Biology subjects

Mukherjee, P.

Publications and source records attributed to Mukherjee, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Disentangling the Crabtree and Warburg effects across syngeneic non-tumor and tumor cells.

Oxidative phosphorylation (OxPhos) and substrate-level phosphorylation (SLP) are the primary biochemical mechanisms for ATP generation in mammalian cells. Increased dependency on SLP can arise from either transient (e.g., Crabtree effect) or chronic (e.g., Warburg effect) suppression of OxPhos. We evaluated the steady-state bioenergetics of quiescent and proliferating mouse cells matched in genetic background and cell biology, including primary thioglycollate-elicited peritoneal macrophages, malignant VM-M3 glioma with macrophage characteristics, and malignant CT-2A astrocytoma. Data are presented showing: 1) OxPhos insufficiency was the primary bioenergetic hallmark of proliferating malignant cells. 2) Dysregulated proliferation was associated with lower metabolic flexibility under nutrient stress than was growth arrest or regulated proliferation. 3) The Crabtree effect was present in macrophages and involved a glucose- and time-dependent suppression of OxPhos coupled to ATP production through SLP and resistance to mitochondrial inhibitors; lowering glucose concentration or substituting glucose with respiratory fuels increased OxPhos and restored sensitivity to mitochondrial inhibitors. 4) The Warburg effect in tumor cells involved a persistent SLP dependency and resistance to mitochondrial inhibitors under equivalent glucose-replete conditions. 5) OxPhos could sustain the regulated proliferation of non-tumor cells in the absence of a Warburg effect. Collectively, our findings emphasize that comparisons between normal and cancer cell metabolism require long-term experimental endpoints that ensure steady-state ATP sufficiency.

cancer 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

eIF3 interactions with miRNAs involved in translational control of early T cell activation

Early T cell activation induces extensive remodeling of the cellular transcriptome and proteome. We previously showed using a transcriptome-wide crosslinking approach that human translation initiation factor eIF3 directly interacts with a select set of immune-related mRNAs shortly after T cell activation. We also found that eIF3 binding to the 3'-untranslated regions (3'-UTRs) of the TCRA and TCRB mRNAs encoding the T cell receptor alpha and beta subunits dynamically regulates a burst in their translation. MicroRNAs (miRNAs) add an additional layer of regulation by fine-tuning both mRNA and protein expression. Although miRNA expression is dynamically regulated during T cell activation, how miRNAs interact with core regulatory pathways in primary T cells remains poorly understood. Here, we reexamined the eIF3-RNA crosslinking experiments in Jurkat cells and probed eIF3 function to investigate miRNA-mediated translational regulation during T cell activation. We found that eIF3 interacts with multiple mature miRNAs in activated Jurkat cells, including members of the miR-17[~]92 cluster. These interactions also occur in primary T cells, as shown by RNA immunoprecipitation followed by qPCR (RIP-qPCR). Knocking out the miR-17[~]92 cluster led to a delay in ILR2A (CD25) cell surface expression in Jurkat cells and reduced activation-associated cell size increase in primary T cells during early T cell activation. In parallel, we used mass spectrometry to identify eIF3-interacting factors in activated primary T cells, and surprisingly found no evidence of Argonaute binding. Together, these findings provide evidence that eIF3-miRNA interactions may play an unappreciated role in translational control during early T cell activation.

Molecular Biology↗

bioRxiv2026

Innate immune sensing of cell traversal by Plasmodium sporozoites drives protective T cell responses

Attenuated Plasmodium sporozoites elicit robust adaptive immune responses that protect against parasite challenge, yet how they engage the innate immune system remains poorly understood. To obtain sterile preparation of sporozoites we used flow cytometry to isolate pure GFP+ P. berghei and P. falciparum sporozoites, which were sorted onto murine bone marrow-derived macrophages (BMDMs) or human PBMCs to assess innate immune activation. Sporozoites induced a distinctive activation pattern resembling cell wounding responses, consistent with their ability to traverse host cells en route to the liver. Traversal-deficient sporozoites failed to activate BMDMs and showed impaired priming of protective CD8+ T cell immunity. This wounding response was independent of inflammasome or Toll-like receptor signaling but required {gamma}{delta} T cells, which are known to support CD8+ T cell responses against Plasmodium. These findings reveal a previously unappreciated innate sensing mechanism triggered by cell traversal that underpins the potent immunogenicity of Plasmodium sporozoites.

