Biology subjects

Chen, L.

Publications and source records attributed to Chen, L..

At least 217 records · Page 12Linked to original sources

bioRxiv2025

Constructing and Using Cell Type Populations of the Human Reference Atlas

The human body contains [~]27-36 trillion cells of up to 10,000 cell types (CTs) within a volume of [~]62-120 liters (males) and 52-89 liters (females). The Human Reference Atlas (HRA) v2.3 provides a quantitative 3D framework of CTs across 73 reference organs and 1,283 3D anatomical structures (ASs). The HRA Cell Type Population (HRApop) effort has quantified CTs per AS using high-quality single-cell datasets processed through scalable, reproducible workflows and cell type annotation (CTann) tools. HRApop v1.0 includes reference CT populations for 73 ASs (112 when sex-specific) using 662 datasets spatially registered to 230 locations across 17 organs (31 when sex-specific). For 558 single-cell (sc-)transcriptomics datasets (11,042,750 cells), CTs and biomarker expressions were computed using Azimuth, CellTypist, and popV. To test generalizability, 104 sc-proteomics datasets (16,576,863 cells) were integrated. In total, HRApop includes 27,619,613 cells and serves as a healthy reference for researchers aiming to elucidate mechanisms underlying cellular interactions, FTU operations, and cellular and tissue level disease progression, which may facilitate advancements in basic discovery and lead to new therapeutic strategies.

bioinformatics↗

bioRxiv2025

SARS-CoV-2 S protein activates the HIV latent reservoir through the mTOR pathway

During the global COVID-19 pandemic, mRNA vaccines using the S protein as antigen were widely used.Vaccine-induced S proteins can persist in vivo for weeks, triggering low-level immune activation. HIV latent reservoir maintenance is a major challenge for ART therapy, especially when immune pressure is waning. This then raises critical questions for HIV-infected patients: does prolonged exposure to S proteins affect HIV latent reservoir stability? Recent studies have pointed out that S proteins may activate the mTOR signaling pathway, which in turn affects the immune response and metabolic processes of cells. And the mTOR pathway is closely related to the maintenance and activation of HIV latent reservoir. However, how S proteins affect the HIV latent reservoir and the mechanism of activation are unclear. To explore the mechanism of how SARS-CoV-2 S proteins regulate the HIV latent reservoir and to explore whether S proteins regulate the HIV latent reservoir through the mTOR pathway, we constructed an in vitro HIV latent reservoir model for our experiments.To evaluate the potential role of S protein in HIV latent reservoir activation, relevant markers of HIV latent reservoir activation were detected using ELISA, flow cytometry, and RT-qPCR; and the relationship between S protein and mTOR was also detected by WB, CO-IP, and IFC.It was found that S proteins activated the HIV latent reservoir while increasing mTOR expression. It was further observed that mTOR inhibitors significantly inhibited S protein-induced activation of the HIV latent reservoir, and mTOR activators reversed the inhibitory effect of mTOR inhibitors on HIV latent reservoir activation. In summary, we found that S proteins activated the HIV latent reservoir through the mTOR pathway. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/670887v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@f6d87aorg.highwire.dtl.DTLVardef@1fe4cc0org.highwire.dtl.DTLVardef@60eefeorg.highwire.dtl.DTLVardef@1ea9a34_HPS_FORMAT_FIGEXP M_FIG S protein interacts with mTOR and activates the mTOR-p-p70S6K-pS6 pathway, which promotes HIV transcription C_FIG

immunology↗

bioRxiv2025

Glycemia Shift Pancreatic Islets Rhythmicity via δ-α Cell in vivo, Impairment in Diabetes

