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Electron paramagnetic resonance (EPR) spectroscopy is a tool that provides sensitive detection of uncoupled electron spins for a variety of applications. This technique enables the specific detection and quantification of radical species while also being able of generating high-contrast, background-free images. However, the EPR labeling and imaging techniques encounter limitations mainly due to the instability of organic radicals from organic probes, which can influence the reliability and scope of the experiment. In that context, the use of nanodiamonds (NDs) in EPR may be a promising route for understanding their unique properties and potential biomedical applications. The ability to perform EPR imaging in combination with the stable intrinsic properties of paramagnetic centers within these particles raises the possibility of extending nanodiamond-based imaging capabilities. Herein, we present a preliminary demonstration of a practical spectroscopy and imaging application using nanosized diamond particles (<18 nm) for electron paramagnetic resonance imaging (EPRI). The discretization of two different nanodiamond production sources among the most studied NDs (HPHT or detonation) allows further characterization of their physicochemical properties. In addition, we have investigated variations in the physicochemical properties of nanodiamonds, including size effects and surface treatments. Finally, we provide experimental evidence of the conditions required for optimal spectroscopic and imaging resolution ( R < 1 mm) as well as achievable EPR sensitivity.

An EPR spectrum or an EPR sinogram for imaging contains information about all the paramagnetic species that are in the analyzed sample. When only one species is present, an image of its spatial repartition can be reconstructed from the sinogram by using the well-known Filtered Back-Projection (FBP). However, in the case of several species, the FBP does not allow the reconstruction of the images of each species from a standard acquisition. One has to use for this spectral-spatial imaging whose acquisition can be very long. A new approach, based on Total Variation minimization, is proposed in order to efficiently extract the spatial repartitions of all the species present in a sample from standard imaging data and therefore drastically reduce the acquisition time. Experiments have been carried out on Tetrathiatriarylmethyl, nitroxide and DPPH., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

This study investigated the relationships between lignin molecular and supramolecular structures and their functional properties within cellulose-based solid matrix, used as a model biodegradable polymer carrier. Two types of derivatives corresponding to distinct structuration levels were prepared from a single technical lignin sample (PB1000): phenol-enriched oligomer fractions and colloidal nanoparticles (CLP). The raw lignin and its derivatives were formulated with cellulose nanocrystals or nanofibrils to prepare films by chemical oxidation or pressure-assisted filtration. The films were tested for their water and lignin retention capacities, radical scavenging capacity (RSC) and antimicrobial properties. A structural investigation was performed by infrared, electron paramagnetic resonance spectroscopy and microscopy. The composite morphology and performance were controlled by both the composition and structuration level of lignin. Phenol-enriched oligomers were the compounds most likely to interact with cellulose, leading to the smoothest film surface. Their RSC in film was 4- to 6-fold higher than that of the other samples. The organization in CLP led to the lowest RSC but showed capacity to trap and stabilize phenoxy radicals. All films were effective against S. aureus (gram negative) whatever the lignin structure. The results show the possibility to tune the performances of these composites by exploiting lignin multi-scale structure., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

A new biobased material based on an original strategy using lignin model compounds as natural grafting additive on a nanocellulose surface through in situ polymerization of coniferyl alcohol by the Fenton reaction at two pH values was investigated. The structural and morphological properties of the materials at the nanoscale were characterized by a combination of analytical methods, including Fourier transform infrared spectroscopy, liquid chromatography combined with mass spectrometry, nuclear molecular resonance spectroscopy, electron paramagnetic resonance spectroscopy, water sorption capacity by dynamic vapor sorption, and atomic force microscopy (topography and indentation modulus measurements). Finally, the usage properties, such as antioxidant properties, were evaluated in solution and the nanostructured casted films by radical 2,2'-diphenyl-1-picrylhydrazyl (DPPH • ) scavenging tests. We demonstrate the structure-function relationships of these advanced CNC-lignin films and describe their dual functionalities and characteristics, namely, their antioxidant properties and the presence of persistent phenoxy radicals within the material.

