Biology subjects

Chakraborty, S.

Publications and source records attributed to Chakraborty, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Rapid femtosecond laser trenching accelerates cryo-electron tomography lamella workflows

Transmission electron microscopy requires electron-transparent specimens, typically below 200 nm thick. For cellular and tissue samples this is typically achieved by focused ion-beam (FIB) thinning to form lamellae. Milling times scale with specimen thickness, and high-pressure-frozen specimens more than 10 m thick can take up to 2 hours per lamella. We present a pre-processing approach in which bulk material is removed by ultrashort-pulse laser ablation prior to milling, rapidly generating large trenches in vitrified grids under cryogenic conditions and leaving material to be finished by conventional milling to yield electron-transparent lamellae. Laser ablation completes in seconds (4 seconds per pattern), compared with minutes to tens of minutes for FIB trench milling, and introduces negligible damage to the final lamellae. By replacing the most time-intensive, contamination-sensitive bulk-removal steps with seconds-long laser trenching, this open-source approach reduces time-to-lamellae, increasing throughput and preserves compatibility with standard cryo-FIB/SEM polishing and cryo-ET data-collection pipelines.

biophysics↗

bioRxiv2026

CiliaBuilder2: a flexible ChimeraX bundle to visualize cilia and centriole structures in 3D

Centrioles and cilia are large organelles essential for cell division, motility, and signalling. They are characterized by distinctive, nine-fold symmetric microtubule doublets or triplets decorated with periodic protein complexes. While cilia and centrioles are highly conserved across eukaryotes, their composition and structure vary considerably between species and tissue types. Advances in cryo-electron tomography and single-particle cryo-electron microscopy have recently led to a surge in structural data for ciliary components. However, visualizing these components within the ciliary context remains challenging due to their scale and the diverse data formats involved, such as density maps, atomic models, and 3D geometric objects. Here, we introduce CiliaBuilder2, a toolset integrated into UCSF ChimeraX that enables simple, flexible visualization of complete ciliary structures from diverse data types using parametric calculations.

biophysics↗

bioRxiv2026

Plakoglobin supports length-dependent force generation and cardiac resistance to fasting

Mechanical activity protects muscle from atrophy, but how contractile forces relate to anabolic signaling remains unclear. We previously showed that plakoglobin promotes insulin-PI3K-AKT-FoxO signaling and that its loss causes skeletal muscle atrophy. Here, super-resolution microscopy revealed that plakoglobin localizes with desmin at the sarcolemma and is lost from the membrane during fasting-induced skeletal muscle atrophy. In skinned muscle fibers, plakoglobin downregulation impaired stretch-induced increase in myofilament activation at long sarcomere length. During fasting, skeletal muscle lost sarcolemmal plakoglobin-containing complexes, insulin signaling, and mass and induced MuRF1 and MAFbx/Atrogin1. In contrast, cardiac muscle retained these complexes and anabolic signaling, and maintained its mass. Cardiac plakoglobin also resisted AAV-shJUP-mediated depletion despite viral delivery. Moreover, MEK inhibition disrupted this cardiac response, reduced AKT signaling, induced atrogenes, and decreased cardiac mass. Thus, plakoglobin supports force generation at increased sarcomere length, and preservation of plakoglobin-containing complexes and MEK-ERK signaling accompany cardiac resistance to fasting-induced atrophy.

cell biology↗

bioRxiv2026

Gut bacterium of Leishmania donovani-non-transmitting sand fly induces IL-23~Th17 axis to protect hosts from drug-resistant visceral leishmaniasis

