Carbon monoxide as a cellular protective agent in a swine model of cardiac arrest protocol.

Out-of-hospital cardiac arrest (OHCA) affects over 360,000 adults in the United States each year with a 50-80% mortality prior to reaching medical care. Despite aggressive supportive care and targeted temperature management (TTM), half of adults do not live to hospital discharge and nearly one-third of survivors have significant neurologic injury. The current treatment approach following cardiac arrest resuscitation consists primarily of supportive care and possible TTM. While these current treatments are commonly used, mortality remains high, and survivors often develop lasting neurologic and cardiac sequela well after resuscitation. Hence, there is a critical need for further therapeutic development of adjunctive therapies. While select therapeutics have been experimentally investigated, one promising agent that has shown benefit is CO. While CO has traditionally been thought of as a cellular poison, there is both experimental and clinical evidence that demonstrate benefit and safety in ischemia with lower doses related to improved cardiac/neurologic outcomes. While CO is well known for its poisonous effects, CO is a generated physiologically in cells through the breakdown of heme oxygenase (HO) enzymes and has potent antioxidant and anti-inflammatory activities. While CO has been studied in myocardial infarction itself, the role of CO in cardiac arrest and post-arrest care as a therapeutic is less defined. Currently, the standard of care for post-arrest patients consists primarily of supportive care and TTM. Despite current standard of care, the neurological prognosis following cardiac arrest and return of spontaneous circulation (ROSC) remains poor with patients often left with severe disability due to brain injury primarily affecting the cortex and hippocampus. Thus, investigations of novel therapies to mitigate post-arrest injury are clearly warranted. The primary objective of this proposed study is to combine our expertise in swine models of CO and cardiac arrest for future investigations on the cellular protective effects of low dose CO. We will combine our innovative multi-modal diagnostic platform to assess cerebral metabolism and changes in mitochondrial function in swine that undergo cardiac arrest with therapeutic application of CO.

The effects of synthesis gas feedstocks and oxygen perturbation on hydrogen production by Parageobacillus thermoglucosidasius.

BACKGROUND: The facultatively anaerobic thermophile Parageobacillus thermoglucosidasius is able to produce hydrogen gas (H2) through the water-gas shift (WGS) reaction. To date this process has been evaluated under controlled conditions, with gas feedstocks comprising carbon monoxide and variable proportions of air, nitrogen and hydrogen. Ultimately, an economically viable hydrogenogenic system would make use of industrial waste/synthesis gases that contain high levels of carbon monoxide, but which may also contain contaminants such as H2, oxygen (O2) and other impurities, which may be toxic to P. thermoglucosidasius. RESULTS: We evaluated the effects of synthesis gas (syngas) mimetic feedstocks on WGS reaction-driven H2 gas production by P. thermoglucosidasius DSM 6285 in small-scale fermentations. Improved H2 gas production yields and faster onset towards hydrogen production were observed when anaerobic synthetic syngas feedstocks were used, at the expense of biomass accumulation. Furthermore, as the WGS reaction is an anoxygenic process, we evaluated the influence of O2 perturbation on P. thermoglucosidasius hydrogenogenesis. O2 supplementation improved biomass accumulation, but reduced hydrogen yields in accordance with the level of oxygen supplied. However, H2 gas production was observed at low O2 levels. Supplementation also induced rapid acetate consumption, likely to sustain growth. CONCLUSION: The utilisation of anaerobic syngas mimetic gas feedstocks to produce H2 and the relative flexibility of the P. thermoglucosidasius WGS reaction system following O2 perturbation further supports its applicability towards more robust and continuous hydrogenogenic operation.

Beneficial Effects of Oral Carbon Monoxide on Doxorubicin-Induced Cardiotoxicity.