The mTOR is a central regulator of cell growth and is highly activated in cancer cells to allow rapid tumor growth. The use of mTOR inhibitors as anticancer therapy has been approved for some types of tumors, albeit with modest results. We recently reported the synthesis of ICSN3250, a halitulin analogue with enhanced cytotoxicity. We report here that ICSN3250 is a specific mTOR inhibitor that operates through a mechanism distinct from those described for previous mTOR inhibitors. ICSN3250 competed with and displaced phosphatidic acid from the FRB domain in mTOR, thus preventing mTOR activation and leading to cytotoxicity. Docking and molecular dynamics simulations evidenced not only the high conformational plasticity of the FRB domain, but also the specific interactions of both ICSN3250 and phosphatidic acid with the FRB domain in mTOR. Furthermore, ICSN3250 toxicity was shown to act specifically in cancer cells, as noncancer cells showed up to 100-fold less sensitivity to ICSN3250, in contrast to other mTOR inhibitors that did not show selectivity. Thus, our results define ICSN3250 as a new class of mTOR inhibitors that specifically targets cancer cells. Significance: ICSN3250 defines a new class of mTORC1 inhibitors that displaces phosphatidic acid at the FRB domain of mTOR, inducing cell death specifically in cancer cells but not in noncancer cells. Cancer Res; 78(18); 5384-97. ©2018 AACR ., (©2018 American Association for Cancer Research.)

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., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

In vivo electron paramagnetic resonance (EPR) imaging and spectroscopy are non-invasive technologies used to specifically detect and quantify paramagnetic species. However, the relative instability of spin probes such as triarylmethyl radicals limits their application to conduct oxygen quantification and mapping. In this study we encapsulated tetrathiatriarylmethyl radical (TAM; known as "Finland" probe) in Pluronic F-127 hydrogel (PF-127) in order to limit its degradation and evaluate its in vitro and in vivo EPR properties as a function of oxygen. Our results show that the EPR signal of encapsulated TAM in PF-127 hydrogel is similar to the one in solution. Although it is less sensitive to oxygen, it is suitable for oximetry. We also demonstrated that the incorporation of TAM in PF-127 hydrogel leads to an improved in vivo EPR stability of the radical under anesthesia. This new formulation enables high quality EPR imaging and oximetry and paves the way for the application of TAM radical-based probes in various biomedical fields., (Copyright © 2016. Published by Elsevier Inc.)

Novel silylated triarylmethyl (TAM) radicals based on TAM core CT-03 and their electron paramagnetic resonance (EPR) spectra are evaluated as a function of oxygen concentration. Combination of peak-to-peak linewidth of the EPR signal and electrochemical determination allows designing a method for oxygen quantification in phosphate buffer, dimethylsulfoxide, and dichloromethane, which can be extended to other solvents.

Reactive oxygen species are by-products of aerobic metabolism involved in the onset and evolution of various pathological conditions. Among them, the superoxide radical is of special interest as the origin of several damaging species such as H2O2, hydroxyl radical, or peroxynitrite (ONOO(-)). Spin trapping coupled with ESR is a method of choice to characterize these species in chemical and biological systems and the metabolic stability of the spin adducts derived from reaction of superoxide and hydroxyl radicals with nitrones is the main limit to the in vivo application of the method. Recently, new cyclic nitrones bearing a triphenylphosphonium or permethylated β-cyclodextrin moiety have been synthesized and their spin adducts demonstrated increased stability in buffer. In this article, we studied the stability of the superoxide adducts of four new cyclic nitrones in the presence of liver subcellular fractions and biologically relevant reductants using an original setup combining a stopped-flow device and an ESR spectrometer. The kinetics of disappearance of the spin adducts were analyzed using an appropriate simulation program. Our results highlight the interest of the new spin trapping agents CD-DEPMPO and CD-DIPPMPO for specific detection of superoxide with high stability of the superoxide adducts in the presence of liver microsomes., (© 2013 Elsevier Inc. All rights reserved.)

