Biology subjects

Das, S.

Publications and source records attributed to Das, S..

At least 73 records · Page 4Linked to original sources

bioRxiv2026

DNA Damage Driven Viability Loss and Transcriptional Reprogramming in Chinese Hamster Ovary Cell Perfusion Culture

Intensified perfusion cultures promise higher yields and consistent product quality, yet extended runs frequently stall due to declining Chinese hamster ovary (CHO) cell viability at high cell density. Here, we identify DNA damage accumulation as a central, previously underappreciated driver of this limitation. Using two antibody-expressing CHO cell lines operated at a control and high cell density in perfusion bioreactors, we combined time-resolved transcriptomics with molecular, biophysical and super-resolution imaging analyses. We observed a progressive, global downregulation of DNA damage response (DDR) pathways accompanied by a time-dependent accumulation of DNA lesions. Notably, {gamma}H2AX signalling declined despite DNA damage, indicating impaired damage sensing and repair. Concomitantly, RNA polymerase II protein levels and transcriptional hub organisation were markedly reduced, consistent with widespread transcriptional dysfunction preceding loss of viability. An extended 21-day perfusion run confirmed continued viability decline beyond day 14, supporting a cumulative damage model. Comparison with HEK293 cells revealed substantially more efficient DNA repair, underscoring an intrinsically attenuated DDR in CHO cells. Together, these findings demonstrate that unrepaired DNA damage fundamentally constrains perfusion culture longevity, exposing limits of CHO genomic plasticity and highlighting DDR pathways as promising targets for host cell engineering to enhance perfusion performance.

bioengineering↗

bioRxiv2026

A structure-based epitope tagging approach identifies vulnerable sites on the malarial P36-P52 protein complex for antibody-mediated neutralization of Plasmodium sporozoites

Malaria is caused by apicomplexan parasites of the genus Plasmodium, which are transmitted through the bite of Anopheles mosquitoes that inject sporozoites (SPZs) into the skin. SPZs migrate to and infect the liver for an initial round of replication. SPZs and liver stages have long been considered as ideal targets for malaria vaccines. The main SPZ surface protein, the circumsporozoite protein (CSP), is the target of currently approved malaria vaccines and prophylactic antibody therapies. Studies in rodent malaria models have shown that anti-CSP antibodies exert their protective effect mainly in the skin, but some of the most potent anti-CSP monoclonal antibodies show additional protective effects in the vasculature and liver. Other SPZ proteins involved at different steps of the infection process may thus represent additional targets for antibody-mediated neutralization. Three 6-cysteine (6-Cys) domain proteins (P36, P52 and B9) play an essential role during SPZ invasion of hepatocytes, yet their molecular function and whether they can be targeted by neutralizing antibodies remains unknown. Here, to fill this gap, we combined an integrative structural biology approach with functional experiments in the P. berghei rodent malaria model. AlphaFold-based structural modeling followed by experimental validation via electron microscopy and small-angle X-ray scattering indicated that the P36-P52 heterodimer displays a head-to-tail architecture with an interaction interface that is largely conserved among Plasmodium species. The structural models supported the rational design of an epitope tagging approach, which, combined with neutralizing assays, revealed that antibodies against P36 and P52 can efficiently block invasion of hepatocytes by SPZs in culture conditions. The data show that the inhibitory activity of antibodies heavily depends on epitope position and revealed that antibody-exposed vulnerable sites lie on the membrane-distal side of the P36-P52 heterodimer. In contrast, antibodies targeting B9 had no inhibitory effect on SPZ invasion, irrespective of epitope positioning. These data show that the invasion step could be targeted by antibodies and indicate that the P36-P52 complex may be considered as a potential target for the development of next generation pre-erythrocytic malaria vaccines or therapeutic antibodies.

