Mitochondria are considered one of the main sites of reactive oxygen species (ROS) production in the eukaryotic cells. For this reason, mitochondrial dysfunction associated with increased ROS production underlies various pathological conditions as well as promotes aging. Chronically increased rates of ROS production contribute to oxidative damage to macromolecules, i.e., DNA, proteins, and lipids. Accumulation of unrepaired oxidative damage may result in progressive cell dysfunction, which can finally trigger cell death. The main by-product of mitochondrial oxidative phosphorylation is superoxide, which is generated by the leak of electrons from the mitochondrial respiratory chain complexes leading to one-electron reduction of oxygen. Mitochondrial superoxide dismutase (MnSOD, SOD2) as well as cytosolic superoxide dismutase (Cu/ZnSOD, SOD1), whose smaller pool is localized in the mitochondrial intermembrane space, converts superoxide to H2O2, which can be then degraded by the catalase to harmless H2O.In this chapter, we focus on the relationship between one of the bioenergetic parameters, which is mitochondrial membrane potential, and the rate of ROS formation. We present a set of various methods enabling the characterization of these parameters applicable to isolated mitochondria or intact cells. We also present examples of experimental data demonstrating that the magnitude and direction (increase or decrease) of a change in mitochondrial ROS production depend on the mitochondrial metabolic state.

AIMS:Nicotine, anatabine, and anabasine are the most prevalent alkaloids in Nicotiana species. While nicotine is the main addictive ingredient in tobacco products, it was also shown to have neuroprotective properties. Mitochondria appear to be one of the targets of nicotine in the cell. These multifunctional organelles are also the first responders to various cellular stresses. Thus, we characterized the impact of tobacco alkaloids on these organelles. METHODS:We investigated the effects of structurally similar alkaloids, anatabine, anabasine, and nicotine, on mitochondrial function in SH-SY5Y neuroblastoma cells under basal conditions and in the presence of rotenone, a mitochondrial stressor commonly used to model the cellular pathology underlying Parkinson's disease. RESULTS:We observed changes in mitochondrial behavior, including hyperpolarization, alterations in mitochondrial network morphology, increased mitochondrial turnover rates, and upregulation of mitochondrial biogenesis regulators. The profiles of changes induced by particular alkaloids slightly differed; however, they shared many features with the stress response observed upon treatment with rotenone. Interestingly, the effects of the alkaloids and rotenone were not additive. Moreover, some parameters altered by rotenone were normalized upon cotreatment with the alkaloids. CONCLUSIONS:The results indicate that the investigated alkaloids stimulate mitochondrial stress adaptation. Despite structural similarity, they act through slightly different mechanisms.

Most cases of Parkinson's disease (PD) are idiopathic, with unknown aetiology and genetic background. However, approximately 10 % of cases are caused by defined genetic mutations, among which mutations in the parkin gene are the most common. There is increasing evidence of the involvement of mitochondrial dysfunction in the development of both idiopathic and genetic PD. However, the data on mitochondrial changes reported by different studies are inconsistent, which can reflect the variability in genetic background of the disease.Mitochondria, as a plastic and dynamic organelles, are the first place in the cell to respond to external and internal stress. In this work, we characterized mitochondrial function and dynamics (network morphology and turnover regulation) in primary fibroblasts from PD patients with parkin mutations.We performed clustering analysis of the obtained data to compare the profiles of mitochondrial parameters in PD patients and healthy donors. This allowed to extract the features characteristic for PD patients fibroblasts, which were a smaller and less complex mitochondrial network and decreased levels of mitochondrial biogenesis regulators and mitophagy mediators. The approach we used allowed a comprehensive characteristics of elements common for mitochondrial dynamics remodelling accompanying pathogenic mutation. This may be helpful in the deciphering key pathomechanisms of the PD disease.

