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Results for “bioengineering”

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bioRxiv2025

Bioengineered iPSC Vessels Recapitulate Human Vascular Physiological Function and Aging Phenotypes

Vascular aging contributes to multisystem diseases and limits health span. Although various animal models have contributed to aging research, their vasculatures poorly recapitulate human physiology. Even existing tissue-engineered blood vessels fail to mimic human vascular function and pathology, hindering translational advances in vascular aging studies. Here, we present a novel human physiological vascular model fabricated via the unique molding-induced circumferential alignment of human induced pluripotent stem cell (iPSC)-derived vascular smooth muscle cells with luminally seeded endothelial cells. This architecture enabled dynamic vasodiameter changes in response to vasoactive stimuli, including hormones and intraluminal pressure. Using iPSCs from a patient with Werner syndrome, the model recapitulated aging-associated phenotypes, such as hypercontractility and increased vascular compliance, possibly due to impaired nitric oxide bioavailability. Transcriptomic and metabolomic analyses revealed age-related dysregulation consistent with vascular senescence. As a key advantage of the vasculature, spatial transcriptomic analysis demonstrated upregulation of the aging marker CDKN1A near the lumen and downregulation of COL6A1 and TPM1 throughout the vessel. Treatment with mitochonic acid 5, a mitochondria-targeted compound, significantly reversed the aging phenotypes. These findings demonstrate that our engineered vascular model recapitulates key aspects of human vascular properties and provides a platform for mechanistic studies of vascular aging and drug discovery aimed at extending health span.

bioengineering↗

bioRxiv2025

A bioengineered human urothelial organoid model reveals the urine-urothelium interplay in tissue resilience and UPEC recurrence in urinary tract infections

Urine is a dynamic and highly variable biofluid. Urine-urothelium interactions are a critical yet underexplored factor in bladder homeostasis and urinary tract infections (UTIs). Here, we report on a human mini-bladder model that exposes a stratified urothelium to urine of defined composition, and incorporates micturition. Prolonged exposure to high-solute concentration urine weakens tight junctions, dysregulates immune responses, and reduces bladder tissue resilience. This increases susceptibility to colonization of the bladder by uropathogenic E. coli (UPEC) which reduces efficacy of antibiotic therapy. In high-solute concentration urine, Fosfomycin monotherapy - prescribed for uncomplicated UTIs, induces the formation of cell wall-deficient (CWD) UPEC in the urine (as observed in patients with recurrent UTIs) but also within deeper urothelial layers. Tissue-associated CWD UPEC directly contributed to recurrence. Our findings expand the conceptual role for CWD UPEC in UTIs, and demonstrate the power of the mini-bladder platform to capture urine-urothelial microenvironment dynamics that actively shape UTI pathogenesis and antibiotic tolerance.

bioengineering↗

bioRxiv2025

Bioengineered human bone-mimetic niche compositions regulatebreast cancer cell quiescence and therapy response

Disseminated tumor cells (DTCs) in the bone are widely regarded as the cellular seeds of late metastatic relapse in estrogen receptor-positive (ER+) breast cancer (BC). However, how distinct bone compartments influence BC cell quiescence and endocrine therapy response remains unclear. Mechanistic insight has been hindered by limited access to human bone samples and by the lack of relevant human models that permit controlled manipulation of stromal compartments. Here, we developed a fully human, 3D bone-mimetic system for modular, controllable assembly of engineered osteoblastic (eON), vascularized (eVN), and vascularized osteoblastic (eVON) niche compositions. These niches were generated by perfusion culture of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) and/or human adipose tissue-derived stromal vascular fraction (hAT-SVF) cells within porous ceramic scaffolds. The resulting tissue microenvironments were then used as a substrate for the culture of an ER+ BC cell line, expressing a mutant reporter of p27 to monitor the quiescent status. We found that the eON enhanced BC cell proliferation, whereas the eVN was enriched in quiescent BC cells positive for NR2F1, a dormancy-associated transcription factor, and located near perivascular elements. Treatment with the selective ER degrader fulvestrant reduced BC cell numbers in vascularized niches (eVN and eVON) but not in eON, despite comparable receptor degradation. In summary, we developed a modular human platform for dissecting niche-specific regulation of BC quiescence, proliferation, and endocrine therapy response. The system can be further used to investigate perivascular niche-dependent mechanisms of BC cell dormancy and to guide the development of therapeutic strategies preventing recurrence in ER+ BC patients.

