Nitric Oxide Toxicity Research Articles

Evaluation of cellular viability in chitosan/L-arginine hydrogels.

There is a lack of studies evaluating the toxicity of nitric oxide (NO) precursors in chitosan/L-arginine hydrogels and their topical administration. However, clarifying the characteristics of these elements is essential for their possible use in non-surgical techniques of tooth movement acceleration. Such characteristics include interaction with different cell types, metabolism and drug safety. This in vitro study aimed to assess the cytotoxicity of chitosan hydrogels on human HeLa cells using different concentrations of L-arginine. The hydrogels were synthesized in a materials engineering laboratory, with a controlled environment, using 4 different L-arginine concentrations of 0%, 10%, 15%, and 20%. Once the hydrogels were prepared, their physical and chemical properties were characterized, and viability analysis was performed using 2 different methods, including a 48-h assay with Artemia salina nauplii and a 24-h cell culture with human HeLa cells followed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assay. Data analysis was performed using a Mann-Whitney U test to evaluate positive and negative controls in the cell culture, with a significance level of 0.01. A Wilcoxon paired test contrasted the 24-h compared to 48-h Artemia salina assays, with a Kruskal-Wallis and post hoc Dunn test used to compare groups using a significance level of 0.05. In the more viscous hydrogels, Artemia salina nauplii decreased drastically in 24 h, while the 15% and 20% hydrogels had no statistical differences from the negative control. The 10% and 20% hydrogels were statistically different from the negative control when comparing cell culture data. Our findings suggest that chitosan/L-arginine hydrogels could be used in humans without toxic effects. However, more trials and tests are needed to evaluate tooth movement rate during orthodontic treatment.

A review of botany, phytochemistry, and pharmacology of the forest Natal mahogany (Trichilia dregeana Sond.)

Trichilia dregeana is an underutilized plant species native to tropical Africa and known for its nutritional and medicinal properties. The present review compiles existing information on botany, traditional uses, and chemical and pharmacological properties of T. dregeana. Multiple searches on existing literature on the traditional, medicinal, phytochemistry, and pharmacological properties of T. dregeana were conducted in online databases such as Scopus®, Google Scholar, SpringerLink®, SciELO, ScienceDirect®, PubMed® and Web of Science, as well as using pre-electronic literature sources obtained from the university library. Trichilia dregeana is used as a medicinal plant against human and animal diseases in seven countries, that is, 33.3% of the countries where the species is indigenous. The phytochemical evaluation of the plant revealed that it contains alcohols, alkanes, amides, esters, lignan, limonoids, sesquiterpenes, steroids, and triterpenoids. The pharmacological assessments showed that the crude extracts and phytochemical compounds isolated from the species demonstrated antibacterial, antifungal, antimycobacterial, antiviral, acetylcholinesterase, anti-inflammatory, antioxidant, insecticidal, prostaglandin-synthesis inhibitory, nitric oxide production inhibitory, wound healing, mutagenicity, and toxicity activities. Detailed studies focusing on toxicity and safety, mechanisms of action in vivo, and clinical research aimed at corroborating the traditional medical applications of T. dregeana are recommended.

Exhaled Nitric Oxide and Pulmonary Oxygen Toxicity Susceptibility.

Individual susceptibility to pulmonary oxygen toxicity (PO2tox) is highly variable and currently lacks a reliable biomarker for predicting pulmonary hyperoxic stress. As nitric oxide (NO) is involved in many respiratory system processes and functions, we aimed to determine if expired nitric oxide (FENO) levels can provide an indication of PO2tox susceptibility in humans. Eight U.S. Navy-trained divers volunteered as subjects. The hyperoxic exposures consisted of six- and eight-hour hyperbaric chamber dives conducted on consecutive days in which subjects breathed 100% oxygen at 202.65 kPa. Subjects' individual variability in pulmonary function and FENO was measured twice daily over five days and compared with their post-dive values to assess susceptibility to PO2tox. Only subjects who showed no decrements in pulmonary function following the six-hour exposure conducted the eight-hour dive. FENO decreased by 55% immediately following the six-hour oxygen exposure (n = 8, p < 0.0001) and by 63% following the eight-hour exposure (n = 4, p < 0.0001). Four subjects showed significant decreases in pulmonary function immediately following the six-hour exposure. These subjects had the lowest baseline FENO, had the lowest post-dive FENO, and had clinical symptoms of PO2tox. Individuals with low FENO were the first to develop PO2tox symptoms and deficits in pulmonary function from the hyperoxic exposures. These data suggest that endogenous levels of NO in the lungs may protect against the development of PO2tox.

