Plants are rich in ascorbic acid (vitamin C) and are a critical source in the human diet. It is often the most abundant primary metabolite in leaves and reaches very high concentration in some fruits. Despite its abundance, poor diets can result in vitamin C deficiency, while poor health may increase the daily requirement. Ascorbate is an effective antioxidant and free radical scavenger and readily reduces iron (for example by acting as a chaperone for 2-oxoglutarate-dependent dioxygenases), so it interacts with metabolism in multiple ways. Plants have specific ascorbate peroxidases which are important for hydrogen peroxide removal. Ascorbate concentration in leaves is strongly light responsive which reflects an important role in scavenging photosynthesis-sourced hydrogen peroxide as well as a role in other photoprotective mechanisms. Plants primarily synthesise ascorbate from L-galactose, which is derived from GDP-L-galactose via GDP-mannose. There is abundant evidence that GDP-L-galactose phosphorylase is the rate controlling enzyme in this pathway. Over-expression of the 6 enzymes on the pathway from mannose 1-P to ascorbate shows that only GDP-L-galactose phosphorylase has a significant effect on ascorbate. This is rationalised by a model of the pathway based on measured kinetic constants. The enzyme is encoded by two paralogues (VTC2 and VTC5) in Arabidopsis thaliana. VTC2 and VTC5 transcription is responsive to light and is repressed as ascorbate accumulates. Additionally, the 5’-UTR of the transcript has a conserved upstream open reading frame (uORF) which appears to have a role in controlling its translation and could contribute to feedback repression of ascorbate synthesis. CRISPR-Cas gene editing of the uORF has caused ascorbate hyper-accumulation in various species including tomato.

The cytosol, chloroplast stroma and mitochondrial matrix contain a regulatory thiol-disulfide redox network consisting of redox input elements, redox transmitters, redox targets, redox sensors and final electron acceptors like H2O2. The network structure is conserved in eukaryotes, and particularly complex in plants as indicated by large gene families encoding, e.g. thioredoxins, glutaredoxins and thiol peroxidases, namely peroxiredoxins (PRX) and glutathione peroxidase-like (GPXL) proteins. Network dynamics in plants is best explored in photosynthesizing chloroplasts. The talk will first elaborate on the role of the chloroplast 2-CysPRX as sensor and regulatory element in adjusting photosynthetic metabolism to the prevailing condition for photosynthetic CO2 fixation. The presence of oxidized 2-CysPRX is required for rapid deactivation of Calvin-Benson cycle enzymes upon lowering of light intensity or darkening. In the case of plant - but also human - 2-CysPRX, the redox-dependent conformational dynamics can be documented by single molecule mass photometry. Mathematical modelling allows for predicting network behavior. The second part will focus on the cytosolic thiol-disulfide redox network. It will be shown how reconstitution of partial networks using recombinant proteins, together with fluorescence sensors for glutathione and H2O2, allows for exploring the redox dynamics upon addition of H2O2 bursts and to explore the contribution of single elements. The talk will conclude with a personal perspective on current and future directions of research on the redox regulatory network.

Background:
Hydroxytyrosol (HT) is a dietary phenol associated with several beneficial health effects in humans. HT main dietary source is extra virgin olive oil. Wine and beer are source of the phenol Tyrosol (Tyr). Pre-clinical and clinical studies had pointed out Tyr as a potential precursor of HT. This reaction is mediated by the cytochrome P450 (CYP450) CYP2A6 and CYP2D6 polymorphic isoforms.

Aims and methods:
We performed a randomized controlled clinical trial to confirm the contribution of HT formation after the administration of Tyr and its effects on the cardiovascular system in individuals at high cardiovascular risk. The trial included 33 volunteers and had a cross-over design with three 4-week interventions: (i) white wine (WW), (ii) WW supplemented with Tyr and (iii) control intervention (no alcohol). Volunteers were genotyped for CYP2A6 and CYP2D6 to predict the activity of both enzymes.

Results:
Our results show that HT urinary recovery was significantly increased at the end of WW+Tyr intervention. CYP2A6 and CYP2D6 genotypes of the volunteers, and their predicted enzyme activity, modulated the production of HT. The following changes in the cardiovascular system were observed at the end of WW+Tyr intervention: an improvement in endothelial function, and lipid profile, an increase in antithrombin III, a decrease in homocysteine and endothelin-1 and a cardioprotective modulation of circulating levels of ceramides. Additionally, we observed a regulatory effect of Tyr supplementation on the expression on several genes associated with the cardiovascular system.
Conclusions:

Tyr was endogenously converted into HT in vivo. This conversion is mediated by polymorphic CYP2A6 and CYP2D6 isoforms of CYP450. Tyr supplementation and its following bioactivation into HT triggered several positive effects on the cardiovascular system.

Background: Our ongoing research on the health benefits of flavonoids from the hops plant (Humulus lupulus) revealed that xanthohumol (XN), the principal prenylated flavonoid in hops, has the potential to improve diet-induced dysfunctional glucose and lipid metabolism in metabolic syndrome. Sphingolipids including ceramides are implicated in the pathogenesis of obesity and insulin resistance. Correspondingly, inhibition of pro-inflammatory and neurotoxic ceramide accumulation prevents obesity-mediated insulin resistance and cognitive impairment. Increasing evidence suggests the farnesoid X receptor (FXR) is involved in ceramide metabolism, as bile acid (BA)-FXR crosstalk controls ceramide levels along the gut-liver axis.
Aims: To determine to which extent XN and structurally related flavonoids improve manifestations of metabolic syndrome through modulation of FXR.
Methods: We examined how XN and its metabolically stable hydrogenated derivatives, dihydroxanthohumol (DXN) and tetrahydroxanthohumol (TXN), improve insulin resistance and cognitive impairment. We analyzed lipid and BA profiles in the liver and hippocampus, measured sphingolipid relative abundance in the hippocampus, and linked them to metabolic and neurocognitive performance in high-fat diet (HFD) fed C57BL/6J mice. Wild-type and liver-specific FXR-null mice (FXRliver-/-) were fed HFD containing XN or the vehicle and liver tissues were examined by histological characterization, lipidomics, and BA profiling.
Results: XN, DXN and TXN (30 mg/kg body weight/day) decreased ceramide content in liver and hippocampus; the latter was linked to improvements in spatial learning and memory. In addition, XN, DXN and TXN decreased hepatic cholesterol content. HFD supplemented with XN (60 mg/kg body weight/day) resulted in amelioration of hepatic steatosis and decreased BA concentrations in FXRliver−/− mice, the effect being stronger in male mice.
Conclusions: XN, DXN and TXN may alleviate obesity-induced metabolic and neurocognitive impairments by targeting the gut-liver-brain axis. The findings indicate a sex-dependent relationship between FXR, lipids and BAs, and suggest that XN ameliorates HFD-induced liver dysfunction via FXR-dependent and independent signaling.

Reactive oxygen species (ROS) are key signalling molecules that enable cells to rapidly respond to different stimuli. In plants, ROS play a crucial role in abiotic and biotic stress sensing, integration of different environmental signals, and activation of stress-response networks, leading to the buildup of defense and acclimation mechanisms and plant resilience. Recent advances in the study of ROS signalling in plants include the identification of ROS receptors and key regulators that connect ROS signalling with other important stress-response signal transduction pathways and hormones, an increased understanding of how ROS are regulated in cells by balancing production, scavenging and transport, and the identification of new roles for ROS in organelle-to-organelle and cell-to-cell signalling. The role of ROS in cell-to-cell signaling, systemic plant responses to abiotic stress, and plant acclimation to climate change-driven multifactorial stress combination will be discussed.

Physiological redox balance: Oxidative eustress
Helmut Sies, Institute for Biochemistry and Molecular Biology I, Faculty of Medicine, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Maintenance of redox homeostasis is a continuously ongoing challenge. In the open metabolic system, redox-related signalling requires monitoring and fine-tuning of the steady state redox set point. The ongoing oxidative metabolism is a persistent challenge, denoted as oxidative eustress (1). This physiological and beneficial oxidative stress is contrasted to non-physiological and detrimental oxidative distress (2). H2O2 is a major redox signalling agent, targeting specific cysteines in proteins which act as molecular redox switches to activate transcription factors (3). Major functions affected by physiological redox signalling in eustress include proliferation, development, cognition, thermogenesis, impacting on global topics such as inflammation, immunity and the aging process. Current research on the control of H2O2 dynamics in subcellular, cellular and intercellular spaces is flourishing. It includes regulation of the sources and sinks of H2O2, its transport via peroxiporins, and the interaction with other signalling pathways. A major challenge is to define the borderline between oxidative eustress and oxidative distress, which is cell type-specific and fluctuates according to metabolic condition.
(1) Sies, H. (2021) Oxidative eustress: On constant alert for redox homeostasis. Redox Biol. 41, 101867
(2) Sies, H., ed. (2020) Oxidative stress: Eustress and distress, pp. 1-844, Academic Press, London
(3) Sies, H., et al (2022) Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology. Nat. Rev. Mol. Cell. Biol. 23, xxx-xxx

Redox mechanisms function in organization of all life forms and have a special role in evolution of multicellular animals due to evolution in response to the dramatic rise in atmospheric O2 about 700 million years ago. Multiple redox mechanisms are used to obtain energy, and redox mechanisms linked to NAD and NADP provide a central logic for metabolic organization while mechanisms linked to H2O2 and thiols provide a central logic for macromolecular structure, signaling and spatial and temporal organization. Advances in omics technologies now enable mapping of functional networks through controlled experimental variations and application of data-driven community detection tools. Results show dynamic responses to environmental such as manganese exposure and highlight adaptive metabolic and transcriptional changes through pleiotropic H2O2 signaling and the redox proteome. Refinement of network models is occurring through incorporation of the metallome into integrated molecular and phenotype analyses, illustrated by recent findings of redox cycling in vanadium-dependent cell senescence. Similarly, incorporation of the gut microbiome into network models has revealed diet-dependent obesity linked to -valerobetaine, a microbial product that inhibits fatty acid -oxidation and increases adiposity. Results suggest that redox mechanisms exist with a hierarchy of metabolic, proteomic and transcriptional responses. Once assembled into atlases with development of appropriate redox systems models, these data will enable redox diagnostics and redox therapeutics for personalized healthcare.

Our understanding of the living world is quite recent. Advances in science and technology during the 20th century show that life is a unit that has changed our planet's environment and transformed its own history in the process. The rise of photosynthetic cyanobacteria extended between 2.9 and 3.4 billion years ago, a slow start long before the great oxygenation event that metamorphosed the Earth. The incorporation of a cyanobacterium by a heterotrophic host was a pivotal horizontal genome transfer event that sustained many ecosystems on our planet. Paradoxically photosynthesis produces reactive oxygen species that are harmful to the host cell. Plants are equipped with complex and multilevel antioxidative system that maintain a redox equilibrium and allows quick adaption to a wide array of stressors, making them the most evolutionarily successful living organisms on earth.
But plants cannot thrive alone; they co-evolve with the microbial world. About 30% of plant’s energy is directed to the root zone to attract and feed surrounding soil microorganisms which, in return, make nutrients bioavailable for plants, produce chemicals to stimulate plant growth, and protect them from pathogens.
Notwithstanding the finetuned survival circuit of natural ecosystems, the plants we need to keep feeding human population while preserving the environment are nowhere to be found. Nature makes plants to survive and thrive, not to excel in productivity. Advances in plant science have enabled new tools for breeding, as well as genetic engineering, editing and synthetic biology. Technically we are ready to make new plants that will meet our needs. But science alone will not be enough for the transformative changes needed to achieve environmental, nutritional, and economic sustainability of human societies. Technological progress always triggers reactions from society, and it is essential to pay attention to how these reactions can influence the acceptance of an innovative product.

Organ fibrosis remains a major challenge affecting 1 in 4 persons globally. Currently there exist very limited therapeutic options, none of them curative. Fibrosis results from an unbalanced response to inflammation and wound healing leading to the activation of specific subpopulations of resident mesenchymal cells, considered precursors of myofibroblasts, which generate extracellular matrix (ECM) that replace the cellular living tissue. Metabolic derangement has been identified as a key culprit in the pathophysiology of fibrogenesis. In the kidney, mitochondrial dysfunction and defective fatty acid oxidation (FAO) in tubular epithelial cells (TECs) play an important role in the development of fibrosis. We have found that conditional overexpression of the enzyme Cpt1a in kidney tubules promotes enhanced FAO, restores mitochondrial homeostasis and protects from fibrosis. In addition, we have identified specific microRNAs that are important to regulate the genesis of fibrosis by targeting specific metabolic routes. Perturbations in the circadian rhythm and the molecular clock have been associated with many human pathologies, including renal disease. We investigated the relationship between the molecular clock and renal damage in experimental models of injury and fibrosis (UUO, FAN and adenine toxicity), employing genetically-modified mice with selective deficiencies of the clock components Bmal1, Clock and Cry. We found that UUO induced a marked increase in the expression of Bmal1. In human TECs, the pro-fibrotic mediator, TGF-, significantly altered the expression of core clock components. We further observed that the absence of Cry drastically aggravated kidney fibrosis, while both Cry and Clock played a role in the neutrophil and macrophage mediated inflammatory responses, respectively. Suppression of Cry1/2 was associated with a major shift in the expression of metabolism-related genes. Overall, our results support that kidney fibrosis is accompanied by a profound metabolic derangement and bioenergetics dysfunction, with specific molecular signatures that may constitute potential therapeutic targets.

Increases in atmospheric CO2 concentrations (eCO2) has a strong impact on the physiology of C3 plants that goes far beyond photosynthesis and carbon metabolism. Accumulating evidence suggests that eCO2 has positive effects on the productivity and yield of C3 plants through effects on photosynthesis and negative impacts on nutrient acquisition and assimilation (see for example Ainsworth & Long, 2021 Global Change Biology 27, 27–49). In addition, eCO2 alters the redox balance and signalling of plant cells in ways that alter biotic and abiotic stress tolerance, as well as the expression of genes involved in nitrate uptake such as NRT2.1. In this talk, I will firstly address how growth under eCO2 might alter cellular redox processes and associated signalling and whether plants perceive eCO2 as a stress in itself.

Ascorbate is a major plant metabolite that plays crucial roles in various processes from reactive oxygen scavenging to epigenetic regulation. However, to what extent and how ascorbate modulates metabolism is largely unknown. To address this, we investigated the consequences of chloroplastic ascorbate deficiency and total cellular ascorbate deficiency, by studying a novel chloroplastic ascorbate transporter PHT4;4 mutant line, and comparing it to the ascorbate-deficient vtc2-4 mutant in Arabidopsis thaliana. Under regular short-day growth conditions, the morphological phenotypes of the mutants and the wild type were indistinguishable, and both deficiencies caused minor alterations in photosynthesis, without being compensated by the accumulation of other antioxidants. In contrast, metabolomics analysis revealed global reorganization of metabolite levels in the vtc2-4 mutant, affecting over 50% of the identified metabolites. Unexpectedly, there was a large overlap in the changes in metabolite levels between the ascorbate-deficient mutant and the chloroplastic ascorbate transporter mutant. The changes concerned the central, amino acid, and nucleotide metabolism and secondary metabolites without the typical symptoms of oxidative stress. Therefore, our data show that upon chloroplastic ascorbate deficiency, the metabolome is rewired independently of oxidative stress, thus ascorbate may possibly act as a metabolic regulator in vascular plants.

Hydrogen sulfide (H2S) is a gaseous effector involved in a wide variety of physiological processes in most organisms including photosynthetic organisms. The oxidative modification of cysteine residues to persulfides is suggested to represent the main way by which H2S exerts its biological functions. Hence, the signaling functions of H2S likely rely on the reactions with specific proteins and cysteines leading to their persulfidation and represents a novel thiol switching mechanism comparable to nitrosylation or glutathionylation.
The widely distributed 3-mercaptopyruvate sulfurtransferases (MSTs) are implicated in the generation of persulfidated molecules and H2S biogenesis through transfer of a sulfane sulfur atom from a suitable donor to an acceptor. Arabidopsis thaliana possesses two MSTs (namely STR1 and STR2) which remain poorly characterized. To learn more about these enzymes, we conducted a series of biochemical experiments combining a variety of sulfur donors and reducing systems as acceptors. Our kinetic studies revealed that both MSTs use 3-mercaptopyruvate efficiently as a sulfur donor while thioredoxins, glutathione, and glutaredoxins all served as high-affinity sulfane sulfur acceptors. Using the redox-sensitive GFP (roGFP2) as a model acceptor protein, we showed that the persulfide-forming MSTs catalyze roGFP2 oxidation and more generally trans-persulfidation reactions. However, a preferential interaction with the thioredoxin system and glutathione was observed in competition assays mixing roGFP2 and these sulfur acceptors. Moreover, while we observed that MST catalytic cysteines are prone to overoxidation, prior persulfidation prevents the irreversible inactivation of MSTs because oxidized persulfides are reduced by thioredoxins or glutathione. This work provides significant insights into Arabidopsis STR1 and STR2 catalytic properties and more specifically emphasizes the roles of cellular reducing systems for the generation of H2S or glutathione persulfide and for the reactivation of oxidatively modified MST forms.

Protein cysteine residues are intrinsically reactive and readily oxidized within cells by electrophilic species such as reactive oxygen and nitrogen species (ROS/RNS). Resulting oxidative post-translational modifications (OxiPTMs) can adjust protein function and thereby act as post-translational regulators in various physiological processes. While various proteomic methods have been developed for identifying and quantifying relative levels of OxiPTMs, assessing the degree of cysteine oxidation remains challenging. Here, we describe a method that quantifies the degree of cysteine oxidation by data-independent acquisition mass spectrometry (DIA-MS). More specifically, isotopically labeled iodoacetamide is used to discriminately label native thiols and those liberated after a reduction step – allowing to monitor absolute ratios within each sample based on isotopically-labeled fragment ions in DIA-MS. Furthermore, as no enrichment step is involved, protein levels can be monitored simultaneously. Taken together, this strategy paves the way towards future reproducible and accurate redox proteome profiling using DIA-MS.

The transcription factor Nrf2 nuclear factor erythroid 2 p45-related factor 2 (Nrf2) and its negative regulator, Kelch-like ECH associated protein 1 (Keap1), regulate the expression of complex networks of genes encoding cytoprotective proteins that provide adaptation to oxidative, electrophilic, inflammatory, and metabolic stress. Using quantitative high-resolution mass-spectrometry approaches, we characterized the proteomes of bone-marrow derived macrophages with graded Nrf2 transcriptional activity at resting and activated (with lipopolysaccharide, LPS) states. We found significant differences among the genotypes in the abundance of proteins that participate in numerous cellular processes, including redox, amino acid, carbohydrate and lipid metabolism, and innate immunity. Complementary metabolomics and respirometry studies supported these findings. Analysis of oxygen consumption rates (OCR) in resting macrophages confirmed a role for Nrf2 in regulating mitochondrial respiration: Nrf2 activation increased the basal respiration rates associated with ATP production, whereas Nrf2 disruption had the opposite effect. Analysis of extracellular acidification rates (ECAR) identified a role for Nrf2 in promoting glycolysis in resting and activated macrophages. In addition to changes in metabolism, we also observed an enrichment in mitochondrial fusion with Nrf2 activation, including the mitochondrial fusion proteins, Opa1, Mfn1 and Mfn2, and a significant increase in the mitochondrial fission factors, Mff and Mief2, with Nrf2 disruption in activated macrophages, suggesting that Nrf2 may play a role in mitochondrial adaptation during inflammation. Confocal microscopy analysis of mitochondrial morphology following immunofluorescence staining of the outer mitochondrial membrane protein Tom20 further showed that prolonged stimulation with LPS caused a switch in mitochondrial morphology, from intermediate to fused/elongated, which was enhanced by Nrf2 activation and suppressed by Nrf2 disruption. Together, these findings show that Nrf2 is a critical factor governing redox and intermediary metabolism and facilitating mitochondrial adaptation in macrophages encountering pro-inflammatory stimuli.

The NRF2 pathway is often constitutively activated in various cancer types, particularly in non-small cell lung cancer (NSCLC), contributing to the malignant phenotype and drug resistance. Unfortunately, direct targeting of NRF2 has remained a challenge, as it is a transcription factor with many structural homologs and functions. Therefore, indirect means to inhibit NRF2 activity by for example targeting factors affecting transcriptional regulation of NRF2 or downstream pathways generating cancer-specific vulnerabilities may provide alternative ways to treat NRF2 overactive cancers. Herein, various multi-omics approaches are used to discover targetable co-dependencies in cancer with constitutive NRF2 activation. It is demonstrated how publicly available genetic and drug repurposing screens can reveal specific vulnerabilities, and how NRF2 activation affects the efficacy of immune therapies. Also, systems proteomics approaches are used to study proteins interacting with the KEAP1-NRF2 system, potentially identifying novel coregulators that can be targeted for therapy.

