Biology subjects

Baral, R.

Publications and source records attributed to Baral, R..

10 recordsLinked to original sources

bioRxiv2026

Breast cancer stem cells mediated CD8+ T cell exhaustion among different molecular subtypes of breast cancer regulated via NOTCH1/RBPJ/PD-L1 axis

BackgroundBreast cancer stem cells (BCSCs) contribute significantly to breast cancer (BC) mortality among women globally. It underpins tumor heterogeneity in BC by driving variations in stemness potential and altering immune microenvironment. However, how BCSCs, subpopulations of breast cancer cells from distinct molecular subtypes differentially modulate CD8 T-cell exhaustion and immune dysfunction remain unclear. MethodsWe conducted our study from patients with BC of four subtypes. MACS sorted (Lin-CD44+CD24-) BCSCs were prepared for mammosphere formation assay from mastectomies samples. Flow-cytometry was used to analyze breast cancer stem cells (BCSCs). Immunofluorescence, immunohistochemistry, Real Time and Reverse Transcriptase PCR array, Chromatin-immunoprecipitation assay, Transwell, ELISA, Western blotting, Cloning, Transfection, Knockdown, chromatin immunoprecipitation approaches were used to investigate the underlying mechanisms. ResultsHere, we report that BCSCs actively participate in tumor progression by modulating effector CD8 T-cells. Triple-negative breast cancer (TNBC), being the subtype with the most adverse outcomes, sustains the enrichment of stem cell regulating transcription factors like NANOG, OCT4 and SOX2 compared to HER2, Luminal B, and Luminal A subtypes. Tumor from TNBC patients exhibited an exhausted phenotype within CD8 T-cell infiltrates with PD1high TIM3high LAG3 high IFN{gamma}low signature. BCSCs induced increased proportion of exhausted CD8 T-cells, predominantly in the TNBC subtype. Cell-surface Notch1 expression was upregulated in BCSCs across all molecular BC subtypes, with the highest elevation observed in TNBC. Knockdown or inhibition of Notch1 downregulated stemness-associated genes and diminished CSC-mediated induction of CD8 T-cell exhaustion. Cumulatively, these findings suggest that assessment of high Notch1 and Nanog frequency within BCSCs can guide Notch1-targeted therapies and may formulate for new combinatorial treatment strategies to improve patient outcomes. Additionally, therapeutic targeting of BCSC-intrinsic NOTCH1-NANOG/NOTCH1-PD-L1 axis could represent an effective strategy to reduce stemness programs and alter BCSC-driven CD8 T-cell exhaustion, majorly in aggressive subtypes such as TNBC. ConclusionsBCSCs aggressiveness is perpetuated through Notch1-mediated axis. Targeting Notch1 would reduce stemness (majorly NANOG), survival, as well as prevent CD8 T-cell exhaustion (upregulating PD-1, TIM3, LAG3), thereby weakening tumor progression.

cancer biology↗

bioRxiv2026

The role of MYB21 in Arabidopsis: transcending flower-specific functions to vegetative tissue

Jasmonic acid (JA) and its derivatives are lipid-derived phytohormones that coordinate plant growth, development, and stress responses through the bioactive conjugate jasmonoyl-isoleucine (JA-Ile). Their role in reproductive development is well established, particularly in stamen maturation through the R2R3-MYB transcription factor MYB21, which has been considered largely flower-specific. Here, we reveal a previously unrecognized role of MYB21 in vegetative tissues of Arabidopsis thaliana. Although basal MYB21 transcript levels in leaves are extremely low and spatially restricted, wounding and exogenous hormone applications induced MYB21 transcription in a JA- and COI1-dependent manner. Transcriptional GUS reporter analyses showed localized MYB21 promoter activity in specialized epidermal cells, including trichomes, hydathodes, and in the vasculature at wound sites. Functional characterization using the myb21-5 mutant indicated roles in germination and vegetative growth, partially phenocopying JA-insensitive mutants despite unaltered JA biosynthesis and signaling. Transcriptome profiling further revealed changes in expression of genes involved in lignin biosynthesis, light-harvesting complex components, cytokinin pathways, and defense-related responses, consistent with reduced resistance of myb21-5 to insect herbivory and infection by Botrytis cinerea. Together, these findings identify MYB21 as a JA-responsive regulator of growth and defense in seedlings and leaves, extending its function beyond reproductive development. HighlightThe transcription factor MYB21, previously considered flower-specific, is induced by jasmonate signaling in Arabidopsis leaves and seedlings, where it regulates growth and defense responses, thereby expanding its role beyond reproductive development.

