Oxidative Stress in Sepsis: A Focus on Cardiac Pathology.

This study aims to analyze post-mortem human cardiac specimens, to verify and evaluate the existence or extent of oxidative stress in subjects whose cause of death has been traced to sepsis, through immunohistological oxidative/nitrosative stress markers. Indeed, in the present study, i-NOS, NOX2, and nitrotyrosine markers were higher expressed in the septic death group when compared to the control group, associated with also a significant increase in 8-OHdG, highlighting the pivotal role of oxidative stress in septic etiopathogenesis. In particular, 70% of cardiomyocyte nuclei from septic death specimens showed positivity for 8-OHdG. Furthermore, intense and massive NOX2-positive myocyte immunoreaction was noticed in the septic group, as nitrotyrosine immunostaining intense reaction was found in the cardiac cells. These results demonstrated a correlation between oxidative and nitrosative stress imbalance and the pathophysiology of cardiac dysfunction documented in cases of sepsis. Therefore, subsequent studies will focus on the expression of oxidative stress markers in other organs and tissues, as well as on the involvement of the intracellular pattern of apoptosis, to better clarify the complex pathogenesis of multi-organ failure, leading to support the rationale for including therapies targeting redox abnormalities in the management of septic patients.

Elevated SLC7A2 expression is associated with an abnormal neuroinflammatory response and nitrosative stress in Huntington's disease.

We previously identified solute carrier family 7 member 2 (SLC7A2) as one of the top upregulated genes when normal Huntingtin was deleted. SLC7A2 has a high affinity for L-arginine. Arginine is implicated in inflammatory responses, and SLC7A2 is an important regulator of innate and adaptive immunity in macrophages. Although neuroinflammation is clearly demonstrated in animal models and patients with Huntington's disease (HD), the question of whether neuroinflammation actively participates in HD pathogenesis is a topic of ongoing research and debate. Here, we studied the role of SLC7A2 in mediating the neuroinflammatory stress response in HD cells. RNA sequencing (RNA-seq), quantitative RT-PCR and data mining of publicly available RNA-seq datasets of human patients were performed to assess the levels of SLC7A2 mRNA in different HD cellular models and patients. Biochemical studies were then conducted on cell lines and primary mouse astrocytes to investigate arginine metabolism and nitrosative stress in response to neuroinflammation. The CRISPR-Cas9 system was used to knock out SLC7A2 in STHdhQ7 and Q111 cells to investigate its role in mediating the neuroinflammatory response. Live-cell imaging was used to measure mitochondrial dynamics. Finally, exploratory studies were performed using the Enroll-HD periodic human patient dataset to analyze the effect of arginine supplements on HD progression. We found that SLC7A2 is selectively upregulated in HD cellular models and patients. HD cells exhibit an overactive response to neuroinflammatory challenges, as demonstrated by abnormally high iNOS induction and NO production, leading to increased protein nitrosylation. Depleting extracellular Arg or knocking out SLC7A2 blocked iNOS induction and NO production in STHdhQ111 cells. We further examined the functional impact of protein nitrosylation on a well-documented protein target, DRP-1, and found that more mitochondria were fragmented in challenged STHdhQ111 cells. Last, analysis of Enroll-HD datasets suggested that HD patients taking arginine supplements progressed more rapidly than others. Our data suggest a novel pathway that links arginine uptake to nitrosative stress via upregulation of SLC7A2 in the pathogenesis and progression of HD. This further implies that arginine supplements may potentially pose a greater risk to HD patients.

Nitrosative Stress in Autism: Supportive Evidence and Implications for Mitochondrial Dysfunction.

A recent study by the Amal team published in this journal in May 2023 proved for the first time the link of nitric oxide (NO) with autism spectrum disorder (ASD), thereby opening new venues for the potential use of neuronal nitric oxide synthase (nNOS) inhibitors as therapeutics for improving the neurological and behavioral symptoms of ASD. The authors conclude that their findings demonstrate that NO plays a significant role in ASD. Indeed, earlier studies support elevated NO and its metabolites, nitrite, and peroxynitrite, in individuals diagnosed with ASD. Dysregulated NOS activity may underlie the well-documented mitochondrial dysfunction in a subset of individuals with ASD. Strategies for treating ASD shall also consider NO effects on mitochondrial respiration in modulating NOS activity. Further experimental evidence and controlled clinical trials with NOS modifiers are required for assessing their therapeutic potential for individuals with ASD.

