Biology subjects

Green, R.

Publications and source records attributed to Green, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Bispecific GD2xB7-H3 Antibody Improves Tumor Targeting and Reduces Toxicity while Maintaining Efficacy for Neuroblastoma

The current treatment regimen for neuroblastoma involves immunotherapy, including a monoclonal antibody that recognizes disialoganglioside (GD2), expressed at high levels on neuroblastoma. GD2 is not present on most normal tissues except for nerves. Thus, anti-GD2 antibody treatment causes substantial, dose-limiting neuropathic pain. B7-H3 is overexpressed on multiple tumor types, including neuroblastoma, and is largely absent on nerves and other normal tissues. We designed a bispecific antibody (bsAb) that requires simultaneous binding of these two tumor antigens to achieve tight binding of tumor cells. Our preclinical research shows that, compared with a monospecific anti-GD2 antibody, the GD2xB7-H3 bsAb has improved tumor specificity, comparable antitumor efficacy, and reduced nerve binding and pain-associated toxicity. Since this bsAb does not bind to nerves, it may permit more tolerable and sustained treatment schedules than are currently feasible with monospecific anti-GD2 antibodies. In addition, its enhanced tumor specificity may support future development as a targeted delivery platform for antibody-drug conjugates or other payload-based therapies, potentially improving both efficacy and quality of life for patients with neuroblastoma.

cancer biology↗

bioRxiv2026

Substrate Binding Relieves Autoinhibition of the Mammalian GCN2 Kinase

Cells respond to amino acid starvation and translational stress through the integrated stress response (ISR) kinase GCN2. Current models maintain that GCN2 senses these stresses either by binding deacylated tRNAs that accumulate during amino acid starvation or by recognizing ribosome collisions that arise from perturbation of translation elongation. As tRNA charging is inherently coupled to translation elongation, assessing the relative contributions of these two pathways has proven difficult. To fully separate the roles of these potential agonists, we reconstituted the regulation of GCN2 in vitro using recombinant proteins. GCN2 has low basal activity, which is stimulated by the addition of ribosomes or the ribosomal P-stalk. By contrast, deacylated tRNAs fail to activate GCN2 both in vitro and in vivo and instead counteract activation by the ribosome and inhibit GCN2 auto- and substrate phosphorylation. This inhibition of autophosphorylation requires the presence of GCN2's substrate, eIF2, suggesting that the substrate plays a role in kinase regulation. Indeed, eIF2 binding to a newly defined autoinhibitory loop in the GCN2 kinase domain relieves its repression and stimulates autophosphorylation of the kinase's activation loop. Together, these data redefine the role of tRNAs in the regulation of GCN2 and reveal a mechanism novel to kinases in general by which substrate binding regulates activation loop phosphorylation.

biochemistry↗

bioRxiv2026

Dendrimer Delivered shRNA Targeting the CCL20-CCR6 Axis Suppresses Complement-Mediated Microglial Synaptic Pruning and Ameliorates Chronic Neuroinflammation After Repetitive Traumatic Brain Injury

Repetitive traumatic brain injury (rTBI) induces persistent microglial activation and chronic neuroinflammation, yet the upstream signals driving long-term synaptic injury remain unclear. In this study, we identify the CCL20-CCR6 chemokine axis as a critical regulator of sustained microglial activation and complement-dependent synaptic loss after rTBI. Proteomic profiling at 30 days post-injury (dpi) showed broad normalization of complement-linked inflammatory and synaptic pathways in the cortex and hippocampus, underscoring a mechanistic link between chemokine signaling, microglial activation, and synaptic vulnerability. To therapeutically target this axis, we developed a dendrimer-based shRNA platform (shCombo-DPX) that simultaneously silences CCL20 and CCR6. Intranasal and intravenous delivery in rTBI mice effectively reduced CCL20-CCR6 expression, attenuated chronic microgliosis and astrogliosis, and suppressed complement activation. Treatment limited microglial synaptic engulfment, preserved synaptic proteins, restored BDNF levels, and improved motor, anxiety-related, and cognitive outcomes. In microglia-neuron coculture systems, CCL20 silencing reduced LPS-induced complement signaling and prevented synaptic loss, neuronal apoptosis, and BDNF depletion. Conversely, exposure to recombinant CCL20 induced dendritic degeneration, caspase-3 activation, microglial reactivity, complement dysregulation, and synaptic injury both in vitro and in vivo. Collectively, these findings establish CCL20-CCR6 as a key upstream driver of chronic complement-mediated synaptic degeneration after rTBI and support dendrimer-delivered shRNA therapy as a targeted strategy to mitigate long-term neurodegeneration.