microbiology↗

bioRxiv2026

ACTIVATED CASO₄-INDUCED VACUOLATION AS A QUANTITATIVE PLATFORM FOR PHAGOCYTOSIS-DRIVEN DRUG SCREENING

Cytoplasmic vacuolization is a fundamental process associated with phagocytosis, lysosomal acidification, and autophagy, yet robust in-vitro models for its quantification and pharmacological screening remainlimitedor insufficiently established. In this study, we demonstrate that thermally activated calcium sulfate (ACS) induces extensive vacuolation across mammalian cell lines including HeLa, RAW 264.7, 3T3-L1, and SH-SY5Y, thereby establishing a versatile platform to study vacuole biogenesis. To ensure reproducibility, particle heterogeneity was addressed using sedimentation-based fractionation, with homogeneous suspensions obtained at the 5th minute producing stable and consistent vacuole formation. Vacuolation was subsequently quantified by Neutral Red (NR) uptake, dose and time dependent response analyses confirmed direct correlation between ACS concentration and vacuole induction. The assay was validated with bafilomycin A1 (BFA1), a selective V-ATPase inhibitor, which served as a positive control and demonstrated concentration and time dependent inhibition of vacuole formation and acidification. Building on this framework, ten commercially available drugs were screened, revealing distinct profiles ranging from early cytotoxicity, strong vacuole inhibition to partial suppression or negligible effects. This dual capacity to discriminate between vacuole inhibition and cytotoxic responses highlights the utility of ACS-induced vacuolization as a sensitive and scalable in vitro platform. Collectively, our findings position this system as a tractable assay for mechanistic studies of vacuole biology and a functional screening tool for identifying modulators of lysosomal and phagocytic pathways relevant to infection, Lysosomal Disorders, and Phagocytotic dysfunction disorders. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/711143v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@e5b920org.highwire.dtl.DTLVardef@1dd1072org.highwire.dtl.DTLVardef@62ae59org.highwire.dtl.DTLVardef@a468bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

Marine Aspergillus terreus produces a chitinase exhibiting a dual mode of enzymatic action

Marine Aspergillus terreus has been explored as an important chitinase-producing fungal strain for-N-Acetyl-D-Glucosamine (GlcNAc) production from chitin substrates. Here, a purified extracellular 45 kDa chitinase of marine Aspergillus terreus (accession number JQ248076) was characterized in terms of substrate specificity. Conventionally, endochitinase cleaves the chitin substrate randomly to produce GlcNAc and its different multimers. So, it requires at least tetramer to characterize the endochitinases; whereas, exochitinases cleaves the chitin substrate from its reducing end and produce either GlcNAc or chitobiose (GlcNAc dimer). In present chitinase characterization, the HPLC followed by HRMS analyses revealed differential product formation from the chitin substrates of varied chain length. With swollen chitin polymer, the enzyme produced GlcNAc as a sole product; whereas with chitohexaose substrate, a mixture of GlcNAc and its oligomers were obtained. Although, mass spectrometry-based proteomics analysis identified the isolated chitinase as an endochitinase 1 precursor (Accession XP_001217186). However, the enzyme kinetic study exhibited higher catalytic efficiency for exochitinase specific dimeric chromogenic substrate in comparison to endochitinase specific tetrameric fluorogenic substrate, which indicated predominantly exochitinase behavior of the enzyme. Further, the in-silico study predicted the differential cleavage pattern of the enzyme, which could be due to different mode of substrate binding and processive mechanism through the tunnel shaped binding cleft of the enzyme. The dual mode of catalytic activity of the present chitinase was further confirmed by a molecular docking study with different lengths of substrates. With the unique dual mode of action, the chitinase of marine Aspergillus terreus offers a great promise towards its utility in the production of GlcNAc.

microbiology↗

bioRxiv2026

Optimizing an avian influenza vaccine using a novel Bacterial Enzymatic Combinatorial Chemistry (BECC) TLR4 adjuvant

The development of broadly protective and dose-sparing influenza vaccines remains a critical challenge, particularly for zoonotic H5N1 strains with pandemic potential. This study evaluates BECC470s, a synthetic TLR4 adjuvant, for its ability to enhance the immunogenicity and protective efficacy of recombinant H5 hemagglutinin (rHA) vaccination in murine models. BECC470s-adjuvanted rHA elicited robust IgG1/IgG2a antibody responses and complete survival following homologous 2004 H5N1 challenge in a prime-boost model. Although BECC470s broadened antibody binding to both variable HA head and conserved stalk domains by ELISA, functional neutralizing antibody responses were restricted to the matched 2004 H5N1 isolate, with no detectable neutralization of H5N1 viruses isolated in 2022 or 2024. These data indicate that BECC470s enhances the magnitude and apparent breadth of binding antibody responses while maintaining strain-specific neutralizing activity, supporting its potential as an adjuvant for next-generation influenza vaccines while underscoring the need for further optimization to achieve true cross-neutralizing protection.

