Biology subjects

Shinde, S.

Publications and source records attributed to Shinde, S..

16 recordsLinked to original sources

bioRxiv2026

Direct CDK1 engagement underlies carbaryl-induced genotoxic cell cycle arrest

Carbaryl, a widely used carbamate pesticide, has been epidemiologically associated with genotoxic and carcinogenic outcomes in exposed human populations, including elevated cancer risk at several tissue sites and, in some agricultural communities, chronic tubulointerstitial kidney disease of unknown etiology (CKDu). However, the cellular mechanisms underlying these associations, and whether carbaryl acts uniformly across tissues, remain poorly defined. Screening a panel of cell lines of distinct species and lineage, we found that carbaryl elicits pronounced, dose-dependent cytotoxicity and cell cycle arrest selectively in a renal tubular epithelial line (IMCD3) and, to a lesser extent, a cervical epithelial line (HeLa), while a fibroblast line (NIH3T3) was unaffected. In IMCD3 cells, carbaryl induces S- and G2/M-phase arrest through direct binding to DNA and consequent DNA damage, triggers Bcl-2-mediated late apoptosis independently of reactive oxygen species, and drives irreversible mitotic arrest. Molecular dynamics simulations, cellular thermal shift assays, and kinase measurements converge on CDK1 as a direct target of carbaryl: carbaryl destabilises the CDK1 active site and inhibits its catalytic activity, whereas CDK2 is engaged through an opposing, rigidifying mode with no functional consequence. This asymmetry provides a coherent mechanistic basis for the combined S-phase and G2/M arrest observed. Together, these findings establish carbaryl as a genotoxic, cell-cycle-disrupting agent with tissue-selective potency acting directly on CDK1, and provide a cell-based mechanistic framework for interpreting its reported associations with renal and other tissue pathologies in exposed human populations.

cell biology↗

bioRxiv2026

Circulating Cell-Free Chromatin Particles Trigger a Unique Biphasic STING Signaling Program that Drives DNA Damage and Inflammation

Cell death, DNA damage, and inflammation are closely interconnected processes implicated in ageing, cancer, and inflammatory disorders, yet the endogenous mechanisms linking them remain unclear. We previously identified cell-free chromatin particles (cfChPs), released from dying cells, as biologically active entities that enter neighboring cells and induce DNA damage and inflammation. Here, we show that serum-derived circulating cfChPs are rapidly internalized by human peripheral blood mononuclear cells and trigger a previously unrecognized biphasic STING signaling response. An early phase involves rapid STING trafficking to the perinuclear region and nucleus, with activation of IRF3 and NF-{kappa}B preceding detectable DNA damage. This is followed by a later phase characterized by STING phosphorylation, puncta formation, persistent DNA damage, and robust inflammatory cytokine production. Pharmacological inhibition or genetic deletion of STING markedly attenuated these responses. These findings identify extracellular cfChPs as endogenous DNA-damaging agents and reveal biphasic STING signaling as a mechanistic link between cell death, DNA damage, and sterile inflammation. One-sentence summaryCirculating cell-free chromatin particles released from dying cells trigger a previously unrecognized biphasic STING signaling program that mechanistically links cell death to DNA damage and sterile inflammation, providing a potential basis for aging, cancer, and inflammatory diseases.

