Late in life, the body is at war with itself. There is a program of self-destruction (phenoptosis) implemented via epigenetic and other changes. I refer to these as type (1) epigenetic changes. But the body retains a deep instinct for survival, and other epigenetic changes unfold in response to a perception of accumulated damage (type (2)). In the past decade, epigenetic clocks have promised to accelerate the search for anti-aging interventions by permitting prompt, reliable, and convenient measurement of their effects on lifespan without having to wait for trial results on mortality and morbidity. However, extant clocks do not distinguish between type (1) and type (2). Reversing type (1) changes extends lifespan, but reversing type (2) shortens lifespan. This is why all extant epigenetic clocks may be misleading. Separation of type (1) and type (2) epigenetic changes will lead to more reliable clock algorithms, but this cannot be done with statistics alone. New experiments are proposed. Epigenetic changes are the means by which the body implements phenoptosis, but they do not embody a clock mechanism, so they cannot be the body's primary timekeeper. The timekeeping mechanism is not yet understood, though there are hints that it may be (partially) located in the hypothalamus. For the future, we expect that the most fundamental measurement of biological age will observe this clock directly, and the most profound anti-aging interventions will manipulate it.

High prevalence of human brain disorders necessitates development of the reliable peripheral biomarkers as diagnostic and disease-monitoring tools. In addition to clinical studies, animal models markedly advance studying of non-brain abnormalities associated with brain pathogenesis. The zebrafish (Danio rerio) is becoming increasingly popular as an animal model organism in translational neuroscience. These fish share some practical advantages over mammalian models together with high genetic homology and evolutionarily conserved biochemical and neurobehavioral phenotypes, thus enabling large-scale modeling of human brain diseases. Here, we review mounting evidence on peripheral biomarkers of brain disorders in zebrafish models, focusing on altered biochemistry (lipids, carbohydrates, proteins, and other non-signal molecules, as well as metabolic reactions and activity of enzymes). Collectively, these data strongly support the utility of zebrafish (from a systems biology standpoint) to study peripheral manifestations of brain disorders, as well as highlight potential applications of biochemical biomarkers in zebrafish models to biomarker-based drug discovery and development.

This paper presents new structural data about mitochondria using correlative light and electron microscopy (CLEM) and cryo-electron tomography. These state-of-the-art structural biology methods allow studying biological objects at nanometer scales under natural conditions. Non-invasiveness of these methods makes them comparable to observing animals in their natural environment on a safari. The paper highlights two areas of research that can only be accomplished using these methods. The study visualized location of the Aβ42 amyloid aggregates in relation to mitochondria to test a hypothesis of development of mitochondrial dysfunction in Alzheimer's disease. The results showed that the Aβ42 aggregates do not interact with mitochondria, although some of them are closely located. Therefore, the study demonstrated that mitochondrial dysfunction is not directly associated with the effects of aggregates on mitochondrial structure. Other processes should be considered as sources of mitochondrial dysfunction. Second unique area presented in this work is high-resolution visualization of the mitochondrial membranes and proteins in them. Analysis of the cryo-ET data reveals toroidal holes in the lamellar structures of cardiac mitochondrial cristae, where ATP synthases are located. The study proposes a new mechanism for sorting and clustering protein complexes in the membrane based on topology. According to this suggestion, position of the OXPHOS system proteins in the membrane is determined by its curvature. High-resolution tomography expands and complements existing ideas about the structural and functional organization of mitochondria. This makes it possible to study the previously inaccessible structural interactions of proteins with each other and with membranes in vivo.

