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Background. The mitochondrial electron transport chain, in particular the respiratory complex I, is one of the main sources of reactive oxygen species (ROS) in living cells. Suppression of the complex I activity in human cells through chemical inhibition or mutations that disrupt the functionality of this complex leads to a significant increase in the content of ROS. The complex I mutants are also known for plants; however, it is still not clear whether the suppression of the activity of this complex leads to an increase in the level of ROS and the development of oxidative stress. Purpose. To study the level of superoxide, hydrogen peroxide and sensitivity to prooxidants in Arabidopsis ndufs4 cells with complex I inactivated by insertional mutagenesis. Materials and methods. A suspension culture of wild-type Arabidopsis cells and a line with knockout the NDUFS4 complex I subunit was used. The level of hydrogen peroxide in the cells was determined using dichlorofluorescein. The superoxide content was estimated by cell staining in the presence of nitroblue tetrazolium. Results. It has been shown that inactivation of the NADH-dehydrogenase complex in Arabidopsis cells due to the absence of one of its subunits leads to a decrease in the content of superoxide and hydrogen peroxide. It was also shown that the level of ROS in cells treated with the menadione and hydrogen peroxide increased several times in wild-type cells, but remained almost unchanged in ndufs4 cells. Conclusion. Suppression of the activity of the NADH dehydrogenase complex in plant cells, but not in animal cells, leads to a decrease in the content of both hydrogen peroxide and superoxide. Cells of ndufs4 line have an increased ability to detoxify ROS, possibly associated with the constant mobilization of antioxidant defense systems.

A new method for extracting the Brillouin frequency shift (BFS) from the Brillouin gain spectrum (BGS), the modified backward correlation method (MBWC), is presented. The possibilities of using MBWC, and MBWC in combination with the Lorentzian curve fitting (LCF) based on Levenberg–Marquardt (LM) method, are studied. The effectiveness of the new method, and its combination with LM, has been demonstrated for processing spectra with a low signal-to-noise ratio (SNR). The experiments, which were in good agreement with the performed simulation, showed that at SNR = 0 dB, the combined (MBWC + LM) method provided the BFS extraction error of less than 4 MHz, while the state-of-the-art LM algorithm extracted it with the error greater than 4.5 MHz. The advantage of correlation methods becomes more significant with the decreasing SNR: at SNR = −2 dB, the LM’s error is 14.3 MHz, and that of the combined one is 8.1 MHz.

Expression of Arabidopsis thaliana glutamate dehydrogenase genes is very low in the light and high in the dark. The molecular signals and mechanisms that provide the light-dependent glutamate dehydrogenase genes regulation remain unknown. The aim of this work was to study a role of redox signals which occur during light shifts in the regulation of the glutamate dehydrogenase genes expression. Using photosynthetic electron transport inhibitors 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl benzoquinone (DBMIB) we demonstrate that transcript levels of the GDH1 and GDH2 genes in Arabidopsis leaves change in accordance with a redox state of plastoquinone pool: they are low when it is highly reduced and high when it is oxidized. Hydrogen peroxide or high light treatment did not result in decreasing of GDH1 or GDH2 expression, so reactive oxygen species cannot be the signals that reduce expression of these genes during dark-to-light shifts. There was no significant difference between the glucose content in the leaves of plants treated with DCMU and the plants treated with DBMIB, so glucose is not the only or the main factor that regulates expression of the studied genes. We presume that expression of Arabidopsis GDH1 and GDH2 genes depends on the plastoquinone pool redox state. Expression of Arabidopsis glutamate dehydrogenase genes during light-to-dark and dark-to-light shifts depends on chloroplast electron transport chain redox state rather than reactive oxygen species or glucose content.

It is known that mitochondrial population of living organisms is heterogeneous. Using the density gradient of Percoll or sucrose, we obtained and characterized two distinct mitochondrial subfractions (named “light” and “heavy” according to their sedimentation) from three plant species (Brassica rapa, Zea mays, and Arabidopsis thaliana). Electron microscopy showed that mitochondria of a light subfraction have a poorly developed cristae structure. The respiratory control of these mitochondria was reduced; however the degree of their outer membrane integrity remained high. These data suggest that mitochondrial subfractions purified in the density gradient differ in their maturity. We carried out DNA import assays to study the ability of these two subfractions for DNA transport. We have shown that the mitochondria of light subfraction in all three plant species uptakes DNA more efficiently than the mitochondria of heavy subfraction. In addition, import of DNA with different sizes into the light and heavy mitochondrial subfractions has different sensitivity to specific inhibitors of the voltage-dependent anion channel and adenine nucleotide translocase, proteins playing a central role in DNA transport across the mitochondrial membrane. The obtained results suggest that DNA import depends on organization of the transport system in the double mitochondrial membrane influenced by the maturity of the mitochondria.

