Atmospheric nitrogen dioxide suppresses the activity of phytochrome interacting factor 4 to suppress hypocotyl elongation.

MAIN CONCLUSION: Ambient concentrations of atmospheric nitrogen dioxide (NO2) inhibit the binding of PIF4 to promoter regions of auxin pathway genes to suppress hypocotyl elongation in Arabidopsis. Ambient concentrations (10-50 ppb) of atmospheric nitrogen dioxide (NO2) positively regulate plant growth to the extent that organ size and shoot biomass can nearly double in various species, including Arabidopsis thaliana (Arabidopsis). However, the precise molecular mechanism underlying NO2-mediated processes in plants, and the involvement of specific molecules in these processes, remain unknown. We measured hypocotyl elongation and the transcript levels of PIF4, encoding a bHLH transcription factor, and its target genes in wild-type (WT) and various pif mutants grown in the presence or absence of 50 ppb NO2. Chromatin immunoprecipitation assays were performed to quantify binding of PIF4 to the promoter regions of its target genes. NO2 suppressed hypocotyl elongation in WT plants, but not in the pifq or pif4 mutants. NO2 suppressed the expression of target genes of PIF4, but did not affect the transcript level of the PIF4 gene itself or the level of PIF4 protein. NO2 inhibited the binding of PIF4 to the promoter regions of two of its target genes, SAUR46 and SAUR67. In conclusion, NO2 inhibits the binding of PIF4 to the promoter regions of genes involved in the auxin pathway to suppress hypocotyl elongation in Arabidopsis. Consequently, PIF4 emerges as a pivotal participant in this regulatory process. This study has further clarified the intricate regulatory mechanisms governing plant responses to environmental pollutants, thereby advancing our understanding of how plants adapt to changing atmospheric conditions.

Nitrous oxide reduction by two partial denitrifying bacteria requires denitrification intermediates that cannot be respired.

Denitrification is a form of anaerobic respiration wherein nitrate (NO3-) is sequentially reduced via nitrite (NO2-), nitric oxide, and nitrous oxide (N2O) to dinitrogen gas (N2) by four reductase enzymes. Partial denitrifying bacteria possess only one or some of these four reductases and use them as independent respiratory modules. However, it is unclear if partial denitrifiers sense and respond to denitrification intermediates outside of their reductase repertoire. Here, we tested the denitrifying capabilities of two purple nonsulfur bacteria, Rhodopseudomonas palustris CGA0092 and Rhodobacter capsulatus SB1003. Each had denitrifying capabilities that matched their genome annotation; CGA0092 reduced NO2- to N2, and SB1003 reduced N2O to N2. For each bacterium, N2O reduction could be used both for electron balance during growth on electron-rich organic compounds in light and for energy transformation via respiration in darkness. However, N2O reduction required supplementation with a denitrification intermediate, including those for which there was no associated denitrification enzyme. For CGA0092, NO3- served as a stable, non-catalyzable molecule that was sufficient to activate N2O reduction. Using a ß-galactosidase reporter, we found that NO3- acted, at least in part, by stimulating N2O reductase gene expression. In SB1003, NO2- but not NO3- activated N2O reduction, but NO2- was slowly removed, likely by a promiscuous enzyme activity. Our findings reveal that partial denitrifiers can still be subject to regulation by denitrification intermediates that they cannot use.IMPORTANCEDenitrification is a form of microbial respiration wherein nitrate is converted via several nitrogen oxide intermediates into harmless dinitrogen gas. Partial denitrifying bacteria, which individually have some but not all denitrifying enzymes, can achieve complete denitrification as a community by cross-feeding nitrogen oxide intermediates. However, the last intermediate, nitrous oxide (N2O), is a potent greenhouse gas that often escapes, motivating efforts to understand and improve the efficiency of denitrification. Here, we found that at least some partial denitrifying N2O reducers can sense and respond to nitrogen oxide intermediates that they cannot otherwise use. The regulatory effects of nitrogen oxides on partial denitrifiers are thus an important consideration in understanding and applying denitrifying bacterial communities to combat greenhouse gas emissions.

Effects of Acute Beetroot Juice and Sodium Nitrate on Selected Blood Metabolites and Response to Transient Ischemia: A Crossover Randomized Clinical Trial.

