Biology subjects

Yang, K.

Publications and source records attributed to Yang, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Integrated Proteomic and Epigenomic Analysis Reveals IGF2 as a Vulnerability in PRC2-Deficient Malignant Peripheral Nerve Sheath Tumors

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with limited therapeutic options. Loss of the Polycomb repressive complex 2 (PRC2), via inactivating mutations in SUZ12 or EED, occurs frequently in MPNSTs and is associated with poor prognosis. However, the downstream chromatin and signaling consequences of these mutations remain incompletely understood. Here, we show that PRC2 deficiency in MPNST cells induces coordinated chromatin remodeling, characterized by loss of repressive H3K27me3 and gain of activating marks, including H3K27ac and H3K36me2. Integrative epigenomic, transcriptomic, and proteomic profiling revealed that this chromatin reprogramming activates a fetal-like growth signature centered on insulin-like growth factor 2 (IGF2) and its post-transcriptional regulators, Insulin-like Growth Factor 2 mRNA-Binding Protein (IGF2BP1-3). Functional studies demonstrate that PRC2-deficient cells are selectively dependent on IGF2 for proliferation, and that restoration of SUZ12 suppresses IGF2 expression and reduces growth. Analysis of human MPNST tumors confirms upregulation of the IGF2-IGF2BP axis in PRC2-deficient tumors, highlighting its clinical relevance. Together, these findings link PRC2 loss to activation of fetal growth factor-driven oncogenic signaling and identify IGF2 and its regulatory network as potential vulnerabilities in this aggressive tumor subtype.

cancer biology↗

bioRxiv2025

MERFISH+, a large-scale, multi-omics spatial technology resolves the molecular holograms of the 3D human developing heart

Hybridization-based spatial transcriptomics technologies have advanced our ability to map cellular and subcellular organization in complex tissues. However, existing methods remain constrained in gene coverage, multimodal compatibility, and scalability. Here, we present MERFISH+, an enhanced version of Multiplexed Error-Robust Fluorescence in Situ Hybridization (MERFISH), which integrates chemical probe anchoring in protective hydrogels with high-throughput microfluidics and microscopy. This optimized design supports robust and repeated hybridization cycles across an entire centimeter-scale tissue sample. MERFISH+ allowed to simultaneously quantify over 1,800 genes and resolve the 3D organization of chromatin loci and their associated epigenomic marks in developing human hearts. Using a generative integration framework for spatial multimodal data (Spateo-VI), we harmonized these MERFISH+ transcriptomic and chromatin data to reconstruct a 3D spatially-resolved multi-omic atlas of the developing human heart at subcellular resolution capturing 3.1 million cells across 34 distinct populations. This 3D atlas provides a holistic view of an entire organ enabling the characterization of 3D cellular neighborhoods and transcriptional gradients of substructures such as the descending arteries. Thus, MERFISH+ offers a robust, large-format platform for spatial multi-omics that enables high resolution mapping of gene expression at subcellular resolution and the characterization of cellular organization within 3D organs. One Sentence SummaryMERFISH+ is an spatial multi-omics platform that integrates hydrogel-based probe anchoring, automated high-throughput microfluidics, and large-format multimodal data production to enable comprehensive, subcellular resolution mapping of gene expression and chromatin organization across millions of cells within complex developing human organs. HighlightsO_LIMERFISH+ expands MERFISH capabilities to measure >1,800 genes and at whole-organ 3D imaging scale C_LIO_LICombines chemical probe anchoring with high-throughput volumetric microscopy and microfluidics C_LIO_LIGenerates a 3D molecular atlas of a developing human heart with > 3.1 million cells at subcellular resolution C_LIO_LIIntroduces Spateo-VI, a novel generative framework integrating 3D multimodal datasets C_LI

genomics↗

bioRxiv2025

Senescent astrocytic deposits drive cognitive decline by disrupting tripartite synapse in the aging brain

