Biology subjects

Wu, X.

Publications and source records attributed to Wu, X..

At least 91 records · Page 5Linked to original sources

bioRxiv2026

Knockdown of the long isoform of the prolactin receptor selectively targets pathogenic immune cells and averts lupus nephritis

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by chronic inflammation in multiple organ systems. While a clinical association between elevated levels of the hormone/cytokine, prolactin (PRL), and exacerbation of SLE has been recognized for some time, little is known about the mechanisms through which PRL affects the course of this disease. Here, we show that immune cells in SLE have aberrant splicing of the prolactin receptor (PRLR) such that the ratio of the long to short splice variants is increased. To determine whether the change in PRLR isoform expression was causal in this disease, we used a splice-modulating oligomer (SMO) that knocked down expression of the long splice variant (LFPRLR). Using patient samples ex vivo, SLE-prone mice in vivo, high-dimensional flow cytometry and single-cell RNA-sequencing, we demonstrate that the aberrant PRLR isoform expression in SLE both directly and indirectly drives production of autoreactive immune cell phenotypes. Thus, LFPRLR knockdown decreased the expression of type I interferon signaling/response genes known to be biomarkers and hub genes for SLE, reduced immunoglobulins with signatures considered autoreactive, and averted glomerular kidney damage in SLE-prone mice. Importantly, LFPRLR knockdown reduced pathogenic B cells and other immune subsets that drive B-cell activation, without negative impact on healthy donor counterparts. Current treatments for SLE adversely affect healthy cells and do not concurrently eradicate multiple pathogenic immune subsets. Since LFPRLR SMO does not share these disadvantages, knockdown of the LFPRLR represents a potential treatment strategy for SLE that merits further investigation. Graphical AbstractThe LFPRLR represents an attractive therapeutic target in SLE. (Left) In addition to increased pituitary/circulating PRL, individuals with SLE exhibit aberrant increases in the production of autocrine/paracrine PRL by immune cells, and in their expression of specifically the long isoform (LF) of the PRLR. (Middle) Expression of the LFPRLR specifically enhances autoreactive immunophenotypes and promotes lupus nephritis. (Right) A splice modulating oligomer (SMO), that prevents synthesis of only the LFPRLR but not the short PRLR isoforms, reduces pathogenic immunophenotypes without affecting the normal counterparts of immune cells.

immunology↗

bioRxiv2026

Learning the All-Atom Equilibrium Distribution of Biomolecular Interactions at Scale

Biomolecular functions are governed by dynamic conformational ensembles rather than static structures. While models like AlphaFold have revolutionized static structure prediction, accurately capturing the equilibrium distribution of all-atom biomolecular interactions remains a significant challenge due to the high computational cost of molecular dynamics (MD). We present AnewSampling, a transferable generative foundation framework designed for the high-fidelity sampling of all-atom equilibrium distributions, which is the first model to faithfully reproduce MD at the all-atom level. It uses a quotient-space generative framework to ensure mathematical consistency and leverages the largest self-curated database of protein-ligand trajectories to date, with over 15 million conformations. Statistically, AnewSampling consistently outperforms all prior generative methods on the ATLAS monomer benchmark, and the all-atom capabilities of AnewSampling enable close statistical alignment with ground-truth MD for evaluating atomic biomolecular interactions in protein-ligand dynamics. Furthermore, AnewSampling successfully recovers coupled ligand and side-chain motions in CDK2 systems, overcoming a major sampling hurdle inherent to conventional MD. AnewSampling enables rapid exploration of conformational landscapes prior to intensive simulations, elucidating fundamental biophysical mechanisms and accelerating the broader design of functional biomolecules.

bioinformatics↗

bioRxiv2026

Recessive POPDC1 Truncation Causes Lethal Short-QT Pattern Arrhythmogenic Cardiomyopathy with Multi-Ion Channel Remodeling and Ankyrin-G Scaffold Disruption

