Biology subjects

Pham, K.

Publications and source records attributed to Pham, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Evaluation of Human Norovirus Replication in Salivary Gland Cells

Human noroviruses (HuNoVs) are the most common cause of non-bacterial foodborne illnesses worldwide. HuNoVs are notoriously difficult to culture, and current cell culture systems, such as human intestinal enteroids have limitations in efficiency and scalability, which has limited their applications in food safety studies. Recently, it has been shown that transformed salivary gland cell lines (including NS-SV-TT-DC) allow for replication and passaging of norovirus GII.4. Herein, we examined whether we can independently observe viral replication in salivary gland cells and if other strains of human norovirus can replicate in these cells. For this purpose, we first evaluated the replication of several norovirus strains in NS-SV-TT-DC cells by either infecting them with vesicle-cloaked viruses or total viruses in stool filtrates. Low levels of viral replication was observed and vesicle isolation did not improve viral replication. Next, we screened for replication of 50 norovirus samples from 9 genotypes, and 14 isolates demonstrated over a two-fold viral replication. We observed that the initial viral load in the samples does not have a significant effect on replication efficiency. We were also able to passage several isolates. Subsequently, we examined whether interferon inhibitors such as Ruxolitinib, TPCA-1, and BX795 would improve viral replication and observed negligible effects. Finally we tested multiple supplements including MgCl2, bile salt, ceramide, and TAK-779. Most supplements had little or no effect on HuNoV replication in NS-SV-TT-DC cells. Bile salt and TAK-779 showed limited, genotype-specific benefits. Altogether these results indicate that salivary gland cells allow limited replication of certain HuNoV isolates.

microbiology↗

bioRxiv2026

A widespread bacterial toxin reveals a deeply divergent DNase family neutralized by diverse immunity proteins.

Bacteria compete for space and resources by delivering toxin proteins into neighboring cells. Many toxins remain uncharacterized because they lack similarity to proteins of known function. Here we identify Rehh as a widespread antibacterial toxin associated with multiple bacterial secretion systems and show that it is used in interbacterial competition. Purified recombinant Rehh functions as a manganese-dependent DNase, consistent with our in-vivo findings that Rehh damages target cell DNA, inducing the SOS response and the accumulation of double-strand breaks. The crystal structure of Rehh in complex with a cognate immunity protein reveals a positively charged histidine-rich metal-binding pocket embedded within a protein scaffold that bears no significant similarity to previously characterized nucleases. However, local structural analysis demonstrates that this pocket preserves the catalytic geometry characteristic of the HNH/His-Me-finger superfamily, revealing Rehh to be a deeply divergent HNH nuclease whose ancestry has been obscured through extensive structural remodeling. The cognate immunity protein neutralizes the toxin via an acidic surface that occludes the catalytic pocket, consistent with mimicry of the electrostatic properties of the DNA phosphate backbone. Comparative genomics further shows that Rehh homologs are associated with multiple unrelated immunity families, suggesting that distinct immunity domains repeatedly converge on a common strategy for inhibition. Together, these findings demonstrate how conflict-driven diversification can obscure enzyme ancestry while preserving catalytic function and reveal a possible case of convergent evolution among immunity proteins that independently exploit the same toxin biochemical vulnerability.

microbiology↗

bioRxiv2026

An RNase Toxin Hijacks Elongation Factor-Tu to Cleave the Ribosomal Sarcin-Ricin Loop

Ribosome-targeting toxins typically recognize and attack their substrates directly. Here, we provide the functional characterization of SRLase1, a widespread member of a recently identified RNase superfamily, and show that it cleaves the sarcin-ricin loop (SRL) of 23S rRNA. Unlike previously described ribosome-targeting toxins, SRLase1 requires the host elongation factor Tu (EF-Tu) for activity. EF-Tu normally engages the ribosome as part of the aminoacyl-tRNA delivery cycle, but SRLase1 instead exploits this factor through a mechanism incompatible with canonical ternary-complex formation. This host-factor-assisted mechanism reveals a distinct strategy for translation inhibition, in which a toxin repurposes a conserved component of the elongation machinery to access a vulnerable ribosomal target. Homologs of SRLase1 are widespread across bacterial phyla and are frequently encoded in conflict-associated genomic loci. Together, these findings define a widespread family of SRL-targeting toxins, expand the mechanistic repertoire of ribosomal antibacterial toxins, and demonstrate how conserved components of the host translation machinery can be co-opted to promote toxin activity.

microbiology↗

bioRxiv2026

A structure-guided classification framework reveals the diversity and catalytic architecture of BECR ribonuclease

