Cardiovascular diseases are the single greatest cause of adult mortality globally, constituting about 31% of all global deaths. Detection of cardiovascular diseases has thus emerged as not only a social and clinical issue but also as an economic one. The current investigation is centered on the development of specific aptamers against human heart type fatty acid binding protein (FABP3), a novel early marker for detection of acute myocardial infarction (AMI). It also encompasses the detection of FABP3 using the developed aptamers on a specially designed paper based microfluidic device (µPAD). Two ss DNA aptamers, N13 and N53 were isolated through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) against human heart-type fatty acid binding-protein (FABP3). The aptamers bound to FABP3 with dissociation constants 0.0743±0.0142 µM and 0.3337±0.1485 µM, respectively. The aptamers displayed stable behavior at different pH, temperature and ionic strength. Considering the large sizes of the aptamers, limited proteolysis of the aptamer-protein complex was performed to map the amino acids involved in binding, which was then used to screen docked structures. The N13 led interaction with stronger affinity, involving more salt bridges and fewer hydrogen bonds, whereas N53 had less number of salt bridges with higher number of hydrogen and hydrophobic interactions. The greater footprint of N53 incited synergistic conformational changes in N53 and FABP3 leading to decrease in binding affinity during the recognition. Nucleic acid aptamers are nucleic acid species (next-gen antibody mimics) having selectivity at par of antibodies for a given target generated via in-vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) ranging from small entities such as heavy metal ions to large entities like cells Acute myocardial infarction is one of the leading causes of death in on the molecular level, aptamers bind to its cognate target through various non-covalent interactions viz., electrostatic interactions, hydrophobic interactions, and induced fitting. The developed world. The prevalence of the disease approaches three million people worldwide, with more than one million deaths in the United States annually. Acute myocardial infarction can be divided into two categories, non-ST-segment elevation MI (NSTEMI) and ST-The aptamers so developed and characterized were then used to detect FABP3 on a paper based microfluidic device designed for the same with leak proof property and low cost. An aptamer modified gold nanoparticle aggregation assay was used as the Yes/No format for the detection of FABP3 with a minimum detection limit of 54 ng per ml. Memoir: Pranab Goswami has completed his PhD degree from Gauhati University during 1994. From 1991 to 2002, he was a Scientist at CSIR, India. He was a BOYSCAST Fellow of DST, India at University of Massachusetts, Boston. He has joined IIT Guwahati in 2002 and became Professor in 2009. He was the Founder Head of CIF Centre during 2004 to 2006 and Head, Biotechnology Department during 2006 to 2009 at IIT Guwahati. Currently he is the Head of Energy Centre at IIT Guwahati. He is working in the field of biosensors with focus on developing novel bio recognition system for various diagnostic applications.
Introduction: 3’-Phosphoadenosine 5’-phosphosulfate synthase (PAPSS) catalyzes the formation of PAPS from inorganic sulfate and ATP. In the first step inorganic sulfate combines with ATP to form adenosine 5’-phosphosulfate (APS) and pyrophosphate (PPi) catalyzed by the ATP sulfurylase domain. In the second step APS kinase catalyzes the phosphorylation of APS at the 3’ hydroxyl of APS to form PAPS and ADP. PAPSS utilizes 2 moles of ATP and cleaves it differently during PAPS formation, a feature that is unique only to PAPSS. ATP sulfurylase domain cleaves the ATP at the alpha-beta position whereas the APS kinase domain splits the ATP between beta-gamma position. The alpha-beta cleavage of ATP among all ATP sulfurylases share the common motif RNPxHxxH and henceforth it is called Venk-Ettrich motif. Site directed mutagenesis and computational modeling confirms the role of HNGH residues on alpha-beta splitting of ATP. The beta-gamma splitting of ATP contains a typical Walker A motif. Further studies are underway to look at the details of the reaction mechanism. Our overall aim is to look at 1. How 3D structure of PAPSS determines the enzyme function? 2. Studying the roles of specific amino acid residues and the dynamics of it in aqueous solution. 3. Making explicit structural and thermodynamic predictions of ligand binding. 4. Understanding the structural/functional consequences of the proteins due to DNA mutations among various human populations. Methods: Homology modelling with YASARA, ligand docking with AUTODOCK, molecular dynamics simulations in GROMACS (for at least 100ns using the AMBER99SB force field) were used. MM-PBSA calculations were performed to calculate free energies. Conclusion & Significance: Experimental site directed mutagenesis studies of key residues on function and molecular simulation results on structure correlates very well. The overall binding energies of the mutants thatexhibited less activity/no enzyme activity required higher free energy of binding for substrates.
