Intracellular Nitric Oxide Levels Research Articles

Asymmetric Dimethylarginine Protects Neurons from Oxygen Glucose Deprivation Insult by Modulating Connexin-36 Expression.

Background Asymmetric dimethylarginine (ADMA) is a nonselective nitric oxide synthase inhibitor. ADMA is thought to inhibit the production of nitric oxide (NO) by neurons after oxygen-glucose deprivation (OGD). The gap junction protein Connexin-36 (cx-36) is involved in the pathophysiology of stroke. We investigated whether ADMA could protect neurons from OGD insults by regulating the expression of cx-36. Methods Cultured rat cortical neuronal cells were used. Neurons were treated with OGD with or without ADMA pretreatment. The lactate dehydrogenase (LDH) release rate was used to assess neuronal injury. Intracellular NO levels were determined using 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate. Western blotting was performed to detect cx-36 expression. Results The LDH release rate increased in the supernatant of neurons after the OGD insult, whereas ADMA treatment reduced the LDH release rate. Intracellular NO levels increased following OGD treatment, and this increase was not inhibited by ADMA treatment. Expression of cx-36 was upregulated in neurons under OGD conditions, and treatment with ADMA downregulated the expression of cx-36. Conclusions ADMA protects neurons from OGD insult, and cx-36 downregulation may be a possible pathway involved in ADMA-mediated neuronal protection.

Endothelial nitric oxide synthase (eNOS)-NO signaling axis functions to promote the growth of prostate cancer stem-like cells

BackgroundAccumulating evidence supports that prostate cancer stem-like cells (PCSCs) play significant roles in therapy resistance and metastasis of prostate cancer. Many studies also show that nitric oxide (NO) synthesized by NO synthases can function to promote tumor progression. However, the exact roles of NOSs and NO signaling in the growth regulation of PCSCs and castration-resistant prostate cancer (CRPC) are still not fully understood.MethodsThe regulatory functions of NOS-NO signaling were evaluated in prostate cancer cells, especially in PCSCs enriched by 3D spheroid culture and CD133/CD44 cell sorting. The molecular mechanisms of NOS-NO signaling in PCSCs growth regulation and tumor metastasis were investigated in PCSCs and mice orthotopic prostate tumor model.ResultsEndothelial NOS (eNOS) exhibited a significant upregulation in high-grade prostate cancer and metastatic CRPC. Xenograft models of CRPC exhibited notable increased eNOS expression and higher intracellular NO levels. PCSCs isolated from various models displayed significant enhanced eNOS-NO signaling. Functional analyses demonstrated that increased eNOS expression could promote in vivo tumorigenicity and metastatic potential of prostate cancer cells. Characterization of eNOS-NO involved downstream pathway which confirmed that enhanced eNOS signaling could promote the growth of PCSCs and antiandrogen-resistant prostate cancer cells via an activated downstream NO-sGC-cGMP-PKG effector signaling pathway. Interestingly, eNOS expression could be co-targeted by nuclear receptor ERRα and transcription factor ERG in prostate cancer cells and PCSCs.ConclusionsEnhanced eNOS-NO signaling could function to promote the growth of PCSCs and also the development of metastatic CRPC. Besides eNOS-NO as potential targets, targeting its upstream regulators (ERRα and ERG) of eNOS-NO signaling could also be the therapeutic strategy for the management of advanced prostate cancer, particularly the aggressive cancer carrying with the TMPRSS2:ERG fusion gene.

Inhibition of interferon-gamma-stimulated melanoma progression by targeting neuronal nitric oxide synthase (nNOS)

