Vitamin E (alpha-tocopherol, VE) is essential to prevent severe neurological symptoms and even death of a genetic form of ataxia associated with vitamin deficiency or AVED. Its essentiality is also proven in secondary deficiencies associated with malnutrition and/or malabsorption syndromes that besides moderate to severe neurological abnormalities can contribute to induce metabolic, musculoskeletal, hematological, and immune dysfunctions, especially in the elderly. VE is the most abundant and ubiquitous fat-soluble nutrient with hydrogen atom donating properties (often described as “antioxidant”) of the plasmalemma; its relative abundance with respect to phospholipid residues is by far the highest among other H donors and its membrane levels influence the flux of lipoperoxyl radicals during both enzymatic and non-enzymatic processes of lipid peroxidation. Consequently, VE directly affects the metabolism and function of membrane fatty acids, also playing a key role in lipid signaling and thus in the indirect control of different enzymes, signal transduction, and transcriptional proteins that connect, under a functional point of view, the VE levels in human tissues with many pathophysiological aspects and deficiency symptoms. Recent evidence strongly supports the participation of the long-chain metabolites of VE in at least some of its “non-antioxidant” properties. Altogether these aspects depict the biological complexity of this vitamin which is far from being comprehensively understood. Last-generation omics technologies make it possible to face such a complexity to represent with unprecedented efficacy both the essentiality aspects and the health-promoting potential of this vitamin in human nutrition studies and clinical trials on deficiency syndromes and other human diseases that may benefit from its biological properties. Transcriptomics and especially metabolomics protocols have been utilized in our laboratories, either separate or in multiomics mode, to develop personalized and precision nutrition (i.e. nutrigenomics) platforms of investigation dedicated to this vitamin, and examples of their potential for innovation in VE research will be given in this presentation, including in vitro studies and clinical trials on hepatic fatty acid metabolism and lipotoxicity, the etiologic factor of non-alcoholic fatty liver disease, and studies in kidney disease patients that develop secondary VE deficiency in the context of severe oxidative stress and lipid peroxidation symptoms.

The physicochemical properties of nitric oxide (NO) as an intercellular messenger, in particular the way it conveys information via volume signaling, translate into advantages of communication in the brain. This becomes apparent when considering neurovascular coupling (NVC), the tightly temporal and spatial functional communication between active neurons and local blood microvessels. That the brain is energetically expensive given its mass and that increased neuronal activity in a region of the brain is associated with a local increase in blood flow (CBF) has been known since the XIX century. In turn, the association between CBF dysregulation and cognitive decline has been consistently established in older adults (brain aging, neurodegenerative diseases, type II DM) and lab rodent models but the neurobiological links are poorly understood. I will discuss the notion that neuronal-derived NO is the key mediator of NVC in the hippocampus and that impairment of NVC is an early and likely causative event leading to cognitive decline. The premise is that by rescuing the functionality of NVC then cognitive enhancement should be observed. This will be experimentally supported on basis of a diet-driven redox mechanism, involving the interaction of nitrite with ascorbate released from active neurons. Data suggest that an operational NVC, allocating energy resources according to neuronal activity, is a most fundamental biochemical process that underlines biological organization to support cognition.   

Supported by project 2022.05454.PTDC (https://doi.org/10.54499/2022.05454.PTDC).

The human skin, being our outermost protective barrier, sustains continuous contact with the environment. As such, its cells must be kept in a state of constant alert against external increased oxidative stress and massive environmental insults (e.g. sunlight and UV radiation, air pollution, and mechanical stress). All these insults ultimately result in an impaired redox balance and increased cellular oxidation. One of the pivotal oxidation regulation mechanisms in the skin is the Nrf2–Keap1 pathway, and its activity leads to cutaneous redox maintenance which evidently sustains the principle of hormesis. We suggest that moderate environmental stressors and skin microbiome can provide the necessary continuous stimuli for the activation of the Nrf2 pathway. We also suggest that endogenous neurotransmitters play a major role in this activation.

A deep understanding of the roles of redox metabolites and pathways in physiology and pathology requires molecular tools that enable both visualization of these processes and their selective modulation. Over the last two decades, a number of genetically encoded fluorescent biosensors for key redox metabolites have been developed, allowing real-time detection in living systems of varying complexity. Recent developments in this area include the ultrasensitive probe HyPer7 and a new fluorogenic probe, HyPerFAST, which enables even more sensitive H2O2 detection across any chosen optical range, from blue to near-infrared. Complementary to imaging with biosensors, chemogenetics offers tunable substrate-dependent modulation of metabolic pathways, allowing the study of normal cell functioning and modeling dysfunctions caused by abnormal pathway activity and/or metabolite levels. We will present recent developments in this area that include insights on oxidative stress brought about by the use of D-amino acid oxidase (DAO) and intriguing details of the Warburg effect brought about by a new mitochondrial "booster," Grubraw, based on bacterial D-amino acid dehydrogenase.

