Improved Detection of Rare HIV-1 Variants using 454 Pyrosequencing

454 pyrosequencing, a massively parallel sequencing (MPS) technology, is often used to study HIV genetic variation. However, the substantial mismatch error rate of the PCR required to prepare HIV-containing samples for pyrosequencing has limited the detection of rare variants within viral populations to those present above ~1%. To improve detection of rare variants, we varied PCR enzymes and conditions to identify those that combined high sensitivity with a low error rate. Substitution errors were found to vary up to 3-fold between the different enzymes tested. The sensitivity of each enzyme, which impacts the number of templates amplified for pyrosequencing, was shown to vary, although not consistently across genes and different samples. We also describe an amplicon-based method to improve the consistency of read coverage over stretches of the HIV-1 genome. Twenty-two primers were designed to amplify 11 overlapping amplicons in the HIV-1 clade B gag-pol and env gp120 coding regions to encompass 4.7 kb of the viral genome per sample at sensitivities as low as 0.01-0.2%.     

Engagement with HIV Prevention Treatment and Care among Female Sex Workers in Zimbabwe: a Respondent Driven Sampling Survey

Objective(S)

To determine the HIV prevalence and extent of engagement with HIV prevention and care among a representative sample of Zimbabwean sex workers working in Victoria Falls, Hwange and Mutare.

Design

Respondent driven sampling (RDS) surveys conducted at each site.

Methods

Sex workers were recruited using respondent driven sampling with each respondent limited to recruiting 2 peers. Participants completed an interviewer-administered questionnaire and provided a finger prick blood sample for HIV antibody testing. Statistical analysis took account of sampling method.

Results

870 women were recruited from the three sites. HIV prevalence was between 50 and 70%. Around half of those confirmed HIV positive were aware of their HIV status and of those 50-70% reported being enrolled in HIV care programmes. Overall only 25-35% of those with laboratory-confirmed HIV were accessing antiretroviral therapy. Among those reporting they were HIV negative, 21-28% reported having an HIV test in the last 6 months. Of those tested HIV negative, most (65-82%) were unaware of their status. Around two-thirds of sex workers reported consistent condom use with their clients. As in other settings, sex workers reported high rates of gender based violence and police harassment.

Conclusions

This survey suggests that prevalence of HIV is high among sex workers in Zimbabwe and that their engagement with prevention, treatment and care is sub-optimal. Intensifying prevention and care interventions for sex workers has the potential to markedly reduce HIV and social risks for sex workers, their clients and the general population in Zimbabwe and elsewhere in the region.     

Characterization of FKGK18 as Inhibitor of Group VIA Ca2+-Independent Phospholipase A2 (iPLA2β): Candidate Drug for Preventing Beta-Cell Apoptosis and Diabetes

Ongoing studies suggest an important role for iPLA₂β in a multitude of biological processes and it has been implicated in neurodegenerative, skeletal and vascular smooth muscle disorders, bone formation, and cardiac arrhythmias. Thus, identifying an iPLA₂βinhibitor that can be reliably and safely used in vivo is warranted. Currently, the mechanism-based inhibitor bromoenol lactone (BEL) is the most widely used to discern the role of iPLA₂β in biological processes. While BEL is recognized as a more potent inhibitor of iPLA₂ than of cPLA₂ or sPLA₂, leading to its designation as a “specific” inhibitor of iPLA₂, it has been shown to also inhibit non-PLA₂ enzymes. A potential complication of its use is that while the S and R enantiomers of BEL exhibit preference for cytosol-associated iPLA₂β and membrane-associated iPLA₂γ, respectively, the selectivity is only 10-fold for both. In addition, BEL is unstable in solution, promotes irreversible inhibition, and may be cytotoxic, making BEL not amenable for in vivo use. Recently, a fluoroketone (FK)-based compound (FKGK18) was described as a potent inhibitor of iPLA₂β. Here we characterized its inhibitory profile in beta-cells and find that FKGK18: (a) inhibits iPLA₂β with a greater potency (100-fold) than iPLA₂γ, (b) inhibition of iPLA₂β is reversible, (c) is an ineffective inhibitor of α-chymotrypsin, and (d) inhibits previously described outcomes of iPLA₂β activation including (i) glucose-stimulated insulin secretion, (ii) arachidonic acid hydrolysis; as reflected by PGE2 release from human islets, (iii) ER stress-induced neutral sphingomyelinase 2 expression, and (iv) ER stress-induced beta-cell apoptosis. These findings suggest that FKGK18 is similar to BEL in its ability to inhibit iPLA₂β. Because, in contrast to BEL, it is reversible and not a non-specific inhibitor of proteases, it is suggested that FKGK18 is more ideal for ex vivo and in vivo assessments of iPLA₂β role in biological functions.     