Microorganisms across all domains of life engage in molecular conflict, deploying toxins to inhibit competitors or respond to biological threats. Among these, ribonuclease toxins are particularly widespread and diverse. A substantial fraction is associated with the BECR fold, a compact /{beta} architecture that supports RNase activity despite extensive divergence. Although several canonical members are well characterized, many BECR-fold proteins remain difficult to identify because of low sequence similarity, variation in catalytic residues, and structural elaborations that obscure evolutionary relationships. The growing availability of high-confidence protein structure predictions provides an opportunity to reassess this deeply divergent protein landscape. Here, we integrate iterative profile-HMM searches, profile-similarity networks, structural analyses, active-site mapping, and genomic context to examine BECR proteins across the tree of life. Our analysis resolves an expanded BECR-fold landscape comprising canonical BECR and BECR-like superfamilies, refines the organization of canonical BECR proteins and identifies previously unrecognized families. We further validate BECR-Tox2 as a toxin neutralized by a cognate immunity protein and show that its homologs occur in both Menshen-like anti-phage systems and polymorphic toxin loci. Together, these findings expand and clarify the BECR-fold landscape and provide a framework for identifying and interpreting highly divergent proteins of this fold.

microbiology↗

bioRxiv2026

Endocrine-Adapted Pituitary Macrophages Regulate Gonadotropin Secretion through CXCL5-CXCR2-MAPK Signaling

Chronic inflammation disrupts hormonal balance in the Hypothalamic-Pituitary-Gonadal (HPG) axis, contributing to reproductive disorders. While immune cells in the hypothalamus and ovaries have been extensively studied, their impact on the pituitary remains largely unexplored. Our research identifies pituitary macrophages (PitMacs) as the dominant pituitary immune cell population with a role in regulating reproductive gonadotropin secretion both in vitro and in vivo. Using a targeted AAV-based depletion strategy, we demonstrate that a reduction of PitMacs decreases serum gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), in female mice. PitMacs are transcriptomically distinct from other tissue-resident macrophages and harbor a unique translational program that reflects the pituitarys endocrine identity, including active translation of growth hormone (Gh) and prolactin (Prl). Cytokine profiling identified CXCL5 and IFN-{gamma} as key PitMac-derived mediators of gonadotropin regulation. Mechanistically, CXCL5 signals through CXCR2 to activate the MAPK pathway, converging with Gonadotropin-Releasing Hormone (GnRH) signaling in a time-dependent manner to regulate LH secretion and GnRH receptor surface expression. These findings establish PitMacs as essential endocrine-immune integrators, opening new avenues for understanding inflammation-driven reproductive disorders. One Sentence SummaryPituitary macrophages are unique hormone-producing immune cells that regulate hormone secretion via cytokine signaling.

immunology↗

bioRxiv2026

How many genes can CRISPR edit to engineer complex adaptations?

Polygenic traits require the coordinated effects of multiple genes. Such complex traits have been a long-term target of study for geneticists, but multiplex CRISPR--the editing of multiple loci in the genome via multiple guide RNAs--is in its infancy. Reviewing 106 plant studies using multiplex CRISPR, we find that the multiplexing capacity has doubled every 5.4 years. Furthermore, a systematic experiment with 8, 16, and 24 simultaneous targets in Arabidopsis thaliana reveals efficiency of up to 75% in 24-plex editing in transformed plants confirmed by sequencing. We surprisingly found that the level of multiplexing, or the number of the targets, causes lesser efficiency reductions than other uncontrolled factors such as gRNA design or natural variation across plants. In fact, mathematical modeling of the decay in editing efficiency as a function of the gRNA numbers, showed a logistic model with sustained efficiency fits the data better than decay due to Cas9 competition or random editing. We then project that editing close to 100 genes in a plant can be feasible with reasonably large plant screens. However, feasible and reliable polygenic genome engineering will need developments outside of the CRISPR editing machinery itself, including innovations in gRNA vector delivery for large cargos, and a broader conceptual shift toward population-level poly-gene editing leveraging large distributions of mutations for breeding, natural selection, or experimental evolution. Author ContributionsM.E.-A. conceived the project and secured funding. M.E.-A. and M.Es. designed the experimental strategy. M.Es. established the multiplex CRISPR and transformation pipelines in the laboratory, propagation through the T1 and T2 generations, and oversaw the first amplicon sequencing. Y.P. established the in-house iSeq amplicon sequencing protocol and contributed to cloning and genotyping pilots. M.Es supervised K.P. to construct cloning, bacterial transformations, plant growth, floral-dip transformations, and selection of T1 plants. E.K. contributed to early amplicon genotyping. J.K. propagated and sampled the T3 and J.K. and J.M. conducted the final amplicon sequencing panel. J.K. and M.E.-A. performed gene editing variant mapping, dataset quality control, and summarized results from published multiplex CRISPR studies. M.E.-A. modeled editing efficiency. J.K and M.E.-A. generated figures and wrote the first draft. All authors revised and improved the manuscript. J.K. and M.Es. contributed equally to this work and are designated as co-first authors.