immunology↗

bioRxiv2026

Formate reduces ischemic injury in the male heart by increasing protein S-nitrosation

Ischemic heart disease is a leading cause of death for both men and women in the United States. We and others have demonstrated that nitric oxide (NO) signaling and associated protein S-nitrosation (SNO) play a key role in reducing ischemic injury in the heart. We also find that while females typically exhibit endogenous protection from ischemic injury, this protection is abrogated with the loss of the formate-generating enzyme alcohol dehydrogenase 5 (ADH5), but formate supplementation provided a rescue. Here, we investigate the cardioprotective efficacy of formate in male hearts. Hearts were Langendorff-perfused and subjected to ischemia/reperfusion (I/R) injury with and without formate. Formate-mediated protection was also examined using an in vitro model of coverslip-induced ischemic injury to identify molecular underpinnings. We found that formate yields protection from I/R injury in ex vivo and in vitro models by increasing post-ischemic protein SNO levels, while NO synthase inhibition blocked this formate-mediated increase in protein SNO in vitro, and attenuated protection from I/R injury ex vivo. Moreover, post-ischemic levels of tetrahydrobiopterin (BH4), a cofactor necessary for NOS function, were preserved in formate-treated hearts. Furthermore, inhibition of dihydrofolate reductase (DHFR), a one-carbon enzyme critical for BH4 recycling, blunted formate-mediated protection ex vivo. Collectively, our findings suggest that formate is a potent cardioprotective agent that confers protection by preserving post-ischemic BH4 levels, and enhancing protein SNO levels through a NOS-dependent mechanism. These findings have significant implications for the clinical prevention and treatment of ischemic heart disease in males.

physiology↗

bioRxiv2026

Dimerization of MilM is essential for catalyzing the pyridoxal-5'-phosphate (PLP)-dependent Cγ-hydroxylation of L-arginine during mildiomycin biosynthesis

MilM from the mildiomycin biosynthetic pathway is a PLP-dependent enzyme, previously annotated as an aminotransferase, but has recently been demonstrated as L-arginine oxidase cum C-hydroxylase. Here, we report detailed biochemical, biophysical, structural modeling, and molecular dynamics simulation-based investigations of MilM from Streptoverticillium rimofaciens B-98891 to elucidate the mechanisms of substrate binding, catalysis, and the role of the active site residues involved in these processes. Our experimental findings confirmed that MilM functions as a stable homodimer, requiring the PLP cofactor and molecular oxygen to transform the L-arginine substrate into 5-guanidino-4-hydroxy-2-oxovaleric acid and 5-guanidino-2-oxovaleric acid through the intermediacy of a possible superoxide radical anion species, while generating H2O2 and NH3 as reaction by-products. Our labeling studies also established that the hydroxyl group in the product is obtained from the solvent, water. The structure-based three-dimensional modeling and simulation of MilM coupled with site-directed mutagenesis further confirmed that, in addition to the catalytic residues Lys232 and His31, active site residues from both the protomers are crucial for stabilization of the PLP cofactor (Ser92, Phe116, Asn164, Asp195, Lys240 from chain A and Tyr89 from chain B) and the substrate (Thr14, Glu17, Asn118, and Arg364 from chain A and Thr259, Ser260 from chain B). Moreover, the molecular dynamics simulation uncovered a dimer-mediated alternating lid mechanism in which large-scale, concerted motions of the dimer interface helices reciprocally expose and occlude the two active sites. This see-saw-like dynamics controls the substrate entry and product release through transiently formed tunnels, while preserving a catalytically protective environment, a critical phenomenon previously left unnoticed in similar PLP-dependent oxidases/hydroxylases. Overall, these findings provide new insights into the substrate/cofactor stabilization and the catalytic mechanism of MilM, a recent member of an emerging family of remarkable PLP-dependent oxidases and help us decode a key puzzle in the mildiomycin biosynthetic pathway.

biochemistry↗

bioRxiv2026

Cell-autonomous Wnt activity promotes transient re-programming and cell cycle re-entry of coronary artery endothelial cells

