SwePub - search: WFRF:(Sanjiv Kumar)

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1.	Kumar, Atul, et al. (author) The structure of Rv3717 reveals a novel amidase from Mycobacterium tuberculosis. 2013 In: Acta Crystallographica Section D. - : Wiley-Blackwell. - 0907-4449 .- 1399-0047. ; 69:Pt 12, s. 2543-54 Journal article (peer-reviewed)abstract Bacterial N-acetylmuramoyl-L-alanine amidases are cell-wall hydrolases that hydrolyze the bond between N-acetylmuramic acid and L-alanine in cell-wall glycopeptides. Rv3717 of Mycobacterium tuberculosis has been identified as a unique autolysin that lacks a cell-wall-binding domain (CBD) and its structure has been determined to 1.7 Å resolution by the Pt-SAD phasing method. Rv3717 possesses an α/β-fold and is a zinc-dependent hydrolase. The structure reveals a short flexible hairpin turn that partially occludes the active site and may be involved in autoregulation. This type of autoregulation of activity of PG hydrolases has been observed in Bartonella henselae amidase (AmiB) and may be a general mechanism used by some of the redundant amidases to regulate cell-wall hydrolase activity in bacteria. Rv3717 utilizes its net positive charge for substrate binding and exhibits activity towards a broad spectrum of substrate cell walls. The enzymatic activity of Rv3717 was confirmed by isolation and identification of its enzymatic products by LC/MS. These studies indicate that Rv3717, an N-acetylmuramoyl-L-alanine amidase from M. tuberculosis, represents a new family of lytic amidases that do not have a separate CBD and are regulated conformationally.
2.	Kumar, Niti, et al. (author) Intrinsically disordered protein from a pathogenic mesophile Mycobacterium tuberculosis adopts structured conformation at high temperature. 2008 In: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 71:3, s. 1123-33 Journal article (peer-reviewed)abstract Compared to eukaryotes, the occurrence of "intrinsically disordered" or "natively unfolded" proteins in prokaryotes has not been explored extensively. Here, we report the occurrence of an intrinsically disordered protein from the mesophilic human pathogen Mycobacterium tuberculosis. The Histidine-tagged recombinant Rv3221c biotin-binding protein is intrinsically disordered at ambient and physiological growth temperatures as revealed by circular dichroism and Fourier transform infrared (FTIR) spectroscopic studies. However, an increase in temperature induces a transition from disordered to structured state with a folding temperature of approximately 53 degrees C. Addition of a structure inducing solvent trifluoroethanol (TFE) causes the protein to fold at lower temperatures suggesting that TFE fosters hydrophobic interactions, which drives protein folding. Differential Scanning Calorimetry studies revealed that folding is endothermic and the transition from a disordered to structured state is continuous (higher-order), implying existence of intermediates during folding process. Secondary structure analysis revealed that the protein has propensity to form beta-sheets. This is in conformity with FTIR spectrum that showed an absorption peak at wave number of 1636 cm(-1), indicative of disordered beta-sheet conformation in the native state. These data suggest that although Rv3221c may be disordered under ambient or optimal growth temperature conditions, it has the potential to fold into ordered structure at high temperature driven by increased hydrophobic interactions. In contrast to the generally known behavior of other intrinsically disordered proteins folding at high temperature, Rv3221c does not appear to oligomerize or aggregate as revealed through numerous experiments including Congo red binding, Thioflavin T-binding, turbidity measurements, and examining molar ellipticity as a function of protein concentration. The amino acid composition of Rv3221c reveals that it has 24% charged and 54.9% hydrophobic amino acid residues. In this respect, this protein, although belonging to the class of intrinsically disordered proteins, has distinct features. The intrinsically disordered state and the biotin-binding feature of this protein suggest that it may participate in many biochemical processes requiring biotin as a cofactor and adopt suitable conformations upon binding other folded targets.
3.	Kumar, Rajender, et al. (author) Biochemical characterization and molecular insights into substrate recognition of pectin methylesterase from Phytophthora infestans 2022 In: Computational and Structural Biotechnology Journal. - : Elsevier BV. - 2001-0370. ; 20, s. 6023-6032 Journal article (peer-reviewed)abstract Pectin methylesterases (PMEs) are a class of carbohydrate-active enzymes that act on the O6-methyl ester groups of the homogalacturonan component of pectins, resulting in de-esterification of the sub-strate polymers and formation of pectate and methanol. PMEs occur in higher plants and microorgan-isms, including fungi, oomycetes, bacteria, and archaea. Microbial PMEs play a crucial role in pathogens' invasion of plant tissues. Here, we have determined the structural and functional properties of Pi-PME, a PME from the oomycete plant pathogen Phytophthora infestans. This enzyme exhibits maxi-mum activity at alkaline pH (8.5) and is active over a wide temperature range (25-50 degrees C). In silico deter-mination of the structure of Pi-PME reveals that the protein consists essentially of three parallel 8-sheets interconnected by loops that adopt an overall 8-helix organization. The loop regions in the vicinity of the active site are extended compared to plant and fungal PMEs, but they are shorter than the corresponding bacterial and insect regions. Molecular dynamic simulations revealed that Pi-PME interacts most strongly with partially de-methylated homogalacturonans, suggesting that it preferentially uses this type of sub-strates. The results are compared and discussed with other known PMEs from different organisms, high-lighting the specific features of Pi-PME.
