Biology subjects

Zhao, B.

Publications and source records attributed to Zhao, B..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Enteric ChAT-expressing neurons as new target for Lactobacillus plantarum ameliorates inflammatory bowel diseases

The gut microbiota plays a crucial role in inflammatory bowel diseases (IBD), yet how specific microbial components influence disease progression remains incompletely understood. Our study reveals that decreased abundance of Lactobacillus species correlates with ulcerative colitis severity in patients. Among tested strains, L. plantarum A736, isolated from traditional fermented foods, demonstrated remarkable efficacy in ameliorating dextran sulfate sodium (DSS)-induced colitis in a mouse model. Strikingly, multi-omics analysis revealed that L. plantarum A736 uniquely enhances choline acetyltransferase-expressing (ChAT+) neurons in the enteric nervous system and elevates acetylcholine (ACh) levels. Further analysis identified that L. plantarum A736 metabolizes tryptophan to produce indole-3-lactic acid (ILA), and supplementation with ILA significantly mimics this strains anti-inflammatory effects by activating ChAT+ neurons. Crucially, selective ablation of colonic ChAT+ neurons completely abolished the therapeutic benefits of both L. plantarum A736 and ILA, establishing their essential role in mediating these effects. These findings demonstrate for the first time that microbial metabolites can directly modulate enteric neuronal circuits to regulate gut immunity. The identified ILA-ChAT+ neuron axis represents a novel neuro-immune pathway that could be targeted for IBD treatment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/673902v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@1e0d3c5org.highwire.dtl.DTLVardef@16f5481org.highwire.dtl.DTLVardef@152283org.highwire.dtl.DTLVardef@18cfad0_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Superresolution imaging of live samples by centroid reassignment microscopy

Superresolution imaging has become one of the most important recent advances in microscopy development. However, most superresolution methods are ill-adapted for live-sample imaging because they are unacceptably slow, susceptible to artifacts, or require the use of specialized fluorophores and labeling protocols. We introduce a superresolution method called centroid reassignment microscopy (CRM) that overcomes these limitations. CRM is a simple variation on confocal microscopy wherein the single-element detector and small pinhole are replaced by a centroid detector and larger pinhole. Superresolution is obtained by reassigning the detected fluorescence to its centroid location of as a function of the scanning excitation focus location. Our method bears resemblance to the method of image scanning microscopy, which involves the use of an array detector, with the advantage that CRM provides improved resolution for the same number of detected photons while being simpler to implement and less sensitive to noise. CRM is light-efficient, fast (single-frame), robust to defocus aberrations, and requires no changes whatsoever in standard fluorescence imaging protocols, making it uniquely attractive for superresolution imaging of live, dynamic samples.

bioengineering↗

bioRxiv2025

Multiparametric Characterization of Individual Suspended Nanoparticles using Confocal Fluorescence and Interferometric Scattering Microscopy with Microfluidic Confinement

Detailed characterization of the size, mass, payload, and structure of suspended mRNA-lipid nanoparticles (LNPs) is necessary to improve our understanding of how these heterogenous properties influence therapeutic efficacy and potency. Methods currently in use face limitations in reporting ensemble-average particle properties or requiring dedicated home-built microscopes that are beyond the reach of nanoparticle developers. In this work, we overcome these limitations by combining a commercially available confocal microscope and a convex lens-induced confinement (CLiC) instrument to achieve simultaneous characterization and correlation of the size, mass, refractive index, and nucleic acid payload of individual LNPs. We established the accuracy and precision of our method using nanosized beads and used it to investigate the size, payload, and water content of LNPs in different solvent pH. By employing readily available microscopy tools, we open the door to widespread adoption of our quantitative, in-solution nanoparticle characterization method.