BACKGROUND: Modification of the nitrate (NO3)-nitrite (NO2)-nitric oxide (NO) pathway can be induced by oral intake of inorganic NO3 (NIT) or NO3-rich products, such as beetroot juice (BRJ). OBJECTIVES: The primary aim of this study was to evaluate the plasma changes in betaine, choline, trimethylamine (TMA), trimethylamine N-oxide (TMAO), and NO3/NO2 (NOx) concentrations over 4 h after single oral ingestion of NIT or BRJ. The flow-mediated skin fluorescence (FMSF) method was applied to measure the changes in nicotinamide adenine dinucleotide reduced form (NADH) in response to transient ischemia and reperfusion. We hypothesized that various sources of NO3 may differently affect endothelial and mitochondrial functions in healthy human subjects. METHODS: In a randomized crossover trial, 8 healthy young adults ingested 800 mg NO3 from either NIT or BRJ on 2 separate days with ≥3 d apart. Venous blood samples were collected every hour, and FMSF determination was applied bihourly. RESULTS: Plasma betaine and choline concentrations peaked at 1 h after BRJ ingestion, and remained significantly higher than baseline values at all time points (P < 0.001 and P < 0.001, compared to preingestion values). Over time, BRJ was more effective in increasing NOx compared with NIT (fixed-trial effect P < 0.001). Baseline fluorescence decreased after both NIT and BRJ consumption (fixed-time effect P = 0.005). Transient ischemia and reperfusion response increased because of NO3 consumption (fixed-time effect P = 0.003), with no differences between trials (P = 0.451; P = 0.912; P = 0.819 at 0, 2, and 4 h, respectively). CONCLUSIONS: Acute ingestion of BRJ elevated plasma betaine and choline, but not TMA and TMAO. Moreover, plasma NOx levels were higher in the BRJ trial than in the NIT trial. Various sources of NO3 positively affected endothelial and mitochondrial functions. This trial was registered at clinicaltrials.gov as NCT05004935.

Effect of nitro-conjugated linoleic acid on the inflammatory response of murine macrophages activated with lipopolysaccharide derived from Prevotella intermedia.

Nitro-conjugated linoleic acid (NO2-CLA) has been observed to manifest salutary signaling responses, including anti-inflammatory and antioxidant properties. Here, the authors have explored the influence and underlying mechanisms of NO2-CLA on the proinflammatory reaction of murine macrophages that were challenged with lipopolysaccharide (LPS) derived from Prevotella intermedia, a putative periodontopathic bacterium. Treatment of LPS-activated RAW264.7 cells with NO2-CLA notably dampened the secretion of iNOS-derived NO, IL-1ß and IL-6 as well as their gene expressions and significantly enhanced the markers for M2 macrophage polarization. NO2-CLA promoted the HO-1 expression in cells challenged with LPS, and tin protoporphyrin IX, an HO-1 inhibitor, significantly reversed the NO2-CLA-mediated attenuation of NO secretion, but not IL-1ß or IL-6. We found that cells treated with NO2-CLA significantly increased mRNA expression of PPAR-γ compared to control cells, and NO2-CLA significantly reverted the decrease in PPAR-γ mRNA caused by LPS. Nonetheless, antagonists to PPAR-γ were unable to reverse the NO2-CLA-mediated suppression of inflammatory mediators. In addition, NO2-CLA did not alter the p38 and JNK activation elicited by LPS. Both NF-κB reporter activity and IκB-α degradation caused by LPS were notably diminished by NO2-CLA. NO2-CLA was observed to interrupt the nuclear translocation and DNA binding of p50 subunits caused by LPS with no obvious alterations in p65 subunits. Further, NO2-CLA attenuated the phosphorylation of STAT1/3 elicited in response to LPS. We propose that NO2-CLA could be considered as a possible strategy for the therapy of periodontal disease, although additional researches are certainly required to confirm this.

Ageing modifies acute resting blood pressure responses to incremental consumption of dietary nitrate: a randomised, cross-over clinical trial.

Beetroot (BR) is a rich source of nitrate (NO3-) that has been shown to reduce blood pressure (BP). Yet, no studies have examined the vascular benefits of BR in whole-food form and whether the effects are modified by age. This study was a four-arm, randomised, open-label, cross-over design in twenty-four healthy adults (young n 12, age 27 ± 4 years, old n 12, age 64 ± 5 years). Participants consumed whole-cooked BR at portions of (NO3- content in brackets) 100 g (272 mg), 200 g (544 mg) and 300 g (816 mg) and a 200-ml solution containing 1000 mg of potassium nitrate (KNO3) on four separate occasions over a 4-week period (≥7-d washout period). BP, plasma NO3- and nitrite (NO2-) concentrations, and post-occlusion reactive hyperaemia via laser Doppler, were measured pre- and up to 5-h post-intervention. Data were analysed by repeated-measures ANOVA. Plasma NO2- concentrations were higher in the young v. old at baseline and post-intervention (P < 0·05). All NO3- interventions decreased systolic and diastolic BP in young participants (P < 0·05), whereas only KNO3 (at 240-300 min post-intake) significantly decreased systolic (-4·8 mmHg, -3·5 %, P = 0·024) and diastolic (-5·4 mmHg, -6·5 %, P = 0·007) BP in older participants. In conclusion, incremental doses of dietary NO3- reduced systolic and diastolic BP in healthy young adults whereas in the older group a significant decrease was only observed with the highest dose. The lower plasma NO2- concentrations in older participants suggest that there may be mechanistic differences in the production of NO from dietary NO3- in young and older populations.