Brain aging involves synapse decline, with astrocytes playing a key role in synapse homeostasis. However, the impact of astrocyte senescence on synaptic dysfunction and cognitive decline remains unclear. Here, we identified a hallmark of aging astrocytes--Senescent Astrocytic Deposits (SAD) observed at aged rodents, macaques, and human hippocampal astrocytic processes --that is associated with tripartite synapse dysfunction and memory decline. Laser capture microdissection-coupled mass spectrometry (LCM-MS), spatial transcriptome analysis and 3D electron microscopy revealed that SAD are abnormal protein deposits at the processes of ApoE-high expression astrocyte subtype and associated with dysfunctional tripartite synapses. Using a transgenic mouse (Nrbf2 knockout) with accelerated SAD formation as a tool for genetic manipulation, we clearly demonstrated that age-dependent defect of phagocytosis at maturation stage in astrocytic drives SAD accumulation, synaptic injury and cognitive deficits. Collectively, our findings establish SAD as a mechanistic link between astrocyte senescence and synaptic damage, underscoring the critical role of astrocytic phagocytic function in preserving synaptic homeostasis and cognitive function during aging. Significance StatementThis study reveals that impaired phagocytic maturation in senescent astrocytes leads to formation of SAD and impaired synaptic plasticity, identifying hippocampal astrocyte senescence as a key contributor to age-related cognitive decline.

neuroscience↗

bioRxiv2025

Macrophages display spatiotemporal specificity in intrahepatic cholangiocarcinoma and drive tumour progression via OSM and THBS1

Tumor-associated macrophages (TAMs) are abundant in intrahepatic cholangiocarcinoma (ICC) and correlate with poor prognosis, but their non-immunosuppressive roles remain unclear. This study aimed to define TAM transcriptional heterogeneity and map their interactions with ICC cells that promote malignancy beyond immune suppression. Single-cell and spatial transcriptome analysis with mouse and human ICC tissues revealed a TAM subpopulation, designated M{varphi}_VEGFA, that emerged as the major macrophage population from the intermediate stage of ICC development onward. Data from Gene Expression Omnibus (GEO) and spatial transcriptome indicate that M{varphi}_VEGFA was associated with poor prognosis and spatially localized to the ICC cells. Cell co-culture and cell-cell communication analyses revealed that M{varphi}_VEGFA promoted ICC cell proliferation via secreted OSM and downstream OSMR-STAT3-CCND1 axis, and enhanced ICC cell invasion through THBS1 and downstream CD47-mTOR/DBN1 signaling. Critically, multiplex immunofluorescence with human ICC tissue microarray indicated that OSM and THBS1 were co-elevated in human ICCs, and dual blockade of both targets inhibited ICC progression in mouse models. This study uncovers the direct and critical roles of M{varphi}_VEGFA in driving ICC progression with paracrine signaling and further highlights the potential of targeting macrophages in ICC treatment.

cancer biology↗

bioRxiv2025

Population-Specific Transcriptomic Shifts Underlie Secondary Metabolic Diversification in Aspergillus flavus and the Domestication of Aspergillus oryzae