neuroscience↗

bioRxiv2026

CCL20-CCR6 Signaling as a Prognostic Biomarker and Therapeutic Target in Temozolomide-Resistant Glioblastoma

Glioblastoma remains highly lethal, with median survival of ~15 months. Resistance to temozolomide is ubiquitous, yet its mechanisms are incompletely understood. Here, we identify the CCL20-CCR6 chemokine axis as a stress-responsive survival pathway limiting therapeutic efficacy. Targeting CCL20-CCR6 in combination with temozolomide and cannabidiol was evaluated using clinical datasets, GBM cell lines, tumor organoids, and a syngeneic CT-2A mouse model integrating proteomic and lipidomic profiling. Low CCL20 expression was associated with improved survival, supporting its prognostic relevance. Across models, TMZ alone or with CBD induced CCL20 expression while exerting limited antitumor activity. Targeted disruption of CCL20-CCR6 signaling using dendrimer-delivered shRNA enhanced therapeutic response in murine models and GBM organoids. Multi-omic analyses revealed that CCL20 inhibition reprograms the tumor microenvironment and induces mitochondrial dysfunction, resulting in elevated reactive oxygen species (ROS) and tumor cell death. This effect was accompanied by accumulation of 17-hydroxydocosahexaenoic acid and activation of oxidative stress-associated cytotoxic pathways. Functional assays confirmed that CCL20 blockade selectively amplifies mitochondrial ROS beyond levels induced by TMZ alone potentiating TMZ efficacy by promoting mitochondrial oxidative stress. Targeting this axis represents a promising strategy to overcome chemoresistance and positions CCL20 as both a prognostic biomarker and a therapeutic vulnerability in GBM.

cancer biology↗

bioRxiv2026

SERBP1 is a master regulator of ribosome interactions

Ribosome function depends on interactions with diverse proteins whose activities determine translational efficiency, impose quality control and activate signaling pathways. These many competing activities must in turn be regulated. SERBP1 is an abundant cellular factor that interacts with ribosomes at multiple functionally critical sites on both dormant and active ribosomes. Here, we perform mass spectrometry across sucrose gradients in untreated and stressed cells and find that SERBP1 modulates ribosome interactions with many factors involved in processes including mRNA degradation, translational control, ribosome quality control and ribosome degradation. We then define the role of SERBP1 in protection of ribosomes against selective 40S degradation in the context of mTOR inhibition through its competition with the E3 ligase RNF10 and the atypical kinase RIOK3. Our work reveals SERBP1 as a key player in maintaining ribosome subunit balance and more broadly in the regulation of diverse ribosome activities.

molecular biology↗

bioRxiv2026

A Deep Learning Framework for Biomarker Segmentation and Classification in Traumatic Brain Injury

Traumatic brain injury (TBI) triggers widespread biomarker activation, including astrocytic markers such as glial fibrillary acidic protein (GFAP) and microglia markers such as ionized calcium-binding adapter molecule 1 (IBA1). Quantifying and analyzing these biomarkers are critical for understanding injury impact; however, current methods are labor-intensive and time-consuming. In this study, we propose an automated deep learning framework for dual-biomarker segmentation and TBI classification using GFAP and IBA1 immunofluorescent images. Four U-Net variants: Baseline U-Net, U-Net++, MANet, and LinkNet were trained for segmentation. Three classification models, ResNet50, Swin_T, and MaxViT, were trained to distinguish TBI from control images under single- and dual-biomarker conditions. The baseline U-Net achieved the highest segmentation Dice score for GFAP (0.9259), while the U-Net++ achieved the highest Dice score for IBA1 (0.9676). Trained segmentation models demonstrated significantly better performance compared to QuPath alternatives. While GFAP alone supported high classification accuracy, IBA1 alone was less effective. Multimodal fusion of GFAP and IBA1 significantly improved classification performance across all models, with Swin_T achieving the highest overall accuracy (0.9489), and ResNet50 achieving the highest F1-score (0.9499). These findings demonstrate that integrating complementary biomarkers enhances automated TBI classification, and deep learning offers a robust alternative to manual analysis for immunofluorescent brain injury imaging. This framework is scalable to additional biomarkers and injury models, offering a reproducible approach to accelerate biomarker research.

