Habitat Preference and Diversity of Anuran in Durgapur, an Industrial City of West Bengal, India

Anuran diversity study was conducted for one year between June 2008 and August 2009 at Durgapur, an industrial city of West Bengal, India. Nine species under four families of the Order Anura were found to occur in Durgapur. They have a wide range of habitat preference like permanent or temporary aquatic bodies, human residential area, forested areas, termite nest, tree hole, under spaces of logs and so forth. Of the nine sp. 8 were confined to Amarabati pond (site-I), 5 were confined to Durgapur Government College campus (site-II) and 7 were confined at Fuljhore (site-III) of Durgapur. Analysis of the collected data on the anuran community of the study area revealed Shannon–Wiener species diversity index minimum (1.312) at site-II and maximum (1.938) at site-III. In contrast Margalef richness index value was minimum (0.627) at site-II and maximum (1.424) at site-III.     

Identification and molecular cytogenetic characterization of a novel complex Y chromosome rearrangement in a boy with disorder of sexual development

Ambiguous genitalia or disorder of the sexual development is a birth defect where the external genitals do not have the typical appearance of either a male or female. Here we report a boy with ambiguous genitalia and short stature. The cytogenetic analysis by G-banding revealed a small Y chromosome and an additional material on the 15p arm. Further, molecular cytogenetic analysis by Fluorescence in situ hybridization (FISH) using whole chromosome paint probes showed the presence of Y sequences on the 15p arm, confirming that it is a Y;15 translocation. Subsequent, FISH with centromere probe Y showed two signals depicting the presence of two centromeres and differing with a balanced translocation. The dicentric nature of the derivative 15 chromosome was confirmed by FISH with both 15 and Y centromeric probes. Further, the delineation of the Y chromosomal DNA was also done by quantitative real time PCR. Additional Y-short tandem repeat typing was performed to find out the extent of deletion on small Y chromosome. Fine mapping was carried out with 8 Y specific BAC clones which helped in defining the breakpoint regions. MLPA was performed to check the presence or absence of subtelomeric regions and SHOX regions on Y. Finally array CGH helped us in confirming the breakpoint regions. In our study we identified and characterized a novel complex Y chromosomal rearrangement with a complete deletion of the Yq region and duplication of the Yp region with one copy being translocated onto the15p arm. This is the first report of novel and unique Y complex rearrangement showing a deletion, duplication and a translocation in the same patient. The possible mechanism of the rearrangement and the phenotype–genotype correlation are discussed.     

158 A molecular dynamics simulation-based prediction of deleterious angiogenin mutations causing amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons leading to paralysis and death between 3–5?years of diagnosis. Through whole genome association studies, several single nucleotide polymorphisms (SNPs) encoding missense mutations in angiogenin (ANG) protein have been identified as one of the primary factors causing ALS. Structural studies of ANG show that catalytic triad comprising His13, Lys40, and His114 residues imparts ribonucleolytic activity while nuclear localization signal residues ³¹RRR³³ are responsible for nuclear translocation activity. Loss of either ribonucleolytic activity or nuclear translocation activity or both of these functions due to mutations cause ALS. However, the mechanisms of loss-of-functions of ANG mutants are not completely understood. Here, we present a cohesive and comprehensive picture of functional loss mechanisms of all known ALS-associated ANG mutants by extensive molecular dynamics (MD) simulations (Padhi, Kumar, Vasaikar, Jayaram, & Gomes, 2012 Padhi, A. K., Kumar, H., Vasaikar, S. V., Jayaram, B. and Gomes, J. 2012. Mechanisms of loss of functions of human angiogenin variants implicated in amyotrophic lateral sclerosis. PLoS One, 7(2): e32479[PubMed] , [Google Scholar]; AK, 2013 Padhi, A.K., Jayaram, B., & Gomes, J. (accepted for publication). Prediction of functional loss of human angiogenin mutants associated with ALS by molecular dynamics simulations. Scientific Reports (NPG).  [Google Scholar]). Our studies show that conformational switching of catalytic residue His114 is responsible for the loss of ribonucleolytic activity while reduction in solvent-accessible surface area (SASA) of ³¹RRR³³ as a result of local folding is responsible for the loss of nuclear translocation activity (Padhi et al., 2012 Padhi, A. K., Kumar, H., Vasaikar, S. V., Jayaram, B. and Gomes, J. 2012. Mechanisms of loss of functions of human angiogenin variants implicated in amyotrophic lateral sclerosis. PLoS One, 7(2): e32479[PubMed] , [Google Scholar]; AK, 2013 Padhi, A.K., Jayaram, B., & Gomes, J. (accepted for publication). Prediction of functional loss of human angiogenin mutants associated with ALS by molecular dynamics simulations. Scientific Reports (NPG).  [Google Scholar]). Our prediction of loss-of-functions of 17 ANG mutants correlated positively with the reported experimental results. We have subsequently developed a fast molecular dynamics method based on certain global attributes / dynamic markers that can be used to determine whether a mutation is deleterious or benign. To make our method accessible to researchers and clinicians, we created a web server-based tool, ANGDelMut, freely available at http://bioschool.iitd.ernet.in/research.htm, where a user can submit new mutations to ascertain whether they cause ALS. We hope that our method will benefit the community at large and will pave the way for the development of a successful therapy for patients suffering from ALS.     