Antigen stasis and airway nitrosative stress in human primary ciliary dyskinesia.

Nasal nitric oxide (nNO) is low in most patients with primary ciliary dyskinesia (PCD). Decreased ciliary motion could lead to antigen stasis, increasing oxidant production and NO oxidation in the airways. This could both decrease gas phase NO and increase nitrosative stress. We studied primary airway epithelial cells from healthy controls (HCs) and patients with PCD with several different genotypes. We measured antigen clearance in fenestrated membranes exposed apically to the fluorescently labeled antigen Dermatophagoides pteronyssinus (Derp1-f). We immunoblotted for 3-nitrotyrosine (3-NT) and for oxidative response enzymes. We measured headspace NO above primary airway cells without and with a PCD-causing genotype. We measured nNO and exhaled breath condensate (EBC) H2O2 in vivo. Apical Derp1-f was cleared from HC better than from PCD cells. DUOX1 expression was lower in HC than in PCD cells at baseline and after 24-h Derp1-f exposure. HC cells had less 3-NT and NO3- than PCD cells. However, NO consumption by HC cells was less than that by PCD cells; NO loss was prevented by superoxide dismutase (SOD) and by apocynin. nNO was higher in HCs than in patients with PCD. EBC H2O2 was lower in HC than in patients with PCD. The PCD airway epithelium does not optimally clear antigens and is subject to oxidative and nitrosative stress. Oxidation associated with antigen stasis could represent a therapeutic target in PCD, one with convenient monitoring biomarkers.NEW & NOTEWORTHY The PCD airway epithelium does not optimally clear antigens, and antigen exposure can lead to NO oxidation and nitrosative stress. Oxidation caused by antigen stasis could represent a therapeutic target in PCD, and there are convenient monitoring biomarkers.

In Vitro anthelmintic efficacy of medicinal plant essential oils against Marshallagia marshalli: Evidence on oxidative/nitrosative stress biomarkers, DNA damage, and egg hatchability.

One of the major public health problems is drug resistance in parasitic diseases. It is therefore important to find new active ingredients to combat parasites. Herbal products such as essential oils (EOs) may show promise in treating infections caused by gastrointestinal nematodes (GINs). This study investigated the in vitro anthelmintic activity of the EOs of Lavandula angustifolia and Quercus infectoria against Marshallagia marshalli. The in vitro study was based on an egg hatch test (EHT), adult and larval motility inhibition tests, DNA damage, and several biomarkers of oxidative/nitrosative stress, including superoxide dismutase [SOD], catalase [CAT], and glutathione peroxidase [GSH -Px], protein carbonylation [PCO], malondialdehyde [MDA], total antioxidant status [TAS], and nitric oxide levels [NO]. Different concentrations of Lavandula angustifolia and Quercus infectoria EOs (1, 5, 10, 25 and 50 mg/ml) were used to determine the anthelmintic effect on three stages of the life cycle of M. marshalli, i.e. eggs, larvae and adult parasites, for 24 hr. The results showed that EOs of L. angustifolia and Q. infectoria play an important role as anthelmintics. These essential oils significantly reduced the egg hatching and motility of larval and adult worms. This anthelmintic effect is dependent on concentration and time. Furthermore, the EOs of L. angustifolia and Q. infectoria caused oxidative/nitrosative stress (reduced SOD, GSH-Px and CAT and increased MDA, PCO and NO) and DNA damage, thereby providing significant antihelminthic effects. Based on the results, it seems that the EOs extracted from L. angustifolia and Q. infectoria may be effective in the control and treatment of M. marshalli infections. Further research is needed to investigate their potential for in vivo use in the treatment of parasitic infections.

