25 results on '"Saini, Deepak Kumar"'
Search Results
2. Imaging cellular signalling: many 'moving tales' in MAP kinase odyssey.
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Jain, Ruchi and Saini, Deepak Kumar
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CELL communication , *MITOGEN-activated protein kinases , *CELL imaging , *BIOSENSORS , *DYNAMICS - Abstract
Cellular signalling events are at the core of every adaptive response. Signalling events link environmental changes to physiological responses, consequently allowing cellular and organismal sustenance and survival. Classical approaches to study cellular signalling have relied on a variety of cell disruptive techniques which yield limited kinetic information, while the underlying events are much more complex. In this article, we discuss how modern live cell imaging microscopy has found increasing utilization in revealing spatio temporal dynamics of various signalling pathways. Utilizing the well studied mitogen-activated protein kinase (MAPK) signalling cascade as a template, the design, construction and utilization of 'mobile' (translocation proficient) biosensors, suitable for studying MAPK signalling in living cells are described in detail. Experimental setup and results obtained from these biosensors, based on different proteins involved in the MAPK signalling cascade, have been described along with the setup of a microscope optimal for live cell imaging applications. Utilizing the ability to activate or deactivate signalling pathways using defined activators and specific pharmacological inhibitors, we also show how these sensors can yield unique spatial and temporal kinetic information of signalling in living cells. [ABSTRACT FROM AUTHOR]
- Published
- 2013
3. Alteration of Golgi structure in senescent cells and its regulation by a G protein γ subunit
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Cho, Joon-Ho, Saini, Deepak Kumar, Karunarathne, W.K. Ajith, Kalyanaraman, Vani, and Gautam, N.
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GOLGI apparatus , *GENETIC regulation , *G proteins , *CELL proliferation , *CELL cycle , *GENE expression , *CHROMOSOMAL translocation , *AGING - Abstract
Abstract: Cellular senescence is a process wherein proliferating cells undergo permanent cell cycle arrest while remaining viable. Senescence results in enhanced secretion of proteins that promote cancer and inflammation. We report here that the structure of the Golgi complex which regulates secretion is altered in senescent cells. In cells where senescence is achieved by replicative exhaustion or in cells wherein senescence has been induced with BrdU treatment dependent stress, the Golgi complex is dispersed. The expression of a G protein γ subunit, γ11, capable of translocation from the plasma membrane to the Golgi complex on receptor activation increases with senescence. Knockdown of γ11 or overexpression of a dominant negative γ3 subunit inhibits Golgi dispersal induced by senescence. Overall these results suggest that in cellular senescence an upregulated G protein gamma subunit mediates alterations in the structure of the Golgi. [Copyright &y& Elsevier]
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- 2011
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4. Regulation of Golgi structure and secretion by receptor-induced G protein βγ complex translocation.
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Saini, Deepak Kumar, Karunarathne, W. K. Ajith, Angaswamy, Nataraju, Saini, Deepti, Joon-Ho Cho, Kalyanaraman, Vani, and Gautam, Narasimhan
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G proteins , *MEMBRANE proteins , *PANCREATIC secretions , *EPITHELIAL cells , *HYPOGLYCEMIC agents - Abstract
We show that receptor induced G protein βγ subunit translocation from the plasma membrane to the Golgi allows a receptor to initiate fragmentation and regulate secretion. A lung epithelial cell line, A549, was shown to contain an endogenous translocating G protein ? subunit and exhibit receptor-induced Golgi fragmentation. Receptor-induced Golgi fragmentation was inhibited by a shRNA specific to the endogenous translocating γ subunit. A kinase defective protein kinase D and a phospholipase C β inhibitor blocked receptor-induced Golgi fragmentation, suggesting a role for this process in secretion. Consistent with βγ translocation dependence, fragmentation induced by receptor activation was inhibited by a dominant negative nontranslocating γ3. Insulin secretion was shown to be induced by muscarinic receptor activation in a pancreatic β cell line, NIT-1. Induction of insulin secretion was also inhibited by the dominant negative γ3 subunit consistent with the Golgi fragmentation induced by βγ complex translocation playing a role in secretion. [ABSTRACT FROM AUTHOR]
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- 2010
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5. Cross talk between DevS sensor kinase homologue, Rv2027c, and DevR response regulator of Mycobacterium tuberculosis
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Saini, Deepak Kumar, Malhotra, Vandana, and Tyagi, Jaya Sivaswami
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MYCOBACTERIUM tuberculosis , *CALCIUM , *CROSSTALK , *MYCOBACTERIAL diseases - Abstract
Rv2027c is a putative orphan histidine sensor kinase that bears strong homology to DevS of the hypoxia-responsive DevR–DevS two-component system in M. tuberculosis. The cytosolic C-terminal domain of Rv2027c protein (Rv2027c194) was overexpressed in E. coli and biochemically characterized. Rv2027c194 underwent autophosphorylation at a conserved His392 residue and engaged in phosphotransfer with DevR response regulator. The rates of autophosphorylation and the stabilities of the phosphorylated species were broadly similar in Rv2027c and DevS. However, unlike DevS, Rv2027c utilized Ca2+ as an alternative divalent ion during autophosphorylation. In contrast to DevS which completed phosphotransfer to DevR in 5–10 min, phosphotransfer from Rv2027c∼P was only partial at 30 min. Unlike devS transcription that was hypoxia-responsive, Rv2027c transcript levels were not upregulated from basal levels during hypoxia. The differential regulation of devS and Rv2027c genes, the ability of Rv2027c to utilize Ca2+ as a divalent cation in autophosphorylation at physiological concentrations and to engage in phosphotransfer with DevR suggests that the DevR regulon could be modulated by more than one environmental cue relayed through DevS and Rv2027c. [Copyright &y& Elsevier]
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- 2004
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6. DevR-DevS is a bona fide two-component system of Mycobacterium tuberculosis that is hypoxia-responsive in the absence of the DNA-binding domain of DevR.
