Component 8 Research & Innovation Colleges
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1) Existing body of the research work in that area – (fresh proposals not allowed). Fit and credibility of past work, Past achievements of proposed centre/concerned researchteam The Project Coordinator’s group has its strong root in unearthing the potential bioactive molecules like 4-phenyl butanoic acid from Bacillus pumilus, Perfluroeicosane, GansidinW from Kocuria marina, Cyclo(L-Leucly- L-Prolyl), a cyclic dipeptide from B. amyloliquefaciens, Curcumin, Eugenol, Morin, Mangiferin, Rosmarinic acid, Chlorogenic acid, Methyl eugenol from various natural resources, Kiwi fruit (Actinidia deliciosa), Mushrooms (Pleurotus florida), Tender Coconut (Cocus nucifera. L) Water and Turmeric (Curcuma longa) against different human opportunistic pathogens. The Project Coordinator’s group has developed expertise in identifying novel bioactive molecules for the better treatment strategies against various bacterial and aquaculture pathogens from natural and marine resources through both culture dependent and culture independent approaches. An editorial about the research work in Nature India (doi:10.1038/nindia.2012.72), (http://www.nature.com/nindia/2012/120515/full/nindia.2012.72.html) explain the importance of the research in discovery of novel anti-pathogenic agents from marine microbes in this part of Indian sub-continent. Detailed preliminary work done by the Investigators group are given in Annexure I
Microbes are ubiquitous and the presence of microbes is well documented in each and every niche of the environment. The unprecedented genomic sequencing data in the databases provides the testament for the presence of millions of bacteria in different environmental locations. Though both beneficial and harmful bacteria are present in the environment, the infectious diseases caused by various pathogens pose a major life threatening effects on human and wreak havoc in aquaculture industries. Antibiotics, which are the magic bullets that fight against these major life threatening diseases, have made significant improvements in the health status worldwide. However, for the past 35 years, scientific communities failed to introduce new classes of antibiotics for the betterment of human health. The overconsumption and uncontrolled utility of antibiotics have created major environmental stress to most of the pathogens that resulted in the evolution of drug resistant pathogens. There are many reports for the emergence of drug resistant pathogens such as Methicillin resistant Staphylococcus auresus (MRSA), Vancomycin resistant Staphylococcus aureus (VRSA), Multidrug resistant Mycobacteria. It was widely known that the source for the present day antibiotics is from the cultivable microorganisms that constitute less than 1% when compared to the remaining 99% of the uncultivable microorganisms. Plethora of reports are available that state the potential application of uncultivable microorganisms in mining novel bioactive molecules that are acting in the following ways viz anti-bacterials, anti-microbials, anti-fungal, anti-protozoans, anti-virals, anti-biofilm and anti-Quorum Sensing agents. The effective way to treat these MDR pathogens other than mining for potential and newer antibiotics is through looking for Novel bioactive molecules that targets Quorum Sensing pathways (cell to cell communication) of the bacterial pathogens. Targeting quorum sensing signals between/among the disease causing pathogens can effectively prevent the virulence factors production that directly affects the host. Hence, identifying novel bioactive molecules from the bacteria of different natural resources will act as a better alternative to antibiotics in treatments that involve the deployment of combinatorial drug therapy. In addition Cancer that comes under the non communicable diseases poses the major life threatening effect in India. The prevalence of cancer in India is estimated to be around 2.5 million, with about 8, 00,000 new cases and 5, 50,000 deaths per annum. Other than cancer, with increasing life span and decreasing cognitive functions in elderly, Alzheimers Disease has become a major health problem in society. Early detection, prevention, and therapeutic intercessions are needed to minimize the ill effects of this disease. Hence, new biocative molecules are needed to combat against these non-communicable diseases. The aquaculture industries in India have been overwhelmed with a wide array of aquatic animal diseases caused by bacteria, fungi and other pathogens. This directs impedes both economic and social development in many countries including India. Hence, identification of novel bioactive molecules that target all the microbial pathogens that pose major life threatening effects to humans and aquatic animals is the only hope. Identification and characterization of Novel bioactive molecules through both Culture Dependent and Independent approaches from natural resources will shed more light on the treatment scenario of microbial pathogenesis. Since only less than 1% of the bacteria can be cultured, remaining 99% of the unknown microbiota needs to be explored through Culture Independent approaches in the name of Metagenomics. Other than marine bacteria, naturally available resources like sea weeds, sea grass, edible fruits, and terrestrial medicinal plants have also proven to be better source for bioactive molecules. This kind of study will surely unearth many novel bioactive molecules which can act as a better alternative for the present day antibiotics and can augment the current treatment strategies of microbial related pathogenesis and other non communicable diseases. In addition, the present study will ameliorate our knowledge of the unexplored microorganisms in a better way and will throw insights to the biotechnological potential of previously unexplored natural resources
Several novel bioactive molecules will be obtained for human and animal health. Process/Product patents will be obtained after the completion of the Project. Technology will be transferred to the collaborating Industrial partners for commercialization. Scientific data obtained through the Project work will be published in peer reviewed International journals of high repute.