The Use of Nitrosative Stress Molecules as Potential Diagnostic Biomarkers in Multiple Sclerosis.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) of still unclear etiology. In recent years, the search for biomarkers facilitating its diagnosis, prognosis, therapy response, and other parameters has gained increasing attention. In this regard, in a previous meta-analysis comprising 22 studies, we found that MS is associated with higher nitrite/nitrate (NOx) levels in the cerebrospinal fluid (CSF) compared to patients with non-inflammatory other neurological diseases (NIOND). However, many of the included studies did not distinguish between the different clinical subtypes of MS, included pre-treated patients, and inclusion criteria varied. As a follow-up to our meta-analysis, we therefore aimed to analyze the serum and CSF NOx levels in clinically well-defined cohorts of treatment-naïve MS patients compared to patients with somatic symptom disorder. To this end, we analyzed the serum and CSF levels of NOx in 117 patients (71 relapsing-remitting (RR) MS, 16 primary progressive (PP) MS, and 30 somatic symptom disorder). We found that RRMS and PPMS patients had higher serum NOx levels compared to somatic symptom disorder patients. This difference remained significant in the subgroup of MRZ-negative RRMS patients. In conclusion, the measurement of NOx in the serum might indeed be a valuable tool in supporting MS diagnosis.

A mitochondrial nexus in major depressive disorder: Integration with the psycho-immune-neuroendocrine network.

Nervous system processes, including cognition and affective state, fundamentally rely on mitochondria. Impaired mitochondrial function is evident in major depressive disorder (MDD), reflecting cumulative detrimental influences of both extrinsic and intrinsic stressors, genetic predisposition, and mutation. Glucocorticoid 'stress' pathways converge on mitochondria; oxidative and nitrosative stresses in MDD are largely mitochondrial in origin; both initiate cascades promoting mitochondrial DNA (mtDNA) damage with disruptions to mitochondrial biogenesis and tryptophan catabolism. Mitochondrial dysfunction facilitates proinflammatory dysbiosis while directly triggering immuno-inflammatory activation via released mtDNA, mitochondrial lipids and mitochondria associated membranes (MAMs), further disrupting mitochondrial function and mitochondrial quality control, promoting the accumulation of abnormal mitochondria (confirmed in autopsy studies). Established and putative mechanisms highlight a mitochondrial nexus within the psycho-immune neuroendocrine (PINE) network implicated in MDD. Whether lowering neuronal resilience and thresholds for disease, or linking mechanistic nodes within the MDD pathogenic network, impaired mitochondrial function emerges as an important risk, a functional biomarker, providing a therapeutic target in MDD. Several treatment modalities have been demonstrated to reset mitochondrial function, which could benefit those with MDD.

Ghrelin misbalance affects mice embryo implantation and pregnancy success by uterine immune dysregulation and nitrosative stress.

Introduction: In a previous study we found that ghrelin (Ghrl) misbalance during the peri-implantation period significantly impaired fetus development. In this study we aimed to evaluate the putative mechanisms underlying these effects, including embryo implantation success, uterine nitric oxide synthase (NOS) activity, nitric oxide synthesis and the inflammatory/immune uterine profile. Methods: Ghrelin misbalance was induced by injecting 4nmol/animal/day of Ghrl (hyperghrelinemia) or 6nmol/animal/day of a Ghrl antagonist (Ant: (D-Lys3)GHRP-6) from day 3 to 8 of pregnancy. Control animals (C) were injected with de vehicle. Females were euthanized at pregnancy day 8 and their uteri excised in order to evaluate: the percentage of reabsorbed embryos (microscopically), eNOS, iNOS and nytrotirosine expression (by immunohistochemistry), nitrite synthesis (by Griess technique), VEGF, IL-10, IL-17, IL-6, MMP9 and GM-CSF expression (by qPCR) and leukocyte infiltration by flow cytometry (evaluating T cells, NK cells, granulocytes, dendritic cells and macrophages). Results: Ant-treatment significantly increased the percentage of reabsorbed embryos and the uterine expression of eNOS, iNOS and nytrotirosine. (D-Lys3)GHRP-6-treatment increased also the expression of the inflammatory cytokines IL-6, IL-17 and MMP9, and decreased that of IL-10 (anti-inflammatory). Moreover, Ant-treatment increased also the NK cells population and that of CD11b+ dendritic cells; and decreased T cells percentages. Similarly, hyperghrelinemia showed a significant increase vs. C on eNOS, iNOS and nytrotirosineuterine expression and a decrease in T cells percentages. Conclusion: Ghrl misbalance during the peri-implantation period induces pro-inflammatory changes and nitrosative stress in the gravid uterus, impairing significantly embryo implantation and/or development.

