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
Sanjeeva Srivastava, Ph.D.
Assistant Professor, Proteomics Lab, IIT-Bombay, India
Identification of Potential Early Diagnostic Biomarkers for Gliomas and Various Infectious Diseases using Proteomic Technologies
The spectacular advancements achieved in the field of proteomics research during the last decade have propelled the growth of proteomics for clinical research. Recently, comprehensive proteomic analyses of different biological samples such as serum or plasma, tissue, CSF, urine, saliva etc. have attracted considerable attention for the identification of protein biomarkers as early detection surrogates for diseases (Ray et al., 2011). Biomarkers are biomolecules that can be used for early disease detection, differentiation between closely related diseases with similar clinical manifestations as well as aid in scrutinizing disease progression. Our research group is performing in-depth analysis of alteration in human proteome in different types of brain tumors and various pathogenic infections to obtain mechanistic insight about the disease pathogenesis and host immune responses, and identification of surrogate protein markers for these fatal human diseases.
Applying 2D-DIGE in combination with MALDI-TOF/TOF MS we have analyzed the serum and tissue proteome profiles of glioblastoma multiforme; the most common and lethal adult malignant brain tumor (Gollapalli et al., 2012) (Figure 1). Results obtained were validated by employing different immunoassay-based approaches. In serum proteomic analysis we have identified some interesting proteins like haptoglobin, ceruloplasmin, vitamin-D binding protein etc. Moreover, proteomic analysis of different grades (grade-I to IV) of gliomas and normal brain tissue was performed and differential expressions of quite a few proteins such as SIRT2, GFAP, SOD, CDC42 have been identified, which have significant correlation with the tumor growth. While proteomic analysis of cerebrospinal fluid from low grade (grade I & II) vs. high grade (grade III & IV) gliomas revealed modulation of CSF levels of apolipoprotein E, dickkopf related protein 3, vitamin D binding protein and albumin in high grade gliomas. The prospective candidates identified in our studies provide a mechanistic insight of glioma pathogenesis and identification of potential biomarkers. We are also studying the role of JAK/STAT interactome and therapeutic potential of STAT3 inhibitors in gliomas using proteomics approach. Several candidates of the JAK/STAT interactome were identified with altered expression and a significant correlation was observed between STAT3 and PDK1 transcript expression level.
We have also investigated the changes in human serum proteome in different infectious diseases including falciparum and vivax malaria (Ray et al., 2012a; Ray et al., 2012b), dengue (Ray et al., 2012c) and leptospirosis (Srivastava et al., 2012). Although, quite a few serum proteins were found to be commonly altered in different infectious diseases and might be a consequence of inflammation mediated acute phase response signaling, uniquely modulated candidates were identified in each pathogenic infection indicating the some inimitable responses. Further, a panel of identified proteins consists of six candidates; serum amyloid A, hemopexin, apolipoprotein E, haptoglobin, retinol-binding protein and apolipoprotein A-I was used to build statistical sample class prediction models employing PLSDA and other classification methods to predict the clinical phenotypic classes and 91.37% overall prediction accuracy was achieved (Figure 2). ROC curve analysis was carried out to evaluate the individual performance of classifier proteins. The excellent discrimination among the different disease groups on the basis of differentially expressed proteins demonstrates the potential diagnostic implications of this analytical approach.
Keywords: Diagnostic biomarkers, Gliomas, Infectious Diseases, Proteomics, Serum proteome
Acknowledgments: This disease biomarker discovery research was supported by Department of Biotechnology, India grant (No. BT/PR14359/MED/30/916/2010), Board of Research in Nuclear Sciences (BRNS) DAE young scientist award (2009/20/37/4/BRNS) and a startup grant 09IRCC007 from the IIT Bombay. The active support from Advanced Center for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Hospital (TMH), and Seth GS Medical College and KEM Hospital Mumbai, India in clinical sample collection process is gratefully acknowledged.
References :
- Ray S, Reddy PJ, Jain R, Gollapalli K. Moiyadi A, Srivastava S. Proteomic technologies for the identification of disease biomarkers in serum: advances and challenges ahead. Proteomics 11: 2139-61, 2011.