4.	Yadav, Sandhya, et al. (author) Comparison and optimization of protein extraction and two-dimensional gel electrophoresis protocols for liverworts 2020 In: BMC Research Notes. - : Springer Nature. - 1756-0500. ; 13:1 Journal article (peer-reviewed)abstract Objective Liverworts possess historical adaptive strategies for abiotic stresses because they were the first plants that shifted from water to land. Proteomics is a state-of-the-art technique that can capture snapshots of events occurring at the protein level in many organisms. Herein, we highlight the comparison and optimization of an effective protein extraction and precipitation protocol for two-dimensional gel electrophoresis (2-DE) of liverworts. Results We compared three different protein extraction methods, i.e.,1.5 M Tris-HCl (pH 8.8), 50 mM Tris-HCl (pH 7.5), and polyvinylpolypyrrolidone (PVPP) extraction, followed by three precipitation methods, i.e., 80% ethanol, 80% acetone, and 20% tricholoroacetic acid (TCA)-acetone, in a liverwort Dumortiera hirsuta. Among these methods, 50 mM Tris-HCl (pH 7.5) extraction, followed by 20% TCA-acetone precipitation, appeared to be more suitable for 2-DE. Furthermore, we performed modifications during protein washing, re-solubilization in rehydration buffer and isoelectric focusing (IEF). The modifications provided us better results in terms of protein yield, resolution, spot numbers, and intensities for 2-DE gels of D. hirsuta and other two liverworts, i.e., Marchantia paleacea and Plagiochasma appendiculatum. Furthermore, we randomly selected spots from the 2-DE gel of D. hirsuta and identified using mass spectrometry, which confirms the applicability of this protocol for liverworts proteomics.
5.	Bonagas, Nadilly, et al. (author) Pharmacological targeting of MTHFD2 suppresses acute myeloid leukemia by inducing thymidine depletion and replication stress 2022 In: NATURE CANCER. - : Springer Science and Business Media LLC. - 2662-1347. ; 3:2, s. 156- Journal article (peer-reviewed)abstract The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors. Helleday and colleagues describe a nanomolar MTHFD2 inhibitor that causes replication stress and DNA damage accumulation in cancer cells via thymidine depletion, demonstrating a potential therapeutic strategy in AML tumors in vivo.
6.	Gad, Helge, et al. (author) MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool 2014 In: Nature. - : Nature Publishing Group. - 0028-0836 .- 1476-4687. ; 508:7495, s. 215-221 Journal article (peer-reviewed)abstract Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bindin the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.
7.	Gupta, Ankit, et al. (author) Reconstruction of Bacterial and Viral Genomes from Multiple Metagenomes. 2016 In: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 7:April Journal article (peer-reviewed)abstract Several metagenomic projects have been accomplished or are in progress. However, in most cases, it is not feasible to generate complete genomic assemblies of species from the metagenomic sequencing of a complex environment. Only a few studies have reported the reconstruction of bacterial genomes from complex metagenomes. In this work, Binning-Assembly approach has been proposed and demonstrated for the reconstruction of bacterial and viral genomes from 72 human gut metagenomic datasets. A total 1156 bacterial genomes belonging to 219 bacterial families and, 279 viral genomes belonging to 84 viral families could be identified. More than 80% complete draft genome sequences could be reconstructed for a total of 126 bacterial and 11 viral genomes. Selected draft assembled genomes could be validated with 99.8% accuracy using their ORFs. The study provides useful information on the assembly expected for a species given its number of reads and abundance. This approach along with spiking was also demonstrated to be useful in improving the draft assembly of a bacterial genome. The Binning-Assembly approach can be successfully used to reconstruct bacterial and viral genomes from multiple metagenomic datasets obtained from similar environments.
8.	Hai, Tao, et al. (author) Task scheduling in cloud environment: optimization, security prioritization and processor selection schemes 2023 In: Journal of Cloud Computing. - : Springer. - 2192-113X. ; 12 Journal article (peer-reviewed)abstract Cloud computing is an extremely important infrastructure used to perform tasks over processing units. Despite its numerous benefits, a cloud platform has several challenges preventing it from carrying out an efficient workflow submission. One of these is linked to task scheduling. An optimization problem related to this is the maximal determination of cloud computing scheduling criteria. Existing methods have been unable to find the quality of service (QoS) limits of users- like meeting the economic restrictions and reduction of the makespan. Of all these methods, the Heterogeneous Earliest Finish Time (HEFT) algorithm produces the maximum outcomes for scheduling tasks in a heterogeneous environment in a reduced time. Reviewed literature proves that HEFT is efficient in terms of execution time and quality of schedule. The HEFT algorithm makes use of average communication and computation costs as weights in the DAG. In some cases, however, the average cost of computation and selecting the first empty slot may not be enough for a good solution to be produced. In this paper, we propose different HEFT algorithm versions altered to produce improved results. In the first stage (rank generation), we execute several methodologies to calculate the ranks, and in the second stage, we alter how the empty slots are selected for the task scheduling. These alterations do not add any cost to the primary HEFT algorithm, and reduce the makespan of the virtual machines’ workflow submissions. Our findings suggest that the altered versions of the HEFT algorithm have a better performance than the basic HEFT algorithm regarding decreased schedule length of the workflow problems.