Antimicrobial and Antibiofilm Activity of Copper-Based Metallic Compounds Against Bacteria Related with Healthcare-Associated Infections.

Health care-associated infections (HAIs) contribute to a significant rate of morbidity, mortality, and financial burden on health systems. These infections are caused by multidrug-resistant bacteria that produce biofilm as the main virulence factor. This study aimed to evaluate the effect of the copper-based metallic compounds [Cu(phen)(pz)NO2]Cl (I), [Cu(bpy)(pz)(NO2)]Cl (II), and [Cu(phen)(INA)NO2]Cl (III), where phen = phenanthroline, bpy = bipyridine, pz = pyrazinamide, and INA = isonicotinic acid, against planktonic cells and biofilms formation of Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli. The susceptibility of the microorganisms was evaluated by minimum inhibitory concentration (MIC), minimum bacterial concentration (MBC), and time-kill curve assay on planktonic cells. The biofilm formation was evaluated by biomass quantification through staining with crystal violet (CV), colony-forming units (CFUs) quantification, and biofilm metabolic activity determination by XTT assay. The compounds showed bacteriostatic and bactericidal activity on all microorganisms analyzed. Regarding the antibiofilm activity, all metallic compounds were able to reduce significantly the biofilm biomass, colony-forming units, and the metabolic activity of remaining cells, varying the efficient concentration according to the strain analyzed. Interestingly, compounds (I), (II) and (III) did not exhibit DNA degradation activity even with up to 100 µM of these metal complexes. On the other hand, complexes (I) and (III) showed a remarkable capacity to cleave DNA upon addition of glutathione, a reducing agent (CuII/CuI) that leads to reactive oxygen species (ROS) formation. The results presented in this study showed promising antimicrobial and antibiofilm effects.

Effect of Allium cepa extract on total and differential WBC, TP level, oxidant and antioxidant biomarkers, and lung pathology in ovalbumin-sensitized rats.

BACKGROUND: Previous studies have shown that Allium cepa (A. cepa) has relaxant and anti-inflammatory effects. In this research, A. cepa extract was examined for its prophylactic effect on lung inflammation and oxidative stress in sensitized rats. METHODS: Total and differential white blood cell (WBC) count in the blood, serum levels of oxidant and antioxidant biomarkers, total protein (TP) in bronchoalveolar lavage fluid (BALF), and lung pathology were investigated in control group (C), sensitized group (S), and sensitized groups treated with A. cepa and dexamethasone. RESULTS: Total and most differential WBC count, TP, NO2, NO3, MDA (malondialdehyde), and lung pathological scores were increased while lymphocytes, superoxide dismutase (SOD), catalase (CAT), and thiol were decreased in sensitized animals compared to controls (p < 0.01 to p < 0.001). Treatment with all concentrations of extract significantly improved total WBC, TP, NO2, NO3, interstitial fibrosis, and emphysema compared to the S group (p < 0.05 to p < 0.001). Two higher concentrations of the extract significantly decreased neutrophil and monocyte count, malondialdehyde, bleeding and epithelial damage but increased lymphocyte, CAT, and thiol compared to the S group (p < 0.05 to p < 0.001). Dexamethasone treatment also substantially improved most measured parameters (p < 0.05 to p < 0.001), but it did not change eosinophil percentage. It was proposed that A. cepa extract could affect lung inflammation and oxidative stress in sensitized rats.

Nitrite Concentration in the Striated Muscles Is Reversely Related to Myoglobin and Mitochondrial Proteins Content in Rats.

Skeletal muscles are an important reservoir of nitric oxide (NO•) stored in the form of nitrite [NO2-] and nitrate [NO3-] (NOx). Nitrite, which can be reduced to NO• under hypoxic and acidotic conditions, is considered a physiologically relevant, direct source of bioactive NO•. The aim of the present study was to determine the basal levels of NOx in striated muscles (including rat heart and locomotory muscles) with varied contents of tissue nitrite reductases, such as myoglobin and mitochondrial electron transport chain proteins (ETC-proteins). Muscle NOx was determined using a high-performance liquid chromatography-based method. Muscle proteins were evaluated using western-immunoblotting. We found that oxidative muscles with a higher content of ETC-proteins and myoglobin (such as the heart and slow-twitch locomotory muscles) have lower [NO2-] compared to fast-twitch muscles with a lower content of those proteins. The muscle type had no observed effect on the [NO3-]. Our results demonstrated that fast-twitch muscles possess greater potential to generate NO• via nitrite reduction than slow-twitch muscles and the heart. This property might be of special importance for fast skeletal muscles during strenuous exercise and/or hypoxia since it might support muscle blood flow via additional NO• provision (acidic/hypoxic vasodilation) and delay muscle fatigue.

Nitrogen-Doped Titanium Dioxide Mixed with Calcium Peroxide and Methylcellulose for Dental Bleaching under Visible Light Activation.