Background: Visceral leishmaniasis (VL) is caused by Leishmania donovani and differentiates under the influence of biotope-specific gut bacteria in the Leishmania donovani-transmitting sand fly vector, Phlebotomus argentipes, which coexists with the Leishmania donovani-non-transmitting Sergentomyia species in the Indian subcontinent. Hypothesis: Sergentomyia are primarily herpetophilic. Interestingly, we identified a specific population of anthropophilic Sergentomyia babu babu, a proven naturally non-competent vector for transmitting Leishmania donovani, with high abundance and human-bite incidences in VL-non-endemic regions, compared with VL-endemic regions of India. Thus, the possibility of egesting gut bacteria to humans from VL-non-transmitting Sergentomyia and inducing downstream immune cascades remains undeniable. Innovation: We characterized gut bacteria from anthropophilic Sergentomyia babu babu in both VL-endemic and VL-non-endemic regions of India and found that a heat-inactivated formulation of Heyndrickxia sporothermodurans [NCBI Sequence Read Archive (SRA): PRJNA1403236], isolated from anthropophilic Sergentomyia babu babu from a VL-non-endemic region, prevented and cured L. donovani infection by inducing IL-23~Th17. More importantly, it also prevented the initial parasite establishment by inducing phagocytosis, ROS, and TNF- in circulatory Ly6G-CD11b+Ly6Chi monocytes. To the best of our knowledge, this is the first report confirming that the gut bacteria of VL-non-transmitting sand flies are critical and need to be investigated in depth for developing therapeutics and/or prophylactics against visceral leishmaniasis (VL), and suggests that possible bite-transfer of gut bacteria from disease-non-transmitting vectors to hosts may restrict disease pathogenicity. Further clinical trials are needed to warrant (Indian patent: 202511041013, April 28, 2025).

immunology↗

bioRxiv2026

A Nanomechanical Fingerprint of Centromeric Methylation Co-Localization in TNBC Chromosomal Instability

Centromeric heterochromatin is maintained by a self-reinforcing loop between DNA methylation (5mC) and histone H3 lysine 9 trimethylation (H3K9me3), yet whether this coupling is preserved at the level of individual chromosomes in cancer has been inaccessible to conventional epigenomic assays, which average signals across cell populations and cannot resolve single-chromosome heterogeneity. Here, we use antibody-functionalized atomic force spectroscopy to simultaneously map 5mC and H3K9me3 at the centromeres of individual metaphase chromosomes from non-tumorigenic, luminal, and triple-negative breast cancer (TNBC) cell models. Co-localized 5mC/H3K9me3 sites are mechanically stiffer than sites carrying either mark alone across the panel, revealing a cooperative biophysical signature of epigenetic crosstalk. Luminal chromosomes acquire the highest mark density, co-localization, and stiffness, whereas TNBC chromosomes show reduced density and diminished, though not absent, co-localization and stiffness, converging toward the low baseline of non-tumorigenic epithelium. TGF-{beta}1-induced epithelial-mesenchymal transition reproduces this attenuated phenotype in luminal cells, linking it directly to a process central to TNBC aggressiveness. These biophysical parameters classify cancer versus non-tumorigenic chromosomes with near-perfect, cross-validated accuracy, and further resolve the two TNBC subtypes using colocalization-derived features alone (macro-F1 = 0.933). These results establish the mechanical consequence of epigenetic mark co-localization as a single-chromosome-resolvable signature of centromeric organization, attenuated in aggressive breast cancer, and demonstrate a force-spectroscopy platform for mechano-epigenetic profiling of cancer chromosomes.

biophysics↗

bioRxiv2026

P-body sequestration of clock transcripts delays repressor synthesis to set circadian period in Drosophila

Negative-feedback oscillators require a delay between the accumulation of a repressor's mRNA and the action of its protein. In the circadian clock, this delay has been attributed largely to post-translational control of PERIOD (PER) stability and nuclear entry. The RNA-binding proteins shown to regulate per translation, ATAXIN2 and its partners, promote it, leaving open whether any step holds clock transcripts back before they are translated. Here, using time-resolved miniTurbo proximity labeling of endogenous PER across four phases of the circadian cycle in Drosophila clock neurons, we define a 252-protein PER proximitome that partitions into a nuclear arm and a cytoplasmic RNA-metabolism arm. A behavioral RNAi screen identified two P-body components, the DEAD-box helicase Me31B (DDX6) and the 5'-3' exonuclease Pacman (Pcm; XRN1), as strong regulators of circadian rhythms. Using single-molecule RNA-FISH, proximity RNA editing and ribosome profiling, we show that as per and tim transcripts accumulate, they localize to Me31B-labeled P-bodies and are poorly translated, most prominently at ZT12. Me31B knockdown disrupts P-bodies and releases per mRNA from them, causing PER to accumulate earlier and to ~2-fold higher levels, whereas Me31B overexpression delays PER accumulation and lengthens the free-running period by ~2 h. Knockdown of Pcm, in contrast, impairs clearance of per mRNA, sustaining PER and TIM accumulation, prolonging the repression phase and abolishing cycling of ~89% of rhythmic transcripts. Together, these findings identify P-body sequestration as a repressive step that delays repressor synthesis, and Pcm-dependent decay as required to end repression on time. Given the deep conservation of DDX6 and XRN1, RNP compartments may provide a conserved means of generating delay in circadian and other negative-feedback circuits.

