Nitro-Oleic Acid (NO2-OA) Improves Systolic Function in Dilated Cardiomyopathy by Attenuating Myocardial Fibrosis

Nitro-oleic acid (NO₂-OA), a nitric oxide (NO)- and nitrite (NO₂⁻)-derived electrophilic fatty acid metabolite, displays anti-inflammatory and anti-fibrotic signaling actions and therapeutic benefit in murine models of ischemia-reperfusion, atrial fibrillation, and pulmonary hypertension. Muscle LIM protein-deficient mice (Mlp^−/−) develop dilated cardiomyopathy (DCM), characterized by impaired left ventricular function and increased ventricular fibrosis at the age of 8 weeks. This study investigated the effects of NO₂-OA on cardiac function in Mlp^−/− mice both in vivo and in vitro. Mlp^−/− mice were treated with NO₂-OA or vehicle for 4 weeks via subcutaneous osmotic minipumps. Wildtype (WT) littermates treated with vehicle served as controls. Mlp^−/− mice exhibited enhanced TGFβ signalling, fibrosis and severely reduced left ventricular systolic function. NO₂-OA treatment attenuated interstitial myocardial fibrosis and substantially improved left ventricular systolic function in Mlp^−/− mice. In vitro studies of TGFβ-stimulated primary cardiac fibroblasts further revealed that the anti-fibrotic effects of NO₂-OA rely on its capability to attenuate fibroblast to myofibroblast transdifferentiation by inhibiting phosphorylation of TGFβ downstream targets. In conclusion, we demonstrate a substantial therapeutic benefit of NO₂-OA in a murine model of DCM, mediated by interfering with endogenously activated TGFβ signaling.     

Xanthine Oxidoreductase-Mediated Superoxide Production Is Not Involved in the Age-Related Pathologies in Sod1-Deficient Mice

Reactive oxygen species (ROS) metabolism is regulated by the oxygen-mediated enzyme reaction and antioxidant mechanism within cells under physiological conditions. Xanthine oxidoreductase (XOR) exhibits two inter-convertible forms (xanthine oxidase (XO) and xanthine dehydrogenase (XDH)), depending on the substrates. XO uses oxygen as a substrate and generates superoxide (O₂^•−) in the catalytic pathway of hypoxanthine. We previously showed that superoxide dismutase 1 (SOD1) loss induced various aging-like pathologies via oxidative damage due to the accumulation of O₂^•− in mice. However, the pathological contribution of XO-derived O₂^•− production to aging-like tissue damage induced by SOD1 loss remains unclear. To investigate the pathological significance of O₂^•− derived from XOR in Sod1^−/− mice, we generated Sod1-null and XO-type- or XDH-type-knock-in (KI) double-mutant mice. Neither XO-type- nor XDH-type KI mutants altered aging-like phenotypes, such as anemia, fatty liver, muscle atrophy, and bone loss, in Sod1^−/− mice. Furthermore, allopurinol, an XO inhibitor, or apocynin, a nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor, failed to improve aging-like tissue degeneration and ROS accumulation in Sod1^−/− mice. These results showed that XOR-mediated O₂^•− production is relatively uninvolved in the age-related pathologies in Sod1^−/− mice.     

Lost-in-Translation of Metabolic Effects of Inorganic Nitrate in Type 2 Diabetes: Is Ascorbic Acid the Answer?

