Biology subjects

Sarkar, A.

Publications and source records attributed to Sarkar, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Design, Amino-Acid-Derived Analogues of FTY-720 and OSU-2S as PP2A Activators: Structure-Activity Relationships and Identification of Lead Compound XML-3D

Protein phosphatase 2A (PP2A) is a critical regulator of oncogenic signaling pathways, and pharmacological reactivation of PP2A through disruption of inhibitory protein interactions has emerged as a promising anticancer strategy. Although the sphingosine analogue FTY-720 and its derivative OSU-2S can restore PP2A activity, further structural diversification is needed to improve potency and expand structure-activity understanding. Here, we report the design, synthesis, and biological evaluation of three series of amino-acid-derived analogues incorporating tyrosine, homotyrosine, and tryptophan scaffolds. Docking models were used qualitatively to guide exploration of head-group functionality, hydrophobic tail variation, and stereochemical configuration; these models were hypothesis-generating and were not experimentally validated. Modular synthetic routes enabled efficient preparation of structurally diverse compounds from naturally occurring starting materials. Biological evaluation in leukemia and lymphoma cell lines revealed that hydrophobic tail length strongly influenced cytotoxic activity, while tryptophan-derived analogues were generally less potent than tyrosine-based scaffolds. Shortening the hydrophobic tail did not improve activity, contrary to the initial docking-based hypothesis. Stereochemical inversion at the amino alcohol head group yielded a lead compound, XML-3D, which demonstrated approximately two-fold enhanced cytotoxicity relative to FTY-720 and OSU-2S across leukemia cell lines (IC50 = 4.1, 1.8, and 1.5 uM in MV4-11, HL-60, and MEC-1 cells, respectively) and showed broader activity in mantle cell lymphoma lines (IC50 = 530 nM, 970 nM, and 110 nM in Jeko-1, Mino-1, and UPN1 cells). XML-3D induced increased PP2A phosphatase activity in cellular assays. Co-immunoprecipitation and pharmacological inhibition experiments were consistent with modulation of the PP2A/SET regulatory axis; however, direct binding measurements were not performed, and the mechanistic interpretation remains provisional. These findings suggest preliminary structure-activity relationships for amino-acid-derived PP2A modulators and identify stereochemical optimization as a productive direction for future development. Ongoing work aims to define molecular target engagement and refine mechanistic understanding to guide next-generation design.

molecular biology↗

bioRxiv2026

Modeling centrosome positioning by actin--microtubule crosstalk and actin anchoring at the cell membrane

Centrosome positioning is governed by mechanical interactions between the actin and microtubule cytoskeletons, yet the contribution of different actin architectures and their attachment to the cell membrane remains unclear. We use numerical simulations to study a centrosomal array of microtubules interacting with cortical, bulk, and centrosome-associated actin networks, including passive cross-linkers, motor proteins, and ERM-mediated coupling of actin to the cell membrane. We find that bulk and centrosome-associated actin alone are insufficient for robust centrosome centering, whereas sufficiently dense cortical actin promotes centering through steric interactions with growing microtubules. ERM-mediated attachment of cortical actin to the cell membrane enhances this effect, allowing stable centering at lower actin densities. In a two-centrosome system, microtubule interactions generate a spindle-like structure, while the organization of actin and its membrane attachment through ERM regulate centrosome separation. Antagonistic microtubule motors further limit centrosome separation and establish a finite spindle length. These results demonstrate that ERM-mediated coupling of actin to the cell membrane provides an important mechanical link between the actin cortex and microtubule-driven centrosome positioning, influencing both centrosome centering and spindle geometry.

biophysics↗

bioRxiv2026

A regenerative fibroblast competence field supports multiple cell fates in adult skin regeneration