The available tooth whitening products in the market contain high concentrations of hydrogen peroxide (H2O2) as an active ingredient. Therefore, in order to curb the high H2O2 concentration and instability of liquid H2O2, this study evaluated the efficacy and cytotoxicity of the bleaching gel composed of 10% calcium peroxide (CaO2) and visible-light-activating nitrogen-doped titanium dioxide (N-TiO2) with methyl cellulose as a thickener. Extracted bovine teeth were discolored using coffee and black tea stain solution and were divided into two groups (n = 6). Bleaching was performed thrice on each tooth specimen in both the groups, with one minute of visible light irradiation during each bleaching time. The CIELAB L*a*b* values were measured pre- and post-bleaching. The N-TiO2 calcinated at 350 °C demonstrated a shift towards the visible light region by narrowing the band gap energy from 3.23 eV to 2.85 eV. The brightness (ΔL) and color difference (ΔE) increased as bleaching progressed each time in both the groups. ANOVA results showed that the number of bleaching significantly affected ΔE (p < 0.05). The formulated bleaching gel exhibits good biocompatibility and non-toxicity upon exposure to 3T3 cells. Our findings showed that CaO2-based bleaching gel at neutral pH could be a stable, safe, and effective substitute for tooth whitening products currently available in the market.

Multicenter Ozone Study in oldEr Subjects (MOSES): Part 2. Effects of Personal and Ambient Concentrations of Ozone and Other Pollutants on Cardiovascular and Pulmonary Function.

INTRODUCTION: The Multicenter Ozone Study of oldEr Subjects (MOSES) was a multi-center study evaluating whether short-term controlled exposure of older, healthy individuals to low levels of ozone (O3) induced acute changes in cardiovascular biomarkers. In MOSES Part 1 (MOSES 1), controlled O3 exposure caused concentration-related reductions in lung function with evidence of airway inflammation and injury, but without convincing evidence of effects on cardiovascular function. However, subjects' prior exposures to indoor and outdoor air pollution in the few hours and days before each MOSES controlled O3 exposure may have independently affected the study biomarkers and/or modified biomarker responses to the MOSES controlled O3 exposures. METHODS: MOSES 1 was conducted at three clinical centers (University of California San Francisco, University of North Carolina, and University of Rochester Medical Center) and included healthy volunteers 55 to 70 years of age. Consented participants who successfully completed the screening and training sessions were enrolled in the study. All three clinical centers adhered to common standard operating procedures and used common tracking and data forms. Each subject was scheduled to participate in a total of 11 visits: screening visit, training visit, and three sets of exposure visits consisting of the pre-exposure day, the exposure day, and the post-exposure day. After completing the pre-exposure day, subjects spent the night in a nearby hotel. On exposure days, the subjects were exposed for 3 hours in random order to 0 ppb O3 (clean air), 70 ppb O3, and 120 ppm O3. During the exposure period the subjects alternated between 15 minutes of moderate exercise and 15 minutes of rest. A suite of cardiovascular and pulmonary endpoints was measured on the day before, the day of, and up to 22 hours after each exposure.In MOSES Part 2 (MOSES 2), we used a longitudinal panel study design, cardiopulmonary biomarker data from MOSES 1, passive cumulative personal exposure samples (PES) of O3 and nitrogen dioxide (NO2) in the 72 hours before the pre-exposure visit, and hourly ambient air pollution and weather measurements in the 96 hours before the pre-exposure visit. We used mixed-effects linear regression and evaluated whether PES O3 and NO2 and these ambient pollutant concentrations in the 96 hours before the pre-exposure visit confounded the MOSES 1 controlled O3 exposure effects on the pre- to post-exposure biomarker changes (Aim 1), whether they modified these pre- to post-exposure biomarker responses to the controlled O3 exposures (Aim 2), whether they were associated with changes in biomarkers measured at the pre-exposure visit or morning of the exposure session (Aim 3), and whether they were associated with differences in the pre- to post-exposure biomarker changes independently of the controlled O3 exposures (Aim 4). RESULTS: Ambient pollutant concentrations at each site were low and were regularly below the National Ambient Air Quality Standard levels. In Aim 1, the controlled O3 exposure effects on the pre- to post-exposure biomarker differences were little changed when PES or ambient pollutant concentrations in the previous 96 hours were included in the model, suggesting these were not confounders of the controlled O3 exposure/biomarker difference associations. In Aim 2, effects of MOSES controlled O3 exposures on forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) were modified by ambient NO2 and carbon monoxide (CO), and PES NO2, with reductions in FEV1 and FVC observed only when these concentrations were "Medium" or "High" in the 72 hours before the pre-exposure visit. There was no such effect modification of the effect of controlled O3 exposure on any other cardiopulmonary biomarker.As hypothesized for Aim 3, increased ambient O3 concentrations were associated with decreased pre-exposure heart rate variability (HRV). For example, high frequency (HF) HRV decreased in association with increased ambient O3 concentrations in the 96 hours before the pre-exposure visit (-0.460 ln[ms2]; 95% CI, -0.743 to -0.177 for each 10.35-ppb increase in O3; P = 0.002). However, in Aim 4 these increases in ambient O3 were also associated with increases in HF and low frequency (LF) HRV from pre- to post-exposure, likely reflecting a "recovery" of HRV during the MOSES O3 exposure sessions. Similar patterns across Aims 3 and 4 were observed for LF (the other primary HRV marker), and standard deviation of normal-to-normal sinus beat intervals (SDNN) and root mean square of successive differences in normal-to-normal sinus beat intervals (RMSSD) (secondary HRV markers).Similar Aim 3 and Aim 4 patterns were observed for FEV1 and FVC in association with increases in ambient PM with an aerodynamic diameter ≤ 2.5 µm (PM2.5), CO, and NO2 in the 96 hours before the pre-exposure visit. For Aim 3, small decreases in pre-exposure FEV1 were significantly associated with interquartile range (IQR) increases in PM2.5 concentrations in the 1 hour before the pre-exposure visit (-0.022 L; 95% CI, -0.037 to -0.006; P = 0.007), CO in the 3 hours before the pre-exposure visit (-0.046 L; 95% CI, -0.076 to -0.016; P = 0.003), and NO2 in the 72 hours before the pre-exposure visit (-0.030 L; 95% CI, -0.052 to -0.008; P = 0.007). However, FEV1 was not associated with ambient O3 or sulfur dioxide (SO2), or PES O3 or NO2 (Aim 3). For Aim 4, increased FEV1 across the exposure session (post-exposure minus pre-exposure) was marginally significantly associated with each 4.1-ppb increase in PES O3 concentration (0.010 L; 95% CI, 0.004 to 0.026; P = 0.010), as well as ambient PM2.5 and CO at all lag times. FVC showed similar associations, with patterns of decreased pre-exposure FVC associated with increased PM2.5, CO, and NO2 at most lag times, and increased FVC across the exposure session also associated with increased concentrations of the same pollutants, reflecting a similar recovery. However, increased pollutant concentrations were not associated with adverse changes in pre-exposure levels or pre- to post-exposure changes in biomarkers of cardiac repolarization, ST segment, vascular function, nitrotyrosine as a measure of oxidative stress, prothrombotic state, systemic inflammation, lung injury, or sputum polymorphonuclear leukocyte (PMN) percentage as a measure of airway inflammation. CONCLUSIONS: Our previous MOSES 1 findings of controlled O3 exposure effects on pulmonary function, but not on any cardiovascular biomarker, were not confounded by ambient or personal O3 or other pollutant exposures in the 96 and 72 hours before the pre-exposure visit. Further, these MOSES 1 O3 effects were generally not modified, blunted, or lessened by these same ambient and personal pollutant exposures. However, the reductions in markers of pulmonary function by the MOSES 1 controlled O3 exposure were modified by ambient NO2 and CO, and PES NO2, with reductions observed only when these pollutant concentrations were elevated in the few hours and days before the pre-exposure visit. Increased ambient O3 concentrations were associated with reduced HRV, with "recovery" during exposure visits. Increased ambient PM2.5, NO2, and CO were associated with reduced pulmonary function, independent of the MOSES-controlled O3 exposures. Increased pollutant concentrations were not associated with pre-exposure or pre- to post-exposure changes in other cardiopulmonary biomarkers. Future controlled exposure studies should consider the effect of ambient pollutants on pre-exposure biomarker levels and whether ambient pollutants modify any health response to a controlled pollutant exposure.

