Molecular Analysis of a Leprosy Immunotherapeutic Bacillus Provides Insights into Mycobacterium Evolution

BACKGROUND: Evolutionary dynamics plays a central role in facilitating the mechanisms of species divergence among pathogenic and saprophytic mycobacteria. The ability of mycobacteria to colonize hosts, to proliferate and to cause diseases has evolved due to its predisposition to various evolutionary forces acting over a period of time. Mycobacterium indicus pranii (MIP), a taxonomically unknown 'generalist' mycobacterium, acts as an immunotherapeutic against leprosy and is approved for use as a vaccine against it. The large-scale field trials of this MIP based leprosy vaccine coupled with its demonstrated immunomodulatory and adjuvant property has led to human clinical evaluations of MIP in interventions against HIV-AIDS, psoriasis and bladder cancer. MIP, commercially available as 'Immuvac', is currently the focus of advanced phase III clinical trials for its antituberculosis efficacy. Thus a comprehensive analysis of MIP vis-à-vis evolutionary path, underpinning its immanent immunomodulating properties is of the highest desiderata. PRINCIPAL FINDINGS: Genome wide comparisons together with molecular phylogenetic analyses by fluorescent amplified fragment length polymorphism (FAFLP), enterobacterial repetitive intergenic consensus (ERIC) based genotyping and candidate orthologues sequencing revealed that MIP has been the predecessor of highly pathogenic Mycobacterium avium intracellulare complex (MAIC) that did not resort to parasitic adaptation by reductional gene evolution and therefore, preferred a free living life-style. Further analysis suggested a shared aquatic phase of MAIC bacilli with the early pathogenic forms of Mycobacterium, well before the latter diverged as 'specialists'. CONCLUSIONS/SIGNIFICANCE: This evolutionary paradigm possibly affirms to marshall our understanding about the acquisition and optimization of virulence in mycobacteria and determinants of boundaries therein

Polyphasic Taxonomic Analysis Establishes Mycobacterium indicus pranii as a Distinct Species

that integrate its phenotypic, chemotaxonomic and molecular phylogenetic attributes..

Dissecting the Role of Critical Residues and Substrate Preference of a Fatty Acyl-CoA Synthetase (FadD13) of Mycobacterium tuberculosis

Newly emerging multi-drug resistant strains of Mycobacterium tuberculosis (M.tb) severely limit the treatment options for tuberculosis (TB); hence, new antitubercular drugs are urgently needed. The mymA operon is essential for the virulence and intracellular survival of M.tb and thus represents an attractive target for the development of new antitubercular drugs. This study is focused on the structure-function relationship of Fatty Acyl-CoA Synthetase (FadD13, Rv3089) belonging to the mymA operon. Eight site-directed mutants of FadD13 were designed, constructed and analyzed for the structural-functional integrity of the enzyme. The study revealed that mutation of Lys487 resulted in ∼95% loss of the activity thus demonstrating its crucial requirement for the enzymatic activity. Comparison of the kinetic parameters showed the residues Lys172 and Ala302 to be involved in the binding of ATP and Ser404 in the binding of CoenzymeA. The influence of mutations of the residues Val209 and Trp377 emphasized their importance in maintaining the structural integrity of FadD13. Besides, we show a synergistic influence of fatty acid and ATP binding on the conformation and rigidity of FadD13. FadD13 represents the first Fatty Acyl-CoA Synthetase to display biphasic kinetics for fatty acids. FadD13 exhibits a distinct preference for C26/C24 fatty acids, which in the light of earlier reported observations further substantiates the role of the mymA operon in remodeling the cell envelope of intracellular M.tb under acidic conditions. A three-dimensional model of FadD13 was generated; the docking of ATP to the active site verified its interaction with Lys172, Ala302 and Lys487 and corresponded well with the results of the mutational studies. Our study provides a significant understanding of the FadD13 protein including the identification of residues important for its activity as well as in the maintenance of structural integrity. We believe that the findings of this study will provide valuable inputs in the development of inhibitors against the mymA operon, an important target for the development of antitubercular drugs

Influence of organic nutrient sources and moisture management on productivity, biofortification and soil health in pearl millet (Pennisetum glaucum) + clusterbean (Cyamopsis tetragonaloba) intercropping system of semi-arid India