immunology↗

bioRxiv2026

Rejuvenation of the Aged Cerebrovascular System via Protein Corona-Guided Fusogenic Liposome Delivery

Brain vascular aging is increasingly recognized as a critical therapeutic target for age-related cognitive decline. Oxidative stress, bioenergetic dysfunction, and molecular damage play central roles in the progression of vascular aging, contributing to cerebrovascular dysfunction and impaired cognitive function. While naturally occurring polyphenols such as resveratrol (RSV) have demonstrated potential in mitigating aging-related pathologies, their poor bioavailability and limited brain targeting efficiency significantly constrain their therapeutic impact. As a result, high doses or advanced drug delivery strategies are necessary to achieve meaningful physiological effects. We introduce a novel nanocarrier system designed to enhance RSV delivery to the cerebral endothelium by leveraging the natural formation of an apolipoprotein E (ApoE)-enriched protein corona around fusogenic liposomes (FL) in vivo. These nanoparticles directly fuse with cytoplasmic cell membranes and thus evade endocytosis. We found that once in the circulation FL spontaneously acquire a protein corona, which is highly enriched in ApoE, a key ligand for brain endothelial low-density lipoprotein receptors (LDLR). Based on this observation, we engineered an ApoE-functionalized protein corona around FL (ApoE-FL) to systematically evaluate whether this mechanism could be exploited for targeted brain delivery. Following optimization and physicochemical characterization, the RSV-loaded liposomes were evaluated in vitro using human cerebral microvascular endothelial cells and in vivo C57BL/6 aged mice to assess their therapeutic potential. Both FL and engineered ApoE-FL liposomal delivery systems exhibited a strong affinity for endothelial cell membranes in vitro. The knockdown of the ApoE receptor, low-density lipoprotein receptor-related protein 1 (LRP1), significantly reduced liposomal docking. Microscopy analysis revealed that both ApoE-FL and non-functionalized FL directly fused with endothelial plasma membranes, thus bypassing intracellular organelles and minimizing lysosomal degradation. This suggests that the naturally formed ApoE corona in vivo may contribute to efficient cerebrovascular targeting, a property successfully replicated by the engineered ApoE corona strategy. In vivo biodistribution and kinetic studies demonstrated that especially ApoE-FL achieved enhanced brain-targeting efficiency, prolonged cerebrovascular retention, and extended targeting distance along the arteriovenous axis. This emphasizes that fusogenic liposomes effectively engage almost the entire microvascular network, including capillaries and post-capillary venules. Functionally, fusogenic liposome-delivered RSV improved blood-brain barrier (BBB) integrity, enhanced neurovascular coupling (NVC) responses, and promoted brain vascularization in aged mice. Single-cell RNA sequencing (scRNA-seq) revealed enhanced endothelial angiogenesis and barrier protective transcriptional profiles in cerebrovascular cells treated with ApoE-FL/RSV, suggesting a molecular basis for the observed vascular benefits. Liposomal RSV delivery achieved near-complete cerebrovascular and cognitive rejuvenation in aged mice applying a 2000-fold lower RSV dose than oral administration used as control sample. Thus, ApoE-FL liposomes exhibited exceptionally high delivery efficiency in deeper brain regions, further expanding their therapeutic potential. These findings underscore the importance of targeted drug delivery in optimizing therapeutic outcomes and establish ApoE-functionalized fusogenic liposomes as a promising strategy for mitigating brain vascular aging and cognitive decline. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/709925v1_ufig1.gif" ALT="Figure 1000"> View larger version (52K): org.highwire.dtl.DTLVardef@acad5borg.highwire.dtl.DTLVardef@a96e3borg.highwire.dtl.DTLVardef@18405aeorg.highwire.dtl.DTLVardef@90e349_HPS_FORMAT_FIGEXP M_FIG C_FIG

animal behavior and cognition↗

bioRxiv2026

Systematic Data-Driven Penalty Calibration for Constrained Quantum Optimization with Application to Molecular Docking