Mitochondrial diseases, characterized by dysfunction in the cellular powerhouse, the mitochondria, present a complex and heterogeneous group of disorders. As research in mitochondrial medicine advances, the need for effective therapies becomes increasingly apparent. Collaborative efforts among researchers, clinicians, regulatory bodies, patient advocacy groups and other stakeholders are crucial to overcome the challenges linked to the complexity of mitochondrial medicine, and to ensure the successful implementation of clinical trials in this field. This lecture explores the key aspects of trial readiness in the context of mitochondrial medicine, emphasizing the challenges and opportunities in designing and executing successful clinical trials. An overview of the ongoing clinical trials will be also provided.

The reduction of intracellular cystine to yield cysteine is critical for protein or glutathione synthesis and many other important biological processes, but its regulation is still unknown. We have shown that the thioredoxin-related protein of 14 kDa (TRP14) is the rate-limiting enzyme for intracellular cystine reduction. Upon TRP14 deficiency, cysteine synthesis through the transsulfuration pathway becomes the major source of cysteine in human cells, and knockout of both pathways is lethal in C. elegans subjected to proteotoxic stress. TRP14 can also reduce protein cysteinylation. However, paradoxically TRP14 knock-out mice were protected in acute pancreatitis through activation of Nrf2 and upregulation of the transsulfuration pathway, thus exhibiting less inflammatory infiltrate and edema. Therefore, TRP14 seems to be the enzyme principally responsible for intracellular cystine reduction, and it is also able to regulate protein cysteinylation together with thioredoxin 1.

Oxysterols are oxidized derivatives of cholesterol initially considered as simple metabolic byproducts, nowadays recognized to play significant roles in various biological and pathological processes. In physiology, they are involved in the regulation of cellular processes beyond cholesterol metabolism, influencing cell proliferation, differentiation, apoptosis, and inflammation through various signaling pathways. In medicine, the study of oxysterols holds promise for understanding and treating various diseases, particularly those associated with dysregulated cholesterol metabolism and inflammation. Indeed, some oxysterols have been associated with adverse health effects, including cytotoxicity, pro-inflammatory effects, and potential contributions to the development of chronic diseases. Dysfunctions in oxysterol metabolism have been implicated in the pathogenesis of cardiovascular diseases, neurodegenerative disorders, and certain cancers. Targeting oxysterol pathways could therefore offer novel therapeutic strategies for these conditions. Oxysterols have potential applications in the pharmaceutical and biotechnology industries. Those generated by cholesterol autoxidation can be used as biomarkers for assessing oxidative stress conditions. Additionally, defined oxysterols of enzymatic origin and/or synthetic oxysterol analogs might be developed as antiviral agents. Oxysterols generated through autoxidation processes can serve as markers of lipid oxidation in cholesterol-containing foods and their quantification can help assess the quality and shelf life of food products, and also for ensuring food safety and consumer health. Finally, with regard to skin health and cosmetics industry, prolonged or excessive exposure to and/or formation of certain toxic oxysterols could potentially damage skin cells and disrupt skin barrier function. Therefore, careful formulation and dosage control are essential to ensure the safety of skincare products. Overall, the study of oxysterols spans molecular biology, medicine, and industry, with implications for understanding fundamental biological processes, developing new medical, industrial, and advancing biotechnological applications.

