Biology subjects

Du, J.

Publications and source records attributed to Du, J..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

UBA1 Mitigates Myocardial Ischemia/Reperfusion Injury by Attenuating Endoplasmic Reticulum-Mitochondria Contacts via Pdzd8 ubiquitination

BACKGROUNDMyocardial ischemia/reperfusion injury (I/RI) represents a serious clinical complication in patients after acute myocardial infarction. Ubiquitin-activating enzyme 1 (UBA1) catalyzes the initial step of ubiquitination and plays a fundamental role in regulating protein homeostasis and related diseases. This study aims to elucidate the functional contribution of UBA1 to the pathogenesis of myocardial I/RI and to uncover its underlying mechanisms. METHODSSingle-cell RNA sequencing was employed to characterize UBA1 expression in human ischemic heart tissues. Myocardial I/R injury was examined in myocardial-specific UBA1 knockout (UBA1cko) mice, UBA1-overexpressing mice (rAAV9-UBA1), and corresponding controls. Neonatal rat cardiomyocytes underwent hypoxia/reoxygenation in vitro. Cardiac function and infarction were evaluated by echocardiography and pathological staining. Protein-protein interactions were analyzed via immunoprecipitation combined with mass spectrometry. The endoplasmic reticulum-mitochondrial contact sites (ERMCSs) and mitochondrial ultrastructure were evaluated through transmission electron microscopy and confocal imaging. RESULTSUBA1 expression was significantly downregulated in human and murine ischemic myocardium, especially in cardiomyocytes. UBA1cko mice exhibited aggravated I/RI with greater infarct size, impaired function, apoptosis, elevated intracellular Ca2+ levels, mitochondrial dysfunction, and ER stress, whereas UBA1 overexpression conferred cardioprotective effects. Mechanistically, UBA1 directly bound to and ubiquitinated Pdzd8, a key ERMCS-tethering protein, thereby promoting its degradation, which inhibited ERMCS formation and improved mitochondrial dysfunction and ER stress. Moreover, knockdown of Pdzd8 via rAAV9-siRNA effectively mitigated UBA1 knockout-induced myocardial damage. Additionally, administration of auranofin (AF), a U.S. Food and Drug Administration-approved drug for treating rheumatoid arthritis, markedly alleviated myocardial I/RI via activating UBA1 in vivo and in vitro. CONCLUSIONSUBA1 confers protection against myocardial I/RI by limiting ERMCS formation through Pdzd8 ubiquitination. Activating UBA1 or targeting Pdzd8 as a potential therapeutic strategy for the treatment of ischemic heart disease. GRAPHIC ABSTRACTA graphic abstract is available for this article. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIUBA1 expression is downregulated in human and murine ischemic myocardium, especially in cardiomyocytes. C_LIO_LICardiac deletion of UBA1 significantly exacerbates myocardial ischemia/reperfusion injury (I/RI), whereas cardiac UBA1 overexpression confers a marked protective effect. C_LIO_LIUBA1 interacts with Pdzd8 (PDZ domain containing 8) and facilitates its ubiquitination and subsequent degradation, which then reduces endoplasmic reticulum-mitochondria contact sites (ERMCSs) and ameliorates mitochondrial dysfunction and ER stress, protecting myocardial I/RI. C_LIO_LIPharmacological activation of UBA1 with the FDA-approved drug auranofin attenuates myocardial I/R injury and improves heart dysfunction. C_LI What Are the Clinical Implications?O_LIUBA1 represents a new therapeutic target for myocardial I/RI. C_LIO_LIActivating UBA1 or targeting Pdzd8 may offer a promising therapeutic strategy for mitigating myocardial I/RI and heart failure, underscoring its potential for clinical translation. C_LI

pathology↗

bioRxiv2026

Healthcare Infrastructure Shapes Evolutionary Trade-offs and Geographic Dissemination of Multidrug-Resistant Acinetobacter baumannii

