Biology subjects

Nandi, D.

Publications and source records attributed to Nandi, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Comparative analysis of immunomodulatory effects of Artemisinin and Parthenolide on activation of immune cells and their roles in Salmonella Typhimurium infection

Plant-derived immunomodulators are of immense therapeutic interest, of which sesquiterpene lactones (SLs) are a promising source. Artemisinin and Parthenolide are two structurally related SLs that are known to inhibit NF-{kappa}B. However, whether this mechanism produces comparable immunomodulatory outcomes has not been directly examined. In this study, we compared the effects of Artemisinin and Parthenolide across different in vitro and in vivo inflammatory contexts: T-cell activation, macrophage activation, and Salmonella Typhimurium infection. Parthenolide inhibited mouse T-cell activation more effectively than Artemisinin in terms of cell cycling, IL-2 production, and induction of activation markers, CD69 and CD44. In addition, Parthenolide suppressed production of LPS-induced nitrite and pro-inflammatory cytokines in primary thioglycolate (TG)-elicited macrophages as well as RAW 264.7 cells. It also reduced ROS production across all tested conditions. In contrast, Artemisinin exhibited comparatively modest effects, although it increased IL-6 in LPS-activated RAW 264.7 cells. Neither compound reduced bacterial burden in an in vitro model of S. Typhimurium infection; however, Parthenolide, but not Artemisinin, lowered TNF- amounts. Together, these findings demonstrate that Parthenolide is a broader and more consistent immunomodulator than Artemisinin. These observations led us to investigate the effects of Parthenolide in an in vivo model of mice orally fed with S. Typhimurium. Parthenolide-treated mice showed higher survival accompanied with lower sera amounts of IL-6 and TNF-, indicating that its protective effects operate by limiting host immunopathology. The implications of these observations on the use of compounds that lower host inflammatory responses during infections are discussed.

immunology↗

bioRxiv2026

TCR-dependent and TCR-independent in-vitro T cell activation generate distinct functional, metabolic, and cytokine programs: Protein kinase C signalling augments anti-CD3+anti-CD28 responses

IntroductionT cell activation is central to the adaptive immune response. In vitro studies on T cell activation often utilize two distinct approaches: first, engaging T cell receptors (TCR) using plate-bound CD3 together with soluble CD28 (TCR-dependent). Second, triggering intracellular signalling cascades using phorbol 12-myristate 13-acetate (PMA) and Ionomycin or P+I (TCR-independent). Both methods are widely used; however, a systematic comparison of the activation methods across a range of stimulation strengths to evaluate their effects on T cell function and metabolism has not been investigated in great detail. In this study, we compared the consequences of engaging T cells using TCR-dependent and TCR-independent activation pathways across varying signal strengths. MethodsT cells from BALB/c mice were isolated and activated under four conditions: CD3, CD3+CD28, PMA with low Ionomycin (P+IL) and PMA with high Ionomycin (P+IH). We studied differences with respect to several parameters: morphology, flow analysis, metabolic activities, cytokines. The roles of Protein kinase C (PKC) and Ca{superscript 2} pathways were addressed by supplementing CD3+CD28 cultures with different doses of exogenous PMA or Ionomycin. ResultsP+I activation outperformed the CD3+CD28 activation system across most readouts by displaying enhanced blasts, higher cycling, greater glucose uptake, increased lactate and ROS production, together with higher upregulation of CD25 and CD44 activation markers. P+IH activation dampened several responses including CD69 expression. CD4 co-receptor was downregulated greatly with P+I activation but not CD3+CD28. Most cytokines followed signal strength comparably between both systems; however, differences were observed with others: P+I stimulation favoured IL-6 and IL-12 induction whereas CD3+CD28 activation preferentially induced CCL2 and IL-1{beta}. Importantly, PKC activity was substantially lower upon CD3+CD28 stimulation and the addition of PMA, but not Ionomycin, to CD3+CD28 cultures enhanced proliferation, metabolism and expression of activation markers. DiscussionTCR-dependent and TCR-independent T cell activation models have clear functional and metabolic differences. The observation that PKC signalling can boost T cell activation with CD3+CD28 is likely to be significant and may have translational implications such as CAR-T cell anti-tumor therapy where CD3+CD28 stimulation is widely used. The implications of our findings with regard to augmenting T cell mediated immunotherapies are discussed.