plant biology↗

bioRxiv2026

H3 dopaminylation and CaMKII modulate diffuse midline glioma response to CDK9 inhibition

Aberrant neurotransmitter signaling and transcriptional dysregulation are hallmarks of gliomagenesis and represent potential therapeutic targets. Monoamine neurotransmitters such as dopamine and serotonin primarily activate GPCRs but can also function epigenetically as histone H3 modifications. Here, we uncover mechanisms of crosstalk between monoamine neurotransmitter signaling, H3 dopaminylation, and RNA polymerase II (Pol2) transcription in diffuse midline glioma (DMG). We find that co-treatment with Pol2-targeting CDK9 inhibitors (CDK9i) and FDA-approved neuropsychiatric drugs, including selective serotonin reuptake inhibitors (SSRIs), synergistically reduces DMG growth. Mechanistically, CDK9i+SSRI treatment alters H3 dopaminylation patterns and represses synaptic and neurodevelopmental gene transcription associated with CDK9i resistance. Further phospho-proteomic analyses show that CDK9i monotherapy activates pro-survival CaMKII signaling, which can be suppressed by co-treatment with neuromodulatory drugs. These studies establish roles for H3 dopaminylation and neurotransmitter signaling in DMG gene regulation and response to CDK9i, suggesting that monoamine neurotransmitter pathways may be exploited as a therapeutic strategy for DMG.

cancer biology↗

bioRxiv2026

Mechanotherapeutic Potential of Survivin in Glioblastoma

Glioblastoma Multiforme (GBM) is a highly aggressive brain cancer characterized by rapid proliferation and extensive remodeling of the extracellular matrix (ECM), leading to progressive tissue stiffening. Although ECM stiffness is known to promote GBM progression, the molecular mechanisms linking mechanical cues to tumor growth remain insufficiently defined. In this study, transcriptomic comparison of GBM tumors and non-neoplastic brain tissue revealed coordinated upregulation of cell cycle regulators and matrisome-associated genes, with survivin (BIRC5) identified as a central node linking proliferative signaling and ECM remodeling networks. Analysis of GBM patient specimens further showed strong nuclear survivin expression in regions with elevated collagen deposition. To directly evaluate stiffness-dependent regulation of survivin, GBM cells were cultured on fibronectin-infused hydrogels with tunable stiffness. Stiff matrices increased survivin expression along with cyclin D1 and cyclin A, consistent with increased cell cycle progression. Pharmacologic inhibition or siRNA-mediated suppression of survivin reduced stiffness-induced proliferation and attenuated expression of matrisome components, including collagens and lysyl oxidase. These findings indicate that survivin functions as a mechanosensitive regulator that coordinates cell cycle progression with ECM production in stiff tumor microenvironments. Collectively, this study identifies survivin as a key mediator linking ECM stiffness to GBM growth and matrisome remodeling. Targeting survivin and its effectors may offer a mechanosensitive strategy to limit GBM growth.

pathology↗

bioRxiv2025

Hepatocyte MLKL Drives Obesity-Driven Hepatocellular Carcinoma Progression via Mitochondrial Dysfunction Independent of Necroptosis in MASLD

Background and AimsMetabolic dysfunction-associated steatotic liver disease (MASLD) is a leading cause of hepatocellular carcinoma (HCC), particularly in obesity, yet mechanisms linking hepatocyte dysfunction to tumorigenesis remain unclear. Mixed lineage kinase domain-like protein (MLKL), the effector of necroptosis, is elevated in MASLD, but its hepatocyte-intrinsic role in obesity-driven MASLD-HCC is unknown. Approach and ResultsUsing a long-term Western diet (WD)-induced MASLD-HCC model in hepatocyte-specific MLKL knockout (MlklHepKO) mice, we defined MLKLs hepatocyte-intrinsic function. WD increased hepatocyte MLKL protein expression without detectable necroptosis activation, indicating a necroptosis-independent role. MLKL deficiency did not alter WD-induced inflammation, fibrosis, or liver injury but increased hepatic lipid accumulation while reducing lipotoxic lipid species and preserving mitochondrial function. WD-fed MlklHepKO mice developed fewer and smaller tumors with reduced incidence, multiplicity, proliferation, and stemness. Transcriptomic analysis revealed upregulation of mitochondrial oxidative phosphorylation pathways in MlklHepKO livers. WD suppressed the mitochondrial fusion protein and tumor suppressor MFN2, whereas MLKL deficiency restored MFN2 expression post-translationally. In HCC cells, MLKL deletion reduced proliferation, improved mitochondrial respiration, and decreased glycolysis; these effects were reversed by MFN2 deletion. MLKL localized to nuclear and mitochondrial compartments, consistent with organelle-intrinsic functions. The human MLKL inhibitor necrosulfonamide (NSA) suppressed HepG2 xenograft growth, and elevated MLKL expression in human HCC correlated with poorer overall survival. ConclusionsHepatocyte MLKL promotes MASLD-associated HCC through a non-necroptotic mechanism involving MFN2 suppression, impaired mitochondrial function, and increased tumor proliferation and stemness. These findings identify MLKL as a potential therapeutic target in MASLD-associated HCC.

