Despite its relevance in several research fields, the regulation of dissolved gas concentration in microfluidic chips remains overlooked. Precise control of dissolved oxygen levels is of importance for life science applications, especially for faithfully replicating in vivo tissue conditions in organ-on-chips. The current methods to control oxygen on-chip rely on the use of chemical scavengers, on the integration of an additional gas channel or on the perfusion of a liquid pre-equilibrated at a set oxygen level. However, for precise oxygen control, these microfluidic devices must be made from gas-impermeable materials. In this regard, glass is a material of choice due to its complete impermeability, but its microfabrication often requires specific clean room processes. Here, we report a low-tech fabrication method for a hybrid glass chip, which involves assembling glass components using an adhesion process. To evaluate this chip's suitability for use under highly controlled oxygen conditions, we developed a two-step assessment protocol. This involved determining the time needed to reach a target oxygen level during perfusion and measuring the reoxygenation time following the cessation of flow. Based on a dual approach of simulations and experiments, we emphasized crucial adhesive properties such as oxygen diffusion and solubility and proposed a range of well-suited adhesive materials. Finally, we demonstrated the interest of this hybrid glass chip for on-chip cell culture and cell respiration measurements. This work paves the way for broader accessibility in producing low tech gas-tight microfluidic chips for diverse applications.

This review arises from the symposium held in honour of Prof Jenny Morgan at UCL in 2024 and the authors would like to acknowledge the outstanding contribution that Prof Morgan has made to the field of translational muscle cell biology. Prof Morgan published a review article in 2010 entitled: Are human and mice satellite cells really the same? In which the authors highlighted differences between species which are still pertinent to skeletal muscle cell culture studies today. To our knowledge there are no comprehensive reviews which outline the considerable work that has been undertaken using human primary skeletal muscle origin cells as the main model system. This review highlights the multitude of muscle biology that has been investigated using human primary cells, as well as discussing the advantages and disadvantages over other cell models. We also discuss future directions for primary cell culture models utilising the latest technologies in cell type specificity and culture systems.

Hyperuricemia, caused by uric acid disequilibrium, is a prevalent metabolic disease that most commonly manifests as gout and is closely associated with a spectrum of other comorbidities such as renal disorders and cardiovascular diseases. While natural and engineered probiotics that promote catabolism of uric acid in the intestine have shown promise in relieving hyperuricemia, limitations in strain efficiency and the requirements for achieving high performance remain major hurdles in the practical application of probiotic-mediated prevention and management. Here, we employed a systematic strategy to engineer a high-efficiency uric acid catabolism pathway in S. cerevisiae. An uricase from Vibrio vulnificus, exhibiting high-level activity in S. cerevisiae, was identified as the uric acid-degrading component. The expression level and stability of urate transporter UapA were improved by constructing a chimera, enabling reliable uric acid import in S. cerevisiae. Additionally, constitutive promoters were selected and combinatorially assembled with the two functional components, creating a collection of pathways that confer varied levels of uric acid catabolic activity to S. cerevisiae. The best-performing pathway can express uric acid-degrading activity up to 365.32 ± 20.54 μmol/h/OD, requiring only simple cultivation steps. Eventually, we took advantage of the genetic similarity between model organism S. cerevisiae and probiotic S. boulardii and integrated the optimized pathway into identified high-expression integration loci in the S. boulardii genome. The activity can be stably maintained under high-density fermentation conditions. Overall, this study provided a high-potential hyperuricemia-managing yeast probiotic strain, demonstrating the capabilities of developing recombinant probiotics.

Beta cell replacement therapy via pancreatic islet transplantation offers a promising treatment for type 1 diabetes as an alternative to insulin injections. However, posttransplantation oxygenation remains a critical challenge; isolated islets from donors lose vascularity and rely on slow oxygen diffusion for survival until revascularization occurs in the host tissue. This often results in significant hypoxia-induced acute graft loss. Overcoming the oxygenation barrier is crucial for advancing islet transplantation. This review is structured in three sections: the first examines oxygen dynamics in islet transplantation, focusing on factors affecting oxygen supply, including vascularity. It highlights oxygen dynamics specific to both transplant sites and islet grafts, with particular attention to extrahepatic sites such as subcutaneous tissue. The second section explores current oxygen delivery strategies, categorized into two main approaches: augmenting oxygen supply and enhancing effective oxygen solubility. The final section addresses key challenges, such as the lack of a clearly defined oxygen threshold for islet survival and the limited precision in measuring oxygen levels within small islet constructs. Recent advancements addressing these challenges are introduced. By deepening the understanding of oxygen dynamics and identifying current obstacles, this review aims to guide the development of innovative strategies for future research and clinical applications. These advancements are anticipated to enhance transplantation outcomes and bring us closer to a cure for type 1 diabetes.

Studying cells exposed to low and controllable oxygen levels is key to investigating various fundamental aspects of pathological states, such as stroke and cancer. At present, available methodologies applied in vitro focus on large groups of cells exposed to low oxygen conditions through slow-time approaches, such as environmental incubators or microfluidic devices. Here, we demonstrate a novel approach for titrating the local oxygen concentration around individual adhered PC12 cells, enabling single cells within a population to be exposed to hypoxic-like conditions. A 25 μm diameter platinum disk microelectrode performing the oxygen reduction reaction (ORR) at constant current (galvanostatic control) is used as a microscale oxygen scavenger that can be positioned precisely over individual cells. By coupling the galvanostatic oxygen challenge with confocal laser scanning microscopy (CLSM) and a commercially available hypoxia dye (Image-iT Green hypoxia reagent), we monitor the response of single cells when exposed to depleted oxygen concentrations over time. Numerical simulations are used to characterize the oxygen and pH gradient imposed by the microelectrode at different cathodic currents, revealing that within seconds, the oxygen depletion zone reaches a steady-state condition, extending a few microelectrode radii into solution, while the corresponding pH gradient is strongly compressed by the buffer solution. Cells under the microelectrode show a marked increase in average fluorescence rate relative to control, reporting their hypoxic conditions and demonstrating the effectiveness of the proposed method. Heterogenous cell response in a challenged group is also observed, highlighting the ability of this approach to investigate the natural heterogeneity in cell populations. This work provides a platform and roadmap for future studies of cellular systems where the ability to control and vary oxygen concentration on a rapid time scale would be beneficial.