plant biology↗

bioRxiv2025

Amyloid β interaction with membranes: Removal of cholesterol from the membranes to catalyze aggregation and amyloid pathology

The interplay between the cholesterol metabolism and assembly of A{beta}42 (the 42- residue form of the amyloid-{beta} peptide) peptides in pathological aggregates is considered as one of the major molecular mechanisms in development of Alzheimers disease (AD). Numerous in vitro studies led to the finding that the high cholesterol levels in membranes accelerate the production of A{beta} aggregates. The molecular mechanisms explaining how cholesterol localized inside the membrane bilayer catalyzes the assembly of A{beta} aggregates above the membrane remain unknown. We addressed this problem by combining different AFM modalities, including imaging and force spectroscopy, with fluorescence spectroscopy. Our combined studies revealed that A{beta}42 was capable of removing cholesterol from the membrane. Importantly, physiologically low concentrations of A{beta}42 demonstrate such ability of monomeric A{beta}42. Extracted cholesterol interacts with A{beta}42 and accelerates its on- membrane aggregation. We propose a model of interaction of A{beta}42 with membranes based on the ability of A{beta}42 to extract cholesterol, which explains several AD associated observations related to cholesterol interplay with A{beta}42 aggregation resulting in the AD onset and progression.

biophysics↗

bioRxiv2025

Tumor-Educated-Platelets interact with Breast Cancer-Stem-Cells via P-selectin- PSGL1 and ensure stemness and metastasis through WNT-β-Catenin-VEGF-VEGFR2 intra-cellular signaling: Therapeutic modulation by aspirin

BackgroundProtagonistic role of platelets promote capillary infiltration of tumors for distant metastasis along with immunosurveillance. Despite existing reports highlighting role of platelets in tumorigenesis, its impact on breast cancer stem cells (BCSCs) remain underexplored. Our first ever report on murine and human system, accentuate that, tumor educated platelets (TEPs) of luminal A and TNBC subtypes are distinct from healthy counterparts, collaborating with BCSCs to generate sub-variants that elevate tumor aggressiveness. MethodsImpact of TEPs on BCSCs was evaluated from primary breast tumor and blood samples of luminal A/TNBC patients along with EC/4T1 murine breast tumor models and MCF-7/MDA-MB-231 cell lines. For downstream assays, TEPs were co-cultured with breast tumor samples or cell lines, followed by magnetic sorting of lin-CD44+CD24- BCSCs. TEP induced alterations of BCSCs were evaluated from 3D tumorsphere, colony formation, transwell migration, scratch-wound healing, matrigel invasion, in-vitro tube formation assays. Fluorescence-confocal microscopy, RT-PCR, flow-cytometry, western-blotting was utilized to decipher the role of genes and protein involved in stemness, metastasis along with the transcription factors in the downstream signaling cascade, followed by verifications by RNAi. ResultsTEPs have elevated expression of P-selectin and interacts with BCSCs via P-selectin and PSGL1 on BCSCs surface. Treatment with aspirin had restorative impact on P-selectin level, converting TEPs from active to resting platelet (RP) state. Under TEPs influence, BCSCs were tumorigenic, clonogenic, multidrug resistant, invasive with numerous invadopodia and remained skewed towards mesenchymal phenotype. Administration of RP reduced TEP associated BCSC virulence both in-vivo and in-vitro. P-selectin-PSGL1 interaction results in binding of WNT to FRIZZLED followed by stabilization and nuclear translocation of {beta}-Catenin. Nuclear {beta}-Catenin promotes stemness-EMT (Epithelial to mesenchymal transition)- metastasis, along with stimulation of autocrine VEGF-VEGFR2 cascade. Inhibition of WNT and VEGFR2 by RNAi confirmed the critical role of this axis in regulating TEPs influence on BCSCs. ConclusionThese insights into TEPs-BCSC interplay, acknowledges TEPs, as-well-as unveils novel receptor-ligand signaling cascade between TEPs and BCSCs, that could be a beneficial therapeutic strategy to target cancer metastasis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/657784v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@11db5d0org.highwire.dtl.DTLVardef@18b7270org.highwire.dtl.DTLVardef@64c2borg.highwire.dtl.DTLVardef@172d68f_HPS_FORMAT_FIGEXP M_FIG C_FIG Cellular interaction between TEPs P-selectin and BCSCs PSGL1 to regulate stemness, EMT, metastasis and angiogenesis via WNT-{beta}-Catenin-VEGF-VEGFR2 cascade: Modulation by pharmacological inhibitor and RNAi.

