Aug
13
Tue
2013
Plenary Talk: Biosensor and Single Cell Manipulation using Nanopipettes @ Amriteshwari Hall
Aug 13 @ 10:06 am – 10:49 am

NaderNader 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.

Invited Talk: Gut microbiome and health- Moving towards the new era of translational medicine @ Acharya Hall
Aug 13 @ 1:30 pm – 1:50 pm

SharmilaSharmila Mande, Ph.D.
Principal Scientist and Head, Bio Sciences R&D, TCS Innovation Labs, Pune


Gut microbiome and health: Moving towards the new era of translational medicine

The microbes inhabiting our body outnumber our own cells by a factor of 10. The genomes of these microbes, called the ‘second genome’ are therefore expected to have great influence on our health and well being. The emerging field of metagenomics is rapidly becoming the method of choice for studying the microbial community (called microbiomes) present in various parts of the human body. Recent studies have implicated the role of gut microbiomes in several diseases and disorders. Studies have indicated gut microbiome’s role in nutrient absorption, immuno-modulation motor-response, and other key physiological processes. However, our understanding of the role of gut microbiota in malnutrition is currently incomplete. Exploration of these aspects are likely to help in understanding the microbial basis for several physiological disorders associated with malnutrition (eg, increased susceptibility to diarrhoeal pathogens) and may finally aid in devising appropriate probiotic strategies addressing this menace. A metagenomic approach was employed for analysing the differences between gut microbial communities obtained from malnourished and healthy children. Results of the analysis using TCS’ ‘Metagenomic Analysis Platform’ were discussed in detail during my talk.

 

Invited Talk: Nanomaterials for ‘enzyme-free’ biosensing @ Amriteshwari Hall
Aug 13 @ 2:17 pm – 2:35 pm

SatheeshSatheesh 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.

Satheesh

Delegate Talk: Novel Cell-Based Biosensors for High Throughput Toxin Detection and Drug Screening Applications @ Amriteshwari Hall
Aug 13 @ 4:08 pm – 4:23 pm
Delegate Talk:  Novel Cell-Based Biosensors for High Throughput Toxin Detection and Drug Screening Applications @ Amriteshwari Hall | Vallikavu | Kerala | India

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)

Delegate Talk: Inflammation Induced Epigenetic Changes in Endothelial Cells: Role in Vascular Insulin Resistance @ Acharya Hall
Aug 13 @ 6:39 pm – 6:49 pm
Delegate Talk: Inflammation Induced Epigenetic Changes in Endothelial Cells: Role in Vascular Insulin Resistance @ Acharya Hall | Vallikavu | Kerala | India

Aswath Balakrishnan, Kapaettu Satyamoorthy and Manjunath B Joshi


Introduction
Insulin resistance is a hall mark of metabolic disorders such as diabetes. Reduced insulin response in vasculature leads to disruption of IR/Akt/eNOS signaling pathway resulting in vasoconstriction and subsequently to cardiovascular diseases. Recent studies have demonstrated that inflammatory regulator interleukin-6 (IL-6), as one of the potential mediators that can link chronic inflammation with insulin resistance. Accumulating evidences suggest a significant role of epigenetic mechanisms such as DNA methylation in progression of metabolic disorders. Hence the present study aimed to understand the role of epigenetic mechanisms involved during IL-6 induced vascular insulin resistance and its consequences in cardiovascular diseases.

Materials and Methods
Human umbilical vein endothelial cells (HUVEC) and Human dermal microvascular endothelial cells (HDMEC) were used for this study. Endothelial cells were treated in presence or absence of IL-6 (20ng/ml) for 36 hours and followed by insulin (100nM) stimulation for 15 minutes. Using immunoblotting, cell lysates were stained for phosphor- and total Akt levels to measure insulin resistance. To investigate changes in DNA methylation, cells were treated with or without neutrophil conditioned medium (NCM) as a physiological source of inflammation or IL-6 (at various concentrations) for 36 hours. Genomic DNA was processed for HPLC analysis for methyl cytosine content and cell lysates were analyzed for DNMT1 (DNA (cytosine-5)-methyltransferase 1) and DNMT3A (DNA (cytosine-5)-methyltransferase 3A) levels using immunoblotting.

Results
Endothelial cells stimulated with insulin exhibited an increase in phosphorylation of Aktser 473 in serum free conditions but such insulin response was not observed in cells treated with IL-6, suggesting chronic exposure of endothelial cells to IL-6 leads to insulin resistance. HPLC analysis for global DNA methylation resulted in decreased levels of 5-methyl cytosine in cells treated with pro-inflammatory molecules (both by NCM and IL-6) as compared to untreated controls. Subsequently, analysis in cells treated with IL-6 showed a significant decrease in DNMT1 levels but not in DNMT3A. Other pro-inflammatory marker such as TNF-α did not exhibit such changes.

Conclusion
Our study suggests: a) Chronic treatment of endothelial cells with IL-6 results in insulin resistance b) Neutrophil conditioned medium and IL-6 decreases methyl cytosine levels c) DNMT1 but not DNMT3a levels are reduced in cells treated with IL-6.

Aug
14
Wed
2013
Plenary Talk: Combined Crystallography and SAXS Methods for Studying Macromolecular Complexes @ Amriteshwari Hall
Aug 14 @ 9:38 am – 10:19 am

JeffPerryJeff Perry, Ph.D.
Assistant Professor, University of California, Riverside


Combined Crystallography and SAXS Methods for Studying Macromolecular Complexes

Recent developments in small angle X-ray scattering (SAXS) are rapidly providing new insights into protein interactions, complexes and conformational states in solution, allowing for detailed biophysical quantification of samples of interest1. Initial analyses provide a judgment of sample quality, revealing the potential presence of aggregation, the overall extent of folding or disorder, the radius of gyration, maximum particle dimensions and oligomerization state. Structural characterizations may include ab initio approaches from SAXS data alone, or enhance structural solutions when combined with previously determined crystal/NMR domains. This combination can provide definitions of architectures, spatial organizations of the protein domains within a complex, including those not yet determined by crystallography or NMR, as well as defining key conformational states. Advantageously, SAXS is not generally constrained by macromolecule size, and rapid collection of data in a 96-well plate format provides methods to screen sample conditions. Such screens include co-factors, substrates, differing protein or nucleotide partners or small molecule inhibitors, to more fully characterize the variations within assembly states and key conformational changes. These analyses are also useful for screening constructs and conditions that are most likely to promote crystal growth. Moreover, these high throughput structural determinations can be leveraged to define how polymorphisms affect assembly formations and activities. Also, SAXS-based technologies may be potentially used for novel structure-based screening, for compounds inducing shape changes or associations/diassociations. This is addition to defining architectural characterizations of complexes and interactions for systems biology-based research, and distinctions in assemblies and interactions in comparative genomics. Thus, SAXS combined with crystallography/NMR and computation provides a unique set of tools that should be considered as being part of one’s repertoire of biophysical analyses, when conducting characterizations of protein and other macromolecular interactions.

1 Perry JJ & Tainer JA. Developing advanced X-ray scattering methods combined with crystallography and computation. Methods. 2013 Mar;59(3):363-71.

Jeff (1)