Excess amino acids from a protein-rich diet are mainly catabolized in the liver. However, it is still unclear to what extent the gut microbiota may be involved in the mechanisms governing this catabolism. Therefore, the aim of this study was to investigate whether consumption of different dietary protein concentrations induces changes in the taxonomy of the gut microbiota, which may contribute to the regulation of hepatic amino acid catabolism. Consumption of a high-protein diet caused overexpression of HIF-1α in the colon and increase in mitochondrial activity, creating a more anaerobic environment that was associated with changes in the taxonomy of the gut microbiota promoting an increase in the synthesis of secondary bile acids, increased secretion of pancreatic glucagon. This effect was demonstrated in pancreatic islets, where secondary bile acids stimulated the expression of the PC2 enzyme that promotes glucagon formation. The increase in circulating glucagon was associated with an induction of the expression of hepatic amino acid-degrading enzymes, an effect attenuated by antibiotics. Thus, high protein intake in mice and humans induced the increase of different species in the gut microbiota with the capacity to produce secondary bile acids leading to an increase in secondary bile acids and glucagon levels, promoting amino acid catabolism.

The gastrointestinal tract is a dynamic biomechanical environment where physical forces, cellular processes, and microbial interactions converge to shape the gut health and disease. In this review, we examine the unique mechanical properties of the gut, including peristalsis, viscoelasticity, shear stress, and tissue stiffness, and their roles in modulating host mechanosignaling and microbial behavior under physiological and pathological conditions. We discuss how these mechanical forces regulate gut epithelial integrity, immune responses, and microbial colonization, leading to distinct ecological niches across different intestinal segments. Furthermore, we highlight recent advancements in 3D culture systems and gut-on-a-chip models that accurately recapitulate the complex interplay between biomechanics and gut microbiota. By elucidating the intricate relationship between mechanobiology and gut function, this review underscores the potential for mechanotherapeutic strategies to modulate host-microbe interactions, offering promising avenues for the prevention and treatment of disorders such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer.

Age-associated changes to the intestinal microbiome may be linked to inflammageing and the development of age-related chronic diseases. Cynomolgus macaques, a common animal model in biomedical research, have strong genetic physiological similarities to humans and may serve as beneficial models for the effect of age on the human microbiome. However, age-associated changes to their intestinal microbiome have previously only been investigated in faecal samples. Here, we have characterised and investigated the effects of age in the cynomolgus macaque intestinal tract in luminal samples from both the small and large intestine.

Whole metagenomic shotgun sequencing was used to analyse the microbial communities in intestinal content obtained from six different intestinal regions, covering the duodenum to distal colon, of 24 healthy, captive-bred cynomolgus macaques, ranging in age from 4 to 20 years. Both reference-based and assembly-based computational profiling approaches were used to analyse changes to intestinal microbiota composition and metabolic potential associated with intestinal biogeography and age. Reference-based computational profiling revealed a significant and progressive increase in both species richness and evenness along the intestinal tract. The microbial community composition also significantly differed between the small intestine, caecum, and colon. Notably, no significant changes in the taxonomic abundance of individual taxa with age were found except when sex was included as a covariate. Additionally, using an assembly-based computational profiling approach, 156 putative novel bacterial and archaeal species were identified.

We observed limited effects of age on the composition of the luminal microbiota in the profiled regions of the intestinal tract except when sex was included as a covariate. The enteric microbial communities of the small and the large intestine were, however, distinct, highlighting the limitations of frequently used faecal microbial profiling as a proxy for the intestinal microbiota. The identification of a number of putative novel microbial taxa contributes to knowledge of the full diversity of the cynomolgus macaque intestinal microbiome.

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 sulfide's reactivity and volatility. Here we develop a transcriptional sulfide biosensor in Escherichiacoli. The sensor relies on enzymatic oxidation of sulfide catalyzed by a sulfide:quinone oxidoreductase (Sqr) to polysulfides, which interact with the repressor SqrR, triggering unbinding from the promoter and transcription of the reporter. Through promoter engineering and improved soluble SqrR expression, we optimized the system to provide an operational range of 50-750 μM and a 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 homologue resulted in an anaerobic H2S response up to 750 μM. 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 noninvasive diagnostic.

Colorectal cancer (CRC) is a significant global health challenge, with rising incidence, particularly among individuals under 50. Increasing evidence highlights the gut microbiota as key contributors to CRC development, with certain oncogenic bacteria influencing cancer initiation, progression, and therapy response. Among these is pks+ Escherichia coli, which produces colibactin, a genotoxic compound that induces DNA damage and leaves a distinct mutational signature in healthy individuals and CRC patients. While research has focused on its genotoxic effects, this review examines the kinetics of colibactin-induced mutations and the epithelial and environmental changes that promote E. coli expansion and colibactin exposure. We also explore the broader role of pks+ E. coli in cancer initiation and progression beyond genotoxicity, and discuss potential therapeutic approaches.

The limitations of conventional animal tests and two-dimensional cell culture hinder their advancement in fundamental research and clinical/translational applications. As an emerging alternative technology, organ-on-a-chip serves as a platform that faithfully simulates the key phenotypical, physiological, and functional features of human tissues/organs through the accurate regulation of the parameters such as physical and biochemical microenvironment, as well as cellular patterns. In this review, we mainly introduce the recent progress in the organ-on-a-chip field, including lung, gut, heart, liver, vasculature and multiorgan studies. Furthermore, we highlight the potential applications in drug screening and personalized medicine. Finally, we conclude the review by addressing the current challenges and future perspective in the technology and commercialization of organ-on-chips. We anticipate that the development of organ-on-a-chip technology will revolutionize the studies on biology and medicine by providing new understanding of mechanisms of diseases and insights into clinical therapeutics.