Evaluation of Nitrosative/Oxidative Stress and Inflammation in Heart Failure with Preserved and Reduced Ejection Fraction.

Heart failure (HF) is a complex syndrome characterized by impaired cardiac function. Two common subtypes of HF include heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF). In this study, we aimed to evaluate and compare the plasma levels of 3-nitrotyrosine (3-NT)-as a marker of nitrosative/oxidative stress and myeloperoxidase (MPO)-as an indicator of inflammation between HFpEF and HFrEF. Twenty-seven patients diagnosed with HFpEF and twenty-two with HFrEF were enrolled in this study. Additionally, forty-one patients were recruited for the control group. An echocardiographic assessment was conducted, followed by the collection of blood samples from all participants. Subsequently, the levels of 3-NT and MPO were quantified using the ELISA method. Comprehensive clinical characteristics and medical histories were obtained. Circulating levels of 3-NT were significantly higher in the HFpEF patients than in the control and the HFrEF groups. Nitrosative/oxidative stress is significantly intensified in HFpEF but not in HFrEF.

Arginase2 mediates contrast-induced acute kidney injury via facilitating nitrosative stress in tubular cells.

Contrast-induced acute kidney injury(CI-AKI) is the third cause of AKI. Although tubular injury has been regarded as an important pathophysiology of CI-AKI, the underlying mechanism remains elusive. Here, we found arginase2(ARG2) accumulated in the tubules of CI-AKI mice, and was upregulated in iohexol treated kidney tubular cells and in blood samples of CI-AKI mice and patients, accompanied by increased nitrosative stress and apoptosis. However, all of the above were reversed in ARG2 knockout mice, as evidenced by the ameliorated kidney dysfunction and the tubular injury, and decreased nitrosative stress and apoptosis. Mechanistically, HO-1 upregulation could alleviate iohexol or ARG2 overexpression mediated nitrosative stress. Silencing and overexpressing ARG2 was able to upregulate and downregulate HO-1 expression, respectively, while HO-1 siRNA had no effect on ARG2 expression, indicating that ARG2 might inhibit HO-1 expression at the transcriptional level, which facilitated nitrosative stress during CI-AKI. Additionally, CREB1, a transcription factor, bound to the promoter region of ARG2 and stimulated its transcription. Similar findings were yielded in cisplatin- or vancomycin-induced AKI models. Taken together, ARG2 is a crucial target of CI-AKI, and activating CREB1/ARG2/HO-1 axis can mediate tubular injury by promoting nitrosative stress, highlighting potential therapeutic strategy for treating CI-AKI.

Effect of red wheat, aleurone, and testa layers on colon cancer biomarkers, nitrosative stress, and gut microbiome composition in rats.