Blood glucose homeostasis relies on the well-coordinated rhythmic activity of millions of islets throughout the pancreas. Islet rhythmicity is triggered by glucose elevation and mediated by paracrine interactions. However, the dynamics of islet population rhythmicity in healthy and diabetic pancreases in vivo remain poorly understood. Using simultaneous multi-islet Ca2+ imaging (20-100 islets per experiment) in both live mice and pancreatic tissue slices, we systematically studied how glycemia fluctuations and intra-islet paracrine signaling collectively shape the islet rhythmicity. In this study, we report that a transition from Hyperglycemia to Euglycemia induces a coordinated shift from slow to fast islet Ca2+ oscillations (HESF) in vivo. HESF is conserved in pancreatic tissue slices and isolated islets, however, not dispersed single cells in vitro, suggesting a mechanistic link with paracrine interactions. We found HESF arises from -cell activation, which is inhibited by {delta} cells upon glucose elevation. The autonomous islets mostly differ in phase and period at high glucose level. Diabetic mice with disrupted glycemic stability lost HESF both in vivo and in vitro. Interestingly, HESF is preserved in {beta}-cell knockout Gcgr transgenetic mice, both in vivo and in vitro, suggesting HESFs dependence on Glp1r. Indeed, HESF was restored in semaglutide-treated diabetic mice with stabilized glycemic stability. These findings offer a comprehensive understanding of how {delta} and cells influence islet rhythmicity and precisely maintain the stability of blood glucose.

physiology↗

bioRxiv2025

A prevalent huge phage clade in human and animal gut microbiomes

Huge phages are widespread in the biosphere, yet their prevalence and ecology in the human gut remain poorly characterized. Here, we report Jug (Jumbo gut) phages with genomes of 360-402 kilobase pairs that comprise [~]1.1% of the reads in human gut metagenomes, and are predicted to infect Bacteroides and/or Phocaeicola. Although three of the four major groups of Jug phages shared >90% genome-wide sequence identity, their large terminase subunits exhibited only 38- 57% identity, suggesting horizontal acquisition from other phages. Over 1,500 genomes of Jug phages were recovered from human and animal gut metagenomes, revealing their broad distribution, with largely shared gene content suggestive of frequent cross-animal-host transmission. Jug phages displayed high gene transcription activities, including the gene for a calcium-translocating P-type ATPase not detected previously in phages. These findings broaden our understanding of huge phages and highlight Jug phages as potential major players in gut microbiome ecology.

microbiology↗

bioRxiv2025

Cytokine Regulation of Human Antibody Responses to Influenza Vaccines

Studies show that human vaccine responses widely vary. Here, we analyzed data measuring 66 cytokines from 4 different inactivated influenza vaccine (IIV) cohorts over 5 seasons (N=581) and identified a significant correlation between baseline/day 0 serum IL-18 and IFN-{beta} concentrations and the vaccine-specific antibody response on day 28, suggesting these factors may have an adjuvant-like effect. To investigate this further, we tested the impact of 19 cytokines on the development of anti-influenza antibody response when administered together with the IIV vaccine in human tonsil and spleen organoids. We found that Type I IFNs (IFN-{beta} and others), IL-21, IL-12, IL-10, but not IL-18 or IFN-{gamma}, enhanced the antibody response. The live attenuated influenza vaccine (LAIV) induced a stronger antibody response than the inactivated one in organoids. Adding a single cytokine, IFN-{beta}, to IIV stimulation recapitulated most of the live vaccine-specific cytokine activation program. It increased the antibody response of the inactivated vaccine to that of the LAIV. Two other antibody-boosting cytokines, IL-12 and IL-21, were induced by LAIV but not by Type I IFNs, indicating different cytokines can affect different pathways leading to a robust antibody response. We then generated cytokine mRNA lipid nanoparticles (LNPs) to test the effect of cytokines on IIV response in a mouse model of immunization. We found that IL-21-LNPs augmented the quantity and breadth of the antibody responses, while IFN-{beta} LNPs enhanced durability. These findings identified parallel cytokine pathways regulating human vaccine responses and provide a rationale for using cytokines as adjuvants to mimic the effectiveness of live-attenuated vaccines without the risk of viral replication.

immunology↗

bioRxiv2025

Single-nucleus transcriptome analyses uncover a dynamic transcriptional landscape of soybean roots in response to soybean cyst nematode infection