Quinone derivatives of triphenylphosphonium have proven themselves to be effective geroprotectors and antioxidants that prevent oxidation of cell components with participation of active free radicals - peroxide (RO2·), alkoxy (RO·), and alkyl (R·) radicals, as well as reactive oxygen species (superoxide anion, singlet oxygen). Their most studied representatives are derivatives of plastoquinone (SkQ1) and ubiquinone (MitoQ), which in addition to antioxidant properties also have a strong antibacterial effect. In this study, we investigated antibacterial properties of other quinone derivatives based on decyltriphenylphosphonium (SkQ3, SkQT, and SkQThy). We have shown that they, just like SkQ1, inhibit growth of various Gram-positive bacteria at micromolar concentrations, while being less effective against Gram-negative bacteria, which is associated with recognition of the triphenylphosphonium derivatives by the main multidrug resistance (MDR) pump of Gram-negative bacteria, AcrAB-TolC. Antibacterial action of SkQ1 itself was found to be dependent on the number of bacterial cells. It is important to note that the cytotoxic effect of SkQ1 on mammalian cells was observed at higher concentrations than the antibacterial action, which can be explained by (i) the presence of a large number of membrane organelles, (ii) lower membrane potential, (iii) spatial separation of the processes of energy generation and transport, and (iv) differences in the composition of MDR pumps. Differences in the cytotoxic effects on different types of eukaryotic cells may be associated with the degree of membrane organelle development, energy status of the cell, and level of the MDR pump expression.

Genes of putative reductases of α,β-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.

Visomitin eye drops are the first and, so far, the only drug based on SkQ1 - the mitochondria-targeted antioxidant 10-(6'-plastoquinonyl) decyltriphenylphosphonium, developed in the laboratories of Moscow State University under the leadership of Academician V. P. Skulachev. SkQ1 is considered as a potential tool to combat the aging program. We have previously shown that it is able to prevent and/or suppress development of all manifestations of accelerated senescence in OXYS rats, including retinopathy, similar to the age-related macular degeneration (AMD). Here, we assessed the effect of Visomitin instillations on progression of the AMD-like pathology and p38 MAPK and ERK1/2 activity in the OXYS rat retina (from the age of 9 to 12 months). Wistar and OXYS rats treated with placebo (composition identical to Visomitin with the exception of SkQ1) were used as controls. Ophthalmological examination showed that in the OXYS rats receiving placebo, retinopathy progressed and severity of clinical manifestations did not differ from the intact OXYS rats. Visomitin suppressed progression of the AMD-like pathology in the OXYS rats and significantly improved structural and functional parameters of the retinal pigment epithelium cells and state of microcirculation in the choroid, which, presumably, contributed to preservation of photoreceptors, associative and ganglion neurons. It was found that the activity of p38 MAPK and ERK1/2 in the retina of 12-month-old OXYS rats is higher than that of the Wistar rats of the same age, as indicated by the increased content of phosphorylated forms of p38 MAPK and ERK1/2 and their target protein tau (at position T181 and S396). Visomitin decreased phosphorylation of p38 MAPK, ERK1/2, and tau indicating suppression of activity of these MAPK signaling cascades. Thus, Visomitin eye drops are able to suppress progression of the AMD-like pathology in the OXYS rats and their effect is associated with the decrease in activity of the MAPK signaling cascades.

An increase in mitochondrial DNA (mtDNA) mutations and an ensuing increase in mitochondrial reactive oxygen species (ROS) production have been suggested to be a cause of the aging process ("the mitochondrial hypothesis of aging"). In agreement with this, mtDNA-mutator mice accumulate a large amount of mtDNA mutations, giving rise to defective mitochondria and an accelerated aging phenotype. However, incongruously, the rates of ROS production in mtDNA mutator mitochondria have generally earlier been reported to be lower - not higher - than in wildtype, thus apparently invalidating the "mitochondrial hypothesis of aging". We have here re-examined ROS production rates in mtDNA-mutator mice mitochondria. Using traditional conditions for measuring ROS (succinate in the absence of rotenone), we indeed found lower ROS in the mtDNA-mutator mitochondria compared to wildtype. This ROS mainly results from reverse electron flow driven by the membrane potential, but the membrane potential reached in the isolated mtDNA-mutator mitochondria was 33 mV lower than that in wildtype mitochondria, due to the feedback inhibition of succinate oxidation by oxaloacetate, and to a lower oxidative capacity in the mtDNA-mutator mice, explaining the lower ROS production. In contrast, in normal forward electron flow systems (pyruvate (or glutamate) + malate or palmitoyl-CoA + carnitine), mitochondrial ROS production was higher in the mtDNA-mutator mitochondria. Particularly, even during active oxidative phosphorylation (as would be ongoing physiologically), higher ROS rates were seen in the mtDNA-mutator mitochondria than in wildtype. Thus, when examined under physiological conditions, mitochondrial ROS production rates are indeed increased in mtDNA-mutator mitochondria. While this does not prove the validity of the mitochondrial hypothesis of aging, it may no longer be said to be negated in this respect. This paper is dedicated to the memory of Professor Vladimir P. Skulachev.