OXIDATIVE MECHANISMS UNDERLYING BRAIN CELL DAMAGE IN RATS UNDER CHANGES IN DURATION OF THE LIGHTDARK CYCLES

One of the main mechanisms underlying cell damage is lipid peroxidation, which is characterized by increased levels of free radical, products of lipid peroxidation chain reactions, and alterations in the activity of the antioxidant system in tissues. Therefore, the objective of this study is to investigate the proand antioxidant balance in rat brain homogenate under conditions of disrupted "light-dark" cycles. The study involved 28 white Wistar rats weighing 150-200 gm, divided into two groups: control (15) and the group of animals exposed to acute desynchronosis simulation (13). To simulate desynchronosis, a normal "light-dark" cycle of 12 hours of light and 12 hours of darkness was initially established for three weeks, followed by a shift of the "light-dark" phases backward by six hours over the next three days. The animals were kept in standard conditions with a balanced diet in the vivarium, and the study adhered to the principles of biomedical ethics.&#x0D; The results obtained indicate a significant increase in the activity of superoxide anion radical production in the brain tissues of the rats with modelled desynchronosis by 75.8% compared to the control group. This increase is attributed to the NADPH-oxidase mechanism by 20.2% and to xanthine oxidase mechanisms by 28%. The concentration of TBA-active products increased by 36.8%, the growth of TBA-active products elevated by 30%, and the activity of the key antioxidant enzymes, superoxide dismutase and catalase, decreased by 53% and 14%, respectively, in the second group of animals. Furthermore, in the brain tissues of rats with disrupted "light-dark" cycles, there were found elevated levels of nitrites by 23.8%, nitrosothiols by 19%, and peroxynitrites by 69%, along with a reduction in the activity of total NO-synthase by 59.1%, inducible NO-synthase by 61.1%, and constitutive NO-synthase by 9% compared to the control group. The activity of ornithine decarboxylase increased by 26.5% in the brain tissues of the rats with simulated desynchronosis compared to the control group.&#x0D; In conclusion, the disruption of "light-dark" cycles leads to the development of oxidative stress in brain tissues of animals and is characterized by increased production of reactive oxygen species and diminished antioxidant protection. Acute desynchronosis decreases nitric oxide production through the NO-synthase pathway, while increasing the accumulation of toxic nitric oxide metabolites in the brain tissues of rats, thereby posing a potential risk of nitrosative stress. Furthermore, alterations in the normal light regimen of the day activate the arginase pathway of L-arginine metabolism and result in increased glutamate production in the brain tissues of rats, potentially leading to oxidative stress.

Post engineering of a chemically stable MOF for selective and sensitive sensing of nitric oxide

A hydrolytically stable luminous metal–organic framework (MOF) sensor was strategically designed for precise dual phase recognition of biologically relevant yet toxic nitric oxide (NO).