neuroscience↗

bioRxiv2026

Host species background, defence systems, and phage tail gene architecture shape phage infectivity in cystic fibrosis-associated Achromobacter

Achromobacter species are emerging multidrug-resistant (MDR) pathogens in people with cystic fibrosis. Their increasing resistance has grown an interest in phage therapy as an alternative treatment strategy. However, the factors governing phage susceptibility remain poorly understood, thereby limiting the rational selection of phage candidates. Using 15 strictly lytic Achromobacter phages and 7 clinical cystic fibrosis isolates representing Achromobacter insolitus and Achromobacter xylosoxidans, we demonstrate substantial variation in infection efficiency across all 105 phage-host combinations, variation that could not be discerned from qualitative plaque assays alone. We integrated complete bacterial and phage genomes with quantitative efficiency-of-plating (EOP) assays and lineage-aware Bayesian mixed-effects modelling to show that phage infectivity in Achromobacter is governed predominantly by bacterial lineage and strain identity, accounting for 90% of total variance in log-normalised EOP, with individual strains varying substantially in permissiveness irrespective of species membership. After accounting for this lineage structure, no individual defence system, antimicrobial resistance gene class, or phage tail cluster retained a statistically significant independent or interaction association with infectivity. Together, these findings demonstrate that bacterial strain identity is the primary driver of infection outcome. Host defence systems and phage tail-associated genes remain biologically plausible contributors; their independent effect could not be resolved after accounting for lineage structure, indicating that infection outcomes are largely strain-dependent. This work shifts the question from which individual traits predict infection to how strain lineage and specific host-phage combinations jointly determine infectivity, and argues that quantitative phenotyping of individual phage-host pairs is essential for guiding phage candidate selection and supporting rational cocktail design against multidrug-resistant Achromobacter infections in cystic fibrosis. Impact statementChronic Achromobacter infections in cystic fibrosis are increasingly difficult to treat due to multidrug resistance and biofilm formation. Although phage therapy is a promising alternative, its development is limited by poorly understood and highly variable infectivity. Here, we show that infectivity within a phage host range spans a broad quantitative continuum spanning several orders of magnitude that cannot be captured by qualitative plaque assays. These infection efficiencies are primarily structured by bacterial lineage and strain identity, while the contributions of individual genomic features remain unresolved, given the current sample size. This work provides a framework for predicting phage-host compatibility and supports a shift from empirical screening toward rational, evidence-based phage selection for MDR Achromobacter infections.

molecular biology↗

bioRxiv2026

Conformational diversity of collided ribosomes determines stress signaling

Ribosome collisions trigger pathways that clear stalled ribosomes, and when sufficiently abundant, the integrated stress response (ISR) through GCN2 and the ribotoxic stress response (RSR) through ZAK. The inhibitors anisomycin (ANS), emetine (EME), and didemnin B (DDB) are commonly used to induce collisions in studying these responses. Here, we demonstrate that these drugs induce distinct signatures: ANS and DDB potently activate ZAK whereas EME does not. We define transcriptional programs induced by these inhibitors, where collisions induce the RSR and general inhibition of translation leads to an RSR-independent response. Surprisingly, we find that collisions induced by EME, unlike ANS, are not cleared by ASCC3. The cryo-EM structure of human disomes stalled by EME reveals its mechanism of inhibition and a conformation distinct from ANS-stalled disomes. These differences in collision geometry explain the different outcomes in quality control and signaling activation, showing how ribosome stalling events can yield distinct cellular responses.

Molecular Biology↗

bioRxiv2026

A conserved dimerization element is required for protein kinase activation by trans-autophosphorylation

