AUTONOMOUS INSTITUTIONS:
The autonomous institutions of the Department have performed well during the year. The scientific excellence of these institutions as reviewed by the Scientific Advisory Committees, Governing Councils and Society meetings was observed to be of highest standards. The data on quality of publications and patents during the year and significant achievements are given below :
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National Institute of Immunology, Delhi
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1 publication IF>30
5 publication IF>15
12 publications IF>5
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National Centre for Cell Science, Pune
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1 publication IF>30
14 publication IF>5
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2 patents granted,
9 patents filed
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Centre for DNA Fingerprinting and Diagnostics, Hyderabad
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1 publication IF>15
9 publication IF>5
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National Brain Research Centre, Manesar
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1 publication IF>30
19 publications IF>5
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National Centre for Plant Genome Research, Delhi
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National Institute of Immunology, New Delhi
The National Institute of Immunology (NII) has made intensive commitments to basic research in areas of modern biology related to the immune system. A wide range of issues have been addressed imaginatively and successfully by the scientists resulting in high impact publications in prestigious journals. Major areas of commitment - immunity and infection, reproduction and development, molecular design, and gene regulation, - are well-placed to provide unity in diversity by their synergism in the broader cause of health and disease. The important contributions are briefly highlighted below:
Immunity and infection
The studies on the analysis of antigen processing and presentation, examining the generation and activation of T, B and antigen-presenting cells, have been actively pursued. While addressing role of Bruton’s tyrosine kinase in myeloid cell development, it was proposed that this enzyme is likely to play significant, although partially redundant, role at multiple points during the development and functioning of the myeloid lineages, affecting the outcome of many infectious as well as non-infectious inflammatory events in vivo. This pleiotropy of functional roles raises the possibility that subtle mutations in Bruton’s tyrosine kinase may contribute significantly to the susceptibility of carriers to both infectious and autoimmune diseases.
While addressing the issues concerning mucosal immune responses, it was concluded that the selection of B cells into the memory pool may not be strictly affinity-based, and the affinity maturation of antibody responses may reflect a cumulative effect of prolonged and overlapping B cell responses to persisting antigen. Towards understanding the regulation of T cell responses, it was shown that soluble antigens given orally or intra peritoneally may lead to T cell commitment to effector rather than proliferative capabilities, necessitating a reassessment of therapeutic modalities for induction of oral tolerance in allergic or autoimmune states.
Studies on the biology of T lymphocytes involved characterisation of signals received by T lymphocytes and antigen presenting cells during activation. Towards characterising the role of T cells as antigen presenting cells for CD4 and CD8 T cells, new data has been generated which confirm and extend earlier findings in the human system showing that both CD4 and CD8 T cells can be turned off when antigens are presented by T cells as priming APCs. These finding have important implications in T-APC mediated allergy in vivo as a potential downmodulatory event in an ongoing infection scenario.
The Salmonella typhii-host cell interaction studies enabled further understanding of the role of Vi capsular polysaccharide during infection with S.typhi. A putative tumor suppressor molecule involved in the regulation of mammalian cell cycle, prohibitin, through which Vi polysaccharide was shown to interact with the host cells was further characterized. It was shown that prohibitin and other related molecules are not only abundant in the mitochondria but have also been localized in the membrane and the nucleus, providing insights into how prohibitins regulate intracellular signaling from the membrane.
Studies on the biology of Japanese encephalitis virus have shown that microparticle-adsorbed DNA induced mixed Th1-Th2 kind of immune responses as opposed to Th1 type of immune responses elicited by the naked DNA, towards developing plasmid DNA-based immunization procedures for vaccination against JEV. Transgenic tobacco plants have been developed as a part of project to eventually develop an oral JEV vaccine.
Molecular design
In a study towards developing systems for intracellular delivery of drugs or pharmacologically active agents exploiting the efficiency of receptormediated endocytosis, cloning and expression of the haemoglobin receptor from leishmania was achieved and the haemoglobin binding activity of the recombinant protein was demonstrated. It has also been shown that the N-terminal end of haemoglobin receptor incorporates the extracellular haemoglobin binding domain and that the C-terminal region is cytoplasmic. It was also shown that the haemoglobin receptor in leishmania is possibly a hexokinase.
The present focus in the project concerning chemical and biosynthesis of GPI cell surface molecules of leishmania parasite, is on the assembly of the four individual domains to construct full-length lipophosphoglycan of L. donovani as the necessary amounts of suitably activated and structurally characterized intermediates have been prepared.
The study addressing the underlying principles of reverse proteolysis, and its application to proteasemediated peptide ligation reactions has provided interesting insights. Currently, the strategy is being used to engineer new and novel haemoglobin molecules to delineate intermolecular interactions in the assembly of the sickle haemoglobin fiber. The role of long-range and non-additive coupling of mutational effects in sickle haemoglobin polymerization has been elucidated.
Continuing the structure, interaction and design studies involving regulatory molecules, thermodynamic analysis of peptide-carbohydrate mimicry using monoclonal antibodies against mannopyranoside has been carried, suggesting that the conformational plasticity associated with a subset of monoclonal antibodies may manifest as mimicry in the antibody response.
Towards development of novel chimeric toxins for targeted therapy by genetic engineering, ribonucleolytic and ribosome-inactivating protein toxins were continued to be subjected to structure-function analyses so that knowledge-based chimeric toxin design could be explored. Several rationally designed mutants of eosinophil-derived neurotoxin were assayed for inhibition of the infectivity of extracellular virions of the paramyxovirus, RSV-B. It was shown that the antiviral activity of eosinophil-derived neurotoxin was dependent on its ribonucleolytic activity.
In order to develop strategies for making sensors and actuators for biological processes, electrical property measurement based systems are being explored. While standardizing the immunization by electroporation, a commercial preparation of hepatitis B surface antigen was used to immunize mice by electroporation. After exploring many diverse designs to optimize electric field strength and current density, it was concluded that the ‘self closing tweezers’ design of electrodes are the most suitable.
The studies associated with the mechanism of survival of salmonella in macrophages led to unambiguous demonstratation that SopE is the major player in the survival mechanism of salmonella in macrophages. SopE mediated recruitment of Rab5 from the host cell on phagosomes promotes fusion with early endosomes. Work has also been initiated to determine the role of SNAREs in the maturation of Salmonella in macrophages wherein the preliminary results suggest that Syntaxin 6 and Syntaxin 13 are recruited more efficiently on late-Salmonella containing phagosome in comparison to early phagosomes.
In a study concerning molecular modelling of peptides and protein-ligand complexes using knowledgebased potentials, substrate specificity of polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) has been studied in silico. It was revealed that the differences between modular and iterative KS domains may originate from the differences in the catalytic pockets of these enzymes. Additionally, a novel computational method has been developed for correct identification of NRPS domains in a query sequence and predicting their catalytic activity or substrate specificity.
Reproduction and development
Towards understanding the biology of cell survival and function, critical components of the early biochemical changes that occur in spermatogenic cells during estrogen exposure were identified. While addressing issues pertaining to cell death, it was suggested that apoptosis is the preferred mode of death for the parasites inside the host cell. Further, concurrent inhibition of respiratory chain complex II with pentamidine administration increased cytotoxicity of the inhibitory drug has emerged as a new possibility providing important insights in the mechanism of oxidative stress-induced apoptotic cell death.