Valorization of quince peel into functional food ingredients: A path towards “zero waste” and sustainable food systems

Quince (Cydonia oblonga Mill.) is an astringent fruit widely processed into marmalade and other sweets through processes that discard the peel as a by-product. Therefore, this study was performed to characterize the quince peel composition in nutrients and phytochemicals and evaluate its in vitro biological activity, following a “zero waste” approach. The quince peel dry powder was particularly rich in fiber (20.2 g/100 g), fructose (34 g/100 g), malic acid (7.2 g/100 g), and potassium (692 mg/100 g). Extracts prepared by dynamic hydroethanolic maceration and hot water extraction yielded 4.70 and 4.27 mg/g of phenolic compounds, respectively, with a prevalence of flavan-3-ols. The hydroethanolic extract was the most effective in inhibiting lipid peroxidation and oxidative hemolysis, and also presented better antimicrobial effects against foodborne pathogens, which agreed with the highest flavan-3-ol contents. The extracts were better than control synthetic food additives against some tested fungal and bacterial strains. On the other hand, no ability to inhibit nitric oxide production or toxicity to the tumor and non-tumor cell lines was observed. Furthermore, the solid residues remaining after extraction contained 35–37 g/100 g of fiber. Overall, quince peel can be upcycled into fiber-rich and bioactive ingredients to endow the value chain with natural food fortifiers, preservatives, and health promoters.

Rationalizing the Use of Polyphenol Nano-formulations in the Therapy of Neurodegenerative Diseases.

Neurodegenerative diseases are a heterogeneous group of disorders among aging populations worldwide characterized by the progressive degeneration of the structure and function of brain cells and the nervous system. Alzheimer's disease and Parkinson's disease are common neurodegenerative diseases (NDs). Classic pathological features of AD are the accumulation of the amyloid betaprotein and aggregates of hyperphosphorylated tau protein around the brain cells. Dopaminergic neuronal death in the midbrain and accumulation of α- synuclein in the neurons are the hallmark of Parkinson's disease. The pathogenesis is multifactorial, and both neurodegenerative disorders have complex etiology. Oxidative stress closely linked with mitochondrial dysfunction, excitotoxicity, nitric oxide toxicity, and neuro-inflammation, is anticipated to trigger neuronal death. Ample evidence has implicated that oxidative stress and inflammation contribute to the pathology of neurodegeneration in AD and PD. Currently, acetylcholinesterase inhibitors are the main treatment option for AD, while L-DOPA is the gold standard therapy for PD. Along with the main therapy, many endogenous antioxidants, like vitamin E, selenium, etc., are also given to the patients to combat oxidative stress. Current treatment for these NDs is limited due to the blood-brain barrier (BBB) that hinders drug targeting towards neurons. In this review, we emphasize adjunct treatment with anti-inflammatory agents that act at the site of the disease and can halt the disease progression by attenuating the effect of ROS triggering neuro-inflammatory response. Polyphenols, either as purified compounds or extracts from various natural plant sources, have been well studied and documented for anti-inflammatory effects, but their use for ND is limited due to their physicochemical attributes. Nanoparticle-mediated drug delivery system exhibits immense potential to overcome these hurdles in drug delivery to the CNS, enabling nanoparticle-based therapies to directly target the inflammation and release bioactive compounds with anti-inflammatory properties to the site of action.

Recent Advancements in CNS Acting Drugs: A Step Towards End of Illness

Progressive degeneration in the morphology and functions of neuronal cells leads to multifactorial pathogenesis conditions of oxidative stress, mitochondrial dysfunction, excitotoxicity, nitric oxide toxicity, and neuro-inflammation to mediate heterogeneous types of neurodegenerative diseases, such as Epilepsy, Alzheimer's (AD) and Parkinson's (PD), more prominently among aging populations. In this editorial, complex mechanisms, challenges, and advancements made in the discovery of new neurotherapeutics, as well as designing approaches being adopted to fabricate brain-targeted delivery systems, are discussed.

