[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.

[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.

[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.

[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.

[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.

[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.

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.

[Circulating microRNAs in lung cancer: prospects for diagnostics, prognosis and prediction of antitumor treatment efficiency].

The major methods of microRNA extraction from different biological fluids (particularly, serum and plasma), approaches to the analysis of microRNA concentration and composition, normalization methods used in data analysis are outlined in the review. The advantages and disadvantages of the described methodological approaches are being highlighted. Special attention is given to microRNAs, circulating in blood, which could be used as the markers for minimally invasive lung cancer diagnostics, prediction of antitumor treatment efficiency and disease prognosis. Prospects and limitations arising from the evaluation of clinical significance of microRNAs as the potential tumor markers, and emerging as roles of various microRNAs in the pathogenesis of lung cancer become known, are discussed.

[Protein Identification of Blood Nucleoprotein Complexes].

Circulating nucleoprotein complexes were isolated-from blood plasma by affinity chromatography using immobilized polyclonal anti-histone antibodies. It was found, that the main part of DNA from histone-contained nucleoprotein complexes have size 170-180 b.p., in blood of breast cancer patients DNA with size 170-180 b.p. and DNA more then 6 k.b.p. are presented in equal quantity. Proteins from circulating nucleoprotein complexes were identified using MALDI-TOF mass-spectrometry. It was shown that nucleoprotein complexes from blood of breast cancer patients contain tumor-specific proteins, such as LDOC1L, ADP/ATP translocase 3 and Lamellipodin. These data indicate, that a part of circulating extracellular DNA have tumor origin.

[Study of patency of vascular grafts manufactured by means of electrospinning].

UNLABELLED: In vivo experiments were carried out to study functioning of vascular grafts manufactured by means of electrospinning from solutions of polycaprolactone (PCL) and hexafluoroisopropanol (HFIP), PCL with 10% gelatine and a low-permeability inner layer (LPIL) 10 µm thick and PCL with 10% gelatine and LPIL (10 µm) wherein as polymeric base instead of PCL copolymer of lactic and hydroxyacetic acids (polylactide-co-glycolide, PLGA) was used. The grafts were implanted into the infrarenal portion of the aorta to 45 rats, 15 rats for each type of the graft. Patency of artificial vessels was assessed by means of magnetic resonance tomography and diagnostic ultrasound Dopplerography at 2, 4 and 20 weeks (5 animals for each time point). The state of the graft and surrounding tissues was analysed by means of intraoperative assessment, survey microscopy and survey fluorescent microscopy. CONCLUSION: The obtained findings demonstrated that vascular grafts made by electrospinning technique with a low-permeability inner layer are less prone to formation of the neointima and stenosing as compared with grafts having no such layer.

[Transcription factor comr acts as a direct activator in the genetic program controlling spermatogenesis in D. melanogaster].

Cardiff University, Cardiff CF10 3AX, UK). In Drosophila melanogaster differentiation of the male germ cells is accompanied by chromatin rearrangement and activation of the specific genes. These processes are regulated by few transcription factors that belong to two classes, can and aly that form distinct functional complexes. Mechanisms of action of aly and can class transcription factors on gene expression and chromatin state remain unclear. To investigate this question we have built the whole genome binding profile of transcription factor Comr belonging to aly class using the tissue-specific DamID method. Resulting datawere correlated with gene expression in comr (aly class) and can (can class) mutant testes. It was shown that Comr is a direct activator for about 300 testis-specific genes. Furthermore a set of genes revealed decreased expression in comr mutants but did not bind Comr protein, suggesting the existence of secondary regulation. Indeed, among the Comr gene targets we found a gene coding an uncharacterized transcription factor that could be a secondary participant in the genetic pathway in spermatocytes. These date allowed us to advance a model of gene activation needed for male gametes differentiation in D. melanogaster.

MIRA analysis of RARß2 gene methylation in DNA circulating in the blood in lung cancer.

Analysis of DNA epigenetic mutations in the blood circulating DNA is a prospective trend for creation of noninvasive methods for the diagnosis and treatment efficiency monitoring in cancer. The methylation status of target genes in circulating DNA was evaluated by methods based on preliminary bisulfite conversion of DNA. We used a different approach based on selection of hypermethylated sequences of circulating DNA by means of DNA-methyl-binding protein (methylated CpG island recovery assay, MIRA). Methylation was evaluated for RARß2 tumor suppression gene in circulating DNA in lung cancer and a trend was detected to higher methylation of this gene in the patients in comparison with healthy donors.

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.

Influence of radical prostatectomy on miRNA dynamics in urine extracellular vesicles.