Antimicrobial-resistant pathogens pose an existential threat to modern medicine, yet the evolutionary forces driving their adaptation in healthcare systems remain largely unexplored. We revealed that hospital network architecture functions as a primary selective pressure, driving pathogen evolution through infrastructure-dependent virulence-transmission trade-offs. Phylogenomic analysis of 5,023 Acinetobacter baumannii isolates across Chinas centralized healthcare system identifies two co-existing evolutionary strategies: a virulence-optimized clade (ESL2.4) that spread slowly (20.4 km per year) in low-connectivity hospitals, and a transmission-optimized clade (ESL2.5) that disseminate rapidly (65.2 km per year) through mega-city healthcare hubs, likely attributed to its capsule conversion and increased upper respiratory colonization. Comparative analysis with European A. baumannii populations demonstrates that healthcare connectivity, not geography, governs pathogen distribution through convergent genomic adaptations. Our simulation suggests competitive asymmetries of the two clades, following the ecotype principle and enabling stable coexistence. The COVID-19 pandemic provided a natural experiment validating these mechanisms: outpatient restrictions reduced transmission-optimized lineage spread by 89%, while virulence-optimized lineage persisted through inpatient networks. These findings establish healthcare infrastructure as a critical evolutionary driver with immediate implications for predicting and controlling antimicrobial resistance emergence across diverse healthcare systems.

microbiology↗

bioRxiv2025

Increased Aperiodic Exponents Track Depression Symptom Severity

Roughly one-third of patients with major depressive disorder (MDD) fail to respond to standard treatments and develop treatment-resistant MDD. For these patients, alternative therapies ofer additional options but yield inconsistent outcomes. Progress has been limited by the absence of objective, brain-based biomarkers to guide target selection or track therapeutic response in real time. Instead, clinicians rely on behavioral assessments that evolve slowly over weeks to months, obscuring the underlying neural dynamics of symptom changes. Here, we test whether the aperiodic exponent of intracranial EEG (iEEG) local field potentials can serve as a neurophysiological marker of depression symptom severity. We leveraged a large iEEG cohort (N = 20) undergoing invasive monitoring for refractory epilepsy, yielding over 1,800 contacts spanning cortical and subcortical zones. For each contact, we estimated the aperiodic exponent (thought to reflect aspects of cortical excitability) of the power spectrum across 10-100 Hz within local brain regions and across distributed cortical association networks. Depressive symptoms were assessed with the Beck Depression Inventory-II (BDI-II) immediately before intracranial resting state recordings. With respect to the BDI-II scale, participants were identified as experiencing minimal (BDI-II [≤] 13) or elevated depression symptoms (BDI-II [≥] 14). Associations between symptom severity (BDI-II total score and Somatic-Afective, Cognitive, and Anhedonia subscales) and region- or network-level exponents were modeled with ordinary least squares (OLS) regression. The whole-brain, mean aperiodic exponent for each participant discriminated symptom status (AUC = 0.82). At the regional level, the orbitofrontal cortex, anterior cingulate cortex, insula, and amygdala showed higher exponents in the elevated depression symptom group (d = 1.18-1.71; p = 0.032-0.004). A post-hoc classification analysis across these four regions misclassified one participant per group (AUC = 0.86; 95% CI 0.64-1.00). In continuous analyses, BDI-II scores correlated positively with exponents in these same four regions (pFDR = 0.019-0.027; partial r=0.61-0.70) and at the network level in the Salience network (pFDR = 0.024; partial r = 0.63) and Default (pFDR = 0.046; partial r = 0.55) network. The Salience network significantly tracked Anhedonia symptoms (p = 0.004; partial r = 0.62). Here we report that intracranial aperiodic exponents within fronto-limbic and insular circuits, overlapping with networks implicated in contemporary accounts of depression pathophysiology, diferentiate depressive symptom status and scale with severity. These findings support the aperiodic exponent as a candidate neurophysiological marker of current depression symptom burden, with potential relevance for individualized neuromodulation in MDD.

neuroscience↗

bioRxiv2025

3' to 5' Translation of Circular RNAs?

