Increased damage by ROS plays a role in the development of neurodegenerative diseases, especially Alzheimer’s Disease and other dementias, and diets rich in antioxidants (high intake of fruits and vegetables) seem neuroprotective (as well as being protective against many other age-related diseases). However, attempts to treat/prevent such diseases by giving high doses of antioxidants such as vitamins E and C and carotenoids have, overall, been unsuccessful. Reasons for this will be discussed. A major focus of our work is a unique diet-derived thiol/thione with antioxidant properties, namely ergothioneine (ET). Low blood levels of ET are a risk factor for the development of neurodegenerative and cardiovascular diseases, frailty, eye disease, pre-eclampsia and age-related diseases generally. We have identified “adequate levels” of plasma ET in humans, levels below which are associated with increased disease occurrence, and the reasons leading to these low levels are under investigation. In animal studies, ET has exhibited the ability to modulate inflammation, scavenge certain ROS, protect against acute respiratory distress syndrome, decrease brain damage in models of Parkinson and Alzheimer diseases and stroke, prevent endothelial dysfunction, protect against ischemia-reperfusion injury, counteract iron dysregulation, hinder lung and liver fibrosis, and mitigate damage to the lungs, kidneys, liver, gastrointestinal tract, and testis. ET may also influence the gut microbiome. There is evidence that ET is specifically accumulated at sites of tissue injury, so we have called it an “adaptive antioxidant” that may not interfere with the normal physiological roles of ROS. But does low ET predispose to age-related diseases or is it a spurious correlation? Extensive cell and animal studies strongly suggest the former. Caveats in the use of ergothioneine supplements to prevent/ameliorate aged-related diseases include its potential to generate trimethylamine-N-oxide by the action of ergothionase enzymes in gut bacteria and its ability to be taken up by many bacteria, a few of which are pathogenic (e.g. H. pylori, M. tuberculosis). These caveats will be discussed.

Mitochondrial diseases are a large family of extremely heterogeneous disorders genetically determined by mutations in either the nuclear genome or the mitochondrial DNA. Most of the mitochondrial disease genes are expressed in all cell types. However, in many conditions, some cell types are more affected than others. However, the reasons for this tissue-specificity remain poorly understood. To investigate the functional basis of the striking tissue-specificity in mitochondrial diseases, we analyzed several bioenergetic parameters, including oxygen consumption rates, Q redox poise, and reactive oxygen species production in mouse brain and liver mitochondria fueled by different substrates. In addition, we determined how these functional parameters are affected by electron transport chain impairment in a tissue-specific manner using pathologically relevant mouse models lacking either Ndufs4 or Ttc19, leading to complex I or III defects, respectively. No cure is currently available for most of the mitochondrial diseases. We previously showed that the coordinated activation of autophagy, lysosomal biogenesis, and mitochondrial biogenesis by rapamycin, ameliorated the myopathic phenotype of a muscle-specific knockout mouse for Cox15 (Cox15sm), encoding an enzyme involved in heme A biosynthesis. However, the role of mitophagy has been poorly investigated. We found that urolithin A, a direct mitophagy inducer, improved motor performance and myopathy in the Cox15sm mice, without increasing the activity of the respiratory chain complexes in a 10 week-treatment. These results indicate that activation of mitophagy can be a suitable treatment to ameliorate mitochondrial myopathies.

In mammals, a multi-oscillator circadian system generates behavioural, metabolic and physiological ~24h rhythms, with tissue-specific physiological cues enabling local circadian phase adjustments. Emerging work has shown musculoskeletal tissue homeostasis and mechanical responses to be under circadian control. Nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the antioxidant response, is a clock-controlled target in several peripheral tissues and also modifies circadian gene expression and rhythmicity. However, the role of NRF2 in mechanical loading-induced changes in musculoskeletal tissues has yet to be elucidated. Wild-type (WT) and Nrf2 KO mice of young (3-6m) or old age (18-20m) harbouring a PER2::luciferase clock reporter were subjected to acute mechanical joint loading of the right leg (peak load 9N, 40 cycles of 10sec) during light phase whilst the contralateral (left) leg served as a non-loaded control. Musculoskeletal tissues were collected for analysis 4 hrs later. Real-time bioluminescence imaging of clock gene reporter activity, protein and mRNA levels of target markers, NRF2/ARE transactivation and genome-wide RNAseq analyses were undertaken. We show that acute mechanical loading in WT mice led to a decrease in gene expression of key members in the negative and auxiliary feedback loops of the molecular clock, associated with the phase-resetting of PER2::luc protein oscillations in the skeletal muscle and a knee joint. This was accompanied by a significant increase in the markers of oxidative burden as well as gene expression and protein abundance levels of antioxidant enzymes. Moreover, acute mechanical loading induced a significant activation of the redox-sensitive energy sensor, AMP-activated kinase (AMPK), known to be involved in molecular clock resetting. We thus examined whether the above acute mechanical responses were dependent on NRF2 activity. Nrf2 KO mice showed an altered response to acute mechanical loading, characterized by blunted circadian resetting and antioxidant responses, and altered AMPK activation. Furthermore, dampened responses to acute mechanical signals were found in ageing WT mouse musculoskeletal tissues, whilst AMPK activator treatments in WT mice induced circadian resetting and antioxidant responses in an NRF2-dependent manner. In conclusion, these data demonstrate that AMPK/NRF2 axis is required for relaying acute mechanical signals to the musculoskeletal system by controlling redox-mediated phase resetting of musculoskeletal clocks and antioxidant protection, which have important implications in understanding biomechanical mechanisms involved in musculoskeletal tissue maintenance in health and with ageing.