The gastric epithelium experiences intermittent hypoxia due to various physiological and pathological conditions. However, the impact of hypoxia and hypoxia-reoxygenation of gastric epithelial cells (GECs) on Helicobacter pylori-mediated gastric cancer (GC) has never been investigated. Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) facilitate H. pylori adhesion onto GECs. We evaluated the effect of hypoxia and hypoxia-reoxygenation on CEACAM6-mediated H. pylori binding, infection, reactive oxygen species (ROS) generation, and GEC proliferation.

Hypoxia-inducible factor 1 (HIF1α) and CEACAM6 levels were assessed in various GECs. ROS were measured using 2',7'-dichlorofluorescin diacetate (DCFDA). Bioinformatics analyses were performed to identify the most prominent stomach adenocarcinoma (STAD)-associated NADPH oxidase (NOX) followed by validation by overexpression/suppression studies and western blotting. GC biopsies were examined by immunofluorescence microscopy. Hypoxia-exposed, reoxygenated, or control cells were compared for ROS generation and H. pylori infection. MTT assay determined cell proliferation.

Hypoxia and HIF1 mediated upregulation of CEACAM6 in GECs. CEACAM6 significantly promoted ROS generation by inducing NOX4 in hypoxic GECs. HIF1α, CEACAM6, and NOX4 upregulation was detected in gastritis and GC tissues. H. pylori infection significantly increased in hypoxia-exposed GECs as compared to normoxic GECs. Infection of hypoxia-reoxygenated GECs also resulted in significantly increased CEACAM6 and NOX4-mediated ROS generation compared to normoxic GECs. In addition, adhesion of H. pylori, cytotoxin-associated gene A (CagA) translocation, and GEC proliferation were significantly enhanced in hypoxia-reoxygenated GECs. Collectively, this study established that hypoxia and hypoxia-reoxygenation of GECs facilitate H. pylori infection and infection-mediated GEC proliferation.

Biomedical research has implicated the bacterial metabolite colibactin as a causal risk factor for several cancer types, in particular, colorectal cancer. Colibactin has been known to drive tumorigenesis by inducing double-strand breaks in the DNA of epithelial cells exposed to colibactin-producing bacteria. Some phylogroup B2 Escherichia coli secrete colibactin during interbacterial warfare, concomitantly exposing the host to an increasing risk of DNA damage. This Personal View reviews the current knowledge about the cancer-colibactin interface and summarises metagenomics-based and population-genomics-based surveys to show that the prevalence of dominant colibactin-producing lineages of E coli varies considerably across geographical regions. The prevalence is further strongly associated with the age-standardised incidences of colorectal cancer, bladder cancer, and prostate cancer, suggesting that the degree of colibactin exposure in a population might contribute to the geographical variation of these cancers. Our observations provide a strong impetus for further research and the development of novel interventions to reduce the risks for colibactin-related cancers.

Microbial metabolism and growth are tightly linked to oxygen (O2). Microbes experience fluctuating O2 levels in natural environments; however, our understanding of how cells respond to fluctuating O2 over various time scales remains limited due to challenges in observing microbial growth at single-cell resolution under controlled O2 conditions and in linking individual cell growth with the specific O2 microenvironment. We performed time-resolved microbial growth analyses at single-cell resolution under a temporally controlled O2 supply. A multilayer microfluidic device was developed, featuring a gas supply above a cultivation layer, separated by a thin membrane enabling efficient gas transfer. This platform allows microbial cultivation under constant, dynamic, and oscillating O2 conditions. Automated time-lapse microscopy and deep-learning-based image analysis provide access to spatiotemporally resolved growth data at the single-cell level. O2 switching within tens of seconds, coupled with precise microenvironment monitoring, allows us to accurately correlate cellular growth with local O2 concentrations. Growing Escherichia coli microcolonies subjected to varying O2 oscillation periods show distinct growth dynamics characterized by response and recovery phases. The comprehensive growth data and insights gained from our unique platform are a crucial step forward to systematically study cell response and adaptation to fluctuating O2 environments at single-cell resolution.

High-altitude environments, characterized by hypobaric and hypoxic conditions, induce acute hypoxia, resulting in decreased blood oxygen saturation. This hypoxic stress perturbs gut microecological homeostasis, significantly contributing to the pathogenesis of acute mountain sickness. Consequently, elucidating the mechanisms by which high altitude affects gut homeostasis is crucial for developing effective interventions.

Oncogenic KRAS mutations are prevalent in colorectal cancer (CRC) and linked to poor prognosis and therapeutic resistance. Emerging evidence suggests that specific KRAS mutations differentially influence treatment responses. In this study, we generate isogenic Apc-null mouse colon epithelial cells with four common KRAS mutations. Transcriptomic and proteomic analyses reveal significant enrichment of cholesterol and lipid metabolism pathways in KRAS G12V cells, driven by increased SREBP1 expression and mTORC1 activation. Furthermore, KRAS G12V cells exhibit elevated ACSS2 expression and greater dependence on ACSS2 for proliferative advantage compared to other mutants. Inhibition of ACSS2 uniquely sensitizes KRAS G12V cells to MEK inhibition, highlighting a distinct therapeutic vulnerability. Finally, ACSS2 plays a critical role in early KRAS G12V adenoma development, unlike in KRAS G12D adenomas. These findings highlight mutation-specific metabolic reprogramming in KRAS-driven CRC and identify ACSS2 as a potential therapeutic target.