cell biology↗

bioRxiv2026

Gasdermin E couples mitochondrial stress to STING-driven neuronal pyroptosis during Chandipura virus encephalitis

Neurotropic RNA viruses are major causes of fatal encephalitis worldwide, yet how infected neurons transition from antiviral defense to inflammatory cell death is not well characterized. Chandipura virus (CHPV), an emerging neurotropic rhabdovirus, causes acute, rapidly progressive encephalitis with high case fatality in children, but the mechanisms underlying its neuropathogenesis remain poorly defined. Here, we demonstrate that CHPV suppresses canonical RNA virus sensing early but subsequently switches to a mitochondria-driven innate immune program that culminates in inflammatory cell death. Early infection of neuronal cells with CHPV was marked by reduced levels of the mitochondrial antiviral adaptor protein, MAVS and attenuation of type I and III interferon responses. As infection progressed, mitochondrial dysfunction promoted accumulation of mtROS, mitochondrial accumulation of cleaved GSDME and cytosolic mtDNA release, triggering STING activation, which coincided with robust neuroinflammation and pyroptotic cell death. Pharmacological inhibition or genetic silencing of STING markedly attenuated inflammatory signaling, prevented pyroptotic membrane rupture and protected neurons from cell death without significantly affecting viral replication. In contrast, GSDME depletion reduced both viral replication and neuronal death. Notably, GSDME depletion markedly attenuated STING phosphorylation, while STING depletion also reduced GSDME activation, revealing functional coupling between these pathways during CHPV-induced neuronal injury. Collectively, our findings identify a mitochondria-GSDME-STING axis linking early immune evasion to neuroinflammation during CHPV infection, revealing a previously unrecognized mechanism of inflammatory neuronal death in viral encephalitis and highlighting STING as a potential therapeutic target in certain CNS viral infections.

immunology↗

bioRxiv2026

Reducing Neutrophil Sialic Acid Residues Alleviates Cerebral Hypoperfusion in Alzheimer's Models

Objective: Dysregulation of the immune system is increasingly recognized as a contributor to Alzheimer's disease (AD) progression, partly through neutrophil adhesion to the cerebral vasculature, which promotes hypoperfusion in AD. Because sialic acid (SA) residues on membrane glycoproteins regulate neutrophil-endothelial interactions, we investigated whether neutrophil sialylation is altered in AD and whether reducing it improves cerebral vascular function. Approach and Results: Lectin blots of isolated neutrophils showed increased SA levels in two AD mouse models, 5xFAD and APP-SAA. We identified 2,3 sialyltransferase-IN-1 as a small-molecule inhibitor that reduces sialylation in vivo. Treating 5xFAD mice with this inhibitor decreased SA on neutrophil membranes, increased cerebral blood flow, and reduced capillary stalling. Leukocytes from patients with preclinical AD and mild cognitive impairment also had higher SA levels than those from age-matched healthy controls. Conclusions: Elevated terminal sialylation of neutrophil glycoproteins contributes to capillary stalling and cerebral hypoperfusion in AD. Neutrophil sialylation may serve as both a biomarker and a therapeutic target for improving cerebral blood flow and slowing disease progression.