9.	Herold, Nikolas, et al. (author) Targeting SAMHD1 with the Vpx protein to improve cytarabine therapy for hematological malignancies 2017 In: Nature Medicine. - : Springer Science and Business Media LLC. - 1078-8956 .- 1546-170X. ; 23:2, s. 256-263 Journal article (peer-reviewed)abstract The cytostatic deoxycytidine analog cytarabine (ara-C) is the most active agent available against acute myelogenous leukemia (AML). Together with anthracyclines, ara-C forms the backbone of AML treatment for children and adults'. In AML, both the cytotoxicity of ara-C in vitro and the clinical response to ara-C therapy are correlated with the ability of AML blasts to accumulate the active metabolite ara-C triphosphate (ara-CTP)(2-5), which causes DNA damage through perturbation of DNA synthesis(6). Differences in expression levels of known transporters or metabolic enzymes relevant to ara-C only partially account for patient-specific differential ara-CTP accumulation in AML blasts and response to ara-C treatment(7-9). Here we demonstrate that the deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) promotes the detoxification of intracellular ara-CTP pools. Recombinant SAMHD1 exhibited ara-CTPase activity in vitro, and cells in which SAMHD1 expression was transiently reduced by treatment with the simian immunodeficiency virus (SIV) protein Vpx were dramatically more sensitive to ara-C-induced cytotoxicity. CRISPR-Cas9-mediated disruption of the gene encoding SAMHD1 sensitized cells to ara-C, and this sensitivity could be abrogated by ectopic expression of wild-type (WT), but not dNTPase-deficient, SAMHD1. Mouse models of AML lacking SAMHD1 were hypersensitive to ara-C, and treatment ex vivo with Vpx sensitized primary patient derived AML blasts to ara-C. Finally, we identified SAMHD1 as a risk factor in cohorts of both pediatric and adult patients with de novo AML who received ara-C treatment. Thus, SAMHD1 expression levels dictate patient sensitivity to ara-C, providing proof-of-concept that the targeting of SAMHD1 by Vpx could be an attractive therapeutic strategy for potentiating ara-C efficacy in hematological malignancies.
10.	Karsten, Stella, et al. (author) MTH1 as a target to alleviate T cell driven diseases by selective suppression of activated T cells 2021 In: Cell Death & Differentiation. - Stockholm : Karolinska Institutet, Dept of Oncology-Pathology. - 1350-9047 .- 1476-5403. Journal article (peer-reviewed)abstract T cell-driven diseases account for considerable morbidity and disability globally and there is an urgent need for new targeted therapies. Both cancer cells and activated T cells have an altered redox balance, and up-regulate the DNA repair protein MTH1 that sanitizes the oxidized nucleotide pool to avoid DNA damage and cell death. Herein we suggest that the up-regulation of MTH1 in activated T cells correlates with their redox status, but occurs before the ROS levels increase, challenging the established conception of MTH1 increasing as a direct response to an increased ROS status. We also propose a heterogeneity in MTH1 levels among activated T cells, where a smaller subset of activated T cells does not upregulate MTH1 despite activation and proliferation. The study suggests that the vast majority of activated T cells have high MTH1 levels and are sensitive to the MTH1 inhibitor TH1579 (Karonudib) via induction of DNA damage and cell cycle arrest. TH1579 further drives the surviving cells to the MTH1[superscript low] phenotype with altered redox status. TH1579 does not affect resting T cells, as opposed to the established immunosuppressor Azathioprine, and no sensitivity among other major immune cell types regarding their function can be observed. Finally, we demonstrate a therapeutic effect in a murine model of experimental autoimmune encephalomyelitis. In conclusion, we show proof of concept of the existence of MTH1[superscript high] and MTH1[superscript low] activated T cells, and that MTH1 inhibition by TH1579 selectively suppresses pro-inflammatory activated T cells. Thus, MTH1 inhibition by TH1579 may serve as a novel treatment option against autoreactive T cells in autoimmune diseases, such as multiple sclerosis.