Studies on molecular characterization of zona pellucida glycoproteins includes addressing the relevance of Y chromosome microdeletion in male infertility. Azoospermia factor located on the long arm of Y-chromosome has been postulated to be important for male germ cell development. Studies aimed at microdeletion in these regions in infertile males with non-obstructive oligospermia or azoospermia are being undertaken.
In a study aimed at developing perfusion bioreactor for culturing hematopoietic stem cells, it was crucial to comprehend the underlying mechanisms governing the cell fate decisions in order to maximize the clinical benefits of human stem cells in tissue regeneration and gene therapy. Towards this end, it was established that the phenotypes of hematopoietic cells grafted in the liver could be changed, although it is yet to be confirmed if these cells acquire hepatocyte specific phenotype and/ or function.
Gene regulation
Studies have been undertaken towards utilizing genome-based approaches to understand the importance of various polyketide synthase gene clusters from Mycobacterium tuberculosis. The molecular basis of the enzymatic crosstalk between FASs and PKSs in mycobacteria through a combination of computational, genetic, biochemical and enzymological studies was established revealing the mechanism involving biosynthesis of complex hybrid metabolites providing insights into how distinct biochemical functions are integrated within a given organism to generate metabolite diversity.
In a study aimed at understanding the role of cytokines and growth factors signaling neuronal apoptosis, a pro-inflammatory cytokine, IL-6, was shown to be upregulated during neuro-degenerative processes and that it may be directly associated with induction of apoptosis.
Studies towards cellular signalling in eukaryotic development have focussed on understanding the role of phosphoinositide mediated cell signaling in malaria parasite-Plasmodium falciparum. It was shown that the protein kinase B from Plasmodium falciparum may play an important role in parasite development and that the N-terminal domain of this protein may negatively regulate the catalytic activity.
Ancillary activities
Work towards production of transgenic and knockout animals have been continuing for the systems that have been developed in various collaborative efforts. Using the earlier developed transgenic mouse model containing CGG repeat, the molecular etiology of fragile- X syndrome in humans leading to mental retardation is being explored. Transgenic mice expressing SMAR1 have been produced. The loss of SMAR1 gene causes prostate, colon and breast cancer. Studies with beta islets of pancreas of transgenic mice indicated that stress to these specialized cells may lead to the over-expression of p53 which in turn lead to their apoptosis. Gereration of transgenic mouse which over-express HABP1 is under progress.
A large number of trainees including both young students and scientists with specific training needs visited the Institute for various periods. The doctoral programme continued to draw nearly twenty highly talented students in a ationwide entrance process. Intense research collaborations have been established with various laboratories both, overseas and within the country.
The scientific strength of the Institute as reflected in publications in highly competitive peer-reviewed international journals and the technological competence as indicated by international patents, continued to show steady growth record. More than a third of the published papers appear in the most prestigious journals like Nature, Nature Structural Biology, EMBO Journal etc.
National Centre for Cell Science, Pune
Efforts continued towards R&D in areas of cell biology, cancer biology, immunology, diabetes, signal transduction and gene regulation. Support was provided for human resource development through teaching and training. The Center serves as a national cell repository for cell lines and hybridomas and during the year supplied 1017 cell lines to 163 scientific institutions within India. Efforts towards establishment and characterization of cell lines, resulted in establishment and characterization of new immortalized and primary cell lines with unique features. The Centre trained 10 visiting researchers at NCCS and also organized onsite training for 70 researchers in institutions located at Allahabad, Guwahati, Baramati, and Kolhapur. One meeting of the Scientific Advisory Committee, two meetings of the Governing Body and one meeting of the Society were held. The annual report and audited statement of accounts were adopted. The highlights of major achievements are as follows:
Cell biology
Cell signaling and cell migration play a critical role in angiogenesis and metastasis. It has been found that Wortmannin, an inhibitor of the PI3K, blocked morphological transition and migration of SiHa cells in wound closure assay. Wortmannin also blocked epidermal growth factor (EGF) induced invasion by SiHa cells in extracellular matrix (ECM) gel assay. Transforming Growth Factor beta (TGF a) controls a wide range of cellular responses including cell proliferation, lineage determination, differentiation, and apoptosis. Studies showed that differential activation of signal transduction pathways by TGFa1 as a function of its concentration underlies its bi-directional effect on hematopoietic cells. It has also been demonstrated that mammalian cells have functionally intact epithelial nitric oxide synthase (eNOS) that is associated with subnuclear structures like spliceosomes, suggesting that it may perform regulatory role in splicing mechanism. Osteopontin (OPN) induces integrin-mediated transcription factor AP1 (AP-1) activation and urokinasetype, plasminogen activator (uPA) secretion by activating extracellular signal regulated protein kinase/epidermal growth factor receptor (c-Src/EGFR/ERK) signaling pathways, which ultimately control the motility and invasiveness of breast cancer cells. This has been reported for the first time.
The bone loss in many important skeletal disorders such as osteoporosis, rheumatoid arthritis, hypercalcemia of malignancy and bone metastases of various tumors occur mainly because of increased osteoclast activity. Studies indicate that IL-3 and granulocyte - macrophage colony stimulating factor (GM-CSF) irreversibly blocks TNF-a-induced osteoclast differentiation by down-regulation of mRNA and surface expression of TNFR1 and TNFR2. It was also found that IL-4 acts directly on mature osteoclasts and abolish bone resorption through inhibition of NFkB activation by IL-4R mediated mechanism.
Signal transduction
Investigations were carried out to determine how -hemolysin (a-HL) establishes the synergistic function with a protein tyrosine phosphatase. It has been observed that the a-HL induced change in Caveolin-1 organization is distinct and clear, probably through a direct interaction with Caveolin-1 beneath the cell surface, present in the lipid rafts. During this oligomerization/assembly of a-HL, Caveolin-1 network is reorganized beneath the cell surface and this is perhaps responsible for an attenuation of receptor mediated signaling events.
Stem cell biology
In a study on bone marrow cryopreservation and revival of stem cells, it has been demonstrated that addition of catalase and trehalose to freezing medium help to preserve the migration capacity of frozen CD34+ cells suggesting that additives improve the in vivo homing of frozen mouse bone marrow. In the neurodegenerative disorders such as Parkinson’s and schizophrenia, the dopaminergic neurons are degenerated in the substantia niagra and the ventral tegmentum respectively. A number of stable embryonic stem cell clones have been successfully generated expressing the live reporter enhanced green fluorescent protein (EGFP) under the regulatory control of dopaminergic neuron specific promoter tyrosine hydroxylase (TH). These clones upon differentiation into neural lineage did exhibit EGFP expression.
Taking note of the significant contributions made by the institute, it has been recommended in the Society meeting that a separate mission-oriented programme on stem cell based research and its applications may be developed and a special unit may be established. NCCS may also co-ordinate and network the research activities in this region of the country. The proposal has been received by the Department and is under active consideration.
Cancer biology
Studies were undertaken to understand the chemosensitivity of cancer cells to drugs, the mechanism of cell death and the role of tumor suppressor p53. It was found that carboplatin mediated inhibition of constitutively active NF-kB leads to Bcl-2 downregulation by direct inhibition of NFkB binding to the response element present in Bcl-2 promoter, thus revealing the molecular mechanism of action for this antineoplastic agent.
