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Natarajan Ganesan, PhD MBA 

Sequence Profiling Tools in Computational Biosciences

Sequence Profiling Tools in bioinformatics and computational biosciences refers to all those software tools (web-based/downloadable) that provide a brief overview on all related information about an input sequence. The advent of post-genomics era has given rise to a range of web based tools and software to handle the deluge of biological information. A simple web-search returns any number of such services and software tools ranging from the popular public domains e.g. NCBI, ExPASy, Ensembl, PDB etc., to private research group sites, e.g. eF-site, Dipole moment server, SuperPose, REBASE. However, most of these remain obscure/under-explored to a bench-top molecular biologist who most often would like to quickly know which record in the BLAST report is useful for further analysis (given that the first few hits are already known) OR like to have a guess at what would a newly found ORF look like after translation. In other words, the user often needs an interface or a gateway, giving him a brief overview about his sequence (DNA/RNA/protein) while still allowing advanced operations without having to leave the browser session. While keyword based profiling is offered by sites/servers Sites like Entrez for text related searches in biomedical field there is very little offered in for sequence input based searches. We have developed some simple interfaces like SEQUEROME  which allow profiling results of a BLAST based sequence alignment report; and InstaSeq which is a Google-powered bioinformatics search engine.
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Transdermal Biosensors

< xml="true" ns="urn:schemas-microsoft-com:vml" prefix="v" namespace="">< xml="true" ns="urn:schemas-microsoft-com:office:office" prefix="o" namespace="">The early stages of the body’s physiological response to either health or environmental trauma can be a useful diagnostic measure for the early detection of stress and a method for determining the appropriate "first response". Recent advances in micro-fabrication technologies allows the creation of novel chip-scale devices capable of non-intrusive, real time sensing of biomedical conditions. The development of the  B-FIT (Bio-Flips Integrable Transdermal Microsystem), promises to be a major advance in this direction, providing a technology for transdermal sampling of molecules that do not normally diffuse across the skin. The device is a micro-fluidic sampling system coupled with a thermal ablation micro-device enabling sampling of the body analyte  at the Stratum Corneum (SC)/Viable Epidermis (VE) interface through extraction of interstitial fluid. The detection of the bioanalyte is limited by its molecular weight, which normally is the limit set by blood-IF barrier (~ 60 Kd). The bioanalytes could vary from monitoring of glucose, lactate to smoking products like nicotine, cotinine etc. Assay of smoking related products gains importance in carrying out field trials, population studies to perform cancer risk assessment in relation to smoking. In some rare diseased conditions antibodies leak into the interstitial fluid (dengue viral infections) cases. The Biochip can be suitably modified for applications proteomics electrochemical DNA biosensors.

Cell Transfection Microdevice
The objective of the proposed research is to develop an integrated, high throughput microfluidic system to efficiently inject functionally useful material within biological cells, and to isolate those cells that underwent successful insertions. The prime motivation for the proposed silicon-based microfluidic system is to develop a simple and efficient method for incorporating specific DNA strands into the cellular genome (transfection) to enable cells to express proteins corresponding to the injected DNA sequence. The need for an improved device that enables the introduction of  biologically useful material (predominantly genetic) arises upon examination of the current techniques of cell transfection, and their respective limitations. A variety of methods exist ranging from chemical based (calcium phosphate, cell membrane permeability (CMP) enhancers like Poly-ethylene Glycol (PEG) ...), Electroporation (applied electric field enhances cell permeability), Microinjection (direct physical injection using micro-needles). While each of these have their advantages, they have other limitations too. Some them include extreme stringency of DNA:Ca2+ (calcium phosphate method), toxicity issues of CMP enhancers, efficiency of transfection, labor intensive and requisition of skilled personnel (micro-injection).

Bio-MEMS or Bio Micro Electromechanical Systems presents an attempt to overcome some of the limitations in the above methods. The device which is of the shape of  a glass slide (click here for device top view), has a bed of micro-needles placed above a reservoir containing the 'to be injected' material. This in turn is supported on a silicon support base (click here for brief presentation). Through the use of physical pressure a group of cells are made to sit on this device wherein each cell sits on an injection device. Then using a syringe like device, the biologically functional material in simultaneously injected into all the cells in a single stroke. The transfected cells would then be released by reverse pressure from their respective needles and flown out into a collection chamber. The device would combine the advantage of micro-injection while performing it on many cells in a single shot and also being minimally invasive and toxic. Some of the pertinent applications of this device include high-throughput gene transfection, in-vitro fertilization, targeted drug delivery apart from basic research in cell biology.

