Biology subjects

Das, S.

Publications and source records attributed to Das, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Tripartite host-parasite-virus interactions reshape chronic visceral leishmaniasis through persistent Leptomonas seymouri co-infection

Background Visceral leishmaniasis (VL), caused by Leishmania donovani (LD), is increasingly associated with the insect-restricted trypanosomatid Leptomonas seymouri (LS), which harbours the RNA virus Leptomonas seymouri narna-like virus 1 (Lepsey NLV1). Our recent study demonstrated that LS co-infection with LD enhances survival of murine (RAW 264.7) and mammalian (THP-1) macrophages and augments LD and LS persistence compared to LD or LS mono-infection in vitro. However, the in vivo fate of LS and its viral endosymbiont during chronic VL remains poorly understood. This study investigated the long-term dynamics of parasite persistence, tissue dissemination and viral maintenance during experimental mono- and co-infection. Methods and Findings BALB/c mice were infected with LD, Lepsey NLV1-positive LS, virus-positive AG83 isolate, or LD: LS co-infections (2:1, 5:1 and 10:1) and monitored for up to seven months. Parasite burden, species composition and viral load were quantified using ITS1 qPCR, densitometry, nested RT-PCR and qRT-PCR, supported by microscopy and immunofluorescence assay. LS established productive visceral infection independently, with parasite burdens exceeding the infecting inoculum, indicating active in vivo replication. Co-infection, particularly at a 10:1 LD: LS ratio, promoted the greatest long-term parasite persistence in visceral organs. Temporal analysis revealed early predominance of LS followed by progressive recovery of LD during chronic infection. Lepsey NLV1 was detected in visceral organs and blood for at least up to five months. Morphological analyses demonstrated intracellular LS amastigote-like forms in murine macrophages and transformation of splenic parasites into promastigotes, confirming parasite viability within mammalian tissues. Conclusions These findings demonstrate sustained visceral persistence of Lepsey NLV1-positive LS in mice and identify dynamic host-parasite-virus interactions that reshape infection during chronic co-infection. This work challenges the conventional view of VL as a strictly mono-parasitic disease and highlights a previously underappreciated tripartite interaction with potential implications of LS and its virus endosymbiont for VL pathogenesis.

microbiology↗

bioRxiv2026

Dietary iron overload enhances susceptibility to Yersinia enterocolitica infection

Hemochromatosis and hemosiderosis are iron overload disorders that cause immune dysfunction and increase susceptibility to bacterial infections. There have been numerous case studies reporting septic-like outcomes for hemochromatosis patients infected with enteric Yersiniae; however, research regarding hemosiderosis and Yersinia infection is limited. Here, we have established a mouse model of hemosiderosis by feeding C57BL/6 mice a high-iron diet. These mice exhibit several indicators of iron overload that are seen clinically, including elevated serum iron levels and iron deposition in various tissues. Characterization of the iron overload mouse model shows that a high-iron diet induces local inflammation in the small intestine and systemic inflammation in a time-dependent manner. Oral infection with Yersinia enterocolitica causes complete mortality in the iron-overloaded mice, while wild-type mice all survive and effectively clear the infection. Lastly, we have observed that iron chelation therapies such as Deferoxamine and Deferisarox are detrimental to iron-overloaded mice during Yersinia infection. This work provides a model to further study iron overload disorders and Yersinia infection.

immunology↗

bioRxiv2026

IRES-mediated translation of delta160p53 regulates p53 functions and fine-tunes cancer homeostasis

Mutations in p53 and its 12 isoforms can alter its functions. As N-terminally truncated isoforms of p53 (delta40p53, delta133p53, and delta160p53) participate in tetramer formation, they are important regulators of cancer fate. Although delta40p53- and delta133p53-mediated regulation of cancer is well reported, the mechanism underlying delta160p53 production and its functional role remains unclear. We investigated the internal ribosomal entry site (IRES)-mediated translation of {Delta}160p53 and its role in cancer regulation. As differential synthesis of delta160p53 was observed under different stress conditions, IRES-mediated translation of this isoform was demonstrated using bicistronic luciferase constructs. No cryptic promoters or splicing sites were detected in the IRES sequence. Cell death and late apoptosis were significantly decreased, while proliferation, the number of cells in the S phase, and drug resistance were induced by delta160p53. Furthermore, delta160p53 did not induce p53-responsive promoters. RNA sequencing analysis of delta160p53 overexpression showed similar results, along with the inhibition of other tumor suppressor genes. Overall, our results provide insights into IRES-mediated translation of delta160p53, which can be considered a novel target for cancer treatment.