11.	Kaur, S., et al. (author) Biochemical and proteomic analysis reveals oxidative stress tolerance strategies of Scenedesmus abundans against allelochemicals released by Microcystis aeruginosa 2019 In: Algal Research. - : Elsevier B.V.. - 2211-9264. ; 41 Journal article (peer-reviewed)abstract We studied the possible survival strategies of a green alga, Scenedesmus abundans, against allelochemicals secreted by Microcystis aeruginosa. We exposed the monoculture of S. abundans to a cell free-filtrate (allelochemicals)of M. aeruginosa at the start of our experiment and measured the growth behaviour, morphological changes and oxidative stress markers. The results suggest that exposure to allelochemicals induced oxidative stress in S. abundans, which had significantly reduced the growth of green alga with certain morphological changes. However, after seven days, S. abundans found ways to reduce oxidative stress by recovering its morphology and growth close to that of control. To understand possible survival strategies of test alga, we measured biochemical as well as protein level changes in S. abundans. Biochemical response of the green alga clearly showed that as a response to allelochemicals, enzymatic and non-enzymatic antioxidants were induced. Proteomic analysis showed that exposure to allelochemicals induced accumulation of 13 proteins on the 2-DE gel of S. abundans, which falls in three functional categories, i.e., (i)energy metabolism (photosynthesis, carbon fixation and respiration), (ii)ROS scavenging enzymes and molecular chaperones, and (iii)amino acid and protein biosynthesis. After chronic oxidative stress, these proteins presumably retained glycolysis, pentose phosphate pathway and turnover rate of the Calvin-Benson cycle. Moreover, these proteins assisted in the adequate detoxification of ROS and played an important role in the damage removal and repair of oxidized proteins, lipids and nucleic acids. Therefore, our study anticipates that S. abundans embraces biochemical and proteomic reprogramming to thrives against allelochemicals released by M. aeruginosa.
12.	Kumar, Sanjiv, et al. (author) Identification of Growth Inhibitors of the Fish Pathogen Saprolegnia parasitica Using in silico Subtractive Proteomics, Computational Modeling, and Biochemical Validation 2020 In: Frontiers in Microbiology. - : FRONTIERS MEDIA SA. - 1664-302X. ; 11 Journal article (peer-reviewed)abstract Many Stramenopile species belonging to oomycetes from the genus Saprolegnia infect fish, amphibians, and crustaceans in aquaculture farms and natural ecosystems. Saprolegnia parasitica is one of the most severe fish pathogens, responsible for high losses in the aquaculture industry worldwide. Most of the molecules reported to date for the control of Saprolegnia infections either are inefficient or have negative impacts on the health of the fish hosts or the environment resulting in substantial economic losses. Until now, the whole proteome of S. parasitica has not been explored for a systematic screening of novel inhibitors against the pathogen. The present study was designed to develop a consensus computational framework for the identification of potential target proteins and their inhibitors and subsequent experimental validation of selected compounds. Comparative analysis between the proteomes of Saprolegnia, humans and fish species identified proteins that are specific and essential for the survival of the pathogen. The DrugBank database was exploited to select food and drug administration (FDA)-approved inhibitors whose high binding affinity to their respective protein targets was confirmed by computational modeling. At least six of the identified compounds significantly inhibited the growth of S. parasitica in vitro. Triclosan was found to be most effective with a minimum inhibitory concentration (MIC100) of 4 mu g/ml. Optical microscopy showed that the inhibitors affect the morphology of hyphal cells, with hyper-branching being commonly observed. The inhibitory effects of the compounds identified in this study on Saprolegnia's mycelial growth indicate that they are potentially usable for disease control against this class of oomycete pathogens. Similar approaches can be easily adopted for the identification of potential inhibitors against other plant and animal pathogenic oomycete infections.
13.	Kumar, Sanjiv, et al. (author) Identification of novel adhesins of M. tuberculosis H37Rv using integrated approach of multiple computational algorithms and experimental analysis. 2013 In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:7 Journal article (peer-reviewed)abstract Pathogenic bacteria interacting with eukaryotic host express adhesins on their surface. These adhesins aid in bacterial attachment to the host cell receptors during colonization. A few adhesins such as Heparin binding hemagglutinin adhesin (HBHA), Apa, Malate Synthase of M. tuberculosis have been identified using specific experimental interaction models based on the biological knowledge of the pathogen. In the present work, we carried out computational screening for adhesins of M. tuberculosis. We used an integrated computational approach using SPAAN for predicting adhesins, PSORTb, SubLoc and LocTree for extracellular localization, and BLAST for verifying non-similarity to human proteins. These steps are among the first of reverse vaccinology. Multiple claims and attacks from different algorithms were processed through argumentative approach. Additional filtration criteria included selection for proteins with low molecular weights and absence of literature reports. We examined binding potential of the selected proteins using an image based ELISA. The protein Rv2599 (membrane protein) binds to human fibronectin, laminin and collagen. Rv3717 (N-acetylmuramoyl-L-alanine amidase) and Rv0309 (L,D-transpeptidase) bind to fibronectin and laminin. We report Rv2599 (membrane protein), Rv0309 and Rv3717 as novel adhesins of M. tuberculosis H37Rv. Our results expand the number of known adhesins of M. tuberculosis and suggest their regulated expression in different stages.
