Design and Functionalization of a µPAD for the Enzymatic Determination of Nitrate in Urine

In this work, the design of a microfluidic paper-based analytical device (μPAD) for the quantification of nitrate in urine samples was described. Nitrate monitoring is highly relevant due to its association to some diseases and health conditions. The nitrate determination was achieved by combining the selectivity of the nitrate reductase enzymatic reaction with the colorimetric detection of nitrite by the well-known Griess reagent. For the optimization of the nitrate determination μPAD, several variables associated with the design and construction of the device were studied. Furthermore, the interference of the urine matrix was evaluated, and stability studies were performed, under different conditions. The developed μPAD enabled us to obtain a limit of detection of 0.04 mM, a limit of quantification of 0.14 mM and a dynamic concentration range of 0.14–1.0 mM. The designed μPAD proved to be stable for 24 h when stored at room temperature in air or vacuum atmosphere, and 60 days when stored in vacuum at −20 °C. The accuracy of the nitrate μPAD measurements was confirmed by analyzing four certified samples (prepared in synthetic urine) and performing recovery studies using urine samples.

Nitric oxide metabolites in complications after open reconstructive procedures in patients with peripheral atherosclerosis

BACKGROUND: Assessing the severity of endothelial dysfunction (ED) and making a prognosis after open reconstructive interventions in patients with peripheral atherosclerosis is complicated due to a limited number of conventional routine methods, including laboratory ones, available for establishing associations between biochemical markers and disease outcomes. AIM: This work is dedicated to the evaluation of nitric oxide (II) metabolites (nitrates and nitrites) in patients with complications following open bypass procedures on the lower extremity arteries using synthetic grafts in order to assess the severity of endothelial dysfunction and its negative sequalae. MATERIALS AND METHODS: The study involved 60 subjects with stage IIb–III chronic lower limb ischemia according to A. V. Pokrovsky–Fontaine classification (categories 3–5 Rutherford) due to atherosclerotic peripheral artery disease. Median age of the subjects was 65 (60–67); the majority of the patients were male (51 (85%). Median value of ankle-brachial index at baseline was 0.32 (0.19–0.42). All participants underwent open bypass grafting procedures using synthetic vascular grafts. Nitric (II) oxide (NO) metabolites level (nitrites and nitrates) was measured before the intervention and three months afterwards by spectrophotocolorimetry using an ELISA analyzer Stat Fax 3200 (Awareness Techonology, Inc., USA) and Griess reagent. The subjects were followed up for a year to determine the rate of development of complications such as restenosis, graft thrombosis, limb loss (major amputation), lethal outcomes, as well as disease progression, myocardial infarction, oncology, and stroke. RESULTS: Performance of a reconstructive procedure on lower extremity arteries was followed by a decrease in NO metabolites level from initial median 76.3 mcmol/ml (48–100.7) to 52.4 mcmol/ml (36.1–93.8) at three months after the intervention (р = 0.015). There was a positive correlation between the NO metabolites level at baseline (0 months) and follow-up visit (3 months) (r = +0.573). Complications within one year after bypass procedures were detected in 66.64% subjects. Baseline level of NO metabolites in patients who required an amputation within one year after the procedure was markedly elevated and reached 116.3 mcmol/ml (90–130.5) (p = 0.025); development of restenosis and lethal outcomes within one year were associated with initially low NO metabolites levels, 35.6 mcmol/ml (p = 0.036) and 33 mcmol/ml (30–36), accordingly (р = 0.043). CONCLUSIONS: Both markedly high and low levels of nitric oxide metabolites (nitrates and nitrites) levels are associated with development of severe complications after open reconstructive procedures in patients with peripheral atherosclerosis. Quantitative determination of the NO metabolites level in serum is a relatively easy and reliable method allowing for the determination of the severity of endothelial dysfunction and its sequalae in subjects after open bypass reconstructive procedures on lower extremity arteries.