Short-Term Exposure to Nitrogen Dioxide Provides Basal Pathogen Resistance.

Nitrogen dioxide (NO2) forms in plants under stress conditions, but little is known about its physiological functions. Here, we explored the physiological functions of NO2 in plant cells using short-term fumigation of Arabidopsis (Arabidopsis thaliana) for 1 h with 10 µL L-1 NO2. Although leaf symptoms were absent, the expression of genes related to pathogen resistance was induced. Fumigated plants developed basal disease resistance, or pattern-triggered immunity, against the necrotrophic fungus Botrytis cinerea and the hemibiotrophic bacterium Pseudomonas syringae Functional salicylic acid and jasmonic acid (JA) signaling pathways were both required for the full expression of NO2-induced resistance against B. cinerea An early peak of salicylic acid accumulation immediately after NO2 exposure was followed by a transient accumulation of oxophytodienoic acid. The simultaneous NO2-induced expression of genes involved in jasmonate biosynthesis and jasmonate catabolism resulted in the complete suppression of JA and JA-isoleucine (JA-Ile) accumulation, which was accompanied by a rise in the levels of their catabolic intermediates 12-OH-JA, 12-OH-JA-Ile, and 12-COOH-JA-Ile. NO2-treated plants emitted the volatile monoterpene α-pinene and the sesquiterpene longifolene (syn. junipene), which could function in signaling or direct defense against pathogens. NO2-triggered B. cinerea resistance was dependent on enhanced early callose deposition and CYTOCHROME P450 79B2 (CYP79B2), CYP79B3, and PHYTOALEXIN DEFICIENT3 gene functions but independent of camalexin, CYP81F2, and 4-OH-indol-3-ylmethylglucosinolate derivatives. In sum, exogenous NO2 triggers basal pathogen resistance, pointing to a possible role for endogenous NO2 in defense signaling. Additionally, this study revealed the involvement of jasmonate catabolism and volatiles in pathogen immunity.

