The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.

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Fatty acid nitroalkenes (NO 2 -FA) are endogenously-generated products of the reaction of metabolic and inflammatory-derived nitrogen dioxide ( . NO 2 ) with unsaturated fatty acids. These species mediate signaling actions and induce adaptive responses in preclinical models of inflammatory and metabolic diseases. The nitroalkene substituent possesses an electrophilic nature, resulting in rapid and reversible reactions with biological nucleophiles such as cysteine, thus supporting post-translational modifications (PTM) of proteins having susceptible nucleophilic centers. These reactions contribute to enzyme regulation, modulation of inflammation and cell proliferation and the regulation of gene expression responses. Herein, focus is placed on the reduction-oxidation (redox) characteristics and stability of specific NO 2 -FA regioisomers having biological and clinical relevance; nitro-oleic acid (NO 2 -OA), bis-allylic nitro-linoleic acid (NO 2 -LA) and the conjugated diene-containing nitro-conjugated linoleic acid (NO 2 -cLA). Cyclic and alternating-current voltammetry and chronopotentiometry were used to the study of reduction potentials of these NO 2 -FA. R–NO 2 reduction was observed around −0.8 V ( vs . Ag/AgCl/3 M KCl) and is related to relative NO 2 -FA electrophilicity. This reduction process could be utilized for the evaluation of NO 2 -FA stability in aqueous milieu, shown herein to be pH dependent. In addition, electron paramagnetic resonance (EPR) spectroscopy was used to define the stability of the nitroalkene moiety under aqueous conditions, specifically under conditions where nitric oxide ( . NO) release could be detected. The experimental data were supported by density functional theory calculations using 6–311++G (d,p) basis set and B3LYP functional. Based on experimental and computational approaches, the relative electrophilicities of these NO 2 -FA are NO 2 -cLA >> NO 2 -LA > NO 2 -OA. Micellarization and vesiculation largely define these biophysical characteristics in aqueous, nucleophile-free conditions. At concentrations below the critical micellar concentration (CMC), monomeric NO 2 -FA predominate, while at greater concentrations a micellar phase consisting of self-assembled lipid structures predominates. The CMC, determined by dynamic light scattering in 0.1 M phosphate buffer (pH 7.4) at 25 °C, was 6.9 (NO 2 -LA) 10.6 (NO 2 -OA) and 42.3 μM (NO 2 -cLA), respectively. In aggregate, this study provides new insight into the biophysical properties of NO 2 -FA that are important for better un...

Electrochemistry of membrane proteins is complicated by the fact that the studied substances are poorly soluble or insoluble in aqueous environment. The solubilization of proteins using surfactants (detergents) affects the electrochemical analysis or even renders it impossible. In the present study, the electrochemistry of the transmembrane protein Na+/K+‐ATPase (NKA) and its water‐soluble isolated cytoplasmic loop C45 is described. The proteins were studied using adsorptive transfer cyclic voltammetry and square‐wave voltammetry on basal‐plane pyrolytic graphite electrode (PGE) as well as constant‐current chronopotentiometric stripping analysis on hanging mercury drop electrode (HMDE). The nonionic surfactant octaethylene glycol monododecyl ether (C12E8) was used for NKA solubilization. Under these conditions the oxidation currents of Tyr and Trp (peak Y: +0.55 V and peak W: +0.7 V, vs. Ag/AgCl/3 M KCl) and catalytic reduction currents (peak H: −1.8 V) of NKA and C45 loop can be observed. Using the experimental procedures suggested in this study, we were able to investigate the oxidation, reduction and adsorption of NKA and C45 at femtomole level without the necessity of labeling by electroactive markers or techniques based on protein immobilization within the lipid bilayer attached to the electrode surface.

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