Cooperatively Support Ciliogenesis and Ciliary Signaling

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2015
Abstract: 

Mutations in genes encoding cilia proteins cause human ciliopathies, diverse disorders affecting many tissues. Individual genes can be linked to ciliopathies with dramatically different phenotypes, suggesting that genetic modifiers may participate in their pathogenesis. The ciliary transition zone contains two protein complexes affected in the ciliopathies Meckel syndrome (MKS) and nephronophthisis (NPHP). The BBSome is a third protein complex, affected in the ciliopathy Bardet-Biedl syndrome (BBS). We tested whether mutations in MKS, NPHP and BBS complex genes modify the phenotypic consequences of one another in both C. elegans and mice. To this end, we identified TCTN-1, the C. elegans ortholog of vertebrate MKS complex components called Tectonics, as an evolutionarily conserved transition zone protein. Neither disruption of TCTN-1 alone or together with MKS complex components abrogated ciliary structure in C. elegans. In contrast, disruption of TCTN-1 together with either of two NPHP complex components, NPHP-1 or NPHP-4, compromised ciliary structure. Similarly, disruption of an NPHP complex component and the BBS complex component BBS-5 individually did not compromise ciliary structure, but together did. As in nematodes, disrupting two components of the mouse MKS complex did not cause additive phenotypes compared to single mutants. However, disrupting both Tctn1 and either Nphp1 or Nphp4 exacerbated defects in ciliogenesis and cilia-associated developmental signaling, as did disrupting both Tctn1 and the BBSome component Bbs1. Thus, we demonstrate that ciliary complexes act in parallel to support ciliary function and suggest that human ciliopathy phenotypes are altered by genetic interactions between different ciliary biochemical complexes.

Document type: 
Article
File(s): 

Screening of the Pan-African Natural Product Library Identifies Ixoratannin A-2 and Boldine as Novel HIV-1 Inhibitors

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2015
Abstract: 

The continued burden of HIV in resource-limited regions such as parts of sub-Saharan Africa, combined with adverse effects and potential risks of resistance to existing antiretroviral therapies, emphasize the need to identify new HIV inhibitors. Here we performed a virtual screen of molecules from the pan-African Natural Product Library, the largest collection of medicinal plant-derived pure compounds on the African continent. We identified eight molecules with structural similarity to reported interactors of Vpu, an HIV-1 accessory protein with reported ion channel activity. Using in vitro HIV-1 replication assays with a CD4+ T cell line and peripheral blood mononuclear cells, we confirmed antiviral activity and minimal cytotoxicity for two compounds, ixoratannin A-2 and boldine. Notably, ixoratannin A-2 retained inhibitory activity against recombinant HIV-1 strains encoding patient-derived mutations that confer resistance to protease, non-nucleoside reverse transcriptase, or integrase inhibitors. Moreover, ixoratannin A-2 was less effective at inhibiting replication of HIV-1 lacking Vpu, supporting this protein as a possible direct or indirect target. In contrast, boldine was less effective against a protease inhibitor-resistant HIV-1 strain. Both ixoratannin A-2 and boldine also inhibited in vitro replication of hepatitis C virus (HCV). However, BIT-225, a previously-reported Vpu inhibitor, demonstrated antiviral activity but also cytotoxicity in HIV-1 and HCV replication assays. Our work identifies pure compounds derived from African plants with potential novel activities against viruses that disproportionately afflict resource-limited regions of the world.

Document type: 
Article
File(s): 

The Homeobox Genes of Caenorhabditis elegans and Insights into Their Spatio-Temporal Expression Dynamics during Embryogenesis

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2015
Abstract: 

Homeobox genes play crucial roles for the development of multicellular eukaryotes. We have generated a revised list of all homeobox genes for Caenorhabditis elegans and provide a nomenclature for the previously unnamed ones. We show that, out of 103 homeobox genes, 70 are co-orthologous to human homeobox genes. 14 are highly divergent, lacking an obvious ortholog even in other Caenorhabditis species. One of these homeobox genes encodes 12 homeodomains, while three other highly divergent homeobox genes encode a novel type of double homeodomain, termed HOCHOB. To understand how transcription factors regulate cell fate during development, precise spatio-temporal expression data need to be obtained. Using a new imaging framework that we developed, Endrov, we have generated spatio-temporal expression profiles during embryogenesis of over 60 homeobox genes, as well as a number of other developmental control genes using GFP reporters. We used dynamic feedback during recording to automatically adjust the camera exposure time in order to increase the dynamic range beyond the limitations of the camera. We have applied the new framework to examine homeobox gene expression patterns and provide an analysis of these patterns. The methods we developed to analyze and quantify expression data are not only suitable for C. elegans, but can be applied to other model systems or even to tissue culture systems.

Document type: 
Article
File(s): 

Genome-Wide Association Study (GWAS) for Growth Rate and Age at Sexual Maturation in Atlantic Salmon (Salmo salar)

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2015
Abstract: 

Early sexual maturation is considered a serious drawback for Atlantic salmon aquaculture as it retards growth, increases production times and affects flesh quality. Although both growth and sexual maturation are thought to be complex processes controlled by several genetic and environmental factors, selection for these traits has been continuously accomplished since the beginning of Atlantic salmon selective breeding programs. In this genome-wide association study (GWAS) we used a 6.5K single-nucleotide polymorphism (SNP) array to genotype ∼480 individuals from the Cermaq Canada broodstock program and search for SNPs associated with growth and age at sexual maturation. Using a mixed model approach we identified markers showing a significant association with growth, grilsing (early sexual maturation) and late sexual maturation. The most significant associations were found for grilsing, with markers located in Ssa10, Ssa02, Ssa13, Ssa25 and Ssa12, and for late maturation with markers located in Ssa28, Ssa01 and Ssa21. A lower level of association was detected with growth on Ssa13. Candidate genes, which were linked to these genetic markers, were identified and some of them show a direct relationship with developmental processes, especially for those in association with sexual maturation. However, the relatively low power to detect genetic markers associated with growth (days to 5 kg) in this GWAS indicates the need to use a higher density SNP array in order to overcome the low levels of linkage disequilibrium observed in Atlantic salmon before the information can be incorporated into a selective breeding program.

Document type: 
Article
File(s): 

Do Housekeeping Genes Exist?

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2015
Abstract: 

The searching of human housekeeping (HK) genes has been a long quest since the emergence of transcriptomics, and is instrumental for us to understand the structure of genome and the fundamentals of biological processes. The resolved genes are frequently used in evolution studies and as normalization standards in quantitative gene-expression analysis. Within the past 20 years, more than a dozen HK-gene studies have been conducted, yet none of them sampled human tissues completely. We believe an integration of these results will help remove false positive genes owing to the inadequate sampling. Surprisingly, we only find one common gene across 15 examined HK-gene datasets comprising 187 different tissue and cell types. Our subsequent analyses suggest that it might not be appropriate to rigidly define HK genes as expressed in all tissue types that have diverse developmental, physiological, and pathological states. It might be beneficial to use more robustly identified HK functions for filtering criteria, in which the representing genes can be a subset of genome. These genes are not necessarily the same, and perhaps need not to be the same, everywhere in our body.

Document type: 
Article
File(s):