Studies on the downstream apoptotic pathways revealed an essential role of volta e dependent anion channel (VDAC) and proapoptotic protein Bax, in altering the membrane potential and release of cytochrome c during apoptosis induced by staurosporine in SK-N-MC human neuroblastoma cell line. Earlier it was shown that high glucose increased mitochondrial superoxide generation from more than one source which initiates cell death. Furthermore, mitochondrial anion channels and cytosolic dismutation to H2O2 seem to be important steps for oxidant induction of high glucoseinduced cardiac cell death.
A novel 600 bp transforming gene, cloned from mouse melanoma cells was identified as a member of non-coding RNAs (NCRs). The NCR was found to be localized to the cytoplasm as a polyadenylated RNA. Transcript analysis by northern hybridization with labeled riboprobe demonstrated the anti-sense expression of the gene with transcript size of 1.8 kb.
Diabetes
In a study towards understanding the molecular basis of both diabetes type I and II, it was observed that unfractionated whole bone marrow derived from experimental diabetic mice when transplanted into other diabetic mice can correct the hyperglycemia. These studies further reveal that transplantation of bone marrow stem cells rescue experimental diabetes in mouse model. Chemical manipulation such as tagging of cell surface with 1-fluoro 2, 4-dinitrobenzene (FDNB) brings about conformational changes on cell surface. Strikingly, immunization of rats with FDNB modified rat insulinoma (RIN) cells produced insulitis.
Insect molecular biology & infection and immunity
In an attempt to understand how the parasites obtain their energy, two complete coding sequences for hexokinase isozymes from Drosophila were cloned. Complete coding sequence for exokinase gene has also been cloned from the parasite Leishmania major. A novel bacterial strain, Aeromonas culicicola was isolated from the midgut of the mosquito Culex quinquefasciatus. Spontaneous development of parasites outside the human host erythrocytes was reported for the first time. Macrophages play host to Leishmania, a parasite that causes leishmaniasis in 0.5 million people annually. It was demonstrated that weak CD40 signals induce extracellular stress-related kinase (ERK)-1/2-dependent IL-10 expression whereas stronger signals induce p38- mitogen activate protein kinase (p38MAPK)-dependent IL-12 production. This data unfolds a novel immune evasion strategy, where a parasite differentially modulates the CD40 engaged reciprocally functioning signaling modules, and a novel strategy for defining drug target(s).
Investigation towards understanding the molecular interaction between host’s complement proteins with various viral proteins, has revealed that like host, complement control proteins also possess factor I cofactor activities for complement proteins C3b and C4b and also possess decay accelerating activities for the classical as well as alternative pathway C3 convertases. Further efforts are being made to determine the functional determinants of viral complement control proteins.
HIV biology
Studies have revealed a direct interaction of the HIV-1 transactivator Tat with NFkB enhancer, a global regulatory sequence for many cellular genes both in vitro and in vivo. This interaction not only provides a novel molecular basis to explain TAR independent transactivation in HIV-1, but also point towards the potential mechanism of Tat mediated modulation of cellular genes. Experiments on DNA immunization using gp120 from subtype C isolate, revealed some startling facts about the elicitation and maturation of cytotoxic T lymphocytes. Anti-HIV activity has been observed in the crude extracts from black clam (Villorita cyprinoids). Studies on fractionation and identification of active molecule were carried out and it was found that crude extract (BCCF) 3.2 had more activity. This was further fractionated into 2 fractions, of which BCCF 3.2b has been found to be more active and is being studied for further characterization. Efforts were made to understand molecular mechanism of inhibition by the black clam extract and it seems the active principle could be a reverse transcriptase inhibitor.
Chromatin architecture and gene regulation
The role of chromatin architecture in fundamental processes involving DNA is emerging. The higher order organization of chromatin in form of loop domains is governed by matrix associated regions (MARs) and their binding proteins. Studies conducted at the Centre have delineated the minimal domain of SMAR1, a candidate tumor suppressor, which is responsible for p53 interaction, activation and stabilization within the nucleus. Further studies using transgenic mice suggest that apart from its role in the control of cell cycle, SMAR1 is also involved in the development of lymphoid organs. The T cell restricted regulator special AT-rich sequence binding protein 1 (SATB1) is known to organize T cell chromatin in a unique three-dimensional architecture that circumscribes heterochromatin. Studies have also unravelled, the primary sequence signature(s) that are embedded into cell type specific chromatin architecture for the first time. It has been proposed that the primary sequence features such as the consensus motifs and repeating hexameric patterns project a unique chromatin context in vivo.
This year NCCS has published 35 papers in reputed peer reviewed journals, obtained 2 US patents and also filed 6 patent applications.
Centre for DNA Fingerprinting and Diagnostics, Hyderabad
CDFD has been providing services related to DNA fingerprinting, diagnostics, screening of new born baby and bioinformatics in addition to basic research in frontier areas of modern biology such as genetics, molecular and cell biology, molecular pathogenesis, bioinformatics etc.
Services
The Centre has been providing DNA fingerprinting services to various government and law enforcement agencies. On an average more than one case per day is received for DNA fingerprinting. The DNA fingerprinting service, given the fact that it has been shown to bring about dramatic increase in the conviction rate, continues to be in much demand. With the crime burden on the society increasing, more and more requests for DNA fingerprinting are naturally anticipated. The Centre has signed MoUs and is working very closely with the State/Central Forensic Science laboratories to popularize this technology for the benefit of society. Proactive measures have been taken which involved setting up of a national level statutory body, the DNA Profiling Advisory Committee (D-PAC), which has been approved by the Union Cabinet. This committee will draft a legislation on DNA which will be put up to the Parliament for enactment as a Law. Several new DNA based services in the areas of seed authentication, certification of genetically modified foods (GM foods) and wildlife and animal identification have been developed.
The range of services provided has increased encompassing cytogenetic, biochemical and molecular diagnosis. Complementing the DNA diagnostic services, the chromosomal diagnostic group has been providing services in the area of molecular cytogenetics. Of the cases referred to the Centre, those for premature ovarian failure comprised the single largest number followed by primary amenorrhea, abnormal sexual development and ambiguous genitalia among others. The primary cause of childhood blindness and the contribution of genetic elements to this abnormality continues to be another area of interest at CDFD.
The Bioinformatics Facility at the Centre is ranked as one of the top centres of its kind in India, which is evident from the recognition given to it by the European Molecular Biology Network (EMBNet). CDFD has been designated as the Indian node for the European Molecular Biology Network and is the only node, other than the one in China, outside Europe. It hosts an unusually large number of software and databases for genome analysis with browsable databases at its website. This includes an indigenously developed database known as the Database of Structural Motifs in Proteins (DSMP). Analysis of the data contained in DSMP will enable the investigator to arrive at an educated guess about the likely structure and therefore function of protein under investigation. The combined expertise of the nodes allow EMBnet to provide state-of-the-art biocomputing services at the click of a button. The website of CDFD EMBnet is available at www@cdfd.org.in. Several data banks, data bases, and the complete genome sequences of Helicobacter pylori, Borrelia burgdorferi, Haemophilus influenzae, Mycoplasma genitalium, Methanococcus jannaschii, Mycobacterium tuberculosis, and Mycoplasma pneumoniae can be accessed from the Web Server. The CDFD website is visited approximately once every minute. The Centre has recently automated and computerized all the case work with plans for the development of complete databases vis-à-vis the appropriateness of a given probe for the Indian population.
