Biology subjects

Hong, S.

Publications and source records attributed to Hong, S..

At least 19 recordsLinked to original sources

bioRxiv2026

A Chemigenetic Fluorescence Lifetime Biosensor for Dopamine Sensing

Measuring the precise dynamics of specific neuromodulators in neurons is key to understanding how information is transmitted and processed in the brain. Despite the growing use of intensity-based neural activity sensors such as dLight in recent years, it remains challenging to monitor the dynamics of multiple neurochemicals simultaneously in live neurons. Unlike intensiometric sensors that are susceptible to artifacts caused by fluctuations in excitation power and rapid photobleaching, fluorescence lifetime-based sensors are less affected by these factors and can provide more stable measurements. Here we introduce dHaloLife635, a novel genetically encoded fluorescence-lifetime-based dopamine (DA) sensor that exhibits a fluorescence lifetime change up to -0.20 ns upon dopamine binding. dHaloLife635 consists of a G protein-coupled dopamine receptor (DRD1), a circularly permuted HaloTag (cpHaloTag), and a cell-permeable dye JF635 functionalized with a HaloTag ligand, a reactive chloroalkane linker (JF635-HTL). When dopamine binds to the dHaloLife635 sensor, it induces a conformational change in the cpHaloTag that alters the local environment of the conjugated dye, thereby changing its fluorescence lifetime. In live HEK293T cells and primary neurons, dHaloLife635 responds selectively to dopamine but not to serotonin, L-DOPA, or GABA, demonstrating excellent specificity. This dHaloLife635 sensor represents an initial step toward the establishment of a broader family of chemigenetic lifetime sensors that exhibit distinct fluorescence lifetime signatures upon binding their respective targets, thereby enabling highly specific and sensitive, real-time monitoring of multiple neurochemical dynamics in vivo.

bioengineering↗

bioRxiv2026

Cascaded Nano-ring Dialyzer for Continuous Regenerative Peritoneal Dialysis

End-stage kidney disease (ESKD) requires lifelong kidney replacement therapy, yet conventional hemodialysis and peritoneal dialysis remain limited by intermittent treatment, restricted mobility, and repeated dialysate exchange. Here, we developed a continuous regenerative peritoneal dialysis platform based on a nano-ring-assisted nanoelectrokinetic dialyzer for portable artificial kidney applications. We first identified key geometric and electrokinetic parameters governing the removal performance of nano-ring meshes, including mesh dimension, nano-ring coating thickness, the number of cascaded nano-rings, and the relative orientation between fluid flow and the applied electric field. We then developed a single nano-ring dialyzer operating at 1.33 mL/min and scaled the treatment capacity to approximately 10 mL min/min through parallel integration of multiple modules. In a canine model, the closed-loop system achieved approximately 10 % reduction in circulating uremic toxin levels during continuous treatment. We further improved the biocompatibility of the regenerated dialysate by integrating plate-type electrodes, activated carbon, bicarbonate buffering, and UV-C treatment. In an unanesthetized canine model, the improved system sustained continuous toxin removal for 4 h while major hepatic and inflammatory markers remained within recoverable ranges. These results demonstrate that cascaded nano-ring enables continuous dialysate regeneration with scalable throughput, reduced dependence on fresh dialysate, and in vivo control of uremic toxins, providing a technological foundation for portable or wearable regenerative peritoneal dialysis systems.

bioengineering↗

bioRxiv2026

TM6SF2 binds cholesterol, interacts with apolipoprotein B, and promotes hepatic lipid secretion

A missense variant in TM6SF2 (transmembrane 6 superfamily member 2, TM6SF2E167K) is a major risk factor for steatotic liver disease1, while protecting against coronary artery disease2. TM6SF2 is a polytopic resident protein of the smooth endoplasmic reticulum (ER) and ER-Golgi intermediate compartment that promotes lipidation of hepatic ApoB-containing lipoproteins before secretion into the circulation. Here, we used cryo-electron microscopy (cryo-EM) to determine the structures of TM6SF2 and TM6SF2E167K at 3.64 [A] and 3.58 [A] resolution, respectively. TM6SF2 comprises 10 transmembrane helices that bind a single cholesterol molecule within a transmembrane cavity. The protein assembles into homodimers and homotetramers that interact with ApoB. Structural and biochemical analyses show that the E167K substitution reduces cholesterol binding and ApoB interaction without disrupting overall protein structure. Expression of wild-type, but not mutant, TM6SF2 restores hepatic triglyceride secretion in TM6SF2-deficient hepatocytes. Together, these findings establish the first structural framework for the bulk lipidation step in hepatic lipoprotein biogenesis, the principal pathway for hepatic triglyceride and cholesterol export into the circulation.