For decades, the 5[->]3 direction of translation has been a central dogma of molecular biology(1). Here we present evidence that eukaryotic circular RNAs (circRNAs) can serve as templates for 3[->]5 backward translation (BT), yielding polypeptides with distinct sequence and structural features not found in canonical proteomes. Mining of more than 6,000 multi-omics datasets identified [~]1 million candidate BT proteins across eukaryotes, including 59,000 high-confidence human BT proteins supported by mass spectrometry. Genetic combinatorial experiments (KO and KI) and cell-free translation of synthetic circRNAs establish BT as a conserved mechanism. Loss-of-function studies of hs.circCAPN15 in human and dm.circROLS in Drosophila underscore the functional importance of BT-derived proteins. Our work challenges the long-standing unidirectional translation paradigm, expands the functional landscape of the genetic code, and reveals a hitherto hidden layer of proteomic complexity with broad implications for biology and therapeutics.

molecular biology↗

bioRxiv2025

Beyond Correlation: An Ultra-Large Physics-Driven Vascularized Tumor Model to Bridge Feature Formation with Underlying Biology

Radiomics provides an appealing, non-invasive approach to probing tumor biology for potential diagnostic and prognostic applications. However, its clinical adoption is limited by challenges in interpretability, which in turn compromise its robustness. To uncover the underlying causation, we developed an ultra-large-scale (ULS) computational model that simulates heterogeneous, vascularized tumor growth under physical constraints to a scale that can be visualized in medical images. Our study revealed the pivotal role of tumor proliferation rate in driving necrosis and tissue heterogeneity and the dominant impact of oxygen consumption rate on vascularization level. Analysis of the resultant tumor Radiomics shows a causal relationship between tumor biophysical parameters and imaging features. Specifically, differences in proliferation and oxygen consumption rates result in distinct changes in radiomic image features, identifying suitable imaging modalities and quantitative imaging metrics for studying these biophysical parameters. This work thus reverse-engineers the building blocks of Radiomics as a means to understand their respective biological underpinnings.

bioinformatics↗

bioRxiv2025

Alphaviral nonstructural protein-host RBP co-condensation as a mechanism to sustain virus replication

It has long been recognized that the intracellular replication of alphaviruses critically relies on several key host RBPs, including G3BP1/2 and FXR1/FXR2/FMR1, but how these RBPs modulate alphaviral replication and whether it would be possible to target these RBPs for antiviral treatment are less explored. Here, using SFV as a model, we report that SFV nsP3 exploits G3BP for its condensation and transforms antiviral stress granules into proviral nsP3-G3BP co-condensates. The gel-like co-condensates of nsP3 and G3BP enrich and protect viral genomic RNAs from host RNase degradation and serve as viral translational hubs to promote viral replication. The mode of nsP3-RBP co-condensation is prevalent across alphaviruses, and disruption of nsP3 condensates is an efficient antiviral approach. Thus, these findings uncover a general anti-alphavirus strategy based on the conserved reliance of nsP3-RBP co-condensation.

cell biology↗

bioRxiv2025

Mechanical Disturbance of Trichomes Enhances Cd2+ Uptake via Malate-Mediated Stomatal Opening in Arabidopsis

Phytoremediation of cadmium-contaminated soils relies on efficient metal uptake and translocation in plants. We previously found that gentle mechanical stimulation of non-glandular trichomes by brushing significantly enhances shoot Cd{superscript 2} accumulation in Arabidopsis thaliana, although the underlying physiological mechanism remained unclear. Here, we demonstrate that trichome brushing specifically induces stomatal opening, elevates transpiration, and promotes Cd{superscript 2} uptake, as evidenced by measurements of stomatal aperture, stomatal conductance, transpiration rate, and Cd{superscript 2} concentration in wild-type and mutant leaves. These responses are absent in the glabrous gl1-1 mutant and are not elicited by brushing epidermal pavement cells, indicating a trichome-specific mechanosensory pathway. Mechanistically, this pathway requires MPK3/MPK6 activity and employs malic acid as a key regulator of stomatal aperture, functioning independently of canonical ABA signaling. Our findings uncover a previously unrecognized trichome-to-stomatal signaling cascade that links trichome mechanostimulation to enhanced heavy metal accumulation.