immunology↗

bioRxiv2026

Real-time, label-free assessment of cell fusion dynamics by high-content imaging

Cell-cell fusion is a fundamental biological process underlying diverse physiological and pathological phenomena, yet its quantitative analysis remains methodologically challenging due to its dynamic, heterogeneous, and multistep nature. Existing approaches to assess fusion largely rely on endpoint assays or manual scoring, limiting temporal resolution, scalability, and reproducibility. Here, we present a label-free, high-content live-cell imaging pipeline for real-time quantification of cell fusion dynamics, developed and validated using trophoblast syncytialization as a model system. The method integrates automated image acquisition with a reproducible, stepwise analysis workflow combining supervised texture-based segmentation, morphology-based measurements, and intensity-independent texture analysis. We define quantitative metrics, including the ratio of total cluster area to the number of detected clusters and cytoplasmic granularity features, that together discriminate bona fide fusion events from non-fusion-related cellular clustering or proliferation. Using canonical pharmacological inducers and inhibitors of fusion, we demonstrate the specificity and sensitivity of these parameters for detecting fusion-associated remodeling over time. We further demonstrate the scalability of the pipeline through high-throughput screening of biologically relevant growth factors, hormones, and inhibitors, enabling classification of modulators based on their independent, synergistic, or antagonistic effects on fusion dynamics. Consistent results obtained in an independent model further support its potential applications to additional fusion systems. By providing a robust, reproducible, and adaptable framework for time-resolved fusion analysis, this methodology bridges the gap between qualitative observation and quantitative kinetic assessment. Thus, the approach could be readily extended to other cell fusion systems following system-specific parameter optimization, offering a versatile platform for both mechanistic studies and discovery-driven screening applications.

developmental biology↗

bioRxiv2025

CrebA regulation of secretory capacity: Genome-wide transcription profiling coupled with in vivo DNA binding studies

DNA binding assays, expression analyses, and binding site mutagenesis revealed that the Drosophila CrebA transcription factor (TF) boosts secretory capacity in the embryonic salivary gland (SG) through direct regulation of secretory pathway component genes (SPCGs). The mammalian orthologues of CrebA, the Creb3L-family of leucine zipper TFs, not only activate SPCG expression in a variety of mammalian tissues but can also activate SPCG expression in Drosophila embryos, suggesting a highly conserved role for this family of proteins in boosting secretory capacity. However, in vivo assays reveal that CrebA binds far more genes than it regulates, and it remains unclear what distinguishes functional binding. It is also unclear if CrebA is the major factor driving SPCG gene expression in all Drosophila embryonic tissues and/or if CrebA also regulates other tissue-specific functions. Thus, we did single cell RNA sequencing (scRNA-seq) of wild-type (WT) and CrebA null embryos to explore the relationship between CrebA binding and gene regulation. We find that CrebA binds the proximal promoters of its targets, that SPCGs are the major class of genes regulated by CrebA across tissues, and that CrebA is sufficient to activate SPCG expression even in cells that do not normally express the protein. A comparison of scRNA-Seq to other methods for capturing regulated transcripts reveals that the different methodologies identify overlapping but distinct sets of CrebA targets.

genomics↗

bioRxiv2025

A Waddingtonian description of the dynamics of Turing patterns

Turing patterns are a well-studied model of reaction-diffusion equations for developmental patterning. Their applicability has often been limited by the difficulty in identifying candidate molecules that satisfy the requisite criteria for patterning. Here, we build on recent work on geometric models to describe Turing patterning as a potential flow. We show how the universal dynamics of Turing patterning is described by a landscape, largely independent of the underlying reaction-diffusion equations. We apply our framework to three-component systems and demonstrate that we can accurately capture the dynamics of any given component. We extend our framework to larger networks and to models of Turing patterns coupled with external morphogens that provide positional information. We provide a quantitative description of the dynamics of chosen markers and apply it to the dynamics of SOX9 expression during digit patterning.

systems biology↗

bioRxiv2024

Induction of pluripotent oncogenic stem cells from mouse fibroblasts

Natural biological agents that can transform normal somatic cells into cancer stem cells, have not been identified. We earlier reported that cell free chromatin particles (cfChPs) that circulate in blood of cancer patients can horizontally transfer themselves to healthy cells to induce dsDNA breaks and inflammation. Here we show that a single cell clone D5 developed from NIH3T3 mouse fibroblast cells treated with cfChPs isolated from sera of cancer patients exhibited upregulation of stemness related transcription factors and surface markers, and the ability to form spheroids in appropriate culture medium. Transcriptome analysis revealed upregulation of cancer related pathways including invasion, metastasis and stemness. Subcutaneous inoculation into SCID mice resulted in development of malignant tumors which expressed all three germline markers. Our results suggest the cfChPs that circulate in blood of cancer patients are oncogenic and can transform susceptible somatic cells into cancer stem cells with the potential to promote metastasis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/622204v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@4288org.highwire.dtl.DTLVardef@1102d4borg.highwire.dtl.DTLVardef@10daaf6org.highwire.dtl.DTLVardef@53084d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