The genomic signatures of microbial domestication remain poorly understood within the context of natural population variation. Here, we demonstrate that Aspergillus oryzae, the filamentous fungus used in soy sauce production, shares more recent ancestry with a predominantly northern, largely non-aflatoxigenic population of Aspergillus flavus (population C). Strikingly, A. oryzae isolates also overlap with the recently described, clinically enriched A. flavus population D, suggesting the possibility of multiple domestication events. Although A. oryzae exhibits reduced virulence compared to A. flavus, all isolates tested retained pathogenicity in a zebrafish infection model. At the transcriptomic level, Aspergillus populations are significantly differentiated, with distinct responses to population density, indicating that population-specific transcriptomes adapt differently to ecological conditions. These differences extend beyond gene content and are not always explained by phylogenetic relationships, suggesting that phenotypic diversification occurs through the rapid reorganization of transcriptomic architectures. For example, A. oryzae displays significantly elevated expression of a module enriched for carbohydrate metabolism. Population-specific variation is also evident among secondary metabolite (SM) gene clusters. While A. oryzae shows markedly reduced expression of specific SM genes, particularly those involved in aflatoxin biosynthesis, this trend does not extend across the entire secondary metabolome. Using machine- learning-based gene regulatory network inference, we identified population-specific transcriptomic differences linked to distinct transcription factors, with evidence for both cis- and trans-acting regulatory divergence, but no changes in global regulators such as laeA. Together, these findings provide new insights into the domestication of A. oryzae, its global significance, and the microevolution of fungal secondary metabolic pathways. Significance StatementAspergillus flavus poses a significant threat to global agriculture by infecting crops and producing aflatoxin, a potent carcinogen that undermines food safety and international trade. The species is also a major human pathogen causing invasive fungal infections. Here, we demonstrate that A. flavus populations exhibit not only variations in their capacity to produce toxins but also in the way they regulate gene expression in response to environmental cues. These regulatory differences played a key role in the domestication of Aspergillus oryzae, a non-aflatoxigenic relative that has been safely used in food fermentation for centuries. Notably, while A. oryzae exhibits reduced aflatoxin biosynthetic gene expression, it expresses other secondary metabolite genes and retains limited opportunistic pathogenic potential. By revealing how transcriptomic rewiring drives population-level divergence, this work provides new insights into the evolution of toxin production, with broad implications for microbial domestication, crop protection, and global food security.

microbiology↗

bioRxiv2025

An SR45-dependent exonic splicing enhancer is essential for regulating alternative splicing in Arabidopsis

Alternative splicing (AS) is a critical gene regulatory mechanism that underpins developmental plasticity and environmental adaptation in eukaryotes. Although numerous cis-elements and trans-factors governing AS have been characterized in animals, their functional roles in plants remain largely unexplored. We previously identified the Arabidopsis sot5 mutant, which exhibits leaf virescence due to a mutation in the conserved 5 splice site (5ss) of intron 7. Here, through characterization of a suppressor of sot5 (E61), we found that E61 harbors a G to A mutation in exon 8 of the SOT5 gene. Interestingly, this mutation creates a synonymous codon but disrupts the exonic splicing enhancer (ESE) motif (AAGAAGA), thereby reducing the splicing efficiency of the adjacent intron 7. Retention of this intron produces a functional protein that rescues the sot5 virescent phenotype. We confirmed the causal role of this ESE by using a cytidine base editor (CBE) to convert the ESE sequence to AAGAAAA or AAAAAAA in sot5. The edited plants phenocopied the suppressor E61. Genetic analyses revealed that mutations of SR45 encoding an RNA-binding protein but not its paralog SR45a similarly rescues the sot5 phenotype by impairing intron 7 splicing. Furthermore, we demonstrated that SR45 directly binds to the ESE motif in vitro, and that mutating the proximal ESEs in intron 2 of another SR45 target, GPDHC1 (Glycerol-3-Phosphate Dehydrogenase C1), recapitulates the splicing defect observed in the sr45 mutant. These findings establish that SR45 specifically recognizes AAG-repeat ESEs to enhance the splicing of adjacent weak introns, revealing a potent strategy for fine-tuning gene expression through targeted editing of splicing regulatory elements for crop improvement.

molecular biology↗

bioRxiv2025

DISC1-PML protein interaction for congenital CMV infection-induced cortical neural progenitor deficit: perturbance of host signaling via viral IE1

Congenital CMV infection is the most common perinatal infection, affecting up to 0.5% of infants. This elicits long-term disabilities that include neuropsychiatric manifestations, such as intellectual disability, microcephaly. Despite its high prevalence, the underlying mechanism of how congenitally acquired CMV infection causes brain pathology remains unknown. Here we discovered the molecular interplay of key host (DISC1 and PML) and viral (IE1) proteins within the neural progenitor cells, which underlay an attenuated neural progenitor proliferation. Abolishing the viral IE1 protein by delivering IE1-targeting CRISPR/Cas9 to fetal brain rescued this progenitor cell deficit, a key pathology in congenital CMV infection. A selective targeting to a viral-specific protein by the CRISPR/Cas9 system is minimal in off-target effects. Therefore, we believe that a pivotal role of IE1 in an attenuated neural progenitor proliferation in the developing cortex through its interfering with interaction between host DISC1 and PML proteins.