immunology↗

bioRxiv2026

Coregulation by arcA and fnr protects Salmonella Typhimurium from bile stress by maintaining redox homeostasis and membrane integrity

Bile salts constitute a major antimicrobial barrier encountered by Salmonella Typhimurium during infections. Here, we identified the interplay of two global regulators, Fnr (fumarate and nitrate reductase) and ArcA (aerobic respiration control protein A), in determining adaptive responses to bile. We utilized WT, {Delta}fnr, {Delta}arcA and {Delta}arcA{Delta}fnr strains and compared the expression and functional responses of S. Typhimurium to bile. The highlights of this study are: First, the {Delta}arcA and {Delta}arcA{Delta}fnr strain form smaller colonies on LB agar plates and the {Delta}arcA{Delta}fnr strain displays lower motility. Second, qRT-PCR expression analysis demonstrates that fnr is induced early with bile, followed by arcA. Also, arcA transcripts are lower in the {Delta}fnr strain and fnr expression is also partially lower in the {Delta}arcA strain. Third, the {Delta}arcA and {Delta}fnr strains display partial sensitivity to bile, whereas the {Delta}arcA{Delta}fnr strain exhibits hypersensitivity to bile. Upon bile exposure, the {Delta}arcA{Delta}fnr strain displays elevated transcripts of the major antioxidant genes (sodA and katG) and outer membrane protein (ompC), higher induction of reactive oxygen species (ROS), and greater membrane damage. Fourth, intracellular nitrite is induced earlier than ROS with bile. Fifth, arcA and fnr protects S. Typhimurium from bile induced stress by activating the nitrate metabolism pathway, which lowers ROS. Functionally, pretreatment of the deletion strains with sodium nitrate reduces ROS, improves membrane integrity and survival with bile. Overall, these findings demonstrate that arcA and fnr function cooperatively to utilize alternate electron acceptors, reduce dependence on aerobic respiration and lower ROS to improve survival of S. Typhimurium during bile stress.

microbiology↗

bioRxiv2026

Proteomic Insights into the Mechanism of Action of Maduramicin, a Novel Ionophore with Potent Antimalarial Activity

Malaria is a common infectious disease in tropical countries and poses serious health burden due to limited treatment choices. In recent years, many of the state of the art anti-malarials such as Artemisinin, Chloroquine and Primaquine have been rendered ineffective due to emergence of resistant parasites. This underscores the urgent need to develop new anti-malarials and elucidate their mechanism of action. Recently, we have demonstrated the potent anti-malarial properties of a known anti-coccidial ionophore, Maduramicin. In this study, we investigated the mechanisms of action of Maduramicin in P. falciparum by assessing its stage-specific activity and time-dependent effect on parasite development. The drug exhibited maximum anti-malarial activity against the schizont stage and was characterised as a slow-acting drug. To gain mechanistic insights, we employed iTRAQ based quantitative proteomics approach to analyse global proteome alterations in P. falciparum following Maduramicin treatment. Analysis of the differentially regulated proteins were carried out by using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway database. The data reflected significant perturbations in protein synthesis, energy metabolism, and other key metabolic pathways in response to Maduramicin treatment. Collectively, our results of the proteomics study were validated by quantitative RT-PCR analysis of 9 representative genes. Our findings provide the basis for understanding the lethal activity of Maduramicin on P.falciparum.

biochemistry↗

bioRxiv2026

Identification of small-molecule adjuvants that enhance the sensitivity of Escherichia coli to nitrofurantoin: Roles of Lon and MarA