AimsBiallelic variants in Popeye domain containing 1 (POPDC1) classically cause limb-girdle muscular dystrophy, but their impact on cardiac system remains unclear. We investigated the functional consequence of a POPDC1 frameshift variant (c.448delT), first identified in a consanguineous family with arrhythmogenic cardiomyopathy (ACM) and sudden death. Methods and resultsComprehensive clinical and genetic evaluation was followed by mechanistic studies in an orthologous Popdc1 knock-in rat model. Functional characterization included biotelemetry and programed electrical stimulation, optical mapping, patch clamp, intracellular Ca2+ imaging, proteomics, and oxidative stress assays. Homozygous mutants (Popdc1fs/fs) exhibited a shortened QTc interval and high incidence of ventricular tachycardia compared to wild-type (Popdc1+/+). Urethane anesthesia provoked second-degree AV block in all 10 Popdc1fs/fs rats but in only 1 of 9 Popdc1+/+ littermates. Optical mapping demonstrated abbreviated action potentials, slowed conduction velocity, and inducible polymorphic VT. Patch clamp confirmed accelerated repolarization, with upregulated transient outward potassium currents (Ito) and L-type calcium currents (ICa, L), but downregulated peak sodium currents (INa). Multi-omics and ultrastructural analyses revealed a post-translational collapse of intercalated disc: POPDC1 loss destabilized Ankyrin-G and its membrane-anchored cargo, Nav1.5, coinciding with enhanced Kv4.3 and Cav1.2 protein levels. These disruptions create a convergence of delayed conduction and shortened refractoriness, forming substrate for malignant re-entry. ConclusionWe defined a recessive short-QT ACM leading to potentially fatal arrhythmias caused by biallelic POPDC1 truncation driving Ankyrin-G disruption, manifesting as a triad: (1) bradycardia/AV block, (2) accelerated repolarization as short-QT pattern/diffuse T-wave flattening or inversion, and (3) progressive cardiomyopathy and sudden death. Whats new?O_LIWe define a novel diagnostic triad for recessive short-QT arrhythmogenic cardiomyopathy (rSQT-ACM) in a loss-of-function POPDC1 truncation: (1) early-onset bradyarrhythmia (sinus bradycardia or AV block); (2) a paradoxical short-QT interval with maladaptive rate response/diffuse T-wave flattening or inversion; and (3) progressive cardiomyopathy accompanied accompanied by with SCD. C_LIO_LIMechanistically, biallelic POPDC1 truncation induces collapse of the POPDC1-AnkG hub, which triggers a unique "opposing remodeling" phenotype (severe INa reduction coupled with paradoxical upregulation of Kv4.3-mediated Ito), and established a unified "slow-conduction, short-wavelength" substrate that underpins malignant re-entry. C_LIO_LIImplications for safety and device therapy: Nav1.5 deficiency indicates that sodium channel blockers may be contraindicated, and early ICD implantation may be preferable to bradycardia pacing. C_LIO_LIMechanism-directed treatments: POPDC1s recessive inheritance and small gene size make it a strong candidate for AAV-mediated gene supplementation aimed at restoring the intercalated disc architecture and ion-channel anchoring. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/710328v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1012d79org.highwire.dtl.DTLVardef@5073dcorg.highwire.dtl.DTLVardef@1e69bc2org.highwire.dtl.DTLVardef@fc15ec_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

genetics↗

bioRxiv2026

MetaSAG: A Tool for Multi-level Exploration and Taxonomic Analysis of Microbial Single-Amplified Genomes

Microbial single-amplified genome (SAG) sequencing technologies have elevated microbial research resolution to the single-cell level. However, neither upstream data processing nor downstream analysis has been fully developed, greatly limiting the research in strain level. Herein, we developed MetaSAG (Multi-level Exploration and Taxonomic Analysis of microbial Single-Amplified Genomes), which enables accurate and rapid taxonomic classification of microbial SAGs. MetaSAG outperforms existing method in species classification certainty, computational efficiency, and sensitivity of low abundance species identification. In addition, MetaSAG enables species-level functional analysis, as well as strain-level evolutionary analysis. With the help of MetaSAG, we discovered the parasitic relationship between phages and bacteria, identifying multiple susceptible bacteria and a broad spectrum of phages. Furthermore, we developed MetaK-Lytic (k-mer-based meta-learning framework to predict phage lytic ability) to achieve accurate prediction of phage lytic activity based on 31-mer short sequences, which is well adapted to the characteristics of incomplete SAG sequences. Overall, we offer a comprehensive integrated tool that can parse microbial SAG data from raw data to the strain level to decipher the functional ecology of microbial dark matter, with broad implications for microbial ecology and phage therapy (https://github.com/liangcheng-hrbmu/MetaSAG). Significance StatementThis work provides a comprehensive framework for high-resolution SAG data analysis. The developed pipeline improves taxonomic annotation sensitivity and speed. Strain-level tracking enables dynamic evolutionary and functional insights, while single-cell bacterial-virus network reconstruction reveals precise interaction patterns. The novel annotation-free short sequence-based MetaK-Lytic facilitates functional prediction of uncharacterized phage sequences. Integrated into the MetaSAG platform, these tools deliver a streamlined, multi-level solution for interpreting SAG data, advancing studies in microbial ecology, evolution, and virology.