plant biology↗

bioRxiv2025

Cysteine availability tunes ubiquitin signaling via inverse stability of LRRC58 E3 ligase and its substrate CDO1

Cellular responses to amino acid fluctuations often hinge on ubiquitin-mediated control of metabolic enzymes, yet the underlying E3 ligase pathways remain incompletely defined. Using quantitative proteomics and active cullin-RING ligase (CRL) profiling, we identify LRRC58 as a cysteine-responsive substrate receptor whose stability increases sharply under cysteine starvation. Proteomics reveals an inverse relationship between LRRC58 and the metabolic enzyme cysteine dioxygenase 1 (CDO1), suggesting a cysteine-linked regulatory axis. Biochemical reconstitution and cryo-EM structures show that LRRC58 forms an active CUL2-or CUL5-based CRL that selectively positions CDO1 for ubiquitylation at Lys8. By contrast, targeted protein degradation via the VHL-based CRL is achieved by recruitment of a distinct surface of CDO1, and more promiscuous ubiquitylation independent of Lys8. Together, our proteomics-guided discovery pipeline, cellular stability studies, and structural analyses uncover a metabolically-tuned LRRC58-CDO1 pathway that links cysteine availability to selective proteasomal turnover, reveals principles of metabolite-regulated CRL activity, and showcases mechanisms distinguishing endogenous and targeted protein degradation.

biochemistry↗

bioRxiv2025

TriLeukeVax: A CD80/IL-15/IL-15Rα Expressing Autologous AML Cell Vaccine Elicits Robust Anti-Leukemic Cytolytic Activity

Acute myeloid leukemia (AML) is the most common acute leukemia in adults and is associated with poor outcomes due to frequent relapse after remission induction. While hematopoietic stem cell transplantation (HSCT) can improve survival, many individuals, especially older patients, are ineligible. Prior immunotherapies have not reliably induced effective anti-leukemic immunity and have been associated with severe and unpredictable toxicities. Thus, there is a need for safe and effective therapies that reduce relapse and increase overall survival (OS). We have developed a universally applicable, patient-specific, lentivirally engineered autologous AML cell vaccine, TriLeukeVax (TLV), designed to stimulate leukemia-specific cytolytic immune responses in AML patients in remission. To generate TLV, AML cells are engineered to express the highly synergistic combination of the co-stimulatory protein CD80 and the IL-15/IL-15-receptor alpha (IL-15R) heterodimer. Prior proof-of-concept (POC) studies demonstrated eradication of disease in >80% of leukemic mice with serial administration of TLV. In the current studies, TLV was generated from 59/60 cryopreserved, diagnostic bone marrow-derived patient AML samples. Ex vivo priming of post-remission patient T-cells by ex vivo co-culture with autologous TLV stimulated robust proliferative and cytotoxic responses. In secondary co-cultures, T-cells previously primed by initial co-culture with TLV, showed greater clonal expansion and leukemia-specific cytolytic activity towards de novo autologous AML blasts than did control, unprimed T-cells. The enhanced anti-leukemic activity of TLV-primed T-cells against de novo AML confirms the potential for vaccine administration to effectively target minimal residual disease (MRD) persisting after chemotherapy and reduce relapse. Key PointsO_LITriLeukeVax induces proliferation, activation, and effective anti-leukemic cytolytic responses in remission T-cells. C_LIO_LIPrimed T-cells show polyclonal expansion and transcription profiles associated with proliferation, memory, and cytotoxicity. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/687000v2_ufig1.gif" ALT="Figure UFIG"> View larger version (38K): org.highwire.dtl.DTLVardef@5c1827org.highwire.dtl.DTLVardef@35b2e2org.highwire.dtl.DTLVardef@1732914org.highwire.dtl.DTLVardef@18df655_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOVisual Abstract.C_FLOATNO TriLeukeVax (TLV) workflow and mechanism of action. Diagnostic AML patient bone marrow aspirates are collected, purified in Ficoll and cryopreserved. Samples are thawed and lentivirally transduced to produce TLV. Vaccination of AML patients in remission with TLV will stimulate the activation and expansion of leukemia-specific T-cells, effector memory cells, and NK cells by combining the co-stimulatory effects of CD80 with immune stimulation by the IL-15/IL-15R heterodimer expressed by the transduced AML cells, thereby targeting MRD and potentially increasing relapse-free survival in AML patients. C_FIG

