A Comprehensive Investigation of Potential Novel Marine Psychrotolerant Actinomycetes sp. Isolated from the Bay-of-Bengal.

BACKGROUND: This study was carried out to classify the diversity of the deep marine psychrotolerant actinomycetes sp. nov., in the Bay of Bengal and exploit the production of cold-active industrial and pharmaceutical biomolecules. OBJECTIVE: 1) Characterization, optimum the growth conditions and classify the diversity of the novel isolated deep marine psychrotolerant actinomycetes sp from the Bay-of-Bengal. 2) Screening for industrially important biocatalysts and determine the antimicrobial activities against the five dreadful pathogens. 3) The differential expression profiling of the candidate genes to regulate the biosynthesis of selected enzymes. METHODS: The cold-adapted actinomycetes were isolated from the deep marine water collections at 1200 mts below the surface in Bay-of-Bengal. The phenotypic and genotypic characterizations have been carried out to understand the persistent diversity of this novel marine psychrotolerant actinomycetes species. The production of cold-active enzymes, such as amylase, cellulase, lipase, pectinase, and L-asparaginase, were screened and the expression profiling genes were determined by using qRT PCR. The antibacterial and antifungal activities have also been investigated. RESULTS: A total number of 37 novel actinomycetes were isolated and the phenotypic and genotypic characterizations identified the genus, dominated by Streptomyces (17 distinct sub-groups) as the major group, followed by Micromonospora, Actinopolyspora, Actinosynnema, Streptoverticillium, Saccharopolyspora, Nocardiopsis, and Nocardia. The optimum growth and abundant mycelium formation are observed at 15°C to 20°C and also capability for thriving at 4°C. All the isolates exhibited a significant role in the production of biocatalysts, and the antagonistic activities were also noted against five major selected pathogens. CONCLUSION: The Streptomyces from the Bay-of-Bengal have high biosynthetic potential and can serve as a good resource for the exploration of bioactive natural products.

Lipid modification of staphylokinase and its implications on stability and activity.

Thrombolytic agents are routinely used to dissolve blood clot by activating fibrinolytic system. Among different thrombolytic agents available, staphylokinase (SAK) is gaining much attention because of their fibrin specificity and reduced inhibition by α2 antiplasmin. Though SAK had exhibited less circulatory half life, they are equipotent to tissue plasminogen activator and streptokinase and had shown more potency for clot dissolution during retracted thrombi. In this study, SAK was lipid modified at the N-terminal by a protein engineering approach to enhance its stability and activity. Native SAK as well as the gene encoding SAK with lipobox was cloned into E. coli GJ1158 using pRSET-B expression vector for higher expression. The lipid modification of SAK was confirmed by a mobility shift of 1.3 kDa against the 15.5 kDa of native SAK using tricine SDS-PAGE. Lipid modification of SAK was confirmed by LC MS/MS. The secondary structure analysis was carried out using circular dichroism and deconvoluted fourier transform infrared spectroscopy. LMSAK was found to have a slightly higher denaturation temperature compared to SAK. The improved stablility and activity of lipid modified SAK was studied by heated plasma agar plate assay and mouse tail bleeding test.

Transformation of animal genomics by next-generation sequencing technologies: a decade of challenges and their impact on genetic architecture.

For more than a quarter of a century, sequencing technologies from Sanger's method to next-generation high-throughput techniques have provided fascinating opportunities in the life sciences. The continuing upward trajectory of sequencing technologies will improve livestock research and expedite the development of various new genomic and technological studies with farm animals. The use of high-throughput technologies in livestock research has increased interest in metagenomics, epigenetics, genome-wide association studies, and identification of single nucleotide polymorphisms and copy number variations. Such studies are beginning to provide revolutionary insights into biological and evolutionary processes. Farm animals, such as cattle, swine, and horses, have played a dual role as economically and agriculturally important animals as well as biomedical research models. The first part of this study explores the current state of sequencing methods, many of which are already used in animal genomic studies, and the second part summarizes the state of cattle, swine, horse, and chicken genome sequencing and illustrates its achievements during the last few years. Finally, we describe several high-throughput sequencing approaches for the improved detection of known, unknown, and emerging infectious agents, leading to better diagnosis of infectious diseases. The insights from viral metagenomics and the advancement of next-generation sequencing will strongly support specific and efficient vaccine development and provide strategies for controlling infectious disease transmission among animal populations and/or between animals and humans. However, prospective sequencing technologies will require further research and in-field testing before reaching the marketplace.

An Integrated In Silico Approach for the Structural and Functional Exploration of Lipocalin 2 and its Functional Insights with Metalloproteinase 9 and Lipoprotein Receptor-Related Protein 2.

Recent evidence demonstrated that Lipocalin 2 (LCN2) is garnering interest from a wide spectrum as biomarker. Here, we present an in silico characterization of LCN2 belonging to prominent organisms focusing for their physicochemical and structural differences. We found significant variations in physicochemical properties between organisms and low sequence similarity based on their amino acid properties alone. However, we identified three main structurally distinct motif regions that are conserved among all variants. Further investigation was carried out to understand the functional insights of LCN2. We selected LCN2 sequence from Gallus gallus as an input query to identify unique scoring-framework based on computational tools and confidence scores of various putative associations. Among all ten proteins associated with LCN2; highest confidence of prediction were seen for the associations between LCN2 and metalloproteinase 9 (MMP9) and lipoprotein receptor-related protein 2 (LRP2) which play vital roles in tumor growth and iron transportation, respectively. We attempted to determine binding affinities of LCN2 with MMP9 and LRP2 through molecular modeling and docking. Selected docked models were examined for complex stability by GROMACS. Alteration of binding affinity between LCN2 with MMP9 and LRP2 proteins that we found in this study may provide new direction for future therapeutic targets.