molecular biology↗

bioRxiv2026

Vibrio cholerae VgrG1-ACD Targets E. coli MreB and Regulates Cellular Morphology

Vibrio cholerae employs the Type VI Secretion System (T6SS) to outcompete neighboring bacteria and establish successful colonization within the polymicrobial gut. The T6SS effector VgrG1 contains a C-terminal Actin Crosslinking Domain (VgrG1-ACD), well known for irreversibly crosslinking eukaryotic actin to disrupt the host cytoskeleton. However, whether VgrG1-ACD also contributes directly to interbacterial competition has remained unexplored. Here, we identify a previously unrecognized bacterial target of VgrG1-ACD: MreB, the bacterial actin homolog essential for cell shape, cell wall synthesis, and cytoskeletal organization. We demonstrate that VgrG1-ACD specifically binds Escherichia coli MreB, resulting in pronounced morphological defects and impaired bacterial growth. Notably, unlike its activity toward mammalian actin, VgrG1-ACD targets MreB without crosslinking it, revealing a distinct mode of action. These findings expand the functional repertoire of VgrG1-ACD beyond host-directed virulence and uncover a dual-targeting strategy through which a single T6SS effector manipulates both mammalian actin and its bacterial homolog, likely enhancing V. cholerae fitness during interbacterial competition and host colonization.

microbiology↗

bioRxiv2026

Thermodynamic Profiling of Glucose Translocation in POPC embedded GLUT-4 protein models

The facilitative transport of hexose through the GLUcose Transporter type 4 (GLUT4) is essential for cellular metabolism and is regulated by allosteric nucleotide interactions. In this study, we conducted a comparative biophysical analysis of glucose translocation using both the native Cryo-EM structure (7WSN) and the AI-predicted AlphaFold model using Steered Molecular Dynamics (SMD) at physiological (310.15 K) and attenuated (303.15 K) temperatures with two different allosteric modulators, ATP and ADP. While the AlphaFold model managed to capture baseline static topologies, dynamic profiling revealed that it intrinsically over-optimizes for static stability, immediately collapsing into a sterically occluded, hyper-packed artifact. Translocation through this constricted geometry forces severe steric solvent exclusion, abruptly stripping the substrates hydration shell and resulting in immediate and immense thermodynamic friction (>300 kJ/mol). Under ATP-bound physiological strain, this hyper-bonded structural clamp prevents functional relaxation, ultimately inducing a catastrophic kinematic failure. Furthermore, ensemble Dynamic Cross-Correlation Matrix (DCCM) analysis demonstrates that the AI-predicted model suffers from persistent rigid-body locking, forcing the transmembrane domain to behave as a kinetically trapped conformation. In contrast, the functionally hydrated Cryo-EM architecture actively maintains a dual-gating pore and successfully isolates the exact mechanical lever driving transport: a native Proline hinge (PRO379) that seamlessly routes allosteric signals from the nucleotide anchor to the pore gate. Within the AlphaFold model, this critical allosteric wiring is disrupted, forcing mechanical coupling through an unphysiological, hyper-correlated aromatic pathway. These findings yield novel atomistic insights into GLUT4 gating mechanics, while definitively establishing that rigid AI-generated architectures lack the essential internal free volume and conformational plasticity required to sustain accurate thermodynamic profiling of dynamic membrane transporters.

biophysics↗

bioRxiv2026

Viral Protease Nsp5 Hijacks La Autoantigen to Orchestrate the Translation to Replication Transition in SARS-CoV-2