Unraveling Nitrogen, Sulfur, and Carbon Metabolic Pathways and Microbial Community Transcriptional Responses to Substrate Deprivation and Toxicity Stresses in a Bioreactor Mimicking Anoxic Brackish Coastal Sediment Conditions

Microbial communities are key drivers of carbon, sulfur, and nitrogen cycling in coastal ecosystems, where they are subjected to dynamic shifts in substrate availability and exposure to toxic compounds. However, how these shifts affect microbial interactions and function is poorly understood. Unraveling such microbial community responses is key to understand their environmental distribution and resilience under current and future disturbances. Here, we used metagenomics and metatranscriptomics to investigate microbial community structure and transcriptional responses to prolonged ammonium deprivation, and sulfide and nitric oxide toxicity stresses in a controlled bioreactor system mimicking coastal sediment conditions. Ca. Nitrobium versatile, identified in this study as a sulfide-oxidizing denitrifier, became a rare community member upon ammonium removal. The ANaerobic Methanotroph (ANME) Ca. Methanoperedens nitroreducens showed remarkable resilience to both experimental conditions, dominating transcriptional activity of dissimilatory nitrate reduction to ammonium (DNRA). During the ammonium removal experiment, increased DNRA was unable to sustain anaerobic ammonium oxidation (anammox) activity. After ammonium was reintroduced, a novel anaerobic bacterial methanotroph species that we have named Ca. Methylomirabilis tolerans outcompeted Ca. Methylomirabilis lanthanidiphila, while the anammox Ca. Kuenenia stuttgartiensis outcompeted Ca. Scalindua rubra. At the end of the sulfide and nitric oxide experiment, a gammaproteobacterium affiliated to the family Thiohalobacteraceae was enriched and dominated transcriptional activity of sulfide:quinone oxidoreductases. Our results indicate that some community members could be more resilient to the tested experimental conditions than others, and that some community functions such as methane and sulfur oxidation coupled to denitrification can remain stable despite large shifts in microbial community structure. Further studies on complex bioreactor enrichments are required to elucidate coastal ecosystem responses to future disturbances.

Review on Parkinson’s Disease, a Neurodegenerative Disorder and The Role of Ceruloplasmin Protein in It

Parkinson’s disease (PD), a neurodegenerative disease is becoming major health concern mainly for elder people of age over 60 years. The main cause of PD is permanent loss/death of dopaminergic nerve cells present in brain part called substantia nigra, which is responsible for dopamine synthesis. MAO-B, monoamine oxidase B, regulates dopamine metabolism and increased activity of MAO-B causes dopamine degradation which in turn promotes the accumulation of glutamate and oxidative stress with free radical liberation. Several factors like oxidative stress, free radical formation, increased cholesterol, mitochondrial dysfunction, nitric oxide toxicity, signal-mediated apoptosis, head trauma, and environmental toxins and gene mutations like VPS35, SNCA, EIF4G1, GBA, CHCHD, LRRK2, PINK1, DNAJC13 and SOD2 are associated with PD. Symptoms of PD include bradykinesia, muscle rigidity, resting tremors, postural instability and shuffling gait, constipation, sleep problems, fatigue, apathy, loss of smell and taste, excessive sweating, frequent nightmares, dream enacting behaviour, anxiety, depression, daytime drowsiness. In PD, low levels of ceruloplasmin were observed in people with early onset of PD. Ceruloplasmin, a ferroxidase enzyme which is synthesized in liver parenchymal cell, regulates iron metabolism and lower level of which causes iron accumulation in brain which is responsible for the early onset of PD. Levodopa-based preparations, Dopamine agonists, Catechol-o-methyltransferase (COMT) inhibitors, MOA-B inhibitors, Adjunctive therapy, Antiglutamatergics drugs are currently used for the treatment of PD.

Integrated omics analyses reveal differential gene expression and potential for cooperation between denitrifying polyphosphate and glycogen accumulating organisms