Adult mammalian wounds typically restore tissue integrity through fibrotic repair rather than regeneration of the original architecture. Here, we compared small non-regenerative and large regenerative skin wounds to investigate how fibroblasts acquire regenerative competence and enter alternative cell-fate programs. Integrated single-cell RNA sequencing identified a transient signaling and extracellular-matrix-remodeling niche enriched in large wounds, together with NCAM1-associated competent fibroblast states. Time-resolved analysis positioned these states within a root-like fibroblast manifold connected to hair dermal-condensate, endothelial, pericyte and glial-associated fate-entry programs. Inferred cell-cell communication networks revealed shared matrix and adhesion signals, superimposed with branch-associated inputs, including VEGF signaling toward NCAM1-positive endothelial-transition cells. Lineage tracing showed that PDGFRA- and NCAM1-expressing lineages contributed to vascular- and nerve/glial-associated regenerative compartments. Functionally, partial NCAM1 perturbation reduced vascular organization while increasing hair follicle neogenesis, whereas stronger inhibition impaired both outcomes. These findings identify NCAM1 as a regulator of regenerative competence and fate specification within a regenerative competence field rather than a marker of a single terminal lineage. We propose that regenerative wounds activate an NCAM1-associated fibroblast competence field whose quantitative modulation biases competent fibroblasts toward alternative outcomes.

cell biology↗

bioRxiv2026

Beta-lactam enhancement against methicillin-resistant Staphylococcus aureus by cell wall blockade is autolysis-dependent: a butyrolactone derivative as case in point

Methicillin-resistant Staphylococcus aureus (MRSA) is non-susceptible to beta-lactams. Blockade of cell wall biosynthesis is a potential target for beta-lactam enhancement but requires further investigation. A butyrolactone derivative enhanced beta-lactams against MRSA strains by reducing the availability of D-Ala-D-Ala. Unlike D-cycloserine, it did not inhibit D-Ala-D-Ala ligase (Ddl). Nor did it show an additive or synergistic effect when combined with cycloserine, indicating a unique mechanism for blocking cell wall precursor production that does not involve the traditional Lipid II pathway. Notably, beta-lactam potentiation by our chemical or D-cycloserine was highly dependent on the intrinsic autolytic ability of the tested MRSA strains. Strains that resisted lysis upon Triton X-100 exposure showed a minimal increase in beta-lactam susceptibility, whereas highly autolytic strains showed significant changes in their beta-lactam MICs. We have thus identified autolytic ability as the Achilles Heel in the strategy of targeting cell wall biosynthesis for beta-lactam potentiation.

microbiology↗

bioRxiv2026

PIAS1 loss and INHBA gain define a TGFβ-driven EMT state in OSCC progression

Oral squamous cell carcinoma (OSCC), a highly aggressive subtype of head and neck cancer, remains a major clinical challenge with limited therapeutic options and poor survival outcomes. The Protein Inhibitor of Activated STAT1 (PIAS1), a SUMO E3 ligase involved in transcriptional regulation, DNA repair, and epithelial-mesenchymal transition (EMT), exhibits cancer type-specific roles, but its function in OSCC has not been well characterized. Here, we investigated the molecular and functional role of PIAS1 in OSCC progression and prognosis. All patient cohorts analysed here comprise HPV-negative disease. Analysis across multiple cohorts revealed that PIAS1 mRNA expression is significantly lower in tumor tissues than in normal oral cavity squamous epithelium, including in a cohort of 60 patient-matched tumor and normal pairs. Within the tumor compartment, protein abundance of PIAS1 correlated positively with patient survival. Functional assays using OSCC cell lines demonstrated that PIAS1 acts in a SUMO E3-dependent manner to suppress TGF{beta}-induced migration and invasion. Transcriptomic profiling of PIAS1-depleted cells revealed alterations in expression of genes with relevance in TGF{beta} signaling and pro-invasive cellular responses. Further analyses showed that 46 of these differentially altered hits intersected with The Cancer Genome Atlas (TCGA)-OSCC datasets, where four of these genes, including INHBA, associated with cell stemness-pathway. Spatial analysis of the mRNA abundance of INHBA within OSCC-derived surgical specimens showed increased abundance at the invasive front, and positively associated with aggressive tumor phenotypes, poor patients' outcomes, and resistance to immunotherapy. Single-cell RNA-seq analyses of INHBA revealed increased levels within tumor cells and cancer-associated fibroblasts (CAFs), suggesting potential tumor-intrinsic and extrinsic (tumor microenvironment) sources and hence actions. Collectively, our findings establish PIAS1 as a tumor suppressor in OSCC that restrains TGF{beta}-driven aggressive behaviour. Furthermore, we identify INHBA as a potential therapeutic target, among other genes characterized by spatially restricted, invasive tumor phenotypes.