Leukemia is driven by complex interactions within the inherently hypoxic bone marrow microenvironment, impacting both disease progression and therapeutic resistance. Co-cultivation of leukemic cells with feeder cells has emerged as a valuable tool to mimic the bone marrow niche. This study explores the interplay between human commercial SD-1 and patient-derived UPF26K leukemic cell lines with feeders - human fibroblasts (NHDF) and mesenchymal stem cells (hMSCs) under normoxic and hypoxic conditions.

Co-cultivation with feeders significantly enhances proliferation and glycolytic activity in the SD-1 cells, improving their viability, while this interaction inhibits the growth and glucose metabolism of the feeders, particularly NHDF. In contrast, UPF26K cells show reduced proliferation when co-cultivated with the feeders while this interaction stimulates NHDF and hMSCs proliferation and glycolysis but reduce their mitochondrial metabolism with hypoxia amplifying these effects.

Cells that switch to glycolysis during co-cultivation, particularly under hypoxia, benefit most from these low oxygen conditions. Due to this leukemic cells' response heterogeneity, targeting microenvironmental interactions and oxygen levels is crucial for personalized leukemia therapy. Advancing co-cultivation models, particularly through innovations like spheroids, can further enhance in vitro studies of primary leukemic cells and support the testing of novel therapies.

Clostridioides difficile, a strict anaerobe, is the major cause of antibiotic-associated diarrhea. This enteropathogen must adapt to oxidative stress mediated by reactive oxygen species (ROS), notably those released by the neutrophils and macrophages recruited to the site of infection or those endogenously produced upon high oxygen (O2) exposure. C. difficile uses a superoxide reductase, Sor, and several peroxidases to detoxify ROS. We showed that Sor has a superoxide reductase activity in vitro and protects the bacterium from exposure to menadione, a superoxide donor. After confirming the peroxidase activity of the rubrerythrin, Rbr, we showed that this enzyme together with the peroxiredoxin, Bcp, plays a central role in the detoxification of H2O2 and promotes the survival of C. difficile in the presence of not only H2O2 but also air or 4% O2. Under high O2 concentrations encountered in the gastrointestinal tract, the bacterium generated endogenous H2O2. The two O2 reductases, RevRbr2 and FdpF, have also a peroxidase activity and participate in H2O2 resistance. The CD0828 gene, which also contributes to H2O2 protection, forms an operon with rbr, sor, and perR encoding a H2O2-sensing repressor. The expression of the genes encoding the ROS reductases and the CD0828 protein was induced upon exposure to either H2O2 or air. We showed that the induction of the rbr operon is mediated not only by PerR but also by OseR, a recently identified O2-responsive regulator of C. difficile, and indirectly by σB, the sigma factor of the stress response, whereas the expression of bcp is only controlled by σB.

ROS plays a fundamental role in intestinal homeostasis, limiting the proliferation of pathogenic bacteria. Clostridioides difficile is an important enteropathogen that induces an intense immune response, characterized by the massive recruitment of immune cells responsible for secreting ROS, mainly H2O2 and superoxide. We showed in this work that ROS exposure leads to the production of an armada of enzymes involved in ROS detoxification. This includes a superoxide reductase and four peroxidases, Rbr, Bcp, revRbr2, and FdpF. These enzymes likely contribute to the survival of vegetative cells of C. difficile in the colon during the host immune response. Distinct regulations are also observed for the genes encoding the ROS detoxification enzymes allowing a fine tuning of the adaptive response to ROS exposure. Understanding the mechanisms of ROS protection during infection could shed light on how C. difficile survives under conditions of an exacerbated inflammatory response.

Background: Capillary tortuosity is a morphological variant of microcirculation. However, the mechanisms by which tortuous vessels meet metabolic requirements in health and disease remain unknown. We recently reported that capillary tortuosity score (CTS) is significantly higher in patients with septic shock than in steady-state individuals, and that CTS is significantly associated with alveolar-to-arterial oxygen (A-a O2) gradient and oxygen debt in septic shock patients. Objective: We aimed to investigate the characteristics of the magnetic fields in the sublingual microcirculation of individuals with normal physiology and patients with septic shock. Methods: Systemic hemodynamics were recorded, and sublingual microcirculation was monitored using sidestream dark field (SDF+) imaging. The number of capillary red blood cells (NRBC), the intensity of the magnetic field of a red blood cell (HRBC), the intensity of the magnetic field of each capillary (HCAP), and the intensity with which the magnetic field of a capillary acts on an RBC (FCAP) were calculated. Results: Significant differences in macro- and microhemodynamic variables were observed between the two groups. Although NRBC was significantly higher in individuals with steady-state physiology [87.4 (87.12) vs. 12.23 (6.9)], HRBC was significantly stronger in patients with septic shock [5.9 × 10-16 (6.9 × 10-16) A m-1 vs. 1.6 × 10-15 (1.4 × 10-15) A m-1]. No significant difference was observed in HCAP [2.16 × 10-14 (2.17 × 10-14) A m-1 vs. 1.34 × 10-14 (1.23 × 10-14) A m-1] and FCAP [1.66 × 10-24 (3.36 × 10-24) A m-1 vs. 6.44 × 10-25 (1.1 × 10-24) A m-1] between the two groups. In patients with septic shock, HRBC was associated with De Backer score (rho = -0.608) and venous-arterial carbon dioxide difference (rho = 0.569). In the same group, HCAP was associated with convective oxygen flow (rho = 0.790) and oxygen extraction ratio (rho = -0.596). Also, FCAP was significantly associated with base deficit (rho = 0.701), A-a O2 gradient (rho = 0.658), and oxygen debt (rho = -0.769). Conclusions: Despite the microcirculatory impairment in patients with septic shock, HRBC was significantly stronger in that group than in steady-state individuals. Also, HCAP and FCAP were comparable between the two groups. Tortuous vessels may function as biomagnetic coils that amplify RBC-induced magnetic fields, enhancing perfusion and oxygenation of adjacent tissues.