Changes in carbohydrate metabolism are a key feature of aging which also manifest in the epidermis. Furthermore, the synthesis and distribution of epidermal lipids changes with age. Both these parameters cannot be investigated with immunohistochemistry, as neither serves as useful epitope. We developed a multimodal analytical histocytometry approach combining modalities that localize lipids and enzymatic activities with immunofluorescent imaging of the skin to localize changes that are correlated with appearance of senescent cells. The activities of key metabolic enzymes were determined on tissue sections of aged and juvenile skin with a formazan-based assay. Lipids were localized and quantified using FTICR MALDI - mass spectrometric imaging. We correlated those modalities with immunofluorescent imaging and analyzed the intensities of the respective signals at single cell level, using Strataquest tissue cytometry. We analyzed skin from donors of young (< 30 y) versus advanced (> 67 y) ages and we investigated epidermal equivalent models containing labeled UV-damaged or senescent keratinocytes. Enzymatic activities displayed specific patterns across the stratifying epidermis, and had diverging trajectories in aging, with a marked decrease in suprabasal glucose-6-phosphate dehydrogenase (G6PD) activity. G6PD, the rate limiting enzyme of the pentose phosphate pathway was also identified as a rapid response pathway activated upon UV damage in the epidermis.  The lipid molecular imaging identified differentiation- and age-related changes of polar lipids in skin biopsies and epidermal equivalents, and pro-senescent stress dependent reactive aldehydophospholipid species in the basal epidermal layers.  While these methodologies are still in development, it is evident that correlative analytical imaging – with the aid of AI driven histocytometry – will continue to yield novel insights into skin and epidermal biology by localizing previously undetectable parameters within the epidermis in the context of aging.

Rett syndrome (RTT), a devastating neurodevelopmental disorder, is caused in 95% of the cases by mutations in the X-chromosome-localized MECP2 gene. RTT manifests as a range of multisystem disturbances including altered lipid profile, subclinical inflammation, and overall OxInflammatory status in which mitochondrial dysfunction acts as central player. To decipher the molecular mechanisms underlying the pathophysiological manifestations affecting patients, we investigated whether mitochondria may play a role in the aberrant immune and oxidative responses of RTT. Recent findings from our and other labs unraveled several abnormalities in RTT mitochondria including atypical mitochondrial structure, deregulated expression of genes encoding oxidative phosphorylation factors and mitochondrial organization factors, impaired mitochondrial quality control, depressed energetic profile, and augmented mt-ROS production. In other brain diseases, mitochondrial dysfunction is a vital event during the activation of NLPR3 inflammasome, a multi-protein complex involved in innate immune response, that represents a common denominator in the crosstalk between inflammation and oxidative stress. Interestingly, using primary fibroblasts and lympho-monocytes isolated from RTT patients, we found a constitutive hyperactivation of NLRP3:ASC inflammasome associated with increased levels of nuclear p65 and ASC proteins, and pro-IL-1β mRNA, without the ability to further respond to the LPS + ATP stimuli. Furthermore, increased circulating levels of ASC, interleukin (IL)-18, and 1β were found in RTT individuals, thus corroborating the aforementioned cellular findings. In order to evaluate NLRP3 involvement in the transition from pre-symptomatic to symptomatic phase of RTT, we detected higher serum levels of IL-1β and IL-18 in symptomatic Het mice compared to WT. Of note, increased gene expression of Il-1b, Nlrp3, and ASC was observed in Het brains at the pre-symptomatic stage, suggesting a likely role of NLRP3 impairment in the early stages of the disease. Preliminary data showed that treatment with resveratrol, known to improve mitochondrial function, ameliorated the RTT mouse phenotype by restoring levels of some NLRP3-related components. Furthermore, mitochondrial dysfunction can result in ferroptosis, a form of cell death characterized by iron-dependent lipid peroxidation and accumulation of reactive oxygen species.  After treatment with two ferroptosis inducers, erastin (GPX4 inhibitor) or RSL3 (inhibitor of the cystine/glutamate antiporter), we found changes in GPx and GR activity, alteration in GPX4 protein levels and increased formation of 4HNE protein adducts. Mitochondrial ROS production and lipid peroxidation levels were higher in RTT after ferroptosis induction, while co-treatment with ferrostatin-1, a well-known inhibitor of ferroptosis, significantly prevented these processes. Interestingly, co-treatment with mito-TEMPO, a mitochondria-targeted superoxide dismutase mimetic, mitigated mitochondrial oxidative burden and prevented ferroptosis cell death in RTT cells. Overall, our results demonstrate the decisive role of mitochondrial dysfunction in RTT OxInflammation. Thus, we can speculate that exposure of RTT cells to any condition affecting the already compromised mitochondrial function could not only hyperactivate the inflammatory status but also precipitate ferroptosis cell death. Targeting mitochondria in RTT could represent a strategic coadjuvant therapy to improve the quality of life of the affected patients.