To investigate the adaptation of hybrid Escherichia coli to the intestinal and extraintestinal milieu, we compared our model hybrid Shiga toxin-producing (STEC) and uropathogenic (UPEC) E. coli O2:H6 strains with non-pathogenic E. coli and canonical UPEC and STEC strains in a carbon source utilization assay testing 95 common carbon sources under aerobic and anaerobic conditions. Comparison of anaerobic to aerobic growth showed a 2-fold decrease and 2.5-fold increase in the growth capacity and lag phase, respectively. While the UPEC and STEC/UPEC hybrids retained the utilization of several organic acids, amino acids, and peptides, the STEC and non-pathogenic strains relied almost exclusively on the utilization of sugar compounds under anaerobic conditions. Cluster analysis indicated a higher degree of difference and separation between all strains under aerobic conditions. The UPEC, hybrids, and STEC strain B2F1 showed high similarities in aerobic carbon utilization following growth patterns observed in previous phenotype assays. Additionally, we observed known UPEC virulence traits, such as the aerobic utilization of D-serine in our model STEC/UPEC hybrids. Combined, these findings suggest that the intestinal STEC/UPEC O2:H6 isolates originated from a UPEC background and acquired the ability to cause intestinal disease with the addition of Shiga toxin as a virulence factor.

Campylobacter jejuni is a common foodborne pathogen worldwide that is associated with high rates of morbidity and mortality among infants in low- to middle-income countries (LMICs). Human milk provides infants with an important source of nutrients and contains antimicrobial components for protection against infection. However, recent studies, including our own, have found significantly higher levels of Campylobacter in diarrheal stool samples collected from breastfed infants compared to non-breastfed infants in LMICs. We hypothesized that C. jejuni has unique strategies to resist the antimicrobial properties of human milk. Transcriptional profiling found human milk exposure induces genes associated with ribosomal function, iron acquisition, and amino acid utilization in C. jejuni strains 81-176 and 11168. However, unidentified proteinaceous components of human milk prevent bacterial growth. Evolving both C. jejuni isolates to survive in human milk resulted in mutations in genes encoding the acyl carrier protein (AcpP) and the major outer membrane porin (PorA). Introduction of the PorA/AcpP amino acid changes into the parental backgrounds followed by electron microscopy showed distinct membrane architectures, and the AcpP changes not only significantly improved growth in human milk, but also yielded cells surrounded with outer membrane vesicles. Analyses of the phospholipid and lipooligosaccharide (LOS) compositions suggest an imbalance in acyl chain distributions. For strain 11168, these changes protect both evolved and 11168∆acpPG33R strains from bacteriophage infection and polymyxin killing. Taken together, this study provides insights into how C. jejuni may evolve to resist the bactericidal activity of human milk and flourish in the hostile environment of the gastrointestinal tract.

In this study, we evolved C. jejuni strains which can grow in the presence of human milk and found that cell membrane alterations may be involved in resistance to the antimicrobial properties of human milk. These bacterial membrane changes are predominantly linked to amino acid substitutions within the acyl carrier protein, AcpP, although other bacterial components, including PorA, are likely involved. This study provides some insights into possible strategies for C. jejuni survival and propagation in the gastrointestinal tract of breastfed infants.

The gut-liver axis plays a crucial role in maintaining metabolic balance and overall human health. It orchestrates various processes, such as blood flow, nutrient transfer, metabolite processing, and immune cell communication between the two organs. Traditional methods, such as animal models and two-dimensional (2D) cell cultures, are insufficient in fully replicating the intricate functions of the gut-liver axis. The emergence of microfluidic technology has revolutionized this field, facilitating the development of organ-on-a-chip (OOC) systems. These systems are capable of mimicking the complex structures and dynamic environments of the gut and liver in vitro and incorporating sensors for real-time monitoring. In this article, we review the latest progress in gut-on-a-chip (GOC) and liver-on-a-chip (LOC) systems, as well as the integrated gut-liver-on-a-chip (GLOC) models. Our focus lies in the simulation of physiological parameters, three-dimensional (3D) structural mimicry, microbiome integration, and multicellular co-culture. All these aspects are essential for constructing accurate in vitro models of the gut and liver. Furthermore, we explore the current applications of OOC technology in the study of the gut and liver, including its use in disease modeling, toxicity testing, and drug screening. Finally, we discuss the challenges that remain and outline potential future directions for advancing GOC and LOC development in vitro.

Inflammation damages the epithelial cell barrier, allowing oxygen to leak into the lumen of the gut. Respiring E. coli and other Enterobacteriaceae produce proinflammatory lipopolysaccharide, exacerbating inflammatory bowel disease. Here we show that respiring membrane vesicles (MV) from E. coli ameliorate symptoms in a mouse model of gut inflammation. Membrane vesicle treatment diminished weight loss and limited shortening of the colon. Notably, oxygenation of the colonic epithelium was significantly decreased in animals receiving wild type MVs, but not MVs from an E. coli mutant lacking cytochromes. Metatranscriptomic analysis of the microbiome shows an increase in anaerobic Lactobacillaceae and a decrease in Enterobacteriaceae, as well as a general shift towards fermentation in MV-treated mice. This is accompanied by a decrease in proinflammatory TNF-α. We report that MVs may lead to the development of a novel type of a therapeutic for dysbiosis, and for treating IBD.

The gut microbiome functions as a metabolic organ, producing numerous enzymes that influence host health; however, their substrates and metabolites remain largely unknown.

We present MicrobeRX, an enzyme-based metabolite prediction tool that employs 5487 human reactions and 4030 unique microbial reactions from 6286 genome-scale models, as well as 3650 drug metabolic reactions from the DrugBank database (v.5.1.12). MicrobeRX includes additional analysis modules for metabolite visualization and enzymatic and taxonomic analyses. When we applied MicrobeRX to 1083 orally administered drugs that have been approved in at least one jurisdiction at some point in time (DrugBank), it predicted metabolites with physicochemical properties and structures similar to metabolites found in biosamples (from MiMeDB). It also outperformed another existing metabolite prediction tool (BioTransformer 3.0) in terms of predictive potential, molecular diversity, reduction of redundant predictions, and enzyme annotation.

