Biology subjects

Walsh, P.

Publications and source records attributed to Walsh, P..

10 recordsLinked to original sources

bioRxiv2025

Immune signaling mediates stromal changes to support epithelial reprogramming in Celiac duodenum

Coeliac Disease (CeD) is a chronic autoimmune disorder affecting 0.5-1% of the general population with a wide geographical distribution. Despite recent efforts to deeply phenotype gluten-specific immune activation at the single cell level, recent clinical studies targeting gluten degradation and other immune tolerance mechanisms have been unsuccessful. To this end, a deeper understanding of immune and non-immune cellular dynamics and interactions are required to characterize tissue-specific mechanisms responsible for CeD pathogenesis, repair, and resolution. Here, we assembled the most comprehensive scRNAseq dataset in Coeliac Disease to date, including 203,555 cells across 21 active CeD and 11 control duodenal samples. Compared to control duodenum, CeD was characterized by single cell differential changes in abundance, gene expression and cell-cell interactions across cellular compartments. In the immune compartments, CeD samples showed expected increases in plasma cell abundance and shifts toward type 1 effector biology (e.g., increase in cycling CD8pos, {gamma}{delta} T cells and IFNG transcriptional shifts) and Tfh-related biology (e.g., increases in IL21 signaling to effector T cells). In addition, activated myeloid subsets, including DC2 and monocytes, were increased in disease and were characterized by increased pro-inflammatory pathway expression, including IL-1{beta}. Non-immune compartments showed increased stem/crypt and secretory enterocytes in CeD samples with a decrease in absorptive enterocytes, reflecting the villus atrophy and crypt hyperplasia hallmarks of CeD epithelial dysfunction. Accompanying the epithelial changes, distinct changes in stromal populations were identified, particularly with increases in abundance and transcriptional activity of NRG1 and SMOC2 fibroblasts. Cell-cell interaction analysis across multiple cellular compartments proposed a distinct increased role of fibroblasts to support the epithelial reprogramming of the increased stem/crypt epithelial fraction in CeD, mediated by myeloid derived IL-1{beta} signal and lymphoid-derived IFN-{gamma}. This dataset reveals a previously unknown role for T-myeloid-stromal-epithelial cell communication in CeD, highlighting key mechanisms of the tissue-level cellular dynamics in response to gluten ingestion.

immunology↗

bioRxiv2024

Profiling Human iPSC-Derived Sensory Neurons for Analgesic Drug Screening Using a Multi-Electrode Array

Chronic pain is a major global health issue, yet effective treatments are limited by poor translation from preclinical studies to humans. To address this, we developed a high-content screening (HCS) platform for analgesic discovery using hiPSC-derived nociceptors. These cells were cultured on multi-well micro-electrode arrays to monitor activity, achieving nearly 100% active electrodes by week two, maintaining stable activity for at least two weeks. After maturation (28 days), we exposed the nociceptors to various drugs, assessing their effects on neuronal activity, with excellent assay performance (Z values >0.5). Pharmacological tests showed responses to analgesic targets, including ion channels (Nav, Cav, Kv, TRPV1), neurotransmitter receptors (AMPAR, GABA-R), and kinase inhibitors (tyrosine, JAK1/2). Transcriptomic analysis confirmed the presence of these drug targets, although expression levels varied compared to primary human dorsal root ganglion cells. This HCS platform facilitates the rapid discovery of novel analgesics, reducing the risk of preclinical-to-human translation failure. MotivationChronic pain affects approximately 1.5 billion people worldwide, yet effective treatments remain elusive. A significant barrier to progress in analgesic drug discovery is the limited translation of preclinical findings to human clinical outcomes. Traditional rodent models, although widely used, often fail to accurately predict human responses, while human primary tissues are limited by scarcity, technical difficulties, and ethical concerns. Recent advancements have identified human induced pluripotent stem cell (hiPSC)-derived nociceptors as promising alternatives; however, current differentiation protocols produce cells with inconsistent and physiologically questionable phenotypes. To address these challenges, our study introduces a novel high-content screening (HCS) platform using hiPSC-derived nociceptors cultured on multi-well micro-electrode arrays (MEAs). The "Anatomic" protocol, used to generate these nociceptors, ensures cells with transcriptomic profiles closely matching human primary sensory neurons. Our platform achieves nearly 100% active electrode yield within two weeks and demonstrates sustained, stable activity over time. Additionally, robust Z factor analysis (exceeding 0.5) confirms the platforms reliability, while pharmacological validation establishes the functional expression of critical analgesic targets. This innovative approach improves both the efficiency and clinical relevance of analgesic drug screening, potentially bridging the translational gap between preclinical studies and human clinical trials, and offering new hope for effective pain management.