We previously found greater reduction of colon cancer (CC) biomarkers for red wheat compared to white wheat regardless of refinement state. In the present study we examined whether the phenolic-rich aleurone and testa layers are drivers of chemoprevention by red wheat and their influence on gut microbiota composition using a 1,2-dimethylhydrazine-induced CC rat model. Rats were fed a low-fat diet (16% of energy as fat), high-fat diet (50% of energy as fat), or high-fat diet containing whole red wheat, refined red wheat, refined white wheat, or aleurone- or testa-enriched fractions for 12 weeks. Morphological markers (aberrant crypt foci, ACF) were assessed after methylene blue staining and biochemical markers (3-nitrotyrosine [3-NT], Dclk1) by immunohistochemical determination of staining positivity within aberrant crypts. Gut microbiota composition was evaluated from 16S rRNA gene sequencing of DNA extracted from cecal contents. Relative to the high-fat diet, the whole and refined red wheat, refined white wheat, and testa-enriched fraction decreased ACF, while only the refined red wheat and aleurone-enriched fraction decreased 3-NT. No significant differences were observed for Dclk1. An increase in microbial diversity was observed for the aleurone-enriched fraction (ACE index) and whole red wheat (Inverse Simpson Index). The diet groups significantly modified overall microbiome composition, including altered abundances of Lactobacillus, Mucispirillum, Phascolarctobacterium, and Blautia coccoides. These results suggest that red wheat may reduce CC risk through modifications to the gut microbiota and nitrosative stress, which may be due, in part, to the influence of dietary fiber and the phenolic-rich aleurone layer.

When nitrosative stress hits the endoplasmic reticulum: Possible implications in oxLDL/oxysterols-induced endothelial dysfunction.

Oxidized LDL (oxLDL) and oxysterols are known to play a crucial role in endothelial dysfunction (ED) by inducing endoplasmic reticulum stress (ERS), inflammation, and apoptosis. However, the precise molecular mechanisms underlying these pathophysiological processes remain incompletely understood. Emerging evidence strongly implicates excessive nitric oxide (NO) production in the progression of various pathological conditions. The accumulation of reactive nitrogen species (RNS) leading to nitrosative stress (NSS) and aberrant protein S-nitrosylation contribute to NO toxicity. Studies have highlighted the involvement of NSS and S-nitrosylation in perturbing ER signaling through the modification of ER sensors and resident isomerases in neurons. This review focuses on the existing evidence that strongly associates NO with ERS and the possible implications in the context of ED induced by oxLDL and oxysterols. The potential effects of perturbed NO synthesis on signaling effectors linking NSS with ERS in endothelial cells are discussed to provide a conceptual framework for further investigations and the development of novel therapeutic strategies targeting ED.

Vascular nitrosative stress in hypertension induced by fetal undernutrition in rats

Fetal undernutrition predisposes to hypertension development. Since nitric oxide (NO) is a key factor in blood pressure control, we aimed to investigate the role of NO alterations in hypertension induced by fetal undernutrition in rats. Male and female offspring from dams exposed to undernutrition during the second half of gestation (MUN) were studied at 21 days (normotensive) and 6 months of age (hypertension developed only in males). In aorta, we analyzed total and phosphorylated endothelial NO synthase (eNOS, p-eNOS), 3-nitrotyrosine (3-NT), and Nrf2 (Western blot). In plasma we assessed L-arginine, asymmetric and symmetric dimethylarginine (ADMA, SDMA; LC–MS/MS), nitrates (NOx, Griess reaction), carbonyl groups, and lipid peroxidation (spectrophotometry). In iliac arteries, we studied superoxide anion production (DHE staining, confocal microscopy) and vasodilatation to acetylcholine (isometric tension). Twenty-one-day-old MUN offspring did not show alterations in vascular e-NOS or 3NT expression, plasma L-Arg/ADMA ratio, or NOx. Compared to control group, 6-month-old MUN rats showed increased aortic expression of p-eNOS/eNOS and 3-NT, being Nrf2 expression lower, elevated plasma L-arginine/ADMA, NOx and carbonyl levels, increased iliac artery DHE staining and reduced acetylcholine-mediated relaxations. These alterations in MUN rats were sex-dependent, affecting males. However, females showed some signs of endothelial dysfunction. We conclude that increased NO production in the context of a pro-oxidative environment, leads to vascular nitrosative damage and dysfunction, which can participate in hypertension development in MUN males. Females show a better adaptation, but signs of endothelial dysfunction, which can explain hypertension in ageing. (AU)

Nitrosative stress-based specific evaluation of creatine use in combination with aerobic running exercise at different speeds: a preclinical study in mice.

