Biology subjects

Ribeiro, N. V.

Publications and source records attributed to Ribeiro, N. V..

4 recordsLinked to original sources

bioRxiv2026

Autologous biopsy-derived co-culture platform for interrogation of intestinal epithelial-T cell crosstalk

Interactions between intraepithelial lymphocytes (IELs) and the intestinal epithelium are central to mucosal homeostasis and disease. However, mechanistic in vitro studies describing their crosstalk in humans are limited by scarceness of primary material and insufficient knowledge about co-culture requirements. Here, we establish an autologous human duodenal IEL-organoid co-culture system encompassing expandable and bankable IEL and organoid protocols, with co-culture conditions that allow viability of both cell types. This system enables successive interrogation of lympho-epithelial interactions starting from minimal biopsy material. Under baseline conditions, CD45CD8CD103TCR{beta} IELs retain tissue-residency and effector features and induce an epithelial interferon response and chemokine production, without overt epithelial apoptosis. IL-15 and IL-21, essential cytokines involved in IEL-activation in intestinal enteropathies like celiac disease, increases granzyme B expression and interferon-{gamma} secretion but do not trigger epithelial cell death. However, enforcing IEL-epithelial contact using an anti-CD3-anti-Ep-CAM bispecific antibody induces epithelial apoptosis accompanied by increased tumor necrosis factor (TNF) and FAS-ligand (FASLG) secretion. These findings validate the platforms ability to resolve non-destructive and cytotoxic lympho-epithelial interaction and provide a tractable system for studying intestinal inflammation and immune-mediated epithelial cell death.

immunology↗

bioRxiv2025

Establishing an in vitro pipeline for the high-throughput quantification of epithelial permeability of gut bacterial metabolites

The epithelium of the human gastrointestinal tract is key for controlling the absorption of small molecules and for forming a tight barrier between the gut microbiota and the host, thereby maintaining metabolic homeostasis. Both the microbiota and the barrier function of the intestinal epithelium have a role in pharmacokinetic variability of medical drugs. In this study, we developed a high-throughput workflow to assess the absorption of bacterially produced (drug) metabolites by intestinal epithelial cells through the combination of anaerobic bacterial cultures and human-intestine derived Caco2 cell cultures in a transwell system. To functionally monitor the barrier integrity during the experiments, we introduced a panel of marker compounds, whose concentration kinetics on either side of the epithelial monolayer indicates barrier integrity and transport. We employed this workflow to systematically probe the effect of different gut bacterial species on the epithelial absorption of 482 drugs and their 172 bacterially produced metabolites. While we could recapitulate known bacterial drug biotransformation reactions and expected drug metabolite absorption profiles, we also identified 33 new bacteria-drug pairs for which bacterial biotransformation alters epithelial permeability. Further, we combined the developed experimental workflow with untargeted metabolomics analysis to systematically study epithelial permeability of metabolites naturally produced by gut bacteria. Tracking the absorption kinetics of 397 bacterially produced metabolites revealed that the majority (>79%) of these metabolites do not pass the Caco2 monolayer, illustrating its role as a physical and metabolic barrier. In summary, we present a highly adaptable high-throughput workflow to quantitatively study the metabolic interactions at the intestinal microbiota-host interface which can impact pharmacokinetics, toxicokinetics, and human physiology.

pharmacology and toxicology↗

bioRxiv2023

Preventing production escape during scale-up using an engineered glucose-inducible genetic circuit

A proper balance of metabolic pathways is crucial for engineering microbial strains that can efficiently produce biochemicals at an industrial scale while maintaining cell fitness. High production loads can negatively impact cell fitness and hinder industrial-scale production. To address this, fine-tuning of gene expression using engineered promoters and genetic circuits can promote control over multiple targets in pathways and reduce the burden. We took advantage of the robust carbon catabolite repression system of Bacillus subtilis to engineer a glucose-inducible genetic circuit that supports growth and production. By simulating cultivation scale-up under repressive conditions, we preserved the production capacity of cells, which could be fully accessed by switching to glucose in the final production step. The circuit is also resilient, enabling a quick switch in the metabolic status of the culture. Furthermore, the simulated scale-up process selected best-growing cells without compromising their production capability, leading to higher product formation at the end of the process. As a pathwayindependent circuit activated by the preferred carbon source, our engineered glucose-inducible genetic circuit is broadly useful and imposes not additional cost to traditional production processes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/530450v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@f02f6corg.highwire.dtl.DTLVardef@b9631dorg.highwire.dtl.DTLVardef@11a2309org.highwire.dtl.DTLVardef@f255ac_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

Characterization of five purine riboswitches in cellular and cell-free expression systems

Bacillus subtilis employs five purine riboswitches for the control of purine de novo synthesis and transport at the transcription level. All of them are formed by a structurally conserved aptamer, and a variable expression platform harboring a rho-independent transcription terminator. In this study, we characterized all five purine riboswitches under the context of active gene expression processes both in vitro and in vivo. We identified transcription pause sites located in the expression platform upstream of the terminator of each riboswitch. Moreover, we defined a correlation between in vitro transcription readthrough and in vivo gene expression. Our in vitro assay demonstrated that the riboswitches operate in the micromolar range of concentration for the cognate metabolite. Our in vivo assay showed the dynamics of control of gene expression by each riboswitch. This study deepens the knowledge of the regulatory mechanism of purine riboswitches.

molecular biology↗