Thymol and Piperine-Loaded Poly(D,L-lactic-co-glycolic acid) Nanoparticles Modulate Inflammatory Mediators and Apoptosis in Murine Macrophages

This study aimed at preparing and characterizing thymol, eugenol, and piperine-loaded poly(D,L-lactic-co-glycolic acid) nanoparticles and evaluating the effect on inflammatory mediators secretion and apoptosis in Raw 264.7 macrophage cells. Nanoparticles were produced by the solvent evaporation technique. Dynamic light scattering and scanning electron microscopy were used to study the physicochemical characteristics. Raw 264.7 macrophage cells were used as a model for in vitro assays. The 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium assay was used to determine the cytotoxicity of the formulated nanoparticles. An annexin V apoptosis detection kit was used to assess apoptosis. Nitric oxide production was determined using the Griess reagent, and the inflammatory mediators level was evaluated with Th1/Th2 cytokine and fluorometric cyclooxygenase kits. The loaded nanoparticles showed a particle size around 190 nm with a low polydispersity between 0.069 and 0.104 and a zeta potential between–1.2 and–9.5 mV. Reduced cytotoxicity of nanoparticles compared to free molecules against Raw 264.7 macrophage cells was observed and seemed to occur through a mechanism associated with apoptosis. A decrease in cyclooxygenase enzyme activity with an increasing concentration was observed. Both free molecules and nanoparticles showed their capacity to modulate the inflammatory process mostly by inhibiting the investigated inflammatory cytokines. The data presented in this study indicate that thymol and piperine-loaded poly(D,L-lactic-co-glycolic acid nanoparticles could serve as a novel anti-inflammatory colloidal drug delivery system with reduced toxicity. However, further study should be considered to optimize the formulation’s loading capacity and thereby probably enhance their bioactivity in treating inflammatory diseases.

Effect of Fractions from Lycopus lucidus Turcz. Leaves on Genomic DNA Oxidation and Matrix Metalloproteinase Activity

Aim and Objective: We investigated the inhibitory effects of fractions from Lycopus lucidus Turcz. leaves on genomic DNA oxidation, nitric oxide (NO) production and matrix metalloproteinase (MMP) activity. Material and Methods: Oxidative damage of genomic DNA was detected after Fenton reaction with H2O2 using DNA electrophoresis. Western blotting was performed to compare the expression levels of MMP-2 in phorbol 12-myristate 13-acetate (PMA)-induced HT-1080 cells. Lipopolysacchride (LPS)-induced NO production in RAW 264.7 cells was measured using Griess reagent. Results: ll fractions (n-Hexane, 85% aq. MeOH, n-BuOH, and water fractions) from the leaves of L. lucidus Turcz. significantly inhibited intracellular production of reactive oxygen species (ROS) (p<0.05). Particularly, 85% aq. MeOH and n-BuOH fractions showed higher ROS inhibitory activity than the other fractions. n-Hexane, 85% aq. MeOH, n-BuOH and water (0.05 mg/mL) fractions significantly inhibited oxidative DNA damage by 57.97%, 68.48%, 58.97%, and 68.39%, respectively (p <0.05). Treatment of RAW 264.7 cells with each fraction reduced LPS-induced NO production in a dose-dependent manner (p<0.05). n-Hexane and 85% aq. MeOH fractions notably reduced MMP-2 secretion levels of in the culture supernatants from HT-1080 cells. Conclusion: Overall, these results indicated that L. lucidus Turcz. leaves can be exploited as plant based sources of antioxidants in the pharmaceutical, cosmetic, nutraceutical and food industries.

Signaling Pathways Associated with Macrophage-Activating Polysaccharide Isolated from Korea Red Ginseng

Background and Objectives: Korean red ginseng (KRG) is known as an immune-enhancing health food and has been approved by the Korea Food and Drug Administration. We analyzed the immune-enhancing activity of KRG and its polysaccharide (KRG-P) using RAW264.7 murine macrophage cells. Materials and Methods: The protein and mRNA expression levels of IL-6 and TNF-α were measured using ELISA and qRT-PCR, respectively. Nitric oxide levels were measured using the Griess reagent. The phosphorylation and total protein levels of ERK, p38, JNK, p65, and GAPDH were determined by immunoblot assay. Results: The polysaccharide (KRG-P), but not KRG, produced nitric oxide, interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) in RAW 264.7 cells. KRG-P increased nitric oxide synthase 2 (NOS2), IL-6, and TNF-α expression in RAW264.7 cells. KRG-P also increased phosphorylation of MAPKs (mitogen-activate protein kinases) including ERK, p38, JNK, and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in a concentration-dependent manner in RAW264.7 cells. Conclusions: The polysaccharide KRG-P is the active component responsible for the immune-enhancing activity of Korean red ginseng and may modulate the systemic immune system in vivo.