BACKGROUND: Doxorubicin and other anthracyclines are crucial cancer treatment drugs. However, they are associated with significant cardiotoxicity, severely affecting patient care and limiting dosage and usage. Previous studies have shown that low carbon monoxide (CO) concentrations protect against doxorubicin toxicity. However, traditional methods of CO delivery pose complex challenges for daily administration, such as dosing and toxicity. To address these challenges, we developed a novel oral liquid drug product containing CO (HBI-002) that can be easily self-administered by patients with cancer undergoing doxorubicin treatment, resulting in CO being delivered through the upper gastrointestinal tract. METHODS AND RESULTS: HBI-002 was tested in a murine model of doxorubicin cardiotoxicity in the presence and absence of lung or breast cancer. The mice received HBI-002 twice daily before doxorubicin administration and experienced increased carboxyhemoglobin levels from a baseline of ≈1% to 7%. Heart tissue from mice treated with HBI-002 had a 6.3-fold increase in CO concentrations and higher expression of the cytoprotective enzyme heme oxygenase-1 compared with placebo control. In both acute and chronic doxorubicin toxicity scenarios, HBI-002 protected the heart from cardiotoxic effects, including limiting tissue damage and cardiac dysfunction and improving survival. In addition, HBI-002 did not compromise the efficacy of doxorubicin in reducing tumor volume, but rather enhanced the sensitivity of breast 4T1 cancer cells to doxorubicin while simultaneously protecting cardiac function. CONCLUSIONS: These findings strongly support using HBI-002 as a cardioprotective agent that maintains the therapeutic benefits of doxorubicin cancer treatment while mitigating cardiac damage.

Physiological basis for atmospheric methane oxidation and methanotrophic growth on air.

Atmospheric methane oxidizing bacteria (atmMOB) constitute the sole biological sink for atmospheric methane. Still, the physiological basis allowing atmMOB to grow on air is not well understood. Here we assess the ability and strategies of seven methanotrophic species to grow with air as sole energy, carbon, and nitrogen source. Four species, including three outside the canonical atmMOB group USCα, enduringly oxidized atmospheric methane, carbon monoxide, and hydrogen during 12 months of growth on air. These four species exhibited distinct substrate preferences implying the existence of multiple metabolic strategies to grow on air. The estimated energy yields of the atmMOB were substantially lower than previously assumed necessary for cellular maintenance in atmMOB and other aerobic microorganisms. Moreover, the atmMOB also covered their nitrogen requirements from air. During growth on air, the atmMOB decreased investments in biosynthesis while increasing investments in trace gas oxidation. Furthermore, we confirm that a high apparent specific affinity for methane is a key characteristic of atmMOB. Our work shows that atmMOB grow on the trace concentrations of methane, carbon monoxide, and hydrogen present in air and outlines the metabolic strategies that enable atmMOB to mitigate greenhouse gases.

Adaptive laboratory evolution of Clostridium autoethanogenum to metabolize CO2 and H2 enhances growth rates in chemostat and unravels proteome and metabolome alterations.

Gas fermentation of CO2 and H2 is an attractive means to sustainably produce fuels and chemicals. Clostridium autoethanogenum is a model organism for industrial CO to ethanol and presents an opportunity for CO2-to-ethanol processes. As we have previously characterized its CO2/H2 chemostat growth, here we use adaptive laboratory evolution (ALE) with the aim of improving growth with CO2/H2. Seven ALE lineages were generated, all with improved specific growth rates. ALE conducted in the presence of 2% CO along with CO2/H2 generated Evolved lineage D, which showed the highest ethanol titres amongst all the ALE lineages during the fermentation of CO2/H2. Chemostat comparison against the parental strain shows no change in acetate or ethanol production, while Evolved D could achieve a higher maximum dilution rate. Multi-omics analyses at steady state revealed that Evolved D has widespread proteome and intracellular metabolome changes. However, the uptake and production rates and titres remain unaltered until investigating their maximum dilution rate. Yet, we provide numerous insights into CO2/H2 metabolism via these multi-omics data and link these results to mutations, suggesting novel targets for metabolic engineering in this bacterium.

