Search

Noncoding RNAs are increasingly being accredited with key roles in gene regulation during development and disease. Here we report the discovery and characterization of a novel long noncoding RNA, Hmrhl, which shares synteny and partial sequence similarity with the mouse lncRNA, Mrhl. The human homolog, Hmrhl, transcribed from intron 14 of phkb gene, is 5.5 kb in size, expressed in all tissues examined and is associated with chromatin. Analysis of Hmrhl locus using ENCODE database revealed that it exhibits hallmarks of enhancers like the open chromatin configuration, binding of transcription factors, enhancer specific histone signature etc. in the K562 Chronic Myelogenous Leukemia (CML) cells. We compared the expression of Hmrhl in the normal lymphoblast cell line, GM12878, with that of K562 cells and lymphoma samples and show that it is highly upregulated in leukemia as well as several cases of lymphoma. Further, we validated the enhancer properties of Hmrhl locus in K562 cells with the help of ChIP-qPCR and Luciferase assay. Moreover, siRNA mediated down-regulation of Hmrhl in K562 cells leads to a concomitant down regulation of its parent gene, phkb, showing that Hmrhl functions as an enhancer RNA and positively regulates its host gene, phkb, in chronic myelogenous leukemia. This study is significant in view of the fact that a better understanding of mechanism of gene regulation under normal conditions and its perturbation in cancer could in turn help in its therapeutic intervention through molecular medicine/RNA based drug discovery.

Noncoding RNAs are increasingly being accredited with key roles in gene regulation during development and disease. Here we report the discovery and characterization of a novel long noncoding RNA, Hmrhl, which shares synteny and partial sequence similarity with the mouse lncRNA, Mrhl. The human homolog, Hmrhl, transcribed from intron 14 ofphkbgene, is 5.5kb in size, expressed in all tissues examined and has acquired additional repeat elements. Analysis of Hmrhl locus using ENCODE database revealed that it is associated with hallmarks of enhancers like the open chromatin configuration, binding of transcription factors, enhancer specific histone signature etc. in the K562 Chronic Myelogenous Leukemia (CML) cells. We compared the expression of Hmrhl in the normal lymphoblast cell line, GM12878, with that of K562 cells and lymphoma samples and show that it is highly upregulated in leukemia as well as several cases of lymphoma. We validated the enhancer properties of Hmrhl locus in K562 cells with the help of Luciferase assay. Moreover, siRNA mediated down-regulation of Hmrhl in K562 cells leads to a concomitant down regulation of its parent gene,phkb, showing that Hmrhl functions as an enhancer RNA and positively regulates its host gene,phkb,in chronic myelogenous leukemia.

Actinomycetes (277 Nos) isolated from marine environment and shrimp culture pond sediments were tested for hydrolytic enzyme production and biogranulation property. Potential isolates were screened for their efficacy in bioremediation of shrimp culture system. Based on the BOD reduction efficiency and water quality parameters, five actinomycete isolates viz., Streptomyces coelicoflavus (A6), Streptomyces diastaticus (A44), Nocardiopsis alba (A55), Streptomyces parvus (A56) and Streptomyces champavatii (R32) were subjected for tertiary screening in Penaeus monodon larval rearing system and the animals were challenged with white spot syndrome virus (WSSV). The bioremediating effect of actinomycete treatments were assessed by analysing the expression profile of five antimicrobial peptide (AMP) genes viz., anti-lipopolysaccharide factor (ALF), crustin-2, crustin-3, penaeidin-3 and penaeidin-5 and eight immune genes viz., alpha-2-macroglobulin (α-2-M), astakine, glutathione-S-transferase, haemocyanin, peroxinectin, pmCathepsinC, prophenol oxidase (proPO) and Rab-7. Expression of eight WSSV genes viz., DNA polymerase, endonuclease, protein kinase, immediate early gene, latency related gene, ribonucleotide reductase, thymidine kinase and VP28 were also analyzed to detect the presence and intensity of viral infection in the experimental animals post-challenge. Theapplication of consortia (1 g/5 L water) yields better results in terms of significant reduction in BOD of shrimp rearing system showing the bioremediation potential of the marine actinomycete strains. The application of marine actinomycetes viz., Streptomyces coelicoflavus (A6), Streptomyces diastaticus (A44), Nocardiopsis alba (A55), Streptomyces parvus (A56) and Streptomyces champavatii (R32) in granulated form were found to be potential bioremediators in shrimp rearing system.

