Browsing by Author "Ajit Kumar Shasany"

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Amplified fragment length polymorphism (AFLP) analysis is useful for distinguishing Leishmania species of visceral and cutaneous forms
(2010) Awanish Kumar; Vijay Raju Boggula; Pragya Misra; Shyam Sundar; Ajit Kumar Shasany; Anuradha Dube
The Leishmania strains belonging to cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) have been reported to possess close homology in genome profiles. To confirm this on genetic basis an attempt was made to differentiate Leishmania major; Leishmania tropica and Leishmania donovani genetically for the first time using amplified fragment length polymorphism (AFLP)-a high throughput DNA fingerprinting technique. The objective of this research work was to identify DNA markers of CL and VL. Ten combinations of selective primers detect a total of 1487 informative AFLP marker. Percentage of polymorphism was 45.12%. Three hundred and thirty-seven unique AFLP markers were also identified in three species of Leishmania. A clear distinction was revealed between L. major and L. donovani. It was inferred by AFLP analysis that a higher rate of polymorphisms occurred among Leishmania species which indicate the distinguished pattern of the disease cause by Leishmania, i.e. VL and CL. Analysis based on polymorphic AFLP markers revealed considerably high genetic variation among the genome of these species which was sufficient to distinguish between CL and VL. © 2009 Elsevier B.V. All rights reserved.
Identification of genetic markers in Sodium Antimony Gluconate (SAG) sensitive and resistant Indian clinical isolates of Leishmania donovani through amplified fragment length polymorphism (AFLP)
(2009) Awanish Kumar; Vijay Raju Boggula; Shyam Sundar; Ajit Kumar Shasany; Anuradha Dube
Sodium Antimony Gluconate (SAG) is currently used worldwide as the first-line drugs for the treatment of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) since 1940s. Unfortunately, the resistance of Leishmania parasite to this drug is increasing in several parts of the world. The mechanism of drug resistance in clinical isolates is still not very clear. Earlier, we have established a differentiation between six clinical isolates as sensitive and resistant on the basis of their sensitivity to SAG in vitro and in vivo as well as expression of proteophosphoglycan contents. In this preliminary study, we have further analyzed these isolates on the basis of their genetic diversity, molecular variance and phylogenetic structure using for the first time, a fingerprinting approach - amplified fragment length polymorphism (AFLP). Altogether 2338 informative AFLP bands were generated using 10 selective primer combinations. Percentage of polymorphism was 55.35%. A number of unique AFLP markers (217) were also identified in these strains. It was deduced that a higher rate of variations occurred among Leishmania clinical isolates which indicate the shifting of drug sensitive nature of parasite towards resistant condition. © 2009 Elsevier B.V. All rights reserved.
Mass spectrometry-based proteomic analysis of Leishmania donovani soluble proteins in Indian clinical isolate
(Blackwell Publishing Ltd, 2014) Awanish Kumar; Pragya Misra; Brijesh Sisodia; Ajit Kumar Shasany; Shyam Sundar; Anuradha Dube
Leishmania donovani, a causative organism of visceral leishmaniasis (VL), is responsible for high mortality throughout the world. Due to the unsatisfactory treatment measures and increasing drug resistance, there has been an urgent need to develop novel drug/vaccine targets against VL. The aim of this study was to identify novel targets in soluble L. donovani (SLD) protein. SLD protein was isolated and resolved by two-dimensional gel electrophoresis and analyzed through MALDI-TOF/TOF-based mass spectrometry. Proteomic results identified several proteins as drug targets, Th1 stimulatory, novel, and hypothetical proteins which could have crucial biological functions in Leishmania pathogenesis. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.