Soybean cyst nematode (SCN, Heterodera glycines) causes substantial yield losses by inducing the formation of specialized feeding structure called syncytia, which arise from extensive reprogramming of host root cells. Despite their pivotal role in SCN parasitism, the molecular mechanisms governing syncytium formation and function remain poorly understood, largely due to the technique challenges in precisely isolating these rare cell types. Here, we employed single-nucleus RNA sequencing (snRNA-seq) to generate a comprehensive, high-resolution transcriptomic atlas of SCN-infected soybean roots, comparing two resistant cultivars (PI 88788 and Forrest) with a susceptible cultivar (Williams 82) across multiple infection stages. By leveraging the well-characterized syncytium marker GmSNAP18, we successfully identified and transcriptionally profiled syncytial cells, and validate the spatial expression of known SCN-resistant genes. Our analyses revealed strong induction of resistance-associated genes specifically enriched in syncytia from resistant cultivars. Functional validation further demonstrated that overexpression of three novel candidate genes significantly suppressed SCN development. Pseudotime trajectory analyses traced syncytium development back to procambial cell origins, uncovering distinct, cultivar-specific developmental pathways. Collectively, this work provides a foundational single cell transcriptomic resource, advances our understanding of syncytial biology and SCN resistance mechanisms, and identifies promising molecular targets for engineering nematode-resistant soybean varieties.

plant biology↗

bioRxiv2025

Clathrin-mediated endocytosis of ERECTA family receptors is essential for proper stomatal development in Arabidopsis

Stomata, specialized structures of plant epidermis, are crucial for gas (e.g. CO2, O2 and H2O) exchange between plants and the environment. Stomatal density and pattern are governed by ERECTA Family (ERf) receptor-like kinases (RLKs)-controlled signaling. Clathrin-mediated endocytosis (CME) is a new mechanism for the internalization of receptor to activate the downstream signaling in animals. Here, we found that mutation of CME components resulted in formation of stomatal clusters and thus increased stomatal index. The adaptor protein 2 {sigma} (AP2{sigma}) subunit of CME interacted with ERf. ERf receptor internalization required CME. Furthermore, the ERf receptor motifs were identified to be recognized by AP2{sigma}. Genetic analysis showed that CME components CHC2, CLC2, and CLC3 acted downstream of EPIDERMAL PATTERNING FACTOR 1/2 (EPF1/2) while upstream of YODA, and worked together with ERf to regulate stomatal development. Consistently, EPF2-induced MAPK activation was significantly reduced in chc2-2 clc2 clc3 and ap2 clc2 clc3 mutants or when the ERf endocytic motifs were mutated, leading to stabilized SPEECHLESS protein and misregulated stomatal lineage progression. Overall, our findings demonstrate that activation of the ERf receptors via internalization is essential for stomatal development, establishing a novel mechanism of CME-mediated receptor internalization activation in plants. Significance StatementStomatal development in Arabidopsis is well characterized, but how EPF-ERf signals activate the MAPK cascade remains unclear. While animals employ clathrin-mediated endocytosis (CME) for receptor internalization and signaling, this mechanism was unknown in plants. We identified CME as a novel regulator of stomatal development. The ERf receptors represent the first identified cargo proteins for AP2{sigma} in plants, undergoing internalization through CME. Clathrin acts downstream of EPF but upstream of YODA, and blocking ERf internalization impairs MAPK activation. Thus, ERf internalization is essential for downstream signaling, revealing a novel CME-dependent activation mechanism in plants.

