The Relationship of p62 Gene Expression with Integrity of Mitochondrial DNA and the Level of Lipid Peroxidation Products in Skeletal Muscles of Rats of Different Ages Exposed to Different Feeding Protocols.

Sequestosome-1 (SQSTM1/p62) is one of the most important multifunctional proteins, which is necessary to maintain mitochondrial stability by eliminating damaged mitochondria through mitophagy. We studied the influence of age and diet on the expression of the p62 gene in the femoral and abdominal muscles of rats, as well as the integrity of some mitochondrial components. In the femoral muscles of 24-month-old rats receiving restricted ration, the expression of the p62 gene increased. We assume that activation of mitophagy contributed to a decrease in the levels of oxidative damage to mitochondrial DNA and LPO intensity in the femoral muscles of 24-month-old rats.

The Effect of Methylene Blue and Its Metabolite-Azure I-on Bioenergetic Parameters of Intact Mouse Brain Mitochondria.

Methylene blue, a phenothiazine dye, that is widely used in medicine and is under clinical trials as an agent for treatment of Alzheimer's disease. One of the factors of the unique therapeutic effect of methylene blue is its redox properties, allowing implementation of alternative electron transport: the dye accepts electrons from reducing equivalents in mitochondria and transfer them to other components of the respiratory chain or molecular oxygen. Azure I, an N-dimethylated metabolite of methylene blue, is potentially a more effective compound than methylene blue, but its ability for alternative electron transport has not been studied yet. We have shown that in contrast to methylene blue, azure I is unable to restore the membrane potential in isolated mouse brain mitochondria, inhibited by rotenone and, therefore, is unable to perform bypass of the respiratory chain complex I. Moreover, addition of azure I does not affect the rate of mitochondrial respiration in contrast to methylene blue, which increases the rate of non-phosphorylation respiration. At the same time, both dyes stimulate an increase in H2O2 production. Thus, only methylene blue is capable of alternative electron transport, while azure I does not produce complex I bypass. This limits its therapeutic application only as a mitochondrial-targeted agent, but does not question its antidepressant effects.

Influence of Gold Nanoparticles on the Tolerance of Wheat to Low Temperature.

It was shown for the first time that the treatment of winter wheat (Triticum aestivum L.) seeds with gold nanoparticles (average diameter 15.3 nm; solution concentration 20 µg/mL) increases plant tolerance to low temperature. It was found that an increase in tolerance under the influence of nanoparticles is accompanied by a number of changes depending on temperature conditions. In optimal temperature conditions, gold nanoparticles stimulated plant growth and the activity of the photosynthetic apparatus, whereas in conditions of low-temperature hardening (2°C, 7 days) they inhibited growth but maintained photosynthetic activity, contributing to the accumulation of soluble sugars (cryoprotectants) in the leaves. It is concluded that gold nanoparticles can be considered as adaptogens that increase plant tolerance to low temperatures; however, their effectiveness in this role and the subtle mechanisms of action require further study.

[Effect of methylene blue and its metabolite - azure I - on bioenergetic parameters of intact mice brain mitochondria]. / Vliianie metilenovogo sinego i ego metabolita ­ azura I ­ na bioénergeticheskie parametry izolirovannykh mitokhondrii mozga myshei.

Methylene blue is a phenothiazine dye that is widely used in medicine and clinical trials for the treatment of Alzheimer's disease. One of the factors of the unique therapeutic effect of methylene blue is its redox properties, allowing implementation of alternative electron transport - the dye accepts electrons from reducing equivalents in the mitochondria and transfer it them to other components of the respiratory chain or molecular oxygen. Azure I, an N-dimethylated metabolite of methylene blue, is potentially a more effective compound than methylene blue, but its ability for alternative electron transport has not been studied. We have shown that azure I, unlike methylene blue, is unable to restore the membrane potential in isolated mouse brain mitochondria, inhibited by rotenone and, therefore, is unable to perform bypass of the respiratory chain Complex I. Moreover, the addition of azure I does not affect the rate of mitochondrial respiration in contrast to methylene blue, which increases the rate of non-phosphorylation respiration. At the same time, both dyes stimulate an increase in H2O2 production. As a consequence, only methylene blue is capable of alternative electron transport, while azure I does not produce complex I bypass. This limits its therapeutic application only as a mitochondrial-targeted drug, but not as a substance with a potentially powerful antidepressant effect.

Study of microbiome changes in patients with ulcerative colitis in the Central European part of Russia.