- Gollapalli K, Ray S, Srivastava R, Renu D, Singh P, Dhali S, Dikshit JB, Srikanth R, Moiyadi A, Srivastava S. Investigation of serum proteome alterations in human glioblastoma multiforme. Proteomics 12(14): 2378-90, 2012.
- Ray S, Renu D, Srivastava R, Gollapalli K, Taur S, Jhaveri T, Dhali S, Chennareddy S, Potla A, Dikshit JB, Srikanth R, Gogtay N, Thatte U, Patankar S, Srivastava S. Proteomic investigation of falciparum and vivax malaria for identification of surrogate protein markers. PLoS One 7(8): e41751, 2012a.
- Ray S, Kamath KS, Srivastava R, Raghu D, Gollapalli K, Jain R, Gupta SV, Ray S, Taur S, Dhali S, Gogtay N, Thatte U, Srikanth R, Patankar S, Srivastava S. Serum proteome analysis of vivax malaria: An insight into the disease pathogenesis and host immune response. J Proteomics 75(10): 3063-80, 2012b.
- Srivastava R, Ray S, Vaibhav V, Gollapalli K, Jhaveri T, Taur S, Dhali S, Gogtay N, Thatte U, Srikanth R, Srivastava S. Serum profiling of leptospirosis patients to investigate proteomic alterations. J Proteomics 76: 56-68, 2012.
- Ray S, Srivastava R, Tripathi K, Vaibhav V, Srivastava S. Serum proteome changes in dengue virus-infected patients from a dengue-endemic area of India: towards new molecular targets? OMICS 16(10): 527-36, 2012c.
* Correspondence: Dr. Sanjeeva Srivastava, Department of Biosciences and Bioengineering, IIT Bombay, Mumbai 400 076, India: E-mail: sanjeeva@iitb.ac.in; Phone: +91-22-2576-7779, Fax: +91-22-2572-3480
Suryaprakash Sambhara, DVM, Ph.D
Chief, Immunology Section, Influenza Division, CDC, Atlanta, USA
Making sense of pathogen sensors of Innate Immunity: Utility of their ligands as antiviral agents and adjuvants for vaccines.
Currently used antiviral agents act by inhibiting viral entry, replication, or release of viral progeny. However, recent emergence of drug-resistant viruses has become a major public health concern as it is limiting our ability to prevent and treat viral diseases. Furthermore, very few antiviral agents with novel modes of action are currently in development. It is well established that the innate immune system is the first line of defense against invading pathogens. The recognition of diverse pathogen-associated molecular patterns (PAMPs) is accomplished by several classes of pattern recognition receptors (PRRs) and the ligand/receptor interactions trigger an effective innate antiviral response. In the past several years, remarkable progress has been made towards understanding both the structural and functional nature of PAMPs and PRRs. As a result of their indispensable role in virus infection, these ligands have become potential pharmacological agents against viral infections. Since their pathways of action are evolutionarily conserved, the likelihood of viruses developing resistance to PRR activation is diminished. I will discuss the recent developments investigating the potential utility of the ligands of innate immune receptors as antiviral agents and molecular adjuvants for vaccines.
Shantikumar Nair, Ph.D.
Professor & Director, Amrita Center for Nanosciences & Molecular Medicine, Amrita University, India
Spatially Distributed and Hierarchical Nanomaterials in Biotechnology
Although nano materials are well investigated in biotechnology in their zero-, one- and two-dimensional forms, three-dimensional nanomaterials are relatively less investigated for their biological applications. Three dimensional nano materials are much more complex with several structural and hierarchical variables controlling their mechanical, chemical and biological functionality. In this talk examples are given of some complex three dimensional systems including, scaffolds, aggregates, fabrics and membranes. Essentially three types of hierarchies are considered: one-dimensional hierarchy, two-dimensional hierarchy and three-dimensional hierarchy each giving rise to unique behaviors.
Nader Pourmand, Ph.D.
