Biology subjects

Owens, R. M.

Publications and source records attributed to Owens, R. M..

7 recordsLinked to original sources

bioRxiv2024

TRPV4 stimulates colonic afferents through mucosal release of ATP and glutamate

Background and PurposeAbdominal pain is a leading cause of morbidity for people living with gastrointestinal disease. While the vanilloid transient receptor potential 4 (TRPV4) ion channel has been implicated in the pathogenesis of abdominal pain, the relative paucity of TRPV4 expression in colon-projecting sensory neurons suggests that non-neuronal cells may also contribute to TRPV4-mediated nociceptor stimulation. Experimental ApproachChanges in murine colonic afferent activity were examined using ex vivo electrophysiology in tissues with the gut mucosa present or removed. ATP and glutamate release were measured by bioluminescence assay from human colon organoid cultures and mouse colon. Dorsal root ganglion sensory neuron activity was evaluated by Ca2+ imaging when cultured alone or co-cultured with colonic mucosal cells. Key ResultsThe TRPV4 agonist GSK1016790A elicited a robust increase in murine colonic afferent activity, which was abolished by removal of the gut mucosa. GSK1016790A promoted ATP and glutamate release from human colon organoid cultures and mouse colon. Inhibition of ATP degradation in mouse colon enhanced the afferent response to GSK1016790A. Pre-treatment with purinoreceptor or glutamate receptor antagonists attenuated and abolished the response to GSK1016790A when given alone or in combination, respectively. Sensory neurons co-cultured with colonic mucosal cells produced a marked increase in intracellular Ca2+ to GSK1016790A compared to neurons cultured alone. Conclusions and ImplicationsOur data indicate that mucosal release of ATP and glutamate is responsible for the stimulation of colonic afferents following TRPV4 activation. These findings highlight an opportunity to target the gut mucosa for the development of new visceral analgesics. Bullet Point SummaryWhat is already known? O_LIActivation of TRPV4 causes visceral hypersensitivity via the stimulation of colonic afferents. C_LI What does this study add? O_LITRPV4-mediated colonic afferent activation is dependent on mucosal release of ATP and glutamate. C_LI What is the clinical significance? O_LIMucosal TRPV4-mediated colonic afferent activation provides a gut restricted target for treating abdominal pain. C_LI

pharmacology and toxicology↗

bioRxiv2024

Implantable Bioelectronics for Real-time in vivo Recordings of Enteric Neural Activity

The gastrointestinal tract is regulated by a complex network of electrically-active cell types that communicate to drive gut function. One of these systems, the enteric nervous system, represents a primary point of contact for a host of factors that influence bodily health and behavior. This division of the autonomic nervous system is unique in both its extensivity, with neurons distributed throughout the gastrointestinal tract from the esophagus to the rectum, and its capability for local information processing. However, the constant intrinsic and extrinsic motion of the gut as well as the sparse distribution of the neurons that constitute the enteric nervous system has made, access and analysis for study of this important component of the gastrointestinal tract exceedingly challenging. Here, we present the construction and validation of a bioelectronic implant for accessing neural information from the distal colon. The implant, constructed for compatibility with a novel surgical approach applicable across multiple species, is designed to be positioned within the colonic wall in close proximity to the enteric nervous plexi. We captured complex multi-frequency electrophysiological responses to neural chemical stimulants and showed that we can record distension activity mimicking gut motility. We also show the feasibility of utilizing this device for recording from putative single neurons in freely-moving rats and examining colonic activity in the context of food intake and stress. This work marks a significant advancement in understanding the complex pathways of the gut-brain axis. Our bioelectronic monitoring system demonstrates the power of real-time electrophysiological monitoring for the distal areas of the autonomic nervous system. Furthermore, direct access to the communication pathways of the enteric nervous system paves the way for novel neuromodulation strategies targeting the gut-brain axis.

neuroscience↗

bioRxiv2023

Protein Language Models Uncover Carbohydrate-Active Enzyme Function in Metagenomic

In metagenomics, the pool of uncharacterized microbial enzymes presents a challenge for functional annotation. Among these, carbohydrate-active enzymes (CAZymes) stand out due to their pivotal roles in various biological processes related to host health and nutrition. Here, we present CAZyLingua, the first tool that harnesses protein language model embeddings to build a deep learning framework that facilitates the annotation of CAZymes in metagenomic datasets. Our benchmarking results showed on average a higher F1 score (reflecting an average of precision and recall) on the annotated genomes of Bacteroides thetaiotaomicron, Eggerthella lenta and Ruminococcus gnavus compared to the traditional sequence homology-based method in dbCAN2. We applied our tool to a paired mother/infant longitudinal dataset and revealed unannotated CAZymes linked to microbial development during infancy. When applied to metagenomic datasets derived from patients affected by fibrosis-prone diseases such as Crohns disease and IgG4-related disease, CAZyLingua uncovered CAZymes associated with disease and healthy states. In each of these metagenomic catalogs, CAZyLingua discovered new annotations that were previously overlooked by traditional sequence homology tools. Overall, the deep learning model CAZyLingua can be applied in combination with existing tools to unravel intricate CAZyme evolutionary profiles and patterns, contributing to a more comprehensive understanding of microbial metabolic dynamics.

bioinformatics↗

bioRxiv2022

Multiparametric sensing of outer membrane vesicle-derived supported lipid bilayers demonstrates the specificity of bacteriophage interactions