cancer biology↗

bioRxiv2025

Butyrate rescues chlorpyrifos-induced social deficits through inhibition of class I histone deacetylases

Chlorpyrifos (CPF) is a widely used organophosphate pesticide effective through inhibiting acetylcholinesterase, which leads to the accumulation of acetylcholine and continuous nerve stimulation. In addition to its well-known acute toxicity, exposure to CPF has also been linked to chronic conditions such as an increasing risk of autism spectrum disorder (ASD) and adverse effects on gut health, including disturbances to the gut microbiome and metabolism. However, the underlying mechanism of CPFs contribution to ASD remains unclear, and the roles of the gut microbiome and gut metabolites in CPF-induced neurodevelopmental toxicity remain elusive. Using a high-throughput social behavior assay, we found that embryonic exposure to CPF induced lasting social deficits in zebrafish. Through a small-scale screen of common health beneficial gut microbiome metabolites, we discovered that butyrate effectively rescued CPF-induced social deficits. RNA sequencing of zebrafish brain tissues revealed that early exposure to CPF induced a lasting suppression of neuronal genes, including many ASD risk genes, and elevated expression of circadian genes. Butyrate partially reversed the suppression of key neuronal genes. Butyrate is a non-selective inhibitor of histone deacetylases (HDACs). Through a series of loss-of-function experiments utilizing CRISPR-Cas9-induced knockouts and selective chemical inhibitors, we found that the class I HDAC, HDAC1, most likely mediates butyrates rescue effect. Metabolomics analysis detected changes in several nitrogen metabolism-related pathways in the zebrafish gut following CPF exposure. Metagenomics analysis revealed an increase in abundance of the denitrifying bacteria Pseudomonas and a reduction in the nitric oxide-sensitive bacteria Aeromonas in the CPF-exposed zebrafish gut microbiome. Our results connect CPF-exposure with changes in the gut microbiome, metabolome, epigenetics, gene expression, and behavior, inspiring a novel hypothesis for the underlying molecular mechanisms of CPF-induced neurodevelopmental toxicity. In the long run, our findings may help elucidate how CPF exposure contributes to autism risk and inspire therapeutic developments.

neuroscience↗

bioRxiv2025

Persistent chromatin loops shape gene expression plasticity upon stimulation and restimulation of human neurons