An Energy Efficient Thermally Regulated Optical Spectroscopy Cell for Lab-on-Chip Devices: Applied to Nitrate Detection

Reagent-based colorimetric analyzers often heat the fluid under analysis for improved reaction kinetics, whilst also aiming to minimize energy use per measurement. Here, a novel method of conserving heat energy on such microfluidic systems is presented. Our design reduces heat transfer to the environment by surrounding the heated optical cell on four sides with integral air pockets, thereby realizing an insulated and suspended bridge structure. Our design was simulated in COMSOL Multiphysics and verified in a polymethyl methacrylate (PMMA) device. We evaluate the effectiveness of the insulated design by comparing it to a non-insulated cell. For temperatures up to 55 °C, the average power consumption was reduced by 49.3% in the simulation and 40.2% in the experiment. The designs were then characterized with the vanadium and Griess reagent assay for nitrate at 35 °C. Nitrate concentrations from 0.25 µM to 50 µM were tested and yielded the expected linear relationship with a limit of detection of 20 nM. We show a reduction in energy consumption from 195 J to 119 J per 10 min measurement using only 4 µL of fluid. Efficient heating on-chip will have broad applicability to numerous colorimetric assays.

Antileishmanial Activity of Flavones-Rich Fraction From Arrabidaea chica Verlot (Bignoniaceae)

Acknowledging the need of identifying new compounds for the treatment of leishmaniasis, this study aimed to evaluate, from in vitro trials, the activity of flavones from Arrabidaea chica against L. amazonensis. The chromatographic profiles of the hydroethanolic extract and a flavone-rich fraction (ACFF) from A. chica were determined by high-performance liquid chromatography coupled with a diode-array UV-Vis detector (HPLC-DAD-UV) and electrospray ionization mass spectrometry in tandem (LC-ESI-MS-MS). The flavones luteolin (1) and apigenin (2), isolated from chromatographic techniques and identified by Nuclear Magnetic Resonance of 1H and 13C, were also quantified in ACFF, showing 190.7 mg/g and apigenin 12.4 mg/g, respectively. The other flavones were identified by comparing their spectroscopic data with those of the literature. The in vitro activity was assayed against promastigotes and intramacrophagic amastigote forms of L. amazonensis. Cytotoxicity tests were performed with peritoneal macrophages of BALB/c mice. Nitrite quantification was performed with Griess reagent. Ultrastructural investigations were obtained by transmission electron microscopy. Anti-Leishmania assays indicated that the IC50 values for ACFF, apigenin, and luteolin were obtained at 40.42 ± 0.10 and 31.51 ± 1.13 μg/mL against promastigotes, respectively. ACFF and luteolin have concentration-dependent cytotoxicity. ACFF and luteolin also inhibited the intra-macrophagic parasite (IC50 3.575 ± 1.13 and 11.78 ± 1.24 μg/mL, respectively), with a selectivity index of 11.44 for ACFF. Promastigotes exposed to ACFF and luteolin exhibited ultrastructural changes, such as intense cytoplasm vacuolization and mitochondrial swelling. These findings data evidence the antileishmanial action of flavone-rich fractions of A. chica against L. amazonensis, encouraging further studies.

Salivary nitrite and systemic biomarkers in obese individuals with periodontitis submitted to FMD

The objective of this 9-month clinical study is to assess the impact of one-stage full-mouth disinfection (FMD) on salivary nitrite levels and systemic biomarkers and its correlation with total subgingival bacterial load in obese and non-obese patients with periodontitis. In total, 94 patients (55 obese and 39 non-obese) were initially evaluated, seven were lost during follow-up, resulting in 87 individuals at the end of the study. Outcomes were assessed at baseline, 3, 6, and 9 months post periodontal treatment by FMD. Salivary nitrite levels were determined using Griess reagent. Blood samples were collected to determine C-Reactive Protein (CRP), alkaline phosphatase and fasting blood glucose. Real-time PCR was used to determine the total subgingival bacterial load. FMD protocol resulted in increased salivary nitrite levels at 6- and 9-months post-treatment in the non-obese group (p<0.05). In obese individuals, FMD treatment led to an increase in salivary nitrite levels at 6 months (p<0.05); however, at 9 months, the nitrite levels returned to baseline levels. For both groups, the highest nitrite values were observed at 6 months. In addition, in both groups, FMD was associated with a decrease in biomarkers related to systemic inflammation and cardiovascular diseases, such as CRP (p<0.05) and alkaline phosphatase (p<0.05), and had no impact on the fasting blood glucose. This study demonstrates that obese patients with periodontitis present similar salivary nitrite levels when compared with non-obese individuals. FMD protocol resulted in increases in salivary nitrite levels and was associated with a positive impact on systemic biomarkers, regardless of obesity status.