Coronary occlusion leads to formation of collateral arteries connecting occluded and perfused arteries. This process, namely artery reassembly, is temporally restricted to the cardiac regenerative window in mice and relies on cell cycle re-entry of post-mitotic artery endothelial cells (ECs). Analyses of single cell RNA sequencing data of ischemic mouse hearts led to identification of Wnt activity in proliferating artery cells. Using mouse genetics and whole organ imaging at cellular resolution, we show that Wntless (Wls)-mediated Wnt secretion and Frizzled (Fzd)4-dependent Wnt reception by artery cells, was essential for development of coronary collaterals. Specifically, Fzd4 promoted proliferation and expression of (progenitor-like) capillary/venous markers in coronary artery ECs. In embryonic hearts, coronary arteries originate from capillaries. Fzd4 in these capillaries was essential for proliferation; depletion of which, accelerated their differentiation into arteries by arterialization. Along these lines, an exogeneous dose of human WNT2-sFRP1 ligand complex reactivated artery cell proliferation and restored artery reassembly in non-regenerative mouse hearts. Thus, ischemia-induced cell-autonomous Wnt signaling promoted developmental reprogramming of coronary artery ECs. Finally, exome sequencing of coronary artery disease patients, revealed unique pathogenic variants of WLS and FZD4. Together, our study provides mechanistic insights into EC plasticity and underscores the significance of WNT pathway in cardiovascular disease.

developmental biology↗

bioRxiv2026

Co-infection with Leptomonas seymouri enhances macrophage survival and promotes intracellular parasite persistence during Leishmania donovani infection

BackgroundLeishmania donovani (LD) is an obligate intracellular parasite that survives and replicates within macrophages. Leptomonas seymouri (LS), a traditionally monoxenous trypanosomatid, has been repeatedly co-isolated with LD from kala-azar cases in India, often together with Leptomonas seymouri narna-like virus 1 (Lepsey NLV1). Whether LS can survive and replicate within mammalian macrophages, and how co-infection influences parasite and virus dynamics, remain unresolved. Methods and FindingsIntracellular survival, replication, and revival of LS alone and during co-infection with LD were systematically evaluated using murine (RAW 264.7) and human (THP-1) macrophages. Quantitative ITS1 PCR demonstrated significant increases in intracellular parasite DNA over 48-168 hours post-infection (h.p.i), indicating active replication rather than persistence. Reduced extracellular count suggested restricted cell lysis and enhanced macrophage survival in co-infection compared to mono-infections. Giemsa staining confirmed intracellular localization of LS. Amastigotes from infected macrophages revived as motile promastigotes upon transformation, whereas extracellular parasites failed to revive beyond 48 hours, confirming macrophages as the exclusive niche for prolonged viability. Co-infection dampened macrophage IL-12 (p40) production, augmenting macrophage survival. MTT assay confirmed the same. In THP-1, co-infection resulted in a marked increase in intracellular LS count and viral load than LS mono-infection at 96 and 168 h.p.i. relative to 48 h.p.i., suggesting that LD co-infection favors higher intracellular virus titres. ConclusionsOur findings demonstrated that LS could replicate within mammalian macrophages and persist during mono-or LD co-infection. The identification of a stable LD-LS-virus interaction highlights a previously underappreciated "triple-pathogen" biology with potential implications for LD pathogenesis.

microbiology↗

bioRxiv2026

Differential effects of two common GVHD prophylaxis regimens on the gut microbiome: Results from the BMT CTN 1801 study

Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative treatment for many hematological malignancies, but graft-versus-host disease (GVHD) is a common complication. Low gut microbiome diversity is associated with higher GVHD risk and shorter survival in multiple studies. Recently, the BMT CTN 1703 clinical trial demonstrated superiority of a GVHD-prophylaxis regimen including post-transplant cyclophosphamide (PTCy) compared to the standard prophylaxis (tacrolimus and methotrexate, Tac/MTX) in terms of GVHD-free, relapse-free survival at one year among reduced intensity conditioning allo-HCT recipients. However, the effect of PTCy on the gut microbiome and its association with clinical outcome have not been described. Here, we report on a companion randomized clinical controlled trial (BMT CTN 1801), which collected 2575 longitudinal stool samples from 304 study participants. Samples were obtained up to weekly up to day 84 post allo-HCT and at less frequent intervals thereafter, up to 2 years. Microbiome diversity and absolute microbial load were lower in the PTCy group compared to the Tac/MTX group on days 14-28 post-HCT. However, diversity at the timepoint closest to neutrophil engraftment was not significantly associated with non-relapse mortality after one year or other clinical outcomes, contrary to expectations from previous studies. Microbial domination events, when a single species exceeds 30% relative abundance, were comparable across treatment arms and reflected both pathogen blooms as well as less severe disruptions of the microbial community. Clostridium scindens and secondary bile acid metabolism pathways were less prevalent in the PTCy arm than in the Tac/MTX arm post-HCT, yet presence of secondary bile acid metabolism pathways was associated with a lower risk of chronic GVHD. Given that PTCy was associated with a greater disruption of the microbiome as measured by diversity, absolute microbial abundance, and bile acid metabolism capability, but better clinical outcomes overall, these data suggest that the importance of the microbiome in modulating the host immune systems after allo-HCT is specific to different types of GVHD prophylaxis.

microbiology↗

bioRxiv2026

Smooth Muscle Dysfunction Drives Cerebrovascular Reserve Failure and End-Organ Brain Injury

BackgroundFailure of cerebrovascular reserve is a fundamental determinant of ischemic vulnerability, yet the mechanisms by which vascular smooth muscle dysfunction compromises reserve and predisposes the brain to injury remain incompletely defined. We therefore tested whether a pathogenic smooth muscle mutation produces a baseline failure of cerebrovascular reserve sufficient to render the brain vulnerable to hypoperfusion, even in the absence of fixed arterial occlusion. MethodsWe examined cerebrovascular structure, hemodynamics, and reserve in a genetically defined mouse model of ACTA2-associated multisystemic smooth muscle dysfunction syndrome with systemic or brain-restricted expression of the mutant allele. Cerebral artery morphology was assessed using magnetic resonance angiography and black ink angiography. Vascular smooth muscle phenotype was evaluated by immunohistochemistry and proliferation assays. Blood pressure reactivity and cerebral blood flow (CBF) were measured simultaneously using femoral arterial catheterization and laser speckle flowmetry during vasoactive challenges and controlled hypotension. Cerebrovascular stress responses were tested using unilateral common carotid artery occlusion. Downstream brain effects were assessed by histology, resting state functional connectivity imaging, and behavioral testing. ResultsImpaired smooth muscle contractility drove rectification and narrowing of major cerebral arteries, downregulation of contractile markers, and increased vascular cell proliferation. These structural changes produced a distinct physiological phenotype: mutant mice exhibited blunted vasoreactivity, diminished spontaneous vasodynamic activity, and a downward shift in the blood pressure-CBF relationship across a wide range of arterial pressures, consistent with loss of cerebrovascular reserve. As a result, CBF was reduced at baseline and could not be maintained during hypotension or acute vascular stress. During carotid occlusion, mutant mice showed impaired compensatory perfusion, greater physiological instability, and worse behavioral outcomes. Chronic reserve failure coincided with white matter loss, reduced neuronal density, disrupted large-scale functional connectivity, and deficits in locomotion, anxiety-related behavior, and working memory. ConclusionsPathogenic smooth muscle dysfunction caused by ACTA2 mutation produces a baseline failure of cerebrovascular reserve that renders the brain vulnerable to hypoperfusion and stress-induced ischemic injury. These findings establish cerebrovascular reserve failure as a central physiological mechanism linking vascular dysfunction to end-organ brain injury and identify reserve preservation as a critical, potentially actionable determinant of brain health in hypotension-prone vascular disease. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABS- ACTA2 smooth muscle dysfunction produces baseline cerebrovascular reserve impairment, with reduced cerebral blood flow and a downward-shifted pressure-flow relationship in the absence of critical large-vessel occlusion. - Vascular tone dysregulation is coupled to end-organ brain injury, including white matter and neuronal loss, disrupted functional connectivity, and behavioral deficits. - The results support complementary disease mechanisms in ACTA2 vasculopathy: baseline reserve limitation and injury-provoked occlusive remodeling. Clinical Implications- Patients with ACTA2 vasculopathy may be vulnerable to ischemic brain injury during hypotension or systemic stress despite the absence of critical stenosis or occlusion on routine imaging. - Peri-procedural and acute-care management should emphasize preserving perfusion pressure and cerebrovascular reserve (e.g., during anesthesia, dehydration, or systemic illness). - More broadly, cerebrovascular reserve is a clinically relevant, potentially modifiable determinant of brain health in hypotension-prone vasculopathies and conditions characterized by impaired vascular reactivity.