biophysics↗

bioRxiv2025

Regulation of YAP activity by nuclear G-actin binding

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/667612v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1d109c2org.highwire.dtl.DTLVardef@d7d6daorg.highwire.dtl.DTLVardef@5ab7fcorg.highwire.dtl.DTLVardef@1e51b37_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO YAP functions as a G-actin binding protein. Three high affinity actin binding sites, L65, L68 and W199 are identified in YAP that when mutated into YAPDDY attenuate YAP/actin interaction as well as YAP activity. Thus, nuclear actin binding to YAP is necessary for its transcriptional activity as a co-activator of TEAD. Incorporation of G-actin into the YAP/TEAD complex is predicted to induce TEAD conformational change involving a 4 interface. Therefore, the formation of a dynamic TEAD/YAP/actin ternary complex necessary for transcription is proposed. C_FIG The Yes-associated protein YAP belongs to the TEAD (TEA/ATTS domain) transcriptional co-activators that shuttle between cytoplasm and nuclear compartment. YAP and its paralog TAZ (transcriptional co-activator with PDZ-binding motif) play essential roles in the Hippo pathway to control tissue and organ size. In addition, YAP is critically involved in numerous cellular processes such as differentiation, proliferation, cell migration and cancer metastasis as well as mechanotransduction and cytoskeletal dynamics. The actin cytoskeleton controls YAP activity in multiple ways via tensile forces, cell density and cell-cell adhesion as well as shear stress or other biomechanical cues. Here we discover YAP as a novel G-actin binding protein. We identify three high affinity YAP actin binding sites involving critical residues within the N-terminus of YAP. Moreover, actin binding to YAP is necessary for its transcriptional activity and function such as during cell density control. Mutation of the three actin binding residues results in a loss of nuclear YAP co-activator function towards TEAD while actin binding to YAP is required for TEAD target gene regulation. Our data point towards the formation of a dynamic TEAD/YAP/actin ternary complex necessary for transcription.

cell biology↗

bioRxiv2025

Systematic investigation of Epstein-Barr virus transcriptional regulator interactions with the human genome

We systematically investigate interactions between Epstein-Barr virus (EBV) transcriptional regulators (vTRs) and the human genome. Starting with 16 known and candidate vTRs, we identify nine whose introduction into human cells results in substantial alterations to host gene expression. Genome-scale determination of vTR genomic binding events and alterations to chromatin accessibility reveals a detailed map of EBVs functional interactions with the human genome, including >100,000 vTR binding events impacting almost a quarter of all human genes. BMRF1 emerges as a potent regulator, impacting >7,000 genes and altering >37,000 chromatin regions. Our results provide new evidence that EBV RTA interacts with and stabilizes the binding of human RBPJ. Network analysis reveals that many human genes are targeted by multiple EBV vTRs, highlighting the vast coordinated impact of EBV on human gene expression. This study provides a valuable, extensive resource for examining EBV-induced alterations to human gene regulation, with data available on multiple platforms.

genomics↗

bioRxiv2025

A Paracrine-to-Autocrine Shunt of GREM1 Fuels Colorectal Cancer Metastasis via ACVR1C

Tumor cells typically rely on paracrine stromal signals to guide malignant behaviors, but whether they acquire signaling autonomy to support metastasis remains unclear. We elucidate this in colorectal cancer (CRC) by uncovering a paracrine-to-autocrine shunt of Gremlin1 (GREM1), a canonical stromal-secreted antagonist of bone morphogenetic proteins (BMPs). We demonstrate that while GREM1 remains restricted to stromal cells in earlier-stage (I-III) CRC, its ectopic expression in tumor epithelium increases markedly in stage IV. Mechanistically, we identify ACVR1C as a novel, high-affinity epithelial receptor for GREM1. Their interaction activates SMAD2/3 signaling, which upregulates SNAI1 and GREM1, establishing a feedback loop that amplifies epithelial-mesenchymal transition (EMT). Disrupting this loop impairs CRC metastasis in vivo. Clinically, epithelial GREM1 or ACVR1C expression predicts metastasis and poor survival. These findings define a paradigm in which tumor cells hijack stromal GREM1 to establish a GREM1-ACVR1C autocrine loop that sustains EMT and metastasis, marking a shift toward signaling autonomy and revealing a targetable vulnerability in advanced CRC.

cancer biology↗

bioRxiv2025

Rational Generation of Monoclonal Antibodies and Intrabodies Selective for Pathogenic TDP-43