developmental biology↗

bioRxiv2025

Real-time visualization of ligand-specific conformational dynamics of GPCR C-terminal domain in living cells

The cytoplasmic tails (C-tails) of membrane receptors are fundamental hubs for signal transductions. Yet directly investigating their dynamics in living cells is challenging. We developed a single-molecule technique to sense C-tail's conformational transitions. It was applied to investigate the ligand-induced activation of {beta}2AR, a prototypical G protein-coupled receptor (GPCR). Our data elucidate how the receptor transduces ligand binding to cell-level outcomes, with the ligand efficacy being interpreted in terms of the association and disassociation rates of the G protein. Moreover, upon G protein binding, not only the C-tail is displaced from the intracellular surface of the receptor but also the eighth helix H8 is dissociated from the plasma membrane. This work establishes a robust framework to directly decipher the activation of membrane receptors without interferences from overexpressed downstream transducers.

biophysics↗

bioRxiv2025

LIN28A-Dependent Kinome and Phosphoproteome Reprogramming Promotes Imatinib Resistance

Chronic myeloid leukemia (CML) resistance to BCR-ABL tyrosine kinase inhibitors (TKIs) can arise from ABL kinase domain mutations, BCR-ABL fusion gene amplification, or kinase-independent mechanisms. To investigate imatinib-resistance, we performed quantitative mass spectrometry comparing the proteome and phosphoproteome of K562 cells (a standard CML model) and ImR cells, an imatinib-resistant K562 derivative that also exhibits cross-resistance to second- and third-generation BCR-ABL TKIs. In addition to revealing global proteome and phosphoproteome changes associated with drug resistance, we identified LIN28A--a multi-functional RNA-binding protein--as a critical mediator of imatinib resistance. LIN28A was significantly overexpressed and hyperphosphorylated in ImR cells. Depleting LIN28A via shRNA restored imatinib sensitivity, while its ectopic expression in parental K562 cells induced imatinib resistance. Mechanistically, LIN28A coordinates an extensive kinase-substrate network regulating proliferation, survival, and metabolism to drive resistance. Notably, pharmacological inhibition of LIN28A-dependent kinases (PKC, AKT, SGK1, and RPS6K) suppressed ImR proliferation. Midostaurin, a clinical PKC/FLT3 inhibitor used in FLT3-ITD--positive AML, potently re-sensitized ImR cells to imatinib. Our findings suggest that targeting LIN28A and its downstream effectors, particularly PKC, could overcome resistance to imatinib and next-generation BCR-ABL inhibitors.

cancer biology↗

bioRxiv2025

NUSAP1 regulates mitotic processes via KIF2C interaction and AURKA phosphorylation in primary microcephaly

Primary microcephaly (PM) is a neurodevelopmental disorder characterized by a significantly smaller head than normal. Despite the identification of several genes associated with PM subtypes, the etiology remains unclear in a significant proportion of patients. Here, we reported two de novo nonsense mutations in the NUSAP1 gene identified in two independent PM families: c.1209 C>G (p.Tyr403*) and c.1213C>T (p.Gln405*). Nusap1-edited mouse models, designed to mimic Human NUSAP1 mutations, replicated the microcephaly phenotype, characterized by a small brain and thinner cerebral cortex. We demonstrated that Nusap1 was specifically expressed in neural stem/progenitor cells (NSPCs), and the number of NSPCs was significantly reduced in Nusap1-edited mice. In vitro experiments revealed that NUSAP1 mutations disrupted mitotic metaphase chromosome alignment, increased polyploidy, induced cell cycle arrest, and promoted apoptosis. Co-IP confirmed direct interactions between NUSAP1 and KIF2C, and showed that truncating mutations impaired their binding. Furthermore, NUSAP1 rescued the cell cycle arrest and increased apoptosis caused by KIF2C overexpression in NUSAP1 KO HEK293T cells, whereas the mutant truncated protein failed to exert such a rescue effect. We also uncovered an interaction between NUSAP1 and Aurora kinase A (AURKA). Intriguingly, subsequent experiments revealed that AURKA-mediated phosphorylation of NUSAP1 modulates its binding affinity to KIF2C. In conclusion, our findings established NUSAP1 as a novel pathogenic gene for PM. We proposed a model in which AURKA-mediated phosphorylation of NUSAP1 regulates its interaction with KIF2C, balancing spindle microtubule stability and depolymerization to ensure proper sister chromatid segregation. Disruptions in this pathway impair NSPCs mitosis, thereby affecting neocortical development.