Lipopolysaccharide (LPS) is an exacerbating factor for allergic airway inflammation at least partly due to the activation of mast cells (MCs). LPS stimulates MCs to express both pro-inflammatory and type 2 cytokines, among which interleukin (IL)-13 is essential for the generation of allergic diseases. LPS also induces the expression of "alarmins" such as IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) from various cell types including epithelial cells, and increased serum IL-33 levels were reported to correlate with disease severity of asthma. MCs reside in peripheral tissues where the oxygen concentration is significantly lower than that in the air and further decreased by inflammation and bronchoconstriction in asthma. However, the effects of hypoxia on LPS-induced cytokine expression in MCs have not been fully elucidated. Here we show that LPS induces Il4, Il6, Il13, Il33, Tnf, and Tslp mRNAs in MCs. Notably, hypoxia robustly enhanced expressions of Il13 and Il33, but not the other cytokines in LPS-stimulated MCs. We also found that this promotive effect is dependent on the presence of hypoxia-inducible factor (HIF) 1α protein. Our study will provide new insight on the role of MCs in the LPS-associated pathogenesis of allergic diseases.

This review addresses oxidative stress and redox signaling in the pancreas under healthy physiological conditions as well as in acute pancreatitis, chronic pancreatitis, pancreatic cancer, and diabetes. Physiological redox homeodynamics is maintained mainly by NRF2/KEAP1, NF-κB, protein tyrosine phosphatases, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α), and normal autophagy. Depletion of reduced glutathione (GSH) in the pancreas is a hallmark of acute pancreatitis and is initially accompanied by disulfide stress, which is characterized by protein cysteinylation without increased glutathione oxidation. A cross talk between oxidative stress, MAPKs, and NF-κB amplifies the inflammatory cascade, with PP2A and PGC1α as key redox regulatory nodes. In acute pancreatitis, nitration of cystathionine-β synthase causes blockade of the transsulfuration pathway leading to increased homocysteine levels, whereas p53 triggers necroptosis in the pancreas through downregulation of sulfiredoxin, PGC1α, and peroxiredoxin 3. Chronic pancreatitis exhibits oxidative distress mediated by NADPH oxidase 1 and/or CYP2E1, which promotes cell death, fibrosis, and inflammation. Oxidative stress cooperates with mutant KRAS to initiate and promote pancreatic adenocarcinoma. Mutant KRAS increases mitochondrial reactive oxygen species (ROS), which trigger acinar-to-ductal metaplasia and progression to pancreatic intraepithelial neoplasia (PanIN). ROS are maintained at a sufficient level to promote cell proliferation, while avoiding cell death or senescence through formation of NADPH and GSH and activation of NRF2, HIF-1/2α, and CREB. Redox signaling also plays a fundamental role in differentiation, proliferation, and insulin secretion of β-cells. However, ROS overproduction promotes β-cell dysfunction and apoptosis in type 1 and type 2 diabetes.

Mitochondrial oxygen consumption, dynamics, and morphology play roles in the occurrence, development, and drug resistance of cancer; thus, they are main targets for many anticancer drugs. Increased mitochondrial oxygen consumption and impaired oxygen delivery creates hypoxia, which influences the balance of metabolic cofactors for biogenesis, disease progression, and response to therapeutics. We therefore investigated the effects of Taxol, a well-known anticancer drug, on mitochondrial respiration (principally via a measure of oxidative phosphorylation versus glycolysis), morphology, and dynamics. The concomitant effects of Taxol on mitochondrial ATP and reactive oxygen species production, mitochondrial membrane potential, radical-induced formation of carbonyl groups, mitochondrial release of cytochrome c, as well as cell cycle were investigated. Cells used in this study include the following: A549 (non-small-cell lung epithelial cancer cell line), A549-ρ0 (mitochondrial DNA-depleted derivative of A549), and BEAS-2B (a noncancer cell line derived from normal bronchial epithelium), as well as PC3 (prostate cancer) and HepG2 (hepatocellular carcinoma); these cell lines are known to have disparate metabolic profiles. Using a multitude of fluorescence-based measurements, we show that Taxol, even at a low dose, still adversely affects mitochondria of actively respiring (aerobic) cancer cells. We find an increase in mitochondrial ROS and cytochrome c release, suppression of ATP production and oxidative phosphorylation, fragmentation of the mitochondrial network, and disruption of mitochondria-microtubule linkage. We find these changes in oxidative, but not glycolytic, cancer cells. Noncancer cells, which are oxidative, do not show these changes.

Inherited cardiac channelopathies are linked to a heightened risk of sudden cardiac death. Despite evolving knowledge on different genes for these inherited conditions, for certain subtypes, such as catecholaminergic polymorphic ventricular tachycardia syndrome, the specific genetic causes remain unidentified. The research of the pathophysiological mechanisms underlying catecholaminergic polymorphic ventricular tachycardia syndrome has been conducted through different in vitro and in vivo models, including genetically modified animal models, cardiac-specific transgenic models, pharmacological interventions in animal models, human-induced pluripotent stem cell-derived cardiomyocytes in 2- and 3-dimensional cardiac models. Recent research predominantly utilizes human-induced pluripotent stem cell-derived cardiomyocytes, focusing on genotype-phenotype correlations and pharmacological screening. The integration of cutting-edge techniques such as clustered regularly interspaced short palindromic repeats/Cas9 genome editing and 3-dimensional-engineered heart tissues has shed new light on the pathophysiological mechanisms of catecholaminergic polymorphic ventricular tachycardia, potentially enhancing drug therapies as part of personalized medicine approaches. This review emphasizes the diverse insights gained from both in vivo and in vitro studies of catecholaminergic polymorphic ventricular tachycardia, along with the application of these models in various research contexts.

Background: The characteristics of hemodynamic coherence in healthy states and disease remain unknown. Capillary tortuosity is a morphologic variant of microcirculatory vessels, but its effects have generally not been considered in the assessment of tissue perfusion and oxygenation. We investigated the role of sublingual capillary tortuosity in the hemodynamic coherence of anesthetized adult individuals with steady-state physiology (ASA 1) and patients with septic shock requiring emergency abdominal surgery (ASA 4E and 5E). Methods: Sublingual macro and microcirculatory variables, oxygen transport, metabolic parameters, and the capillary tortuosity score (CTS) were assessed. Results: Mean (SD) CTS was 0.55 (0.76) and 3.31 (0.86) in the steady-state and septic shock group, respectively (p < 0.001). In patients with septic shock, CTS was significantly associated with alveolar-to-arterial oxygen gradient (r = 0.658, p = 0.015) and oxygen debt (r = -0.769, p = 0.002). Significant differences were also observed in Consensus Proportion of Perfused Vessels (PPV; p < 0.001), Consensus PPV (small) (p < 0.001), Microvascular Flow Index (p < 0.001), vessel diameter (p < 0.001) and length (p < 0.001), wall shear stress (p < 0.001), lactate (p < 0.001), oxygen extraction ratio (p = 0.001), arterial oxygen content (p < 0.001), venous oxygen content (p < 0.001), oxygen delivery (p < 0.001), oxygen consumption (p < 0.001), and oxygen debt (p = 0.002) between the two groups. Conclusions: Sublingual tortuosity was essentially absent in individuals with steady-state physiology. In contrast, it was significantly increased and associated with Alveolar-to-arterial oxygen gradient and oxygen debt in critically ill patients with septic shock.