microbiology↗

bioRxiv2026

Dissecting Gap Junctional and Ephaptic Contributions to Electrical Conduction in a Novel Cardiomyocyte Pair Model

Electrical communication between excitable cells depends on both direct gap junction (GJ) currents and field-mediated ephaptic interactions, but their relative contributions have remained difficult to quantify experimentally, limiting mechanistic insight into arrhythmia and other disorders of bioelectric signaling in excitable tissues. Building on the concept of a nanoscale, sodium channel-rich perinexus at the cardiac intercalated disc, we developed a Single-on-Paired (SoP) preparation in which whole-cell sodium current is recorded from one adult ventricular myocyte that remains end-to-end coupled to an intact partner. This configuration revealed a composite two-cell sodium current characterized by a unique pre-peak waveform which exhibits two slopes in the rising phase, and a pronounced activation jump in sodium current amplitude between closely spaced voltage steps. This feature was absent in isolated single myocytes and interpretable as an Intercalated Disc Signature of intercellular activation. By combining graded GJ inhibition, perinexal widening via a Scn1b-derived competitive adhesion peptide, and controlled modulation of extracellular sodium, we show that low sodium conditions favor GJ-dominated activation, whereas at higher, more physiological sodium levels, perinexus-centered ephaptic mechanisms provide substantial support for intercellular activation. A complementary two-cell computational model, simulating our SoP model and incorporating lateral and junctional sodium channels, reproduces the two-cell Intercalated Disc Signature and predicts a shift from GJ-dominated activation at low sodium to ephaptic-dominated support at higher, more physiological sodium concentrations when GJ conductance is reduced. Together, these results provide direct experimental evidence for a specific structural and molecular substrate of ephaptic coupling in the heart and establish a framework for dissecting how nanoscale extracellular cleft geometry, channel organization, sodium level, and GJ conductance jointly tune electric-field-based mechanisms of activation in excitable tissues, with implications for paradoxical clinical responses to anti-arrhythmic interventions.

biophysics↗

bioRxiv2026

HuR inhibition attenuates hypertension and fibrosis in chronic kidney disease

BackgroundElevated RNA-binding protein HuR has been reported in patients with chronic kidney disease (CKD), but its specific pathogenic role remains unclear. Here, we investigated HuR involvement in progressive CKD induced by deoxycorticosterone acetate (DOCA) plus angiotensin II (Ang II) in mice and evaluated the therapeutic efficacy and mechanisms of the HuR inhibitor KH3. MethodsAdult male mice underwent uninephrectomy and were subjected to DOCA + Ang II infusion with 1% NaCl in drinking water. Mice were then treated with KH3 or vehicle for 3 weeks. Control mice received saline injections without DOCA and Ang II infusion. ResultsDOCA + Ang II infusion markedly increased HuR expression in circulating exosomes and kidney tissues, which was attenuated by KH3. KH3 halted the progression of albuminuria and improved renal function, and reduced kidney hypertrophy and glomerular and tubulointerstitial fibrosis compared with untreated DOCA + Ang II mice. These improvements were associated with reduced podocyte and tubular injury. KH3 also decreased renal macrophage infiltration and suppressed NF-{kappa}Bp65, Nox2, AKT phosphorylation, TGF-{beta}1, and Wisp1, consistent with reduced inflammation, oxidative stress, and fibrosis. In addition, KH3 partially lowered arterial blood pressure in DOCA + Ang II-infused mice, an effect that may involve suppression of SGLT2-associated profibrotic vascular responses, as supported by studies in cultured VSMCs and mesenteric resistance arteries. ConclusionsHuR is upregulated in DOCA + Ang II-induced renal and vascular injury and contributes to hypertensive, inflammatory, oxidative, and fibrotic responses in CKD. Pharmacologic inhibition of HuR-RNA interactions represents a promising therapeutic strategy for CKD. NOVELTY AND RELEVANCEO_ST_ABSWhat Is New?C_ST_ABSThis study identifies the RNA-binding protein HuR (ELAVL1) as a previously unrecognized upstream post-transcriptional regulator of blood pressure in hypertensive chronic kidney disease. We demonstrate for the first time that pharmacologic disruption of HuR-RNA interactions lowers arterial blood pressure in vivo. In addition, we uncover a novel HuR-SGLT2-vascular smooth muscle cell (VSMC) signaling axis, revealing that HuR regulates inducible vascular SGLT2 expression and Ang II-mediated vasoconstrictive responses. What Is Relevant?Hypertension in CKD arises from integrated renal and vascular dysfunction that is incompletely controlled by current therapies. Our findings are highly relevant because we identify HuR as a nodal post-transcriptional regulator that coordinates renal injury, vascular inflammation, and smooth muscle contractility, rather than acting within a single cell type or signaling pathway. Clinical and Pathophysiological ImplicationThese data support a model in which HuR-driven RNA regulatory programs amplify Ang II-dependent vascular hypercontractility and hypertension in CKD. Therapeutic targeting of HuR-RNA interactions represents a novel antihypertensive strategy that may complement renin-angiotensin-aldosterone system (RAAS) blockade and provides mechanistic insight into the blood pressure-lowering and vascular protective effects of SGLT2 inhibitors, including in non-diabetic CKD.