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Saini, Deepak Kumar, Malhotra, Vandana, Dey, Deepanwita, Pant, Neha, Das, Taposh K., and Tyagi, Jaya Sivaswami
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MYCOBACTERIUM tuberculosis , *DNA , *HYPOXEMIA , *TISSUES , *GENES , *CLONING - Abstract
Two-component systems play a central role in the adaptation of pathogenic bacteria to the environment prevailing within host tissues. The genes encoding the response regulator DevR (Rv3133c/DosR) and the cytoplasmic portion (DevS201) of the histidine kinase DevS (Rv3132c/DosS), a putative two-component system of Mycobacterium tuberculosis, were cloned and the protein products were overexpressed, purified and refolded as N-terminally His6-tagged proteins from Escherichia coil. DevS201 underwent autophosphorylation and participated in rapid phosphotransfer to DevR in a Mg2+-dependent manner. Chemical stability analysis and site-directed mutagenesis implicated the highly conserved residues His395 and Asp54 as the sites of phosphorylation in DevS and DevR, respectively. Mutations in Asp8 and Asp9 residues, postulated to form the acidic Mg2+-binding pocket, and the invariant Lys104 of DevR, abrogated phosphoryl transfer from DevS201 to DevR. DevR-DevS was thus established as a typical two-component regulatory system based on His-to-Asp phosphoryl transfer. Expression of the Rv3134c-devR-devS operon was induced at the RNA level in hypoxic cultures of M. tuberculosis H37Rv and was associated with an increase in the level of DevR protein. However, in a devR mutant strain expressing the N-terminal domain of DevR, induction was observed at the level of RNA expression but not at that of protein. DevS was translated independently of DevR and induction of devS transcripts was not associated with an increase in protein level in either wild-type or mutant strains, reflecting differential regulation of this locus during hypoxia. [ABSTRACT FROM AUTHOR]
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- 2004
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7. Cloning, Overexpression, Purification, and Matrix-Assisted Refolding of DevS (Rv 3132c) Histidine Protein Kinase of Mycobacterium tuberculosis
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Saini, Deepak Kumar, Pant, Neha, Das, Taposh K., and Tyagi, Jaya Sivaswami
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MYCOBACTERIUM tuberculosis , *PROTEIN kinases , *PROTEIN folding - Abstract
The devR-devS (Rv 3133c-Rv 3132c) two-component system of Mycobacterium tuberculosis was identified in our laboratory by RNA subtractive hybridization. This genetic system was predicted to encode a response regulator and histidine protein kinase, respectively. The putative histidine kinase protein DevS was overexpressed to high levels in Escherichia coli as a fusion protein with a hexahistidine tag, His(6)-DevS201, in the form of inclusion bodies. Here we report a “redox-based” method of matrix-bound renaturation of DevS protein. The refolded protein was biochemically active in an autophosphorylation reaction characteristic of histidine kinases and was suitable for the generation of polyclonal antibodies and as an antigen in ELISA. [Copyright &y& Elsevier]
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- 2002
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8. A Platinum(II) Boron‐dipyrromethene Complex for Cellular Imaging and Mitochondria‐targeted Photodynamic Therapy in Red Light.
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Upadhyay, Aarti, Nepalia, Amrita, Bera, Arpan, Saini, Deepak Kumar, and Chakravarty, Akhil R.