14.	Kumar, Sanjiv, et al. (author) Sequence variation of rare outer membrane protein β-barrel domains in clinical strains provides insights into the evolution of treponema pallidum subsp. Pallidum, the syphilis spirochete : QC 20181121 2018 In: mBio. - : American Society for Microbiology. - 2161-2129 .- 2150-7511. ; 9:3 Journal article (peer-reviewed)abstract In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum, the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions of tprC, tprD, and bamA in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used T. pallidum 0548 (tp0548) genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants in T. pallidum subsp. pallidum reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane β-strands of T. pallidum repeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive T. pallidum subsp. pallidum OMP diversity and that genetic exchange contributes to the evolutionary biology of T. pallidum subsp. pallidum. They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development. IMPORTANCE Despite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum, little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci, tprC, tprD, and bamA, in T. pallidum subsp. pallidum isolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations drive T. pallidum subsp. pallidum OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of T. pallidum subsp. pallidum and frame strategies for vaccine development based upon conserved OMP extracellular loops.
15.	Mittal, P., et al. (author) Metagenome of a polluted river reveals a reservoir of metabolic and antibiotic resistance genes 2019 In: Environmental Microbiomes. - : BioMed Central. - 2524-6372. ; 14:1 Journal article (peer-reviewed)abstract Background: Yamuna, a major tributary of Ganga, which flows through the national capital region of Delhi, is among the major polluted rivers in India. The accumulation of various effluents, toxic chemicals, heavy metals, and increased organic load in the Yamuna directly affects the organisms that thrive inside or around this river. It also makes it an ideal site for studying the impact of pollution on the river microflora, which are sentinels of the water quality. Results: In this study, the microbial community structure and functional diversity of the Yamuna river water was assessed from the New Delhi region. The community structure of Yamuna during pre-monsoon (June) was found to be significantly different from the post-monsoon (November) time, with Acinetobacter being the most abundant genus during June, and Aeromonas during November. The functional characterization revealed the higher abundance of Methyl-accepting chemotaxis protein in the river water, which could be important for the microbial chemosensory adaptation in the environment. A higher abundance of genes related to nitrogen and sulfur metabolism, metal tolerance, and xenobiotic degradation, and complete degradation pathways of aromatic compounds such as toluene, xylene, benzene and phenol were identified. Further, the results showed the presence of a pool of antibiotic resistance genes in the bacterial microbiome in the Yamuna alongside a large number of broad-spectrum antibiotics, such as carbapenemases and metallo-β-lactamases. Efflux mechanism of resistance was found to dominate among these microbes conferring multi-drug resistance. The Principal Coordinate Analysis of the taxonomic composition of the Yamuna River water with publicly available freshwater and sewage datasets revealed significant differences in the two Yamuna samples and a greater resemblance of pre-monsoon Yamuna sample to sewage sample owing to the higher pollution levels in Yamuna in the pre-monsoon time. Conclusion: The metagenomic study of the Yamuna river provides the first insights on the bacterial microbiome composition of this large polluted river, and also helps to understand the dynamics in the community structure and functions due to seasonal variations. The presence of antibiotic resistance genes and functional insights on the metabolic potential of a polluted river microbiome are likely to have several applications in health, biotechnology and bioremediation.
16.	Natarajan Arul, Murugan, et al. (author) Searching for target-specific and multi-targeting organics for Covid-19 in the Drugbank database with a double scoring approach 2020 In: Scientific Reports. - : Nature Research. - 2045-2322. ; 10:1 Journal article (peer-reviewed)abstract The current outbreak of Covid-19 infection due to SARS-CoV-2, a virus from the coronavirus family, has become a major threat to human healthcare. The virus has already infected more than 44 M people and the number of deaths reported has reached more than 1.1 M which may be attributed to lack of medicine. The traditional drug discovery approach involves many years of rigorous research and development and demands for a huge investment which cannot be adopted for the ongoing pandemic infection. Rather we need a swift and cost-effective approach to inhibit and control the viral infection. With the help of computational screening approaches and by choosing appropriate chemical space, it is possible to identify lead drug-like compounds for Covid-19. In this study, we have used the Drugbank database to screen compounds against the most important viral targets namely 3C-like protease (3CLpro), papain-like protease (PLpro), RNA-dependent RNA polymerase (RdRp) and the spike (S) protein. These targets play a major role in the replication/transcription and host cell recognition, therefore, are vital for the viral reproduction and spread of infection. As the structure based computational screening approaches are more reliable, we used the crystal structures for 3C-like main protease and spike protein. For the remaining targets, we used the structures based on homology modeling. Further, we employed two scoring methods based on binding free energies implemented in AutoDock Vina and molecular mechanics-generalized Born surface area approach. Based on these results, we propose drug cocktails active against the three viral targets namely 3CLpro, PLpro and RdRp. Interestingly, one of the identified compounds in this study i.e. Baloxavir marboxil has been under clinical trial for the treatment of Covid-19 infection. In addition, we have identified a few compounds such as Phthalocyanine, Tadalafil, Lonafarnib, Nilotinib, Dihydroergotamine, R-428 which can bind to all three targets simultaneously and can serve as multi-targeting drugs. Our study also included calculation of binding energies for various compounds currently under drug trials. Among these compounds, it is found that Remdesivir binds to targets, 3CLpro and RdRp with high binding affinity. Moreover, Baricitinib and Umifenovir were found to have superior target-specific binding while Darunavir is found to be a potential multi-targeting drug. As far as we know this is the first study where the compounds from the Drugbank database are screened against four vital targets of SARS-CoV-2 and illustrates that the computational screening using a double scoring approach can yield potential drug-like compounds against Covid-19 infection.