physiology↗

bioRxiv2026

Metabolic reallocation in spinal cord oligodendrocytes drives chronic pain via neuronal β-amyloid production

Peripheral injury reprograms metabolism in spinal cord oligodendrocytes, initiating a molecular cascade that drives chronic pain via neuronal {beta}-amyloid (A{beta}) release. After injury, mouse spinal oligodendrocytes downregulate myelin protein synthesis and upregulate lipid biosynthesis--but reroute lipids toward neuroplastic remodeling and away from myelin maintenance. This metabolic reallocation disrupts myelin integrity and axonal function, causing neuronal accumulation of amyloid precursor protein, enhanced expression of its processing {beta}-secretase BACE1, and local release of A{beta} peptides. Blocking A{beta} production or clearing A{beta} deposits stops the transition to pain chronicity. Deleting the lysosomal lipid hydrolase NAAA in oligodendrocytes prevents both injury-induced A{beta} production and chronic pain development. The findings identify an unexpected mechanistic link between chronic pain and Alzheimers-like neurodegeneration, positioning A{beta} as a target for therapeutic intervention.

neuroscience↗

bioRxiv2026

ISG15 orchestrates dynamic crosstalk between mitochondrial fat oxidation and type 1 interferon in myeloid cells

In contrast to Krebs cycle intermediates, the role of mitochondrial fatty acid oxidation (FAO) in immunometabolism remains incompletely characterized. Studying primary bone marrow-derived macrophages (BMDMs), we show that IFN-{beta} and STING activation augments FAO and associated enzymes carnitine palmitoyltransferase 1a (CPT1a) and acetyl-CoA acetyltransferase 1 (ACAT1). Depleting BMDM Cpt1a reduces FAO and dampens type 1 interferon (IFN) signaling due to decreased histone H3-K9/K14 acetylation, supporting the prior finding of an epigenetic role of FAO in sustaining type 1 IFN. Interestingly, FAO induction by IFN was dynamic as a heightened IFN response suppressed FAO, suggesting a concurrent negative-feedback mechanism. This FAO blunting correlated with increased expression of interferon-stimulated gene 15 (ISG15), a ubiquitin-like modifier known to modulate metabolic proteins through ISGylation. This immune-metabolic signature was similarly operational in mice infected with lymphocytic choriomenigitis virus (LCMV) with temporal discordance between ISG15 levels and FAO. The role of ISG15 in this negative feedback was shown with increased FAO and type 1 IFN response in Isg15 knockout BMDMs. Parallely, endogenous co-immunoprecipitation showed interactions between ISG15 and CPT1a/ACAT1. This ISG15-FAO regulatory interaction was also evident in systemic lupus erythematosus (SLE)-associated interferonopathy and in primary monocytes from SLE individuals that exhibited increased ISG15 levels and reduced FAO rates. Collectively, these findings support a model in which type 1 IFN initially enhances FAO to amplify interferon production, but excessive IFN-signaling induces ISG15-mediated inhibition of FAO, a putative feedback loop that restrains inflammation and preserves immune homeostasis. Together these data identify a novel biphasic FAO-dependent immunometabolic regulatory program. Graphical Summary O_FIG O_LINKSMALLFIG WIDTH=190 HEIGHT=200 SRC="FIGDIR/small/700051v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@13f572eorg.highwire.dtl.DTLVardef@174b6aeorg.highwire.dtl.DTLVardef@1856483org.highwire.dtl.DTLVardef@89e5a6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIType 1 IFN rewires myeloid cell metabolism towards FAO. C_LIO_LIFAO epigenetically fuels Type 1 1IFN response. C_LIO_LIEffect of IFN on FAO is dynamic: inducing FAO at low concentrations while suppressing it during a heightened IFN response. C_LIO_LIEnhanced Type 1 IFN response produces ISG15 which ISGylates key enzymes of FAO altering their stability C_LIO_LIISG15 negatively regulates FAO to control Type 1 IFN homeostasis in a negative feedback loop. C_LI

immunology↗

bioRxiv2026

Illuminating spatial dynamics of glutamine metabolism with a sensitive genetically encoded biosensor