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PHOTODYNAMIC therapy , *CELL imaging , *TUBULINS , *PHOTOTHERAPY , *FLUORESCENCE yield , *PLATINUM , *LIGANDS (Chemistry) - Abstract
Cisplatin‐derived platinum(II) complexes [Pt(NH3)2(pacac)](NO3) (1, DPP‐Pt) and [Pt(NH3)2(Acac‐RB)](NO3) (2, Acacplatin‐RB), where Hpacac is 1,3‐diphenyl‐1,3‐propanedione and HAcac‐RB is a red‐light active distyryl‐BODIPY‐appended acetylacetone ligand, are prepared, characterized and their photodynamic therapy (PDT) activity studied (RB abbreviated for red‐light BODIPY). Complex 2 displayed an intense absorption band at λ=652 nm (ϵ=7.3×104 M−1 cm−1) and 601 nm (ϵ=3.1×104 M−1 cm−1) in 1 : 1 DMSO‐DPBS (Dulbecco's Phosphate Buffered Saline). Its emission profile includes a broad maximum at ~673 nm (λex=630 nm). The fluorescence quantum yield (ΦF) of HAcac‐RB and 2 are 0.19 and 0.07, respectively. Dichlorodihydrofluorescein diacetate and 1,3‐diphenylisobenzofuran assay of complex 2 indicated photogeneration of singlet oxygen (ΦΔ: 0.36) as reactive oxygen species (ROS). Light irradiation caused only minor extent of ligand release forming chemo‐active cisplatin analogue. The complex showed ~70–100 fold enhancement in cytotoxicity on light exposure in A549 lung cancer cells and MDA‐MB‐231 multidrug resistant breast cancer cells, giving half maximal inhibitory concentration (IC50) of 0.9–1.8 μM. Confocal imaging showed its mitochondrial localization and complex 2 exhibited anti‐metastasis properties. Immunostaining of β‐tubulin and Annexin V‐FITC/propidium iodide staining displayed complex 2 induced photo‐selective microtubule rupture and cellular apoptosis, respectively. [ABSTRACT FROM AUTHOR]
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- 2023
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9. BCG∆BCG1419c and BCG differ in induction of autophagy, c-di-GMP content, proteome, and progression of lung pathology in Mycobacterium tuberculosis HN878-infected male BALB/c mice.
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Aceves-Sánchez, Michel de Jesús, Barrios-Payán, Jorge Alberto, Segura-Cerda, Cristian Alfredo, Flores-Valdez, Mario Alberto, Mata-Espinosa, Dulce, Pedroza-Roldán, César, Yadav, Rahul, Saini, Deepak Kumar, de la Cruz, Miguel Angel, Ares, Miguel A., Bielefeldt-Ohmann, Helle, Baay-Guzmán, Guillermina, Vergne, Isabelle, Velázquez-Fernández, Jesús Bernardino, Barba León, Jeannette, and Hernández-Pando, Rogelio
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MYCOBACTERIUM tuberculosis , *LUNGS , *MICE , *AUTOPHAGY , *BCG vaccines , *PATHOLOGY , *VACCINE effectiveness - Abstract
• BCG△BCG1419c increased autophagy in murine macrophages compared with BCG. • BCG△BCG1419c differs in c-di-GMP content compared with BCG. • BCG△BCG1419c has a different proteome compared with BCG. • BCG△BCG1419c delayed progression of lung pathology in Mtb HN878-infected mice. The efficacy of BCG vaccines against Mycobacterium tuberculosis (Mtb) strains of lineage 2 (Beijing) in preclinical models and humans has been questioned. We have developed BCG∆BCG1419c, by deletion of BCG1419c in BCG Pasteur, which improved control of tuberculosis (TB) in preclinical models. Here, we compared the capacity of BCG and BCG∆BCG1419c to induce autophagy in murine macrophages, modify c-di-GMP content and transcript levels of BCG1416c , encoding the enzyme responsible for c-di-GMP synthesis/degradation, and of BCG1419c , encoding the phosphodiesterase involved in c-di-GMP degradation. Furthermore, we evaluated proteomic differences in vitro and compared protection against TB produced by a low dose of the HN878-Beijing strain at 3- and 6-months post-infection. We found that BCG∆BCG1419c induced more autophagy and produced different levels of c-di-GMP as well as different transcription of BCG1416c with no expression of BCG1419c. BCG∆BCG1419c differentially produced several proteins, including some involved in interaction with host cells. Vaccination with either BCG strain led to control of bacillary burden in lungs and spleen at 3- but not 6-months post-infection, whereas it reduced pneumonic areas compared with unvaccinated controls at 6 months post-infection. Vaccination with BCG∆BCG1419c delayed progression of lung necrosis as this was observed only at 6 months post-infection. Taken together, compared with BCG, BCG∆BCG1419c increased autophagy, presented different levels of c-di-GMP and transcription of BCG1416c in vitro in a growth-phase dependent manner, modified its proteome and delayed progression of lung pathology produced by a highly virulent Beijing strain. [ABSTRACT FROM AUTHOR]
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- 2023
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10. A small molecule for theraNOstic targeting of cancer cells.
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Ravikumar, Govindan, Bagheri, Meisam, Saini, Deepak Kumar, and Chakrapani, Harinath
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HYDROGEN peroxide , *SMALL molecules , *NITRIC oxide - Abstract
Thera/NO – a small molecule that is activated by hydrogen peroxide to generate nitric oxide (NO) and a fluorescence signal is reported. Using cancer and primary cells, we show that Thera/NO preferentially releases NO in cancer cells, which can trigger DNA damage and cell death in them. The coupled fluorescence signal facilitated tracking the NO release in living cells without collateral consumption of NO. [ABSTRACT FROM AUTHOR]
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- 2017
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11. ERK activated by Histamine H1 receptor is anti-proliferative through spatial restriction in the cytosol.