neuroscience↗

bioRxiv2026

A dopaminergic visuomotor gateway to the zebrafish optic tectum

Dopamine (DA) is widely known as a neuromodulator essential for reward, motivation and learning, yet it also modifies rapid sensorimotor transformations. Understanding how dopaminergic neurons process sensory input and motor-related signals is therefore critical for elucidating their role in sensorimotor control. In vertebrates, a conserved visual center under dopaminergic influence is the superior colliculus, or optic tectum in fish. Here, using anatomical and molecular analyses in larval zebrafish, we identify a defined cluster of pretectal DA (PrDA) neurons whose axons densely innervate the tectum, predominantly in its deep neuropil. Combining functional Ca2+ imaging with visual stimulation and motor recordings, we show that PrDA neurons respond reliably to visual stimulation. However, most PrDA neurons exhibit pronounced activity also during spontaneous locomotion, and enhanced activity when visual stimuli and motor output co-occur, indicating that PrDA neurons integrate convergent input from visual and motor centers. Furthermore, spontaneous PrDA neuron activity was synchronized and the synchrony was even stronger when visual or motor activity contributed to their activation. Notably, as visual stimuli differed in their efficacy to drive swim activity, motor-associated responsiveness of PrDA neurons produced apparent direction selectivity to stimulus motion when motor activity was not accounted for. This apparent neural response bias disappeared once motor activity was taken into account. Together, these findings suggest that PrDA neurons provide rapid, visuomotor-related dopaminergic modulation of tectal circuits that transform visual information into context-dependent motor commands, a principle that may extend to homologous mammalian midbrain circuits.

neuroscience↗

bioRxiv2026

An Ultrasensitive Luciferase Reporter Reveals Low-Level Extrahepatic mRNA Expression for Enhanced Apparent Biodistribution of Lipid Nanoparticles

Reporter mRNAs are widely used to screen lipid nanoparticle (LNP) formulations and characterize functional mRNA expression in vivo, yet conventional firefly luciferase (FLuc) imaging fails to detect low-level expression. Consequently, weak or absent FLuc signal can be misinterpreted as failed LNP delivery or translation, potentially excluding formulations and overlooking extrahepatic or off-target expression. Here, we compared FLuc/D-luciferin with an ultrasensitive NLuc-GFP bioluminescence resonance energy transfer luciferase reporter detected with fluorofurimazine (FFz), using SM-102 and U105 LNPs following intravenous administration in mice. Both reporters showed predominantly hepatic and splenic expression. However, NLuc-GFP/FFz produced substantially greater photon output and detected reporter activity in the brain, heart, kidney, lymph nodes and other tissue where FLuc activity was weak or undetectable. Collectively, we show traditional FLuc imaging can underappreciate mRNA delivery, not necessarily because LNPs fail to reach or deliver mRNA to the tissue but because low-level FLuc expression may fall below its assay sensitivity. This limitation can underappreciate mRNA delivery and unintended protein expression in non-target organs. Therefore, we describe a reproducible workflow encompassing reporter design, LNP formulation and characterization, controlled reporter comparison, ex vivo organ imaging, and a cost-effective DSPE-PEG2000-micelle FFz formulation. This provides a more sensitive framework that improves detection and interpretation of low-level functional expression during preclinical mRNA-LNP screening and safety assessment.

bioengineering↗

bioRxiv2026

Mapping the Nonhistone Interactome of the TRIM24 Bromodomain Using Genetically Encoded Complementary Photocrosslinkers