Yeast Dre2 is an essential Fe-S cluster-containing protein that has been implicated in cytosolic Fe-S protein biogenesis and in cell death regulation in response to oxidative stress. Its absence in yeast can be complemented by the human homologous antiapoptotic protein cytokine-induced apoptosis inhibitor 1 (also known as anamorsin), suggesting at least one common function. Using complementary techniques, we have investigated the biochemical and biophysical properties of Dre2. We show that it contains an N-terminal domain whose structure in solution consists of a stable well-structured monomer with an overall typical S-adenosylmethionine methyltransferase fold lacking two α-helices and a β-strand. The highly conserved C-terminus of Dre2, containing two Fe-S clusters, influences the flexibility of the N-terminal domain. We discuss the hypotheses that the activity of the N-terminal domain could be modulated by the redox activity of Fe-S clusters containing the C-terminus domain in vivo., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Although laboratory data clearly suggest a role for oxidants (dioxygen and free radicals derived from dioxygen) in the pathogenesis of many age-related and degenerative diseases (such as arthrosis and arthritis), methods to image such species in vivo are still very limited. This methodological problem limits physiopathologic studies about the role of those species in vivo, the effects of their regulation using various drugs, and the evaluation of their levels for diagnosis of degenerative diseases. In vivo electron paramagnetic resonance (EPR) imaging and spectroscopy are unique, noninvasive methods used to specifically detect and quantify paramagnetic species. However, two problems limit their application: the anatomic location of the EPR image in the animal body and the relative instability of the EPR probes. Our aim is to use EPR imaging to obtain physiologic and pathologic information on the mouse knee joint. This article reports the first in vivo EPR image of a small tissue, the mouse knee joint, with good resolution (≈ 160 μm) after intra-articular injection of a triarylmethyl radical EPR probe. It was obtained by combining EPR and x-ray micro-computed tomography for the first time and by taking into account the disappearance kinetics of the EPR probe during image acquisition to reconstruct the image. This multidisciplinary approach opens the way to high-resolution EPR imaging and local metabolism studies of radical species in vivo in different physiologic and pathologic situations.

The complete oxidation sequence of a model for ferrociphenols, a new class of anticancer drug candidate, is reported. Cyclic voltammetry was used to monitor the formation of oxidation intermediates on different timescales, thereby allowing the electrochemical characterization of both the short-lived and stable species obtained from the successive electron-transfer and deprotonation steps. The electrochemical preparation of the ferrocenium intermediate enabled a stepwise voltammetric determination of the stable oxidation compounds obtained upon addition of a base as well as the electron stoichiometry observed for the overall oxidation process. A mechanism has been established from the electrochemical data, which involves a base-promoted intramolecular electron transfer between the phenol and the ferrocenium cation. The resulting species is further oxidized then deprotonated to yield a stable quinone methide. To further characterize the transient species successively formed during the two-electron oxidation of the ferrociphenol to its quinone methide, EPR was used to monitor the fate of the paramagnetic species generated upon addition of imidazole to the electrogenerated ferrocenium. The study revealed the passage from an iron-centered to a carbon-centered radical, which is then oxidized to yield the quinone methide, namely, the species that interacts with proteins and so forth under biological conditions., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tah18-Dre2 is a recently identified yeast protein complex, which is highly conserved in human and has been implicated in the regulation of oxidative stress induced cell death and in cytosolic Fe-S proteins synthesis. Tah18 is a diflavin oxido-reductase with binding sites for flavin mononucleotide, flavin adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, which is able to transfer electrons to Dre2 Fe-S clusters. In this work we characterized in details the interaction between Tah18 and Dre2, and analysed how it conditions yeast viability. We show that Dre2 C-terminus interacts in vivo and in vitro with the flavin mononucleotide- and flavin adenine dinucleotide-binding sites of Tah18. Neither the absence of the electron donor nicotinamide adenine dinucleotide phosphate-binding domain in purified Tah18 nor the absence of Fe-S in aerobically purified Dre2 prevents the binding in vitro. In vivo, when this interaction is affected in a dre2 mutant, yeast viability is reduced. Conversely, enhancing artificially the interaction between mutated Dre2 and Tah18 restores cellular viability despite still reduced cytosolic Fe-S cluster biosynthesis. We conclude that Tah18-Dre2 interaction in vivo is essential for yeast viability. Our study may provide new insight into the survival/death switch involving this complex in yeast and in human cells., (© 2011 Blackwell Publishing Ltd.)