bioengineering↗

bioRxiv2026

Bioengineering of a Chimeric Metarhizium anisopliae cPr1A Protease with Enhanced Binding and Enzyme Activity Through C-terminal Fusion of Bombyx mori Chitin-Binding Domain

AbstractMetarhizium anisopliae is an important entomopathogenic fungi used in biological control of agricultural pests, but its commercial application is limited by relatively slow host mortality. This study aimed to engineer a chimeric protease (cPr1A) with enhanced binding affinity and protease activity against insect cuticle. We hypothesized that stronger cuticle binding would increase local enzyme concentration at the cuticle surface and thereby enhance cuticle degradation. To achieve this, the Bombyx mori chitin-binding domain (BmCBD) was fused to the C-terminus of the Pr1A protease from M. anisopliae. Recombinant Pr1A and cPr1A were expressed in Escherichia coli, purified by Ni-NTA affinity chromatography. Binding and protease activity were assayed in triplicate using Samia ricini cuticle powder as substrate. Results are presented as mean +/- SEM. The chimeric protease cPr1A showed a 28.9% increase in cuticle binding compared to wild-type Pr1A (15.81 +/- 1.97 vs. 12.27 +/- 2.13 g bound protein/mg cuticle powder; p < 0.002) and a 35% increase in protease activity (0.343 +/- 0.08 U/mg vs. 0.254 +/- 0.06 U/mg; p < 0.03). These results indicate that cPr1A is a promising candidate for overexpression in M. anisopliae to enhance cuticle degradation and potentially improve fungal virulence against insect pests.

bioengineering↗

bioRxiv2020

CRISPR-based bioengineering of the Transferrin Receptor revealed a role for Rab7 in the biosynthetic secretory pathway

The regulated secretory trafficking of neosynthesized transmembrane receptors is particularly challenging to investigate as it is under-represented at steady state compared to the abundance of the other trafficking routes. Here, we combined the retention using selective hook (RUSH) system to a CRISPR/Cas9 gene editing approach (eRUSH) to identify molecular players involved in the trafficking of neosynthesized Transferrin Receptor (TfR) en route to the plasma membrane (PM). TfR-eRUSH monoclonal cells expressing endogenous, ER-retainable and fluorescent TfR were engineered and characterized. Spatiotemporal quantitative proteomics of TfR-eRUSH cells allowed the identification of molecular partners associated with TfR-containing membranes and provided a comprehensive list of potential regulators, co-trafficking cargos, and enriched pathways. Furthermore, we chose to focus our attention on the Rab GTPase family members for their function as vesicle trafficking regulators and performed a Rab-targeted siRNA screen that we correlated to our proteomics data. Unexpectedly, we identified Rab7-harboring vesicles as an intermediate compartment of the Golgi-to-PM transport of the neosynthetic TfR. These vesicles did not exhibit degradative properties and were not associated to Rab6A-harboring vesicles, also involved in Golgi-to-PM transport. However, Rab6A-TfR vesicles delivered TfR directly to the PM, while in contrast, Rab7A was transiently associated to neosynthetic TfR-containing post-Golgi vesicles but dissociated before PM vesicle fusion. Together, our study proposes the eRUSH as a powerful tool to further study the secretory pathway and reveals an unforeseen role for Rab7 in the neosynthetic transport of the TfR, highlighting the diversity of the secretory vesicles nature for a given cargo.

cell biology↗

bioRxiv2020

Bioengineered ciprofloxacin loaded chitosan nanoparticles for the treatment of Bovine Mastitis

Mastitis is the most devastating economic disease of dairy cattle. Mastitis in dairy cattle frequently occurs during the dry period or in early lactation. E. coli. and S. aureus is the main causative agent of mastitis. S. aureus has an ability to form encapsulated focuses in the udder and develop subclinical form of mastitis. This property of bacteria hinders the effective cure during lactation period. Antibiotics used for treatments have a short half-life span at the site of action because of frequent milking, therefore unable to maintain desired drug concentration for effective clearance of bacteria. We demonstrated the potential of ciprofloxacin encapsulated nanocarriers which can improve availability of drugs and could provide effective means of the treatment of mastitis. These drug loaded nanoparticles show low toxicity & slow clearance from the site of action. Antimicrobial studied against clinical strain of E.coli. and Staphylococcus aureus zone of inhibition depend on dose 0.5 mg to 2 mg/ml nanoparticles solution from 11.6 to 14.5 mm and 15 to 18mm. These nanoparticles show good antimicrobial activity in broth culture as well against bacteria.