Association between short- and medium-term air pollution exposure and risk of mortality after intravenous thrombolysis for stroke.

The exposure to air pollutants may increase both incidence and mortality of stroke. We aimed to investigate the association of short- and medium-term exposure to particulate matter (PM) and nitrogen dioxide (NO2) with the outcome of intravenous thrombolysis (IVT) for stroke. We conducted a retrospective analysis based on data prospectively collected from 944 consecutive IVT-treated stroke patients. The main outcome measure was 3-month mortality. The secondary outcome measures were causes of neurological deterioration (≥ 1 NIHSS point from baseline or death < 7 days), including intracerebral hemorrhage, cerebral edema (CED), and persistence or new appearance of hyperdense cerebral artery sign. In the adjusted model, higher PM2.5 and PM10 values in the last 3 days and 4 weeks before stroke were independently associated with higher mortality rate [hazard ratio (HR) 1.014, 95% confidence intervals (CI) 1.005-1.024, p = 0.003; HR 1.079, 95% CI 1.055-1.103, p = 0.001; HR 1.019, 95% CI 1.005-1.032, p = 0.008; and HR 1.015, 95% CI 1.004-1.027, p = 0.007; respectively]. Higher PM2.5 and PM10 values in the last 4 weeks were associated with higher CED rate [odd ratio (OR) 1.023, 95% CI 1.007-1.040, p = 0.006; and OR 1.017, 95% CI 1.003-1.032, p = 0.021; respectively]. No significant association between PM or NO2 and other causes of neurological deterioration was observed. Higher exposure to PM in the last 3 days and 4 weeks before stroke may be independently associated with 3-month mortality after IVT. Higher exposure to PM in the last 4 weeks before stroke may also be independently associated with CED after IVT.

Nitro-Oleic Acid Regulates Endothelin Signaling in Human Endothelial Cells.

Nitro-fatty acids are reactive signaling mediators that are formed when unsaturated fatty acids react with nitric oxide or nitric oxide-derived species. Nitro-fatty acids can modify specific signaling pathways via post-translational modifications of Cys residues in key regulatory proteins. One of the signaling cascades activated by nitro-fatty acids is the Keap1-Nrf2 pathway. We have previously studied the effects of nitro-oleic acid (OA-NO2) on the human endothelial cell transcriptome. We observed that endothelin receptor B [ET-B (gene name EDNRB)], the receptor mediating the vasodilatory effects of endothelin-1 (ET-1) is induced by OA-NO2 Inasmuch as ET-1 is one of the key regulators of vascular tone, we chose to examine in more detail the effect of OA-NO2 on endothelin signaling in human endothelial cells. Nrf2 was found to regulate the OA-NO2-induced transcription of ET-B in human and mouse endothelial cells. Furthermore, chromatin immunoprecipitation analysis revealed that OA-NO2 increased the binding of Nrf2 to an antioxidant response element in the enhancer region of the EDNRB gene. In addition, we show that the overexpression of both OA-NO2 and Nrf2 substantially decreased and that Nrf2 silencing increased the ET-1 concentration in the culture media of endothelial cells. The change in the extracellular ET-1 concentration was dependent on ET-B receptor expression. These data suggest that OA-NO2 modulates endothelin signaling by increasing Nrf2-dependent expression of the ET-B receptor in endothelial cells, which in turn mediates the decrease in extracellular ET-1 concentration. Based on these results, we propose that OA-NO2 and Nrf2 may alleviate the vasoconstrictive effects of ET-1 by removing it from the circulation.

The Dose-Response Association between Nitrogen Dioxide Exposure and Serum Interleukin-6 Concentrations.

Systemic inflammation is an integral part of chronic obstructive pulmonary disease (COPD), and air pollution is associated with cardiorespiratory mortality, yet the interrelationships are not fully defined. We examined associations between nitrogen dioxide (NO2) exposure (as a marker of traffic-related air pollution) and pro-inflammatory cytokines, and investigated effect modification and mediation by post-bronchodilator airflow obstruction (post-BD-AO) and cardiovascular risk. Data from middle-aged participants in the Tasmanian Longitudinal Health Study (TAHS, n = 1389) were analyzed by multivariable logistic regression, using serum interleukin (IL)-6, IL-8 and tumor necrosis factor-α (TNF-α) as the outcome. Mean annual NO2 exposure was estimated at residential addresses using a validated satellite-based land-use regression model. Post-BD-AO was defined by post-BD forced expiratory ratio (FEV1/FVC) < lower limit of normal, and cardiovascular risk by a history of either cerebrovascular or ischaemic heart disease. We found a positive association with increasing serum IL-6 concentration (geometric mean 1.20 (95% CI: 1.1 to 1.3, p = 0.001) per quartile increase in NO2). This was predominantly a direct relationship, with little evidence for either effect modification or mediation via post-BD-AO, or for the small subgroup who reported cardiovascular events. However, there was some evidence consistent with serum IL-6 being on the causal pathway between NO2 and cardiovascular risk. These findings raise the possibility that the interplay between air pollution and systemic inflammation may differ between post-BD airflow obstruction and cardiovascular diseases.

