Purpose: Aging is a complex biological process that begins at the cellular level, disrupting energy homeostasis. This study investigated the role of Sirt3, major mitochondrial deacetylase involved in metabolic pathways, in sex-dependent changes in energy homeostasis during aging in kidney of Sirt3 WT and KO mice.

Methods: Enzymatic activity, lipid peroxidation, protein carbonylation with Western blot and metabolomic analyses were performed to assess physiological and metabolic parameters.

Results: Higher Sirt3 expression in male WT mice leads to increased vulnerability to its deficiency, as reflected in the shorter lifespan of male KO mice. This is further supported by distinct metabolomic clustering in male KO mice, highlighting significant metabolic disruptions. Male-specific declines in metabolites such as creatine, phosphorylcholine, trimethylamine-N-oxide, and L-carnitine, along with reduced trifunctional multienzyme complex subunit β (HADHB) expression, point to impaired fatty acid metabolism and mitochondrial dysfunction.

Conclusions: The findings emphasize the sex-specific function of Sirt3 in regulating mitochondrial activity, energy metabolism, and oxidative stress in the murine kidney, with male mice exhibiting a greater reliance on Sirt3 for metabolic stability.

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.

Mechanisms and factors that lead to the formation of lipomas, benign tumors of adipose tissue, are still insufficiently elucidated. Mesenchymal stem cells (MSCs) isolated from lipomas have some similar characteristics to MSCs isolated from white adipose tissue but differ at the molecular level and in their differentiation potential. Considering histological appearance of lipomas, it is not clear to what extent lipomas share common characteristics with other adipose tissue type, brown adipose tissue. Therefore, the aim of this study was to examine the level of uncoupling protein 1 (UCP1), a marker of brown adipose tissue, expression in lipoma tissue as well as in MSCs isolated from lipomas, i.e. lipoma-derived mesenchymal stem cells (LDSCs). LDSCs were grown in standard cell culture conditions and subjected to adipogenic differentiation. UCP1 expression was examined at the RNA level, using Real-Time PCR, and at the protein level, using immunohistochemistry and immunogold staining. Expression of UCP1 in lipoma tissue and LDSCs was compared with the expression of UCP1 in subcutaneous white adipose tissue (scWAT) and adipose-derived mesenchymal stem cells (ADSCs) grown and differentiated in the same cell culture conditions. Differences were observed in UCP1 expression at both RNA and protein levels in lipomas compared to scWAT directing the future research towards the potential of browning mechanisms of adipose tissue involved in lipoma tissue formation.

This research was financially supported by the Science Fund of the Republic of Serbia, PROMIS, #6066747, WARMED and the Ministry of Science, Technological Development and Innovations of the Republic of Serbia, Contract No. 451-03-65/2024-03/200113.

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.

Succinate dehydrogenase (SDH) connects the tricarboxylic acid (TCA) cycle and the respiratory chain. Mutations in SDH subunits have been associated with tumorigenesis and mitochondrial disease. In this project, we focused on subunit A of SDH (SDHA), primarily associated with inherited mitochondrial disease, and investigated the consequences of its loss or re-expression of mutant variants in HEK cells (SDHA KO). Lack of SDHA led to a downregulation of all SDH subunits and a secondary downregulation of the majority of mitochondrial complex I and IV subunits. Cellular respiratory capacity was severely decreased in the model, SDH-dependent respiration completely abolished and complex I-dependent respiration attenuated, reflecting the downregulation of respiratory chain complexes in general. Finally, the NAD⁺/NADH ratio was increased in SDHA KO, indicating complex rearrangement of the TCA. It resulted in higher glycolytic activity and lipid accumulation.

Supported by Czech Science Foundation (21-18993S), Grant Agency of Charles University (283423) and Czech Health Research Council (NU22-01-00499).