Trans-autophosphorylation is the most common mode of protein kinase activation and involves two copies of the same kinase dimerizing so that one can phosphorylate the activation loop of the other. The diversity among structures of trans-autophosphorylation dimers supported the view that each kinase evolved a unique mode of recognition. We screened all human kinase crystal structures and identified an expanded set of dimers compatible with trans-autophosphorylation (655 dimers from 143 kinases). These dimers share no conserved structural arrangement, but 85% bury the same helix, G, at the dimer interface. We validate G-mediated dimerization by mutagenesis in kinases from each group of the kinome activated by trans-autophosphorylation. G substitution impaired or abolished activation of full-length proteins, in cells, in every case. In purified kinase domains, G substitution disrupted dimerization and autophosphorylation. These data establish that dimerization during trans-autophosphorylation is conserved and is mediated by a common structural element that, surprisingly, does not impose a specific arrangement of the two kinase domains relative to each other. G is the least conserved element in the kinase fold, yet is required for activation across both the human kinome and other species, suggesting an ancestral function of the kinase fold. Significance StatementProtein kinases are the largest enzyme family in the human genome and common pharmaceutical targets. Most are activated by trans-autophosphorylation, during which two copies of the same kinase dimerize and one phosphorylates the activation loop of the other. How this is achieved remains poorly understood. We find dimerization during activation is conserved, but in an unexpected way. An unbiased structural screen of all human kinase crystal structures reveals kinases across the kinome bury the same helix, G, at the dimer interface. G-mediated dimerization extends to other species suggesting an ancestral function of the kinase fold. Substitution of G disrupts activation of every kinase tested. Despite this conservation, G-mediated dimers share no common arrangement, representing an unusual mode of protein-protein interaction.

biochemistry↗

bioRxiv2026

Mechanism of nucleolytic degradation of human ribosomes

Stresses like starvation trigger degradation of mature 40S ribosomes, requiring the coordinated breakdown of large and stable RNA-protein complexes. The atypical kinase RIOK3 orchestrates degradation by binding ubiquitylated 40S ribosomes and promoting rRNA decay. However, the mechanisms and factors that mediate rRNA decay remain unknown. Here we find that in response to starvation, RIOK3 recruits the terminal uridylyl-transferase TUT7 and the exonuclease DIS3L2 to 40S ribosomes. Sequencing analyses show that TUT7 adds oligo(uridine) tails to the 3' end of the 18S rRNA in these ribosomes. DIS3L2 subsequently recognizes uridylated 18S rRNA and carries out 3'-5' decay. We identify major decay intermediates that undergo further uridylation in a process of iterative uridylation and decay. Loss of DIS3L2 impairs 18S rRNA decay during starvation and leads to accumulation of uridylated 18S rRNA. Together these findings define a mechanism for ribosome degradation in which 3' oligo(uridine) tailing drives decay of rRNA from ribosomes.

molecular biology↗

bioRxiv2026

The LARP1 RRM functions as a ribosome responsive regulator of TOP mRNAs

Ribosome concentration varies across cell type, state, and disease, yet the mechanism by which mammalian cells sense ribosome levels to regulate ribosome synthesis remains unknown. Previous studies implicate LARP1 in binding ribosomes and, separately, in repressing mRNAs containing TOP motifs (TOPs), which encode all ribosomal proteins as well as several translation factors. Here, we show that ribosome binding is the essential licensing event for LARP1-mediated TOP repression. Phylogenetic analysis reveals that LARP1's ribosome binding region is part of a highly conserved RNA recognition motif (RRM) which is predicted to directly interact with its TOP-binding HEAT repeat domain. Ribosome binding activates the HEAT repeats to bind and repress TOPs, thereby repressing TOP translation and ribosome synthesis. Mutants which uncouple the coordinated functions of these domains constitutively repress TOPs and compromise cell fitness. These data reveal an unprecedented ribosome sensing function of LARP1, orchestrated through the unique coordinating role of its RRM, unveiling the mechanism by which mammalian cells tune ribosome synthesis to demand across diverse contexts.

molecular biology↗

bioRxiv2025

Large-scale culturing of the tree microbiome enables targeted disease suppression

The tree microbiome is essential for host health and pathogen suppression. Synthetic microbial communities (SynComs) are emerging as important tools to understand microbiome dynamics and engineer microbiomes to harness beneficial properties. However, while the rational design, assembly and application of SynComs requires representative microbiota isolate collections combined with functional information, microbial culture collections from tree species such as oak (Quercus) are critically lacking. Here, we generated an oak microbiota culture collection comprising >30,000 isolates from 150 oak trees across Britain, belonging to key bacterial and fungal taxa that represented 61% of the total bacterial sequences and 87% of total fungal sequences as determined by culture-independent sequencing. Over 22,000 isolates were screened for suppression of bacterial species associated with degradation of live stem tissue in trees affected by Acute Oak Decline (AOD), identifying 341 bacterial isolates that suppressed oak pathogens. In vitro screening of 40 randomly assembled SynComs demonstrated that oak microbiota SynComs can suppress oak pathogenic bacteria associated with AOD. Inoculation of a disease-suppressive SynCom into the stem of oak seedlings and logs prior to pathogen challenge reduced the quantities of the bacteria, Brenneria goodwinii and Gibbsiella quercinecans, by 56% and 87%, respectively, in seedlings, and 71% and 95% in logs. This work demonstrates that the tree microbiome can be engineered using disease suppressive SynComs.