Drought Responses of Foliar Metabolites in Three Maize Hybrids Differing in Water Stress Tolerance

Maize (Zea mays L.) hybrids varying in drought tolerance were treated with water stress in controlled environments. Experiments were performed during vegetative growth and water was withheld for 19 days beginning 17 days after sowing. Genotypic comparisons used measured changes of leaf water potential or results were expressed by time of treatment. Total dry matter of the drought tolerant hybrid on the final harvest was 53% less than that of the intermediate and susceptible maize hybrids when plants were water sufficient. This showed that maize hybrids selected for extreme drought tolerance possessed a dwarf phenotype that affected soil water contents and leaf water potentials. Changes of shoot and root growth, leaf water potential, net photosynthesis and stomatal conductance in response to the time of water stress treatment were diminished when comparing the drought tolerant to the intermediate or susceptible maize hybrids. Genotypic differences were observed in 26 of 40 total foliar metabolites during water stress treatments. Hierarchical clustering revealed that the tolerant maize hybrid initiated the accumulation of stress related metabolites at higher leaf water potentials than either the susceptible or intermediate hybrids. Opposite results occurred when changes of metabolites in maize leaves were expressed temporally. The above results demonstrated that genotypic differences were readily observed by comparing maize hybrids differing in drought tolerance based on either time of treatment or measured leaf water potential. Current findings provided new and potentially important insights into the mechanisms of drought tolerance in maize.     

piggyBac Transposon plus Insulators Overcome Epigenetic Silencing to Provide for Stable Signaling Pathway Reporter Cell Lines

Genetically modified hematopoietic progenitors represent an important testing platform for a variety of cell-based therapies, pharmaceuticals, diagnostics and other applications. Stable expression of a transfected gene of interest in the cells is often obstructed by its silencing. DNA transposons offer an attractive non-viral alternative of transgene integration into the host genome, but their broad applicability to leukocytes and other “transgene unfriendly” cells has not been fully demonstrated. Here we assess stability of piggyBac transposon-based reporter expression in murine prostate adenocarcinoma TRAMP-C2, human monocyte THP-1 and erythroleukemia K562 cell lines, along with macrophages and dendritic cells (DCs) that have differentiated from the THP-1 transfects. The most efficient and stable reporter activity was observed for combinations of the transposon inverted terminal repeats and one 5’- or two cHS4 core insulators flanking a green fluorescent protein reporter construct, with no detectable silencing over 10 months of continuous cell culture in absence of any selective pressure. In monocytic THP-1 cells, the functional activity of luciferase reporters for NF-κB, Nrf2, or HIF-1α has not decreased over time and was retained following differentiation into macrophages and DCs, as well. These results imply pB as a versatile tool for gene integration in monocytic cells in general, and as a convenient access route to DC-based signaling pathway reporters suitable for high-throughput assays, in particular.     

Degradation of chemical alarm cues and assessment of risk throughout the day

The use of chemical information in assessment of predation risk is pervasive across animal taxa. However, by its very nature, chemical information can be temporally unreliable. Chemical cues persist for some period of time after they are released into the environment. Yet, we know surprisingly little about the rate of degradation of chemical cues under natural conditions and hence little about how they function in temporal risk assessment under natural conditions. Here, we conducted an experiment to identify a concentration of fresh alarm cues that evoke a strong antipredator response in coral reef damselfish, Pomacentrus ambonensis. We then tested the rate at which these alarm cues degraded under natural conditions in ocean water, paying attention to whether the rate of degradation varied throughout the day and whether the temporal pattern correlated with physicochemical factors that could influence the rate of degradation. Fresh alarm cues released into ocean water evoke strong avoidance responses in juvenile fish, while those aged for 30 min no longer evoke antipredator responses. Fish exposed to cues aged for 10 or 20 min show intermediate avoidance responses. We found a marked temporal pattern of response throughout the day, with much faster degradation in early to mid‐afternoon, the time of day when solar radiation, temperature, dissolved oxygen, and pH are nearing their peak. Ecologists have spent considerable effort elucidating the role of chemical information in mediating predator–prey interactions, yet we know almost nothing about the temporal dynamics of risk assessment using chemical information. We are in dire need of additional comparative field experiments on the rate of breakdown of chemical cues, particularly given that global change in UV radiation, temperature, and water chemistry could be altering the rates of degradation and the potential use of this information in risk assessment.     

The bacterial parasite Pasteuria ramosa is not killed if it fails to infect: implications for coevolution

Strong selection on parasites, as well as on hosts, is crucial for fueling coevolutionary dynamics. Selection will be especially strong if parasites that encounter resistant hosts are destroyed and diluted from the local environment. We tested whether spores of the bacterial parasite Pasteuria ramosa were passed through the gut (the route of infection) of their host, Daphnia magna, and whether passaged spores remained viable for a “second chance” at infecting a new host. In particular, we tested if this viability (estimated via infectivity) depended on host genotype, whether or not the genotype was susceptible, and on initial parasite dose. Our results show that Pasteuria spores generally remain viable after passage through both susceptible and resistant Daphnia. Furthermore, these spores remained infectious even after being frozen for several weeks. If parasites can get a second chance at infecting hosts in the wild, selection for infection success in the first instance will be reduced. This could also weaken reciprocal selection on hosts and slow the coevolutionary process.