Interferon-gamma (IFN-γ) is shown to stimulate melanoma development and progression. However, the underlying mechanism has not been completely defined. Our study aimed to determine the role of neuronal nitric oxide synthase (nNOS)-mediated signaling in IFN-γ-stimulated melanoma progression and the anti-melanoma effects of novel nNOS inhibitors. Our study shows that IFN-γ markedly induced the expression levels of nNOS in melanoma cells associated with increased intracellular nitric oxide (NO) levels. Co-treatment with novel nNOS inhibitors effectively alleviated IFN-γ-activated STAT1/3. Further, reverse phase protein array (RPPA) analysis demonstrated that IFN-γ induced the expression of HIF1α, c-Myc, and programmed death-ligand 1 (PD-L1), in contrast to IFN-α. Blocking the nNOS-mediated signaling pathway using nNOS-selective inhibitors was shown to effectively diminish IFN-γ-induced PD-L1 expression in melanoma cells. Using a human melanoma xenograft mouse model, the in vivo studies revealed that IFN-γ increased tumor growth compared to control, which was inhibited by the co-administration of nNOS inhibitor MAC-3-190. Another nNOS inhibitor, HH044, was shown to effectively inhibit in vivo tumor growth and was associated with reduced PD-L1 expression levels in melanoma xenografts. Our study demonstrates the important role of nNOS-mediated NO signaling in IFN-γ-stimulated melanoma progression. Targeting nNOS using highly selective small molecular inhibitors is a unique and effective strategy to improve melanoma treatment.

The toxicity of cooking oil fumes on human bronchial epithelial cells through ROS-mediated MAPK, NF-κB signaling pathways and NLRP3 inflammasome.

Cooking oil fumes (COFs) are the main pollutants in kitchen and indoor air, which threaten human health. Exposure to COFs may lead to respiratory diseases and impair pulmonary function. To investigate the toxicity of COFs on human bronchial epithelial cells (Beas-2B) and explore the underlying mechanisms, MTT assay was conducted to detect the viability of Beas-2B. Intracellular reactive oxygen species (ROS) levels and nitric oxide (NO) levels were determined with DCFH-DA assay and DAF-FM assay. The expression of genes involved in inflammation were measured with quantitative real-time PCR (qRT-PCR). The phosphorylation and the expression of proteins related to Mitogen-activated protein kinase (MAPK), NF-κB signaling pathways were measured with western blot. Our results revealed that COFs decreased cell viability, increased the ROS levels and NO levels and induced apoptosis in Beas-2B cells. The results of qRT-PCR and western blot showed that the expression of NLRP3, p65, iNOS, IL-1β, and the factors related to oxidative stress and inflammation increased, NF-κB signaling pathway and MAPK signaling pathway were activated. This study provided some useful information to evaluate the toxicity of COFs and revealed the possible mechanism for the damage on respiratory system induced by COFs.

Rheum tanguticum Alleviates Cognitive Impairment in APP/PS1 Mice by Regulating Drug-Responsive Bacteria and Their Corresponding Microbial Metabolites.

Drugs targeting intestinal bacteria have shown great efficacy for alleviating symptoms of Alzheimer’s disease (AD), and microbial metabolites are important messengers. Our previous work indicated that Rheum tanguticum effectively improved cognitive function and reshaped the gut microbial homeostasis in AD rats. However, its therapeutic mechanisms remain unclear. Herein, this study aimed to elaborate the mechanisms of rhubarb for the treatment of AD by identifying effective metabolites associated with rhubarb-responsive bacteria. The results found that rhubarb reduced hippocampal inflammation and neuronal damage in APP/PS1 transgenic (Tg) mice. 16S rRNA sequencing and metabolomic analysis revealed that gut microbiota and their metabolism in Tg mice were disturbed in an age-dependent manner. Rhubarb-responsive bacteria were further identified by real-time polymerase chain reaction (RT-PCR) sequencing. Four different metabolites reversed by rhubarb were found in the position of the important nodes on rhubarb-responsive bacteria and their corresponding metabolites combined with pathological indicators co-network. Furthermore, in vitro experiments demonstrated o-tyrosine not only inhibited the viabilities of primary neurons as well as BV-2 cells, but also increased the levels of intracellular reactive oxygen species and nitric oxide. In the end, the results suggest that rhubarb ameliorates cognitive impairment in Tg mice through decreasing the abundance of o-tyrosine in the gut owing to the regulation of rhubarb-responsive bacteria. Our study provides a promising strategy for elaborating therapeutic mechanisms of bacteria-targeted drugs for AD.

Knockdown of the inducible nitric oxide synthase (NOS2) splicing variant S3 promotes autophagic cell death from nitrosative stress in SW480 human colon cancer cells.