cell biology↗

bioRxiv2026

Proteomic Profiling Identifies CLDN3 as a Tumor-Selective Therapeutic Target in Small Cell Lung Cancer

Small cell lung cancer (SCLC) remains a highly lethal disease with limited targetable surface antigens beyond delta-like ligand 3 (DLL3). We sought to systematically identify and validate tumor-selective cell-surface targets in relapsed SCLC. To this end, we developed an integrated proteogenomic pipeline combining single-cell RNA sequencing, combinatorial optimization, mass spectrometry-based proteomics, and immunohistochemistry to systematically map the SCLC surfaceome of 49 tumors across 25 patients with relapsed SCLC. Using this approach we found claudin-3 (CLDN3) to be a consistently expressed and tumor-selective antigen, with broader coverage than DLL3, which is the current clinical benchmark. CLDN3 was highly expressed across various treatment states, while maintaining low expression in most nonmalignant tissues. Functional validation using a novel CLDN3-specific monoclonal antibody (ABN501) demonstrated NK cell-mediated cytotoxicity in vitro and tumor regression in vivo, as well as a favorable safety profile. These findings support clinical development of CLDN3-directed therapies and demonstrate the utility of integrated proteogenomic approaches for antigen discovery in solid tumors.

cancer biology↗

bioRxiv2026

A Modular T7 RNAP Expression Architecture for Orthogonal Multigene Expression in Yeast

Orthogonal gene expression systems based on bacteriophage-derived T7 RNA polymerase (T7 RNAP) offer a promising strategy for decoupling engineered transcription from host regulatory networks. Although recent advances have enabled productive T7 RNAP-mediated expression in Saccharomyces cerevisiae, strategies for independently controlling multiple genes within a shared T7 transcriptional framework remain limited. Here, we developed a modular pYTK-compatible T7 RNAP expression architecture that separates overall transcriptional capacity from gene-specific control of protein output. We established interchangeable T7 promoter, terminator, polyadenylation signal, and 5 untranslated region (UTR) parts for combinatorial assembly. Analysis of expression cassette dosage showed that transcript abundance and expression output scale with cassette copy number, with high-copy constructs producing transcript levels comparable to those driven by the GAL1 promoter. To enable gene-specific tuning, we constructed a library of fourteen 18 nt UTR elements that generated a [~]38-fold range of protein output from a common T7 promoter. Application of this library to a three-gene {beta}-carotene biosynthesis pathway enabled [~]20-fold variation in product titer through UTR assignment alone, without altering promoter identity or gene copy number. Together, these results establish a modular T7 RNAP expression framework in which cassette dosage defines overall transcriptional capacity while interchangeable UTR elements enable gene-specific tuning of expression output, providing a practical strategy for multigene expression control in yeast.

Synthetic Biology↗

bioRxiv2026

Killer-cell dominance dichotomy governs tumor immune networks and stratifies inflamed cancers

Cancer immunotherapy benefits remain limited, even among "hot" tumors with high killer lymphocyte infiltration. Here, we investigated the population-level architecture of killer cells based on nearly 5,000 pan-cancer scRNA-seq samples, together with orthogonal validation by spectral cytometry and spatial transcriptomics. Unlike the prevailing "hot-cold" paradigm, which assumes coordinated infiltration of multiple cytotoxic lineages, we uncovered a conserved framework wherein terminal cytotoxic immunity in individuals or malignancies diverges into states dominated by either exhausted CD8+ T cells (Tex) or CD56dimCD16hi NK (NK1) cells. Despite the complexity of the tumor microenvironment, Tex-NK1 divergence governs the primary axis of tumor-intrinsic and tumor-extrinsic variance. Distinct from the conventional view that NK cells positively contribute to immunotherapy efficacy, NK1-skewed tumors, although highly cytolytic, are refractory to current immune checkpoint blockade regimens. This killer divergence defines a foundational axis of cancer immunity and provides a resource for prioritizing next-generation targets for NK-directed immunotherapy.

