Kinetics of fluoride after brushing with the no-rinse method.

BACKGROUND: Fluoride plays a vital role in preventing dental caries, with its addition to oral care products significantly promoting oral hygiene. A no-rinse brushing method aims to increase fluoride retention in the oral cavity, as rinsing with water decreases fluoride levels in saliva, which could affect remineralization. While the no-rinse brushing method holds promise for improving fluoride retention in the oral cavity, critical inquiries persist regarding its safety. This study investigated the kinetics of oral fluoride and potential risks to fully assess its effectiveness and implications for oral health. METHODS: Ten healthy adults participated in a crossover study comparing the no-rinse with the rinse method. All subjects followed American Dental Association (ADA) brushing guidelines. Levels of fluoride in saliva (supernatant and sediment) and urine were measured over time, and plasma fluoride was measured one hour after brushing. Pharmacokinetic parameters were also calculated from the data. RESULTS: Participants using the no-rinse method had higher fluoride levels in supernatant immediately and up to 30 min post-brushing compared to the rinse method. Fluoride levels in sediment were higher only immediately after brushing. The total fluoride concentration in saliva remained elevated for up to 5 min with the no-rinse method. Systemic fluoride absorption showed no significant difference between the two methods based on blood and urine analysis. CONCLUSION: This research indicates that the no-rinse method can enhance fluoride retention in the oral cavity for up to 30 min after a single brushing. In addition, our findings suggest that this method does not significantly influence systemic fluoride levels or toxicity. REGISTRY: Thai Clinical Trials Registry, TCTR ( http://thaiclinicaltrials.org ). CLINICAL TRIAL REGISTRATION NUMBER: TCTR20231104001 (4/11/2023).

Biocompatibility and biofilm formation on conventional and CAD/CAM provisional implant restorations.

Dental implant treatment is a complex and sophisticated process, and implant provisional restorations play a vital role in ensuring its success. The advent of computer-aided design and computer-aided manufacturing (CAD/CAM) technology has revolutionized the field of implant restorations by providing improved precision leading to a reduction in chair time and more predictable treatment outcomes. This technology offers a promising solution to the drawbacks of conventional methods and has the potential to transform the way implant procedures are approached. Despite the clear advantages of CAD/CAM over conventional provisional implant restorations including higher accuracy of fit and superior mechanical properties, little research has been conducted on the biological aspect of these novel restorations. This study aims to fill that gap, comprehensively assessing the biocompatibility, gingival tissue attachment and biofilm formation of a range of provisional implant restorations using CAD/CAM technology through milling and 3-D printing processes compared to conventional fabrication. The biocompatibility of the tested restorations was assessed by MTT assay, Calcein-AM assay as well as SEM analysis. The surface roughness of the tested samples was evaluated, alongside the attachment of Human Gingival Fibroblasts (HGF) cells as well as biofilm formation, and estimated Porphyromonas gingivalis (P. gingivalis) cell count from DNA detection.The results showed all tested provisional implant restorations were non-toxic and good HGF cell attachment but differed in their quantity of biofilm formation, with surface texture influenced by the material and fabrication technique, playing a role. Within the limitation of this study, the findings suggest that CAD/CAM-fabricated provisional implant restorations using a milling technique may be the most favourable among tested groups in terms of biocompatibility and periodontal-related biofilm formation.

Nitrite in paraffin-stimulated saliva correlates with blood nitrite.

Nitrite anion (NO2-) is a circulating nitric oxide (NO) metabolite considered an endothelial function marker. Nitrite can be produced from nitrate (NO3-) secreted from plasma into saliva. The nitrate reductase of oral bacteria converts salivary nitrate to nitrite, which is swallowed and absorbed into circulation. In this study, we aimed to examine the relevance between these species' salivary and blood levels. We collected three whole saliva samples (unstimulated, paraffin-stimulated, and post-chlorhexidine mouthwash stimulated saliva) and blood from 75 healthy volunteers. We measured the nitrite and nitrate by the chemiluminescence method. The nitrite levels in stimulated saliva and post-mouthwash stimulated saliva exhibited weak correlations with blood nitrite. There was no correlation between nitrite in unstimulated saliva with blood nitrite. The baseline platelet activity, determined as P-selectin expression, negatively correlated with nitrite in plasma and post-mouthwash stimulated saliva. The salivary nitrate in all saliva samples showed correlations with its plasma levels. We conclude that nitrite in stimulated saliva correlates with blood nitrite.