17.	Pudelko, Linda, et al. (author) Glioblastoma and glioblastoma stem cells are dependent on functional MTH1 2017 In: Oncotarget. - : Impact Journals LLC. - 1949-2553. ; 8:49, s. 84671-84684 Journal article (peer-reviewed)abstract Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with poor prognosis. Cancer cells are characterized by a specific redox environment that adjusts metabolism to its specific needs and allows the tumor to grow and metastasize. As a consequence, cancer cells and especially GBM cells suffer from elevated oxidative pressure which requires antioxidant-defense and other sanitation enzymes to be upregulated. MTH1, which degrades oxidized nucleotides, is one of these defense enzymes and represents a promising cancer target. We found MTH1 expression levels elevated and correlated with GBM aggressiveness and discovered that siRNA knock-down or inhibition of MTH1 with small molecules efficiently reduced viability of patient-derived GBM cultures. The effect of MTH1 loss on GBM viability was likely mediated through incorporation of oxidized nucleotides and subsequent DNA damage. We revealed that MTH1 inhibition targets GBM independent of aggressiveness as well as potently kills putative GBM stem cells in vitro. We used an orthotopic zebrafish model to confirm our results in vivo and light-sheet microscopy to follow the effect of MTH1 inhibition in GBM in real time. In conclusion, MTH1 represents a promising target for GBM therapy and MTH1 inhibitors may also be effective in patients that suffer from recurring disease.
18.	Puniya, Bhanwar Lal, et al. (author) Integrated gene co-expression network analysis in the growth phase of Mycobacterium tuberculosis reveals new potential drug targets 2013 In: Molecular Biosystems. - : Royal Society of Chemistry. - 1742-206X .- 1742-2051. ; 9:11, s. 2798-2815 Journal article (peer-reviewed)abstract We have carried out weighted gene co-expression network analysis of Mycobacterium tuberculosis to gain insights into gene expression architecture during log phase growth. The differentially expressed genes between at least one pair of 11 different M. tuberculosis strains as source of biological variability were used for co-expression network analysis. This data included genes with highest coefficient of variation in expression. Five distinct modules were identified using topological overlap based clustering. All the modules together showed significant enrichment in biological processes: fatty acid biosynthesis, cell membrane, intracellular membrane bound organelle, DNA replication, Quinone biosynthesis, cell shape and peptidoglycan biosynthesis, ribosome and structural constituents of ribosome and transposition. We then extracted the co-expressed connections which were supported either by transcriptional regulatory network or STRING database or high edge weight of topological overlap. The genes trpC, nadC, pitA, Rv3404c, atpA, pknA, Rv0996, purB, Rv2106 and Rv0796 emerged as top hub genes. After overlaying this network on the iNJ661 metabolic network, the reactions catalyzed by 15 highly connected metabolic genes were knocked down in silico and evaluated by Flux Balance Analysis. The results showed that in 12 out of 15 cases, in 11 more than 50% of reactions catalyzed by genes connected through co-expressed connections also had altered fluxes. The modules 'Turquoise', 'Blue' and 'Red' also showed enrichment in essential genes. We could map 152 of the previously known or proposed drug targets in these modules and identified 15 new potential drug targets based on their high degree of co-expressed connections and strong correlation with module eigengenes.
19.	Puthenveetil, Robbins, et al. (author) The major outer sheath protein forms distinct conformers and multimeric complexes in the outer membrane and periplasm of Treponema denticola. 2017 In: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7:1 Journal article (peer-reviewed)abstract The major outer sheath protein (MOSP) is a prominent constituent of the cell envelope of Treponema denticola (TDE) and one of its principal virulence determinants. Bioinformatics predicts that MOSP consists of N-and C-terminal domains, MOSPN and MOSPC. Biophysical analysis of constructs refolded in vitro demonstrated that MOSPC, previously shown to possess porin activity, forms amphiphilic trimers, while MOSPN forms an extended hydrophilic monomer. In TDE and E. coli expressing MOSP with a PelB signal sequence (PelB-MOSP), MOSPC is OM-embedded and surface-exposed, while MOSPN resides in the periplasm. Immunofluorescence assay, surface proteolysis, and novel cell fractionation schemes revealed that MOSP in TDE exists as outer membrane (OM) and periplasmic trimeric conformers; PelB-MOSP, in contrast, formed only OM-MOSP trimers. Although both conformers form hetero-oligomeric complexes in TDE, only OM-MOSP associates with dentilisin. Mass spectrometry (MS) indicated that OM-MOSP interacts with proteins in addition to dentilisin, most notably, oligopeptide-binding proteins (OBPs) and the beta-barrel of BamA. MS also identified candidate partners for periplasmic MOSP, including TDE1658, a spirochete-specific SurA/PrsA ortholog. Collectively, our data suggest that MOSP destined for the TDE OM follows the canonical BAM pathway, while formation of a stable periplasmic conformer involves an export-related, folding pathway not present in E. coli.