Viruses genetically encode proteins to regulate their life cycle. To translate this genetic information, most viruses exploit host translation machinery. Host dependency leads to competition between viral RNA and host mRNAs. Most viruses overcome it by inhibiting cap-dependent translation of host mRNAs and shifting its own translation into cap-independent mode. This switch is largely orchestrated by host RNA binding proteins (RBPs) by binding to viral 5UTRs. Here we demonstrate that human La autoantigen (La protein) specifically binds to GCAC sequence near the initiator AUG within the 5UTR of SARS-CoV-2 RNA promoting viral non-structural protein synthesis by cap independent translation, while suppressing replication early in infection. Interestingly, around 18-24 h post-infection, the viral protease Nsp5 cleaves La at a conserved LQ site, abrogating its 5'UTR interaction, which correlates with reduced polysome association and increased viral RNA availability for replication. MD simulation and docking study suggests that the cleavage disrupts La dimerization and truncates its C-terminal intrinsically disordered region, reducing RNA-binding affinity and conformational stability required for translation-competent ribonucleoprotein complexes. Results put forward a unique mechanism by which Nsp5-mediated proteolysis of La drives the temporal switch from translation to replication of SARS-CoV-2.

microbiology↗

bioRxiv2026

A Computational Foundation Toward Targeting the ELMO1/DOCK2 Complex

Engulfment and Cell Motility protein 1 (ELMO1) regulates cell migration, phagocytosis, and cytoskeletal remodeling, positioning it as a compelling therapeutic target across kidney diseases, oncology, enteric infections and inflammation. Despite this potential, no approved therapeutics or clinically validated small molecule modulators of ELMO1 currently exist. ELMO1 functions by forming a complex with DOCK180 (or DOCK2) to activate the small GTPase Rac1, and the recent structural resolution of the ELMO1/DOCK2 complex now provides an opportunity to target this protein protein interface directly. Here, we present the first investigation into the druggability of the ELMO1/DOCK2 complex and report the initial virtual screening to identify small molecule inhibitors of this interaction. Molecular dynamics (MD) and free energy level (FEL) studies were carried out to validate the potential of the predicted hits. This work establishes a computational framework for the development of the first generation of ELMO1 targeted therapeutics. In addition to demonstrating the drugability of ELMO1, this work introduces two open source Python tools for the rapid analysis and visualization of protein protein interaction and ligand protein MD trajectories from DESMOND output files. These tools are designed to be broadly accessible, offering practical utility to the wider DESMOND user community.

cell biology↗

bioRxiv2026

LINE-1 transposon derepression and epigenetic remodeling in Retinoblastoma

Retinoblastoma (RB), the most common pediatric intraocular malignancy, is initiated by biallelic inactivation of the RB1 tumor suppressor gene. Although LINE-1 (L1) retrotransposon activation has been linked to genomic instability and tumor evolution in several adult cancers, its contribution to pediatric malignancies remains poorly understood. Here, we investigated L1 expression in retinoblastoma samples from an Indian pediatric cohort by integrating transcriptomic, locus-specific, and immunohistochemistry. Transcriptomic profiling revealed increased L1 expression in RB tumors compared with control retinal tissues, which was validated by ORF1p immunostaining in tumor sections. Locus-specific analysis identified a subset of transcriptionally active L1 loci, suggesting selective activation of young L1 elements. We also observed altered expression of genes associated with proliferation, chromatin regulation, and oncogenic signaling, including MYCN, MDM2, E2F3, SOX4 and CREBBP. Our findings identify previously underexplored L1 activation as a molecular feature associated with retinoblastoma and suggest that disruption of transposon silencing accompanies the RB transcriptional landscape. Validation in larger patient cohorts will be important; however, this study provides a foundation for exploring L1 activity as a potential molecular marker and future therapeutic vulnerability in retinoblastoma. HighlightsO_LITranscriptomic profiling reveals increased LINE-1 expression in retinoblastoma. C_LIO_LIORF1p protein is robustly expressed in retinoblastoma. C_LIO_LIA subset of LINE-1 loci exhibits transcriptional activation, suggesting locus-specific derepression. C_LI In BriefRetinoblastoma is initiated by RB1 loss, but the contribution of transposable elements to its molecular landscape remains poorly understood. Here, we show that retinoblastoma is associated with LINE-1 promoter hypomethylation, increased LINE-1 transcript expression, ORF1p accumulation, and preferential activation of selected LINE-1 loci. These changes occur together with altered expression of oncogenic signalling pathways and host factors involved in transposable element repression, identifying dysregulated LINE-1 control as a molecular feature of this paediatric cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/740688v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@197e89org.highwire.dtl.DTLVardef@1c26c04org.highwire.dtl.DTLVardef@14180a4org.highwire.dtl.DTLVardef@16493bb_HPS_FORMAT_FIGEXP M_FIG C_FIG Retinoblastoma exhibits epigenetic activation of LINE-1 characterized by promoter hypomethylation, increased expression of young LINE-1 elements, and ORF1p accumulation. This is accompanied by coordinated transcriptomic remodeling and disruption of host mechanisms that normally restrain transposable elements, linking LINE-1 activation to the molecular landscape of retinoblastoma.