pharmacology and toxicology↗

bioRxiv2020

Bioengineering a light-responsive encapsulin nanoreactor: a potential tool for photodynamic therapy

Encapsulins, a prokaryotic class of self-assembling protein nanocompartments, are being re-engineered to serve as ‘nanoreactors’ for the augmentation or creation of key biochemical reactions. However, approaches that allow encapsulin nanoreactors to be functionally activated with spatial and temporal precision is lacking. We report the construction of a light-responsive encapsulin nanoreactor for “on-demand” production of reactive oxygen species (ROS). Herein, encapsulins were loaded with the fluorescent flavoprotein mini-Singlet Oxygen Generator (miniSOG), a biological photosensitizer that is activated by blue-light to generate ROS, primarily singlet oxygen (1O2). We established that the nanocompartments stably encased miniSOG, and in response to blue-light were able to mediate the photoconversion of molecular oxygen into ROS. Using an in vitro model of lung cancer, ROS generated by the nanoreactor was shown to trigger photosensitized oxidation reactions that exerted a toxic effect on tumour cells, suggesting utility in photodynamic therapy. This encapsulin nanoreactor thus represents a platform for the light-controlled initiation and/or modulation of ROS-driven processes in biomedicine and biotechnology.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2020

Engineered Tissue Interfaces for in vitro and in vivo Bioengineering

In regenerative medicine, the healing of the interfacial zone between tissues is a major challenge, yet approaches for studying the complex microenvironment of this interface remain lacking. Here, we study these complex living interfaces by manufacturing modular "blocks" of naturally porous decellularized plant-derived scaffolds with a computer numerical controlled mill. We demonstrate how each scaffold can be seeded with different cell types and easily assembled in a manner akin to LEGO bricks to create an engineered tissue interface (ETI). Cells migrate across the interface formed between an empty scaffold and a scaffold pre-seeded with cells. However, when both scaffolds contain cells, only a shallow cross-over zone of cell infiltration forms at the interface. As a proof-of-concept study, we use ETIs to investigate the interaction between lab grown bone and connective tissues. Consistent with the above, a cross-over zone of the two distinct cell types forms at the interface between scaffolds, otherwise the populations remain distinct. Finally, we demonstrate how ETIs are biocompatible in vivo, becoming vascularized and integrated into surrounding tissue after implantation. This work creates new tissue design avenues for understanding biological processes or the development of synthetic artificial tissues.

biophysics↗

bioRxiv2021

Global anti-tumor immunity after localized, bioengineered Treg depletion

Over 90% of deaths from cancer occur due to solid tumors, occurring at a rate of [~]1,500 deaths per day in the US, highlighting a profound and unmet need for new therapies. Solid tumors evade clearance by T cells due to a variety of immunosuppressive properties of the tumor microenvironment. However, this immunosuppression cannot be easily blocked on a global level because systemic activation of the immune system elicits a host of complications. An ideal therapy for solid tumors would act locally to activate the immune response without evoking global adverse effects. Here we present a biodegradable, macroporous scaffold that is implanted adjacent to the tumor and suppresses the main obstacle to cancer immunosurveillance: intratumoral regulatory T cells. The scaffold also promotes the recruitment and activation of T cell effectors into the tumor, resulting in clearance of otherwise aggressive and fatal tumors in mice. Unexpectedly, the local depletion of Tregs results in an "immunological abscopal effect" acting on distant tumors. We demonstrate that this versatile platform can also deliver tumor-antigen-specific T cells directly to the peri-tumoral environment, bypassing difficulties in intravenous delivery including the environmental barriers imposed by the tumors vasculature. By orchestrating multiple local immunomodulatory treatments, this scaffold offers a general approach to engineer T-cell responses to solid tumors without systemic toxicities.

immunology↗

bioRxiv2023

Bioengineering of a Lactococcus lactis subsp. lactis strain enhances nisin production and bioactivity.