miR-744/eNOS/NO axis: A novel target to halt triple negative breast cancer progression

BACKGROUND: Nitric oxide (NO) may have a dual role in cancer. At low concentrations, endogenous NO promotes tumor growth and proliferation. However, at very high concentrations, it mediates cancer cell apoptosis and inhibits cancer growth. High levels of NO have been observed in blood of breast cancer (BC) patients, which increases tumor blood flow and promotes angiogenesis. To date, the regulation of NO-synthesizing enzyme, eNOS, by miRNAs has not been adequately investigated in BC. Therefore, the main aim of this study is to unravel the possible regulation of eNOS by miRNAs in BC and to examine their influence on NO production and BC progression. METHODS: Expression profile of eNOS in Egyptian BC patients and MDA-MB-231 cell lines was investigated using qRT-PCR. In-silico analysis was performed to predict a putative upstream regulator of eNOS. miR-744-5p was selected and its expression was quantified in BC tissues using qRT-PCR. MDA-MB-231 cells were cultured and transfected with miR-744-5p using lipofection method. NO levels were determined using Griess Reagent. Cellular viability and colony-forming ability were assessed using MTT and colony-forming assays; respectively. RESULTS: eNOS and miR-744-5p were significantly up-regulated in BC tissues compared to paired normal tissues. In-silico analysis revealed that miR-744-5p putatively binds to eNOS transcript with high binding scores. Transfection of MDA-MB-231 cells with miR-744-5p mimics resulted in a significant up-regulation of eNOS and consequently NO levels. In addition, miR-744-5p transfection led to an increase in cellular viability and colony-forming ability of the MDA-MB-231. CONCLUSION: miR-744-5p acts as an upstream positive regulator of the NO synthesizing enzyme, eNOS which in turn elevates NO levels. Furthermore, miR-744-5p is a novel oncogenic miRNA in BC. Thus, targeting miR-744/eNOS/NO axis may act as a therapeutic tool in TNBC.

Assessment of Immunotoxicity and Oxidative Stress Induced by Zinc Selenium/Zinc Sulphide Quantum Dots

Although ZnSe/ZnS quantum dots (QDs) have emerged as apparently less hazardous substitute to cadmium-based QDs, their toxicity has not been fully understood. Huge levels of ROS production and associated difficulties comprise the underlying reason for nanomaterial toxicity in cells. This will cause both immunotoxicity and genotoxicity. In the current work, Zinc Selenium/Zinc Sulphide (ZnSe/ZnS) QDs was synthesized, characterized and analyzed for its role in oxidative stress induction in two cell lines (HepG2 and HEK) and Swiss Albino mice. ROS production and influence of catalase activity in ROS production measured by DCFHDA assay in both HepG2 and HEK cells after exposure to ZnSe/ZnS QDs. Assessment of nitrile radical formation carried out by griess reagent. Level of GSH is assessed as a marker for oxidative stress induced by QDs. Cell death induced after exposure to ZnSe/ZnS QDs investigated by Calcein AM-PI live dead assay. Apoptotic DNA ladder assay carried out for studying the potential of ZnSe/ZnS QDs to induce DNA fragmentation. In vivo bio-nano interaction was studied by exposing Swiss Albino mice to ZnSe/ZnS QDs via i.v. and i.p. injection. Antioxidant assays were carried out in brain and liver homogenates to study the oxidative stress. LPO, GSH, GPx, GR and SOD are considered as biomarkers for the stress analysis. Blood brain barrier (BBB) integrity also studied. Spleenocytes proliferation assay was carried out to study the immunotoxicity response. ZnSe/ZnS QDs do not induce visible oxidative stress upto a concentration of 50 μg/ml. Cell death occurs at higher concentration (100 μg/ml) caused by ROS production. Overall study apparently provide attentive information that ZnSe/ZnS QDs is not capable of eliciting any serious damages to liver and brain tissues which in turn substantiates its applicability in biomedical applications.