immunology↗

bioRxiv2026

HLA-G engineering reprograms CAR-T cells with an immune privilege

Personalized T cell therapy empowered by chimeric antigen receptor (CAR) that recognizes specific tumor antigen has cured numerous blood cancer patients since its initial approval in 2017. However, its access to a broader population has been limited by the unavailability of an off-the-shelf product derived from an allogeneic donor that can evade immune rejection, which is mediated by polymorphic class I and class II human leukocyte antigens (HLAs). Since class II HLAs are only expressed in specialized antigen-presenting cells but not T cells, it might suffice to evade T cells by deleting the common class I HLA light chain Beta-2 Microglobulin (B2M) (1). However, B2M-deficient cells can trigger a "missing-self" response to activate natural killer (NK) cells (2), a second function that was evolved to compensate loss of T cell response. Inserting a less polymorphic class I HLA gene encoding a known NK inhibitory ligand, namely HLA-E or HLA-G (3), into the B2M locus so that the endogenous B2M expression is disrupted could theoretically allow evasion of both T and NK cells. Despite being a seemingly better candidate in that HLA-G is uniquely expressed in immune-privileged sites such as the placenta with a believed function in protecting the fetus from immune rejection by the pregnant mother, whereas ubiquitously-expressing HLA-E is known to bind both inhibitory and activating NK receptors (4, 5), only HLA-E engineering has been attempted yet without convincing success in vivo (6, 7). Here, we generate an off-the-shelf CAR-T product with B2M replaced by a gene fusion encoding an HLA-G single-chain trimer under minimally impacted B2M epigenetic landscape, and observe its immune evasion property and a tumor-inhibitory function that is equivalent to its autologous control using a humanized mouse model for the first time with T and NK cells reconstituted from a donor with a distant HLA haplotype. HLA-G engineering may thus reprogram T cells into an immune-privileged state that can be utilized for all cell-based therapies.

immunology↗

bioRxiv2026

A High-Throughput Platform for Rapidly Adapting DNA Aptamers to SARS-CoV-2 Evolution

Rapid pathogen evolution threatens public health by eroding the effectiveness of vaccines, therapeutics, and diagnostic tools. Although spike protein targeting monoclonal antibodies (mAbs) were developed within 10-12 months of the initial outbreak to serve as key theranostic agents, their redesign has struggled to keep pace with viral evolution, rendering many neutralizing antibodies ineffective. Here we demonstrate a novel platform that combines a random-rational hybrid library diversification with high-throughput MiSeq screening to rapidly reprogram aptamers against emerging SARS-CoV-2 spike variants. Interactions between 3 different spike proteins and 11,806 unique aptamer variant designs were profiled within a few days. Starting from a 40-nt aptamer originally selected against wild-type (WT) spike protein, our screen identified a Delta-binding mutant with a 4-fold affinity improvement and an Omicron-binding mutant that converted undetectable binding into nanomolar affinity. We also identified a WT-selective mutant with substantially reduced affinity for Delta, as well as previously unrecognized bases that critically contribute to spike recognition. Integrating high-throughput binding data with molecular dynamics simulations further revealed sequence-dependent structural features underlying variant-specific aptamer-spike interactions. Finally, we developed a sensor based on the identified WT-selective aptamer mutant, enabling highly specific detection of the WT spike protein with robust performance. Together, this work establishes a rapid and adaptable aptamer engineering platform for rapid adaptation of aptamers to evolving pathogens in future pandemics.

