Functional analysis of the role of GCN2 kinase in longevity and amino acid homeostasis in Drosophila melanogaster

The eIF2α kinase, General Control Non-derepressible-2 (GCN2), senses amino acid starvation and coordinates cellular translation with amino acid availability allowing cells to cope with the nutritional stress. Accumulating evidence shows an important role of amino acids in modulation of longevity and healthspan in diverse species, however the underlying mechanisms remain elusive. With its function as an evolutionarily conserved amino acid starvation sensor, GCN2 kinase is a potential candidate to be involved in regulation of amino-acid-dependent physiological responses. In the first part of this study, I have analyzed the in vivo functions of GCN2 kinase under deprivation of each of the 10 essential amino acids (EAAs) using a Drosophila Gcn2 null mutant and a fully defined chemical diet. The present study shows that GCN2 function is essential for ensuring fly development and survival under deprivation of individual EAAs. Furthermore, this study suggests that GCN2 induces a compensatory feeding response in flies under long-term nutritional deprivation of an EAA, leading to increased lipid level, which is probably causal for the increased resistance of flies under full starvation. Interestingly, Gcn2 null mutant and wild-type control flies exhibit similar physiological responses upon methionine deprivation, suggesting that methionine is the only EAA whose deprivation is sensed by a GCN2-independent mechanism. Furthermore, I tested whether enhanced expression of the downstream effector of GCN2 kinase, activating transcription factor-4 (ATF4), in the absence of GCN2 is sufficient to rescue the phenotypes of Gcn2 null mutant under starvation of different EAAs. The present study shows that ATF4 activation partially rescues GCN2 mediated functions under deprivation of specific EAAs and that the rescue efficiency upon ATF4 activation depends on which EAA is being deprived from the diet. Another important finding of this study is that an EAA starvation is different from full amino acid starvation and probably sensed by GCN2-independent mechanisms. In addition, this study shows that in contrast to worms, GCN2 function in flies is not essential for lifespan extension conferred by rapamycin-mediated Target of Rapamycin (TOR) inhibition. In the second part of this study, I have analyzed the role of GCN2 kinase in dietary restriction (DR) and the transsulfuration pathway (TSP). Amino acids, particularly methionine and cysteine, play an important role in mediating longevity and health benefits of DR. Recently, it has been suggested that the TSP, responsible for methionine and cysteine metabolism, controls DR-mediated longevity through one of its metabolite, H2S. An increase in the TSP-mediated H2S production was seen in yeast, worm, fruit fly, and rodent models of DR. Interestingly, the present study shows that DR- and methionine and/or cysteine restriction (M/C-R)- induced longevity is GCN2-independent in flies but TSP-mediated H2S production, at least in parts, is dependent on GCN2. I, therefore re-examined the connection between DR-induced longevity and TSP-mediated H2S production and surprisingly found that increased lifespan do not correlate with increased H2S production capacity in flies

MGDD: Mycobacterium tuberculosis Genome Divergence Database

<p>Abstract</p> <p>Background</p> <p>Variation in genomes among different closely-related organisms can be linked to phenotypic differences. A number of mechanisms, such as replication error, repeat expansion and contraction, recombination and transposition can contribute to genomic differences. These processes lead to generation of SNPs, different types of repeat-based and transposons or IS-element-based polymorphisms, inversions and duplications and changes in synteny. A database of all the variations in a group of organisms is not only useful for understanding genotype-phenotype relationship but also in clinical applications. There is no database available at present that provides information about detailed genomic variations among different strains and species of <it>Mycobacterium tuberculosis </it>complex, organisms responsible for human diseases.</p> <p>Description</p> <p>MGDD is a free web-based database that allows quick user friendly search to find different types of genomic variations among a group of fully sequenced organisms belonging to <it>M. tuberculosis </it>complex. The searches are based on data generated by pair wise comparison using a tool that has already been described. Different types of variations that can be searched are SNPs, indels, tandem repeats and divergent regions. The searches can be designed to find specific variations either in a given gene or any given location of the query genome with respect to any other genome currently available.</p> <p>Conclusion</p> <p>Web-based database MGDD can help to find all the possible differences that exists between two strains or species of <it>M. tuberculosis </it>complex. The search tool is very user-friendly and can be used by anyone not familiar with computational methods and will be useful to both clinicians and researchers working on tuberculosis and other Mycobacterial diseases.</p

A self assembled monolayer based microfluidic sensor for urea detection

Urease (Urs) and glutamate dehydrogenase (GLDH) have been covalently co-immobilized onto a self-assembled monolayer (SAM) comprising of 10-carboxy-1-decanthiol (CDT) via EDC–NHS chemistry deposited onto one of the two patterned gold (Au) electrodes for estimation of urea using poly(dimethylsiloxane) based microfluidic channels (2 cm × 200 μm × 200 μm). The CDT/Au and Urs-GLDH/CDT/Au electrodes have been characterized using Fourier transform infrared (FTIR) spectroscopy, contact angle (CA), atomic force microscopy (AFM) and electrochemical cyclic voltammetry (CV) techniques. The electrochemical response measurement of a Urs-GLDH/CDT/Au bioelectrode obtained as a function of urea concentration using CV yield linearity as 10 to 100 mg dl−1, detection limit as 9 mg dl−1 and high sensitivity as 7.5 μA mM−1 cm−2

Analysis of gut bacteriome of in utero arsenic-exposed mice using 16S rRNA-based metagenomic approach

IntroductionApproximately 200 million people worldwide are affected by arsenic toxicity emanating from the consumption of drinking water containing inorganic arsenic above the prescribed maximum contaminant level. The current investigation deals with the role of prenatal arsenic exposure in modulating the gut microbial community and functional pathways of the host.Method16S rRNA-based next-generation sequencing was carried out to understand the effects of in utero 0.04 mg/kg (LD) and 0.4 mg/kg (HD) of arsenic exposure. This was carried out from gestational day 15 (GD-15) until the birth of pups to understand the alterations in bacterial diversity.ResultsThe study focused on gestational exposure to arsenic and the altered gut microbial community at phyla and genus levels, along with diversity indices. A significant decrease in firmicutes was observed in the gut microbiome of mice treated with arsenic. Functional analysis revealed that a shift in genes involved in crucial pathways such as insulin signaling and non-alcoholic fatty liver disease pathways may lead to metabolic diseases in the host.DiscussionThe present investigation may hypothesize that in utero arsenic exposure can perturb the gut bacterial composition significantly as well as the functional pathways of the gestationally treated pups. This research paves the way to further investigate the probable mechanistic insights in the field of maternal exposure environments, which may play a key role in epigenetic modulations in developing various disease endpoints in the progeny