S. Ramaswamy, Ph.D.
CEO of c-CAMP, Dean, inStem, NCBS, Bangalore, India
Discovery, engineering and applications of Blue Fish Protein with Red Fluorescence
Swagatha Ghosh, Chi-Li Yu, Daniel Ferraro, Sai Sudha, Wayne Schaefer, David T Gibson and S. Ramaswamy
Fluorescent proteins and their applications have revolutionized our understanding of biology significantly. In spite of several years since the discovery of the classic GFP, proteins of this class are used as the standard flag bearers. We have recently discovered a protein from the fish Sanders vitrius that shows interesting fluorescent properties – including a 280 nm stoke shift and infrared emission. The crystal structure of the wild type protein shows that it is a tetramer. We have engineered mutations to make a monomer with very similar fluorescent properties. We have used this protein for tissue imaging as well as for in cell-fluorescence successfully
Krishnakumar Menon, Ph.D.
Associate Professor, Centre for Nanosciences & Molecular Medicine, Amrita University, Kochi, India
A Far-Western Clinical Proteomics Approach to Detect Molecules of Clinical and Pathological Significance in the Neurodegenerative Disease Multiple Sclerosis.
Multiple Sclerosis (MS), an autoimmune neurodegenerative disorder of the central nervous system. The disease affects young adults at their prime age leading to severe debilitation over several years. Despite advances in MS research, the cause of the disease remains elusive. Thus, our objective is to identify novel molecules of pathological and diagnostic significance important in the understanding, early diagnosis and treatment of MS. Biological fluids such as cerebrospinal fluid (CSF), that bathe the brain serve as a potential source for the identification of pathologically significant autoantibody reactivity in MS. In this regard, we report the development of an unbiased clinical proteomics approach for the detection of reactive CSF molecules that target brain proteins from patients with MS. Proteins of myelin and myelin-axolemmal complexes were separated by two-dimensional gel electrophoresis, blotted onto membranes and probed separately with biotinylated unprocessed CSF samples. Protein spots that reacted specifically to MS-CSF’s were further analyzed by matrix assisted laser desorption ionization-time-of-flight time-of-flight mass spectrometry. In addition to previously reported proteins found in MS, we have identified several additional molecules involved in mitochondrial and energy metabolism, myelin gene expression and/or cytoskeletal organization. Among these identified molecules, the cellular expression pattern of collapsin response mediator protein-2 and ubiquitin carboxy-terminal hydrolase L1 were investigated in human chronic-active MS lesions by immunohistochemistry. The observation that in multiple sclerosis lesions phosphorylated collapsin response mediator protein-2 was increased, whereas Ubiquitin carboxy-terminal hydrolase L1 was down-regulated, not only highlights the importance of these molecules in the pathology of this disease, but also illustrates the use of our approach in attempting to decipher the complex pathological processes leading to multiple sclerosis and other neurodegenerative diseases. Further, we show that in clinicaly isolated syndrome (CIS), we could identify important molecules that could serve as an early diagnostic biomarker in MS.
Bharat B. Chattoo, Ph.D.
Professor, Faculty of Science M.S.University of Baroda, India
Biology of plant infection by Magnaporthe oryzae
The rice blast disease caused by the ascomycetous fungus Magnaporthe oryzae is a major constraint in rice production. Rice-M.oryzae is also emerging as a good model patho-system to investigate how the fungus invades and propagates within the host. Identification and characterisation of genes critical for fungal pathogenesis provides opportunities to explore their use as possible targets for development of strategies for combating fungal infection and to better understand the complex process of host-pathogen interaction.