Ulcerative colitis (UC) is an inflammatory disease that affects the colon and rectum. Recently, evidence has emerged about the influence of microbiota on the development of this disease. However, studies on the role of intestinal microbiota in the pathogenesis of UC have been incomplete. In addition, there are no comprehensive studies of the causes of ulcerative colitis and data on the microbiological composition of the intestines of patients with ulcerative colitis in Russia. We carried out a study of the microbiological composition of the intestines of patients with ulcerative colitis and healthy individuals. We found significant changes in the bacteria genera and species in patients with UC compared with the control group using sequencing on the IonTorrent PGM system and subsequent data analysis. In our study we observed a significant increase of the genus Haemophilus, Olsenella, Prevotella, Cedecea, Peptostreptococcus, Faecalibacterium, Lachnospira, Negativibacillus, Butyrivibrio, and the species Bacteroides coprocola, Phascolarctobacterium succinatutens, Dialister succinatiphilus, Sutterella wadsworthensis, Faecalibacterium prausnitzii in patients with ulcerative colitis. In addition, in patients with ulcerative colitis there was a significant decrease in the genus Fusicatenibacter, Butyricimonas, Lactococcus, Eisenbergiella, Coprobacter, Cutibacterium, Falsochrobactrum, Brevundimonas, Yersinia, Leuconostoc and in the species Fusicatenibacter saccharivorans. We found confirmation of our data with literary sources and studies of UC. In addition, we discovered a few taxa such as Negativibacillus spp. and Falsochrobactrum spp. that have not been previously found in human stool samples. Our data confirm that more research is needed to understand the role of microbiome changes in the development of UC in different people populations.

[The effect of L-carnitine depletion induced by long-term therapy of mice with meldonium on brain mitochondrial balance]. / Vliianie istoshcheniia L-karnitina pri dlitel'noi terapii mel'doniem na mitokhondrial'nyi balans v mozge myshei.

Meldonium is a metabolic drug used for treatment of coronary heart disease. The effect of the drug lies in its ability to inhibit synthesis and transport of L-carnitine. At the same time, a long-term deficiency of L-carnitine can theoretically negatively affect the activity of the transcription factor Nrf2, which is extremely important for maintaining mitochondrial balance in cells. We have shown that meldonium therapy for 3 months at a dose of 100 mg/kg in mice causes a decrease in the expression of the Nrf2 gene in the brain. A decrease in the Nrf2 level causes suppression of mitochondrial biogenesis, which is manifested in a decrease in the level of mtDNA and the level of Cox1 expression. However, no negative effect of meldonium on the bioenergetics parameters of mitochondria was found, as evidenced by the maintenance of a stable mitochondrial potential and the level of production of reactive oxygen species. Jne mohth after the end of the meldonium therapy, expression of the genes responsible for mitochondrial biogenesis and mitophagy (p62, Pink1, Tfam) was observed and the expression level of genes responsible for mitochondrial fusion returned to control values. These changes may be associated with the normalization of the level of L-carnitine in brain cells.

[The fenofibrate effect on genotoxicity in brain and liver and on the expression of genes regulating fatty acids metabolism of mice]. / Vliianie fenofibrata na genotoksichnost' v mozge i pecheni i na ékspressiiu genov, reguliruiushchikh metabolizm zhirnykh kislot, u myshei.

Fibrates are well-known agonists of the PPAR family (peroxisome proliferator-activated receptors). This class of drugs is used for the treatment of dyslipidemia and atherosclerosis. Fenofibrate is one of the members of this class of synthetic PPARα receptor ligands. The oral administration of 0.3% fenofibrate caused a decrease in strength due to loss of body weight in laboratory animals when improving behavioural features. Analysis of the mitochondrial DNA of liver cells showed a genotoxic effect of fenofibrate, due to accumulation of reactive oxygen species, which could be attributed to activation of peroxisomal ß-oxidation processes, as well as to the lack of increase in the expression of genes encoding antioxidant defense proteins. Treatment with fenofibrate did not cause brain mtDNA damage. It has been shown that fenofibrate induced mitochondrial ß-oxidation in the brain, as indicated by the increased expression of the Acadm and Cpt1a and Ppargc1a and Ppara. The study found no effect of fenofibrate on the increase of mitochondrial biogenesis in brain and liver cells. Thus, we can conclude that fenofibrate significantly affects lipid metabolism in the liver and brain, but in the liver it is associated with an increase of oxidative stress, resulting in mtDNA oxidative damage. However, fenofibrate-induced increase in the expression of Ppargc1a is not associated with an increase of mitochondrial biogenesis. This is consistent with the recent suggestion that PGC-1α might not be a master regulator of mitochondrial biogenesis.