Common ragweed (Ambrosia artemisiifolia L.): allergenicity and molecular characterization of pollen after plant exposure to elevated NO2.

Ragweed pollen is the main cause of allergenic diseases in Northern America, and the weed has become a spreading neophyte in Europe. Climate change and air pollution are speculated to affect the allergenic potential of pollen. The objective of this study was to investigate the effects of NO2 , a major air pollutant, under controlled conditions, on the allergenicity of ragweed pollen. Ragweed was exposed to different levels of NO2 throughout the entire growing season, and its pollen further analysed. Spectroscopic analysis showed increased outer cell wall polymers and decreased amounts of pectin. Proteome studies using two-dimensional difference gel electrophoresis and liquid chromatography-tandem mass spectrometry indicated increased amounts of several Amb a 1 isoforms and of another allergen with great homology to enolase Hev b 9 from rubber tree. Analysis of protein S-nitrosylation identified nitrosylated proteins in pollen from both conditions, including Amb a 1 isoforms. However, elevated NO2 significantly enhanced the overall nitrosylation. Finally, we demonstrated increased overall pollen allergenicity by immunoblotting using ragweed antisera, showing a significantly higher allergenicity for Amb a 1. The data highlight a direct influence of elevated NO2 on the increased allergenicity of ragweed pollen and a direct correlation with an increased risk for human health.

Nitrite is the driver, phytohormones are modulators while NO and H2O2 act as promoters of NO2-induced cell death.

This study aimed to understand the molecular mechanisms of nitrogen dioxide (NO2)-induced toxicity and cell death in plants. Exposure of Arabidopsis to high concentrations of NO2 induced cell death in a dose-dependent manner. No leaf symptoms were visible after fumigation for 1 h with 10 parts per million (ppm) NO2 However, 20 ppm NO2 caused necrotic lesion formation and 30 ppm NO2 complete leaf collapse, which had already started during the 1 h fumigation period. NO2 fumigation resulted in a massive accumulation of nitrite and in protein modifications by S-nitrosylation and tyrosine nitration. Nitric oxide (NO) at 30 ppm did not trigger leaf damage or any of the effects observed after NO2 fumigation. The onset of NO2-induced cell death correlated with NO and hydrogen peroxide (H2O2) signaling and a decrease in antioxidants. NO- and H2O2-accumulating mutants were more sensitive to NO2 than wild-type plants. Accordingly, experiments with specific scavengers confirmed that NO and H2O2 are essential promoters of NO2-induced cell death. Leaf injection of 100 mM nitrite caused an increase in S-nitrosylation, NO, H2O2, and cell death suggesting that nitrite functioned as a mediator of NO2-induced effects. A targeted screening of phytohormone mutants revealed a protective role of salicylic acid (SA) signaling in response to NO2 It was also shown that phytohormones were modulators rather than inducers of NO2-induced cell death. The established experimental set-up is a suitable system to investigate NO2 and cell death signaling in large-scale mutant screens.

Nitrogen dioxide regulates organ growth by controlling cell proliferation and enlargement in Arabidopsis.

• To gain more insight into the physiological function of nitrogen dioxide (NO2), we investigated the effects of exogenous NO2 on growth in Arabidopsis thaliana. • Plants were grown in air without NO2 for 1 wk after sowing and then grown for 1-4 wk in air with (designated treated plants) or without (control plants) NO2. Plants were irrigated semiweekly with a nutrient solution containing 19.7 mM nitrate and 10.3 mM ammonium. • Five-week-old plants treated with 50 ppb NO2 showed a ≤ 2.8-fold increase in biomass relative to controls. Treated plants also showed early flowering. The magnitude of the effects of NO2 on leaf expansion, cell proliferation and enlargement was greater in developing than in maturing leaves. Leaf areas were 1.3-8.4 times larger on treated plants than corresponding leaves on control plants. The NO2-induced increase in leaf size was largely attributable to cell proliferation in developing leaves, but was attributable to both cell proliferation and enlargement in maturing leaves. The expression of different sets of genes for cell proliferation and/or enlargement was induced by NO2, but depended on the leaf developmental stage. • Collectively, these results indicated that NO2 regulates organ growth by controlling cell proliferation and enlargement.