This paper describes MMP, a three-stage framework for systematic quantum optimization of constrained molecular docking problems. The protocol addresses the "formulation bottleneck"--the critical challenge of translating constrained optimization problems into valid QUBO (Quadratic Unconstrained Binary Optimization) formulations for quantum solvers. MMP replaces heuristic penalty tuning with data-driven calibration through: (1) classical solution-space analysis to validate fragment libraries before quantum deployment, (2) systematic penalty sweeps to identify optimal "Goldilocks Zone" coefficients, and (3) MAC-QAOA (MMP Adaptive Constraint QAOA) with layer-dependent penalty decay. Preliminary benchmarks on synthetic constrained optimization problems demonstrate 99.7% solution validity at identified elbow points and 25.5% improvement in solution quality over static-penalty QAOA. MMP is hardware-agnostic but designed for near-term devices including Pasqals Orion Gamma (140+ qubits). The theoretical framework, algorithmic details, and preliminary validation results of the protocol are discussed, establishing a systematic methodology for quantum-augmented optimization workflows for drug discovery. All benchmarks are conducted on synthetic constrained optimization instances that reproduce structural features of docking formulations; application to real molecular docking targets is left for future work.

bioinformatics↗

bioRxiv2025

Cystathionine Beta-Synthase Promotes Anoikis Resistance and Transcoelomic Metastasis in Ovarian Cancer via the SP1-ITGB1 axis

Anoikis resistance is crucial for ovarian cancer (OvCa) transcoelomic metastasis, during which exfoliated OvCa cells survive as spheroids before invading the omentum. Here, we demonstrate that cystathionine {beta}-synthase (CBS), an H2S-producing transsulfuration pathway enzyme, is a key determinant of OvCa spheroidal viability and metastatic potential. Analysis of publicly available patient datasets, as well as an in-house tissue microarray, revealed that high CBS expression positively correlates with both poor progression-free survival and clinically observed peritoneal/omental metastasis. Integrated functional and proteomic analyses indicated that CBS silencing induces apoptosis in 2D monolayers. Consistent with this, CBS silencing in spheroids caused apoptosis and disrupted spheroid architecture. Mechanistically, this phenotype was associated with downregulation of oncogenic stemness and epithelial-mesenchymal transition. Further, through proteomic and bioinformatic analyses, we identified ITGB1 to be the hub protein in OvCa spheroidogenesis. Interestingly, knockdown of CBS led to abrogation of the ITGB1-mediated downstream pathway. Moreover, by proteomic and network analyses, we identified SP1 as a key transcriptional regulator of CBS-induced pro-spheroidal transcriptional programs of stemness and invasiveness. Stabilization of SP1 through persulfidation by H2S supplementation restored spheroidal viability and underlying protein signaling. Further, our results reveal that loss of CBS, through ITGB1 repression, disrupts spheroid architecture, leading to reduced metastatic docking on the murine omental surface in vivo. Collectively, these findings identify CBS as a central regulator of anoikis resistance and OvCa transcoelomic metastasis, highlighting the therapeutic potential of targeting CBS-dependent SP1-ITGB1 regulation to control metastatic spread.

cancer biology↗

bioRxiv2025

Orthogonal and Robust Analytics Enable Reproducible and Scalable Manufacturing of High Purity Extracellular Vesicles Derived from Mesenchymal Stromal Cells

The development of extracellular vesicle (EV) based therapeutics requires robust analytical assays and scalable downstream processing (DSP) strategies to ensure product quality and reproducibility. In this study, we established an analytical toolbox comprising scattering and fluorescence mode nanoparticle tracking analysis (NTA), fluorescence-based flow cytometry (Fl-FC), and multi-detector analytical size exclusion chromatography. Liposomes, selected for their physicochemical similarity to EVs, were used as reference materials to optimize assay parameters, fluorescence labeling conditions, and dynamic range, minimizing artifacts such as photobleaching and masking effects. Using these optimized analytical tools, we designed a scalable DSP workflow for human bone marrow mesenchymal stromal cell (hBM-MSC) derived EVs, incorporating clarification, tangential flow filtration (TFF), ion exchange chromatography (IEX), buffer exchange, and sterile filtration. IEX chromatography resulted in the elution of two cell-secreted populations, with similar scattering signals while eluate 1 showed approximately 30 times higher absorbance signal compared to eluate 2. Transmission electron microscopy revealed that eluate 1 contained non-vesicular extracellular particles (NVEPs), and eluate 2 was enriched in EVs and showed higher expression of positive markers such as CD81 and CD73 using Simple Western. Additionally, the two IEX eluates showed different proteomic and lipidomic profiles. Then the analytical toolbox was utilized to monitor the DSP process in terms of particle recovery and impurity removal throughout the process determining the high purity level of the final EV preparation. Together, these results demonstrate that orthogonal analytics coupled to a scalable DSP yield reproducible MSC-EV preparations while depleting commonly co-isolated NVEPs. This practical framework advances process analytics of MSC-EV manufacturing and supports reporting of identity, purity, and function aligned with existing guidelines in the field. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/672476v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@142ff8aorg.highwire.dtl.DTLVardef@196c0bforg.highwire.dtl.DTLVardef@485c36org.highwire.dtl.DTLVardef@9dff39_HPS_FORMAT_FIGEXP M_FIG C_FIG Liposomes were used as reference materials to optimize single particle analytics as well as multi detector analytical chromatography to assess particle recovery and impurity removal. This optimized analytical toolbox enabled accurate monitoring of downstream processing steps, establishing a robust workflow for reproducible and scalable mesenchymal stromal derived extracellular vesicles bioprocessing.