Unusually high accumulation of the potent greenhouse gas nitrous oxide (N2 O) has previously been documented in denitrifying biological phosphorus (P) removal bioprocesses, but the roles of differential denitrification gene expression patterns and ecological interactions between key functional groups in driving these emissions are not well understood. To address these knowledge gaps, we applied genome-resolved metagenomics and metatranscriptomics to a denitrifying bioprocess enriched in as-yet-uncultivated denitrifying polyphosphate accumulating organisms (PAOs) affiliated with Candidatus Accumulibacter. The six transcriptionally most active populations in the community included three co-occurring Accumulibacter strains affiliated with clades IF (a novel clade identified in this study), IA and IC, a competing glycogen accumulating organism (GAO) affiliated with Competibacteraceae (GAO1), a Gammaproteobacteria PR6 and an Anaerolineae CH7. Strongly elevated expression of nitrite reductase genes compared to nitrous oxide reductase genes was observed in the overall community and in Accumulibacter populations, suggesting a strong role for differential gene expression in driving N2 O accumulation. Surprisingly, while ~90% of the nirS gene transcripts were expressed by the three co-occurring PAO populations, ~93% of the norB gene transcripts were expressed by GAO1 and ~75% of the norZ gene transcripts were mapped to PR6 and several other non-PAO flanking populations. This suggests the potential for cooperation between flanking populations and PAOs in reducing denitrification intermediates. Such cooperation may benefit the community by reducing the accumulation of toxic nitric oxide.

Heuristics in Role of Human Glutathione S-transferase Mu 1 as Nitric Oxide Carrier and its Engineered Variants for Enhanced Activity.

Human glutathione S-transferases (hGSTs) are phase-II detoxification enzymes that catalyze the conjugation of electrophilic compounds and glutathione. Anomalous excess production of NO in the cellular environment under diseased or stressed condition results in lethal effects to the cell. Studies have reported that the evolution of tyrosine-based GSTs as a defense mechanism by the cell to mitigate Nitric Oxide (NO) toxicity. The dual role of hGSTP1 as NO carrier and scavenger is a prelude for the research forthwith. A plausible role of hGSTM1 as NO carrier is considered. Being a prominent cellular messenger and secondary metabolite, excess production of NO is lethal to the cell. Moreover, hGSTM1 polymorphisms lead to diminished catalytic activity that promotes a diseased state. Hence, it is compelling to generate hGSTM1 mutants that have more catalytic efficacy compared to Wild Type (WT). hGSTM1 mutants with enhanced efficiency were generated using in silico and in vitro Site-Directed Mutagenesis (SDM). WT and mutant proteins were overexpressed and purified using affinity chromatography. The catalytic activity and binding efficiency of WT and mutant proteins towards CDNB (1-chloro-2, 4-dinitrobenzene) & NO were determined. NO assay reveals the probable interaction of WT hGSTM1 with NO. In silico, SDM studies provided E129K and Q109K mutants with superior NO binding efficiency as compared to WT. The catalytic activity (GST and NO assays) of the mutants corroborate the in silico results. WT hGSTM1 is recognized as a positive NO carrier. The novel mutant enzymes E129K and Q109K are inferred to possess superior NO carrying capacity.

Synthesis of MnO2/Mg-Al layered double hydroxide and evaluation of its NO-removal performance

The removal of toxic nitric oxide (NO) from exhaust gas is challenging and expensive. MnO2/Mg-Al LDH, a composite of manganese dioxide and Mg-Al layered double hydroxide, was synthesized via the rehydration method for potential application in NO removal. MnO2/Mg-Al LDH was found to be complexed with MnO2, pillared between the LDH layers and intercalated with CO32− and OH−. We investigated the performance of a MnO2/Mg-Al LDH treatment in removing NO from exhaust gas. The exhaust gas was circulated in a reaction tube filled with MnO2/Mg-Al LDH, CO3･Mg-Al LDH, or a MnO2 + CO3･Mg-Al LDH mixture, and the NO removal efficiency of the MnO2/Mg-Al LDH composite was compared with that of the other compounds. When NO gas (150 ppm, linear velocity 1.0 m/min) was distributed at 170 °C, treatment with the MnO2･Mg-Al LDH composite and CO3･Mg-Al LDH yielded removal rates of 83.9% and 0%, respectively. The significantly higher NO removal rate of MnO2/Mg-Al LDH was ascribed to the transformation of NO to NO2 via oxidation over MnO2. Further, the NO removal rate after 90 min through the application of a simple mixture of MnO2 and CO3･Mg-Al LDH (Mn: 4.2 wt%) was only 1.4%, suggesting that a synergistic effect was generated by the MnO2/Mg-Al LDH composite. Though NO removal by MnO2/Mg-Al LDH was reduced at temperatures of 140 °C and higher, the results suggest that the synthesized compound can be much more effective than those commonly used for NO removal from exhaust gas.