A deep learning convolutional neural network distinguishes neuronal models of Parkinson's disease from matched controls

Parkinsons disease (PD) is a neurodegenerative disorder that results in the loss of dopaminergic neurons in the substantia nigra pars compacta. Despite advances in understanding PD, there is a critical need for novel therapeutics that can slow or halt its progression. Induced pluripotent stem cell (iPSC)-derived dopaminergic neurons have been used to model PD but measuring differences between PD and control cells in a robust, reproducible, and scalable manner remains a challenge. In this study, we developed a binary classifier convolutional neural network (CNN) to accurately classify microscopy images of PD models and matched control cells. We acquired images of iPSC-derived neural precursor cells (NPCs) and dopaminergic (DANs) and trained multiple CNN models comparing control cells to genetic and chemical models of PD. Our CNN accurately predicted whether control NPC cells were treated with the PD-inducing pesticide rotenone with 97.60% accuracy. We also compared control to a genetic model of PD (deletion of the Parkin gene) and found a predictive accuracy of 86.77% and 95.47% for NPC and DAN CNNs, respectively. Our cells were stained for nuclei, mitochondria, and plasma membrane, and we compared the contribution of each to the CNNs accuracy. Using all three features together produced the best accuracy, but nuclear staining alone produced a highly predictive CNN. Our study demonstrates the power of deep learning and computer vision for analyzing complex PD-related phenotypes in DANs and suggests that these tools hold promise for identifying new targets for therapy and improving our understanding of PD.

neuroscience↗

bioRxiv2023

Metastases arise as new cancers from cells of target organs transformed by cell-free chromatin particles released from dying cancer cells

IntroductionBased on our earlier finding that cell-free chromatin particles (cfChPs) released from dying cancer cells are potentially oncogenic, we hypothesised that metastases arise as new cancers from the cells of target organs transformed by cfChPs released from dying cancer cells. MethodsWe fluorescently dually-labelled MDA-MB-231 human breast cancer cells and A-375 human melanoma cells in their DNA with BrdU and in their histones with CellLight(R) Histone 2BGFP. One hundred thousand dually-labelled cells were intravenously injected into SCID mice. In other experiments unlabelled cells were injected for detection of lung metastasis. Also intravenously injected were 700 ng of purified cfChPs isolated from radiation-killed MDA-MB-231 cells. ResultsWe observed that the fluorescently dually-labelled MDA-MB-231 and A-375 cells died upon reaching the lungs and released dually-labelled fluorescent chromatin particles that accumulated in the nuclei of lung cells at 48 h. Injection of unlabelled cfChPs led to the activation of 10 hallmarks of cancer and immune checkpoints in lung cells at 72 h, suggesting that the lung cells had rapidly transformed into incipient cancer cells. Fluorescent in situ hybridisation analysis of lung metastases that subsequently developed using mouse and human specific DNA probes revealed that the tumour cells contained both mouse and human DNA in almost equal proportions. Similarly, immune-fluorescence analysis using species specific mAbs revealed that the tumour cells co-expressed mouse and human specific proteins. Metaphase preparations and single-cell clones developed from cell cultures of lung metastases were found to contain chimeric chromosomes containing both mouse and human DNA, and the cells to co-express both human- and mouse-specific proteins. The Intravenously injected purified cfChPs isolated from radiation-killed MDA-MB-231 cells also induced lung metastasis which predominantly contained mouse DNA strongly suggesting that the metastatic tumours had arisen from the mouse lung cells. ConclusionThese results provide strong evidence that cfChPs released from dying cancer cells integrate into genomes of target lung cells to transform them into new cancers that masquerade as metastasis. They support our hypothesis that metastases arise from the cells of target organs and not from those of the primary tumour. These findings have implications for the principles of cancer therapy.

cancer biology↗

bioRxiv2023

Cell-free chromatin particles activate immune checkpoints in human T cells: Implications for cancer therapy