Isolation and identification of carbon monoxide producing microorganisms from compost.

Carbon monoxide (CO) formation has been observed during composting of various fractions of organic waste. It was reported that this production can be biotic, associated with the activity of microorganisms. However, there are no sources considering the microbial communities producing CO production in compost. This preliminary research aimed to isolate and identify microorganisms potentially responsible for the CO production in compost collected from two areas of the biowaste pile: with low (118 ppm) and high CO concentration (785 ppm). Study proved that all isolates were bacterial strains with the majority of rod-shaped Gram-positive bacteria. Both places can be inhabited by the same bacterial strains, e.g. Bacillus licheniformis and Paenibacillus lactis. The most common were Bacillus (B. licheniformis, B. haynesii, B. paralicheniformis, and B. thermolactis). After incubation of isolates in sealed bioreactors for 4 days, the highest CO levels in the headspace were recorded for B. paralicheniformis (>1000 ppm), B. licheniformis (>800 ppm), and G. thermodenitrificans (∼600 ppm). High CO concentrations were accompanied by low O2 (<6%) and high CO2 levels (>8%). It is recommended to analyze the expression of the gene encoding CODH to confirm or exclude the ability of the identified strains to convert CO2 to CO.

Prevalence of trace gas-oxidizing soil bacteria increases with radial distance from Polloquere hot spring within a high-elevation Andean cold desert.

High-elevation arid regions harbor microbial communities reliant on metabolic niches and flexibility to survive under biologically stressful conditions, including nutrient limitation that necessitates the utilization of atmospheric trace gases as electron donors. Geothermal springs present "oases" of microbial activity, diversity, and abundance by delivering water and substrates, including reduced gases. However, it is unknown whether these springs exhibit a gradient of effects, increasing their impact on trace gas-oxidizers in the surrounding soils. We assessed whether proximity to Polloquere, a high-altitude geothermal spring in an Andean salt flat, alters the diversity and metabolic structure of nearby soil bacterial populations compared to the surrounding cold desert. Recovered DNA and metagenomic analyses indicate that the spring represents an oasis for microbes in this challenging environment, supporting greater biomass with more diverse metabolic functions in proximal soils that declines sharply with radial distance from the spring. Despite the sharp decrease in biomass, potential rates of atmospheric hydrogen (H2) and carbon monoxide (CO) uptake increase away from the spring. Kinetic estimates suggest this activity is due to high-affinity trace gas consumption, likely as a survival strategy for energy/carbon acquisition. These results demonstrate that Polloquere regulates a gradient of diverse microbial communities and metabolisms, culminating in increased activity of trace gas-oxidizers as the influence of the spring yields to that of the regional salt flat environment. This suggests the spring holds local importance within the context of the broader salt flat and potentially represents a model ecosystem for other geothermal systems in high-altitude desert environments.

An orally active carbon monoxide-releasing molecule enhances beneficial gut microbial species to combat obesity in mice.

Carbon monoxide (CO), a gaseous signaling molecule, has shown promise in preventing body weight gain and metabolic dysfunction induced by high fat diet (HFD), but the mechanisms underlying these effects are largely unknown. An essential component in response to HFD is the gut microbiome, which is significantly altered during obesity and represents a target for developing new therapeutic interventions to fight metabolic diseases. Here, we show that CO delivered to the gut by oral administration with a CO-releasing molecule (CORM-401) accumulates in faeces and enriches a variety of microbial species that were perturbed by a HFD regimen. Notably, Akkermansia muciniphila, which exerts salutary metabolic effects in mice and humans, was strongly depleted by HFD but was the most abundant gut species detected after CORM-401 treatment. Analysis of bacterial transcripts revealed a restoration of microbial functional activity, with partial or full recovery of the Krebs cycle, ß-oxidation, respiratory chain and glycolysis. Mice treated with CORM-401 exhibited normalization of several plasma and fecal metabolites that were disrupted by HFD and are dependent on Akkermansia muciniphila's metabolic activity, including indoles and tryptophan derivatives. Finally, CORM-401 treatment led to an improvement in gut morphology as well as reduction of inflammatory markers in colon and cecum and restoration of metabolic profiles in these tissues. Our findings provide therapeutic insights on the efficacy of CO as a potential prebiotic to combat obesity, identifying the gut microbiota as a crucial target for CO-mediated pharmacological activities against metabolic disorders.

