Browsing by Author "Awanish Kumar"

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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.
Characterization of Swarna Bhasma and Swarnaprash and Determination of Antimicrobial Properties Against Gut-Pathobiont and Symbiont
(Springer, 2024) Kavita Singh; Prashant Kumar Gupta; Awanish Kumar; Brij Mohan Singh
Ayurveda has a long-standing tradition of healthcare in Southeast Asia. Swarnaprash, a classical Ayurveda preparation, is commonly given as the pre-lacteal feed to neonates to prevent labor-related complications and infections. It comprises incinerated gold particles (InAuP/Swarna Bhasma), honey (Madhu), and clarified butter oil (CBO/Cow Ghrita). This in vitro study aimed to evaluate the therapeutic potential of the individual ingredients and combinations of Swarnaprash against selected neonatal gut pathobionts and symbionts. The study employed sophisticated instruments, including SEM with EDAX and X-ray diffraction analysis, to investigate the shape and structural disparities in the ingredients of Swarnaprash. The reported size of gold particles in Swarnaprash ranges from 0.6 to 9.5 µm. These particles are relatively smaller than those in Swarna Bhasma but larger than synthetic gold particles. Swarnaprash demonstrated both bactericidal and bacteriostatic activity against selected neonatal gut pathobionts, with the largest inhibition zones observed for P. aeruginosa and S. Typhi. It surpassed the individual efficacy of its components—Prash, InAuPs, honey, or CBO alone. Notably, Swarnaprash did not affect the selected beneficial gut bacteria. The results warrant further in vivo and clinical studies to explore the effects of Swarnaprash on neonatal gut flora, which would provide vital information for research in neonatal healthcare. © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2024.
Characterization of the proliferating cell nuclear antigen of leishmania donovani clinical isolates and its association with antimony resistance
(American Society for Microbiology, 2014) Rati Tandon; Sharat Chandra; Rajendra Kumar Baharia; Sanchita Das; Pragya Misra; Awanish Kumar; Mohammad Imran Siddiqi; Shyam Sundar; Anuradha Dube
Previously, through a proteomic analysis, proliferating cell nuclear antigen (PCNA) was found to be overexpressed in the sodium antimony gluconate (SAG)-resistant clinical isolate compared to that in the SAG-sensitive clinical isolate of Leishmania donovani. The present study was designed to explore the potential role of the PCNA protein in SAG resistance in L. donovani. For this purpose, the protein was cloned, overexpressed, purified, and modeled. Western blot (WB) and real-time PCR (RT-PCR) analyses confirmed that PCNA was overexpressed by≥3-fold in the log phase, stationary phase, and peanut agglutinin isolated procyclic and metacyclic stages of the promastigote form and by5-fold in the amastigote form of the SAG-resistant isolate compared to that in the SAG-sensitive isolate. L. donovani PCNA (LdPCNA) was overexpressed as a green fluorescent protein (GFP) fusion protein in a SAG-sensitive clinical isolate of L. donovani, and modulation of the sensitivities of the transfectants to pentavalent antimonial (SbV) and trivalent antimonial (Sb III) drugs was assessed in vitro against promastigotes and intracellular (J774A.1 cell line) amastigotes, respectively. Overexpression of LdPCNA in the SAG-sensitive isolate resulted in an increase in the 50% inhibitory concentrations (IC50) of SbV (from 41.2±0.6 μg/ml to 66.5±3.9 μg/ml) and SbIII (from 24.0±0.3 μg/ml to 43.4±1.8 μg/ml). Moreover, PCNA-overexpressing promastigote transfectants exhibited less DNA fragmentation compared to that of wild-type SAG-sensitive parasites upon SbIII treatment. In addition, SAG-induced nitric oxide (NO) production was found to be significantly inhibited in the macrophages infected with the transfectants compared with that in wild-type SAG-sensitive parasites. Consequently, we infer that LdPCNA has a significant role in SAG resistance in L. donovani clinical isolates, which warrants detailed investigations regarding its mechanism. © 2014, American Society for Microbiology. 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.