Beneficial metabolic effects of inorganic nitrate (NO₃⁻) and nitrite (NO₂⁻) in type 2 diabetes mellitus (T2DM) have been documented in animal experiments; however, this is not the case for humans. Although it has remained an open question, the redox environment affecting the conversion of NO₃⁻ to NO₂⁻ and then to NO is suggested as a potential reason for this lost-in-translation. Ascorbic acid (AA) has a critical role in the gastric conversion of NO₂⁻ to NO following ingestion of NO₃⁻. In contrast to AA-synthesizing species like rats, the lack of ability to synthesize AA and a lower AA body pool and plasma concentrations may partly explain why humans with T2DM do not benefit from NO₃⁻/NO₂⁻ supplementation. Rats also have higher AA concentrations in their stomach tissue and gastric juice that can significantly potentiate gastric NO₂⁻-to-NO conversion. Here, we hypothesized that the lack of beneficial metabolic effects of inorganic NO₃⁻ in patients with T2DM may be at least in part attributed to species differences in AA metabolism and also abnormal metabolism of AA in patients with T2DM. If this hypothesis is proved to be correct, then patients with T2DM may need supplementation of AA to attain the beneficial metabolic effects of inorganic NO₃⁻ therapy.     

Simultaneous Monitoring of pH and Chloride (Cl−) in Brain Slices of Transgenic Mice

Optosensorics is the direction of research possessing the possibility of non-invasive monitoring of the concentration of intracellular ions or activity of intracellular components using specific biosensors. In recent years, genetically encoded proteins have been used as effective optosensory means. These probes possess fluorophore groups capable of changing fluorescence when interacting with certain ions or molecules. For monitoring of intracellular concentrations of chloride ([Cl⁻]_i) and hydrogen ([H⁺] _i) the construct, called ClopHensor, which consists of a H⁺- and Cl⁻-sensitive variant of the enhanced green fluorescent protein (E²GFP) fused with a monomeric red fluorescent protein (mDsRed) has been proposed. We recently developed a line of transgenic mice expressing ClopHensor in neurons and obtained the map of its expression in different areas of the brain. The purpose of this study was to examine the effectiveness of transgenic mice expressing ClopHensor for estimation of [H⁺]_i and [Cl⁻]_i concentrations in neurons of brain slices. We performed simultaneous monitoring of [H⁺]_i and [Cl⁻]_i under different experimental conditions including changing of external concentrations of ions (Ca²⁺, Cl⁻, K⁺, Na⁺) and synaptic stimulation of Shaffer’s collaterals of hippocampal slices. The results obtained illuminate different pathways of regulation of Cl⁻ and pH equilibrium in neurons and demonstrate that transgenic mice expressing ClopHensor represent a reliable tool for non-invasive simultaneous monitoring of intracellular Cl⁻ and pH.     

FeIII,CuII and ZnII Complexes of the Rigid 9-Oxido-phenalenone Ligand—Spectroscopy,Electrochemistry, and Cytotoxic Properties

The three complexes [Fe(opo)₃], [Cu(opo)₂], and [Zn(opo)₂] containing the non-innocent anionic ligand opo⁻ (opo⁻ = 9-oxido-phenalenone, Hopo = 9-hydroxyphenalonone) were synthesised from the corresponding acetylacetonates. [Zn(opo)₂] was characterised using ¹H nuclear magnetic resonance (NMR) spectroscopy, the paramagnetic [Fe(opo)₃] and [Cu(opo)₂] by electron paramagnetic resonance (EPR) spectroscopy. While the EPR spectra of [Cu(opo)₂] and [Cu(acac)₂] in dimethylformamide (DMF) solution are very similar, a rather narrow spectrum was observed for [Fe(opo)₃] in tetrahydrofuran (THF) solution in contrast to the very broad spectrum of [Fe(acac)₃] in THF (Hacac = acetylacetone, 2,4-pentanedione; acac⁻ = acetylacetonate). The narrow, completely isotropic signal of [Fe(opo)₃] disagrees with a metal-centred S = 5/2 spin system that is observed in the solid state. We assume spin-delocalisation to the opo ligand in the sense of an opo⁻ to Fe^III electron transfer. All compounds show several electrochemical opo-centred reduction waves in the range of −1 to −3 V vs. the ferrocene/ferrocenium couple. However, for Cu^II and Fe^III the very first one-electron reductions are metal-centred. Electronic absorption in the UV to vis range are due to π–π* transitions in the opo core, giving Hopo and [Zn(opo)₂] a yellow to orange colour. The structured bands ranging from 400 to 500 for all compounds are assigned to the lowest energy π−π* transitions. They show markedly higher intensities and slight shifts for the Cu^II (brown) and Fe^III (red) complexes and we assume admixing metal contributions (MLCT for Cu^II, LMCT for Fe^III). For both complexes long-wavelength absorptions assignable to d–d transitions were detected. Detailed spectroelectrochemical experiments confirm both the electrochemical and the optical assignments. Hopo and the complexes [Cu(opo)₂], [Zn(opo)₂], and [Fe(opo)₃] show antiproliferative activities against HT-29 (colon cancer) and MCF-7 (breast cancer) cell lines in the range of a few µM, comparable to cisplatin under the same conditions.     

