Deepti D. Deobagkar

Potential application of protease isolated from Pseudomonas aeruginosa PD100

A protease isolated from Pseudomonas aeruginosa PD100 could act in the presence of SDS and Tween 80. This protease could be useful for degradation of protein in the presence of solvent, dehairing of cow skin and degradation of natural proteins. The immobilized protease showed 15-20% increases in temperature stability and the entrapped enzyme retained 83% of its initial activity after six cycles. With respect to properties of the enzyme and its capability for degradation of different protein sources, this protease finds potential application for waste treatment, used in detergents and leather industry. Proteases execute a large variety of functions and have important biotechnological applications. Proteases represent one of the three largest groups of industrial enzymes and find application in detergents, leather industry, food industry, pharmaceutical industry and bioremediation processes (Anwar and Saleemuddin, 1998; Gupta et al. 2002). Probably the largest application of proteases is in laundry detergents, where they help removing protein based stains from clothing (Banerjee et al. 1999). For an enzyme to be used as an detergent additive it should be stable and active in the presence of typical

A novel halotolerant xylanase from marine isolate Bacillus subtilis cho40: gene cloning and sequencing

Although several xylanases have been studied, only few xylanases from marine micro-organisms have been reported. We report here a novel halotolerant xylanase from marine bacterium Bacillus subtilis cho40 isolated from Chorao island of mandovi estuary Goa, India. Extracellular xylanase was produced by using agricultural residue such as wheat bran as carbon source under solid-state fermentation (SSF). The optimal pH and temperature of xylanase were reported to be 6.0 and 60°C, respectively. Xyn40 was highly salt-tolerant, and showed highest activity at 0.5M NaCl. Xylanase activity was greatly induced (140%) when pre-incubated with 0.5M NaCl for 4h. The xylanase gene, xyn40, from marine bacterium B. subtilis cho40 was cloned, and expressed in Escherichia coli. The xylanase gene was 645 bp long and had a 215 amino acid ORF protein with a molecular mass of 22.9 kDa. It had all features of xylanase enzyme and showed homology to xylanases reported from B. subtilis. It differs from the earlier reported xylanase sequences by the presence of more serine residues compared to threonine and also by the presence of polar (hydrophilic) amino acids in higher abundance (61%) than non-polar amino acids (39%). The novel xylanase, reported in this study is a halotolerant enzyme from marine isolate and can play a very important role in bioethanol production from marine seaweeds.

A novel method to assess the full genome methylation profile using monoclonal antibody combined with the high throughput based microarray approach.

In this work we demonstrate a novel method of methylation detection that utilises immunoaffinity to detect the presence of methylated DNA hybridised to a cDNA microarray. We use a monoclonal antibody specific to 5 methyl cytidine to detect the presence of 5 methyl cytosine in genomic DNA from human fibroblasts bearing the karyotype 45 XO. We report that over 2900 genes show the presence of methylation in this condition. We also report that 165 genes are consistently methylated in all replicates of these experiments. The methylated genes show a uniform distribution over all the chromosomes. The gene ontology of these also indicates no functional correlation between the genes that are methylated. We detect the presence of methylation in IGF2, an imprinted gene and thus known to harbour DNA methylation. The method is extremely specific and offers a quick and efficient way to analyse the methylation landscape on a high throughput scale. This method uses existing technology to assess methylation and thus can integrate very efficiently into any platform used.

Synthesis and extracellular accumulation of silver nanoparticles by employing radiation-resistant Deinococcus radiodurans , their characterization, and determination of bioactivity

There has been rapid progress in exploring microorganisms for green synthesis of nanoparticles since microbes show extraordinary diversity in terms of species richness and niche localization. Microorganisms are easy to culture using relatively inexpensive and simple nutrients under varied conditions of temperature, pressure, pH, etc. In this work, Deinococcus radiodurans that possesses the ability to withstand extremely high radiation and desiccation stress has been employed for the synthesis of silver nanoparticles (AgNPs). D. radiodurans was able to accumulate AgNPs in medium under various conditions, and process optimization was carried out with respect to time, temperature, pH, and concentration of silver salt. AgNPs were characterized using UV/vis spectroscopy, scanning electron microscopy, transmission electron microscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, and Fourier transform infrared spectroscopy. The microbially synthesized AgNPs exhibited good antimicrobial activity against both Gram-negative and Gram-positive organisms and anti-biofouling activity. Their ability to inhibit growth and proliferation of cancer cell line was also examined, and it could be seen that AgNPs synthesized using D. radiodurans exhibited excellent anticancer activity.

Human 45,X fibroblast transcriptome reveals distinct differentially expressed genes including long noncoding RNAs potentially associated with the pathophysiology of Turner syndrome.

Turner syndrome is a chromosomal abnormality characterized by the absence of whole or part of the X chromosome in females. This X aneuploidy condition is associated with a diverse set of clinical phenotypes such as gonadal dysfunction, short stature, osteoporosis and Type II diabetes mellitus, among others. These phenotypes differ in their severity and penetrance among the affected individuals. Haploinsufficiency for a few X linked genes has been associated with some of these disease phenotypes. RNA sequencing can provide valuable insights to understand molecular mechanism of disease process. In the current study, we have analysed the transcriptome profiles of human untransformed 45,X and 46,XX fibroblast cells and identified differential expression of genes in these two karyotypes. Functional analysis revealed that these differentially expressing genes are associated with bone differentiation, glucose metabolism and gonadal development pathways. We also report differential expression of lincRNAs in X monosomic cells. Our observations provide a basis for evaluation of cellular and molecular mechanism(s) in the establishment of Turner syndrome phenotypes.

