Biology subjects

Carrier, R. L.

Publications and source records attributed to Carrier, R. L..

5 recordsLinked to original sources

bioRxiv2025

Development of a Transcriptional Biosensor for Hydrogen Sulfide that Functions under Aerobic and Anaerobic Conditions

Hydrogen sulfide (H2S) is a gaseous gut metabolite with disputed effects on gastrointestinal health. Monitoring H2S concentration in the gut would provide insight into its role in disease, but is complicated by sulfides reactivity and volatility. Here we develop a transcriptional sulfide biosensor in E. coli. The sensor relies on enzymatic oxidation of sulfide catalyzed by a sulfide:quinone reductase (Sqr) to polysulfides, which bind to the repressor SqrR, triggering unbinding from the promoter and transcription of the reporter. Through promoter engineering and improving soluble SqrR expression, we optimized the system to provide an operational range of 50 {micro}M - 750 {micro}M and dynamic range of 18 aerobically. To enable sensing in anaerobic environments, we identified an Sqr from Wolinella succinogenes that uses menaquinone, facilitating reoxidation through the anaerobic electron transport chain by fumarate or nitrate. Use of this homolog resulted in an anaerobic H2S response up to 750 {micro}M. This sensor could ultimately enable spatially and temporally resolved measurements of H2S in the gastrointestinal tract to elucidate the role of this metabolite in disease, and potentially as a non-invasive diagnostic.

synthetic biology↗

bioRxiv2024

Human Mesofluidic Intestinal Model for Studying Transport of Drug Carriers and Bacteria Through a Live Mucosal Barrier

The intestinal mucosal barrier forms a critical interface between lumen contents such as bacteria, drugs, and drug carriers and the underlying tissue. Current in vitro intestinal models, while recapitulating certain aspects of this barrier, generally present challenges with respect to imaging transport across mucus and uptake into enterocytes. A human mesofluidic small intestinal chip was designed to enable facile visualization of a mucosal interface created by growing primary human intestinal cells on a vertical hydrogel wall separating channels representing the intestinal lumen and circulatory flow. Type I collagen, fortified via cross-linking to prevent deformation and leaking during culture, was identified as a suitable gel wall material for supporting primary organoid-derived human duodenal epithelial cell attachment and monolayer formation. Addition of DAPT and PGE2 to culture medium paired with air-liquid interface culture increased the thickness of the mucus layer on epithelium grown within the device for 5 days from approximately 5 mm to 50 m, making the model suitable for revealing intriguing features of interactions between luminal contents and the mucus barrier using live cell imaging. Time-lapse imaging of nanoparticle diffusion within mucus revealed a zone adjacent to the epithelium largely devoid of nanoparticles up to 4.5 hr after introduction to the lumen channel, as well as pockets of dimly lectin-stained mucus within which particles freely diffused, and apparent clumping of particles by mucus components. Multiple particle tracking conducted on the intact mucus layer in the chip revealed significant size-dependent differences in measured diffusion coefficients. E. coli introduced to the lumen channel were freely mobile within the mucus layer and appeared to intermittently contact the epithelial surface over 30 minute periods of culture. Mucus shedding into the lumen and turnover of mucus components within cells were visualized. Taken together, this system represents a powerful tool for visualization of interactions between luminal contents and an intact live mucosal barrier.

