Biology subjects

Sharma, D. K.

Publications and source records attributed to Sharma, D. K..

12 recordsLinked to original sources

bioRxiv2026

Exploiting NDH-2 Vulnerability: Quinolines as Antitubercular Agents

Mycobacterium tuberculosis possesses a flexible metabolic system helping it to survive inside the host. The type II NADH dehydrogenase, composed of Ndh and NdhA, essential for bacilli, is a promising drug target. Based on ATP depletion values, two quinoline scaffolds were shortlisted after screening of a library of drug like molecules. Structurally, both 64-9C and 64-9D carry ester moieties at the 5- and 8-positions of the quinoline core, respectively. Ease to re-synthesise 64-9D resulted in synthesis of a focused library of compounds, with MIC values of 0.25-4 g/mL, consistent with ATP depletion. These compounds exhibited bactericidal activity against non-replicating mycobacteria, and showed potent efficacy against multidrug-resistant isolates. Altered, intracellular NADH/NAD+ ratio and reduced respiration was indicative of oxidative phosphorylation inhibition. Inhibition of the purified recombinant NDH protein uncompetitively, SNPs in gene encoding NDH-2 for selected one step mutants and, molecular modelling of 4FQN and 2FQN validated NDH-2 as a target for these compounds. The derivative 2FQN exhibited dose-dependent bactericidal efficacy in mice, underscoring the potential of the series as a promising anti-tuberculosis candidates.

microbiology↗

bioRxiv2026

Operon-driven antagonism and novel modulation of DNA damage sensor STPK specific PP2C phosphatase in Deinococcus radiodurans

This study identifies a novel operon-driven signaling module in Deinococcus radiodurans. The operon includes a von Willebrand A domain protein (DRA0331), a Ser/Thr protein kinase (DRA0332), a canonical FHA-domain protein (DRA0333), and a PP2C-type phosphatase (DRA0334). DRA0334 shows Mn{superscript 2}{square}-dependent phosphatase activity and has a unique dual-domain structure that combines a Kinase-Interacting FHA (KI-FHA) domain with a PP2C catalytic domain. Functional assays show that FHA-domain protein, DRA0333 boosts the phosphorylation of STPKs like RqkA and DR1243 while operonic partner PP2C-type phosphatase, DRA0334 counteracts this through targeted dephosphorylation, establishing a phospho-regulatory antagonism. Notably, the KI-FHA domain of the DRA0334 phosphatase competitively interacts with the FHA domain to modulate the radiation-responsive RqkA kinase, thereby maintaining kinase-phosphatase balance. This KI-FHA domain also imparts substrate specificity and enables feedback regulation. Additionally, DRA0334 modular variants confirm separate roles of catalytic and docking modules, and STRING analyses link DRA0334 functions to DNA repair and stress recovery. Collectively, the findings suggest an operonic connection between DRA0333 and DRA0334, indicating that the KI-FHA and FHA domains may act as phospho-docking switches. These switches can regulate both kinase and phosphatase activities in a push-pull regulatory mechanism within the phosphorylation-dephosphorylation cycle of signal transduction, depending on their association with the type of catalytic domain.

molecular biology↗

bioRxiv2024

Surviving the Storm: Exploring the Role of Natural Transformation in Nutrition and DNA Repair of Stressed Deinococcus radiodurans

