Recombinant trehalose-6-phosphate phosphatase of Brugia malayi cross-reacts with human Wuchereria bancrofti immune sera and engenders a robust protective outcome in mice

Trehalose-6-phosphate phosphatase of Brugia malayi (Bm-TPP) represents an attractive vaccine candidate because it is present in all the major life stages of parasite, but is absent in mammals. We have previously cloned, purified and biochemically characterized Bm-TPP. In the present study, we investigated the cross-reactivity of recombinant Bm-TPP (r-Bm-TPP) with the sera of human bancroftian patients belonging to different disease categories. In silico study using bioinformatics tool demonstrated that Bm-TPP is highly immunogenic in nature. BALB/c mice administered with r-Bm-TPP alone or in combination with Freund's complete adjuvant (FCA) generated a strong IgG response. Further investigations on the antibody isotypes showed generation of a mixed T helper cell response which was marginally biased towards Th1 phenotype. r-Bm-TPP with or without adjuvant lead to significantly increased accumulation of CD4+ and CD8+ T cells in the spleen of infected mice and increased the activation of peritoneal macrophages. Additionally, r-Bm-TPP enhanced the production of both proinflammatory (IL-2, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines and mice immunized with r-Bm-TPP alone or in combination with FCA showed 54.5% and 67% protection respectively against B. malayi infective larvae challenge. Taken together, our findings suggest that Bm-TPP is protective in nature and might be a potential candidate for development of vaccine against lymphatic filarial infections.     

Feasibility and Effectiveness of Provider Initiated HIV Testing and Counseling of TB Suspects in Vizianagaram District, South India

Background

Though internationally recommended, provider initiated HIV testing and counseling (PITC) of persons suspected of tuberculosis (TB) is not a policy in India; HIV seroprevalence among TB suspects has never been reported. The current policy of PITC for diagnosed TB cases may limit opportunities of early HIV diagnosis and treatment. We determined HIV seroprevalence among persons suspected of TB and assessed feasibility and effectiveness of PITC implementation at this earlier stage in the TB diagnostic pathway.

Methods

All adults examined for diagnostic sputum microscopy (TB suspects) in Vizianagaram district (population 2.5 million), in November-December 2010, were offered voluntary HIV counseling and testing (VCT) and assessed for TB diagnosis.

Results

Of 2918 eligible TB suspects, 2465(85%) consented to VCT. Among these, 246(10%) were HIV-positive. Of the 246, 84(34%) were newly diagnosed as HIV (HIV status not known previously). To detect a new case of HIV infection, the number needed to screen (NNS) was 26 among ‘TB suspects’, comparable to that among ‘TB patients’. Among suspects aged 25–54 years, not diagnosed as TB, the NNS was 17.

Conclusion

The seroprevalence of HIV among ‘TB suspects’ was as high as that among ‘TB patients’. Implementation of PITC among TB suspects was feasible and effective, detecting a large number of new HIV cases with minimal additional workload on staff of HIV testing centre. HIV testing of TB suspects aged 25–54 years demonstrated higher yield for a given effort, and should be considered by policy makers at least in settings with high HIV prevalence.     

1H, 15N, 13C resonance assignment of folded and 8 M urea-denatured state of SUMO from Drosophila melanogaster

SUMO, an important post-translational modifier of variety of substrate proteins, regulates different cellular functions. Here, we report the NMR resonance assignment of the folded and 8 M urea-denatured state of SUMO from Drosophila melanogaster (dsmt3).     

Reversible monomer-oligomer transition in human prion protein

The structure and the dissociation reaction of oligomers PrP^oligo from reduced human prion huPrP^C(23–231) have been studied by ¹H-NMR and tryptophan fluorescence spectroscopy at varying pressure, along with circular dichroism and atomic force microscopy. The ¹H-NMR and fluorescence spectral feature of the oligomer is consistent with the notion that the N-terminal residues including all seven Trp residues, are free and mobile, while residues 105∼210, comprising the AGAAAAGA motif and S1-Loop-HelixA-Loop-S2-Loop-HelixC, are engaged in intra- and/or inter-molecular interactions. By increasing pressure to 200 MPa, the oligomers tend to dissociate into monomers which may be identified with PrP^C*, a rare metastable form of PrP^C stabilized at high pressure (Kachel et al., BMC Struct Biol 6:16). The results strongly suggest that the oligomeric form PrP^oligo is in dynamic equilibrium with the monomeric forms via PrP^C*, namely huPrP^C ⇆ huPrP^C* ⇆ huPrP^oligo.Key words: human prion, oligomer structure, pressure dissociation, reversible monomer-oligomer transition, circular dichroism, high pressure NMR, atomic force microscopy     

Dissecting the functional role of polyketide synthases in Dictyostelium discoideum: biosynthesis of the differentiation regulating factor 4-methyl-5-pentylbenzene-1,3-diol

Dictyostelium discoideum exhibits the largest repository of polyketide synthase (PKS) proteins of all known genomes. However, the functional relevance of these proteins in the biology of this organism remains largely obscure. On the basis of computational, biochemical, and gene expression studies, we propose that the multifunctional Dictyostelium PKS (DiPKS) protein DiPKS1 could be involved in the biosynthesis of the differentiation regulating factor 4-methyl-5-pentylbenzene-1,3-diol (MPBD). Our cell-free reconstitution studies of a novel acyl carrier protein Type III PKS didomain from DiPKS1 revealed a crucial role of protein-protein interactions in determining the final biosynthetic product. Whereas the Type III PKS domain by itself primarily produces acyl pyrones, the presence of the interacting acyl carrier protein domain modulates the catalytic activity to produce the alkyl resorcinol scaffold of MPBD. Furthermore, we have characterized an O-methyltransferase (OMT12) from Dictyostelium with the capability to modify this resorcinol ring to synthesize a variant of MPBD. We propose that such a modification in vivo could in fact provide subtle variations in biological function and specificity. In addition, we have performed systematic computational analysis of 45 multidomain PKSs, which revealed several unique features in DiPKS proteins. Our studies provide a new perspective in understanding mechanisms by which metabolic diversity could be generated by combining existing functional scaffolds.     

Biosynthesis of rubradirin as an ansamycin antibiotic from Streptomyces achromogenes var. rubradiris NRRL3061

The four overlapping cosmids from the rubradirin producer, Streptomyces achromogenes var rubradiris NRRL 3061, have 58 ORFs within a 105.6 kb fragment. These ORFs harbored essential genes responsible for the formation and attachment of four distinct moieties, along with the genes associated with regulatory, resistance, and transport functions. The PKS (rubA) and glycosyltransferase (rubG2) genes were disrupted in order to demonstrate a complete elimination of rubradirin production. The rubradirin biosynthetic pathway was proposed based on the putative functions of the gene products, the functional identification of sugar genes, and the mutant strains. The GeneBank accession number for the sequence reported in this paper is AJ871581.     

Synthesis and antibacterial activity of the C-7 side chain of 3-aminoquinazolinediones

A novel series of bacterial topoisomerase (3-aminoquinazolinediones) inhibitors are described. The side-chain SAR against Gram-positive and Gram-negative organisms as well as DNA gyrase activity is reported.