pathology↗

bioRxiv2026

Universal physical principles govern the deterministic genesis of protein structure

Protein structure is usually treated as the endpoint of sequence by most AI models, yet its true biological emergence is a process of ordered change, whose logic remains hidden in opaque black box. ProtGenesis creates a bidirectional mirror world: it maps amino acid assembly, elongation and mutation in biological space into quantitative trajectories and ensembles in protein language model representation space, then translates spatial geometry into testable biophysical hypotheses. Across peptides, reporter proteins and protein families, three universal general principles emerged: hierarchical directional assembly (Principle I), quantitative and reproducible structural-emergence trajectories (Principle II) and discrete topological transitions (Principle III) from short- to long-range order. Three novel metrics of spatial features [D,{rho} ,{delta} ], complemented by squared Gaussian Wasserstein-2 measures, located structural anchors, sensitive regions and state transitions, enabling split-protein engineering and programmable protein design. ProtGenesis makes latent representations mechanistically interpretable, offering AI a route to discovering, rather than merely predicting, scientific principles. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=173 HEIGHT=200 SRC="FIGDIR/small/706798v2_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@18f690org.highwire.dtl.DTLVardef@e36903org.highwire.dtl.DTLVardef@35f15org.highwire.dtl.DTLVardef@1577b81_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2026

NanoHIVSeq: A Long-Read Bioinformatics Pipeline for High-Throughput Processing of HIV Env Sequences

High-throughput sequencing of the HIV-1 envelope (Env) gene from viral quasispecies is essential for epidemiology, virus-antibody coevolution studies, and evaluating therapeutics, but the conventional single-genome amplification (SGA) coupled with Sanger sequencing is labor-intensive and low-throughput. Oxford Nanopore Technology (ONT) offers long-read sequencing advantages, but high error rates (1-7%) poses a challenge in identifying biological variants from sequencing artifacts. Without unique molecular identifiers (UMIs), which lose DNA template and add complexity in library preparation, here we introduce NanoHIVSeq, a UMI-free and reference-free bioinformatics pipeline that processes ONT data from bulk Env PCR amplicons through multistep clustering, consensus polishing, indel correction, denoising, and genotyping to recover functional full-length Env variants. By leveraging advanced ONT duplex sequencing technology, NanoHIVSeq was assessed using plasmid env and bulk HIV reservoir datasets, demonstrating high robustness, recovery rate, reproducibility, and accuracy (>99.9% or >Q30) comparable to UMI approaches. Our findings indicated that NanoHIVSeq allows flexible and simplified ONT library preparation for reproducible and efficient Env sequencing especially for large cohorts.

bioinformatics↗

bioRxiv2026

Detection of Somatic Point Mutations Directly from Spatial Transcriptomics Enables in vivo Spatiotemporal Lineage Tracing