Research
The R&D group on molecular genetics has been able to initiate several novel findings relating to water stress adaptation and spontaneous mutation in E. coli. The Centre has created data base of microsatellites and has looked at the distribution and mutation of such microsatellites in the silkworm Bombyx mori and other silkmoths. Working together with the L V Prasad Eye Institute and the Hyderabad Eye Research Foundation, the Centre has been looking at the molecular basis of primary congenital glaucoma. In the process, inexpensive diagnostic methods have been developed for rapid detection of this disease at the genetic level leading to early diagnosis and treatment. These diagnostic technologies also enable carrier detection and help to provide genetic counselling to the affected population.
The comparative and functional genomics group has been focussing on Mycobacterium tuberculosis, and H.pylori. CDFD has networked with hospitals, tuberculosis centres and tuberculosis research institutes in and outside the country to set up the first of its kind a National Epidemiological Databank of Mycobacterium tuberculosis genotypes based on whole genome fingerprinting of clinical isolates of tuberculosis.
The structural computational biology group has been able to perform the computational analysis of biomolecules and genomes while the structural biology group has been able to determine the structure and biochemical characterization of quite a few mycobacterium proteins. Automated DNA sequencing, comprehensive in silico modeling and microarray based approaches have already been initiated. Knowledge based tools in computational genomics are being developed towards effective utilization of the data generated from human and microbial sequencing projects. It is expected that this will revolutionize and redefine conventional time-scales, and methods related to the discovery of drugs and drug targets.
Human resource development
The Centre has been imparting training of personnel for providing state-of-art services in DNA fingerprinting. Further, an active summer training and project training programme attracts and enables young scholars to enter the Ph.D. programme in different institutions in the country. The students enrolled at the Centre register for Ph.D degree at the University of Hyderabad.
New initiatives
New activities have been launched, which include studies on improved methodologies for high throughput STR based DNA fingerprinting; new diagnostics tools development; DNA fingerprinting of ethnic populations in India of particular interest in forensics and analysis of human genome diversity; computational biology of pathogenic mutations etc. Programmes have been initiated to study host-parasite interactions during cellular signaling; setting up of specialized laboratory for plant genetic fingerprinting; sequencing of the Mycobacterium W; studies of the biology of M.tuberculosis using genetic, immunological and structural aspects; genetics and epidemiology of leptospirosis. Capacity has been developed and the centre is setting up National DNA Training Academy to train the members of the Law Enforcement Agencies and the Judiciary on DNA technologies as part of the new activities.
National Brain Research Centre, Manesar
The National Brain Research Center established in 1999 as an “Apex Coordination Centre” for neuroscience research has the mandate to create state-of-art facilities in the country through a networking approach and generate highly trained human resource. Major areas identified for research include computational neuroscience, system and cognitive neuroscience, stem cell research, developmental neurobiology and basic research towards understanding of neurological and psychiatric disorders.
The year 2004-05 has been a year of growth and consolidation for the Centre, now located at its permanent campus at Manesar. A one-of-its-kind functional magnetic resonance imaging (fMRI) facility for neuroscience research is currently being developed. A confocal microscope and a laser microdissection system have been added to the imaging facility and are being utilized for experiments. Ph.D. programme has been improved and a M.Sc. programme in neuroscience has been initiated. Networking with existing neuroscience groups/institutions in the country to promote multidisciplinary research has grown to 44 centres. The institute also continues to provide the facility of a digital library, which it shares with scientists throughout the country.
Molecular and cellular neuroscience
The molecular mechanisms underlying neurodegeneration is being studied with particular reference to mitochondrial dysfunction that is commonly seen in several of these disorders. Research has focused on identification of the components of the mitochondrial electron transport chain, which are specifically affected and interplay between protein thiol homeostasis and mitochondrial dysfunction. The recovery mechanisms involved in the maintenance of protein thiol homeostasis and restoration of mitochondrial dysfunction have been identified. Collectively, it has been demonstrated that perturbations in redox-status of thiols underlies the mitochondrial dysfunction seen in some of the neurodegenerative disorders and maintenance of redoxstatus of thiols is important for maintaining and restoring mitochondrial function. Further, it has been discovered that estrogen regulates the expression of certain critical genes involved in the maintenance and recovery of mitochondrial function and thus affords neuroprotection following a toxic insult.
The underlying cause of the neurodegenerative disorders Huntington’s disease (HD) and spinocerebellar ataxia type 3 (SCA3) is the expression of proteins containing expanded polyglutamine tracts. However, the mechanism by which the expanded polyglutamine proteins cause neurodegeneration is not well understood. The research aims to investigate the possible role of ubiquitin-proteosome system (UPS) in the pathogenesis of HD and SCA3 by using cellular models of these diseases. It has been observed that oxidative stressinduced UPS dysfunction is linked with expanded polyglutamine protein induced cell death. A protein, CHIP (C-terminus of hsp70 interacting protein), has been identified that interacts specifically with polyglutamineexpanded protein and is responsible for their ubiquitination and degradation. The effect of curcumin on polyglutamine protein induced cell death has been investigated and it has been found that the curcumininduced apoptosis is mediated through impairment of ubiquitin-proteasome system. The role of NFkB pathway and unfolded protein responses in the polyglutamine diseases pathogenesis is being studied.
Transcription factors such as neural retina leucine zipper (NRL) and photoreceptor specific nuclear receptor (PNR) have been suggested to have key regulatory roles in the differentiation of photoreceptors (rods and cones) and the normal differentiation of theretina. Mutations of both NRL and PNR genes are known to be associated with retinal degeneration. The factors involved in photoreceptor (rod) differentiation pathway are being identified with a view to understand their role in disease. Several proteins (including the TATA Box Binding protein, CREB-1, two retinal proteins of the 30-45 kDA range), have been identified which interact with NRL. A kinase has also been dentified, which phosphorylates NRL and may play a role in NRL mediated transactivation of retinal genes.
Stem cell research
Growth associated protein 43 (GAP-43) is a nervous system specific actin modulatory protein that is required for cell-adhesion mediated signalling. All GAP-43 knock out mice have abnormal cerebellar development such that by P0, the day of birth, abnormal foliation pattern is evident. The role of GAP-43 in coordinating cell cycle responses of granule neuronal precursors is being investigated. The function of transcription factors, which contain the basic helix-loop-helix (bHLH) motif and underlie commitment to neuronal lineage, neuronal differentiation and cell type specification, is also being elucidated. The function of different types of proneural and neural differentiation bHLH genes is being studied using Embryonic stem (ES) cells as a model system for studying neuronal differentiation. The group has found that in GAP-43 knock outs cells from the developing cerebellum have different cell cycle characteristics when compared to wild type animals. Further proliferation response to basic fibroblast growth factor and sonic hedgehog is greater in wild type cerebellar cultures than for the GAP-43 knockout cultures. The group also defined conditions under which differentiated ES cells express the neurotransmitter serotonin and are now looking at the pattern of bHLH expression by quantitative PCR.