A field experimentation was carried out at New Delhi, India for three years (2010-12) under rainfed semi-arid conditions to assess the effect of three organic nutrient sources and two moisture management practices on productivity, biofortification and soil health under pearl millet [Pennisetum glaucum (L.) R. Br. emend Stuntz] + clusterbean [Cyamopsis tetragonaloba (L.) Taub] intercropping system (PCIS). In current study, the pearl millet grain yield under sole pearl millet was at par with PCIS while its stover yield reduced by 5%. Under PCIS, inclusion of clusterbean led to significant reduction in clusterbean yield compared to its sole stand, however, PCIS system productivity was more over their sole crop stands, indicating that additive series of PCIS may bring more net economic gains over their sole stands in semi-arid ecology of India. The coarse cereal-legume interactions under PCIS have also led to improved protein content in pearl millet (11.2%) over its sole stand (10.7%). Zinc (Zn) and iron (Fe) deficiency is a common feature in semi-arid India and the crops grown as well. Study revealed that Zn and Fe content in pearl millet were increased by 6.5 and 1.6%, respectively, by the inclusion of clusterbean in PCIS. Plant growth, yield attributes and yield of pearl millet and clusterbean crops showed an improvement under flat sowing + organic mulching (FS+OM) over ridge and furrow sowing (RFS) but the differences were non-significant. Among organic nutrient sources, leaf compost (LC) @ 10 t/ha and FYM @ 10 t/ha produced significantly more system productivity in terms of pearl millet-equivalent-yield (PEY) than 100% RDF and control. Protein content both in pearl millet and clusterbean, and Zn and Fe biofortification in pearl millet grains was higher under FYM treated plots closely followed by LC, and leaf + cowdung mixture compost. Application of organics especially FYM @ 10 t/ha also resulted in improved soil health in terms of bulk density, soil organic carbon and NPK status after three years experimentation

Electrochemical sensing of hydrogen peroxide based on nano γ-Fe2O3 modified glassy carbon electrode

Maghemite (γ-Fe2O3) nanoparticles were prepared using chemical synthesis method and used for sensing the hydrogen peroxide. The morphology of the γ-Fe2O3 nanoparticles was characterized by scanning electron microscopy. The γ-Fe2O3 nanoparticles were used to modify glassy carbon electrode (GCE) to form nano γ-Fe2O3 modified GC electrode for electrochemical sensing of hydrogen peroxide (H2O2). A potential controlled cyclic voltammetric (CV) technique was performed to sense hydrogen peroxide using nano γ-Fe2O3 modified GC electrode. The nano γ-Fe2O3 modified GC electrode showed excellent electrocatalytic ability towards H2O2 in 0.1M KCl supporting electrolyte. A significant enhancement in anodic peak current was observed for the nano γ-Fe2O3 modified GC electrode than those of bare electrode. The electrochemical characteristics of hydrogen peroxide on nano γ-Fe2O3 modified GC electrode  had been explored in this research communication

Electrochemical sensing of hydrogen peroxide based on nano γ-Fe2O3 modified glassy carbon electrode

162-166Maghemite (γ-Fe2O3) nanoparticles were prepared using chemical synthesis method and used for sensing the hydrogen peroxide. The morphology of the γ-Fe2O3 nanoparticles was characterized by scanning electron microscopy. The γ-Fe2O3 nanoparticles were used to modify glassy carbon electrode (GCE) to form nano γ-Fe2O3 modified GC electrode for electrochemical sensing of hydrogen peroxide (H2O2). A potential controlled cyclic voltammetric (CV) technique was performed to sense hydrogen peroxide using nano γ-Fe2O3 modified GC electrode. The nano γ-Fe2O3 modified GC electrode showed excellent electrocatalytic ability towards H2O2 in 0.1M KCl supporting electrolyte. A significant enhancement in anodic peak current was observed for the nano γ-Fe2O3 modified GC electrode than those of bare electrode. The electrochemical characteristics of hydrogen peroxide on nano γ-Fe2O3 modified GC electrode  had been explored in this research communication