TAR DNA-binding protein 43 (TDP-43), encoded by the TARDBP gene, is a ribonucleoprotein associated with the pathogenesis of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimers disease (AD). Under physiological conditions, TDP-43 is predominantly localized in the nucleus, where it participates in a variety of cellular functions related to RNA splicing, transport, and stability, as well as miRNA biogenesis. In disease, it is disproportionately mislocalized to the cytoplasm where it forms aggregates, which contribute to neurotoxicity and prion-like cell-to-cell propagation of pathogenic TDP-43. Targeting of misfolded aggregates of TDP-43 represents an attractive therapeutic strategy. However, development of effective immunotherapeutic agents remains a challenge, as they require stringent selectivity for misfolded TDP-43 in order to maintain the essential functions of physiologically native TDP-43. To address this issue, monoclonal antibodies (mAbs) and intrabodies were generated against an epitope in the N-terminal domain of TDP-43 that is only exposed when the protein is misfolded, but not in its properly folded form. We show that mouse and rabbit mAbs against this epitope displayed high binding affinities by surface plasmon resonance analysis and selectively reacted with pathological TDP-43 in post-mortem tissues from ALS, FTD, and AD patients. In a cell line system, human embryonic kidney (HEK) 293T cells, mAbs and corresponding intrabodies specifically reacted with cytoplasmic aggregates of transfected misfolded TDP-43 lacking the nuclear localization signal, TDP-43{Delta}NLS. Functionally, mAbs inhibited cell-to-cell transmission of misfolded TDP-43 and the seeding activity of misfolded TDP-43 from FTLD brain homogenates by a novel RT-QuIC assay. Intrabodies promoted the degradation of intracellular aggregates of TDP-43 in HEK293T cells and in induced pluripotent stem cell-derived motor neurons (iPSC-MNs) from ALS patients. The results provide proof-of-concept evidence that supports selective targeting of misfolded toxic aggregates of TDP-43 as a potentially safe and effective avenue to treat neurodegenerative diseases associated with TDP-43 proteinopathy.

immunology↗

bioRxiv2025

Human meibomian gland organoids to study epithelial homeostasis and dysfunction

Meibomian glands (MGs) are holocrine glands that secrete lipids to maintain the homeostasis of ocular surface, and their dysfunction leads to dry eye disease. Herein, we established long-term 3D organoid culture for murine and human MGs, which retained the cell lineages and lipid-producing ability. The organoids mimicked the drug treatment responses and generated functional MGs after orthotopic transplantation. Inspired by organoid cultures, we found FGF10 eye drops could rescue all-trans retinoic acid-induced MG dysfunction in mice. Besides, nicotinamide uniquely hampered the human MG organoid expansion by inhibiting FGF10 signaling. Single-cell atlas and lipidome not only aligned the delineated cell types and featured lipids between human MGs and organoids, but highlighting MAPK signaling inhibition enhanced acinar cell differentiation and functional maturation of MG organoids. In summary, this study established an organoid platform to explore epithelial homeostasis and dysfunction of MGs, facilitating drug development and regenerative medicine for dry eye disease.

cell biology↗

bioRxiv2025

A Radiomic Approach to Clinical MRI Refines the Thalamus-Cognition Link in Multiple Sclerosis