In vitro cell culture models of immortalized human astrocytes (SVG cells; SV40 Glial cells) as well as the human fetal primary astrocytes are used in an attempt to understand how viral proteins from HIV-1 induce pathology in brain. These cells have now been successfully sub-cultured at NBRC cell culture facilities and are used for transfections experiments in an attempt to modify these astrocytes to express HIV-1 transactivating viral protein, Tat. Samples are being collected from the astocytes that were transected with expression vectors for HIV-1 Tat B and Tat C to analyze them for the levels of inflammatory cytokines and cell death markers at gene – and protein-expression levels.
Research is being carried out to understand how microglia/macrophages participate in the overall process of inflammation in Japanese encephalitis. The primary focus of this project is to understand how JEV modulates (i) the CNS immune response and inflammation pattern and (ii) the plausible role played by microglia and astrocytes in this process. The role of cytokines in CNS inflammation and their contribution to physiological regulation and disease pathology is being examined.
System neuroscience
Studies have been initiated to understand how the primate sensorimotor system processes sensory information to enable tactile perception and motor control, and how peripheral and central nervous system injuries in adults and during early development affect the functional organization of the system. Experiments have shown that animals with unilateral lesions of the dorsal columns could use their deafferented hands for most of the normal activities except to pick up small food items but had difficulty in picking up large objects which tended to slip from their grasp. Another goal is to determine how injuries early in development affect organization of sensorimotor areas by using the same experimental paradigm. In experiments carried out in collaboration with Vanderbilt University, USA it was found that unlike in adult animals, there was very limited expansion of the face representation in the deafferented area 3b in newborn monkeys. The representations of the digits in the primary motor cortex were considerably smaller in proportion as compared to normal animals while representation of the shoulder and upper trunk was expanded.
Studies have been carried out to understand the mechanisms that underlie injury-induced neuronal plasticity in the central nervous system and to develop novel therapeutic approaches for recovery of function. Unilateral lesions of the rat somatic sensory cortex was made to determine the effect of injury on the expression of plasticity-related molecules that regulate neurotransmission. Excitatory amino acid receptors, including both NMDA and AMPA subtypes, were reduced in the penumbra of the lesions by 24 hours. Degenerative changes are also seen in the ipsilateral thalamus. These observations suggest that there could be changes in neurotransmission around the lesion. To address this question electrophysiological studies and in addition behavioural analysis have been initiated to determine the onset and extent of the deficits.
Research on the visual system involved understanding how the brain plans and initiated actions, how partially prepared actions are canceled and how the consequences of actions are registered. Understanding how the normal brain controls action is necessary to understand the causes underlying various psychopathologies and motor abnormalities where there is a failure of control, such as observed in Parkinson’s and Huntington’s disease. These questions are addressed by recording eye movements from normal subjects during cognitive tasks that are designed to induce errors. The results of these experiments suggest that the correction of errors maybe predictive in nature, occurring before error is made. These results, although counterintuitive, suggest that our brains possess the capacity to predict the consequences of actions before they are executed.
In order to understand whether non-primary auditory areas which perceive speech-related sounds are present early in development, research has been initiated to study the cytoarchitecture of primary and non-primary auditory cortical areas during late fetal and early postnatal ages in humans using different histochemical and immunohistochemical staining methods on post-mortem brain tissue. In addition, an animal model (zebra finches) will be used to study the pathways involved in the acquisition of learned vocalization (song). Findings from these studies will be related to comparable regions in humans (such as the Broca’s area).
Computational neuroscience
The differential diagnosis of MRI scans of brain tumour recurrence vis-à-vis post-radiation necrosis is a confounding neurological problem. Another problem underlying the use of MRI scans is the use of high-priced gadolinium agents for increasing contrast in neuroimaging. Stochaostic resonance has been used to produce enhanced images from non-contrast scans that compare favourably with gadolinium enhanced scans. The enhanced images obtained by this method can also be used to differentiate between tumours and postradiation necrosis in the brain. Neurocomputational methods have been applied towards understanding of spatiotemporal information processing in neural system and it has been found that multiplexed information transmission is a basic operation, whether in neuronal transmission, cortical transmission or linguistic transmission. In addition, a vector-tensor matrix model has been developed to relate spatiotemporal information flux with the activation induced by the neuromodulator. The empirical data confirm the non-equilibrium information dynamics model, that is, more multiplexed channelling opens up as a neural system operates under increasingly intensive conditions.
A novel computational approach has been initiated to study acoustic features of human speech by setting up the modulation spectrum. The speech and language laboratory is looking at the development of different spectral and temporal features of speech in normal developing children. Speech samples from different schools in Gurgaon have been obtained and the data is being analyzed. Voice samples from children who have speech impairments are also being obtained. After analysis, these will be compared with the data obtained from normal children.
Academic activities
As a Deemed University, NBRC is continuing with its Ph.D. programme for Research Fellows with a comprehensive coursework for the first year, covering a diverse range of topics related to neuroscience. A M.Sc. programme in neuroscience has also been initiated. The teaching programme aims at developing a solid foundation in the basic areas of neuroscience in an interdisciplinary manner and to expose the students to the latest research in different areas.
Networking - national and international collaboration
The Centre continued to expand and strengthen the existing national network of universities and the total number currently stands at 44. It also shared its digital library with its networked centres and all facilities are open to the scientists and students from networked centres.
Several research projects have been awarded to the scientists from the International and the National agencies. Two research grants have been awarded from the Wellcome Trust, UK, two from National Institutes of Health (NIH), USA, one each from FIRCA, USA, RIKEN Brain Research Institute, Japan, the US-India Fund for Cultural, Educational and Scientific Cooperation and the Third World Academy of Sciences (TWAS). The collaborations also continued with National Institute of Mental Health (NIMH), USA and Pavlov institute in Russia, in addition to the research grants from the Department of Biotechnology and Department of Science and Technology. The 6th China- India-Japan-Korea Joint Workshop on Neurobiology and Neuroinformatics was organized on November 18-19, 2004, with an aim to bring together experimental and computational neuroscientists and mathematicians from these countries to interact with each other.
Physical progress
The student’s hostel at the institute’s permanent campus at Manesar is complete and fully operational. The foundation stone of the fMRI research facility was laid by the Hon’ble Minister for Science and Technology, Shri Kapil Sibal on September 29, 2004 and its construction will begin soon. Necessary steps have been initiated to construct the phase-II campus of NBRC.
National Centre for Plant Genome Research, New Delhi
The research programme aimed to contribute to the understanding of the structure, expression and function of genes along with arrangement of genes on plant genomes and manipulation of plant genes/genomes to breed improved varieties of food and industrial crops for high yields and of better quality products. The Centre was established to contribute in the achievement of such crops as a part of national effort for meeting the challenges in the midst of fast pace of international genomic research and grasping of opportunities on longterm basis.