bioengineering↗

bioRxiv2026

Cholesterol-dependent lysosomal docking of mTORC1 mediated by TM6SF1

The Transmembrane 6 Superfamily (TM6SF) comprises two members: TM6SF1, a ubiquitously expressed lysosomal membrane protein of unknown function, and TM6SF2, an endoplasmic reticulum protein required for bulk lipidation of Apolipoprotein B-containing lipoproteins. Here we used cryo-electron microscopy (cryo-EM) to determine the structure of human TM6SF1 at 2.9- [A] resolution. TM6SF1 forms a polytopic homodimer, with each protomer comprising ten transmembrane helices (TMs). TMs 1-6 form a pocket that accommodates a cholesterol molecule. Cell-based assays revealed that loss of TM6SF1 perturbs mTORC1 signaling, resulting in reduced phosphorylation of S6 kinase 1 and 4E-BP1 and constitutive activation of Transcription Factor EB (TFEB) and that cholesterol is required for these effects. Biochemical analyses support the model that TM6SF1 directly engages LAMTOR1, a component of Ragulator complex, in a cholesterol-dependent manner. Together, these findings identify TM6SF1 as a lysosomal cholesterol binding protein involved in regulating mTORC1 signaling. Significance StatementWe determine the cryo-EM structure of TM6SF1 and show that it is a lysosomal membrane protein that associates with LAMTOR1, a component of Ragulator complex, in a cholesterol-dependent manner. Loss of TM6SF1 impairs mTORC1 signaling and promotes nuclear accumulation of TFEB, suggesting a role for TM6SF1 in lysosome-dependent nutrient sensing and signaling. Together, our findings suggest that TM6SF1 is involved in the regulation of mTORC1 activity and provide insight into the functional diversification of the TM6SF protein family.

cell biology↗

bioRxiv2026

A novel fracture lattice in spiny mouse skin facilitates tissue autotomy and regeneration

Autotomy is a unique phenotype whereby an animal sheds a body part to escape predation1-3. The timing and location of autotomy are tightly regulated by preformed planes of weakness (aka fracture planes) which facilitate tissue loss. While autotomy is often followed by regeneration, these phenotypes are rarely reported in mammals4-9. A notable exception are spiny mice (Acomys) which exhibit skin autotomy and more remarkably, complete tissue regeneration10-14. Presently, mechanisms underlying autotomy and complete regeneration in Acomys skin remain elusive. Here, we report the discovery of a honeycomb-like fracture lattice in Acomys skin whose design directs tissue destruction but also facilitates regenerative healing. Unlike the single continuous surface of a fracture plane, this fracture lattice consists of a three-dimensional array of hexagonal units whose boundaries guide tissue breakage. Moreover, we identify collagen VI as the main constituent of the fracture lattice and find that it is distinctly arranged to initiate fracturing and propagation of skin tearing. By preconditioning the tissue for autotomy, the fracture lattice dampens the damage-induced inflammatory response but also upregulates a pro-regenerative gene signature, accelerating skin appendage regeneration. Lastly, we discovered the key role of spiny hairs in fracture lattice formation, as inhibiting their development leads to abnormal pattern formation and changes in skin fracture mechanics. Our results present a novel example of a uniquely evolved structural adaptation in mammalian skin that links tissue patterning, autotomy and regeneration. We expect that the application of a modular compartment structure to artificial skin and other organ engineering may enhance resilience to injury and facilitate efficient regeneration.

developmental biology↗

bioRxiv2026

Mechanistic insights into the color transformation of a non-FRET substrate for RNase activity detection

DNA-templated silver nanoclusters (DNA/AgNCs) have created a new class of non-FRET DNase substrates, termed Subak, that exhibits a color change upon DNase digestion. Although Subak substrates offer advantages such as ratiometric readouts and low manufacturing costs over traditional FRET substrates, the mechanism governing AgNC color switching remains unclear. Here, using a site-specific cleavage strategy, we identify color-switching hotspots and demonstrate that AgNC transformation can be controlled by the cleavage positions within the nucleic acid host. Our data support a cleavage-driven reorganization of the AgNC coordination environment, converting a non-emissive precursor into a red-emitting cluster, rather than direct enzyme-cluster interactions. Leveraging this insight, we engineer rSubak, an RNA-incorporated Subak that displays 95 nm red shift (530 to 625 nm) upon RNase cleavage. In amplification-free CRISPR/Cas13 assays for SARS-CoV-2, influenza A (A/H5N1), and measles viruses (MV) detection, rSubak achieved a limit of detection of 0.3 pM, superior to that of the commercial RNaseAlert ([~]250 pM). Collectively, our results establish Subak as a generalizable, non-FRET platform for sensitive ratiometric reporting the activities of diverse nucleases.