Spatial transcriptomics reveals tissue organization but lacks in vivo lineage-tracing methods applicable to humans. We introduce SpaceTracer, a computational framework that accurately detects somatic single-nucleotide variants (SNVs) directly from spatial transcriptomics data. By leveraging naturally occurring somatic SNVs, SpaceTracer reconstructs cellular phylogenies within native tissue architecture, enabling the mapping of lineage spread, migration, lineage-coupled expression changes and lineage-aware local interactions. Applied to human cutaneous squamous cell carcinoma, it traced tumor initiation and progressions, uncovered widespread pre-invasive migration of dedifferentiated epithelial cells and characterized mutant B cells migrating from tertiary lymphoid structures (TLS) into the tumor boundary. The framework also reconstructed developmental lineages across multiple tissues and identified tissue-resident mutant immune cells. SpaceTracer thus provides a perturbation-free platform for high-resolution spatiotemporal lineage tracing, offering a transformative tool for elucidating complex biological systems--especially tumor-immune ecosystems--with direct implications for advancing cancer immunotherapy.

bioinformatics↗

bioRxiv2026

PhyloSOLID: Robust phylogeny reconstruction from single-cell data despite inherent error and sparsity

While lineage tracing based on somatic mutations in single-cell sequencing data offers a powerful approach to reconstructing cellular histories in vivo, its reliability is fundamentally limited by pervasive technical artifacts--specifically, high error rates and data sparsity. These issues introduce false phylogenetic signals that corrupt tree topology and lead to spurious evolutionary conclusions. To overcome these limitations, we present PhyloSOLID, a phylogenetic algorithm designed to be inherently robust to these data imperfections. PhyloSOLID employs a progressive scaffolding strategy that begins with graph-based construction of a low-resolution, high-confidence backbone tree from reliable and uniformly covered mutations. This scaffold is then refined through the iterative integration of remaining data, guided by a Bayesian statistical model that penalizes phylogenetic inconsistencies to effectively separate the true evolutionary signal from technical artifacts. Benchmarking on both simulated datasets and multiple ground-truth datasets demonstrates that PhyloSOLID achieves superior lineage reconstruction accuracy over existing methods, for both single-cell RNA-seq and DNA-seq data. Additionally, a user-friendly web interface enables customized quality assessment, artifact removal, and interpretation of lineage structures. PhyloSOLID provides a powerful solution for decoding cellular evolution in developmental and disease contexts.

bioinformatics↗

bioRxiv2026

Dysregulation of xenobiotic metabolism and mitochondrial dysfunction exacerbate acetaminophen-induced hepatotoxicity in human antigen R-deficient male mice

Acetaminophen (APAP) overdose is a leading cause of acute liver failure worldwide. The RNA-binding protein Human antigen R (HuR) is a multifunctional post-transcriptional regulator that plays a pivotal role in cellular stress responses, including those triggered by APAP toxicity. This study investigated the mechanisms by which HuR protects against APAP-induced hepatotoxicity in male mice. Hepatocyte-specific HuR-deficient (HuRHep-/-) male mice on a C57BL/6N background and wild-type (WT) littermates were treated with 200 mg/kg APAP, and liver tissues were collected at 2, 6, and 24 hours post-treatment. APAP administration increased hepatic HuR mRNA expression and induced HuR cleavage and the formation of a higher-molecular weight HuR-immunoreactive band, with the latter two correlating with injury severity. Compared with WT controls, HuRHep-/- mice exhibited markedly increased susceptibility to hepatotoxicity at both 2 and 6 hours. Metabolite profiling revealed altered APAP metabolism and reduced glutathione S-transferase (Gst) expression in HuRHep-/- livers, consistent with impaired APAP detoxification and increased APAP-protein adduct formation. Fourier-transform infrared (FTIR) spectroscopy further identified early biochemical differences between WT and HuRHep-/- livers as early as 2 hours after APAP exposure. Additionally, HuR deficiency resulted in pronounced mitochondrial structural abnormalities and dysfunction at 2 and 6 hours, accompanied by reduced expression of the mitochondrial fission and fusion proteins Drp1 and Mfn2, increased mitochondrial protein release, and enhanced hepatocyte death. Although pro-inflammatory cytokine levels were elevated in HuRHep-/- mice relative to WT controls at 24 hours, hepatocyte proliferation was similarly blunted in both genotypes, consistent with severe liver injury and delayed recovery. Collectively, these findings identify hepatocyte HuR as a critical regulator of xenobiotic metabolism and mitochondrial integrity and establish its essential role in early protection against APAP-induced hepatotoxicity in male mice.