PURPOSE: Cancer statistics demonstrate leading growth of prostate cancer. As a rule, radical prostatectomy (RP) is a mandatory option in the treatment of localized prostate cancer (PCa). Over 30% of patients develop biochemical resistance after the surgery and over 30% of these patients experience prostate cancer recurrence and metastasis. Currently used PCa patient's diagnostic features fail to identify PCa recurrence. To identify the risk group of PCa patients after RP we attempt to apply miRNAs which were shown as promising liquid biopsy markers for PCa diagnosis and prognosis. MATERIALS AND METHODS: Expression of 14 miRNAs closely involved in the development of prostate cancer from urine extracellular vesicles (uEV) of PCa patients before as well as 3, 6 and 12 months after radical prostatectomy was assessed using RT PCR and compared with their expression from uEV of healthy donors in the current study. RESULTS: It was shown that 22 miRNA pairs prognostic ratios (MPPRs) significantly changed after radical prostatectomy. MPPRs the most promising in terms of evaluating the effectiveness of radical prostatectomy have been identified. These include two groups: MPPRs significantly changed after surgery towards that in healthy donors; and MPPRs, which divided PCa patients into two significantly different subgroups 3 or 6 months after radical prostatectomy. CONCLUSIONS: The obtained data indicate that urine EVs represent a valuable source of both MPDR and MPPR for prostate cancer.

[Domain regulation of gene expression in the intercalary heterochromatin of Drosophila melanogaster].

A significant part of Drosophila genome is repressed in most cells. These areas, called intercalary heterochromatin regions, contain a significant amount of genes. Most of these genes work in well-defined cell types, that is, are tissue-specific. The most numerous class of genes in the intercalary heterochromatin are testis-specific genes. These genes are activated only in maturing spermatocytes and their coordinated activation is necessary for the normal spermatogenesis. Our work aims to study the mechanism of activation of testis-specific genes. We have found that Comr, one of the factors required for their transcription, is associated with extensive regions of chromosomes, which are often coextensive with the repressed parts of the salivary glands chromosomes. However, Comr binding to the large chromatin domains leads to the selective activation of testis-specific genes only. Our results suggest that, at the initial stages, activation of the testis-specific genes involves the entire domains of intercalary heterochromatin.

[A genetic system for somatic and germinal lineage tracing in the Drosophila melanogaster gonads].

Significant progress in the developmental biology of Drosophila is largely due to the improvement of methods of genetic manipulation and, in particular, development of ways to create mosaic organisms. The main characteristic of the mosaic organisms is the presence of genetically different populations of cells. For example, some tissues express a transgenic reporter gene that is absent in other cells of the body. This principle is used in a variety of the methods with the common name lineage tracing. The essence of these approaches is to perform the targeted changes in the genetic apparatus of progenitor cells that give rise to cell lines or organs and tissues. Genetic modification in progenitor cells, such as the ability to express a fluorescent protein, will be inherited by the next cell generations, and, as a result, the entire cell line or tissue will have a tag, which distinguishes it from the rest of the body. The lineage tracing methods allow tracking the cell generations, studying the cell proliferation process, tracing their origin and investigating the function of genes of interest in the development of a single tissue or organ. We have designed an approach to selectively label germ line or somatic cells in the gonads of Drosophila.

[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.

A method for generating selective DNA probes for the analysis of C-negative regions in human chromosomes.

Linker-adapter polymerase chain reaction (LA-PCR) is among the most efficient techniques for whole genome DNA amplification. The key stage in LA-PCR is the hydrolysis of a DNA sample with restriction endonucleases, and the choice of a restriction endonuclease (or several endonucleases) determines the composition of DNA probes generated in LA-PCR. Computer analysis of the localization of the restriction sites in human genome has allowed us to propose an efficient technique for generating DNA probes by LA-PCR using the restriction endonucleases HaeIII and RsaI. In silico hydrolysis of human genomic DNA with endonucleases HaeIII and RsaI demonstrate that 100- to 1,000-bp DNA fragments are more abundant in the gene-rich regions. Applying in situ hybridization to metaphase chromosomes, we demonstrated that the produced DNA probes predominantly hybridized to the C-negative chromosomal regions, whereas the FISH signal was almost absent in the C-positive regions. The described protocol for generating DNA probes may be successfully used in subsequent cytogenetic analysis of the C-negative chromosomal regions.

[Molecular-genetic markers in lung cancer diagnostics].

The major approaches to different lung cancer marker development are outlined in the review, including genetic, epigenetic, protein, transcryptomic, proteomic, metabolic, and miRNA markers. As far as epigenetic changes are among the earliest events in malignant transformation, methylated markers are thoroughly discussed. Special attention is given to minimally invasive tumor markers, which could be detected in easily accessible biological fluids, because they can be useful for screening and early diagnostics of cancer (before its clinical manifestation) as well as for verification of standard methods of diagnostics. Extracellular nucleic acids, circulating in blood (cirNA), are highlighted as the potential source of material for the early lung cancer diagnostics, prediction of antitumor treatment efficiency, post-treatment monitoring and disease prognosis.