cancer biology↗

bioRxiv2026

Multimodal Sensitivity Enhancement of Binding-based Diagnostic Assays Using Functional Hydrogels

Immunoassays are critical for clinical diagnostics, yet their performance is restricted by the physical constraints of flat surfaces used to immobilize capture molecules which limit loading capacity and induce protein denaturation. Hydrogels provide immense volumetric molecular loading capacity but translating them across diverse assay formats and readout modalities without compromising analyte permeability remains challenging. Here, we present a universal strategy for hydrogel integration into immunoassay platforms for enhanced optical and electronic biosensing. Utilizing bio-orthogonal photo-click chemistry, we developed a modular and tuneable poly(ethylene glycol) (PEG) matrix that achieves a 6,000-fold increase in molecular loading and a large mesh size for analyte diffusion. This 3D architecture demonstrates broad multimodal utility with easy integration into a range of binding-based assays enabling higher sensitivity, repeatability and multiplexability. It achieves high sensitivity enhancement for fluorescence detection in microarrays (38-fold) and microtiter plates (25-fold) and enables multiplexed readout in single wells, including from clinical serum samples. In silver metallization-based assays it boosts densitometric optical detection sensitivity (18-fold) and enables inexpensive electronic detection. In lateral flow assays, it generates 20-fold signal enhancement while enabling multiplexability and higher repeatability. By standardizing high-capacity volumetric biosensing, this scalable, plug-and-play technology empowers next generation highly sensitive, field-deployable point-of-care diagnostics.

bioengineering↗

bioRxiv2026

TRPV1-cardiac afferent ablation after completed myocardial infarction prevents arrhythmogenic remodeling

Background Therapies to prevent ventricular arrhythmias after completed myocardial infarction (MI) remain limited. Although TRPV1 afferent ablation at acute MI improves cardiac remodeling, the effect of delayed subacute targeting remains unknown. Objective We investigated whether ablating cardiac TRPV1 afferents during the subacute post-MI window mitigates structural, electrophysiological, and neuro-cardiac axis remodeling to suppress ventricular arrhythmias. Methods Yorkshire pigs underwent sham surgery or anterior MI creation. Two weeks post-MI, animals were randomized to percutaneous epicardial resiniferatoxin (RTX, for cardiac-selective TRPV1 afferent depletion) or vehicle administration. Four weeks later, terminal studies assessed the effects of RTX on cardiac structure and function, ventricular arrhythmogenesis, and neuro-cardiac axis remodeling using in vivo electrophysiologic mapping, real-time neurotransmitter sensing, immunohistochemistry, and transcriptomic profiling. Results Cardiac TRPV1 afferent depletion was confirmed by blunted responses to TRPV1 agonists. RTX-treated animals exhibited improved left ventricular function, reduced end-diastolic diameter, and suppressed ventricular tachycardia/fibrillation (VT/VF) inducibility. Endocardial electroanatomic mapping revealed improved VT/VF electrophysiologic correlates in RTX-treated animals, including fewer deceleration zones and late potentials. Epicardial multielectrode mapping demonstrated reduced electrophysiologic heterogeneity in the scar border zone. Real-time release of adrenergic neurotransmitters (noradrenaline and neuropeptide Y) was normalized during sympathoexcitation in RTX-treated animals. Histologically, RTX treatment attenuated scar border zone myocardial fibrosis and sympathetic nerve sprouting, while suppressing T cell infiltration in cardiac sensory ganglia. Bulk RNA-sequencing of stellate ganglia revealed downregulation of adrenergic genes. Conclusion Cardiac TRPV1 afferent ablation post-completed MI alters disease trajectory by mitigating structural, functional, and neuro-cardiac axis remodeling. Targeting cardiac TRPV1 afferents represents a promising subacute post-MI therapeutic strategy.