pharmacology and toxicology↗

bioRxiv2026

Decoding the MYC locus reveals a druggable ultraconserved RNA element

The human genome is dominated by noncoding sequences, most of which are poorly conserved across species. How genetic information is distributed between coding and noncoding regions remains a fundamental unresolved question. Using CRISPR saturation mutagenesis at base-pair resolution, we mapped the functional fitness landscape of the 10-kb human MYC locus with a near-PAMless, high-fidelity SpRY-Cas9. This unbiased interrogation revealed that the majority (67%) of functionally essential base-pairs in this locus are noncoding. Paradoxically, the phenotypic impact of noncoding sequences correlates inversely with evolutionary conservation, driven in part by rapidly diverging cis-regulatory DNA elements that remain functionally constrained in humans. Within this landscape, we identified an ultraconserved RNA element in the 3 untranslated region (UTR) that is indispensable for MYC-dependent cancer cells. Remarkably, steric-blocking antisense oligos targeting this RNA element selectively eliminate MYC-addicted cancer cells by suppressing MYC function without reducing MYC abundance. Mechanistically, this 3' UTR element promotes perinuclear localization of MYC mRNA and efficient nuclear import of the short-lived MYC protein, enabling its function as a nuclear transcription factor. Together, these findings highlight noncoding sequences as major carriers of functional genetic information, provide a comprehensive fitness map of the MYC locus, and uncover a therapeutically actionable RNA element that disables MYC-driven cancer.

genomics↗

bioRxiv2026

Disruption of the VEGFR3-HSPG2 induces lymphatic reflux dysfunction and sepsis

The lymphatic system plays a central role in fluid transport and immune regulation, yet its contribution to sepsis remains incompletely understood. Analysis of more than 500,000 individuals from the UK Biobank reveals that lymphatic system disorders and genetic variants in FLT4 (VEGFR3) are strongly associated with increased sepsis risk. Single-cell RNA sequencing further shows a marked reduction of VEGFR3^high lymphatic endothelial cells during sepsis. LEC-specific deletion of VEGFR3 impairs lymphatic reflux without disrupting vessel architecture, leading to barrier failure, systemic inflammation, bacterial dissemination, and sepsis. Mechanistically, VEGFR3 sustains lymphatic glycocalyx integrity by promoting SOX18-dependent HSPG2 transcription and heparan sulfate biosynthesis. Consistently, LEC-specific HSPG2 knockdown or pharmacological inhibition of SOX18 phenocopies VEGFR3 deficiency, whereas heparan sulfate supplementation alleviates sepsis-like pathology. Moreover, HSPG2 genetic variants are significantly associated with both lymphatic disorders and sepsis. Collectively, these findings identify lymphatic reflux dysfunction as a pathogenic determinant of sepsis and establish disruption of the VEGFR3-SOX18-HSPG2-heparan sulfate axis as an active driver of disease.

molecular biology↗

bioRxiv2026

A dendrite-resolved, in vivo transfer function from spike patterns to dendritic Ca2+

Dendrites transform local electrical activity into intracellular Ca2+ signals that drive plasticity1,2, yet the voltage[->]Ca2+ mapping during natural behavior remains poorly defined. Here, we measure this transfer function via simultaneous voltage and Ca2+ imaging throughout the dendritic arbors of hippocampal CA2 pyramidal neurons in behaving mice. Dendritic Ca2+ exhibited a hierarchical activation pattern dominated by back-propagating action potentials: simple spikes primarily drove somatic and proximal Ca2+, whereas complex spikes produced larger somatic Ca2+ signals and propagated farther into distal dendrites, sometimes in a branch-selective manner. Dendrite-restricted co-activation of voltage and Ca2+ without concurrent somatic events was rare. A biophysics-inspired model accurately predicted local Ca2+ transients from local voltage waveforms. Our data and model provide a quantitative understanding of when - and why - dendritic Ca2+ signals in CA2 pyramidal cells arise during behavior.

neuroscience↗

bioRxiv2026

Site-specific integration of transposon via engineered piggyBac transposase

The precise integration of large DNA fragments into the human genome holds significant therapeutic potential. Here, we demonstrate that combining engineered piggyBac (PB) transposase with CRISPR/Cas9 enables targeted integration of PB transposons into specified genomic loci. Our engineered PB transposase (PBase) retains high excision activity while substantially reducing endogenous integration activity. In the developed Cas9-PBase fusion system, PBase excises the transposon to generate linear DNA fragments, while Cas9 introduces site-specific double-strand breaks (DSBs), facilitating insertion of the excised fragment at the target locus. The optimized tool achieves 6.1-7.3 kb transposon integration at multiple genomic sites with 10-15% efficiency, demonstrating 60-80% targeted integration specificity. As a proof of concept, we inserted a 7.1 kb transposon encoding three genes into the {beta}2M locus of human induced pluripotent stem cells (iPSCs), conferring protection against allogeneic natural killer (NK) cell-mediated cytotoxicity in derived iNK cells. These results establish Cas9-PBase as a precise and programmable platform for large DNA sequence insertion with potential clinical applications.

