Biology subjects

Majumdar, D.

Publications and source records attributed to Majumdar, D..

12 recordsLinked to original sources

bioRxiv2026

Spatial uncoupling of endothelial calcium signaling from phospha-tidylserine externalization in vascular injury

Ca2+ is both the signal that spreads injury information through the endothelium and the trigger for phospholipid scrambling that makes a membrane procoagulant. How a blood vessel can disseminate the first without broadcasting the second is unknown. Here, we combine single-cell laser injury in endothelial monolayers with imaging of intact mesenteric vessels in vitro and intravital Ca2+ imaging in vivo. Injury generated a coherent Ca2+ wavefront that spread rapidly before progressively slowing and terminating at a reproducible boundary 197 {+/-} 7 m from the wound, engaging 50 {+/-} 8 cells. The first ring of neighboring cells exhibited a distinct response that was prevented by carbenoxolone, whereas propagation beyond this ring required extracellular ATP, purinergic receptor activation, and IP3-dependent Ca2+ release. Wavefront kinematics were consistent with ATP diffusion. In vivo, injury elicited vasoconstriction and bidirectional Ca2+ waves propagating 205 {+/-} 28 m before terminating. Remarkably, despite widespread Ca2+ elevations, phosphatidylserine (PS) externalization remained confined to the wounded cell in monolayers and intact vessels in vitro. In contrast, ionomycin-induced Ca2+ overload exposed phosphatidylserine in nearly all endothelial cells. These findings reveal an unexpected spatial uncoupling between endothelial Ca2+ signaling and phospholipid scrambling, demonstrating that Ca2+ dependent scramblase activation is subject to additional spatial regulation. The endothelium can therefore propagate injury signals across a broad field of cells while maintaining a highly localized procoagulant membrane response.

physiology↗

bioRxiv2025

RAPMS 2.0 Improves Specificity and Throughput for Proteomic Identification of RNA Binding Proteins

RNA-protein complexes are critical factors in development, homeostasis, and disease. RNA proteomics methods are essential for characterizing these complexes but suffer from high levels of background, which hinders identification of ribonucleoprotein (RNP) components. Here, we present RNA Antisense Purification followed by Mass Spectrometry 2.0 (RAP-MS 2.0), an updated version our original RAP-MS protocol with innovations in bead preparation, RNA capture, and peptide purification. RAP-MS 2.0 has lower background than our original protocol, and allows lysate to be reused to capture multiple RNAs. We demonstrate that RAP-MS 2.0 recapitulates known RNPs for 7SL, 7SK, RMRP, U1, U2, U6, U7, and Xist. Additionally, we use RAP-MS 2.0 to identify novel RNA-protein interactions between Xist and TREX components and U1 with FET family transcriptional regulators.

molecular biology↗

bioRxiv2025

SEASONAL PM2.5 DIFFERENTIALLY REGULATES JAK2/STAT3 SIGNALING IN RURAL AND URBAN COHORTS

Ambient particulate matter (PM2.5) is a major environmental carcinogen linked to lung cancer, yet its molecular insights on asymptomatic non-smokers remains unclear. This study examined the effect of seasonal fluctuations of PM2.5 on oxidative stress and pro-oncogenic signaling in rural (RU) and urban (UR) cohorts from West Bengal, India. Environmental monitoring revealed higher PM2.5 and associated benzo[]pyrene in UR, during winter, induced oxidative stress (elevated ROS, 8-OHdG), reduced antioxidants (SOD, catalase, GPx), and promoted airway inflammation. Transcriptomic and bioinformatic analyses identified activation of IL-6/EGFR-driven JAK2/STAT3 signaling and its crosstalk with Ras/Raf/MAPK, leading to increased expression of downstream effectors (BCL-2, MCL-1, c-MYC, cyclin D1) and repression of tumor suppressors (BAX, p21). Notably, downregulation of JAK/STAT inhibitors PIAS2 and SOCS2 suggested persistent activation of oncogenic signaling. Linear mixed-effects models linked winter PM2.5 surges to oxidative stress, inflammation, and altered JAK2/STAT3 signaling, while regression models showed stronger responses in UR. Risk modeling predicted significantly higher lung cancer mortality in UR, underscoring the role of seasonal PM2.5 surges in JAK2/STAT3-driven carcinogenic susceptibility and the urgent need for targeted interventions.