As the war in Ukraine enters its fourth month, Russian forces continue to destroy the nation's scientific institutions and infrastructure, signaling Russia's intent to obliterate the future for Ukraine. In Kharkiv, for instance, the renowned Institute of Physics and Technology and its newly built Neutron Source nuclear facility have been heavily damaged. Even the Plant Production Institute with its underground national seed bank-one of the world's largest-has been bombed. At the Chernobyl nuclear labs, Russian forces have looted or destroyed hundreds of computers, radiation dosimeters, and irreplaceable software and equipment. Although the response to each international science crisis is necessarily unique, the US National Academy of Sciences is once again joining with international and regional partners to support beleaguered colleagues, as it did last year in the successful extraction and resettlement of Afghanistan scientists at risk from the Taliban. To that end, the national science academies of Poland, Ukraine, and the United States recently convened a meeting of leaders from several national science academies (including the presidents of Germany's Leopoldina science academy, the Royal Danish Academy of Sciences and Letters, and the ALLEA European Federation of Academies of Sciences and Humanities, and leaders from the Royal Society of the United Kingdom) to explore how the global science community can best help Ukraine. The resulting 10-point action plan for the world's research community aims to help meet several immediate needs and also provide the building blocks for revitalizing Ukrainian science in the future.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. Only 10% of all cases are familial form, the remaining 90% are sporadic form with unknown genetic background. The etiology of sporadic AD is still not fully understood. Pathogenesis and pathobiology of this disease are limited due to the limited number of experimental models. We used primary culture of fibroblasts derived from patients diagnosed with sporadic form of AD for investigation of dynamic properties of mitochondria, including fission-fusion process and localization of mitochondria within the cell. We observed differences in mitochondrial network organization with decreased mitochondrial transport velocity, and a drop in the frequency of fusion-fission events. These studies show how mitochondrial dynamics adapt to the conditions of long-term mitochondrial stress that prevails in cells of sporadic form of AD.

Mitochondria are among the first responders to various stress factors that challenge cell and tissue homeostasis. Various plant alkaloids have been investigated for their capacity to modulate mitochondrial activities. In this study, we used isolated mitochondria from mouse brain and liver tissues to assess nicotine, anatabine and anabasine, three alkaloids found in tobacco plant, for potential modulatory activity on mitochondrial bioenergetics parameters. All alkaloids decreased basal oxygen consumption of mouse brain mitochondria in a dosedependent manner without any effect on the ADP-stimulated respiration. None of the alkaloids, at 1 nM or 1.25 mu M concentrations, influenced the maximal rate of swelling of brain mitochondria. In contrast to brain mitochondria, 1.25 mu M anatabine, anabasine and nicotine increased maximal rate of swelling of liver mitochondria suggesting a toxic effect. Only at 1 mM concentration, anatabine slowed down the maximal rate of Ca2+-induced swelling and increased the time needed to reach the maximal rate of swelling. The observed mitochondrial bioenergetic effects are probably mediated through a pathway independent of nicotinic acetylcholine receptors, as quantitative proteomic analysis could not confirm their expression in pure mitochondrial fractions isolated from mouse brain tissue.

In a recent prospective study, we reported an association between a low serum selenium level and five-year survival among breast cancer patients. We now have updated the cohort to include 10-year survival rates. A blood sample was obtained from 538 women diagnosed with first primary invasive breast cancer between 2008 and 2015 in the region of Szczecin, Poland. Blood was collected before initiation of treatment. Serum selenium levels were quantified by mass spectroscopy. Each patient was assigned to one of four quartiles based on the distribution of serum selenium levels in the whole cohort. Patients were followed from diagnosis until death or last known alive (mean follow-up 7.9 years). The 10-year actuarial cumulative survival was 65.1% for women in the lowest quartile of serum selenium, compared to 86.7% for women in the highest quartile (p < 0.001 for difference). Further studies are needed to confirm the protective effect of selenium on breast cancer survival. If confirmed this may lead to an investigation of selenium supplementation on survival of breast cancer patients.

Glycogen storage disease type IV (GSD IV) is caused by mutations in the glycogen branching enzyme gene (GBE1) that lead to the accumulation of aberrant glycogen in affected tissues, mostly in the liver. To determine whether dysfunctional glycogen metabolism in GSD IV affects other components of cellular bioenergetics, we studied mitochondrial function in heterozygous Gbe1 knockout (Gbe1+/−) mice. Mitochondria isolated from the livers of Gbe1+/− mice showed elevated respiratory complex I activity and increased reactive oxygen species production, particularly by respiratory chain complex III. These observations indicate that GBE1 deficiency leads to broader rearrangements in energy metabolism and that the mechanisms underlying GSD IV pathogenesis may include more than merely mechanical cell damage caused by the presence of glycogen aggregates.