20.	Radolf, Justin D, et al. (author) The Treponema pallidum Outer Membrane 2017 In: Spirochete Biology. - Cham : Springer. ; , s. 1-38 Book chapter (peer-reviewed)abstract The outer membrane (OM) of Treponema pallidum, the uncultivatable agent of venereal syphilis, has long been the subject of misconceptions and controversy. Decades ago, researchers postulated that T. pallidum's poor surface antigenicity is the basis for its ability to cause persistent infection, but they mistakenly attributed this enigmatic property to the presence of a protective outer coat of serum proteins and mucopolysaccharides. Subsequent studies revealed that the OM is the barrier to antibody binding, that it contains a paucity of integral membrane proteins, and that the preponderance of the spirochete's immunogenic lipoproteins is periplasmic. Since the advent of recombinant DNA technology, the fragility of the OM, its low protein content, and the lack of sequence relatedness between T. pallidum and Gram-negative outer membrane proteins (OMPs) have complicated efforts to characterize molecules residing at the host-pathogen interface. We have overcome these hurdles using the genomic sequence in concert with computational tools to identify proteins predicted to form β-barrels, the hallmark conformation of OMPs in double-membrane organisms and evolutionarily related eukaryotic organelles. We also have employed diverse methodologies to confirm that some candidate OMPs do, in fact, form amphiphilic β-barrels and are surface-exposed in T. pallidum. These studies have led to a structural homology model for BamA and established the bipartite topology of the T. pallidum repeat (Tpr) family of proteins. Recent bioinformatics has identified several structural orthologs for well-characterized Gram-negative OMPs, suggesting that the T. pallidum OMP repertoire is more Gram-negative-like than previously supposed. Lipoprotein adhesins and proteases on the spirochete surface also may contribute to disease pathogenesis and protective immunity.
21.	Rudd, Sean, et al. (author) Ribonucleotide reductase inhibitors suppress SAMHD1 ara-CTPase activity enhancing cytarabine efficacy 2020 In: EMBO Molecular Medicine. - : Blackwell Publishing Ltd. - 1757-4676 .- 1757-4684. Journal article (peer-reviewed)abstract The deoxycytidine analogue cytarabine (ara-C) remains the backbone treatment of acute myeloid leukaemia (AML) as well as other haematological and lymphoid malignancies, but must be combined with other chemotherapeutics to achieve cure. Yet, the underlying mechanism dictating synergistic efficacy of combination chemotherapy remains largely unknown. The dNTPase SAMHD1, which regulates dNTP homoeostasis antagonistically to ribonucleotide reductase (RNR), limits ara-C efficacy by hydrolysing the active triphosphate metabolite ara-CTP. Here, we report that clinically used inhibitors of RNR, such as gemcitabine and hydroxyurea, overcome the SAMHD1-mediated barrier to ara-C efficacy in primary blasts and mouse models of AML, displaying SAMHD1-dependent synergy with ara-C. We present evidence that this is mediated by dNTP pool imbalances leading to allosteric reduction of SAMHD1 ara-CTPase activity. Thus, SAMHD1 constitutes a novel biomarker for combination therapies of ara-C and RNR inhibitors with immediate consequences for clinical practice to improve treatment of AML. © 2020 The Authors. Published under the terms of the CC BY 4.0 license
22.	Sanjiv, Kumar, et al. (author) MTH1 Inhibitor TH1579 Induces Oxidative DNA Damage and Mitotic Arrest in Acute Myeloid Leukemia 2021 In: Cancer Research. - : American Association For Cancer Research (AACR). - 0008-5472 .- 1538-7445. ; 81:22, s. 5733-5744 Journal article (peer-reviewed)abstract Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, exhibiting high levels of reactive oxygen species (ROS). ROS levels have been suggested to drive leukemogenesis and is thus a potential novel target for treating AML. MTH1 prevents incorporation of oxidized nucleotides into the DNA to maintain genome integrity and is upregulated in many cancers. Here we demonstrate that hematologic cancers are highly sensitive to MTH1 inhibitor TH1579 (karonudib). A functional precision medicine ex vivo screen in primary AML bone marrow samples demonstrated a broad response profile of TH1579, independent of the genomic alteration of AML, resembling the response profile of the standard-of-care treatments cytarabine and doxorubicin. Furthermore, TH1579 killed primary human AML blast cells (CD45+) as well as chemotherapy resistance leukemic stem cells (CD45+Lin−CD34+CD38−), which are often responsible for AML progression. TH1579 killed AML cells by causing mitotic arrest, elevating intracellular ROS levels, and enhancing oxidative DNA damage. TH1579 showed a significant therapeutic window, was well tolerated in animals, and could be combined with standard-of-care treatments to further improve efficacy. TH1579 significantly improved survival in two different AML disease models in vivo. In conclusion, the preclinical data presented here support that TH1579 is a promising novel anticancer agent for AML, providing a rationale to investigate the clinical usefulness of TH1579 in AML in an ongoing clinical phase I trial.