The progress made is summarized briefly hereunder :
Structural and functional genomics of chickpea and Catharanthus structural genomics
Genome structure analysis in chickpea:
Considerable progress has been made in the analysis of genome structure of the important legume crop chickpea Cicer arietinum. A microsatellite enriched genomic library has been constructed and from it 600 microsatellite containing clones have been isolated and sequence characterized. Of these 300 have been used to design STMS primer pairs to study polymorphism among a large number of chickpea accessions and the wild species. The markers used have been found to be conserved in the cultivated and wild species of Cicer. The hierarchical relationships between the accessions have been revealed. A set of 126 recombinant inbred lines (RILs) of chickpea is being used to construct the linkage map and tag traits for certain grain quality and yield in C. arietinum. In addition, STMS and AFLP markers have been used to tag Fusarium-resistance locus on two RIL populations. Currently, these markers have been put on the chickpea linkage map with a long term goal to clone the ‘R’ gene for fusarium wilt in chickpea. STMS markers have also been used to reveal linkages between them and the resistance genes. Genetic and mapping analyses using STMS, AFLP, RGA, SCAR and ISSR markers are in progress.
Genomic structure studies in Catharanthus roseus: Several genomic libraries of C. roseus have been constructed in order to develop RFLP and STMS markers. These have been used to reveal hierarchical relationships between its accessions having diverse geographical origins. Dendograms have been constructed based on DNA, biochemical and morphological markers, which show presence of enormous variability in the genetic resources available in the gene bank. The mapping population developed from a cross involving the genetically distant accessions have been used to study segregation pattern of morphological, biochemical and DNA markers. The first draft of the genetic map of C. roseus has been developed.
Functional Genomics
Genome Function Analysis in Chickpea
Genomics of wilt disease:
To elucidate, the signal transduction mechanism(s) involved in plant fungal interaction during compatible and incompatible reaction, subtractive hybridization libraries have been constructed from Fusarium infected chickpea tissues for DNA array analysis. In total 8500 recombinant clones were obtained. Transcript profiling by macroarray analysis of 2184 clones identified a set of 1284 differentially expressed ones. Putative functions were assigned to all these clones after DNA sequencing and computational data analysis. In total 706 unigene set have been identified.
Genomics of blight disease:
Transcript profiling has been carried out in chickpea crop infected with Ascochyta rabiei to identify genes of three categories namely ‘late’, ‘early’ and ‘immediate early’ genes using plants after 9 days, 24 hrs and 3 hrs of fungal spore spray respectively. Several genes showed induced expression in response to infection. Some of the important genes like WRKY and bHLH transcription factors, HS1 Pro1 –like resistance gene, protein kinase and MAP kinase genes are now being selected and characterized in the laboratory. 5’-upsteam region of these genes were also isolated and presently been characterized in transgenic plants. The transgenic tobacco plants harbouring 5’ upstream sequence of HS1 Pro1 –like resistance gene showed localized expression in trichomes.
Genomics of drought tolerance;
i) Proteomic approach for detection of water-stress responsive genes: Studies have been initiated to develop subcellular proteomes of legumes and cereals under water stress in order to identify the fungal transduction pathway involved in the stress and to clone novel drought response protein genes. A detailed cell-wall proteome of chickpea has been developed. Differentially expressed CW proteins in chickpea and ABA-dependent and ABAindependent cytoplasmic proteins in grasspea have been identified. Molecular characterization of these proteins by mass spectroscopy and differential subcellular proteomes in rice, chickpea and grasspea under waterstress is in progress.
ii) Transcriptome approaches to drought tolerance in chickpea: As part of chickpea genomics program, an EST database of dehydration-inducible transcripts of chickpea has been constructed. Temperate grain legumes such as pea, fava bean, lentil, chickpea and others share similar gene arrangements. It is expected that this data base will benefit the study of other legume plants by comparing the expression patterns of the transcripts. One hundred and one dehydration-inducible transcripts of chickpea have been identified by repetitive rounds of cDNA subtraction (a subtractive library of 3000 clones); differential DNA-array hybridization followed by northern-blot analysis and analyzed their responses to exogenous application of Abscisic acid (ABA). Steadystate expression levels of the dehydration-induced transcripts were monitored during the recovery period between two consecutive dehydration stresses. Cool season crops face intermittent drought. Exposure to drought and other abiotic stresses is known to increase tolerance of the plants against subsequent exposure to such stresses. Storage of environmental signals is also proposed. Pre-exposure to a dehydration shock improved adaptive response during subsequent dehydration treatment in a cool season crop Chickpea. This observation of the longer period of abundance of those transcripts in the recovery period has been correlated with the improved adaptation of the plants to subsequent dehydration stress.
In another approach the Chickpea homologue of DREB2 (CaDREB2), the master regulator of Arabidopsis drought-response has been cloned from a Chickpea full length cDNA library. The functional characterization of CaDREB2 is under progress.
Chickpea genes expressed in dark:
Two novel genes called, CaDIN1 for dark regulated expression, which is upregulated in dark, but also expressed in light and the other one, CaLIN1, which is downregulated in dark have been cloned from chickpea. It has been found that this gene is expressed in cotyledons and root but not in hypocotyl of light grown chickpea seedlings. In contrast the DRE1 gene is expressed in cotyledon and hypocotyls and not in root of dark grown chickpea seedlings.
Genome function analysis in Catharanthus roseus
Relationship between glycophytic response and secondary metabolism: Chemical induced salinity / drought resistant mutants isolated in C. roseus was characterised. By complementation analysis, thirteen GSR-1 to GSR-13 genes have been identified which upon loss of function give salt and drought tolerance. All the mutants harbour pleiotropic, morphological, biochemical and alkaloid phenotypes much distinct from wild type. They synthesize and accumulate proline and glycinebetaine in higher concentrations than the wild type. Several of these hyperaccumulate certain TIA alkaloids. The mechanism by which pleiotropy arises in these mutants is under investigation. The pyramiding of GSR mutants has been in progress and several lines harbouring two mutants in various permutations have been constructed. Partial function of a gsr mutant has been revealed.
Cloning and expression of TIA pathway gene/s and alkaloids in vivo and in vitro grown tissue of Catharanthus roseus:
Transcript analysis was done for three important TIA biosynthetic pathway genes in several accessions that differed in accumulation of pharmaceutically important alkaloids quantitatively. Correspondence was noted between transcriptional expression of genes and accumulation of TIA alkaloids in these cultivars, which revealed a possibility of using this technique for screening of germplasm for high yielding cultivars. Several clones of hairy roots, generated from C. roseus var. Prabal, were characterized with regard to Ri T-DNA gene integration. Among these, those found to express catharanthine in high amounts were identified.
In order to clone a novel peroxidase gene from C. roseus, which catalyses the coupling of the monomeric precursors - catharanthine and vindoline to yield anhydrovinblastine (AVLB), a leaf specific cDNA library was prepared. Five clones showed homology with peroxidase genes from different plant species in different degrees. RT-PCR based methods using gene specific primers from heterologous species were also used toclone the peroxidase gene from C. roseus. The amplifiedband of expected size was obtained which is now being sequenced.
Nutritional genomics
AmA1 protein rich food crops:
Nutritionally enriched potato lines developed by transfer of Ama1 gene of Amaranthus hypochondriacus into elite cultivars of potato have now reached advanced stage. The selected Kufri AmA1+ potato transgenics accumulate proteins in large amounts (30 – 60% increase) with considerable increase in the amount of essential amino acids in transgenic tubers. The regeneration of rice, sweet potato and cassava has been achieved. Transformation protocols using marker gene and AmA1 in these systems are being standardized.