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Jain, Ruchi, Watson, Uchenna, and Saini, Deepak Kumar
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HISTAMINE receptors , *CYTOSOL , *PHOSPHORYLATION , *MITOGEN-activated protein kinases , *FUNCTIONAL analysis - Abstract
Histamine, a primary mediator of allergic responses, elicits its effects by activating specific receptors belonging to the GPCR family in target cells. Activation of histamine receptor can activate MAP kinases as recorded by monitoring the phosphorylation of extracellular signal regulated kinase (ERK). Despite this, ERK phosphorylation does not translate into pro-proliferative changes after histamine stimulation in HeLa cells. Here we show that histamine H1 receptor activation mediates MAPK activation through PLCβ, Src, PKCδ and MEK pathway, but does not lead to nuclear relocalization of phospho-ERK (pERK), classically associated with pro-proliferative changes. Live cell imaging, FRET and FRAP measurements along with functional analysis reveal that pERK generated by histamine activation is physically and functionally restricted in the cytosol and the findings report a spatial regulation of MAPK cascade activated non-canonically through GPCRs unlike its canonical activation by EGF. [ABSTRACT FROM AUTHOR]
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- 2016
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12. Erratum to "BCGΔBCG1419c and BCG differ in induction of autophagy, c-di-GMP content, proteome, and progression of lung pathology in Mycobacterium tuberculosis HN878-infected male BALB/c mice" [Vaccine 41(26) (2023) 3824–3835].
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Aceves-Sánchez, Michel de Jesús, Barrios-Payán, Jorge Alberto, Segura-Cerda, Cristian Alfredo, Flores-Valdez, Mario Alberto, Mata-Espinosa, Dulce, Pedroza-Roldán, César, Yadav, Rahul, Saini, Deepak Kumar, de la Cruz, Miguel Angel, Ares, Miguel A., Bielefeldt-Ohmann, Helle, Baay-Guzmán, Guillermina, Vergne, Isabelle, Velázquez-Fernández, Jesús Bernardino, Barba León, Jeannette, and Hernández-Pando, Rogelio
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MYCOBACTERIUM tuberculosis , *AUTOPHAGY , *LUNGS , *PATHOLOGY , *MICE , *VACCINES , *IMMUNOGLOBULINS - Published
- 2024
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13. A high-frequency single nucleotide polymorphism in the MtrB sensor kinase in clinical strains of Mycobacterium tuberculosis alters its biochemical and physiological properties.
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Waturuocha, Uchenna Watson, P. J., Athira, Singh, Krishna Kumar, Malhotra, Vandana, Krishna, M. S., and Saini, Deepak Kumar
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SINGLE nucleotide polymorphisms , *MYCOBACTERIUM tuberculosis , *PATHOGENIC bacteria , *DETECTORS , *CELL division , *CELLULAR signal transduction - Abstract
The DNA polymorphisms found in clinical strains of Mycobacterium tuberculosis drive altered physiology, virulence, and pathogenesis in them. Although the lineages of these clinical strains can be traced back to common ancestor/s, there exists a plethora of difference between them, compared to those that have evolved in the laboratory. We identify a mutation present in ~80% of clinical strains, which maps in the HATPase domain of the sensor kinase MtrB and alters kinase and phosphatase activities, and affects its physiological role. The changes conferred by the mutation were probed by in-vitro biochemical assays which revealed changes in signaling properties of the sensor kinase. These changes also affect bacterial cell division rates, size and membrane properties. The study highlights the impact of DNA polymorphisms on the pathophysiology of clinical strains and provides insights into underlying mechanisms that drive signal transduction in pathogenic bacteria. [ABSTRACT FROM AUTHOR]
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- 2021
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14. Publisher's Note: Temporally distinct roles of ATM and ROS in genotoxic-stress-dependent induction and maintenance of cellular senescence.
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Nair, Raji R., Bagheri, Meisam, and Saini, Deepak Kumar
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AUTOMATED teller machines , *CELLULAR aging - Published
- 2021
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15. Cyclic di‐GMP sensing histidine kinase PdtaS controls mycobacterial adaptation to carbon sources.