Real-world safety and effectiveness of inhaled nitric oxide therapy for pulmonary hypertension during the perioperative period of cardiac surgery: a post-marketing study of 2817 patients in Japan.

OBJECTIVE: To evaluate the real-world safety and effectiveness of inhaled nitric oxide (INOflo® for Inhalation 800 ppm) for perioperative pulmonary hypertension associated with cardiac surgery in Japan. METHODS: This was a prospective, non-interventional, all-case, post-marketing study of pediatric and adult patients who received perioperative INOflo with cardiac surgery from November 2015-December 2020. Safety and effectiveness were monitored from INOflo initiation to 48 h after treatment completion or withdrawal. Safety outcomes included adverse drug reactions, blood methemoglobin concentrations, and inspired nitrogen dioxide concentrations over time. Effectiveness outcomes included changes in central venous pressure among pediatrics, mean pulmonary arterial pressure among adults, and the partial pressure of arterial oxygen/fraction of inspired oxygen ratio (PaO2/FiO2) in both populations. RESULTS: The safety analysis population included 2,817 Japanese patients registered from 253 clinical sites (pediatrics, n = 1375; adults, n = 1442). INOflo was generally well tolerated; 15 and 20 adverse drug reactions were reported in 14 pediatrics (1.0%) and 18 adults (1.2%), respectively. No clinically significant elevations in blood methemoglobin and inspired nitrogen dioxide concentrations were observed. INOflo treatment was associated with significant reductions in both central venous pressure among pediatrics and mean pulmonary arterial pressure among adults, and significant improvements in PaO2/FiO2 among pediatrics and adults with PaO2/FiO2 ≤ 200 at baseline. CONCLUSIONS: Perioperative INOflo treatment was a safe and effective strategy to improve hemodynamics and oxygenation in patients with pulmonary hypertension during cardiac surgery. These data support the use of INOflo for this indication in Japanese clinical practice.

Protective Effect of NO2-OA on Oxidative Stress, Gliosis, and Pro-Angiogenic Response in Müller Glial Cells.

Inflammation and oxidative and nitrosative stress are involved in the pathogenesis of proliferative retinopathies (PR). In PR, a loss of balance between pro-angiogenic and anti-angiogenic factors favors the secretion of vascular endothelial growth factor (VEGF). This vascular change results in alterations in the blood-retinal barrier, with extravasation of plasma proteins such as α2-macroglobulin (α2M) and gliosis in Müller glial cells (MGCs, such as MIO-M1). It is well known that MGCs play important roles in healthy and sick retinas, including in PR. Nitro-fatty acids are electrophilic lipid mediators with anti-inflammatory and cytoprotective properties. Our aim was to investigate whether nitro-oleic acid (NO2-OA) is beneficial against oxidative stress, gliosis, and the pro-angiogenic response in MGCs. Pure synthetic NO2-OA increased HO-1 expression in a time- and concentration-dependent manner, which was abrogated by the Nrf2 inhibitor trigonelline. In response to phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS), NO2-OA prevented the ROS increase and reduced the gliosis induced by α2M. Finally, when hypoxic MGCs were incubated with NO2-OA, the increase in VEGF mRNA expression was not affected, but under hypoxia and inflammation (IL-1ß), NO2-OA significantly reduced VEGF mRNA levels. Furthermore, NO2-OA inhibited endothelial cell (BAEC) tubulogenesis. Our results highlight NO2-OA's protective effect on oxidative damage, gliosis; and the exacerbated pro-angiogenic response in MGCs.

Effects of nitrogen dioxide and anoxia on global gene and protein expression in long-term continuous cultures of Nitrosomonas eutropha C91.

Nitrosomonas eutropha is an ammonia-oxidizing betaproteobacterium found in environments with high ammonium levels, such as wastewater treatment plants. The effects of NO(2) on gene and protein expression under oxic and anoxic conditions were determined by maintaining N. eutropha strain C91 in a chemostat fed with ammonium under oxic, oxic-plus-NO(2), and anoxic-plus-NO(2) culture conditions. Cells remained viable but ceased growing under anoxia; hence, the chemostat was switched from continuous to batch cultivation to retain biomass. After several weeks under each condition, biomass was harvested for total mRNA and protein isolation. Exposure of N. eutropha C91 to NO(2) under either oxic or anoxic conditions led to a decrease in proteins involved in N and C assimilation and storage and an increase in proteins involved in energy conservation, including ammonia monooxygenase (AmoCAB). Exposure to anoxia plus NO(2) resulted in increased representation of proteins and transcripts reflective of an energy-deprived state. Several proteins implicated in N-oxide metabolism were expressed and remained unchanged throughout the experiment, except for NorCB nitric oxide reductase, which was not detected in the proteome. Rather, NorY nitric oxide reductase was expressed under oxic-plus-NO(2) and anoxic-plus-NO(2) conditions. The results indicate that exposure to NO(2) results in an energy-deprived state of N. eutropha C91 and that anaerobic growth could not be supported with NO(2) as an oxidant.