neuroscience↗

bioRxiv2024

Transcriptomic analysis and high throughput functional characterization of human induced pluripotent stem cell derived sensory neurons

Peripheral sensory neurons are a primary effector in pain neurotransmission, and have become a useful cellular model for the study of pain. While rodent tissue has historically served as a source of these neurons, it has become increasingly clear that pain mechanisms in rodents and humans are substantially divergent. Sensory neurons harvested from cadaveric human tissue serve as a superior translational model for studying pain mechanisms, however their relative paucity limits their widespread utility. Theoretically, sensory neurons manufactured from human pluripotent stem cells (hPSCs) could help bridge this translational gap given their relative abundance and potential similarity to primary human tissue. However, hPSC-derived sensory neurons manufactured with the most common methodologies correlate poorly to human tissue both transcriptionally and functionally. In the present work, we compare a novel population of hPSC-derived sensory neurons to previously published datasets and find this novel population to more closely resemble human primary dorsal root ganglia transcriptionally. Furthermore, we evaluate the heterogeneity of this novel population via single nucleus RNA sequencing and find it resembles specific nociceptor and mechanoreceptor subsets found in vivo. Finally, we assay the functionality of this population with high throughput automated patch clamp electrophysiology with respect to voltage-gated sodium (Nav) and potassium channels (Kv), and ligand-gated ionotropic GABA and P2X receptors. Overall, we find this population of hPSC-derived sensory neurons to be of relatively high fidelity, and suitable for interrogating numerous potential pain targets on a fully humanized platform.

cell biology↗

bioRxiv2024

High-Resolution Multimodal Profiling of Human Epileptic Brain Activity via Explanted Depth Electrodes

Understanding neurological disorders necessitates systems-level approaches that integrate multimodal data, but progress has been hindered by limited sample availability, and the absence of combined electrophysiological and molecular data from live patients. Here, we demonstrate that intracranial stereoelectroencephalography (SEEG) electrodes used for identifying the seizure focus in epilepsy patients enable the integration of RNA sequencing, genomic variants and epigenome maps with in vivo recordings and brain imaging. Specifically, we report a method, MoPEDE (Multimodal Profiling of Epileptic Brain Activity via Explanted Depth Electrodes) that recovers extensive protein-coding transcripts, DNA methylation and mutation profiles from explanted SEEG electrodes matched with electrophysiological and radiological data allowing for high-resolution reconstructions of brain structure and function in human patients. Our study shows that epilepsies of different aetiologies have distinct molecular landscapes and identify transcripts correlating with neurophysiological signals, including immediate early genes, inflammation markers, and axon guidance molecules. Additionally, we identify DNA methylation profiles indicative of transcriptionally permissive or restrictive chromatin states. While gene expression gradients corresponded with the assigned epileptogenicity index, we found outlier molecular fingerprints in some electrodes, potentially indicating seizure generation or propagation zones not detected during electroclinical assessments. These findings validate that RNA profiles, genetic variation and genome-wide epigenetic data from explanted SEEG electrodes offer high-resolution surrogate molecular landscapes of brain activity. Our transformative MoPEDE approach has the potential to enhance diagnostic decisions and deepen our understanding of epileptogenic network processes in the human brain.

genomics↗

bioRxiv2024

Parameterizing Pantherinae: de novo mutation rate estimates from Panthera and Neofelis pedigrees