Inflammation plays an important role in the pathology of most diseases, including non-alcoholic steatohepatitis (NASH) for which there is no currently approved drug to stop disease progression. Transcription factor NRF2 has been proposed recently as a promising target to stop NASH, but the most widely analyzed compounds are electrophiles that, in addition to inhibiting its main repressor KEAP1, display many off-target effects and also elicit a very strong supra-physiological NRF2 activation. As an alternative, we screened a chemical library of ~ 1 M small molecules to identify disrupters of the interaction between NRF2 and the E3 ligase adapter beta-TrCP. In vitro ubiquitination and cell culture experiments demonstrated that our hit compound is a beta-TrCP/NRF2 interaction inhibitor that activates NRF2 within physiological levels. This compound is specific for NRF2, as it does not disrupt the interaction between beta-TrCP and other substrates, such as beta-catenin. Pharmacodynamics studies demonstrated selective exposure and NRF2 activation in the liver. In mice submitted to LPS-induced acute liver inflammation, the compound greatly attenuated Kupfer cells’ activation and the NFkB-mediated inflammatory response. We further analyzed the effect of this compound in the STAM model of progressive liver damage by NMR, measuring the fat/water ratio, and histochemistry of oil red (fat) and Sirius red (fibrosis), and correlated inflammatory and metabolic parameters with NRF2 activation. We found that this PPI inhibitor prevented NASH onset. Importantly, mice which were allowed to develop NASH and were then submitted to chronic treatment with this compound for 4 weeks exhibited a significant protection against the development of fibrosis. Our results report an innovative mechanism to activate NRF2 and protect the liver from NASH and fibrosis.

Air pollution fine particulate matter (PM2.5) is a risk factor for the development of cardiometabolic disorders, by yet unclear mechanisms. In this study, we aimed to evaluate the impact of PM2.5 exposure, with a special focus on unravelling redox, inflammatory, and metabolic pathways. First, male 8-week-old C57BL/6 mice received 1 mg/kg body weight of a PM2.5 surrogate (ROFA, Residual Oil Fly Ash) or PBS (control) by intranasal instillation. A biphasic lung inflammatory cell recruitment was observed in ROFA-exposed mice with neutrophils peaking at 6 h post-exposure and macrophages peaking at 72 h, together with increased pro-inflammatory gene expression and cytokine levels (TNF-α, IL-6, CCL2). Bulk mRNA sequencing of sorted alveolar macrophages revealed a pro-inflammatory gene expression signature and altered pathways for redox and lipid metabolism. Upregulated differentially expressed genes were validated by a customised cytokine bead assay in BAL and plasma, which showed a sustained increase for up to 72 h in ROFA-exposed mice. In parallel, decreased metabolic gene expression (Ucp1, Elovl3, Adrb3) in brown adipose tissue suggests reduced lipolysis and thermogenesis, despite ongoing white adipose tissue inflammation. To further explore this observation, another set of mice were exposed to ROFA or PBS and monitored in metabolic cages for 48 h. Despite enhanced physical activity and lowered caloric intake, ROFA-exposed mice showed significantly reduced heat production. Lastly, consequences of PM2.5 inhalation were evaluated in a real-life mice model of exposure to polluted urban air for 16 weeks. Increased weight gain, impaired glucose homeostasis, and adipose tissue inflammation were observed in mice breathing urban air (27±8 µg PM2.5/m3) versus filtered air (2±1 µg PM2.5/m3), together with altered metabolic gene expression in adipose tissue. Our findings indicate that air pollution PM2.5 exposure blunts metabolic pathways in adipose tissue and promotes obesity, likely due to pulmonary and systemic inflammation.

Emerging evidence suggests that the presence of oxidative stress per se does not rationalize the use of antioxidants indiscriminately, emphasizing the need to identify responsive phenotypes for personalized interventions. This partially originates from the fact that all people inherit and acquire different or even unique biological and behavioural characteristics. As a result, the impact of any, for example nutritional or pharmacological, redox treatment that aims to regulate our physiology may be differentiated, resulting in beneficial, harmful or neutral outcomes. On this basis, the issue of individual responsiveness attracted the interest of researchers across diverse scientific fields, while ‘personalized’ - also known as ‘precision’ or ‘subject-tailored’ - treatments became the ultimate translational goal. Unfortunately, the ‘personalized treatment’ still remains more of a buzzword than a substantive and applicable concept, at least in redox biology. A potential explanation for this issue is the lack of specificity in our methodological and statistical approaches. Noteworthy, this is in stark contrast to the great advances in analytical redox chemistry, which aims to identify and quantify the precise reactive species involved in particular settings. In fact, many studies on the topic follow suboptimal methodological approaches to quantify individual responses as well as to specify statistical or clinical thresholds of effectiveness for precise redox-dependent measures (i.e., ‘minimal clinically important difference’ or ‘smallest worthwhile change’). Hence, the interpretational potential of any finding at the individual level commonly lies in the eye of the beholder, while any causative association between redox biology and physiology becomes tricky. Methodological and statistical practices from other fields that seem to provide a more straightforward approach for personalized studies will be presented. Hopefully, similar approaches will be applied in a redox biology context in future studies as well.

Necroptosis activation is highly dependent on the generation of mitochondrial reactive oxygen species. In this study, we assess the contribution of p53 and sulfiredoxin to necroptosis in pancreas with acute pancreatitis. Acute pancreatitis was induced by seven hourly intraperitoneal injections of cerulein in C57BL6/J wild-type mice, p53 knockout mice and sulfiredoxin knockout mice. Necroptosis occurred intensely in pancreas of wild-type mice 24 h after the first cerulein injection. At this time point p53 was upregulated and translocated into pancreatic mitochondria. However, necroptosis was abrogated in p53 knockout mice with pancreatitis. In the pancreas of p53 knockout mice, PGC-1α protein levels as well as those of its transcriptional target peroxiredoxin 3 increased and remained high upon pancreatitis induction. In addition, sulfiredoxin protein levels also increased in p53-deficient mice with pancreatitis, which prevented peroxiredoxin-3 hyperoxidation. During the early stages of pancreatitis, when necroptosis was still absent in the pancreas of wild-type mice, sulfiredoxin was upregulated and located into the mitochondria, protecting peroxiredoxin 3 from hyperoxidation. The absence of sulfirredoxin caused peroxiredoxin 3 hyperoxidation, p53 mitochondrial translocation and necroptosis early in the course of acute pancreatitis. Mito-TEMPO treatment in acute pancreatitis abrogated p53 mitochondrial translocation and necroptosis in pancreas. In conclusion, p53 is required for necroptosis in pancreas during acute pancreatitis through loss of sulfiredoxin and peroxiredoxin 3.

Treatments available for ischaemic stroke remain limited due to failures in clinical translation and to improve this, physiological oxygen encountered in vivo need to be considered in cell culture. As cells in vivo experience O2 levels ranging from ~13kPa to ~1kPa, cells cultured under room air – 18kPa O2 – are hyperoxic. Using an oxygen-sensitive probe (MitoXpress-INTRA, Agilent), we have identified that long-term culture under 5kPa O2 is needed to recapitulate reported intracellular O2 levels in the brain. Long-term culture under 5kPa O2 demonstrates a phenotype different to cultures under 18kPa O2, as evidenced by downregulation of specific Nrf2 target antioxidant genes. Superoxide production measured using luminescent L-012 and mitochondrial-specific superoxide indicator MitoSOX™ corroborated findings that long-term culture under 5kPa O2 prevented superoxide production associated with hypoxia-reoxygenation injury. Similarly, real-time labile Fe2+ measurements revealed less Fe2+ release following reoxygenation in bEnd.3 cells adapted to 5kPa O2. Exaggerated superoxide production in cultures exposed to hyperoxic environment may create misleading insights in screening potential therapies for pathology involving hypoxia-reoxygenation injury. The present study provides evidence that adapting cells to physiological normoxia has direct consequence for hypoxia-reoxygenation injury. Future in vitro studies should consider a paradigm shift by conducting cell culture studies under their respective physiological O2 levels to enhance translation to the clinic.

Interscapular brown adipose tissue (IBAT) is a highly metabolically active, thermogenic tissue essential for the maintenance of total energy homeostasis, with a remarkable ability for remodelling in response to exogenous stimuli. Given that nuclear factor erythroid 2-related factor 2 (NRF2) has a pivotal role in redox-metabolic homeostasis, we aimed to investigate its role in IBAT homeostasis under basal conditions or upon cold stimulation. Therefore, we analysed structural and redox-metabolic profiles of IBAT in wild-type (WT) and mice lacking functional Nrf2 (Nrf2KO) maintained at room (RT, 24±1°C) or low temperature (4±1°C). Our results show that both WT and Nrf2KO mice appear to be acclimated to cold, showing characteristics of thermogenically active IBAT, including increased gene and protein expression of uncoupling protein 1 (UCP1). Surprisingly, light and electron microscopy revealed that Nrf2KO mice at RT displayed distinct structural features of activated IBAT, together with the presence of vasodilated blood vessels, while the expression of thermogenic marker UCP1 did not show a corresponding cold-induced change, thus indicating IBAT functional inactivity. This lack of IBAT thermogenic activity in Nrf2KO mice at RT is consistent with its altered redox-metabolic profile, whereby protein expression of the main antioxidant defence and key metabolic enzymes either remained the same or was decreased compared to WT mice at RT. Accordingly, circulatory levels of triglycerides and cholesterol were decreased while glucose, urea and creatinine remained unchanged. Moreover, gene and/or protein expression of important redox-metabolic transcriptional factors - erythroid NRF1, NFkB, PGC-1α and PPARγ, as well as eNOS and AMPKα were increased, suggesting compensatory molecular mechanisms leading to altered IBAT phenotype in Nrf2KO mice at RT. In conclusion, the lack of functional Nrf2 leads to marked structural characteristics of active IBAT in Nrf2KO mice at RT, which are only followed by its functional activation through distinct redox-metabolic reprogramming after cold stimulation.

Cardiovascular diseases (CVDs) result in several conditions such as increase in ROS level that lead to decrease nitric oxide availability and vasoconstriction, promoting arterial hypertension. Physical exercise (PE) has been shown to be protective against CVD. PE can lead to a maintenance of redox homeostasis, with a decrease of ROS by increased expression of antioxidant enzymes and HSPs modulation. These molecules can be shuttled between the cells by Extracellular Vesicle (EVs). EVs are lipids bound vesicles secreted by cells with a crosstalk function, carrying bioactive molecules (i.g.proteins). Our preliminary studies, show the presence of Hsp70, Hsp27 and SOD2 in plasma EVs isolated from trained and untrained healthy young males, with higher enrichment of SOD2 in untrained respect trained. Moreover, Hsp27 phosphorylation increased by acute endurance exercise (70% HRmax for 30’). These proteins are of particular interest for their protective role in cardiomyocyte (Vicencio et al., Bartz et al.) thus the aim of this work is to test the cardioprotective effect of plasma-EVs isolated from 2 groups of healthy young males with different VO2max levels (Untrained: 44.35 ± 3.0, Trained 52.27 ± 4.21) before and after a single bout of endurance exercise. Trained subject showed a significant decrease in EVs number (p=0,05) and an increase in size (p=0,0001) after training, the same trend is observed in untrained. Isolated small and large EVs were used to treat human iPS-derived cardiomyocyte exposed to a prooxidant condition (Doxorubicin, 0.2 uM for 3h and H2O2, 100 uM for 24h). ROS level (ROS detection kit far-red), apoptosis (caspase-3 detection) and senescence phenotype (p16 and Beta-galactosidase detection) were used as readout for cardioprotection (Milano et al.). Protein cargo of plasma EVs are also in depth characterized with proteomic (Mass spectrometry) and target specific (western blot) approach in order to identify the putative PE-related proteins involved in cardioprotection.

Hypoxia, a condition of limited oxygen availability, occurs naturally in the context of flooding and negatively affects the viability of plants. Various cellular signals generated under hypoxia must be integrated in order to induce a stress-specific response. Changes in the cellular redox status in the presence of oxygen deprivation are not yet associated with important transcription cascades required for adaptation. We found that RADICAL-INDUCED CELL DEATH1 (RCD1), a redox-sensitive interactor of several transcription factor families in Arabidopsis thaliana, is mandatory for the regulation of the most prominent transcriptional regulators under low-oxygen stress. In addition to RCD1, one of its homologues is also involved in the repression of transcription factors. To unravel the molecular mechanism of repression by RCD1, a variety of genetic, biochemical and molecular biological approaches is applied. In addition, the influence of redox signals generated under hypoxia on the function of RCD1 is examined in detail. Novel regulators of transcription under hypoxia that are controlled in an RCD1-dependent manner are also identified. Taken together, a significant expansion of our understanding of the integration of redox signals in signaling processes leading to low-oxygen stress tolerance is presented.

During evolution, plants have developed different resistance mechanisms against biotic and abiotic stress. Reactive oxygen and nitrogen species (ROS/RNS) are signaling molecules being involved in many physiological processes, including defence response during plant-pathogen interactions. Especially, it is well known that NO produced after plant recognition of pathogens is part of the signaling cascades that trigger the expression of defence genes, the production of secondary metabolites and finally, hypersensitive response (HR) and systemic acquired resistance (SAR).

One of the major economically relevant plant pathogen is Fusarium spp. Root-colonizing fungi Fusarium oxysporum causes vascular wilt disease that leads to devastating yield losses. Indeed, the eradication is very tough, due to its persistence and colonizing capacity. In addition, phytoglobin 1 (Glb1) is an important player regulating NO concentration protecting organisms from oxidative and nitrosative stress. Recently, the role of Glb1 in F. oxysporum-tomato interaction has been assessed. In order to get a deeper insight onto NO signaling and Glb1 role in plant defence, in this work, we have studied the response of a model plant Arabidopsis thaliana, wild type (WT) and lines with altered levels of Glb1, to F. oxysporum. Both, the antisense and the overexpressor line showed a more resistant phenotype than WT to the fungus. The mutants also present differences in defence gene expression and RNS production among others, suggesting that Glb1 may be able to regulate NO level and to enhance the defence response.

Plants are essential for life on Earth converting light into chemical energy in the form of sugars. To adjust for changes in light intensity and quality, and to become as efficient as possible in harnessing light, plants utilize multiple light receptors, signaling, and acclimation mechanisms. In addition to altering plant metabolism, development and growth, light cues sensed by some photoreceptors, such as phytochromes, impact many plant responses to biotic and abiotic stresses. Central for plant responses to different stresses are reactive oxygen species (ROS) that function as key signaling molecules. Recent studies demonstrated that respiratory burst oxidase homolog (RBOH) proteins that reside at the plasma membrane and produce ROS at the apoplast play a key role in plant responses to different biotic and abiotic stresses. Here we reveal that phytochrome B (phyB) and RBOHs function as part of a key regulatory module that controls ROS production, transcript expression, and plant acclimation to excess light stress. We further show that phyB can regulate ROS production during stress even if it is restricted to the cytosol, and that phyB, RBOHD and RBOHF co-regulate thousands of transcripts in response to light stress. Surprisingly, we found that phyB is also required for ROS accumulation in response to heat, wounding, cold, and bacterial infection. Taken together, our findings reveal that phyB plays a canonical role in plant responses to biotic and abiotic stresses, regulating apoplastic ROS production, and that phyB and RBOHs function in the same pathway.

To ensure reproductive success, flowering plants produce an excess of pollen to fertilize a limited number of ovules. However, in many flowering plants the male gametophyte is considered to be the most sensitive tissue to high temperatures, with even a single hot day being able to cause male sterility. Using flow cytometry approach, we recently showed that pollen grains mature into two distinct subpopulations – those that display high metabolic activity and elevated reactive oxygen species (ROS) levels immediately after hydration (high-ROS/active), and those that maintain an extended period of dormancy with low metabolic activity (low-ROS/dormant). We show that while the high-ROS pollen grains are highly susceptible to heat stress, low-ROS pollen grains may survive even extreme temperatures. We propose that the dormant pollen serves as a backup to provide a second chance for successful fertilization when the 'first wave' of pollen encounters an unpredictable growth condition such as heat stress. Pollen grains expressing redox-sensitive ro-GFP further reveal two distinct subpopulations with the majority being highly reduced. While the relationship between the redox status and the active/dormant state of the pollen awaits further investigation, a recently developed ro-mCherry, is now being employed in Arabidopsis to simultaneously determine the redox and ROS status within the pollen population. In addition, FACS-purified active and dormant pollen revealed different ribosomal RNA and transcriptional profiles, pointing to mechanisms involved in dormancy initiation/release and resilience. Our findings suggest that by regulating the activity state of pollen, thus modulating the composition of the pollen population, flowering plants may optimize their reproductive success to best-fit changes in their environment.

Background: An injured leaf (e.g., by insect herbivory or excess light) generates electric signals (ES) that spread to tissues, leaves, and organs of the entire plant. ES are mediated by changes in the activity of ion channels and are accompanied by waves of reactive oxygen species (ROS) and nonphotochemical quenching (NPQ). These waves are interdependent and propagate systemically throughout the plant. This process is essential for priming specific changes in gene expression and plant acclimation (e.g., cellular light memory). As a result, the entire plant enters a state of Systemic Acquired Acclimation (SAA). Question: Could ES, ROS and NPQ waves spread from an injured plant to neighbouring plants, providing their leaves touch each other? Up to now, only indirect signalling routes like underground ES mediated by mycorrhizal networks between roots of different plants, or aboveground plant volatiles, were found to connect different plants. Findings: Under humid conditions, an injured plant can directly communicate a danger signal to other plants that touch it within a community of plants, like a meadow of dandelion. ES and ROS waves serve as plant-to-plant signals propagating on and in the leaf with a velocity of several mm per s or cm per min, respectively. These signals can induce changes in NPQ that are prerequisite for chloroplast retrograde signalling, genes expression, hormones, ROS signalling, and acclimation responses, in neighbouring plants. Majority of these complex communication responses can also be induced between two plants connected by a copper wire circuit indicating that ES is the main player of plant-to-plant communication. We show that ES, NPQ and ROS induce a new acclimation phenomenon termed “Network Acquired Acclimation (NAA)”, necessary for SAA induction within a plant community. Next steps: Could plants be capable of using ES as communication or warning signals between a plant and animals (e.g. insects)?

Vimentin is a type III intermediate filament protein with multiple functions in the maintenance of cell homeostasis, cytoskeletal crosstalk and signaling, but also in the extracellular milieu. We have previously shown that vimentin single cysteine residue, C328, is the target of diverse posttranslational modifications that impact the organization of the vimentin network in a structure-dependent manner, thus acting as a regulatory element of vimentin assembly (1,2). Here we have explored the downstream cellular consequences of C328 modifications by subjecting cells expressing vimentin wild type (wt) or various C328 mutants to electrophilic stress. We have observed that treatment with the electrophiles 4-hydroxynonenal (HNE) or dinitro-imidazole (DNI) elicits a marked reorganization of vimentin wt, with disruption of filamentous structures. In parallel, both electrophiles potently induce actin remodeling with the formation of robust stress fibers. Remarkably, expression of a C328H vimentin mutant, which can assemble into filaments but is resistant to electrophiles, blunts actin remodeling. Both, full length and GFP-fusion constructs of vimentin C328H prevent electrophile-induced stress fiber formation. In contrast, two GFP-fusion constructs which cannot form filamentous structures, namely, GFP-vimentin C328D and GFP-vimentin C328S, do not preclude the formation of stress fibers in response to DNI. Our results indicate that disruption of vimentin filamentous structures through modification of C328 may play a permissive role in actin remodeling in response to certain electrophiles. Therefore, vimentin could play a key role as a sensor and effector of electrophilic stress through the modulation of the actin cytoskeleton.
(1) Mónico et al., 2019, Redox Biol. 23:101098. Doi: 0.1016/j.redox.2019.101098
(2) González-Jiménez et al., 2021, Free Rad Biol Med 165, Suppl. 1: 26. Doi: 10.1016/j.freeradbiomed.2020.12.319
Funding: Micinn/ERDF RTI2018-097624-B-I00 and PRE2019-088194; RETIC Aradyal ISCIII/EDRF RD16/0006/0021

Animal and clinical association studies have shown that phospholipids containing oxidized PUFA residues (oxidized phospholipids, OxPLs) play an important causative role in different pathologies. The data justify the need for deciphering protective mechanisms that destroy pathogenic OxPLs or prevent their interaction with cellular targets. We have developed an immune assay for quantification of total OxPL-inactivating activity of blood plasma. The method, which we call a “masking assay”, is based on two monoclonal antibodies (mAbs) that detect OxPLs that are present in OxLDL particles. We found that preincubation of OxLDL with diluted human plasma significantly inhibited binding of anti-OxPL mAbs, thus suggesting that OxPLs were either cleaved or physically masked by plasma components. In support of this possibility, treatment with serum reduced pro-inflammatory effects of OxPLs on endothelial cells. A pilot clinical study demonstrated reduced anti-OxPL masking activity in patients with coronary artery disease, hypertension and diabetes. We hypothesize that inadequate protection against OxPLs may contribute to the pathogenesis of cardiovascular disease. Furthermore, quantification of protective activity potentially may become a biomarker for risk stratification. We further started characterization of individual plasma proteins and enzymes responsible for the masking effect. Using a PAF-acetylhydrolase inhibitor darapladib, as well as a recombinant PAF-acetylhydrolase, we found that this enzyme, which is known to cleave OxPLs, constitutes only a small part of masking activity present in plasma. We further established a pull-down assay using liposomes containing oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC) or non-oxidized PAPC. Bound proteins were identified by mass spectrometry. Several plasma proteins that selectively bound OxPAPC as compared to PAPC were SAA1, SHBG, SFTPB, and CETP. Two purified proteins (SHBG and SFTPB) demonstrated masking activity comparable with that of physiological levels of PAF-AH. The data suggest that the masking effect can be explained by simultaneous action of multiple plasma proteins that cleave or bind OxPLs.