23.	Sharma, Ashok K., et al. (author) Prediction of peptidoglycan hydrolases- a new class of antibacterial proteins. 2016 In: BMC Genomics. - : BioMed Central. - 1471-2164. ; 17:1 Journal article (peer-reviewed)abstract BACKGROUND: The efficacy of antibiotics against bacterial infections is decreasing due to the development of resistance in bacteria, and thus, there is a need to search for potential alternatives to antibiotics. In this scenario, peptidoglycan hydrolases can be used as alternate antibacterial agents due to their unique property of cleaving peptidoglycan cell wall present in both gram-positive and gram-negative bacteria. Along with a role in maintaining overall peptidoglycan turnover in a cell and in daughter cell separation, peptidoglycan hydrolases also play crucial role in bacterial pathophysiology requiring development of a computational tool for the identification and classification of novel peptidoglycan hydrolases from genomic and metagenomic data.RESULTS: In this study, the known peptidoglycan hydrolases were divided into multiple classes based on their site of action and were used for the development of a computational tool 'HyPe' for identification and classification of novel peptidoglycan hydrolases from genomic and metagenomic data. Various classification models were developed using amino acid and dipeptide composition features by training and optimization of Random Forest and Support Vector Machines. Random Forest multiclass model was selected for the development of HyPe tool as it showed up to 71.12 % sensitivity, 99.98 % specificity, 99.55 % accuracy and 0.80 MCC in four different classes of peptidoglycan hydrolases. The tool was validated on 24 independent genomic datasets and showed up to 100 % sensitivity and 0.94 MCC. The ability of HyPe to identify novel peptidoglycan hydrolases was also demonstrated on 24 metagenomic datasets.CONCLUSIONS: The present tool helps in the identification and classification of novel peptidoglycan hydrolases from complete genomic or metagenomic ORFs. To our knowledge, this is the only tool available for the prediction of peptidoglycan hydrolases from genomic and metagenomic data.AVAILABILITY: http://metagenomics.iiserb.ac.in/hype/ and http://metabiosys.iiserb.ac.in/hype/ .
24.	Sharma, Ashok K., et al. (author) Woods : A fast and accurate functional annotator and classifier of genomic and metagenomic sequences 2015 In: Genomics. - : Academic Press. - 0888-7543 .- 1089-8646. ; 106:1, s. 1-6 Journal article (peer-reviewed)abstract Functional annotation of the gigantic metagenomic data is one of the major time-consuming and computationally demanding tasks, which is currently a bottleneck for the efficient analysis. The commonly used homology-based methods to functionally annotate and classify proteins are extremely slow. Therefore, to achieve faster and accurate functional annotation, we have developed an orthology-based functional classifier 'Woods' by using a combination of machine learning and similarity-based approaches. Woods displayed a precision of 98.79% on independent genomic dataset, 96.66% on simulated metagenomic dataset and >97% on two real metagenomic datasets. In addition, it performed >87 times faster than BLAST on the two real metagenomic datasets. Woods can be used as a highly efficient and accurate classifier with high-throughput capability which facilitates its usability on large metagenomic datasets.
25.	Srivastava, Akanksha, et al. (author) Acute cadmium toxicity and post-stress recovery : Insights into coordinated and integrated response/recovery strategies of Anabaena sp. PCC 7120 2021 In: Journal of Hazardous Materials. - : Elsevier BV. - 0304-3894 .- 1873-3336. ; 411 Journal article (peer-reviewed)abstract Cyanobacteria, the first photoautotrophs have remarkable adaptive capabilities against most abiotic stresses, including Cd. A model cyanobacterium, Anabaena sp. PCC 7120 has been commonly used to understand cyanobacterial plasticity under different environmental stresses. However, very few studies have focused on the acute Cd toxicity. In this context, Anabaena was subjected to 100 ?M Cd for 48 h (acute Cd stress, ACdS) and then transferred into the fresh medium for post-stress recovery (PSR). We further investigated the dynamics of morpho-ultrastructure, physiology, cytosolic proteome, thylakoidal complexes, chelators, and transporters after ACdS, as well as during early (ER), mid (MR), and late (LR) phases of PSR. The findings revealed that ACdS induced intracellular Cd accumulation and ROS production, altered morpho-ultrastructure, reduced photosynthetic pigments, and affected the structural organization of PSII, which subsequently hindered photosynthetic efficiency. Anabaena responded to ACdS and recovered during PSR by reprogramming the expression pattern of proteins/genes involved in cellular defense and repair; CO2 access, Calvin-Benson cycle, glycolysis, and pentose phosphate pathway; protein biosynthesis, folding, and degradation; regulatory functions; PSI-based cyclic electron flow; Cd chelation; and efflux. These modulations occurred in an integrated and coordinated manner that facilitated Anabaena to detoxify Cd and repair ACdS-induced cellular damage.
26.	Visnes, Torkild, et al. (author) Small-molecule inhibitor of OGG1 suppresses proinflammatory gene expression and inflammation 2018 In: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 362:6416, s. 834- Journal article (peer-reviewed)abstract The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor-alpha-induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor kappa B and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo.
27.	Visnes, Torkild, et al. (author) Targeting OGG1 arrests cancer cell proliferation by inducing replication stress 2020 In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 48:21, s. 12234-12251 Journal article (peer-reviewed)abstract Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.

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