Heme oxygenase activates calcium release from the endoplasmic reticulum of bovine mammary epithelial cells to promote TFEB entry into the nucleus to reduce the intracellular load of Mycoplasma bovis.

Heme oxygenase HO-1 (HMOX) regulates cellular inflammation and apoptosis, but its role in regulation of autophagy in Mycoplasma bovis infection is unknown. The objective was to determine how the HO-1/CO- Protein kinase RNA-like endoplasmic reticulum kinase (PERK)-Ca2+- transcription factor EB (TFEB) signaling axis induces autophagy and regulates clearance of M. bovis by bovine mammary epithelial cells (bMECs). M. bovis inhibited autophagy and lysosomal biogenesis in bMECs and suppressed HO-1 protein and expression of related proteins, namely nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (keap1). Activation of HO-1 and its production of carbon monoxide (CO) were required for induction of autophagy and clearance of intracellular M. bovis. Furthermore, when HO-1 was deficient, CO sustained cellular autophagy. HO-1 activation increased intracellular calcium (Ca2+) and cytosolic localization activity of TFEB via PERK. Knockdown of PERK or chelation of intracellular Ca2+ inhibited HO-1-induced M. bovis autophagy and clearance. M. bovis infection affected nuclear localization of lysosomal TFEB in the MiT/TFE transcription factor subfamily, whereas activation of HO-1 mediated dephosphorylation and intranuclear localization of TFEB, promoting autophagy, lysosomal biogenesis and autophagic clearance of M. bovis. Nuclear translocation of TFEB in HO-1 was critical to induce M. bovis transport and survival of infected bMECs. Furthermore, the HO-1/CO-PERK-Ca2+-TFEB signaling axis induced autophagy and M. bovis clearance, providing a viable approach to treat persistent M. bovis infections.

Pulmonary diffusing capacity for carbon monoxide and nitric oxide after COVID-19: A prospective cohort study (the SECURe study).

Many patients exhibit persistently reduced pulmonary diffusing capacity after coronavirus disease 2019 (COVID-19). In this study, dual test gas diffusing capacity for carbon monoxide and nitric oxide (DL,CO,NO) metrics and their relationship to disease severity and physical performance were examined in patients who previously had COVID-19. An initial cohort of 148 patients diagnosed with COVID-19 of all severities between March 2020 and March 2021 had a DL,CO,NO measurement performed using the single-breath method at 5.7 months follow-up. All patients with at least one abnormal DL,CO,NO metric (n = 87) were revaluated at 12.5 months follow-up. The DL,CO,NO was used to provide the pulmonary diffusing capacity for nitric oxide (DL,NO), the pulmonary diffusing capacity for carbon monoxide (DL,CO,5s), the alveolar-capillary membrane diffusing capacity and the pulmonary capillary blood volume. At both 5.7 and 12.5 months, physical performance was assessed using a 30 s sit-to-stand test and the 6 min walk test. Approximately 60% of patients exhibited a severity-dependent decline in at least one DL,CO,NO metric at 5.7 months follow-up. At 12.5 months, both DL,NO and DL,CO,5s had returned towards normal but still remained abnormal in two-thirds of the patients. Concurrently, improvements in physical performance were observed, but with no apparent relationship to any DL,CO,NO metric. The severity-dependent decline in DL,NO and DL,CO observed at 5.7 months after COVID-19 appears to be reduced consistently at 12.5 months follow-up in the majority of patients, despite marked improvements in physical performance.