Background and ObjectivesRadiomics extracts imaging features that may not be detectable through conventional volumetric analyses. Given their role in multiple sclerosis (MS), we applied radiomics to thalamic nuclei and examined their associations with cognitive performance. MethodsA total of 601 individuals were included (342 people with MS-PwMS from two cohorts, and 259 healthy controls-HC). Radiomic features (RF) and volumes were extracted from the whole thalamus, five thalamic nuclei, and the putamen segmented on 3D T1-weighted images. Cognitive performance was assessed using the Symbol Digit Modalities Test (SDMT) and Paced Auditory Serial Addition Test (PASAT) in PwMS, and the Digit Symbol Substitution Test (DSST) in HC. In the first MS cohort, multivariate linear regression in a discovery set (N=103) identified thalamus-derived RF associated with SDMT, which were retested in a replication set (N=63). Their associations with PASAT in a second MS cohort (N=176) and DSST in HC were also evaluated. We then tested whether the same RF, when extracted from the putamen, were associated with SDMT. LASSO models assessed the combined predictive value of RF and volumes. Figure 2 presents an overview of the involvement of different cohorts in the study, outlining the specific study objectives and the statistical analysis approaches employed. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/657698v1_fig2.gif" ALT="Figure 2"> View larger version (40K): org.highwire.dtl.DTLVardef@6f1dd8org.highwire.dtl.DTLVardef@11069c6org.highwire.dtl.DTLVardef@905512org.highwire.dtl.DTLVardef@1c12171_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2.C_FLOATNO Overview of the involvement of different cohorts in the study, specific study objectives and the statistical analysis approaches employed. Abbreviations: SDMT: Symbol Digit Modalities Test, PASAT: Paced Auditory Serial Addition Test, DSST: Digit Symbol Substitution Test, LASSO: Least Absolute Shrinkage and Selection Operator. C_FIG ResultsTwenty-eight RF-ROI pairs were associated with SDMT in the replication set (FDR<0.05). Of these, 24 were also associated with PASAT (FDR[≤]0.03), and 2 with DSST. Only ventral nuclei volume showed replicated associations among volumetrics. Only 4 putamen-derived pairs were associated with SDMT (FDR=0.04). LASSO results confirmed RF outperformed volumes. DiscussionRF extracted from the thalamus are strongly associated with cognitive performance in PwMS, outperforming volumetric measures and supporting their potential as sensitive imaging biomarkers.

neuroscience↗

bioRxiv2025

Cell fate specification during respiratory development requires ARID1A-containing canonical BAF complex activity

Development of the mammalian lung requires formation of a definitive lung bud from the foregut endoderm, branching morphogenesis, specification of a proximal to distal axis, and differentiation of extensive specialized airway and alveolar epithelial lineages. These steps require coordinated temporal and regional gene expression to define the lung epithelium in preparation for the first breath at birth. While the transcriptional and signaling regulators required for lung development are increasingly well known, the key epigenetic complexes that interact with lineage transcription factors for cell-specific control of gene expression remain to be defined. Here, we identify a key role for the canonical BAF complex, an ATP-dependent chromatin remodeling complex, during lung epithelial development. Loss of canonical BAF activity throughout the foregut endoderm leads to complete failure of lung formation, and selective deletion of a single key subunit, ARID1A, leads to failure of proximal-distal axis specification, with dilated airway-like structures lined by ectopic basal cells found throughout the distal lung, and failure of specification of alveolar type 1 (AT1) cells in the distal saccular epithelium. In place of AT1 cells, we identified a highly proliferative epithelial cell state defined by joint activation of YAP and WNT signaling and loss of BMP signaling response, leading to increased proliferation and failure of appropriate epithelial differentiation. These changes resulted in secondary failure of mesenchymal and endothelial specification, leading to broad loss of patterning of the distal lung further disrupting peripheral lung morphogenesis. Using embryonic lung organoids, we demonstrate that exogenous BMP4 signaling is sufficient to rescue AT1 and AT2 cell differentiation in ARID1A-null epithelium, while WNT and YAP signaling require functional BAF complex. Together, these data demonstrate a requirement for the BAF complex for lung formation, proximal-distal patterning, and cell fate acquisition, and reveal surprising differential specificity between signaling pathways during lung development. One-Sentence SummaryFunctional BAF complex containing ARID1A is required for lung development.

developmental biology↗

bioRxiv2025

Biological characteristics of rabbit KLF12 and its regulation of proliferation and apoptosis of ovarian granulosa cells