cell biology↗

bioRxiv2025

The DDIT4L-TOM40-ATP5A pathway suppresses glioblastoma oncogenesis

The characteristics of glioblastoma (GBM), including resistance to cell death and aberrant energy metabolism, are associated with the function of mitochondria. To date, the inherent suppressors that couple mitochondrial function with cell death remain unclear. DNA damage-inducible transcript 4-like (DDIT4L) expression in human gliomas and its association with patient survival was determined by public databases, western-blot and immunostaining. The role of DDIT4L in regulating tumor growth was analyzed using lentivirus and PDX model. The mechanism of DDIT4L on GBM mitochondrial function was determined by different assays. Here, we show that DDIT4L is an endogenous inhibitor of GBM via suppressing mitochondrial function. DDIT4L was expressed at high levels in GBM and transported into mitochondria via single import channel TOM40 other than the import receptors TOM22, TOM20 or TOM70. Then, DDIT4L interacted with the subunit of ATP synthase, inhibited mitochondrial function and induced tumor cell apoptosis. Furthermore, the synthetic peptide DDIT4LV125-P132 suppressed GBM oncogenesis. Based on these results, DDIT4L suppresses GBM oncogenesis through both TOM40- and ATP5A-dependent mechanisms to curb mitochondrial dynamics and induce cell apoptosis, and the DDIT4LV125-P132 peptide is a potential therapy for GBM.

cancer biology↗

bioRxiv2025

Engineering light robustness: Adaptive evolution uncovers new genetic determinants of HL tolerance in Synechocystis

Excess light absorption causes severe photo-oxidative damage and limits photosynthetic efficiency. Enhancing high-light (HL) tolerance and energy utilization in photosynthetic cyanobacteria holds great potential for sustainable biomanufacturing. Here, using Synechocystis sp. PCC 6803 (Syn6803) as a model, we generated eight independently evolved strains tolerant to hyper-HL (2000 mol photons/m2/s) through approximately two years of natural adaptive laboratory evolution (ALE). Remarkably, these strains circumvented common evolutionary trade-offs; all demonstrated markedly enhanced quantum yields and photosynthetic performance, with four strains exhibiting a 121.71%-168.36% increase in biomass accumulation under HL. When equipped with the heterologous sucrose transporter CscB, sucrose productivity under HL increased by up to 208.33%. Whole-genome resequencing unveiled a total of 77 mutations across the eight lineages, uncovering a diverse polygenic landscape for HL adaptation. Reverse genetics combined with structural modeling highlighted pivotal roles for regulatory and structural loci, including slr0758 (KaiC1) and slr1513 (SbtB), in modulating circadian rhythmicity and inorganic carbon uptake to prevent photo-oxidative overload. Overall, our findings uncover diverse evolutionary routes toward high-light acclimation and provide superior cyanobacterial chassis for photosynthetic biotechnology.

microbiology↗

bioRxiv2025

Palmitoylation-dependent activation of NADK promotes NADP+ synthesis and tumorigenesis

NAD kinase (NADK) is the sole cytosolic enzyme that catalyzes the synthesis of nicotinamide adenine dinucleotide phosphate (NADP+) from NAD+. NADP+ is essential for anabolic reactions and redox balance. Here, we show that palmitate facilitates NADP+ synthesis by enhancing NADK palmitoylation and activity. NADK is post-translationally S-palmitoylated on three cysteine residues (Cys22, Cys23, and Cys26) within the amino-terminal domain by the protein-acyl transferase ZDHHC5, which stimulates NADK activity. Fatty acids activate NADK by enhancing its palmitoylation and relief of an autoinhibitory function inherent to its amino terminus. Furthermore, ZDHHC5-/- mice showed defect in NADK palmitoylation and NADP+ production. Clinically, elevated expression of ZDHHC5 in pancreatic cancer patients was associated with increased NADK palmitoylation and correlated with poor prognoses for patients. These data reveal that fatty-acid and ZDHHC5-mediated palmitoylation has a critical role in NADP+ synthesis and cancers.

