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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Free radicals, particularly reactive oxygen species (ROS), are involved in various pathologies, injuries related to radiation, ischemia-reperfusion or ageing. Unfortunately, it is virtually impossible to directly detect free radicals in vivo, but the redox status of the whole organism or particular organ can be studied in vivo by using magnetic resonance techniques (EPR and MRI) and paramagnetic stable free radicals – nitroxides. Here we review results obtained in vivo following the pharmacokinetics of nitroxides on experimental animals (and a few in humans) under various conditions. The focus was on conditions where the redox status has been altered by induced diseases or harmful agents, clearly demonstrating that various EPR/MRI/nitroxide combinations can reliably detect metabolically induced changes in the redox status of organs. These findings can improve our understanding of oxidative stress and provide a basis for studying the effectiveness of interventions aimed to modulate oxidative stress. Also, we anticipate that the in vivo EPR/MRI approach in studying the redox status can play a vital role in the clinical management of various pathologies in the years to come providing the development of adequate equipment and probes.

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...

Nitro-fatty acids modulate inflammatory and metabolic stress responses, thus displaying potential as new drug candidates. Herein, we evaluate the redox behavior of nitro-oleic acid (NO 2 -OA) and its ability to bind to the fatty acid transporter human serum albumin (HSA). The nitro group of NO 2 -OA underwent electrochemical reduction at −0.75 V at pH 7.4 in an aqueous milieu. Based on observations of the R–NO 2 reduction process, the stability and reactivity of NO 2 -OA was measured in comparison to oleic acid (OA) as the negative control. These electrochemically-based results were reinforced by computational quantum mechanical modeling. DFT calculations indicated that both the C9-NO 2 and C10-NO 2 positional isomers of NO 2 -OA occurred in two conformers with different internal angles (69° and 110°) between the methyl- and carboxylate termini. Both NO 2 -OA positional isomers have LUMO energies of around −0.7 eV, affirming the electrophilic properties of fatty acid nitroalkenes. In addition, the binding of NO 2 -OA and OA with HSA revealed a molar ratio of ~7:1 [NO 2 -OA]:[HSA]. These binding experiments were performed using both an electrocatalytic approach and electron paramagnetic resonance (EPR) spectroscopy using 16-doxyl stearic acid. Using a Fe(DTCS) 2 spin-trap, EPR studies also showed that the release of the nitro moiety of NO 2 -OA resulted in the formation of nitric oxide radical. Finally, the interaction of NO 2 -OA with HSA was monitored via Tyr and Trp residue electro-oxidation. The results indicate that not only non-covalent binding but also NO 2 -OA-HSA adduction mechanisms should be taken into consideration. This study of the redox properties of NO 2 -OA is applicable to the characterization of other electrophilic mediators of biological and pharmacological relevance.