The selenoperoxidase GPx4, discovered in 1982, plays a pivotal role in preventing ferroptosis. In a moonlighting function, GPx4, in its mitochondrial and nuclear forms, also contributes to spermatogenesis. The critical advantage of Selenium vs. Sulfur catalysis is the stability of the oxidized form of the chalcogen in the catalytic cycle. While the mechanisms of catalytic cycle are understood, its regulation remains largely unknown. Existing evidence supports the notion that ferroptosis is activated when GPx4 is inhibited, glutathione (GSH) concentration is lowered, or the labile iron pool is expanded. The outcome is framed in the context of oxygen toxicity playing the physiological function of controlling cell death. GPx4 stands out as the sole peroxidase indispensable to aerobic life. Moreover, a recent study exploring the role of the residue Arg152 in GPx4, linked to a fatal although not embryonically lethal disease, revealed that the wild-type enzyme exhibits surface-sensing and positive cooperativity in the presence of cardiolipin. This adds complexity to the mechanism of physiological function encompassing the interaction with acidic phospholipids in mitochondrial membranes. Ferroptosis is implicated in both physio-pathological conditions, including embryogenesis, cancer suppression, neurodegenerations, inflammatory disorders, metabolic syndrome, heart and kidney diseases. No antioxidant enzymatic system can substitute for GPx4 in inhibiting ferroptosis, emphasizing the vital role of selenium. Phenolic antioxidants, which reduce lipid hydroperoxyl radicals, can only inhibit lipid peroxidation under physiological conditions, and thus ferroptosis, when the lipid hydroperoxides formed are immediately reduced by GPx4. In contrast, the ferroptosis inhibitor Ferrostatin-1 (Fer-1) proves to be significantly more efficient than phenolic antioxidants. Analytical and computational evidence supports the notion of a pseudo-catalytic cycle where the ferrostatin-iron complex, both produces and reduces lipid alkoxyl radicals from lipid hydroperoxides. This discloses the roadmap for the identification of innovative antioxidants competent for preventing ferroptosis.

A high proliferation rate and the malignancy of cancer cells are favoured by redox and metabolic plasticity, which is determined by the co-evolution of cancer cells with their host microenvironment. The tight functional connections between the mammary glands' epithelium and adipose tissue (AT) allow breast cancer cells to subjugate the AT and form a protumorigenic cancer-associated adipose tissue (CAAT). Our findings in luminal invasive ductal carcinomas in premenopausal women confirmed key cancer cell strategies - the Warburg effect, increased mitochondrial metabolism and redox adaptability, which are associated with a specific shift in the metabolic and redox phenotype of CAAT. Notably, the upregulated master redox-sensitive transcription factor Nrf2 appears to be responsible for the cancer cell-induced redox and metabolic shift of CAAT. We also investigated the role of Nrf2 in the metabolic co-evolution of cancer cells and CAAT during disease progression. Our results in the orthotopic breast cancer mouse model and in the co-culture of breast cancer cells with adipocytes confirmed the different spatiotemporal redox and metabolic properties of cancer cells and CAAT, established with respect to the Nrf2-coupled/uncoupled tumour microenvironment. The uncovered metabolic and redox strategies adopted by breast cancer cells according to CAAT properties and at different disease stages have helped to better understand the biology of the aggressive disease and to identify breast cancer vulnerabilities that could become therapeutic targets.

This research was supported by the Science Fund of the Republic of Serbia, #7750238, Exploring new avenues in breast cancer research: Redox and metabolic reprogramming of cancer and associated adipose tissue - REFRAME.

Friedreich's ataxia (FRDA) (OMIM #229300, ORPHA95) is a rare hereditary disease with a prevalence of 1/20,000 to 1/50,000 in the European population. It is classified as a hereditary peripheral neuropathy of a sensory type, with autosomal recessive inheritance. This disease is caused by the deficiency of a mitochondrial protein called frataxin. Lack of expression of this protein produces accumulation of iron, alterations in the biogenesis of iron-sulfur clusters, failures in complexes I, II and III of the respiratory chain and in the activity of the aconitase enzyme, and a reduction in the biosynthesis of the heme groups. As a consequence, finally, an overload of ROS derived from the Fenton reaction occurs. Together with the movement impairment, 60% of FRDA patients suffer cardiomyopathy, which is the most common cause of death in these patients and has no clear explanation of its physiopathological cause. Two iPSC cell lines from FRDA patients with cardiomyopathy) and a control line were differentiated to ventricular cardiomyocytes in our lab.  Both FRDA cell lines showed changes in heartbeat parameters, such as heart rate and amplitude when compared to the control cell line. Also, calcium homeostasis measured by immunofluorescence showed important differences when compared to the control cell line. RT-PCR analyses of miRNAs related to myocardial function also showed clear differences, especially for miR-323-3p and miR-142-3p. Using EM, we found differences in the mitochondrial size, shape and in mitochondrial cristae organization. These results also correlate with changes in the cardiomyocytes cytoskeleton and in the structure of the sarcomeres using confocal microscopy techniques. Our results showed the correlation between mitochondrial changes and the impairment in ventricular cardiomyocytes activity derived from FRDA’s iPS cells.