Leishmania is an intracellular protozoan parasite which causes Leishmaniasis, a global health problem affecting millions of people throughout 89 different countries in the world. The current treatment which includes use of amphotericin B, antimonials and others has major drawbacks due to toxicity, resistance and extraordinary high cost. So there is an urgent need of development of new drug targets to fight against leishmaniasis. In this regard we have selected Leishmania donovani Ca²⁺ ion channel (Ld-CC) as potential drug target. Ld-CC regulates concentration of Ca²⁺ ions which is involved in several functions like flagellar motion, mitochondrial oxidative metabolism and entry inside the macrophages. Since Ld-CC has not been characterized yet, we performed homology modeling of Leishmania donovani Ca²⁺ ion channel (Ld-CC) and docking studies of ligand library against this channel. 542 compound library of National Cancer Institute (NCI) diversity 3 dataset selected for screening studies. The ligands ZINC17287336 and ZINC29590262 were selected as best energy conformers because they show highest binding affinity towards its target (Ld-CC). They interact with the active site residues in the pocket of Ld-CC which suggests that the docked conformations are good and acceptable. Moreover these two selected compounds also have relatively high binding affinity than nifedipine and verapamil, known human calcium channel blockers which had been reported to have mild anti-leishmanial activity. Among these two top screened inhibitors the ligand ZINC29590262 shows poor binding affinity towards the Human voltage-dependent L-type calcium channel subunit alpha-1C in comparison to the Ld-CC. Therefore we proposed this ligand as the best inhibitor which shows 40% more binding affinity with Ld-CC than the human-VDCC. These results suggest that our screened ligand ZINC29590262 could act as novel drug and may show much better anti-leishmanial activity.

Primary immunodeficiency (PID), a rare group of genetic disorders, is characterized by increased susceptibility to various infections, growth failure, eczema and malignancy. Hyper-IgE Syndromes (HIES) are a heterogeneous group of heritable inborn errors of immune function and include deficiencies in DOCK8, PGM3, STAT3 and severe atopic dermatitis. Lymphoblastoid cell lines were established from patients with HIES. Comprehensive “multi-OMICs” profiling using LC-MS/MS (metabolomics, proteomics), and RNA-Seq (transcriptomics) were performed on these cell lines. Multi-OMICs data was analyzed using differential expression, Ingenuity Pathway Analysis (IPA) as well as the Genomatix Pathway system tool which creates a network functionally related genes. Multiple canonical pathways, especially EIF2 signaling,       P13/AKT signaling, epithelial adherences junction signaling and its remodeling, protein ubiquitination pathway, glycolysis as well as pathways of pyrimidine ribonucleotides were significantly perturbed with an impact on cellular growth and proliferation; amino Acid metabolism; DNA replication, recombination, and repair. Adenosine and homocysteine were most up-regulated while acetyl-L-carnitine, glutathione and several pyrimidine ribonucleotides. Infectious diseases and cancer were the top diseases identified by IPA analysis with MYC, 5-FU and sirolimus as the upstream regulators. Several interacting immune-modulators including IL6, 7, 17 and IFNG. DOCK8, STAT3 and ARHGEF2 isoforms were differentially expressed and appeared to be disease specific. Combined “multi-OMICs” analysis in DOCK8 deficiency identified apparently unique perturbations in multiple cellular processes and pathways leading to predisposition to infection and cancer.

Poor sperm motility remains predominantly a clinical sign of infertility, rather than a true diagnosis of the cause of infertility. Considering that sperm motility is one of the essential factors for normal fertility, research on proteins or protein functions related to sperm motility is sparse. However, our current understanding of spermatozoa, including their different physiological and pathological aspects, is still limited and not well defined. Therefore, to explore the biological display of the human genome, the physiological functions of each protein should be understood first. Proteomics involves the comprehensive study of proteins with their particular structural and functional aspects. As such, proteomics has the potential to transform our understanding of how mature sperm cells work. Hence, we proposed to identify all the protein molecules that differ in the normal and non-motile conditions of sperm using proteomics methods and validated by immunocytochemistry. The diagnosis of asthenozoospemia was made in the andrology lab of the department of reproductive biology when the sperm motility was observed to be less than 20% as per WHO recommendations. 50 samples each of non-motile and 50 normal samples of semen were used in this study. Proteomics analysis of asthnozoospermia and fertile individuals were done using standard protocol. After identification of proteins were validated by immunocytochemistry. Our results and reports from others suggest that mammalian spermatozoa need local ATP production for flagellar movement and control of the acrosomal reaction. Hence, we demonstrate that glycolysis is very important for human sperm motility. Glyceraldehyde-3-Phosphate dehydrogenase (GAPDH), Enolase and Pyruvate Kinase (PK) can serve as potential targets for the development of biomarkers and can be used as diagnostic indicators and their potential implications to assist fertility or to develop a male contraceptive strategy.