Persistent molecular correlates of long-term memory storage remain an open question. Here, we stimulate and re-stimulate human neurons and use multi-modal single-nucleus technologies to query DNA methylation, higher-order chromatin folding, and gene expression. We find enduring traces of activity-gained and activity-lost chromatin loops. Genes anchoring persistent activity-gained loops exhibit activity-upregulated expression, whereas persistent activity-lost loops anchor activity-downregulated genes that remain repressed five days post-stimulation. CTCF-bound looped enhancers and promoters are refractory to activity-dynamic DNA methylation. Looped enhancers bound by CTCF can exhibit memory of activity-induced histone modifications and persistent expression of activity-upregulated genes. Upon second stimulation, activity-upregulated genes are robustly re-induced when unlooped but remain nonresponsive at persistent loops akin to habituation. Activity-independent gene expression can be downregulated when unlooped but protected from homeostatic downscaling when anchored in persistent loops. Our data reveal long-term genome folding persistence linked to plasticity of activity-dependent gene expression during recall in human neurons. Structured AbstractO_ST_ABSIntroductionC_ST_ABSA long-standing question in neuroscience is how memories of previous experiences are stored in the mammalian brain to facilitate recall over the lifetime of an individual. Classic models of memory posit that both synapse-specific events and cell-wide transcriptional programs are required for encoding, consolidation, and long-term storage of memory (1, 2). Learning involves synapse strengthening, and synapse weakening has been linked to memory loss (3, 4). Nascent transcription and protein synthesis also occur in response to neural activation in vitro and in vivo (5-7). A leading hypothesis, the synaptic tagging and capture model, asserts that specific synapses are biochemically marked during encoding and newly made cell-wide RNA/proteins act specifically on tagged synapses to maintain long-term memory (8). Multiple historic studies have pursued the identification of proteins linked to potentiated synapses (9-13), but few explore the possibility of persistent, activity-dependent patterns of DNA, chromatin, higher-order chromatin folding, or RNA in human models of long-term memory. Over the last decade, the molecular technique of Chromatin-Conformation-Capture has been employed to discover that the mammalian genome folds into thousands loops of (5, 14-28). Loops bring distal non-coding cis regulatory elements into contact with their target genes to influence gene expression (29). They form via the processive motion of the cohesin ring along chromatin until it stalls at the architectural protein CTCF, thus extruding out the intervening DNA (22, 30-32). Loops can markedly reconfigure during lineage commitment in development and in response to genetic perturbations, and their formation is critical for spatiotemporal regulation of expression (22, 30, 33-41). RationaleMultiple recent works suggest that loops connecting activity-dependent enhancers to distal genes and can be induced during neural stimulation in vitro and behavior paradigms in vivo (5, 27, 42, 43). Genetic elimination of the architectural proteins CTCF and cohesin prior to learning substantially impairs memory encoding across multiple behavior tasks in vivo (42, 44, 45). Cohesin-mediated loops are necessary for the establishment of new gene expression programs in post-mitotic neurons, including the upregulation of genes encoding axon guidance, dendritic spine morphology, and synaptic plasticity during neuron maturation in vivo and activity-dependent gene expression during neural stimulation in vitro (27). Fear conditioning recruits epigenetically plastic neurons to form the memory engram (46) and induces chromatin accessibility changes at enhancers that might persist at least five days after fear conditioning (43). Together, these data provide the rationale for our hypothesis that key structural features of higher-order chromatin folding could be persistent long after the exposure and removal of a pharmacological, environmental, or behavioral stimulus that causes neurons to fire action potentials. ResultsHere, we apply cutting-edge single-nucleus multi-modal technologies toward the goal of ascertaining the extent to which there are enduring traces of higher-order chromatin folding, DNA methylation, histone modifications at activity-dependent enhancers, and/or mRNA levels in single human neurons upon exposure to a repetitive stimulation and re-stimulation paradigm. We find that pharmacological stimulation of human induced pluripotent stem cell (iPSC)-derived neurons induces chromatin loop plasticity, including persistent activity-gained (PG) and persistent activity-lost (PL) loops that are dependent on CREB phosphorylation. We also query activity-dependent patterns of DNA methylation genome-wide and demonstrate that the majority of looped enhancers and promoters are negligibly differentially methylated in human neurons in response to pharmacological stimulation. CTCF binding to looped enhancers and promoters further protects such regulatory elements from activity-stimulated changes in DNA methylation. What are the potential functional effects of persistent chromatin loops? We find that activity-lost persistent loops anchor activity-downregulated genes that remain repressed five days post-stimulation. By contrast, activity-gained persistent loops are enriched for activity-induced gene expression at 2-5 hours post-stimulation, but mRNA levels resume baseline levels by five days after removal of the stimulus. A subset of promoter-enhancer loops bound by CTCF can exhibit enduring traces of the histone modification H3K27ac. Promoters persistently looped to CTCF-bound persistent activity-induced enhancers can exhibit memory of activity-upregulated gene expression for at least five days after stimulation. Finally, we re-stimulated our human neurons five days after the original stimulation event and assessed gene expression with single-nucleus RNA-seq. Upon second stimulation, we unexpectedly find that activity-upregulated genes can be robustly re-induced when unlooped but remain nonresponsive at persistent loops akin to habituation. We also unexpectedly find that activity-independent, invariant gene expression can be downregulated at second stimulation when unlooped but protected from homeostatic downscaling when anchored in persistent loops. ConclusionTaken together, our data reveal enduring traces of higher-order chromatin loops in human neurons after activity-simulation and their link to gene expression plasticity and habituation during re-stimulation and re-stimulation of human neurons. Our work sheds light on the role for chromatin and gene expression in an important unsolved paradox in neuroscience: How is information encoded in neural circuits on the timescale of years despite the rapid turnover of synaptic proteins/RNAs in hours to weeks? Persistent chromatin loops shape plasticity in cell-wide RNA levels, thus laying the foundation for future studies linking genes identified here to their subcellular localization and possible functional role at the synapse.

neuroscience↗

bioRxiv2025

Learning induces persistent chromatin loops underlying robust gene expression during memory recall