Dysregulation of the redox system and the interconnected low-level inflammation (LLI) act as a driving force of damaging mechanisms in gestational diabetes mellitus (GDM) and are strongly related to severe obstetric and neonatal complications of hyperglycaemic pregnancies. Major disturbances in microRNA-based mechanism accompany (glyco)oxidative stress ((g)OS), for which reason we hypothesized that microRNAs may serve as sensors and/or effectors of (g)OS/LLI in GDM and we chose candidates for GDM biomarker analysis among known (g)OS/LLI-associated microRNAs. The aim of the study was to analyze the properties of miR-146a-5p and miR-21-5p as redox status indicators in GDM, as well as to compare two different biological samples as sources of potentially relevant GDM biomarkers.  miR-146a-5p and miR-21-5p were quantified by real-time polymerase chain reaction in peripheral blood mononuclear cells of patients with GDM and normoglycaemic pregnant controls (n=40 each), as well as in paired samples of extracellular vesicles (EVs) extracted from serum. Correlation analysis was conducted for the expression levels of tested microRNAs and the activities of glutathione reductase (GR), total superoxide dismutase (SOD), catalase (CAT), concentration of serum thiol groups and the level of Nrf2 mRNA. In both samples, tested microRNAs were upregulated in GDM group, with a more pronounced increase in expression in EVs, compared to peripheral blood mononuclear cells (PBMCs) (1.81 vs. 1.52 fold for miR-146a-5p and 1.98 vs. 1.58 fold for miR-21-5p). There was a significant positive correlation between the expression of miR-21-5p from PBMCs and Nrf2 in both GDM patients and controls, as well as a positive correlation with the activity of total SOD in GDM patients. On the other hand, miR-146a-5p from EVs demonstrated negative correlation with Nrf2 expression and the activity of total SOD. These data demonstrate the potential of (g)OS/LLI-related microRNAs miR-146a-5p and miR-21-5p to serve as indicators of GDM and the associated (g)OS-related changes.

Aging is a natural process characterized by a decline in organic structure-function and an increase in mortality over time. While many exogenous and endogenous factors contribute to aging, the long-term effects of low environmental temperature have been poorly described. To address this, our study compared 24-month-old male Mill Hill hybrid hooded rats raised at a standard temperature of 22±1°C with age-matched rats that were kept in a cold room (4±1°C) from the age of 6 to 24 months. 3- and 6-month-old rats raised at 22±1°C were included as room temperature controls. We examined two metabolically active organs, interscapular brown adipose tissue (iBAT) and liver. It was found that 24-month-old rats chronically exposed to cold exhibit increased food consumption, which may be attributed to a higher metabolic demand. Chronic exposure of aged rats to low environmental temperature led to an increase in iBAT relative mass, total glutathione (GSH) content, and antioxidant defense (AD) enzyme activity: CuZn superoxide dismutase, Mn superoxide dismutase, catalase, glutathione peroxidase, and thioredoxin reductase. Respirometric analysis further demonstrated an increase in mitochondrial uncoupling in iBAT in 24-month-old rats kept at 4±1°C. Conversely, there was no change of the same parameters in the liver, which maintained consistent AD enzyme activity and GSH content across all experimental groups. Our study confirms that iBAT of aged rats remains responsive to stimulation by low environmental temperature, supporting thermogenic processes through uncoupling and a robust increase in the AD system. These results highlight tissue-specific effects of chronic cold exposure on aged rats underlying acclimation-driven physiological changes.