Granulosa cells (GCs), as the largest somatic cell group in follicles, provide nutrients and microenvironment for oocyte growth and maturation. GC proliferation and apoptosis are influenced by several genes, with the Kruppel Like Factor 12 (KLF12) gene contributing to the modulation of several cellular activities. Nevertheless, the precise modulatory function of this gene within the context of ovarian GCs remains unelucidated. We cloned the KLF12 gene of New Zealand female rabbits, and evaluated its expression and localization in the ovaries of female rabbits of different ages by quantitative real-time PCR (qRT-PCR) and fluorescence in situ hybridization (FISH). In order to determine the role of KLF12 in ovarian GCs, we used qRT-PCR, WB, CCK-8 and Annexin V-FITC/PI to detect the effect of KLF12 on GCs proliferation and apoptosis. The coding sequence of KLF12 gene of New Zealand female rabbit was predicted and cloned for the first time in this study. The coding region was 1209 bp, encoding 402 amino acids. KLF12 overexpression has a negative effect on the proliferation and cell cycle progression of rabbit GCs. KLF12 knockdown partially rescues GCs proliferation, accelerates GCs cycle progression, and promotes Estradiol (E2) and Progesterone (P) secretion. In addition, KLF12 was also proved to be a key regulator of PI3K/Akt signaling pathway in GCs. In conclusion, the study provides evidence that KLF12 plays a pivotal role in the maturation and proliferation of ovarian GCs in female rabbits. This work establishes a conceptual foundation for investigating deeper into the possible regulatory mechanisms underlying follicular development and growth. Author summaryGCs are essential for oocyte growth, not only providing necessary nutritional support for oocyte growth and maturation, but also constructing a key microenvironment. In this study, the biological function of KLF12 gene was systematically elucidated in rabbit for the first time. It was found that KLF12 inhibited follicular development by negatively regulating GCs proliferation and cell cycle. Combined with its effect on E2 and P secretion, the important regulatory role of KLF12 in follicular maturation and hormone balance was revealed. These innovative findings not only deepen the understanding of the regulatory network of ovarian development, but also provide a new theoretical basis for the basic research of reproductive biology. More importantly, it provides a potential molecular target for the development of new technologies to improve the reproductive efficiency of livestock. It also provides a valuable reference model for the study of the mechanism of human reproductive dysfunction related diseases.

genetics↗

bioRxiv2025

Oncogenic drivers shape the tumor microenvironment in human gliomas

Gliomas are aggressive and heterogeneous brain tumors with limited treatment options. While oncogenic mutations in gliomas have been well-characterized, their impact on the tumor microenvironment remains poorly understood. To investigate how genomic alterations may influence the glioma microenvironment, we performed an integrative multiomic and spatial transcriptomic analysis of 93 glioma samples (46 IDH-mutant gliomas and 47 IDH-wildtype glioblastoma) from 69 patients representing both primary and recurrent stages. Using whole-genome sequencing, chromatin conformation capture (Hi-C), RNA-seq, and single-cell spatial transcriptomics (Xenium), we defined how major driver mutations influence spatial tumor organization. We found that IDH-mutant gliomas frequently harbored inflammatory microglia expressing CX3CR1 specifically within their astrocyte-like malignant neighborhoods. In contrast, glioblastomas demonstrated relatively higher T-cell infiltration and enrichment of immunosuppressive myeloid cell populations. We further compared glioblastomas harboring EGFR amplifications due to extrachromosomal DNA (ecDNA) amplifications versus linear chromosomal 7 gains. Tumors with EGFR ecDNA displayed increased presence of mesenchymal-like malignant cells, and higher interactions between pericytes and mesenchymal-like malignant cells, likely driven by hypoxia-associated vascular proliferation. Our findings reveal that the mode of oncogene amplification--linear versus ecDNA--shapes distinct tumor architectures and transcriptional dynamics. Taken together, our study highlights the role of oncogenic drivers in shaping the glioma microenvironment, revealing subtype-specific cellular ecosystems that could inform targeted therapeutic strategies.

genomics↗

bioRxiv2025

Structural basis for L-isoaspartyl-containing protein recognition by the PCMTD1 cullin-RING E3 ubiquitin ligase

A major type of spontaneous protein damage that accumulates with age is the formation of kinked polypeptide chains with L-isoaspartyl residues. Mitigating this damage is necessary for maintaining proteome stability and prolonging organismal survival. While repair through methylation by PCMT1 has been previously shown to suppress L-isoaspartyl accumulation, we provide an additional mechanism for L-isoaspartyl maintenance through PCMTD1, a cullin-RING ligase (CRL). We combined cryo-EM, native mass spectrometry, and biochemical assays to provide insight on how the assembly and architecture of PCMTD1 in the context of a CRL complex fulfils this alternative mechanism. We show that the PCMTD1 CRL complex specifically binds L-isoaspartyl residues when bound to AdoMet. This work provides evidence for a growing class of E3 ubiquitin ligases that recognize spontaneous covalent modifications as potential substrates for ubiquitylation and subsequent proteasomal degradation. eTOC BlurbLimiting the accrual of L-isoaspartyl damaged proteins is essential during aging. While this is thought to be mediated solely by the repair activity of the protein, PCMT1, Pang et al. now demonstrate that a related protein, PCMTD1, functions as a cullin-RING ligase to selectively target L-isoaspartyl-damaged substrates for potential regulation by the ubiquitylation-proteosomal system. HighlightsO_LIAtomic cryo-EM structure of CRL5-PCMTD1 determined C_LIO_LIArchitecture of PCMTD1 when complexed as a CRL supports ubiquitylation activity C_LIO_LIPCMTD1 recognizes L-isoaspartyl residues as a recruitment motif for potential CRL activity C_LIO_LIRecognition of L-isoaspartyl residues is dependent on cofactor engagement C_LI