Long-term memories are stored in neuronal ensembles called engrams, but the existence of persistent molecular traces in nuclei of engram neurons remain unknown. Using activity-dependent nuclear tagging in vivo, we profiled higher-order chromatin folding and DNA methylation in thousands of single hippocampal neurons up to a month after contextual fear conditioning (CFC). We find CFC-induced chromatin loop plasticity genome-wide, including persistently gained and lost loops with enduring traces in vivo. DNA methylation showed minimal CFC-induced persistence at promoters and enhancers. Persistently gained and lost loops connect distinct enhancers and promoters in excitatory and inhibitory subtypes and correlate with robust CFC-upregulated and -downregulated gene expression, respectively, upon recall. Synaptic genes associated with post-traumatic stress disorder and autism anchor neuronal subtype-specific persistent loops, suggesting relevance to neuropsychiatric dysfunction. We harness the power and sensitivity of multi-modal single-cell measurements to find enduring chromatin traces linked to robust gene expression during fear memory recall. Structured AbstractO_ST_ABSIntroductionC_ST_ABSLong-term memory is thought to be stored in specific neural circuits called engrams (1). Mechanisms centered at the synapse have been proposed, but what is altered within each engram cell that persists for the duration of the memory is unknown. Neural circuits fire action potentials in response to experiences. Such electrochemical signals are converted to molecular signaling pathways which travel from the synapse to the nucleus to activate new gene expression programs. However, experience-dependent RNA and protein molecules as well as synaptic plasticity phenomena are short-lived, lasting for only a few hours to several days (2). Thus, the extent to which chromatin and gene expression changes induced by behavior persist and functionally contribute to memory encoding, consolidation, and retrieval remains an important answered question. DNA is folded in the mammalian nucleus into higher-order long-range chromatin looping interactions (3). Loops form mechanistically through extrusion in which cohesin subunits form a ring that shuttles along chromatin and extrudes out the intervening DNA until it stalls at boundaries occupied by architectural proteins such as CTCF (4, 5). CTCF-independent looping mechanisms have also been uncovered (6-10). A subset of loops regulates gene expression by bringing distant enhancers into spatial proximity with their target promoters. Loops connect activity-dependent enhancers to their distal target genes to govern gene expression during in vitro neural stimulation and in vivo behavior paradigms (11-14). However, the extent to which chromatin changes persist on time scales to support long-term memory storage in vivo remains unclear. RationaleMultiple studies support the idea that higher-order chromatin architecture is plastic and can undergo activity- and experience-dependent remodeling linked to gene expression. Electron microscopy measurements provide direct evidence of chromatin architecture plasticity within minutes, with some changes persisting for up to an hour after potassium chloride stimulation of primary hippocampal neurons (15). Perturbing loops by selectively eliminating either CTCF or cohesin subunits impairs memory encoding in multiple behavior models (11, 14, 16-18), disrupts long-term potentiation (17, 18), and alters dendritic morphology (13, 19, 20). Cohesin-mediated loops are necessary for the establishment of new gene expression programs in post-mitotic neurons, including the upregulation of genes encoding axon guidance, dendritic spine morphology, and synaptic plasticity during neuron maturation in vivo as well as activity-dependent regulation of secondary response genes during neural stimulation in vitro (12, 13). The state of histone modifications before learning influences which neurons are recruited into memory traces, suggesting chromatin carries long-lasting yet adaptable information during memory encoding and long-term storage (21). Moreover, a subset of enhancers retains chromatin accessibility after learning in vivo (11). Together, these observations suggest that chromatin might provide a durable regulatory scaffold that persists long after the initial experience to govern gene expression programs required for the aspects of long-term memory storage. ResultsHere, we employ activity-dependent nuclear labeling in TRAP2 (targeted recombination in active populations) mice to isolate nuclei from neurons stimulated during contextual fear conditioning (CFC) (22, 23). Using single-nucleus methyl 3C-sequencing (snm3C-seq3), we simultaneously profiled DNA methylation and 3D genome folding in the same single tagged neurons from the hippocampus in a time course up to 28 days after CFC and during fear memory recall. Our multi-modal inquiry offered ability to group neurons by their neuronal subtype-specific DNA methylation profile, and thereby computationally generate pseudobulk Chromosome Conformation Capture heatmaps per each sorted hippocampal neuron subtype. We find CFC-induced chromatin loop plasticity genome-wide, including persistently gained and persistently lost loops with enduring structural traces up to 28 days after training. Persistent loops connect distinct distal enhancers to target genes encoding synaptic plasticity and neurotransmitter signaling pathways unique to each excitatory and inhibitory neuron cell type. By contrast to loops, we observe negligible persistence of CFC-induced changes in DNA methylation at enhancers and promoters. Persistently gained and lost loops show upregulation and downregulation of gene expression upon recall, respectively, with the functional effect focused on genes with mRNA levels influenced during training. We uncovered a strong enrichment for genes associated with post-traumatic stress disorder and autism spectrum disorder anchoring the base of persistent loops, suggesting the relevance of enduring chromatin architecture traces to dysregulation of fear, anxiety, and stress in neuropsychiatric disorders. ConclusionOur findings reveal that learning induces neuronal subtype-specific changes in chromatin loops, a subset of which remain durable for at least a month in vivo. Persistent chromatin loops are linked to expression of disease-associated synaptic genes during fear memory recall, thus highlighting relevance to neuropsychiatric and neurodevelopmental disorders. Our work opens up new avenues for investigating enduring chromatin traces for their role in governing cell-wide mRNA levels of genes that impact synaptic plasticity during fear learning and long-term memory storage.