Role of Herbal Medicines in the Management of Brain Injury.

Brainis susceptible to oxidative stress due to its increased oxygen consumption and low antioxidant levels. Oxidative stress plays a crucial role in the pathogenesis of various neurological diseases. This review on the role of herbal medicines in the management of brain injury was performed by searching Web of Science, PubMed, Google Scholar, Scopus, and Iran Medex between 1976 to January 2020. The search words contained brain injury, and the total number of publications for the review study was 32. Studies with various medicinal plants such as Acanthopanax senticosus, Bacopa monnieri, carnosol, Cassia mimosoides, Centella asiatica, Crocus sativus, Cuminum cyminum, curcumin, Feronia limonia, Gardenia jasminoides, Ginkgo biloba, Kaempferia parviflora, Mentha longifolia, Nigella sativa, olive, orientin, pomegranate, quercetin, rice bran, Rosa damascena, Thymus vulgaris, Viola odorata, Withania coagulans, Zingiber officinale, and Ziziphus spina-christi show a significant improvement in brain injury. The different mechanisms for improvement in brain injury by these medicinal plants include HIF-1 (hypoxia-inducible factor 1) signaling, free-radical scavenging, reduction of nitric oxide (NO) toxicity and acetylcholine esterase (AChE) activity, decrease of pAkt and its downstream targets, downregulation of the aquaporin-4 (AQP-4) and TLR4/NF-ĸB/TNF-α signal, reduction in malondialdehyde and NO levels, increasing neuronal density in the hippocampus, and inhibition of oxidative stress. In this review, the neuroprotective actions and molecular mechanisms of herbal medicines are evaluated by reviewing available studies.

The relationship between nitric oxide and cadmium toxicity in wheat (triticum aestivum L.) seedlings

In this study, biochemical responds against different cadmium concentrations (25 µM, 50 µM and 75 µM) in seedlings belonging to three wheat (Triticum aestivum L.) varieties applied to different SNP (25 µM and 50 µM) concentrations. As the material of the study, fifteen days old seedlings of wheat (Triticum aestivum L.) were used. In all applications carried out to the seedlings, hydroponic method was preferred. The seedlings were divided into three groups in which pretreatment of SNP for 48 hours were done. After that, different concentrations of cadmium were applied to these three groups to except controls (pure water and SNPs). In addition, reduced glutathione (GSH) / oxidized glutathione (GSSG) ratio, catalase (CAT) with superokside glutathione (SOD) activities were detected in the leaves. &#x0D; According to the obtained results, (GSH) / (GSSG) ratio reduced in all three varieties; CAT activity was reduced in Bayraktar and Ikizce, but it was increased in Tosunbey. SOD activity was increased all three varieties. The most prominent responses of SOD enzyme activity in the leaves of wheat seedlings were determined in Tosunbey wheats. When the results are evaluated, generally, 50 µM of SNP pre-application was found as more successful than 25 µM of SNP application in terms of attenuating Cd toxicity. SNP was found to have a mitigating effect against Cd depending on the dose.