Ethylene-Insensitive Arabidopsis Mutants etr1-1 and ein2-1 Have a Decreased Freezing Tolerance.

The freezing tolerance of Arabidopsis thaliana (L.) Heynh. was studied in relation to functioning of the ethylene signaling pathway. Constitutive freezing tolerance was compared in wild-type plants (ecotype Col-0) and ethylene-insensitive mutants etr1-1 and ein2-1. For the first time it was established that the ethylene-insensitive mutants had a 25-30% lower net photosynthesis rate, a decreased content of soluble sugars, and, as a result, a lower freezing tolerance. Our work provides evidence that the perception and transduction of ethylene signal are necessary for constitutive tolerance of Arabidopsis to low temperature.

Quality Control of Bee-Collected Pollen Using Bumblebee Microcolonies and Molecular Approaches Reveals No Correlation Between Pollen Quality and Pathogen Presence.

Bee-collected pollen is an essential protein source for honey bee and bumblebee colonies. Its quality directly affects bee health. We estimated the quality of pollen samples using bumblebee microcolonies and high-throughput sequencing for the presence of microorganisms. The tested samples of bee-collected pollen were of different quality, as estimated from their effect on the development of bumblebee microcolonies. Based on the pollen quality, we selected a subset of high-quality and low-quality pollen samples to further analyze them for the presence of microorganisms and pathogens. High-throughput sequencing revealed that the most common microorganisms in the bee-collected pollen were Acinetobacter spp. and bacteria of the genera Lactobacillus and Lactococcus. No pathogenic bacteria infectious for honey bees (e.g., those causing American and European foulbrood) or bumblebees have been identified in the analyzed pollen samples. Among potentially harmful microorganisms, there were bacteria from the Enterobacteriaceae family. The fungal pathogens Nosema apis and Nosema ceranae were detected in four samples; Ascosphaera sp. was found in six samples. Several viruses were found in the pollen samples, such as chronic bee paralysis virus, Israeli acute paralysis virus, deformed wing virus, sacbrood virus, and Kashmir bee virus. No correlation between the presence of these microorganisms or viruses and the impact of low-quality pollen samples on the bumblebee development was found. It is possible that factors affecting pollen quality are the absence of certain biologically active compounds or the presence of pesticides.

Development and validation of a TaqMan RT-PCR method for identification of mayonnaise spoilage yeast Pichia kudriavzevii.

Food spoilage and its contamination with yeast and mold is a serious problem of food industry. Despite the high fat content, mayonnaise is an attractive substrate for food spoilage microorganisms. The aim of this study was to develop a method for yeast identification in mayonnaise and to test commercially available mayonnaises for the presence of these contaminating microorganisms. Based on the sequencing of intergenic regions ITS1 and ITS2, we identified a yeast microorganism that causes mayonnaise spoilage. We found that DNA sequences were more than 99% identical to the GenBank DNA sequences from Pichia kudriavzevii. We developed a specific to P. kudriavzevii TaqMan probe and primers. The reaction conditions were optimized regarding to the components concentration and temperature cycle. The minimum amount of P. kudriavzevii DNA that could be detected by developed method was 50 fg. The minimal number of P. kudriavzevii cells that could be detected by developed method without pre-enrichment was 50. We tested verified method with DNAs from microorganisms of different taxonomic groups that were obtained from three collections of microorganisms. Finally, we analyzed 20 different brands of mayonnaise from 14 producers and 10 different brands of mayonnaise sauce from seven producers. We determined the Cq parameter that characterizes transition of the fluorescence curve to the logarithmic phase and, therefore, correlates with the extent of sample contamination with P. kudriavzevii yeast. P. kudriavzevii was detected in six analyzed samples of mayonnaise and one sample of mayonnaise sauce.

A simple molecular method for rapid identification of commercially used Amblyseius and Neoseiulus species (Acari: Phytoseiidae).