cancer biology↗

bioRxiv2025

Endothelial Trauma Depends on Surface Charge and Extracellular Calcium Levels

We tested the hypothesis that the ubiquitous store-operated Ca2+ entry (SOCE) pathway contributes to histone-induced endothelial Ca2+ events. We also considered an alternate hypothesis: cationic electrostatic interactions between histones and negatively charged phospholipids deform endothelial membranes and thereby allow extracellular Ca2+ entry. A role for SOCE in histone responses was ruled out by genetic ablation of the ORAI1/2/3 channel trio; yet, histone effects were blocked by application of the multivalent cation gadolinium Gd3+. Using live cell video microscopy of endothelial cells labeled with membrane dye FM1-43, we recorded plasma membrane movements including vesiculation, blebbing, and ruffling of lamellipodia over 60 minutes following histone exposure. These cell membrane theatrics were markedly different from the uniform pattern of exocytosis and subsequent blebbing produced by calcium overload with ionomycin. The membrane permeabilization produced by histones, and not ionomycin, was transient and a subset of cells recovered membrane integrity within 1 hour. Removal of extracellular Ca2+ prevented histone-induced intracellular Ca2+ overload while surprisingly exacerbating plasma membrane deformation. Conversely, decreasing the density of the negative charge surface by adding calcium or or increasing extracellular Ca2+ levels effectively screened common membrane phospholipids from interactions with labeled histones and prevented endothelial damage in cells exposed to histones. Collectively these results indicate that low extracellular Ca2+ levels enhance interactions between histones and endothelial cell membrane phospholipids to increase cytotoxicity. Importantly, this supports the concept of aggressive Ca2+ repletion during resuscitation to prevent hypocalcemia, stabilize endothelial cell membranes and improve cardiovascular recovery from shock. SignificanceIn acute critical illness, the rapid collapse of vascular endothelial functions drives aberrant blood clotting and organ failure through mechanisms that are not understood. Emerging evidence that early administration of donor plasma improves survival of trauma patients has transformed the massive transfusion protocols used in surgical settings, but the sodium citrate included in transfused blood products to prevent coagulation often produces significant and severe hypocalcemia. Here, we demonstrate that cytotoxic trauma factors that are elevated in the blood during resuscitation interact electrostatically with endothelial cell phospholipids, and that low Ca2+ exacerbates toxicity by increasing this interaction. Using high speed video imaging, we demonstrate fast endothelial cell membrane movements in response to injury, including protrusion and ruffling of lamellipodia, release and reuptake of extracellular vesicles, and blebbing. These findings provide important insights into the nature of shock-induced endotheliopathy and highlight the potential cardiovascular risk associated with chelation-induced hypocalcemia during resuscitation.

physiology↗

bioRxiv2024

Novel preclinical model of human lung cancer cachexia

Cancer cachexia (CC), a syndrome of skeletal muscle and adipose wasting, reduces responsiveness to therapies and increases mortality. There are no approved treatments for CC, which may relate to discordance between pre-clinical models and human CC. To address the need for clinically relevant models of lung CC, we generated inducible, lung epithelial cell specific KrasG12D/+ (G12D) mice. G12D mice develop CC over a protracted time course and phenocopy tissue and tumor, cellular, mutational, transcriptomic, and metabolic characteristics of human lung CC. G12D mice demonstrate early loss of adipose, a phenotype that was apparent across numerous models of CC and translates to patients with lung cancer. Tumor-released factors promote adipocyte lipolysis, a driver of adipose wasting in CC, and adipose wasting was inversely related to tumor burden. Thus, G12D mice model key features of human lung CC and highlight a role for early tumor metabolic reprogramming of adipose tissue in CC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/615385v3_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@66d87dorg.highwire.dtl.DTLVardef@f1cb4org.highwire.dtl.DTLVardef@2580f1org.highwire.dtl.DTLVardef@3463d5_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Identification of Human Pathways Acting on Nuclear Non-Coding RNAs Using the Mirror Forward Genetic Approach