Oxidized phospholipids (OxPLs) containing enzymatically or non-enzymatically oxidized fatty acids (oxylipins) are increasingly recognized as lipid mediators involved in disease pathogenesis. Further understanding of structure-activity relationship and molecular mechanisms activated by OxPLs is hampered by the complexity of synthesis of individual molecular species. Although dozens of individual free oxylipins are commercially available, their attachment to the phospholipid scaffold requires relatively harsh conditions during activation of carboxyl groups, which may lead to decomposition of unstable oxylipins. Furthermore, additional protection-deprotection steps are required for oxylipins containing hydroxyl groups. In this work we describe synthesis of OxPLs containing oxylipins bound at the sn-2-position via an amide bond that is characteristic of sphingophospholipids. Activation of oxylipins and attachment to the phospholipid scaffold are performed under mild conditions and characterized by high yield. Hydroxyl groups of oxylipins do not interfere with reactions and therefore no protection/deprotection steps are needed. In order to prevent oxylipin migration, the sn-1 residue is bound through an alkyl bond, which is a common bond present in a large proportion of naturally occurring phospholipids. An additional advantage of combining alkyl and amide bonds in a single phospholipid molecule is that both types of bonds are phospholipase A1/A2-resistant, which may be expected to improve biological stability of OxPLs and thus simplify analysis of their effects. As a proof of principle, several alkyl-amide oxidized phosphatidylcholines (OxPCs) containing either linear or prostane cycle oxylipins have been synthesized. Importantly, we show here that alkyl-amide OxPCs demonstrated biological activities similar to those of diacyl OxPCs. Alkyl-amide OxPCs inhibited pro-inflammatory action of LPS and increased endothelial cellular barrier in vitro and in mouse models. The effects of alkyl-amide and diacyl OxPCs developed in a similar range of concentrations. We hypothesize that alkyl-amide OxPLs may become a useful tool for deeper analysis of the structure-activity relationship of OxPLs.

As reported recently, 233 nm radiation emitted by a spectrally pure UVC LED source shows sufficient bactericidal properties at an applied dose between 20 and 80 mJ/cm2. In ex vivo human skin and skin models, the formation of epidermal DNA lesions at bactericidal doses was minor compared to one tenth of the minimal erythema dose of UVB light. This can be attributed to the strong absorption for wavelengths below 240 nm in the upper non-nucleated dermal cell layers. Furthermore, the radical formation was far lower than for a dose equivalent to a stay of 20 min outdoors, which can be compensated by the antioxidant defense system.
To assess the influence of skin barrier disruption on radiation-related skin damage, we detached the stratum corneum from the viable epidermis in ex vivo human skin mechanically. After irradiation of the skin with a wavelength of 233 nm, we screened the tissue for the formation of CPD and 6,4-PP positive cells via immunohistochemistry. An increased formation of DNA lesions was determined in barrier-disrupted skin as compared to intact skin after irradiation. However, this effect was absent if artificial wound exudate was applied on the disrupted skin surface before irradiation. Interestingly, intact skin and barrier-disrupted skin showed no significant differences in radical formation as quantified by EPR spectroscopy.
We further compared the formation of DNA damage in different skin types using ex vivo human skin. After irradiation with a wavelength of 233 nm, less epidermal DNA lesions were found in dark skin types than in fair skin types. In contrast, irradiation with a wavelength of 222 nm induced no skin type-dependent differences due to the lower penetration depth of photons.
In conclusion, 233nm LED irradiation at the studied dose could be suitable for skin antisepsis and indoor air decontamination in the presence of humans.

Skeletal muscle is one of the most dynamic tissues in the body, inheriting the ability to fine tune its response to environmental and physiological stimuli, including exercise, diet, disuse, and disease. To achieve skeletal muscle adaptations, transcriptional and post-transcriptional programs are carried out by muscle cells. In addition, a complex network of RNA binding proteins is engaged to achieve alternative splicing programs, thus increasing muscle proteome diversity.
Exercise activates signaling molecules to promote physiological adaptations, such as fiber type transformation, angiogenesis, and mitochondrial biogenesis. The underlying mechanisms involve a complex interplay of signaling pathways and downstream regulators, sensing the energy state and promoting glucose metabolism and fatty acid utilization.
Glucose uptake is a crucial event for energy supply, and GLUT4 protein is the main actor in glucose transport in brain and muscle. GLUT4 pre-mRNA, encoded by Slc25a4 gene, is affected by alternative splicing: in addition to the main full-length isoform, a shorter transcript is produced, which leads to a truncated protein. We found that the RNA binding protein Sam68 modulates GLUT4 pre-mRNA processing. Sam68 expression promotes the full length GLUT4 isoform, whereas its depletion leads to the truncated GLUT4. Mechanistically, we found that Sam68 binds to and inhibits the recognition of an alternative poly-adenylation signal located in the intron 10 of the pre-mRNA.
Accordingly, Sam68-/- muscle biopsies, which are enriched in type I fibers, displayed increased level of the truncated Slc25a4 isoform. Cross-linking and immunoprecipitation experiments in mouse myoblasts document that stimulation of the IGF1 signaling promotes Sam68 tyrosine phosphorylation and inhibits Slc25a4 binding.
Collectively, our results identify Sam68 as a novel regulator of glucose homeostasis in the skeletal muscle, and highlights an unprecedented link between Sam68, GLUT4 and muscle glycolytic pathway.

Well known for its important involvement in oxidized protein elimination within the mitochondrial matrix, Lon protease also intervenes with the mitochondrial DNA. In addition to a maintenance action on this genome, Lon contributes to the regulation of its replication and transcription. Therefore, a Lon depletion can be expected to induce effects on the mitochondrial DNA and, from there, on the mitochondrial function.
This is why we decided to deepen the missions provided by Lon by looking for possible damage to mitochondrial DNA associated with its underexpression. We have therefore investigated whether or not the integrity of mitochondrial DNA is preserved in a HeLa cell line stably transfected with an inducible shRNA directed against Lon.
To this end, we carried out qPCR experiments based on the principle that DNA lesions (abasic sites, strand breaks, thymine nucleic acids oxidation,…) can slow down or even block the progression of DNA polymerase. Therefore, the amplification of a damaged sequence will be less efficient than that of an undamaged sequence. To localize the sites of possible lesions, the mitochondrial DNA was divided into nine consecutive sequences and nine pairs of primers were used to amplify these sequences separately.
According to our results, Lon deficiency results in less amplification of the mitochondrial genome with differences between the nine sequences. Thus, Lon depletion results in mitochondrial DNA alterations. What is the nature of these damages ? Due to the oxidative stress (ROS and protein carbonylation) observed in our previous works on the effects of a Lon knockdown, we speculated that nucleotide oxidation could be one of these mtDNA damages. Regarding the guanine tendency to oxidation and the fact that Lon protease binds mt DNA on guanine-rich sequences, we are now working on the evaluation of 8-hydroxy-2'-deoxyguanosine levels with and without Lon.

Free radicals play important roles in biological systems. These ubiquitous chemical species are formed by all live eukaryotic cells and are involved both in normal cell metabolism and pathological processes. It is vital to know where, when and in what quantities free radicals are formed. However, commonly used techniques often suffer from low sensitivity or selectivity, making direct in situ detection of free radicals a challenging task.

Our group has been developing a new approach for free radical sensing in biological samples – nanodiamond magnetometry. This method is based on using fluorescent diamond nanoparticles (FNDs). As free radicals, by definition, have unpaired electrons, they are paramagnetic. FND fluorescence strongly and reversibly depends on the magnetic properties of the particle’s vicinity. Changing concentration of free radicals will affect the fluorescent signal of FNDs, which can be read out with an all-optical setup, based on a simple confocal microscope. This method does not require external magnets, like MRI. The measurements can be done at room temperature, and the cells are preserved in the process. This allows for repeated measurements on the same cell over the course of several days, or using complementary techniques to obtain additional context for the recorded signals.

We have used this technology in several biological models. Recently, we have applied nanodiamond magnetometry to detect the initial response of primary bronchial epithelial cells obtained from healthy donors or the donors with pulmonary disease to the cigarette smoke extract. We show that exposure to this stressor results in an altered free radical load, and that the health status of the donor influences this response. This study illustrates the excellent sensitivity of our method and paves the way for the potential applications of nanodiamond magnetometry in the (pre-)clinical setting.

Cellular senescence -a process characterized by the irreversible cell cycle arrest- affects the function of eukaryotic cells, including stem cells, leading to tissue regeneration impairment, and mediating organismal aging and the development of age-related pathologies. Senescence can occur due to telomere shortening (replicative senescence), but can also be the result of damage, induced by internal or external stressors, such as oxidative stress (OS), leading to stress induced premature senescence (SIPS). Several plants and plant derived compounds could serve as potential anti-SIPS agents, possibly due to their antioxidant abilities. Here, we aimed at investigating the effects of Platanus derived compounds on Mesenchymal Stem Cells from the Wharton’s Jelly (WJ-MSCs) in the context of cellular senescence. Early passage WJ-MSCs treated or not with H2O2 in order to induce premature senescence were exposed to 3 different concentrations (0,1%, 1%, 10%) of compounds derived from various Platanus parts, extracted with different organic solvents and water. The activity of the senescence associated marker β-galactosidase (SA-β-gal) was then assessed. Our preliminary results showed that while some compounds, at higher concentrations exhibit a pro-senescence or even a cytotoxic function, at lower concentrations have the ability to protect from the establishment of cellular senescence. Compounds derived from Platanus have previously been reported to possess an antioxidative ability and given the well established relationship between oxidative stress and cellular senescence’s onset, it would be of utmost importance to analyze the underlying mechanisms of the observed outcome. We have previously reported that caveolin-1 -a component of the caveolae and recently recognized as major regulator of cellular senescence- plays an important role in the cells’ ability to repair OS induced DNA damage, and consequently to the development of WJ-MSCs premature senescence. Therefore, the involvement of caveolin-1-depended signaling in these compounds’ protective function against SIPS is currently extensively studied.

Background. Free radicals play a significant role in physiological processes and pathological conditions such as degenerative diseases and the aging process. We have developed a new technology which allows nanoscale MRI measurements for quantifying these with subcellular resolution. We make use of diamond nanoparticles, which change their optical properties based on magnetic surrounding. While the method have already been proven in physics we demonstrate measurements in life cells for the first time. In this study we could measure radical production after provoking Saccharomyces cerevisiae . We were also able to clearly differentiate production rates between young and old as well as wild type and knock out strains. Additionally, we are able to follow how an antioxidant protects the cells from radical damage.
Methods. Saccharomyces cerevisiae BY4741 were grown in synthetic defined (SD) complete medium supplemented with 2% glucose and treated with zymolyase for creating spheroplasts which facilitated FNDs uptake. The cells were placed in 96-well plates and 4 compartments cell culture dishes then triggered with 1, 3, and 10% of hydrogen peroxide. They were analyzed by using MTT assay for evaluating metabolic activity and H2DCFDA for measuring ROS production. 0,1 M of L-ascorbic acid had been used for antioxidant during the measurements. Free radicals level during stress condition has been monitored by using diamond magnetometry technique with 50 µwatt laser power. All the measurements were performed triplicates.
Results. With our newly developed technique we are able to measure free radical generation with nanoscale spatial resolution.

Excessive or too small amounts of reactive oxygen species (ROS) lead to oxidative imbalance, named oxidative stress or antioxidative/reductive stress, respectively, while moderate concentrations of ROS are required for normal cell function. H2O2 is the main redox signalling and redox regulation molecule. Increased levels of H2O2 are a part of a normal response to moderate stress in primary liver cells (hepatocytes) and trigger a stress response that prevents apoptosis triggering through caspase-9, called preapoptotic cell stress response (PACOS).

An increased amount of ROS production and lower apoptosis triggering are reversible in primary hepatocytes and their function is preserved at all times, in both, stress adapted and normal cells. Antioxidants, like N-acetylcysteine (NAC), annul the PACOS stress response and its protective role against apoptosis. A moderate increase of H2O2 can induce a stress response that prevents the cells from unnecessary apoptosis.

Delaying fatherhood is a global trend. Aging affects the male germline decreasing the reproductive fitness and increasing the frequency of congenital anomalies in the progeny. Oxidative stress (OS) may play a leading role in the mechanisms behind age-related male reproductive problems. Understanding how OS affects the precursor cells of male gametes is critical to connect aging with inheritable genomic alterations that may affect the next generation. Nevertheless, most available methods to measure OS show several drawbacks that hinder the completion of such an ambitious goal. Diamond magnetometry (DM) is a promising technique that allows the measurement of free radical levels in real-time with subcellular resolution. DM uses fluorescent nanodiamonds (FNDs) with NV centers to translate the magnetic noise generated by free radicals into easily detectable fluorescent signals. Performing DM requires cell immobilization on a suitable coating and the optimization of FNDs concentration and incubation times to achieve the cellular internalization of the particles. Using a mouse model of aging we studied representative stages of spermatogenesis. We tested different coating conditions (0.4% gelatin, 0.01% poly-L-lysine, and 1 mg/ml hyaluronic acid) and concentrations of FNDs (2, 4, and 8 µg/ml) at three incubation times (2h, 4h, and 6h). Finally, we performed diamond magnetometry for the first time in male germ cells. Our results show that 0.4% gelatin, 4 µg/ml FNDs, and 6 hours of incubation are the optimal conditions to achieve more than 80% of attachment and at least 50% of cells with 3-5 internalized FNDs for all populations. Free radicals measurements allowed us to differentiate between germ cells from aged and control animals and to compare between different populations from the same group. In conclusion, we optimized the utilization of a novel tool to study OS in the male germline during aging, opening a myriad of potential applications in Reproductive Medicine.

Breast cancer (BC) is one of the most commonly diagnosed types of cancer in women. Oxidative
stress may contribute to cancer aetiology through several mechanisms involving damage to DNA,
proteins and lipids leading to genetic mutations and genomic instability. The literature indicates that
physical activity (PA) has positive effects on every aspect of breast cancer evolution, including
negative effects from treatment. Specifically, how the beneficial association
between PA and BC survival are partially related to its influence on antioxidant status of the body.
Fifteen newly diagnosed BC patients (40-60 years), who underwent the same surgery and before
beginning cancer-related treatments, were recruited and divided randomly into a control group
(CG,n=5) undergoing usual care, and an exercise group (EG,n=10), which additionally participated in
a PA program. With the aim to verify the ability of PA to counteract the negative effects on systemic
redox-homeostasis induced by the BC treatment, we examined the impact of a 4-month exercise
program on the modulation of plasma markers of oxidative stress, inflammation and the stress
response, such as superoxide dismutase (SOD) and catalase (CAT) activity, total-glutathione (tGSH),
lipid-oxidation (TBARs), total-antioxidant-capacity (TAC) and total-free-thiols (tFTH), as well as
interleukin-6 (IL6), interleukin-8 (IL8), interleukin-10 (IL10), and tumor-necrosis-factor-alpha (TNFα).
Even in the absence of significant changes in CAT activity, TAC, tFTH and TBARs levels (p > 0.05),
exercise maintained SOD activity and tGSH levels in EG whereas in CG they were significantly
decreased (p < 0.05). Moreover, we found a significant decrease of IL8 in both groups, whereas only in
EG we observed a significant reduction in the pro-inflammatory IL6 and an increase of antiinflammatory IL10 (p < 0.05).
These results highlighted the importance of PA as a potential coadjuvant therapy, alongside usual
care of BC, able to counteract the chemotherapy-induced negative effects on an already compromised
redox homeostasis.

Altered redox homeostasis is recognized as a hallmark of neoplastic transformation. However, data from various in vitro and in vivo studies often show increased or decreased transcriptional and translational levels of antioxidant defense (AD) enzymes. One of the underlying causes for such conflicting reports is cell heterogeneity within the complex tumor microenvironment, especially in breast cancer. To overcome barriers associated with bulk tissue gene and protein expression analysis, we choose an immunohistochemical approach. We cross-examined serial tissue sections of tumor and adipose tissue from premenopausal women with malignant invasive ductal carcinoma and benign fibroadenoma to gain a comprehensive overview of cell-specific AD enzymes expression and localization patterns. At the level of overall tissue architecture, malignant tumor tissue shows significantly higher immunopositivity for copper, zinc- and manganese- superoxide dismutase, catalase, and glutathione peroxidase compared to benign tumor tissue. Generally, AD enzymes are specifically localized in the cytoplasm (copper, zinc superoxide dismutase, catalase, glutathione peroxidase) and mitochondria (manganese superoxide dismutase, glutathione peroxidase) of cancer cells and cancer-associated adipocytes. Detailed analysis of different regions of the tumor tissue revealed significant heterogeneity in the degree of immunopositivity along the axis of tumor center–invasive front–adipose tissue. Clusters of cancer cells at the invasive front of the tumor often show a higher degree of immunopositivity for AD enzymes compared to cancer cells in the center of the tumor mass. Similarly, cancer-associated adipocytes that are in close proximity to cancer cells at the invasive front of the tumor show a higher degree of immunopositivity for AD enzymes compared to adipocytes from distant peritumoral adipose tissue. In conclusion, immunohistochemical approach confirms high AD enzymes expression in breast cancer and further reveals distinct regional mosaicism consistent with cell heterogeneity within the tumor microenvironment.
This research was supported by the Science Fund of the Republic of Serbia, #7750238-REFRAME.

Non-Alcoholic Fatty Liver Disease (NAFLD) is a common chronic liver disease (CLD) highly prevalent in obese patients. This is now becoming the first cause of liver transplantation, and its evolution to steatohepatitis increases the risk of cirrhosis and hepatocellular carcinoma (HCC). The latter is the second cause of cancer deaths worldwide, and the first one in CLD patients.
Drug-resistance (DR) limits the efficacy of chemotherapy in HCC. The oncogene Glutathione S-transferase P (GSTP) is involved in DR. Its expression increases in association with cancerogenic transformation of the liver tissue. In human HCC cell lines, we recently observed a possible functional interaction between GSTP and Nrf2, a transcription factor involved in the stress response to cellular electrophiles and in the carcinogenesis process.
Such interaction was investigated in the present study using the mouse model of N-nitrosodiethylamine (DEN)-induced HCC. In this model, the liver expression of GSTP and Nrf2 increased during tumor development in association with progressive desensitization to apoptotic stimuli and DR gene induction. Co-immunoprecipitation experiments showed a physical interaction of these proteins, that in HCC specifically involves the dimeric form of GSTP. Also, a nuclear translocation of GSTP was observed during tumor development in association with β−TrCP protease expression, suggesting a dynamic distribution of GSTP during the feedback response to Nrf2 nuclear translocation.
In conclusion, a physical (protein-protein) interaction of the chaperone GSTP with the transcription factors Nrf2 is confirmed in the DEN model of HCC. Its role in carcinogenesis and DR is worth investigating.

Pepper (Capsicum annuum L.) fruit is characterized by its high amounts of antioxidants (vitamins C and A), polyphenols and capsaicinoids. It has been reported that capsaicin, which is exclusive from pepper fruits, shows anti-inflammatory, antiproliferative and analgesic activities. Recently, by the use of untargeted metabolomic approaches, it has been found that pepper (Capsicum annuum) fruits contain a series of compounds with potential therapeutic properties due to the presence, among others, of quercetin and its derivatives, with their content being modulated by nitric oxide (NO) (Guevara et al., 2021, IJMS 22, 4476). In this work, the anti-proliferative activity of crude extracts from four pepper fruits varieties (Melchor from type California, Padrón, Piquillo and Alegría riojana) against HEPG2 (hepatoma) and MIA Paca-2 (pancreas) cells was investigated. Interestingly, it was was demonstrated that pepper fruit tissues from the four varieties which contained the lower capsaicin content displayed the higher anti-proliferative activity in both cell lines. The antioxidant profile of the two tumor cell lines incubated with pepper fruit extracts from Alegría riojana was then investigated, and the activity profile of catalase, superoxide dismutase, glutathione peroxidase and several NADPH-generating enzyme systems was followed. As the most highlighting results, it has to be remarked that the anti-proliferative pattern of the pepper fruit extracts was linked to an altered profile of the (iso)enzymatic activity of catalase and the glucose-6-phosphate dehydrogenase. This research opens new windows on the pepper fruit’s bioactive compounds with nutraceutical and biomedical potentiality, as well as on their posible targets in the tumor cells.
[Supported by a European Regional Development Fund cofinanced grants from Junta de Andalucía (P18-FR-1359) and the Ministry of Science and Innovation (PID2019-103924GB-I00), Spain]

The treatment of sweet pepper (Capsicum annuum) fruits with nitric oxide (NO) provokes ripening delay and increases in the ascorbate content, linked to a moderate lipid peroxidation and protein nitration, as nitro-oxidative stress markers. This situation was considered as some sort of “phystress” (physiological stress). It has been also demonstrated that the NO treatment triggered changes at both transcriptional and proteomic levels in pepper fruits. Based on these data, a metabolomic study was conducted to investigate the metabolomic profiles in pepper fruits at different ripening stages (ripe red versus green immature) and as a consequence of the NO treatment, with the aim of searching for compounds which may have therapeutic potential, that boosting the nutraceutical value of this vegetable. Thus, high performance liquid chromatography (HPLC) coupled to metabolite identification by high resolution mass spectrometry (HRMS) was achieved, and different platforms and databases were used to characterize the metabolites. Twelve differential bioactive compounds were identified in sweet pepper fruits, including quercetin and its derivatives, L-tryptophan, phytosphingosine, FAD, gingerglycolipid A, tetrahydropentoxylin, blumenol C glucoside, colnelenic acid and capsoside A. The abundance of these metabolites varied depending on the ripening stage and on the presence of NO. Besides, the potential anti-proliferative activity of crude extracts from pericarp and placenta of pepper fruits with different pungency levels (capsaicin content) was investigated against seven tumor cell lines. Results proved that tissues with the highest capsaicin levels did not correlate with the greatest anti-proliferative capacity. Altogether, these data open new research perspectives on the pepper fruit’s bioactive compounds with therapeutic potentiality, where biotechnological strategies can be applied for optimizing the level of these beneficial molecules.