genetics↗

bioRxiv2026

Curcumin Promotes Myelin Repair after Spinal Cord Injury via Spatially Selective Regulation of CLASP2 Phosphorylation

Spinal cord injury (SCI) remains a devastating neurological disorder, where limited axonal regeneration and inefficient remyelination severely restrict recovery. Although curcumin has recognized neuroprotective properties, its mechanism in myelin repair is unclear. Here, we identify a phosphorylation-dependent cytoskeletal pathway as a key target of curcumin. In a mouse spinal cord transection model, curcumin treatment improved hindlimb motor function, reduced fibrotic scarring, and increased myelin basic protein (MBP) in the injured region. Phosphoproteomic profiling revealed cytoskeletal regulation as a major process affected by curcumin, with CLASP2 emerging as a critical target. Curcumin enhanced CLASP2 phosphorylation at Ser1025, a modification that strengthened Golgi association and increased EB1 distribution at the cell periphery, thereby promoting microtubule anchoring without altering global stability. This spatially selective regulation provides a novel mechanism by which curcumin fine-tunes cytoskeletal organization to support remyelination. Analysis of published single-cell sequencing data further showed CLASP2 enrichment in myelin-forming cells, underscoring its relevance. Unlike prior studies emphasizing anti-inflammatory or antioxidant effects, our findings reveal a defined molecular mechanism linking curcumin to cytoskeletal remodeling and myelin repair, highlighting its potential as a safe and accessible therapeutic candidate for SCI.

cell biology↗

bioRxiv2026

Microhomology-mediated end joining acts directly on replication forks to repair single-ended double strand breaks

Replication stress, intrinsic to oncogenesis, often leads to fork breakage and double-strand break (DSB) formation. Conventionally, break-induced replication (BIR) is considered the primary mechanism for repairing replication-associated single-ended DSBs (seDSBs). Here, we demonstrate that microhomology-mediated end joining (MMEJ) acts directly to repair seDSBs at broken replication forks (fork-MMEJ), preferentially on the leading strands, and functions cooperatively with BIR. We also showed that while fork-MMEJ is promoted by Pol{theta}, it operates independently of MRE11/CtIP-mediated end resection, relies on RPA, and produces asymmetric deletion patterns, which is distinct from canonical MMEJ (cMMEJ) defined at replication-independent double-ended DSBs (deDSBs). ATR, activated as end resection proceeds, serves as a pivotal switch to suppress fork-MMEJ while promoting BIR. Combined inactivation of ATR and Pol{theta} synergistically kills cancer cells under high replication stress with minimal toxicity to normal cells. Together, our study provides fundamental insights into the MMEJ mechanism and offers new strategies for cancer treatment.

molecular biology↗

bioRxiv2026

THIK-1 channel mediates microglial glucose sensing and modulates AgRP neurons

Microglia play essential roles in maintaining energy homeostasis, and their dysfunction contributes to metabolic disease. Although high-fat diet (HFD) exposure induces microglial activation, the underlying mechanisms remain poorly defined. Here, we identified a previously unrecognized role for THIK-1 channel in mediating glucose sensing of microglia in arcuate nucleus of the hypothalamus (ARH), during HFD-induced obesity. Pharmacological inhibition of THIK-1 channel with tetrapentylammonium (TPA) suppresses feeding and attenuates body-weight gain in diet induced obese mice. Mechanistically, inhibition of agouti-related peptide (AgRP) neurons is indispensable for TPA-induced hypophagia. Moreover, THIK-1 inhibition promotes microglial phagocytosis of perineuronal nets (PNNs), leading to reduced AgRP neuronal activity and feeding suppression. Together, these findings establish THIK-1 as a critical glucose sensor in hypothalamic microglia and uncover a microglia-dependent pathway through which overnutrition modulates AgRP neuronal activity via PNN remodeling to regulate energy balance, highlighting THIK-1 as a potential therapeutic target for treatment of diet-induced obesity.

neuroscience↗