Electron transfer chains of plant organelles (both chloroplasts and mitochondria) contain their own special set of ferredoxins. The relatively recently described adrenodoxin-like [2Fe-2S]-ferredoxins MFDX1 and MFDX2 of plant mitochondria are among the least studied of these. Until now, the only established function for them is participation in the final stage of biotin biosynthesis. In this work, using genetic and biochemical approaches, we searched for possible partners of these proteins in the genomes and proteomes of tobacco (Nicotiana tabacum L.) and foxglove (Digitalis purpurea L.) plants. MORF9 protein, one of the auxiliary components of the RNA editing complex of organelles (editosome), was found among the most prominent protein partners of adrenodoxin-like [2Fe-2S] tobacco ferredoxins. According to the results obtained from the yeast two-hybrid system, NtMFDX1 and NtMFDX2 of tobacco also bind and interact productively with the previously uncharacterised long non-coding polyadenylated RNA, which, based on its structural features, is capable of regulating the function of a number of components of complexes I (Nad1, Nad5) and III (protein of the cytochrome c synthesis system CcmF) and contributes to the formation of Fe/S-clusters in the corresponding protein complexes of the respiratory chain of plant mitochondria. We found one of the main components of the thiazol synthase complex (mitochondrial protein DpTHI1) to be the partner of ferredoxin DpMFDX2 of Digitalis purpurea. Finally, additional arguments were obtained in favour of the possible participation of MFDX1 and MFDX2 in the very ancient, but only recently described ‘progesterone’ steroid hormonal regulatory system: in leaves of the previously constructed CYP11A1-transgenic tomato plants, only the mature form of mitochondrial cytochrome P450scc (CYP11A1) of mammals is able to enter the mitochondria, where the above-mentioned components of the electron transport chain are localised. In summary, all of the newly revealed interactions of adrenodoxin-like [2Fe-2S] ferredoxins MFDX1 and MFDX2 indicate their participation in a wide range of functions in plant mitochondria.

«Genome uncoupled» (gun) is a molecular phenotype manifesting in a high expression of a number of nuclear photosynthesis associated genes in plants with arrested chloroplast biogenesis. The chloroplast biogenesis arrest could be achieved by growing of plants in presence of lincomycin (plastid translation inhibitor) or norflurazon (carotenoid biosynthesis inhibitor). We have found that Arabidopsis thaliana gun1-1gun5-1 double mutant seedlings with gun phenotype associated with both lincomycin and norflurazon demonstrate a repressed root growth when germinated on lincomycin but not norflurazon-containing media. Statistical analysis has demonstrated a high negative correlation between root length and gun phenotype evaluated on both LHCB1.2 and HEMA1 genes expression in three gun mutants of Arabidopsis: gun1-1, cch1-1 and the double mutant gun1-1gun5-1. Our results demonstrate that regulatory signals of plastid origin might participate in root development.

Recombinant proteins are the most important product of current industrial biotechnology. They are indispensable in medicine (for diagnostics and treatment), food and chemical industries, and research. Plant cells combine advantages of the eukaryotic protein production system with simplicity and efficacy of the bacterial one. The use of plants for the production of recombinant proteins is an economically important and promising area that has emerged as an alternative to traditional approaches. This review discusses advantages of plant systems for the expression of recombinant proteins using nuclear, plastid, and mitochondrial genomes. Possibilities, problems, and prospects of modifications of the three parts of the genome in light of obtaining producer plants are examined. Examples of successful use of the nuclear expression platform for production of various biopharmaceuticals, veterinary drugs, and technologically important proteins are described, as are examples of a high yield of recombinant proteins upon modification of the chloroplast genome. Potential utility of plant mitochondria as an expression system for the production of recombinant proteins and its advantages over the nucleus and chloroplasts are substantiated. Although these opportunities have not yet been exploited, potential utility of plant mitochondria as an expression system for the production of recombinant proteins and its advantages over the nucleus and chloroplasts are substantiated.