cancer biology↗

bioRxiv2025

Variants in Cas9 and nanos regulatory elements modulate activity and reduce resistance allele formation in homing gene drive

Gene drive is a novel approach for controlling vector borne disease via either population modification or suppression. Even with high efficiency, though, overall drive performance can be reduced by somatic Cas9 expression and by maternal deposition of Cas9, leading to resistance allele formation. The nanos promoter for Cas9 shows very little leaky somatic expression, but it causes high rates of embryo resistance allele formation in Drosophila melanogaster. By truncating the promoter, we reduced rates of embryo resistance to undetectable levels, but germline cutting in females decreased by over half. Germline cutting and successful drive conversion was eventually lost when only the 5' UTR was present, though males still retained moderate germline drive efficiency. Several additional methods were tested to improve performance, including additional suppressor elements to the 3' UTR and introns to increase expression level. The most successful of these was the addition of a second nuclear localization signal, which substantially increased activity when coupled with a full-length or truncated nanos promoter. Overall, these experiments show the potential to modulate Cas9 regulatory elements to achieve desired expression for gene drive applications, while also showcasing the difficulty of obtaining an optimal activity profile.

genetics↗

bioRxiv2025

Development and validation of an ultra-low-cost, open source normothermic ex vivo organ perfusion platform

BackgroundNormothermic ex vivo organ perfusion (NEVOP) promises to catalyze organ preservation, therapeutic discovery, and organ-specific disease modeling. Existing technology platforms remain inaccessible for research due to restricted access to commercial organ perfusion devices, high costs of both devices and proprietary consumables, and steep technical learning curves. Additionally, the available technology is not optimized to perfuse smaller organs such as the kidney. MethodsTo overcome these barriers, a custom NEVOP circuit was developed using recycled, repurposed, and low-cost components. Porcine kidneys and autologous blood were used to iteratively optimize circuit design. A porcine kidney autotransplantation protocol was adapted to evaluate in vivo kidney function after ex vivo perfusion. To pilot the flexibility of this system as a multi-organ platform for ex vivo human biology, non-transplantable human donor kidney, spleen, and pancreas specimens were stably perfused using human blood products and analyzed. ResultsAn ultra low-cost NEVOP system engineered to perfuse porcine kidneys and diverse human organs (kidney, pancreas, and spleen) sustained viable organs for up to 24 hours with evidence of both function and viability. Key innovations included a parallel flow resistor to facilitate low-flow perfusion in non-heparinized organs and a containment bag with adjustable magnets to provide vascular stability and recycling of venous overflow. The circuit costs less than 1,500USD to construct, and porcine kidneys perfused for 24 hours on this platform demonstrated healthy in vivo function upon autotransplantation. ConclusionsCustom NEVOP platforms constitute novel and potentially transformative research platforms which use low-cost and readily available materials. Paired with access to non-transplantable research organs from altruistic donors, this model provides a road map for investigators to advance biomedical discovery and human ex vivo biology.