Estimates of de novo mutation rates are essential for phylogenetic and demographic analyses, but their inference has previously been impeded by high error rates in sequence data and uncertainty in the fossil record. Here, we directly estimate de novo germline mutation rates for all extant members of Panthera, as well as the closely related outgroup Neofelis nebulosa, using pedigrees. We use a previously validated pipeline (RatesTools) to calculate mutation rate for each species and subsequently explore the impacts of the novel rates on historic effective population size estimates in each of these charismatic felids of conservation concern. Importantly, we find that the choice of reference genome, the data type and coverage, and the individual all impact estimates of the mutation rate. Despite these stochastic effects, we inferred that base pair mutation rates for all species fell between 0.5 and 1.4e-08 per generation per base pair (mean 0.81e-08 {+/-} 0.35-08 across Pantherinae). Our results provide a cautionary view on inter-species mutation rate comparisons, given the error associated with the reference genome choice and sequencing depth of coverage of the individuals.

evolutionary biology↗

bioRxiv2023

Food availability early in life impacts among and within individual variation in behaviour

O_LIThe availability of food during early life has been proposed as a key proximate mechanism for the development of variation in behaviour among and within individuals. C_LIO_LIIndividuals can vary amongst each other in their personality, plasticity and predictability and if an individuals behaviour is correlated across contexts this can lead to behavioural, plasticity and predictability syndromes. C_LIO_LIIn this study, we used a split brood design to raise African clawed frog tadpoles (Xenopus laevis) on a high or low diet in food availability and measured the distance they swam in a familiar and unfamiliar context eight times during their development. C_LIO_LIIn a familiar context, we found that there was an increase in among individual variance in plasticity and predictability in the high food treatment. This shows that when resources are not restricted, individuals are not constrained in the expression of their behaviour at certain phenotypic levels. C_LIO_LIIn an unfamiliar context, we found a different response, with an increase in individual variance in personality in the low but not the high feed tadpoles. As unfamiliar contexts may be riskier, our results highlight that individuals receiving less food may take greater foraging risks in novel contexts. C_LIO_LIAcross contexts, we found a predictability syndrome in the high but not the low feed tadpoles, highlighting that cross-context behaviours can become decoupled in some developmental conditions but remain intact in others. C_LIO_LITogether our findings show that early life conditions contribute to among individual variation in behaviour but that these may only impact the phenotype at specific phenotypic levels and are context specific. C_LIO_LIWe emphasise that having a fundamental understanding of how early development may promote or constrain individual variation can provide a greater understanding of how individuals and populations may respond to novel conditions brought about by anthropogenic activity. C_LI

evolutionary biology↗

bioRxiv2022

A unified peptide array platform for antibody epitope binning, mapping, specificity and predictive off-target binding

Therapeutic antibody efficacy is largely determined by the target epitope. In addition, off-target binding can result in unanticipated side-effects. Therefore, characterization of the epitope and binding specificity are critical in antibody discovery. Epitope binning provides low-resolution of an antibody epitope and is typically performed as a cross-blocking assay to group antibodies into overlapping or non-overlapping bins. Epitope mapping identifies the epitope with high resolution but requires low throughput methods. In addition to binning and mapping, there is a need for a scalable and predictive approach to reveal off-target binding early in antibody discovery to reduce the risk of in vivo side effects. Peptide microarrays are an information-rich platform for antibody characterization. However, the potential of peptide microarrays in early-stage antibody discovery has not been realized because they are not produced at the scale, quality and format needed for reliable high-throughput antibody characterization. A unified, peptide library platform for high-resolution antibody epitope binning, mapping and predictive off-target binding characterization is described here. This platform uses highly scalable array synthesis and photolithography to synthesize more than 3 million addressable peptides. These arrays conform to a microplate format and each synthesis is qualified with mass spectrometry. Using this platform, a scalable approach to early-stage epitope and specificity characterization, with prediction of off-target interaction(s), is demonstrated using a panel of anti-HER2 monoclonal antibodies. This study highlights the prospect of this platform to improve antibody discovery productivity by generating epitope and specificity information much earlier with potentially hundreds of antibody clones.

bioengineering↗

bioRxiv2022

The effect of heterospecific and conspecific competition on inter-individual differences in tungara frog tadpole (Engystomops pustulosus) behaviour