cancer biology↗

bioRxiv2024

Docking interactions determine substrate specificity of members of a widespread family of protein phosphatases

How protein phosphatases achieve specificity for their substrates is a major outstanding question. PPM family serine/threonine phosphatases are widespread in bacteria and eukaryotes, where they dephosphorylate target proteins with a high degree of specificity. In bacteria, PPM phosphatases control diverse transcriptional responses by dephosphorylating anti-anti-sigma factors of the STAS domain family, exemplified by B. subtilis phosphatases SpoIIE, which controls cell-fate during endospore formation, and RsbU, which initiates the General Stress Response. Using a combination of forward genetics, biochemical reconstitution, and AlphaFold2 structure prediction, we identified a conserved, tripartite substrate docking interface comprised of three variable loops on the surface of the PPM phosphatase domain that recognize the three-dimensional structure of the substrate protein. Non-conserved amino acids in these loops facilitate the accommodation of the cognate substrate and prevent dephosphorylation of the non-cognate substrate. Together, single-amino acid substitutions in these three elements cause an over five-hundred fold change in specificity. Our data additionally suggest that substrate-docking interactions regulate phosphatase specificity through a conserved allosteric switch element that controls the catalytic efficiency of the phosphatase by positioning the metal cofactor and substrate. We hypothesize that this is a generalizable mechanistic model for PPM family phosphatase substrate specificity. Importantly, the substrate docking interface with the phosphatase is only partially overlapping with the much more extensive interface with the upstream kinase, suggesting the possibility that kinase and phosphatase specificity evolved independently.

biochemistry↗

bioRxiv2024

Experimental type 1 diabetes metabolically rejuvenates CD8+ T cells for improved control of tumor growth through an IGF1-IGF1R axis

AbstractEpidemiological studies suggest that patients with pre-existing type 1 diabetes (T1D) have a decreased risk of developing melanoma, prostate cancer, and breast cancer, although the underlying mechanism remains to be elucidated. In translational modelling, we observed that streptozotocin (STZ) induced T1D mice exhibited restricted melanoma and carcinoma (mammary, lung and colon) growth in association with extended overall survival. Tumor-infiltrating CD8+ T cells were found to be responsible for tumor growth restriction. Tumor infiltrating CD8+ T cells but not tumor cells themselves exhibited higher glycolytic and cytotoxic activities in T1D hosts. Such improved anti-tumor T cell function was linked to selective upregulated expression of insulin-like growth factor 1, insulin-like growth factor 1 receptor, and phospho-mTOR in CD8+ T cells in the TME. T1D patient derived CD8+ T cells displayed superior activation in vitro after tumor antigen stimulation vs. non-diabetic CD8+ T cells. Activation of T1D patient derived CD8+ T cells was sensitive to targeted antagonism of IGF1R and mTOR, supporting the operational involvement of the IGF1R-mTOR signaling axis. Our results suggest that selective activation of the intrinsic IGF1R-mTOR signaling axis in CD8+ T cells represents a preferred endpoint to achieving more effective immunotherapy outcomes and improved cancer patient management. SignificanceExperimental type 1 diabetes decelerates tumor growth through metabolic activation of cytotoxic T cells dependent on an IGF1R-mTOR signaling pathway. CD8+IGF1R+IGF1+ T cells play a crucial role in T1D dependent tumor control.

cancer biology↗

bioRxiv2024

Unraveling the Role of 12-cis-Oxo-Phytodienoic Acid in the Wound Response of Arabidopsis thaliana: Insights from Transcriptomic and Complementation Studies

12-cis-Oxo-phytodienoic acid (OPDA), the main precursor of the key plant growth and defense hormone jasmonoyl-isoleucine (JA-Ile), is believed to have distinct signaling roles in plants responses to stress. In Arabidopsis, insights into OPDA functions have been gained from studying a mutant, which is affected in the conversion of OPDA by missing OPDA REDUCTASE3 (OPR3). opr3 mutants, however, accumulate JA-Ile through a cytosolic bypass mediated by OPR2. Therefore, wound-induced transcriptome of opr2opr3 in comparison to wild-type and allene oxide synthase mutant was analyzed to unravel OPDA signaling. Results showed that OPDA lacked a distinct transcriptional signature, whereas known OPDA-response genes were wound-induced independently of OPDA. The application of OPDA to opr2opr3 resulted in a distinct transcriptional response compared to the endogenous rise of OPDA in the same mutant, with the activation of the sulfur assimilation pathway genes occurring only with the external application of the compound. These findings suggested a compartmentalization of endogenously produced OPDA, investigated further through trans-organellar complementation. OPR3 complemented opr2opr3 mutants in fertility and wound-induced JA-Ile production regardless of its localization. Since in vitro enzymatic studies revealed OPR3s activity on both OPDA and 4,5-ddh-JA, conclusions on translocation of OPDA were not unequivocal. Dissecting the conversion of either OPDA or 4,5-ddh-JA by OPR2 and OPR1 organelle variants pointed, however, to a strong OPDA compartmentalization supporting its lacking signaling function.