bioengineering↗

bioRxiv2025

Enhancing HIPEC for Ovarian Cancer using Adjunctive Biomaterials

Ovarian cancer is one of the most lethal gynecological malignancies, with high mortality rates primarily due to late-stage diagnoses and extensive peritoneal metastases. Despite improvements in surgical and chemotherapeutic treatments, the prognosis for advanced ovarian cancer remains poor, highlighting the urgent need for innovative therapeutic approaches. Hyperthermic intraperitoneal chemotherapy (HIPEC) has emerged as a promising treatment, delivering heated chemotherapeutic agents directly into the peritoneal cavity post-cytoreductive surgery. However, HIPEC adoption is limited by three critical complications: suboptimal therapeutic efficacy in resistant tumors; abdominal adhesion formation; and systemic toxicity, including cisplatin-induced nephrotoxicity. This study investigates carbon monoxide gas- entrapping materials (CO-GEMs) as a novel multifunctional adjunctive therapy to address the three HIPEC limitations simultaneously. CO-GEMs effectively encapsulate and deliver carbon monoxide, leveraging the differential effects of CO in cancerous where it has been shown to reduce tumor burden. In ovarian cancer models, CO-GEMs significantly enhanced cisplatin efficacy, reducing the metastatic tumor burden by 46.6% through the downregulation of drug resistance pathways, including the IL-17, TNF, and NF-{kappa}B pathways; ECM receptor interaction, and VEGF signaling. CO-GEMs also prevented peritoneal adhesion formation by suppressing inflammatory cell infiltration and collagen deposition, with a significant reduction in adhesion severity scores. Additionally, enteral CO-GEMs provided significant nephroprotection against cisplatin-induced acute kidney injury, as demonstrated by reduced blood urea nitrogen levels. CO-GEMs represent a promising innovation that simultaneously improves HIPEC therapeutic efficacy, prevents surgical complications, and reduces systemic toxicity. This multifunctional approach addresses multiple clinical limitations of HIPEC, potentially transforming treatment outcomes for patients with advanced ovarian cancer through an enhanced therapeutic index and improved safety profile.

bioengineering↗

bioRxiv2025

OCA-B/Pou2af1 Expression in Mouse T Cells Promotes PD-1 Blockade-Induced Autoimmunity but is Dispensable for Anti-Tumor Immunity

The transcription coregulator OCA-B promotes CD4+ T cell memory recall responses and autoimmunity. OCA-B T cell deletion prevents spontaneous type-1 diabetes (T1D) onset in non-obese diabetic (NOD) mice and blunts T1D in a subset of more aggressive models. However, the role of OCA-B in diabetes induced by treatment with immune checkpoint inhibitors (ICIs), and the role of OCA-B in the control of tumors with and without ICI treatment, has not been studied. Here we show that islet and pancreatic lymph node T cells from T1D individuals express measurable POU2AF1 mRNA. Deletion of OCA-B in T cells fully insulates 8-week-old non-obese diabetic (NOD) mice against ICI-induced diabetes and partially protects 12-week-old mice. Salivary and lacrimal gland infiltration and inflammation were also reduced. Protection was associated with a block in the differentiation of progenitor exhausted CD8+ T cells (TPEX) into terminally exhausted CD8+ T cells (TEX). We show that OCA-B T cell loss preserves anti-tumor immune responses following PD-1 blockade in different tumors and mouse strains. These findings point to a potential therapeutic window in which pharmaceuticals targeting OCA-B could be used to block the emergence of both spontaneous and ICI-induced autoimmunity while sparing anti-tumor immunity. We develop first-in-class small molecule inhibitors of Oct1/OCA-B transcription complexes and show that administration into NOD mice also blocks diabetes emergence following PD-1 blockade. These results identify OCA-B as a promising therapeutic target for the prevention of autoimmunity and immune-related adverse events (irAEs).

immunology↗

bioRxiv2025

A cytosolic function of DNMT1 controls neuronal morphogenesis via microtubule regulation