Focus area: Health- New Bioactive molecules for human and animal health Aim: To screen, identify, characterize bioactive molecules (anti-QS, anti-biofilm, therapeutic agents and Probiotics) against various human and aquaculture microbial pathogens and non communicable diseases such as cancer, neurodegenerative diseases (Alzheimer’s, Rett Syndrome & Autism Spectrum Disorder), Malaria and HIV.
Objectives for Functional Materials
Objectives for Healthcare Management Examination of Healthcare Management systems of India in comparison to Global standards
Plan of work, methods and techniques to be used Sample collection Choice of the collection locations We will collect marine samples ranging from near-shore and to a distance of 500 meters. Marine sediments, Rhizosphere sediment, Seawater, Sea weeds, Sea grasses and Marine Microalgae will be collected from Palk Bay and Gulf of Mannar coastalregions. Extraction of Metagenome from Environmental Samples Metagenome will be extracted from environmental samples (Marine sediments, sea weeds, sea grasses and sediments of rhizosphere region from mangroves) by using Ultraclean TM Soil DNA Isolation Kit (MO BIO Laboratories Inc., Solana Beach,CA). Construction of Metagenomic Library from Marine metagenomic DNA Metagenomic Library will be constructed from all the metagenomic DNA using pCC1FOS, a copy control vector which has an insert size of approximately 40kb. 40kb insert is enough to accommodate a complete set of genes/operons that code for novel bioactive compounds. Screening of marine bacteria and metagenomic library for quorum quenchingactivity Quorum quenchers or quorum sensing inhibitors will be screened from the metagenomic library according to Riaz et al., (2008) and Quorum Quenching activity of marine bacteria from different marine sources will be screened according to Nithya et al., (2010). Structural elucidation and characterisation of active bioactivemolecules Structural elucidation and characterization of anti-QS and anti-biofilm agents from marine bacteria and metagenomic clones will be identified through the followingapproaches
Protein-Ligand Interaction studies; Molecular docking against receptor Protein of various human microbial and aquaculture pathogens using computational approach. In vivo analysis In vivo analysis of the effect of the identified anti-QS and anti-biofilm agents will be assessed by the well established model systems Caenorhabditis elegans and Artemia. C. elegans is an effective animal model system to study the effect of bioactive molecules against various human bacterial pathogens. In addition, toxicity analysis of the bioactive molecules will also be carried out in vivo using C. elegans and Artemia as modelsystems. Effect of bioactive molecules on the proteome and excretome of the test pathogens To study the effect of anti-QS and anti-biofilm agents on the expression of various genes more completely, proteomic approach will be employed. Two dimensional gel electrophoresis followed by the identification of differentially expressed proteins through mass spectrometry (MALDI-ToF-ToF & Nano-LC approaches) will be done to study the impact of bioactive molecules on the proteome and excretome (total secreted or excreted proteins) of the test pathogens. Interlink between the proteins differentially expressed upon exposure to bioactive molecules will be predicted to determine the global network involved in the antibiofilm activity. The proteomic studies are expected to unveil valuable data which is required to predict the target proteins and pathways and this approach could be used for design and discovery of novel drugs against drug resistant pathogens (Ong et al., 2009; Kopec et al., 2005). Screening and identification and Characterization of Therapeutic agents against Cancer In vitro toxicity testing In vitro toxicity testing will be performed by using the followingassays
Anti proliferative activity
Screening for Mode of action
Screening and identification and Characterization of Therapeutic agents against Alzheimer’s Disease