Exploring Rhodospirillum rubrum response to high doses of carbon monoxide under light and dark conditions.

Environmental concerns about residues and the traditional disposal methods are driving the search for more environmentally conscious processes, such as pyrolysis and gasification. Their main final product is synthesis gas (syngas) composed of CO, CO2, H2, and methane. Syngas can be converted into various products using CO-tolerant microorganisms. Among them, Rhodospirillum rubrum is highlighted for its biotechnological potential. However, the extent to which high doses of CO affect its physiology is still opaque. For this reason, we have studied R. rubrum behavior under high levels of this gas (up to 2.5 bar), revealing a profound dependence on the presence or absence of light. In darkness, the key variable affected was the lag phase, where the highest levels of CO retarded growth to more than 20 days. Under light, R. rubrum ability to convert CO into CO2 and H2 depended on the presence of an additional carbon source, such as acetate. In those conditions where CO was completely exhausted, CO2 fixation was unblocked, leading to a diauxic growth. To enhance R. rubrum tolerance to CO in darkness, a UV-accelerated adaptive laboratory evolution (UVa-ALE) trial was conducted to isolate clones with shorter lag phases, resulting in the isolation of clones 1.4-2B and 1.7-2A. The adaptation of 1.4-2B was mainly based on mutated enzymes with a metabolic function, while 1.7-3A was mostly affected at regulatory genes, including the anti-repressor PpaA/AerR. Despite these mutations having slight effects on biomass and pigment levels, they successfully provoked a significant reduction in the lag phase (-50%). KEYPOINTS: • CO affects principally R. rubrum lag phase (darkness) and growth rate (light) • CO is converted to CO2/H2 during acetate uptake and inhibits CO2 fixation (light) • UVa-ALE clones showed a 50% reduction in the lag phase (darkness).

Carbon monoxide is involved in melatonin-enhanced drought resistance in tomato seedlings by enhancing chlorophyll synthesis pathway.

BACKGROUND: Drought is thought to be a major abiotic stress that dramatically limits tomato growth and production. As signal molecule, melatonin (MT) and carbon monoxide (CO) can enhance plant stress resistance. However, the effect and underlying mechanism of CO involving MT-mediated drought resistance in seedling growth remains unknown. In this study, tomato (Solanum lycopersicum L. 'Micro-Tom') seedlings were used to investigate the interaction and mechanism of MT and CO in response to drought stress. RESULTS: The growth of tomato seedlings was inhibited significantly under drought stress. Exogenous MT or CO mitigated the drought-induced impairment in a dose-dependent manner, with the greatest efficiency provided by 100 and 500 µM, respectively. But application of hemoglobin (Hb, a CO scavenger) restrained the positive effects of MT on the growth of tomato seedlings under drought stress. MT and CO treatment promoted chlorophyll a (Chl a) and chlorophyll a (Chl b) accumulations. Under drought stress, the intermediate products of chlorophyll biosynthesis such as protoporphyrin IX (Proto IX), Mg-protoporphyrin IX (Mg-Proto IX), potochlorophyllide (Pchlide) and heme were increased by MT or CO, but uroporphyrinogen III (Uro III) content decreased in MT-treated or CO-treated tomato seedlings. Meanwhile, MT or CO up-regulated the expression of chlorophyll and heme synthetic-related genes SlUROD, SlPPOX, SlMGMT, SlFECH, SlPOR, SlChlS, and SlCAO. However, the effects of MT on chlorophyll biosynthesis were almost reversed by Hb. CONCLUSION: The results suggested that MT and CO can alleviate drought stress and facilitate the synthesis of Chl and heme in tomato seedlings. CO played an essential role in MT-enhanced drought resistance via facilitating chlorophyll biosynthesis pathway.

Neuroprotection via Carbon Monoxide Depends on the Circadian Regulation of CD36-Mediated Microglial Erythrophagocytosis in Hemorrhagic Stroke.