Director, UCSC Genome Technology Center,University of California, Santa Cruz
Biosensor and Single Cell Manipulation using Nanopipettes
Approaching sub-cellular biological problems from an engineering perspective begs for the incorporation of electronic readouts. With their high sensitivity and low invasiveness, nanotechnology-based tools hold great promise for biochemical sensing and single-cell manipulation. During my talk I will discuss the incorporation of electrical measurements into nanopipette technology and present results showing the rapid and reversible response of these subcellular sensors to different analytes such as antigens, ions and carbohydrates. In addition, I will present the development of a single-cell manipulation platform that uses a nanopipette in a scanning ion-conductive microscopy technique. We use this newly developed technology to position the nanopipette with nanoscale precision, and to inject and/or aspirate a minute amount of material to and from individual cells or organelle without comprising cell viability. Furthermore, if time permits, I will show our strategy for a new, single-cell DNA/ RNA sequencing technology that will potentially use nanopipette technology to analyze the minute amount of aspirated cellular material.
Satheesh Babu T. G., Ph.D.
Associate Professor, Department of Sciences, School of Engineering, Amrita University, Coimbatore, India
Nanomaterials for ‘enzyme-free’ biosensing
Enzyme based sensors have many draw backs such as poor storage stability, easily affected by the change in pH and temperature and involves complicated enzyme immobilization procedures. To address this limitation, an alternative approach without the use of enzyme, “non-enzymatic” has been tried recently. Choosing the right catalyst for direct electrochemical oxidation / reduction of a target molecule is the key step in the fabrication of non-enzymatic sensors.
Non-enzymatic sensors for glucose, creatinine, vitamins and cholesterol are fabricated using different nanomaterials, such as nanotubes, nanowires and nanoparticles of copper oxide, titanium dioxide, tantalum oxide, platinum, gold and graphenes. These sensors selectively catalyse the targeted analyte with very high sensitivity. These nanomaterials based sensors combat the drawbacks of enzymatic sensors.
Arathy R and Binoy B Nair
PC based heart sound monitoring system
Heart diseases caused by disorders of the heart and blood vessels, are world’s largest killers. Early detection and monitoring of heart abnormalities is essential for diagnosis and effective treatment of heart diseases. Severalmethodologies are used for screening and diagnosing heart diseases. They are auscultation, electrocardiogram (ECG), echo-cardiogram, ultrasound etc. The effectiveness and applicability of all these diagnostic methods are highly dependent on the equipment cost and size as well as skill of the physician. This paper presents the design and development of a low cost portable wireless/tubeless digital stethoscope which can be used by the physician for monitoring the patient from a distance. The stethoscope system interfaces to a PC and is also capable of analyzing the heart sounds and identifying abnormalities in the heart sound and its classification. Storage of heart sound for later analysis is also possible.This advanced functionality increases the physician’s diagnostic capability, and such a PCG is not still available in most hospitals. Acoustic stethoscope can be changed into a digital stethoscope by inserting an electric capacity microphone into its diaphragm (Wang, Chen and Samjin, 2009).
Anupama Natarajan, James Hickman and Peter Molnar
Novel Cell-Based Biosensors for High Throughput Toxin Detection and Drug Screening Applications
Over the last decade there has been focus on the development of cellbased biosensors to detect environmental toxins or to combat the threats of biological warfare. These sensors have been shown to have multiple applications including understanding function and behaviour at the cellular and tissue levels, in cell electrophysiology and as drug screening tools that can eliminate animal testing. These factors make the development of cell-based biosensors into high throughput systems a priority in pharmacological, environmental and defence industries (Pancrazio J J et al. 1999, Kang G et al. 2009, Krinke D et al. 2009). We have developed a high through-put in vitro cell-silicon hybrid platform that could be used to analyze both cell function and response to various toxins and drugs. Our hypothesis was that by utilizing surface modification to provide external guidance cues as well as optimal growth conditions for different cell types (Cardiac and Neuronal), we could enhance the information output and content of such a system. An intrinsic part of this study was to create ordered or patterned functional networks of cells on Micro-electrode arrays (MEA). Such engineered networks had a two-fold purpose in that they not only aided in a more accurate analysis of cell response and cell and tissue behaviour, but also increased the efficiency of the system by increasing the connectivity and placement of the cells over the recording electrodes. Here we show the response of this system to various toxins and drugs and the measurement of several vital cardiac parameters like conduction velocity and refractory period (Natarajan A et al. 2011)