bioengineering↗

bioRxiv2025

DNA Polymerase α has pyrimidine dimer translesion activity that is suppressed during normal replication

To ensure rapid and accurate DNA replication during S-phase, the cell uses DNA damage tolerance (DDT) pathways, leaving lesions to be repaired after completion of replication. One established DDT pathway is Translesion Synthesis (TLS), in which lesions are bypassed by specialized TLS Polymerases that work in conjunction with the replisome. Here we demonstrate that DNA Polymerase alpha (Pol ), the replicative primase/polymerase, can also unexpectedly replicate through bulky lesions in vitro. We use biochemical and single-molecule fluorescence assays to characterize cyclobutane pyrimidine dimer (CPD) TLS activity of Pol . We observe that Pol {varepsilon}, the leading strand replicative polymerase, and RPA, a single-stranded DNA binding protein complex, both strongly inhibit CPD TLS activity of Pol . In contrast, Pol , the canonical TLS Pol for pyrimidine dimers, is unaffected by Pol {varepsilon} and is conversely stimulated by RPA. Finally, we demonstrate with single-molecule Fluorescence Resonance Energy Transfer (FRET) that the DNA binding cleft of Pol must remain in the open state to accommodate a bulky CPD lesion during TLS, possibly accounting for the relatively slow kinetics of CPD bypass that we observe. The results suggest that the intrinsic bulky TLS activity of Pol is likely suppressed at the replication fork by the replisome itself during normal replication. RESEARCH HIGHLIGHTSO_LIPolymerase has translesion synthesis (TLS) activity past a CPD lesion, the first demonstration of TLS activity at a bulky lesion by a replicative polymerase at physiological nucleotide levels. C_LIO_LIIntrinsic Pol TLS activity is suppressed during normal replication by replisome components Pol {varepsilon} and RPA. C_LIO_LIPol {varepsilon} suppresses TLS incorporation by Pol by proofreading C_LIO_LIThe binding cleft of Pol stays open to accommodate a pyrimidine dimer. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/662766v2_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@1750e05org.highwire.dtl.DTLVardef@1eb8cdcorg.highwire.dtl.DTLVardef@16cdd39org.highwire.dtl.DTLVardef@2fd532_HPS_FORMAT_FIGEXP M_FIG Pol harbors CPD TLS activity that is suppressed by RPA and Pol {varepsilon} at the replication fork during normal replication. C_FIG

biochemistry↗

bioRxiv2025

miRNA-Mediated Regulation of Gene Expression During Early Activation in Jurkat Cells

BackgroundT cell activation induces substantial changes in gene expression by rapidly increasing transcription and translation. Additionally, microRNAs play a crucial role in regulating protein expression in T cell physiology, adding a layer of complexity by fine-tuning protein levels. While various miRNAs have been implicated in T cell function, a systematic analysis of differentially expressed miRNAs during early T cell activation and identification of their mRNA targets remains mostly unknown. ResultsWe investigated dynamic changes in global gene expression during early T cell activation using a multi-omics approach combining small RNA-seq, mRNA-seq and ribosome profiling. Our results show that most differential expression changes occur by 5 hours post-activation, with translational upregulation predominating over downregulation. From 5 to 12 hours, we observed modest transcriptional and translational reprogramming. We identified 9 miRNAs that are differentially expressed (DE) during early activation, with most changes occurring as early as 5 hours. We calculated translation efficiency (TE) and classified genes based on changes in both mRNA abundance and ribosome-protected fragments (RPFs). By integrating TE and miRNA expression data, we examined the relationship between TE group-specific regulation patterns and the number of miRNA binding sites. Interestingly, rather than observing a uniform downregulation of targets with 4 or more predicted DE miRNA binding sites, we found distinct regulatory patterns that varied with both activation time point and TE category. ConclusionsOur data provide new insights into how genes associated with key events in T cell activation such as translation, cell proliferation, and immune signaling are regulated at both the transcriptional and translational levels. The observation that most regulatory changes occur within the first 5 hours post-activation highlights the rapid and coordinated nature of T cell responses. The differential patterns of target regulation, based on translation efficiency groups and miRNA binding site density, suggest a context-dependent role for miRNAs in shaping protein output. Future experiments will be required to functionally validate specific miRNA-target interactions and to explore their relevance in primary T cells in vivo. This study also lays the groundwork for identifying miRNA-based regulatory circuits for therapeutic modulation of T cell activity.