Methylation profile of genes on the human X chromosome

X chromosome inactivation occurs in female mammals for the purpose of equalisation of dosage of X linked genes between the two sexes. In eutherian mammals, one of the two copies of the X chromosome present in female individuals is silenced. Epigenetic modifications of both DNA and histones have been implicated to play a crucial role in this inactivation phenomenon. In this work, we have employed a novel method published earlier by us, to assess the DNA methylation levels of genes on the inactive X chromosome in the human system. We have used genomic DNA from cells with the following karyotype namely, 47,XXX and 45,X to compare methylation levels from the active and inactive X. We report differential methylation of genes from the active and the inactive X chromosome with higher number of methylated genes being present on the inactive X chromosome. Our work has also led to identification of motifs that show a significant similarity to microRNA sequences which are enriched in methylated regions specific to the inactive X.

An immunochemical method for detection and analysis of changes in methylome.

DNA methylation is an important epigenetic modification involved in the ability of an organism to respond to stress and adaptation. It has been implicated in development, differentiation, oncogenesis, chromatin remodelling, nutrigenomics, and appears to play a pivotal role in many regulatory and adaptive functions. It is therefore important to analyze the status of DNA methylation and its changes under various developmental, carcinogenic, pharmacological, and environmental conditions. In this report we describe an immunochemical method for the detection of genome wide DNA methylation and its alterations under various conditions along with the analysis of DNA methyltransferase activity. The ability of this approach to detect and provide a map of methylomic changes in a genome facilitates assessment of various agents and conditions which can alter this important epigenetic signal. This experimental system permits rapid evaluation of potential target genes which would be modulated by DNA methylation changes and thus the gene networks that govern the processes.

Epigenetic modulation upon exposure of lung fibroblasts to TiO 2 and ZnO nanoparticles: alterations in DNA methylation

Titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NPs) are promising candidates for numerous applications in consumer products. This will lead to increased human exposure, thus posing a threat to human health. Both these types of NPs have been studied for their cell toxicity, immunotoxicity, and genotoxicity. However, effects of these NPs on epigenetic modulations have not been studied. Epigenetics is an important link in the genotype and phenotype modulation and misregulation can often lead to lifestyle diseases. In this study, we have evaluated the DNA methylation-based epigenetic changes upon exposure to various concentrations of NPs. The investigation was designed to evaluate global DNA methylation, estimating the corresponding methyltransferase activity and expression of Dnmt gene using lung fibroblast (MRC5) cell line as lungs are the primary route of entry and target of occupational exposure to TiO2 and ZnO NPs. Enzyme-linked immunosorbent assay-based immunochemical assay revealed dose-related decrease in global DNA methylation and DNA methyltransferase activity. We also found direct correlation between the concentration of NPs, global methylation levels, and expression levels of Dnmt1, 3A, and 3B genes upon exposure. This is the first study to investigate effect of exposure to TiO2 and ZnO on DNA methylation levels in MRC5 cells. Epigenetic processes are known to play an important role in reprogramming and adaptation ability of an organism and can have long-term consequences. We suggest that changes in DNA methylation can serve as good biomarkers for early exposure to NPs since they occur at concentrations well below the sublethal levels. Our results demonstrate a clear epigenetic alteration in response to metal oxide NPs and that this effect was dose-dependent.

Evidence for Epigenetic Alterations in Turner Syndrome Opens up Feasibility of New Pharmaceutical Interventions

DNA methylation is an important regulatory component which influences phenotypes by modulating gene expression. Changes in DNA methylation may lead to altered phenotypes and ability of an organism to respond to stress leading to subsequent manifestation of life style diseases, cancer, etc. The human X chromosome represents a classical model for epigenetic processes governing differential regulation of homologous chromosomes. X monosomy (45, XO) leads to Turners syndrome in human with mild to severe phenotypes. Using a novel cDNA based high throughput approach of assessing genome wide methylation; we have examined the methylation landscape in human fibroblasts in 45, XO and 46, XX individuals. We report here that as expected methylation of X linked genes is different in these two situations. It was observed that methylation of several autosomal genes is also affected in this X monosomy state. Genes involved in bone remodeling, glucose sensitivity and ovarian function appear to be altered in addition to genes involved in epigenetic regulatory processes. This opens up interesting possibility of misregulation of DNA methylation in the X monosomy state resulting in altered gene expression and altered phenotypes. This may be one of the reasons for the variance, differential severity and penetrance in case of Turners syndrome. We propose that a systematic analysis of the molecular genetic mechanisms governing this epigenetic regulation will open up new therapeutic interventions which will certainly help in reducing severity of the disease and help in better management of X monosomy (Turners syndrome).

Ensemble-Based Virtual Screening and Experimental Validation of Inhibitors Targeting a Novel Site of Human DNMT1

Human DNA methyltransferase1 (hDNMT1) is responsible for preserving DNA methylation patterns that play important regulatory roles in differentiation and development. Misregulation of DNA methylation has thus been linked to many syndromes, life style diseases, and cancers. Developing specific inhibitors of hDNMT1 is an important challenge in the area since the currently targeted cofactor and substrate binding site share structural features with various proteins. In this work, we generated a structural model of the active form of hDNMT1 and identified that the 5‐methylcytosine (5‐mC) binding site of the hDNMT1 is structurally unique to the protein. This site has been previously demonstrated to be critical for methylation activity. We further performed multiple nanosecond time scale atomistic molecular dynamics simulations of the structural model followed by virtual screening of the Asinex database to identify inhibitors targeting the 5‐mC site. Two compounds were discovered that inhibited hDNMT1 in vitro, one of which also showed inhibition in vivo corroborating the screening procedure. This study thus identifies and attempts to validate for the first time a unique site of hDNMT1 that could be harnessed for rationally designing highly selective and potent hypomethylating agents.