Efficacy and safety of inhaled nebulized sodium nitrite in asthmatic patients.

BACKGROUND: Nitrite is a physiologic nitric oxide (NO) derivative that can be bioactivated to NO. NO has been shown to attenuate airway inflammation and enhance the anti-inflammatory effect of corticosteroids in the animal model of asthma. Here, we aimed to investigate the efficacy and safety of inhaled sodium nitrite as add-on therapy with inhaled corticosteroid (ICS) in adult patients with persistent asthma. METHODS: In protocol 1, 10 asthmatic patients were administered a single dose of nebulized 15-mg sodium nitrite to assess safety, effect on lung function, and pharmacokinetics of nitrite within 120 min. In protocol 2, 20 patients were randomly assigned to a nitrite (15 mg twice daily) group or a placebo group to assess the efficacy over 12 weeks. The primary outcome was the forced expiratory volume in 1 s (FEV1). The secondary outcomes were other lung function parameters, unplanned asthma-related visits at the emergency department (ED) or outpatient department (OPD), admission days, asthma control test (ACT), and safety. RESULTS: Nebulized sodium nitrite had neither acute adverse effect nor effect on lung function test within 120 min. No blood pressure change was seen. At week 12, FEV1 increased in the nitrite group, whereas there was no change in the placebo group. There were 5 events of asthma exacerbation, 4 ED visits, and one unplanned OPD visit in the placebo group, but none of these was noted in the nitrite group. There was no change in ACT scores in both groups. No adverse event was reported during 12 weeks in the nitrite group. There was no change in methemoglobin levels and sputum inflammatory markers. CONCLUSION: From our pilot trial, nebulized sodium nitrite is safe in asthmatic patients, and shows the potential to reduce asthma exacerbation compared with placebo.

Nitrate uptake and metabolism in human skeletal muscle cell cultures.

Several studies show that dietary nitrate enhances exercise performance, presumably by increasing muscle blood flow and improving oxygen utilization. These effects are likely mediated by nitrate metabolites, including nitrite and nitric oxide (NO). However, the mechanisms of nitrate production, storage, and metabolism to nitrite and NO in skeletal muscle cells are still unclear. We hypothesized that exogenous nitrate can be taken up and metabolized to nitrite/NO inside the skeletal muscle. We found rapid uptake of exogeneous nitrate in both myoblasts and myotubes, increasing nitrite levels in myotubes, but not myoblasts. During differentiation we found increased expression of molybdenum containing proteins, such as xanthine oxidoreductase (XOR) and the mitochondrial amidoxime-reducing component (MARC); nitrate and nitrite reductases. Sialin, a known nitrate transporter, was detected in myoblasts; nitrate uptake decreased after sialin knockdown. Inhibition of chloride channel 1 (CLC1) also led to significantly decreased uptake of nitrate. Addition of exogenous nitrite, which resulted in higher intracellular nitrite levels, increased intracellular cGMP levels in myotubes. In summary, our results demonstrate for the first time the presence of the nitrate/nitrite/NO pathway in skeletal muscle cells, namely the existence of strong uptake of exogenous nitrate into cells and conversion of intracellular nitrate to nitrite and NO. Our results further support our previously formulated hypothesis about the importance of the nitrate to nitrite to NO intrinsic reduction pathways in skeletal muscles, which likely contributes to improved exercise tolerance after nitrate ingestion.

Pharmacokinetics and pharmacodynamics of single dose of inhaled nebulized sodium nitrite in healthy and hemoglobin E/ß-thalassemia subjects.