genomics↗

bioRxiv2025

Lactate cannot replace glucose for maintaining the viability of mouse and human glioma cells

ObjectivesAerobic lactic acid fermentation (the "Warburg effect") is associated with OxPhos insufficiency and altered energy metabolism in most cancers. Whether lactate is a major fuel in cancer cells remains debated. This study investigated whether lactate could serve as a metabolic fuel in glioma cells and replace glucose to support viability. MethodsA bioluminescence ATP assay and calcein-AM/EthD-III double-staining were used to measure ATP content and viability in mouse (VM-M3, CT-2A) and human (U-87MG) glioma cells differing in cell biology and genetic background. Viability was assessed in thioglycollate-elicited peritoneal macrophages (TPMs) from VM/Dk and C57BL/6J mice, used as syngeneic non-neoplastic controls for VM-M3 and CT-2A gliomas, respectively. Oxygen consumption rate (OCR) was determined using the Resipher system. ResultsLactate alone failed to sustain ATP content and viability in all glioma cell lines. ATP content and viability were lower in cancer cells cultured in glutamine and lactate than in glucose and glutamine. In contrast, lactate alone sustained over 45% viability in both TPM models. Moreover, viability was similar between TPMs cultured in glutamine and lactate and in glucose and glutamine. In human U-87MG, lactate addition under severe glucose restriction increased OCR and viability. A low dose of the glycolysis inhibitor 2-deoxy-D-glucose abolished both increases. ConclusionsOur results suggest non-neoplastic mouse TPMs utilize lactate more effectively than mouse glioma cells. In U-87MG, lactate utilization appears glycolysis-dependent, given its sensitivity to 2-deoxy-D-glucose. In conclusion, our data does not support lactate as a major oxidative fuel for viability in mouse and human glioma cells.

cancer biology↗

bioRxiv2025

The differential effect of glutamine supplementation on the orthotopic and subcutaneous growth of two syngeneic murine models of glioma

Glutamine serves as a major fuel source for tumor cell proliferation while simultaneously playing an essential role in maintaining gastrointestinal health and immune function. Controversy exists regarding glutamine supplementation for cancer patients undergoing chemotherapy and radiation, with concerns that it may stimulate cancer growth. The present study is the first to examine the effects of dietary glutamine supplementation (0.4g/kg/day) on the growth of malignant gliomas, which utilize large amounts of glutamine to satisfy metabolic demands. Brain bioluminescence and subcutaneous tumor volumes were used to assess the influence of glutamine supplementation on the growth of the syngeneic VM-M3 and CT-2A preclinical models of glioma. Glutamine supplementation had no significant effect on the orthotopic growth of the VM-M3 or the CT-2A gliomas when compared to non-supplemented controls. However, glutamine supplementation significantly increased tumor volume by 28% in the VM-M3 and by 166% in the CT-2A tumors when grown subcutaneously outside the central nervous system (CNS) relative to controls. Additionally, glutamine supplementation increased serum glutamine despite a localized decrease in intratumoral glutamine concentrations. Caution is warranted when considering glutamine supplementation in patients with glutamine-dependent malignancies. Further studies are needed to better understand the potential risks and benefits of glutamine supplementation in cancer therapy.