Imatinib, a tyrosine kinase inhibitor (TKI) is used as a standard treatment in chronic myeloid leukemia (CML) patients. Increased levels of BCR-ABL1 expression in CML cells are associated with oxidative stress induction due to overproduction of reactive oxygen species (ROS) or by deficient antioxidant system, disease progression, and imatinib resistance. Current scientific research confirms that oxidative stress is involved in CML pathogenesis and response to TKI treatment. Moreover, recent findings suggest that the antioxidant properties of some natural compounds can provide benefits to patients with CML. To determine the effect of adjuvant treatment with polyphenols on the oxidative stress markers in imatinib-treated CML patients. 40 CML patients at the University Clinic of Hematology, Skopje, who received imatinib longer than 1 month were included in the study. 20 patients were additionally treated with Aronia melanocarpa extract and 20 patients received only imatinib (control group). Besides the regular clinical laboratory analysis for these patients, total antioxidant power (PAT) and plasma peroxides (d-ROMs) were measured at initial visit and after 21 and 42 days of treatment using FRAS5 analytical photometric system and the oxidative stress index (OSI) was automatically calculated. Oxidative stress parameters (d-ROM and OSI) were significantly higher at initial visit in both groups. In group of patients who received adjuvant polyphenols values for d-ROM and OSI were significantly lower after 21 and 42 days of treatment (p<0.05). Also, total antioxidant capacity (PAT) was significantly higher after 21 and 42 days of treatment initiation in comparison with the pretreatment values. In the control group, no significant differences were obtained between investigated parameters at any time of measurement. Adjuvant treatment with Aronia melanocarpa extract after 21 and 42 days led to significant reduction of oxidative stress parameters in patients with CML treated with imatinib. 

The World Health Organization (WHO) estimates that noise pollution leads to the loss of 1.6 million healthy life years annually in Western Europe alone, primarily due to night-time noise exposure which disrupts sleep and triggers stress responses. This study investigates the adverse health effects of aircraft noise on the brain-heart-vessel axis, combining cardiovascular and neuropsychiatric approaches. We aim to characterize the functional and biochemical consequences of both short-term and long-term noise exposure utilizing an established mouse model. Behavioural changes in exposed mice, including cognition, anxiety, depression, and social behaviour were assessed alongside cardiovascular parameters such as blood pressure, endothelial function tests, and analyses of oxidative stress and inflammation markers. Short-term noise exposure did not lead to any significant differences in the behaviour of the noise-exposed mice, whereas long-term noise-exposure leads to reduced social interaction and working memory as behavioural markers of depression. Functional cardiovascular parameters point to hypertension and impaired endothelial function in both short-term and long-term noise exposure, as well as oxidative stress and inflammation. These findings underscore previously reported cardiovascular impact of noise exposure while adding the suspected behavioural changes and metabolic markers of the affected brain-heart axis. The observed behavioural changes and cardiovascular impairments emphasize the complex interplay between environmental stressors and health, suggesting that long-term noise exposure can have profound effects on both mental and cardiovascular health. This study provides a comprehensive framework for future research aimed at reducing the adverse effects of noise pollution on the brain-heart-vessel axis.