Mitochondria possess transport mechanisms for import of RNA and DNA. Based on import into isolated Solanum tuberosum mitochondria in the presence of competitors, inhibitors or effectors, we show that DNA fragments of different size classes are taken up into plant organelles through distinct channels. Alternative channels can also be activated according to the amount of DNA substrate of a given size class. Analyses of Arabidopsis thaliana knockout lines pointed out a differential involvement of individual voltage-dependent anion channel (VDAC) isoforms in the formation of alternative channels. We propose several outer and inner membrane proteins as VDAC partners in these pathways.

A new method of Brillouin spectra post-processing, which could be applied in modern distributed optical sensors: Brillouin optical time domain analyzers/reflectometers (BOTDA/BOTDR), has been demonstrated. It operates by means of the correlation analysis performed with special technique (&laquo, backward-correlation&raquo, ). It does not need any additional data for time or space averaging and operates with the single spectrum only. We have simulated the method accuracy dependence on signal-to-noise ratio (SNR) and other parameters. It is shown that the new method produces better results at low SNRs than conventional technique, based on finding of Brillouin spectrum maximum, do. These results are in a good agreement with the experiment. Finally, we have estimated the performance of the new method for its application in polarization-BOTDA set-up for a polarization maintaining (PM) fiber modal birefringence distributed study.

In the nuclear genome of Arabidopsis thaliana some cas genes and CRISPR cassettes were found by in silico methods. It is hypothesized that CRISPR-Cas-like elements appeared in plant genome as a result of endosymbiosis of ancient alphaproteobacteria.

We studied capabilities of confocal laser scanning microscopy in the analysis of lipid droplets volume and of quantity of functional mitochondria and reactive oxygen species production in liver cells for early diagnosis of cytochemical disturbances at dyslipoproteinemia (16 days of experiment). The results showed the increase of lipid droplets volume in hepatocytes, decrease of functional mitochondria and increase of reactive oxygen species production. We evaluated the potential of real-time PCR method in the analysis of mitochondrial DNA of blood plasma at early stages of dyslipoproteinemia and in experimental infarction. On the background of registered blood lipid metabolism disorders and structural and functional changes in liver cells, we determined the tendency to three-time increase in concentration of circulating cell-free mtDNA on the 16th day of dyslipoproteinemia as compared to the control data. We used a model of myocardial infarction to show statistically significant increase in the level of circulating cell-free blood mtDNA from 48 hours after adrenaline injection and we found that this level maintained up to 144 hours after adrenaline injection. Obtained data can serve as a basis for creation of technologies for diagnostic monitoring of atherosclerosis and myocardial infarction severity.

Mitochondria have retained indispensable but limited genetic information and they import both proteins and nucleic acids from the cytosol. RNA import is essential for gene expression and regulation, whereas competence for DNA uptake is likely to contribute to organellar genome dynamics and evolution. Contrary to protein import mechanisms, the way nucleic acids cross the mitochondrial membranes remains poorly understood. Using proteomic, genetic and biochemical approaches with both plant and yeast organelles, we develop here a model for DNA uptake into mitochondria. The first step includes the voltage-dependent anion channel and an outer membrane-located precursor fraction of a protein normally located in the inner membrane. To proceed, the DNA is then potentially recruited in the intermembrane space by an accessible subunit of one of the respiratory chain complexes. Final translocation through the inner membrane remains the most versatile but points to the components considered to make the mitochondrial permeability transition pore. Depending on the size, DNA and RNA cooperate or compete for mitochondrial uptake, which shows that they share import mechanisms. On the other hand, our results imply the existence of more than one route for nucleic acid translocation into mitochondria.

