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 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.

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.

Individual complexes of the mitochondrial oxidative phosphorylation system (OXPHOS) are not linked solely by their function; they also share dependencies at the maintenance/assembly level, where one complex depends on the presence of a different individual complex. Despite the relevance of this ‘interdependence’ behavior for mitochondrial diseases, its true nature remains elusive. To understand the mechanism that can explain this phenomenon, we examined the consequences of the aberration of different OXPHOS complexes in human cells. We demonstrate here that complete disruption of each of the OXPHOS complexes resulted in a perturbation in energy deficiency sensing pathways, including the integrated stress response (ISR) pathway. The secondary decrease of complex I (cI) level was triggered by both complex IV and complex V deficiency, and it was independent of ISR signaling. On the other hand, we identified the unifying mechanism behind cI downregulation in the downregulation of mitochondrial ribosomal proteins and, thus, mitochondrial translation. We conclude that the secondary cI defect is due to mitochondrial protein synthesis attenuation, while the responsible signaling pathways could differ based on the origin of the OXPHOS defect.

Estuarine and intertidal bivalve mollusks frequently experience salinity fluctuations that may drive oxidative stress (OS) in the organism. Here we investigated OS markers and histopathological changes in gills and hemolymph of Mediterranean mussels Mytilus galloprovincialis acclimated to a wide range of salinities (6, 10, 14, 24, and 30 ‰). Mussels were captured at the shellfish farm with the salinity of 18% and then acclimated to hypo- and hypersaline conditions in the laboratory at the speed of 1.5±0.5‰ per day. Indicators of redox balance in hemocytes (intracellular reactive oxygen species (ROS) levels, DNA damage) and gills (thiobarbituric acid reactive substances (TBARS), protein carbonyls (PC), activity of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured. The results revealed induction of OS in tissues and cells of mussels for both experimental increase and decrease salinity modeling. Hemocytes showed higher sensitivity to oxidative damage from salinity stress compared to gills, as DNA damage and elevated ROS levels were observed in all experimental groups except 14‰. A decrease in environmental salinity to 10 ‰ was likely within the physiological norm for mussels, as minor oxidative damage was noted. At a salinity of 6 ‰, the most significant signs of redox imbalance, including DNA damage, increased ROS production levels in hemocytes, and suppressed activity of SOD in gills were observed, along with elevated PC levels. An increase in environmental salinity up to 30 ‰ led to the enhancement of the activity of antioxidant enzymes in the gills, which may be attributed to the high capacity of the antioxidant system in this organ. The study provides new insights into the effects of salinity stress on the tissue and cellular redox balance of bivalves, which is crucial for better understanding the potential consequences of the global transformation of coastal ecosystems.

The metastasis of breast cancer to the axillary lymph nodes represents a crucial aspect of disease progression and prognostic evaluation. The presence of metastases in the axillary lymph nodes is a key indicator that breast cancer is in an advanced stage, which can influence the therapeutic approach and the patient's prognosis. For this reason, we conducted a study aimed at examining the factors that contribute to the presence of metastases in lymph nodes in our female population. This research represents a prospective study conducted at the Institute of Oncology of Vojvodina in Sremska Kamenica. The study included 72 female participants diagnosed with breast cancer who underwent surgery at the Institute of Oncology of Vojvodina and had not received preoperative chemotherapy or radiation therapy. Initially, anamnestic data were collected from the participants, followed by a pathohistological analysis of the tumor tissue samples, including immunohistochemical analysis. We examined the influence of age, tumor size, activity of estrogen, progesterone, and HER2 receptors (human epidermal growth factor receptor-2)  in tumors, as well as the occurrence of menarche and breastfeeding duration, on the presence of metastases in axillary lymph nodes. The results of binary logistic regression showed that the only significant predictor for the presence of metastases in axillary lymph nodes was tumor size (p=0.01, Wald=6.57, and Exp(B)=1.11), while the other examined predictors were not statistically significant (p>0.05). In our study population, the size of the breast cancer was crucial for the presence of metastases in the axillary lymph nodes.

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.

AMPylation (adenylation) is one of the post-translational protein modifications (PTM) leading to the diversification of protein functions and activity. With our collaborators, we discovered that the SelO family members of humans, yeast, and E. coli have AMPylase activity. The yeast SelO – Fmp40 – was identified in the proteome of the inter-membrane space of mitochondria. We have shown that Fmp40 is involved in the response of cells to hydrogen peroxide (H2O2) and menadione treatment: cells lacking the Fmp40 AMPylase grow sensitivity upon H2O2 and menadione treatment. E. coli SelO AMPylates glutaredoxin GrxA and the s-glutathionylation level of proteins is reduced in bacterial and yeast cells lacking SelO1. The objective of the study is to reveal the biological functions of Fmp40 in mitochondrial redox regulation. The decreased survival of fmp40Δ cells, observed in survival tests, depends on the oxidation of Trx3 upon oxidative stress. In contrast, we verified that fmp40Δ cells are resistant upon exposure to high concentrations of the hydrogen peroxide - phenotype dependent on the presence of the Glutaredoxin Grx2, Thioredoxin Trx3, Peroxiredoxin Prx1, Oxidation Resistance Oxr1, and Apoptotic inducing factor Aif1 basing on qPCR analysis. We found multidimensional genetic interactions of FMP40 with other known redox genes upon low or high oxidative stress. We revealed that Fmp40 AMPylates Prx1, Trx3, and Grx2 in vitro and it has a matrix-localized echo form. We discovered that Fmp40 is critical for the efficient reduction of Prx1 upon high oxidative stress. Moreover, Grx2 is involved in the Prx1 reduction directly and at the level of Trx3 reduction in vivo. Fmp40 regulates its function on Trx3 protein, most probably through Threonine66 which is AMPylated in vivo. In addition, Fmp40 is necessary to maintain the balance of cellular redox buffers GSH and NADPH. Overall Fmp40 regulates redox gene expression for efficient ROS neutralization and signaling which eventually determines the fate of cell survival upon oxidative stress.