Involvement of Cathepsins Protein in Mycobacterial Infection and Its Future Prospect as a Therapeutic Target
(Springer Science and Business Media B.V., 2022) Rajat Anand; Shivendra K. Chaurasiya; Ambuj Kumar Kushwaha; Kishan Kumar Nyati; Awanish Kumar
Tuberculosis (TB), the second most fatal infectious disease after HIV, is caused by Mycobacterium tuberculosis (M. tuberculosis or MTB). It is a facultative intracellular pathogen that persists inside the host macrophage by blocking their maturation into phagolysosomes and preventing an acidic environment rich in proteases. TB infections are challenging to treat with the current arsenal of drugs due to the emergence of multi and extensive drug-resistant MTB strains. Another primary reason for its intracellular survival within macrophages is its ability to resist some degradative mechanisms of the endocytic pathways. Several studies have emphasized the critical role of host cathepsins (proteolytic enzymes found in the endolysosomal system) in MTB infection. The cathepsins actively take part in the suppression of MTB, but it evades their enzymatic activity by downregulating their expression in infected cells. Cathepsin targeting is not fully explored in the context of MTB infection, and cathepsins could be considered as emerging drug targets for the therapy of MTB infection. The first clinical results from the targeting of cathepsins have paved the way for the next generation molecular diagnosis of TB. Therefore, it is imminent to review the role of cathepsins and their inhibitors as a promising target in discovering effective anti-MTB molecules. From the perspective of basic science and pharmacological research, this article summarizes the in-depth roles of individual cathepsins in TB, their mode of action, their role in mediating the killing of MTB, and how MTB modulates their expression and activity to favor its intracellular survival. It also discusses how MTB interacts with cathepsins and their inhibitors and a series of events that occur upon its invasion. Further, it is concluded by suggesting various novel strategies to control TB infection and a few reported inhibitors that could serve as potential targets for MTB therapy. The information will extend novel avenues for designing innovative strategies for the theranostic intervention of TB. © 2022, The Author(s), under exclusive licence to Springer Nature B.V.
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.
Proteomic approaches for discovery of new targets for vaccine and therapeutics against visceral leishmaniasis
(2008) Shraddha Kumari; Awanish Kumar; Mukesh Samant; Shyam Sundar; Neeloo Singh; Anuradha Dube
Visceral leishmaniasis (VL) is the most devastating type caused by Leishmania donovani, Leishmania infantum, and Leishmania chagasi. The therapeutic mainstay is still based on the antiquated pentavalent antimonial against which resistance is now increasing. Unfortunately, due to the digenetic life cycle of parasite, there is significant antigenic diversity. There is an urgent need to develop novel drug/ vaccine targets against VL for which the primary goal should be to identify and characterize the structural and functional proteins. Proteomics, being widely employed in the study of Leishmania seems to be a suitable strategy as the availability of annotated sequenced genome of Leishmania major has opened the door for dissection of both protein expression/regulation and function. Advances in clinical proteomic technologies have enable to enhance our mechanistic understanding of virulence/pathogenicity/host-pathogen interactions, drug resistance thereby defining novel therapeutic/vaccine targets. Expression proteomics exploits the differential expression of leishmanial proteins as biomarkers for application towards early diagnosis. Further using immunoproteomics efforts were also focused on evaluating responses to define parasite T-cell epitopes as vaccine/diagnostic targets. This review has highlighted some of the relevant developments in the rapidly emerging field of leishmanial proteomics and focus on its future applications in drug and vaccine discovery against VL. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Tocopherol from seeds of Cucurbita pepo against diabetes: Validation by invivo experiments supported by computational docking
(2013) Sudhanshu Kumar Bharti; Amit Kumar; Neeraj Kumar Sharma; Om Prakash; Sudhir Kumar Jaiswal; Supriya Krishnan; Ashok Kumar Gupta; Awanish Kumar
Background/purpose: Tocopherol from raw pumpkin seeds has been reported to be effective in the alleviation of diabetes through its antioxidant activities. This study evaluates the antidiabetic activities of the tocopherol fraction of raw seeds of Cucurbita pepo L. (CPSE) in a diabetic rat model. In addition, the putative action mechanisms of its botanicals were computationally investigated. Methods: Seed water activity (Aw) was assessed. Tocopherol was extracted and quantified from raw seed oil. The effect of CPSE was studied in poloxamer-407 (PX-407)-induced type 2 diabetic Wistar rats. Glycemic, insulinemic, and lipid profiles, as well as lipid peroxidation status, were evaluated. Glucagon like peptide-1 (GLP-1) content in the cecum was evaluated and histopathological analysis of the pancreas was performed. Further, HYBRID and FRED docking were performed for 10 documented CPSE botanicals, for putative action mechanisms concerning three proteins [protein-tyrosine phosphatase 1B (PTP-1B), peroxisome proliferator-activated receptor gamma (PPAR-γ), and dipeptidyl peptidase IV (DPP-IV)] known to have diabetic therapeutic potential. Results: The Aw of raw seeds was found to be 0.544±0.002. Using tocopherol standards, HPLC determination of CPSE revealed the presence of tocopherol isomers (α, β, γ, and δ). The tocopherol content was found to be 107.4±2.9mg/100g of CPSE. When compared to diabetic control (DC) rats, the CPSE-treated diabetic rats presented a significant amelioration of glycemia, insulinemia, and lipid dysmetabolism. A remarkable reduction in oxidative markers and improved cecal and pancreatic characteristics were also observed. Tocopherol isomers have shown a considerable interaction potential with the aforesaid proteins in docking. Conclusion: The results provide pharmacological evidence of CPSE as an antihyperglycemic mediated by the interaction of various botanicals with multiple targets operating in diabetes mellitus (DM). © 2013.