TRIM24 is a tandem epigenetic reader containing plant homeodomain (PHD) and bromodomain modules in its C-terminal region. The PHD domain recognizes unmodified lysine 4 of histone H3, whereas the bromodomain recognizes acetylated histones H3 and H4. However, whether the TRIM24 bromodomain also engages acetylated nonhistone proteins remains poorly understood. To address this question, we developed site-specific photocrosslinking probes for the TRIM24 bromodomain using interaction-based protein profiling (IBPP). 4-azido-L-phenylalanine (AzF) and 4-benzoyl-L-phenylalanine (BzF) were genetically incorporated at L922, a residue positioned within the acetyllysine-binding pocket. Both variants preserved recognition of acetylated histone peptides and underwent efficient UV-dependent photocrosslinking. Quantitative proteomics of photocaptured proteins revealed distinct profiles for the two probes, identifying NPM1 among the L922AzF-enriched proteins and CDK9 among the L922BzF-enriched proteins. Additional candidate proteins included MAP4, RAN, and SND1. ChIP-seq analysis showed overlapping genomic enrichment of TRIM24, CDK9, and NPM1 near transcription start sites of cancer-associated genes. KEGG analysis further placed the identified proteins in several signalling pathways. Collectively, this study demonstrates that site-specific photocrosslinking can expand interactome analysis of epigenetic reader domains and provides candidate nonhistone proteins associated with the TRIM24 bromodomain for further investigation.

biochemistry↗

bioRxiv2026

Understanding cephalopod regeneration: impact of sexual maturation, injury severity and general anesthesia on octopus arm restoration

Octopuses exhibit remarkable regenerative capacity, as shown by their ability to fully restore a whole arm. During this process, a complex appendage containing muscle, connective tissue, vasculature, suckers, sensory structures, peripheral nerves and the axial nerve cord (ANC) is regrown. Although the morphology of octopus regeneration is increasingly well described, the physiological variables that determine regenerative speed, including the potential contribution of ion-channel-dependent processes, remain poorly understood. In this work we examined the influence of reproductive state, injury severity, biological sex and magnesium chloride (MgCl2) anesthesia, on the rate of longitudinal arm regrowth. Reproductive stage was the strongest determinant of regenerative performance: pre-reproductive stage (pre-RS) animals showed robust regrowth, whereas reproductive stage (RS) animals regenerated significantly slower. In pre-RS animals, regeneration speed scaled strongly with injury severity, whereas this relationship was absent in RS animals. Biological sex did not independently predict regeneration speed, and MgCl2 exposure showed no long-term inhibitory effect on regeneration. Our final model explained 81.4% of variance in regeneration speed, with reproductive state and injury severity having major effects. These findings demonstrate that sexual maturation and optic-gland-mediated senescence act as an irreversible physiological block against somatic regeneration, and provide empirical support for mechanosensory and bioelectric-dependent scaling models of cephalopod limb restoration.

neuroscience↗

bioRxiv2026

Evaluating the Spatial Overlap of Cattle and Wildlife on Buck Island Ranch, Florida

Spatial overlap of domestic animals and wildlife can have positive effects such as increased biodiversity in some areas and reduction in forest fires but it can also have negative consequences such as pathogen spillover, vector and pathogen propagation into new areas among others. Thus, understanding the interactions between wildlife and domestic animals on shared landscapes is critical for conservation and management purposes. To investigate these cattle-wildlife interactions, we chose Buck Island Ranch (BIR) in southcentral Florida. We studied spatial overlap between Domestic Cattle (Bos Taurus) and multiple wildlife species such as Wild Boar (Sus scrofa), White-tailed Deer (Odocoileus virginianus), Rabbits (Sylvilagus spp) and others in pasture, semi-pasture, and hammock habitats/vegetation types at BIR. Five management blocks were selected that had pasture and hammock habitats and within each block we established matched pairs of 100m transects. Animal dung samples were surveyed and identified in 13 pairs of transects. We used Wilcoxon signed rank test to determine differences in dung counts between the habitats and calculated Simpson diversity index to analyze the differences in diversity between the habitat types. We found that there was greater dung count of white-tailed deer in hammock habitats compared to pastures (p=0.048) and greater diversity of wildlife in hammocks than pastures (p=0.028). Cattle dung was found in high density in both hammocks and pastures. These results indicate that hammocks are areas shared between cattle and wildlife at BIR and depending on the extent of direct and indirect contacts between these animals there can be a risk for pathogen spillover. We recommend further research on local land and cattle management practices that can facilitate the co-existence of livestock and wildlife.

ecology↗

bioRxiv2026

Oligomannose Fc Glycans Reprogram the Energetic and Conformational Basis of CD16a Recognition