Our analysis revealed both unique and overlapping metabolic capabilities in human and microbial metabolism and chemo- and taxa-specific microbial biotransformations. MicrobeRX bridges the genomic and chemical spaces of the gut microbiome, making it a valuable tool for unlocking the chemical potential of the gut microbiome in human health, the food and pharmaceutical industries, and environmental safety. Video Abstract.

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 sulfide's 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 μM - 750 μM 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 μM. 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.

Restoration of the intestinal epithelial barrier is crucial for achieving mucosal healing, the therapeutic goal for inflammatory bowel disease (IBD). During homeostasis, epithelial renewal is maintained by crypt stem cells and progenitors that cease to divide as they differentiate into mature colonocytes. Inflammation is a major effector of mucosal damage in IBD and has been found to affect epithelial stemness, regeneration and cellular functions. However, the impact of immune cell-modulating IBD drugs on epithelial homeostasis and repair is poorly understood. It is likely that these drugs will have distinct mechanisms of action (MOA) in intestinal epithelium relevant for homeostasis that will vary among patients. We investigated cellular effects of pan-Janus Kinase (JAK) inhibitor tofacitinib and the corticosteroid budesonide on uninflamed and TNF + Poly(I:C) stimulated human colon organoids (colonoids) from healthy donors and IBD-patients. Our findings reveal that although both tofacitinib and budesonide exhibit anti-inflammatory effects, tofacitinib increased colonoid size and proliferation during differentiation, and promoted epithelial stemness. In contrast, budesonide decreased colonoid size and showed no consistent effect on proliferation or stemness. Our study demonstrates the value of employing human colonoids to investigate how IBD drugs affect intestinal epithelial cells and inter-individual variations relevant to mucosal healing and personalized IBD treatment.

This research seeks to address the gap in past studies by examining the role of the Nrf2 (nuclear factor erythroid 2-related factor 2) and HO-1 (heme oxygenase-1) signaling pathways in hypoxia and the potential effects of alpha-pinene on these factors. Wistar rats were divided into 7 experimental groups (n = 7): 1) control, 2 and 3) groups receiving alpha-pinene 5 and 10 mg/kg (i.p., for 21 days), 4) hypoxia group (7% O2 and 93 N2, 3 h, GD14 to GD18), 5 and 6) groups receiving alpha-pinene 5 and 10 after hypoxia. Memory and cognition were evaluated using the Morris water maze and novel object recognition tests. Inflammation was assessed by ELISA method and Nrf2 and HO-1 Nrf2 gene expression was evaluated using real-time PCR in the hippocampus. Recognition index, spatial memory, and Nrf2-H0-1 gene expression significantly reduced in the hypoxia group compared to the control group, and alpha-pinene injection in the offspring improved cognition, memory, and Nrf2- HO-1 gene expression in the groups were affected by hypoxia. Inflammation factors in the hypoxia group were higher than the control, but alpha-pinene significantly decreased inflammation ( all cases p < 0.05). Based on the results, it seems that alpha-pinene prevents cognitive and memory loss by increasing Nrf2 and H0-1 gene expression and reducing inflammation.

Necrotizing enterocolitis (NEC) is an often-lethal disease of the premature infant intestinal tract, exacerbated by significant diagnostic difficulties. In NEC, the intestine exhibits hypoperfusion and dysmotility, contributing to disease pathogenesis. However, these features cannot be accurately and quantitively assessed with current imaging modalities. We have previously demonstrated the ability of photoacoustic imaging (PAI) to non-invasively assess intestinal tissue oxygenation and motility in a healthy neonatal rat model.

In this first-in-disease application, we evaluated NEC using PAI to assess intestinal health biomarkers in an experimental model of NEC. NEC was induced in neonatal rats from birth to 4-days. Healthy breastfed (BF) and NEC rat pups were imaged at 2- and 4-days.

Intestinal tissue oxygen saturation was measured with PAI, and NEC pups showed significant decreases at 2- and 4-days. Ultrasound and PAI cine recordings were used to capture intestinal peristalsis and contrast agent transit within the intestine. Intestinal motility, assessed using computational intestinal deformation analysis, demonstrated significant reductions in both early and established NEC. NEC damage was confirmed with histology and dysmotility was confirmed by small intestinal transit assay.

This preclinical study presents PAI as an emerging diagnostic imaging modality for intestinal disease assessment in premature infants.

Necrotizing enterocolitis (NEC) is a devastating intestinal disease affecting premature infants with significant mortality. NEC presents significant clinical diagnostic difficulties, with limited diagnostic confidence complicating timely and effective interventional efforts. This study is an important foundational first-in-disease preclinical study that establishes the utility for PAI to detect changes in intestinal tissue oxygenation and intestinal motility with NEC disease induction and progression. This study demonstrates the feasibility and exceptional promise for the use of PAI to non-invasively assess oxygenation and motility in the healthy and diseased infant intestine.

The small intestinal microbiota has a crucial role in gastrointestinal health, affecting digestion, immune function, bile acid homeostasis and nutrient metabolism. The challenges of accessibility at this site mean that our knowledge of the small intestinal microbiota is less developed than of the colonic or faecal microbiota. Here, we summarize the features and fluctuations of the microbiota along the small intestinal tract, focusing on humans, and discuss physicochemical factors and assessment methods, including the technical challenges of investigating the low microbial biomass of the proximal small bowel. We highlight the essential protective mechanisms of the small intestine, including motility, the paracellular barrier and mucus, and secretory immunity, to show their roles in limiting excessive exposure of host tissues to microbial metabolites. We address current knowledge gaps, particularly the variability among individuals, the effects of dysbiosis of the small intestinal microbiota on health and how different taxa in small intestinal microbiota could compensate for each other functionally.