Low levels of nitric oxide (NO) produced by constitutively expressed inducible NO synthase (NOS2) in tumor cells may be an important factor in their development. NOS2 expression is associated with high mortality rates for various cancers. Alternative splicing of NOS2 down-regulates its enzymatic activity, resulting in decreased intracellular NO concentrations. Specific probes to detect alternative splicing of NOS2 were used in two isogenic human colon cancer cell lines derived either from the primary tumor (SW480) or from a lymph node metastasis (SW620). Splicing variant of NOS2 S3, lacking exons 9, 10, and 11, was overexpressed in SW480 cells. NOS2 S3 was silenced in SW480 cells. Flow-cytometry analysis was used to estimate the intracellular NO levels and to analyze the cell cycle of the studied cell lines. Western blot analysis and quantitativereal-time polymerase chain reaction(qRT-PCR) were used to determine apoptosis and autophagy markers. SW480 and SW620 cells expressed NOS2 S3. Overexpression of the NOS2 S3 in SW480 cells downregulated intracellular NO levels. SW480 cells with knocked down NOS2 S3 (referred to as S3C9 cells) had higher intracellular levels of NO compared to the wild-type SW480 cells under serum restriction. Higher NO levels resulted in the loss of viability of S3C9 cells, which was associated with autophagy. Induction of autophagy by elevated intracellular NO levels in S3C9 cells under serum restriction, suggests that autophagy operates as a cytotoxic response to nitrosative stress. The expression of NOS2 S3 plays an important role in regulating intracellular NO production and maintaining viability in SW480 cells under serum restriction. These findings may prove significant in the design of NOS2/NO-based therapies for colon cancer.

Thrombin-Induced Oxidative Phosphorylation in Platelets Is Repressed By Dengue Virus through Inhibition of the PI3K/Akt Signaling Pathway

Background:There are many very important events that condition the progression to severe dengue, these include cytokine storm and a profound thrombocytopenia. Still, the molecular mechanisms underlying these events are not fully understood. Increasing evidence supports the fact that platelet dysfunction, in patients with dengue, could be due to direct interaction between platelets and dengue virus. This event induces changes in platelet function and contributes to pathogenesis. Recent results from our laboratory have shown that thrombin induces oxygen consumption through activation of glycoprotein Ib and subsequent phosphorylation of PI3K and Akt enzymes. Here we want to evaluate changes in these signalling pathways as well as their impact on mitochondrial function.Aims: To evaluate DENV2 effects on platelet function, PI3K/Akt signaling, oxidative phosphorylation and GPIb expression.Methods:Blood samples from healthy volunteers were collected and platelet fraction was obtained. Purified DENV2 particles were incubated with platelets for 2 h at 37ºC. Then, morphological changes, activation and aggregation, mitochondrial function and surface markers expression were tested in platelets.Results:We found that DENV2 induces conspicuous morphological changes on platelets not induced by other arboviruses; increases CD41 (p<0.05), CD62P (8979 ± 1189 vs1723 ± 160 MFI; p<0.05) and reduces CD42b (p<0.5) expressions on platelet surface; reduces agonist-induced platelet aggregation (30 ± 10 vs 71 ± 9%; p<0.05) through increases intracellular NO production (0.4 ± 0.2 vs 0.07 ± 0.01; p<0.05); inhibits thrombin-induced oxidative phosphorylation (45 ± 6.69 vs 348 ± 26.9 ngatomO/min/10 9 platelets; p<0.0001) through PI3K/Akt signaling pathways inhibition (p<0.001). Surprisingly, rDC-SIGN treatment reverts DENV2 effects on platelet activation (9693 ± 1033 vs 2379 ± 390 MFI; p<0.01).Conclusions:DENV2 inhibits thrombin-induced oxidative phosphorylation through PI3K/Akt signaling pathway inhibition. Dengue virus inhibits thrombin-induced phosphorylation at different levels in the platelet. First, by reducing the expression of the GPIb molecule on the platelet surface. This molecule is important for thrombin-induced intracellular signalling. Second, dengue virus inhibits the PI3K/Akat signalling pathway, which is important for the induction of oxidative phosphorylation in mitochondria. And third, dengue virus increases intracellular nitric oxide levels and promotes its excretion into the extracellular milieu, this mechanism could be involved in the inhibition of platelet aggregation processes.These changes induce by short-term interaction of platelets with DENV2 has a direct effect over platelet function and may be correlated with the clinical manifestations observed during severe dengue stage, such as thrombocytopenia and hemorrhages. The overall impact of these changes in signaling pathways related to immune activity remains to be determinate, as does the potential to develop new pharmacological treatments. DisclosuresNo relevant conflicts of interest to declare.