Introduction: The exploration of the natural diversity, through data mining, functional genomics and/or metagenomics is an efficient mean to discover enzymes showing new functions or improved performances. These approaches can be further completed or run in parallel with semirational protein engineering based on structure/function studies or directed molecular evolution inspired from nature. Which of these alternatives are the best ones, in terms of effort, rapidity and efficiency? This is an open question to which a definite answer can be hardly formulated a priori. For illustration, we will take a few examples from our most recent work on glucansucrases from GH13 and GH70 families. These enzymes are naturally very efficient transglucosylases. They use sucrose as substrate and catalyze polymerization of its glucosyl units as a main reaction. Depending on their specificity, structures varying in size as well as in glycosidic linkage types can be obtained, thus giving access to an interesting panel of biopolymers. A campaign of genome sequencing and data mining allowed the isolation of atypical enzymes with new product specificities. In particular, a hyper efficient polymerase producing a gel-like polymer and, in contrast an enzyme synthesizing directly from sucrose a polymer of wellcontrolled low molar mass could be characterized. Structure-function studies combined with mutagenesis assays allowed us to decipher some of the molecular mechanisms behind the control of the polymer size and enzyme processivity. Another key property of these catalysts is coming from their ability to glucosylate a broad spectrum of hydroxylated molecules. Computational protein design, structurally-guided engineering and also random approaches such as neutral evolution was implemented for a fine tuning of their acceptor specificity toward non-natural acceptors such chemically protected disaccharides for vaccinal applications, polyol, flavonoids, or various chemicals. These various approaches will be described and discussed with regard to the engineering objectives.Recent Publications 1. Claverie M et al. (2017). Investigations on the determinants responsible for low molar mass dextran formation by DSR-M dextransucrase. ACS Catal. 7(10):7106-7119. 2. Vuillemin M et al. (2017) A dextran with unique rheological properties produced by the dextransucrase from Oenococcus kitaharae DSM 17330. Carbohydr. Polym. 179:10-18. 3. M Vuillemin et al. (2016). Characterization of the first α-(1→ 3) branching sucrases of GH70 family. J Biol Chem. 291(14):7687- 702. 4. Salamone S et al. (2015) .Programmed chemoenzymatic synthesis of the oligosaccharide component of a carbohydratebased antibacterial vaccine candidate. Chem. Comm. 51(13):2581-2584. 5. Verges A et al. (2015) Computer-aided engineering of a transglucosylase for the glucosylation of an unnatural disaccharide of relevance for bacterial antigen synthesis. ACS Catalysis. 5(2):1186-1198
The furcation defect is defined as the pathological reabsorption of interadicular bone that occurs in multi-rootedand bi-rooted teeth in advanced stages of periodontal disease, it represents a great challenge for dentists andspecialists when treating them, due to their different anatomical variations such as: root trunk, furcation opening,root relationship, interdental and interadicular morphologic features, the interadicular separation and the angle ofroot distance, that can interfere in the response to treatment.Some surgical strategies to cover furcation defects and exposed roots include free gingival grafts, pedicle flaps,sub epithelial connective tissue grafts, and application of different biomaterial-based grafts. We present a Casereport of a 49 year-old patient who was diagnosed with grade II furcation defects on teeth 46 and 47, which weretreated with Platelet- Rich Fibrin (PRF) is an autograft obtained from a blood sample of the patient undergoingprocessing in a centrifuge machine plus a coronal displacement flap surgical technique.A mucoperiosteal partial superficial thickness flap was lifted up from affected teeth and PRF was obtainedfrom a patient blood sample (10mL) in a glass tube without anticoagulant, which was immediately processedon centrifuge machine. The flap was repositioned to coronal level beyond the cement enamel line with 2 PRFmembranes placed on the root surfaces and sutured. Morphometric, tomography and clinical measurement wasperformed 6 months after the procedure to analyze the interadicular molar zones.The surgery showed presence of hard and soft tissues evaluated clinically and tomographically with a significantcoverage of p <0.05 in the fornix zones of the molars. On the interadicular area morphometric values shows thattooth 46 there was a decrease (0.0005) of -1.127 (2.104 ± 0.06 vs 0.977 ± 0.07) of defect and tooth 47 (0.0047)of -0.850 (1.891 ± 0.04) vs 1.041 ± 0.05) CONCLUSION Use of CDF together with PRF can be considered asa treatment option because it achieves a ostensibly osteoconductive, biocompatible function and reduces patientrecovery time improving the prognosis of established defects.