cancer biology↗

bioRxiv2026

Targeted measurement of blood flow in the Anterior Cerebral Artery using Diffuse Correlation Spectroscopy

SignificanceDiffuse Correlation Spectroscopy (DCS) is an established technique for non-invasive monitoring of Cerebral Blood Flow (CBF), but existing applications primarily measure CBF changes averaged over cortical tissue volumes. A method capable of targeting blood flow within a particular intracranial artery would expand the utility of DCS for vessel-specific cerebral perfusion monitoring and continuous bedside assessment. AimWe aim to investigate the feasibility of targeted, non-invasive monitoring of blood flow in the Anterior Cerebral Artery (ACA) using DCS through optimization of probe geometry and placement. ApproachA custom-built DCS system operating at 785 nm was used to probe ACA from the glabellar region. Source-detector (SD) separation, probe orientation, and probe location were systematically optimized using lower-limb motor tasks and a mental arithmetic task. The optimized configuration was evaluated using an ACA-mimicking multilayer phantom and validated in forty healthy volunteers during lower-limb activation tasks and postural changes. ResultsAn SD separation of 17 mm, vertical probe orientation, and glabellar placement provided the highest sensitivity to ACA-related blood flow changes. Phantom experiments demonstrated sensitivity to flow changes in a vessel located 45 mm beneath the scalp and showed that the frontal sinus, cerebrospinal fluid, and the absence of cortical tissue beneath the glabella along the longitudinal fissure together provide the best optical window for probing deep ACA flow. Using the optimized probe configuration, significant increases in relative CBF were observed during standing leg marching (66.4 {+/-} 38.6%) and supine leg crunches (39.4 {+/-} 32.2%) (p < 0.01), with consistent responses during supine-to-stand postural transitions. ConclusionThe proposed DCS approach enables targeted measurement of blood flow within the ACA territory. This technique provides a framework for continuous, non-invasive monitoring of ACA perfusion and has potential applications in cerebrovascular monitoring and stroke care.

bioengineering↗

bioRxiv2026

Kruppel-like factors KLF5 & KLF8 emerge as master transcriptional regulators of Alzheimers disease, as revealed on cell fate regulomes in human brain organoids

In addition to the well described beta-amyloid plates accumulation and tau hyper-phosphorylation, Alzheimers disease (AD) is accompanied by major changes in gene expression. Herein, we aimed at revealing master transcription factors (TFs) responsible for gene expression changes during AD progression. For this, we have used human brain organoids (BORGs) harbouring AD-related genetic mutations (APP-Swedish, PSEN1-M146V), which were traced over multiple time-points by bulk and spatially-resolved transcriptomics. By reconstructing gene regulatory networks (GRNs) that recapitulate BORG development, we have identified a subset of 110 AD-specific master TFs, and for 75 of them we retrieved KLF5 and/or KLF8 binding motifs within their promoters. Furthermore, 64 of the AD-specific TFs found in BORGs are significantly over-expressed on AD human patients samples, confirming the relevance of these factors beyond the context of the familial genetic mutations. Finally, we have demonstrated that this AD-specific regulome is at least partially controlled by the aberrant CREB3L2-ATF4 heterodimer previously described as being induced by the beta-amyloid plates deposition. Overall, these findings reconstitute the regulome behind the progression of AD and highlights key TFs as potential druggable targets for the treatment of the disease.