Although the sporadic form of Alzheimer’s disease (AD) is the prevalent form, the cellular events underlying the disease pathogenesis have not been fully characterized. Accumulating evidence points to mitochondrial dysfunction as one of the events responsible for AD progression. We investigated mitochondrial function in fibroblasts collected from patients diagnosed with the sporadic form of AD (sAD), placing a particular focus on mitochondrial turnover. We measured mitochondrial biogenesis and autophagic clearance, and evaluated the presence of bioenergetic stress in sAD cells. The mitochondrial turnover was clearly lower in the fibroblasts from sAD patients than in the fibroblasts from the control subjects, and the levels of many proteins regulating mitochondrial biogenesis, autophagy and mitophagy were decreased in patient cells. Additionally, the sAD fibroblasts had slightly higher mitochondrial superoxide levels and impaired antioxidant defense. Mitochondrial turnover undergoes feedback regulation through mitochondrial retrograde signaling, which is responsible for the maintenance of optimal mitochondrial functioning, and mitochondria-derived ROS participate as signaling molecules in this process. Our results showed that in sAD patients cells, there is a shift in the balance of mitochondrial function, possibly in response to the presence of cellular stress related to disease development.

The familial form of Parkinson's disease (PD) is linked to mutations in specific genes. The mutations in parkin are one of the most common causes of early-onset PD. Mitochondrial dysfunction is an emerging active player in the pathology of neurodegenerative diseases, because mitochondria are highly dynamic structures integrated with many cellular functions. Herein, we overview and discuss the role of the parkin protein product, Parkin E3 ubiquitin ligase, in the cellular processes related to mitochondrial function, and how parkin mutations can result in pathology in vitro and in vivo.

Glycogen branching enzyme (GBE1) introduces branching points in the glycogen molecule during its synthesis. Pathogenic GBE1 gene mutations lead to glycogen storage disease type IV (GSD IV), which is characterized by excessive intracellular accumulation of abnormal, poorly branched glycogen in affected tissues and organs, mostly in the liver. Using heterozygous Gbe1 knock-out mice (Gbe1(+/)), we analyzed the effects of moderate GBE1 deficiency on oxidative stress in the liver. The livers of aged Gbe1(+/) mice (22 months old) had decreased GBE1 protein levels, which caused a mild decrease in the degree of glycogen branching, but did not affect the tissue glycogen content. GBE1 deficiency was accompanied by increased protein carbonylation and elevated oxidation of the glutathione pool, indicating the existence of oxidative stress. Furthermore, we have observed increased levels of glutathione peroxidase and decreased activity of respiratory complex I in Gbe1(+/) livers. Our data indicate that even mild changes in the degree of glycogen branching, which did not lead to excessive glycogen accumulation, may have broader effects on cellular bioenergetics and redox homeostasis. In young animals cellular homeostatic mechanisms are able to counteract those changes, while in aged tissues the changes may lead to increased oxidative stress.

Metabolic reactions in living cells are limited by diffusion of reagents in the cytoplasm. Any attempt to quantify the kinetics of biochemical reactions in the cytosol should be preceded by careful measurements of the physical properties of the cellular interior. The cytoplasm is a complex, crowded fluid characterized by effective viscosity dependent on its structure at a nanoscopic length scale. In this work, we present and validate the model describing the cytoplasmic nanoviscosity, based on measurements in seven human cell lines, for nanoprobes ranging in diameters from 1 to 150 nm. Irrespective of cell line origin (epithelial–mesenchymal, cancerous–noncancerous, male–female, young–adult), we obtained a similar dependence of the viscosity on the size of the nanoprobes, with characteristic length-scales of 20 ± 11 nm (hydrodynamic radii of major crowders in the cytoplasm) and 4.6 ± 0.7 nm (radii of intercrowder gaps). Moreover, we revealed that the cytoplasm behaves as a liquid for length scales smaller than 100 nm and as a physical gel for larger length scales.