OBJECTIVE: In acute and chronic aerobic exercise, skeletal muscle and liver are the main organs that adapt and regulate metabolic activity. The levels of nitrosative stress caused by exercise in these organs are extremely important in the continuity of exercise, its health-promoting benefits, and the evaluation of therapeutic efficacy. In this study, nitrosative stress levels were investigated in musculus quadriceps femoris tissue and liver tissue of mice that were given low and high-speed aerobic running exercise and also received Cr supplementation. MATERIALS AND METHODS: In this study, nitrosative stress levels were investigated in the muscle/liver tissue of 42 BALB/c mice that were given low and high-speed aerobic running exercise and creatine monohydrate (Cr) (40 mg/kg of diet daily) supplementation with exercise. The study included six groups with and without Cr supplementation, low-speed aerobic running, high-speed aerobic running, and no exercise. The mice in groups with low-speed and high-speed aerobic exercise with and without Cr supplementation were run on the treadmill for 8 weeks. Then, nitric oxide (NO·), nitric oxide synthase (NOS), and peroxynitrite (ONOO-) levels in muscle/liver tissue were measured by spectrophotometric method. RESULTS: It was found that the nitrosative stress level in the groups that did low and high-speed aerobic running exercises increased compared to the group that did not exercise. It was found that NO· decreased NOS activity and ONOO- level increased in muscle tissues of low and high-speed aerobic exercise groups that received Cr supplementation compared to those that did not. However, NO· and ONOO- levels in liver tissue decreased while NOS activity did not change. The lowest level of nitrosative stress in both muscle and liver tissue was found in the low-speed exercise group receiving Cr supplementation. CONCLUSIONS: Although supplements in exercise are an important component, the simultaneously measured nitrosative stress level is critical in determining the optimal exercise.

Modulation of the activation of endothelial nitric oxide synthase and nitrosative stress biomarkers by aspirin triggered lipoxins: A possible mechanism of action of aspirin in the antiphospholipid syndrome.

PROBLEM: Antiphospholipid syndrome (APS) is characterized by the clinical manifestation of vascular thrombosis (VT) or pregnancy morbidity (PM) and antiphospholipid antibodies (aPL) that can modify the nitric oxide production. Low-dose aspirin is used in the prevention and treatment of diverse alterations of pregnancy. One of the mechanisms of action of aspirin is to induce the production of aspirin-triggered-lipoxins (ATL). The aim of this study was to evaluate the modulatory effect of ATL over the activation of endothelial nitric oxide synthase (eNOS) and nitrosative stress biomarkers induced by aPL. METHODS: We used polyclonal IgG and sera from women with aPL and PM/VT or VT only, and from women with PM only and positive for non-criteria aPL (SN-OAPS). In these sera, biomarkers of nitrosative stress (nitrites and nitrotyrosine) were measured. The protein expression of nitrotyrosine and the phosphorylation of eNOS (at Ser1177) were estimated in human umbilical vein endothelial cells (HUVECs) stimulated with polyclonal IgG with or without ATL. RESULTS: Women with SN-OAPS showed increased circulating levels of nitrites and nitrotyrosine. Likewise, polyclonal IgG from either SN-OAPS or VT patients stimulated nitrotyrosine expression in HUVECs. ATL decreased the nitrotyrosine expression induced by polyclonal IgG from the SN-OAPS group. ATL also recovered the reduced eNOS phosphorylation at Ser1177 in HUVECs stimulated with polyclonal IgG from women with PM/VT or SN-OAPS. CONCLUSIONS: Increased nitrosative stress present in serum of women with SN-OAPS is associated with IgG-mediated impaired endothelial NO synthesis in endothelial cells. ATL prevent these cellular changes.

Staphylococcus epidermidis biofilms undergo metabolic and matrix remodeling under nitrosative stress.