In a number of dicotyledonous plants, including Arabidopsis, the transcription of organellar genes is performed by three nuclear-encoded RNA polymerases, RPOTm, RPOTmp, and RPOTp. RPOTmp is a protein with a dual targeting, which is presumably involved in the control of gene expression in both mitochondria and chloroplasts. A previous study of the Arabidopsis insertion rpotmp mutant showed that it has retarded growth and development, altered leaf morphology, changed expression of mitochondrial and probably some chloroplast genes, and decreased activities of the mitochondrial respiratory complexes. To date, there is no clear evidence as to which of these disorders are associated with a lack of RPOTmp in each of the two organelles. The aim of this study was to elucidate the role that this RNA polymerase specifically plays in mitochondria and chloroplasts. Two sets of Arabidopsis transgenic lines with complementation of RPOTmp function in either mitochondria or chloroplasts were obtained. It was found that the recovery of RPOTmp RNA polymerase activity in chloroplasts, although restoring the transcription from the RPOTmp-specific PC promoter, did not lead to compensation of the mutant growth defects. In contrast, the rpotmp plants expressing RPOTmp with mitochondrial targeting restored the level of mitochondrial transcripts and exhibit a phenotype resembling that of the wild-type plants. We conclude that despite its localization in two cell compartments, Arabidopsis RPOTmp plays an important role in mitochondria, but not in chloroplasts.

Mitochondria are competent for DNA uptake in vitro, a mechanism which may support delivery of therapeutic DNA to complement organelle DNA mutations. We document here key aspects of the DNA import process, so as to further lay the ground for mitochondrial transfection in intact cells.We developed DNA import assays with isolated mitochondria from different organisms, using DNA substrates of various sequences and sizes. Further import experiments investigated the possible role of ATP and protein phosphorylation in the uptake process. The fate of adenine nucleotides and the formation of phosphorylated proteins were analyzed.We demonstrate that the efficiency of mitochondrial uptake depends on the sequence of the DNA to be translocated. The process becomes sequence-selective for large DNA substrates. Assays run with a natural mitochondrial plasmid identified sequence elements which promote organellar uptake. ATP enhances DNA import and allows tight integration of the exogenous DNA into mitochondrial nucleoids. ATP hydrolysis has to occur during the DNA uptake process and might trigger phosphorylation of co-factors.Our data contribute critical information to optimize DNA delivery into mitochondria and open the prospect of targeting whole mitochondrial genomes or complex constructs into mammalian organelles in vitro and in vivo.

Mitochondrial gene products are essential for the viability of eukaryote obligate aerobes. Consequently, mutations of the mitochondrial genome cause severe diseases in man and generate traits widely used in plant breeding. Pathogenic mutations can often be identified but direct genetic rescue remains impossible because mitochondrial transformation is still to be achieved in higher eukaryotes. Along this line, it has been shown that isolated plant and mammalian mitochondria are naturally competent for importing linear DNA. However, it has proven difficult to understand how such large polyanions cross the mitochondrial membranes. The genetic tractability of Saccharomyces cerevisae could be a powerful tool to unravel this molecular mechanism. Here we show that isolated S. cerevisiae mitochondria can import linear DNA in a process sharing similar characteristics to plant and mammalian mitochondria. Based on biochemical data, translocation through the outer membrane is believed to be mediated by voltage-dependent anion channel (VDAC) isoforms in higher eukaryotes. Both confirming this hypothesis and validating the yeast model, we illustrate that mitochondria from S. cerevisiae strains deleted for the VDAC-1 or VDAC-2 gene are severely compromised in DNA import. The prospect is now open to screen further mutant yeast strains to identify the elusive inner membrane DNA transporter.

Plant mitochondria are remarkable with respect to their content in foreign, alien and plasmid-like DNA, raising the question of the transfer of this information into the organelles. We demonstrate the existence of an active, transmembrane potential-dependent mechanism of DNA uptake into plant mitochondria. The process is restricted to double-strand DNA, but has no obvious sequence specificity. It is most efficient with linear fragments up to a few kilobase pairs. When containing appropriate information, imported sequences are transcribed within the organelles. The uptake likely involves the voltage-dependent anion channel and the adenine nucleotide translocator, i.e. the core components of the mitochondrial permeability transition pore complex in animal cells, but it does not rely on known mitochondrial membrane permeabilization processes. We conclude that DNA import into plant mitochondria might represent a physiological phenomenon with some functional relevance.