Investigation of NO Adsorption and NO/O2 Co-adsorption on NOx-Storage-Components by DRIFT-Spectroscopy

NO adsorption and NO/O₂ co-adsorption on CeO₂ at different temperatures was studied by DRIFT-Spectroscopy. The results indicate that this oxide plays an important role in storing NO _x . FTIR studies show that NO adsorption is dominated by the formation of nitrite species. Furthermore, cis- and trans hyponitrite species are detected. Co-adsorption of NO/O₂ leads to the formation of nitrates. The experimental data show that the formation of nitrates is a consecutive reaction: adsorption of NO to form nitrite species (NO₂ ^–), followed by an oxidation to form nitrate species (NO₃ ^–).     

Sod1 Loss Induces Intrinsic Superoxide Accumulation Leading to p53-Mediated Growth Arrest and Apoptosis

Oxidative damages induced by a redox imbalance cause age-related changes in cells and tissues. Superoxide dismutase (SOD) enzymes play a major role in the antioxidant system and they also catalyze superoxide radicals (O₂^•−). Since the loss of cytoplasmic SOD (SOD1) resulted in aging-like phenotypes in several types of mouse tissue, SOD1 is essential for the maintenance of tissue homeostasis. To clarify the cellular function of SOD1, we investigated the cellular phenotypes of Sod1-deficient fibroblasts. We demonstrated that Sod1 deficiency impaired proliferation and induced apoptosis associated with O₂^•− accumulation in the cytoplasm and mitochondria in fibroblasts. Sod1 loss also decreased the mitochondrial membrane potential and led to DNA damage-mediated p53 activation. Antioxidant treatments effectively improved the cellular phenotypes through suppression of both intracellular O₂^•− accumulation and p53 activation in Sod1-deficient fibroblasts. In vivo experiments revealed that transdermal treatment with a vitamin C derivative significantly reversed the skin thinning commonly associated with the upregulated p53 action in the skin. Our findings revealed that intrinsic O₂^•− accumulation promoted p53-mediated growth arrest and apoptosis as well as mitochondrial disfunction in the fibroblasts.     

Electrochemical Determination of Food Preservative Nitrite with Gold Nanoparticles/p-Aminothiophenol-Modified Gold Electrode