cancer biology↗

bioRxiv2025

Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows

Activation-induced marker (AIM) assays are a promising tool to track antigen-specific T cells, but methodological heterogeneity between research groups hinders their clinical utility. To evaluate AIM assay reproducibility, we conducted a multi-site study of SARS-CoV-2 and cytomegalovirus AIMs. We found inherent variability in AIM assays and optimized approaches to enhance reproducibility, including a standardized workflow to minimize technical variability and a generalizable Box-Cox transformation-based statistical method to optimize calculation of AIM stimulation responses. We further standardized AIM data analysis through development of automated flow cytometric gating software, which had superior reproducibility compared to manual analysis. We also characterized antigen-specific Tregs, finding that gating on a combination of CD134, CD137, FOXP3 and HELIOS optimally detects antigen-specific cells. The combined methodology results in a high degree of reproducibility within and between research groups and provides a comprehensive foundation from which standardized AIM assays can be implemented across diverse scientific and clinical settings. MOTIVATIONReliable detection of antigen-specific T cells is critical to understand immune responses to infection and vaccination, and has translational potential to monitor T cell responses across diverse clinical settings. Activation-induced marker (AIM) assays offer a variety of advantages over methods such as ELISPOT and tetramers, but are limited by methodological heterogeneity between research groups and a lack of standardized protocols. As such, the degree to which AIM assay results are reproducible is unknown. Key variables such as cell source, media, stimulation time, marker selection for CD4+ T cells, CD8+ T cells and regulatory T cells; as well as data analysis parameters such as flow cytometric gating strategies and mathematical correction for background AIM+ frequencies in unstimulated control samples, have not been rigorously studied. To address this, we comprehensively characterized variability in AIM assays, including within and between operators and across multiple research centres, and sought to optimize a standard AIM workflow to enhance reproducibility at both the experimental and analytical levels.

immunology↗

bioRxiv2025

Scar-associated endothelial-stellate cellular crosstalk drives fibrosis resolution in MASH

Fibrosis, or scarring, can affect many organs including liver, lung, heart, kidney, intestines etc. and is responsible for [~]40% of mortality in the industrialized world. Compared to other organs, fibrosis in the liver typically resolves when the source of injury is extinguished. Elucidating the molecular mechanisms that underlie spontaneous fibrosis resolution in the liver may lead to novel antifibrotic strategies for all organs. In this study we established a robust mouse model of fibrosis regression in MASH (Metabolic dysfunction-Associated Steatohepatitis), a highly prevalent chronic liver diseases worldwide, and performed single cell and in situ molecular profiling of the liver to define novel drivers of fibrosis regression. As fibrosis regressed, we detected a reduction of inflammatory cells and an expansion of endothelial cells. Prediction of cell-cell communication using the Calligraphy pipeline identified a Wnt9b-Sfrp2 crosstalk that emerges as fibrosis resolved in our model. To establish the Wnt9b-Sfrp2 crosstalk as a driver of fibrosis resolution we treated mice with recombinant Sfrp2, which slowed spontaneous fibrosis regression compared to vehicle treated mice. From our single cell datasets we identified a subset of endothelial cells, termed "Endo4", as the source of Wnt9b. Immunostaining of the Endo4 marker VWF using tissue clearing and 3D imaging revealed VWF+ vasculature enveloped by activated hepatic stellate cells (HSCs) that penetrated deep into the fibrotic septa, establishing Endo4 as de facto scar-associated endothelial cells and providing a structural basis of their cellular crosstalk with HSCs. Finally, using a recently developed in situ protease activity screen, prominent serine protease activity co-localized with both scar-associated Endo4 cells and HSCs. In summary, we uncovered an WNT-dependent endo-stellate crosstalk within the fibrotic niche as a novel regulatory node underlying murine MASH fibrosis regression, and a promising therapeutic target.

molecular biology↗

bioRxiv2025

ODFormer: a Virtual Organoid for Predicting Personalized Therapeutic Responses in Pancreatic Cancer

Pancreatic cancer (PC) patient-derived organoids (PDOs) faithfully recapitulate therapeutic responses but face clinical translation barriers, including high costs and technical complexity. To address these problems and the lack of frameworks for PDO-based drug-response assays, we developed ODFormer, a computational framework that simulates PC PDOs to predict clinically actionable, patient-specific drug responses by integrating transcriptomic and mutational profiles. ODFormer first employed two encoders, pretrained on 30,000 pan-cancer bulk transcriptomics and 1 million PC single-cell profiles respectively, to distil tissue-and organoid-specific representations. Then, trained on our curated 14,000 PDO drug-response assay (across 183 PDOs and 98 drugs) using a transformer-augmented hybrid contrastive network, ODFormer significantly outperformed state-of-the-art methods, notably achieving a PCC >0.9 in predicting standardized drug response. Multi-cohort retrospective analyses further demonstrated that ODFormer-guided personalized therapy significantly improves clinical outcomes, without requiring physical organoid assays. Furthermore, ODFormer identified novel clinico-biological PC subtypes and revealed therapy resistance biomarkers by stratifying predicted responders and non-responders. These were validated using independent datasets including TCGA-PDAC. Notably, ODFormer-guided treatment efficacy showed high concordance with prospective clinical responses by CA19-9.