physiology↗

bioRxiv2026

Extracting deep learning based morphology segmentation footprint for boar sperm cells

Reliable delineation of the head and tail of swine spermatozoa supports automated assessment of boar semen quality, from morphometric measurement to the quality control of insemination doses. In practice this relies on fluorescent staining, which adds chemistry, cost, and delay to every acquisition and labels only the nucleus. Recent work coupling imaging flow cytometry with machine learning has advanced rapidly, yet the segmentation stage still depends on a stained channel at inference and resolves the head alone. We present a supervised encoder decoder network that segments boar spermatozoa from brightfield images acquired on an Amnis ImageStream Mark II with no stain at inference. Training labels derive from the Hoechst 33342 nuclear channel (Ch7), recorded in registration with brightfield (Ch1); the dye serves only as an annotation source, and the network sees Ch1 alone. The best semantic segmentation model reaches a Dice coefficient of 0.940 on held-out cells. For comparison we evaluate a classical morphological pipeline, four further semantic segmentation models spanning three decoder families and two ImageNet-pretrained backbones, and two zero-shot pipelines built on the Segment Anything Model 2 (SAM 2), prompted either by a dilated box around the predicted head mask or by head and tail boxes emitted by a Gemma 4 Vision Language Model (VLM). The zero-shot route scores 0.637 against Ch7 but labels the tail, which the fluorescence protocol cannot. Cells scoring worst under the supervised model proved to be mostly registration failures rather than segmentation failures, as Ch7 is displaced relative to Ch1. Manual screening for this drift is infeasible at dataset scale, so we propose a flagging system that marks any Dice below 0.792, two standard deviations below the mean, and pairs it with a zero-shot pipeline in which a VLM l and SAM 2 cross-check the flagged cell before human review.

bioinformatics↗

bioRxiv2026

Evolutionary trade-offs between growth and reproduction under obesogenic conditions: sex-biased skeletal and gonadal maturation in mice

Adolescence is a brief period during which finite energy resources are reallocated from linear growth toward reproductive maturation. Rising childhood obesity and earlier onset of puberty suggest that modern, energy rich diets may distort these evolved energy allocation rules, but causal mechanisms remain unclear. Here, we expose male and female wild-type and leptin receptor deficient (Db/Db) to either high fat or normal chow diets. We longitudinally analyze metabolic, skeletal, gonadal, endocrine and insulin-receptor phenotypes from 4-10 weeks of age (sexual maturation window in mice). In WT males, HFDs increased adiposity and impaired glucose tolerance, and selectively remodeled joint morphology, advanced gonadal maturation, and shifted insulin receptor expression from growth plates to testes. Together, these changes indicate a rebalancing of energy use toward reproduction at the expense of skeletal and metabolic health. WT females showed subtler systemic metabolic disruption but clear diet-responsive changes in growth plate and ovarian maturation. Our findings indicate that energy-rich diets during adolescence shift evolved allocation rules to prioritize reproductive readiness over skeletal robustness in a sex-dependent manner, with potential consequences for precocious puberty and bone health in humans.

evolutionary biology↗

bioRxiv2026

Additive Effects of Sleep Loss, Psychological Distress and Physical Inactivity on Cognitive Failures in Young Adults