Staphylococcus epidermidis is a commensal skin bacterium that forms host- and antibiotic-resistant biofilms that are a major cause of implant-associated infections. Most research has focused on studying the responses to host-imposed stresses on planktonic bacteria. In this work, we addressed the open question of how S. epidermidis thrives on toxic concentrations of nitric oxide (NO) produced by host innate immune cells during biofilm assembly. We analyzed alterations of gene expression, metabolism, and matrix structure of biofilms of two clinical isolates of S. epidermidis, namely, 1457 and RP62A, formed under NO stress conditions. In both strains, NO lowers the amount of biofilm mass and causes increased production of lactate and decreased acetate excretion from biofilm glucose metabolism. Transcriptional analysis revealed that NO induces icaA, which is directly involved in polysaccharide intercellular adhesion (PIA) production, and genes encoding proteins of the amino sugar pathway (glmM and glmU) that link glycolysis to PIA synthesis. However, the strains seem to have distinct regulatory mechanisms to boost lactate production, as NO causes a substantial upregulation of ldh gene in strain RP62A but not in strain 1457. The analysis of the matrix components of the staphylococcal biofilms, assessed by confocal laser scanning microscopy (CLSM), showed that NO stimulates PIA and protein production and interferes with biofilm structure in a strain-dependent manner, but independently of the Ldh level. Thus, NO resistance is attained by remodeling the staphylococcal matrix architecture and adaptation of main metabolic processes, likely providing in vivo fitness of S. epidermidis biofilms contacting NO-proficient macrophages.

Biomarkers of nitrosative stress in exhaled breath condensate and serum among patients with cystic fibrosis.

PURPOSE: The measurement of biomarkers in exhaled breath condensate (EBC) offers a non-invasive way to assess airway disease and can be easily done in a clinical setting among patients with cystic fibrosis (CF). The role of oxidative and nitrosative stress in the complex pathophysiology of CF is widely accepted and biomarkers of oxidative and nitrosative stress can be measured in the serum and EBC. To our knowledge, this is the first study to assess markers of nitrosative stress in EBC and serum, collected simultaneously from the CF patients. PATIENTS AND METHODS: Paired EBC and serum samples were collected from 36 stable patients with CF and 14 healthy controls. Markers of nitrosative stress ‒ 3-nitrotyrosine and nitrate/nitrite were measured in the EBC and serum using an enzyme-linked immunosorbent assay. RESULTS: We found no differences in 3-nitrotyrosine and nitrate/nitrite in the EBC of patients with CF as compared to healthy controls (125.37 â€‹± â€‹3.29 vs. 126.24 â€‹± â€‹2.21 â€‹nmol/L, p â€‹= â€‹0.218; 12.66 â€‹± â€‹7.23 vs. 8.79 â€‹± â€‹4.83 â€‹µmol/L, p â€‹= â€‹0.133, respectively). Furthermore, 3-nitrotyrosine and nitrate/nitrite were significantly higher in the serum of patients with CF as compared to the healthy controls (0.13 â€‹± â€‹0.02 vs. 0.11 â€‹± â€‹0.01 â€‹nmol/mg protein, p â€‹= â€‹0.003; 70.78 â€‹± â€‹22.55 vs. 53.08 â€‹± â€‹8.5 â€‹µmol/L, p â€‹= â€‹0.009, respectively). No correlations were found between the markers determined in the EBC and serum. CONCLUSIONS: The results of the EBC nitrosative stress biomarkers should be interpreted with caution, especially in patients with stable disease, as the EBC values may be independent on levels of circulating markers that are elevated in the serum of patients with stable CF.

Swamp eel aldehyde reductase is involved in response to nitrosative stress via regulating NO/GSH levels.

Aldehyde reductase (ALR) plays key roles in the detoxification of toxic aldehyde. In this study, the authors cloned the swamp eel ALR gene using rapid amplification of cDNA ends-PCR (RACE-PCR). The recombinant protein (rALR) was expressed in Escherichia coli and purified using a Ni2+ -NTA chelating column. The rALR protein exhibited efficient reductive activity towards several aldehydes, ketones and S-nitrosoglutathione (GSNO). A spot assay suggested that the recombinant E. coli strain expressing rALR showed better resistance to formaldehyde, sodium nitrite and GSNO stress, suggesting that swamp eel ALR is crucial for redox homeostasis in vivo. Consequently, the authors investigated the effect of rALR on the oxidative parameters of the liver in swamp eels challenged with Aeromonas hydrophila. The hepatic glutathione (GSH) content significantly increased, and the hepatic NO content and levels of reactive oxygen species and reactive nitrogen species significantly decreased when rALR was administered. In addition, the mRNA expression of hepatic Alr, HO1 and Nrf2 was significantly upregulated, whereas the expression levels of NF-κB, IL-1ß and NOS1 were significantly downregulated in the rALR-administered group. Collectively, these results suggest that ALR is involved in the response to nitrosative stress by regulating GSH/NO levels in the swamp eel.