Our recent observations show that both resistant and stem-like cancer cells predominantly responsible for metastasis differ from chemotherapy-sensitive cells. We have shown bioinformatically and experimentally that mitochondria of such cells are much more prone to oxidative phosphorylation (OXPHOS) than radio- or chemotherapy-sensitive cancer cells from which they evolved during therapeutic interventions. Specifically, in triple-negative breast cancer models, we observed that such resistant cells exhibit higher mitochondrial membrane potential, higher OXPHOS and respiration, and increased resistance to oxidative stress, allowing them to survive chemo-radiotherapy.  These findings of increased expression of OXPHOS-associated genes and proteins in chemoresistant cells and biopsies of relapsed tumors suggest an alternative druggable target. Our in vitro and in vivo (nude mice and Artemia salina) data suggest that certain antibiotics, inducers of mitochondrial dysfunction, create additive oxidative stress and can reduce the growth rate of tumors developed from resistant or stem-like cancer cells. Such repurposed drugs, selected from a chemical library, are also able to resensitize resistant tumors, allowing reuse of chemotherapeutic agents. In addition, their modification with a specific moiety (TPP) allows for increased delivery to mitochondria to reduce cytotoxic pressure on normal cells. Thus, research from our laboratory offers an alternative strategy for anticancer therapy of resistant tumors.

Oxidative stress (OS) has a significant impact on the lifespan and physical fitness of living organisms. It is commonly associated with ageing and can lead to changes in the functionality of red blood cells (RBCs). The precise mechanisms underlying these changes are not fully understood. Unlike mammals, avian RBCs have a nucleus and functional mitochondria that regulate the cellular response to oxidative stress. In this study, we examined the effects of OS on red blood cells from adult female quail (Coturnix japonica, n=12). We used flow cytometry to analyze the formation of OS-induced microparticles and RBC transformation. We also evaluated band 3 clustering and phosphatidylserine externalization at the cell surface using eosin-5-maleimide and Annexin-V fluorescent probes, respectively. In addition, we analyzed band 3 clustering using confocal microscopy. We used a laser diffraction-based method to analyze cell deformability, and we characterized hemoglobin species spectrophotometrically. We found that OS caused band 3 clustering, microparticle formation, and phosphatidylserine release onto the cell membrane. The microparticles formed under the influence of oxidants differed from those formed under the influence of A23187 (calcium ionophore). The rate of microparticle formation and the onset of osmotic rigidity depended on the oxidant concentration. Erythrocyte-derived microparticles contained hemoglobin oxidized to hemichrome (HbChr). Overall, these findings demonstrate that avian erythrocytes undergo different processes during oxidative stress, depending on the level of oxidation. These differences are due to variations in cellular transformations and the formation of different types of microparticles. This research was supported by the Russian Fund for Basic Researches (grant no. 23-15-00142)

Small cell lung cancer (SCLC) is the leading cause of cancer-related deaths worldwide and is characterized by rapid growth, early metastasis, and high mortality rates. This study investigated the prognostic potential of leukocyte telomere length (LTL) and paraoxonase 1 (PON1) activity in 60 SCLC patients treated with a cisplatin/etoposide (PE) regimen. Patients were observed at baseline, after 2 cycles, and after 4 cycles of chemotherapy. The primary objective was to evaluate the prognostic potential of these biomarkers for patient survival. LTL was measured from isolated genomic DNA using real-time quantitative polymerase chain reaction (RTq-PCR), while PON1 activity was determined using a spectrophotometric method. A Kaplan-Meier survival analysis was performed with cut-off values below the 25^th percentile for LTL and PON1 activity to determine their prognostic power for overall survival. The analysis revealed that both LTL and PON1 are significant predictors of patient survival, suggesting that patients with levels below the 25^th percentile have a higher risk of death (Log Rank = 3.956, p = 0.047; Log Rank = 3.834, p = 0.050, respectively). Telomeres, the protective caps at the ends of chromosomes, shorten with each cell division and reflect cell aging and genomic stability. Shorter telomere lengths in leukocytes have been associated with a poorer prognosis and lower survival rates in SCLC patients. Similarly, reduced PON1 activity is associated with increased oxidative stress, which contributes to cancer progression and poorer clinical outcomes. Monitoring PON1 activity could help in assessing patient prognosis and adjusting treatment strategies. These findings suggest that LTL and PON1 activity have significant prognostic value in SCLC and serve as useful indicators for identifying high-risk patients and guiding treatment decisions to improve outcomes.