bioinformatics↗

bioRxiv2025

BrainBeacon: A Cross-Species Foundation Model for Single-cell Resolved Brain Spatial Transcriptomics

The brains functional complexity emerges from spatiotemporal heterogeneity in molecular, cellular, and circuit organization, shaped by evolution, aging, and disease. Single-cell spatial transcriptomics provides a transformative framework to decode this complexity, resolving cell-type-specific expression patterns, region-specific regulation, evolutionary divergence, and disease-associated disruptions. Despite recent advances in spatial transcriptomics, a unified foundation model capturing cross-species, whole-brain cellular architecture remains lacking. Here, we introduce BrainBeacon, the first cross-species foundation model for whole-brain spatial transcriptomics. Trained on 133 million spatially resolved cells covering a total area of 210194 mm2 from human, macaque, marmoset and mouse brains profiled across five major platforms, BrainBeacon integrates gene expression rankings, spatial cellular organization, and evolutionarily conserved genetic relationships into a unified transformer-based architecture. A two-stage training strategy captures both intra-cellular transcriptional dependencies and inter-cellular spatial interactions, yielding biologically grounded and interpretable representations of brain structure. Subsequently, BrainBeacon enables cross-species alignment of brain cell types and anatomical regions, facilitating integrative analyses of conserved and divergent cellular architectures. Moreover, it uncovers spatial regulatory mechanisms of aging by predicting bidirectional perturbation responses between cells and their local niche. Together, BrainBeacon serves as a foundational cross-species model for whole-brain spatial transcriptomics, enabling virtual brain atlas construction, systematic in silico perturbation studies, and mechanistic insights into brain disease pathogenesis.

bioinformatics↗

bioRxiv2025

A Compact Two-Photon Module for Simultaneous Dual-Depth, Multi-Region in vivo Brain Imaging

Understanding how distributed brain regions interact across cortical depths is crucial for uncovering the neural basis of behavior and cognition. Two-photon microscopy (2PM) excels at imaging the superficial layer, while three-photon microscopy (3PM) penetrates deep tissues but lacks sufficient speed for capturing fast superficial dynamics. Here, we present a compact two-photon imaging module (2PIM) that seamlessly integrates with existing 3PM to achieve simultaneous, high-speed imaging of superficial and deep brain regions, while preserving the deep tissue penetration of 3PM. This dual-modality system allows for remote and independent control of imaging planes in both axial and lateral dimensions. Leveraging this technology, we discovered context-dependent modulation of hippocampal CA1 and visual cortex responses to identical tactile stimuli, layer-specific temporal dynamics in sensory cortex during air puff stimulation, and depth-divergent neuronal activity patterns in motor cortex across acute-to-chronic pain. These results highlight the systems potential for dissecting the spatiotemporal coordination of distributed brain circuits.

bioengineering↗

bioRxiv2025

Ligand-induced ubiquitination regulates endocytosis and homeostasis of the ERECTA receptor kinase for stomatal development