Deinococcus radiodurans, a natural transformation (NT) enabled bacterium renowned for its exceptional radiation resistance, employs unique DNA repair and oxidative stress mitigation mechanisms as a strategic response to DNA damage. This study excavate into the intricate roles of NT machinery in the stressed D. radiodurans, focusing on the genes comEA, comEC, endA, pilT and dprA, which are instrumental in the uptake and processing of extracellular DNA (eDNA). Our data reveals that NT not only supports the nutritional needs of D. radiodurans under stress but also have roles in DNA repair. The study findings establish that NT-specific proteins (ComEA, ComEC, and EndA) might contribute to support the nutritional requirements in unstressed and heavily DNA-damaged cells while DprA contribute differently and in a context-dependent manner to navigating through the DNA damage storm. Thus, this dual functionality of NT-specific genes is proposed to be one of factor in D. radiodurans remarkable ability to survive and thrive in environments characterized by high levels of DNA-damaging agents. Author SummaryDeinococcus radiodurans, a bacterium known for its extraordinary radiation resistance. This study explores the roles of natural transformation (NT) machinery in the radiation-resistant bacterium Deinococcus radiodurans, focusing on the genes comEA, comEC, endA, pilT, and dprA. These genes are crucial for the uptake and processing of extracellular DNA (eDNA) and contribute to the bacterium nutritional needs and DNA repair under stress. The findings suggest that the NT-specific proteins ComEA, ComEC, and EndA may help meet the nutritional needs of unstressed and heavily DNA-damaged cells, whereas DprA plays a distinct role that varies depending on the context in aiding cells to cope with DNA damage. The functionality of NT genes is proposed to enhance D. radiodurans survival in environments with high levels of DNA-damaging agents.

molecular biology↗

bioRxiv2024

Eukaryotic initiation factor 3F (eIF3F) regulates the IRES-mediated translation of Bcl-xL via its interaction with programmed cell death 4 (PDCD4) protein

Programmed cell death 4 (PDCD4) protein is a well-characterized tumor suppressor protein. PDCD4 inhibits mRNA translation by inhibiting the activity of an RNA helicase, eukaryotic initiation factor 4A (eIF4A). We have previously reported that PDCD4 interacts with the internal ribosome entry site (IRES) element that is found within the 5 untranslated region (UTR) of mRNA encoding B-cell lymphoma extra-large (Bcl-xL) protein. PDCD4s interaction with the Bcl-xL IRES element inhibits the IRES-mediated translation initiation on Bcl-xL mRNA. However, S6 kinase (S6K)-mediated phosphorylation of PDCD4 activates its degradation by proteasomal degradation pathway and derepress IRES-mediated translation initiation of Bcl-xL mRNA. Interestingly, eIF3F (one of the 13 subunits of eIF3) was reported to recruit S6K to phosphorylate eIF3. Therefore, we were intrigued by the possibility of co-regulation of PDCD4 and eIF3F by S6K and the regulation of IRES-mediated translation initiation by PDCD4-eIF3F. To this end, using co-immunoprecipitation (co-IP), we demonstrated that PDCD4 interacts with several subunits of eIF3. Reciprocal co-IP, endogenous IP, and in vitro pull-down assays demonstrated that eIF3F directly interacts with PDCD4 in an RNA-independent manner. In order to functionally characterize the PDCD4-eIF3F complex, we depleted PDCD4 from the glioblastoma (GBM) cells, which resulted in decreased levels of eIF3F. Also, depletion of eIF3F from GBM cells reduced the levels of PDCD4 protein. However, this was not observed in non-cancer cells. Overexpression of PDCD4 resulted in enhanced levels of eIF3F, and vice versa. We further confirmed that the interaction of eIF3F and PDCD4 proteins prevents each others proteasomal degradation. By performing RNA-IP, we showed that PDCD4 and eIF3F interact with Bcl-xL RNA independently. Moreover, our IRES-bi-cistronic reporter assay and polysome profiling experiments demonstrated that eIF3F regulates IRES-mediated translation of Bcl-xL mRNA, likely via its interaction with PDCD4. SignificanceThis study uncovers the fundamental mechanism of the internal ribosome entry site (IRES)- mediated translation regulation of B-cell lymphoma extra-large (Bcl-xL) mRNA by programmed cell death 4 (PDCD4) protein, and the eukaryotic initiation factor 3F (eIF3F). Our results show that PDCD4 and eIF3F interact with each other directly and they also interact with Bcl-xL mRNA independently. We show that PDCD4 works via eIF3F to regulate Bcl-xL levels. We also show that the PDCD4-eIF3F-dependent mechanism of Bcl-xL mRNA translation is implicated in glioblastoma (GBM) cells, including the primary brain tumor stem cells (BTSCs), and would likely affect the GBM pathophysiology.

molecular biology↗

bioRxiv2023

Natural transformation specific DprA coordinate DNA double strand break repair pathways in heavily irradiated D. radiodurans