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Hariharan, Vignesh Narayan, Yadav, Rahul, Thakur, Chandrani, Singh, Albel, Gopinathan, Renu, Singh, Devendra Pratap, Sankhe, Gaurav, Malhotra, Vandana, Chandra, Nagasuma, Bhatt, Apoorva, and Saini, Deepak Kumar
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Cell signaling relies on second messengers to transduce signals from the sensory apparatus to downstream signaling pathway components. In bacteria, one of the most important and ubiquitous second messenger is the small molecule cyclic diguanosine monophosphate (c‐di‐GMP). While the biosynthesis, degradation, and regulatory pathways controlled by c‐di‐GMP are well characterized, the mechanisms through which c‐di‐GMP controls these processes are not entirely understood. Herein we present the report of a c‐di‐GMP sensing sensor histidine kinase PdtaS (Rv3220c), which binds to c‐di‐GMP at submicromolar concentrations, subsequently perturbing signaling of the PdtaS‐PdtaR (Rv1626) two‐component system. Aided by biochemical analysis, genetics, molecular docking, FRET microscopy, and structural modelling, we have characterized the binding of c‐di‐GMP in the GAF domain of PdtaS. We show that a pdtaS knockout in Mycobacterium smegmatis is severely compromised in growth on amino acid deficient media and exhibits global transcriptional dysregulation. The perturbation of the c‐di‐GMP‐PdtaS‐PdtaR axis results in a cascade of cellular changes recorded by a multiparametric systems’ approach of transcriptomics, unbiased metabolomics, and lipid analyses. [ABSTRACT FROM AUTHOR]
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- 2021
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16. Rapid detection of bacterial infection and viability assessment with high specificity and sensitivity using Raman microspectroscopy.
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Kumar, Srividya, Gopinathan, Renu, Chandra, Goutam Kumar, Umapathy, Siva, and Saini, Deepak Kumar
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BACTERIAL diseases , *BACTERIAL typing , *PATHOGENIC bacteria , *AUTOMATIC classification , *COMMUNICABLE diseases - Abstract
Infectious diseases caused by bacteria still pose major diagnostic challenges in spite of the availability of various molecular approaches. Irrespective of the type of infection, rapid identification of the causative pathogen with a high degree of sensitivity and specificity is essential for initiating appropriate treatment. While existing methods like PCR possess high sensitivity, they are incapable of identifying the viability status of the pathogen and those which can, like culturing, are inherently slow. To overcome these limitations, we developed a diagnostic platform based on Raman microspectroscopy, capable of detecting biochemical signatures from a single bacterium for identification as well as viability assessment. The study also establishes a decontamination protocol for handling live pathogenic bacteria which does not affect identification and viability testing, showing applicability in the analysis of sputum samples containing pathogenic mycobacterial strains. The minimal sample processing along with multivariate analysis of spectroscopic signatures provides an interface for automatic classification, allowing the prediction of unknown samples by mapping signatures onto available datasets. Also, the novelty of the current work is the demonstration of simultaneous identification and viability assessment at a single bacterial level for pathogenic bacteria. [ABSTRACT FROM AUTHOR]
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- 2020
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17. A universal stress protein in Mycobacterium smegmatis sequesters the cAMP-regulated lysine acyltransferase and is essential for biofilm formation.
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Samanta, Sintu, Biswas, Priyanka, Banerjee, Arka, Bose, Avipsa, Siddiqui, Nida, Nambi, Subhalaxmi, Saini, Deepak Kumar, and Visweswariah, Sandhya S.
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HEAT shock proteins , *MYCOBACTERIUM smegmatis , *ACYLTRANSFERASES , *REGULATOR genes , *ACYLATION , *ACETYLATION , *MYCOBACTERIA - Abstract
Universal stress proteins (USPs) are present in many bacteria, and their expression is enhanced under various environmental stresses. We have previously identified a USP in Mycobacterium smegmatis that is a product of the msmeg_4207 gene and is a substrate for a cAMP-regulated protein lysine acyltransferase (KATms; MSMEG_5458). Here, we explored the role of this USP (USP4207) in M. smegmatis and found that its gene is present in an operon that also contains genes predicted to encode a putative tripartite tricarboxylate transporter (TTT). Transcription of the TTT-usp4207 operon was induced in the presence of citrate and tartrate, perhaps by the activity of a divergent histidine kinase-response regulator gene pair. A usp4207-deleted strain had rough colony morphology and reduced biofilm formation compared with the WT strain; however, both normal colony morphology and biofilm formation were restored in a usp4207 Δkatms strain. We identified several proteins whose acetylation was lost in the Δkatms strain, and whose transcript levels increased in M. smegmatis biofilms along with that of USP4207, suggesting that USP4207 insulates KATms from its other substrates in the cell. We propose that USP4207 sequesters KATms from diverse substrates whose activities are down-regulated by acylation but are required for biofilm formation, thus providing a defined role for this USP in mycobacterial physiology and stress responses. [ABSTRACT FROM AUTHOR]
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- 2020
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18. High throughput screening identifies auranofin and pentamidine as potent compounds that lower IFN-γ-induced Nitric Oxide and inflammatory responses in mice: DSS-induced colitis and Salmonella Typhimurium-induced sepsis.