An advanced draft genome assembly of a desi type chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) is an important pulse legume crop. We previously reported a draft genome assembly of the desi chickpea cultivar ICC 4958. Here we report an advanced version of the ICC 4958 genome assembly (version 2.0) generated using additional sequence data and an improved genetic map. This resulted in 2.7-fold increase in the length of the pseudomolecules and substantial reduction of sequence gaps. The genome assembly covered more than 94% of the estimated gene space and predicted the presence of 30,257 protein-coding genes including 2230 and 133 genes encoding potential transcription factors (TF) and resistance gene homologs, respectively. Gene expression analysis identified several TF and chickpea-specific genes with tissue-specific expression and displayed functional diversification of the paralogous genes. Pairwise comparison of pseudomolecules in the desi (ICC 4958) and the earlier reported kabuli (CDC Frontier) chickpea assemblies showed an extensive local collinearity with incongruity in the placement of large sequence blocks along the linkage groups, apparently due to use of different genetic maps. Single nucleotide polymorphism (SNP)-based mining of intra-specific polymorphism identified more than four thousand SNPs differentiating a desi group and a kabuli group of chickpea genotypes

Mitochondria in epithelial ovarian carcinoma exhibit abnormal phenotypes and blunted associations with biobehavioral factors

Malignant tumor cells exhibit mitochondrial alterations and are also influenced by biobehavioral processes, but the intersection of biobehavioral factors and mitochondria in malignant tumors remains unexplored. Here we examined multiple biochemical and molecular markers of mitochondrial content and function in benign tissue and in high-grade epithelial ovarian carcinoma (EOC) in parallel with exploratory analyses of biobehavioral factors. First, analysis of a publicly-available database (n = 1435) showed that gene expression of specific mitochondrial proteins in EOC is associated with survival. Quantifying multiple biochemical and molecular markers of mitochondrial content and function in tissue from 51 patients with benign ovarian masses and 128 patients with high-grade EOC revealed that compared to benign tissue, EOCs exhibit 3.3–8.4-fold higher mitochondrial content and respiratory chain enzymatic activities (P < 0.001) but similar mitochondrial DNA (mtDNA) levels (− 3.1%), documenting abnormal mitochondrial phenotypes in EOC. Mitochondrial respiratory chain activity was also associated with interleukin-6 (IL-6) levels in ascites. In benign tissue, negative biobehavioral factors were inversely correlated with mitochondrial content and respiratory chain activities, whereas positive biobehavioral factors tended to be positively correlated with mitochondrial measures, although effect sizes were small to medium (r = − 0.43 to 0.47). In contrast, serous EOCs showed less pronounced biobehavioral-mitochondrial correlations. These results document abnormal mitochondrial functional phenotypes in EOC and warrant further research on the link between biobehavioral factors and mitochondria in cancer

Tetrahydropyrazolo[1,5-a]Pyrimidine-3-Carboxamide and N-Benzyl-6′,7′-Dihydrospiro[Piperidine-4,4′-Thieno[3,2-c]Pyran] analogues with bactericidal efficacy against Mycobacterium tuberculosis targeting MmpL3

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-ca​rboxamide(THPP) and N-benzyl-6′,7′-dihydrospiro[piperidine-4,​4′-thieno[3,2-c]pyran](Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This ‘genetic phenotype’ was further confirmed by a ‘chemical phenotype’, whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice

Detection of Pathogenic Mycobacteria Based on Functionalized Quantum Dots Coupled with Immunomagnetic Separation

Mycobacteria have always proven difficult to identify due to their low growth rate and fastidious nature. Therefore molecular biology and more recently nanotechnology, have been exploited from early on for the detection of these pathogens. Here we present the first stage of development of an assay incorporating cadmium selenide quantum dots (QDs) for the detection of mycobacterial surface antigens. The principle of the assay is the separation of bacterial cells using magnetic beads coupled with genus-specific polyclonal antibodies and monoclonal antibodies for heparin-binding hemagglutinin. These complexes are then tagged with anti-mouse biotinylated antibody and finally streptavidin-conjugated QDs which leads to the detection of a fluorescent signal. For the evaluation of performance, the method under study was applied on Mycobacterium bovis BCG and Mycobacterium tuberculosis (positive controls), as well as E. coli and Salmonella spp. that constituted the negative controls. The direct observation of the latter category of samples did not reveal fluorescence as opposed to the mycobacteria mentioned above. The minimum detection limit of the assay was defined to 104 bacteria/ml, which could be further decreased by a 1 log when fluorescence was measured with a spectrofluorometer. The method described here can be easily adjusted for any other protein target of either the pathogen or the host, and once fully developed it will be directly applicable on clinical samples