Despite critical roles in diseases, human pathways acting on strictly nuclear non-coding RNAs have been refractory to forward genetics. To enable their forward genetic discovery, we developed a single-cell approach that "Mirrors" activities of nuclear pathways with cytoplasmic fluorescence. Application of Mirror to two nuclear pathways targeting MALAT1s 3' end, the pathway of its maturation and the other, the degradation pathway blocked by the triple-helical Element for Nuclear Expression (ENE), identified nearly all components of three complexes: Ribonuclease P and the RNA Exosome, including nuclear DIS3, EXOSC10, and C1D, as well as the Nuclear Exosome Targeting (NEXT) complex. Additionally, Mirror identified DEAD-box helicase DDX59 associated with the genetic disorder Oral-Facial-Digital syndrome (OFD), yet lacking known substrates or roles in nuclear RNA degradation. Knockout of DDX59 exhibits stabilization of the full-length MALAT1 with a stability-compromised ENE and increases levels of 3'-extended forms of small nuclear RNAs. It also exhibits extensive retention of minor introns, including in OFD-associated genes, suggesting a mechanism for DDX59 association with OFD. Mirror efficiently identifies pathways acting on strictly nuclear non-coding RNAs, including essential and indirectly-acting components, and, as a result, uncovers unexpected links to human disease.

genetics↗

bioRxiv2024

Altered polyadenylation site usage in SERPINA1 3'UTR in response to cellular stress affects A1AT protein expression

Alternative polyadenylation results in different 3 isoforms of messenger RNA (mRNA) transcripts. Alternative polyadenylation in the 3 untranslated region (3UTR) can alter RNA localization, stability and translational efficiency. The SERPINA1 mRNA has two distinct 3 UTR isoforms, both of which express the protease inhibitor -1-antitrypsin (A1AT). A1AT is an acute phase protein that is expressed and secreted from liver hepatocytes and upregulated during inflammation. Low levels of A1AT in the lung contributes to chronic obstructive pulmonary disease, while misfolding of A1AT in the liver contributes to liver cirrhosis. We analyzed the dynamics of alternative polyadenylation during cellular stress by treating the liver cell line HepG2 with the cytokine interleukin 6 (IL-6), ethanol or peroxide. SERPINA1 is transcriptionally upregulated after IL-6 treatment and has altered polyadenylation, resulting in an increase in long 3UTR isoforms. We find that the long 3UTR represses endogenous A1AT protein expression even with high levels of SERPINA1 mRNA. SERPINA1 expression and 3 end processing were not affected by ethanol or peroxide. IL-6-induced changes in transcriptome-wide transcriptional regulation suggest changes to the endoplasmic reticulum and in secretory protein processing. Our data suggest that inflammation influences polyA site choice for SERPINA1 transcripts, resulting in reduced A1AT protein expression.

molecular biology↗

bioRxiv2024

Mapping the developmental trajectories of SAP-dependent innate-like gamma delta (γδ) T cells