Immune checkpoint blockade is an exciting breakthrough in cancer therapy, but how immune checkpoints are activated is unknown. We have earlier reported that cell-free chromatin particles (cfChPs) that circulate in the blood, or those that are released locally from dying cells, are readily internalized by healthy cells with biological consequences. Here we show that treatment of human lymphocytes with cfChPs isolated from sera of cancer patients led to marked activation of immune checkpoints viz. PD-1, CTLA-4, LAG-3, NKG2A, and TIM-3. Concurrently activated were stress-related markers cJun, cFos, JunB, FosB, NF[Kcy]B, and EGR1. The above immune checkpoints were also activated when lymphocytes were treated with cfChPs released from dying HeLa cells; the latter could be abrogated by three cfChPs deactivating agents. These results suggest that immune checkpoints are activated by lymphocytes as stress response to cfChPs. Simultaneous downregulation of multiple immune checkpoints may herald a new approach to immunotherapy of cancer. Statement of SignificanceWe show that cell-free chromatin particles (cfChPs) that circulate in the blood of cancer patients, or those released from dying cancer cells, simultaneously activate five immune checkpoints as a stress response by human lymphocytes. Activation of checkpoints was abrogated by cfChPs deactivating agents suggesting a novel approach to cancer treatment.

immunology↗

bioRxiv2023

Therapeutic interventions on human xenografts promote systemic dissemination of oncogenes

We generated xenografts of human cancer cells in mice, and using immuno-FISH analysis detected multiple co-localizing signals of human DNA and eight human oncoproteins in brain cells. Signals increased dramatically five days after treatment with chemotherapy, localized radiotherapy or surgery, which could be minimized by concurrent treatment with cell-free chromatin deactivating agents. These results suggest that therapeutic interventions may potentially encourage metastatic spread which is preventable by deactivating cell-free chromatin.

cancer biology↗

bioRxiv2021

Effect of Human Mesenchymal Stem Cells on Freund's adjuvant-induced Rheumatoid Arthritis in Sprague Dawley Rats

IntroductionMesenchymal stem cells (MSC) therapy is a new approach to treat RA. Studies evaluating anti-inflammatory effects of MSCs per RA severity are scarce. Our primary objective was to evaluate anti-inflammatory effects, change in cytokine levels and cartilage regeneration of two different MSC preparations delivered through two different routes of administration in three RA stages: mild, moderate and severe. MethodsHuman-derived umbilical cord tissue MSCs (hUCT-MSCs) and human bone marrow-derived MSCs (hBM-MSCs) delivered via intra-plantar and intravenous routes were tested in Freunds adjuvant-induced arthritis in rats. Arthritis severity was based on the arthritis score (<3=mild, 3=moderate and 4=severe). Assessments included changes in arthritis scoring, paw swelling, haematology parameters, biomarkers (TNF and IL-10) and histopathology analysis. ResultsMSC treatment significantly reduced arthritis scores in all treatment groups. IL-10 levels increased 30 days after treatment with (IP)hUCT-MSCs (P=0.0241), (IV)hUCT-MSCs (P=0.0095) and (IP)hBM-MSCs (P=0.0002). TNF- levels reduced compared to positive control at 30 days: (IP)hUCT-MSCs (P=0.0060), (IV)hUCT-MSCs (P=0.0003), (IP)hBM-MSCs (P=0.0005), (IV)hBM-MSCs (P<0.0001) and continued through 30-60 days. Microscopic examination showed regenerative changes in animal joints treated with both intra-plantar or intravenous MSCs. Arthritis scores reduced in all RA severity groups while benefits (changes in IL-10 and TNF-alpha) were more pronounced in moderate and severe RA. Haematology parameters remained similar among all animal groups at baseline, 30 days and 60 days indicating safety of MSCs. ConclusionTreatment with hUCT MSCs and hBM MSCs were safe, well-tolerated and effectively reduced joint inflammation, synovial cellularity and pro-inflammatory cytokine levels in CFA-induced RA rat model.

cell biology↗

bioRxiv2020

The biphasic and age-dependent impact of Klotho on hallmarks of aging and skeletal muscle function