The gastrointestinal tract can be deranged by ailments including sepsis, trauma and haemorrhage. Ischaemic injury provokes a common constellation of microscopic and macroscopic changes that, together with the paradoxical exacerbation of cellular dysfunction and death following restoration of blood flow, are collectively known as ischaemia-reperfusion injury (IRI). Although much of the gastrointestinal tract is normally hypoxemic, intestinal IRI results when there is inadequate oxygen availability due to poor supply (pathological hypoxia) or abnormal tissue oxygen use and metabolism (dysoxia). Intestinal oxygen uptake usually remains constant over a wide range of blood flows and pressures, with cellular function being substantively compromised when ischaemia leads to a >50% decline in intestinal oxygen consumption. Restoration of perfusion and oxygenation provokes additional injury, resulting in mucosal damage and disruption of intestinal barrier function. The primary cellular mechanism for sensing hypoxia and for activating a cascade of cellular responses to mitigate the injury is a family of heterodimer proteins called hypoxia-inducible factors (HIFs). The HIF system is connected to numerous biochemical and immunologic pathways induced by IRI and the concentration of those proteins increases during hypoxia and dysoxia. Activation of the HIF system leads to augmented transcription of specific genes in various types of affected cells, but may also augment apoptotic and inflammatory processes, thus aggravating gut injury. KEY POINTS: During intestinal ischaemia, mitochondrial oxygen uptake is reduced when cellular oxygen partial pressure decreases to below the threshold required to maintain normal oxidative metabolism. Upon reperfusion, intestinal hypoxia may persist because microcirculatory flow remains impaired and/or because available oxygen is consumed by enzymes, intestinal cells and neutrophils.

Iron deficiency is the top leading cause of anaemia, whose treatment has been shown to deteriorate gut health. However, a comprehensive analysis of the intestinal barrier and the gut microbiome during iron deficiency anemia (IDA) has not been performed to date. This study aims to delve further into the analysis of these two aspects, which will mean a step forward minimising the negative impact of iron supplements on intestinal health.

IDA was experimentally induced in an animal model. Shotgun sequencing was used to analyse the gut microbiome in the colonic region, while the intestinal barrier was studied through histological analyses, mRNA sequencing (RNA-Seq), qPCR and immunofluorescence assays. Determinations of lipopolysaccharide (LPS) and bacteria-specific immunoglobulins were performed to assess microbial translocation.

Microbial metabolism in the colon shifted towards an increased production of certain amino acids, short chain fatty acids and nucleotides, with Clostridium species being enriched during IDA. Structural alterations of the colonic epithelium were shown by histological analysis. RNA-Seq revealed a downregulation of extracellular matrix-associated genes and proteins and an overall underdeveloped epithelium. Increased levels of serum LPS and an increased immune response against dysbiotic bacteria support an impairment in the integrity of the gut barrier during IDA.

IDA negatively impacts the gut microbiome and the intestinal barrier, triggering an increased microbial translocation. This study emphasizes the deterioration of gut health during IDA and the fact that it should be addressed when treating the disease.

The prolyl hydroxylase domain-containing (PHD or EGL9-homologs) enzyme family is mainly known for its role in the cellular response to hypoxia. HIF-PH inhibitors can stabilize hypoxia-inducible factors (HIFs), activating transcriptional programs that promote processes such as angiogenesis and erythropoiesis to adapt to changes in oxygen levels. HIF-PH inhibitors have been clinically approved for treating several types of anaemia. While most discussions of the HIF-PH signalling axis focus on hypoxia, there is a growing recognition of its importance under normoxic conditions. Recent advances in PHD biology have highlighted the potential of targeting this pathway therapeutically for a range of aging-related diseases. In this article, we review these recent discoveries, situate them within the broader context of aging and disease, and explore current therapeutic strategies that target PHD enzymes for these indications.

Mitochondrial membranes are defined by their diverse functions, complex geometries, and unique lipidomes. In the inner mitochondrial membrane, highly curved membrane folds known as cristae house the electron transport chain and are the primary sites of cellular energy production. The outer mitochondrial membrane is flat by contrast, but is critical for the initiation and mediation of processes key to mitochondrial physiology: mitophagy, interorganelle contacts, fission and fusion dynamics, and metabolite transport. While the lipid composition of both the inner mitochondrial membrane and outer mitochondrial membrane have been characterized across a variety of cell types, a mechanistic understanding for how individual lipid classes contribute to mitochondrial structure and function remains nebulous. In this review, we address the biophysical properties of mitochondrial lipids and their related functional roles. We highlight the intrinsic curvature of the bulk mitochondrial phospholipid pool, with an emphasis on the nuances surrounding the mitochondrially-synthesized cardiolipin. We also outline emerging questions about other lipid classes - ether lipids, and sterols - with potential roles in mitochondrial physiology. We propose that further investigation is warranted to elucidate the specific properties of these lipids and their influence on mitochondrial architecture and function.