Carcinogen-DNA-Adduct dosimetry
Quantification of low levels of irreversible DNA damage (carcinogen-DNA adducts) is important in selectively identifying individuals as high risk cancer subjects. As a prototype, we have demonstrated the feasibility of  a new assay for the detection of 4-aminobiphenyl-DNA adducts in human samples and plan to validate the same by proving its utility in carrying out epidemiological studies. The novelty of this assay is based on a modified 14C postlabelling method using the enzyme N-acetyltransferase (NAT) (E.C.- with labeled acetyl-CoA as the 14C donor. 4-ABP-DNA standards were prepared by reacting N-hydroxy-4-aminobiphenyl (synthesized from 4-Nitrobiphenyl) with calf thymus DNA. The prepared 4-ABP-DNA standard was subjected to an alkaline hydrolysis procedure, releasing 4-ABP from DNA. After extraction into hexane and HPLC purification, the released 4-aminobiphenyl (confirmed by mass spectrometry) was subjected to enzymatic acetylation using 3H Acetyl CoA as the acetyl donor. The reaction mix was subjected to a single step clean-up procedure using a C18 SEPAK column. The reaction products were collected in methanol, dried and re-dissolved in water/acetonitrile mix for analysis by HPLC. Un-reacted acetyl-CoA eluted out at the solvent front while the acetylated product eluted before the starting material during the separation. The 3H label was distributed between acetyl CoA and the acetylated product without spillover into other byproducts. For very low levels of adduction, the samples would be subjected to an additional purification step involving immuno-affinity chromatography. The acetylated product will be detected and quantified using accelerator mass spectrometry (in collaboration with Turteltaub KW, CA). Following the documented sensitivity of AMS procedure, we expect a thousand-fold increase in adduct detection sensitivity over the existing procedures. 

DNA binding Anti-Cancer Platinum(II) complexes
Cancer chemotherapy has come a long way since the serendipitous discovery of Cisplatin in the '60s by Rosenberg and his co-workers. Progress in the field of platinum(II) drug discovery has been greatly aided by biophysical and physicochemical studies carried out to study the mechanisms of interaction of platinum(II) complexes. This has led to development of second generation Pt(II) analogs that have vastly reduced side effects.

Carboplatin, was introduced in the late eighties and has since then gained popularity in clinical treatment due to its vastly reduced side effects. Two theories exist to explain the molecular mechanism of action of the anti-tumor drug carboplatin with DNA i.e. 1) Aquation, or the like cisplatin hypothesis and 2) Activation, or the unlike cisplatin hypothesis. The former is more accepted owing to the similarity of the leaving groups with its predecessor cisplatin, while the latter hypothesis envisages a biologically activation mechanism to release the active Pt2+ species. Carboplatin differs from cisplatin in that it has a closed cyclobutane dicarboxylate(CBDCA) moiety on its leaving arm in contrast to the readily leaving chloro groups. This results in very different DNA binding kinetics, though it forms the same reaction products in vitro at equivalent doses with cisplatin. However, recent studies provide a new caveat on the DNA binding molecular mechanisms  with the possibility of being activated by nucleophiles (as opposed to cisplatin), before forming the toxic adducts. There are also results to show that cisplatin and carboplatin cause different morphological changes in MCF-7 cell lines while exerting their cytotoxic behaviour.

Oxaliplatin is another example where a closed group in the form of oxalate covers the chloro arm of Pt(II). However there is little information on DNA binding activities of this drug. Given the emergence of similar Pt(II) analogs in clinical trials, it would be interesting to carry out biophysical and physicochemical studies on the DNA binding properties of Pt(II) analogs with similar slow leaving side groups on the chloro arm. These results are likely to aid in a better design of specific side groups on the leaving arm, leading to reduced side effects during treatment..

One of the crucial aspects of anti-cancer platinum(II) drug design is controlling the release of the active Pt2+ species before binding to its target sites. Extending the theory of slow leaving side groups further, assume a bunch of Pt2+ to be coupled to a polymer consisting of multiple COO- sites. Poly(β-L-malate)(PMLA)is a polyanionic polyester, isolated from the plasmodia of Physarum polycephalum. PMLA consists of repeating units of L-malate (a common metabolite), joined by β-ester linkages. Since its discovery, this bio-resorbable polymer has aroused interest primarily for its biomedical applications, especially as drug delivery systems.  As a result of its structure, PMLA is a reservoir of carboxylate groups, protruding from the polyester chain, and providing liganding groups similarly as CBDCA in carboplatin. A polymer-Pt complex has been synthesized by coupling polymalate to the diaqua Pt2+ species of cisplatin. The DNA binding properties of this complex show interesting results, suggesting unconventional DNA binding mechanisms and cytotoxic activities by PMLA-Pt(II) that are dictated by the polyanionic structure of PMLA.