[Supported by a European Regional Development Fund cofinanced grants from Junta de Andalucía (P18-FR-1359) and the Ministry of Science and Innovation (PID2019-103924GB-I00), Spain]

There have been several studies that have tried to build a ‘biological age’ predictor using metabolomics data. The conclusion from these studies is that metabolomics-based ‘biological age’ predictors are less capable of predicting chronological age in comparison to, for example, epigenetic-based ‘clocks’, but they are still able to capture part of an individual’s ‘biological age’. As healthy nutrition and antioxidant micronutrients are crucial in maintaining health and robustness, we aimed at identifying metabolites and lipophilic micronutrients associated with age-related diseases and their outcomes (mortality, multimorbidity, general health, physical and multidimensional (pre-)frailty and nursing needs). To do so, within a large prospective study with one year follow up in the Emergency Department (ED) (>1,000 participants until time of submission), a blood withdrawal and a deep clinical phenotyping were obtained from a subgroup of 265 patients (140 M, age range 65-96 yeras). High‐throughput NMR-spectroscopy metabolomic analyses (Nightingale Health Ltd., Helsinki, Finland) and HPLC measurements of carotenoids, retinol and tocopherols were performed in serum as well as the comprehensive geriatric assessment (CGA) – based Multidimensional Prognostic Index (MPI) as a surrogate marker of biological age. The simultaneous quantification of routine lipids, lipoprotein subclass profiling with lipid concentrations within 14 subclasses, fatty acid composition, and various low-molecular metabolites, including amino acids, ketone bodies, and gluconeogenesis-related metabolites yielded the preliminary results of a differential association of specific carotenoids and tocopherols with lipid classes. Furthermore, selected carotenoids appear to be preferentially associated to the metabolomics mortality score and selected geriatric syndromes and comorbidities. Although analyses are ongoing at the time of submission and the final results will be presented at the Redox Biology Congress, preliminary observations suggest a metabolomics and frailty signature of lipophilic micronutrients which may disclose pathophysiological mechanisms of age-related diseases and new therapeutic options.

The amount of air pollutants is increasing globally and causing serious health problems; according to the World Health Organization, more than 7 million people die each year from air pollution. Pollution not only damages the lining of the lungs; it also affects skin health and has been linked to the development of skin diseases. Promotion of the development of oxidative stress can lead to premature skin aging, impaired skin barrier, pigment disturbances, and cellular damage caused by free radicals. Existing symptoms may also worsen. To date, there is no established method to clearly assess the degree of risk of skin contact or the protective effect of the substances used.
With the help of electron paramagnetic resonance (EPR) spectroscopy, a new method was established to assess the potential risk of air pollution in the skin and to verify the effectiveness of products designed to protect the skin. Spin-labeled PCA (3-(carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy) was used to study free radical formation, with cigarette smoke as a model pollutant. To make the stress effect measurable, UVA light was used as an additional external stressor.
In addition, the method of confocal Raman microscopy was applied which allows an assessment without the use of an additional marker and external stressor.
For subjecting excised pig ear skin samples reproducibly to cigarette smoke, an exposure chamber for ex vivo and in vivo studies was developed. For quantification of the smoke exposure, the deposit of the nicotine concentration next to the investigated area was used as a marker substance. Initial studies have shown that cigarette smoke promotes the production of free radicals in the skin, and there is a positive correlation between nicotine concentration and free radical production. These results will help to establish a new way to measure pollutant effectiveness and preventive measures.

Melatonin (MLT) is a cytoprotection agent holding potential to prevent cadmium (Cd) toxicity, a pro-oxidant heavy metal reported to interfere with testicular function and fertility. However, its efficacy in Sertoli cells remains unexplored. Porcine Sertoli cells (SCs) were used in the present study to explore this cytoprotection function of MLT during Cd toxicity; investigated parameters included cellular levels of H2O2, redox-sensitive transcription factors such as Nrf2, c-Jun and NFkB, and downstream detoxification and antioxidant effectors, such as the H2O2-scavenging enzyme catalase (CAT) and some inducible components of the glutathione stress response system.
Cd toxicity in SCs resulted in impaired viability and function, that was associated with increased H2O2 generation and induction of reductive stress by the upregulation of Nrf2 expression and activity, cystine uptake, cellular glutathione biosynthesis and efflux, and GSTP expression, whereas cellular protein glutathionylation decreased. MLT produced a potent cytoprotection effect in Cd-exposed SCs, with significant restoration of cell viability and function, and inhibition of H2O2 generation and efflux. Mechanistically, these effects of MLT were associated with increased CAT activity and NFkB phosphorylation, and with marked reduction of Nrf2 activation and GSTP expression to confirm a restoration effect on the cellular redox; at the higher dose of Cd investigated in this study, MLT significantly induced c-Jun, xCT protein expression, and GSH biosynthesis and efflux.
In conclusion, MLT is a cytoprotective in SCs exposed to Cd toxicity with efficient activity in modulating Nrf2 and other genes important to control the cellular flux of H2O2 and the reductive stress response.

Particulate matter (PM2.5) exposure aggravates cardiorespiratory diseases by inflammatory cytokine secretion from alveolar macrophages (AMs). Inhalation of silica particles and asbestos triggers NLRP3 inflammasome activation and IL-1β release. However, the mechanisms involved in NLRP3 engagement by PM2.5 remain unclear. To study this process, THP1-ASC-GFP cells were incubated for 6 or 24 h with 0, 1, 10, or 100 µg/mL of PM2.5 surrogates of variable chemical composition, including ROFA, CAPs, SRM1648a, and SRM2975. NLRP3 priming and specks formation was detected by flow cytometry after incubation with ROFA and SRM1648a, which was confirmed by imaging ROFA-exposed bone marrow derived macrophages (BMDMs) from C57BL/6 ASC-Citrine transgenic mice. No LDH release following PM2.5 stimulation indicated conserved cell viability in our experimental conditions. Increased IL-1β was detected by ELISA in cell culture supernatants of ROFA-exposed BMDMs and AMs from wild type mice, but not from Nlrp3-/- or Casp1-/- mice, or after pre-incubation with the NLRP3-specific inhibitor MCC950. Upregulation of Tnf gene expression and increased TNF-α levels in cell culture supernatants were also observed. Pre-incubation with anti-TNF-α antibody resulted in decreased IL-1β release from ROFA-exposed AMs. Moreover, increased mitochondrial O2●- production by MitoSOX fluorescence was found in ROFA-exposed BMDMs, together with decreased maximal respiration by the Seahorse MitoStress Test. Accordingly, inhibition of mitochondrial complex I site responsible for O2●- production by S1QEL resulted in decreased IL-1β levels in ROFA-exposed BMDMs. K+ efflux contribution on NLRP3 activation was evident in ROFA-exposed BMDMs incubated with increasing concentrations of KCl. Lysosomal leakage in BMDMs was also observed after ROFA exposure. In conclusion, PM2.5 induces NLRP3 inflammasome priming and activation in macrophages, leading to inflammatory mediator release. TNF-α, mitochondrial O2●- production, K+ efflux, and lysosomal disruption were identified as potential drivers of inflammasome engagement after PM2.5 exposure. These findings unravel the mechanisms by which PM2.5 promotes cardiorespiratory inflammation and disease.

Ultraviolet radiation (UVR) plays a role in skin photodamage through triggering various biological responses of skin cells including apoptosis, oxidative stress and melanogenesis. The microenvironment created by epidermal keratinocytes (KC) potentially influences the survival and function of melanocytes (MC) in response to various exogenous insults including UVB. In this study, we identified the candidate paracrine factors derived from UVB-irradiated KC that exerted the regulatory roles in cellular responses of MC to UVB irradiation using in vitro and in vivo models. We observed that conditioned media (CM) from UVB-irradiated KC potentially mitigated apoptosis and oxidative stress as well as stimulated melanogenesis in UVB-irradiated MC. RNA-sequening and functional experiments revealed that, among the upregulated transcripts in UVB-irradiated KC, the GCSF (granulocyte colony stimulating factor) and CCL20 (chemokine (C-C Motif) Ligand 20) mRNA were highly expressed in correlation with the KC’s pararince effects on the stress responses of MC to UVB. Then, treatment of MC with the recombinant GCSF and CCL20 revealed the strongest modulatory effects on UVB-induced MC responses including apoptosis, ROS formation and melanogenesis. Exposure of KC to UVB led to a substantial increase in secretion of both GCSF and CCL20. To demonstrate the in vivo relevance of the in vitro findings, immunofluorescence analysis revealed a correlation between protein expressions of the GCSF and CCL20, in KC and tyrosinase in MC in mouse skin exposed to UVB irradiation. In conclusion, GCSF and CCL20 might be the candidate paracrine factors secreted from KC that play a regulatory role in the responses of MC to UVB. Our findings might give novel insight into development of the UVB-responsive genes as epidermal biomarkers for predicting susceptibility of the skin to photodamage.

There is a strong relation between early life environment and age-related diseases in later life, including sarcopenia. Skeletal muscle development is especially prone to nutritional deficiency. With age, skeletal muscle has higher generation of reactive oxygen species that can cause oxidative stress and can potentially cause muscle atrophy. Also, mitochondrial dysfunction is among the most frequently reported mechanisms of aging muscle atrophy. Hence, we aimed to investigate the association between suboptimal early life nutrition and oxidative stress and mitochondrial turnover in skeletal muscle.
Female mice were fed either a normal (20%) or a low-protein (8%) diet prior to mating and during pregnancy. New-born pups were cross-fostered to different lactating dams (maintained on a 20% or 8% diet) within 24h after birth. At 21 days, they moved on to either a 20% or 8% protein diet, creating 3 groups: control (NNN), Normal-Low-Normal (NLN), and Normal-Low-Low (NLL). Mice were maintained for up to 3 months. Antioxidant defence capacity and mitochondrial biogenesis-related targets were measured by Western blotting and qPCR. Citrate synthase activity also measured.
NLL mice were smaller than control mice. PGC-1α content (P < 0.0001) and mRNA expression (P=0.0074) and NRF1 mRNA expression (P=0.0030) were significantly decreased in NLL and NLN mice compared to controls, suggesting decreased mitochondrial biogenesis. Muscles from these mice also had reduced SOD2 content (P=0.0002) and mRNA expression (P=0.0079) and Prx3 content (P=0.0080) and mRNA expression (P=0.0031). Cat mRNA expression was only reduced in NLL mice (P=0.0007). These mice also had a significantly decreased (P=0.0065) citrate synthase activity suggesting a reduction in mitochondrial number. These observations suggest that early life protein restriction can affect mitochondrial biogenesis and antioxidant defence capacity at a young age which may explain the impact of maternal diet on skeletal muscle aging.

The aim of this study is to assess the cytoprotection function and the corresponding transcriptomics fingerprint of vitamin D (VD; 10 nM) in human hepatocytes (HepaRG) exposed to steatosis and lipotoxicity by means of oleic and palmitic acids (OA-PA) supplementation. A natural source of vitamin D (a Shiitake Mushroom extract) was compared with a synthetic form (DIBASE) and the active metabolite (1,25(OH)2D) of the vitamin in protecting and the presence of lipotoxicity was assessed by the cellular production of the reactive oxygen species (ROS) and induction of specific transcriptomic changes.
Cell viability data demonstrated that the in vitro model of steatosis produced conditions of sub-maximal lipotoxicity and cellular damage. Transcriptional modifications indicated the modulation of genes associated with long-chain fatty acid -oxidation, ROS production, cholesterol synthesis, AMPK activity, and hepatocyte apoptosis, liver fibrosis and damage. the different VD formulations showed similar efficacy in improving both the levels of steatosis and ROS. However, formulation-specific modifications of the cellular transcriptome were observed. Transcriptional fingerprints and biological functions identified by “Ingenuity Pathway Analysis” (IPA) showed higher similarities when the mushroom extract was compared with 1,25(OH)2-D metabolite than DIBASE. In conclusion, VD showed efficient protection against lipotoxicity in HepaRG cells. Comparable cytoprotection activity was observed for the natural and synthetic formulations, but transcriptomics data highlighted some specificities in the mechanism of action.

In the present study, we have performed an ex vivo blood culture and challenging assay with different inducers or inflammation and oxidative stress conditions, optimized to assess the anti-inflammatory and anti-oxidative response of individuals participants of a trial. The trial encompasses the raw intake of olive oils differing in their composition, in comparison to a standard virgin olive oil. Up to date, additional health benefits based on this trial have been determined and described on inflammatory, metabolic syndrome and endothelial function biomarkers, mainly in vivo.
Now, we have determined several oxidative markers including iNOS, presence of Tyr-nitrated proteins and production of NO. This study strongly confirms the first level of evidence already obtained in vivo regarding the health benefits of olive oil components in healthy humans, now in a inflammatory and oxidative scenario induced ex vivo. As discussed, the experimental system also provides an excellent tool to dissect the effects of individual componentes present in the oil in a individual manner.

This research was funded by research projects BFU-2016-77243-P, PID2020-113324GB-100 and STED202100X129616SV0 of the Spanish Ministry of Science, Innovation and Universities (MICIIN)/ State Research Agency (AGE)/ European Regional Development Fund (ERDF)/ European Union (EU).

Natural products often contain compounds, which may be cytotoxic for cancer cells. The aim of this study was to compare the cytotoxic action of 1:10 (w/v) phosphate-buffered saline extracts of several vegetables, teas and medicinal plants on PEO1 and SKOV3 cancer ovary cells and to examine whether oxidative stress contributes to their effects. We found that the extracts of green tea, garlic, and black tea were the most cytotoxic to PEO1 cells (IC50 of 1.66, 2.01 and 3.25 vol%, respectively), while extracts of green tea, horseradish and black tea were the most cytotoxic to SKOV3 cells (IC50 of 1.66, 4.34 and 10.56 vol% when estimated with Neutral Red). The presence of catalase (10 µg/ml) in the cell medium partly protected against the cytotoxicity of the extracts of teas, garlic, curly kale, Cistus incanus, Gingko biloba, and Betula pendula, evidencing the contribution of hydrogen peroxide formed by the extracts to their cytotoxic effects. Tea and horseradish but not garlic extracts increased the intracellular ROS levels detected with d5hydroethidine. These results indicate that oxidative stress, due mainly to the generation of hydrogen peroxide, participated in the cytotoxic effects of the extracts but its contribution depended on the kind of the extract.

Scope
Recent studies suggest that lipid metabolism has strong associated with many cellular stress responses including cell survival as well as cell death. For this reason, compounds targeting intracellular lipid homeostasis are of high interest. α-13′-carboxychromanol (α-13‘-COOH), a long-chain metabolite (LCM) of vitamin E, has emerged as a new regulatory molecule exhibiting more potent or even different effects compared with its metabolic precursor α-tocopherol. Here, we present new insight into the interaction of α-13‘-COOH with the cellular lipid homeostasis in murine macrophages RAW264.7 cells.
Methods and results
Using cell extraction assays and Western Blot analyses the translocation of the transcriptional factor sterol regulatory element-binding protein-1 (SREBP1) we found that the treatment with α-13'-COOH suppressed significantly the formation of SREBP1 active fragment in the cytoplasmic fraction and also decreased its translocation into the nucleus. We also observed that the level of the enzyme stearoyl-CoA desaturase-1 (SCD1), a target gene of SREBP1, was significantly downregulated by the LCM. Considering the function of SCD1 as a desaturase enzyme, we hypothesized that the lipid desaturation ratio could be changed by the LCM. Using UPLC-MS/MS-based lipidomics, we could show that α-13'-COOH modulates the intracellular lipid composition of macrophages: In both, triglycerides and phospholipids, the amount of species with saturated fatty acid was increased, and the amount of species with mono-unsaturated fatty acid decreased. Next, we observed that α-13‘-COOH modulated cell proliferation, cell cycle arrest and cell apoptosis/necrosis using photometric and FACS analyses, with effects that are consistent with the SCD1 antagonist, CAY10566.
Conclusions
Our results provide evidence that the LCM α-13‘-COOH can modulate the viability of macrophages through the regulation of cellular lipid desaturation via SREBP1/SCD1.

Functional molecules from natural extracts may possess specific properties capable of improving health and reducing disease. Incorporation of such molecules into foods is a promising strategy for the food industry. The olive growing sector, widely spread in Mediterranean countries, generates large amounts of byproducts which include seeds with a high agri-food potential due to their high nutritional value and the presence of bioactive compounds. Zebrafish is a well-established model, widely used in various fields, including the study of human pathologies and immune responses. Also, oxidative and inflammatory responses can be easily induced in a reproducible manner, and visualized both in early developmental stages and in adult fishes. The main objective of this work is to evaluate the anti-inflammatory capacity of olive seeds, by optimizing commercial diets enriched with this material, in an experimental model of inflammation by LPS treatment in adult zebrafish. A control fish group was fed three times a day (once with Artemia and twice with standard fish chow, while the olive seed fish group was fed with standard fish chow enriched with olive seeds at 20%. After 8 weeks, inflammation was induced with lipopolysaccharide (LPS) for 8h. After the inductions, liver was extracted, and expression of inflammation and oxidative biomarkers were analyzed. Results exhibited a drastic drop in the levels of inflammation-related biomarkers in the group of animals fed with the olive seed-enriched diet, compared to the control group. Results point to a possible protective and anti-inflammatory effect of the olive seed-enriched diet. This research was funded by research projects BFU-2016-77243-P, PID2020-113324GB-100 and STED202100X129616SV0 of the Spanish Ministry of Science, Innovation and Universities (MICIIN)/ State Research Agency (AGE)/ European Regional Development Fund (ERDF)/ European Union (EU).

Recent years have seen the field of redox biology evolve its understanding of free radicals from the toxic by-products of cellular metabolism to hormetic metabolites with a potent second messenger capacity. An emerging area of interest regarding free radical hormesis is that of growth factor stimulus, particularly insulin signalling in relation to both physiology and pathophysiology. This interest is driven by numerous growth factors driving a cellular ‘oxidative shift’ upon signal transduction and suggests a dynamic role for thiol-based free radical signalling in signalling pathways and their dysregulated, diseased states. Insulin Resistance, a pathophysiological state defined as a loss of insulin sensitivity in several tissues, is defined on the molecular level as the perturbation of glucose transporter GLUT4 trafficking to the plasma membrane by the insulin signal cascade. Indeed, we predict a complex network of cysteine-based thiol groups exists that comes under oxidation and reduction upon insulin stimulus, fine-tuning the activity of target proteins and eliciting the canonical insulin response. Our study utilises a protocol that labels all oxidised cysteines in the proteome with Cysteine-reactive Phosphate Tags (CPTs), phospho-tagging thiols of interest. From here, we utilise the EasyPhos phosphoproteomic workflow developed in house by Dr Sean Humphrey to enrich for never-before-seen coverage of the redox proteome. We apply this technical leap forward to newly optimised models of insulin resistance, designed around chronic exposure of adipocytes to subtle concentrations of perturbations to minimise off-target effects, revealing the hypothesised network of protein thiols responsive to acute insulin stimulus and characterising a reorganisation of this network under insulin resistant conditions. In future, our findings will serve as a launching pad for describing the underlying crossplay between canonical insulin signalling and redox signalling, hopefully contributing a better understanding of insulin resistance that will one day progress to therapeutic applications within a clinical setting.

The chronic intake of second generation antipsychotics (SGAs) has been related to increased risk of cardiovascular diseases. Taking into account the importance of redox balance in vascular function, we analysed the effect of Ari and Ola in the bioenergetics of primary bovine aortic endothelial vascular cells (BAEC). The results indicate that both Ola and Ari can interfere with mitochondrial function after 3h treatment, increasing the mitochondrial production of O2-. After 24h, we observed a higher recovery capacity in Ola than of Ari treated cells, suggesting a higher mitochondrial toxicity or a blunted capacity to induce compensatory systems in Ari treated cells. The effects of these drugs on mitochondrial respiration were also measured in in peripheral blood mononuclear cells of healthy volunteers treated with Ari or Ola. We found a stronger effect on respiration, ROS production and deficient ATP-linked respiration with Ari treatment than for Ola, consistent with previous results. Next, we analysed whether this SGAs can accumulate in mitochondria. We found that both Ari and Ola accumulated in the mitochondrial membranes, with Ola showing a trend for higher levels but also faster turnover. To evaluate the physiological effects of this inhibition we treated a mouse model of mitochondrial dysfunction (PGC-1-/-) with Ari and Ola. At 5 days treatment, the data seem to indicate a better compensatory response to Ola's treatment than Ari's in the heart. After 6 months, we observed a reduction in O2 consumption, cardiac fibrosis, left ventricular remodeling and exacerbated cardiac I/R Injury, with all parameters being more evident in Ari than in Ola treated mice. Remarkably, Ola treated mice showed increased mitochondrial content, suggesting that Ola allows a partial compensation by increasing mitochondrial mass. These results suggested that both Ari and Ola interfered with mitochondrial function, and thus lead to increased risk of cardiovascular disease.