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Chattopadhyay, Avik, Joseph, Joel P., Jagdish, Sirisha, Chaudhuri, Somak, Ramteke, Nikita S., Karhale, Aagosh Kishore, Waturuocha, Uchenna, Saini, Deepak Kumar, and Nandi, Dipankar
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SEPSIS , *HIGH throughput screening (Drug development) , *INFLAMMATORY bowel diseases , *INTERFERON gamma , *AURANOFIN , *KILLER cells - Abstract
[Display omitted] • High throughput screening against IFN-γ-induced NO production identified four leads. • All the leads reduce IFN-γ-induced NO, and ROS formation. • Auranofin most potently inhibits NO and IL-6 production from IFN-γ-activated peritoneal macrophages. • Pentamidine and auranofin are effective in two mice models: colitis and sepsis. Interferon-gamma (IFN-γ) is a type II interferon produced primarily by T cells and natural killer cells. IFN-γ induces the expression of inducible nitric oxide synthase (NOS2) to catalyze Nitric Oxide (NO) production in various immune and non-immune cells. Excessive IFN-γ-activated NO production is implicated in several inflammatory diseases, including peritonitis and inflammatory bowel diseases. In this study, we screened the LOPAC®1280 library in vitro on the H6 mouse hepatoma cell line to identify novel non-steroidal small molecule inhibitors of IFN-γ-induced NO production. Compounds with the highest inhibitory activity were validated, which led to identifying the lead compounds: pentamidine, azithromycin, rolipram, and auranofin. Auranofin was the most potent compound determined based on IC 50 and goodness of fit analyses. Mechanistic investigations revealed that majority of the lead compounds suppress the IFN-γ-induced transcription of Nos2 without negatively affecting NO-independent processes, such as the IFN-γ-induced transcription of Irf1 , Socs1 and MHC class 1 surface expression. However, all four compounds lower IFN-γ-induced reactive oxygen species amounts. In addition, auranofin significantly reduced IFN-γ-mediated NO and IL6 production in resident as well as thioglycolate-elicited peritoneal macrophages (PMs). Finally, in vivo testing of the lead compounds in the pre-clinical DSS-induced ulcerative colitis mice model revealed pentamidine and auranofin to be the most potent and protective lead compounds. Also, pentamidine and auranofin greatly increase the survival of mice in another inflammatory model: Salmonella Typhimurium-induced sepsis. Overall, this study identifies novel anti-inflammatory compounds targeting IFN-γ-induced NO-dependent processes to alleviate two distinct inflammatory models of disease. [ABSTRACT FROM AUTHOR]
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- 2023
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19. Acetylation of Response Regulator Proteins, TcrX and MtrA in M. tuberculosis Tunes their Phosphotransfer Ability and Modulates Two-Component Signaling Crosstalk.
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Singh, Krishna Kumar, Bhardwaj, Neerupma, Sankhe, Gaurav D., Udaykumar, Niveda, Singh, Rambir, Malhotra, Vandana, and Saini, Deepak Kumar
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ACETYLATION , *CROSSTALK , *PHOSPHORYLATION , *DNA-binding proteins , *DEPHOSPHORYLATION - Abstract
Abstract Two-component signal transduction (TCS) cascades involve stimulus-dependent activation and phosphorylation of a sensor kinase (SK), which then transfers the phosphoryl moiety to the response regulator (RR) protein. The fidelity of this phosphotransfer reaction from the SK to the RR provides specificity to TCS signaling. In the present study, we show that for TcrX, a transcriptionally autoregulated RR of Mycobacterium tuberculosis , acetylation enhances its net phosphorylation from cognate SK TcrY and lowers it from a non-cognate SK MtrB. Similar acetylation mediated increase in phosphorylation was also observed for another RR MtrA from cognate SK MtrB. Thus, we establish a novel TCS signaling design wherein acetylation of RRs results in enhanced cognate phosphorylation and suppresses non-cognate phosphorylation. Using wild-type or acetylation-deficient TcrX proteins in M. tuberculosis H37Ra, we demonstrate that non-acetylated TcrX acts as a "phosphate sink" for MtrB and suppressing signal propagation from MtrB to MtrA in vivo , linking metabolism to TCS signaling. Overall, we report that acetylation of RRs shields TCSs from crosstalk, modulates the phosphatase activities and alters the DNA-binding activities of RRs, all of which are non-intuitive behavior of TCS systems. Graphical Abstract Unlabelled Image Highlights • Two-component systems of bacteria can be specific or participate in crosstalk. • Phosphotransfer ability between SK and RR is tuned by acetylation of response regulator proteins. • Acetylated RRs are more specific, while non-acetylated RR are amenable to crosstalk. • Acetylation of RR also alters their DNA-binding ability and dephosphorylation rates • Design links signaling fidelity to nutritional status in the bacterium. [ABSTRACT FROM AUTHOR]
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- 2019
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20. H2S contributed from CSE during cellular senescence suppresses inflammation and nitrosative stress.