The increasing cases of resistance among UTI pathogens pose a significant threat to the continued clinical use of nitrofurantoin. In this study, we explored the molecular mechanisms underlying nitrofurantoin resistance and investigated the potential of synergistic activity of salicylates in enhancing the antibacterial activity of nitrofurantoin. In our initial observation, deletion of lon ({Delta}lon) conferred enhanced susceptibility to nitrofurantoin. We identified the critical role of Lon protease in regulating the sensitivity to nitrofurantoin. Investigation into the mechanisms revealed that the lon deletion strains show a higher level of marA and nfsA, which is likely to facilitate the conversion of nitrofurantoin from its pro form to its active form. The {Delta}lon strains displayed an elevated level of ROS, membrane alteration and filamentation upon treatment with nitrofurantoin. Higher ROS levels and membrane alteration were reversed upon treatment with glutathione, further confirming the role of oxidative stress in mediating the sensitivity to nitrofurantoin. Building on these mechanistic insights, we tested salicylates to synergistically enhance the efficacy of nitrofurantoin by indirectly inducing marA through the repression of the mar operon, thereby enhancing nfsA transcription. Both sodium salicylate and acetyl salicylate enhanced the efficacy of nitrofurantoin and lowered the dose of nitrofurantoin required to inhibit the growth of the WT strain. Importantly, this synergistic effect with acetyl salicylate was also observed in nitrofurantoin-resistant clinical isolates, where the combination reduced the effective nitrofurantoin concentration required for growth inhibition. This work provides novel insights into the roles of transcriptional regulators and proteolysis in antibiotic susceptibility, advancing the notion that antibiotic adjuvants are a reliable means of reviving the efficacy of antibiotics. ImportanceThis study unravels the uncharacterised role of Lon protease in nitrofurantoin susceptibility and illustrates the enhanced efficacy of nitrofurantoin-salicylate combinations as a promising therapeutic strategy to overcome emerging resistance in UTI pathogens. This study highlights the importance of investigating the repurposing of other FDA-approved molecules to combat resistance.

microbiology↗

bioRxiv2025

Scratcher: An automated machine-vision tool for dissecting the neural basis of itch

Itch or pruritus invokes a specific reflexive and repetitive directed nocifensive behavioural response, known as scratching. Recent decades have revealed neural circuits that are involved in the sensory and affective-motivational aspects of itch-induced scratching. However, most of these studies relied on manual subjective methods of quantifying scratching in laboratory mice and rats. Recent advances in deep learning have opened avenues for the development of computational tools to analyze animal behaviour in a reliable and automated manner. Further, combined with optogenetic and chemogenetic strategies, these tools can accelerate our understanding of neural circuits underlying itch and scratching. To that end, we have developed Scratcher, a GUI-based computational tool based on a real-time object detection algorithm that allows semi-supervised automated analysis of scratching behaviour in mice in a computationally inexpensive manner. We recorded chloroquine-induced acute itch as it developed and determined the consequence of nail-trimming on acute-itch induced scratching with Scratcher. To probe the neural mechanisms underlying itch, we combined Scratcher with genetic circuit dissection using the Fos-TRAP mouse line. By targeting itch-activated neurons in the lateral parabrachial nucleus (LPBN) - a key brainstem hub for pruritic signal transmission - we demonstrated that LPBN activity modulates itch-evoked scratching. Together, we present a novel, easy-to-use computational tool to dissect molecular, cellular, and circuit mechanisms of itch and scratching.

neuroscience↗

bioRxiv2025

Acoustic individual identification in a species of field cricket using deep learning

Individual animal identification is essential for wildlife conservation and management, helping to estimate animal abundance and related parameters. We assessed the feasibility of identifying individual field crickets (Plebeiogryllus guttiventris) from their calls using deep learning. In a closed population of known individuals, the best models recognized crickets with up to 99.9% accuracy when trained and tested on calls from the same night, and 63.2% when tested on calls from nights not used in training. When matching pairs of calls, without knowledge of all individuals in the population, models identified pairs of calls from the same night with 97% accuracy, falling to 87.1% if calls were from different nights. Accuracy remained high when tested on individuals not observed during training (within night: 94.1%; across nights 78.8%). Pooling training data across multiple nights improved test accuracy for all models. Deep learning outperformed random forests, particularly on harder tasks, although both were able to discriminate individuals. Analysis of temperature effects on cricket calling behavior showed that higher temperatures were associated with shorter chirp durations and higher carrier frequencies. However, adjusting spectrograms for chirp duration alone, or for both duration and frequency, did not improve performance. The results are the first to demonstrate AIID using wild recordings of an insect species, and further highlight the potential of deep learning-based AIID for non-invasive monitoring of animal populations.