The hypoxic response in animals is mediated via the transcription factor hypoxia-inducible factor (HIF). An oxygen-sensing component of the HIF system is provided by Fe(II) and 2-oxoglutarate-dependent oxygenases that catalyse the posttranslational hydroxylation of the HIF-α subunit. It is proposed that the activity of the HIF hydroxylases can be regulated by their reaction with nitric oxide. We describe biochemical and biophysical studies on the reaction of prolyl hydroxylase domain-containing enzyme (PHD) isoform 2 (EGLN1) with nitric oxide and a nitric oxide transfer reagent. The combined results reveal the potential for the catalytic domain of PHD2 to react with nitric oxide both at its Fe(II) and at cysteine residues. Although the biological significance is unclear, the results suggest that the reaction of PHD2 with nitric oxide has the potential to be complex and are consistent with proposals based on cellular studies that nitric oxide may regulate the hypoxic response by direct reaction with the HIF hydroxylases., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The formation of hydroxo complexes based on a calix[6]trisimidazole ligand is described. Deprotonation of the mononuclear Cu(II)-aqua complex gives rise to a dinuclear bis(mu-hydroxo) complex that has been characterized by X-ray diffraction analysis. Spectroscopic studies (EPR and UV-vis), conducted in dichloromethane solutions in the presence of various coordinating cosolvents (DMF, EtOH, or RCN) or with acetamide, revealed the coexistence of a mononuclear hydroxo species. The latter could be trapped by acetic acid to provide an acetato-Cu(II) complex that presents close spectroscopic features. An EXAFS study first conducted on the hydroxo-Cu(II) complex led to an excellent fit for the dinuclear core. It then allowed for the characterization of the mononuclear acetato complex with an acetamide guest included in the calixarene cavity. Hence, this study illustrates the flexibility of calixarene-based ligands and the role of the second coordination sphere in the stabilization of acidic or basic Cu(II) complexes.

Duox2 (and probably Duox1) is a glycoflavoprotein involved in thyroid hormone biosynthesis, as the thyroid H2O2 generator functionally associated with Tpo (thyroperoxidase). So far, because of the impairment of maturation and of the targeting process, transfecting DUOX into nonthyroid cell lines has not led to the expression of a functional H2O2-generating system at the plasma membrane. For the first time, we investigated the H2O2-generating activity in the particulate fractions from DUOX2- and DUOX1-transfected HEK293 and Chinese hamster ovary cells. The particulate fractions of these cells stably or transiently transfected with human or porcine DUOX cDNA demonstrate a functional NADPH/Ca2+-dependent H2O2-generating activity. The immature Duox proteins had less activity than pig thyrocyte particulate fractions, and their activity depended on their primary structures. Human Duox2 seemed to be more active than human Duox1 but only half as active as its porcine counterpart. TPO co-transfection produced a slight increase in the enzymatic activity, whereas p22(phox), the 22-kDa subunit of the leukocyte NADPH oxidase, had no effect. In previous studies on the mechanism of H2O2 formation, it was shown that mature thyroid NADPH oxidase does not release O2*- but H2O2. Using a spin-trapping technique combined with electron paramagnetic resonance spectroscopy, we confirmed this result but also demonstrated that the partially glycosylated form of Duox2, located in the endoplasmic reticulum, generates superoxide in a calcium-dependent manner. These results suggest that post-translational modifications during the maturation process of Duox2 could be implicated in the mechanism of H2O2 formation by favoring intramolecular superoxide dismutation.

Reactive oxygen species (ROS) are implicated in the pathophysiology of various diseases, including cancer. In this study, we show that JunD, a member of the AP-1 family of transcription factors, reduces tumor angiogenesis by limiting Ras-mediated production of ROS. Using junD-deficient cells, we demonstrate that JunD regulates genes involved in antioxidant defense, H2O2 production, and angiogenesis. The accumulation of H2O2 in junD-/- cells decreases the availability of FeII and reduces the activity of HIF prolyl hydroxylases (PHDs) that target hypoxia-inducible factors-alpha (HIFalpha) for degradation. Subsequently, HIF-alpha proteins accumulate and enhance the transcription of VEGF-A, a potent proangiogenic factor. Our study uncovers the mechanism by which JunD protects cells from oxidative stress and exerts an antiangiogenic effect. Furthermore, we provide new insights into the regulation of PHD activity, allowing immediate reactive adaptation to changes in O2 or iron levels in the cell.

Electron transfer from tetrahydropterins to iron porphyrins, with formation of intermediate tetrahydropterin cation radicals, is a very general reaction that was shown to occur not only with tetrahydrobiopterin, as originally found in NO-synthases, but also with another important biological cofactor, tetrahydrofolate, and various iron porphyrins, either in their ferric state, or in the Fe(II)O(2) state, as in the first model of the corresponding NO-synthase reaction described in this paper.