For most of their lifespan, the probability of death for many animal species increases with age. Gompertz law states that this increase is exponential. In this work, we have compared previously published data on the survival kinetics of different lines of progeric mice. Visual analysis showed that in six lines of these rapidly aging mutants, the probability of death did not strictly depend on age. In contrast, ten lines of progeric mice have survival curves similar to those of the control animals, that is, in agreement with Gompertz law, similar to the shape of an exponential curve upside down. Interestingly, these ten mutations cause completely different cell malfunctions. We speculate that what these mutations have in common is a reduction in the lifespan of cells and/or an acceleration of the transition to the state of cell senescence. Thus, our analysis, similar to the conclusions of many previously published works, indicates that the aging of an organism is a consequence of the aging of individual cells.

A decrease in muscle mass and its functionality (strength, endurance, and insulin sensitivity) is one of the integral signs of aging. One of the triggers of aging is an increase in the production of mitochondrial reactive oxygen species. Our study was the first to examine age-dependent changes in the production of mitochondrial reactive oxygen species related to a decrease in the proportion of mitochondria-associated hexokinase-2 in human skeletal muscle. For this purpose, a biopsy was taken from m. vastus lateralis in 10 young healthy volunteers and 70 patients (26-85 years old) with long-term primary arthrosis of the knee/hip joint. It turned out that aging (comparing different groups of patients), in contrast to inactivity/chronic inflammation (comparing young healthy people and young patients), causes a pronounced increase in peroxide production by isolated mitochondria. This correlated with the age-dependent distribution of hexokinase-2 between mitochondrial and cytosolic fractions, a decrease in the rate of coupled respiration of isolated mitochondria and respiration when stimulated with glucose (a hexokinase substrate). It is discussed that these changes may be caused by an age-dependent decrease in the content of cardiolipin, a potential regulator of the mitochondrial microcompartment containing hexokinase. The results obtained contribute to a deeper understanding of age-related pathogenetic processes in skeletal muscles and open prospects for the search for pharmacological/physiological approaches to the correction of these pathologies.

Worldwide, interest in mitochondria is constantly growing, as evidenced by scientific statistics, and studies of the functioning of these organelles are becoming more prevalent than studies of other cellular structures. In this analytical review, mitochondria are conditionally placed in a certain cellular center, which is responsible for both energy production and other non-energetic functions, without which the existence of not only the eukaryotic cell itself, but also the entire organism is impossible. Taking into account the high multifunctionality of mitochondria, such a fundamentally new scheme of cell functioning organization, including mitochondrial management of processes that determine cell survival and death, may be justified. Considering that this issue is dedicated to the memory of V. P. Skulachev, who can be called mitocentric, due to the history of his scientific activity almost entirely aimed at studying mitochondria, this work examines those aspects of mitochondrial functioning that were directly or indirectly the focus of attention of this outstanding scientist. We list all possible known mitochondrial functions, including membrane potential generation, synthesis of Fe-S clusters, steroid hormones, heme, fatty acids, and CO2. Special attention is paid to the participation of mitochondria in the formation and transport of water, as a powerful biochemical cellular and mitochondrial regulator. The history of research on reactive oxygen species that generate mitochondria is subject to significant analysis. In the section "Mitochondria in the center of death", special emphasis is placed on the analysis of what role and how mitochondria can play and determine the program of death of an organism (phenoptosis) and the contribution made to these studies by V. P. Skulachev.