Starch hydrolyzing isolate GUVSC8 is obtained from commercial salt crystals of Vedaranyam, Tamil Nadu, India. GUVSC8 is categorized as an extreme halophile based on its growth on complex medium containing 25% (w/v) NaCl. Morphological, chemotaxonomic, and 16S rRNA gene sequencing reveal the cells to be coccus, producing bright orange C‐50 bacterioruberin pigmentation with 95.95% similar to Halococcus hamelinensis DQ017835. The strain GUVSC8 grown in complex medium devoid of starch does not exhibit amylolytic activity, thereby confirming the induction of amylase production in presence of starch. On incubation of the starch with crude enzyme, numerous pores are observed on the starch granules after 24 h of incubation, indicating vigorous degradation and thereby confirming the amylolytic activity. Thin‐layer chromatography (TLC) and liquid chromatography‐electrospray ionization‐mass spectrometry (LC‐ESI‐MS) analysis reveal that the major end product obtained after amylolytic activity are glucose, maltose, maltotriose, maltotetrose, maltopentose, and other maltooligosaccharides, thus confirming it to be an alpha‐amylase. Effects of NaCl, pH, and temperature, on activity of partially purified amylase, reveal best amylase activity at 2M NaCl, pH6, and 45 °C. The amylase is stable and active in presence of divalent cations such as Mg²⁺, Ni²⁺, Zn²⁺, Ca²⁺, and Co²⁺. Enzyme activity significantly reduces with Cu²⁺ and increases in presence of Mn²⁺. The amylase is characterized as α‐amylase enzyme as it lost 87% of its activity in presence of EDTA. To the best of the author's knowledge, this is the first report on alpha‐amylase production by salt crystal isolate belonging to the genus Halococcus.

A marine yeast Candida aquaetextoris S527 as a source of immunostimulant in Penaeus monodon was studied. Yeast diet was prepared by incorporating 10% C. aquaetextoris S527 biomass into a standard shrimp diet and administered in P. monodon at different frequencies (daily, once in three days, once in seven days and once in ten days) followed by challenge with white spot syndrome virus (WSSV). Immune parameters such as total protein, total hemocyte count, pro-phenoloxidase, nitroblue tetrazolium reduction, alkaline phosphatase activity and acid phosphatase activity were tested. Expression profile of antimicrobial peptide (AMP) genes viz., anti-lipopolysaccharide factor (ALF), crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5; immune genes viz., alpha-2-macroglobulin (α-2-M), astakine, peroxinectin, prophenol oxidase (proPO) and transglutaminase, and WSSV genes viz., DNA polymerase, endonuclease, protein kinase, immediate early gene, latency related gene, ribonucleotide reductase, thymidine kinase and VP28 were analyzed. The study demonstrated that marine yeast diet administered once every seven days conferred better protection to P. monodon against WSSV infection, supported by the hematological and immune gene expression profiles analyzed.

The serotype-specific glucosyltransferase, GtrV, is responsible for glucosylation of the O-antigen repeating unit of Shigella flexneri serotype 5a strains. GtrV is an integral inner membrane protein with two essential periplasmic loops: the large Loop 2 and the C-terminal Loop 10. In this study, the full length of the Loop 2 was shown to be necessary for GtrV function. Site-directed mutagenesis within this loop revealed that conserved aromatic and charged amino acids have a critical role in the formation of the active site. Sequential deletions of the C-terminal end indicated that this region may be essential for assembly of the protein in the cytoplasmic membrane. The highly conserved FWAED motif is thought to form the substrate-binding site and was found to be critical in GtrV and GtrX, a serotype-specific glucosyltransferase with homology to GtrV. The data presented constitutes a targeted analysis of the formation of the GtrV active site and highlights the essential role of the large periplasmic Loop 2 in its function.