The molecular basis for circadian dependency in stroke due to subarachnoid hemorrhagic stroke (SAH) remains unclear. We reasoned that microglial erythrophagocytosis, crucial for SAH response, follows a circadian pattern involving carbon monoxide (CO) and CD36 surface expression. The microglial BV-2 cell line and primary microglia (PMG) under a clocked medium change were exposed to blood ± CO (250 ppm, 1 h) in vitro. Circadian dependency and the involvement of CD36 were analyzed in PMG isolated from control mice and CD36-/- mice and by RNA interference targeting Per-2. In vivo investigations, including phagocytosis, vasospasm, microglia activation and spatial memory, were conducted in an SAH model using control and CD36-/- mice at different zeitgeber times (ZT). In vitro, the surface expression of CD36 and its dependency on CO and phagocytosis occurred with changed circadian gene expression. CD36-/- PMG exhibited altered circadian gene expression, phagocytosis and impaired responsiveness to CO. In vivo, control mice with SAH demonstrated circadian dependency in microglia activation, erythrophagocytosis and CO-mediated protection at ZT2, in contrast to CD36-/- mice. Our study indicates that circadian rhythmicity modulates microglial activation and subsequent CD36-dependent phagocytosis. CO altered circadian-dependent neuroprotection and CD36 induction, determining the functional outcome in a hemorrhagic stroke model. This study emphasizes how circadian rhythmicity influences neuronal damage after neurovascular events.

On the Question of CO's Ability to Induce HO-1 Expression in Cell Culture: A Comparative Study Using Different CO Sources.

With the recognition of the endogenous signaling roles and pharmacological functions of carbon monoxide (CO), there is an increasing need to understand CO's mechanism of actions. Along this line, chemical donors have been introduced as CO surrogates for ease of delivery, dosage control, and sometimes the ability to target. Among all of the donors, two ruthenium-carbonyl complexes, CORM-2 and -3, are arguably the most commonly used tools for about 20 years in studying the mechanism of actions of CO. Largely based on data using these two CORMs, there has been a widely accepted inference that the upregulation of heme oxygenase-1 (HO-1) expression is one of the key mechanisms for CO's actions. However, recent years have seen reports of very pronounced chemical reactivities and CO-independent activities of these CORMs. We are interested in examining this question by conducting comparative studies using CO gas, CORM-2/-3, and organic CO donors in RAW264.7, HeLa, and HepG2 cell cultures. CORM-2 and CORM-3 treatment showed significant dose-dependent induction of HO-1 compared to "controls," while incubation for 6 h with 250-500 ppm CO gas did not increase the HO-1 protein expression and mRNA transcription level. A further increase of the CO concentration to 5% did not lead to HO-1 expression either. Additionally, we demonstrate that CORM-2/-3 releases minimal amounts of CO under the experimental conditions. These results indicate that the HO-1 induction effects of CORM-2/-3 are not attributable to CO. We also assessed two organic CO prodrugs, BW-CO-103 and BW-CO-111. BW-CO-111 but not BW-CO-103 dose-dependently increased HO-1 levels in RAW264.7 and HeLa cells. We subsequently studied the mechanism of induction with an Nrf2-luciferase reporter assay, showing that the HO-1 induction activity is likely due to the activation of Nrf2 by the CO donors. Overall, CO alone is unable to induce HO-1 or activate Nrf2 under various conditions in vitro. As such, there is no evidence to support attributing the HO-1 induction effect of the CO donors such as CORM-2/-3 and BW-CO-111 in cell culture to CO. This comparative study demonstrates the critical need to consider possible CO-independent effects of a chemical CO donor before attributing the observed biological effects to CO. It is also important to note that such in vitro results cannot be directly extrapolated to in vivo studies because of the increased level of complexity and the likelihood of secondary and/or synergistic effects in the latter.