Exploiting the Anti-Inflammatory Potential of White Capsicum Extract by the Nanoformulation in Phospholipid Vesicles.

The peppers of the Capsicum species are exploited in many fields, as flavoring agents in food industry, or as decorative and therapeutic plants. Peppers show a diversified phytochemical content responsible for different biological activities. Synergic activity exerted by high levels of antioxidant compounds is responsible for their important anti-inflammatory property. A methanolic extract was obtained from a new pepper genotype and tested for anti-inflammatory activity. The extract was incorporated into phospholipid vesicles to increase the bioavailability of its bioactive components. Two types of phospholipid vesicles were produced, conventional liposomes and Penetration Enhancer containing Vesicles (PEVs). They were tested in human monoblastic leukemia U937 cell line, showing no cytotoxic effect. The intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels were measured to value the in vitro efficacy of the vesicles in regulating inflammatory responses. Liposomal incorporation significantly reduced ROS levels in extract-treated LPS-activated cells. Furthermore, LC-MS/MS analyses demonstrated that liposomes facilitated the transport of the extract components across the cell membrane and their accumulation into the cytoplasm.

Measurement of Intracellular Nitric Oxide with a Quantitative Mass Spectrometry Probe Approach.

Nitric oxide (NO) is a molecule of physiological importance, and the function of NO depends on its concentration in biological systems, particularly in cells. Concentration-based analysis of intracellular NO can provide insight into its precise role in health and disease. However, current methods for detecting intracellular NO are still inadequate for quantitative analysis. In this study, we report a quantitative mass spectrometry probe approach to measure NO levels in cells. The probe, Amlodipine (AML), comprises a Hantzsch ester group that reacts with NO to form a pyridine, Dehydro Amlodipine (DAM). Quantification of DAM by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) allows specific measurement of intracellular NO levels. Notably, the AML/NO reaction proceeds rapidly (within 1 s), which is favorable for NO detection considering its large diffusivity and short half-life. Meanwhile, studies under simulated physiological conditions revealed that the AML response to NO is proportional and selective. The presented UPLC-MS/MS method showed high sensitivity (LLOQ = 0.24 nM) and low matrix interference (less than 15%) in DAM quantification. Furthermore, the mass spectrometry probe approach was demonstrated by enabling the measurement of endogenous and exogenous NO in cells. Hence, the quantitative UPLC-MS/MS method developed using AML as a probe is expected to be a new method for intracellular NO analysis.

Mechanisms of angiogenic incompetence in Hutchinson-Gilford progeria via downregulation of endothelial NOS.

Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disorder with features of accelerated aging. The majority of HGPS cases are caused by a de novo point mutation in the LMNA gene (c.1824C>T; p.G608G) resulting in progerin, a toxic lamin A protein variant. Children with HGPS typically die from coronary artery diseases or strokes at an average age of 14.6 years. Endothelial dysfunction is a known driver of cardiovascular pathogenesis; however, it is currently unknown how progerin antagonizes normal angiogenic function in HGPS. Here, we use human iPSC‐derived endothelial cell (iPSC‐EC) models to study angiogenesis in HGPS. We cultured normal and HGPS iPSC‐ECs under both static and fluidic culture conditions. HGPS iPSC‐ECs show reduced endothelial nitric oxide synthase (eNOS) expression and activity compared with normal controls and concomitant decreases in intracellular nitric oxide (NO) level, which result in deficits in capillary‐like microvascular network formation. Furthermore, the expression of matrix metalloproteinase 9 (MMP‐9) was reduced in HGPS iPSC‐ECs, while the expression of tissue inhibitor metalloproteinases 1 and 2 (TIMP1 and TIMP2) was upregulated relative to healthy controls. Finally, we used an adenine base editor (ABE7.10max‐VRQR) to correct the pathogenic c.1824C>T allele in HGPS iPSC‐ECs. Remarkably, ABE7.10max‐VRQR correction of the HGPS mutation significantly reduced progerin expression to a basal level, rescued nuclear blebbing, increased intracellular NO level, normalized the misregulated TIMPs, and restored angiogenic competence in HGPS iPSC‐ECs. Together, these results provide molecular insights of endothelial dysfunction in HGPS and suggest that ABE could be a promising therapeutic approach for correcting HGPS‐related cardiovascular phenotypes.