neuroscience↗

bioRxiv2025

A programmable genetic platform for engineering noninvasive biosensors

Creating genetic sensors for noninvasive visualization of biological activities in deep, optically opaque tissues holds immense potential for basic research and the development of genetic and cell-based therapies. MRI stands out among deep-tissue imaging methods for its ability to generate high-resolution images without ionizing radiation. However, the adoption of MRI as a mainstream biomolecular technology has been hindered by the lack of adaptable methods to link molecular events with genetically encodable MRI contrast. To address this challenge, we introduce universal reporter circuit-based activatable sensors (URCAS), a highly programmable platform for the systematic creation of genetic sensors for MRI. In developing URCAS, we engineered protease-activatable MRI reporters using two distinct approaches: protein stabilization and subcellular trafficking. We established the applicability of URCAS in five diverse mammalian cell types and showcased its versatility by assembling a toolkit of genetic sensors for viral proteins, small-molecule drugs, logic gates, protein-protein interactions, and calcium, without requiring new customization for each target. Our findings suggest that URCAS provides a modular, programmable platform for streamlining the development of noninvasive, nonionizing, and genetically encoded sensors for biomedical research and in vivo diagnostics.

bioengineering↗

bioRxiv2025

Protocol to isolate oligodendrocytes, microglia, endothelial cells, astrocytes, and neurons from a single mouse brain using magnetic-activated cell sorting

The isolation of specific cell types of the brain is essential to study cell-type-specific differences in complex neurological diseases such as Alzheimers disease. This protocol isolates oligodendrocytes, microglia, endothelial cells, astrocytes, and neurons from a single mouse brain. The process involves gentle tissue homogenization, debris removal, and sequential sorting of five distinct cell types. We validate cell purity and viability using flow cytometry and RT-qPCR. This protocol is well-suited for a range of downstream applications, including genomics, transcriptomics, and proteomics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/666877v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1426b97org.highwire.dtl.DTLVardef@1a5bb77org.highwire.dtl.DTLVardef@1b69f32org.highwire.dtl.DTLVardef@8da416_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Mimicking the night shift: Hypothalamic-pituitary-adrenal (HPA) axis regulation, energy homeostasis, and oxidative stress in a model diurnal songbird

Everchanging socioeconomic demands, coupled with modern day technological advancements have lifted constraints on human activity patterns, enabling us to take advantage of the 24-h period. However, varying patterns of light at night and sleep/wake may come at the cost of disrupting endogenous rhythms that govern biological processes and maintain homeostasis. To this end, we exposed zebra finches (Taeniopygia guttata castanotis), a diurnal songbird, to a consistent light/dark cycle of 12L:12D or alternating lighting patterns of 12D:12L and 12L:12D every three days for 66 days to mimic night shift work conditions. Surprisingly, we found that corticosterone profiles were inverted in birds exposed to simulated night shift work, with a slight trend indicating the inversion of melatonin profiles. Moreover, they had poor HPA axis reactivity in response to a standardized capture and restraint challenge and a significant reduction in corticosterone levels at ZT0 after simulated night shift work. Yet, we did not detect changes in body mass, subcutaneous fat deposits, or 4-hydroxynonaneal damage in the brain or liver. Our study underscores the need to capitalize on unique model organisms to evaluate consequences and resilience of simulated night shift work to inform public health decisions and the role of circadian plasticity.

physiology↗

bioRxiv2025

Mosaic H3K9me3 at BREACHes predicts synaptic gene expression associated with fragile X syndrome cognitive severity

Diseases vary in clinical presentation across individuals despite the same molecular diagnosis. In fragile X syndrome (FXS), mutation-length expansion of a CGG short tandem repeat (STR) in FMR1 causes reduced gene expression and FMRP loss. Nevertheless, FMR1 and FMRP are limited predictors of adaptive functioning and cognition in FXS patients, suggesting that molecular correlates of clinical measures would add diagnostic value. We recently uncovered Megabase-scale domains of heterochromatin (BREACHes) in FXS patient-derived iPSCs. Here, we identify BREACHes in FXS brain tissue (N=4) and absent from sex/age-matched neurotypical controls (N=4). BREACHes span >250 genes and exhibit patient-specific H3K9me3 variation. Using N=4 FXS iPSC lines and N=7 single-cell isogenic FXS iPSC subclones, we observe a strong correlation between inter-sample H3K9me3 variation and heterogeneous BREACH gene repression. We demonstrate improved prediction of cognitive metrics in FXS patients with an additive model of blood FMRP and mRNA levels of H3K9me3-mosaic, but not H3K9me3-invariant, BREACH genes. Our results highlight the utility of H3K9me3 variation at BREACHes for identifying genes associated with FXS clinical metrics.

genomics↗

bioRxiv2024

The CoREST complex inhibitor, corin, leads to decreased tumor growth, increased cellular differentiation and extended lifespan in atypical teratoid rhabdoid tumor xenograft models