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Gupta, Kavya, Mathew, Abraham Binoy, Chakrapani, Harinath, and Saini, Deepak Kumar
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CELLULAR aging , *NF-kappa B , *CYSTATHIONINE gamma-lyase , *GENE expression , *REACTIVE oxygen species , *CARBOXYHEMOGLOBIN - Abstract
Aging involves the time-dependent deterioration of physiological functions attributed to various intracellular and extracellular factors. Cellular senescence is akin to aging and involves alteration in redox homeostasis. This is primarily marked by increased reactive oxygen/nitrogen species (ROS/RNS), inflammatory gene expression, and senescence-associated beta-galactosidase activity, all hallmarks of aging. It is proposed that gasotransmitters which include hydrogen sulfide (H 2 S), carbon monoxide (CO), and nitric oxide (NO), may affect redox homeostasis during senescence. H 2 S has been independently shown to induce DNA damage and suppress oxidative stress. While an increase in NO levels during aging is well established, the role of H 2 S has remained controversial. To understand the role of H 2 S during aging, we evaluated H 2 S homeostasis in non-senescent and senescent cells, using a combination of direct measurements with a fluorescent reporter dye (WSP-5) and protein sulfhydration analysis. The free intracellular H 2 S and total protein sulfhydration levels are high during senescence, concomitant to cystathionine gamma-lyase (CSE) expression induction. Using lentiviral shRNA-mediated expression knockdown, we identified that H 2 S contributed by CSE alters global gene expression, which regulates key inflammatory processes during cellular senescence. We propose that H 2 S decreases inflammation during cellular senescence by reducing phosphorylation of IκBα and the p65 subunit of nuclear factor kappa B (NF-κB). H 2 S was also found to reduce NO levels, a significant source of nitrosative stress during cellular senescence. Overall, we establish H 2 S as a key gasotransmitter molecule that regulates inflammatory phenotype and nitrosative stress during cellular senescence. • H 2 S and protein sulfhydration levels are elevated in senescent cells. • This is driven by increased expression of CSE in senescence. • Elevated H 2 S keeps a check on NO and inflammation in senescence. • H 2 S levels do not affect the ROS levels but affect peroxynitrite levels. • Transcriptome analysis reveals enhanced expression of the inflammatory axis in CSE KD cells. [ABSTRACT FROM AUTHOR]
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- 2023
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21. ATM-ROS-iNOS axis regulates nitric oxide mediated cellular senescence.
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Bagheri, Meisam, Nair, Raji R., Singh, Krishna Kumar, and Saini, Deepak Kumar
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NITRIC oxide analysis , *DNA damage , *BIOCHEMICAL genetics , *GENETIC toxicology , *REACTIVE oxygen species - Abstract
Cellular senescence is an outcome of the accumulation of DNA damage which induces the growth arrest in cells. Physiologically, it is presumed to be mediated by accumulation of reactive oxygen species (ROS). Here, we show that another free radical, nitric oxide (NO) produced during inflammation or present as an environmental pollutant can also induce cellular senescence. In primary cells and various immortalized cell lines, exposure to chronic NO, through external addition or internally generated by iNOS expression, leads to the activation of DNA damage response and causes cellular senescence. The phenotype generated by NO includes robust growth arrest, increase in the levels of the DNA damage foci, ROS, SAβ-gal staining, and inflammatory cytokines like IL-6 and IL-8, all hallmarks of cellular senescence similar to replicative senescence. Mechanistically, inhibitor and knockdown analysis revealed that NO mediates senescence through ATM kinase activation and the viability of cells is dependent on both ROS and ATM kinase involving the ATM-ROS-iNOS axis. Overall, we demonstrate that nitric oxide mediates cellular senescence through a novel free radical dependent genotoxic stress pathway. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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22. A molecular beacon-based DNA switch for reversible pH sensing in vesicles and live cells.
- Author
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Narayanaswamy, Nagarjun, Nair, Raji R., Suseela, Y. V., Saini, Deepak Kumar, and Govindaraju, T.
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DNA structure , *HYDROGEN-ion concentration , *VESICLES (Cytology) , *GENE transfection , *CELL proliferation , *APOPTOSIS - Abstract
In this Communication, a molecular beacon-based DNA switch (LMB) is developed as an efficient and reversible pH sensing probe. Remarkably, LMB exhibited reversible structural transition between the closed (molecular beacon) and open (A-motif) states very efficiently in synthetic vesicles and live cells without the need for any transfection agents. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