Lactococcus lactis subsp. lactis is a food bacterium that has been utilized for decades in food fermentation and the development of high-value industrial goods. Among these, nisin is produced by several strains of L. lactis subsp. lactis plays a crucial role as a food bio-preservative. The relative expression of the gene cluster involved in nisin production was evaluated using qPCR analysis. Additionally, a series of re-transformations of the strain introducing multi copies of the nisA and nisRK genes involved in nisin biosynthesis were developed. The simultaneous expression of nisA and nisZ genes was used to potentiate the effective inhibition of foodborne pathogens. Further, qPCR analysis revealed a low expression of the nisA and nisRK genes in wild-type L. lactis subsp. lactis. After several re-transformations of the strain with the nisA and nisRK genes, a high expression of these genes was obtained, contributing to improved nisin production. Also, the co-expression of nisA and nisZ genes results in highly efficient antimicrobial activity. Hence, this study would supply an approach to enhancing nisin production during industrial processes and antimicrobial activity.

microbiology↗

bioRxiv2024

Nanoliter-scale selection of optimized bioengineered peptide antibiotics that rescue mice with bacterial lung infection

Increasing numbers of multi-drug resistant pathogens call for new chemical scaffolds, addressing novel targets, that can serve as lead structures for the development of life-saving drugs. For antibiotics, natural product-inspired molecules represent a most promising resource. Natural products evolved to high chemical complexity and occupy a chemical space different than synthetic libraries. However, clinical translation of promising natural products is often impeded by their relative inaccessibility to medicinal chemistry optimization, e.g. iterative synthesis of large series of derivatives. Here, this limitation is addressed with a randomized library of bicyclic heptapeptides based on the natural product darobactin that hits the clinically not addressed target BamA. Variants of the ribosomally synthesized and post-translationally modified peptides were generated using heterologous mutasynthesis. A parallelized screening assay is adapted in nanoliter-scale beads to test the darobactin derivatives against our sensor strain. Loss of fluorescence sorting prioritized 563 events out of the analyzed [~]500k beads. Re-testing confirmed 48 hit events, of which 40 proved to produce distinct darobactin-type molecules. Most promising structures were isolated and the growth inhibitory effects against Gram-negative pathogens validated. One of our current frontrunner compounds (i.e., darobactin B) was reinforced by the randomized screen. While microbiological investigations of the new derivatives is ongoing, darobactin B was profiled in later tier assays and compared to another promising, rationally-designed analog (i.e., darobactin B9, "D22"). Early ADMET profiling and efficacy tests in a mouse pneumonia model were performed. Darobactin B reduced bacterial load of Pseudomonas aeruginosa and Klebsiella pneumoniae by intraperitoneal, as well as intratracheal administration. Our study showcases the potential of mutasynthetic libraries for high-throughput screening and identification of functional peptides for drug lead discovery.

microbiology↗

bioRxiv2024

SubtiToolKit - a bioengineering kit for Bacillus subtilis and Gram-positive bacteria.

Building DNA constructs of increasing complexity is key to synthetic biology. Golden Gate methods led to the creation of cloning toolkits - collections of modular standardized DNA parts hosted on hierarchic plasmids, developed for yeast, plants, Gram-negative bacteria, and human cells. However, Gram-positive bacteria have been neglected. Bacillus subtilis is a Gram-positive model organism and a workhorse in the bioindustry. Here, we present the SubtiToolKit, a high- efficiency cloning toolkit for B. subtilis and Gram-positive bacteria. Its design permits DNA constructs for transcriptional units, operons, knock-in and knock-out applications. It contains libraries of promoters, RBSs, fluorescent proteins, protein tags, terminators, genome integration parts, a no-leakage genetic device to control the expression of toxic products during E. coli assembly, and a toolbox for industrially relevant strains of Geobacillus and Parageobacillus as an example of SubtiToolKit versatility for other Gram-positive bacteria and its future perspective as a reference toolkit.

synthetic biology↗

bioRxiv2024

Bioengineered yeast tethered respiratory supercomplexes reveal mechanisms governing efficient substrate utilization