bioengineering↗

bioRxiv2026

Intermittent AP-1 activation in muscles contributes to exercise-induced health benefits

Regular physical exercise extends healthspan, yet the molecular mechanisms that translate intermittent contractile stress into lasting benefit remain incompletely understood. Using global nuclear run-on (GRO-seq) in mouse skeletal muscle after treadmill running, we profiled enhancer RNA (eRNA), a sensitive marker of enhancer activity. Activation protein-1 (AP-1), a family of pioneering factors for senescence, emerged as the top transcription factor with motif enrichment in exercise-activated enhancers. Our screen in the contracting C2C12 myotubes pinpointed cFos/JunD as the primary AP-1 factor responsible for contraction-induced transcriptional changes. Muscle-specific overexpression of A-Fos, a dominant-negative mutant of cFos, disrupted transcriptomic responses to exercise and attenuated exercise-mediated improvement in muscle functions. Interestingly, intermittent but not continuous overexpression of cFos/JunD in mouse muscles mimicked exercise-induced transcriptomic changes, increased mitochondrial volume density, enhanced muscle strength and fatigue resistance, and improved glucose tolerance. These results define a transcriptional regulatory signaling pathway linking exercise intermittency to beneficial adaptations and highlight the necessary recovery cycles in training. The paradoxical anti- and pro-aging roles of AP-1 offer insights into the timing and dynamics of stressors and stress responses in shaping senescence and healthspan.

molecular biology↗

bioRxiv2025

A Machine Learning- 3D Microvessel Platform Identifies Kinase Targets Restoring Blood-Brain-Barrier Endothelial Integrity.

Disruption of the brain endothelial barrier is a hallmark of traumatic brain injury (TBI), and contributes to cerebral edema, coagulopathy, and delayed neurological deficits, yet there are no mechanism-guided therapies that directly stabilize human brain vessels. Here we used a three-dimensional (3D) human brain endothelial vessel platform with quantifiable macromolecular barrier function to assess responses to thrombin and plasma from patients with TBI. This endothelial-focused 3D system isolates the central cellular regulator of blood-brain barrier function and enables mechanistic analysis of endothelial signaling and drug responses as a foundation for personalized vascular therapeutics. By integrating this platform with machine learning-guided kinase analysis, we identified kinase pathways associated with barrier stabilization versus disruption and nominated inhibitors that reversed thrombin- and TBI plasma-induced leakage as well as compounds that exacerbated endothelial loss. This work establishes an important first step toward precision vascular therapeutics in TBI, by linking patient-derived plasma phenotypes to specific endothelial barrier responses and pharmacologically tractable kinase signaling, and it provides a generalizable framework for mechanistic drug discovery across neurovascular disorders. SIGNIFICANCE STATEMENTBlood-brain barrier (BBB) disruption drives cerebral edema, coagulopathy, ischemia, and neurological deficits after TBI. Targeting kinases as therapeutics is promising but challenged by complex inhibitor profiles and patient variability. We present a machine learning-guided platform built on 3D perfusable human brain endothelial-centric microvessels to systematically screen and select kinase inhibitors that reverse TBI-induced endothelial barrier breakdown. By pinpointing key kinase and gene regulators and demonstrating restoration of endothelial barrier integrity in patient-derived plasma models, our approach provides a transformative strategy for stabilizing the endothelial barrier after TBI and offers a versatile framework for therapeutic discovery across neurovascular disorders.

bioengineering↗

bioRxiv2025

Enhanced liver regeneration via targeted mRNA delivery for partial in vivo reprogramming