biochemistry↗

bioRxiv2025

Control of leaflet movement pattern by a novel PP2C phosphatase DLM1 in Medicago truncatula

Sessile plants exhibit diverse movement behaviors that have long intrigued the scientists. The legume plants display a rhythmic leaflet movement pattern characterized by horizontal opening during the day and vertical closure at night. However, the underlying mechanisms remain largely enigmatic. Here, we isolated a mutant designated as dlm1 (downward leaflet movement1) from Medicago truncatula that displays leaflets downward opening during daytime while upward closure at night. Cellular analyses reveal that this aberrant phenotype correlates with abnormal volume changes of motor cells within the pulvinus. DLM1 exhibits high expression level in the motor organ pulvinus and encodes an unreported nuclear-localized PP2C phosphatase. Notably, phylogenetic analysis demonstrates that species exhibiting rhythmic leaflet movements consistently retain DLM1 homologs, suggesting the possibility of its functionally conserved role in regulating leaflets movement pattern. Structural characterization reveals that DLM1 possesses both phosphatase and kinase domains. Functional complementation assays demonstrate that the phosphatase domain is necessary and sufficient for maintaining the leaflet movement pattern. Collectively, our work uncovers a novel PP2C protein that governs the leaflet movement, providing mechanistic insights into this intriguing plant behavior.

plant biology↗

bioRxiv2025

PASTA: Versatile Tyramine-oligonucleotide Amplification for Multi-modal Spatial Biology

Spatial proteomics techniques have revolutionized our understanding of tissue architecture, but are frequently limited by detection sensitivity, bioconjugation limitations, multiplexing capacity, and multi-modal integration. Here we present Protein and nucleic Acid Serial Tyramine Amplification (PASTA), a novel signal amplification approach that significantly enhances detection sensitivity while maintaining compatibility with diverse spatial profiling methodologies. PASTA utilizes horseradish peroxidase (HRP) recruitment pathways to generate tyramine radicals that deposit oligonucleotides, enabling adaptable signal amplification across multiple biomarkers at high-plex via cyclical imaging using complementary fluorophore-labeled oligonucleotides. We demonstrate that PASTA achieves up to 100-fold signal enhancement for markers with minimal background in blank controls. The method is compatible with in situ hybridization for DNA/RNA detection, proximity ligation assays for protein-protein interactions, sequential antibody staining protocols, or any modular combination thereof. PASTA enables antibody rescue of markers with suboptimal signal-to-noise ratios and is versatile in its applications to unconjugated antibodies, and multi-round probe-based RNA detection systems beyond current capabilities. This technique addresses key limitations in spatial-omics by enhancing sensitivity for challenging targets while maintaining compatibility with established multiplexing strategies, providing a versatile, cost-efficient, and valuable tool for comprehensive spatial tissue analysis in both research and clinical applications.