Inhaled sodium nitrite has been reported to decrease pulmonary artery pressure in hemoglobin E/ß-thalassemia (HbE/ß-thal) patients with pulmonary hypertension. This study investigated the pharmacokinetics and pharmacodynamics of inhaled nebulized sodium nitrite in 10 healthy subjects and 8 HbE/ß-thal patients with high estimated pulmonary artery pressure. Nitrite pharmacokinetics, fraction exhaled nitric oxide (FENO), estimated right ventricular systolic pressure (eRVSP) measured by echocardiography, and platelet activation were determined. Nebulized sodium nitrite at doses used in this study (37.5 and 75 mg for healthy subjects and 15 mg for HbE/ß-thal patients) was well tolerated and did not cause changes in methemoglobin levels and systemic blood pressure. Absorption of inhaled nitrite was rapid with the absolute bioavailability of 18%. In whole blood, nitrite exhibited the dose-independent pharmacokinetics with clearance (CL) of 1.5 l/h/kg, volume of distribution (Vd) of 1.3 l/kg and half-life (t1/2) of 0.6 h. CL and Vd of nitrite was higher in red blood cells (RBC) than whole blood and plasma. HbE/ß-thal patients had lower nitrite CL and longer t1/2 in RBC than healthy subjects. FENO increased immediately after inhalation. Following nitrite inhalation, eRVSP remained unchanged but platelet activation was suppressed as evidenced by inhibition of adenosine diphosphate (ADP)-induced P-selectin expression and increase in phosphorylated vasodilator-stimulated phosphoprotein (P-VASPSer239) in platelets. There were no changes in markers of oxidative and nitrosative stress after inhalation. Our results support further development of inhaled nebulized sodium nitrite for treatment of pulmonary hypertension in ß-thalassemia.

Inhaled nebulized sodium nitrite decreases pulmonary artery pressure in ß-thalassemia patients with pulmonary hypertension.

Pulmonary hypertension is a life-threatening complication in ß-thalassemia. Inhaled sodium nitrite has vasodilatory effect on pulmonary vasculature. However, its effect on pulmonary artery pressure (PAP) in ß-thalassemia subjects with pulmonary hypertension has never been reported. In this study, we investigated the change in PAP during inhalation of sodium nitrite in 5 ß-thalassemia patients. We demonstrated that sodium nitrite administered by nebulization rapidly decreased PAP as measured by echocardiography and right heart catheterization. The effect of nitrite was short as PAP returned to baseline at end of inhalation. Our findings support acute pulmonary vasodilation effect of nitrite in ß-thalassemia with pulmonary hypertension.

Cysteinyl leukotriene receptor antagonists induce apoptosis and inhibit proliferation of human glioblastoma cells by downregulating B-cell lymphoma 2 and inducing cell cycle arrest.

Glioblastoma is the most aggressive type of brain cancer with the highest proliferation, invasion, and migration. Montelukast and zafirlukast, 2 widely used leukotriene receptor antagonists (LTRAs) for asthma treatment, inhibited invasion and migration of glioblastoma cell lines. Montelukast induces apoptosis and inhibits cell proliferation of various cancer cells. Herein, apoptotic and antiproliferative effects of montelukast and zafirlukast were investigated in 2 glioblastoma cell lines, A172 and U-87 MG. Both LTRAs induced apoptosis and inhibited cell proliferation of glioblastoma cells in a concentration-dependent manner. Montelukast was more cytotoxic and induced higher levels of apoptosis than zafirlukast in A172 cells, but not in U-87 MG cells. Both drugs decreased expression of B-cell lymphoma 2 (Bcl-2) protein without affecting Bcl-2-associated X (Bax) levels. LTRAs also reduced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). In contrast, zafirlukast showed a greater antiproliferative effect than montelukast and induced G0/G1 cell cycle arrest by upregulating p53 and p21 expression. These results suggested the therapeutic potential of LTRAs in glioblastoma.

Deferiprone increases endothelial nitric oxide synthase phosphorylation and nitric oxide production.