genetics↗

bioRxiv2025

Spatial profiling of chromatin accessibility reveals alteration of glial cells in Alzheimer's disease mouse brain

Abnormal epigenetic modifications, including altered chromatin accessibility, have been implicated in the development and progression of Alzheimers disease (AD). In this study, we applied spatially resolved chromatin accessibility profiling by performing spatial assay for transposase-accessible chromatin using sequencing (ATAC-seq) to analyze brain tissues from 5xFAD AD model and C57BL/6J control mice. Our analysis identified seven major cell types across 11 brain regions and further characterized glial subtypes--microglia and astrocytes-- revealing subtype-specific chromatin accessibility changes in 5xFAD mice relative to controls. These alterations were associated with AD-related pathways, including neuroinflammation and immune dysregulation, and synaptic dysfunction and neuronal signaling in microglia, as well as lysosomal and proteasomal activity, lipoprotein metabolism, and mitochondrial dysfunction in astrocytes. We also characterized cell-type-specific enrichments of motifs and transcription factors, including enrichment of Bcl11a in microglia. Furthermore, we linked chromatin accessibility changes in 5xFAD mice to human AD risk genes, highlighting altered epigenetic signatures in genes such as Trem2. Supporting the spatial ATAC-seq findings, flow cytometry validated a selective increase in VISTA (encoded by Vsir) protein expression in 5xFAD microglia, supporting the spatial ATAC-seq findings and indicating a shift toward a pro-inflammatory, disease-associated microglial (DAM-like) phenotype. These findings underscore the utility of spatial chromatin accessibility profiling for uncovering brain-region specific cell identities and epigenetic mechanisms underlying AD pathogenesis.

bioinformatics↗

bioRxiv2025

13C tracing in synaptosomes reveals that SGLT2 inhibition with dapagliflozin prevents metabolic deficits in the 5X-FAD model of Alzheimer's Disease

Metabolic dysfunction is linked to several forms of age-related neurodegeneration including Alzheimers Disease (AD), and targeting brain energy metabolism is an increasingly attractive mode of therapeutic intervention. However, commonly used in vitro methods to identify specific metabolic pathways of interest in preclinical models of neurodegenerative disease have considerable limitations. They are prone to subselection of sample material, unable to identify cell type-specific effects, or cannot identify metabolic defects upstream of mitochondria. Here we address these challenges by validating a method for stable isotope tracing with isolated synaptic nerve terminals, or synaptosomes. We further applied this approach to study glucose metabolism in synaptosomes isolated from the 5X-FAD mouse model of AD treated with the antidiabetic sodium-glucose linked transporter-2 (SGLT-2) inhibitor Dapagliflozin. Treatment with Dapagliflozin preserved steady-state levels of synaptosomal metabolites and enrichment from labeled glucose into citrate that was reduced in the 5X-FAD model. These changes correlated with trends towards improved spatial working memory but not amyloid burden. The results highlight the utility of stable isotope tracing in synaptosomes to identify precise sites of metabolic dysfunction and mechanisms of action for metabolic drug candidates in preclinical models of neurodegeneration.

biochemistry↗

bioRxiv2025

Ex vivo recapitulation of intramuscular mRNA vaccination with naive and recall antigens using a human Lymphoid Follicle Chip platform

