Role of sirtuins in epigenetic regulation and aging control.

Advances in modern healthcare in developed countries make it possible to extend the human lifespan, which is why maintaining active longevity is becoming increasingly important. After the sirtuin (SIRT) protein family was discovered, it started to be considered as a significant regulator of the physiological processes associated with aging. SIRT has deacetylase, deacylase, and ADP-ribosyltransferase activity and modifies a variety of protein substrates, including chromatin components and regulatory proteins. This multifactorial regulatory system affects many processes: cellular metabolism, mitochondrial functions, epigenetic regulation, DNA repair and more. As is expected, the activity of sirtuin proteins affects the manifestation of classic signs of aging in the body, such as cellular senescence, metabolic disorders, mitochondrial dysfunction, genomic instability, and the disruption of epigenetic regulation. Changes in the SIRT activity in human cells can also be considered a marker of aging and are involved in the genesis of various age-dependent disorders. Additionally, experimental data obtained in animal models, as well as data from population genomic studies, suggest a SIRT effect on life expectancy. At the same time, the diversity of sirtuin functions and biochemical substrates makes it extremely complicated to identify cause-and-effect relationships and the direct role of SIRT in controlling the functional state of the body. However, the SIRT influence on the epigenetic regulation of gene expression during the aging process and the development of disorders is one of the most important aspects of maintaining the homeostasis of organs and tissues. The presented review centers on the diversity of SIRT in humans and model animals. In addition to a brief description of the main SIRT enzymatic and biological activity, the review discusses its role in the epigenetic regulation of chromatin structure, including the context of the development of genome instability associated with aging. Studies on the functional connection between SIRT and longevity, as well as its effect on pathological processes associated with aging, such as chronic inflammation, fibrosis, and neuroinflammation, have been critically analyzed.

[Insulator Protein CP190 Regulates Expression of Spermatocyte Differentiation Genes in Drosophila melanogaster Male Germline].

CP190 protein is one of the key components of Drosophila insulator complexes, and its study is important for understanding the mechanisms of gene regulation during cell differentiation. However, Cp190 mutants die before reaching adulthood, which significantly complicates the study of its functions in imago. To overcome this problem and to investigate the regulatory effects of CP190 in adult tissues development, we have designed a conditional rescue system for Cp190 mutants. Using Cre/loxP-mediated recombination, the rescue construct containing Cp190 coding sequence is effectively eliminated specifically in spermatocytes, allowing us to study the effect of the mutation in male germ cells. Using high-throughput transcriptome analysis we determined the function of CP190 on gene expression in germline cells. Cp190 mutation was found to have opposite effects on tissue-specific genes, which expression is repressed by CP190, and housekeeping genes, that require CP190 for activation. Mutation of Cp190 also promoted expression of a set of spermatocyte differentiation genes that are regulated by tMAC transcriptional complex. Our results indicate that the main function of CP190 in the process of spermatogenesis is the coordination of interactions between differentiation genes and their specific transcriptional activators.

[Changes of a2-macroglobulin activity and endothelin-1 concentration in tears of rabbits after transplantation of retinal pigment epithelium cells derived from the induced pluripotent stem cells]. / Izmenenie urovnia a2-makroglobulina i éndotelina-1 v sleznoi zhidkosti krolikov posle transplantatsii kletok retinal'nogo pigmentnogo épiteliia, differentsirovannykh iz indutsirovannykh pliuripotentnykh stvolovykh kletok.

Retinal diseases accompanied with the dysfunction or death of the retinal pigment epithelial (RPE) cells are widespread, hard to treat, and appear to be a leading case of visual loss and blindness among the persons older than 55 years. Transplantation of RPE cells derived from the induced pluripotent stem cells (IPSC-RPE) is a promising method of therapy for these diseases. To ensure the transplant survival instant follow-up is required. It can be based on biochemical analyses of tear fluid that can be easily non-invasively collected. For the post-transplantation process monitoring we have choosen such polyfunctional bioregulators as α2-macroglobulin (α2-MG) and endothelin-1 (ET-1). RPE atrophy in New Zealand Albino rabbits was modeled via the subretinal injection of bevacizumab. IPSC-RPE in suspension or as a monolayer on the scaffold were transplanted subretinally 1 month after the injection. α2-MG activity and ET-1 concentration in tears were estimated during the first month and after 2, 3 and 7 months after transplantation. On the 7-14 days after transplantation α2-MG activity increased in tears of the both operated and controlateral eye probably as a reaction on the corticosteroid therapy. In 50% rabbits there was one more increase after 2-3 months that could be due to the immune inflammation. Concentration of ET-1 in tears decreased dramatically on the 7-14 days and 7 months after transplantation, and it could have an influence upon the retinal vassal tone. The data obtained show that estimation of bioregulators in tears can help monitoring local metabolic processes after RPE transplantation that is necessary for the opportune, reasonable and focused medicamental correction of post-transplantation process.