systems biology↗

bioRxiv2026

Integrative structure determination of a human mitochondrial contact site and cristae organizing system (MICOS) sub-assembly

The Mitochondrial contact site and Cristae Organizing System (MICOS) complex is an inner mitochondrial membrane (IMM) assembly present at the cristae junction. It is responsible for regulating cristae formation and remodeling. However, its structure is not known. We applied Bayesian integrative structure determination to characterize the structure of the Mic60, Mic19, Mic10, and Mic13-containing MICOS complex combining AlphaFold predictions with data from crosslinking mass spectrometry, biochemical assays, electron tomography, homology modeling, and sequence alignments. The integrative structure revealed novel mutual interfaces among Mic10N,C, Mic60LBS1,LBS2,mitofilin, and Mic13central,C, which were experimentally validated. Several likely-pathogenic missense mutations also localize to these novel interfaces, highlighting their importance. Our results indicate that Mic13 likely facilitates MICOS assembly by binding Mic10 in the IMM-proximal region and Mic60 in the intermembrane space. Taken together, our integrative approach sheds light on the structure and assembly of the MICOS complex.

bioinformatics↗

bioRxiv2026

A new set of DNA methylation variants in the human genome show predominant tissue specificity and sensitivity to reprogramming with a potential for disease susceptibility.

Analyses of 3,370 normal human tissues of ectodermal, endodermal and mesodermal origins identified 12,587 regions averaging [~]585 bp with significant differences in DNA methylation levels within identical tissues. These methylation variants (MeVars) occurred in 8,037 genes enriched in neurological disorders and cancers of which, majority were tissue-specific rather than being systemic. This somatic variation was reduced by reprogramming in vitro into iPSCs and in vivo during spermatogenesis. Analysis of prefrontal cortices showed a higher incidence of MeVars in the candidate genes in controls than schizophrenia patients wherein a subset showed significantly altered transcript levels. Similar effects were observed for oral tissues and skin fibroblast cells. MeVars showed significant association with SINE1, simple and low complexity repeats, H3K27me3, H3k9me3 and H3K4me1 modifications and EZH2, SUZ12 and REST binding sites. Collectively, MeVars have postzygotic origins with an ability to reset during reprogramming, adding a new dimension in the form of epigenetic diversity and its relevance to disease susceptibility in humans.

bioinformatics↗

bioRxiv2026

Morphological and molecular diversity of rice blast isolates from Terai foothills of the Himalayas

Rice blast disease, caused by Magnaporthe oryzae (formerly as Pyricularia grisea), is a major threat to rice production globally, causing devastating yield losses up to 30-50% of rice production annually. Here, we analysed 48 isolates collected from rice fields in the Terai foothills of the Himalayas, India, to assess pathogen occurrence in a new agroecological zone, potentially influenced by climate change and favourable environmental conditions. All isolates were screened for virulence and pathogenicity, and two highly virulent isolates (UBKV1 and UBKV2) were selected for detailed characterization of their morphological, growth, and genetic variability. Significant differences (p-value=4.16 x 10-) were observed in conidial dimensions, with UBKV2 producing larger spores (33.61 {micro}m) compared to UBKV1 (28.07 {micro}m). In terms of growth, mycelial biomass (fresh weight) and sporulation intensity was also higher in UBKV2 (22.98 g and 2315.5) than UBKV1 (15.82 g, and 1812.3) when they grew under the same conditions. Distinct colony growth patterns were observed on different media, particularly on Mathurs medium and Rice Straw Extract Dextrose Oatmeal Agar, where UBKV2 exhibited suppressed growth and unique pigmentation. Phylogenetic analysis of the ITS region revealed sequence similarities ranging from 95.11% to 100% among the isolates. UBKV2 showed closer genetic relatedness to isolates from Odisha (96.95-97.23%) than to UBKV1 (95.57%), highlighting significant genetic differentiation. These findings demonstrate substantial morphological, cultural, and genetic variation within M. oryzae populations in the Terai foothills, providing important insights into pathogen evolution, virulence mechanisms, and implications for region-specific resistance breeding strategies.