Nitric oxide synthases (NOSs) are flavohemeproteins that catalyze the oxidation of l-arginine to l-citrulline with formation of the widespread signal molecule NO. Beside their fundamental role in NO biosynthesis, these enzymes are also involved in the formation of reactive oxygen species and in the interactions with some xenobiotic compounds. Nilutamide is a nonsteroidal antiandrogen that behaves as a competitive antagonist of the androgen receptors and is proposed in the treatment of metastatic prostatic carcinoma. However, therapeutic effects of nilutamide are overshadowed by the occurrence of several adverse reactions mediated by toxic mechanism(s), which remain(s) poorly investigated. Here, we studied the interaction of NOSs with nilutamide. Our results show that the purified recombinant neuronal NOS reduced the nitroaromatic nilutamide to the corresponding hydroxylamine. The reduction of nilutamide catalyzed by neuronal NOS proceeded with intermediate formation of a nitro anion free radical easily observed by EPR, was insensitive to the addition of the usual heme ligands and l-arginine analogues, but strongly inhibited by O(2) and a flavin/NADPH binding inhibitor. Involvement of the reductase domain of nNOS in the reduction of nilutamide was confirmed by (i) the ability of the isolated reductase domain of nNOS to catalyze the reaction and (ii) the stimulating effect of Ca(2+)/calmodulin on the accumulation of hydroxylamine and nitro anion radical. In a similar manner, the recombinant inducible and endothelial NOS isoforms also displayed nitroreductase activity, albeit with lower yields. The selective reduction of nilutamide to its hydroxylamino derivative by the NOSs could explain some of the toxic effects of this drug.

A new tripod N(3) ligand (L), containing three imidazole rings, was synthesized in good yield. At variance with usual aromatic ligands with N(2) or N(3) donor sets such as pyridine or pyrazole derivatives, L stabilizes the Fe(III) oxidation state. The corresponding iron(III) complexes [Fe(L)Cl(3)] (1) and [Fe(L)(2)](ClO(4))(3) (2) were prepared and characterized by X-ray structural analysis and spectroscopic methods. The coordination environment around all the Fe(III) centers has a distorted octahedral geometry. [Fe(L)Cl(3)] (1) belongs to the monoclinic system, space group P2(1)/n, a = 9.7406(5) A, b = 17.207(2) A, c = 14.615(2) A, beta = 104.448(9)(o) Z = 4, V = 2372.1(4) A(3); R = 0.044, R(w) = 0.055. [Fe(L)(2)](ClO(4))(3) (2) belongs to the monoclinic system, space group P2(1)/c, a = 16.1057(15) A, b = 11.1079(12) A, c = 26.283(2) A, beta = 102.062(10)(o), Z = 4, V = 4598.2(8) A(3); R = 0.0465, R(w) = 0.0902. The Fe-N((i)PrIm) bond lengths are systematically longer than the Fe-N(MeIm) ones. Compound 2 is a highly anisotropic low-spin Fe(III) complex displaying a rather unusual EPR spectrum with a sharp signal at g = 3.5 and a broad one at g approximately 1.6. The fitting of this EPR spectrum is discussed.

Nitric oxide (NO) is a potent intra- and intercellular messenger involved in the control of vascular tone, neuronal signalling and host response to infection. In mammals, NO is synthesized by oxidation of l-arginine catalysed by hemeproteins called NO-synthases with intermediate formation of Nomega-hydroxy-l-arginine (NOHA). NOHA and some hydroxyguanidines have been shown to be able to deliver nitrogen oxides including NO in the presence of various oxidative systems. In this study, NOHA and a model compound, N-(4-chlorophenyl)-N'-hydroxyguanidine, were tested for their ability to generate NO in the presence of a haemprotein model, microperoxidase 8 (MP8), and hydrogen peroxide. Nitrite and nitrate production along with selective formation of 4-chlorophenylcyanamide was observed from incubations of N-(4-chlorophenyl)-N'-hydroxyguanidine in the presence of MP8 and hydrogen peroxide. In the case of NOHA, the corresponding cyanamide, Ndelta-cyano-L-ornithine, was too unstable under the conditions used and l-citrulline was the only product identified. A NO-specific conversion of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide to 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl and formation of MP8-Fe-NO complexes were observed by EPR spectroscopy and low-temperature UV/visible spectroscopy, respectively. These results clearly demonstrate the formation of nitrogen oxides including NO from the oxidation of exogenous hydroxyguanidines by hydrogen peroxide in the presence of a minienzyme such as MP8. The importance of the bioactivation of endogenous (NOHA) or exogenous N-hydroxyguanidines by peroxidases of physiological interest remains to be established in vivo.