cancer biology↗

bioRxiv2025

Estimating Brain Similarity Networks with Diffusion MRI

Structural similarity has emerged as a promising tool in mapping the network organization of an individual, living human brain. Here, we propose diffusion similarity networks (DSNs), which employ rotationally invariant spherical harmonic features derived from diffusion magnetic resonance imaging (dMRI), to map gray matter structural organization. Compared to prior approaches, DSNs showed clearer laminar, cytoarchitectural, and micro-architectural organization; greater sensitivity to age, cognition, and sex; higher heritability in a large dataset of healthy young adults; and straightforward extension to non-cortical regions. We show DSNs are correlated with functional, structural, and gene expression connectomes and their gradients align with the sensory-fugal and sensorimotor-association axes of the cerebral cortex, including neuronal oscillatory dynamics, metabolism, immunity, and dopaminergic and glutaminergic receptor densities. DSNs can be easily integrated into conventional dMRI analysis, adding information complementary to structural white matter connectivity, and could prove useful in investigating a wide array of neurological and psychiatric conditions.

neuroscience↗

bioRxiv2025

Tubulin hyperacetylation drives HMGB1 nuclear exit via the ROS-PARP1 axis leading to rotenone-induced G2/M Arrest

Rotenone, a lipophilic pesticide, is strongly linked to dopaminergic neuronal loss in Parkinsons disease (PD), primarily through mitochondrial complex I inhibition. While rotenone induces G2/M arrest in dividing cells, the underlying molecular events remain unclear. We identify HMGB1 as a key player in this process. HMGB1, known for its roles in genomic integrity and inflammation, exits the nucleus during rotenone-induced G2/M arrest, whereas its nuclear retention protects against mitotic DNA damage. We further reveal that tubulin hyperacetylation precedes HMGB1 nuclear release. Notably, reducing tubulin acetyltransferase lowers mitochondrial ROS (mtROS), preventing HMGB1 nuclear exit and mitotic DNA damage. Additionally, inhibiting PARP1 hyperactivation with PJ34 blocks HMGB1 exit and prevents G2/M arrest. These findings suggest that ROS-induced DNA damage elevates PARP1 activity, promoting HMGB1 nuclear exit and interfering with DNA repair. This tubulin acetylation/mtROS/HMGB1 axis may underlie rotenone-induced neurotoxicity, contributing to dopaminergic neuron vulnerability in PD and offering potential neuroprotective targets.

cell biology↗

bioRxiv2025

E2F1 Suppresses EBV Lytic Reactivation through Cellular and Viral Transcriptional Networks

Latent EBV infection is causally associated with various B-cell malignancies, while periodic lytic-cycle replication is essential for sustaining viral progeny. Lytic cycle induction represents a promising therapeutic strategy for EBV-associated neoplasms. Therefore, uncovering the mechanisms that regulate EBV lytic-cycle reactivation is pivotal for understanding viral pathogenesis and advancing novel therapies. Our genome-wide transcriptomic analysis reveals that E2F1 expression is transcriptionally activated during EBV latent infection in B-lymphocytes but significantly suppressed during lytic cycle reactivation. While ectopic E2F1 expression suppresses lytic replication, E2F1 depletion markedly accelerates this process. Mechanistically, we establish that E2F1 and the lytic transactivator BZLF1 form a negative transcriptional feedback loop, tightly controlling viral lytic replication. Furthermore, E2F1 positively regulates c-Myc expression and together they repress the leaky BZLF1 expression during latency. Importantly, c-Myc does not influence E2F1 expression, nor does BZLF1 modulate c-Myc transcription, underlining a distinct regulatory hierarchy. In sum, our findings reveal that EBV tightly controls the latent-to-lytic switch through precise regulation of E2F1 expression, positioning E2F1 as a pivotal regulator of both cellular and viral gene expression. SynopsisEBV coordinates the latent-to-lytic switch by sensing E2F1 abundance, which acts as a crucial transcriptional regulator of both cellular and viral gene expressions. O_LIDuring EBV latent infection, E2F1 promotes c-Myc transcription, and together they suppress EBV lytic cycle transactivator BZLF1 expression. C_LIO_LIE2F1 and BZLF1 form a negative feedback loop in order to control each others transcriptions. C_LIO_LIBZLF1-driven controlled E2F1 expression successively inhibits c-Myc level, thereby stimulating EBV lytic cycle reactivation. C_LIO_LIBZLF1 does not regulate c-Myc, nor does c-Myc reciprocally regulate E2F1, emphasizing a unidirectional regulatory hierarchy. C_LI

cancer biology↗

bioRxiv2025

Atomic resolution structures of key enzyme MCR in anaerobic methanotrophy reveal novel and extensive post-translational modifications.