Mycobacterium tuberculosis (Mtb), a leading cause of death from infection, completes its life cycle entirely in humans except for transmission through the air. To begin to understand how Mtb survives aerosolization, we mimicked liquid and atmospheric conditions experienced by Mtb before and after exhalation using a model aerosol fluid (MAF) based on the water-soluble, lipidic, and cellular constituents of necrotic tuberculosis lesions. MAF induced drug tolerance in Mtb, remodeled its transcriptome, and protected Mtb from dying in microdroplets desiccating in air. Yet survival was not passive: Mtb appeared to rely on hundreds of genes to survive conditions associated with transmission. Essential genes subserving proteostasis offered most protection. A large number of conventionally nonessential genes appeared to contribute as well, including genes encoding proteins that resemble antidesiccants. The candidate transmission survival genome of Mtb may offer opportunities to reduce transmission of tuberculosis.

Most eukaryotes require oxygen for their survival and, with increasing multicellular complexity, oxygen availability and delivery rates vary across the tissues of complex organisms. In humans, healthy tissues have markedly different oxygen gradients, ranging from the hypoxic environment of the bone marrow (where our haematopoietic stem cells reside) to the lungs and their alveoli, which are among the most oxygenated areas of the body. Immune cells are therefore required to adapt to varying oxygen availability as they move from the bone marrow to peripheral organs to mediate their effector functions. These changing oxygen gradients are exaggerated during inflammation, where oxygenation is often depleted owing to alterations in tissue perfusion and increased cellular activity. As such, it is important to consider the effects of oxygenation on shaping the immune response during tissue homeostasis and disease conditions. In this Review, we address the relevance of both physiological oxygenation (physioxia) and disease-associated hypoxia (where cellular oxygen demand outstrips supply) for immune cell functions, discussing the relevance of hypoxia for immune responses in the settings of tissue homeostasis, inflammation, infection, cancer and disease immunotherapy.

Storage of platelets as platelet concentrates for transfusion is limited to 7 days in the United Kingdom due to deleterious effects on platelet quality and function that occur over time. Oxygen (O2) availability and sufficient gaseous exchange are known to be essential in maintaining the viability and function of platelets stored for transfusion. Despite this, there is a paucity of studies undertaking direct measures of O2 and optimization of conditions throughout storage. We address this and modulate the storage conditions to improve platelet quality and function.

Electron paramagnetic resonance oximetry was implemented to directly measure the [O2] experienced by stored platelet concentrates and the O2 consumption rate under standard blood banking conditions. From these direct measures the mathematical modeling was then applied to predict the main parameters contributing to effective O2 distribution throughout the unit.

This study demonstrates reducing the storage [O2] to reflect near physiological levels significantly alters O2 distribution within the unit and negatively impacts platelet functionality and quality, and therefore is not a viable storage option.

We show the reduction of platelet concentration within a unit improves O2 availability and pH, promotes a more uniform distribution of O2 throughout prolonged storage, and maintains platelet agonist-induced aggregation comparable to 100% platelet concentration. This may be a viable option and could potentially lead to reduced donor demand.

To investigate the effects of hypoxia on tooth germ development in mice and explore the underlying mechanisms.

Tooth germs were extracted from E14.5 mouse embryos and divided into the control and hypoxia groups for organ culture. The hypoxia group was exposed to hypoxia (0% oxygen) for 3 h, followed by normoxia for 21 h. After 2 or 7 days, samples were collected for morphometric analysis, reverse transcription-quantitative polymerase chain reaction, immunohistochemistry (IHC), and immunofluorescent staining (IF). Additionally, superoxide dismutase 3 (SOD3) expression patterns in mandibular molar tooth germs from C57BL/6 mouse embryos were analyzed using IHC. The SOD inhibitor sodium N, N-diethyldithiocarbamate trihydrate (DETC; 400 μM) was applied under normoxia for 3 days, followed by morphometry, IHC, and IF.

After 7 days, the hypoxia group exhibited significantly smaller tooth size, fewer cusps, reduced cell proliferation, and increased apoptosis in the epithelium compared to the control group. Sod3 mRNA expression was higher than other Sod family member expressions in the control group. In the hypoxia group, Sod3 mRNA and SOD3 protein expression were significantly decreased, whereas hypoxia-inducible factor-1 expression and reactive oxygen species levels were increased. SOD3 was primarily expressed in the dental epithelium from E12.5 to E17.5. DETC impaired tooth germ development in the control group, resulting in a phenotype similar to that of the hypoxia group, and significantly reduced amelogenin and msh homeobox 2 expression in the epithelium.

Hypoxia impairs tooth germ development. SOD3 probably plays a protective role during this process.

Reactive oxygen species, particularly hydrogen peroxide (H2O2), play crucial roles in cellular signaling, with Nrf2 serving as a key transcription factor in maintaining redox homeostasis. However, the precise influence of H2O2 on Nrf2 activity under physiological normoxia remains unclear due to the limitations of oxygen-sensitive imaging methods. To address this, we developed and validated an oxygen-insensitive Nrf2 reporter named pericellular oxygen-insensitive Nrf2 transcriptional performance reporter (POINTER). We employed this reporter in human cerebral microvascular endothelial cells (hCMEC/D3). Using POINTER, we investigated how varying intracellular H2O2 concentrations affect Nrf2 regulation under normoxia (5 kPa O2) compared to hyperoxia (ambient air, 21 kPa O2). We manipulated intracellular H2O2 levels through exogenous application, chemogenetic production using a modified amino acid oxidase, and pharmacological induction with Auranofin. Our findings reveal that Nrf2 transcriptional activity is significantly lower under normoxia than under hyperoxia, supporting previous literature and expectations. Using POINTER, we found that both antioxidant pathway inhibition and sustained H2O2 elevation are essential for modulating Nrf2 activity. These findings provide new insights into the regulation of Nrf2 by H2O2.