BackgroundAtypical teratoid rhabdoid tumor (ATRT) is the most common malignant brain tumor in infants, and more than 60% of children with ATRT die from their tumor. ATRT is associated with mutational inactivation/deletion of SMARCB1, a member of the SWI/SNF chromatin remodeling complex, suggesting that epigenetic events play a critical role in tumor development and progression. Moreover, disruption of SWI/SNF allows unopposed activity of epigenetic repressors, which contribute to tumorigenicity. We therefore explored the role of the CoREST repressor complex in ATRT. MethodsWe evaluated the effects of the bifunctional LSD1/HDAC1/2 small molecule CoREST inhibitor, corin, on ATRT tumor cell growth, apoptosis, differentiation, gene expression and chromatin accessibility. ResultsWe found that corin inhibited the growth of ATRT cells regardless of their epigenetic subgroup, and was associated with increased tumor cell apoptosis and differentiation. ATAC-seq showed increases in chromatin accessibility in corin-treated ATRT cells, with changes seen at genes associated with neuronal differentiation and synaptic function. RNA-seq confirmed increased expression of neuronal differentiation genes and decreased DNA replication/cell cycle-associated genes in ATRT cells treated with corin. Corin suppressed orthotopic ATRT tumor growth, leading to significant extension of lifespan. In addition, increased histone acetylation (H3K9ac, H3K27ac) and methylation (H3K4Me1) was seen in corin-treated ATRT orthotopic xenografts, consistent with on-target pharmacodynamics. ConclusionThe CoREST inhibitor, corin, suppresses tumor growth, induces differentiation, and promotes apoptosis in ATRT, leading to significantly increased survival of mice bearing ATRT orthotopic xenografts. Our results suggest a potential application of CoREST complex inhibitors in patients with ATRT. Key PointsO_LICoREST complex inhibition by corin leads to decreased cell growth and increased apoptosis in ATRT C_LIO_LICorin promotes chromatin accessibility and neuronal differentiation in ATRT C_LIO_LICorin inhibits tumor growth and extends lifespan in ATRT animal models C_LI Importance of the StudyLoss of function of SMARCB1 is a hallmark of ATRT which leads to dysfunction of the mammalian SWI/SNF complex and an inability to counteract epigenetic repressor complexes. The CoREST complex functions as a chromatin remodeling complex that represses neuronal differentiation genes during development. Inhibition of the CoREST complex by corin in ATRT leads to decreased tumor cell growth, induction of apoptosis and increased survival of mice bearing ATRT orthotopic xenografts. These changes are associated with increased chromatin accessibility and expression of genes associated with neuronal differentiation. Corin therefore reverses the primary block of differentiation that maintains a stem cell state in ATRT, which contributes to tumorigenesis. These studies significantly improve our understanding of how to therapeutically address the underlying epigenetic drivers of ATRT and support further development of corin for ATRT.

cancer biology↗

bioRxiv2024

Survivin modulates stiffness-induced vascular smooth muscle cell motility

Arterial stiffness is a key contributor to cardiovascular diseases, including atherosclerosis, restenosis, and coronary artery disease, it has been characterized to be associated with the aberrant migration of vascular smooth muscle cells (VSMCs). However, the underlying molecular mechanisms driving VSMC migration in stiff environments remain incompletely understood. We recently demonstrated that survivin, a member of the inhibitor of apoptosis protein family, is highly expressed in both mouse and human VSMCs cultured on stiff polyacrylamide hydrogels, where it modulates stiffness-mediated cell cycle progression and proliferation. However, its role in stiffness-dependent VSMC migration remains unknown. To assess its impact on migration, we performed time-lapse video microscopy on VSMCs seeded on fibronectin-coated soft and stiff polyacrylamide hydrogels, mimicking the physiological stiffness of normal and diseased arteries, with either survivin inhibition or overexpression. We observed that VSMC motility increased under stiff conditions, while pharmacologic or siRNA-mediated inhibition of survivin reduced stiffness-stimulated migration to rates similar to those observed under soft conditions. Further investigation revealed that cells on stiff hydrogels exhibited greater directional movement and robust lamellipodial protrusion compared to those on soft hydrogels. Interestingly, survivin-inhibited cells on stiff hydrogels showed reduced directional persistence and lamellipodial protrusion compared to control cells. We also examined whether survivin overexpression alone is sufficient to induce cell migration on soft hydrogels, and found that survivin overexpression modestly increased cell motility and partially rescued the lack of directional persistence compared to GFP-expressing control VSMCs on soft hydrogels. In conclusion, our findings demonstrate that survivin plays a key role in regulating stiffness-induced VSMC migration, suggesting that targeting survivin and its signaling pathways could offer therapeutic strategies for addressing arterial stiffness in cardiovascular diseases.

cell biology↗