bioinformatics↗

bioRxiv2025

Identification of 4-Amino-2-Nitrophenol as a novel inducer of phenotypic antibiotic resistance in Escherichia coli: roles of Lon protease and its substrate MarA

In prokaryotes, the energy-dependent protein degradation is controlled primarily by two ATP-dependent proteases, Lon and Clp. This study investigates the roles of the Lon protease in the metabolism of 2,4-dinitrophenol (2,4-DNP), a toxic industrial compound, in Escherichia coli (E. coli). During the study, an observation was made that the absence of Lon protease resulted in an enhanced conversion of yellow coloured 2,4-DNP to a reddish-brown product. This study aims to characterise the compound observed in the media with wild type (WT) and {Delta}lon strains, understand the mechanisms of 2,4-DNP conversion and decipher the roles of Lon protease in the conversion of 2,4-DNP. UV-visible and LC-MS analyses revealed differences in the conversion products between the WT and {Delta}lon strains. One of the substrates of Lon protease is MarA, a transcription factor. Growth studies with different mutants and trans-complemented strains demonstrated MarA-dependent conversion. The bathochromic shift of spectral peaks suggested a reduction process and possible involvement of nitroreductase enzymes. Indeed, the expression of two nitroreductases, nfsA and nfsB, increased with 2,4-DNP and was dependent on MarA. Importantly, the production of the reddish-brown product was lower in strains lacking nfsA or nfsB. Finally, LC-MS analysis identified one of the conversion products of 2,4-DNP to be 4-Amino-2-nitrophenol (4,2-ANP). Dose studies with purified 4,2-ANP demonstrated that it did not lower the growth of E. coli (unlike 2,4-DNP) but induced phenotypic antibiotic resistance (like 2,4-DNP). This study contributes to our understanding of biological treatment of nitroaromatics and may offer insights into environmental pollution mitigation strategies. ImportanceThis study identifies the roles of Lon protease and its substrate MarA in inducing nitroreductases, NfsA and NfsB, in reducing toxic 2,4-DNP to less toxic 4,2-ANP, a novel inducer of phenotypic antibiotic resistance. This study contributes to understanding the biological treatment of nitroaromatics, offering insights into environmental pollution mitigation strategies and the development of efficient bioremediation techniques.

microbiology↗

bioRxiv2025

Global transcriptome analysis identifies nicotinamide metabolism to play key roles in IFN-gamma and nitric oxide modulated responses

Interferon-gamma (IFN-{gamma}) is a key regulator of immune responses. A hallmark of the IFN-{gamma} response is inducible nitric oxide (NO) production, driven primarily by nitric oxide synthase 2 (NOS2). In this study, we investigated the influence of NO on the IFN-{gamma}-induced transcriptomic and metabolic changes in the RAW 264.7 macrophage cell line. IFN-{gamma} activation led to NO-dependent lactate production and lower cell survival. Bulk RNA sequencing analysis identified genes differentially expressed early by IFN-{gamma} that were either NO-independent or NO-dependent. Inhibition of NO modulated a minor subset of the transcriptome, notably affecting Klf6 (a tumor suppressor) and Zfp36 (a regulator of pro-inflammatory cytokines). Interestingly, both Klf6 and Zfp36 correlatively showed high expression in most cancers. The PPI network exhibited dense clustering with scale-free and small-world properties, identifying Stat1, Irf7, Irf1, Cxcl10, and Isg15 as top five hubs. The top IFN-{gamma} signalling genes exhibited deficient expression in the brain but were highly expressed in lung, spleen, and EBV-transformed lymphocytes. Gene-disease associations linked the IFN-{gamma}-regulated genes to immunodeficiencies, chronic inflammatory responses and malignancies. Interestingly, IFN-{gamma} upregulated genes were involved in nicotinamide metabolism, suggesting a transcriptional basis for modulation of metabolic pathways. This novel aspect was experimentally tested to show that IFN-{gamma} induced NAD amounts. Importantly, the inhibition of purine nucleoside phosphorylase (using Forodesine hydrochloride) which is involved in the endogenous pathway for NAD generation, lowered IFN-{gamma} induced nitrite and increased cell survival in vitro. Functionally, enriched nicotinamide metabolism by IFN-{gamma} may regulate inflammatory responses and the implications of our findings are discussed.