Due to the negative impact of nitrate and nitrite on human health, their presence exceeding acceptable levels is not desired in foodstuffs. Thus, nitrite determination at low concentrations is a major challenge in electroanalytical chemistry, which can be achieved by fast, cheap, and safe electrochemical sensors. In this work, the working electrode (Au) was functionalized with p-aminothiophenol (p-ATP) and modified with gold nanoparticles (Au-NPs) to manufacture the final (Au/p-ATP-Au_nano) electrode in a two-step procedure. In the first step, p-ATP was electropolymerized on the electrode surface to obtain a polyaminothiophenol (PATP) coating. In the second step, Au/p-ATP-Au_nano working electrode was prepared by coating the surface with the use of HAuCl₄ solution and cyclic voltammetry. Determination of aqueous nitrite samples was performed with the proposed electrode (Au/p-ATP-Au_nano) using square wave voltammetry (SWV) in pH 4 buffer medium. Characteristic peak potential of nitrite samples was 0.76 V, and linear calibration curves of current intensity versus concentration was linear in the range of 0.5–50 mg·L⁻¹ nitrite with a limit of detection (LOD) of 0.12 mg·L⁻¹. Alternatively, nitrite in sausage samples could be colorimetrically determined with high sensitivity by means of p-ATP‒modified gold nanoparticles (AuNPs) and naphthylethylene diamine as coupling agents for azo-dye formation due to enhanced charge-transfer interactions with the AuNPs surface. The slopes of the calibration lines in pure NO₂⁻ solution and in sausage sample solution, to which different concentrations of NO₂⁻ standards were added, were not significantly different from each other, confirming the robustness and interference tolerance of the method. The proposed voltammetric sensing method was validated against the colorimetric nanosensing method in sausage samples.     

Tissue Printing and Dual Excitation Flow Cytometry for Oxidative Stress—New Tools for Reactive Oxygen Species Research in Seed Biology

The intracellular homeostasis of reactive oxygen species (ROS) and especially of superoxide anion and hydrogen peroxide participate in signaling cascades which dictate developmental processes and reactions to stresses. ROS are also biological molecules that play important roles in seed dormancy and germination. Because of their rapid reactivity, short half-life and low concentration, ROS are difficult to measure directly with high accuracy and precision. In presented work tissue printing method with image analysis and dual excitation flow cytometry (FCM) were developed for rapid detection and localization of O₂^•− and H₂O₂ in different part of seed. Tissue printing and FCM detection of ROS showed that germination of wild oat seeds was associated with the accumulation of O₂^•− and H₂O₂ in embryo (coleorhiza, radicle and scutellum), aleurone layer and coat. To verify if printing and FCM signals were specified, the detection of O₂^•− and H₂O₂ in seeds incubated in presence of O₂^•− generation inhibitor (DPI) or H₂O₂ scavenger (CAT) were examined. All results were a high level of agreement among the level of ROS derived from presented procedures with the ones created from spectrophotometric measured data. In view of the data obtained, tissue printing with image analysis and FCM are recommended as a simple and fast methods, which could help researchers to detection and level determination of ROS in the external and inner parts of the seeds.     

Oxidase Reactivity of CuII Bound to N-Truncated Aβ Peptides Promoted by Dopamine

The redox chemistry of copper(II) is strongly modulated by the coordination to amyloid-β peptides and by the stability of the resulting complexes. Amino-terminal copper and nickel binding motifs (ATCUN) identified in truncated Aβ sequences starting with Phe4 show very high affinity for copper(II) ions. Herein, we study the oxidase activity of [Cu–Aβ_4−x] and [Cu–Aβ_1−x] complexes toward dopamine and other catechols. The results show that the Cu^II–ATCUN site is not redox-inert; the reduction of the metal is induced by coordination of catechol to the metal and occurs through an inner sphere reaction. The generation of a ternary [Cu^II–Aβ–catechol] species determines the efficiency of the oxidation, although the reaction rate is ruled by reoxidation of the Cu^I complex. In addition to the N-terminal coordination site, the two vicinal histidines, His13 and His14, provide a second Cu-binding motif. Catechol oxidation studies together with structural insight from the mixed dinuclear complexes Ni/Cu–Aβ_4−x reveal that the His-tandem is able to bind Cu^II ions independently of the ATCUN site, but the N-terminal metal complexation reduces the conformational mobility of the peptide chain, preventing the binding and oxidative reactivity toward catechol of Cu^II bound to the secondary site.     