Aging is accompanied by a disrupted information flow, which results from accumulation of molecular mistakes. These mistakes ultimately give rise to debilitating disorders such as skeletal muscle wasting, or sarcopenia. To estimate the growing "disorderliness" of the aging muscle system, we employed a statistical physics approach to estimate the state parameter, entropy, as a function of genes associated with hallmarks of aging. Although the most prominent structural and functional alterations were observed in the oldest old mice (27-29 months), we found that the escalating network entropy reached an inflection point at old age (22-24 months). To probe the potential for restoration of molecular "order" and reversal of the sarcopenic phenotype, we overexpressed the longevity protein, -Klotho. Klotho overexpression modulated genes representing all hallmarks of aging in both old and oldest-old mice. However, whereas Klotho improved strength in old mice, intervention failed to induce a benefit beyond the entropic tipping point.

physiology↗

bioRxiv2019

Antinociceptive and Antioxidant Activities of Methanolic Extract of Leaves of Azadirachta Indica (Neem).

Graphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC=\"FIGDIR/small/694505v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (33K):\norg.highwire.dtl.DTLVardef@a4a41dorg.highwire.dtl.DTLVardef@1127160org.highwire.dtl.DTLVardef@1d175f9org.highwire.dtl.DTLVardef@98596c_HPS_FORMAT_FIGEXP M_FIG C_FIG BackgroundAzadirachta indica (Neem) is a communal plant of Meliaceae family called Neem Or Kadunimb in Maharashtra, India Neem stated anti-inflammatory through regulation of proinflammatory enzyme activities with COX and LOX enzyme. Previous studies show that Azadirachta Indica (neem) and its chief constituents play essential role in anticancer management via the modulation of different molecular pathways including NF-{kappa}B, p53, PI3K/Akt, Bcl-2, pTEN and VEGF. Many parts of the plant are traditionally used in the treatment of various pharmacological action, the analgesic activity of Neem Seed Oil has already reported but Neem Leaves.\n\nMethodsThe antinociceptive activity of Azadirachta Indica Leaves (AZIL) was examined using heat-induced-mechanical (hot-plate and tail-immersion test) and chemical-induced (acetic acid, formalin, glutamic acid, cinnamaldehyde) nociception models in mice at 50,100, and 200 mg/kg doses. ATP-sensitive K+ channel pathway, cyclic guanosine monophosphate (cGMP) pathway and involvement of opioid system was also tested using glibenclamide, methylene blue and naloxone/morphine respectively. The methanolic extract of leaves of A.Indica was assessed by using different in vitro antioxidant models of screening like scavenging of 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical, nitric oxide radical, superoxide anion radical, and hydroxyl radical.\n\nResultsAZIL showed antinociceptive activity and antioxidant activity. In both hot plate and tail immersion tests AZIL significantly increases the latency to the thermal stimuli. In acetic acid-induced writhing test the extract repressed the number of abdominal writhing. Similarly, AZIL produced substantial dose-dependent inhibition of paw licking in both neurogenic and inflammatory pain induced by intraplanar injection of formalin. As well, AZIL also expressively withdrawn cinnamaldehyde-induced pain and the glutamate-induced pain in mice. It was also proved that pretreatment with naloxone significantly reversed the antinociception produced by AZIL in mechanical tests signifying the involvement of opioid system in its effect. Furthermore, administration of methylene blue, enhanced AZIL induced antinociception while glibenclamide, an ATP-sensitive K+ channel antagonist, could not converse antinociceptive activity induced by AZIL.\n\nConclusionBased on the results of the present study it can be said that AZIL keeps significant antinociceptive activity which acts in both central and peripheral mechanisms.\n\nEthnopharmacological relevanceAzadiracta Indica having family Melliases is one of the common medicinal plant in United states and south Asia including India, Pakistan, Bangladesh. Various parts of the plants used in treatment of many inflammatory conditions, skin diseases, tooth protection, antidiabetic in the form of oil and herbal porridge.

pharmacology and toxicology↗

bioRxiv2019

Glycogen metabolism jump-starts photosynthesis through the oxidative pentose phosphate pathway (OPPP) in cyanobacteria