Deinococcus radiodurans exhibits remarkable survival under extreme conditions, including ionizing radiation, desiccation, and various DNA-damaging agents. It employs unique repair mechanisms, such as single-strand annealing (SSA) and extended synthesis-dependent strand annealing (ESDSA), to efficiently restore damaged DNA fragments. In this study, we investigate the regulatory role of the NT-specific protein DprA in DNA repair pathways following acute gamma radiation exposure. Our findings demonstrate that the absence of DprA leads to rapid repair of gamma radiation-induced DNA double-strand breaks (DSBs), with diminished involvement of the ESDSA pathway. Furthermore, our data suggest that the SSA pathway becomes the primary mechanism for DNA DSB repair in the absence of DprA. Overall, our results highlight the regulatory function of DprA in modulating the choice between SSA and ESDSA pathways for DNA repair in the radiation-resistant bacterium D. radioduransx.

cell biology↗

bioRxiv2022

Evaluation of the safety, immunogenicity and efficacy of a new live-attenuated lumpy skin disease vaccine in India

Lumpy skin disease (LSD) was reported for the first time in India in 2019 and since then, it has become endemic. Since a homologous (LSD-virus based) vaccine was not available in the country, goatpox virus (GPV)-based heterologous vaccine was authorized for mass immunization against LSD in cattle. This study describes the evaluation of safety, immunogenicity and efficacy of a new live-attenuated LSD vaccine developed using an Indian field strain (LSDV/India/2019/Ranchi). The virus was attenuated by continuous passage (P=50) in Vero cells. The vaccine (50th LSDV passage in Vero cells, named as Lumpi-ProVacInd) did not induce any local or systemic reaction upon its experimental inoculation in calves (n=10). At day 30 post-vaccination (pv), the vaccinated animals were shown to develop antibody- and cell-mediated immune response and exhibited complete protection upon virulent LSDV challenge. We observed a minimum Neethling response (0.018% animals; 5 out of 26940 animals) of the vaccine in field trials among 26940 animals. There was no significant reduction in the milk yield in lactating animals (n=10108), besides there was no abortion or any other reproductive disorder in the pregnant animals (n=2889). Sero-conversion was observed in 85.18% animals in the field by day 30 pv.

microbiology↗

bioRxiv2022

Characterization of DNA processing protein A (DprA) of the radiation-resistant bacterium Deinococcus radiodurans

The uptake of environmental DNA (eDNA) by certain bacteria and its integration into their genome creates genetic diversity and new phenotypes. A DNA processing protein A (DprA) is part of a multiprotein complex and facilitate natural transformation (NT) phenotype in most bacteria. The Deinococcus radiodurans, an extremely radioresistant bacterium, is efficient in N T, and its genome encodes nearly all the components of the natural competence complex. Here, we have characterized the DprA of this bacterium (DrDprA) for the known characteristics of DprA proteins in other bacteria and the mechanisms underlying its roles in the transformation of eDNA into the bacterial genome. DrDprA is found to be a unique domain organization implicating some unique functions compared with DprA of other bacteria. In vitro studies showed that the purified recombinant DrDprA binds to both ssDNA and dsDNA with nearly equal affinity and protects ssDNA from nucleolytic degradation. DrDprA showed a strong interaction with DrRecA indicating its role in RecA catalyzed functions in vivo. Mutational studies identified amino acid residues responsible for its oligomerization, interaction with DrRecA, and DNA binding characteristics of DrDprA. Further, we demonstrated that both oligomerization and DNA binding properties of DrDprA are integral to its support in DrRecA catalyzed strand exchange reaction (SER) in vitro. These results suggested that DrDprA is largely structurally conserved with DprA homologs but showed some unique structure-function features like additional domain, the same affinity to ss/ds DNA and both oligomerization and DNA binding domains collectively contribute to its support in DrRecA functions.

molecular biology↗

bioRxiv2022

Development of duplex real time PCR for quick detection of Cryptosporidia in goats