Anaerobic methanotrophic archaea (ANME) are crucial to planetary carbon cycling. They oxidise methane in anoxic niches by transferring electrons directly to nitrate or metal oxides and alternatively to sulfate-reducing bacteria. Due to their physiological complexity, no ANME species have been isolated, hampering the biochemical investigation of the enzymatic processes involved in anaerobic methane oxidation. To study the methane-capturing enzyme of these microorganisms, we circumvented the isolation barrier by exploiting microbial enrichments of freshwater nitrate-reducing ANME-2d grown in bioreactors, and marine ANME-2c in syntrophy with bacterial partners. The crystal structures of their Methyl-Coenzyme M Reductases (MCRs), refined to true atomic resolution, provided the most precise image of the enzyme to date. Despite their physiological differences, these ANMEs have extremely conserved MCR structures, similar to homologs from methanogenic Methanosarcinales, rather than the phylogenetically distant MCR of ANME-1 isolated from Black Sea mats. The three studied MCRs are highly modified, with seven post-translational modifications. Among them was a novel 3(S)-methylhistidine on the {gamma}-chain of both ANME-2d MCRs. Labelling with gaseous krypton did not reveal any internal channels that would facilitate alkane diffusion to the active site as observed in the ethane-specialized enzyme. Based on our data, the methanotrophic MCRs should follow the same radical reaction mechanism proposed for the methane-generating homologues. The described pattern of post-translational modifications underscores the importance of native purification as a powerful approach to discovering intrinsic enzymatic features in uncultivated microorganisms existing in nature.

microbiology↗

bioRxiv2024

Controlling reactogenicity while preserving immunogenicity from a self-amplifying RNA vaccine by modulating nucleocytoplasmic transport

Self-amplifying RNA (saRNA)-based vaccines have emerged as a potent and durable RNA vaccine platform. However, RNA vaccine platforms trigger undesirable side effects at protective doses, underscoring the need for improved tolerability. To address this, we leveraged the Cardiovirus leader protein, which is well-characterized to dampen host innate signaling by modulating nucleocytoplasmic transport (NCT). Co-administration of a leader-protein-encoding mRNA (which we have named "RNAx") delivered alongside vaccine cargo saRNA reduced interferon production while enhancing Influenza hemagglutinin (HA) expression in human primary cells and murine models. RNAx potently decreased serum biomarkers of reactogenicity after immunizations with an HA-expressing saRNA-LNP vaccine while maintaining the magnitude of the antibody and cellular response. RNAx also consistently enhanced binding antibody titers after a single injection and in some conditions enhanced binding antibody and neutralization titers post-boost. These findings support RNAx as a promising platform approach for improving tolerability of saRNA-LNP vaccines while preserving or enhancing immunogenicity.

immunology↗

bioRxiv2024

TRPV4 calcium-permeable channel contributes to valve stiffening in aortic stenosis

Aortic valve stenosis (AVS) is a progressive disease marked by fibrosis, inflammation, calcification, and stiffening of the aortic valve leaflets, leading to disrupted blood flow and left ventricular pressure overload. AVS can result in heart failure and death within 2 to 5 years if left untreated, highlighting its high mortality rate. Understanding the molecular mechanisms of AVS is essential for developing noninvasive treatments. Emerging data suggest that extracellular and intracellular matrix stiffness influences gene expression, inflammation, and cell differentiation. Myofibroblast activation of valvular interstitial cells (VICs) along with excess extracellular matrix (ECM) accumulation and remodeling are primary drivers of AVS progression. Inflammation also plays a critical role, with macrophages accumulating in valve leaflets from AVS patients, promoting inflammation, activating VICs, and synthesizing and remodeling the ECM. Our lab and others have reported that macrophage and fibroblast activities, including migration, inflammatory gene expression, and myofibroblast activation, are sensitive to matrix stiffness, indicating that valve leaflet stiffening may regulate AVS progression via a cellular stiffness sensor. Our published work shows that mechanosensitive Ca2+-permeable transient receptor potential vanilloid 4 (TRPV4) channels regulate fibrosis in other organs and control macrophage and fibroblast activation, suggesting TRPV4 as the potential stiffness sensor in AVS. This implies that fibrosis and tissue stiffening may reinforce each other, creating a vicious cycle in AVS development, with VICs and macrophages playing central roles. Here, we identify the cellular stiffness sensor mediating the link between stiffness and AVS development using human aortic valve tissues, a murine model of aortic valve stenosis, and atomic force microscopy analysis.

pathology↗