The intestinal mucosa is a dynamic surface that facilitates interactions between the host and an outside world that includes trillions of microbes, collectively termed the microbiota. This fine balance is regulated by an energetically demanding physical and biochemical barrier that is formed by the intestinal epithelial cells. In addition, this homeostasis exists at an interface between the anaerobic colonic lumen and a highly oxygenated, vascularized lamina propria. The resultant oxygen gradient within the intestine establishes "physiologic hypoxia" as a central metabolic feature of the mucosa. Although oxygen is vital for energy production to meet cellular metabolism needs, the availability of oxygen has far-reaching influences beyond just energy provision. Recent studies have shown that the intestinal mucosa has purposefully adapted to use differential oxygen levels largely through the presence of short-chain fatty acids (SCFAs), particularly butyrate (BA). Intestinal epithelial cells use butyrate for a multitude of functions that promote mucosal homeostasis. In this review, we explore how the physiologic hypoxia profile interfaces with SCFAs to benefit host mucosal tissues.

Glucagon-like peptide (GLP)-1 is a hormone released by enteroendocrine L-cells after food ingestion. L-cells express various receptors for nutrient sensing including G protein-coupled receptors (GPRs). Intestinal epithelial cells near the lumen have a lower O2 tension than at the base of the crypts, which leads to hypoxia in L-cells. We hypothesized that hypoxia affects nutrient-stimulated GLP-1 secretion from the enteroendocrine cell line STC-1, the most commonly used model. In this study, we investigated the effect of hypoxia (1% O2) on alpha-linolenic acid (αLA) stimulated GLP-1 secretion and their receptor expressions. STC-1 cells were incubated for 12 h under hypoxia (1% O2) and treated with αLA to stimulate GLP-1 secretion. 12 h of hypoxia did not change basal GLP-1 secretion, but significantly reduced nutrient (αLA) stimulated GLP-1 secretion. In normoxia, αLA (12.5 μM) significantly stimulated (~ 5 times) GLP-1 secretion compared to control, but under hypoxia, GLP-1 secretion was reduced by 45% compared to normoxia. αLA upregulated GPR120, also termed free fatty acid receptor 4 (FFAR4), expressions under normoxia as well as hypoxia. Hypoxia downregulated GPR120 and GPR40 expression by 50% and 60%, respectively, compared to normoxia. These findings demonstrate that hypoxia does not affect the basal GLP-1 secretion but decreases nutrient-stimulated GLP-1 secretion. The decrease in nutrient-stimulated GLP-1 secretion was due to decreased GPR120 and GPR40 receptors expression. Changes in the gut environment and inflammation might contribute to the hypoxia of the epithelial and L-cells.

Hepatic clearance of lipid nanoparticles (LNP) with encapsulated nucleic acids restricts their therapeutic applicability. Therefore, tools for regulating hepatic clearance are of high interest for nucleic acid delivery. To this end, this work employs wild-type (WT) and low-density lipoprotein receptor (Ldlr)-/- mice pretreated with either a leukotriene B4 receptor inhibitor (BLT1i) or a high-density lipoprotein receptor inhibitor (HDLRi) prior to the injection of siRNA-LNP. This work is able to demonstrate significantly increased hepatic uptake of siRNA-LNP by the BLT1i in Ldlr-/- mice by in vivo imaging and discover an induction of specific uptake-related proteins. Irrespective of the inhibitors and Ldlr deficiency, the siRNA-LNP induced RNA-binding and transport-related proteins in liver, including haptoglobin (HP) that is also identified as most upregulated serum protein. This work observes a downregulation of proteins functioning in hepatic detoxification and of serum opsonins. Most strikingly, the HDLRi reduces hepatic uptake and increases siRNA accumulation in spleen and myeloid immune cells of blood and liver. RNA sequencing demonstrates leukocyte recruitment by the siRNA-LNP and the HDLRi through induction of chemokine ligands in liver tissue. The data provide insights into key mechanisms of siRNA-LNP biodistribution and indicate that the HDLRi has potential for extrahepatic and leukocyte targeting.

We establish an in vitro perfusion intestinal tissue bioreactor system tailored to study drug responses related to inflammatory bowel disease (IBD). The system includes key components including multiple human intestinal cell types (colonoids, myofibroblasts, and macrophages), a three-dimensional (3D) intestinal architecture, and fluid flow. Inclusion of myofibroblasts resulted in increased secretion of cytokines such as glypican-1 (GCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 1-α (IL-1α), whereas inclusion of macrophages resulted in increased secretion of monocyte chemoattractant proteins (MCPs) demonstrating a significant role of both stromal and immune cell types in intestinal inflammation. The system is responsive to drug treatments, as reflected in the reduction of pro-inflammatory cytokine production in tissue in some treatment scenarios. While future studies are needed to evaluate more nuanced responses in an IBD context, the present study demonstrates the ability to establish a 3D intestinal model with multiple relevant cell types and flow that is responsive to both inflammatory cues and various drug treatment options.

Streptococcus agalactiae (Group B Streptococcus, GBS) is a Gram-positive bacterial species that causes disease in humans across the lifespan. While antibiotics are used to mitigate GBS infections, it is evident that antibiotics disrupt human microbiomes (which can predispose people to other diseases later in life), and antibiotic resistance in GBS is on the rise. Taken together, these unintended negative impacts of antibiotics highlight the need for precision approaches for minimizing GBS disease. One possible approach involves selectively depleting GBS in its commensal niches before it can cause disease at other body sites or be transmitted to at-risk individuals. One understudied commensal niche of GBS is the adult gastrointestinal (GI) tract, which may predispose colonization at other body sites in individuals at risk for GBS disease. However, a better understanding of the host-, microbiome-, and GBS-determined variables that dictate GBS GI carriage is needed before precise GI decolonization approaches can be developed. In this review, we synthesize current knowledge of the diverse body sites occupied by GBS as a pathogen and as a commensal. We summarize key molecular factors GBS utilizes to colonize different host-associated niches to inform future efforts to study GBS in the GI tract. We also discuss other GI commensals that are pathogenic in other body sites to emphasize the broader utility of precise de-colonization approaches for mitigating infections by GBS and other bacterial pathogens. Finally, we highlight how GBS treatments could be improved with a more holistic understanding of GBS enabled by continued GI-focused study.