Methods of massive parallel reporter assays for investigation of enhancers.

The correct deployment of genetic programs for development and differentiation relies on finely coordinated regulation of specific gene sets. Genomic regulatory elements play an exceptional role in this process. There are few types of gene regulatory elements, including promoters, enhancers, insulators and silencers. Alterations of gene regulatory elements may cause various pathologies, including cancer, congenital disorders and autoimmune diseases. The development of high-throughput genomic assays has made it possible to significantly accelerate the accumulation of information about the characteristic epigenetic properties of regulatory elements. In combination with high-throughput studies focused on the genome-wide distribution of epigenetic marks, regulatory proteins and the spatial structure of chromatin, this significantly expands the understanding of the principles of epigenetic regulation of genes and allows potential regulatory elements to be searched for in silico. However, common experimental approaches used to study the local characteristics of chromatin have a number of technical limitations that may reduce the reliability of computational identification of genomic regulatory sequences. Taking into account the variability of the functions of epigenetic determinants and complex multicomponent regulation of genomic elements activity, their functional verification is often required. A plethora of methods have been developed to study the functional role of regulatory elements on the genome scale. Common experimental approaches for in silico identification of regulatory elements and their inherent technical limitations will be described. The present review is focused on original high-throughput methods of enhancer activity reporter analysis that are currently used to validate predicted regulatory elements and to perform de novo searches. The methods described allow assessing the functional role of the nucleotide sequence of a regulatory element, to determine its exact boundaries and to assess the influence of the local state of chromatin on the activity of enhancers and gene expression. These approaches have contributed substantially to the understanding of the fundamental principles of gene regulation.

Concentration of cell-free DNA in different tumor types.

Introduction: Cell-free DNA (cfDNA) circulates in the blood for a long time. The levels of cfDNA in the blood are assayed in cancer diagnostics because they are closely related to the tumor burden of patients.Areas covered: cfDNA escapes the action of DNA-hydrolyzing enzymes, being a part of supramolecular complexes or interacting with the plasma membrane of blood cells. cfDNA has heterogeneous size and composition, which impose various restrictions on both isolation methods and subsequent analysis. cfDNA concentration and structural changes with the development of diseases highlight the high potential of cfDNA as a diagnostic and prognostic marker. The concentration of cfDNA released in the blood by tumor cells determines the specificity of such diagnostics and the required blood volume. The present review aimed to synthesize the available data on cfDNA concentration in the cancer patient's blood as well as pre-analytical, analytical, and biological factors, which interfere with cfDNA concentration.Expert opinion: The concentration of cfDNA and tumor cell DNA (ctDNA), and the over-presentation of DNA loci in cfDNA must be considered when looking for tumor markers. Some inconsistent data on cfDNA concentrations (like those obtained by different methods) suggest that the study of cfDNA should be continued.

Data analysis algorithm for the development of extracellular miRNA-based diagnostic systems for prostate cancer.

Urine of prostate cancer (PCa) carries miRNAs originated from prostate cancer cells as a part of both nucleoprotein complexes and cell-secreted extracellular vesicles. The analysis of such miRNA-markers in urine can be a convenient option for PCa screening. The aims of this study were to reveal miRNA-markers of PCa in urine and design a robust and precise diagnostic test, based on miRNA expression analysis. The expression analysis of the 84 miRNAs in paired urine extracellular vesicles (EVs) and cell free urine supernatant samples from healthy donors, patients with benign and malignant prostate tumours was done using miRCURY LNA miRNA qPCR Panels (Exiqon, Denmark). Sets of miRNAs differentially expressed between the donor groups were found in urine EVs and urine supernatant. Diagnostically significant miRNAs were selected and algorithm of data analysis, based on expression data on 24-miRNA in urine and obtained using 17 analytical systems, was designed. The developed algorithm of data analysis describes a series of steps necessary to define cut-off values and sequentially analyze miRNA expression data according to the cut-offs to facilitate classification of subjects in case/control groups and allows to detect PCa patients with 97.5% accuracy.

[Vascular stents: Approaches used to increase their clinical efficacy]. / Stenty sosudov: podkhody, ispol'zuemye dlia povysheniia ikh klinicheskoi éffektivnosti.