Impedance study on calcium nitrite as a penetrating corrosion inhibitor for steel in concrete

Penetrating corrosion inhibitors are thought to be able to penetrate through the capillary structure of concrete to reinforcing steel and to reduce the already initiated corrosion of steel. In this work the ability of calcium nitrite to inhibit the chloride induced corrosion of steel was studied. The test protocol was adjusted to simulate the performance of penetrating corrosion inhibitors in concrete structures. Steel samples were first prepassivated in saturated solution of Ca(OH)₂ and then exposed to the same solution with 1% NaCl addition, simulating pore liquid in chloride contaminated concrete. After the initiation of steel corrosion, the first dose of calcium nitrite was added, and then its concentration was gradually increased and the inhibition effect was related to the molar ratio of chloride to nitrite ions [Cl⁻]/[NO₂⁻]. Different rates of the increase in the inhibitor concentration were applied.Electrochemical impedance spectroscopy was used to follow the behaviour of steel at different stages of the corrosion process. The evolution of acquired spectra reflected the initiation of localized corrosion of steel and then the gradual inhibition with increasing concentration of the inhibitor. It was found, that calcium nitrite is able to inhibit the initiated corrosion (pitting) of steel and the optimum inhibitor efficiency was observed for the [Cl⁻]/[NO₂⁻] ratio below 1. The inhibition efficiency was larger, when this value of the [Cl⁻]/[NO₂⁻] ratio was reached in early stages of the corrosion development. Calcium nitrite can be effective as a penetrating corrosion inhibitor for steel in concrete, if it will be present in the sufficient concentration at the steel surface in early stages of the corrosion development.     

Involvement of the MetO/Msr System in Two Acer Species That Display Contrasting Characteristics during Germination

The levels of methionine sulfoxide (MetO) and the abundances of methionine sulfoxide reductases (Msrs) were reported as important for the desiccation tolerance of Acer seeds. To determine whether the MetO/Msrs system is related to reactive oxygen species (ROS) and involved in the regulation of germination in orthodox and recalcitrant seeds, Norway maple and sycamore were investigated. Changes in water content, MetO content, the abundance of MsrB1 and MsrB2 in relation to ROS content and the activity of reductases depending on nicotinamide adenine dinucleotides were monitored. Acer seeds differed in germination speed—substantially higher in sycamore—hydration dynamics, levels of hydrogen peroxide, superoxide anion radicals (O₂^•−) and hydroxyl radicals (•OH), which exhibited peaks at different stages of germination. The MetO level dynamically changed, particularly in sycamore embryonic axes, where it was positively correlated with the levels of O₂^•− and the abundance of MsrB1 and negatively with the levels of •OH and the abundance of MsrB2. The MsrB2 abundance increased upon sycamore germination; in contrast, it markedly decreased in Norway maple. We propose that the ROS–MetO–Msr redox system, allowing balanced Met redox homeostasis, participates in the germination process in sycamore, which is characterized by a much higher speed compared to Norway maple.     

Effects of NO2 ions on localised corrosion of steel in NaHCO3 + NaCl electrolytes

Pitting corrosion of carbon steel electrodes in 0.1 mol L⁻¹ NaHCO₃ + 0.02 mol L⁻¹ NaCl solutions was induced by anodic polarisation. The evolution of the breakdown potential E_b with NO₂⁻ concentration was investigated by linear voltammetry. E_b increased from −15 ± 5 mV/SCE for [NO₂⁻] = 0 up to 400 ± 50 mV/SCE for [NO₂⁻] = 0.1 mol L⁻¹. During anodic polarisation at potentials comprised between E_b([NO₂⁻] = 0) and E_b([NO₂⁻] ≠ 0), the behaviour of the whole electrode surface, followed by chronoamperometry, was compared to the behaviour of one single pit, followed via scanning vibrating electrode technique (SVET). Addition of a NaNO₂ solution after the beginning of the polarisation led to a rapid repassivation of pre-existing well-grown pits. In situ micro-Raman spectroscopy was then used to identify the corrosion products forming inside the pits. The first species to be detected in the presence of NO₂⁻ were mainly dissolved Fe(III) species, more likely [Fe^III(H₂O)₆]³⁺ complexes. Iron(II) carbonate FeCO₃, siderite, and carbonated green rust GR(CO₃²⁻) were also detected in the active pits, as in the absence of nitrite. But they were accompanied by maghemite γ-Fe₂O₃, a phase structurally similar to the passive film, that forms from the Fe(III) complexes. The Raman analyses then correlate with the SVET observations and confirm that the main effect of nitrite ions is to oxidize iron(II) into iron(III). The passive film would then form from the Fe(III) species still bound to the steel surface.     