Almost all eukaryotic mRNAs have a poly (A) tail at the 3′-end. Canonical PAPs (PAPα/γ) polyadenylate nuclear pre-mRNAs. The recent identification of the non-canonical Star-PAP revealed specificity of nuclear PAPs for pre-mRNAs, yet the mechanism how Star-PAP selects mRNA targets is still elusive. Moreover, how Star-PAP target mRNAs having canonical AAUAAA signal are not regulated by PAPα is unclear. We investigate specificity mechanisms of Star-PAP that selects pre-mRNA targets for polyadenylation. Star-PAP assembles distinct 3′-end processing complex and controls pre-mRNAs independent of PAPα. We identified a Star-PAP recognition nucleotide motif and showed that suboptimal DSE on Star-PAP target pre-mRNA 3′-UTRs inhibit CstF-64 binding, thus preventing PAPα recruitment onto it. Altering 3′-UTR cis-elements on a Star-PAP target pre-mRNA can switch the regulatory PAP from Star-PAP to PAPα. Our results suggest a mechanism of poly (A) site selection that has potential implication on the regulation of alternative polyadenylation.

The Gram-negative bacterium Shigella flexneri is the causative agent of shigellosis, a diarrhoeal disease also known as bacillary dysentery. S. flexneri infects the colonic and rectal epithelia of its primate host and induces a cascade of inflammatory responses that culminates in the destruction of the host intestinal lining. Molecular characterization of host-pathogen interactions in this infection has been challenging due to the host specificity of S. flexneri strains, as it strictly infects humans and non-human primates. Recent studies have shown that S. flexneri infects the soil dwelling nematode Caenorhabditis elegans, however, the interactions between S. flexneri and C. elegans at the cellular level and the cause of nematode death are unknown. Here we attempt to gain insight into the complex host-pathogen interactions between S. flexneri and C. elegans. Using transmission electron microscopy, we show that live S. flexneri cells accumulate in the nematode intestinal lumen, produce outer membrane vesicles and invade nematode intestinal cells. Using two-dimensional differential in-gel electrophoresis we identified host proteins that are differentially expressed in response to S. flexneri infection. Four of the identified genes, aco-1, cct-2, daf-19 and hsp-60, were knocked down using RNAi and ACO-1, CCT-2 and DAF-19, which were identified as up-regulated in response to S. flexneri infection, were found to be involved in the infection process. aco-1 RNAi worms were more resistant to S. flexneri infection, suggesting S. flexneri-mediated disruption of host iron homeostasis. cct-2 and daf-19 RNAi worms were more susceptible to infection, suggesting that these genes are induced as a protective mechanism by C. elegans. These observations further our understanding of the processes involved in S. flexneri infection of C. elegans, which is immensely beneficial to the routine use of this new in vivo model to study S. flexneri pathogenesis.

S. flexneri strains, most frequently linked with endemic outbreaks of shigellosis, invade the colonic and rectal epithelium of their host and cause severe tissue damage. Here we have attempted to elucidate the contribution of the periplasmic enzyme, L-asparaginase (AnsB) to the pathogenesis of S. flexneri. Using a reverse genetic approach we found that ansB mutants showed reduced adherence to epithelial cells in vitro and attenuation in two in vivo models of shigellosis, the Caenorhabditis elegans and the murine pulmonary model. To investigate how AnsB affects bacterial adherence, we compared the proteomes of the ansB mutant with its wild type parental strain using two dimensional differential in-gel electrophoresis and identified the outer membrane protein, OmpA as up-regulated in ansB mutant cells. Bacterial OmpA, is a prominent outer membrane protein whose activity has been found to be required for bacterial pathogenesis. Overexpression of OmpA in wild type S. flexneri serotype 3b resulted in decreasing the adherence of this virulent strain, suggesting that the up-regulation of OmpA in ansB mutants contributes to the reduced adherence of this mutant strain. The data presented here is the first report that links the metabolic enzyme AnsB to S. flexneri pathogenesis.

SfII is a serotype-converting temperate bacteriophage of the highly prevalent Shigella flexneri serotype 2a. We isolated the SfII phage from a wild-type strain of S. flexneri serotype 2a. Here, we present the complete genome sequence of this phage.