The use of bacteriophage, viruses that specifically infect bacteria, as antibiotics has become an area of great interest in recent years as the effectiveness of conventional antibiotics recedes. The detection of phage interactions with specific bacteria in a rapid and quantitative way is key for identifying phage of interest for novel antimicrobials. Outer membrane vesicles (OMVs) derived from gram-negative bacteria can be used to make supported lipid bilayers (SLBs) and therefore in vitro membrane models that contain naturally occurring components of the bacterial outer membrane. In this study, we used Escherichia coli OMV derived SLBs and use both fluorescent imaging and surface sensitive techniques to show their interactions with T4 phage. We also integrate these bilayers with microelectrode arrays (MEAs) functionalised with the conducting polymer PEDOT:PSS and show that the pore forming interactions of the phage with the SLBs can be monitored using electrical impedance spectroscopy. To highlight our ability to detect specific phage interactions, we also generate SLBs using OMVs derived from Citrobacter rodentium, which is resistant to T4 phage infection, and identify their lack of interaction with phage. The work presented here shows how interactions occurring between phage and these complex SLB systems can be monitored using a range of experimental techniques. We believe this approach can be used to identify phage against bacterial strains of interest, as well as more generally to monitor any pore forming structure (such as defensins) interacting with bacterial outer membranes, and thus aid in the development of next generation antimicrobials.

bioengineering↗

bioRxiv2022

3D Bioelectronics with a Remodellable Matrix for Long-term Tissue Integration and Recording

Bioelectronics hold the key for understanding and treating disease. However, achieving stable, long-term interfaces between electronics and the body remains a challenge. Implantation of a bioelectronic device typically initiates a foreign body response, which can limit long-term recording and stimulation efficacy. Techniques from regenerative medicine have shown a high propensity for promoting integration of implants with surrounding tissue, but these implants lack the capabilities for the sophisticated recording and actuation afforded by electronics. Combining these two fields can achieve the best of both worlds. Here, we show the construction of a hybrid implant system for creating long-term interfaces with tissue. We create implants by combining a microelectrode array with a bioresorbable and remodellable gel. These implants are shown to produce a minimal foreign body response when placed into musculature, allowing us to record long-term electromyographic signals with high spatial resolution. This device platform drives the possibility for a new generation of implantable electronics for long-term interfacing.

bioengineering↗

bioRxiv2022

3D Organic Bioelectronics for Monitoring In Vitro Stem Cell Cultures

Three-dimensional in vitro stem cell models has enabled a fundamental understanding of cues that direct stem cell fate and be used to develop novel stem cell treatments. While sophisticated 3D tissues can be generated, technology that can accurately monitor these complex models in a high-throughput and non-invasive manner is not well adapted. Here we show the development of 3D bioelectronic devices based on the electroactive polymer poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) - PEDOT:PSS and their use for non-invasive, electrical monitoring of stem cell growth. We show that the electrical, mechanical and wetting properties as well as the pore size/architecture of 3D PEDOT:PSS scaffolds can be fine-tuned simply by changing the processing crosslinker additive. We present a comprehensive characterization of both 2D PEDOT:PSS thin films of controlled thicknesses, and 3D porous PEDOT:PSS structures made by the freeze-drying technique. By slicing the bulky scaffolds we show that homogeneous, porous 250 um thick PEDOT:PSS slices are produced, generating biocompatible 3D constructs able to support stem cell cultures. These multifunctional membranes are attached on Indium-Tin oxide substrates (ITO) with the help of an adhesion layer that is used to minimize the interface charge resistance. The optimum electrical contact result in 3D devices with a characteristic and reproducible, frequency dependent impedance response. This response changes drastically when human adipose derived stem cells grow within the porous PEDOT:PSS network as revealed by fluorescence microscopy. The increase of these stem cell population within the PEDOT:PSS porous network impedes the charge flow at the interface between PEDOT:PSS and ITO, enabling the interface resistance to be extracted by equivalent circuit modelling, used here as a figure of merit to monitor the proliferation of stem cells. The strategy of controlling important properties of 3D PEDOT:PSS structures simply by altering processing parameters can be applied for development of a number of stem cell in vitro models. We believe the results presented here will advance 3D bioelectronic technology for both fundamental understanding of in vitro stem cell cultures as well as the development of personalized therapies.

bioengineering↗

bioRxiv2018

Nanoscale investigation in 3D scaffolds of cell-material interactions for tissue-engineering

Cell fate is largely determined by interactions that occur at the interface between cells and their surrounding microenvironment. For this reason, especially in the field of cell- and tissue-engineering, there is a growing interest in developing characterization techniques that allow a deep evaluation of cell-material interaction at the nanoscale, particularly focusing on cell adhesion processes. While for 2D culturing systems a consolidated series of tools already satisfy this need, in 3D environments, more closely recapitulating complex in vivo structures, there is still a lack of procedure furthering the comprehension of cell-material interactions. Here, we report for the first time the use of a SEM/FIB system for the characterization of cellular adhesion in 3D scaffolds fabricated by means of different techniques. Our results clearly show the capability of the developed approach to finely resolve both scaffold-cells interface and nanometer scale features of cell bodies involved in the upregulation of cellular behavior. These results are relevant for studying cellular guidance strategies and for the consequent design of more efficient cell-instructive platforms for tissue-engineering applications as well as for in vitro 3D models.

bioengineering↗