Cryptosporidium spp. is the most important foodborne and waterborne pathogens and the leading cause of mortality from foodborne and waterborne gastrointestinal disease. In neonates of domestic animals it is associated with consistent diarrhoea and dehydration. Cryptosporidium infection begins with the ingestion of sporulated oocytes disseminated by carrier animals that consistently contaminate the environment. Many diagnostic tests are available including microscopy, antigen trap ELISA, but none of the diagnostic tests available currently cannot differentiate between active and passive infection in the host. In the current study, to address this challenge an mRNA based duplex TaqMan(R) probe PCR (dRT-qPCR) was developed to target the Cryptosporidium oocyst wall protein (COWP) gene and 18ssu rRNA gene in a single tube that can detect metabolically active Cryptosporidial oocysts. The mRNA transcripts are the direct indicator of any actively replicating cell and it will help decipher the active stages of its lifecycle in host. This diagnostic assay was standardized by computing transcript copy number-based limit of detection. For COWP and 188ssu rRNA genes the limit of detection was 7.08x1004 and 5.95x1005 respectively. During active infections the oocyst wall protein will be active and so its COWP gene transcripts will act as marker for active infection. While transcripts for 18SSU rRNA are constitutively expressing in Cryptosporidial life cycle. This current diagnostic assay will be a quantitative marker that will help assess active stages of Cryptosporidium infection in neonates. The disease dynamics will help better understand to formulate the control strategies and contain infection among the healthy animals. ImportanceCryptosporidiosis is an important neonatal disease affecting goats causing diarrhoea, dehydration and stunted growth. For diagnosing this condition, many diagnostic tests are available including microscopy, immunological tests, but none of the diagnostic tests available currently can differentiate between active and passive infection in the host. The mRNA transcripts are the direct indicator of any actively replicating cell and especially in intracellular parasites it will help decipher the infective stages of a lifecycle in the host, and hence the test was developed in a reverse transcriptional format in a duplex mode. The currently developed diagnostic assay for cryptosporidiosis was evaluated for sensitivity using Limit of detection (LOD). This diagnostic test will act as a quantitative marker to aid in detecting active stages of Cryptosporidium infection in neonatal goats and will eventually lead to better control strategies for managing cryptosporidial infections in the future.

molecular biology↗

bioRxiv2021

Comparative evaluation of plasma protein purification and 2-D gel electrophoresis protocols for analysis of HIV-1 infected human plasma proteins

Purification of proteins from human plasma is a herculean task to perform 2-D gel electrophoresis. Human plasma contains nearly 70% albumin and globulin. The removal of such high abundance high molecular weight proteins is very difficult before performing 2-D gel electrophoresis. It becomes more difficult when we intent to investigate in infectious diseases like HIV/AIDS. We tried to the best of our efforts adopting various organic and non-organic based protocols based on various published papers. After failure of these protocols in results of 2-D gel-electrophoresis Aurum serum mini kit (Bio-Rad, USA) was adopted for plasma protein purification for performing 2-D gel electrophoresis. The low-abundance proteins were better resolved by 10% SDS-PAGE in 2-D gel-electrophoresis. Then,we extended the MALDI-TOF/TOF analysis of low-abundance proteins in human plasma by adopting the Aurum serum mini kit (Bio-Rad, USA) for 2-D gel electrophoresis. Thus, we concluded that, depletion of high abundant proteins like albumin and globulin, the use of the Aurum serum mini kit (Bio-Rad, USA) is the protocol of choice to perform the 2-D gel electrophoresis of HIV-1 infected human plasma.

biochemistry↗

bioRxiv2020

Foraging analysis of Endangered Greater Adjutant Stork Leptotilus dubios Gemlin in certain habitat of Assam, India.

The endangered Greater Adjutant Stok Leptotilus dubius Gemlin confined only in some pockets in Assam and Bihar in India and in certain areas of Laos and Cambodia has poorly understood in its foraging activities in its natural habitats. Attempt has been made to address the forage pattern in its natural habitat wetlands in the Kamup district and in a city garbage dump in Guwahati in Assam, India. The various forage methods like walking, visual tactile etc and their relation with the body metrices like beak length, tibia tarsus length and its mass were assessed in terms of foraging habits. Forage actvities were evaluated during the breeding (May to September) and non breeding (October to April) time of the study period of 2012-2017 at different water level of the wetland(s). The foraging range of this stork assessed at an confinement of aerial distance of 15 km from the nesting sites appears as resident non migratory birds by habits. Types of diet composition obtained from the regurgitated food at the ground of the nesting sites were mostly the fish group channa and cyprinids. Profitability index was determined at the captive stork showed in favour of these two groups within the size group of 5 to 15 cm. Larger food item showed lesser profitability index. PCA analysis showed negative foraging correlation with the Prey size greater than 8cm and 15 cm, while the captivity study was conducted Thus the present findings on the foraging assessment of Greater Adjutant might be the baseline information for conservation action plan.Competing Interest StatementThe authors have declared no competing interest.View Full Text