BACKGROUND/AIMSAdverse effects of cigarette smoke on health are widely known. Heating rather than combusting tobacco is one of strategies to reduce the formation of toxicants. The sensitive nature of mitochondrial dynamics makes the mitochondria an early indicator of cellular stress. For this reason, we studied the morphology and dynamics of the mitochondrial network in human bronchial epithelial cells (BEAS-2B) exposed to total particulate matter (TPM) generated from 3R4F reference cigarette smoke and from aerosol from a new candidate modified risk tobacco product, the Tobacco Heating System (THS 2.2).METHODSCells were subjected to short (1 week) and chronic (12 weeks) exposure to a low (7.5 µg/mL) concentration of 3R4F TPM and low (7.5 µg/mL), medium (37.5 µg/mL), and high (150 µg/mL) concentrations of TPM from THS 2.2. Confocal microscopy was applied to assess cellular and mitochondrial morphology. Cytosolic Ca2+ levels, mitochondrial membrane potential and mitochondrial mass were measured with appropriate fluorescent probes on laser scanning cytometer. The levels of proteins regulating mitochondrial dynamics and biogenesis were determined by Western blot.RESULTSIn BEAS-2B cells exposed for one week to the low concentration of 3R4F TPM and the high concentration of THS 2.2 TPM we observed clear changes in cell morphology, mitochondrial network fragmentation, altered levels of mitochondrial fusion and fission proteins and decreased biogenesis markers. Also cellular proliferation was slowed down. Upon chronic exposure (12 weeks) many parameters were affected in the opposite way comparing to short exposure. We observed strong increase of NRF2 protein level, reorganization of mitochondrial network and activation of the mitochondrial biogenesis process.CONCLUSIONComparison of the effects of TPMs from 3R4F and from THS 2.2 revealed, that similar extent of alterations in mitochondrial dynamics and biogenesis is observed at 7.5 µg/mL of 3R4F TPM and 150 µg/mL of THS 2.2 TPM. 7 days exposure to the investigated components of cigarette smoke evoke mitochondrial stress, while upon chronic, 12 weeks exposure the hallmarks of cellular adaptation to the stressor were visible. The results also suggest that mitochondrial stress signaling is involved in the process of cellular adaptation under conditions of chronic stress caused by 3R4F and high concentration of THS 2.2.

Biochemistry in living cells is an emerging field of science. Current quantitative bioassays are performed ex vivo , thus equilibrium constants and reaction rates of reactions occurring in human cells are still unknown. To address this issue, we present a non-invasive method to quantitatively characterize interactions (equilibrium constants, K D ) directly within the cytosol of living cells. We reveal that cytosolic hydrodynamic drag depends exponentially on a probe’s size, and provide a model for its determination for different protein sizes (1–70 nm). We analysed oligomerization of dynamin-related protein 1 (Drp1, wild type and mutants: K668E, G363D, C505A) in HeLa cells. We detected the coexistence of wt-Drp1 dimers and tetramers in cytosol, and determined that K D for tetramers was 0.7 ± 0.5 μM. Drp1 kinetics was modelled by independent simulations, giving computational results which matched experimental data. This robust method can be applied to in vivo determination of K D for other protein-protein complexes, or drug-target interactions.

Mitochondria are multifunctional and dynamic organelles deeply integrated into cellular physiology and metabolism. Disturbances in mitochondrial function are involved in several disorders such as neurodegeneration, cardiovascular diseases, metabolic diseases, and also in the aging process. Nicotine is a natural alkaloid present in the tobacco plant which has been well studied as a constituent of cigarette smoke. It has also been reported to influence mitochondrial function both in vitro and in vivo. This review presents a comprehensive overview of the present knowledge of nicotine action on mitochondrial function. Observed effects of nicotine exposure on the mitochondrial respiratory chain, oxidative stress, calcium homeostasis, mitochondrial dynamics, biogenesis, and mitophagy are discussed, considering the context of the experimental design. The potential action of nicotine on cellular adaptation and cell survival is also examined through its interaction with mitochondria. Although a large number of studies have demonstrated the impact of nicotine on various mitochondrial activities, elucidating its mechanism of action requires further investigation.