Proteome mapping of overexpressed membrane-enriched and cytosolic proteins in sodium antimony gluconate (SAG) resistant clinical isolate of Leishmania donovani
(2010) Awanish Kumar; Brijesh Sisodia; Pragya Misra; Shyam Sundar; Ajit Kumar Shasany; Anuradha Dube
WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT Over 60% of patients with visceral leishmaniasis (VL) in India and Sudan have become unresponsive to treatment with pentavalent antimonials, the first line of drugs for over 60 years. The drug resistance mechanism, studied so far in in vitro selected laboratory strains, has been attributed to various biochemical parameters. The resistance to Sb (V) in Leishmania field isolates is still unexplored. WHAT THIS STUDY ADDS In order to elucidate for the first time the mechanism of drug resistance in field isolates, this study was done in those clinically relevant field isolates which were either responsive or non responsive to SAG. A comparison of proteome profiles of membrane-enriched as well as cytosolic protein fractions of these isolates has pinpointed the multiple overexpressed proteins in resistant isolates. This study has indicated their possible essential role in antimony resistance of the parasite and provides a vast field to be exploited to find much needed novel treatment strategies against VL. AIMS This study aimed to identify differentially overexpressed membrane-enriched as well as cytosolic proteins in SAG sensitive and resistant clinical strains of L. donovani isolated from VL patients which are involved in the drug resistance mechanism. METHODS The proteins in the membrane-enriched as well as cytosolic fractions of drug-sensitive as well as drug-resistant clinical isolates were separated using two-dimensional gel electrophoresis and overexpressed identified protein spots of interest were excised and analysed using MALDI-TOF/TOF. RESULTS Six out of 12 overexpressed proteins were identified in the membrane-enriched fraction of the SAG resistant strain of L. donovani whereas 14 out of 18 spots were identified in the cytosolic fraction as compared with the SAG sensitive strain. The major proteins in the membrane-enriched fraction were ABC transporter, HSP-83, GPI protein transamidase, cysteine-leucine rich protein and 60S ribosomal protein L23a whereas in the cytosolic fraction proliferative cell nuclear antigen (PCNA), proteasome alpha 5 subunit, carboxypeptidase, HSP-70, enolase, fructose-1,6-bisphosphate aldolase, tubulin-beta chain have been identified. Most of these proteins have been reported as potential drug targets, except 60S ribosomal protein L23a and PCNA which have not been reported to date for their possible involvement in drug resistance against VL. CONCLUSION This study for the first time provided a cumulative proteomic analysis of proteins overexpressed in drug resistant clinical isolates of L. donovani indicating their possible role in antimony resistance of the parasite. Identified proteins provide a vast field to be exploited for novel treatment strategies against VL such as cloning and overexpression of these targets to produce recombinant therapeutic/prophylactic proteins. © 2010 The British Pharmacological Society.
Proteomic analyses of membrane enriched proteins of Leishmania donovani Indian clinical isolate by mass spectrometry
(Elsevier Ireland Ltd, 2015) Awanish Kumar; Pragya Misra; Brijesh Sisodia; Ajit Kumar Shasany; Shyam Sundar; Anuradha Dube
Visceral leishmaniasis (VL) is a major fatal disease prevalent in North-East India, caused by a protozoan parasite Leishmania donovani. The parasite multiplies and thrives inside mammalian macrophages and is transmitted by the bite of the sandfly. Due to the unsatisfactory treatment measures, increasing drug resistance and the advent of HIV- Leishmania co-infection there has been an urgent need to develop novel drug/vaccine targets against VL. Target identification is the key step in drug discovery and proteomics seems to be a suitable strategy for it due to the availability of Leishmania major, Leishmania infantum, Leishmania braziliensis, Leishmania donovani, Leishmania mexicana and Leishmania tarentolae genome sequence. Since, majority of proteome analyses of Leishmania have, so far, been performed on whole-cell extracts; this study is dealing with the sub-proteome analysis of the membrane-enriched protein (MEP) fractions of L. donovani. The analysis of 95 protein spots of the MEPs from two dimensional (2-D) gel image through matrix asserted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) endorsed the identification of various relevant functional proteins. Out of 95 the MEP spots 72 have been identified and were classified on the basis of their biological function. Several proteins of unknown function that belong to different classes like cell signaling, transmembrane receptors, and transporters have been identified which could be the new potential therapeutic targets against VL in future. The proteome array of the MEPs contributes to further elucidation of the biological system of L. donovani as well as host-parasite relationships which may be further investigated for their crucial biological role in L. donovani for disease management. © 2015 Elsevier Ireland Ltd.
Whole-genome sequences of four Indian isolates of azospirillum brasilense
(American Society for Microbiology, 2019) Chhaya Singh; Parul Pandey; Durgesh Narain Singh; Rachna Pandey; Ajit Kumar Shasany; Anil Kumar Tripathi
Azospirillum brasilense is used worldwide as a plant growth-promoting inoculant for agricultural crops. To understand how the genomes of Indian strains of A. brasilense compare with their South American counterparts, we determined the whole-genome sequences of four strains of A. brasilense isolated from the rhizosphere of grasses from India. Copyright © 2019 Singh et al.