A novel experimental method to determine substrate uptake kinetics of gaseous substrates applied to the carbon monoxide-fermenting Clostridium autoethanogenum.

Syngas fermentation has gained momentum over the last decades. The cost-efficient design of industrial-scale bioprocesses is highly dependent on quantitative microbial growth data. Kinetic and stoichiometric models for syngas-converting microbes exist, but accurate experimental validation of the derived parameters is lacking. Here, we describe a novel experimental approach for measuring substrate uptake kinetics of gas-fermenting microbes using the model microorganism Clostridium autoethanogenum. One-hour disturbances of a steady-state chemostat bioreactor with increased CO partial pressures (up to 1.2 bar) allowed for measurement of biomass-specific CO uptake- and CO2 production rates ( q CO ${q}_{{CO}}$ , q CO 2 ${q}_{{{CO}}_{2}}$ ) using off-gas analysis. At a pCO of 1.2 bar, a q CO ${q}_{{CO}}$ of -119 ± 1 mmol g-1 X h-1 was measured. This value is 1.8-3.5-fold higher than previously reported experimental and kinetic modeling results for syngas fermenters. Analysis of the catabolic flux distribution reveals a metabolic shift towards ethanol production at the expense of acetate at pCO ≥ $\ge $ 0.6 atm, likely to be mediated by acetate availability and cellular redox state. We characterized this metabolic shift as acetogenic overflow metabolism. These results provide key mechanistic understanding of the factors steering the product spectrum of CO fermentation in C. autoethanogenum and emphasize the importance of dedicated experimental validation of kinetic parameters.

Membrane-Bound Redox Enzyme Cytochrome bd-I Promotes Carbon Monoxide-Resistant Escherichia coli Growth and Respiration.

The terminal oxidases of bacterial aerobic respiratory chains are redox-active electrogenic enzymes that catalyze the four-electron reduction of O2 to 2H2O taking out electrons from quinol or cytochrome c. Living bacteria often deal with carbon monoxide (CO) which can act as both a signaling molecule and a poison. Bacterial terminal oxidases contain hemes; therefore, they are potential targets for CO. However, our knowledge of this issue is limited and contradictory. Here, we investigated the effect of CO on the cell growth and aerobic respiration of three different Escherichia coli mutants, each expressing only one terminal quinol oxidase: cytochrome bd-I, cytochrome bd-II, or cytochrome bo3. We found that following the addition of CO to bd-I-only cells, a minimal effect on growth was observed, whereas the growth of both bd-II-only and bo3-only strains was severely impaired. Consistently, the degree of resistance of aerobic respiration of bd-I-only cells to CO is high, as opposed to high CO sensitivity displayed by bd-II-only and bo3-only cells consuming O2. Such a difference between the oxidases in sensitivity to CO was also observed with isolated membranes of the mutants. Accordingly, O2 consumption of wild-type cells showed relatively low CO sensitivity under conditions favoring the expression of a bd-type oxidase.

Unraveling the Interplay of Dopamine, Carbon Monoxide, and Heme Oxygenase in Neuromodulation and Cognition.

The dopaminergic system plays important roles in neuromodulation, including prominent roles in complex neurological functions such as cognition, reward, motivation, and memory. Understandably, the highly complex nature of such physiological functions means that their regulation is intertwined with other signaling pathways, as has been demonstrated by numerous studies. Contrary to its public perception of being poisonous at all concentrations, carbon monoxide (CO) is produced endogenously from heme degradation by heme oxygenase (HO) as part of the physiological process of red blood cell turnover. Physiological concentrations of CO can reach high micromolar ranges in the hemoglobin bound form. Low-dose CO has shown therapeutic effects in numerous animal models, including traumatic brain injury via engaging various hemoprotein targets. As such, the HO-CO axis has been shown to offer beneficial effects in organ protection, anti-inflammation, and neuroprotection, among many others. Further, a large number of publications have shown the interactions among CO, HO, and the dopaminergic system. In this review, we critically examine such experimental evidence in a holistic fashion and in the context of a possible dopamine-HO-CO signaling axis. We hope that this Perspective will stimulate additional investigations into the molecular connectivity related to this possible axis and open doors to the development of novel therapeutics that impact the dopaminergic system.