During thymic development, most {gamma}{delta} T cells acquire innate-like characteristics that are critical for their function in tumor surveillance, infectious disease, and tissue repair. The mechanisms, however, that regulate {gamma}{delta} T cell developmental programming remain unclear. Recently, we demonstrated that the SLAM-SAP signaling pathway regulates the development and function of multiple innate-like {gamma}{delta} T cell subsets. Here, we used a single-cell proteogenomics approach to identify SAP-dependent developmental checkpoints and to define the SAP-dependent {gamma}{delta} TCR repertoire. SAP deficiency resulted in both a significant loss of an immature Gzma+Blk+Etv5+Tox2+{gamma}{delta}T17 precursor population, and a significant increase in Cd4+Cd8+ Rorc+Ptcra+Rag1+ thymic {gamma}{delta} T cells. SAP-dependent diversion of embryonic day 17 thymic {gamma}{delta} T cell clonotypes into the {beta} T cell developmental pathway was associated with a decreased frequency of mature clonotypes in neonatal thymus, and an altered {gamma}{delta} TCR repertoire in the periphery. Finally, we identify TRGV4/TRAV13-4(DV7)-expressing T cells as a novel, SAP-dependent V{gamma}4 {gamma}{delta}T1 subset. Together, the data suggest that SAP-dependent {gamma}{delta}/{beta} T cell lineage commitment regulates {gamma}{delta} T cell developmental programming and shapes the {gamma}{delta} TCR repertoire. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/575073v3_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1d8b5aeorg.highwire.dtl.DTLVardef@7fd02dorg.highwire.dtl.DTLVardef@13c5c76org.highwire.dtl.DTLVardef@1da72b1_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

The polyanionic drug suramin neutralizes histones and prevents endotheliopathy

Drugs are needed to protect against the neutrophil-derived histones responsible for endothelial injury in acute inflammatory conditions such as trauma and sepsis. Heparin and other polyanions can neutralize histones but may cause secondary, deleterious effects such as excessive bleeding. Here, we demonstrate that suramin--a widely available polyanionic drug--completely neutralizes the toxic effects of histones. The sulfate groups on suramin form stable electrostatic interactions with hydrogen bonds in the histone octamer with a dissociation constant of 250 nM. In cultured endothelial cells (Ea.Hy926), histone-induced thrombin generation was significantly decreased by suramin. In isolated murine blood vessels, suramin abolished aberrant endothelial cell calcium signals and rescued impaired endothelial-dependent vasodilation caused by histones. Suramin significantly decreased pulmonary endothelial cell ICAM-1 expression and neutrophil recruitment caused by infusion of sub-lethal doses of histones in vivo. Suramin also prevented lung edema, intra-alveolar hemorrhage and mortality in mice receiving a lethal dose of histones. Protection of vascular endothelial function from histone-induced damage is a novel mechanism of action for suramin with therapeutic implications for conditions characterized by elevated histone levels. Significance StatementPathologic levels of circulating histones cause acute endotheliopathy, characterized by widespread disruption of critical endothelial functions and thromboinflammation. We discovered that suramin binds histones and prevents histone-induced endothelial dysfunction, thrombin generation, lung injury, and death. Histone binding is a novel mechanism of action for suramin, considered among the safest and most effective drugs by the World Health Organization. These results support the use of suramin for protection of blood vessels in conditions exacerbated by circulating histones including trauma and sepsis.

immunology↗

bioRxiv2021

Increased Histone-DNA Complexes and Endothelial-Dependent Thrombin Generation in Severe COVID-19