immunology↗

bioRxiv2024

High intracellular calcium amounts inhibit activation-induced proliferation of mouse T cells

Optimal T cell activation is critical to orchestrate adaptive immune responses. Calcium is critical for T cell activation and integrates signaling pathways necessary to activate key transcription factors. In fact, patients with calcium channelopathies are immunodeficient. Here, we investigated the effects of different concentrations of intracellular calcium on activation of mouse T cells. High intracellular calcium amounts inhibited in vitro T cell proliferation as evidenced by a decreased cell cycling-to-hypodiploidy ratio in two models of activation: the combination of phorbol 12-myristate 13-acetate (PMA) and Ionomycin (an ionophore)/Thapsigargin (a SERCA inhibitor) or plate bound anti-CD3 and anti-CD28. High intracellular calcium amounts increased the production of reactive oxygen species (ROS) in T cells activated with PMA and Ionomycin and scavenging excess ROS using N-acetyl cysteine (NAC) or PEGylated superoxide dismutase (PEG-SOD) rescued the decrease in cycling-to-hypodiploidy ratio. To test the universality of our observations, we studied the effects of tert-Butylhydroquinone (tBHQ), a SERCA inhibitor and Nrf2 activator. tBHQ alone did not increase intracellular calcium amounts but increase was observed along with PMA. Also, tBHQ inhibited T cell activation in a dose-dependent manner in both in vitro models of T cell activation. Importantly, intraperitoneal injection of tBHQ ameliorated Dextran Sodium Sulfate (DSS)-induced colitis in mice as evidenced by rescue of colon length shortening and lower disease activity index. Overall, this study identifies high calcium amounts as a potential target to lower T cell activation. The implications of these observations are discussed as this strategy may be important in treatment of some autoimmune diseases.

immunology↗

bioRxiv2023

Interferon-γ lowers tumour growth by increasing glycolysis and lactate production in a nitric oxide-dependent manner: implications for cancer immunotherapy

Interferon-gamma (IFN-{gamma}), the sole member of the type-II interferon family, is well known to protect the host from infectious diseases as well as mount anti-tumour responses. The amounts of IFN-{gamma} in the tumour microenvironment determine the host responses against tumours; however, several tumours employ evasive strategies by responding to low IFN-{gamma} signalling. In this study, the response of various tumour cell lines to IFN-{gamma} was studied in vitro. IFN-{gamma}-activation increases glycolytic flux and reduces mitochondrial function in a nitric oxide (NO)- and reactive oxygen species (ROS)-dependent manner in the H6 hepatoma tumour cell line. The higher glycolysis further fuelled NO and ROS production, indicating a reciprocal regulation. These processes are accompanied by Hypoxia inducing factor (HIF)-1 stabilization and HIF-1-dependent augmentation of the glycolytic flux. The IFN-{gamma} enhancement of lactate production also occurred in other NO-producing cell lines: RAW 264.7 monocyte/macrophage and Renca renal adenocarcinoma. However, two other tumour cell lines, CT26 colon carcinoma and B16F10 melanoma, did not produce NO and lactate upon IFN-{gamma}-activation. HIF-1 stabilization upon IFN-{gamma}-activation led to lower cell growth of B16F10 but not CT26 cells. Importantly, the IFN-{gamma}-activation of both CT26 and B16F10 cells demonstrated significant cellular growth reduction upon metabolic rewiring by exogenous administration of potassium lactate. Clinical studies have shown the crucial roles of IFN-{gamma} for successful cancer immunotherapies involving checkpoint inhibitors and chimeric antigen receptor T cells. The positive implications of this study on the metabolic modulation of IFN-{gamma} activation on heterogeneous tumour cells are discussed.