Using stents for endovascular restoration of blood flow made a revolution in vascular surgery, however, despite numerous variants of stents presented on the pharmacological market, there are no stents which would completely solve the problem of restenosis in the area of stent placement. In order to decrease growth of the neointima of the stented portions of vessels, stents coated with cytostatic and cytotoxic agents were worked out. To optimize the rate of drug release it was suggested to apply them in a mixture with biodegradable or biostable polymers. Placement of drug-eluting stents in a combination with dual antiplatelet therapy made it possible to decrease frequency of restenosis and reocclusion of the restored vascular lumen in patients, however it did not solve the problem of the development of thromboses and neointimal hyperplasia in the remote postoperative period. The article provides an overview of various modifications of vascular stents, clinical studies of stents of various manufacturers, as well as modern developments in manufacturing polymer/drug coatings and methods of applying them onto the stent. This is followed by analyzing the contribution of coatings to clinical efficacy of stents and prospects of increasing efficacy of vascular stents.

[Modern polyurethanes in cardiovascular surgery]. / Sovremennye poliuretany v serdechno-sosudistoi khirurgii.

Currently, there is great clinical demand for synthetic tissue-engineered cardiovascular prostheses with good long-term patency. Polyurethanes belong to the class of polymers with excellent bio- and hemocompatibility. They are known to possess good mechanical properties, but are prone to processes of degradation in conditions of functioning in living organisms. Attempts at solving this problem have resulted in the development of various new subclasses of polyurethanes such as thermoplastic polyether polyurethanes, polyurethanes with a silicone segment, polycarbonate polyurethanes and nanocomposite polyurethanes. This was accompanied and followed by offering a series of new technologies of production of implantable medical devices such as vascular grafts, heart valves and others. In the presented review, we discuss biological and mechanical properties of modern subclasses of polyurethanes, as well as modern methods of manufacturing implantable medical devices made of polyurethanes, especially small-diameter vascular prostheses.

[Maturation and Antigen Loading Protocols Influence Activity of Anticancer Dendritic Cells].

The practical use of dendritic cell-based vaccines in anticancer therapy is limited by a lack of standards for dendritic cell (DC) generation, as well as standard procedures for controlling their activation and the technique of DC loading with nucleic acids encoding tumor antigens. Analyzing the currently available data, the most promising cocktails for DC maturation were selected and a comparative study of the cocktails and time of maturation on the capacity of DC to activate T-cell immune response has been performed. A study of the expression of surface markers and the production of IL-12, IL-6, and IL-10 cytokines, as well as the efficacy of T-cell activation showed that the use of the standard 7-day maturation protocol is preferable to the 4-day maturation protocol. Cocktails composed of TNF-α, IL-lß, IFN-α, IFN-γ, and poly(I:C), as well as TNF-α, IL-lß, IFN-γ, R848, and PGE2 were shown to be the most efficient activators of DCs. A comparison of the efficacy of different methods of DNA transfection into DCs and RNA delivery using alphavirus vectors demonstrated the superiority of magnet-assisted transfection (MATra) to other protocols.

[Representation analysis of miRNA from clarified urine and urine microvesicles in prostate malignancies and non-malignant neoplasms]. / Analiz predstavlennosti mikroRNK v mikrovezikulakh mochi i beskletochnoi moche pri zabolevaniiakh predstatel'noi zhelezy.

Urine of prostate cancer patients contains tumor-specific biopolymers, including protein- and microvesiclesassociated miRNAs that can potentially be used as oncomarkers. Previously we have characterized urine extracellular vesicles and demonstrated diagnostic potential of their miRNA cargo. In this study, we have performed a comparative analysis of the expression of 84 miRNA in paired samples of urine microvesicles and clarified urine from healthy men, patients with benign hyperplasia and cancer of the prostate using miRCURY LNA miRNA qPCR Panels. Subsets of miRNAs with differences in expression between the fractions of the urine were found in all three groups. Two groups of miRNA were identified based on the patterns of their differential expression. They regulate several key signaling pathways associated with prostate cancer development.

[Comparative sub-population analysis of exosomes from blood plasma of cancer patients]. / Sravnitel'nyi subpopuliatsionnyi analiz ékzosom plazmy krovi bol'nykh zlokachestvennymi novoobrazovaniiami.

To increase the sensitivity and specificity of the developed methods for diagnosis of oncological diseases using exosomes of blood, a stage of pre-selection of tumor exosomes from a common pool of circulating microvesicles is required. In the present work, universal proteins have been identified, their expression has been increased in the exosomes of patients with colorectal cancer, head and neck squamous cell carcinomas, and lung cancer. The use of antibodies against major exosomal proteins will further develop a simple and high-performance method of affinity isolation of tumor exosomes.