Young adults frequently report cognitive complaints often attributed to sleep loss alone. However, subjective cognitive functioning is shaped by broader lifestyle and affective factors. Cross-sectional data were analyzed from 530 young adults (mean age 22.1 {+/-} 2.3 years) to examine the independent, interactive, and cumulative associations of short sleep duration, low physical activity, and psychological distress with everyday cognitive failures. Cognitive failures were strongly associated with sleep duration, physical activity, sleep quality, and distress in univariate analyses. However, hierarchical regression revealed that psychological distress, poor sleep quality, and short sleep duration were the dominant independent correlates of cognitive failures, collectively explaining a substantial proportion of variance in Cognitive Failures Questionnaire scores (R2 = 0.585, p < 0.001). In contrast, the apparent protective effect of physical activity was not observed after adjustment for sleep and distress (p = 0.976), and no significant sleep-by-physical activity interaction was observed. Further, cumulative risk modeling demonstrated a robust dose-dependent relationship, with cognitive failures increasing progressively as behavioral and psychological risk factors accumulated (ptrend < 0.001). Individuals exposed to all three risk factors exhibited more than double the cognitive failure burden observed in individuals with no risk factors. These results indicate that the cognitive burden in young adults can best be described by a additive increase of behavioral and psychological risk factors as a function of the co-occurrence, rather than by the presence of compensatory effects of lifestyle risk factors. Interventions aimed at preserving cognitive function may therefore benefit from simultaneously targeting sleep health and psychological well-being rather than relying on physical activity alone to offset cognitive burden.

neuroscience↗

bioRxiv2026

The TIRR–53BP1 axis controls PLK1 spatiotemporal regulation during mitosis

p53-binding protein 1 (53BP1) is a key mediator of the DNA damage response and genome stability. While its interphase function is well-characterized, its mitotic role remains less understood. Here we show that aberrant activation of 53BP1 through the loss of its negative regulator, TIRR, leads to mitotic abnormalities including altered spindle geometry, kinetochore-microtubule (k-MT) attachment errors and whole chromosome missegregation. We demonstrate that loss of TIRR results in excess interaction between 53BP1 and the key mitotic kinase Polo-like kinase 1 (PLK1), altering PLK1s activation, spatial distribution, and its interaction with known PLK1 substrates at multiple mitotic stages. Moreover, due to PLK1s established role in CENP-A loading, hyperactivation of 53BP1 compromises CENP-A loading, triggers gradual loss of CENP-A from centromeres and generates severe kinetochore assembly defects. These findings uncover a non-canonical mitotic function of 53BP1 as a key regulator of PLK1 activity and chromosome segregation fidelity.

Cell Biology↗

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

Anisotropic subdiffusion in the cytoplasm of living cells

The cytoplasm of eukaryotic cells is a complex fluid that harbors a variety of membrane-enveloped organelles with distinct spatial distributions and duties. Peroxisomes, for example, are vesicular entities with radii in the sub-micron range, dispersed all over the cytoplasm to provide oxidative reaction vessels across the cell. Here, we have tracked peroxisomes as biologically relevant tracer particles to explore the local structure of the cytoplasm. Focusing on the vast majority of (sub)diffusively moving peroxisomes, we show that these display a significant motion anisotropy even in the absence of cytoskeletal filaments or when the endoplasmic reticulum (ER) is disrupted. In untreated cells, the local preference direction aligns to a global axis that correlates with the cells long axis; disrupting the cytoskeleton or the ER progressively randomizes this alignment. Altogether, our data indicate that the cytoplasm features a nematic-like sub-structure rather than being an isotropic fluid.

biophysics↗

bioRxiv2026

Stretching mucins: revealing the complex rheology of a natural gly coprotein network

Flow and extensional deformation of mucin networks are fundamental in mucus biophysics, governing how mucus functions as a protective and lubricating, and transport-facilitating layer. While the shear and oscillatory rheology of mucin solutions have been characterized in considerable detail, their behavior under extensional deformation remains comparatively understudied. Here, we report a concentration-dependent transition in extensional flow response of mucin solutions using a bespoke dripping-onto-substrate extensional rheometer. We show that mucin solutions at the lower concentrations undergo linear filament thinning, whereas semidilute mucin solutions form highly extensible filaments, with radius decaying exponentially in time, consistent with the elastocapillary thinning observed in solutions of high molecular weight synthetic polymers. Remarkably, at higher mucin concentrations inter-chain mucin associations produce a sudden reduction in the apparent elastocapillary relaxation time. We demonstrate how increasing macromolecular concentration redistributes the balance between viscous and elastic stresses during capillary thinning in a biopolymer network and reveal a concentration-driven reduction in mucin filament extensibility. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/725541v2_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1f593acorg.highwire.dtl.DTLVardef@1b23686org.highwire.dtl.DTLVardef@119add3org.highwire.dtl.DTLVardef@e31908_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