Carnosine Protects Macrophages against the Toxicity of Aβ1-42 Oligomers by Decreasing Oxidative Stress.

Carnosine (β-alanyl-L-histidine) is a naturally occurring endogenous peptide widely distributed in excitable tissues such as the brain. This dipeptide has well-known antioxidant, anti-inflammatory, and anti-aggregation activities, and it may be useful for treatment of neurodegenerative disorders such as Alzheimer’s disease (AD). In this disease, peripheral infiltrating macrophages play a substantial role in the clearance of amyloid beta (Aβ) peptides from the brain. Correspondingly, in patients suffering from AD, defects in the capacity of peripheral macrophages to engulf Aβ have been reported. The effects of carnosine on macrophages and oxidative stress associated with AD are consequently of substantial interest for drug discovery in this field. In the present work, a model of stress induced by Aβ1-42 oligomers was investigated using a combination of methods including trypan blue exclusion, microchip electrophoresis with laser-induced fluorescence, flow cytometry, fluorescence microscopy, and high-throughput quantitative real-time PCR. These assays were used to assess the ability of carnosine to protect macrophage cells, modulate oxidative stress, and profile the expression of genes related to inflammation and pro- and antioxidant systems. We found that pre-treatment of RAW 264.7 macrophages with carnosine counteracted cell death and apoptosis induced by Aβ1-42 oligomers by decreasing oxidative stress as measured by levels of intracellular nitric oxide (NO)/reactive oxygen species (ROS) and production of peroxynitrite. This protective activity of carnosine was not mediated by modulation of the canonical inflammatory pathway but instead can be explained by the well-known antioxidant and free-radical scavenging activities of carnosine, enhanced macrophage phagocytic activity, and the rescue of fractalkine receptor CX3CR1. These new findings obtained with macrophages challenged with Aβ1-42 oligomers, along with the well-known multimodal mechanism of action of carnosine in vitro and in vivo, substantiate the therapeutic potential of this dipeptide in the context of AD pathology.

Naphthalimide-4-(4-nitrophenyl)thiosemicarbazide: A Fluorescent Probe for Simultaneous Monitoring of Viscosity and Nitric Oxide in Living Cells.

Intracellular viscosity is a physicochemical factor that determines the outcomes of various biological processes, while nitric oxide (NO) is an essential signaling molecule that controls many cellular processes, including oxidative stress. Anticipating that both may be interrelated with a variety of pathologies, their simultaneous measurement would be highly valuable for the investigation of the pathological condition of cells. However, the development of a sensor for such simultaneous detection has not been attempted yet. Herein, we present the synthesis of naphthalimide-4-(4-nitrophenyl)thiosemicarbazide, probe 1, and its application to living cells under conditions of lipopolysaccharide or nystatin treatment, adopted as oxidative stress and altered intracellular viscosity models, respectively. The probe showed increased fluorescence in response to elevation of viscosity and NO levels at 470 and 550 nm, respectively, in the solution studies. When the probe was used for a confocal microscopic study of HeLa cells under stressed conditions, simultaneous monitoring of viscosity and NO level elevations was possible through fluorescence imaging using band-pass filters of 420-475 and 505-600 nm, respectively, upon excitation at a wavelength of 405 nm. Interestingly, both the cellular viscosity and NO levels increased together under lipopolysaccharide or nystatin treatment. Therefore, we suggest that probe 1 is a fluorescent chemical probe that enables the monitoring of alterations in intracellular viscosity and NO levels in living cells, which would be valuable in studies of various cellular damage models.

Protective effects of luteolin on the venous endothelium.