IgG1 Fc recognition by Fc{gamma}RIIIa/CD16a is a central determinant of antibody-dependent cellular cytotoxicity and is strongly regulated by Fc N297 glycosylation. While afucosylation and galactosylation have been extensively studied, the structural basis by which oligomannosidic Fc glycans modulate CD16a binding remains less clear, despite their prevalence in therapeutic antibodies and association with accelerated serum clearance. Here, we use all-atom molecular dynamics simulations to investigate how mannose-5 (M5) Fc glycosylation alters IgG1 Fc-CD16a recognition across Paired Biantennary (complex glycans on both Fc), asymmetric Unpaired (complex glycan on one Fc arm and M5 on the other), and Paired M5 glycoforms. Computed interaction energies reproduce the experimental trend that Paired M5 glycoforms bind CD16a less favorably than complex-type paired glycans, supporting the use of the simulations to interrogate the structural origin of this energetic hierarchy. Residue-wise energetic decomposition and contact analyses show that Paired M5 glycosylation redistributes energetic contributions away from the productive Fc-CD16a interface and reduces both protein-mediated and glycan-mediated physical contacts. Free energy surface analyses further reveal that Paired M5 systems sample broader, less stable receptor-bound conformational ensembles, while dynamic cross-correlation analysis shows reduced intra-domain and inter-domain coupling across the complex. Importantly, a single M5 glycan is sufficient to perturb productive recognition by increasing Fc-arm separation heterogeneity, reducing high-frequency protein contacts, and weakening long-range dynamic communication. Glycan identity on the receptor-proximal Fc arm emerges as a decisive determinant of binding, indicating that Fc glycan composition, pairing, and receptor-bound placement jointly encode CD16a recognition. Together, these findings provide a mechanistic framework for understanding how oligomannose Fc glycans remodel antibody-receptor engagement and suggest that asymmetric Fc glycosylation, combined with residue-level interface engineering, may offer new strategies for tuning therapeutic antibody effector function.

immunology↗

bioRxiv2026

Targeting glucocorticoid signalling in dendritic cells for glioblastoma treatment

Glioblastoma (GBM) remains a devastating disease with few meaningful therapeutic advances over the past three decades. Dendritic cell (DC) vaccination is a promising immunotherapeutic strategy for GBM, but its efficacy is limited by the clinical use of dexamethasone to control cerebral oedema and associated symptoms. Here we show that steroid signalling is a central regulator of DC dysfunction in GBM. Through targeted metabolomics of primary GBM samples, we identified a steroid-rich tumour microenvironment in which dexamethasone is present at high levels. Across bulk and single-cell transcriptomic and epigenomic datasets, NR3C1 emerged as the dominant steroid receptor in GBM immune cells and was negatively associated with activated DC states. In patient-derived DCs, dexamethasone altered NR3C1 chromatin occupancy, induced broad transcriptional and chromatin remodelling, and suppressed co-stimulatory antigen-presentation and cytokine programmes. DC-specific deletion of Nr3c1 restricted syngeneic glioblastoma growth, enhanced DC activation, promoted cytotoxic CD8+ T cell responses and remodelled myeloid states in vivo. In GBM patient-derived DCs, pharmacological or non-viral CRISPR-mediated disruption of NR3C1 restored inflammatory, antigen presentation and T cell-stimulatory programmes, enhanced antigen-specific CD8+ T cell priming, and improved tumour lysate-loaded DC vaccination. Together, these findings identify glucocorticoid signalling as a key barrier to DC immunotherapy in GBM and establish NR3C1-targeted, steroid-resistant DCs as a potential therapeutic strategy.

immunology↗

bioRxiv2026

Amaranthus viridis-derived phytopriming reprograms redox homeostasis and limits arsenic accumulation in rice