Lipid mobilization establishes metabolic tolerance and prevents autonomic collapse in infection

Survival during infection depends on both pathogen clearance and the ability to tolerate infection-induced physiological changes. Metabolic adaptations are a central component of this tolerance, but the mechanisms underlying these responses remain incompletely defined. Here, we identify white adipose tissue (WAT) lipolysis as a central regulator of metabolic tolerance to infection. In patients with sepsis, higher circulating non-esterified fatty acid (NEFA) levels were associated with reduced mortality. In mouse models of polymicrobial sepsis, infection induced robust adipose lipolysis and increased circulating NEFAs. Genetic ablation of adipose triglyceride lipase (ATGL) in adipose tissue impaired lipolysis, leading to hypothermia, bradycardia, and increased mortality without altering immune cell populations or pathogen burden, consistent with a defect in tolerance rather than resistance. Mechanistically, lipolysis-derived NEFAs, but not glycerol, were required for protection, as restoring circulating NEFAs rescued autonomic stability and survival in adipose tissue ATGL-deficient mice. Infection-induced lipolysis was redundantly regulated and did not depend on any single upstream signaling pathway. Both pharmacologic activation of lipolysis using a {beta}3-adrenergic agonist and exogenous fatty acid supplementation increased circulating NEFAs, improved survival, and promoted tolerance in mice. Consistent with these findings, analysis of real-world electronic health record data demonstrated that septic patients receiving FDA-approved {beta}3-adrenergic agonists had reduced mortality or hospice discharge in a propensity-matched cohort. Together, these results identify WAT lipolysis and circulating fatty acids as key mediators of tolerance to infection and support a therapeutic strategy based on repurposing clinically available {beta}3-adrenergic agonists to improve outcomes in sepsis. One Sentence SummaryWhite adipose tissue lipolysis promotes metabolic tolerance to infection through circulating fatty acids and is associated with improved survival in sepsis

physiology↗

bioRxiv2026

Serial vaccination expands and refines human CD4+ T cell memory

CD4+ T cells coordinate protective immunity against pathogens. However, a major unresolved question is how human CD4+ T cell memory is established and evolves following primary and repeated vaccination. Using COVID-19 mRNA vaccination as a model, we tracked 50 distinct antigen-specific populations directly ex vivo with peptide-MHC class II tetramers in eight SARS-CoV-2-naive individuals from pre-vaccine baseline through memory time points after three mRNA doses. Our findings identify the primary vaccine series as the main driver of memory pool size. It leverages pre-existing memory while preferentially recruiting high-avidity T cells, establishing an immunodominance hierarchy dominated by a small subset of precursors. Booster vaccination refines both the magnitude and quality of T cell memory. It increases select populations and enhances differentiation of subdominant CD4+ T cells. Populations that did not become more abundant after boosting retained their polyfunctional potential. Beyond establishing memory to the ancestral spike, vaccinations broadened responses by recruiting cross-reactive T cells recognizing viral variants. Collectively, these findings reveal how human CD4+ T cell memory evolves through sequential immunizations to generate a functionally diverse and broadly responsive memory repertoire against future viral challenges.

immunology↗

bioRxiv2026

Increases in BCL2L1 and ID1 dosage synergistically drive fate bias and competitive advantage in human pluripotent stem cells