We have used insertional mutagenesis and RNAi based approaches to identify pathogenesis related genes in this fungus. A large number of mutants were isolated using Agrobacterium tumefaciens mediated transformation (ATMT). Characterisation of several interesting mutants is in progress. We have identified a novel gene, MGA1, required for the development of appressoria. The mutant mga1 is unable to infect and is impaired in glycogen and lipid mobilization required for appressorium development. The glycerol content in the mycelia of the mutant was significantly lower as compared to wild type and it was unable to tolerate hyperosmotic stress. A novel ABC transporter was identified in this fungus. The abc4 mutant did not form functional appressoria, was non-pathogenic and showed increased sensitivity to certain antifungal molecules implying the role of ABC4 in multidrug resistance (MDR). Another mutant MoSUMO (MGG_05737) was isolated using a Split Marker technique; the mutant showed defects in growth, germination and infection. Immuno-fluorescence microscopy revealed that MoSumo is localized to septa in mycelia and nucleus as well as septa in spores. Two Dimensional Gel Electrophoresis showed differences in patterns of protein expression between Wild Type B157 and MoΔSumo mutant. We also isolated and charaterised mutants in MoALR2 (MGG_08843) and MoMNR2 (MGG_09884). Our results indicate that both MoALR2 and MoMNR2 are Mg2+ transporters, and the reduction in the levels of CorA transporters caused defects in surface hydrophobicity, cell wall stress tolerance, sporulation, appressorium formation and infection are mediated through changes in the key signaling cascades in the knock-down transformants. (Work supported by the Department of Biotechnology, Government of India)
Terry Hermiston, Ph.D.
Vice President, US Biologics Research Site Head, US Innovation Center Bayer Healthcare, USA
ColoAd1 – An oncolytic adenovirus derived by directed evolution
Attempts at developing oncolytic viruses have been primarily based on rational design. However, this approach has been met with limited success. An alternative approach employs directed evolution as a means of producing highly selective and potent anticancer viruses. In this method, viruses are grown under conditions that enrich and maximize viral diversity and then passaged under conditions meant to mimic those encountered in the human cancer microenvironment. Using the “Directed Evolution” methodology, we have generated ColoAd1, a novel chimeric oncolytic adenovirus. In vitro, this virus demonstrated a >2 log increase in both potency and selectivity when compared to ONYX-015 on colon cancer cells. These results were further supported by in vivo and ex vivo studies. Importantly, these results have validated this methodology as a new general approach for deriving clinically-relevant, highly potent anti-cancer virotherapies. This virus is currently in clinical trials as a novel treatment for cancer.
V. Nagaraja Ph.D.
Professor, Indian Institute of Science, Bengaluru, India
Perturbation of DNA topology in mycobacteria
To maintain the topological homeostasis of the genome in the cell, DNA topoisomerases catalyse DNA cleavage, strand passage and rejoining of the ends. Thus, although they are essential house- keeping enzymes, they are the most vulnerable targets; arrest of the reaction after the first trans-esterification step leads to breaks in DNA and cell death. Some of the successful antibacterial or anticancer drugs target the step ie arrest the reaction or stabilize the topo -DNA covalent complex. I will describe our efforts in this direction – to target DNA gyrase and also topoisomerase1 from mycobacteria. The latter, although essential, has no inhibitors described so far. The new inhibitors being characterized are also used to probe topoisomerase control of gene expression.
In the biological warfare between the organisms, a diverse set of molecules encoded by invading genomes target the above mentioned most vulnerable step of topoisomerase reaction, leading to the accumulation of double strand breaks. Bacteria, on their part appear to have developed defense strategies to protect the cells from genomic double strand breaks. I will describe a mechanism involving three distinct gyrase interacting proteins which inhibit the enzyme in vitro. However, in vivo all these topology modulators protect DNA gyrase from poisoning effect by sequestering the enzyme away from DNA.
Next, we have targeted a topology modulator protein, a nucleoid associated protein(NAP) from Mycobacterium tuberculosis to develop small molecule inhibitors by structure based design. Over expression of HU leads to alteration in the nucleoid architecture. The crystal structure of the N-terminal half of HU reveals a cleft that accommodates duplex DNA. Based on the structural feature, we have designed inhibitors which bind to the protein and affect its interaction with DNA, de-compact the nucleoid and inhibit cell growth. Chemical probing with the inhibitors reveal the importance of HU regulon in M.tuberculosis.