Introduction: Ischemic cardiomyopathy leads to inflammation and left ventricular (LV) dysfunction. Animal studies provided evidence for cardioprotective effects of the endocannabinoid system, including cardiomyocyte adaptation, inflammation and remodeling. Since endocannabinoid receptor CB2-deficiency led to increased apoptosis and infarct size with worsened LV-function, we investigated the impact of elevated level of the endocannabinoid anandamide in fatty acid amide hydrolase (FAAH)-/--mice undergoing repetitive I/R.
Methods: Repetitive daily 15 min. left anterior descending artery occlusion over 1, 3 and 7d in C57/Bl6 (WT)- and FAAH-/--mice (n≥8). Possible PPAR-α mediated effects of anandamide in FAAH-/--mice were eliminated with selective PPAR-α antagonist GW6471 i.v. LV-function was assessed using M-mode echocardiography. Immunohistochemical analysis revealed collagen deposition, macrophage accumulation and remodeling. Hypertrophy was determined by cardiomyocyte area and heart weight/tibia length. Molecular analyses involved Taqman® RT-qPCR and ELISA.
Results: FAAH-/--mice showed cardiomyocyte loss after 7dI/R, accompanied by scar formation with persistent LV-dysfunction 60d after discontinuation of I/R, while WT-mice recovered after 60d. Collagen deposition was reduced to WT-levels when FAAH-/--mice were treated with GW6471. CCL2-expression was significantly higher in FAAH-/--mice, followed by higher macrophage infiltration in infarcted areas, which was reversed by GW6471. Further, abolished HMOX-1 induction in FAAH-/--mice, as well as enhanced hypertrophy and adverse remodeling, were normalized by PPAR-α antagonism. Finally, FAAH-/- -mice showed stronger downregulation of PPAR-α when compared to WT, suggesting a compensatory mechanism as endocannabinoids are also ligands for PPAR-α, and its activation causes lipotoxicity leading to cardiomyocyte apoptosis.
Discussion: Our study gives novel insights into the role of endocannabinoids acting via PPAR-α. We hypothesize that increase in endocannabinoids may have partially detrimental effects on cardiomyocyte survival due to PPAR-α activation.

Uncoupling protein 1 (UCP1) is a molecular hallmark of thermogenic adipocytes. It can also reside in the unilocular adipocytes of white adipose tissue (WAT) that do not possess almost any oxidation and thermogenesis capacity. Therefore, the significance of UCP1 in WAT is vague. To clarify the physiological role of UCP1 in white adipocytes we aimed to investigate the relation of UCP1 expression with the components of redox-adaptive homeostasis and metabolic function in WAT. Toward this, we investigated the expression pattern of UCP1 with Nrf2 and its downstream targets, glutathione (GSH), and lipid peroxidation levels during extensive lipolysis induced by long-term cold exposure and during reversal, when lipid deposits in adipocytes recover, upon re-acclimation from cold to room temperature (RT). To this end, stated molecular targets were investigated at different time points during 45 days of cold acclimation and re-acclimation in the rat retroperitoneal WAT (rpWAT) and compared to respective RT and cold-acclimated controls. The results have shown that in response to cold-induced lipid mobilization transient induction of UCP1 precedes Nrf2 expression and upregulation of downstream antioxidant enzymes (such as MnSOD and GST). The reverse sequence of molecular events was observed during the early (1-12. days) and late (12-45. days) periods of re-acclimation from cold to RT. Namely, in the initial days of re-acclimation high lipogenesis and redox threshold (GSH, and expression of CuZnSOD and MnSOD) correspond to lower UCP1 levels. From the moment of restitution of lipid reserves (revealed by rpWAT mass) and on, UCP1, GSH, and most antioxidant enzymes return to their RT control values. The results emphasize that UCP1 and Nrf2 represent levers of redox-metabolic seesaw fine-tuning of redox homeostasis for optimal regulation of lipid mobilization and deposition in white adipocytes.
Research is supported by the Science Fund of the Republic of Serbia, PROMIS, #6066747-WARMED.

The prevalence and clinical importance of arterial hypertension is still growing. Inorganic nitrite (NO2-) represents an attractive dietary antihypertensive agent but its metabolism and mode of action are not completely understood, which we aimed to investigate with the present study.
Isolated aortic rings from rats were treated ex vivo with oxidants or rats were infused in vivo with angiotensin-II. Vascular responses to acetylcholine (ACh) and nitrite were assessed by isometric tension recording. The loss of vasodilatory potency in response to oxidants (but not in vivo angiotensin-II) was much more pronounced for ACh as compared to nitrite. This effect may be caused by the redox regulation of conversion to xanthine oxidase (XO). Conventionally-raised and germ-free mice were treated with nitrite by gavage, which did not improve ACh-mediated vasodilation but increased plasma levels of S-nitros(yl)ated proteins in the conventionally-raised but not in the germ-free mice.
In conclusion, inorganic nitrite represents a dietary drug option to treat arterial hypertension in addition to already established pharmacological treatment. Short-term oxidative stress did not impair the vasodilatory properties of nitrite, which may be beneficial in cardiovascular disease patients. The gastrointestinal microbiome appears to play a key role in nitrite metabolism and bioactivation.

Background: Diabetes leads to endothelial dysfunction and thrombus formation in a context of oxidative stress. Our group has shown that peri/epicellular (pec) protein disulphide isomerase A1 (pecPDI), a thiol isomerase protein, influences vascular cell adhesion and that platelet PDI is positively correlated with glycaemia. Therefore, we investigated the impact of pecPDI in platelet-endothelium interactions upon the exposure to high glucose levels.
Methods: Human umbilical vein endothelial cells (HUVECs) were cultured in a normo- (5.5 mM) or high-glucose (25 mM) medium for 48 hours in the presence or absence of PDI inhibitors while platelets were isolated from healthy donors. Hydrogen peroxide was measured through amplex red and cell adhesion was assessed through immunofluorescence.
Results: Platelets adhered more onto HUVECs exposed to high glucose levels, which also presented thicker actin fibres when compared to those of normo-glucose HUVECs. High-glucose HUVECs produced more hydrogen peroxide and were more adhesive onto a collagen matrix. High-glucose cells also showed an augmented fractal dimension, suggestive of increased spreading. Interestingly, there was an increased co-localization between PDIA1 and collagen receptor integrin beta 1 in high-glucose cells when compared to normal HUVECs. Rac1 and RhoA, which are a key GTPases downstream of integrin activation, were markedly translocated to the cell membrane when HUVECs were exposed to 25 mM glucose. PecPDI inhibitors impaired all of the abovementioned processes.
Conclusion: Platelets adhere more onto HUVECs exposed to high glucose levels, in a process largely mediated by pecPDI. Mechanistically, pecPDI was shown to regulate oxidant production, cell adhesion, cytoskeleton organization and activation of RhoGTPases. We propose that pecPDI is a master regulator of platelet-endothelium interactions in high-glucose conditions, thus fostering the development of improved targets to treat cardiovascular diseases of diabetic individuals.

Recently, fibroblast growth factor 21 (FGF21) has been recognized as a stress-responsive hormone that plays a key role in glucose and lipid metabolism and in the control of energy balance. It is also considered as a key regulator of the oxidative stress cell responses, due to relations of the FGF21 gene with NRF2, UCP3, SOD2, ERK and others. Its expression is induced by oxidative stress, and it also exerts protective effects, e.g. by inhibiting inflammation in response to oxidative stress.
We hypothesized that circulating levels of FGF21 are affected in health-to-disease transition and focused on early stages of metabolic disturbances in NCDs in comparison to full health.
Apparently healthy subjects with mildly impaired renal function (eGFR 30-60 ml/min/1.73 m2) (group A), mildly impaired glucose tolerance (HOMA index >2.5 and HbA1c 38.8-44 mmol/mol) (group B) or early stages of arteriosclerotic lesions (carotid intima-media thickness left and right >75th percentile) (group C) were studied and compared to subjects with clinically and biochemically proven full health. Overlaps between groups were excluded to allow for identifying FGF21 behavior in different types of disturbances. Along with FGF21, a comprehensive panel of metabolic and oxidative stress biomarkers was assessed.
Both groups A and B showed higher FGF21 concentrations compared to subjects with full health and group C; they also showed higher levels of branched-chain amino acid-derived C5-acylcarnitine (suggesting incomplete fatty acid oxidation), leptin, triglycerides and fatty liver index, BMI and waist circumference, and lower HDL-cholesterol, vitamin C and β-cryptoxanthin; human mercaptalbumin (reduced:oxidized form) was impaired in group A (all P < 0.001). Glutathione peroxidase, myeloperoxidase and malondialdehyde did not differ.
These results demonstrate that circulating FGF21 levels are elevated in early stages of metabolic disturbances in insulin resistance and impaired kidney function, but not in early vascular changes in the absence of such disturbances.

The incidence and prevalence of non-alcoholic fatty liver disease (NAFLD) are rapidly rising worldwide due to the global obesity epidemic. Obesity-induced oxidative stress plays an important role during the progression of NAFLD through different mechanisms, including oxidative modifications of protein thiols, stimulation of transcription pathways, and promoting the recruitment of inflammatory cells. Indeed, it is known that obesogenic environments in NAFLD, characterized by high levels of glucose, fatty acids, insulin, and pro-inflammatory cytokines (i.e., IFN-γ, TNF-α and IL-6), increase the generation of reactive oxygen species like hydrogen peroxide (H2O2). Chronic hepatic oxidative stress in obesity is mainly attributed to palmitate-induced mitochondrial and peroxisomal H2O2 generation, UPR/ER stress, and an increased flux of the DAG-PKC-NOX signaling pathway. However, the contribution of H2O2 originating from different cellular compartments during NAFLD development remains unclear. Likewise, it remains to be elucidated which environmental compounds contribute to H2O2 generation from each of the contributing compartments. To address this, we express the latest ultrasensitive pH-independent H2O2 probe HyPer7 in different cellular compartments of hepatocytes including the cytosol, mitochondrial matrix, peroxisomes, and nucleus. This allows us to monitor H2O2 levels and dynamics live in different cellular compartments with spatial and temporal resolution. By exposing the HyPer7-expressing hepatocytes to obesogenic environments, we attempt to identify the subcellular source(s) of H2O2 responsible for the progression of NAFLD. In the long term, the results of this study can be used to design targeted therapeutic options to curb NAFLD progression.

In mammals, DNA is generally confined to the cell nucleus and mitochondria. Its presence in the cytosol is abnormal and its efficient detection is fundamental to control cellular homeostasis. One of pivotal post-translational modifications governing cellular fate is
S-nitrosation, which protects against oxidative stress endothelial cells, however predisposes them to premature aging and promotes protein aggregation. Moreover, our preliminary studies indicate that increased S-nitrosation results in the appearance of cytosolic DNA inclusions, which fully colocalise with protein aggregates. We aimed to elucidate the molecular mechanisms governing the release and further fate of cytosolic DNA. Therefore, we combined the molecular biology and the biophysical methods to address this issue.
We observed that the cytosolic inclusions are composed of dsDNA, which is not related to the mitochondria content and does not activate canonical pathways related to the recognition of cytosolic DNA. Mechanistically, S-nitrosation causes DNA damage and alters nuclear envelope architecture and permeability. Concomitantly, atomic force microscopy revealed very dense actin cytoskeleton over the nucleus. Live cell actin staining revealed rapid changes in actin cytoskeleton which correlated with the DNA expulsion from the cell. The instantaneous release of dsDNA from cells can be further intensified by induction of autophagy. The secretion is executed by large spheric SNO-positive components and the accumulation of protein aggregates presumably serves as a signal for autophagy activation to enable the disposal of cytoplasmic DNA.
To sum up, here we propose a S-nitrosation related mechanistic model of nuclear DNA release, which relies on concomitant occurrence of DNA damage, increased permeability and actin confinement of the nucleus. Interplay between autophagy and S-nitrosated proteins orchestrate the disposal of DNA. Finally, this observations can give valuable insights into understanding the basis of tumorigenicity and autoimmune diseases.

Hydrogen peroxide (H2O2) signaling is a process by which particular thiols on particular proteins are reversibly oxidized to modulate protein function in a dynamic manner. H2O2 can be generated from various intracellular sources, but their identities and relative contributions are often unknown. To identify endogenous ‘hotspots’ of H2O2 generation on the scale of individual proteins and protein complexes, we generated a yeast library in which the H2O2 sensor HyPer7 was fused to the C-terminus of all protein-coding open reading frames (ORFs). We also generated a control library in which a redox-insensitive mutant of HyPer7 (SypHer7) was fused to all ORFs. Both libraries were screened side-by-side to identify proteins located within H2O2-generating environments. Screening under a variety of different metabolic conditions revealed dynamic changes in H2O2 availability which were highly specific to individual proteins and protein complexes. For example, amino acid deficiencies cause H2O2 to increase in proximity to certain proteins, but to decrease in proximity to certain other proteins, even if they are in the same cellular compartment. These findings suggest that H2O2 generation and impact is much more localized and differentiated than previously recognized.

Background: Oxidative stress is closely associated with atherosclerosis and acute coronary syndromes.
Aims: In this study, we aimed to evaluate proportional oxidative stress parameters in ST segment elevation myocardial infarction (STEMI) patients and also to investigate their effects on in-hospital prognosis.
Methods: This is a single centre, prospective, and cross-sectional case-control study. Total oxidative status (TOS), total antioxidant status (TAS), oxidative stress index (OSI), ischemia modified albumin (IMA), myeloperoxidase (MPO), paraoxonase-1 (PON-1) and arylesterase (ARES) enzyme activities as well as MPO/PON-1, MPO/ARES and MPO/HDL ratios were studied in 107 patients. Short-term in-hospital prognosis markers used in this study were; in-hospital mortality, early systolic dysfunction and spontaneous complete revascularization which is Thrombolysis in Myocardial Infarction (TIMI) grade 3 flow.
Results: Both patient (n=63) and control (n=44) groups were similar in terms of age and gender. TOS, OSI, IMA and MPO values, as well as, MPO/PON-1 and MPO/HDL ratios were significantly higher and PON-1 and ARES values were significantly lower in STEMI patients compared to the control group. In terms of in-hospital short-term prognostic markers, a significant relationship was found only between OSI value and spontaneous complete revascularization status. The OSI value was higher in the group with TIMI grade 3 flow than in the group with TIMI grade 0-2 flow (2.42 [0.81-4.49] vs 1.63 [0.33-6.07], p = 0.016).
Conclusions: As a result of this study, MPO/PON-1 and MPO/HDL ratios are considered as new oxidative stress parameters that are found to be elevated in STEMI patients. Among in-hospital short term prognostic markers, only an association between spontaneous complete revascularization and OSI values was found.

The release of neutrophil extracellular traps (NETs) is a key innate immune defense to combat infection. NETs consist of a mesh of DNA and histones, and contain other neutrophil proteins, including myeloperoxidase (MPO). Although NETs have important anti-bactericidal properties, they are also implicated in the development of numerous inflammatory diseases, particularly atherosclerosis. However, the mechanisms involved in the pathological effects of NETs are not well understood. Enzymatically active MPO is present on the NET backbone and produces the potent oxidant hypochlorous acid (HOCl). In this study, we examine how the modification of NET-associated histones by HOCl influences vascular smooth muscle cell function. Experiments were performed with a preparation of histones containing both linker (H1) and core (H2A, H2B, H3 and H4) histones incubated in the absence or presence of HOCl for 24 h, to allow decomposition of unstable N-chloramines. Exposure of primary human coronary artery smooth muscle cells to native and HOCl-modified histones resulted in a dose-dependent loss of viability, consistent with the known toxicity of extracellular histones. Interestingly, less cell death was apparent on pre-treatment of the histones with HOCl compared to the non-modified histones. In addition, the change in cell viability observed with the HOCl-modified histones was dependent on the extent of oxidative modification. The HOCl-modified histones altered the redox balance in the cells, as reflected by a decrease in reduced thiol concentration, to a greater extent than that seen with the native histones. Similarly, only the HOCl-modified histones increased the expression of different pro-inflammatory mediators, including vascular cell adhesion molecule 1. Together, these results suggest that HOCl-induced modification could perturb the extracellular reactivity of histones to favour activation of inflammatory signaling. This could have implications for the development of diseases linked to aberrant NET release, particularly atherosclerosis.

Reactive oxygen species (ROS) regulates cellular homeostasis and acts as a modulator of cellular dysfunction. Exacerbated production of ROS is reported in inflammatory disorders however contribution of free radical-mediated processes in both physiological and pathological conditions has not received much attention. ROS randomly damage lipids, proteins and nucleic acids giving rise to the formation of macromolecule centered radicals. Being highly reactive and with increased abundance within a cellular environment, proteins remain a major target for intermediate radical formation. As macrophage plays a vital role in the progression of metabolic and inflammatory disorders, our study aimed at deciphering the role of ROS in macrophage polarization and subsequent protein centered radicals formation. Our study employs THP-1 as a model system to study and evaluate free radical-mediated protein oxidation in differentiated macrophages. The study utilizes immuno spin trapping techniques in the identification of ROS type, source and its corresponding protein radical intermediate formation along the process of macrophage polarization. Results from this study help in the identification of protein targets undergone modification and their subcellular localization thereby serving as promising biomarkers of free radical-mediated oxidative stress. Herein, we provide a methodological platform for elucidation of redox regulation within a cellular environment and processes involved in metabolic and inflammation-driven tissue dysfunction.

The silkworm and its main product, the cocoon, contain proteins, such as sericin and fibroin, along with enzymes, such as serrapeptase, which can help reduce inflammation and promote skin healing. Previous relative studies have indicated a possible positive in vivo effect of the silkworm products on the healing of 2nd degree burns.

The first aim of this study was to examine the action of three different silkworm products (silkworm body, glands, and cocoon) in higher doses than those having already been used. Hairless, female mice, type SKH-hr2, brown and black were used, to which 2nd degree burns were induced. The animals were separated into 9 groups with two control groups. For the treatment groups, gels with silkworm body extract and silkworm gland extract were used, while silkworm cocoons were applied as patches. Histopathological and clinical evaluations were performed, along with measurements of biophysical parameters, thickness, and surface of the burn area. The cocoon and the higher dose of silkworm body showed the best results, regarding the clinical observations and the histopathological findings.

Based on these results a second study was conducted to examine the synergistic action of the cocoon and the higher dose of the silkworm body extract, compared with the effect of each treatment separately. The same type of materials was used. The animals were separated into 5 groups with two control groups. For the treatment groups, gel with silkworm body extract was used while silkworm cocoons were applied as patches, either along with the silkworm body-extract gel or without it. After the evaluation of skin parameters, a slightly better action of the combination group was observed according to the histopathological results.

The incorporation of drug substances into the cocoon is proposed as a next step to examine the effect of such product on the healing of burn injuries.

By affecting mitochondrial bioenergetics and protein persulfidation, H2S acts as a redox regulator that may modulate the redox metabolome and the thiol proteome. H2S exerts both stimulatory and inhibitory effects, for example on cellular proliferation and differentiation. Particularly large amounts of H2S are produced within the human gut, by commensal bacteria and by intestinal epithelial cells. CBS, CTH and MPST have long been known as human H2S-producing enzymes; recently, selenium-binding protein 1 (SELENBP1) has been reported to generate H2S as well, by means of its methanethiol oxidase activity. Here, we provide the first comparative analysis of the four H2S-producing enzymes and the H2S-catalyzing enzyme SQOR in differentiating Caco-2 human intestinal epithelial cells. Protein and mRNA levels were determined by immunoblotting and qRT-PCR during spontaneous and butyrate-induced differentiation. H2S production deriving from the enzymatic substrates methanethiol, cysteine and/or homocysteine was assessed in an assay based on lead sulfide precipitation. Both spontaneous and butyrate-induced differentiation resulted in pronounced increases in gene expression and enzymatic activity of the differentiation marker ALPI. The levels of the H2S-modulating enzymes were differentially altered during spontaneous differentiation of Caco-2 cells: While SELENBP1 and SQOR were strongly upregulated in differentiated as compared to proliferating cells, CBS was downregulated, and CTH as well as MPST remained largely unaffected. Methanethiol- and homocysteine-derived H2S production was strongly elevated in differentiated cells. Treatment with butyrate also resulted in upregulation of SELENBP1 and SQOR; however, the effects were less pronounced. In contrast, butyrate treatment did not mimic the downregulation of CBS during spontaneous differentiation. In conclusion, CBS and SELENBP1 are reversely regulated during spontaneous differentiation of Caco-2 cells. Moreover, SELENBP1 represents the H2S-producing enzyme whose upregulation was most pronounced during differentiation. Finally, butyrate exposure, while imitating some aspects of spontaneous differentiation, does not elicit the same expression patterns of genes encoding H2S-generating enzymes.