BackgroundGains of chromosome 20q11.21 are among the most common culture-acquired abnormalities in human pluripotent stem cells (hPSC), conferring a well-defined survival advantage while altering differentiation capacity. However, it remains unclear whether this advantage persists during differentiation, how the aneuploidy alters ectodermal and retinal pigment epithelium (RPE) lineage specification, and which genes within the minimal amplicon drive these effects. MethodsWe used three isogenic human embryonic stem cell line pairs (wild-type and 20q11.21 gain) and assessed their behaviour in two neuroectoderm differentiation systems: directed neuroectoderm induction (dual SMAD inhibition) and long-term spontaneous RPE differentiation. Competitive dynamics were measured in mixed cultures, and lineage outcomes were analysed using immunostaining, gene expression profiling and single-cell RNA sequencing. To identify driver genes, we generated BCL2L1 and ID1 overexpression lines and tested their effects under both directed and spontaneous differentiation conditions. ResultsAcross all lines and conditions, 20q cells expanded from a minor fraction to dominate mixed cultures, indicating that their competitive advantage persists beyond the undifferentiated state. Despite this dominance, pure 20q cells failed to specify to neuroectoderm or RPE. Single-cell transcriptomics revealed consistent diversion toward non-neural ectodermal and extraembryonic fates. Mechanistically, overexpression of BCL2L1 and ID1 alone or in combination impaired neuroectoderm specification, while synergistic effect of both genes promoted non-neural ectodermal outcomes under directed differentiation conditions. In spontaneous differentiation, both genes could disrupt differentiation. ConclusionsThe 20q11.21 gain couples a persistent survival advantage with a disruption of neural and RPE lineage competence, redirecting cells toward alternative ectodermal and extraembryonic fates. These effects arise from the combined action of two dosage-sensitive genes BCL2L1 and ID1 within the amplicon, illustrating how regional gene dosage can reshape developmental signalling responses in hPSC.

developmental biology↗

bioRxiv2026

GP130 Y814 SIGNALING IS REQUIRED FOR THE DYNAMIN-MEDIATED ENDOCYTOSIS, MAPK/P38 ACTIVATION AND PERSISTENCE OF CHRONIC SYSTEMIC INFLAMMATION INDUCED BY HIGH FAT DIET

In chronic inflammatory diseases such as obesity, tissue and cellular homeostasis are disrupted by persistent low-grade inflammation, where one of the most prominent pro-inflammatory cytokines that correlates with age and body mass index (BMI) is IL-6. All members of the IL-6 family of cytokines signal through their obligate co-receptor gp130, in whichsignaling tyrosines on the intracellular portion of gp130 activate multiple downstream pathways such as the canonical STAT and MAPK pathways. However, non-canonical gp130 pathways such as SRC family of kinases (SFKs) signaling have emerged as drivers of cellular stress response. Our recently published mutant mouse model carrying a constitutive inactivation of gp130-Y814 (F814 mice), which impairs SFK activation, exhibited an enhanced resolution of inflammatory responses and improved regenerative outcomes in both acute skin wound healing and post-traumatic osteoarthritis models. The current study was designed to explore whether the gp130-Y814 mutation reduces systemic chronic inflammation and multimorbidity in a high-fat diet (HFD)-induced model and to interrogate the possible downstream cellular mechanisms that are affected. In response to HFD, F814 mice showed significantly reduced systemic and tissue-specific inflammatory responses and protection from obesity-induced bone loss and osteoarthritis compared to wild type (WT) mice. After extensive characterization, the role of gp130-Y814 in monocytes/macrophages appeared to be dispensable, but we discovered that the F814 mutation blunts gp130 receptor internalization, p38/MAPK activation and the release of matrix metalloproteinases (MMPs) in chondrocytes. Finally, we showed that F814 chondrocytes had markedly reduced activation of the SFK-dependent GTPase dynamin 2 (Dyn2) in response to catabolic IL-6 cytokines. Introduction of a dominant-negative Dyn2 mutant into WT chondrocytes phenocopied the effects of the F814 mutation, resulting in attenuated p38/MAPK signaling and reduced MMP activation following stimulation with IL-6 cytokines. This study demonstrates, for the first time, that the Y814 residue is directly implicated in Dyn2-mediated internalization of the gp130 receptor, thereby modulating downstream signaling and contributing to pathological outcomes in chronic inflammatory and degenerative diseases in a cell type-specific manner.

cell biology↗