Background: Acute myeloid leukemia (AML) is a heterogeneous disease highly resistant to chemotherapeutic agents. Leukemia stem cells (LSCs) can enter a dormant state and avoid apoptosis in the protective niche of the bone marrow (BM) microenvironment. Moreover, bone marrow stromal cells protect leukemia cells by promoting pro-survival signaling pathways and drug resistance. Therefore, attenuating interactions between leukemia cells and BM cells may have a positive therapeutic effect. Objectives: In this work, we hypothesized that sondages may inhibit the adhesion of leukemia cells to the bone marrow by inhibiting the Hedgehog (Hh) signaling pathway. The Hedgehog pathway is a key therapeutic target in AML due to its role in leukemic cell growth and survival. Methods: We investigated the effects of sonidegib on the adhesion of individual OCI-AML3 cells to a bone marrow stromal spheroid derived from the HS-5 cell line. For this purpose, we precisely determined the minimum cell-to-cell adhesion time using optical tweezers under normoxic (21% of O2) and hypoxic (1% of O2) conditions. Results: Our results demonstrated that sonidegib significantly increased the minimum cell-to-cell adhesion time necessary for leukemic cells to establish adhesive bonds with bone marrow stromal cells, thereby indicating a reduction in their adhesive properties. Additionally, we showed that sonidegib is particularly effective at hypoxic oxygen concentrations. Conclusions: The results obtained in this study suggest that sonidegib, through its modulation of the Hedgehog signaling pathway, holds promise as a potential therapeutic approach to target leukemic cell adhesion within the bone marrow microenvironment.

Adequate oxygen supply is crucial for proper cellular function. The emergence of high-throughput (HT) expansion of human stem-cell-derived cells and HT in vitro cellular assays for drug testing necessitate monitoring and understanding of the oxygenation conditions, yet virtually no data exists for such settings. For metabolically active cells like cardiomyocytes, variations in oxygenation may significantly impact their maturation and function; conversely, electromechanical activity can drive oxygen demands. We used HT label-free optical measurements and computational modeling to gain insights about oxygen availability (peri-cellular oxygen dynamics) in syncytia of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) and human cardiac fibroblasts (cFB) grown in glass-bottom 96-well plates under static conditions. Our experimental results highlight the critical role of cell density and solution height (oxygen delivery path) in peri-cellular oxygen dynamics. The developed 3D reaction-diffusion model with Michaelis-Menten kinetics, trained on the obtained comprehensive data set, revealed that time-variant maximum oxygen consumption rate, Vmax, is needed to faithfully capture the complex peri-cellular oxygen dynamics in the excitable hiPSC-CMs, but not in the cFB. For the latter, accounting for cell proliferation was needed. Interestingly, we found both hypoxic (< 2%) and hyperoxic (> 7%) conditions can easily emerge in these standard HT plates in static culture and that peri-cellular oxygen dynamics evolves with days in culture. Our results and the developed computational model can directly be used to optimize cardiac cell growth in HT plates to achieve desired physiological conditions, important in cellular assays for cardiotoxicity, drug development, personalized medicine and heart regeneration applications.

Ubiquinone (UQ), the only known electron carrier in the mammalian electron transport chain (ETC), preferentially delivers electrons to the terminal electron acceptor oxygen (O2). In hypoxia, ubiquinol (UQH2) diverts these electrons onto fumarate instead. Here, we identify rhodoquinone (RQ), an electron carrier detected in mitochondria purified from certain mouse and human tissues that preferentially delivers electrons to fumarate through the reversal of succinate dehydrogenase, independent of environmental O2 levels. The RQ/fumarate ETC is strictly present in vivo and is undetectable in cultured mammalian cells. Using genetic and pharmacologic tools that reprogram the ETC from the UQ/O2 to the RQ/fumarate pathway, we establish that these distinct ETCs support unique programs of mitochondrial function and that RQ confers protection upon hypoxia exposure in vitro and in vivo. Thus, in discovering the presence of RQ in mammals, we unveil a tractable therapeutic strategy that exploits flexibility in the ETC to ameliorate hypoxia-related conditions.

Dendritic cells (DCs) are specialized antigen-presenting cells crucial for initiating and regulating adaptive immune responses, making them promising candidates for therapeutic interventions in various immune-mediated diseases. Increasing evidence suggests that the microenvironment in which cells are cultured, as well as the milieu in which they perform their functions, significantly impact their immunomodulatory properties. Among these environmental factors, the role of oxygen in DC biology and its significance for both their in vitro generation and in vivo therapeutic application require investigation. Unlike the atmospheric oxygen level of 21 % commonly used in in vitro assays, physiological oxygen levels are much lower (3-9 %), and hypoxia (<1.3 %) is a prevalent condition of both healthy tissues and disease states. This mismatch between laboratory and physiological conditions underscores the critical need to culture and evaluate therapeutic cells under physiologically relevant oxygen levels to improve their translational relevance and clinical outcomes. This review explores the characteristic hallmarks of human DCs that are influenced by oxygen-dependent pathways, including metabolism, phenotype, cytokine secretion, and migration. Furthermore, we discuss the potential of manipulating oxygen levels to refine the generation and functionality of DCs for therapeutic purposes.

The lack of oxygen (O2) causes changes in the cell functioning. Modeling hypoxic conditions in vitro is challenging given that different cell types exhibit different sensitivities to tissue O2 levels. We present an effective in vivo platform for assessing various tissue and organ parameters in Danio rerio larvae under acute hypoxic conditions. Our system allows simultaneous positioning of multiple individuals within a chamber where O2 level in the water can be precisely and promptly regulated, all while conducting microscopy. We applied this approach in combination with a genetically encoded pH-biosensor SypHer3s and a highly H2O2-sensitive HyPer7 biosensor. Hypoxia causes H2O2 production in areas of brain, heart, and skeletal muscles, exclusively in the mitochondrial matrix; it is noteworthy that H2O2 does not penetrate into the cytosol and is neutralized in the matrix upon reoxygenation. Hypoxia causes pronounced tissue acidosis, expressed by a decrease in pH by 0.4-0.6 units everywhere. Using imaging photoplethysmography, we measured in D. rerio larvae real-time heart rate decrease under conditions of hypoxia and subsequent reoxygenation. Our observations in this experimental system lead to the hypothesis that mitochondria are the only source of H2O2 in cells of D. rerio under hypoxia. Antioxid. Redox Signal. 42, 292-300.