microbiology↗

bioRxiv2025

Neuromodulation of a peripheral nerve using fully polymeric cuff electrodes: Understanding predictability of selective stimulation

Peripheral nerve stimulation (PNS) offers therapeutic benefits across numerous clinical applications but suffers from limitations in high resolution spatial selectivity, especially in mixed nerves. This study presents a fully polymeric, transverse, multipolar nerve cuff made from a conductive elastomer (CE), designed for selective activation of individual fascicles. Fabricated with laser-based manufacturing techniques, the CE nerve cuff offers mechanical conformity and high charge injection capacity. Ex vivo experiments on the rat sciatic nerve demonstrate reliable compound action potential recordings and fascicular selectivity (SI > 0.65). The non-metal electrodes enable microCT-aided 3D reconstruction of nerve-electrode geometries without imaging artefacts, informing anatomically accurate simulations via the ASCENT pipeline. While in silico simulations predict some selective fascicular activation, discrepancies were observed between predicted and experimental selectivity magnitudes and electrode positions, particularly for the sural and tibial fascicles. The model was more sensitive to neuroanatomical variation than the experimental data, indicating limitations in current perineurium and CE electrode modelling assumptions. This work validates CE-based cuffs as viable alternatives to metallic devices for selective fascicular peripheral nerve activation and highlights the potential of imaging-informed simulations to optimize nerve interface design. Future improvements in electrode and nerve tissue modelling are needed to enhance in silico prediction accuracy and further advance spatially selective PNS technologies.

bioengineering↗

bioRxiv2025

Astrocyte-Guided Maturation of Neural Constructs in a Modular Biosynthetic Hydrogel for Biohybrid Neurotechnologies

Bionic implants are increasingly used to restore neural function yet achieving a chronically stable neural interface remains challenging. Biohybrid neurotechnologies aim to overcome this limitation by integrating living tissue components that can promote long-term performance and functional integration with the nervous system. However, existing biomaterial coatings often lack the physiological complexity, cellular heterogeneity, and neurotrophic support required to sustain neural network formation. Here, we present a modular strategy which leverages astrocyte-guided mechanisms of neural network development. A biosynthetic hydrogel was developed using norbornene-functionalized poly(vinyl alcohol) and gelatin (PVA-GEL), crosslinked via visible light-triggered thiol-ene chemistry, to yield bioactive and highly tuneable scaffolds. A systematic characterization varying gelatin content and polymer weight enabled the identification an optimal formulation to promote astrocytic growth, while maintaining mechanical stability and degradation profiles that are compatible with brain implants. Co-encapsulation with neural progenitors promoted neuronal differentiation, neurite outgrowth, and the formation of synaptically competent networks. The construct developed into functional interfaces when in contact with brain tissue ex vivo, highlighting its potential as a biohybrid electrode coating. This work lays the foundation for the development of biologically guided biohybrid interfaces, towards seamless integration with the nervous system.

bioengineering↗

bioRxiv2025

Protein Structure Informed Bacteriophage Genome Annotation with Phold

Bacteriophage (phage) genome annotation is essential for understanding their functional potential and suitability for use as therapeutic agents. Here we introduce Phold, an annotation framework utilising protein structural information that combines the ProstT5 protein language model and structural alignment tool Foldseek. Phold assigns annotations using a database of over 1.36 million predicted phage protein structures with high quality functional labels. Benchmarking reveals that Phold outperforms existing sequence-based homology approaches in functional annotation sensitivity whilst maintaining speed, consistency and scalability. Applying Phold to diverse cultured and metagenomic phage genomes shows it consistently annotates over 50% of genes on an average phage and 40% on an average archaeal virus. Comparisons of phage protein structures to other protein structures across the tree of life reveals that phage proteins commonly have structural homology to proteins shared across the tree of life, particularly those that have nucleic acid metabolism and enzymatic functions. Phold is available as free and open-source software at https://github.com/gbouras13/phold.

bioinformatics↗

bioRxiv2025

Identifying rare spontaneous mutations through wildtype E. coli population sequencing