Chandipura Virus Induces Neuronal Death through Fas-Mediated Extrinsic Apoptotic Pathway

Chandipura virus (CHPV; genus Vesiculovirus, family Rhabdoviridae) is an emerging tropical pathogen with a case fatality rate of 55 to 75% that predominantly affects children in the age group of 2 to 16 years. Although it has been established as a neurotropic virus causing encephalitis, the molecular pathology leading to neuronal death is unknown. The present study elucidates for the first time the mechanism of cell death in neurons after CHPV infection that answers the basic cause of CHPV-mediated neurodegeneration. Through various cell death assays in vitro and in vivo, a relationship between viral replication within neuron and neuronal apoptosis has been established. We report that expression of CHPV phosphoprotein increases up to 6 h postinfection and diminishes thereafter in neuronal cell lines, signifying the replicative phase of CHPV. Various analyses conducted during the investigation established that CHPV-infected neurons are undergoing apoptosis through an extrinsic pathway mediated through the Fas-associated death domain (FADD) following activation of caspase-8 and -3 and prominent cleavage of poly(ADP-ribose) polymerase (PARP). Knocking down the expression of caspase-3, the final executioner of apoptosis, in a neuronal cell line by endoribonuclease-prepared small interfering RNA (siRNA) validated its pivotal role in CHPV-mediated neurodegeneration by showing reduction in apoptosis after CHPV infection.     

Kinetic Enrichment of 34S during Proteobacterial Thiosulfate Oxidation and the Conserved Role of SoxB in S-S Bond Breaking

During chemolithoautotrophic thiosulfate oxidation, the phylogenetically diverged proteobacteria Paracoccus pantotrophus, Tetrathiobacter kashmirensis, and Thiomicrospira crunogena rendered steady enrichment of ³⁴S in the end product sulfate, with overall fractionation ranging between −4.6‰ and +5.8‰. The fractionation kinetics of T. crunogena was essentially similar to that of P. pantotrophus, albeit the former had a slightly higher magnitude and rate of ³⁴S enrichment. In the case of T. kashmirensis, the only significant departure of its fractionation curve from that of P. pantotrophus was observed during the first 36 h of thiosulfate-dependent growth, in the course of which tetrathionate intermediate formation is completed and sulfate production starts. The almost-identical ³⁴S enrichment rates observed during the peak sulfate-producing stage of all three processes indicated the potential involvement of identical S-S bond-breaking enzymes. Concurrent proteomic analyses detected the hydrolase SoxB (which is known to cleave terminal sulfone groups from SoxYZ-bound cysteine S-thiosulfonates, as well as cysteine S-sulfonates, in P. pantotrophus) in the actively sulfate-producing cells of all three species. The inducible expression of soxB during tetrathionate oxidation, as well as the second leg of thiosulfate oxidation, by T. kashmirensis is significant because the current Sox pathway does not accommodate tetrathionate as one of its substrates. Notably, however, no other Sox protein except SoxB could be detected upon matrix-assisted laser desorption ionization mass spectrometry analysis of all such T. kashmirensis proteins as appeared to be thiosulfate inducible in 2-dimensional gel electrophoresis. Instead, several other redox proteins were found to be at least 2-fold overexpressed during thiosulfate- or tetrathionate-dependent growth, thereby indicating that there is more to tetrathionate oxidation than SoxB alone.     

Episodic positive selection during the evolution of naphthalene dioxygenase to nitroarene dioxygenase

Using different maximum-likelihood models of adaptive evolution, signatures of natural selective pressure, operating across the naphthalene family of dioxygenases, were examined. A lineage- and branch-site specific combined analysis revealed that purifying selection pressure dominated the evolutionary history of the enzyme family. Specifically, episodic positive Darwinian selection pressure, affecting only a few sites in a subset of lineages, was found to be responsible for the evolution of nitroarene dioxygenases (NArDO) from naphthalene dioxygenase (NDO). Site-specific analysis confirmed the absence of diversifying selection pressure at any particular site. Different sets of positively selected residues, obtained from branch-site specific analysis, were detected for the evolution of each NArDO. They were mainly located around the active site, the catalytic pocket and their adjacent regions, when mapped onto the 3D structure of the α-subunit of NDO. The present analysis enriches the current understanding of adaptive evolution and also broadens the scope for rational alteration of substrate specificity of enzyme by directed evolution.     