Stomata, valves on the plant epidermis, control gas and water vapor exchange. The patterning and spacing of stomata are primarily regulated by the ERECTA leucine-rich repeat receptor kinase (LRR-RK). Ubiquitination is a crucial mechanism to regulate the homeostasis of RKs by impacting their protein stability and localization. It has been shown that plant U-box ubiquitin E3 ligases, PUB30 and PUB31, act as key attenuators of the ERECTA signaling pathway. However, the molecular link between ubiquitination and function of ERECTA remains unclear. Here, we reveal that perception of the peptide ligand EPIDERMAL PATTERNING FACTOR2 (EPF2) by ERECTA induces the K63-linked ubiquitination of ERECTA mediated by PUB30 and PUB31. We further identify the specific ubiquitination sites within the juxtamembrane and kinase domains of ERECTA. Importantly, the site-directed mutagenesis of these K63-linked ubiquitination sites overly inhibited stomatal development, indicating that the ubiquitination-deficient ERECTA is hyperactive. The ubiquitination-deficient ERECTA stably accumulates at the plasma membrane and fails to internalize upon EPF2 application. Our findings thus uncover a mechanism by which ligand-induced ubiquitination orchestrates receptor homeostasis and endocytosis for proper stomatal patterning and differentiation.

plant biology↗

bioRxiv2025

Structure and iron-transporting mechanism of brain organic cation transporter 1

Brain organic cation transporter 1 (BOCT1), also known as the solute carrier family 22 member 17 (SLC22A17) or the receptor for lipocalin-2 (LCN2), plays critical roles in health and disease. Its deficiency in mice results in early postnatal mortality and severe neurogenesis impairments. Despite its importance in physiology and pathophysiology, BOCT1s structure and transport mechanism remain elusive. Here, we integrate cryo-electron microscopy (cryo-EM), functional assays, biochemical experiments, and molecular dynamics simulations to elucidate the structure, substrate recognition, and transport mechanism of mouse BOCT1 (mBOCT1). The high-resolution cryo-EM structure reveals a distinctive N-terminal domain with a unique folding pattern dominated by a transmembrane loop atop TM6 (TML6), diverging from both known structures of SLC22 transporters and AlphaFold predictions. Notably, mBOCT1 functions as a high-capacity, low-affinity iron transporter independent of LCN2 binding. Iron transport is facilitated by a substrate gating mechanism involving TML6. These findings establish a structural basis for BOCT1s role as an independent iron transporter, enhancing our understanding of the transport mechanisms within major facilitator superfamily (MFS) transporters and providing new insights into brain iron homeostasis.

biophysics↗

bioRxiv2025

Electric fields trigger ceramide-dependent budding of exosome vesicles into multivesicular endosomes and boost the generation of exosomes

Exosomes hold immense therapeutic and diagnostic potential, yet their clinical translation remains constrained by low yields. Physiological electric fields (EFs), naturally occurring during wound healing and embryo development, have unexplored roles in exosome biogenesis. Here, we demonstrate that physiological-strength EFs (Direct Current, 50-200 mV/mm) dramatically enhance cellular exosome secretion, achieving a nearly 100-fold increase in a strength-dependent manner. Mechanistically, EFs augmented intraluminal vesicle formation within multivesicular bodies. We proposed a novel EF-driven membrane electroassembly and vesiculogenesis hypothesis and tested it using giant plasma membrane vesicles. We reconstructed EF-induced membrane remodeling and observed EFs driving inward budding of nanoscale exosome-like vesicles. Notably, alternating current EFs (50-400 Hz) exhibited significantly diminished efficacy compared to direct current EFs, highlighting membrane electropolarization-dependent modulation. Pharmacological inhibition of the lipid raft and ceramide almost abolishes EF-induced exosome secretion. Inhibition of the PI3K partially attenuated EF-triggered exosome release. Our findings not only unveil EFs as a potent physiological regulator of exosome secretion but also establish a novel, high-yield production strategy leveraging bioelectric cues. Significant statementWe reported that the bodys natural bioelectricity, the faint electric currents in healing wounds and embryo development, can trigger cells to release 100 times more "natural nanoscale vesicle couriers" called exosomes. This pure physical method is far more efficient than current available technologies, paving the way for breakthroughs in cancer early detection, stem cell therapies, and precision medicine. We found that gentle electric fields can "blow" the cell membrane to release exosomes like "soap bubbles". This explains how bioelectricity controls cell communication during development and wound healing. In short, harnessing the bodys hidden "electric language" unlocks a fast and natural way to mass-produce exosomes.

cell biology↗