Understanding the rate and types of mutations occurring in different populations is fundamental to our ability to predict and potentially manipulate evolution. Common techniques for microbial mutation rate estimation fall into 2 camps; rapid, locus specific, generally liquid-culture based fluctuation assays; and slow, genome-wide, generally solid-culture based mutation accumulation experiments. This constrains the hypotheses which can be tested, notably the mutagenic effects of liquid environments cannot be rapidly quantified at a genome-wide scale. One example of such effect is the negative association previously observed between population density and mutation rates at many marker loci using the fluctuation assay. Because homogeneous population density is specifically relevant in liquid culture, this association has not been tested at a truly genome-wide scale. We fill this methodological gap by developing a novel pipeline that relies on population sequencing to capture how mutation rates are affected by population density in liquid cultures. We simulate expected mutation counts in a growing population along with random sampling during sequencing to estimate the necessary sequencing coverage as [≥]1,000-fold. We then carry out PCR-free sequencing of 95 wildtype E. coli populations at this coverage, calling rare variants with both reference-based and reference-free methods. These variant-calling methods are prone to different sources of error which can be minimised by considering only mutations called by both pipelines. This approach identifies 119 mutations across all cultures, with (non)coding/(non)synonymous and mutational spectrum profiles consistent with being samples of spontaneous mutation unbiased by selection. The distribution of these mutations also supports the motivating hypothesis, finding that mutation counts across the genome are strongly negatively associated with population density. This demonstrates the utility of population sequencing for the rapid testing of many previously inaccessible evolutionary biology hypotheses.

evolutionary biology↗

bioRxiv2025

CTx001 for geographic atrophy: a gene therapy expressing soluble, truncated complement receptor 1 (mini-CR1)

Over-activation of the complement system is strongly associated with geographic atrophy (GA), a late-stage form of age-related macular degeneration (AMD) and major cause of blindness. Here we report the development of a candidate GA treatment that is a potent complement modifier (mini-CR1) that addresses consequences of complement over-activation including membrane attack complex (MAC) formation. Mini-CR1 acts a potent cofactor for factor I driven proteolysis of C3b leading to iC3b and then C3dg formation; iC3b itself remains a potent opsonin. As well as inhibiting the alternative complement pathway, mini-CR1 acts as a cofactor for C4b degradation thereby inhibiting the classical pathway. Mini-CR1 prevents MAC formation in activated human serum with an IC50 of 125 nM. Mini-CR1 was shown to cross human Bruchs membrane ex vivo, implying the ability to cross into the choroidal space. Transduction of RPE cell lines with rAAV2-mini-CR1 (CTx001) resulted in dose-dependent transcription, and both basolateral and apical secretion of mini-CR1 by monolayers of human iPSC derived RPE cells. Mini-CR1 transduction of ARPE19 cells resulted in increased consumption of C3b and iC3b1 in activated culture media and decreased MAC formation on the cell surfaces. Subretinal injection of CTx001 in rats resulted in dose-dependent mini-CR1 production as demonstrated by solid-phase immunoassay. MAC formation following laser induced CNV in a rat model was reduced by 75% in CTx001-treated animals relative to null vector (p<0.01). This first generation of CTx001 represents a potent single administration complement modifier capable of effectively addressing pathologic complement amplification in the retina/choroid.

biochemistry↗

bioRxiv2025

Excess prenatal folic acid supplementation alters cortical gene expression networks and electrophysiology

Folate is crucial for various biological processes, with deficiencies during pregnancy being linked to increased risk for neural tube defects and neurodevelopmental disorders. As a proactive measure, folic acid fortification in foods has been mandated in many countries, in addition to dietary supplementation recommendations during pregnancy. However, the risks of excess prenatal folic acid supply have yet to be fully understood. To better appreciate in utero molecular changes in mouse brain exposed to 5-fold folic acid excess over normal supplementation, we investigated the transcriptome and methylome for alterations in gene networks. RNA-seq analysis of cerebral cortex collected at birth, revealed significant expression differences in 646 genes with major roles in protein translation. Whole genome bisulfite sequencing revealed 910 significantly differentially methylated regions with functions enriched in glutamatergic synapse and glutathione pathways. To explore the physiological consequences of excess prenatal folic acid exposure, we applied high-density microelectrode arrays to record network-level firing patterns of dissociated cortical neurons. Folic acid excess-derived cortical neurons exhibited significantly altered network activity, characterized by reduced burst amplitude and increased burst frequency, indicating compromised network synchronization. These functional deficits align with the observed molecular alterations in glutamatergic synapse pathways, underscoring the potential for excess prenatal folic acid exposure to disrupt developing metabolic and neurological pathways.

neuroscience↗