Predicting the efficacy and toxicity of intramuscular mRNA vaccines remains challenging. Here, we describe an ex vivo human cell-based model that replicates immune responses to lipid nanoparticle (LNP)-based mRNA vaccines that require intramuscular injection. Vaccines are administered into a biomimetic muscle module containing human skeletal myoblasts and antigen-presenting cells (APCs) to mimic intramuscular vaccination, followed by transfer of the APCs and soluble factors to a microfluidic human lymphoid follicle chip (LF Chip) to mimic lymphatic drainage. Non-replicating mRNA vaccines directly induce antigen expression in APCs, whereas self-amplifying mRNA vaccines require muscle cell-APC contact within the intramuscular vaccination module. Transfer of APCs and soluble factors to the LF Chip induces LF expansion, de novo antigen-specific IgG production against a naive antigen (rabies virus glycoprotein), and cytokine release, with responses varying depending on LNP type. Vaccination of LF chips against SARS- COV-2 Spike recall antigen using the Moderna Spikevax vaccine generates neutralizing antibodies and induces somatic hypermutation. This biomimetic platform offers an all-human alternative for evaluating vaccine-induced immunity, potentially obviating the need for non-human primates and accelerating vaccine development.

immunology↗

bioRxiv2025

Au18(L-NIBC)14 targets ASC oligomerization for autoimmune disease treatment

Gold nanoclusters (AuNCs), unlike conventional nanoparticles, possess molecular characteristics besides ultrasmall nano-features. Recently, we and others showed that AuNCs are promising in treatments of various major diseases. However, the AuNCs used were usually mixtures, and the specific target and their relationship with AuNC structures are unclear, which largely restrict their druggability. Multiple sclerosis (MS) is an autoimmune disease (AID) implicating central nervous system (CNS), in which drug discovery is challenging. Here we used Au18(L-N-isobutyryl-L-cysteine)14 (Au18(L-NIBC)14) and Au25(L-NIBC)18, two AuNCs with the same ligand, to report their much different therapeutic effects in experimental autoimmune encephalomyelitis (EAE). We show that, Au18(L-NIBC)14, but not Au25(L-NIBC)18, specifically targets apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization, thus inhibit the activation of ASC-dependent inflammasomes, resulting in comprehensive restoration of cytokine homeostasis in the CNS of EAE mice. Au18(L-NIBC)14 significantly prevents axon demyelination, protects blood-brain barrier, blocks immune cell infiltration into CNS, and completely prevents motor deficits and relieve the early-cognitive impairments of EAE mice. Remarkable efficacies were also observed in animal models of inflammatory bowel disease, psoriasis, systemic lupus erythematosus, indicating a broad prospect in AIDs treatments. Especially, definite molecular structure, specific target, clear mechanism, and exact therapeutic effects imply a good druggability of Au18(L-NIBC)14.

pharmacology and toxicology↗

bioRxiv2025

Delphy: scalable, near-real-time Bayesian phylogenetics for outbreaks

Pathogen genomic analysis is central to tracking, understanding, and containing outbreaks, but complexity and high costs of state-of-the-art (SOTA) phylogenetic tools limit global access and impact. We introduce Delphy, an exact reformulation of Bayesian phylogenetics designed to transform its speed, scalability and accessibility while retaining SOTA accuracy. Delphys central data structure, an Explicit Mutation Annotated Tree, exploits the high sequence similarity in large-scale epidemic datasets for efficient tree exploration and convergence. By reproducing key analyses from recent major epidemics (Ebola, Zika, SARS-CoV-2, mpox, and H5N1), we demonstrate SOTA accuracy with up to 1,000x speedups. Assessing Delphys scalability, we show that a simulated dataset of 100,000 sequences can be analyzed in under a day-the largest such computation to date. We distribute Delphy as a client-side web application, enabling users worldwide to turn raw data into interactive results within minutes, without the data ever leaving the users machine. Delphy automatically identifies key viral lineages and mutations, as well as their emergence and prevalence through time, all with quantified uncertainties derived from a solid theoretical foundation. Delphy shows the power of Bayesian phylogenetics as a fast, accessible frontline tool for tackling future outbreaks.