Financed by National Science Centre of Poland: 2018/31/B/NZ3/01117.

Human activity and rapid urbanization created disturbance factors that drastically changed natural habitats. The introduction of artificial light at night changed natural light-dark regimes affecting a range of biological processes. Disruption of circadian rhythm is linked with changes in endocrine and neurobiological systems that control hormonal regulation, food intake, metabolism, reproduction, and behavior of animals. Oxidative stress was suggested as a possible mechanism through which artificial light could affect an organism’s physiology and health. We examined the oxidative status of tree frog (Hyla arborea) under two artificial night light intensities 20 lux and 90 lux. Artificial light affects the antioxidant system of both larval and juvenile stages. Larvae had higher activity for glutathione peroxidase only for 90 lux, while greater lipid damage was observed in individuals under both light regimes compared to control. Juvenile individuals showed boosted antioxidant response seen through higher activities of superoxide dismutase, catalase, and glutathione peroxidase. Finally, development under artificial light led to higher levels of protein damage in juveniles. Artificial light at night acts primarily through direct effects and can persist across life stages. Overall results point out that exposure to artificial light alters physiological traits in amphibians, such as oxidative status that could have various consequences on individuals in natural populations.

Particulate matter (PM) is well recognized as the major contributor to the air pollution disease burden. Presently, the data pointing to the direct effects of PM on the cardiovascular health are numerous, but the mitigation strategies are still at the level of reduction of exposure. In the present study, we used a mouse model of real-life PM2.5 exposure treated with either a statin (atorvastatin) or an ACE inhibitor (captopril) in order to observe the potentially protective effects of cardiovascular drug treatment on the underlying mechanisms of detrimental, PM2.5-induced, cardiovascular effects. Captopril treatment mitigated the PM2.5-induced blood pressure while both drugs reduced selected markers of oxidative stress in the vasculature and heart. Both drugs were successful in mitigating the vascular oxidative stress by reducing the activation of the NADPH oxidase enzyme. In addition, both drugs were able to reverse the PM2.5-induced increase in vascular endothelin-1. The treatment also reduced the level of 3-NT positive proteins in the lung and mitigated the effects on dysregulated eNOS expression. Drugs did not mitigate the inflammatory response in the lung and in circulation with only captopril reducing the pulmonary IL-6, but not CD68 expression. In summary, ACE inhibitors can potentially mitigate the effects of PM2.5 on the vascular function and oxidative stress by lowering blood pressure and statins have a known antioxidant effect, e.g. via inhibition of NADPH oxidase. Our present data provide novel insights into possible mitigation strategies for PM2.5-induced cardiovascular disease. Since statins and ACE inhibitors represent first-line therapies for cardiovascular disease, CVD patients, e.g. with coronary artery disease, ischemic heart disease, and hypertension representing highly vulnerable groups for air pollution health effects, may benefit from pre-established therapies with these drugs to prevent additive cardiovascular damage by PM2.5 exposure.

Particulate matter (PM) air pollution presents a major environmental and public health challenge because of its non-uniform size distribution and chemical composition. Air quality regulations generally categorize particulate matter (PM) size into PM10, PM2.5, and ultrafine particles (UFPs) with aerodynamic diameters smaller than 10, 2.5, and 0.1 µm, respectively. We examined the differential impact of particle size per se on selected organ systems using a custom whole-body mouse exposure system using synthetic PM (SPM). The micrometer-sized SPM accumulated in the lungs as the primary entry organ, while ultrafine SPM showed less accumulation, implying a transition into circulation. Micro SPM-exposed mice exhibited inflammation and NADPH oxidase-derived oxidative stress in the lungs. Ultrafine SPM-exposed mice did not show oxidative stress in the lungs but rather at the brain, heart, and vasculature levels. Endothelial dysfunction and blood pressure increase were more pronounced in ultrafine SPM exposed mice, supported by increased endothelin-1 and decreased endothelial nitric oxide synthase expression, enhancing constriction and reducing vasodilation. To derive a preliminary estimate of the cardiovascular disease burden of UFPs in humans, we used new high-resolution exposure data at a global scale, and applied hazard ratios from an epidemiological cohort study. We derived a UFP-associated incidence of 419 (95% CI 78–712) thousand cardiovascular disease cases per year in the European Union and 5.6 (95% CI 1.1–9.3) million globally. This work provides novel insights into the different toxicological profiles of inhaled ultrafine particles and public health consequences of exposure, guiding future studies.