Calcium influx through glutamate receptors upon neuronal activity plays an essential role in neurotransmission, but an excessive activation of the pathway leads to calcium overload and neuronal death in a process termed excitotoxity. Together with calcium imbalance, mitochondrial dysfunction and oxidative stress are often involved in the mechanism of neurodegeneration in disorders such as tau-related frontotemporal dementia (FTD). We have employed iPSC-derived neurons of healthy controls and patients of FTD to understand the interaction between these pathways in physiology and pathology.

We have found that reactive oxygen species (ROS) produced at the mitochondria (mROS) play an essential signalling role in neurotransmission. mROS, via oxidation of proteins involved in their trafficking, control the surface expression of specific glutamate receptor subunits in neurons, therefore modulating the calcium entry through them. In FTD, tau-induced mitochondrial dysfunction leads to an overproduction of mROS, which in turns upregulates the surface expression of glutamate receptors, leading to excessive calcium influx and promoting excitotoxic neuronal death. Importantly, mitochondrial antioxidants are neuroprotective and prevent the glutamate-induced calcium overload and mPTP opening, caspase-3 activation and neuronal death triggered by mitochondrial calcium overload.

These results highlight a) the role of mitochondria in cellular signalling in physiology and pathology, by modulating calcium entry through glutamate receptors via mitochondrial ROS; b) the reversible redox regulation of glutamatergic signalling, which might serve as a target for indirect pharmacological strategies to modulate calcium entry through glutamate receptors in neurodegenerative disorders.

BACKGROUND
Although oxidative stress (OS) and neuroinflammation are thought to play a role in the etiology of type-C hepatic encephalopathy (C-HE), their involvement and synergistic action is not well understood. Using in-vivo-longitudinal 1H-MRS we have shown an impaired antioxidant system, an indirect presence of OS, together with ex-vivo EPR detected increase of CNS superoxide-anion concentrations in bile-duct-ligated (BDL) rat-model of C-HE. The immunohistochemistry was used to highlight the OS findings as well as to analyze the potential synergistic involvement of OS and neuroinflammation in C-HE.
METHODS
In-vivo-1H-MRS: Cerebellum/hippocampus of adult rats-scanned before and post-BDL every 2-weeks up to week6 (n=18)(9.4T-MR-Varian/Magnex-Scientific).
Ex-vivo-ESR: ESP300E(Bruker-BioSpin)-CMH-spin-probe: intracellular superoxide-anion detection, week 2(BDL n=2, sham n=2), 4(BDL n=2,sham n=2), 6(BDL n=5,sham n=5) and 8(BDL n=2, sham n=2).
Immunohistochemistry: BDL/sham-rats (BDL:4-weeks n=3,8-weeks n=3,sham:n=3).
Oxidative stress: Oxo-8-dG,GPX1,SOD1/SOD2-relative immunofluorescence quantification.
Neuroinflammation: IL-6- accumulation and UV-Vis-spectroscopy- quantitative evaluation(TC5600-microscope-MEIJI-TECHNO) coupled with Ocean-HDX-UV-VIS-spectrometer.
RESULTS: Chronic-liver-disease was validated by blood biochemistry(increased bilirubin and ammonium).
For sham-rats, ESR revealed differences in redox-state between hippocampus and cerebellum (~31%,p=0.004). However, the relative OS increase in BDL-rats vs. shams at week-6 was similar ~42%. The significant increase of hippocampal/cerebellar OS in BDL(p=0.01,p=0.001) corroborate the 1H-MRS findings of decreased Asc concentrations(Fig1A).
BDL-rats exhibited a strong increase in Oxo-8-dG-immunoreactivity in the hippocampus and cerebellum(p=0.001 vs. sham,Fig.1A). The cytoplasmic localization revealed that OS affected the mitochondrial and cytosolic-nucleic-acids. BDL-rats exhibited a significantly increased GPX-1 synthesis(p=0.001) in the hippocampus(Fig.1B). OS-driven regulation of SOD1/SOD2 resulted in enhanced immunoreactivity of both(p=0.001,Fig.1C). SOD1 localization changed from predominantly cytoplasmic to prominently nuclear(Fig.1C). Sham-rats showed a weak IL-6 immunofluorescence while BDL-rats displayed a significant increase(p=0.001) and upregulation in all brain regions(Fig.1D).
CONCLUSIONS
This work proves the importance of OS and neuroinflammation in C-HE and highlight a possible vicious-circle between OS and neuroinflammation in C-HE in addition to the well-known contributions of ammonium and brain glutamine.

Protein homeostasis (proteostasis) refers to the molecular mechanisms that are responsible for the maintenance of the cellular protein network. Proteostatic mechanisms tend to decline with age and this often leads to accumulation of toxic protein aggregates. The Aβ peptide that has been causally related to Alzheimer’s disease (AD) onset and progression represents one of these aggregation-prone proteins. Plant secondary metabolites have been shown to be beneficial for proteostasis maintenance and/or restoration. Here, we have searched for natural products with anti-aggregating properties from the Greek flora, using various C. elegans AD models for screening. We have identified a mountain tea extract with anti-aggregation properties derived from the Greek endemic Sideritis clandestina subsp. Peloponnesiaca (SCP). We have further fractionated the extract to identify the specific bioactive compounds that are responsible for these properties. We show that the identified compounds may decelerate: (1) the progression of the AD phenotype in CL4176 nematode strain, a strain expressing the human Aβ1-42 in its body wall muscle cells that undergoes paralysis upon temperature upshift due to Aβ aggregation, as well as, (2) the accumulation of Aβ aggregates in CL2331 nematode strain, a strain expressing the human Aβ3-42 peptide fused to green fluorescent protein (GFP) in its body wall muscle cells where Aβ aggregates can be visualized in vivo. Our study uncovers the need to identify bioactive compounds (that ideally are part of our diet) with anti-aggregation properties.

Acknowledgements
This research has been co‐financed by the European Union and Greek national funds through the Operational Program “Competitiveness, Entrepreneurship and Innovation”, under the call “RESEARCH – CREATE – INNOVATE” (project code: T1EDK-00353).

Skin aging is influenced by several factors including environmental exposure and hormonal changes. Reactive oxygen species (ROS), which mediate many of the effects of these factors, can be formed by extrinsic factors, such as sun exposure, or can result from mitochondrial dysfunction which occurs during ageing. ROS activate the nuclear factor-kappa B (NFƙB) transcription systems leading to inflammatory processes and increased production of matrix metalloproteinase (MMPs) by skin cells, which leads to collagen degradation. Several studies have shown the protective role of estrogens and of various phytonutrients including carotenoids and polyphenols on skin health. The aim of the current study was to examine the damage caused by ROS that originate in the mitochondria due to its dysfunction, and to examine the protective role of tomato extract containing lycopene, rosemary extract and estradiol. Human dermal fibroblasts and keratinocyte were used to determine ROS levels and their effect on cell viability, MMP1 and pro-collagen secretion as markers of skin damage. Rotenone was used to cause mitochondrial dysfunction and reduction in oxygen consumption, which causes accumulation of mitochondrial and cytosolic ROS, apoptotic cell death by activating caspase 3 activity, upregulation of MMP1 secretion and decreased collagen secretion. This was accompanied by activation of the antioxidant response element/Nrf2 (ARE/Nrf2) and NFƙB transcriptional activity. Pretreatment with dietary compounds such as tomato extract and rosemary extract and estradiol reduced ROS level and MMP1 secretion and increased cell viability. These effects can be partially explained by the increased synergistic activity of ARE/Nrf2, and the decreased activity of NFƙB transcriptional activities. This study indicates that phytonutrients and sex hormones protect skin cells from damage caused by mitochondria generated ROS and may delay skin aging and improve skin health and appearance.

There is increasing evidence that mitochondrial dysfunction is associated with many diseases, including Alzheimer’s Disease and vascular disorders, as well as toxicities of common drugs such as fluoroquinolones. Unlike the mixed results from trials involving the use of non-specific antioxidants in alleviating mitochondrial toxicity, mitochondrial-targeted antioxidant therapies such as MitoQ have shown some promise. L-ergothioneine (ET) is a naturally occurring thiol-thione that is found in humans wholly via dietary absorption using a highly specific and almost ubiquitously expressed transporter, OCTN1. ET accumulates at high concentrations in humans with a slow excretion rate. While numerous studies have evaluated its antioxidant abilities in vitro and in vivo, its physiological function remains unknown. Preliminary evidence suggests that L-ergothioneine may be accumulated within the mitochondria, although this has yet to be conclusively established. In this study, we verified the intracellular localisation of L-ergothioneine within the mitochondria in both in vitro cell and in vivo mouse models using LC-MS, identifying a dose-dependent increase in L-ergothioneine levels within the mitochondria. Subsequently, we explored the relationship between L-ergothioneine and complexes of the electron transport chain using isolated mitochondrial activity assays, identifying L-ergothioneine’s potential role in preserving mitochondrial complex I function. As a compound demonstrating antioxidant properties with likely mitochondrial localisation, L-ergothioneine presents as a potential mitochondrial-targeted antioxidant therapy without need for any further structural modifications. In addition, its excellent safety profile combined with slow excretion further illustrates L-ergothioneine’s potential as a pharmacological intervention for mitochondrial toxicity. Our present findings suggest a role for L-ergothioneine within the mitochondria, although more work is necessary to elucidate specific therapeutic areas where its pharmacological potential can be explored in relation to mitochondrial toxicity.

Since its introduction over 20 years ago (1), the method of assay of total antioxidant capacity based on the reduction of pre-formed radical of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•) has become one of the most often used method of estimation of total antioxidant activity. According to Google Scholar, the paper presenting this method1 has been cited 23913 times. The aim of this study was to study this reaction in a more detail and to propose a convenient adaptation of the method for a plate reader.
We diluted stock ABTS• solution with 10 mM phosphate, pH 7.4, to the final concentration providing absorbance of a 200-µl sample in a well of a Greiner 96-well plate (fluid layer of 6.25 mm) of 1.0; it corresponds to ABTS• concentration of 106.7 µM. ABTS• reactivity with Trolox did not change significantly with pH in the range of 2.0-7.4; the reactivity with Trolox and blood plasma was lowered with increasing ionic strength.
Various antioxidants differed in the rates of reaction with ABTS•. In the concentration range not exceeding the total reduction capacity of ABTS•, reactions of some antioxidants (e.g. Trolox) were completed within seconds (“fast antioxidants”) while the reactions other compounds (“slow antioxidants”) and complex materials like blood plasma or mushroom extracts lasted for minutes and could be not completed after 6 and even 30 min. The reasonable time for the measurement of total ABTS• reduction capacity in such cases was found to be 60 min.
The Area Under Curve approach was found to be equivalent to the measurement of final absorbance decrease. For calculation of the Trolox Equivalent Antioxidant Capacity, comparison of the slopes of dependence of ABTS• reduction capacity on the concentration were found superior to the comparison of decreases in absorbance.

(1) Re et al., Free Radic. Biol. Med. 1999; 26:1231.

HyPer7 is a fluorescent protein-based biosensor for hydrogen peroxide that has distinguished itself from its predecessors in the HyPer family by displaying nanomolar sensitivity, ratiometric pH-independence and increased brightness. HyPer7 has already contributed to the elucidation of H2O2 signalling in a wide variety of organisms. Nevertheless, many mechanistic details about the functioning of the biosensor remain unexplored. These include the kinetics of its reaction with H2O2, as well as of its reduction by the cellular reducing systems such as the thioredoxin and glutaredoxin systems. Further, it is not understood how the conformational coupling between the sensing moiety (OxyR), and the SYG chromophore of cpYFP occurs. Finally, the basis of the pH-independence compared to other HyPer biosensors remains unclear. Using a combination of artificial intelligence predictions and various biophysical and biochemical techniques here we address these questions and provide a mechanistic dissection of HyPer7. Our results will on the one hand contribute to a more informed interpretation and planning of experiments on HyPer7 and on the other hand serve as a basis for the development of further biosensors.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, mainly in older adults, characterized by uncontrolled growth of immature myeloid blasts. Despite initial responses to standard chemotherapy, prognosis remains grim for most patients. Differentiation therapy of AML is an alternative to cytotoxic chemotherapy. Natural and synthetic vitamin D derivatives (VDDs) are powerful inducers of monocytic differentiation of AML cells in culture; however, their differentiation-inducing concentrations can be lethal in vivo due to severe hypercalcemia. We have previously shown that fumaric acid esters (FAEs), such as the clinically approved drug dimethyl fumarate and its in-vivo metabolite monomethyl fumarate (MMF), can synergistically enhance the prodifferentiation effects of near-physiologic concentrations of different VDDs [PMID: 30508646]. Since FAEs are known activators of the transcription factor Nrf2, we hypothesized that Nrf2 may mediate the enhancing effects of these agents on the differentiation of AML cells induced by 19-nor-1,25-(OH)2-vitamin D2 (paricalcitol). Here, we demonstrate that in non-transfected and empty vector-transfected HL60 human AML cells, the differentiation-inducing effect of paricalcitol was markedly potentiated by MMF, monoethyl fumarate (MEF) or the Nrf2-activating phenolic diterpene carnosic acid (CA). This potentiation was associated with a marked upregulation of the vitamin D receptor (VDR) protein levels and mRNA expression of VDR target genes, e.g. CAMP and CYP24A1. However, these enhancing effects of the Nrf2 activators were dramatically reduced in HL60 cells stably expressing a dominant-negative Nrf2 (dnNrf2) mutant that lacks the transactivation domain. Notably, co-treatment of dnNrf2-expressing cells with the glutathione precursor N-acetyl cysteine or cell-permeant glutathione ethyl ester partially restored the synergy between paricalcitol and Nrf2 activators. These data suggest that the differentiation-enhancing effects of FAEs and CA are mediated by the transcriptionally active Nrf2, possibly through the Nrf2-dependent elevation of cellular glutathione levels.

Background: Several studies point to the anti-inflammatory, antithrombotic and antioxidant effects of vitamin D, which could have a beneficial impact in the SARS-Cov-2 infection treatment. Aims: This study was performed in order to evaluate the vitamin D status in relation to oxidative stress parameters and disease outcome in hospitalized COVID-19 patients. Methods: Thirty-three hospitalized patients with COVID-19 were included in this study. Serum vitamin D levels, as well as selected laboratory parameters (blood cell count, neutrophil to lymphocyte ratio, C reactive protein, creatine kinase, lactate dehydrogenase and D-dimer) were analyzed at admission. Oxidative stress markers (PAT (total antioxidant power, iron reducing) and d-ROMs (plasma peroxides)) were measured on analytical photometric system. Results: Twenty-four patients had vitamin D levels below 30 ng/mL, whereas the remaining 9 had vitamin D levels above 30 ng/mL. Vitamin D deficient patients had increased oxidative stress, as determined by significantly higher levels of d-ROMs (414.9 ± 15.82 U.Carr vs. 352.4 ± 18.77 U.Carr) and Oxidative Stress Index-OSI (92.25 ± 6.60 vs. 51.89 ± 6.45), but without a difference in the antioxidant capacity between the two study groups (PAT=2665 ± 133.5 U.Carr vs. 2868 ± 160.9 U.Carr). This was accompanied by significantly higher LDH (604.8 ± 76.98 IU/mL vs. 261.57+47.33 IU/mL) and the D-dimer (5978 ± 2028 ng/mL vs. 977.7 ± 172 ng/mL). A significant inverse correlation was detected between vitamin D and d-ROM, OSI and LDH, whereas the correlation between vitamin D and D-dimers was found to be not statistically significant. Regarding the outcome, the group of patients with lower vitamin D levels had a higher mortality rate (37.5%) compared to 22.2% mortality rate in the group with vitamin D >30 ng/mL. Conclusions: Our findings suggest a potential benefit from vitamin D supplementation in the supportive treatment

COVID-19 is a disease in several stages starting with virus replication to dysregulation in immune system response, organ failure and recovery/death. Our aim was to determine the effect of combination extract of Ganoderma lucidum, lycopene, sulforaphane, royal jelly and resveratrol on the markers of oxidative stress, inflammation (IL-6, IL-8 and TNF-α), routine laboratory analyses and duration of symptoms in patients infected with SARS-CoV-2.
The oxidative stress parameters (d-ROM, PAT, OS index) and IL6, IL8, TNF-a were determined in order to estimate the antioxidant and the anti-inflammatory effect of the product using a spectrophotometric and a magnetic bead-based multiplex assay in serum of 35 patients with mild form of COVID-19. Ge132+ Natural™ was used as a product of interest.
We have obtained statistically significant differences for all investigated parameters between the treated patients and the control group. Moreover, significantly differences were observed for leukocytes, NLR and iron. The average duration of the symptoms was 9.4±0.487 days versus 13.1±0.483 days in the treatment and the control group, respectively (p=0.0003). The evidence presented herein regarding the anti-inflammatory and the antioxidant effect of the product suggest it could be used as a potent an adjuvant therapy in diseases accompanied by increased oxidative stress and inflammation.
Our results demonstrated the promising effect of the product on reducing the oxidative stress and the IL-6, IL-8 and TNF-a levels, and duration of the symptoms in ambulatory COVID-19 patients. The evidence presented herein regarding the anti-inflammatory and the antioxidant effect of Ge132+Natural™ (combination of Ganoderma lucidum extract, lycopene, sulforaphane, royal jelly and resveratrol) suggest it could be used as a potent an adjuvant therapy in diseases accompanied by increased oxidative stress and inflammation.

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Halophytes are known to accumulate excess salts in tissues, removing them from the immediate environment. This makes them suitable candidates for haloremetiation. In this two-phase experiment, we explored the feasibility of intercropping tomato (Solanum lycopersicum var. Sargento) with the halophyte Arhtrocaulon macrostachyum, native of South East Spain, trying to mitigate the negative effects of saline soil while providing a value-added crop.
Two consecutive greenhouse experiments, from March to July 2021 and from October 2021 to February 2022, were conducted under soil Na+ and Cl- contents of 950 ppm and 1500 ppm, respectively. Plots were arranged in randomised block designs with three replicates. In the first season, three types of plots – halophyte and tomato in monocultures and mix cultivation – were arranged, whereas in the second season tomato was additionally cultivated where halophyte was previously grown (sequential cropping).
Overall, intercropping and sequential cropping affected the activity of antioxidants enzymes in leaf extracts. This was reflected on an enhanced accumulation of reactive oxygen species, which may lead to the establishment of a moderate oxidative stress. In parallel, changes on chlorophyll fluorescence parameters were recorded. In this sense, non-photochemical quenching (NPQ) and electron transport rate (ETR) were higher in intercropping and sequential cropping than in monoculture. With respect to the halophyte, both leaves and roots showed higher Na and Cl than in monoculture, while in tomato the opposite behaviour occurred. Intercropping did not affect tomato production. However, sequential cropping significantly increased (up to 20%) both tomato number and weight. On the other hand, sequential cropping decreased soluble solids (measured as °Brix) and acidity (as total acidity to citric acid equivalents) for tomato in comparison to both tomato in monoculture and in intercropping. Further experiment replications will be conducted, which will also involve valorization of the halophyte for ulterior uses.

The hasher environments resulted by climate change cause oxidative stress to plants, triggering reactive oxygen species (ROS) production in plant cells, which are potent signaling molecules prone to activate defense responses. Relatively stable ROS, such as hydrogen peroxide (H2O2), can provoke reversible and irreversible oxidative post-translational modifications (Ox-PTMs) on protein cysteine (Cys), which might act in diverse signaling pathways in plant stress adaption. Protein Cys thiols (–SH) are very susceptible to H2O2. The initial reaction of H2O2 with –SH forms sulfenic acid (–SOH) that is intrinsically unstable and an intermediary en route to other OxiPTMs, such as the relatively more stable sulfinic (–SO2H) and sulfonic (–SO3H) acids, which are consider as overoxidation. The –SO3H formation is irreversible, whereas –SO2H can be recycled in an ATP-dependent manner by sulfiredoxin (Huang et al. 2018; Willems et al. 2021).
To get insight into Cys-mediated redox switches in plants, we have generated an unprecedented view on the –SOH landscape using variou proteomic approaches that facilitate further functional redox studies (Huang et al. 2019; Wei et al. 2020). To better understand the protection mechanism from Cys overoxidations, our current ongoing work also focuses on –SO2H and its reduction enzyme, sulfiredoxin. We aim to uncover the Cys-based redox switches in plants under oxidative stresses. The identified Cys-based redox mechanisms could be extrapolated and contribute to optimize crops and vegetables to be more resilient against climate change. Here, the general strategy for studying Cys oxidation will be outlined, and several examples for functional studies including ongoing work will be presented.