Glutamine is the most abundant amino acid in serum, used as a key nutrient by cells for protein synthesis, energy production, carbon and nitrogen metabolism, and cellular redox balance. The use of glutamine in the cell is highly compartmentalized, but the dynamics of glutamine metabolism across organelles and individual cells are not fully understood. To illuminate subcellular glutamine dynamics, we developed a green fluorescent protein-based intracellular glutamine optical reporter, iGlo. We find iGlo is sensitive and specific for glutamine and can be used to measure glutamine uptake, production, and consumption with high spatiotemporal resolution in multiple cell types. Furthermore, multiplexed imaging of iGlo with a lactate biosensor in single cells reveals the temporal crosstalk between glucose and glutamine metabolism to maintain energy homeostasis. Thus, iGlo enables the sensitive and precise study of compartmentalized glutamine dynamics and represents a new and enhanced tool for studying the spatiotemporal dynamics and regulation of metabolism.

biochemistry↗

bioRxiv2026

Interplay of microRNA-20a and HuR in the regulation of beclin1 during Withaferin-A mediated impaired autophagy in Breast Cancer cell-line, MCF-7

Dysfunctional autophagy is connected to multiple diseases. Withaferin-A, a biologically active withanolide, has been shown to impair autophagy in the breast cancer cell line, MCF-7; however, the underlying mechanism remains unclear. Here while uncovering the mechanism, it was found that treatment of MCF-7 cells with WA declines Beclin1 protein synthesis by restricting association of its mRNA to polysomes with no significant reduction in its mRNA level. Reporter assay established that WA-treatment enhanced the expression level of mature miR-20a which directly interacts with the 3UTR of beclin1 mRNA. RNA affinity chromatography revealed association of mRNA stabilizing protein, HuR with the 3UTR of beclin1 mRNA. Additionally, co-immunoprecipitation assay determined the interactions of GW182, an essential component of GW-body and Ago2, a crucial member of miRNA related silencing complex (RISC) with the 3UTR of beclin1 mRNA. In parallel, it was found that knocking down of HuR eliminates the interaction of GW182 with beclin1 3UTR, while the association of miRNA (Ago2) remains unaffected. These results suggested simultaneous association of HuR, GW182 and microRNA with beclin1 mRNA which could be pivotal for sequestration of beclin1 mRNA in GW-bodies. Thus, our findings culminated in identifying an unconventional mechanism of regulation of Beclin1 expression by the dual interplay between hsa-miR-20a and HuR during WA-induced impaired autophagy in MCF-7 cells.

molecular biology↗

bioRxiv2026

Quantitative Deciphering of Mammalian Histamine Receptors through Mathematical Genomics

Histamine receptors (HRH1-HRH4) are G-protein coupled receptors (GPCRs) that mediate essential physiological functions, including neurotransmission, gastric acid secretion, immune regulation, and presynaptic autoregulation. Despite their importance, systematic comparative analyses across mammalian histamine receptor sequences remain scarce. In this study, we performed a comprehensive evaluation of HRH1-HRH4 across multiple mammalian species, integrating sequence homology, invariant residue mapping, amino acid substitutions, compositional frequencies, Shannon entropy, polystring distributions, intrinsic disorder profiling, and phylogenetic clustering. HRH2 and HRH1 were highly conserved among primates, HRH3 showed strong cohesion within rodents, while HRH4 exhibited pronounced divergence consistent with immune-related specialization. Invariant residues localized to transmembrane helices and activation motifs (D107, W428/Y431, NPxxY), underscoring strict evolutionary constraints on ligand binding, receptor stability, and G-protein coupling. Substitutions were confined to non-essential lipid-facing and loop regions, predominantly conservative in nature, enabling diversification without disrupting the GPCR fold. Amino acid frequency and entropy analyses revealed hydrophobic dominance with subtype-specific enrichment of polar residues, while disorder profiling identified HRH1 as the most dynamic and HRH2 as the most structurally constrained. Polystring analysis highlighted conserved motifs (WWW, PP) alongside subtype- and species-specific repeats, reflecting evolutionary strategies balancing receptor stability with adaptive flexibility. Phylogenetic clustering confirmed subtype-specific cohesion, with HRH3 and HRH4 forming compact clades, HRH1 showing moderate dispersion, and HRH2 forming the most isolated cluster. Collectively, these findings demonstrate that mammalian histamine receptor evolution is governed by conserved biophysical cores and selective variability, offering insights into structural conservation, functional diversification, and translational relevance for drug design and model selection.