Development of low oxalate fungal tolerant vegetable and grain crops using OXDC gene:
Oxalic acid is known to act as signal for fungal pathogenecity in wide range of phtyopathogenic fungi. In order to develop low oxalate, fungal tolerant tomato, OXDC gene was genetically engineered in this crop. OXDC expressing tomato plants were found to be resistant to fungal infection.
High concentrations of oxalates in fruits and vegetables are toxic and lead to several kinds of disease conditions including kidney stone formation among consumers. To remediate oxalate toxicity in vegetables and grain crops, considerable progress has been made to develop transgenics using OXDC gene in tomato which has very low content of oxalate and tolerant to fungal infection. OXDC tomato transgenic lines are now in final stage of field trial. Protocols for the introduction of OXDC gene into groundnut, soybean, Lathyrus and spinach are at different stages of standardization.
Functional genomics of other plant systems
Genetics of compound leaf morphogenesis in Pisum sativum:
A new gain of function dominant mutation in a gene, multifoliate pinna, has been described. The mutation causes increased ramification singly and in combination with the mutations in AF and TL genes.
Analysis of light signal transduction pathway in Arabidopsis thaliana: Work is in progress to reveal the new features of light signaling pathway generally operating in plants by use of A. thaliana as a model system. In this regard CAB1 and Z-GATA containing promoters have been shown to be downregulated in hy5 mutants in the light. Moreover a promoter with Z-box alone has been shown to be downregulated in hy5 mutants both in dark and light conditions suggesting identical signal regulation in skotomorphogenic and photomorphogenic pathways. Two transcription factors that can specifically interact with Z-box have been identified. The promoter of TOP2 has been dissected to identify a minimal region in it, which responds to phytochrome mediated light signaling.
Genes for increasing shelf life in fruits and vegetables: The á-mannosidase has been identified for genetic engineering to increase shelf life of fruits and vegetables. The á-mannosidase enzyme has been purified to homogeneity from capsicum and tomato. Antibodies specific to á-mannosidase have been raised. Currently, the cDNA library construction in order to clone the gene is in progress.
Expressed sequence tag (EST) sequencing
Progress has been made in the EST database development by sequencing of 3500 cDNA clones in chickpea and periwinkle.
The Centre has developed a high artemisinin content & high leaf yielding variety of Artemisia annua CV Jeevanraksha-2 and transferred the know-how for commercial utilization of the variety to a company. The Centre’s scientists have published a number of papers in high impact journals developed linkages with national and international institutions.
The Centre has made rapid progress in construction of its new campus, a few kilometers away from the present interim premises. Many of the buildings like the Service block, Transit Houses, Essential staff quarters, Director’s residence, Underground Reservoir, Overhead Tank, etc. already completed in all respects have been taken over. The Main Institutional Building comprising laboratory complex and other related activities is complete and final finishing work is being carried out and would be occupied very soon.
The work pertaining to ‘treated effluent re-use’ has also been completed and the treated effluent is being used for the horticulture purposes on the campus. Steady progress has been made in the work relating to horticulture, landscaping and development of experimental fields.
Institute of Bioresources and Sustainable Development, Imphal.
The scientific progress of the institute was reviewed in the third meeting of the Scientific Advisory Committee (SAC) of the IBSD held in September, 2004. Meetings of the Governing Council and IBSD Society were also held in September, 2004 and November, 2004 respectively. A ‘National Consultation on Conservation and Sustainable Utilization of Bioresources of North- East India – Priorities for Networking’ was organized during December, 2004 mainly to identify the priorities of research for the institute and developing network projects in the North-East region. As a result of the national consultation, five network projects are being developed by the institute. The focus of the institute is bioresource development and their sustainable use through biotechnological interventions for the socioeconomic growth of the North-East region. The institute at present operates with three scientific divisions viz bioresources data base unit, medicinal plant and horticultural resources division and microbial resources division. Salient features of the work accomplished during the year are given below :
Bioresources data base unit
A model-digitized database of bioresources with more than 3000 species record including 575 floras and 2466 fauna of North-East region has been developed. Separate databases of orchids, medicinal and aromatic plants, Zingibers and legumes of Manipur have also been developed. Survey, collection and evaluation of the bioresource wealth in the Indo-Burma region has been initiated.
Medicinal plant and horticultural resources
Phytochemical analysis of Cinnamomum tamala and Centella asiatica has been carried out. High percentage of eugenol was found to be present in C. tamala leaves collected from the region. In vitro protocorm development and seedling formation have been achieved in two popular orchids viz, Aerides odoratum and Dendrobium transparens. In vitro cultures have been established using rhizome buds of various Zingiberales (Kaempferia galanga, Curcuma longa var. lakadan, C. amada, Hedychium coronarium, H. coccineum, H.maximum, Zingiber officinale, Z. cassumunar, Z.zerumbat), embryos of Citrus species (C. sinensis, C aurentium, C. aurentifolia, C. macroptera, C. maxima), bulbs of Allium species (A. hookerii, A. ascalonicum, A. odorum), Pogostemon parviflorus, Elsholtizia blanda and Centella asiatica. Grains of 13 local Manipur rice cultivars and seed of 11 wild and cultivated legumes were collected and preserved. Herbarium specimens of about 20 species and illustrated diagrams of eight species have been prepared and maintained.
Microbial resources
Based on the colony and cell morphology, 11 predominant Bacillus groups (156 strains) were characterized from traditional fermented soybean (Hawaijar) samples. About 160 microbial resources viz. Mucor, Rhizopus, Aspergillus, Saccharomyces, Candida and other fungal groups associated with a traditional starter used for rice wine production (Hamie) were partially characterized.
Survey has been carried out at rice fields and acidic soils of Manipur for collection of phosphate solubilising microorganisms. Out of 150 isolates collected so far, 60 are fluorescent Pseudomonas sp., 28 are fungal isolates and the remaining 62 isolates are other bacterial isolates. Physiological and biochemical features of nitrogenfixing cultures from wild leguminous plants were analyzed and a few efficient strains were identified as Rhizobium phaseoleae, R. leguminosarum, R. legumins, Bradyhizobium japonicum and B. leguminous. In pot culture experiments, significant effect was observed by the combined treatment of R.phaseoleae, Pseudomonas striata and P. aeruginosa in the plants in the plants. Attempts have been made to systematically survey and isolate cyanobacterial flora from rice growing areas of manipur including Loktak Lake and other freshwater reservoirs. A total of 80 strains belonging to 13 genera have been isolated in pure cultures from different locations of manipur.
Other activities
The institute co-sponsored a workshop on “Rice Heritage of the North-East - Challenges, Opportunities and Strategies for the Future” organized by M. S. Swaminathan Research Foundation, Chennai during November 5-6, 2004 at Shillong to commemorate the International Year of Rice. A two-day training programme on “Biotechnology Tools for Conservation and Sustainable Utilization of Bioresources” was organized by the institute during June 25-26, 2004. About 40 young scientists of the region participated in the training programme. The IBSD has also started shortterm training for M.Sc students of the region. Eleven students from eight institutes from outside the state have been given training. The scientists of the IBSD also participated in various seminars / workshops / training courses relevant to their areas as a part of in-house human resource development.
Institute of Life Sciences, Bhubaneswar
The Institute continued its activities in the areas of molecular biology of aging and cancer, infectious diseases including malaria, filariasis, cholera; bioresource development and utilization including molecular aspects to stress adaptation, plant microbe interaction and microbial prospecting, plant molecular biology and functional genomics; and environmental biotechnology.
