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.

Delegate Talk: A Novel Versatile Human Cell Based In Vitro High Throughput Genotoxicity Screen @ Acharya Hall
Aug 13 @ 6:50 pm – 7:00 pm
Delegate Talk: A Novel Versatile Human Cell Based In Vitro High Throughput Genotoxicity Screen @ Acharya Hall | Vallikavu | Kerala | India

Sunilkumar Sukumaran, Ayyappan Nair, Madhuri Subbiah, Gunja Gupta, Lakshmi Rajakrishna, Pradeep Savanoor Raghavendra, Subbulakshmi Karthikeyan, Salini Krishnan Unni and Ganesh Sambasivam


Genotoxicity is defined as DNA damage that leads to gene mutations which can become tumorigenic. Genotoxicity testing is important to ensure drug safety and is mandatory prior to Phase I/II clinical trials of new drugs. The results from genetic toxicology studies help to identify hazardous drugs and environmental genotoxins. Currently, among others there are four tests recommended by regulatory authorities (Ames test-bacterial, chromosome aberrations; in vitro gene mutation-eukaryotic cells and in vivo test). These assays are laborious, time consuming, require large quantities of test compounds and limited by throughput challenges. The site and mechanism of genotoxicity are not revealed by these assays and data obtained from bacterial tests might not translate the same in mammals. To address these we have developed a novel, versatile, human cell based, high throughput, reporter based genotoxicity screen (Anthem’s Genotox screen). This screen is performed on genetically engineered human cell lines that express 3 reporter genes under transcriptional control of ‘early DNA damage sensors’ (p53, p21 and GADD153). These genes are involved in DNA repair, cell cycle arrest and/or apoptosis. p21 and GADD are also known to be induced in a p53 independent manner. p53 blocks G1/S transition of cell cycle while the p53 independent DNA damage block G2/M transition. Identification of the mechanism of genotoxicity helps in rational drug designing. Additionally, the platform can be used to screen other potential genotoxins from cosmetics, food and environment. Initial validation studies of the Genotox screen was performed with over 60 compounds chosen from a variety of chemical classes. The genotoxic potential of metabolites was tested using rat liver S9 fractions. The results demonstrated a sensitivity of 86.7–92.3% and a specificity of 70–78.6% when compared with currently available in vitro genotoxicity assays. This Genotox screen would prove to be an invaluable human cell based tool to weed out potential genotoxins in various industries.

Aug
14
Wed
2013
Invited Talk: Electrospray ionization ion trap mass spectrometry for cyclic peptide characterization @ Amriteshwari Hall
Aug 14 @ 12:14 pm – 12:43 pm

SudarslalSudarslal S, Ph.D.
Associate Professor, School of Biotechnology, Amrita University


Electrospray ionization ion trap mass spectrometry for cyclic peptide characterization

There has been considerable interest in the isolation and structural characterization of bioactive peptides produced by bacteria and fungi. Most of the peptides are cyclic depsipeptides characterized by the presence of lactone linkages and β-hydroxy fatty acids. Occurrence of microheterogeneity is another remarkable property of these peptides. Even if tandem mass spectrometers are good analytical tools to structurally characterize peptides and proteins, sequence analysis of cyclic peptides is often ambiguous due to the random ring opening of the peptides and subsequent generation of a set of linear precursor ions with the same m/z. Here we report combined use of chemical derivatization and multistage fragmentation capability of ion trap mass spectrometers to determine primary sequences of a series of closely related cyclic peptides.

Sudars (1) Sudars (2)

 

Delegate Talk: Bioanalytical Characterization of Therapeutic Proteins @ Amriteshwari Hall
Aug 14 @ 12:44 pm – 12:54 pm
Delegate Talk: Bioanalytical Characterization of Therapeutic Proteins @ Amriteshwari Hall | Vallikavu | Kerala | India

Ravindra Gudihal, Suresh Babu C V


Bioanalytical Characterization of Therapeutic Proteins

The characterization of therapeutic proteins such as monoclonal antibody (mAb) during different stages of manufacturing is crucial for timely and successful product release. Regulatory agencies require a variety of analytical technologies for comprehensive and efficient protein analysis. Electrophoresis-based techniques and liquid chromatography (LC) either standalone or coupled to mass spectrometry (MS) are at the forefront for the in-depth analysis of protein purity, isoforms, stability, aggregation, posttranslational modifications, PEGylation, etc. In this presentation, a combination of various chromatographic and electrophoretic techniques such as liquid-phase isoelectric focusing, microfluidic and capillary-based electrophoresis (CE), liquid chromatography (LC) and combinations of those with mass spectrometry techniques will be discussed. We present a workflow based approach to the analysis of therapeutic proteins. In successive steps critical parameters like purity, accurate mass, aggregation, peptide sequence, glycopeptide and glycan analysis are analyzed. In brief, the workflow involved proteolytic digestion of therapeutic protein for peptide mapping, N-Glycanase and chemical labeling reaction for glycan analysis, liquid-phase isoelectric focusing for enrichment of charge variants followed by a very detailed analysis using state of the art methods such as CE-MS and LC-MS. For the analysis of glycans, we use combinations of CE-MS and LC-MS to highlight the sweet spots of these techniques. CE-MS is found to be more useful in analysis of highly sialylated glycans (charged glycans) while nano LC-MS seems to be better adapted for analysis of neutral glycans. These two techniques can be used to get complementary data to profile all the glycans present in a given protein. In addition, microfluidic electrophoresis was used as a QC tool in initial screening for product purity, analysis of papain digestion fragments of mAb, protein PEGylation products, etc. The described workflow involves multiple platforms, provides an end to end solution for comprehensive protein characterization and aims at reducing the total product development time.