[Dynamics of LINE-1 Retrotransposon Methylation Levels in Circulating DNA from Lung Cancer Patients Undergoing Antitumor Therapy].

Malignant cell transformation is accompanied with abnormal DNA methylation, such as the hypermethylation of certain gene promoters and hypomethylation of retrotransposons. In particular, the hypomethylation of the human-specific family of LINE-1 retrotransposons was observed in lung cancer tissues. It is also known that the circulating DNA (cirDNA) of blood plasma and cell-surface-bound circulating DNA (csb-cirDNA) of cancer patients accumulate tumor-specific aberrantly methylated DNA fragments, which are currently considered to be valuable cancer markers. This work compares LINE-1 retrotransposon methylation patterns in cirDNA of 16 lung cancer patients before and after treatment. CirDNA was isolated from blood plasma, and csb-cirDNA fractions were obtained by successive elution with EDTA-containing phosphate buffered saline and trypsin. Concentrations of methylated LINE-1 region 1 copies (LINE-1-met) were assayed by real-time methylation-specific PCR. LINE-1 methylation levels were normalized to the concentration of LINE-1 region 2, which was independent of the methylation status (LINE-1-Ind). The concentrations of LINE-1-met and LINE-1-Ind in csb-cirDNA of lung cancer patients exhibited correlations before treatment (r = 0.54), after chemotherapy (r = 0.72), and after surgery (r = 0.83) (P < 0.05, Spearman rank test). In the total group of patients, the level of LINE-1 methylation (determined as the LINE-1-met/LINE-1-Ind ratio) was shown to increase significantly during the follow-up after chemotherapy (P < 0.05, paired t test) and after surgery compared to the level of methylation before treatment (P < 0.05, paired t test). The revealed association between the level of LINE-1 methylation and the effect of antitumor therapy was more pronounced in squamous cell lung cancer than in adenocarcinoma (P < 0.05 and P > 0.05, respectively). These results suggest a need for the further investigation of dynamic changes in levels of LINE-1 methylation depending on the antitumor therapy.

[Current methods of extracellular DNA methylation analysis].

The discovery of the enormous role methylated cytosine plays in regulating gene expression has led to the development of a variety of techniques for detecting cytosine modification. A majority of these techniques are geared towards analyzing genomic DNA, which is typically available in large quantities. The concentration of cell-free DNAs (cfDNA) extracted from biological fluids including plasma, saliva, tears, or urine is relatively low and their degree of the fragmentation is high. Moreover, for noninvasive diagnostics of cancer, methylation patterns must be studied in minor cancer-specific fractions of DNA molecules substantially diluted by excess unmethylated molecules. The above limitations complicate the application of traditional techniques for cfDNA methylation analysis. In this manuscript, we review the state-of-art analysis of cfDNA methylation, hydroxymethylation, and noncanonical methylation (outside of CpG islands). The review covers methodological approaches to studying individual CpGs and genomic loci, as well as techniques for the large-scale analysis of methylation.

[Isolation and characterization of exosomes from blood plasma of breast cancer and colorectal cancer patients]. / Vydelenie i kharakterizatsiia ékzosom plazmy krovi bol'nykh rakom molochnoi zhelezy i kolorektal'nym rakom.

A simple approach for isolation of exosomes from the blood plasma, which allows to obtain highly purified preparations of microvesicles no larger than 100 nm has been proposed. The presence of different subpopulations of exosomes in the blood plasma of healthy donors and cancer patients has been recognized. We found the presence of the universal markers CD9, CD24 and CD81 on exosomes isolated from blood plasma that can be used to their routine typing.

[Investigation of the surface layer of 3D-matrices for tissue engineering]. / Issledovanie poverkhnostnogo sloia 3D-matriksov dlia tkanevoi inzhenerii.

Electrospinning is a convenient and promising manufacturing method a variety of materials for tissue engineering. 3D matrices fabricated by electrospinning from solutions of polycaprolactone with human serum albumin or gelatin in 1,1,1,3,3,3-hexafluoroisopropanol were studied. The microstructure of the 3D matrices and surface of the fibers were investigated using scanning electron microscopy. Protein distribution in the surface layer was studied by modification of protein amino groups with N-(2-hydroxyethyl)phenazine and X-ray photoelectron spectroscopy. It was shown, that concentration of the proteins in the surface layer of fibers exceeded their concentration in the initial electrospun solution up to 12 times and the surface layer was enriched in the protein inversely to the concentration of the protein in solution. The minor part of the proteins was released from fibers during first 30-60 min after swelling in water. Treatment of matrices with proteinase K hydrolyzed about 1/3 of the surface exposed human serum albumin. Thus, both methods can be used to study the surface content of the materials produced by electrospinning from blends of synthetic and natural polymers, however X-ray photoelectron spectroscopy appears to be more convenient and informative.