Repeated social interactions with conspecifics and/or heterospecifics during early development may drive the differentiation of behaviour among individuals. This behavioural differentiation may occur through individuals behaving more different from each other on average and/or individuals behaving more consistently. Competition is a major form of social interaction and its impacts can depend on whether interactions occur between conspecifics or heterospecifics and the directionality of a response could be specific to different behavioural traits. To test this, we reared tungara frog tadpoles (Engystomops pustulosus) either in isolation, with a conspecific tadpole or with an aggressive heterospecific tadpole, the whistling frog tadpole, Leptodactylus fuscus. In each treatment, we measured the body size, activity, exploration and risk taking in the presence of a predator in focal E. pustulosus tadpoles six times during development. We used univariate and multivariate hierarchical mixed effect models to investigate the effect of treatment on mean behaviour and on among individual variance between and within individuals across behavioural traits. There was a strong effect of competition on behaviour, with different population and individual level responses across social treatments. Within their home tank, individuals were more consistent in their movements under conspecific competition but heterospecific competition caused more variance in the average movement among individuals. Behavioural responses were also trait specific as conspecific competition caused greater variability in movements among individuals in a novel environment. The results highlight that the impact of competition on inter-individual differences in behaviour is dependent on competitor species identity and is trait specific.

animal behavior and cognition↗

bioRxiv2020

Sensory and motor electrophysiological mapping of the cerebellum in humans.

Damage to the cerebellum during posterior fossa surgery can lead to ataxia and in paediatric cases, the risk of cerebellar mutism syndrome. Animal electrophysiological and human imaging studies have shown compartmentalisation of sensorimotor and cognitive functions within the cerebellum. In the present study, electrophysiological monitoring of sensory and motor pathways was carried out to assess the location of limb sensorimotor representation within the human cerebellum, as a potential approach for real time assessment of neurophysiological integrity to reduce the incidence of cerebellar surgical morbidities. Thirteen adult and paediatric patients undergoing posterior fossa surgery were recruited. For sensory mapping (n=8), electrical stimulation was applied to the median nerves, the posterior tibial nerves, or proximal and distal limb muscles and evoked field potential responses were sought on the cerebellar surface. For motor mapping (n=5), electrical stimulation was applied to the surface of the cerebellum and evoked EMG responses were sought in facial and limb muscles. Evoked potentials on the cerebellar surface were found in two patients (25% of cases). In one patient, the evoked response was located on the surface of the right inferior posterior cerebellum in response to stimulation of the right leg. In the second patient, stimulation of the extensor digitorum muscle in the left forearm evoked a response on the surface of the left inferior posterior lobe. In the motor mapping cases no evoked EMG responses could be found. Intraoperative electrophysiological mapping, therefore, indicates it is possible to record evoked potentials on the surface of the human cerebellum in response to peripheral stimulation.

neuroscience↗

bioRxiv2017

Enabling Precision Medicine via standard communication of NGS provenance, analysis, and results

A personalized approach based on a patients or pathogens unique genomic sequence is the foundation of precision medicine. Genomic findings must be robust and reproducible, and experimental data capture should adhere to FAIR guiding principles. Moreover, effective precision medicine requires standardized reporting that extends beyond wet lab procedures to computational methods. The BioCompute framework (https://osf.io/zm97b/) enables standardized reporting of genomic sequence data provenance, including provenance domain, usability domain, execution domain, verification kit, and error domain. This framework facilitates communication and promotes interoperability. Bioinformatics computation instances that employ the BioCompute framework are easily relayed, repeated if needed and compared by scientists, regulators, test developers, and clinicians. Easing the burden of performing the aforementioned tasks greatly extends the range of practical application. Large clinical trials, precision medicine, and regulatory submissions require a set of agreed upon standards that ensures efficient communication and documentation of genomic analyses. The BioCompute paradigm and the resulting BioCompute Objects (BCO) offer that standard, and are freely accessible as a GitHub organization (https://github.com/biocompute-objects) following the \"Open-Stand.org principles for collaborative open standards development\". By communication of high-throughput sequencing studies using a BCO, regulatory agencies (e.g., FDA), diagnostic test developers, researchers, and clinicians can expand collaboration to drive innovation in precision medicine, potentially decreasing the time and cost associated with next generation sequencing workflow exchange, reporting, and regulatory reviews.

scientific communication and education↗