Decline in nitrosative stress drives antibiotic persister regrowth during infection.

Internalization of pathogenic bacteria by macrophages results in formation of antibiotic-tolerant persisters. These cells are maintained in a non-growing state for extended periods of time, and it is assumed that their growth resumption causes infection relapse after cessation of antibiotic treatment. Despite this clinical relevance, the signals and conditions that drive persister regrowth during infection are not yet understood. Here, we found that after persister formation in macrophages, host reactive nitrogen species (RNS) produced in response to Salmonella infection lock persisters in growth arrest by intoxicating their TCA cycle, lowering cellular respiration and ATP production. Intracellular persisters resume growth when macrophage RNS production subsides and functionality of their TCA cycle is regained. Persister growth resumption within macrophages is slow and heterogeneous, dramatically extending the time the persister reservoir feeds infection relapse. Using an inhibitor of RNS production, we can force recalcitrant bacteria to regrow during antibiotic treatment, thereby facilitating their eradication.

The influence of ambient temperature and polyphenols from plant leaves on growth and the response to oxidative and nitrosative stress in African nightcrawler earthworm, Eudrilus eugeniae (Kinberg, 1867).

Earthworms are one of the organisms that may be affected by climate change. Finding ways to help them deal with this problem is, therefore, important and necessary. The objective of this experiment was to understand the influence of ambient temperature and polyphenols from mulberry (Morus alba L.), almond (Terminalia catappa L.) and cassava (Manihot esculenta (L.) Crantz) leaves on growth, ferric reducing antioxidant power (FRAP), malondialdehyde (MDA), hydrogen peroxide (H2O2) and nitric oxide (NO) concentration of the African night crawler, Eudrilus eugeniae (Kinberg, 1867) earthworm. The earthworms were cultured in 2 different conditions of ambient temperature, and in 4 types of substrate i.e. dairy cow faeces (BS), dairy cow faeces + mulberry leaves (BS + MA), almond leaves (BS + TC), and cassava leaves (BS + ME), respectively. At week 2 of the experiment, body weight, FRAP, MDA, H2O2 and NO were measured in the earthworms. It was found that the body weight gain (BWG) of the earthworms cultured in BS at cyclical temperature (26 + 1oC - 34 + 1oC - 26 + 1oC, CyT) was higher than the constant temperature (26 ± 1 °C, CoT) (P < 0.05). FRAP of earthworms cultured in BS + TC was higher than in other groups (P < 0.05). MDA of earthworms cultured at CyT was higher than ambient temperature at CoT (P < 0.05). At CyT, the MDA of earthworms cultured in BS + MA was higher than that of those cultured in BS, BS + TC and BS + ME (P < 0.05). NO of earthworms at CoT was higher than at CyT(P < 0.05). At CoT, the NO of earthworms cultured in BS + TC was lower than that of those cultured in BS + MA and BS + ME (P < 0.05). H2O2 of earthworms at CoT was higher than those at CyT (P < 0.05). The level of H2O2 of the earthworms cultured in BS + ME at CoT was higher than at CyT (P < 0.05). In addition, the H2O2 of earthworms cultured in both ambient temperatures and cultured in BS + MA was higher than the other groups (P < 0.05). These phenomena indicated that low and high ambient temperatures induced nitrosative and oxidative stress in earthworms, respectively. Mulberry leaves are toxic to earthworms. On the other hand, almond leaves could reduce nitrosative stress in earthworms. While at the CoT, cassava leaves induced the production of H2O2 in the earthworms.