Frontline Health Service Providers’ Perspectives on HIV Vaccine Trials among Female Sex Workers and Men Who Have Sex with Men in Karnataka,South India

Background

Little qualitative research is available on the role of frontline health service providers (FHSPs) in the implementation of clinical trials, particularly in developing countries. This paper presents findings from a qualitative study about the perspectives of FHSPs on future HIV vaccine trials involving female sex workers (FSWs) and men who have sex with men (MSM) in three districts of Karnataka, India. In particular, we explore FHSPs’ knowledge of and views on clinical trials in general, and examine their potential willingness to play a role if such trials were introduced or implemented in the region.

Methods

A field team of four researchers from Karnataka—two of whom self-identified with FSW or MSM communities (“community researchers”) and two with backgrounds in social work—conducted in-depth interviews with FHSPs. Including community researchers in the study helped to build rapport with FSW and MSM participants and facilitate in-depth discussions. A coding scheme for transcribed and translated data was developed using a framework analysis approach. Data was then analysed thematically using a combination of a priori and emergent codes.

Results

Over half of FHSPs demonstrated limited knowledge or understanding of clinical trials. Despite reported skepticism around the testing of HIV vaccines in developing countries and concerns around potential side effects, most FHSPs strongly advocated for the implementation of HIV vaccine clinical trials in Karnataka. Further, most FHSPs expressed their willingness to be involved in future HIV vaccine clinical trials in varying capacities.

Conclusion

Given that FHSPs are often directly involved in the promotion of health and well-being of FSWs and MSM, they are well-positioned to play leadership, ethical, and communicative roles in future HIV vaccine trials. However, our findings reveal a lack of awareness of clinical trials among FHSP participants, suggesting an important area for capacity building and staff development before viable and ethical clinical trials can be set up in the region.     

Molecular Disruption of the Power Stroke in the ATP-binding Cassette Transport Protein MsbA

ATP-binding cassette transporters affect drug pharmacokinetics and are associated with inherited human diseases and impaired chemotherapeutic treatment of cancers and microbial infections. Current alternating access models for ATP-binding cassette exporter activity suggest that ATP binding at the two cytosolic nucleotide-binding domains provides a power stroke for the conformational switch of the two membrane domains from the inward-facing conformation to the outward-facing conformation. In outward-facing crystal structures of the bacterial homodimeric ATP-binding cassette transporters MsbA from Gram-negative bacteria and Sav1866 from Staphylococcus aureus, two transmembrane helices (3 and 4) in the membrane domains have their cytoplasmic extensions in close proximity, forming a tetrahelix bundle interface. In biochemical experiments on MsbA from Escherichia coli, we show for the first time that a robust network of inter-monomer interactions in the tetrahelix bundle is crucial for the transmission of nucleotide-dependent conformational changes to the extracellular side of the membrane domains. Our observations are the first to suggest that modulation of tetrahelix bundle interactions in ATP-binding cassette exporters might offer a potent strategy to alter their transport activity.     

Synthesis and anticonvulsant activity of new N-1',N-3'-disubstituted-2'H,3H,5'H-spiro-(2-benzofuran-1,4'-imidazolidine)-2',3,5'-triones

Thirteen new N-1',N-3'-disubstituted-2'H,3H,5'H-spiro-(2-benzofuran-1,4'-imidazolidine)-2',3,5'-triones were synthesized and their pharmacological activity determined with the objective to better understand their SAR for anticonvulsant activity. The anticonvulsant effects of these compounds were evaluated by standard pentylenetetrazol (scPTZ test) and maximum electroshock seizure (MES test) models in mice. Most of the compounds showed ability to protect against the pentylenetetrazol-induced convulsions. Compound 3o (the N-1'-p-nitrophenyl, N-3'-ethyl derivative) in the N-1'-aryl, N-3'-alkyl disubstituted series exhibited maximum activity with ED(50) of 41.8 mg/kg in scPTZ convulsion model.     

Efficient and repetitive production of hematopoietic and endothelial cells from feeder-free monolayer culture system of primate embryonic stem cells

We have established an innovative culture system for the efficient differentiation of hematopoietic and endothelial cells from primate embryonic stem (ES) cells without feeder cells, embryoid bodies, or cell-sorting processes. After several days' culture in murine stromal OP9-conditioned medium supplemented with a cytokine cocktail on collagen-coated dishes, ES cells differentiated into a very unique population of cells with a finger-like appearance. These finger-like cells were positive for mesodermal and/or hemangioblastic markers of kinase insert domain receptor (KDR) and T-cell acute lymphocytic leukemia 1 (TAL1), and produced large amounts of protein tyrosine phosphatase, receptor type, C-positive hematopoietic cells. These hematopoietic cells showed the morphology of immature hematopoietic cells, formed blast cell colonies with high efficiency, and were positive for CD34 antigen, KDR, TAL1, and GATA binding protein 1, suggesting that these blast cells are equivalent to the multipotent hematopoietic progenitor cells. Moreover, they produced functional macrophages in murine stromal MS-5-conditioned medium and primitive erythroblasts in the presence of erythropoietin. The finger-like cells, putative mesodermal progenitors and/or hemangioblasts, actively proliferated and repetitively produced hematopoietic cells as long as they were maintained on the original dish. By contrast, the majority of the finger-like cells differentiated into endothelial cells with specific markers and specific functions after transfer to fresh dishes, indicating that conditions established in the original dish supported the proliferation and hematopoietic differentiation of the finger-like cells. Our method provides a highly controllable culture protocol for repetitive production of hematopoietic and endothelial cells from feeder-free monolayer cultivation of primate ES cells.     

