Transitioning a Large Scale HIV/AIDS Prevention Program to Local Stakeholders: Findings from the Avahan Transition Evaluation

Background

Between 2009–2013 the Bill and Melinda Gates Foundation transitioned its HIV/AIDS prevention initiative in India from being a stand-alone program outside of government, to being fully government funded and implemented. We present an independent prospective evaluation of the transition.

Methods

The evaluation drew upon (1) a structured survey of transition readiness in a sample of 80 targeted HIV prevention programs prior to transition; (2) a structured survey assessing institutionalization of program features in a sample of 70 targeted intervention (TI) programs, one year post-transition; and (3) case studies of 15 TI programs.

Findings

Transition was conducted in 3 rounds. While the 2009 transition round was problematic, subsequent rounds were implemented more smoothly. In the 2011 and 2012 transition rounds, Avahan programs were well prepared for transition with the large majority of TI program staff trained for transition, high alignment with government clinical, financial and managerial norms, and strong government commitment to the program. One year post transition there were significant program changes, but these were largely perceived positively. Notable negative changes were: limited flexibility in program management, delays in funding, commodity stock outs, and community member perceptions of a narrowing in program focus. Service coverage outcomes were sustained at least six months post-transition.

Interpretation

The study suggests that significant investments in transition preparation contributed to a smooth transition and sustained service coverage. Notwithstanding, there were substantive program changes post-transition. Five key lessons for transition design and implementation are identified.     

DOTA-amide lanthanide tag for reliable generation of pseudocontact shifts in protein NMR spectra

Structural studies of proteins and protein-ligand complexes by nuclear magnetic resonance (NMR) spectroscopy can be greatly enhanced by site-specific attachment of lanthanide ions to create paramagnetic centers. In particular, pseudocontact shifts (PCS) generated by paramagnetic lanthanides contain important and unique long-range structure information. Here, we present a high-affinity lanthanide binding tag that can be attached to single cysteine residues of proteins. The new tag has many advantageous features that are not available in this combination from previously published tags: (i) it binds lanthanide ions very tightly, minimizing the generation of nonspecific effects, (ii) it produces PCSs with high reliability as its bulkiness prevents complete motional averaging of PCSs, (iii) it can be attached to single cysteine residues, alleviating the need of detailed prior knowledge of the 3D structure of the target protein, and (iv) it does not display conformational exchange phenomena that would increase the number of signals in the NMR spectrum. The performance of the tag is demonstrated with the N-terminal domain of the E. coli arginine repressor and the A28C mutant of human ubiquitin.     

Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases

Expression of foreign pathways often results in suboptimal performance due to unintended factors such as introduction of toxic metabolites, cofactor imbalances or poor expression of pathway components. In this study we report a 120% improvement in the production of the isoprenoid-derived sesquiterpene, amorphadiene, produced by an engineered strain of Escherichia coli developed to express the native seven-gene mevalonate pathway from Saccharomyces cerevisiae (Martin et al. 2003). This substantial improvement was made by varying only a single component of the pathway (HMG-CoA reductase) and subsequent host optimization to improve cofactor availability. We characterized and tested five variant HMG-CoA reductases obtained from publicly available genome databases with differing kinetic properties and cofactor requirements. The results of our in vitro and in vivo analyses of these enzymes implicate substrate inhibition of mevalonate kinase as an important factor in optimization of the engineered mevalonate pathway. Consequently, the NADH-dependent HMG-CoA reductase from Delftia acidovorans, which appeared to have the optimal kinetic parameters to balance HMG-CoA levels below the cellular toxicity threshold of E. coli and those of mevalonate below inhibitory concentrations for mevalonate kinase, was identified as the best producer for amorphadiene (54% improvement over the native pathway enzyme, resulting in 2.5 mM or 520 mg/L of amorphadiene after 48 h). We further enhanced performance of the strain bearing the D. acidovorans HMG-CoA reductase by increasing the intracellular levels of its preferred cofactor (NADH) using a NAD⁺-dependent formate dehydrogenase from Candida boidinii, along with formate supplementation. This resulted in an overall improvement of the system by 120% resulting in 3.5 mM or 700 mg/L amorphadiene after 48 h of fermentation. This comprehensive study incorporated analysis of several key parameters for metabolic design such as in vitro and in vivo kinetic performance of variant enzymes, intracellular levels of protein expression, in-pathway substrate inhibition and cofactor management to enable the observed improvements. These metrics may be applied to a broad range of heterologous pathways for improving the production of biologically derived compounds.     

Genetic transformation of Indian isolate of Lemna minor mediated by Agrobacterium tumefaciens and recovery of transgenic plants

Transgenic plants of an Indian isolate of Lemna minor have been developed for the first time using Agrobacterium tumefaciens and hard nodular cell masses ‘nodular calli’ developed on the BAP - pretreated daughter frond explants in B₅ medium containing sucrose (1.0 %) with 2,4-D (5.0 μM) and 2-iP (50.0 μM) or 2,4-D (50.0 μM) and TDZ (5.0 μM) under light conditions. These calli were co-cultured with A. tumefaciens strain EHA105 harboring a binary vector that contained genes for β-glucuronidase with intron and neomycin phosphortransferase. Transformed cells selected on kanamycin selection medium were regenerated into fronds whose transgenic nature was confirmed by histochemical assay for GUS activity, PCR analysis and Southern hybridization. The frequency of transformation obtained was 3.8 % and a period of 11–13 weeks was required from initiation of cultures from explants to fully grown transgenic fronds. The pretreatment of daughter fronds with BAP, use of non-ionic surfactant, presence of acetosyringone in co-cultivation medium, co-culture duration of 3 d and 16 h photoperiod during culture were found crucial for callus induction, frond regeneration and transformation of L. minor. This transformation system can be used for the production of pharmaceutically important protein and in bioremediation.     