Mutational Analysis of the Nitrogenase Carbon Monoxide Protective Protein CowN Reveals That a Conserved C-Terminal Glutamic Acid Residue Is Necessary for Its Activity.

Nitrogenase is the only enzyme that catalyzes the reduction of nitrogen gas into ammonia. Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Many nitrogen fixing bacteria protect nitrogenase from CO inhibition using the protective protein CowN. This work demonstrates that a conserved glutamic acid residue near the C-terminus of Gluconacetobacter diazotrophicus CowN is necessary for its function. Mutation of the glutamic acid residue abolishes both CowN's protection against CO inhibition and the ability of CowN to bind to nitrogenase. In contrast, a conserved C-terminal cysteine residue is not important for CO protection by CowN. Overall, this work uncovers structural features in CowN that are required for its function and provides new insights into its nitrogenase binding and CO protection mechanism.

Investigation of Cerebral Mitochondrial Injury in a Porcine Survivor Model of Carbon Monoxide Poisoning.

INTRODUCTION: Carbon monoxide (CO) is a colorless and odorless gas that is a leading cause of environmental poisoning in the USA with substantial mortality and morbidity. The mechanism of CO poisoning is complex and includes hypoxia, inflammation, and leukocyte sequestration in brain microvessel segments leading to increased reactive oxygen species. Another important pathway is the effects of CO on the mitochondria, specifically at cytochrome c oxidase, also known as Complex IV (CIV). One of the glaring gaps is the lack of rigorous experimental models that may recapitulate survivors of acute CO poisoning in the early phase. The primary objective of this preliminary study is to use our advanced swine platform of acute CO poisoning to develop a clinically relevant survivor model to perform behavioral assessment and MRI imaging that will allow future development of biomarkers and therapeutics. METHODS: Four swine (10 kg) were divided into two groups: control (n = 2) and CO (n = 2). The CO group received CO at 2000 ppm for over 120 min followed by 30 min of re-oxygenation at room air for one swine and 150 min followed by 30 min of re-oxygenation for another swine. The two swine in the sham group received room air for 150 min. Cerebral microdialysis was performed to obtain semi real-time measurements of cerebral metabolic status. Following exposures, all surviving animals were observed for a 24-h period with neurobehavioral assessment and imaging. At the end of the 24-h period, fresh brain tissue (cortical and hippocampal) was immediately harvested to measure mitochondrial respiration. RESULTS: While a preliminary ongoing study, animals in the CO group showed alterations in cerebral metabolism and cellular function in the acute exposure phase with possible sustained mitochondrial changes 24 h after the CO exposure ended. CONCLUSIONS: This preliminary research further establishes a large animal swine model investigating survivors of CO poisoning to measure translational metrics relevant to clinical medicine that includes a basic neurobehavioral assessment and post exposure cellular measures.

Comparing in vitro nitric oxide blood uptake to its pulmonary diffusing capacity.

Whether endothelium derived Nitric Oxide (NO) uptake by the blood is limited by a boundary layer, the red cell membrane or its interior is the subject of continued debate. Whether lung uptake of NO in the single-breath DLNO test is limited by blood or not is also debated. To understand which processes are limiting blood NO uptake we have modelled NO chemical kinetics and we have derived a shrinking core model, Thiele Modulus and FTCS (Euler) numerical solution. In a rapid reaction apparatus, NO uptake appears limited by a boundary layer, and throughout the red cell, by diffusion. In the single breath situation, and arguably with endogenous NO in vivo, NO uptake appears limited by a boundary layer and a pseudo first order chemical reaction in the outer molecular layers of the red cell. We have not found evidence to support red cell membrane limitation.