molecular biology↗

bioRxiv2025

MiR-126 Improves the Therapeutic Effect of EPC in Hypertensive Ischemic Stroke

Background and PurposeHypertension promotes circulatory endothelial inflammation, reduces endothelial progenitor cell (EPC) function, and impairs their therapeutic potential following ischemic stroke. MiR-126 is known to regulate vascular development and angiogenesis. This study investigates the therapeutic potential of miR-126 by overexpressing it in EPCs to enhance their efficacy in hypertensive stroke conditions. MethodsAdult spontaneously hypertensive rats (SHRs, n=118) underwent permanent suture middle cerebral artery occlusion (MCAO) to induce ischemic stroke. One week post-stroke, animals were injected with EPCs overexpressing miR-126 or control EPCs. Treatment effects were evaluated over 35 days, using neurological scoring, infarct volume measurements, behavioral testing, and assessments of neuroinflammation, blood pressure, and angiogenesis. Exosomes from miR-126 overexpressing EPCs were isolated and analyzed for mechanistic studies. ResultsIn vitro, miR-126 enhanced EPC angiogenic function under stress. In vivo, miR-126 modified EPC treatment improved functional recovery, reduced infarct volume, and promoted angiogenesis compared to controls in SHRs. Furthermore, miR-126 treatment preserved the blood-brain barrier, reduced peripheral immune cell infiltration, and modulated neuroinflammation. Exosomes derived from miR-126 overexpression EPCs also promoted angiogenesis and reduced endothelial activation under stress conditions. ConclusionsEPC overexpressing miR-126 provides neuroprotection by enhancing angiogenesis, reducing ischemic injury, and preserving blood-brain barrier integrity in hypertensive stroke models. This approach modulates neuroinflammation and improves neurological outcomes, suggesting gene-modified EPCs as a promising strategy for ischemic stroke therapy, particularly under hypertensive conditions.

neuroscience↗

bioRxiv2025

Inverted Alu repeats in loop-out exon skipping across hominoid evolution

BackgroundChanges in RNA splicing over the course of evolution have profoundly diversified the functional landscape of the human genome. While DNA sequences proximal to intron-exon junctions are known to be critical for RNA splicing, the impact of distal intronic sequences remains underexplored. Emerging evidence suggests that inverted pairs of intronic Alu elements can promote exon skipping by forming RNA stem-loop structures. However, their prevalence and influence throughout evolution remain unknown. ResultsHere, we present a systematic analysis of inverted Alu pairs across the human genome to assess their impact on exon skipping through predicted RNA stem-loop formation and their relevance to hominoid evolution. We found that inverted Alu pairs, particularly pairs of AluY-AluSx1 and AluSz-AluSx, are enriched in the flanking regions of skippable exons genome-wide and are predicted to form stable stem-loop structures. Exons defined by weak 3' acceptor and strong 5' donor splice sites appear especially prone to this skipping mechanism. Through comparative genome analysis across nine primate species, we identified 67,126 hominoid-specific Alu insertions, primarily from AluY and AluS subfamilies, which form inverted pairs enriched across skippable exons in genes of ubiquitination-related pathways. Experimental validation of exon skipping among several hominoid-specific inverted Alu pairs further reinforced their potential evolutionary significance. ConclusionThis work extends our current knowledge of the roles of RNA secondary structure formed by inverted Alu pairs and details a newly emerging mechanism through which transposable elements have contributed to genomic innovation across hominoid evolution at the transcriptomic level.

genomics↗

bioRxiv2025

Multi-omic analysis of the ciliogenic transcription factor RFX3 reveals a role in promoting activity-dependent responses via enhancing CREB binding in human neurons

Heterozygous loss-of-function (LoF) variants in RFX3, a transcription factor known to play key roles in ciliogenesis, result in autism spectrum disorder (ASD) and neurodevelopmental delay. RFX binding motifs are also enriched upstream of genes found to be commonly dysregulated in transcriptomic analyses of brain tissue from individuals with idiopathic ASD. Still, the precise functions of RFX3 in the human brain is unknown. Here, we studied the impact of RFX3 deficiency using human iPSC-derived neurons and forebrain organoids. Biallelic loss of RFX3 disrupted ciliary gene expression and delayed neuronal differentiation, while monoallelic loss of RFX3 did not. Instead, transcriptomic and DNA binding analyses demonstrated that monoallelic RFX3 loss disrupted synaptic target gene expression and diminished neuronal activity-dependent gene expression. RFX3 binding sites co-localized with CREB binding sites near activity-dependent genes, and RFX3 deficiency led to decreased CREB binding and impaired induction of CREB targets in response to neuronal depolarization. This study demonstrates a novel role of the ASD-associated gene RFX3 in shaping neuronal synaptic development and plasticity.

neuroscience↗