Improved catalytic efficiency of a monomeric γ-glutamyl transpeptidase from Bacillus licheniformis in presence of subtilisin

Monomeric 30 kDa γ-glutamyl transpeptidase (GGT₃₀) was purified from culture broth of Bacillus licheniformis ER-15 along with a heterodimeric 67 kDa GGT (GGT₆₇). In presence of subtilisin, GGT₃₀ had improved catalytic efficiency (V_max/K_m) of 59 min^?1, altered pH and temperature optima of pH 11 and 70°C.and had salt-tolerant glutaminase activity. Glutaminase activity was retained even in protease-inhibited condition in presence of 2 mM PMSF. GGT₃₀ and subtilisin complexation was also confirmed by relative electrophoretic mobility and fluorescence quenching experiment.     

The FLIGHT Drosophila RNAi database: 2010 update

FLIGHT (http://flight.icr.ac.uk/) is an online resource compiling data from high-throughput Drosophila in vivo and in vitro RNAi screens. FLIGHT includes details of RNAi reagents and their predicted off-target effects, alongside RNAi screen hits, scores and phenotypes, including images from high-content screens. The latest release of FLIGHT is designed to enable users to upload, analyze, integrate and share their own RNAi screens. Users can perform multiple normalizations, view quality control plots, detect and assign screen hits and compare hits from multiple screens using a variety of methods including hierarchical clustering. FLIGHT integrates RNAi screen data with microarray gene expression as well as genomic annotations and genetic/physical interaction datasets to provide a single interface for RNAi screen analysis and datamining in Drosophila.Key words: RNAi, database, integration, bioinformatics, phenotype     

The changing epidemiology of dengue in Delhi, India

The distribution of deformed wing virus infection within the honey bee reproductive castes (queens, drones) was investigated by in situ hybridization and immunohistology from paraffin embedded sections. Digoxygenin or CY5.5 fluorochrome end-labelled nucleotide probes hybridizing to the 3' portion of the DWV genome were used to identify DWV RNA, while a monospecific antibody to the DWV-VP1 structural protein was used to identify viral proteins and particles. The histological data were confirmed by quantitative RT-PCR of dissected organs. Results showed that DWV infection is not restricted to the digestive tract of the bee but spread in the whole body, including queen ovaries, queen fat body and drone seminal vesicles.     

Role of highly central residues of P-loop and it's flanking region in preserving the archetypal conformation of Walker A motif of diverse P-loop NTPases

P-loop NTPases represent a large and highly diverse protein family that is involved in variety of cellular functions. Walker A motif forms a typical arched conformation, necessary to accommodate the phosphate moiety of the nucleoside tri (or di-) phosphate in Ploop NTPases. The feature that maintains the ancient architecture of P-loop is unidentified and uncharacterized. Here, using a well established global network parameter, closeness centrality, we identify that Walker A and its flanking regions (N- and C-terminal) have high density of globally connected residue positions. We find that closeness centrality of these residue positions are conserved across common structural core of diverse domains of P-loop NTPase fold. Our results suggest the potential role of globally connected residues in maintaining the local conformation of P-loop.     

Interpretation of Genomic Variants Using a Unified Biological Network Approach

The decreasing cost of sequencing is leading to a growing repertoire of personal genomes. However, we are lagging behind in understanding the functional consequences of the millions of variants obtained from sequencing. Global system-wide effects of variants in coding genes are particularly poorly understood. It is known that while variants in some genes can lead to diseases, complete disruption of other genes, called ‘loss-of-function tolerant’, is possible with no obvious effect. Here, we build a systems-based classifier to quantitatively estimate the global perturbation caused by deleterious mutations in each gene. We first survey the degree to which gene centrality in various individual networks and a unified ‘Multinet’ correlates with the tolerance to loss-of-function mutations and evolutionary conservation. We find that functionally significant and highly conserved genes tend to be more central in physical protein-protein and regulatory networks. However, this is not the case for metabolic pathways, where the highly central genes have more duplicated copies and are more tolerant to loss-of-function mutations. Integration of three-dimensional protein structures reveals that the correlation with centrality in the protein-protein interaction network is also seen in terms of the number of interaction interfaces used. Finally, combining all the network and evolutionary properties allows us to build a classifier distinguishing functionally essential and loss-of-function tolerant genes with higher accuracy (AUC = 0.91) than any individual property. Application of the classifier to the whole genome shows its strong potential for interpretation of variants involved in Mendelian diseases and in complex disorders probed by genome-wide association studies.     