ObjectiveCoagulopathy in severe COVID-19 is common but poorly understood. The purpose of this study was to determine how SARS-CoV-2 infection impacts histone levels, fibrin structure, and endogenous thrombin potential in the presence and absence of endothelial cells. ApproachWe studied individuals with SARS-CoV-2 infection and acute respiratory distress syndrome at the time of initiation of mechanical ventilation compared to healthy controls. Blood samples were assayed for levels of histone-DNA complexes. Confocal microscopy was used to evaluate fibrin structure in clots formed from recalcified plasma samples using fluorescently-labeled fibrinogen. Endogenous thrombin potential was measured by calibrated automated thrombin assays in the presence of tissue factor and phospholipid (PCPS) or cultured human endothelial cells. ResultsCirculating nucleosomes were elevated in the plasma of COVID-19 patients relative to healthy controls (n=6, each group). COVID-19 patient plasma thrombin generation was also altered. Despite having an increased endogenous thrombin potential, patient plasma samples exhibited prolonged lag times and times to peak thrombin in the presence of added tissue factor and PCPS. Strikingly different results were observed when endothelial cells were used in place of tissue factor and PCPS. Control plasma samples did not generate measurable thrombin (lag time >60 min); in contrast, plasma samples from COVID-19+ patients generated thrombin (mean lag time [~]20 min). Consistent with the observed alterations in thrombin generation, clots from COVID-19 subjects exhibited a denser fibrin network, thinner fibers and lower fibrin resolvability. ConclusionsElevated histones, aberrant fibrin formation, and increased endothelial-dependent thrombin generation in COVID-19 may contribute to coagulopathy. HIGHLIGHTSO_LIHistone-DNA complexes are significantly elevated in the plasma of patients with severe SARS-CoV-2 infection. C_LIO_LIMeasures of thrombin generation by calibrated automated thrombography and fibrin clots formed in situ are altered in severe COVID-19. C_LIO_LIPlasma from COVID-19 patients promotes thrombin generation on cultured endothelial cells in the absence of added tissue factor or phospholipids. C_LIO_LIThe additive effects of histones on thrombin generation and endothelial cell function may play a major role in the thrombotic complications observed in severe SARS-CoV-2 infection. C_LI

biochemistry↗

bioRxiv2018

Programmed Delayed Splicing: A Mechanism for Timed Inflammatory Gene Expression

Inflammation involves timed gene expression, suggesting that the fine-tuned onset, amplitude, and termination of expression of hundreds of genes is of critical importance to organismal homeostasis. Recent study of post-transcriptional regulation of inflammatory gene expression led to the suggestion of a regulatory role for pre-mRNA splicing. Here, using a hybrid capture approach to purify incompletely spliced, chromatin-associated pre-mRNAs, we use deep sequencing to study pre-mRNA splicing of the NF-B transcriptome. By freezing transcription and examining subsequent splicing of complete transcripts, we find many introns splice tens to hundreds of times slower than average. Investigating the basis of these delays, we focused on evolutionarily conserved introns with suboptimal splice donor sequences and found that strengthening these donor sites by as few as two nucleotides in minigene reporter assays markedly increased gene expression for several targets. This suggests that such sites can act as timing elements that both delay mRNA production and limit expression amplitude. To broaden this mechanistic view, we applied deep learning sequence-to-function models with feature attribution to identify additional regulatory sequences--both intronic and exonic--that may contribute to delayed splicing through mechanisms independent of donor site strength. This integrated approach revealed non-canonical motifs enriched in slow-splicing introns, pointing to a broader repertoire of cis-elements that can fine-tune transcript maturation during inflammation. Together, these findings support a model in which the temporal regulation of pre-mRNA splicing serves as a layer of control in inflammatory gene expression, and raise the possibility that similar timing mechanisms operate in other rapid-response transcriptional programs.

immunology↗

bioRxiv2018

Bud13 Promotes a Type I Interferon Response By Countering Intron Retention in Irf7

Intron retention (IR) has emerged as an important mechanism of gene expression control. Despite this, the factors that control IR events remain poorly understood. We observed consistent IR in one intron of the Irf7 gene and identified Bud13 as an RNA-binding protein that acts at this intron to increase the amount of successful splicing. Deficiency in Bud13 led to increased IR, decreased mature Irf7 transcript and protein levels, and consequently to a dampened type I interferon response. This impairment of Irf7 production in Bud13-deficient cells compromised their ability to withstand VSV infection. Global analysis of Bud13 knockdown and BUD13 cross-linking to RNA revealed a subset of introns that share many characteristics with the one found in Irf7 and are spliced in a Bud13-dependent manner. Deficiency of Bud13 led to decreased mature transcript from genes containing such introns. Thus, by acting as an antagonist to IR, Bud13 facilitates the expression of genes at which IR occurs.

molecular biology↗