Proteins traditionally confined to a single cellular compartment are increasingly recognized to exert non-canonical functions in alternative domains. The DNA methyltransferase 1 (DNMT1), classically defined as the maintenance methyltransferase that preserves DNA methylation patterns during replication, exemplifies this versatility. Beyond its canonical role, DNMT1 is highly expressed in the developing and adult brain, where it contributes to transcriptional regulation in postmitotic neurons. Notably, cytoplasmic DNMT1 localization has been observed in neural cells, and emerging evidence links DNMT1 to mitochondrial function with implications for neurodegenerative disease, whereby the underlying functional mechanisms remain to be fully elucidated. Here, we identify a previously unrecognized cytosolic function of DNMT1 in developing cortical excitatory neurons. Through genetic perturbation, proteomics, and high-resolution imaging, we show that DNMT1 regulates dendritic and axonal branching independently of its catalytic activity and nuclear localization. Instead, DNMT1 operates as a cytosolic scaffold interacting with the polarity regulator DOCK7 to modulate Rac1-STMN1 signaling, microtubule dynamics, and organelle trafficking. These findings expand the conceptual framework of DNMT1 from a genome guardian to a dual-compartment regulator that coordinates cytoskeletal remodeling and mitochondrial positioning. Beyond advancing our understanding of neuronal morphogenesis, this work provides mechanistic insight into how DNMT1 mutations may lead to neurodegenerative diseases.

neuroscience↗

bioRxiv2025

Absolute quantification of TCA cycle intermediates in mouse ocular tissues reveals distinct tissue- and sex-specific mitochondrial metabolism

ObjectiveMitochondrial tricarboxylic acid (TCA) cycle is central to energy production and redox balance in the eye, which must sustain high metabolic activity to support vision. Retinal neurons, the retinal pigment epithelium (RPE), cornea, and lens each have distinct physiological roles and metabolic demands, yet the absolute concentrations of key TCA intermediates and their variation by tissue, sex, and time of day are not well-defined. MethodsTargeted gas chromatography-mass spectrometry was employed to quantify the absolute concentrations of TCA cycle metabolites in mouse ocular tissues collected at 10 AM and 2 PM to capture diurnal variations. Key metabolite ratios were subsequently calculated to provide insight into TCA cycle dynamics across eye tissues. ResultsThe retina showed the highest concentrations of TCA metabolites among all ocular tissues, particularly succinate, citrate, and malate, consistent with its high energy demands. The RPE/choroid demonstrated well-balanced intermediates with the highest -ketoglutarate (-KG)/Isocitrate ratio, reflecting its efficient mitochondrial oxidation and reductive carboxylation. Corneal metabolism was featured by dominant malate, especially in females, suggesting a metabolic adaptation for redox regulation and oxidative stress defense. The lens had uniformly low metabolite levels except for succinate, indicating minimal mitochondrial activity under physiologically low oxygen conditions. Notably, both the cornea and lens showed significant sex-dependent and diurnal variations in TCA cycle intermediates. ConclusionThis study demonstrates distinct tissue-specific mitochondrial metabolism in the eye, reflecting the unique functional and biochemical demands of each tissue. These metabolic signatures may underlie their susceptibility to mitochondrial dysfunction in various ocular diseases.

biochemistry↗

bioRxiv2025

Loss of PIKfyve in Rod Photoreceptors and RPE Cells Leads to Endolysosomal Dysfunction and Retinal Degeneration

