Showing papers by "I. S. Bright Singh published in 2016"

Pyocyanin induced in vitro oxidative damage and its toxicity level in human, fish and insect cell lines for its selective biological applications.

Abstract: Pyocyanin is a redox active phenazine pigment produced by Pseudomonas aeruginosa, with broad antibiotic activity having pharmacological, aquaculture, agriculture and industrial applications. In the present work cytotoxicity induced by pyocyanin is demonstrated in a human embryonic lung epithelial cell line (L-132), a rainbow trout gonad cell line (RTG-2) and a Spodoptera frugiperda pupal ovarian cell line (Sf9). For toxicity evaluation, cellular morphology, mitochondrial function (XTT), membrane leakage of lactate dehydrogenase, neutral red uptake, affinity of electrostatic binding of protein with sulforhodamine B dyes, glucose metabolism, and reactive oxygen species, were assessed. Results showed that higher pyocyanin concentration is required for eliciting cytotoxicity in L-132, RTG-2 and Sf9. The microscopic studies demonstrated that the cell lines exposed to pyocyanin at higher concentrations alone showed morphological changes such as clumping and necrosis. Among the three cell lines L-132 showed the highest response to pyocyanin than the others. In short, pyocyanin application at concentrations ranging from 5 to 10 mg l−1 were not having any pathological effect in eukaryotic systems and can be used as drug of choice in aquaculture against vibrios in lieu of conventional antibiotics and as biocontrol agent against fungal and bacterial pathogens in agriculture. This is besides its industrial and pharmacological applications.

Fluorescence detection of the pathogenic bacteria Vibrio harveyi in solution and animal cells using semiconductor quantum dots

Abstract: Validation of microbial infection pathways in eukaryotic cells is challenging in the control of various infectious diseases. Semiconductor nanocrystals, also called quantum dots (QD), due to their exceptional brightness and photostability can be exploited in the long term monitoring of pathogens in host cells. However, the limited information about interactions of QDs and their bioconjugates with microorganisms confines the microbiological applications of QDs. Here we investigate the binding and toxicity of CdSe/ZnS QDs to the free-swimming marine pathogenic bacteria Vibrio harveyi using fluorescence microscopy, elastase assay, polyacrylamide gel electrophoresis (PAGE), and comet assay. The electrostatic binding of QDs to the cell surface has been found effective for the detection of the bacteria in aqueous solutions and bacteria-infected mammalian cells. The electrostatic binding is evaluated by the transient reversal of the cell surface charge contributed by macromolecules such as heparan sulfate proteoglycan (HSPG). Essentially, no fluorescence is detected for those bacteria treated with NiCl2 that reverses the cell surface charge. On the other hand, the efficiency of the cell surface to adsorb QDs remains intact even after treatment with elastase, which denatures the outer membrane proteins (Omps), suggesting HSPG-based binding of QD to cell surface and subsequently QDs are internalized. PAGE and comet assays show that the interactions of QDs with V. harveyi do not impart any cytotoxicity or genotoxicity. Further, we evaluate the integrity of adsorbed QDs for the detection of bacterial infection to mammalian cells by taking mouse fibroblast L929 as the model. Here, the stable fluorescence of QDs present in V. harveyi enables us for identifying the infected host cells. In short, the current study shows the potentials of for the detection of pathogens but without causing any toxic effects, which can be a promising method for not only the detection of the progression or regression of pathogenic infections but also phototherapy of microbial infections.

High-quality metagenomic DNA from marine sediment samples for genomic studies through a preprocessing approach

Abstract: Recent advances in culture-independent studies of microbes had proved to be more reliable and efficient than the conventional ones. The isolation of good quality and quantity of total community DNA are one of the major hurdles in this endeavour. Shearing of DNA during the extraction process and the co-extraction of inhibitory compounds reduce the quality of the isolated nucleic acids making it unsuitable for the construction of large insert metagenomic libraries. In the present study, a multi-level filtration step was brought in which efficiently isolated total bacterial DNA from three different environment samples. The preprocessing method could efficiently improve the 260/230 ratio of the isolated DNA by 2.3–45 % and decreased the protein contamination by 22.5–34.5 % on saltpan and arctic sediment samples, respectively. The more significant part of the experiment was that the DNA obtained was of high quality with minimal shearing making it most suitable for the construction of large insert genomic libraries. PCR amplification of 16S rRNA gene confirmed that the filtration method was effective in the isolation of high-quality DNA.