With the aim of carrying out a long-term medical follow-up with radiation dose reconstruction, a cohort of subjects was selected among inhabitants of the most contaminated area in Russia following the Chernobyl accident (the western districts of Bryansk region). The cohort is comprised of 1065 subjects who were under 10 years old at the time of the accident. Most of them were examined on health status in the Chernobyl Sasakawa Health and Medical Cooperation Project. Since the main findings of studies in the project were thyroid abnormalities, selection of subjects was conducted on the basis of the plausible estimates of radiation dose to the thyroid. To estimate thyroid doses, the data from direct measurements of 131I in the thyroid and questionnaire data on individual dietary habits in May 1986 were used. Reasonable approximations were applied to reconstruct individual doses from available data, including doses for those persons who had not been measured for thyroidal radioiodine. The distribution of internal radiation dose to the thyroid among cohort members was obtained. The individual doses to particular subjects are estimated with inevitably essential degree of uncertainty. However, the distribution of subjects into wide dose intervals, from under 200 mGy to over 2 Gy, seems to be an acceptable approach for cohort study in radiation epidemiology.

Aim. To understand the mechanism(s) underlying mitochondrial competence for DNA uptake and to exploit these pathways for the development of in vivo models of gene therapy. Methods. DNA uptake into isolated mitochondria from plant or from mutant Saccharomyces cerevisiae defective for mitochondrial proteins and carriers, biochemical approaches and transfection of mammalian cells with DNA bound to mitochondriotropic liposomes. Results. Special focus on the inner membrane showed the involvement of isoforms of the adenine nucleotide translocator and the contribution of proteins controlling mitochondrial morphology in DNA uptake into yeast organelles. Transfection assays led to significant incorporation of a mitochondrial construct into mammalian cells and expression of a marker gene. Conclusions. The data imply that there are multiple mitochondrial DNA import pathways. On the other hand, preliminary results suggest that mitochondriotropic liposomes can deliver DNA to mitochondria in live mammalian cells. Мета. Визначити механізми поглинання ДНК мітохондріями і використати їх для удосконалення існуючих моделей генної терапії in vivo. Методи. Поглинання ДНК ізольованими мітохондріями рослин або мітохондріями мутантних ліній Saccharomyces cerevisiae, дефектних за мітохондріальними білками та переносниками, біохімічні підходи і трансфекція в клітини ссавців ДНК, зв’язаної з мітохондріотропними ліпосомами. Результати. Основним підсумком вивчення внутрішньої мембрани виявилося встановлення того факта, що до процесу перенесення ДНК дріжджовими органелами залучені кілька ізоформ аденіннуклеотидтранслокази, а також білки, які контролюють мітохондріальну морфологію. В експериментах з трансфекції ДНК у клітини ссавців виявлено вбудовування в них мітохондріальної конструкції і експресію маркерного гена. Висновки. Отримані дані дозволяють припустити існування декількох механизмів імпорту ДНК у мітохондрії. Проте є попередні результати, які показують, що мітохондріотропні ліпосоми можуть бути використані для доставки ДНК у мітохондрії клітин ссавців in vivo. Цель. Выяснить механизмы поглощения ДНК митохондриями и использовать их для усовершенствования существующих моделей генной терапии in vivo. Методы. Поглощение ДНК изолированными митохондриями растений или митохондриями мутантных линий Saccharomyces cerevisiae, дефектных по митохондриальным белкам и переносчикам, биохимические подходы и трансфекция в клетки млекопитающих ДНК, связанной с митохондриотропными липосомами. Результаты. Основным итогом изучения внутренней мембраны оказалось установление того факта, что в процесс переноса ДНК дрожжевыми органеллами вовлечены несколько изоформ адениннуклеотидтранслоказы, а также белки, контролирующие митохондриальную морфологию. В экспериментах по трансфекции ДНК в клетки млекопитающих выявлены встраивание в них митохондриальной конструкции и экспрессию маркерного гена. Выводы. Полученные данные позволяют предположить существование нескольких механизмов импорта ДНК в митохондрии. Однако есть предварительные результаты, показывающие, что митохондриотропные липосомы могут быть использованы для доставки ДНК в митохондрии клеток млекопитающих in vivo.