Recent studies suggest that injury-induced dedifferentiation, which leads to the formation of injury-responsive cells, contributes significantly to tissue repair across various organs, including the liver. Utilizing Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc: OSKM) for in vivo partial reprogramming generates injury-responsive cells in the intestine, mirroring those derived from injury-induced dedifferentiation. Thus, the transgene induction of OSKM or viral delivery of Oct4, Sox2, and Klf4 shows promise in facilitating tissue regeneration in the intestine, liver, skeletal muscle, and retina. Herein, we demonstrated that transient OSKM induction produces two distinct liver progenitor-like cell populations. One of these populations resembles liver progenitor-like cells (LPLCs) generated by acute acetaminophen (APAP) injury without triggering immune responses. To explore in vivo reprogramming as a viable strategy for tissue regeneration, we employed lipid nanoparticles (LNP) carrying OSKM mRNA (OSKM mRNA-LNP) to stimulate LPLCs formation. Notably, the production of Sox9+ LPLCs, and OSKM-induced dedifferentiation, was closely correlated with successful tissue regeneration in the liver post APAP injury. Thus, the OSKM mRNA-LNP approach represents a promising therapeutic intervention for the repair of acute liver injuries.

physiology↗

bioRxiv2025

Lipid peroxidation intrinsically induces mitochondrial iron overload via Bach1-HO-1 signaling to promote cardiac ferroptosis

BackgroundLipid peroxidation and iron accumulation are hallmarks of ferroptosis, a form of cell death characterized by iron-dependent oxidative damage to cellular membranes. However, the molecular link between lipid peroxidation and iron overload in the execution of ferroptosis remains elusive. Moreover, the pathophysiological implications of the interaction between lipid peroxidation and iron overload in cardiac homeostasis and remodeling are also unknown. MethodsWe assessed the role of lipid peroxidation in mediating cardiac iron overload and ferroptosis using genetic mouse models. We also performed molecular and cellular biology studies to elucidate the mechanisms by which lipid peroxidation regulates iron homeostasis and ferroptosis signaling in cardiomyocytes. ResultsCardiomyocyte-specific ablation of Gpx4 (glutathione peroxidase 4), a key suppressor of lipid peroxidation, promoted iron overload and ferroptosis in the heart, leading to dilated cardiomyopathy. Mice with heterozygous Gpx4 knockout were also predisposed to adverse cardiac remodeling and dysfunction following pressure overload. Mechanistically, elevated lipid peroxidation due to GPX4 inactivation intrinsically induced iron overload by promoting the nuclear export of Bach1 and subsequent induction of heme oxygenase-1 (HO-1). Genetic and pharmacologic inhibition of HO-1 markedly attenuated iron overload and ferroptosis in cardiomyocytes and rescued dilated cardiomyopathy associated with Gpx4 deficiency. Moreover, we identified HO-1 mitochondrial translocation as a key mechanism driving mitochondrial iron overload and ferroptosis. Targeted inhibition of mitochondrial iron overload or lipid peroxidation abrogated cardiac ferroptosis and pathological remodeling induced by Gpx4 deficiency. ConclusionsThese findings identified a mechanistic link between lipid peroxidation and iron overload via the Bach1-HO-1 signaling pathway, revealing new regulators and molecular targets for cardiac ferroptosis.

cell biology↗

bioRxiv2025

CerS2 is a druggable target in triple-negative breast cancer

Triple-negative breast cancer (TNBC) remains a significant clinical challenge due to its aggressive nature and lack of effective targeted therapies. The enzyme ceramide synthase 2 (CerS2), which synthesizes pro-apoptotic very long-chain ceramides (VLCCs), represents a promising therapeutic target. Here, we identify and characterize DH20931, a novel, first-in-class small-molecule agonist of CerS2. We demonstrate that DH20931 directly activates CerS2 with nanomolar potency, leading to significant VLCC accumulation in breast cancer cells. This lipotoxic event induces endoplasmic reticulum (ER) stress and triggers apoptosis via the canonical ATF4/CHOP/PUMA signaling pathway. Mechanistically, we uncover a novel interaction between CerS2 and the ER calcium channel, Inositol 1,4,5-trisphosphate receptor 1 (IP3R1). We demonstrate that DH20931 promotes this interaction, enhancing ER-mitochondria proximity and facilitating a CerS2-dependent flux of calcium (Ca{superscript 2}) from the ER into mitochondria. This subsequent mitochondrial Ca{superscript 2} overload serves as a critical trigger for apoptosis. In preclinical evaluations, DH20931 potently inhibited the growth of TNBC cells in 2D and 3D cultures and significantly suppressed tumor progression in orthotopic and patient-derived xenograft (PDX) models, all while exhibiting a favorable safety profile. Our findings validate CerS2 as a druggable target in TNBC and establish a novel therapeutic strategy that leverages a coordinated attack on cancer cells through ER stress and calcium-mediated mitochondrial dysfunction. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/670525v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@2db377org.highwire.dtl.DTLVardef@6175d4org.highwire.dtl.DTLVardef@7edb8corg.highwire.dtl.DTLVardef@80042e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDH20931 is a first-in-class, potent agonist of Ceramide Synthase 2 (CerS2). C_LIO_LICerS2 activation induces ER stress and engages the ATF4/CHOP/PUMA apoptotic pathway. C_LIO_LIDH20931 promotes a novel CerS2-IP3R1 interaction, causing lethal mitochondrial calcium overload. C_LIO_LITargeting CerS2 shows significant preclinical efficacy against triple-negative breast cancer. C_LI

