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Protein interactions of paralogous proteins: a proxy for protein function evolution (Summer 2010)

Gene duplication is one of the main sources of functional divergence and evolutionary innovation in organisms. Duplicates also called paralogs, may be the result of small scale duplication or WGD. Duplication provides redundant genetic material thus relaxing purifying selection and allowing mutations. This can lead to accumulation of changes that may result in one copy becoming a pseudogene or acquiring a new function. It is also possible that both the copies of the gene loose a subset of function. These evolutionary processes manifest as changes in interactions between proteins. Gene duplicates will immediately after duplication have identical protein sequences and thus the same interactions. As sequences diverge, duplicates will share fewer interactions with loss or gain of interactions. This difference in interacting partners among paralogues can be used as an indicator of functional divergence. Thus PPI study can be used to explore the fate of duplicated genes and be used to determine extent of functional divergence among duplicated genes. To examine the extent of functional divergence of paralogues, a list of paralogous proteins and their known interactions were examined by a common method Y2H. Several different platforms have been previously used to determine interactions of paralogous proteins. However there is minimal overlap between different methods and no one method is able to detect all the interactions. Interaction study using a unified platform can thus help determine extent of functional divergence between paralogs on basis of shared interacting partners and understand the fate of paralogs. The study involved:

1. Cloning E.coli ORFeome library using gateway cloning
2. Shortlisting E.coli paralogous proteins of interest using MPIDB and literature survey
3. Studying E.coli protein interactions using Yeast 2 hybrid assay in matrix format

Gastric Tissue Gene Expression Associated with Obesity-related Non-alcoholic Fatty Liver Disease (NAFLD) (2010)

Morbid obesity and the chronic diseases associated with metabolic syndrome have, in recent years, taken the lead in national public health concerns. Obesity is multisystem disorder strongly associated with Type 2 Diabetes, coronary heart disease, and particularly, non- alcoholic fatty liver disease (NAFLD). The increasing toll on individuals and public health resources alike has spurred the scientific community to investigate the causes and consequences of this emerging disease. Increasingly, research from various institutions and study centers is showing that morbid obesity is a chronic inflammatory condition seemingly due to inflammatory cytokines produced in adipose tissue (Csendes et al 2008). Further in energy homeostasis there is interplay of substances produced by various organs, including visceral adipose, liver, muscle and gastric tissues. It is thus plausible that in obesity which is result of deregulation of energy homeostasis, soluble molecules produced by gastric tissues and their receptors may play a role in the pathogenesis of obesity-related disorders. The study involved:

1. Total RNA extraction from human gastric tissue
2. qRT-PCR for obesity related genes and human inflammatory genes and receptors
3. Statistical analysis of data using MATLAB, S plus

Differential gene expression profiling of brown adipose tissue specific genes in visceral adipose tissue of lean and obese individuals (2009)

Obesity currently is one of the leading problems overburdening our national public health system. Morbid obesity has been linked to a variety of severe, progressively degenerating conditions such as NASH, NAFLD, reproductive and cardiovascular disorders (Baranova et al. 2007). The increasing toll on individuals and public health resources alike has spurred the scientific community to investigate the intricate properties of fat formation and metabolism. Significant efforts have been made to understand the properties and functions of white adipose tissue (WAT) and its contribution to fat metabolism. Currently, another type of fat, namely brown fat (BAT) has drawn the attention of researchers. Brown adipose tissue (BAT) is thought to aid in the control of body temperature in hibernating animals and newborn infants. recent studies have shown that not only is there active BAT cells in adult humans, but that the mechanisms of adaptive thermogenesis, thought to be a major function of BAT, may indeed be a factor to be considered when dealing with obesity (Lowell et al 2000). So far no research has been done on the detection of BAT in omental tissue from adult humans with varying degrees of obesity. Using previously collected fat samples, we plan to profile the gene expression of several genes integral to the formation and maintenance of BAT. The study involved:

1. Shortlisting BAT specific genes from literature survey
2. Designing primers
3. Total RNA extraction from human omental adipose tissue
4. qRT-PCR
5. Statistical analysis of data involving Mann Whitney U test

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose tissue (summer 2009)

Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals. The study involved

1. Shortlisting putative reference genes from literature survey for omental adipose tissue
2. Designing primers
3. Total RNA extraction from human omental adipose tissue
4. qRT-PCR
5. Statistical analysis of data using GeNorm, NormFinder and BestKeeper algorithms

Publication: 1. Rohini Mehta et al. Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples. 2010, 11:39, BMC Molecular Biology.

Influence of genetic variability in a study on 'Effect of physical activity on cognition' (2009)

The human and non-human animal research suggests that physical activity, and aerobic fitness training in particular, can have a positive effect on multiple aspects of brain function and cognition. This study is part of cognitive intervention study being done at the University of Illinois. In this project, the role of genetic polymorphisms in the study of exercise effects on cognition is being studied. Given that single nucleotide polymorphisms exist on a number of genes that influence proteins implicated in fitness-training effects ( Eg: BDNF ) examination of the moderating influence of genetic variability on relevant target systems will be useful.

Brain-derived neurotrophic factor also known as BDNF is a protein encoded by the BDNF gene. BDNF is a member of the "neurotrophin" family of growth factors. In the brain, BDNF has been implicated in development, neural regeneration, synaptic transmission,synaptic plasticity and neurogenesis. BDNF has been implicated having correlation with some psychiatric disorders, including depression , obsessive compulsive disorder , attention deficit hyperactivity disorder (ADHD) , and anxiety-related personality traits .