immunology↗

bioRxiv2023

Functional loss of rffG and rfbB, encoding dTDP-glucose 4,6-dehydratase, changes colony morphology, cell shape, motility and virulence in Salmonella Typhimurium

Lipopolysaccharide (LPS) O-antigen and enterobacterial common antigen (ECA) play crucial roles in maintaining the structural integrity of the outer membrane in Gram-negative bacteria. Previous studies conducted with either LPS or ECA mutants have highlighted the importance of these cell surface polysaccharides in the physiology of Salmonella enterica serovar Typhimurium (S. Typhimurium). However, the functional consequences resulting from the abrogation of both O-antigen and ECA synthesis in S. Typhimurium are not well studied. In the present study, we generated single and double gene-deleted mutants of rffG and rfbB, which are paralogs, encoding dTDP-glucose 4,6-dehydratase that catalyze steps in the synthesis of both O-antigen and ECA. The functional loss of both rffG and rfbB ({Delta}rffG{Delta}rfbB), but not in single gene-deleted strains, results in a round cell morphology, smaller colony formation and altered LPS profile. In addition, the {Delta}rffG{Delta}rfbB strain displays defects in outer membrane permeability, causing hypersensitivity to bile and cell wall targeting antibiotics, e.g., meropenem and polymyxin B. Transcriptomic analysis identified flagellar and SPI-1 pathway to be highly down-regulated in the {Delta}rffG{Delta}rfbB strain which leads to impaired swimming and swarming motility and lower adhesion and invasion of HeLa cells. Importantly, the {Delta}rffG{Delta}rfbB strain is less proficient in colonizing Peyers patches, spleen and liver, is unable to induce pro-inflammatory cytokines and is attenuated in both the oral and intra-peritoneal models of S. Typhimurium infection in mice. Overall, this study highlights the importance of rffG and rfbB in maintaining cell wall integrity, colony and cellular morphology, motility and virulence in S. Typhimurium.

microbiology↗

bioRxiv2023

Global transcriptome analysis reveals Salmonella Typhimurium employs the nitrate-dependent anaerobic pathway to combat bile stress

Salmonella Typhimurium is an enteric pathogen that is highly tolerant to bile. Next-generation mRNA sequencing was performed to analyse the stress and adaptive responses of S. Typhimurium to bile. We identified the cellular pathways affected during bile stress in wild type (WT) and a mutant lacking cspE ({Delta}cspE), which plays an essential role in protection from bile stress. We observed transcriptional upregulation of several genes involved in nitrate metabolism, in response to bile stress. These genes were also differentially expressed between the bile-resilient WT and the bile-sensitive {Delta}cspE strain. To understand the role of nitrate metabolism in bile stress response, we generated a strain lacking fnr ({Delta}fnr), which is the global regulator of nitrate metabolism in S. Typhimurium. fnr was highly induced in the bile treated WT strain but not in the {Delta}cspE strain. Notably, the {Delta}fnr strain was susceptible to bile-mediated killing. Our studies revealed a new role for fnr in mediating the bile stress response. In addition, a strain lacking arcA ({Delta}arcA), a two-component system response regulator involved in anaerobic metabolism, also showed a marked reduction in growth in presence of bile. This corroborated the significance of anaerobic metabolism in S. Typhimurium bile tolerance. Importantly, overexpression of fnr and arcA lowered reactive oxygen species and significantly enhanced the survival of the bile-sensitive {Delta}cspE strain. We also observed that S. Typhimurium pre-treated with nitrate displayed better growth in the presence of bile. Together, these results demonstrate that nitrate-dependent anaerobic metabolism promotes adaptation of S. Typhimurium to bile. ImportanceSalmonella Typhimurium, as an enteric pathogen, manifests an extreme example of bile tolerance. This study describes the diverse metabolic changes at the level of transcriptome in S. Typhimurium exposed to bile. We identified the differential expression of several genes involved in anaerobic metabolism between bile-tolerant WT and bile-sensitive {Delta}cspE strains. Two major regulators of anaerobic metabolism, fnr and arcA, support the growth of S. Typhimurium in bile. Our results highlight that, in presence of bile, S. Typhimurium activates genes involved in anaerobic metabolism, specifically nitrate metabolism, that improves survival of bacteria during bile stress.