Cyanobacteria experience drastic changes in their carbon metabolism under daily light-dark cycles. In the light, the Calvin-Benson cycle fixes CO2 and divert excess carbon into glycogen storage. At night, glycogen is degraded to support cellular respiration. Dark-light transition represents a universal environmental stress for cyanobacteria and other photosynthetic lifeforms. Recent studies in the field revealed the essential genetic background necessary for the fitness of cyanobacteria during diurnal growth. However, the metabolic engagement behind the dark-light transition is not well understood. In this study, we discovered that glycogen metabolism can jump-start photosynthesis in the cyanobacterium Synechococcus elongatus PCC 7942 when photosynthesis reactions start upon light. Compared to the wild type, the glycogen mutant ({Delta}glgC) showed much lower photosystem II efficiency and slower photosystem I-mediated cyclic electron flow rate when photosynthesis starts. Proteomics analyses indicated that glycogen is degraded through the oxidative pentose phosphate pathway (OPPP) during dark-light transition. We confirmed that the OPPP is essential for the initiation of photosynthesis, and further showed that glycogen degradation through the OPPP is likely to contribute to the activation of key Calvin-Benson cycle enzymes by modulating NADPH levels during the transition period. This ingenious strategy helps jump-start photosynthesis in cyanobacteria following dark respiration, and stabilize the Calvin-Benson cycle under fluctuating environmental conditions. It has evolutionary advantages for the survival of photosynthetic organisms using the Calvin-Benson cycle for carbon fixation.

plant biology↗

bioRxiv2019

Predictive markers for Parkinson’s disease using deep neural nets on neuromelanin sensitive MRI

Neuromelanin sensitive magnetic resonance imaging (NMS-MRI) has been crucial in identifying abnormalities in the substantia nigra pars compacta (SNc) in Parkinsons disease (PD). PD is characterized by loss of dopaminergic neurons in SNc and current techniques employ estimation of contrast ratios of the SNc visualized on NMS-MRI to discern PD patients from the healthy controls. However, the extraction of these features is time-consuming and laborious and moreover provides lower prediction accuracies. Furthermore, these do not account for patterns of subtle changes in PD in the SNc. To mitigate this, our work establishes a computer-based analysis technique based on convolutional neural networks (CNNs) to create prognostic and diagnostic biomarkers of PD from NMS-MRI. The technique not only performs with a superior cross-validation accuracy (83.7%) as well as testing accuracy (80%) as compared to contrast ratio-based classification (52.7% cross-validation and 56.5% testing accuracy) and radiomics based classifier (81.1% cross-validation and 60.3% testing accuracy); but also locates the most discriminative regions on the neuromelanin contrast images. These discriminative activations demonstrate that the left SNc plays a key role in the classification in comparison to the right SNc, and are in agreement with the concept of asymmetry in PD. Overall, the proposed technique has the potential to support radiological diagnosis of PD while facilitating deeper understanding into the abnormalities in SNc.

neuroscience↗

bioRxiv2018

RMI-chip: a microfluidics setup for functional imaging of microbial interactions with tree roots

Coupling microfludics with microscopy has emerged as a powerful approach to study at cellular resolution the dynamics in plant physiology and root-microbe interactions. Most devices have been designed to study the model plant Arabidopsis thaliana at higher throughput than conventional methods. However, there is a need for microfluidic devices which enable in vivo studies of root development and root-microbe interactions in woody plants. Here, we developed the RMI-chip, a simple microfluidic setup in which Populus tremuloides (aspen tree) seedlings can grow for over a month, allowing continuous microscopic observation of interactions between live roots and rhizobacteria. We find that the colonization of growing aspen roots by Pseudomonas fluorescens in the RMI-chip involves dynamic biofilm formation and dispersal, in keeping with previous observations in a different experimental set-up. Also, we find that whole-cell biosensors based on the rhizobacterium Bacillus subtilis can be used to monitor compositional changes in the rhizosphere but that the application of these biosensors is limited by their efficiency at colonizing aspen roots and persisting. These results indicate that functional imaging of dynamic root-bacteria interactions in the RMI-chip requires careful matching between the host plant and the bacterial root colonizer.

physiology↗