A common SNP in the Brain-Derived Neurotrophic Factor (BDNF) gene coding the Val66Met substitution in the pro-BDNF protein has been associated with a number of behavioural and neuroanatomical phenotypes. This polymorphism has been reported to be associated with obsessive compulsive disorder , attention deficit hyperactivity disorder (ADHD), anxiety-related personality traits and other childhood-onset mood disorder. This SNP has also been suggested to have a role in the hippocampal and prefrontal cortex functions involved in human memory and learning. My contribution towards the achievement of the objectives involved

1. Isolation of genomic DNA from human blood samples
2. BDNF gene SNP Val66Met( rs6265 ) detection using Real-Time PCR

References:

1. Arthur F. Kramer, Kirk I. Erickson and Stanley J. Colcombe. J. Appl. Physiol., 101:1237-1242, 2006.
2. Charles H. Hillman, Kirk I. Erickson and Arthur F. Kramer. Nature., 9:58-65, 2008.

Development and validation of analytical methods for food safety evaluation (2006-2008)

Legume proteins constitute a major component of the dietary protein in developing countries. Grasspea ( Lathyrus sativus ) an annual winter legume crop grown for stock feed and human consumption is considered as the poor mans meat in the central region of India valued primarily as a nutritious staple food due to its high protein content ( 18-34% of the dry seed weight ). Its use has been restricted due to the presence of neurotoxic non-protein amino acid ( beta-ODAP ). Beta - ODAP is known to cause neuronal over-excitation and eventual cytotoxicity of neurons manifesting as spastic paralysis of the leg muscles together with muscular rigidity and weakness. A valid concern in food safety is the intentional or unintentional presence of grasspea in the more expensive edible legumes. The objectives of this study were to develop biochemical and molecular techniques for detection and quantification of Lathyrus sativus in food samples.

Biochemical technique:

First objective of the project was to develop rapid, sensitive and cost effective biochemical method to detect and quantify adulteration of legume based products with grasspea. My contributions in this study were:

1. Development of a sensitive RP-HPLC method for detection of beta-ODAP.
2. Evaluation of the efficacy of this method in detecting and quantifying the presence of grasspea in legume based traditional Indian foods subjected to different types of thermal processing viz roasting, deep frying, fermentation and steaming.
3. Assessment of sensitivity of this method : lower limit of detection.
4. Study of effect of thermal processing on beta-ODAP content.

The sensitivity and reproducibility of this HPLC method has rendered it suitable for qualitative and quantitative analysis of beta-ODAP in thermally processed foods.

References:

1. R. Thippeswamya, Asha Martin and Lalitha R. Gowda. Food Chemistry., 101 (3) :1290-1295, 2007.
2. Rohini Misra, Asha Martin and Lalitha R. Gowda. Journal of Food Composition and Analysis., xx:xx-xx, 2009.

Molecular technique:

Second objective was to develop a robust, reliable method for species molecular identification of Lathyrus sativus without ambiguity. This was based on PCR amplifications of DNA and subsequent DNA analysis. My contributions toward the achievement of the goal include:

1. Optimization of PCR for amplification of targeted gene by:
a.Designing universal primers for amplification of desired gene universal to legumes.
b.Designing species-specific primers internal to universal primers and optimization of PCR for amplification of species specific region of the targeted gene.
c.Optimization of PCR conditions for amplification from highly processed samples.
d.Assessment of sensitivity of optimized PCR:lower limit of detection.


2. Species molecular identification of Lathyrus sativus by:
a.Direct Sequencing of the product to confirm the identity of PCR product.
b.Restriction digestion of the product to delineate from closely related species on basis of digestion pattern.
c.Successful development and optimization of Southern hybridization using species specific probe to identify Lathyrus sativus from other closely related species: Short DNA probe was designed within the targeted gene region specific to L.sativus The specificity of the probe was enhanced by incorporating locked nucleic acid (LNA) nucleotides for accurate and sensitive discrimination.
d.Successful development and optimization of Direct DNA detection using labeled species specific primers.

DNA based methods were extended to processed household food items and found to give a very good amplification even in 1% spiked samples and highly processed samples. Thus a common gene was successfully used to uniquely identify the adulterant Lathyrus sativus from an admixture of legumes as well to develop a species specific PCR.

Analysis of genetically modified soya and genetically modified maize and related GM products (2005-2006)

Genetically modified organisms (GMOs) have distinct characteristics with altered genome to produce novel protein(s) of desired trait. The commercial marketing of GMOs and of the products, produced by these organisms could provide consumers with new options to improve diets and reduce food prices. However, the use of GMOs in products gives rise to a number of questions regarding food safety, nutrition, environmental manipulation and governmental regulations. As the issues surrounding the safety of GM foods awash the world, health conscious consumers demand foods containing modified components to be labeled. The major objectives of the study were to develop DNA based analytical methods for the detection and quantification of GMOs in food materials and processed foods. My contribution towards the achievement of goal include:

1. Designing of primers specific and unique for GM soy (RUR soy) and GM maize (MON 810).
2. Confirmation of the specificity of the amplified products by restriction digestion and direct DNA sequencing of the products.
3. Assessment of sensitivity of the developed methods – lower limit of detection.
4. Successful application of the developed methods for the detection and quantification of GMOs in highly processed foods and market sample.
5. Screening of GMO by PCR-ELISA method:
a.Primer pairs with biotinylation at 5 end were designed. The amplified product was transferred to a nylon membrane and detected using streptavidin linked alkaline phosphatase system.
b.Improvement of the sensitivity of PCR-ELISA method by incorporation of biotin-incorporated nucleotide ( 11-biotin dUTP ).

References:

1. K.R. Vijayakumar, Asha Martin, Lalitha R. Gowda ,V. Prakash. Food Chemistry., 117 (3) :514-521, 2009.