Self‐rescue of an EXTENSIN mutant reveals alternative gene expression programs and candidate proteins for new cell wall assembly in Arabidopsis

Plants encode a poorly understood superfamily of developmentally expressed cell wall hydroxyproline‐rich glycoproteins (HRGPs). One, EXTENSIN3 (EXT3) of the 168 putative HRGPs, is critical in the first steps of new wall assembly, demonstrated by broken and misplaced walls in its lethal homozygous mutant. Here we report the findings of phenotypic (not genotypic) revertants of the ext3 mutant and in‐depth analysis including microarray and qRT‐PCR (polymerase chain reaction). The aim was to identify EXT3 substitute(s), thus gaining a deeper understanding of new wall assembly. The data show differential expression in the ext3 mutant that included 61% (P ≤ 0.05) of the HRGP genes, and ability to self‐rescue by reprogramming expression. Independent revertants had reproducible expression networks, largely heritable over the four generations tested, with some genes displaying transgenerational drift towards wild‐type expression levels. Genes for nine candidate regulatory proteins as well as eight candidate HRGP building materials and/or facilitators of new wall assembly or maintenance, in the (near) absence of EXT3 expression, were identified. Seven of the HRGP fit the current model of EXT function. In conclusion, the data on phenotype comparisons and on differential expression of the genes‐of‐focus provide strong evidence that different combinations of HRGPs regulated by alternative gene expression networks, can make functioning cell walls, resulting in (apparently) normal plant growth and development. More broadly, this has implications for interpreting the cause of any mutant phenotype, assigning gene function, and genetically modifying plants for utilitarian purposes.     

Identification of Penicillin-binding proteins employing support vector machines and random forest

Penicillin-Binding Proteins are peptidases that play an important role in cell-wall biogenesis in bacteria and thus maintaining bacterial infections. A wide class of β-lactam drugs are known to act on these proteins and inhibit bacterial infections by disrupting the cell-wall biogenesis pathway. Penicillin-Binding proteins have recently gained importance with the increase in the number of multi-drug resistant bacteria. In this work, we have collected a dataset of over 700 Penicillin-Binding and non-Penicillin Binding Proteins and extracted various sequence-related features. We then created models to classify the proteins into Penicillin-Binding and non-binding using supervised machine learning algorithms such as Support Vector Machines and Random Forest. We obtain a good classification performance for both the models using both the methods.     

Within and between Population Variation in Epidermal Club Cell Investment in a Freshwater Prey Fish: A Cautionary Tale for Evolutionary Ecologists

Many prey fishes possess large club cells in their epidermis. The role of these cells has garnered considerable attention from evolutionary ecologists. These cells likely form part of the innate immune system of fishes, however, they also have an alarm function, releasing chemical cues that serve to warn nearby conspecifics of danger. Experiments aimed at understanding the selection pressures leading to the evolution of these cells have been hampered by a surprisingly large intraspecific variation in epidermal club cell (ECC) investment. The goal of our current work was to explore the magnitude and nature of this variation in ECC investment. In a field survey, we documented large differences in ECC investment both within and between several populations of minnows. We then tested whether we could experimentally reduce variation in mean ECC number by raising fish under standard laboratory conditions for 4 weeks. Fish from different populations responded very differently to being held under standard laboratory conditions; some populations showed an increase in ECC investment while others remained unchanged. More importantly, we found some evidence that we could reduce within population variation in ECC investment through time, but could not reduce among-population variation in mean ECC investment. Given the large variation we observed in wild fish and our limited ability to converge mean cell number by holding the fish under standard conditions, we caution that future studies may be hard pressed to find subtle effects of various experimental manipulations; this will make elucidating the selection pressures leading to the evolution of the cells challenging.     

A proteomics approach to identifying key protein targets involved in VEGF inhibitor mediated attenuation of bleomycin‐induced pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a life expectancy of less than 5 years post diagnosis for most patients. Poor molecular characterization of IPF has led to insufficient understanding of the pathogenesis of the disease, resulting in lack of effective therapies. In this study, we have integrated a label‐free LC‐MS based approach with systems biology to identify signaling pathways and regulatory nodes within protein interaction networks that govern phenotypic changes that may lead to IPF. Ingenuity Pathway Analysis of proteins modulated in response to bleomycin treatment identified PI3K/Akt and Wnt signaling as the most significant profibrotic pathways. Similar analysis of proteins modulated in response to vascular endothelial growth factor (VEGF) inhibitor (CBO‐P11) treatment identified natural killer cell signaling and PTEN signaling as the most significant antifibrotic pathways. Mechanistic/mammalian target of rapamycin (mTOR) and extracellular signal‐regulated kinase (ERK) were identified to be key mediators of pro‐ and antifibrotic response, where bleomycin (BLM) treatment resulted in increased expression and VEGF inhibitor treatment attenuated expression of mTOR and ERK. Using a BLM mouse model of pulmonary fibrosis and VEGF inhibitor CBO‐P11 as a therapeutic measure, we identified a comprehensive set of signaling pathways and proteins that contribute to the pathogenesis of pulmonary fibrosis that can be targeted for therapy against this fatal disease.