plant biology↗

bioRxiv2024

Forage conservation is a neglected source of nitrous oxide

Agricultural activities are the major anthropogenic source of nitrous oxide (N2O), an important greenhouse gas and ozone-depleting substance. However, the role of forage conservation as a potential source of N2O has rarely been studied. We investigated N2O production from the simulated silage of the three major crops--maize, alfalfa, and sorghum--used for silage in the US, which comprises over 90% of the total silage production. Our findings revealed a substantial N2O could be generated, which potentially places forage conservation as the third largest N2O source in the agricultural sector. Notably, the application of chlorate as an additive significantly reduced N2O production, but neither acetylene nor intermittent exposure to oxygen showed any impact. Overall, the results highlight that denitrifiers, rather than nitrifiers, are responsible for N2O production from silage, which was confirmed by molecular analyses. Our study reveals a previously unexplored source of N2O and provides a crucial mechanistic understanding for effective mitigation strategies. Significance StatementN2O is the third most important greenhouse gas (GHG) and agriculture contributes 80% of the total anthropogenic emissions in the US. The major sources of N2O in the agricultural sector identified by the USEPA include agricultural land management, manure management, and the field burning of agricultural residues. Here, we show that forage conservation could be a significant unaccounted source of N2O, surpassing the field burning by 30. Our study provides a mechanistic understanding of N2O production and a simple and effective remedy for reducing N2O emissions. The findings have substantial implications for mitigating climate change, informing policy-makers, and guiding future research on reducing greenhouse gas emissions from livestock production.

bioengineering↗

bioRxiv2024

A General Mechanism for the General Stress Response in Bacteria

The General Stress Response promotes survival of bacteria in adverse conditions, but how sensor proteins transduce species-specific signals to initiate the response is not known. The serine/threonine phosphatase RsbU initiates the General Stress Response in B. subtilis upon binding a partner protein (RsbT) that is released from sequestration by environmental stresses. We report that RsbT activates RsbU by inducing otherwise flexible linkers of RsbU to form a short coiled-coil that dimerizes and activates the phosphatase domains. Importantly, we present evidence that related coiled-coil linkers and phosphatase dimers transduce signals from diverse sensor domains to control the General Stress Response and other signaling across bacterial phyla. This coiled-coil linker transduction mechanism additionally suggests a resolution to the mystery of how shared sensory domains control serine/threonine phosphatases, diguanylate cyclases and histidine kinases. We propose that this provides bacteria with a modularly exchangeable toolkit for the evolution of diverse signaling pathways.

microbiology↗

bioRxiv2018

Telomere DNA G-quadruplex folding within actively extending human telomerase

Telomerase maintains telomere length by reverse transcribing short G-rich DNA repeat sequences from its internal RNA template. G-rich telomere DNA repeats readily fold into G-quadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using K+ versus Li+ salts yield changes in DNA product profiles consistent with formation of GQ structure within the telomerase-DNA complex. Single-molecule FRET experiments reveal complex DNA structural dynamics during real-time catalysis, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we fit telomerase time series data to a global kinetic model that converges to a unique set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis and provide a detailed characterization of GQ modulation of polymerase function.\n\nSIGNIFICANCETelomeres protect the ends of linear chromosomes from illicit DNA processing events that can threaten genome stability. Telomere structure is built upon repetitive G-rich DNA repeat sequences that have the ability to fold into stable secondary structures called G-quadruplexes (GQs). In rapidly dividing cells, including the majority of human cancers, telomeres are maintained by the specialized telomerase enzyme. Thus, telomerase and its telomere DNA substrates represent important targets for developing novel cancer drugs. In this work, we provide evidence for GQ folding within the newly synthesized DNA product of an actively extending telomerase enzyme. Our results highlight the delicate interplay between the structural properties of telomere DNA and telomerase function.

biochemistry↗