Cystine/Glutamate Antiporter in Schizophrenia: From Molecular Mechanism to Novel Biomarker and Treatment

Glutamate, a crucial excitatory neurotransmitter, plays a major role in the modulation of schizophrenia’s pathogenesis. New drug developments for schizophrenia have been prompted by the hypoglutamatergic hypothesis of schizophrenia. The cystine/glutamate antiporter system x_c⁻ is related to glutamate-release regulation. Patients with schizophrenia were recently discovered to exhibit downregulation of x_c⁻ subunits—the solute carrier (SLC) family 3 member 2 and the SLC family 7 member 11. We searched for relevant studies from 1980, when Bannai and Kitamura first identified the protein subunit system x_c⁻ in lung fibroblasts, with the aim of compiling the biological, functional, and pharmacological characteristics of antiporter x_c⁻, which consists of several subunits. Some of them can significantly stimulate the human brain through the glutamate pathway. Initially, extracellular cysteine activates neuronal x_c⁻, causing glutamate efflux. Next, excitatory amino acid transporters enhance the unidirectional transportation of glutamate and sodium. These two biochemical pathways are also crucial to the production of glutathione, a protective agent for neural and glial cells and astrocytes. Investigation of the expression of system x_c⁻ genes in the peripheral white blood cells of patients with schizophrenia can facilitate better understanding of the mental disorder and future development of novel biomarkers and treatments for schizophrenia. In addition, the findings further support the hypoglutamatergic hypothesis of schizophrenia.     

Sorption–Desorption of NOx from a Lean Gas Mixture on H3PW12O40·6H2O Supported on Carbon Nanotubes

NO _x sorption capacities and efficiencies were measured on a new type of sorbent formed by 12-tungstophosphoric acid (HPW) supported on carbon nanotubes. On such a system, the sorption of both NO and NO₂ was observed but compared with HPW alone, a complementary sorption of NO _x is possible leading to a capacity of 25 mg/g_HPW at 300 °C with an efficiency of 50%. The sorption results from the formation of a [H⁺(NO₂ ^–,NO⁺)] complex on HPW and an additional mode of adsorption by a free-nitrate which was identified by the bands at 2261, 1384 and 1295 cm^–1 using infrared spectroscopy.     

Influence of Incorporation of Different dn-Electron Metal Cations into Biologically Active System on Its Biological and Physicochemical Properties

Three new compounds, namely [HL]₂⁺[CuCl₄]²⁻, [HL]₂⁺[ZnCl₄]²⁻, and [HL]₂⁺[CdCl₄]²⁻ (where L: imipramine) were synthesized and their physicochemical and biological properties were thoroughly investigated. All three compounds form isostructural, crystalline systems, which have been studied using Single-Crystal X-ray diffraction analysis (SC-XRD) and Fourier-transform infrared spectroscopy (FTIR). The thermal stability was investigated using thermogravimetric analysis (TGA) and melting points for all compounds have been determined. Magnetic measurements were performed in order to study the magnetic properties of the compounds. The above mentioned techniques allowed us to comprehensively examine the physicochemical properties of the newly obtained compounds. The biological activity was investigated using the number of Zebrafish tests, as it is one of the most common models for studying the impact of newly synthesized compounds on the central nervous system (CNS), since this model is very similar to the human CNS.