Some of the salient research achievements are:
Molecular biology of aging and cancer
Aging : Research is being pursued to identify factors responsible for aging process.
Molecular analysis of age-dependent alterations in the activity of transcription factor NFkB and androgen receptor (AR): Most of the transcription factors interacting with AR promoter are down regulated; few are up-regulated while others show no change in DNA binding during aging. Correlation of increased phosphorelation and degradation of IkBá with higher DNA binding activity of NFkB during aging has been demonstrated.
Cloning, sequencing and characterization of senescence marker protein (SMP 30): Sequencing was done up to -3 kb of SMP30 promoter. Promoter sequence has been analyzed on Transcription factor data base. Several 5’ deleted SMP30 promoter fragments have been generated.
Identification, cloning and characterization of agedependent genes: eight cDNA bands were reproducible and they were differentially expressed. These include few important genes involved in stress pathways and general metabolism. For the first time, it has been reported that in the rat liver, the expression of pancreatic secretory trypsin inhibitor -II (PSTI-II) gene declines during aging.
Cancer : Role of potent cell cycle regulators in tobacco chewing mediated oral cancer progression and the regulation of Cdk4 and cyclin D1 genes in oral carcinoma are being investigated.
Role of different cell cycle regulators such as, cyclin D1 and Cdks in tobacco chewing mediated Oral Squamous Cell Carcinoma has been identified that may be used as a diagnostic marker for detection of oral cancer. Role of p53 in leukemia has been established. Any inhibitor or drug targeting these sites that can inhibit over expression of these genes, may be very useful in control of cancer.
Infectious diseases
The common locally prevalent infectious diseases are malaria, filariasis, diarrhoeal diseases, respiratory tract infection, middle ear infection, viral hepatitis etc. The focus of research in infectious diseases is to understand (a) molecular epidemiology and mechanism of drug resistance in malaria, development of transmission blocking vaccines (b) development of diagnostic tests for filariasis and to understand the mechanism of innate immunity in mosquito vectors (c) ecology, mechanism of survival, development of virulence, and genetic diversity of cholera pathogen (d) genetic diversity, identification of virulence genes and their correlation with symptoms of Helicobacter pylori infection (e) monitoring emergence of multi-drug resistance clones of Streptococcus pneumoniae/pyogenes responsible for pneumonia and otitis media, elucidation of the infection mechanism, identification and characterization of regulatory network, and virulence associated genes.
Cholera : A cholera toxin gene-negative strain of Vibrio cholerae O139 Bengal was characterized, which was devoid of the core of the CTX genetic element, failed to produce cholera toxin, showed resistance to ampicillin, cotrimoxazole, furazolidone, streptomycin and belongs to ribotype I and ERIC type-IV, not described earlier among cholera toxin gene-negative strains.
Malaria : Mutation in drug resistance PfCRT gene in Plasmodium falciparum in endemic areas of Orissa was demonstrated. Polymorphism in variable regions and mutation in a conserved region (block-17) of MSA-1 gene was demonstrated in P. falciparum. Telomerase activity has been demonstrated in gametocytes, a sexual stage of the malaria, a possible drug target that may mimic the growth of parasite in human.
Filariasis : A molecular diagnostic method for detection of filariasis worms Brugia malayi and Wochereria bancrofti present in blood and mosquitoes has been developed. Prevalence of Wolbachia in different mosquito species and clearance of Wolbachia from Aedes aegypti has been studied. An HPLC method for determination of invermectin in plasma lymphatic filariasis patients has also been developed Infrastructure development of Institute of Life Sciences, Bhubaneswar
Bioresource development and utilization
The institute is undertaking studies on (a) molecular aspects to stress adaptation including the role of stress hormone and photosynthesis in wheat plant to decipher the mechanism underlying the salicylic acid dependent stimulation in photosynthetic activity, study of the effect of light on seed germination and seedling establishment: an assessment of antioxidant enzyme activities, and photosynthetic adaptation of selected aquatic angiosperm to varied light regimes and (b) plant microbe interaction including identification of the various genetic determinants in Mesorhizobium ciceri during symbiotic nitrogen fixation in chickpea and over expression of some of the identified genes for enhanced biological nitrogen fixation, and characterization of microbial culture isolated from extreme environment, and detection and expression of the industrial important enzymes viz. proteases, lipases, antimicrobial activity and lithotrophic genes from these microbial strains.
Molecular aspects to stress adaptation:
The mechanism underlying the salicylic acid dependent stimulation in photosynthetic activity has been developed. A thermostable SOD enzyme has been purified and its physicochemical properties studied. The protein was purified to nearly 3000 fold by using solventsolvent extraction protocol (less cost effective). The semi-purified protein was further extracted by acrylamide gel electrophoresis and the eluted protein was studied for its thermo-stability. The enzyme was found to be fully functional at 100oC heated for 10 min. A octameric catalase has also been purified and its further characterization is in progress.
Purification of the catalase enzyme and its further biochemical characterization:
Catalase enzyme have been purified and polyclonal antibodies have been prepared.
Photosynthetic adaptation of Hydrilla verticillata:
In order to identify any novel gene expression, the genomic DNA and total RNA has been purified for which protocol has been standardized. The protocol is useful for purification of DNA and RNA for restriction enzyme digestion and for PCR amplification. Using random primers different fragments have been amplified in variable sizes from Hydrilla genomic DNA.
Molecular microbiology-plant microbe interaction
Role of different metabolic pathways in biological nitrogen fixation of Mesorhizobium ciceri:
Plant microbe interaction: Role of different metabolic pathways in biological nitrogen fixation of Mesorhizobium ciceri has been identified. Transposon Tn5 insertion mutagenesis was employed to Mesorhizobium ciceri for isolating mutants defective in various metabolic pathways. Mutants’ requiring different amino acids were identified. Mutants unable to grow on aspartic acid and fumaric acid have also been characterized. In doing so, Tn5 containing flanking region from one of such mutant’s had been cloned into the pBlueScript vector. Primary sequencing result indicated the insertion of Tn5 into the Sigma rpoN factor. It appeared that rpoN gene is involved in aspartate and fumarate uptake and symbiotic nitrogen fixation.
Environmental biotechnology
Studies have been undertaken to identify and characterize enzymes and proteins involved in ion transport involved in homeostasis, osmotic adjustment and its regulation, molecular aspects of uptake, transport and sequestration of heavy metals, and oxidative stress build-up under stress and its regulation. Isoenzymes of SOD and peroxidases induced under salt stress have been identified. Work is also in progress on understanding Cr toxicity and tolerance mechanism(s) in plants growing in the chromites mining areas of Orissa. Protective role of proline against metal toxicity has been studied. A relationship between oxidative stress and salt-tolerance was found with few phytoplankton species like Skeletonema costatum, Nitzschia sigma, Oscillatoria erythraea, Achnanthes compacta, etc.
During the year, the Governing Body and Scientific Advisory Committee met twice. One meeting of the Society was held under the Chairmanship of Hon’ble Minister S&T. The SAC has identified specific thrust areas. Doctoral committee for research students have been constituted. Future plans for infrastructure development have been worked out. A new animal house, and a green house would be constructed shortly.