Iron chelation can improve endothelial function. However, effect on endothelial function of deferiprone has not been reported. We hypothesized deferiprone could promote nitric oxide (NO) production in endothelial cells. We studied effects of deferiprone on blood nitrite and blood pressure after single oral dose (25 mg/kg) in healthy subjects and hemoglobin E/ß-thalassemia patients. Further, effects of deferiprone on NO production and endothelial NO synthase (eNOS) phosphorylation in primary human pulmonary artery endothelial cells (HPAEC) were investigated in vitro. Blood nitrite levels were higher in patients with deferiprone therapy than those without deferiprone (P = 0.023, n = 16 each). Deferiprone increased nitrite in plasma and whole blood of healthy subjects (P = 0.002 and 0.044) and thalassemia patients (P = 0.003 and 0.046) at time 180 min (n = 20 each). Asymptomatic reduction in diastolic blood pressure (P = 0.005) and increase in heart rate (P = 0.009) were observed in healthy subjects, but not in thalassemia patients. In HPAEC, deferiprone increased cellular nitrite and phospho-eNOS (Ser1177) (P = 0.012 and 0.035, n = 6) without alteration in total eNOS protein and mRNA. We conclude that deferiprone can induce NO production by enhancing eNOS phosphorylation in endothelial cells.

Platelet inhibition and increased phosphorylated vasodilator-stimulated phosphoprotein following sodium nitrite inhalation.

In the presence of red blood cells (RBCs), nitrite inhibits platelets through its conversion to nitric oxide (NO) by the reductase activity of partially deoxygenated hemoglobin. Inhaled sodium nitrite is being investigated as a therapy for pulmonary hypertension. Here, we measured platelet aggregation, P-selectin expression, platelet-leukocyte aggregates and phosphorylated vasodilator-stimulated phosphoprotein (P-VASPSer239) following sodium nitrite inhalation in healthy subjects. In vitro incubation of nitrite with deoxygenated whole blood showed an increase in P-VASPSer239, which was inhibited by ODQ, a soluble guanylyl cyclase (sGC) inhibitor. Immediately and 60 min after nitrite inhalation, P-VASPSer239 increased in platelets. Platelet aggregation, P-selectin expression, platelet-monocyte and platelet-lymphocyte aggregates decreased after inhalation. In conclusion, sodium nitrite administered to healthy subjects by inhalation can inhibit platelet activation and increase P-VASPSer239 in platelets. Platelet inhibition by nitrite administration may be useful in disorders associated with platelet hyperactivity.

Increased platelet activation in subjects chronically exposed to cadmium: A pilot study.

Cadmium exposure has been reported to be associated with the risk of vascular disorders. Here, we investigated platelet activity in subjects with chronic cadmium exposure. Eighteen and 15 women participated in this study as chronically cadmium-exposed and control non-exposed subjects, respectively. Plasma P-selectin and CD40 ligand (CD40L), soluble markers of platelet activation, were measured. Platelet aggregation in whole blood, P-selectin and activated glycoprotein (aGP) IIb/IIIa expression on platelets and platelet-leukocyte aggregates were determined. The levels of plasma P-selectin and CD40L increased in subjects with chronic cadmium exposure compared with control subjects. Platelet aggregation induced by adenosine diphosphate (ADP) was higher in cadmium-exposed subjects than control subjects. Cadmium-exposed subjects had higher baseline and ADP-induced aGPIIb/IIIa expression on platelets than control subjects. Platelet-neutrophil aggregates also increased in cadmium-exposed subjects. Blood cadmium correlated with ADP-induced aggregation, aGPIIb/IIIa expression and platelet-neutrophil aggregates, while urinary cadmium correlated with soluble P-selectin. However, cadmium only at high concentration (15 µM) could potentiate ADP-induced platelet activation in vitro. In conclusion, our pilot data show that cadmium-exposed subjects have increased baseline platelet activation and reactivity.

Hydroxychavicol Inhibits In Vitro Osteoclastogenesis via the Suppression of NF-κB Signaling Pathway.