microbiology↗

bioRxiv2023

Functional characterization of Salmonella Typhimurium encoded YciF, a domain of unknown function (DUF892) family protein, and its role in protection during bile and oxidative stress

YciF (STM14_2092) is a member of domain of unknown function (DUF892) family. It is an uncharacterized protein, involved in stress responses in Salmonella Typhimurium. In this study, we investigated the significance of YciF and its DUF892 domain during bile and oxidative stress responses of S. Typhimurium. Purified wild type YciF forms higher order oligomers, binds to iron and displays ferroxidase activity. Studies on the site-specific mutants revealed that the ferroxidase activity of YciF is dependent on the two metal binding sites present within the DUF892 domain. Transcriptional analysis displayed that the {Delta}cspE strain, which has compromised expression of YciF, encounters iron toxicity due to dysregulation of iron homeostasis in presence of bile. Utilizing this observation, we demonstrate that the bile mediated iron toxicity in {Delta}cspE causes lethality, primarily through the generation of reactive oxygen species (ROS). Expression of wild type YciF, but not the three mutants of the DUF892 domain, in {Delta}cspE alleviate ROS in presence of bile. Our results establish the role of YciF as a ferroxidase that can sequester excess iron in the cellular milieu to counter ROS-associated cell death. In fact, pre-treatment with an iron chelator mitigates the hypersensitivity of {Delta}cspE to bile. ImportanceThe DUF892 domain has a wide taxonomic distribution encompassing several bacterial pathogens. This domain belongs to the ferritin-like superfamily; however, it has not been biochemically and functionally characterized. This is the first report of characterization of a member of this family. In this study, we demonstrate that S. Typhimurium YciF is an iron binding protein with ferroxidase activity, which is dependent on the metal binding sites present within the DUF892 domain. It combats iron toxicity and oxidative damage caused due to exposure to bile. The functional characterization of YciF delineates the significance of the DUF892 domain in bacteria. In addition, our studies on S. Typhimurium bile stress response divulged the importance of comprehensive iron homeostasis in bacteria in presence of bactericidal compounds that tend to generate ROS irrespective of their primary targets.

microbiology↗

bioRxiv2022

Cytoplasmic membrane thinning observed by interfacial dyes is likely a common effect of bactericidal antibiotics

The lipid membrane is a fundamental part of life. However, the effects of different stresses on membranal integrity and physiology are less understood. Using novel 4-aminophthalimide-based membrane-specific dyes (4AP-Cn: n is carbon chain-length), aided with confocal microscopy, fluorescence spectroscopy, molecular dynamics simulations, and flow cytometry, we have studied stress-mediated changes in E. coli membranes. By exploiting the depth-dependent positioning and subsequent environmental sensitivity of the dyes, we have proposed a measure of antibiotic-induced membrane damage: the fluorescence Peak Maxima Difference (PMD) between 4AP-C9 and 4AP-C13. The ROS-influenced PMD quantifies cytoplasmic membrane thickness and measures sensitivity against most bactericidal antibiotics, depending upon the extent of lipid peroxidation. Importantly, we have verified this observation using antibiotic-sensitive and resistant clinical isolates of E. coli and ESKAPE pathogens like K. pneumoniae and Enterobacter spp. This study underscores the role of antibiotic-induced membrane changes; a critical issue given the increasing global antimicrobial resistance.

microbiology↗

bioRxiv2021

The Anaphase Promoting Complex/ cyclosome co-activator, Cdh1, is a novel target of human Papillomavirus 16 E7 oncoprotein in cervical oncogenesis