[Characteristics of exosomes andmicroparticles discovered in human tears].

Exosomes represent a sort of extracellular vesicles, which transfer molecular signals in organism and possess markers of producing cells. Our study was aimed at search of exosomes in the tears of healthy humans, confirmation of their nature and examination of exosome morphological and molecular-biological characteristics. The tears (110-340 ml) were collected from 24 healthy donors (aged 46-60 years); individual probes were centrifuged at 20000 g for 15 min to pellet cell debris. The supernatants were examined in electron microscope using negative staining; and they were also used for isolation and purification of the exosomes by filtration (100 nm pore-size) and double ultracentrifugation (90 min at 100000 g, 4°C). The "pellets" were subjected to electron microscopy, immunolabeling. The RNA and DNA were isolated from the samples, and their sizes were evaluated by capillary electrophoresis, the concentration and localization of nucleic acids were determined. Sequencing of DNA was performed using MiSeq ("Illumina", USA), data were analyzed using CLC GW 7.5 ("Qiagen", USA). Sequences were mapped on human genome (hg19). Electron microscopy revealed in supernatants of the tears cell debris, spherical microparticles (20-40 nm), membrane vesicles and macromolecular aggregates. The "pellets" obtained after ultracentrifugation, contained microparticles (17%), spherical and cup-shaped EVs (40-100 nm, 83%), which were positive for CD63, CD9 and CD24 receptors (specific markers of exosomes). Our study showed presence of high amount of exosomes in human tears, and relation of the exosomes with RNA (size less than 200 nt) and DNA (size was 3-9 kb). Sequencing of the DNA showed that about 92% of the reads mapped to human genome.

Design of Polyepitope DNA Vaccine against Breast Carcinoma Cells and Analysis of Its Expression in Dendritic Cells.

Polyepitope DNA vaccine inducing T-cell-mediated immune response against cancer-specific antigens is a promising tool for selective elimination of tumor cells. Breast cancer-specific polyepitope DNA vaccine was designed using TEpredict and PolyCTLDesigner software on the basis of immunogenic peptides of HER2 and Mammaglobin-1 (Mam) tumor antigens. LPS-free preparations of plasmid DNA encoding polyepitope T-cell antigen and full-length copies of HER2 and Mam antigens were obtained. TaqMan-PCR systems for evaluation of the expression of immunogens in cells were created. The protocol of vaccine DNA delivery into dendritic cells was optimized. Expression of the target immunogens in dendritic cells derived from human peripheral blood mononuclear fraction after transfection with plasmid DNA preparations is demonstrated.

Extracellular Nucleic Acids in Urine: Sources, Structure, Diagnostic Potential.

Cell-free nucleic acids (cfNA) may reach the urine through cell necrosis or apoptosis, active secretion of nucleic acids by healthy and tumor cells of the urinary tract, and transport of circulating nucleic acids (cir- NA) from the blood into primary urine. Even though urinary DNA and RNA are fragmented, they can be used to detect marker sequences. MicroRNAs are also of interest as diagnostic probes. The stability of cfNA in the urine is determined by their structure and packaging into supramolecular complexes and by nuclease activity in the urine. This review summarizes current data on the sources of urinary cfNA, their structural features, diagnostic potential and factors affecting their stability.

[Generation of blood circulating DNA: the sources, peculiarities of circulation and structure].

Extracellular nucleic acids (exNA) were described in blood of both healthy and illness people as early as in 1948, but staied overlooked until middle 60th. Starting from the beginning of new millennium and mainly in the last 5 years exNA are intensively studied. Main attention is directed to investigation of exNA as the source of diagnostic material whereas the mechanisms of their generation, as well as mechanisms to providing long-term circulation of exNA in the bloodstream are not established unambiguously. According to some authors, the main source of circulating nucleic acids in blood are the processes of apoptosis and necrosis, while others refer to the possible nucleic acid secretion by healthy and tumor cells. Circulating DNA were found to be stable in the blood for a long time, escaping from the action of DNA hydrolyzing enzymes and are apparently packed in different supramolecular complexes. This review presents the opinions of various authors and evidence in favor of all the theories describingappearance of extracellular DNA, the features of the circulation and structure of the extracellular DNA and factors affecting the time of DNA circulation in blood.