The global pandemic crisis affected almost every society and economy, challenged almost every health system worldwide. Above all, governments and non-governmental organizations had to fight the misinformation and conspiracy theories placed by the social and mass media. All of this had a profound impact on the public in terms of vaccine safety and the advantages of vitamin use in fighting the virus. This fear has opened doors to alternative medicines such as supplements (vitamins, minerals, herbal products, oils) that may have profound effects on the immune system. To determine the pattern of use of supplements during the pandemic in healthy individuals who tested negative for SARS-CoV-2. The 33 healthy individuals tested negative for SARS-CoV-2 in the pandemic period were included (Group 1). Total antioxidant power, iron-reducing (PAT), and plasma peroxides (d-ROMs) were measured using FRAS5 analytical photometric system and are reported in equivalents of ascorbic acid and H2O2, respectively. The oxidative stress index (OSI) was automatically calculated by the software. The obtained values were compared with 30 healthy individuals analyzed prior to the pandemic (Group 2). The mean values for oxidative stress parameters in Group 1 vs Group 2 were: d-ROMs 418 vs 266 U. Carr, PAT 3862 vs 2554 U. Carr, and OSI 111 vs 36. In all comparisons, a statistically significant difference was obtained (p<0.05, t-test). Individuals belonging to Group 1 had reported that they have consumed daily doses of Zinc (30 mg), Vitamin C (at least 1000 mg) and Vitamin D (at least 2000 IU) in a period of >1 month. Several of them have also used Isoprinosine, magnesium, and selenium. Uncontrolled intake of supplements can have a profound effect on the pro- and antioxidant balance resulting in interruption of the phycological balance and leading to increased oxidative stress index in otherwise healthy individuals. 

Colorectal cancer presents a significant global health challenge, with a high mortality rate. It is the third most commonly diagnosed cancer and is therefore a major cause for concern. The development of colorectal cancer is multifaceted, involving a combination of genetic predispositions and lifestyle factors. The redox and metabolic states may influence the intricate process of colon cancer development. To gain a deeper understanding of the redox-metabolic profiles associated with colon cancer, a human study was conducted. In biopsies from patients with colon cancer, the antioxidant status: copper, zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutamate-cysteine ligase (GCL), thioredoxin (Trx) and lactate metabolism were examined in tumor and unaffected colon tissue (remote 15-20 cm) as well as in adipose tissue: proximal (near the tumour tissue), distal (remote 6 cm) and unaffected (remote over 6 cm). The protein levels of CuZnSOD, MnSOD, GSH-Px, and Trx are increased in the tumor tissue compared to the unaffected colon tissue. In addition, the expression of the lactate dehydrogenase (LDH) A isoform, the total activity of LDH and the lactate concentration are higher in transformed tumor tissue than in normal colon tissue. On the other hand, lactate concentration increases and several AD components (CuZnSOD, MnSOD, CAT, GSH-Px, GCL and Trx) decrease in adipose tissue with tumor proximity. Shifts in redox and lactate metabolism in tumor tissue associated with spatial changes in lactate and antioxidant enzymes gradients in adjacent adipose tissue clearly indicate a local redox metabolic interaction between tumor and tumor-associated adipose tissue in shaping the malignant phenotype in human colorectal cancer.

Ionising radiation damages DNA directly, or indirectly, causing water radiolysis and formation of free radicals. Indirect irradiation effects could be diminished in the presence of free radical scavengers, such as dimethyl sulfoxide (DMSO). Such conditions would allow the evaluation of direct radiation effects and provide a better understanding of cellular response to irradiation-induced damages. The goal of this study was to investigate the effects of low (γ-rays) and high linear energy transfer (LET) radiation (carbon ions) in non-small lung cancer cells HTB177. Cells were pre-treated with DMSO and irradiated with 60Co γ-rays and 62 MeV/u carbon ions, with doses ranging from 1-5 Gy. Results obtained by clonogenic survival and γ-H2AX foci assay showed that DMSO increased cell survival and decreased number of DNA damages, which points to radioprotective effect of DMSO. The contribution of direct and indirect radiation effects was estimated by the degree of protection (DP) in presence of DMSO. The values of DP rose in a concentration-dependent manner in all irradiated samples. In cells irradiated with γ-rays, 35% of damages were caused directly, while 65% of lesions could be attributed to indirect radiation actions. In presence of carbon ions, contribution of direct effects was 49%, while 51% of damage resulted from indirect radiation effects, showing that free radicals attain an important role in both low and high LET irradiations. The obtained results showed that DMSO can be used as a free radical scavenger to examine the direct and indirect effects on human cancer cells. The numerical Monte Carlo simulations allow modelling of direct and indirect irradiation actions in cancer cells with photons and hadrons. Therefore, this data will be used for validation and further improvement of numerical simulations in comparison to the data collected on different cell lines and irradiation energies, with the goal to improve therapeutic protocols.