Hydroxychavicol, a primary active phenolic compound of betel leaves, previously inhibited bone loss in vivo by stimulating osteogenesis. However, the effect of hydroxychavicol on bone remodeling induced by osteoclasts is unknown. In this study, the anti-osteoclastogenic effects of hydroxychavicol and its mechanism were investigated in receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclasts. Hydroxychavicol reduced the number of tartrate resistance acid phosphatase (TRAP)-positive multinucleated, F-actin ring formation and bone-resorbing activity of osteoclasts differentiated from RAW264.7 cells in a concentration-dependent manner. Furthermore, hydroxychavicol decreased the expression of osteoclast-specific genes, including cathepsin K, MMP-9, and dendritic cell-specific transmembrane protein (DC-STAMP). For mechanistic studies, hydroxychavicol suppressed RANKL-induced expression of major transcription factors, including the nuclear factor of activated T-cells 1 (NFATc1), c-Fos, and c-Jun. At the early stage of osteoclast differentiation, hydroxychavicol blocked the phosphorylation of NF-κB subunits (p65 and Iκßα). This blockade led to the decrease of nuclear translocation of p65 induced by RANKL. In addition, the anti-osteoclastogenic effect of hydroxychavicol was confirmed by the inhibition of TRAP-positive multinucleated differentiation from human peripheral mononuclear cells (PBMCs). In conclusion, hydroxychavicol inhibits osteoclastogenesis by abrogating RANKL-induced NFATc1 expression by suppressing the NF-κB signaling pathway in vitro.

Decreased nitrite levels in erythrocytes of children with ß-thalassemia/hemoglobin E.

Nitrite anion is bioactive nitric oxide (NO) species circulating in blood, and represents the NO bioavailability and endothelial function. In this study, we aimed to investigate the nitrite levels and the correlation with hemolysis and severity in ß-thalassemia/hemoglobin E (ß-thal/HbE). 38 Children (12.0±1.9 years of age) with a diagnosis of mild, moderate and severe ß-thalassemia were enrolled in the study. The blood nitrite levels and potential plasma NO consumption were measured by the chemiluminescence method. The nitrite levels in whole blood and erythrocytes of the severe thalassemia subjects were lower than those of the control subjects. At day 7 after transfusion of packed erythrocytes, the nitrite levels in erythrocytes increased. The plasma hemoglobin and NO consumption increased in the severe thalassemia subjects. The nitrite levels in erythrocytes inversely correlated with plasma hemoglobin, lactate dehydrogenase activity, potential NO consumption, and lipid peroxidation. Our studies demonstrate the decreased NO bioavailability in thalassemia, which could result from endothelial dysfunction, the increased potential NO consumption in plasma by cell-free hemoglobin and oxidative stress.

Gene Profiling of Cannabis-sativa-mediated Apoptosis in Human Melanoma Cells.

BACKGROUND/AIM: Malignant melanoma is an aggressive skin cancer, accounting for the majority of skin cancer deaths. Prognosis is often poor and finding effective treatment remains a challenge. Tetrahydrocannabinol (THC) and cannabidiol (CBD) are main bioactive components of Cannabis sativa plant extracts that have been shown to exert anti-tumor effects. In this study, we aimed to perform gene expression analysis of human melanoma A375 cells following stimulation with C. sativa extracts. MATERIALS AND METHODS: Gene expression profiles of A375 human melanoma and Vero (control) cell lines were evaluated by RNA sequencing and quantitative real-time PCR. RESULTS: Flow cytometry showed that the THC+CBD cannabis fractions induced apoptosis on A375 cells. Induction of apoptosis was accompanied by a notable up-regulation of DNA damage inducible transcript 3 (DDIT), nerve growth factor receptor (NGFR), colony-stimulating factor 2 (CSF2), growth arrest and DNA damage inducible beta (GADD45B), and thymic stromal lymphopoietin (TSLP) genes and down-regulation of aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), cyclin E2 (CCNE2), integrin subunit alpha 9 (ITGA9), proliferating cell nuclear antigen (PCNA) and E2F transcription factor 1 (E2F1) genes. Treatment of A375 cells with the THC+CBD fraction inhibited the phosphorylation of ERK1/2 signaling pathway, which regulates melanoma cell proliferation. We showed that the THC+CBD combination disrupted melanoma cell migration. CONCLUSION: Use of C. sativa-derived extracts containing equal amounts of THC and CBD is proposed as a potential treatment of melanoma.