Arsenic (As) poses a major threat to rice productivity and food safety due to its high bioaccumulation potential and subsequent entry into the human food chain. In rice, As impairs seed germination, disrupts morpho-anatomical development, and induces oxidative stress. This study evaluates seed priming with an aqueous extract of the agricultural weed Amaranthus viridis (AvE) as a sustainable strategy to alleviate As-induced phytotoxicity. AvE priming significantly improved germination (71-75%) and morpho-physiological performance under As stress. It reduced oxidative stress markers, including H2O2 (21-38%), malondialdehyde (13-26%), and proline (18.9-44.7%), while increasing antioxidant metabolites, polyphenols and glutathione by up to 2.34-fold and 41%, respectively. Microscopy confirmed restoration of cellular integrity and anatomical organisation in primed seedlings. ICP-OES analysis showed that AvE priming reduced root As uptake by up to 39%, root-to-shoot translocation by up to 58%, and grain As accumulation by up to 95% compared with unprimed plants. qRT-PCR revealed modulation of genes involved in As homeostasis, indicating coordinated physiological and transcriptional responses. Importantly, improved agronomic performance further demonstrated the translational potential of this approach. This study provides the first evidence that A. viridis extract is a cost-effective, sustainable biostimulant for producing low-As rice in contaminated regions.

plant biology↗

bioRxiv2026

BCL11B targeting in tumor CD8+ T cells amplifies anti-tumor response by blocking exhaustion while promoting stemness and cytotoxicity

Tumor infiltrating CD8+ T cells (TILs) progress to a state of terminal exhaustion (Ttex) which have impaired functionality and are nonrenewable. However their precursors (Tpex) are renewable and can generate efficient effector cells. We started from the observation that melanoma patients undergoing therapy with checkpoint inhibitors show increased survival when their T cells have low BCL11B mRNA. In line with this, ablation of Bcl11b in CD8+ TILs conferred a superior anti-tumor response in murine melanoma and ovarian cancer models. Bcl11b KO TILs failed to progress to the Ttex state and retained elevated stemness. Bcl11b exerted its role by repressing expression of essential transcription factors (TF) controlling stemness, and conversely by promoting expression of exhaustion-associated TFs and inhibitory receptor genes, through complex epigenetic control. In addition, Bcl11b KO CD8+ T cells showed increased Ag-specific cytolytic activity and elevated Gzmb and Prf1 proteins, but no increase in their mRNAs, however presented higher expression of genes with role in translation. Furthermore, CRISPR-CAS9-mediated deletion of BCL11B in human TILs from a patient with poor response to adoptive cell therapy with autologous TILs, improved their cytolytic activity and promoted expression of the stemness-associated TF TCF1, underlying its potential therapeutic use. HIGHLIGHTS- Adoptive transfer of Bcl11b KO CD8+ TILs surpasses WT in tumor burden reduction - Bcl11b ablation reprograms TILs and impairs the progression to Ttex state - Bcl11b KO CD8+ T cells have elevated cytotoxicity and kill only Ag-MHCI targets - BCL11B deletion in nonresponder ACT-TIL improves cytolytic activity and elevates TCF1 GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/742578v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@c3b965org.highwire.dtl.DTLVardef@1c09d75org.highwire.dtl.DTLVardef@a19748org.highwire.dtl.DTLVardef@d2fd6b_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Reverse DTNB assay: a novel in vitrobiochemical approach to detect oxidized thiol modifications

The participation of sulfhydryl or thiol functions in a multitude of protein posttranslational modifications, although reflects on the redox versatility of cysteine residues, but their assessment in a dynamic cellular milieu involving the facile inter-conversion of SH to SSG, S-S, SNO, and S-R has been overwhelmingly difficult despite theirimplications in protein folding, enzyme structure and function, signalling and detoxification pathways, and pathophysiological ramifications.The current methodology, in contrast to a wide variety of cumbersome and prolonged techniques,repurposes the conventional DTNB assay for a hassle-free qualitative and quantitative analysisof redox-modified single or multiple susceptible thiol residues of cysteines in pure proteins as well as in a complex mixture of proteins.In this study, we document the thiol content, bearing the susceptibility to undergo reversible, oxidative thiol modifications, utilizing reverse DTNB assay in cell-free lysates and purified proteins that might provide a possible framework for dissecting the physiological phenomena behind the concealment of the susceptible cysteines through their redox-modified forms.

biochemistry↗