Transgene expression in Penaeus monodon cells: evaluation of recombinant baculoviral vectors with shrimp specific hybrid promoters

Abstract: It has been realized that shrimp cell immortalization may not be accomplished without in vitro transformation by expressing immortalizing gene in cells. In this process, efficiency of transgene expression is confined to the ability of vectors to transmit gene of interests to the genome. Over the years, unavailability of such vectors has been hampering application of such a strategy in shrimp cells. We report the use of recombinant baculovirus mediated transduction using hybrid promoter system for transgene expression in lymphoid cells of Penaeus monodon. Two recombinant baculovirus vectors with shrimp viral promoters (WSSV-Ie1 and IHHNV-P2) were constructed (BacIe1-GFP and BacP2-GFP) and green fluorescent protein (GFP) used as the transgene. The GFP expression in cells under the control of hybrid promoters, PH-Ie1 or PH-P2, were analyzed and confirmed in shrimp cells. The results indicate that the recombinant baculovirus with shrimp specific viral promoters (hybrid) can be employed for delivery of foreign genes to shrimp cells for in vitro transformation.

Molecular Characterization of a Newly Identified Subfamily Member of Penaeidin from two Penaeid Shrimps, Fenneropenaeus indicus and Metapenaeus monoceros.

Abstract: Penaeidins are a major group of antimicrobial peptides found in penaeid shrimps. This study reports a new isoform of penaeidin from the hemocytes of Indian white shrimp, Fenneropenaeus indicus (Fi-PEN, JX657680), and the pink shrimp, Metapenaeus monoceros (Mm-PEN, KF275674). Mm-PEN is also the first antimicrobial peptide to be identified from M. monoceros. The complete coding sequences of the newly identified Fi-PEN and Mm-PEN consisted of an ORF of 338 bp encoding 71 amino acids with a predicted molecular weight of 5.66 kDa and a pI of 9.38. The penaeidins had its characteristic signal peptide region (19 amino acids), which was followed by a mature peptide with a proline-rich domain (24 amino acids) at the N-terminal region and a cysteine-rich domain (28 amino acids) at the C-terminal region, designating it to penaeidin-3 subgroup. Structural analysis revealed an alpha-helix in its secondary structure and an extended structure at the proline-rich domain. The newly identified penaeidin isoform showed maximum similarity of 63 % to a penaeidin-3 isoform of P. monodon, which further proves it to be a new isoform. Phylogenetic analysis showed that it possessed similar evolutionary status like other penaeidins, which has subsequently diverged at different phases of evolution. The wide distribution of penaeidins in penaeid shrimps indicates the importance of these AMPs in the innate immunity.

Impaired telomerase activity hinders proliferation and in vitro transformation of Penaeus monodon lymphoid cells

Abstract: Retaining terminal transferase activity of telomerase, the ribonucleoprotein enzyme which add telomeric repeats on chromosome end is thought to be required to prevent cellular ageing. Additionally, telomerase considered as a marker for cell proliferation and immortalization in eukaryotes. We examined telomerase activity in tissues and lymphoid cell culture of Penaeus monodon. Along with telomerase activity, telomere repeats and an attempt on identification of telomerase reverse transcriptase (PmTERT) were made. Telomeric repeat amplification protocol revealed that telomerase-dependent telomeric lengthening has been taking place in P. monodon and the adult tissues were retaining this capacity throughout their lifespan with the highest activity in ovary, testis and lymphoid organ. However, telomerase activity could not be detected in lymphoid cells in culture. The canonical telomeric repeats added by telomerase of lymphoid tissue extract were identified as TTAGG, but pentameric repeats GGTTA and AGGTT were also added by the telomerase. PmTERT protein sequence (partial) shared 100 % identity with the TERT sequence of Daphnia pulex, 27 % sequence identity with Purple sea urchin and 24–25 % with Zebra fish. Undetectable telomerase activity in lymphoid cell culture supports the hypothesis that the inadequate telomerase activity or gene expression may be a reason that prevents neoplastic transformation and spontaneous immortalization of the cells in vitro. Thus, it is envisaged that telomerase activation in lymphoid cells may surmount cellular ageing for in vitro transformation and cell line establishment.