A protein is a sequence of amino-acids which forms a long and thin string-like molecule. This string twists and turns to fold into a ball that is suspended within the gel-like cell cytoplasm, for instance. Different proteins fold differently. Moreover, folding is consistent with a protein giving it a specific ball shape which determines its function. This is one of the most prominent cases where form dictates function. Predicting the 3D structure into which a sequence of amino-acids will fold is important. In fact, that is the so called protein folding problem or PFP. To illustrate, we are given the sequence of amino-acids ITIHSILDWI EDNLESPLSL EKVSERSGYS KWHLQRMFKK ETGHSLQYI RSRKMTEIAQ KLKESNEPIL YLAERYGFES QQTLTRFKN YFDVPPHKYR MTNMQGESRF LHPLNHYNS MTMSRRNTDA. The objective of this talk is to look at the problem of structure prediction, formulate it and highlight the difficulties and recent advances in its solution with some ideas for further research.

New Delhi metallo-b-lactamase-1 (NDM-1) is expressed by various members of Enterobacteriaceae as a defense mechanism to hydrolyze b-lactam antibiotics. Despite various studies showing the significance of active site residues in the catalytic mechanism, there is paucity of reports addressing the role of non-active site residues in the structure and function of NDM-1. Here, we investigated the significance of non-active site residue Trp-93 in the structure and function of NDM-1. We cloned bla_NDM-1 from E. cloacae clinical strain (EC-15) and introduced Trp93Ala mutation by PCR based site-directed mutagenesis. Proteins were expressed and purified to homogeneity by affinity chromatography. The MICs of Trp93Ala mutant were reduced by 4-8 folds against ampicillin, cefotaxime, ceftazidime, cefoxitin, imipenem and meropenem. The poor hydrolytic activity of Trp93Ala mutant was also reflected by its reduced catalytic efficiency. The overall catalytic efficiency of Trp93Ala was reduced by 40-55% (K_m was reduced while k_cat was similar to that of wtNDM-1). Heat-induced denaturation showed that ΔG_D^o and T_m of Trp93Ala mutant was reduced by 1.8 kcal/mol and 4.8 ^oC, respectively. Far-UV CD analysis showed that the α-helical content of Trp93Ala mutant was reduced by 2.9 %. The decrease in stability and catalytic efficiency of Trp93Ala mutant was due to the loss of two hydrogen bonds with Ser-63 and Val-73 and hydrophobic interactions with Leu-65, Val-73, Gln-123 and Asp-124. The study provides an insight into the role of non-active site amino acid residues in the hydrolytic mechanism of NDM-1.

Introduction & Objectives: Adenocarcinomas are the cancers originating from the gland forming cells of the colon and rectal lining and are known to be the most common type of colorectal cancer. The current diagnosis options for colorectal cancers are limited to biopsy, stool tests and other laboratory tests, barium enema based imaging and other imaging techniques, colonoscopy and other endoscopic procedures which are time consuming. In this study, we used proteomics approach with an aim to identify protein biomarkers which can aid in early detection of colon adenocarcinomas to be precise.

Methods: Proteins from tumor tissue of colon adenocarcinoma subjects (n=11) and their matched controls were subjected to 4-plex iTRAQ labeling followed by off-gel fractionation prior to LC-MS/MS run. The mass spectrometry data were analyzed independently using two different analysis software: Spectrum mill (SM) and Trans proteome pipeline (TPP). The proteins identified using either SM and/or TPP were subjected to pathway analysis using the database for annotation, visualization and integrated discovery (DAVID) v6.8 and the proteins common between the two analyses were compared with the data from CPTAC portal and human protein atlas. The expression level of the shortlisted panel of proteins was studied in brain cancers as a non-colon adenocarcinoma control group and validated using MRM approach.

Results: A list of 285 unique proteins was identified to be significantly dys-regulated in colon adenocarcinoma as compared to its matched controls. These proteins were found to be involved in glycolysis, pentose phosphate pathway, biosynthesis of amino acids, protein processing, spliceosome, proteosome, focal adhesion and proteoglycans in cancer. 94 of the 285 proteins were identified by both SM and TPP. 34 of these 94 proteins were found to be dysregulated with same trend as that in data reported on CPTAC portal and 9 of these 34 proteins were validated using MRM approach. Further, to shortlist the proteins specific to colon adenocarcinoma, the list of 34 proteins was compared for their expression levels in brain tumors and 10 proteins were found to be dysregulated with same trend in colon adenocarcinoma vs. its controls as that in colon adenocarcinoma vs. brain tumors.

Conclusion: The proteins identified from this study could be validated further to investigate the role of these proteins as potential biomarkers for early detection of colon adenocarcinoma.