cancer biology↗

bioRxiv2025

Epilepsy-Associated SCN2A-L1342P Mutation Drives Network Hyperexcitability and Widespread Transcriptomic Changes in Human Cortical Organoids

ObjectiveSCN2A pathogenic mutations, such as the recurrent heterozygous Nav1.2-L1342P, are monogenic causes of epilepsy. In this human-induced pluripotent stem cell model system, we aim to investigate the molecular and cellular mechanisms underlying the SCN2A-L1342P-associated pathology. MethodsUsing a human male iPSC reference line (KOLF) carrying the Nav1.2-L1342P mutation, we generated 3D cortical organoids for functional studies. Patch clamp and multi-electrode array (MEA) recordings, immunocytochemistry, and RNA sequencing were used to characterize the disease phenotypes. ResultsNav1.2-L1342P organoid neurons displayed increased intrinsic excitability, and amplified excitatory post-synaptic currents, which are consistent with an increase in excitatory synapse formation revealed by PSD95/SYN1 immunostaining. Moreover, elevated network firing activity, as demonstrated by MEA, indicates a pronounced network hyperexcitability. Transcriptomic profiling of organoids carrying the Nav1.2-L1342P mutation further revealed significant alterations in synaptic, glutamatergic, and developmental pathways. SignificanceOur findings demonstrate that the Nav1.2-L1342P mutation drives a multifaceted disease phenotype, including network hyperexcitability and disruption of pathways related to neuronal and synaptic functions. These results advance our understanding of SCN2A-related Developmental and Epileptic Encephalopathy (DEE), laying a foundation for personalized interventions. Key PointsO_LIKey Point 1: Human neurons carrying epilepsy-causing SCN2A-L1342P display intrinsic hyperexcitability in cortical organoids. C_LIO_LIKey Point 2: Synaptic transmission is enhanced in organoids carrying the SCN2A-L1324P mutation, consistent with the presence of elevated excitatory synapses, which leads to increased network hyperexcitability. C_LIO_LIKey Point 3: Transcriptomics analysis reveals that the SCN2A-L1342P mutation causes significant differential changes in forebrain developmental genes, synaptic and glutamatergic signaling pathways, and enhances cellular senescence and apoptosis. C_LI

neuroscience↗

bioRxiv2025

A handheld microfluidic manifold for massively multiplexed CRISPR-based nucleic acid detection

Multiplexed methods for nucleic acid detection are immensely challenging to deploy outside of laboratory settings. Conversely, field-deployable methods are limited to low levels of multiplexing. Here, we introduce Scalable On-site Nucleic Acid Testing Architecture (SONATA), enabling >100-chamber reaction partitioning for multiplexed nucleic acid amplification and detection with a portable microfluidic manifold. The manifold directs a diluted sample into individual reaction chambers, each of which contains lyophilized reagents and a small stir bar or bead for mixing. Samples can be loaded using a syringe by hand, greatly simplifying the testing process. We demonstrate the integration of the platform with Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), a sensitive and deployable CRISPR-based detection technology. We show that deployed with SONATA, SHINE retains its sensitivity, enabling highly multiplexed pathogen detection in [≤] 1 hour. In addition, we demonstrate the detection of single-nucleotide variants, including mutations associated with drug susceptibility.

bioengineering↗