Increased platelet activation and lower platelet-monocyte aggregates in COVID-19 patients with severe pneumonia.

BACKGROUND: The increased procoagulant platelets and platelet activation are associated with thrombosis in COVID-19. In this study, we investigated platelet activation in COVID-19 patients and their association with other disease markers. METHODS: COVID-19 patients were classified into three severity groups: no pneumonia, mild-to-moderate pneumonia, and severe pneumonia. The expression of P-selectin and activated glycoprotein (aGP) IIb/IIIa on the platelet surface and platelet-leukocyte aggregates were measured prospectively on admission days 1, 7, and 10 by flow cytometry. RESULTS: P-selectin expression, platelet-neutrophil, platelet-lymphocyte, and platelet-monocyte aggregates were higher in COVID-19 patients than in uninfected control individuals. In contrast, aGPIIb/IIIa expression was not different between patients and controls. Severe pneumonia patients had lower platelet-monocyte aggregates than patients without pneumonia and patients with mild-to-moderate pneumonia. Platelet-neutrophil and platelet-lymphocyte aggregates were not different among groups. There was no change in platelet-leukocyte aggregates and P-selectin expression on days 1, 7, and 10. aGPIIb/IIIa expression was not different among patient groups. Still, adenosine diphosphate (ADP)-induced aGPIIb/IIIa expression was lower in severe pneumonia than in patients without and with mild-to-moderate pneumonia. Platelet-monocyte aggregates exhibited a weak positive correlation with lymphocyte count and weak negative correlations with interleukin-6, D-dimer, lactate dehydrogenase, and nitrite. CONCLUSION: COVID-19 patients have higher platelet-leukocyte aggregates and P-selectin expression than controls, indicating increased platelet activation. Compared within patient groups, platelet-monocyte aggregates were lower in severe pneumonia patients.

Extracellular heme enhances the antimalarial activity of artemisinin.

Artemisinin exerts the antimalarial activity through activation by heme. The hemolysis in malaria results in the elevated levels of plasma heme which may affect the activity of artemisinin. We hypothesized that the extracellular heme would potentiate the antimalarial activity of artemisinin. Hemin (ferric heme) at the pathologic concentrations enhanced the activity of artemisinin against Plasmodium falciparum in vitro and increased the levels of the lipid peroxidation products in the presence of artemisinin. The antimalarial activity of artemisinin and potentiation by hemin was decreased by vitamin E. Hemin had no effect on the activity of quinoline drugs (chloroquine, quinine and mefloquine). Furthermore, the oxidative effect of hemin in the presence of artemisinin or quinoline drugs was studied using low-density lipoprotein (LDL) oxidation as a model. Artemisinin enhanced the effects of hemin on lipid peroxidation and a decrease of tryptophan fluorescence in LDL whereas the quinoline drugs inhibited the oxidation by hemin. In conclusion, the extracellular hemin enhances the antimalarial activity of artemisinin as a result of the increasing oxidative effect of hemin.

Hemoglobin-bound platelets correlate with the increased platelet activity in hemoglobin E/ß-thalassemia.