Coculture of intestinal bacteria with primary human intestinal epithelium provides a valuable tool for investigating host-colon bacterial interactions and for testing and screening therapeutics. However, most current intestinal model systems lack key physiological features of the in vivo colon, such as both a proper oxygen microenvironment and a mucus layer. In this work, a new in vitro colonic microphysiological system is demonstrated with a cell-derived, functional mucus that closely resembles the in vivo colonic mucosa and apical microenvironment by employing an anaerobic air-liquid interface culture. The human primary colon epithelial cells in this new in vitro system exhibit high cell viability (>98%) with ≈100 μm thick functional mucus layer on average. Successful coculture of model anaerobic gut bacterial strains Lactobacillus rhamnosus GG and Anaerobutyricum hallii without loss in human cell viability demonstrates that this new model can be an invaluable tool for future studies of the impact of commensal and pathogenic bacteria on the colon.

From the moment of birth, the newborn gastrointestinal tract is infiltrated by various bacteria originating from both maternal and environmental sources. These colonizing bacteria form a complex microbiota community that undergoes continuous changes until adulthood and plays an important role in infant health. The maturation of the infant gut microbiota is driven by many factors and follows a distinct patterned trajectory, with specific bacterial taxa establish in the intestine in accordance with developmental milestones as the infant grows. In this review, we highlight how elements such as diet and host physiology select for specific microbial functions and shape the composition of the bacterial community in the large intestine.

Recent advancements in organoid technology have heralded a transformative era in biomedical research, characterized by the emergence of gut organoids that replicate the structural and functional complexity of the human intestines. These stem cell-derived structures provide a dynamic platform for investigating intestinal physiology, disease pathogenesis, and therapeutic interventions. This model outperforms traditional two-dimensional cell cultures in replicating cell interactions and tissue dynamics. Gut organoids represent a significant leap towards personalized medicine. They provide a predictive model for human drug responses, thereby minimizing reliance on animal models and paving the path for more ethical and relevant research approaches. However, the transition from basic organoid models to more sophisticated, biomimetic systems that encapsulate the gut's multifaceted environment-including its interactions with microbial communities, immune cells, and neural networks-presents significant scientific challenges. This review concentrates on recent technological strides in overcoming these barriers, emphasizing innovative engineering approaches for integrating diverse cell types to replicate the gut's immune and neural components. It also explores the application of advanced fabrication techniques, such as 3D bioprinting and microfluidics, to construct organoids that more accurately replicate human tissue architecture. They provide insights into the intricate workings of the human gut, fostering the development of targeted, effective treatments. These advancements hold promise in revolutionizing disease modeling and drug discovery. Future research directions aim at refining these models further, making them more accessible and scalable for wider applications in scientific inquiry and clinical practice, thus heralding a new era of personalized and predictive medicine.

Helicobacter pylori is a microaerophilic Gram-negative bacterium that resides in the human stomach and is classified as a class I carcinogen for gastric cancer. Numerous studies have demonstrated that H. pylori infection plays a role in regulating the function of host cells, thereby contributing to the malignant transformation of these cells. However, H. pylori infection is a chronic process, and short-term cellular experiments may not provide a comprehensive understanding of the in vivo situation, especially when considering the lower oxygen levels in the human stomach. In this study, we aimed to investigate the mechanisms underlying gastric cell dysfunction after prolonged exposure to H. pylori under hypoxic conditions. We conducted a co-culture experiment using the gastric cell line GES-1 and H. pylori for 30 generations under intermittent hypoxic conditions. By closely monitoring cell proliferation, migration, invasion, autophagy, and apoptosis, we revealed that sustained H. pylori stimulation under hypoxic conditions significantly influences the function of GES-1 cells. This stimulation induces epithelial-mesenchymal transition and contributes to the propensity for malignant transformation of gastric cells. To confirm the in vitro results, we conducted an experiment involving Mongolian gerbils infected with H. pylori for 85 weeks. All the results strongly suggest that the Nod1 receptor signaling pathway plays a crucial role in H. pylori-related apoptosis and autophagy. In summary, continuous stimulation by H. pylori affects the functioning of gastric cells through the Nod1 receptor signaling pathway, increasing the likelihood of cell carcinogenesis. The presence of hypoxic conditions further exacerbates this process.IMPORTANCEDeciphering the collaborative effects of Helicobacter pylori infection on gastric epithelial cell function is key to unraveling the development mechanisms of gastric cancer. Prior research has solely examined the outcomes of short-term H. pylori stimulation on gastric epithelial cells under aerobic conditions, neglecting the bacterium's nature as a microaerophilic organism that leads to cancer following prolonged stomach colonization. This study mimics a more genuine in vivo infection scenario by repeatedly exposing gastric epithelial cells to H. pylori under hypoxic conditions for up to 30 generations. The results show that chronic exposure to H. pylori in hypoxia substantially increases cell migration, invasion, and epithelial-mesenchymal transition, while suppressing autophagy and apoptosis. This highlights the significance of hypoxic conditions in intensifying the carcinogenic impact of H. pylori infection. By accurately replicating the in vivo gastric environment, this study enhances our comprehension of H. pylori's pathogenic mechanisms in gastric cancer.