The mitochondrial respiratory chain (MRC) enzymatic complexes, essential for aerobic energy transduction in eukaryotic cells, are organized into evolutionarily conserved higher-order structures known as supercomplexes (SCs). The elucidation of the physiological relevance of respiratory SCs is essential for our understanding of mitochondrial function and cellular bioenergetics, yet it has been severely hampered by the limited availability of experimental models isolating SC formation as the sole variable. In the yeast Saccharomyces cerevisiae, where SCs are formed by the association of complexes III and IV into III2IV1 and III2IV2 configurations, compelling evidence suggests that SCs confer a competitive advantage by facilitating cytochrome c diffusion along the SC surface and enhancing respiratory rates. However, the significance of the proposed MRC plasticity and the role of distinct SC conformations in substrate utilization remain unresolved, leaving critical gaps in our understanding of mitochondrial bioenergetics and the adaptive evolution of energy transduction. To address these open questions, we engineered a yeast strain expressing a covalently linked III2IV2 SC, whose high-resolution structure is virtually identical to wild-type. Exclusive expression of this tethered SC supports robust overall respiratory activity but selectively affects mitochondrial respiration of cytosolically-generated NADH. This is attributable to the preferential interaction of distinct SC species with mitochondrial NADH dehydrogenases. We propose that in yeast mitochondria, substrate-driven formation of defined respirasome-like SC organizations contributes to the optimization of electron fluxes across the MRC and support metabolic plasticity.

biochemistry↗

bioRxiv2025

Fibroblasts drive differential response in immunotherapy via IL-6 in bioengineered 3D cancer constructs

Despite various therapeutic advancements in cancer, heterogeneity in the tumor microenvironment is a major hindrance to the overall efficacy of treatment. In this study, we focus on the most abundantly found cellular stromal components - fibroblasts - which can be tumor-promoting or tumor-suppressing based on the extent of disease progression and numerous additional factors. In the studies described herein, we evaluated their role in influencing chemotherapy as well as in immunotherapy using colorectal cancer (CRC) cell line-derived 3D tumor constructs. Our initial efforts centered on common chemotherapeutic agents utilized in CRC. However, while the presence of fibroblasts often did influence chemotherapy efficacy, a clear and consistent increase or decrease in efficacy was not obvious. We then focused subsequent studies on natural killer (NK) cell-based immunotherapy efficacy. There are numerous components and pathways involved in tumorigenesis and tumor-immune cell interactions, and the crosstalk between fibroblasts and immune cells like NK cells is still not well-defined. Fibroblasts have been documented to be a major player with respect to immune modulation and understanding this in greater detail can elucidate more options for anti-tumor immunity. Specifically, we used an extracellular matrix-based hydrogel platform to generate 3D CRC constructs to evaluate how fibroblasts affect tumor cell viability in presence and absence of NK cells. In general, fibroblasts aid the tumor cells in our models. We then identify a cytokine-based pathway (interleukin 6 [IL-6]) via which activated fibroblasts engage with the tumor cells and show how NK cell effector function can be restored when the IL-6 pathway is blocked in a melanoma-based tumor-on-a-chip platform. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/660473v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@53797forg.highwire.dtl.DTLVardef@4dbc83org.highwire.dtl.DTLVardef@19346deorg.highwire.dtl.DTLVardef@11d834_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Renewable Self-Folding Origami Constructed from Bioengineered Bacterial Cellulose

This work presents the first genetically engineered cellulose actuator where the structural basis for actuation is directly encoded in the producing organisms DNA. By modifying Komagataeibacter rhaeticus to secrete BslA protein and treating with NaOH, we create surface-activated bacterial cellulose that exhibits water contact angles 2.4x greater than and water retention 8.2 x less than unmodified cellulose. Layering this hydrophobic BslA-activated cellulose with untreated bacterial cellulose produces a biofabricated actuator driven by differential strain during dehydration. The actuation follows the Timoshenko bilayer beam equation adapted for hydrogels and can be fabricated either by shaping mature pellicles or through direct growth in 3D-printed autoclavable molds. We demonstrate two applications with a self-folding bacterial cellulose origami crane and a biomimetic bacterial cellulose gripper capable of supporting more than 360 times its own weight. This approach represents a significant advance in sustainable soft robotics through the creation of fully renewable, biodegradable, and genetically programmable actuators.

synthetic biology↗

bioRxiv2025

Bioengineered 3D hPSC-cholangiocyte ducts with physiological signals for biliary disease modelling