Luteolin is a flavonoid with antioxidant properties already demonstrated in studies related to inflammation, tumor, and cardiovascular processes; however, there are no available information regarding its antioxidant effects at the venous endothelial site. We investigated the effects of luteolin (10, 20, and 50μmol/L) in cultures of rat venous endothelial cells. Nitric oxide (NO) and reactive oxygen species (ROS) were analyzed by fluorimetry; 3-nitrotyrosine (3-NT) residues were evaluated by immunofluorescence, and prostacyclin (PGI2) release was investigated by colorimetry. Intracellular NO levels were significantly enhanced after 10min of luteolin incubation, with a parallel decrease in ROS generation. These results were accompanied by a significant reduction in the expression of 3-NT residues and enhanced PGI2 rates. Therefore, luteolin is effective in reducing ROS thereby improving NO availability in venous endothelial cells. Besides, luteolin-induced decrease in 3-NT residues may correlate with the enhancement in endothelial PGI2 bioavailability. These findings suggest the future application of this flavonoid as a protective agent by improving endothelial function in several circulatory disorders related to venous insufficiency.

Dimethyl Fumarate Attenuates Lung Inflammation and Oxidative Stress Induced by Chronic Exposure to Diesel Exhaust Particles in Mice.

Air pollution is mainly caused by burning of fossil fuels, such as diesel, and is associated with increased morbidity and mortality due to adverse health effects induced by inflammation and oxidative stress. Dimethyl fumarate (DMF) is a fumaric acid ester and acts as an antioxidant and anti-inflammatory agent. We investigated the potential therapeutic effects of DMF on pulmonary damage caused by chronic exposure to diesel exhaust particles (DEPs). Mice were challenged with DEPs (30 μg per mice) by intranasal instillation for 60 consecutive days. After the first 30 days, the animals were treated daily with 30 mg/kg of DMF by gavage for the remainder of the experimental period. We demonstrated a reduction in total inflammatory cell number in the bronchoalveolar lavage (BAL) of mice subjected to DEP + DMF as compared to those exposed to DEPs alone. Importantly, DMF treatment was able to reduce lung injury caused by DEP exposure. Intracellular total reactive oxygen species (ROS), peroxynitrite (OONO), and nitric oxide (NO) levels were significantly lower in the DEP + DMF than in the DEP group. In addition, DMF treatment reduced the protein expression of kelch-like ECH-associated protein 1 (Keap-1) in lung lysates from DEP-exposed mice, whereas total nuclear factor κB (NF-κB) p65 expression was decreased below baseline in the DEP + DMF group compared to both the control and DEP groups. Lastly, DMF markedly reduced DEP-induced expression of nitrotyrosine, glutathione peroxidase-1/2 (Gpx-1/2), and catalase in mouse lungs. In summary, DMF treatment effectively reduced lung injury, inflammation, and oxidative and nitrosative stress induced by chronic DEP exposure. Consequently, it may lead to new therapies to diminish lung injury caused by air pollutants.

Oxidative and Nitrosative Pattern in Circulating Leukocytes of Very Early/Early Hepatocellular Carcinoma Patients.

In chronic liver disease, various immune cell subsets exert pro or anti-tumour effects by releasing reactive oxygen and nitrogen species (ROS, RNS). Here, we evaluated the oxidative and nitrosative pattern in peripheral blood leukocyte subpopulations of early hepatocellular carcinoma (HCC) patients compared with HCC-free cirrhotic patients. Venous blood samples from 18 HCC-free cirrhotic patients and 17 early stage HCC patients were collected to determine ROS, RNS and reduced glutathione levels in isolated leukocytes analyzed by flow cytometry. Intracellular levels of ROS and glutathione were higher in lymphocytes, monocytes, and neutrophils from HCC patients as well as mitochondrial superoxide in neutrophils and monocytes whereas intracellular levels of nitric oxide were lower in lymphocytes, monocytes, and neutrophils. Early HCC alters intracellular levels of ROS and RNS of some circulating leukocytes subsets. This finding may represent a potential area of interest concerning the development of new treatments and prognostic markers.

Nitrate reductase-dependent nitric oxide plays a key role on MeJA-induced ganoderic acid biosynthesis in Ganoderma lucidum.