Predatory mites from the Amblyseius (Neoseiulus) genus (Family Phytoseiidae, Order Parasitiformes) are widely used for protecting plants against pests, especially thrips. The differentiation of Amblyseius and Neoseiulus species by their morphological features is problematic despite the fact that they are taxonomically different genera. The Phytoseiidae family includes a lot of species that are extremely difficult to distinguish from each other. The discovery of new molecular genetic markers might considerably facilitate the express identification of commercial mite species. Despite their high morphological similarity, the three common commercial Amblyseius and Neoseiulus mite species (Neoseiulus cucumeris, Amblyseius swirskii, and Neoseiulus barkeri) differed significantly in the nucleotide sequences of DNA fragments containing the ITS1 and ITS2 internal transcribed spacers. We found that when PCR products of the amplified DNA fragments from A. swirskii, N. cucumeris, and N. barkeri were treated with a combination of AccB1I, AspLEI, and SspI endonucleases and the resulting products were separated by electrophoresis in agarose gel, the obtained picture was sufficiently specific to provide accurate identification of the analyzed mite species. The lengths of the digestion products were different enough to allow their resolution in agarose gels. The PCR-RFLP method developed by us allows the rapid and accurate identification of commercially used Amblyseius and Neoseiulus species without DNA sequencing. The combination of the three endonucleases (AccB1I, AspLEI, and SspI) and FaeI can be used for the differentiation of all other but less frequently commercially used Phytoseiidae mites.

Changes in levels of metabolic pathway gene expression under conditions of clear cell renal carcinoma.

A whole-transcriptome analysis of gene expression in six samples of clear cell renal cancer was performed. Using bioinformatics methods, we established a relationship between gene expression data and changes in activity of metabolic pathways: in this cancer type, the expression of genes involved in the metabolism of carbohydrates, lipids, and amino acids as well as in energy metabolism changed most significantly.

[The effect of fenofibrate on expression of genes involved in fatty acids beta-oxidation and associated free-radical processes]. / Vliianie fenofibrata na ékspressiiu genov beta-okisleniia zhirnykh kislot i sviazannye s nim svobodnoradikal'nye protsessy.

Fenofibrate is a synthetic ligand for peroxisome proliferator-activated receptors subtype alpha (PPARa); it is used for the treatment of a wide range of metabolic diseases such as hypertriglyceridemia, dyslipidemia, diabetes and various neurodegenerative diseases. We have studied the effect of fenofibrate on b-oxidation of fatty acids and related free-radical processes. The most effective concentration of fenofibrate (0.3%) added to the chow caused a significant decrease of the body weight of mice. The data obtained by quantitative PCR demonstrated increased hepatic gene expression responsible for b-oxidation of fatty acids in peroxisomes and mitochondria. Enhancement of oxidative processes caused a 2-fold increase in the rate of reactive oxygen species (ROS) production, as evidenced by determination of the level of lipid peroxidation (LPO) products in the liver. Mitochondrial antioxidant systems are more sensitive to elevated ROS production, as they respond by increased expression of SOD2 and PRDX3 genes, than cytoplasmic and peroxisomal antioxidant systems, where expression of CAT1, SOD1, PRDX5 genes remained unaltered.

[ISSR analysis of some species of angustifoliate fescue].

Using ISSR analyses genetic relationships were studied between the populations of the next species of angustifoliate fescue: F. macutrensis, F. rupicola, F. arietina, F. valesiaca, F. pallens, F. psammophila and F. brevipila. High level of ISSR loci polymorphism (average 92.9%) was shown. The species specific amplicon was revealed for F. valesiaca as well as the common fragment was identified for two related species--F. pallens and F. psammophila. On the basis of NJ analyses the differentiation of species was confirmed on such large aggregates as F. glauca agg. and F. valesiaca agg. More clear differentiation of the species was shown for F. glauca agg., while for F. valesiaca agg. we have not found any regularity of genetic relations among the species that represent this aggregate.

Mitochondrial energy-dissipating systems (alternative oxidase, uncoupling proteins, and external NADH dehydrogenase) are involved in development of frost-resistance of winter wheat seedlings.