genomics↗

bioRxiv2025

Scalable and cost-efficient custom gene library assembly from oligopools

Advances in metagenomics, deep learning, and generative protein design have enabled broad in silico exploration of sequence space, but experimental characterization is still constrained by the cost and scalability of DNA synthesis. Here, we present OMEGA (Oligo-based Multiplexed Efficient Gene Assembly), a low-cost, accessible method for assembling hundreds to thousands of full-length genes in parallel using standard laboratory techniques. OMEGA computationally fragments target genes into short, high-fidelity Golden Gate-compatible oligonucleotides that can be ordered as a pooled library and assembled across multiplexed subpools. We systematically optimized the number of fragments per gene and orthogonal ligation sites per reaction and determine that OMEGA can assemble up to 2.6 kb constructs using as many as 70 Golden Gate sites. To validate the approach, we assembled and functionally screened a library of 810 natural and synthetic GFP variants, recovering 94-97% of target sequences with high uniformity. OMEGA enables precision library construction at scale, with per-gene costs as low as $1.50, and offers a broadly applicable solution for bridging computational protein design with high-throughput experimental validation. We have developed OMEGA as an open-source software package and an easy-to-use Colab notebook available at https://github.com/RomeroLab/omega.

synthetic biology↗

bioRxiv2025

Benchmarking porcine pancreatic ductal organoids for drug screening applications

Primary human pancreatic ductal organoids (HPDO) have emerged as a model to study pancreas biology and disease. Yet, donor material availability, and a lack of extensive benchmarking limits the range of applications. To address this gap, we established porcine pancreatic ductal organoids (PPDO) as a system from an easily obtainable source to model pancreatic ductal/progenitor biology. We benchmarked PPDO to HPDO and primary porcine pancreas using single-cell RNA sequencing (scRNA-Seq). We observed no overt phenotypic differences in PPDO derived from distinct developmental stages, with a WNT signaling enriched population characterizing PPDO. PPDO exhibited differentiation potential towards mature ductal cells and limited potential towards endocrine lineages. We used PPDO as a platform to assess the safety of FDA-approved drugs and showed conserved toxicity of statins and -adrenergic receptor inhibitors between PPDO and HPDO cultures. Overall, our results highlight the PPDO as a model for mammalian duct/progenitor applications.

developmental biology↗

bioRxiv2025

Paradoxical ventral tegmental area GABA signaling drives enhanced morphine reward after adolescent nicotine

BackgroundAn important yet poorly understood risk factor for opioid use disorder is adolescent nicotine use. We investigated the neural mechanisms underlying this understudied interaction. MethodsMale and female adolescent mice received two-weeks of nicotine water (Adol Nic) or plain water (Adol Water). In adulthood, mice underwent three morphine tests: conditioned place preference (CPP), locomotor sensitization, and two-bottle choice. Ex vivo ventral tegmental area (VTA) brain slices were assessed via patch clamp for GABA and dopamine (DA) neuron morphine responses. Finally, VTA GABA neurons were chemogenetically inhibited during morphine CPP. ResultsIn adulthood, Adol Nic mice had greater morphine CPP, more morphine locomotor sensitization, and more choice-based oral morphine consumption vs. Adol Water mice. In contrast, adult mice given nicotine vs. water had similar morphine CPP. Patch clamp analysis of VTA neurons from adult Adol Water mice showed canonical cell-type responses to bath-applied morphine: fewer action potentials in GABA neurons and more in DA neurons. Paradoxically, VTA GABA and DA neurons from adult Adol Nic mice did not show these morphine responses. In support of a causal relationship between GABA neuron firing and reward behavior, chemogenetic inhibition of VTA GABA neurons in Adol Water mice during pairing increased morphine CPP. In contrast, inhibition of VTA GABA neurons in Adol Nic mice brought morphine CPP down to control levels. ConclusionsThese data reveal an electrophysiological mechanism by which adolescent nicotine intake promotes morphine reward later in life, showing that adolescent nicotine exposure alters reward circuitry well into adulthood.

neuroscience↗