Ganoderma lucidum, which contains numerous biologically active compounds, is known worldwide as a medicinal basidiomycete. Because of its application for the prevention and treatment of various diseases, most of artificially cultivated G. lucidum is output to many countries as food, tea, and dietary supplements for further processing. Methyl jasmonate (MeJA) has been reported as a compound that can induce ganoderic acid (GA) biosynthesis, an important secondary metabolite of G. lucidum. Herein, MeJA was found to increase the intracellular level of nitric oxide (NO). In addition, upregulation of GA biosynthesis in the presence of MeJA was abolished when NO was depleted from the culture. This result demonstrated that MeJA-regulated GA biosynthesis might occur via NO signaling. To elucidate the underlying mechanism, we used gene-silenced strains of nitrate reductase (NR) and the inhibitor of NR to illustrate the role of NO in MeJA induction. The results indicated that the increase in GA biosynthesis induced by MeJA was activated by NR-generated NO. Furthermore, the findings indicated that the reduction of NO could induce GA levels in the control group, but NO could also activate GA biosynthesis upon MeJA treatment. Further results indicated that NR silencing reversed the increased enzymatic activity of NOX to generate ROS due to MeJA induction. Importantly, our results highlight the NR-generated NO functions in signaling crosstalk between reactive oxygen species and MeJA. These results provide a good opportunity to determine the potential pathway linking NO to the ROS signaling pathway in fungi treated with MeJA. KEY POINTS: • MeJA increased the intracellular level of nitric oxide (NO) in G. lucidum. • The increase in GA biosynthesis induced by MeJA is activated by NR-generated NO. • NO acts as a signaling molecule between reactive oxygen species (ROS) and MeJA.

Silent hypoxia: higher NO in red blood cells of COVID-19 patients

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19) has spread to almost 100 countries, infected over 31 M patients and resulted in 961 K deaths worldwide as of 21st September 2020. The major clinical feature of severe COVID-19 requiring ventilation is acute respiratory distress syndrome (ARDS) with multi-functional failure as a result of a cytokine storm with increased serum levels of cytokines. The pathogenesis of the respiratory failure in COVID-19 is yet unknown, but diffuse alveolar damage with interstitial thickening leading to compromised gas exchange is a plausible mechanism. Hypoxia is seen in the COVID-19 patients, however, patients present with a distinct phenotype. Intracellular levels of nitric oxide (NO) play an important role in the vasodilation of small vessels. To elucidate the intracellular levels of NO inside of RBCs in COVID-19 patients compared with that of healthy control subjects.MethodsWe recruited 14 COVID-19 infected cases who had pulmonary involvement of their disease, 4 non-COVID-19 healthy controls (without pulmonary involvement and were not hypoxic) and 2 hypoxic non-COVID-19 patients subjects who presented at the Masih Daneshvari Hospital of Tehran, Iran between March–May 2020. Whole blood samples were harvested from patients and intracellular NO levels in 1 × 106 red blood cells (RBC) was measured by DAF staining using flow cytometry (FACS Calibour, BD, CA, USA).ResultsThe Mean florescent of intensity for NO was significantly enhanced in COVID-19 patients compared with healthy control subjects (P ≤ 0.05). As a further control for whether hypoxia induced this higher intracellular NO, we evaluated the levels of NO inside RBC of hypoxic patients. No significant differences in NO levels were seen between the hypoxic and non-hypoxic control group.ConclusionsThis pilot study demonstrates increased levels of intracellular NO in RBCs from COVID-19 patients. Future multi-centre studies should examine whether this is seen in a larger number of COVID-19 patients and whether NO therapy may be of use in these severe COVID-19 patients.

SIRT1-dependent effects of resveratrol and grape juice in an in vitro model of preeclampsia