Gene expression, protein synthesis, and activities of alternative oxidase (AOX), uncoupling proteins (UCP), adenine nucleotide translocator (ANT), and non-coupled NAD(P)H dehydrogenases (NDex, NDPex, and NDin) were studied in shoots of etiolated winter wheat (Triticum aestivum L.) seedlings after exposure to hardening low positive (2°C for 7 days) and freezing (-2°C for 2 days) temperatures. The cold hardening efficiently increased frost-resistance of the seedlings and decreased the generation of reactive oxygen species (ROS) during further cold shock. Functioning of mitochondrial energy-dissipating systems can represent a mechanism responsible for the decrease in ROS under these conditions. These systems are different in their response to the action of the hardening low positive and freezing temperatures. The functioning of the first system causes induction of AOX and UCP synthesis associated with an increase in electron transfer via AOX in the mitochondrial respiratory chain and also with an increase in the sensitivity of mitochondrial non-phosphorylating respiration to linoleic and palmitic acids. The increase in electron transfer via AOX upon exposure of seedlings to hardening freezing temperature is associated with retention of a high activity of NDex. It seems that NDex but not the NDPex and NDin can play an important role in maintaining the functional state of mitochondria in heterotrophic tissues of plants under the influence of freezing temperatures. The involvement of the mitochondrial energy-dissipating systems and their possible physiological role in the adaptation of winter crops to cold and frost are discussed.

Isolation and properties of flight muscle mitochondria of the bumblebee Bombus terrestris (L.).

This report describes the isolation procedure and properties of tightly coupled flight muscle mitochondria of the bumblebee Bombus terrestris (L.). The highest respiratory control index was observed upon oxidation of pyruvate, whereas the highest respiration rates were registered upon oxidation of a combination of the following substrates: pyruvate + malate, pyruvate + proline, or pyruvate + glutamate. The respiration rates upon oxidation of malate, glutamate, glutamate + malate, or succinate were very low. At variance with flight muscle mitochondria of a number of other insects reported earlier, B. terrestris mitochondria did not show high rates of respiration supported by oxidation of proline. The maximal respiration rates were observed upon oxidation of α-glycerophosphate. Bumblebee mitochondria are capable of maintaining high membrane potential in the absence of added respiratory substrates, which was completely dissipated by the addition of rotenone, suggesting high amount of intramitochondrial NAD-linked oxidative substrates. Pyruvate and α-glycerophosphate appear to be the optimal oxidative substrates for maintaining the high rates of oxidative metabolism of the bumblebee mitochondria.

[Characteristics of functioning of succinate dehydrogenase from flight muscles of the bumblebee Bombus terrestris (L.)].

It was found that the succinate oxidation rate in mitochondria of flight muscles of Bombus terrestris L. in- creased by a factor of 2.15 after flying for 1 h. An electrophoretically homogenous preparation of succinate dehydrogenase with a specific activity of 7.14 U/mg protein and 81.55-fold purity was isolated from B. terrestris flight muscles. It is shown that this enzyme is represented in the muscle tissue by only one isoform with R,f = 0.24. The molecular weight of the native molecule and its subunits A and B was determined. The kinetic characteristics ofsuccinate dehydrogenase (Km = 0.33 mM) and the optimal concentration of hydrogen ions (pH 7.8) were established, and the effect of salts on the enzyme activity was studied. The role of succinate as a respiratory substrate in stress and the structural and functional characteristics of the succinate dehydrogenase system in the flight muscles of insects are discussed.

[Polymorphism of microsatellite loci of European chickpea cultivars].

Polymorphism of 13 microsatellite loci in 61 chickpea varieties was studied. The 47 alleles were detected. Three loci were monomorphic, the other ones showed polymorphism. Genetic diversity of chickpea varieties in all loci was 0,41 according to Nei polymorphism index (D). It was concluded that chickpea varieties from Europe had insignificant microsatellite loci diversity. The most polymorphic group was chickpea varieties from Russia (D = 0,38), the least polymorphic - Spanish varieties (D = 0,25). Consensus tree representing the most credible divergence of Europe chickpea varieties was constructed. The possible causes of clustering chickpea varieties in a common cluster are discussed.

Experimental evidence of a mechanical coupling between layers in an individual double-walled carbon nanotube.

We perform transmission electron microscopy, electron diffraction, and Raman scattering experiments on an individual suspended double-walled carbon nanotube (DWCNT). The first two techniques allow the unambiguous determination of the DWCNT structure: (12,8)@(16,14). However, the low-frequency features in the Raman spectra cannot be connected to the derived layer diameters d by means of the 1/d power law, widely used for the diameter dependence of the radial-breathing mode of single-walled nanotubes. We discuss this disagreement in terms of mechanical coupling between the layers of the DWCNT, which results in collective vibrational modes. Theoretical predictions for the breathing-like modes of the DWCNT, originating from the radial-breathing modes of the layers, are in a very good agreement with the observed Raman spectra. Moreover, the mechanical coupling qualitatively explains the observation of Raman lines of breathing-like modes, whenever only one of the layers is in resonance with the laser energy.