Dynamics, energetics, and structure in protein folding

For many decades, protein folding experimentalists have worked with no information about the time scales of relevant protein folding motions and without methods for estimating the height of folding barriers. Protein folding experiments have been interpreted using chemical models in which the folding process is characterized as a series of equilibria between two or more distinct states that interconvert with activated kinetics. Accordingly, the information to be extracted from experiments was circumscribed to apparent equilibrium constants and relative folding rates. Recent developments are changing this situation dramatically. The combination of fast-folding experiments with the development of analytical methods more closely connected to physical theory reveals that folding barriers in native conditions range from minimally high (approximately 14RT for the very slow folder AcP) to nonexistent. While slow-folding (i.e., > or = 1 ms) single-domain proteins are expected to fold in a two-state fashion, microsecond-folding proteins should exhibit complex behavior arising from crossing marginal or negligible folding barriers. This realization opens a realm of exciting opportunities for experimentalists. The free energy surface of a protein with a marginal (or no) barrier can be mapped using equilibrium experiments, which could resolve energetic factors from structural factors in folding. Kinetic experiments on these proteins provide the unique opportunity to measure folding dynamics directly. Furthermore, the complex distributions of time-dependent folding behaviors expected for these proteins might be accessible to single-molecule measurements. Here, we discuss some of these recent developments in protein folding, emphasizing aspects that can serve as a guide for experimentalists interested in exploiting this new avenue of research.     

Spectroscopic and Scanning Electron Microscopy Studies of Bioaccumulation of Pollutants by Algae

The ability of Chlorella species and two other algal blooms collected locally to take up Cu⁺² and Ni⁺² was investigated using infrared and scanning electron microscopy (SEM) data. The percentage of metal uptake was determined with atomic absorption spectroscopy. The effects of pH and initial concentrations of metal ions on bioaccumulation were examined. The uptake of methyl orange dye by Chlorella species was also studied using Langmuir and Freundlich adsorption isotherms.     

A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements

We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2′deoxycytidine (DAC), where we found a 1–16% decrease in Alu element and 18–60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation.     

Heart rate measures in blind cave crayfish during environmental disturbances and social interactions

Most animals continually assess the environment in which they live and alter their behavior according to various stimuli. As an observer, one looks for changes in a behavior indicating that an animal responded to a particular event. When the animal does not make significant behavioral changes as measured by bodily movements, the animal may be characterized as unresponsive to a given stimulus. This study demonstrates that when behavioral body movements can not be observed an internal physiological measure of heart rate (HR) shows dramatic changes following presentation of defined stimuli. This study used the blind cave crayfish and examined their responsiveness to the following stimuli: light (infrared, dim red, and white), water-borne vibrations, removal of water, olfactory cues, and social interaction with partners. This study demonstrates that there is substantial individual variation of HR at basal levels and with the intensity of an social interaction. We find HR is a reasonable measure of the responsiveness of blind cave crayfish to given stimuli even in the absence of observable behavioral changes. This enables the observer to determine if an individual is responsive to and making an assessment of particular cues.     

Role for the alpha-helix in aberrant protein aggregation

Is the alpha-helix structure capable of triggering the formation of aberrant protein aggregates? To answer this question, we investigate the in vitro aggregation of tau protein in the presence of the helix-inducing agent TFE. Tau is a natively unfolded protein that binds to microtubules and forms aggregates in Alzheimer's disease. We find that full-length tau has residual alpha-helix structure, which is further enhanced by three mutations involved in genetic neurological disorders. TFE concentrations matching an alpha-helical content of 40% in full-length tau and the triple mutant induce the formation of aggregates that are morphologically and structurally heterogeneous. A simple dilution experiment reveals that heterogeneity results from the competition between alpha-helical fibrillar aggregates and more classical amyloid-like aggregates. The alpha-helical aggregates are more resilient to dilution and have the spectroscopic features of alpha-helical coiled coils. We propose a general mechanism by which intrinsically stable alpha-helices can associate into aggregates with only coarse coiled-coil symmetry. In tau, high intrinsic alpha-helix stability and coarse coiled-coil symmetry could be byproducts of its biological function.     

Robustness of protein folding kinetics to surface hydrophobic substitutions

We use both combinatorial and site-directed mutagenesis to explore the consequences of surface hydrophobic substitutions for the folding of two small single domain proteins, the src SH3 domain, and the IgG binding domain of Peptostreptococcal protein L. We find that in almost every case, destabilizing surface hydrophobic substitutions have much larger effects on the rate of unfolding than on the rate of folding, suggesting that nonnative hydrophobic interactions do not significantly interfere with the rate of core assembly.