Establishment of callus culture Friable callus will be initiated from in vitro leaf explants of selected medicinal plants by using different combinations and concentrations of plant growth regulators in MS solid medium. Initiation and establishment of fine cell suspensions In order to establish cell suspension cultures, friable calli will be inoculated to liquid MS medium supplemented with particular plant growth regulators. Fine suspensions will be obtained on frequent sub culturing and repeated sieving of callus cells growing in liquid medium. Growth index (GI) and packed cell volume (PCV) of suspensions will be monitored at regular time intervals (Verma et al. 2014). Agrobacterium rhizogenes - mediated transformation Hairy roots will be induced from in vitro leaf explants using four different strains of Agrobacterium rhizogenes such as MTCC 532, A4, MTCC 2364 and R1000. Various parameters such as culture O.D., co cultivation duration, acetosyringone concentration, age of explants will be assessed for achieving maximum transformation frequency. Induced roots will be excised and grown in MS basal medium augmented with suitable antibiotic concentrations and will be maintained as individual root clones. For mass propagation, hairy roots will be cultured in liquid medium in shake flasks. Molecular analysis of hairy roots In order to confirm the transgenic nature of hairy roots, PCR amplification of rol genes present in TL-DNA of Ri plasmid will be carried out. Genomic DNA will be isolated from the hairy roots by Hi Pur A Plant Genomic DNA isolation kit (Himedia, Mumbai). The presence of rol genes will be checked by PCR amplification of rol A, rol B and rol C genes with respective primers. Elicitation of cell suspensions and hairy rootcultures Various marine seaweed extracts will be used for elicitation experiments. Different parameters such as elicitor concentration, exposure duration and age of cultures for elicitation will be analysed to achieve maximum product accumulation. The effect of different seaweed extracts on biomass accumulation will also be studied. In addition, total phenolic content, total flavanoid content and antioxidant potential of elicited cultures will be determined by biochemical assays as mentioned below.
The total phenolics content of the methanolic extracts of elicited cell suspension and hairy root cultures will be estimated by Folin Ciocalteau method by using Gallic acid as reference standard and expressed as milligram Gallic acid equivalents per gram dry weight of the sample (mg GAE/g DW) (Shah et al. 2012).
The total flavanoids content of the methanolic extracts of elicited cultures will be estimated by Aluminium chloride method by using quercetin as reference standard and expressed as milligram Quercetin equivalents per gram dry weight of the sample (mg QRE/g DW) (Shah et al. 2012).
The antioxidant potential of the methanolic extracts of elicited cultures will be estimated and compared by means of DPPH (1, 1-diphenyl-2-picrylhydrazyl) assay (Itidel et al. 2013; Kamaladevi et al. 2013). HPLC analysis of elicited cultures Elicited cell suspensions and hairy root cultures will be harvested after specific time period and subjected to HPLC analysis for the quantification of major phytochemicals such as Bacoside A and Andrographolide in their respective extracts. The retention time of the compounds will be compared with their reference standards. Evaluation of in vivo cytotoxicity of elicited cultures in animal models Caenorhabditis elegans and Zebra fish The extracts of elicited and unelicited hairy roots and cell suspensions of above mentioned medicinal plants will be tested for their in vivo toxicity using the animal model Caenorhabditis elegans. Various assays such as nematode liquid killing assay, full lawn solid plate killing assay, pharyngeal pumping assay, C. elegans short-time exposure assays (Jebamercy et al. 2012) will be performed in order to examine the cytotoxicity of the therapeutic compounds produced in vitro. Similarly the in vivo toxicity will also be assessed in Zebra fish animal model. Evaluation of antioxidant potential of elicited cultures in animal model Caenorhabditis elegans The extracts of elicited and unelicited hairy roots and cell suspensions will be tested for their antioxidant potential using the animal model C. elegans. C. elegans exposed to the plant extracts will be examined for antioxidant potential by assessing oxidative stress markers including, reactive oxygen species and hydrogen peroxide. In addition, the levels of antioxidative enzymes such as catalase, superoxide dismutase, glutathione reductase, glutathione peroxidase, glutathione-S-transferase and reduced glutathione will also be assessed by following the protocol of Kamaladevi et al. (2013). In addition, the impact of plant extract on the innate immune regulatory players of C. elegans system will be assessed at the transcriptomics and proteomic levels (Durai et al. 2013 & 2014) to understand the immune regulatory pathways involved during the normal development andreproduction. Roles and Responsibility of the Investigators A. Alagappa University
B) Annamalai University
c) Bharathidasan University
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