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.

Cell models play a central role in preclinical research aimed at the mechanism of disease and drug discovery. The outside environment of the cells, including levels of nutrients and oxygen tension, regulates cellular stress response pathways. Routinely used in vitro disease models often overlook cell growth conditions. This study aimed to evaluate the effect of substituting classic cell media (DMEM) with media matching the nutrient composition of human plasma (Plasmax) on cell viability, the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), glutathione (GSH), and malondialdehyde (MDA) levels by different pharmacological inducers of cell stress. The cells were grown at ambient (~19%) and reduced (5%) oxygen levels. The activation of Nrf2 by ferroptosis activators (erastin and RSL3) was dependent on cell media and oxygen tension. The induction of Nrf2 by an inducer of endoplasmic reticulum stress, thapsigargin, was observable only in cells grown in DMEM and at low oxygen tension. GSH and MDA levels were elevated in Plasmax media. Results indicate that stress tolerance and the activation of Nrf2 in the HepG2ARE cell line depend on the growth conditions, including cell media and oxygen. Cell culture conditions should be critically considered when designing in vitro models of diseases involving oxidative stress.

Oxygen is essential for human life, yet a growing body of preclinical research is demonstrating that chronic continuous hypoxia can be beneficial in models of mitochondrial disease, autoimmunity, ischemia, and aging. This research is revealing exciting new and unexpected facets of oxygen biology, but translating these findings to patients poses major challenges, because hypoxia can be dangerous. Overcoming these barriers will require integrating insights from basic science, high-altitude physiology, clinical medicine, and sports technology. Here, we explore the foundations of this nascent field and outline a path to determine how chronic continuous hypoxia can be safely, effectively, and practically delivered to patients.

In vitro and ex vivo studies are crucial for mitochondrial research, offering valuable insights into cellular mechanisms and aiding in diagnostic and therapeutic strategies. Accurate in vitro models rely on adequate cell culture conditions, such as the composition of culture media and oxygenation levels. These conditions can influence energy metabolism and mitochondrial activities, thus impacting studies involving mitochondrial components, such as the effectiveness of anticancer drugs. This chapter focuses on practical guidance for creating setups that replicate in vivo microenvironments, capturing the original metabolic context of cells. We explore protocols to better mimic the physiological cell environment, promote cellular reconfiguration, and prime cells according to the modeled context. The first part is dedicated to the use of human dermal fibroblasts, which are a promising model for pre-clinical mitochondrial research due to their adaptability and relevance to human mitochondrial physiology. We present an optimized protocol for gradually adjusting extracellular glucose levels, which demonstrated significant mitochondrial, metabolic, and redox remodeling in normal adult dermal fibroblasts. The second part is dedicated to replication of tumor microenvironments, which are relevant for studies targeting cellular energy metabolism to inhibit tumor growth. Currently available physiological media can mimic blood plasma metabolome but not the specific tumor microenvironment. To address this, we describe optimized media formulation and oxygenation protocols, which can simulate the tumor microenvironment in cell culture experiments. Replicating in vivo microenvironments in in vitro and ex vivo studies can enhance our understanding of cellular processes, facilitate drug development, and advance personalized therapeutics in mitochondrial medicine.

Oxygen (O2)-controlled cell culture has been pivotal in studying mammalian mechanisms of O2 sensing, regulation, and utilization. We posit, however, that O2-controlled cell culture is paradoxically not controlling O2. There is overwhelming evidence that the pericellular O2 is lower than the surrounding gas phase due to cellular O2 consumption. Standard hypoxic cell culture is at high risk of inducing pericellular anoxia. We discuss the implications of poor O2 control for cellular O2 regulation mechanisms, bioenergetics, and redox signaling. We also highlight the evidence of frequent under-oxygenation in standard (i.e., normoxic) cell culture. This issue has been largely overlooked because strategies to control pericellular O2 have been lacking. Here, we propose a framework to control pericellular O2 based on our recent investigation into the nature of the gas/pericellular O2 gradient. Implementing this framework into standard practice will unlock quantitative O2 control in vitro, improving our ability to understand the role of O2 in biology.

Osteoporosis, a prevalent metabolic bone disorder, is characterized by reduced bone density and increased fracture risk. The pathogenesis of osteoporosis is closely associated with an imbalance in bone remodeling, in which the resorption function of osteoclasts exceeds the formation function of osteoblasts. Hypoxia has been implicated in the promotion of osteoclast differentiation and the subsequent development of osteoporosis. The ubiquitin-proteasome system (UPS) and its regulatory enzymes, deubiquitinating enzymes (DUBs), play a significant role in bone homeostasis. In this study, we investigated the contribution and mechanism of Ubiquitin-specific protease 18 (USP18), a DUB, in osteoclast differentiation under hypoxic conditions. BMDMs and RAW264.7 cells were treated with RANKL to induce osteoclastogenesis and were subjected to overexpression or knockdown of USP18 under normoxic or hypoxia conditions. Osteoclast formation was assessed using TRAP staining, and the expression of osteoclast marker genes was determined using qRT-PCR. The activation of the NF-κB signaling pathway was evaluated using immunoblotting. We found that hypoxia significantly enhanced the differentiation of BMDMs and RAW264.7 cells into osteoclasts, accompanied by a notable downregulation of USP18 expression. The overexpression of USP18 inhibited RANKL-induced osteoclast differentiation, while the knockdown of USP18 promoted that process, unveiling the inhibitory effect of USP18 in osteoclastogenesis. Furthermore, the overexpression of USP18 rescued the hypoxia-induced increase in osteoclast differentiation. Mechanistic insights revealed that USP18 inhibits osteoclastogenesis by suppressing the NF-κB signaling pathway, with a potential target on TAK1 or its upstream molecules. This study indicates that hypoxia promotes osteoclast differentiation through the downregulation of USP18, which, in turn, relieves the suppression of the activation of the NF-κB signaling pathway. The USP18 emerges as a potential therapeutic target for osteoporosis treatment, highlighting the importance of the hypoxia-DUB axis in the pathogenesis of the disease.