The progression of intrahepatic biliary diseases remains poorly understood, underscoring the urgent need to develop physiologically relevant human intrahepatic cholangiocyte disease models. Current approaches lack the complexity and throughput to capture how diverse biliary microenvironmental signals shape cholangiocyte behavior. To address this gap, we created a fully epithelialized and perfusible 3D bile duct from human pluripotent stem cell-derived cholangiocytes characterized by robust primary ciliation, apical-basal polarity and CFTR-mediated chloride conductance. Our results revealed physiologically relevant fluid flow and biliary stroma cells as essential components for sustaining cholangiocyte epithelial barrier integrity and ciliation. From here, we interrogated a broad spectrum of biliary signals to study bile acid toxicity and cytokine-driven injury, offering an unprecedented view into intrahepatic cholangiocyte stress responses and potential pathological mechanisms. By integrating physiologically relevant signals, fidelity and functional resolution, this platform provides the foundation needed to precisely decode pathogenic drivers and accelerate therapeutic development for devastating cholangiopathies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/686855v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@11c1255org.highwire.dtl.DTLVardef@1f78d11org.highwire.dtl.DTLVardef@14e9398org.highwire.dtl.DTLVardef@1bc08d5_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2019

Bioengineering a novel 3D in-vitro model to recreate physiological oxygen levels and tumor-immune interactions

Oxygen deprivation within tumors is one of the most prevalent causes of resilient cancer cell survival and increased immune evasion in breast cancer (BCa). Current in vitro models do not adequately mimic physiological oxygen levels relevant to breast tissue and its tumor-immune interactions. Here, we propose an approach to engineer a three-dimensional (3D) model (named 3D engineered oxygen, 3D-O) that supports growth of BCa cells and generates physio- and pathophysiological oxygen levels. Low oxygen-induced changes within the 3D-O model supported known tumor hypoxia characteristics such as reduced BCa cell proliferation, increased extracellular matrix protein expression, increased extracellular vesicle secretion and enhanced immune surface marker expression on BCa cells. We further demonstrated that low oxygen-induced changes mimicked tumor-immune interactions leading to immune evasion mechanisms. CD8+ T cell infiltration was significantly impaired under pathophysiological oxygen levels and we were able to establish that hypoxia inhibition re-sensitize BCa cells to cytotoxic CD8+ T cells. Therefore, our novel 3D-O model could serve as a promising platform for the evaluation of immunological events and as a drug-screening platform tool to overcome hypoxia-driven immune evasion.

cancer biology↗

bioRxiv2026

A Bioengineered Live Biotherapeutic Exploits Inflammation to Restore Gut Liver Brain Axis Function under Diet-Induced Stress

Systemic inflammatory diseases can be influenced by dietary intake, with gastrointestinal dysfunction driving both metabolic and behavioural changes mirroring the altered inflammatory profile. Additionally, the use of live biotherapeutic products (LBPs) shows promise for treating metabolic and inflammatory diseases, but their efficacy is limited by poor persistence in inflamed gut environments. Designed to utilize inflammatory byproducts, the LBP EcN::ttr has proven efficacy in the treatment of acute and chronic colitis, however its effects on the metabolic and behavioural patterns remain uncharacterized. We evaluated the effects of EcN::ttr on mice fed a proinflammatory omega-6 PUFA-rich diet. EcN::ttr-treated mice exhibited notable changes in the gut, including an improved expression of tight junction protein occludin, accompanied by reduced serum lipopolysaccharide (LPS) - binding protein, indicating protection against endotoxemia. EcN::ttr improved insulin sensitivity compared to the parental strain, associated with increased hepatic insulin receptor expression and reduced GSK3{beta} activation and endoplasmic reticulum stress. Secondary bile acids in mice treated with EcN::ttr were more abundant, with increases in those associated with resolving diarrhea and bile acid detoxification. Behavioural assessment highlighted a normalization of long-term memory along with a reduction of stress management behaviours. Altogether, EcN::ttr restores gut-liver-brain axis function through coordinated modulation of inflammation, barrier integrity, and bile acid metabolism. HighlightsO_LILive Biotherapeutic Product EcN::ttr, designed with a fitness advantage to survive inflammation, and provides protection against a proinflammatory omega 6-rich diet C_LIO_LIAdministration of EcN::ttr improved metabolic outcomes including increasing insulin sensitivity C_LIO_LIEcN::ttr increased the abundance of secondary bile acids including those that modulate bile acid detoxification C_LIO_LIBehavioural parameters were normalized in mice given EcN::ttr C_LIO_LIEcN::ttr partially normalizes gut-liver-brain axis through restoring barrier integrity, modulating inflammation and improving secondary bile acid metabolism C_LI

systems biology↗