This study aimed to evaluate the adverse effects of particulate matter (PM) exposure on endometrial cells and fertility and to identify possible underlying mechanisms. Thirteen women (aged 15-52 years) were included in this study. Enrolled patients underwent laparoscopic surgery at Gangnam Severance Hospital between 1 January and 31 December 2021. For in vivo experiments, 36 female and nine male C57BL/6 mice were randomly divided into control(vehicle), low-dose(10 mg/kg/d), and high-dose exposure groups(20 mg/kg/d). PM was inhaled nasally for four weeks and natural mating was performed. NIST® SRM® 1648a was used for PM exposure. qRT-PCR, western blotting and Masson's trichrome staining were performed. PM treatment in human endometrial stromal cells induced inflammation with significant upregulation of IL-1β, p-NF-kB, and p-c-Jun compared to those of controls. Additionally, PM treatment significantly increased apoptosis in human endometrial stromal cells by downregulating p-AKT and upregulating p-p53/p53, Cas-3, BAX/Bcl-2, p-AMPK, and p-ERK. After PM treatment, the relative expression of IL-1β, IL-6, TNF-α, p-NF-κB, p-c-Jun, and p-Nrf2/Nrf2 significantly increased in murine endometrium compared to those of the controls. Expression of apoptotic proteins p53, p27, and Cas-3, was also significantly elevated in murine endometrium of the PM exposure group compared to that of the controls. A significant increase in expression of procollagen Ⅰ, and Masson's trichrome staining scores in the murine endometrium was noted after PM treatment. PM treatment significantly decreased ERα expression. After natural mating, all 3 female mice in the control group gave birth to 25 offspring (mean 8.1), whereas in the low-dose PM treatment group, two of three female mice gave birth to nine offspring (mean 4.5). No pregnant mice or offspring was present in the high-dose PM treatment group. PM exposure induces adverse effects on the endometrium through aberrant activation of inflammatory and apoptotic pathways and is associated with detrimental effects on murine fertility.

In this study, we investigated the role of hypoxia in the development of chronic inflammatory bowel disease (IBD), focusing on its impact on the HIF-1α signaling pathway through the upregulation of lipocalin 2 (LCN2). Using a murine model of colitis induced by sodium dextran sulfate (DSS) under hypoxic conditions, transcriptome sequencing revealed LCN2 as a key gene involved in hypoxia-mediated exacerbation of colitis. Bioinformatics analysis highlighted the involvement of crucial pathways, including HIF-1α and glycolysis, in the inflammatory process. Immune infiltration analysis demonstrated the polarization of M1 macrophages in response to hypoxic stimulation. In vitro studies using RAW264.7 cells further elucidated the exacerbation of inflammation and its impact on M1 macrophage polarization under hypoxic conditions. LCN2 knockout cells reversed hypoxia-induced inflammatory responses, and the HIF-1α pathway activator dimethyloxaloylglycine (DMOG) confirmed LCN2's role in mediating inflammation via the HIF-1α-induced glycolysis pathway. In a DSS-induced colitis mouse model, oral administration of LCN2-silencing lentivirus and DMOG under hypoxic conditions validated the exacerbation of colitis. Evaluation of colonic tissues revealed altered macrophage polarization, increased levels of inflammatory factors, and activation of the HIF-1α and glycolysis pathways. In conclusion, our findings suggest that hypoxia exacerbates colitis by modulating the HIF-1α pathway through LCN2, influencing M1 macrophage polarization in glycolysis. This study contributes to a better understanding of the mechanisms underlying IBD, providing potential therapeutic targets for intervention.

Gut microbiota is a diverse community of microorganisms that constantly work to protect the gut against pathogens. Salmonella stands out as a notorious foodborne pathogen that interacts with gut microbes, causing an imbalance in the overall composition of microbiota and leading to dysbiosis. This review focuses on the interactions between Salmonella and the key commensal bacteria such as E. coli, Lactobacillus, Clostridium, Akkermansia, and Bacteroides. The review highlights the role of these gut bacteria and their synergy in combating Salmonella through several mechanistic interactions. These include the production of siderophores, which compete with Salmonella for essential iron; the synthesis of short-chain fatty acids (SCFAs), which exert antimicrobial effects and modulate the gut environment; the secretion of bacteriocins, which directly inhibit Salmonella growth; and the modulation of cytokine responses, which influences the host's immune reaction to infection. While much research has explored Salmonella, this review aims to better understand how specific gut bacteria engage with the pathogen, revealing distinct defense mechanisms tailored to each species and how their synergy may lead to enhanced protection against Salmonella. Furthermore, the combination of these commensal bacteria could offer promising avenues for bacteria-mediated therapy during Salmonella-induced gut infections in the future.

Mechanistic investigation of host-microbe interactions in the human gut are hindered by difficulty of co-culturing microbes with intestinal epithelial cells. On one hand the gut bacteria are a mix of facultative, aerotolerant or obligate anaerobes, while the intestinal epithelium requires oxygen for growth and function. Thus, a coculture system that can recreate these contrasting oxygen requirements is critical step towards our understanding microbial-host interactions in the human gut. Here, we demonstrate Intestinal Organoid Physoxic Coculture (IOPC) system, a simple and cost-effective method for coculturing anaerobic intestinal bacteria with human intestinal organoids (HIOs). Using commensal anaerobes with varying degrees of oxygen tolerance, such as nano-aerobe Bacteroides thetaiotaomicron and strict anaerobe Blautia sp., we demonstrate that IOPC can successfully support 24-48 hours HIO-microbe coculture. The IOPC recapitulates the contrasting oxygen conditions across the intestinal epithelium seen in vivo. The IOPC cultured HIOs showed increased barrier integrity, and induced expression of immunomodulatory genes. A transcriptomic analysis suggests that HIOs from different donors show differences in the magnitude of their response to coculture with anaerobic bacteria. Thus, the IOPC system provides a robust coculture setup for investigating host-microbe interactions in complex, patient-derived intestinal tissues, that can facilitate the study of mechanisms underlying the role of the microbiome in health and disease.