Bone fractures are associated with hypoxia, but no longitudinal studies of perfusion measurements in human patients have been reported despite the clinical and research potential. In this longitudinal observational cohort study, the near-infrared spectroscopy (NIRS) device PortaMon was used to assess oxy-(O2Hb), deoxy-(HHb) and total (tHb) haemoglobin, as well as the differences between O2Hb and HHb (HbDiff) and the tissue saturation index (TSI) at three different depths in the fracture gap. Linear mixed effect models were fitted to analyse time effects. One-way ANOVAs were conducted to compare groups. The time points corresponding to minima were calculated via linear regression. In this study, 11 patients with tibial shaft fractures underwent longitudinal measurements. Additionally, 9 patients with diagnosed tibial shaft nonunion and 23 age-matched controls were measured once. In the longitudinal group, all fractures healed, and decreases in O2Hb and HbDiff (all p < 0.05) were observed, with minima occurring 19-21 days after fracture. O2Hb values in nonunion patients did not differ from the minima in longitudinally measured union patients, whereas differences in HHb and tHb were significant (all p < 0.05). Previously, the onset of hypoxia has been assumed to be much faster. The characteristic trajectories of the NIRS parameters O2Hb and HbDiff can be used to fulfil the need for a non-invasive method to monitor fracture healing. These results suggest that NIRS could supplement radiographs and clinical impressions in daily clinical practice and may enable earlier diagnosis of nonunion.

Hyperoxic exposure lasting days alters mitochondrial bioenergetic and dynamic functions in pulmonary cells as indices of oxygen toxicity. The aim of this study was to examine effects of short duration hyperbaric and hyperoxic exposures to induce oxygen toxicity similarly. Cultured human lung microvascular endothelial cells, human pulmonary artery endothelial cells and A549 cells were exposed to hyperoxia (∼5 % CO2 equivalent, balance O2) under hyperbaric conditions (4.8 ATA) for 1 and 4 h. Measures of mitochondrial dynamics, inner membrane potential, mitochondrial respiration, the intracellular distribution of bioenergetic capacity and respiration complex protein levels were then quantified. Exposures resulted in altered mitochondrial motility, presence of inhomogeneities in respiration parameters, loss of inner membrane potential, and changes in intracellular partitioning of ATP-linked respiration. Changes in the levels of respiration complex protein levels were also found. The combination of hyperoxic exposure with hyperbaric conditions rapidly produced changes in mitochondrial dynamics and bioenergetics in pulmonary cells. These changes are consistent with the onset of pulmonary oxygen toxicity previously known to result from long duration exposure to hyperoxia alone. These findings suggest health caution is warranted in environmental settings in which both hyperoxic and hyperbaric conditions are present. The synergism of hyperoxia and hyperbaria for rapid induction of oxygen toxicity in cellular models has utility for the study of mechanistic determinants of oxygen toxicity, testing of putative therapeutics, and associated investigations of mitochondrial dysfunction.

Oxygen is essential for normal cellular functions. Hypoxia impacts various cellular processes, such as metabolism, growth, proliferation, angiogenesis, metastasis, tumorigenesis, microbial infection, and immune response, mediated by hypoxia-inducible factors (HIFs). Hypoxia contributes to the progression and development of cancer, cardiovascular diseases, metabolic disorders, kidney diseases, and infections. The potential alleviation of hypoxia has been explored through the enzymatic in situ decomposition of hydrogen peroxide, leading to the generation of oxygen. However, challenges such as limited stability restrict the effectiveness of enzymes such as catalase in biomedical and in vivo applications. To overcome these limitations, targeted delivery of the enzymes has been proposed. This review offers a critical comparison of i) current approaches to enhance the in vivo stability of catalase; and ii) the structure, mechanism of action, and kinetics of catalase and catalase-like nanozymes.

A novel radiotherapy (RT) approach termed FLASH-RT, which irradiates areas at ultra-high dose rates, is of current interest to medical researchers. FLASH-RT can maintain equivalent antitumor effects while sparing healthy tissue compared with conventional RT (CONV-RT), which uses low dose rates. The sparing effect on healthy tissue after FLASH-RT is known as the FLASH effect. Owing to the FLASH effect, FLASH-RT can raise the maximum tolerable dose to control tumor growth or eradicate the tumor and provide a new strategy for clinical RT. However, definitive irradiation conditions for reproducing the FLASH effect and the biological mechanism of the FLASH effect have not yet been fully elucidated. The efficacy of FLASH-RT is controversial despite its successful application in clinical RT. The present review recapitulates the progression of FLASH-RT and critically comments on the hypothesis of the FLASH effect. In addition, the review expounds on the current issues with regard to the differential phenomena between in vitro and in vivo studies, and elaborates on the challenges for the application of FLASH-RT that need to be addressed in the future.

Cell culture is a fundamental experimental tool for understanding cell physiology. However, translating these findings to in vivo settings has proven challenging. Replicating donor tissue conditions, including oxygen levels, is crucial for achieving meaningful results. Nevertheless, oxygen culture conditions are often overlooked, particularly in the context of chemotherapy-induced neurotoxicity.

In this study, we investigated the role of oxygen levels in primary neuronal cultures by comparing neuronal performance under cisplatin exposure (1 μg/mL) in supraphysiological normoxia (representing atmospheric conditions in a standard incubator; 18.5% O2) and physioxia (representing physiologic oxygen conditions in nervous tissue; 5% O2). Experiments were also conducted to assess survival, neurite development, senescence marker expression, and proinflammatory cytokine secretion.

Under control conditions, both oxygen concentration conditions exhibited similar behaviors. However, after cisplatin administration, sensory neurons cultured under supraphysiological normoxic conditions show higher mortality, exhibit an evolutionarily proinflammatory cytokine profile over time, and activate apoptotic-regulated neuron death markers. In contrast, under physiological conditions, neurons treated with cisplatin exhibited senescence marker expression and an attenuated inflammatory secretome.

These results underscore the critical role of oxygen in neuronal culture, particularly in studying compounds where neuronal damage is mechanistically linked to oxidative stress. Even at identical doses of evaluated neurotoxic drugs, distinct cellular phenotypic fates can emerge, impacting translatability to the in vivo setting.