Inside-out inner mitochondrial membranes free of matrix proteins were isolated from purified potato tuber (Solanum tuberosum L.) mitochondria and incubated with (Q- 32 P)ATP. Proteins were separated by SDS^PAGE and visualized by autoradiography. Phosphorylation of inner mem- brane proteins, including ATPase subunits, was strongly inhibited by the phosphoprotein phosphatase inhibitor NaF. We propose that an inner membrane phosphoprotein phosphatase is required for activation of the inner membrane protein kinase. When prelabelled inner membranes were incubated in the absence of (Q Q- 32 P)ATP, there was no phosphoprotein dephosphorylation unless a soluble matrix fraction was added. This dephosphoryla- tion was inhibited by NaF, but not by okadaic acid. We conclude that the mitochondrial matrix contains a phosphoprotein phosphatase that is responsible for dephosphorylation of inner membrane phosphoproteins. fl 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Mitochondrial transcription rate and RNA steady-state levels were examined in shoots of Arabidopsis seedlings. The shoots were treated with inhibitors of complex III and IV of the cytochrome pathway (CP) and with an inhibitor of the alternative oxidase (AOX) of the mitochondrial electron transport chain. The inhibition of AOX and CP complexes III and IV affected transcription and transcript levels in different ways. CP and AOX inhibitors had opposite effects. Our data support the idea that the redox state of the electron transport chain is involved in the regulation of mitochondrial gene expression at transcriptional and post-transcriptional levels.

Measurements of 137Cs body burden were carried out in 1991-1996 for children residing in the western part of Bryansk Oblast (Russia) where area contamination with 137Cs following the Chernobyl accident varied from 0.03-3.7 MBq m(-2). The mean and median values of 137Cs specific activity (SA) in the bodies of children 5-15 y old for the whole period of surveillance averaged for 26,029 measurements was 85 Bq kg(-1) and 49 Bq kg(-1), respectively. Mean values in different places of residence varied from 30-342 Bq kg(-1) for the settlements where the number of examinees was not less than 50. There is a moderately strong relationship between mean SA in the settlement and the level of area contamination with 137Cs. A strong seasonal effect on 137Cs body burden was found. The ratio of average SA values is 1:1.75 for spring to autumn 1992-1995 in the settlement where the largest number of children was examined. This effect might be attributed to a seasonal change in diet. Based on questionnaire information on individual consumption of locally produced foods, the descending rank of contribution of food items to 137Cs intake by children was found to be: meat-mushrooms-milk-vegetables. Assuming that the dose distribution would follow the distribution of SA values, internal doses to the whole body from ingested 137Cs were assessed. The mean value of annual internal dose averaged for the whole set of measurements is 0.21 mSv, and the median of the individual dose distribution is 0.12 mSv y(-1) For 2% of the total sample the annual dose exceeded 1 mSv, with the highest individual dose being 9 mSv in 1994 for a 7-y-old girl.

Subunit 9 of the mitochondrial ATPase, but also other hydrophobic mitochondrially encoded proteins, contains a high frequency of the leucine motif, -Leu-X9-Leu-, which is highly conserved through RNA editing. The leucine motif may provide specific recognition sites between membrane-spanning domains of the F0-ATPase and between other hydrophobic subunits during the assembly of multienzyme complexes in the inner mitochondrial membrane.

Background The efficacy of treating acute myocardial ischemic damages depends, to a large extent, on the development of technologies for predicting their course and outcome. The aim of this paper was to explore whether it would be possible to consider the content of free circulating mitochondrial DNA as a danger-associated molecular pattern for assessing the probability of death from myocardial infarction. Methods We have analyzed the clinical outcomes based on discharge summaries and autopsy reports obtained in the course of the PROTOCOL observational trial. This study was approved by the Irkutsk Scientific Center of Surgery and Traumatology ethics committee (protocol No. 3, 10.08.2015). To examine whether the assessment of the level of free circulating mtDNA in acute coronary syndrome can help predicting clinical outcomes, all patients were divided into two groups: group 1, involving those who survived during 30 days after hospitalization, and group 2, involving those who died during this time. A quantitative analysis of the free circulating mtDNA was conducted using the PCR method in situ. Results The analysis showed that in patients who survived the level of freely circulating mtDNA (36.0 copies/ml) was 164 times lower than in those who died (5900 copies/ml, p = 0.049). It should be mentioned that according to the logistic regression analysis, the probability of death of patients with the increased level of blood plasma mtDNA (more than 4000 copies/ml) is 50%. Conclusions Thus, the PROTOCOL observational trial proved that the increase in the content of free circulating mtDNA in blood is a predictor of lethal outcome in patients with acute coronary syndrome. Trial registration The observational studies (those in which the assignment of the medical intervention is not at the discretion of the investigator) do not require registration.