Preeclampsia (PE) is a multifactorial hypertensive disorder of pregnancy that is partly responsible for both maternal and fetal morbidity and mortality levels worldwide. It has been recently discovered that sirtuin-1 (SIRT1) is reduced in the circulation and in an in vitro model of PE. Therefore, in this study, we investigated the effects of trans-resveratrol, a potent antioxidant and activator of SIRT1, on oxidative stress and nitric oxide (NO) production in an in vitro model of PE compared to gestational hypertensive (GH) and healthy pregnant (HP) women. Furthermore, we also evaluated the effects of an acute intake of grape juice on women with PE to assess whether it could mimic in vitro trans-resveratrol supplementation.(1) In the GH group, resveratrol decreased intracellular reactive oxygen species (ROS) and increased their antioxidant capacity, while inhibiting SIRT1 reestablished previous levels. (2) In PE, inhibition of SIRT1 increased antioxidant activity. (3) Intracellular NO and supernatant nitrite levels were increased by inhibiting SIRT1 in the PE group. (4) Grape juice intake increased intracellular NO levels versus before grape juice intake control; however, the inhibition of SIRT1 before grape juice intake initially increased NO, but decreased it 1 h after grape juice intake.In conclusion, activating SIRT1 by using resveratrol alone may not be beneficial to women with PE, and GH and PE seem to have different responsive mechanisms to this molecule. Furthermore, grape juice intake seems to have different effects compared to resveratrol supplementation alone in this in vitro model of PE, demonstrating the potential of the combination of other biologically active molecules from grape juice over the SIRT1-eNOS-NO in PE treatment.

Wavelength- and irradiance-dependent changes in intracellular nitric oxide level.

.Significance: Photobiomodulation (PBM) refers to the beneficial effects of low-energy light absorption. Although there is a large body of literature describing downstream physiological benefits of PBM, there is a limited understanding of the molecular mechanisms underlying these effects. At present, the most popular hypothesis is that light absorption induces release of nitric oxide (NO) from the active site of cytochrome oxidase (COX), allowing it to bind instead. This is believed to increase mitochondrial respiration, and result in greater overall health of the cell due to increased adenosine triphosphate production.Aim: Although NO itself is a powerful signaling molecule involved in a host of biological responses, less attention has been devoted to NO mechanisms in the context of PBM. The purpose of our work is to investigate wavelength-specific effects on intracellular NO release in living cells.Approach: We have conducted in-depth dosimetry analyses of NO production and function in an in vitro retinal model in response to low-energy exposure to one or more wavelengths of laser light.Results: We found statistically significant wavelength-dependent elevations (10% to 30%) in intracellular NO levels following laser exposures at 447, 532, 635, or 808 nm. Sequential or simultaneous exposures to light at two different wavelengths enhanced the NO modulation up to 50% of unexposed controls. Additionally, the immediate increases in cellular NO levels were independent of the function of NO synthase, depended greatly on the substrate source of electrons entering the electron transport chain, and did not result in increased levels of cyclic guanosine monophosphate.Conclusions: Our study concludes the simple model of light-mediated release of NO from COX is unlikely to explain the wide variety of PBM effects reported in the literature. Our multiwavelength method provides a novel tool for studying immediate and early mechanisms of PBM as well as exploring intracellular NO signaling networks.

A compact pulse-modulation air plasma jet for the inactivation of chronic wound bacteria: Bactericidal effects & host safety

Previously, a novel compact low-temperature plasma jet device with adjustable pulse-parameters was developed to use ambient air as a plasma source for the inactivation of chronic wound bacteria. Its adjustable burst-mode pulse was proved to be highly beneficial for the controllable generation of the desired reactive oxygen and nitrogen species. In this study, further bactericidal effects and its safety on human dermal fibroblasts (HDF) were investigated. The generated plasma inactivated Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) effectively in a time- and dose-dependent manner. Quantification of colony-forming units from bacterial biofilms confirmed the elimination of biofilms formed by PA or SA. Next, HDF cells were plasma-treated and the morphology was observed using an inverted microscope and a scanning electron microscope. No particular morphological changes were observed on cell membrane and cell attachment. MTT assay of the HDF cells after plasma exposure also confirmed the functional enzymatic activity of the cells up to 5 min of treatment. Flow cytometric assay for annexin V and 7-AAD showed that plasma exposure did not significantly induce apoptosis or necrosis of the cell. Intracellular nitric oxide, reactive oxygen species, and Ki-67 protein, a marker for cell proliferation, were also measured by flow cytometry. Intracellular levels of nitric oxide significantly increased, while reactive oxygen species and Ki-67 protein showed no significant alteration after the plasma exposure. Taken together, we demonstrated that the newly developed adjustable-pulse low-temperature air plasma jet device was an effective tool for the elimination of bacteria especially on chronic wound, where the treatment is needed to be least invasive and safe for the surrounding tissue.