Ammonia oxidation at pH 2.5 by a new gammaproteobacterial ammonia-oxidizing bacterium

Ammonia oxidation was considered impossible under highly acidic conditions, as the protonation of ammonia leads to decreased substrate availability and formation of toxic nitrogenous compounds. Recently, some studies described archaeal and bacterial ammonia oxidizers growing at pH as low as 4, while environmental studies observed nitrification at even lower pH values. In this work, we report on the discovery, cultivation, and physiological, genomic, and transcriptomic characterization of a novel gammaproteobacterial ammonia-oxidizing bacterium enriched via continuous bioreactor cultivation from an acidic air biofilter that was able to grow and oxidize ammonia at pH 2.5. This microorganism has a chemolithoautotrophic lifestyle, using ammonia as energy source. The observed growth rate on ammonia was 0.196 day−1, with a doubling time of 3.5 days. The strain also displayed ureolytic activity and cultivation with urea as ammonia source resulted in a growth rate of 0.104 day−1 and a doubling time of 6.7 days. A high ammonia affinity (Km(app) = 147 ± 14 nM) and high tolerance to toxic nitric oxide could represent an adaptation to acidic environments. Electron microscopic analysis showed coccoid cell morphology with a large amount of intracytoplasmic membrane stacks, typical of gammaproteobacterial ammonia oxidizers. Furthermore, genome and transcriptome analysis showed the presence and expression of diagnostic genes for nitrifiers (amoCAB, hao, nor, ure, cbbLS), but no nirK was identified. Phylogenetic analysis revealed that this strain belonged to a novel bacterial genus, for which we propose the name “Candidatus Nitrosacidococcus tergens” sp. RJ19.

Sustainable NO removal and its sensitive monitoring at room temperature by electrogenerated Ni (I) electron mediator

Online monitoring of gas pollutants in the gas phase at room temperature using an electrochemical macro gas flow sensor is challenging and important for the pollutant treatment process. In this work, for the first time, we tried to explore the homogeneous and heterogeneous application of Ni(II) (CN)42− in the KOH environment for the removal and monitoring of toxic nitric oxide gas. The homogeneous electrogenerated Ni(I) (CN)43− was effectively removing the toxic nitric oxide gas by electro scrubbing method and the novel Ni(II) (CN)42− and KOH modified electrode used for heterogeneous sensor application with high sensitivity, and reliability toward Nitric oxide gas. The sensor showed enhanced gas diffusion and high sensitivity. Scanning electron microscopy and X-ray diffraction confirmed the modification of the carbon felt electrode. In a high concentrated KOH environment, the active mediator stabilized the sensor for a long time compared to the neutral environment. The Ni(II) (CN)42− fabricated carbon felt was used to monitor the concentration of nitric oxide gas pollutant; the calculated sensitivity was approximately −0.33 mA ppm−1 cm−2. The current increased linearly with increasing nitric oxide concentration up to 12 ppm and was validated by online gas chromatography. The developed electrochemical gas flow sensor successfully monitored the unremoved nitric oxide gas at the exit from the MER electro-scrubbing process; the concentration was calculated using a calibration plot.

Two interacting ATPases protect Mycobacterium tuberculosis from glycerol and nitric oxide toxicity.

The Mycobacterium tuberculosis H37Rv genome has been sequenced and annotated over 20 years ago, yet roughly half of the protein-coding genes still lack a predicted function. We characterized two genes of unknown function, rv3679 and rv3680, for which inconsistent findings regarding their importance for virulence in mice have been reported. We confirmed that a rv3679-80 deletion mutant (Δrv3679-80) was virulent in mice and discovered that Δrv3679-80 suffered from a glycerol-dependent recovery defect on agar plates following mouse infection. Glycerol also exacerbated killing of Δrv3679-80 by nitric oxide. Rv3679-Rv3680 have previously been shown to form a complex with ATPase activity and we demonstrate that the ability of M. tuberculosis to cope with elevated levels of glycerol and nitric oxide requires intact ATP-binding motifs in both Rv3679 and Rv3680. Inactivation of glycerol kinase or Rv2370c, a protein of unknown function, suppressed glycerol mediated toxicity in Δrv3679-80 Glycerol catabolism led to increased intracellular methylglyoxal pools and Δrv3679-80 was hypersusceptible to extracellular methylglyoxal suggesting that glycerol toxicity in Δrv3679-80 is caused by methylglyoxal. Rv3679 and Rv3680 interacted with Rv1509, and Rv3679 had numerous additional interactors including proteins of the type II fatty acid synthase (FASII) pathway and mycolic acid modifying enzymes linking Rv3679 to fatty acid or lipid synthesis. This work provides experimentally determined roles for Rv3679 and Rv3680 and stimulates future research on these and other proteins of unknown function.Importance A better understanding of the pathogenesis of tuberculosis requires a better understanding of gene function in M. tuberculosis This work provides the first functional insight into the Rv3679/Rv3680 ATPase complex. We demonstrate that M. tuberculosis requires this complex and specifically its ATPase activity to resist glycerol and nitric oxide toxicity. We provide evidence that the glycerol-derived metabolite methylglyoxal causes toxicity in the absence of Rv3679/Rv3680. We further show that glycerol-dependent toxicity is reversed when glycerol kinase (GlpK) is inactivated. Our work uncovered other genes of unknown function that interact with Rv3679 and/or Rv3680 genetically or physically, underscoring the importance of understanding uncharacterized genes.