Comparative analysis of human matrix metalloproteinases: Emerging therapeutic targets in diseases

The identification of specific target proteins for any diseased condition involves extensive characterization of the potentially involved proteins. Members of a protein family demonstrating comparable features may show certain unusual features when implicated in a pathological condition. Advancements in the field of computational biology and the use of various bioinformatics tools for analysis can aid researchers to comprehend their system of work in primary stages of research. This initial screening can help to reduce time and cost of testing and experimentation in laboratory. Human matrix metalloproteinase (MMP) family of endopeptidases is one such family of 23 members responsible for the remodeling of extracellular matrix (ECM) by degradation of the ECM proteins. Though their role has been implicated in various pathological conditions such as arthritis, atherosclerosis, cancer, liver fibrosis, cardio-vascular and neurodegenerative disorders, little is known about the specific involvement of members of the large MMP family in diseases. A comparative in silico characterization of the MMP protein family has been carried out to analyze their physico-chemical, secondary structural and functional properties. Based on the observed patterns of occurrence of atypical features, we hypothesize that cysteine rich and highly thermostable MMPs might be key players in diseased conditions. Thus, a plausible grouping of disease responsive MMPs that might be considered as promising clinical targets may be done. This study can be used as a fundamental approach to characterize, analyze and screen large protein families for the identification of signature patterns.     

<Emphasis Type="Italic">N</Emphasis>-acyl homoserine lactones are degraded via an amidolytic activity in <Emphasis Type="Italic">Comamonas</Emphasis> sp. strain D1

Abtsract Comamonas strain D1 enzymatically inactivates quorum-sensing (QS) signal molecules of the N-acyl homoserine lactone (N-AHSL) family, and exhibits the broadest inactivation range of known bacteria. It degrades N-AHSL with acyl-side chains ranging from 4 to 16 carbons, with or without 3-oxo or 3-hydroxy substitutions. N-AHSL degradation yields HSL but not N-acyl homoserine: strain D1 therefore harbors an amidohydrolase activity. Strain D1 is the fifth bacterium species in which an N-AHSL amidohydrolase is described. Consistent with its N-AHSL degradation ability, strain D1 efficiently quenches various QS-dependent functions in other bacteria, such as violacein production by Chromobacterium violaceum and pathogenicity and antibiotic production in Pectobacterium.     

Probiotic Potential of a Lactobacillus Bacterium of Canine Faecal-Origin and Its Impact on Select Gut Health Indices and Immune Response of Dogs

The objective of the present study was to develop a probiotic of canine-origin for its potential application in pet nutrition. Accordingly, 32 lactic acid bacteria (LAB) strains were isolated from faeces of dogs, out of which 9 strains were short-listed for further in vitro testing based on the aggregation time and cell surface hydrophobicity. The results of acid-, bile- and phenol-tolerance tests indicated that out of the nine, isolate cPRO23 was having better resistance to these adverse conditions likely to be encountered in the gastrointestinal tract. The isolate also showed optimal enzymatic activities for amylase, lipase and protease. Further assessments also indicated its superiority in terms of co-aggregation and antagonistic activity against pathogenic strains of Salmonella typhimurium and Salmonella enteritidis. Subsequently, the isolate was identified through 16S rRNA sequencing and sequence homology, and designated as Lactobacillus johnsonii CPN23. The candidate probiotic was then evaluated in vivo using 15 adult Labrador dogs, divided into 3 groups, viz. CON (with no probiotics), dPRO (with Lactobacillus acidophilus NCDC 15 as a conventional dairy-origin probiotic) and cPRO (with L. johnsonii CPN23 as a canine-origin probiotic). Results of the 9-week study indicated that supplementation of cPRO improved (P < 0.05) the faecal concentration of acetate and butyrate with a concomitant reduction (P < 0.05) in faecal ammonia. The cell-mediated immune response, assessed as delayed-type hypersensitivity reaction to phytohaemagglutinin-P, was better (P < 0.05) in dogs fed cPRO as compared to the CON dogs. There were, however, no variations evident in the antibody response to sheep-erythrocytes among the three groups. It is concluded that the canine-origin L. johnsonii CPN23, in addition to possessing all the in vitro functional attributes of a candidate probiotic, also has the potential to be used as a probiotic in pet nutrition programs.     

Molecular mapping of the rf1 gene for pollen fertility restoration in sorghum (Sorghum bicolor L.)

We report the molecular mapping of a gene for pollen fertility in A1 (milo) type cytoplasm of sorghum using AFLP and SSR marker analysis. DNA from an F₂ population comprised of 84 individuals was screened with AFLP genetic markers to detect polymorphic DNAs linked to fertility restoration. Fifteen AFLP markers were linked to fertility restoration from the initial screening with 49 unique AFLP primer combinations (+3/+3 selective bases). As many of these AFLP markers had been previously mapped to a high-density genetic map of sorghum, the target gene (rf1) could be mapped to linkage group H. Confirmation of the map location of rf1 was obtained by demonstrating that additional linkage group-H markers (SSR, STS, AFLP) were linked to fertility restoration. The closest marker, AFLP Xtxa2582, mapped within 2.4 cM of the target loci while two SSRs, Xtxp18 and Xtxp250, flanked the rf1 locus at 12 cM and 10.8 cM, respectively. The availability of molecular markers will facilitate the selection of pollen fertility restoration in sorghum inbred-line development and provide the foundation for map-based gene isolation. Received: 22 August 2000 / Accepted: 18 October 2000