zoology↗

bioRxiv2020

In silico Proteome analysis of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (2019-nCoV), is a positive-sense, single-stranded RNA coronavirus. The virus is the causative agent of coronavirus disease 2019 (COVID-19) and is contagious through human-to-human transmission. The present study reports sequence analysis, complete coordinate tertiary structure prediction and in silico sequence-based and structure-based functional characterization of full SARS-CoV-2 proteome based on the NCBI reference sequence NC_045512 (29903 bp ss-RNA) which is identical to GenBank entry MN908947 and MT415321. The proteome includes 12 major proteins namely orf1ab polyprotein (includes 15 proteins), surface glycoprotein, ORF3a protein, envelope protein, membrane glycoprotein, ORF6 protein, ORF7a protein, orf7b, ORF8, Nucleocapsid phosphoprotein and ORF10 protein. Each protein of orf1ab polyprotein group has been studied separately. A total of 25 polypeptides have been analyzed out of which 15 proteins are not yet having experimental structures and only 10 are having experimental structures with known PDB IDs. Out of 15 newly predicted structures six (6) were predicted using comparative modeling and nine (09) proteins having no significant similarity with so far available PDB structures were modeled using ab-initio modeling. Structure verification using recent tools QMEANDisCo 4.0.0 and ProQ3 for global and local (per-residue) quality estimates indicate that the all-atom model of tertiary structure of high quality and may be useful for structure-based drug designing targets. The study has identified nine major targets (spike protein, envelop protein, membrane protein, nucleocapsid protein, 2-O-ribose methyltransferase, endoRNAse, 3-to-5 exonuclease, RNA-dependent RNA polymerase and helicase) for which drug design targets could be considered. There are other 16 nonstructural proteins (NSPs), which may also be percieved from the drug design angle. The protein structures have been deposited to ModelArchive. Tunnel analysis revealed the presence of large number of tunnels in NSP3, ORF 6 protein and membrane glycoprotein indicating a large number of transport pathways for small ligands influencing their reactivity.

bioinformatics↗

bioRxiv2020

WD 40 domain of RqkA regulates its kinase activity and role in extraordinary radioresistance in Deinococcus radiodurans

RqkA, a DNA damage responsive Serine / Threonine kinase is characterized for its role in DNA repair and cell division in D. radiodurans. It has a unique combination of a kinase domain at N-terminus and a WD40 type domain at C-terminus joined through a linker. WD40 domain is comprised of eight {beta} propeller repeats held together via "tryptophan-docking motifs" and forming a typical velcro closure structure. RqkA mutants lacking the WD40 region (hereafter referred to as WD mutant) could not complement RqkA loss in {gamma} radiation resistance in D. radiodurans and lacked {gamma} radiation mediated activation of kinase activity in vivo. WD mutants failed to phosphorylate its cognate substrate (e.g. DrRecA) in surrogate E. coli cells. Further, unlike wild type enzyme, the kinase activity of its WD40 mutants was not stimulated by Pyrroloquinoline quinine (PQQ) indicating the role of the WD motifs in PQQ interaction and stimulation of its kinase activity. Together, results highlighted the importance of the WD40 domain in the regulation of RqkA kinase signaling functions in vivo and thus the role of WD40 domain in the regulation of any STPK is the first time demonstrated in bacteria. ImportanceThis study highlights the importance of the WD40 domain in activity regulation and signaling activity of bacterial serine/ threonine kinase for the first time in the bacterial response to gamma radiation and DNA damage.

molecular biology↗