bioinformatics↗

bioRxiv2026

Distinct TAF15 amyloid filament folds define multiple subtypes of FTLD-TAF15

Neurodegenerative diseases are characterised by the assembly of a limited number of disease-specific proteins into amyloid filaments, which form intracellular inclusions or extracellular deposits in the central nervous system (CNS)1,2. We previously found that amyloid filaments of TATA-binding protein-associated factor 15 (TAF15) characterise a subtype of frontotemporal lobar degeneration with FET protein-immunoreactive inclusions (FTLD-FET)3, termed atypical FTLD with ubiquitin-positive inclusions (aFTLD-U)4, which causes early-onset, rapidly progressive behavioural variant frontotemporal dementia (FTD). However, it was not clear if TAF15 proteinopathy was more widespread in neurodegenerative diseases. Two additional FTLD-FET subtypes have been proposed, neuronal intermediate filament inclusion body disease (NIFID) and basophilic inclusion body disease (BIBD)5,6, which have more heterogenous clinical presentations including FTD, motor neuron diseases (MND) and movement disorders. Here, we used electron cryo-microscopy (cryo-EM) to determine a total of 32 amyloid filament structures from the brains of 17 individuals encompassing all three proposed subtypes of FTLD-FET and their diverse clinical presentations. All cases were characterised by TAF15 filaments, in the absence of filaments of the other FET proteins, fused in sarcoma (FUS) and Ewings sarcoma (EWS). All three aFTLD-U cases had the previously-reported TAF15 fold3. Unexpectedly, we found four distinct TAF15 folds among 11 NIFID cases. Eight of these cases shared a common fold, while the remaining three were each distinct. Furthermore, we found distinct TAF15 folds for each of the three BIBD cases. Neuropathological reassessment of the neocortical TAF15 inclusion pathology of these cases distinguished the NIFID cases with the common fold from the others. Thus, TAF15 filament structures form the basis of a new, expanded classification of FTLD-FET subtypes. Moreover, we discovered a TAF15 Y38C variant in the filament fold of one of the individuals with BIBD. The structure is unable to incorporate wild-type TAF15, despite the individual being heterozygous, suggesting that this variant drives TAF15 filament assembly. This study provides structural and genetic evidence that TAF15 amyloid filaments underlie the diverse group of neurodegenerative diseases currently termed FTLD-FET, which we therefore rename FTLD-TAF15.

molecular biology↗

bioRxiv2026

Dual Regulatory Role of Nuclear FNBP4 in Actin Binding and Formin FMN1 Inhibition

Nuclear actin dynamics are increasingly recognized as central to genome regulation, yet the identity and function of dedicated nuclear actin regulators remain elusive. Here, we uncover formin-binding protein 4 (FNBP4) as a previously uncharacterized nuclear actin-binding protein that integrates dual regulatory functions. We identify a conserved basic KRRK motif within its C-terminal that directly interacts actin and coincides with a non-canonical nuclear localization signal. In parallel, FNBP4 interacts with the formin FMN1 via its N-terminal WW domains, potently suppressing FMN1-mediated actin nucleation. Strikingly, full-length FNBP4, which unites both the FMN1- and actin-binding modules, displays synergistic inhibition far exceeding that of truncated fragments. Furthermore, confocal imaging confirms nuclear colocalization of FNBP4 with actin, highlighting its role in shaping nuclear actin architecture. Our findings position FNBP4 as the first example of a nuclear-resident formin inhibitor that simultaneously binds actin and a formin. This dual functionality establishes a new regulatory paradigm in which a single protein integrates direct actin interaction with formin inhibition to fine-tune nuclear actin organization.

biochemistry↗