plant biology↗

bioRxiv2026

Head-to-head organized segmental paralogs AtOFP2 and AtOFP17 exhibit differential, spatio-temporal partitioning of function, and negative regulation of multiple developmental traits including seed-yield and root architecture

Ovate Family Proteins (OFPs) are a class of plant-specific, negative nuclear transcriptional regulators characterized by conserved C-terminal OVATE domain. This study on comparative functional characterization of two head-to-head arranged OFPs - AtOFP2 (Ovate-OFP with full ovate domain) and AtOFP17 (Ovate-Like OFP with partial ovate domain) provides critical insight into how structural variations in ovate domain leads to functional divergence. Detailed phenotypic analysis of 28 physical and physiological traits of loss- and gain-of-function mutants revealed that both genes act as broad, pleotropic repressors of plant growth and development. Removal of repression in knock-down mutants of both genes exhibited reduced duration of seed dormancy, faster rate of germination and growth, bigger plants and significantly higher seed yield. In contrast, constitutive over-expression showed a generalized repressive nature of both genes, with nuanced differences for fine tuning of specific traits. For example, both genes showed antagonistic behaviours on root hair architecture. AtOFP2 act as a strong repressor of root hair development whereas AtOFP17 is a stronger repressor of hypocotyl and root cell architecture. AtOFP17 owing to partial ovate domain exerts a mild level of repression throughout life span as indicated by smaller plants and lesser yield in knock-down AtOFP17 mutants. On the contrary, AtOFP2 exerted a much stronger repressor effect in which > 90% over-expression mutants died at the juvenile stage ; the survival of remaining 10% is probably owing to activation of dosage-dependent feedback loop mechanism as indicated by normal growth of mature plants, and is also evident by transcriptome data. Transcriptome analysis of roots of 7-day old seedling of knock-down and over-expression mutants of AtOFP2 showed downregulation of OFP2 in over-expressed mutants. However, severely stunted phenotype indicated presence of stable OFP2 protein to exert effects. Analysis of DEGs in OFP2 mutants revealed that it acts as an important regulator working at intersection of hormonal signalling affecting critical genes required for auxin, cytokinin, GA, BR and ABA functioning. Perturbations across hormonal signalling pathways affects cell wall remodelling factors such as EXPANSINS, Xyloglucan hydrolases (XTHs) and cellulose synthases (CSLs) causing overall stunted growth; and epidermal patterning genes such as WER, GL1, EGL3, TTG1 leading to severely reduced root length and root hairs. Significantly, functional analysis of this master regulator highlighted a significant economic potential. Knockdown of both these genes relieves their natural repression on reproductive traits, leading to longer siliques, bigger and heavier seeds, and substantially increased overall seed yield, positioning AtOFP2 and AtOFP17 as highly valuable targets for agricultural crop improvement.

plant biology↗

bioRxiv2026

Cerebrovascular Single-Nucleus RNA-Seq Reveals Heat Shock Activation and Vascular Remodeling in Alzheimer's Disease and Primary Tauopathies

Cerebrovascular alterations are widely observed in both Alzheimers Disease (AD) and primary tauopathies. Here, we hypothesized that mechanisms of cerebrovascular alterations are shared between AD and primary tauopathies. We performed single-nucleus RNA sequencing of postmortem human inferior temporal gyrus to characterize transcriptomic changes across cerebrovascular cell types in AD and primary tauopathies (Corticobasal Degeneration, Picks disease, and Progressive Supranuclear Palsy). Differential gene expression analyses revealed disease-specific transcriptional programs across vascular cell populations. However, genes involved in the heat-shock response were consistently upregulated across all diseases, suggesting a conserved cerebrovascular stress response during neurodegeneration. We further identified marked cerebrovascular remodeling in AD relative to primary tauopathies, along with dysregulation of genes mapping to AD risk loci in endothelial cells. Transcriptomic findings were validated using tissue clearing, light-sheet microscopy, and immunofluorescence quantification of vascular markers. These results define a conserved vascular stress program alongside AD-specific remodeling, highlighting the vasculature as a therapeutic target in neurodegeneration.