Gene regulation is a fundamental process during plant growth and development, but is also important for plant responses to external stimuli. Gene regulatory network plays important roles in different physiological responses and pathways to help plant adapt to the environments. However, our global knowledge about the complexity of TF control for different genes and biological processes is incomplete. To enhance our global understanding of regulatory interactions in Arabidopsis thaliana, different regulatory input networks capturing complementary information about DNA motifs, open chromatin, TF binding and expression-based regulatory interactions, were combined using a supervised learning approach, resulting in an integrated gene regulatory network (iGRN) covering 1,491 TFs and 31,393 target genes (1.7 million interactions) in this work. The iGRN correctly inferred known functions for 681 TFs and predicted new gene functions for hundreds of unknown TFs. For regulators predicted to be involved in reactive oxygen species stress regulation, we confirmed in total 75% of TFs with a function in ROS and/or physiological stress responses. This includes 13 novel ROS regulators, previously not connected to any ROS or stress function, that were experimentally validated in our ROS-specific phenotypic assays of loss- or gain-of-function lines (MV, H2O2 or 3-AT). In conclusion, the presented iGRN offers a high-quality starting point to give us a better understanding for gene regulation in plants by integrating different experimental data types at the network level.

The evolutionary conserved Spt–Ada–Gcn5–Acetyltransferase (SAGA) complex is a transcriptional coactivator that regulates a myriad of cellular processes through modulation of chromatin structure and transcription. As a part of its histone acetylation module, the histone acetyltransferase GENERAL CONTROL NONDEREPRESSIBLE 5 (GCN5) acetylates lysine 14 of histone 3 (H3K14) in the promoter regions of its target genes. GCN5 has been implicated in stress responses and development processes thus functioning at the crosstalk between environmental and stress programs. Intriguingly, despite its profound impact on gene expression, the molecular mechanisms regulating the acetylation activity of the SAGA complex are still unknown. Here, we report that GCN5 is sulfenylated at an evolutionary conserved cysteine residue and this modification is implicated in mounting an effective stress response. Complementation of the gcn5 mutant with a GCN5 variant carrying a cysteine to serine mutation reverted the dwarfed gcn5 phenotype under control conditions, whereas it did not impact the sensitivity to oxidative stress observed in gcn5 mutant plants. Our results suggest that GCN5 is an important redox sensitive component of the SAGA complex that might play a role in novel molecular mechanisms integrating redox signaling and chromatin remodeling.

The antioxidants, ascorbate and glutathione, which are regenerated by the ascorbate-glutathione cycle, and carotenoids, are required for oxidative stress defense in photosynthetic organisms. The euglenophyte Euglena gracilis is a model microalga, which is used for both biological studies and industrial applications. Previous study has shown that treatment of E. gracilis with carotenoid biosynthesis inhibitor, norflurazon, suppresses photo-induction of ascorbate peroxidase enzyme (APX), suggesting the functional relationship between carotenoids and the ascorbate-glutathione cycle in this alga. In this study, using RNAi-mediated knockdown cells of carotenoid biosynthetic gene lycopene cyclase (EgLCY), we investigated the effects of suppressed carotenoid biosynthesis on the ascorbate-glutathione cycle and oxidative stress status in E. gracilis.
The KD-lcy cells, which were introduced double-stranded RNA, showed colorless appearance and 81% decrease in the total carotenoid contents compared to wild-type cells. The APX and superoxide dismutase (SOD) activities of KD-lcy cells decreased by 52% and increased 7.9 times, respectively, compared to wild-type cells. Among the ascorbate-glutathione cycle, the dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MADR) activities of KD-lcy cells increased by 77 and 45%, respectively, compared to wild-type cells and their glutathione reductase (GR) activity decreased by 29%. Correlating with the altered enzyme activities of the ascorbate-glutathione cycle enzymes, the ascorbate contents of KD-lcy cells increased by 55%, whereas their glutathione contents decreased by 38% compared to wild-type cells. The imaging analysis using H2O2-specific fluorescent reagent demonstrated that KD-lcy cells accumulated significantly higher level of H2O2 than that of wild-type cells. These results reveal that carotenoid biosynthesis contributes to chloroplast development and the ascorbate-glutathione cycle homeostasis, resulting oxidative stress defense in E. gracilis. To our knowledge, this study clearly reports for the first time the possible relationship between major antioxidant systems carotenoid biosynthesis and the ascorbate-glutathione cycle in photosynthetic organism.

Glyphosate, the most used herbicide worldwide, inhibits the 5-enolpiruvyl-3-phosphate synthase (EPSPS) in the shikimate pathway. Acetolactate synthase (ALS) inhibitors are a diverse herbicide group that inhibit ALS in the branched-chain amino acid biosynthesis pathway. Besides their different enzymatic target, glyphosate and ALS inhibitors trigger common physiological effects, including oxidative stress. However, the linkage between EPSPS/ALS inhibition and oxidative stress is not completely elucidated. Additionally, the massive usage of these herbicides has led to the development of weed resistant populations, as in Amaranthus palmeri.

Physiological effects of herbicides in resistant populations are poorly studied. The objective is to get new insights in the oxidative stress triggered by glyphosate and ALS inhibitors comparing sensitive and resistant plants with resistance mechanisms related to the target enzyme. To this purpose, glyphosate-resistant and ALS inhibitor-resistant populations and their sensitive reference populations were grown and treated with different doses of glyphosate or the ALS inhibitor nicosulfuron, respectively: untreated, field rate and 3 times field rate. Hydrogen peroxide content and gene expression levels of the antioxidant enzymes superoxide dismutase (CuZnSOD) and glutathione reductase (GR1, GR2) were measured as oxidative stress parameters.

All herbicide treatments were lethal for sensitive individuals, while resistant individuals survived. Early upon herbicide treatment, H2O2 highly accumulated but only in sensitive plants. Superoxide dismutase gene expression was induced in both sensitive and resistant populations after exposure to both herbicides. The GR1 expression level increased with glyphosate, especially in the sensitive population; while it did not change with nicosulfuron. The GR2 expression was unaltered by herbicide treatments.

In conclusion, the increase in antioxidant gene expression induced by glyphosate and ALS inhibitors in sensitive plants was not enough to avoid H2O2 accumulation in contrast to resistant populations. Probably their resistant physiology and mild increase in CuZnSOD and GR1 gene expression is sufficient to avoid oxidative stress.

Reactive oxygen and nitrogen species play a key role for the development of cardiovascular, metabolic and neurodegenerative disease, but they are also involved in cellular functions via redox signalling. We have previously identified an efficient mechanism of S-nitrosation by low levels of nitric oxide and superoxide (3:1 ratio) with potential formation of N2O3. Here, we elucidated whether S-nitrosation (as observed under hypoxic conditions) could prevent sulfoxidation and thereby oxidative inactivation of enzymes by superoxide/hydrogen peroxide (as observed during reoxygenation). We found that increasing concentrations of xanthine oxidase/hypoxanthine caused conversion of S-nitrosoglutathione (GSNO) to reduced glutathione (GSH) up to a certain concentration of xanthine oxidase, indicating that superoxide can induce denitrosation of GSNO. This finding was unexpected since in the presence of excess superoxide one would not expect regeneration of reduced GSH from GSNO. Also, the observed substantial dihydrorhodamine oxidation that was prevented by uric acid and tyrosine nitration of albumin during denitrosation of GSNO by superoxide, clearly pointed to intermediary formation of peroxynitrite. As a proof-of-concept, we demonstrate that isocitrate dehydrogenase (ICDH2) can be S-nitrosated / inactivated by spermine NONOate and denitrosated / partially reactivated by superoxide formed by xanthine oxidase. In summary, we propose that S-nitrosation of (mitochondrial) proteins during ischemia represents a protective mechanism to prevent irreversible overoxidation of thiols during the reperfusion phase and to re-establish reduced thiol state in (mitochondrial) key enzymes of energy metabolism and cell survival. Wide-spread mitochondrial protein S-nitrosation may represent a central feature of the protective preconditioning effects of nitric oxide.

Pollution of soils with metals such as cadmium (Cd) inhibits plant growth, thereby hindering their economic validation. Cadmium induces an imbalance between reactive oxygen species (ROS) and antioxidants at the cellular level, which can damage macromolecules. Damaged cellular components can be engulfed by vesicles (i.e. autophagosomes) that transport them into the vacuole for nutrient recycling in a process called autophagy. Autophagy is increasingly put forward as a protective mechanism during various stresses that may be intertwined with the redox environment. Therefore, this research aims to investigate autophagy in leaves and roots of Arabidopsis thaliana exposed to Cd and its connection to the Cd-induced oxidative challenge.

One important protein that is involved in autophagosome formation is ATG8, which is encoded by nine isoforms in A. thaliana. At the transcript (RT-qPCR) and protein levels (western blot), A. thaliana plants exposed to 5 μM Cd for 24 h in hydroponics show an increased expression of ATG8. Confocal microscopy analyses are currently performed in Cd-stressed GFP-ATG8 reporter lines to further confirm autophagy induction. To investigate the link between autophagy and the redox environment during Cd stress, the expression of all ATG8 isoforms was compared between wild-type and glutathione (GSH)-deficient cad2 plants. Overall, no clear difference could be observed between both genotypes under control conditions. However, an overall lower induction could be observed for Cd-responsive ATG8 isoforms in the leaves of mutant plants following Cd exposure. In contrast, the overall expression level in the roots was rather higher in Cd-exposed mutants compared to wild-type plants, which was mainly attributed to ATG8E. This indicates that the redox environment differentially affects ATG8 expression and potentially autophagy in both plant organs during Cd stress.

Cellular redox signaling is triggered by accumulation of various reactive oxygen species (ROS) that integrate with other signaling cascades to enable plants to ultimately respond to (a)biotic stresses. The identification of key regulators underlying redox signaling networks is therefore of high priority. We improved an mRNA interactome capture method that allows to systematically detect oxidative stress responsive regulators in the post-transcriptional gene regulation (PTGR) pathway. The protocol includes Arabidopsis cell cultures, setup of oxidative stress conditions, short-term exposure to UV irradiation, cell lysis, pull-down and purification of crosslinked messenger ribonucleoproteins, their mass spectrometric analyses and identification of the proteome by statistical analyses. As a result, a comprehensive inventory of the functional oxidative stress responsive RBPome (OxRBPome) is generated. Novel candidate proteins with previously unlinked functions to RNA biology, were further explored to pave the way towards new insights into PTGR processes in redox signaling. The obtained comprehensive view of the RBPome under oxidative stress provides us a more detailed insight and understanding of post-transcriptional regulatory processes in redox signaling in plants.

Global climate change cause various adverse environmental conditions, such as drought, salinity, high temperature, and toxic metal accumulation, which affect plants growth and may affect food security. Rice (Oryza sativa) feeds more than one half of the world’s population and is the model system for monocotyledonous. However, rice is the most salt sensitive cereal crop. To ensure rice production and preserve the biodiversity, in salt-affected soils, is crucial to understand the stress-induced metabolic alterations in tolerant and resistant plants, in order to identify interesting traits for improving plant resilience toward unfavorable environmental conditions. We have investigated on the mechanisms underpinning tolerance towards salinity, focusing on root system of two rice varieties showing contrasting resistance to salt, Baldo and Vialone Nano. Plants have evolved several interconnected molecular pathways to defend themselves against different abiotic stresses. A common theme during plants’ stress responses and adaptation is the production of reactive oxygen species (ROS) and the redox signaling plays a pivotal role in determining plant tolerance and survival to stress. A detailed analysis of the salt stress-dependent modulation of the redox network is here presented. The different phenotypes observed after salt exposure in the two rice varieties is coherent with a differential regulation of cell cycle progression and cell death patterns observed at root level. Baldo showed a highly responsive antioxidative capacity, and different pattern of H2O2 accumulation compared to Vialone Nano. Moreover, glutathione metabolism was analyzed at transcriptional, post-transcriptional and post-translational level in both varieties. These results contribute to highlight the role of ROS and antioxidative pathways as a part of a complex redox network activated in rice toward salt stress.

Tobacco mosaic virus (TMV) replicates in non-host barley at prolonged high temperatures (30 °C), especially if heat-exposed barley is infected by an adapted virus, e.g. Barley stripe mosaic virus (BSMV) (Dodds and Hamilton, 1972, Virology 59, 418). We have shown recently that non-host resistance to TMV in BSMV-infected barley can be also suppressed by heat shock pre-treatments (Király et al., 2021, FESPB-EPSO Plant Biology Europe Congress). In the present study we aimed at elucidating 1/ How heat shock influences non-host resistance of barley to TMV in absence of an adapted virus like BSMV, 2/ Does an early burst of reactive oxygen species (ROS) like superoxide (O2.-) play a role in the non-host resistance of barley to TMV?
In TMV-inoculated barley (cv. Ingrid), heat shock pre-treatments (30 °C for 3 hours, before TMV inoculation; 49 °C for 20 seconds, at 2 hours before TMV inoculation) resulted in significantly (50-100%) higher TMV accumulation, compared to non-pretreated controls kept at constant 20 °C (assayed by RT-qPCR). It seems that heat shock may impair non-host resistance of barley to TMV even in absence of an adapted virus like BSMV. Expression of a NADPH oxidase gene (HvRBOHF2) governing superoxide production and host disease resistance, and HvSOD1 (superoxide dismutase) and HvBI-1 (BAX-inhibitor), genes encoding for an antioxidant and a cell death regulator, respectively, displayed an inverse correlation with TMV levels, even at relatively late time points (1 to 7 DAI). This implies a pivotal role of these defense-related genes in maintaining non-host resistance to TMV. Furthermore, we found that an early (2 HAI) burst of superoxide is essentially absent in TMV-infected barley exposed to heat shock pre-treatments (assayed by nitro blue tetrazolium chloride /NBT/ staining), pointing to the role of ROS (superoxide) in non-host resistance of barley to TMV.

Acknowledgement: A grant from NKFIH (K128868)

It has been reported that hydrogen peroxide is generated in such beverages as tea and coffee, due to autoxidation of polyphenols present in the extracts. As polyphenols are present also in other beverages and food of plant origin, this study was aimed at checking whether H2O2 can be present in other commonly consumed products. We estimated the formation of hydrogen peroxide in wine, herbal infusions and cooked vegetables using the Xylenol Orange method, enhancing its specificity by assaying two parallel samples, one of them preincubated with catalase.
Variable low concentrations of hydrogen peroxide were found to accumulate in freshly opened red wines during 3-h incubation, from 0 to 6.6 µM, depending on the type and batch. Higher concentrations accumulated during 3-day storage of opened wines (up to ca 30 µM and ca 20 µM in a red wine and a white wine studied, respectively). Like in the tea, hydrogen peroxide was generated in infusions of medicinal herbs. From among 16 herbs studied, the highest concentrations of H2O2 were found in fresh 1% (w/v) infusions of the birch Betula pendula leaves (34.3±3.9 µM), of inflorescence of the lime Tilia cordata (25.9±1.2 µM) and of leaves of the plantain Plantago lanceolata (22.3±0.6 µM). Hydrogen peroxide was also found in the homogenates of cooked vegetables. The highest concentration of hydrogen peroxide in 1:2 (w/v) homogenates was found for the broad bean (73.4±9.0 µM) followed by broccoli (18.6±0.3 µM), onion (10.4±1.6 µM) and leek (10.0±0.3 µM) at pH of about 7; H2O2 concentrations were lower at lowered pH.
These results show that our nutritional exposure to hydrogen peroxide may be higher than estimated to date. These small concentrations of H2O2 do not seem to have deleterious health effects and may be even beneficial due to their bactericidal and virucidal action.

In eukaryotes, poly(A) binding proteins (PABPs) bind to the poly(A) tail of transcripts and thereby influence the RNA stability, nucleo-cytoplasmic transport and translation. Three members of PABPs (PABP2, 4 and 8) in Arabidopsis, which form the class II PABPs, play various roles in plant growth and development as loss-of-function double mutants (pabp2/4, pabp2/8 and pabp4/8) exhibit phenotypic abnormalities in leaf shape, flowering time and plant height [1]. In previous redox proteomics studies, we charted the sulfenomic landscapes in Arabidopsis. There, we demonstrated that upon H2O2 stimulation of cells, class II PABPs underwent thiol-dependent oxidative post-translational modifications on Cysteine (Cys) residues, which reside in their RNA recognition motif (RRM) domains [2, 3]. Therefore, we hypothesized that Cys oxidation might affect the RNA binding activity of class II PABP. First, by means of Electrophoretic Mobility Shift Assays (EMSA) with recombinantly expressed proteins and biotinylated poly(A), we found that the poly(A) binding capacity of class II PABP proteins decreased under elevated H2O2 concentrations. Mutated isoforms in which the Cys residues were replaced with Serine residues, were insensitive to H2O2 dose-dependent decrease of poly(A) binding activity. In order to functionally characterize the thiol-based redox regulation of Class II PABPs, we are currently generating transgenic plants in which both wildtype and Cys mutant PABPs are ectopically expressed into double mutants (pabp2/4 and pabp2/8) for future phenotypic analysis under normal growth and oxidative stress conditions.

References
1. Gallie, D.R., Class II members of the poly(A) binding protein family exhibit distinct functions during Arabidopsis growth and development. Translation, 2017. 5.
2. Huang, J., et al., Mining for protein S-sulfenylation in Arabidopsis uncovers redox-sensitive sites. Proc Natl Acad Sci U S A, 2019. 116.
3. Wei, B., et al., Identification of Sulfenylated Cysteines in Arabidopsis thaliana Proteins Using a Disulfide-Linked Peptide Reporter. Frontiers in plant science, 2020.11

The alterations induced by the toxicity of thallium (Tl) in the roots and leaves of Dittrichia viscosa plants were determined. The plants were grown hydroponically with different concentrations of Tl (0, 10, 50 and 100 µM) during 7 days, a metal which reduces biomass production and growth, and the relative water content decrease. The leaves shown venal chlorosis. Tl is accumulated mostly in the roots, with the concentrations in the leaves being much lower. ICP-MS analysis showed that the K content decreased both roots and leaves. Fe, Cu, Mn concentrations was altered.
Chlorophyll a and b content declined, and the ratio chl a/chl b increased, but the carotenoid content increase (x2.2). The photosynthetic efficiency decrease for higher Tl concentrations (0.779, 0.731, 0.547 and 0.522 for control, 10 µM, 50 and 100 µM Tl, respectively). These results would indicate that Tl causes an alteration of the photosynthetic apparatus, probably by increasing ROS despite the increase in carotenoids.
Increases were observed in the amount of lipid peroxidation in roots, but not in leaves. The O2.-, H2O2, NO increase, specially in roots. The H2S content increases in leaves, but in roots only with the 10 µM Tl. The induced oxidative stress leads to a strong increase in the superoxide dismutase (SOD).
Tl induced alterations in ascorbate homeostasis, the AsA increase and DHA decrease. The ascorbate pool only decrease with 100 µM Tl. However, the glutation pool decrease depending of Tl concentrations. The GSH content decrease and almost all glutathione is GSH.
In summary, in Dittrichia the Tl accumulates more in roots than in leaves. Tl toxicity induces oxidative stress, with redox homeostasis imbalance. These alterations are greater in roots than in leaves, where the antioxidant system is more efficient.

Acknowledgments: This study was made possible thanks to the Junta de Extremadura/FEDER (GR21112).

H2S has acquired great attention in plant research because it has signaling functions under physiological and stress conditions. However, the direct detection of endogenous H2S and its potential emission is still a challenge in higher plants. Using the fresh extract of different plant species with agronomical interest including pepper fruits, broccoli, ginger, and different members of the genus Allium including garlic, leek, welsh and purple onion, it was determined the endogenous H2S and its emission using an ion-selective microelectrode and a specific gas detector, respectively. The data show that endogenous H2S content range from pmol to μmol H2S · g-1 fresh weight whereas the H2S emission of fresh-cut vegetables was only detected in the different species of the genus Allium with a maximum of 9 ppm in garlic cloves. Additionally, it was characterized the activity and isozymes of the L-cysteine desulfhydrase (LCD), which is one of the main enzymatic sources of H2S, being the different species of the genus Allium which showed the highest activities. Using non-denaturing gel electrophoresis, the data indicated the presence of up to 9 isoenzymes different LCD isozymes from one in ginger to four in onion, leek, and broccoli. In summary, the data indicate a correlation between higher LCD activity with the endogenous H2S content and its emission in the analyzed horticultural species.
[Supported by a European Regional Development Fund cofinanced grants from the Ministry of Science and Innovation (PID2019-103924GB-I00) and Junta de Andalucía (P18-FR-1359), Spain]

Given the current Climate Change scenario, a deeper understanding of abiotic stress resistance in crops is a priority towards securing food and feed supplies. Plants have evolved diverse strategies to avoid and/or cope with the increasingly frequent drought and flooding events and the various stresses associated with them, namely oxidative stress.
Studying reactive oxygen species (ROS) homeostasis within the context of water stress in plants is important, not only because antioxidant defense plays a central role in preventing the impacts of oxidative stress, but also, because complex and highly articulate signal transduction networks involving ROS are required for sensing and adapting to new environmental conjectures.
The legume Lathyrus sativus L. (grass pea) is of high economic importance for food and feed in Asian and African developing countries. Interest has been raising also in the Mediterranean region, where this crop is part of cultural heritage of more marginal areas, due to its outstanding robustness under adverse environmental conditions, like salt, temperature, and water stress, compared with other legume species.
The aim of this work is to unravel some of the molecular mechanisms underlying grass pea’ water and oxidative stress response by comparing tolerant and susceptible grass pea accessions, previously identified by an extensive phenotyping of a worldwide collection of germplasm of this species, evaluated under three water treatments (well-watered, waterlogging and water deficit).
Some preliminary metabolite quantification results are presented, namely of glutathione and ascorbate oxidized and reduced forms, as an attempt to unravel contrasting redox conjectures among susceptible and tolerant plants, subject to different water treatments.
Future biochemical and transcriptomic studies will deepen the understanding of the potential role of ROS in response mechanisms to water deficit and waterlogging in grass pea.