ClpC-Mediated Sporulation Regulation at Engulfment Stage in Bacillus anthracis

Bacterial sporulation is a conserved process utilized by members of Bacillus genus and Clostridium in response to stress such as nutrient or temperature. Sporulation initiation is triggered by stress signals perceived by bacterial cell that leads to shutdown of metabolic pathways of bacterial cells. The mechanism of sporulation involves a complex network that is regulated at various checkpoints to form the viable bacterial spore. Engulfment is one such check point that drives the required cellular rearrangement necessary for the spore assembly and is mediated by bacterial proteolytic machinery that involves association of various Clp ATPases and ClpP protease. The present study highlights the importance of degradation of an anti-sigma factor F, SpoIIAB by ClpCP proteolytic machinery playing a crucial role in culmination of engulfment process during the sporulation in Bacillus anthracis.     

LARVA: an integrative framework for large-scale analysis of recurrent variants in noncoding annotations

In cancer research, background models for mutation rates have been extensively calibrated in coding regions, leading to the identification of many driver genes, recurrently mutated more than expected. Noncoding regions are also associated with disease; however, background models for them have not been investigated in as much detail. This is partially due to limited noncoding functional annotation. Also, great mutation heterogeneity and potential correlations between neighboring sites give rise to substantial overdispersion in mutation count, resulting in problematic background rate estimation. Here, we address these issues with a new computational framework called LARVA. It integrates variants with a comprehensive set of noncoding functional elements, modeling the mutation counts of the elements with a β-binomial distribution to handle overdispersion. LARVA, moreover, uses regional genomic features such as replication timing to better estimate local mutation rates and mutational hotspots. We demonstrate LARVA''s effectiveness on 760 whole-genome tumor sequences, showing that it identifies well-known noncoding drivers, such as mutations in the TERT promoter. Furthermore, LARVA highlights several novel highly mutated regulatory sites that could potentially be noncoding drivers. We make LARVA available as a software tool and release our highly mutated annotations as an online resource (larva.gersteinlab.org).     

Insulin Regulates Nitric Oxide Production in the Kidney Collecting Duct Cells

The kidney is an important organ for arterial blood pressure (BP) maintenance. Reduced NO generation in the kidney is associated with hypertension in insulin resistance. NO is a critical regulator of vascular tone; however, whether insulin regulates NO production in the renal inner medullary collecting duct (IMCD), the segment with the greatest enzymatic activity for NO production in kidney, is not clear. Using an NO-sensitive 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) fluorescent dye, we found that insulin increased NO production in mouse IMCD cells (mIMCD) in a time- and dose-dependent manner. A concomitant dose-dependent increase in the NO metabolite (NOx) was also observed in the medium from insulin-stimulated cells. NO production peaked in mIMCD cells at a dose of 100 nm insulin with simultaneously increased NOx levels in the medium. At this dose, insulin significantly increased p-eNOS^Ser1177 levels in mIMCD cells. Pretreatment of cells with a PI 3-kinase inhibitor or insulin receptor silencing with RNA interference abolished these effects of insulin, whereas insulin-like growth factor-1 receptor (IGF-1R) silencing had no effect. We also showed that chronic insulin infusion to normal C57BL/6J mice resulted in increased endothelial NOS (eNOS) protein levels and NO production in the inner medulla. However, insulin-infused IRKO mice, with targeted deletion of insulin receptor from tubule epithelial cells of the kidney, had ∼50% reduced eNOS protein levels in their inner medulla along with a significant rise in BP relative to WT littermates. We have previously reported increased baseline BP and reduced urine NOx in IRKO mice. Thus, reduced insulin receptor signaling in IMCD could contribute to hypertension in the insulin-resistant state.