Photoreceptor outer segment (OS) degradation is primarily mediated by retinal pigment epithelial (RPE) cells through daily phagocytosis of shed distal OS tips. In contrast, much less is understood about the cell-autonomous mechanisms photoreceptors use to clear mislocalized molecules caused by protein misfolding or trafficking defects. Mislocalized or excess rhodopsin that fails to reach the OS is retained in the inner segment or cell body, where it is presumably degraded via the endolysosomal system. We identify PIKfyve, a phosphoinositide kinase that generates PI(3,5)P2, as a key regulator of this pathway. Using Translating Ribosome Affinity Purification (TRAP), we find that PIKfyve is highly expressed in rod photoreceptors. Rod-specific PIKfyve deletion causes progressive retinal degeneration, marked by inner segment vacuolation, elevated LAMP1/2, thinning of the outer nuclear layer, and eventual loss of rod and cone function. Loss of one copy of PIKfyve in rod photoreceptors accelerates degeneration in P23H rhodopsin mutant mice. In RPE cells, PIKfyve loss disrupts phagocytosis and autophagy, leading to accumulation of rhodopsin, LAMP1, LC3A/B, and lipid droplets, along with metabolic disturbances. These findings demonstrate that PIKfyve is essential for photoreceptor and RPE health by regulating lysosomal function, phagocytosis, autophagy and metabolism, and suggest that enhancing PIKfyve activity could be a therapeutic strategy for retinal degenerative diseases.

neuroscience↗

bioRxiv2025

Beyond Ornithine Metabolism in Gyrate Atrophy: Tissue-Specific Proteomic Insights into Neonatal and Adult OAT Deficiency

Ornithine aminotransferase (OAT) links the urea cycle, TCA cycle, and amino acid metabolism by interconverting ornithine to pyrroline-5-carboxylate and glutamate. Mutations in OAT cause hyperornithinemia and predominantly affect the eye, leading to gyrate atrophy of the choroid and retina (GA), a rare inherited blinding disorder. To understand the early molecular changes that make the eye susceptible to damage, we performed quantitative proteomic and metabolomic profiling of liver, retina, and retinal pigment epithelium and choroid (RPE/Cho) from OAT-deficient (Oatrhg) mice prior to detectable vision impairment. In addition to reduced OAT expression and elevated ornithine, methylation-related metabolites such as N(6)-methyl-lysine were altered in all examined tissues of Oatrhgmice. In the liver, ornithine disposal through the urea cycle was enhanced, together with altered expression of detoxification enzymes and histone H2B proteins. In contrast, the retina had minimal proteomic changes but pronounced alterations in amino acid pathways supporting glutamate homeostasis. The RPE/Cho demonstrated the most extensive proteomic changes, particularly in mitochondrial metabolism, cytoskeleton, and extracellular matrix, along with reductions in metabolites involved energy metabolism and antioxidant capacity. Together, these findings highlight common and tissue-specific impacts of OAT on the liver and ocular tissues and provide insight into early molecular changes that contribute to the selective vulnerability of the eye in GA. Proteomics data are available via ProteomeXchange (PXD063614) and metabolomics data via MassIVE repository (MSV000101103).

biochemistry↗

bioRxiv2025

Rapid elicitation of a new class of neutralizing N332-glycan independent V3-glycan antibodies against HIV-1 in nonhuman primates.

Sequential immunization is a promising approach to elicit broadly neutralizing antibodies (bNAbs) against the HIV-1 Envelope (Env). However, available protocols are inefficient and involve multiple immunizations over long periods of time. Here, we present WIN332, a new engineered Env-immunogen that induces a new class of neutralizing N332-glycan-independent antibodies to the conserved V3-glycan epitope of Env after a single bolus immunization in nonhuman primates. WIN332 binds to precursors of canonical human N332-glycan-dependent (Type-I) V3-glycan bNAbs but also of a first-of-its-class N332-glycan-independent (Type-II) V3-glycan bNAb. A single immunization elicits neutralizing serum and monoclonal antibodies that are boosted and affinity matured with a heterologous immunogen. EMPEM analysis of serum antibodies, antibody cloning and cryo-EM analysis reveal that WIN332 elicits N332-glycan-independent antibodies with remarkable sequence and binding similarities with the most potent human type-I and type-II V3-glycan bNAbs. Thus, WIN332 is a promising vaccine candidate to streamline V3-glycan bNAb elicitation.

immunology↗