bioengineering↗

bioRxiv2024

Mathematical model of intestinal lipolysis of a long-chain triglyceride

Lipids are an important component of food and oral drug formulations. Upon release into gastrointestinal fluids, triglycerides, common components of foods and drug delivery systems, form emulsions and are digested into simpler amphiphilic lipids (e.g., fatty acids) that can associate with intestinal bile micelles and impact their drug solubilization capacity. Digestion of triglycerides is dynamic and dependent on lipid quantity and type, and quantities of other components in the intestinal environment (e.g., bile salts, lipases). The ability to predict lipid digestion kinetics in the intestine could enhance understanding of lipid impact on the fate of co-administered compounds (e.g., drugs, nutrients). In this study, we present a kinetic model that can predict the lipolysis of emulsions of triolein, a model long-chain triglyceride, as a function of triglyceride amount, droplet size, and quantity of pancreatic lipase in an intestinal environment containing bile micelles. The model is based on a Ping Pong Bi Bi mechanism coupled with quantitative analysis of partitioning of lipolysis products in colloids, including bile micelles, in solution. The agreement of lipolysis model predictions with experimental data suggests that the mechanism and proposed assumptions adequately represent triglyceride digestion in a simulated intestinal environment. In addition, we demonstrate the value of such a model over simpler, semi-mechanistic models reported in the literature. This lipolysis framework can serve as a basis for modeling digestion kinetics of different classes of triglycerides and other complex lipids as relevant in food and drug delivery systems.

bioengineering↗

bioRxiv2022

Regenerating axolotl retinas regrow diverse cell types with modulation by Notch signaling and reconnect to the brain

Some species successfully repair retinal injuries in contrast to non-regenerative mammalian retina. We show here that the Mexican axolotl salamander regrows its excised retina even in adulthood. During early regeneration, cell proliferation occurred in the retinal pigment epithelium (RPE). All dividing cells expressed Vimentin, and some also expressed Muller glia and neural progenitor cell marker Glast (Slc1a3), suggesting that regeneration is driven by RPE-derived retinal progenitor cells. Bulk RNA sequencing showed that genes associated with the extracellular matrix and angiogenesis were upregulated in early-to-mid retinal regeneration. The fully regenerated retina re-established nerve projections to the brain and contained all the original retinal cell types, including Muller glia. Regeneration of cellular diversity may be modulated by Notch signaling, as inhibiting Notch signaling in early regeneration promoted production of rod photoreceptors. Our study highlights the axolotl salamander as an advantageous model of adult tetrapod retinal regeneration and provides insights into its mechanisms. SummaryWe demonstrate that adult Mexican axolotl salamanders regenerate retinas after a retinectomy. We also show some cellular and molecular mechanisms that drive axolotl retinal regeneration.

neuroscience↗

bioRxiv2020

Primary human colonic mucosal barrier crosstalk with super oxygen-sensitive Faecalibacterium prausnitzii in continuous culture

The gut microbiome plays an important role in human health and disease. Gnotobiotic animal and in vitro cell-based models provide some informative insights into mechanistic crosstalk. However, there is no existing system for a chronic co-culture of a human colonic mucosal barrier with super oxygen-sensitive commensal microbes, hindering the study of human-microbe interactions in a controlled manner. Here, we investigated the effects of an abundant super oxygen-sensitive commensal anaerobe, Faecalibacterium prausnitzii, on a primary human mucosal barrier using a Gut-MIcrobiome (GuMI) physiome platform that we designed and fabricated. Chronic continuous co-culture of F. prausnitzii for two days with colon epithelia, enabled by continuous flow of completely anoxic apical media and aerobic basal media, resulted in a strictly anaerobic apical environment fostering growth of and butyrate production by F. prausnitzii, while maintaining a stable colon epithelial barrier. We identified elevated differentiation and hypoxia-responsive genes and pathways in the platform compared with conventional aerobic static culture of the colon epithelia, attributable to a combination of anaerobic environment and continuous medium replenishment. Furthermore, we demonstrated anti-inflammatory effects of F. prausnitzii through HDAC and the TLR-NFKB axis. Finally, we identified that butyrate largely contributes to the anti-inflammatory effects by downregulating TLR3 and TLR4. Our results are consistent with some clinical observations regarding F. prausnitzii, thus motivating further studies employing this platform with more complex engineered colon tissues for understanding the interaction between the human colonic mucosal barrier and microbiota, pathogens, or engineered bacteria.Competing Interest StatementA provisional patent was filed.View Full Text

bioengineering↗