23. FRET reveals multiple interaction states between two component signalling system proteins of M. tuberculosis.
- Author
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Agrawal, Ruchi, Kumar V., Prem, Ramanan, Harini, and Saini, Deepak Kumar
- Subjects
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FLUORESCENCE resonance energy transfer , *PHOSPHORYLATION , *MYCOBACTERIUM tuberculosis , *BIOLOGICAL crosstalk , *PHOSPHOTRANSFERASES , *PROTEIN kinases - Abstract
Background Two component signalling involves interaction between sensor kinase (SK) and response regulator (RR) proteins which depends on their phosphorylation status. Methods In this study we report the development of an in vitro FRET assay for studying interaction between fluorescently tagged SK and RR proteins. Results Using TCS proteins of Mycobacterium tuberculosis , we demonstrate that phosphorylation status of SK affects the SK–RR interaction, which varies from one TCS to another. The observation was strengthened by recordings from mutant SK and RR proteins. The assay retained the specificity/crosstalk potential of the participating proteins and reflected the inherent phosphotransfer potentials. Conclusions SK and RR proteins interact with each other in unphosphorylated state and the phosphorylation affects the interaction between SK and RR, which was reflected as reduction in FRET ratio. General significance A non-radioactive, in vitro FRET based assay is reported, which can be utilized for studying genome-wide partner screening, identifying crosstalk or specificity in TCSs. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
24. Evaluation of spike protein antigens for SARS-CoV-2 serology.
- Author
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Jagtap, Suraj, K, Ratnasri, Valloly, Priyanka, Sharma, Rakhi, Maurya, Satyaghosh, Gaigore, Anushree, Ardhya, Chitra, Biligi, Dayananda S., Desiraju, Bapu Koundinya, Natchu, Uma Chandra Mouli, Saini, Deepak Kumar, and Roy, Rahul
- Subjects
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SARS-CoV-2 , *COVID-19 , *ANTIGENS , *SEROLOGY , *PROTEIN domains , *IMMUNOGLOBULIN G - Abstract
• Spike trimer displays the highest antibody titer in SARS-CoV-2 infections among spike protein antigens. • Spike trimer IgG ELISA displays a sensitivity of 50 % within six days and 86.2 % after 14 days from onset of symptoms. • IgA and IgG responses to spike trimer antigen were comparable and concomitant in time after infection. • 16 % (IgG) and 15 % (IgA) of COVID-19 RT-PCR positive patients did not seroconvert even after 21 days from onset of symptoms. Spike protein domains are being used in various serology-based assays to detect prior exposure to SARS-CoV-2 virus. However, there has been limited comparison of antibody titers against various spike protein antigens among COVID-19 infected patients. We compared four spike proteins (RBD, S1, S2 and a stabilized spike trimer (ST)) representing commonly used antigens for their reactivity to human IgG antibodies using indirect ELISA in serum from COVID-19 patients and pre-2020 samples. ST ELISA was also compared against the EUROIMMUN IgG ELISA test. Further, we estimated time appropriate IgG and IgA seropositivity rates in COVID-19 patients using a panel of sera samples collected longitudinally from the day of onset of symptoms (DOS). Among the four spike antigens tested, the ST demonstrated the highest sensitivity (86.2 %; 95 % CI: 77.8–91.7 %), while all four antigens showed high specificity to COVID-19 sera (94.7–96.8 %). 13.8 % (13/94) of the samples did not show seroconversion in any of the four antigen-based assays. In a double-blinded head-to-head comparison, ST based IgG ELISA displayed a better sensitivity (87.5 %, 95 % CI: 76.4–93.8 %) than the EUROIMMUN IgG ELISA (67.9 %, 95 % CI: 54.8–78.6 %). Further, in ST-based assays, we found 48 % and 50 % seroconversion in the first six days (from DOS) for IgG and IgA antibodies, respectively, which increased to 84 % (IgG) and 85 % (IgA) for samples collected ≥22 days from DOS. Comparison of spike antigens demonstrates that spike trimer protein is a superior option as an ELISA antigen for COVID-19 serology. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
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25. Aging associated altered response to intracellular bacterial infections and its implication on the host.
- Author
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Fernandes, Sheryl Erica, Alakesh, Alakesh, Rajmani, R.S., Jhunjhunwala, Siddharth, and Saini, Deepak Kumar
- Subjects
- *
BACTERIAL diseases , *EPITHELIAL cells , *NITRIC oxide , *IMMUNOSENESCENCE , *MITOGEN-activated protein kinases , *OLDER people - Abstract
The effects of senescence on geriatric disorders are well explored, but how it influences infections in the elderly is poorly addressed. Here, we show that several anti-microbial responses are elevated in senescent epithelial cells and old mice, which results in decreased bacterial survival in the host after infection. We identify higher levels of iNOS as a crucial host response and show that p38 MAPK in senescent epithelial cells acts as a negative regulator of iNOS transcription. However, in older mice, the ability to impede bacterial infection does not result in enhanced survival, possibly because elevated pro-inflammatory responses are not countered by a robust host protective anti-inflammatory response. Overall, while addressing an alternate advantage of senescent cells, our study demonstrates that infection-associated morbidity in the elderly may not be the sole outcome of pathogen loads but may also be influenced by the host's ability to resolve inflammation-induced damage. [Display omitted] • Several anti-microbial factors are elevated in senescent cells resulting in reduced intracellular bacterial. • Nitric Oxide plays a pivotal role in limiting infection in senescent cells and is negatively regulated by p38 MAPK. • Similarly, high nitrosative and pro-inflammatory responses decrease bacterial infection in old mice. • But a deficient host-protective anti-inflammatory response results in infection-induced mortality of old mice. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
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