Plant cells sense fluctuations in the levels of cellular polyamines (PAs), namely, putrescine (Put), spermidine (Spd) and spermine (Spm). PA cellular titer regulates biological processes, including senescence. Changes in PA concentration in properly functioning cells are unlikely. Induced senescence disturbs their homeostasis which leads to senescence-dependent metabolic changes. PA catabolism was proposed as process promoting mainly by H2O2 production. Here, we show that darkness-induced plant senescence is associated with changes in the PA levels together with the changes of signaling molecules such as nitric oxide (NO) and hydrogen peroxide (H2O2). Upon blocking Put catabolism, the senescing cells generated lesser amounts of NO and more of H2O2 and, in turn, accelerated senescence. We opine that a possible linkage exists between PAs and signaling molecules such as NO and H2O2. Thus, the balance in the triad network made of NO↔PA↔H2O2 signaling may be involved in re-programming metabolism and regulate the direction in which a plant may be ushered into - growth, senescence or cell death.

This work was supported by Polish National Science Centre in the frame of the project 2017/27/N/NZ9/02135 (EPL)

The study presents new insights into the role of nitric oxide metabolism in senescing Arabidopsis leaf defined as the dynamics of the nitration phenomenon. Detection of reactive oxygen and nitrogen species in dark-induced leaf senescence (DILS) model allows us to confirm that senescence-like phenomena are accompanied by a gradual decrease in NO emission and indicated a transient wave of peroxynitrite (ONOO–) formation on day 3 of DILS. To assess the metabolic fate of the time-dependent ONOO– bioavailability in individually darkened leaves (leaf 7 of the rosette), nitration levels at protein and nucleic acids level were determined on the following days of DILS (from day 1 to day 7). As evidenced by immunoassay the boosted ONOO– formation did not promote protein tryptophan nitration and progress of senescence depleted the pool of nitrotryptophan containing proteins. On the contrary, nitration of tyrosine containing proteins was intensified twice on day 3 of DILS overlapping with nitrating agent overaccumulation. Also, the abundance of 8-nitroguanine, a marker of nitrative modification of RNA and DNA, increased significantly on days 3 and 7 of DILS, respectively. Taken together ONOO– could be considered as a novel positive regulator of DILS fine-tuning redox environment to selective nitrative modification of biotatrgets such as tyrosine containing proteins and RNA.

This work was supported by Polish National Science Centre in the frame of the project UMO-2017/26/E/NZ4/00226

Phytophthora infestans (Mont.) de Bary is one of the most important plant pathogens in agriculture. The presence of a large number of acetyltransferase and deacetylase orthologs in the genome of Phytophthora species suggests that lysine acetylation of various proteins may be crucial in these fungal-like organisms. The pathogen is also able to synthesize nitric oxide, a signaling molecule that could be engaged in molecular reprogramming via changes in the histone acetylation status.
To gain insight into the acetylation status in the phytopathogen structures we performed the experiments on the avirulent (avr MP946) and virulent (vr MP977) P. infestans isolates in reference to the potato (Solanum tuberosum L.) cv. Sarpo Mira. In order to verify whether and to what extent reactive nitrogen species (RNS) affect nuclear histone deacetylases (HDACs) and acetyltransferases (HATs) at transcript and enzyme activity levels, the pathogen was treated with specific donors to mimic nitrosative stress conditions to which the pathogen is exposed during in planta growth.
The results indicated a RNS-dependent induction of HDACs activity at 2nd h after donors application; however, it reduced starting from 24h after S-nitrosoglutathione and SIN-1 (3-morpholino-sydnonimine) treatments. Among the 5 analyzed genes encoding nuclear HDACs, RNS provoked a significant increase of the transcript accumulation for HDAC1, HDAC3, and HDAC5. Analyses of HATs activity showed the strongest enzyme induction at 48h after donors treatment. Among the 13 analyzed genes encoding HATs, 6 showed altered expression via RNS. Moreover, different patterns of the gene expression were observed in both isolates.
Summing up, nitrosative stress modified HDACs and HATs at transcript and enzyme activity levels in both P. infestans isolates. Thus, the interaction of RNS-HDACs/HATs can be crucial in controlling the expression of a plethora of P. infestans genes. This research was funded by National Science Centre – project no. UMO-2018/31/B/NZ9/00355.

In recent years, the development of fluorescent probes has revolutionized our experimental access to physiological parameters in live cells. Genetically-encoded-probes based on redox-sensitive yellow fluorescent protein (rxYFP) and green fluorescent proteins (roGFPs), allow real-time monitoring of thiol redox dynamics, combined with the option of precise targeting to specific subcellular locations in any cellular systems or organisms including plants. For instance, the redox-sensitive GFP (roGFP2) has been fused to glutaredoxins and thiol peroxidases to allow dynamic imaging of the glutathione redox potential (EGSH) or H2O2, respectively.
Cysteine is an essential metabolite, that is required for protein synthesis but also for the production of many important sulfur-containing molecules, for the biosynthesis of cofactors such as iron-sulfur centers or molybdenum cofactors but also for the biogenesis of hydrogen sulfide. To broaden our understanding of redox biology in a cellular and physiological context, biosensors able to detect cysteine levels and its degradation/derived products are needed.
In principle, any protein domain with a catalytic cysteine undergoing a specific and reversible oxidative modification may oxidize roGFP2 unless this is sterically/structurally hampered. Cysteine desulfurases (CDs) and sulfurtransferases (STRs) catalyze the transfer of a sulfur atom from sulfur donors to nucleophilic sulfur acceptors by forming an intermediate persulfide on a single reactive catalytic cysteine. Thus, they represent good candidates to establish genetically-encoded probes as they seem perfectly suited to transfer an efficient and selective oxidation of roGFP2. In this work, we first confirmed the ability of a natural CD-STR fusion protein to oxidize the roGFP2 in the presence of cysteine. Then, using in vitro fluorescence assays, we investigated the specificity, sensitivity and reduction (once oxidized) of a CD-STR-roGFP2 fusion. These results prompted us to express the CD-STR-roGFP2 fusion in Escherichia coli, Saccharomyces cerevisiae and Arabidopsis thaliana to perfom in vivo measurements.

Due to climate change, plant tolerance to environmental stresses is an important objective of breeding efforts. Combination of extreme conditions such as drought and high temperature can affect plants more than individual stresses. Our previous research revealed that the mitochondrial pentatricopeptide (PPR) domain protein 40 (PPR40), connects mitochondrial electron transport and of stress responses by influencing redox balance and ROS signaling in Arabidopsis thaliana (Zsigmond et al., 2008, Plant Physiol. 146:1721-1737). In a subsequent study influence of PPR40 on drought tolerance was characterized with T-DNA insertion mutants ppr40-1, ppr40-2 and PPR40 overexpression plants. Plant growth and survival rates as well as stress-related physiological parameters (chlorophyll fluorescence changes, relative water content, ROS accumulation and oxidative damage and proline levels) were analysed in plants subjected to drought, heat stresses and their combinations. Physiological studies were completed by complex phenotyping of the ppr40 mutants. Our results revealed that T-DNA insertion in the PPR40 gene could enhance drought tolerance by reducing water loss, stabilizing photosynthetic electron transport rates and improving viability of the mutants. Our results suggest that genes implicated in mitochondrial electron transport can be potential candidates for engineering drought tolerance in crop plants.
This research was supported by NKFI FK-128920.

Ectomycorrhizal fungi (ECM) are an important group of organisms that ensure that trees can flourish in all types of environmental conditions. By providing nutrients, water and protection against stress conditions in exchange for sugars, they allow their host trees to grow. The stress response of plants has already been studied in detail. However, stress response signalling remains poorly understood in ECM fungi. In this study, we assess the potential of H2O2 to regulate a putative transcription factor, LbAP1, using a bio-informatic approach, and localisation of heterologously expressed eGFP-fused LbAP1 upon H2O2 treatment. LbAP1 is a homolog of YAP1, a key regulator of ROS responses in Saccharomyces cerevisiae. Oxidation of two cysteines in YAP1, located in the N- and C-terminal CRD, results in nuclear accumulation through masking of the NES by disulfide bond formation and transcriptional activation of target genes. In contrast to YAP1, results obtained by fluorescence microscopy showed that an LbAP1-eGFP fusion protein did not accumulate in the nucleus upon treatment of transformed Saccharomyces cerevisiae BY4741 with 0.4 mM H2O2. As C-terminal eGFP could be interfering with disulfide bond formation, the H2O2 sensitivity of eGFP-LbAP1 is currently being examined. Upon analysis of both protein sequence and structure of YAP1 and LbAP1, it was revealed that LbAP1 contains 2 Cys less than YAP1. Furthermore, the Cys responsible for nuclear localisation upon oxidation in YAP1 are not conserved in LbAP1. The NLS and NES, however, are partially conserved. Structural predictions of LbAP1 also showed that the helix structures containing these important cysteines are not conserved, possibly indicating that LbAP1 nuclear localisation is not H2O2 dependent. From these results, we can conclude that LbAP1 regulation might be H2O2 independent, and that the cellular mechanisms of ECM fungi should be investigated separately to other fungi.

Chestnut (Castanea sativa Miller) is an important species throughout Europe, particularly in Portugal. However, it is highly sensitive to episodes of high temperatures and water scarcity scenarios that are increasingly common due to climate change. Given the need and importance of understanding the impact of these stress factors on the physiology of C. sativa, this study focused on the evaluation of the response of young chestnut plants (3 months old) to the combination of drought and heat (4 h/d 42 ºC). The exposure to high temperatures did not substantially affect the physiological performance of these plants, as no major impacts were found on growth and oxidative metabolism. On the other hand, after 21 days of exposure to drought, individually and in combination, this condition was the one that most affected plant growth. This effect was accompanied, in the case of individual stress exposure, by a decrease in the production of reactive oxygen species, namely superoxide anion (O2.-) and hydrogen peroxide (H2O2), as well as by an increase in lipid peroxidation and proline levels. A prevalence of the effect of drought in the co-exposure treatment was observed, although the plants subjected to the combination of stresses showed an increase in carotenoids and O2.- contents. Ongoing studies are focused on the evaluation of the photosynthetic and antioxidant response of C. sativa to these abiotic stresses ensure a holistic view on the impact of climate change on chestnut plants. Additionally, and recognizing the imperative need to develop new effective and eco-friendly approaches to overcome the harmful consequences of these unfavorable conditions, studies are also underway to assess the potential of mycorrhization as a strategy to mitigate the effects of climate change on this species.

Currently, climate change is affecting crop production worldwide. For instance, in the Mediterranean basin, comprising the most important European producers and exporters of tomato, productivity has been rapidly declining, mostly due to the increased temperatures and soil salinization, two conditions that frequently occur together in the environment. Indeed, recent studies carried out by our team showed that the combination of these two stressors affects tomato plants more severely than the sum of the individual effects. In this sense, it becomes highly important to develop and establish sustainable strategies to improve crop growth under this scenario of climate instability. Thus, the goal of this study was to assess how the exogenous application of two phytohormones [brassinosteroids (BRs; 1 µM 24-epibrassinolide) and strigolactones (SLs; 5 µM GR24)] and a beneficial element (silicon – Si; 2 mM) can modulate the response of tomato plants (Solanum lycopersicum L. var. cerasiforme) to heat and salinity. After 7 d of acclimation to growth chamber conditions, salt stress was applied through NaCl irrigation (100 mM) every alternate day during 28 d, with heat stress being applied in the last 21 d, through exposure to 42 ºC for 4 h every other day. SLs, BRs, and Si treatments were given individually as a foliar spray, twice per week, throughout the experiment. After the growth period, the obtained data indicated that regardless of the applied compound, the macroscopic effects observed in the stress situation were maintained, as well as those related to the oxidative (hydrogen peroxide and lipid peroxidation) and photosynthetic (chlorophyll and carotenoids) metabolism. However, it is important to highlight that there was a small tendency for higher biomass in all sprayed plants, as well as increased glutathione and ascorbate, and reduced proline levels in the case of plants treated with SLs, suggesting the activation of protective pathways.

While nanomaterials (NMs) offer wide-ranging solutions, their intensified use has been resulting in the contamination of the environment, posing ecotoxicological risks to diverse organisms, including plants. In this sense, it becomes important not only to understand the phytotoxicity of NMs, but also to find efficient and sustainable strategies to enhance plant tolerance to these emergent contaminants. Thus, this study aimed at assessing the potential of ascorbic acid (AsA), a powerful antioxidant (AOX), in enhancing the tolerance of in vitro grown tomato seedlings to nickel oxide NMs (nano-NiO). For this purpose, seeds of Solanum lycopersicum L. cv. Micro-Tom were germinated in half-strength MS medium supplemented with 30 mg L-1 nano-NiO alone or in combination with 150 mg L-1 AsA. A control situation, without nano-NiO nor AsA, was also included. After 28 days of exposure, the growth and development of S. lycopersicum was severely repressed by nano-NiO, with evident phytotoxicity symptoms (leaf chlorosis and necrosis), that did not translate, however, into severe redox disorders. Even so, proline levels, SOD and DHAR activities were diminished in shoots of nano-NiO-exposed plants, while glutathione, phenols, CAT and GR activities were increased. In response to the co-exposure to AsA, nano-NiO-induced growth inhibition was efficiently counteracted, being accompanied by a more notorious response of the AOX network, especially of glutathione, phenolics, SOD and GR. Surprisingly, the solo AsA administration in the culture medium suppressed growth and development of tomato seedlings, increased the lipid peroxidation of membranes and inhibited key enzymes of the AsA-glutathione cycle, in spite of enhancing the total AOX capacity, with increased levels of proline, phenols and glutathione. Overall, exposure to nano-NiO negatively impacted tomato seedlings’ growth, and co-application of exogenous AsA had stress-ameliorative effects by enhancing the AOX response to counteract nano-NiO-induced growth inhibitory effects.

Hydrogen peroxide (H2O2) is a major reactive oxygen species (ROS) whose level must be carefully controlled to allow optimal cellular signaling. One important plant antioxidant enzyme that removes excess H2O2 is ascorbate peroxidase (APX), which reduces this reactive molecule to water. Ascorbate peroxidase requires ascorbate and, in reducing H2O2, produces monodehydroascorbate (MDHA), which must be regenerated to ascorbate by reductases.

One of the regenerating enzymes is MDHA reductase, which converts MDHA directly to ascorbate.
In Arabidopsis, several MDHAR isoforms exist and they are found in different subcellular compartments. While there are two peroxisomal isoforms (MDHAR1 and MDHAR4), MDHAR2 and MDHAR3 are found in the cytosol, and dual targeting of MDHAR5/6 allows expression in mitochondria and chloroplasts, respectively. It is widely thought that MDHAR plays an important role in the response of plants to oxidative stress by maintaining the redox state of intracellular ascorbate, but few specific detailed studies of the different isoforms have been conducted.

We are performing a detailed comparison of the different MDHAR isoforms. To establish how these enzymes work within the cellular redox network, a comparison of their affinities for NADH and NADPH is being conducted using recombinant enzymes while a reverse genetics approach using specific mutants aims at assigning specific biological functions to each gene. To this end, single and double mutant lines are being produced. Questions we aim to answer include whether the different MDHARs show specificity in biochemical properties as well as the importance of each for the functioning of plants under optimal and stress conditions.

Plant-parasitic nematodes produce effectors to overcome plant immunity and finetune plant cellular processes. Molecular mechanisms of how effector proteins co-opt plant processes, especially plant immunity to support nematode survival, have been intensively investigated, but they are still poorly understood. Identifying protein-protein interactions is crucial for understanding this cross-kingdom network.
Using the high throughput screening technique Y2H-seq, we have identified tomato proteins involved in various cellular processes interacting with M. javanica effectors. Among those, Mj-NEROSS (Nematodes effector involved in ROS suppression, previously referred to as 4D01 or Msp3) was found to interact with a Heavy metal transport/detoxification superfamily protein and a Rieske iron-sulphur protein (ISP), one of the putative subunits of the cytochrome b6f complex. Furthermore, we confirmed direct interaction in planta and showed that ISP is a conserved dicot target of Mj-NEROSS.
We showed that the plastid localization of Mj-NEROSS plays a crucial role in its interaction with ISP. Once this interaction has been established, a significant decrease in electron transport rate and subsequently in the host's reactive oxygen species production is observed. Furthermore, we reveal that the presence of the effector in the plastids leads to changes in genes expression. Importantly, we show that the differentially expressed genes (DEGs) are involved in ROS production, protein folding recognition, and upregulation of oxidative phosphorylation.
We hypothesized that DEGs are most likely subsequent consequences of interaction of Mj-NEROSS with ISP and biochemical communication between plastids and the nucleus, leading to suppression of plant basal defense and attenuation of host resistance to the nematode infection.

Our functional analysis of MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) for hybrid aspen (Populus tremula × tremuloides) grown under natural field conditions for several seasons provided evidence of the role of MPK4 in the genetic and environmental regulation of stomatal formation, differentiation, signalling, and function; control of the photosynthetic and thermal status of leaves; and growth and acclimation responses. Divergence between absorbed energy and assimilated energy is a bottleneck, and MPK4 can participate in the control of energy dissipation (thermal effects). Furthermore, MPK4 can participate in balancing the photosynthetic energy distribution via its effective use in growth or redirection to acclimation/defence responses.
The vast majority of chloroplast proteins are nuclear-encoded, and must be imported into the organelle after synthesis in the cytoplasm. This import is essential for the development of fully functional chloroplasts. On the other hand, functional chloroplasts act as sensors of environmental changes and can trigger acclimatory responses (e.g. SAA and SAR) that influence nuclear gene expression. Signalling via mobile transcription factors (TFs) has been recently recognized as a way of communication between organelles and the nucleus. In this study, we performed a targeted reverse genetic screen to identify dual-localized TFs involved in chloroplast retrograde signalling during stress responses. We found that CHLOROPLAST IMPORT APPARATUS 2 (CIA2) has a functional plastid transit peptide, and can be located both in chloroplasts and the nucleus. Further, we found that CIA2, along with its homologue CIA2-like (CIL) are involved in the regulation of Arabidopsis responses to UV-AB, high light and heat shock. Finally, our results suggest that both CIA2 and CIL are crucial for chloroplast translation. Our results contribute to a deeper understanding of signalling events in the chloroplast-nucleus cross-talk.
1. Witoń et al., Plant Phys. 2021, 186: 2190-2204; 2. Gawroński et al., Plant J. 2021 105: 619–638.

Reactive Oxygen Species (ROS) are compounds derived from oxygen with important implications in biological processes in plants, some of them related to reproduction. Among ROS, superoxide is the primary oxidant, since an array of other ROS are eventually derived from this anion. There-fore, analysis of the molecular systems able to generate this molecule and the cellular compart-mentalization of these events is of paramount importance. We have used the fluorochrome DCFH2-DA and the chromogenic substrate NBT in association with DPI (a specific inhibitor of Rboh enzymes generating superoxide in plants) in combination with confocal microscopy and stereomicroscopy, respectively to identify cell localization of ROS in general, and superoxide ac-cumulation in olive reproductive tissues. A significant production of both ROS and superoxide has been described, showing a fairly precise spatial and temporal location throughout olive flower development. The reduction of the NBT signal after the addition of DPI suggests that the generation of superoxide is largely due to Rboh or other flavin oxidase activity. At the subcellular level, accumulation of O2.- has been located in the plasma membrane of mature pollen and germi-nated pollen, as well as in the rough endoplasmic reticulum and in mitochondria.
This research was funded by research projects BFU-2016-77243-P, PID2020-113324GB-100 and STED202100X129616SV0 of the Spanish Ministry of Science, Innovation and Universities (MICIIN)/ State Research Agency (AGE)/ European Regional Development Fund (ERDF)/ European Union (EU).

The involvement of NADPH oxidases in the regulation of K and Cd flux and photosynthesis was assessed in cadmium-challenged wild type and three RBOHs (Respiratory Burst Oxidase Homologues) mutants of Arabidopsis plants (AtrbohC, AtrbohD, and AtrbohF) using different approaches. Plants were grown under hydroponic conditions supplemented or not with 50 µM for 24 h. Results showed that Cd differentially affect photosynthesis and trasnpiration in WT and Atrbohs mutants. Electrophysiological experiments revealed that knocking out RBOHs induced a more Cd2+ influx compared to WT with differential trend in mature and elongation zone, being observed in the Atrboh mutants. Under Cd stress the three Atrbohs mutants had a more ability to retain K+ in the elongation root zone compared to WT, while in leaves, a dramatic reduction in K+ influx was observed in Atrbohs mutants and a change from efflux to intensive influx was registered in WT. The analysis of expression of several K+ and Cd2+ genes transporters and transcription factors suggest that RBOH-dependent H2O2 regulates ion homeostasis and Cd in a highly complex process involving multilevel regulation from transpirational water flow to transcriptional and posttranslational modifications of K/metals transporters.

This work was supported by FEDER Funds (A1123060E00013), Junta deAndalucía I+D+I grants Ref PY20_00364, and Spanish Ministry of Science and Innovation Ref PGC2018-098372-B-I00.