Studies on Deimmunization of Antileukaemic L-Asparaginase to have Reduced Clinical Immunogenicity--An in silico Approach.

Protein therapeutics, particularly of heterologous origin are shown to elicit immunogenic responses which result in adverse allergic reactions in spite of their promising clinical benefit. L-Asparaginase is one such well known chemotherapeutic agent that has enhanced the survival rates to 90 % in the treatment of acute lymphoblastic leukaemia for past 30 years. But the use of this enzyme is accompanied by hypersensitive reactions ranging from allergy to anaphylactic shock which have a drastic influence in treatment outcomes. Numerous attempts have been made to minimize the problems of immunogenicity, which remained as a major bottleneck in the treatment protocols. Conjugating the enzyme L- Asparaginase with PEG was successful as it has reduced the complications in therapy and frequency of injections (dosages), and thus became prominent in reducing the immunogenicity up to a certain extent. Keeping the bottlenecks in consideration during the development of therapeutics, the present study concentrates on engineering of protein as an alternative to the PEGylated enzyme, having reduced immunogenicity as an inbuilt character of protein by using in silico approaches. L-Asparaginase from Escherichia coli and Pectobacterium carotovorum were selected for the present study. The methodology consists of (i) locating the B and CD4+ T cell epitopes of enzyme by in silico tools (ii) generating point mutations of these epitopes to alter or reduce the immunogenicity of protein (iii) generating enzyme models by molecular modelling (iv) assessing the binding affinity of the substrate with L-Asparaginase variants by in silico docking methods using Autodock 4.2 and (v) validating the mutated model for stability by molecular dynamics simulation studies using Gromacs.

Studies on lipidification of streptokinase: a novel strategy to enhance the stability and activity.

Thrombotic disorders and their associated problems are extensively prevalent in developed and developing countries. Streptokinase (SK) is a well-known thrombolytic agent, which is very useful in treating coronary thrombosis and acute myocardial infarction. Several attempts have been made to date to make improvements of this wonderful molecule in terms of reducing or eliminating the problems of eliciting immunogenicity and enhancing the half-life of the molecule. The present research is focused to produce a recombinant SK with enhanced stability and biological activity by the methodology of lipid modification. SK was targeted successfully to the membrane with the help of modified apyrase signal sequence. Higher expression was reported for GJ1158 strain in LBON medium when compared with BL21 (DE3). The obtained recombinant SK was tested for its biological activity by the method of caseinolytic assay. The higher clearance zone was observed in recombinant lipid-modified streptokinase, which denotes the enhanced activity of the protein. The present trial of lipid modification of therapeutics, particularly SK, could help for its superior use as a thrombolytic agent and also paves way for many of the other clinical applications.

Cloning and expression of recombinant human GMCSF from Pichia pastoris GS115--a progressive strategy for economic production.

Human granulocyte-macrophage colony-stimulating factor (hGMCSF) is a proinflammatory cytokine and hematopoietic growth factor. Recombinant human granulocyte-macrophage colony-stimulating factor (rhGMCSF) serves as a biotherapeutic agent in bone marrow stimulations, vaccine development, gene therapy approaches, and stem cell mobilization. The objective of the present study includes construction of rhGMCSF having N-terminal intein tag, expression of protein both extracellularly and intracellularly from yeast expression system followed by its purification in a single step by affinity chromatography. The soluble and biologically active rhGMCSF was obtained from Pichia pastoris GS115. About 122 g DCW/L of final yield was obtained for both cytosolic and secretory expression of Pichia GS115 strain. Purified intracellular hGMCSF was 420 mg/L with a specific activity of 2.1×108 IU/mg, and the purified extracellular recombinant protein was 360 mg/L with a specific activity of 1.9×108 IU/mg. The data presented here indicate the possibilities of exploring the economic ways of producing the rhGMCSF.

Evaluation of a multifunctional staphylokinase variant with thrombin inhibition and antiplatelet aggregation activities produced from salt-inducible E. coli GJ1158.

Reocclusion is one of the major root causes for secondary complications that arise during thrombolytic therapy. A multifunctional staphylokinase variant SRH (staphylokinase (SAK) linked with tripeptide RGD and didecapeptide Hirulog) with antiplatelet and antithrombin activities in addition to clot specific thrombolytic function, was developed to address the reocclusion problem. We preferred to use Escherichia coli GJ1158 as the host in this study for economic production of SRH by osmotic (0.3 mol/L sodium chloride) induction, to overcome the problems associated with the yeast expression system. The therapeutic potential of SRH was evaluated in the murine model of vascular thrombosis. The SAK protein (1 mg/kg body mass) and SRH protein (1 mg/kg and 2 mg/kg) were administered intravenously to the different treatment groups. The results have shown a dose-dependent antithrombotic effect in carrageenan-induced mouse tail thrombosis. The thrombin time, activated partial thromboplastin time, and prothrombin time were significantly prolonged (p < 0.05) in the SRH-infused groups. Moreover, SRH inhibited platelet aggregation in a dose-dependent manner (p < 0.05), while the bleeding time was significantly (p < 0.05) prolonged. All of these results inferred that the osmotically produced multifunctional fusion protein SRH (SAK-RGD-Hirulog) is a promising thrombolytic agent, and one which sustained its multifunctionality in the animal models.