INTRODUCTION: An increase in platelet activity is a contributing factor to vascular complications in hemoglobin E/ß-thalassemia (HbE/ß-thal). Plasma-free hemoglobin (Hb) increases in HbE/ß-thal patients and correlates with platelet activation, but the levels of Hb-bound platelets have never been reported. In this study, we aimed to investigate the levels of Hb-bound platelets and its association with platelet activity in HbE/ß-thal patients. METHODS: Hb-bound platelets were measured by flow cytometry in 22 healthy subjects and 26 HbE/ß-thal patients (16 nonsplenectomized and 10 splenectomized HbE/ß-thal patients). Plasma Hb was measured by the chemiluminescence method based on the consumption of nitric oxide (NO) by Hb. Expression of P-selectin and activated glycoprotein (aGP) IIb/IIIa on platelets was measured by flow cytometry as a marker of platelet activity. RESULTS: Both nonsplenectomized and splenectomized HbE/ß-thal patients had higher levels of Hb-bound platelets and plasma Hb than healthy subjects. In vitro incubation of dialyzed Hb from patients with platelets of healthy subjects caused an increase in Hb-bound platelets, which was partially inhibited by anti-GPIbα antibody. Plasma Hb positively correlated with Hb-bound platelets. Platelet P-selectin expression at baseline and in response to adenosine diphosphate (ADP, 1 µM) stimulation was higher in nonsplenectomized and splenectomized HbE/ß-thal patients than healthy subjects. The ADP-induced aGPIIb/IIIa expression on platelets was also higher in HbE/ß-thal patients than healthy subjects. Hb-bound platelets correlated with baseline P-selectin expression and ADP-induced P-selectin expression. CONCLUSION: HbE/ß-thal patients have increased Hb-bound platelets, which is associated with increased baseline platelet activation and reactivity.

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation.

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Phosphorylated vasodilator-stimulated phosphoprotein (P-VASPSer239) in platelets is increased by nitrite and partially deoxygenated erythrocytes.

Nitrite is recognized as a bioactive nitric oxide (NO) metabolite. We have shown that nitrite inhibits platelet activation and increases platelet cGMP levels in the presence of partially deoxygenated erythrocytes. In this study, we investigated the effect of nitrite on phosphorylation of vasodilator-stimulated phosphoprotein on residue serine 239 (P-VASPSer239), a marker of protein kinase G (PKG) activation, in human platelets. In platelet-rich plasma (PRP), nitrite itself had no effect on levels of P-VASPSer239 while DEANONOate increased P-VASPSer239. Deoxygenation of PRP + erythrocytes (20% hematocrit) raised baseline P-VASPSer239 in platelets. At 20% hematocrit, nitrite (10 µM) increased P-VASPSer239 in platelets about 31% at 10-20 minutes of incubation while the levels of P-VASPSer157, a marker of protein kinase A (PKA) activation, were not changed. Nitrite increased P-VASPSer239 in platelets in the presence of deoxygenated erythrocytes at 20-40% hematocrit, but the effects were slightly greater at 20% hematocrit. In conclusion, our data confirm that nitrite increases P-VASPSer239 in platelets in the presence of deoxygenated erythrocytes. They also further support the idea that partially deoxygenated erythrocytes may modulate platelet activity, at least in part, via the NO/sGC/PKG pathway from NO formed by reduction of circulating nitrite ions.

Leukotriene Receptor Antagonists Inhibit Mitogenic Activity in Triple Negative Breast Cancer Cells

Despite a discovery of hormonal pathways regulating breast cancer, a definitive cure for the disease requires further identification of alternative targets that provide a hormone-independent support. Apart from their role in inflammatory diseases, cysteinyl leukotriene (CysLT) receptor antagonists (LTRAs) decrease the risk of lung cancer in asthma patients and inhibit tumor progression in several malignancies. In the present study, we evaluate the effects of two chemically different, clinically relevant LTRAs (montelukast and zafirlukast) in a triple negative breast cancer cell line, MDAMB- 231. We found that these two LTRAs reduced breast cancer cell viability in a dose-dependent manner with the 50% inhibitory concentration (IC50) between 5-10 µM. Although both LTRAs have several pharmacological properties in common, we noticed that montelukast mainly induced apoptosis, while zafirlukast mainly exerted its action on cell cycle. However, the precise mechanisms responsible for such different effects remain unclear. In summary, our results suggest that CysLT plays a role in proliferation and survivability of breast cancer cells in the absence of hormonal stimuli.