Lalitha Subramanian, Ph.D.
Chief Scientific Officer & VP, Services at Scienomics, USA
Nanoscale Simulations – Tackling Form and Formulation Challenges in Drug Development and Drug Delivery
Lalitha Subramanian, Dora Spyriouni, Andreas Bick, Sabine Schweizer, and Xenophon Krokidis Scienomics
The discovery of a compound which is potent in activity against a target is a major milestone in Pharmaceutical and Biotech industry. However, a potent compound is only effective as a therapeutic agent when it can be administered such that the optimal quantity is transported to the site of action at an optimal rate. The active pharmaceutical ingredient (API) has to be tested for its physicochemical properties before the appropriate dosage form and formulation can be designed. Some of the commonly evaluated parameters are crystal forms and polymorphs, solubility, dissolution behavior, stability, partition coefficient, water sorption behavior, surface properties, particle size and shape, etc. Pharmaceutical development teams face the challenge of quickly and efficiently determining a number of properties with small quantities of the expensive candidate compounds. Recently the trend has been to screen these properties as early as possible and often the candidate compounds are not available in sufficient quantities. Increasingly, these teams are leveraging nanoscale simulations similar to those employed by drug discovery teams for several decades. Nanoscale simulations are used to predict the behavior using very little experimental data and only if this is promising further experiments are done. Another aspect where nanoscale simulations are being used in drug development and drug delivery is to get insights into the behavior of the system so that process failures can be remediated and formulation performance can be improved. Thus, the predictive screening and the in-depth understanding leads to experimental efficiency resulting in far-reaching business impacts.
With specific examples, this talk will focus on the different types of nanoscale simulations used to predict properties of the API in excipients and also provide insight into system behavior as a function of shelf life, temperature, mechanical stress, etc.
![Delegate Talk: Intrinsic modulation of cytokine response by mycobacteria @ Acharya Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/btindia.jpg)
Sukhithasri V, Nisha N, Vivek V and Raja Biswas
The host innate immune system acts as the first line of defense against invading pathogens. During an infection, the host innate immune cells recognize unique conserved molecules on the pathogen known as Pathogen Associated Molecular Patterns (PAMPs). This recognition of PAMPs helps the host mount an innate immune response leading to the production of cytokines (Akira et al. 2006). Peptidoglycan, one of the most conserved and essential component of the bacterial cell wall is one such PAMP. Peptidoglycan is known to have potent proinflammatory properties (Gust et al. 2007). Host recognize peptidoglycan using Nucleotide oligomerization domain proteins (NODs). This recognition of peptidoglycan activates the NODs and triggers downstream signaling leading to the nuclear translocation of NF-κB and production of cytokines (McDonald et al. 2005). Pathogenic bacteria modify their peptidoglycan as a strategy to evade innate immune recognition, which helps it to establish infection in the host. These peptidoglycan modifications include O-acetylation and N-glycolylation of muramic acid and N-deacetylation of N-acetylglucosamine (Davis et al. 2011). Modification of mycobacterial peptidoglycan by N-glycolylation prevents the catalytic activity of lysozyme (Raymond et al. 2005). Additionally, mycobacterial peptidoglycan is modified by amidation for unknown reasons.
Here, we have investigated the role of amidated peptidoglycan in Mycobacterium sp in modulating the innate immune response. We isolated amidated peptidoglycan from Mycobacterium sp and non-amidated peptidoglycan from Escherichia coli. We made a comparative analysis of the cytokine response produced on stimulation of innate immune cells by peptidoglycan from E. Coli and Mycobacterium sp. Macrophages and whole blood were treated with peptidoglycan and the cytokines secreted into spent medium and plasma respectively were analyzed using ELISA. Our results show that peptidoglycan from Mycobacterium sp is less effective in stimulating innate immune cells to produce cytokines. This intrinsic modulation of the cytokine response suggests that mycobacteria modify their peptidoglycan by amidation to evade innate immune response.