Cultured brain cells are used conventionally to investigate fundamental neurobiology and identify therapeutic targets against neural diseases. However, standard culture conditions do not simulate the natural cell microenvironment, thus hampering in vivo translational insight. Major weaknesses include atmospheric (21%) O2 tension and lack of intercellular communication, the two factors likely impacting metabolism and signaling. Here, we addressed this issue in mouse neurons and astrocytes in primary culture. We found that the signs of cellular and mitochondrial integrity were optimal when these cells were acclimated to grow in coculture, to emulate intercellular coupling, under physiologic (5%) O2 tension. Transcriptomic scrutiny, performed to elucidate the adaptive mechanism involved, revealed that the vast majority of differentially expressed transcripts were downregulated in both astrocytes and neurons. Gene ontology evaluation unveiled that the largest group of altered transcripts was glycolysis, which was experimentally validated by metabolic flux analyses. This protocol and database resource for neural cells grown under in vivo-like microenvironment may move forward the translation of basic into applied neurobiological research.

Epidermis is one of the most rapidly proliferating tissues in the body with high demands for adenosine triphosphate and cellular building blocks. In this study, we show that to meet these requirements, keratinocytes constitutively express HIF-1α, even in the presence of oxygen levels sufficient for HIF-1α hydroxylation. We previously reported that mice with severe epidermal mitochondrial dysfunction actually showed a hyperproliferative epidermis but rapidly died of systemic lactic acidosis and hypoglycemia, indicating excessive glycolysis. In this work, we interrogated HIF-1α function in glycolysis by its epidermal ablation combined with mitochondrial dysfunction, which resulted in decreased proliferation but even earlier lethality due to a severe barrier defect. Our data demonstrate that HIF-1α is indispensable for maintaining a high aerobic glycolytic flux necessary for supplying energy but also for synthetizing cellular building blocks such as lipids, which are both essential for proliferation as well as barrier formation. HIF-1α is stabilized in keratinocytes in the presence of oxygen by high levels of HIF-1α transcripts, low levels of prolyl-4-hydroxylases (PHD2 and PHD3), and a low cellular a-ketoglutarate/lactate ratio, likely inhibiting prolyl-4-hydroxylase activity. Our data suggest a key role for constitutive HIF-1α expression allowing a Warburg-like metabolism in healthy, highly proliferative keratinocytes, similar to that in tumor cells.

Despite the overwhelming number of sports supplements on the market, only seven are currently recognized as effective. Biological functions are largely regulated through redox reactions, yet no comprehensive analysis of the redox properties of these supplements has been compiled. Here, we analyze the redox characteristics of these seven supplements: bicarbonates, beta-alanine, caffeine, creatine, nitrates, carbohydrates, and proteins. Our findings suggest that all sports supplements exhibit some degree of redox activity. However, the precise physiological implications of these redox properties remain unclear. Future research, employing unconventional perspectives and methodologies, will reveal new redox pixels of the exercise physiology and sports nutrition picture.

Hypoxia (insufficient O2) is a pivotal factor in cancer progression, triggering genetic, transcriptional, translational and epigenetic adaptations associated to therapy resistance, metastasis and patient mortality. In this review, we outline the microenvironmental origins and molecular mechanisms responsible for hypoxic cancer cell adaptations in situ and in vitro, whilst outlining current approaches to stratify, quantify and therapeutically target hypoxia in the context of precision oncology.

Variations in oxygen level affect the phenotype of cells and extracellular vesicles (EVs). Depending on the metabolic oxygen demand of cells, hypoxic cell culture can produce conditions more like those found in vivo, and with appropriate oxygen levels, mimic hypoxic tumours. However, most previous experiments studying both EVs and the effects of hypoxia on cells use periods of 72 h or less of hypoxia. We hypothesised that this was insufficient time for adaptation to hypoxic conditions both for EVs and cells which may skew the results of such studies. In this study, the effects of acute (72 h) and chronic hypoxia (> 2 weeks) on the phenotype of HepG2 and PC3 cells and their EVs were examined. Cells could be cultured normally under chronic hypoxic conditions and cryopreserved and recovered. The effects of hypoxia on EV phenotype are slow to establish and dependent on cell line. In PC3 cells, the greatest change in phenotype and increase in EV production occurred only with chronic hypoxic culture. In HepG2 cells, the number of EVs produced was insensitive to hypoxic culture and the greatest changes in protein expression were observed after acute hypoxic culture. Nonetheless, biphasic changes in EV phenotype were detected in both cell types in response to either acute or chronic hypoxia. These results indicate that for cells which do not induce consumptive oxygen depletion, prolonged hypoxic culture is required for complete adaptation.

Clostridioides difficile, the major cause of antibiotic-associated diarrhea, is a strict anaerobic, sporulating Firmicutes. However, during its infectious cycle, this anaerobe is exposed to low oxygen (O2) tensions, with a longitudinal decreasing gradient along the gastrointestinal tract and a second lateral gradient with higher O2 tensions in the vicinity of the cells. A plethora of enzymes involved in oxidative stress detoxication has been identified in C. difficile, including four O2-reducing enzymes: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). Here, we investigated the role of the four O2-reducing enzymes in the tolerance to increasing physiological O2 tensions and air. The four enzymes have different, yet overlapping, spectra of activity. revRbr2 is specific to low O2 tensions (<0.4%), FdpA to low and intermediate O2 tensions (0.4%-1%), revRbr1 has a wider spectrum of activity (0.1%-4%), and finally FdpF is more specific to tensions > 4% and air. These different O2 ranges of action partly arise from differences in regulation of expression of the genes encoding those enzymes. Indeed, we showed that revrbr2 is under the dual control of σA and σB. We also identified a regulator of the Spx family that plays a role in the induction of fdp and revrbr genes upon O2 exposure. Finally, fdpF is regulated by Rex, a regulator sensing the NADH/NAD+ ratio. Our results demonstrate that the multiplicity of O2-reducing enzymes of C. difficile is associated with different roles depending on the environmental conditions, stemming from a complex multi-leveled network of regulation.

The gastrointestinal tract is a hypoxic environment, with the existence of two gradients of O2 along the gut, one longitudinal anteroposterior decreasing gradient and one proximodistal increasing from the lumen to the epithelial cells. O2 is a major source of stress for an obligate anaerobe such as the enteropathogen C. difficile. This bacterium possesses a plethora of enzymes capable of scavenging O2 and reducing it to H2O. In this work, we identified the role of the four O2-reducing enzymes in the tolerance to the physiological O2 tensions faced by C. difficile during its infectious cycle. These four enzymes have different spectra of action and protect the vegetative cells over a large range of O2 tensions. These differences are associated with a distinct regulation of each gene encoding those enzymes. The complex network of regulation is crucial for C. difficile to adapt to the various O2 tensions encountered during infection.