neuroscience↗

bioRxiv2026

Raman spectral signatures reflect altered metabolic state of exhausted T cells

T cell exhaustion within the tumor microenvironment drives CD8+ T cells into a dysfunctional state characterized by progressive loss of proliferative capacity and effector functions, thereby limiting anti-tumor immunity and therapeutic efficacy. To investigate the biochemical alterations associated with exhaustion, an in vitro model of CD8+ T cell exhaustion was established through chronic antigenic stimulation of murine OT-1 CD8+ T cells. Phenotypic, functional, metabolic, and transcriptional characterization confirmed the acquisition of an exhausted state. Single-cell Raman spectroscopy was subsequently employed to generate biochemical signatures of activated, and exhausted CD8+ T cells. Principal component analysis (PCA) of the Raman spectral data revealed distinct separation of these two cell subsets, reflecting underlying biochemical differences associated with their functional states. Differential Raman spectral features corresponding to nucleic acids, carbohydrates, proteins, and lipids contributed significantly to this segregation, reflecting altered metabolic and biosynthetic states during exhaustion progression. Classification of the spectral data using machine-learning algorithms enabled accurate segregation of activated and exhausted T cells. Collectively, this study demonstrates that single-cell Raman spectroscopy can distinguish exhausted CD8+ T cells in a label-free and non-destructive manner, highlighting its potential as a platform for immune profiling and monitoring T cell dysfunction.

immunology↗

bioRxiv2026

Stochasticity and heterogeneity restrict tipping points and alternative stable states

Gradual environmental stress may trigger abrupt tipping points, trapping ecosystems in degraded states that are difficult to reverse. This possibility has strongly influenced ecosystem management, yet empirical evidence for such behavior remains mixed. A major unresolved question is how broadly stochasticity and heterogeneity restrict hysteresis, and whether their effects accumulate. To address it, we reconstructed full phase diagrams for four canonical ecological models (dryland desertification, grazing, insect outbreaks, and lake eutrophication) across gradients of stress, demographic stochasticity, environmental fluctuations, spatial heterogeneity, and dispersal. In every model, stochasticity and heterogeneity narrowed the region supporting alternative stable states and made the remaining transition less abrupt. Dispersal set their effective strength by averaging across space. These complexities eroded hysteresis along different routes -toward the degraded state, through a continuous transition, or, counterintuitively, toward the healthy state. Effects were largely additive, so several moderate complexities could eliminate hysteresis even when no single factor did. Tipping-point claims should therefore be tested under stochastic, heterogeneous conditions.

ecology↗

bioRxiv2026

PAD4-mediated histone citrullination contributes to enhanced NETosis in type 2 diabetes but not in type 1 diabetes

Neutrophil extracellular traps (NETs) are web-like structures released by activated neutrophils through a process known as NETosis, which help immobilize, trap, and eliminate invading pathogens. While NETs play a critical role in host defense, excessive or dysregulated NET formation can contribute to chronic inflammatory diseases such as diabetes and its complications. Elevated levels of NETs and increased expression of its mediator, protein-arginine deiminase type 4 (PAD4), have been reported in diabetes. However, the underlying molecular mechanisms governing the enhanced NETosis in type 1 (T1D) and type 2 diabetes (T2D) remain unclear. Using mouse models and human patient samples, we show that neutrophils undergo enhanced NETosis in T1D and T2D through distinct pathways. We found enhanced NETosis in T1D occurs in a PAD4-independent manner, driven by robust cytosolic ROS production by NADPH oxidase (NOX). This leads to myeloperoxidase activation and chromatin decondensation. We further confirm the PAD4 independent mechanism in neutrophils from STZ-induced PAD4-/- mice. In contrast, in T2D, neutrophils undergo NOX-independent NETosis, which relies on calcium-mediated mitochondrial ROS production, PAD4 activation, and hyper-histone citrullination. Taken together, our findings reveal previously uncharacterized distinct mechanisms of enhanced NETosis in T1D and T2D.

Molecular Biology↗