The transforming properties of the high risk human papillomavirus E7 oncoprotein are indispensable for driving the virus life cycle and pathogenesis. Besides inactivation of retinoblastoma (Rb) family of tumor suppressors as part of its oncogenic endeavors, E7-mediated perturbations of eminent cell cycle regulators, checkpoint proteins and proto-oncogenes are considered to be the tricks of its transformative traits. However, many such critical interactions are still unknown. In the present study, we have identified the anaphase promoting complex/ cyclosome (APC/C) co-activator, Cdh1, as a novel interacting partner and a degradation target of E7. We found that HPV16 E7-induced inactivation of Cdh1 promoted abnormal accumulation of multiple Cdh1 substrates. Such a mode of deregulation possibly contributes to HPV-mediated cervical oncogenesis. Our mapping studies recognized the carboxyl-terminal zinc finger motif of E7 to associate with Cdh1 and interfere with the timely degradation of FoxM1, a bona fide Cdh1 substrate and a potent oncogene. Importantly, the E7 mutant with impaired interaction with Cdh1 exhibited defects in its ability for overriding typical cell cycle transition and oncogenic transformation, thereby validating the functional and pathological significance of the E7-Cdh1 axis during cervical carcinoma progression. Altogether, the findings from our study discover a unique nexus between E7 and APC/C-Cdh1, thereby adding to our understanding of the mechanism of E7-induced carcinogenesis and provide a promising target for the management of cervical carcinoma.

biochemistry↗

bioRxiv2021

Particle Uptake Driven Phagocytosis in Macrophages and Neutrophils Enhances Bacterial Clearance

Humans are exposed to numerous synthetic foreign particulates in the form of environmental pollutants and diagnostic or therapeutic agents. Specialized immune cells (phagocytes) clear these particulates by phagocytosing and attempting to degrade them. The process of recognition and internalization of the particulates may trigger changes in the function of phagocytes. Some of these changes, especially the ability of a particle-loaded phagocyte to take up and neutralize pathogens, remains poorly studied. Herein, we demonstrate that the uptake of non-stimulatory cargo-free particles enhances the phagocytic ability of monocytes, macrophages and neutrophils. The enhancement in phagocytic ability was independent of particle properties, such as size or the base material constituting the particle. Additionally, we show that the increased phagocytosis was not a result of cellular activation or cellular heterogeneity but was driven by changes in cell membrane fluidity and cellular compliance. A consequence of the enhanced phagocytic activity was that particulate-laden immune cells neutralize E. coli faster in culture. Moreover, when administered in mice as a prophylactic, particulates enable faster clearance of E. coli and S. epidermidis. Together, we demonstrate that the process of uptake induces cellular changes that favor additional phagocytic events. This study provides insights into using non-stimulatory cargo-free particles to engineer immune cell functions for applications involving faster clearance of phagocytosable particulates.

immunology↗

bioRxiv2020

Highly accurate random DNA sequencing using inherent interlayer potential traps of bilayer MoS2 nanopores

Solid-state MoS2 nanopores are emerging as potential real-time DNA sequencers due to their ultra-thinness and pore stability. One of the major challenges in determining random nucleotide sequence (unlike polynucleotide strands) is the non-homogeneity of the charge interaction and velocity during DNA translocation. This results in varying blockade current for the same nucleotide, reducing the sequencing confidence. In this work, we studied the inherent impedance-tunability (due to vertical interlayer potential gradient and ion accumulation) of multilayered MoS2 nanopores along with its effect on improving analyte capture and charge interaction, for more sensitive and confident sensing. Experimentally we demonstrate that 2-3 nm diameter bilayer MoS2 pores are best suited for high accuracy (~90%) sequencing of mixed nucleotides with signal-to-noise-ratio greater than 11 in picomolar concentration solutions. High temporal resolution demonstrated by bilayer MoS2 nanopores can help detect neutral proteins in future. The high accuracy detection in low concentration analyte can hence be applied for control and prevention of hereditary diseases and understanding health effects of rare microbial strains.

bioengineering↗