Functional interactions between nitrite reductase and nitric oxide reductase from Paracoccus denitrificans

Denitrification is a microbial pathway that constitutes an important part of the nitrogen cycle on earth. Denitrifying organisms use nitrate as a terminal electron acceptor and reduce it stepwise to nitrogen gas, a process that produces the toxic nitric oxide (NO) molecule as an intermediate. In this work, we have investigated the possible functional interaction between the enzyme that produces NO; the cd1 nitrite reductase (cd1NiR) and the enzyme that reduces NO; the c-type nitric oxide reductase (cNOR), from the model soil bacterium P. denitrificans. Such an interaction was observed previously between purified components from P. aeruginosa and could help channeling the NO (directly from the site of formation to the side of reduction), in order to protect the cell from this toxic intermediate. We find that electron donation to cNOR is inhibited in the presence of cd1NiR, presumably because cd1NiR binds cNOR at the same location as the electron donor. We further find that the presence of cNOR influences the dimerization of cd1NiR. Overall, although we find no evidence for a high-affinity, constant interaction between the two enzymes, our data supports transient interactions between cd1NiR and cNOR that influence enzymatic properties of cNOR and oligomerization properties of cd1NiR. We speculate that this could be of particular importance in vivo during metabolic switches between aerobic and denitrifying conditions.

Clustering as a Means To Control Nitrate Respiration Efficiency and Toxicity in Escherichia coli.

Respiration is a fundamental process that has to optimally respond to metabolic demand and environmental changes. We previously showed that nitrate respiration, crucial for gut colonization by enterobacteria, is controlled by polar clustering of the nitrate reductase increasing the electron flux through the complex. Here, we show that the formate dehydrogenase electron-donating complex, FdnGHI, also clusters at the cell poles under nitrate-respiring conditions. Its proximity to the nitrate reductase complex was confirmed by its identification in the interactome of the latter, which appears to be specific to the nitrate-respiring condition. Interestingly, we have identified a multiprotein complex dedicated to handle nitric oxide resulting from the enhanced activity of the electron transport chain terminated by nitrate reductase. We demonstrated that the cytoplasmic NADH-dependent nitrite reductase NirBD and the hybrid cluster protein Hcp are key contributors to regulation of the nitric oxide level during nitrate respiration. Thus, gathering of actors involved in respiration and NO homeostasis seems to be critical to balancing maximization of electron flux and the resulting toxicity.IMPORTANCE Most bacteria rely on the redox activity of respiratory complexes embedded in the cytoplasmic membrane to gain energy in the form of ATP and of an electrochemical gradient established across the membrane. Nevertheless, production of harmful and toxic nitric oxide by actively growing bacteria as either an intermediate or side-product of nitrate respiration challenges how homeostasis control is exerted. Here, we show that components of the nitrate electron transport chain are clustered, likely influencing the kinetics of the process. Nitric oxide production from this respiratory chain is controlled and handled through a multiprotein complex, including detoxifying systems. These findings point to an essential role of compartmentalization of respiratory components in bacterial cell growth.