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A series of polyurethanes, polyureas, and poly(urethane ureas) with flexible blocks based on α,ω-bis(hydroxyalkyl)polydimethylsiloxane and α,ω-bis(aminopropyl)polydimethylsiloxane with molecular weights (5.6 and 0.9) × 103, respectively, is synthesized to obtain artificial vessels by electrospinning. The structure and properties of the polymers are characterized by gel permeation chromatography, differential scanning calorimetry, thermogravimetry, and dynamic mechanical analysis. It is found that polydimethylsiloxane segments in the polymer structure form their phase with a glass transition temperature characteristic of polydimethylsiloxane. The features of fiber spinning from solutions of the synthesized elastomers are studied. It is shown that the storage modulus of the polymers has a great influence on the morphology of the presented fibers.

Epigenetic regulation of gene expression plays a key role in controlling many cellular processes. One essential element of such regulation are miRNAs—a class of short non-coding RNAs. The miRNAs were first discovered in 1993, but their active investigation was not started until early 2000s. Recent data show that miRNAs regulate the expression of at least half of human genes. Being involved in regulation of numerous target genes implicated in cell functioning, miRNA activity is critical for normal human development and function, while dysregulation of miRNA is a hallmark of various pathophysiological processes. Current review introduces the reader to the main stages of miRNA life cycle in human cells. The origins of cellular miRNAs, their biogenesis, functions, and regulation of their activity are discussed. The publication is aimed at readers looking for first insights into the intricacies of miRNA biology, but can also serve as a source of reference material for specialists in the area.

To improve the endothelization of 3D matrices produced by electrospinning we propose adding cyclo(RGDfC) peptide conjugated to human serum albumin (HSA) (HSA-c(RGDfC)) to the electrospinning solution. HSA-c(RGDfC) has been prepared using a bifunctional linker with N-hydroxysuccinimide and maleimide groups, and the product has been confirmed by 1Н NMR spectroscopy. Electrospun matrices have been fabricated from the solutions of 3% polyurethane Tecoflex EG-80A with 10% HSA and 0.01–1.3% HSA–c(RGDfC) (relative to Tec) in 1,1,1,3,3,3-hexafluoro-2-propanol. The presence of HSA and the cyclo(RGDfC) peptide on the surface of electrospun matrices has been confirmed by FTIR spectroscopy. The structure of matrices has been examined by scanning electron microscopy. The ability of the matrices to facilitate the adhesion of primary human umbilical vein endotheliocytes (HUVECs) has been studied. The presence of HSA–c(RGDfC) conjugate in the matrices enhanced cellular adhesion in a dose dependent manner with a maximum effect at 1% HSA-c(RGDfC).

Pre-selection of tumor exosomes from a total pool of circulating microvesicles is an important stage required to increase sensitivity and specificity of the developing methods for diagnostics of oncological diseases. In this study, universal proteins characterized by increased expression in plasma exosomes have been identified in patients with colorectal cancer, head and neck squamous cell carcinomas, and lung cancer. The use of antibodies against major exosomal proteins will help to develop a simple and high-performance method of affinity isolation of tumor exosomes.

Urine of prostate cancer patients contains tumor-specific biopolymers, including protein- and microvesicles-associated miRNAs that can potentially be used as oncomarkers. Previously we have characterized urinary extracellular vesicles and demonstrated diagnostic potential of their miRNAs. In this study, we have performed a comparative analysis of 84 miRNA in paired samples of urine microvesicles and urine supernatant from healthy men, patients with benign prostate hyperplasia, and prostate cancer patients using miRCURY LNA miRNA qPCR Panels. In all groups of patients, miRNA subsets characterized by different distribution between the urinary fractions have been found. In this context, two groups of miRNAs have been identified, which are involved in several signaling pathways including those associated with prostate cancer development.

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-1β, IFN-α, IFN-γ, and poly(I:C), as well as TNF-α, IL-1β, 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.

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.Predlozhen uproshchennyĭ sposob vydeleniia ékzosom iz plazmy krovi, pozvoliaiushchiĭ poluchit' vysokoochishchennye preparaty mikrovezikul razmerom ne bolee 100 nm. Ustanovleno nalichie razlichnykh subpopuliatsiĭ ékzosom kak v plazme krovi zdorovykh donorov, tak i onkologicheskikh bol'nykh. Universal'nye markery CD9, CD24 i CD81 mogut byt' ispol'zovany dlia rutinnogo tipirovaniia ékzosom.

Matrices fabricated by electrospinning from polycaprolactone solutions with human albumin or gelatin to 1,1,1,3,3,3-hexafluoroisopropanol have been investigated. Microstructure of matriх surface was analyzed using scanning electron microscopy. Protein distribution in the surface layer was studied by modification with N-(2-hydroxyethyl)phenazine and X-ray photoelectron spectroscopy. The protein concentration in the surface layer of matrices was up to 12 times higher than in the initial solution, and the lower the protein concentration in the solution, the higher the relative protein content is on the surface of matrices. During incubation of matrices in aqueous solutions protein concentration in the surface layer decreased by less than 10% during the first 30–60 min and remained at this level for a long time (seven days). Treatment with proteinase K resulted in about one-third decrease in protein concentration in the surface layer. Thus, both methods used in this study are applicable for analysis of the surface layer of materials fabricated by electrospinning from mixtures of synthetic and natural polymers; however, X-ray photoelectron spectroscopy appears to be a more informative and convenient method.

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.

Microvesicles were isolated from blood plasma and total blood of healthy females and breast cancer patients by filtration and ultracentrifugation. According to flow cytometry, different subpopulations of exosomes were represented in blood of healthy donors and cancer patients at different levels with median fluorescence intensity (MFI) values in both groups arranged in the following order: CD24/СD9 > СD9/СD81 > CD9/CD63 = CD24/CD63. Concentration of exosomes in blood plasma of healthy females estimated by nanoparticle tracking analysis (NTA) did not exceed (3.71 ± 1.15) × 107 particles/mL of blood and did not differ from that in plasma of breast cancer patients, which averaged (3.99 ± 1.03) × 107 particles/mL of blood. Concentration of total exosomes in blood (including exosomes from plasma and blood cell surface-bound exosomes) did not depend on the presence/absence of a tumor; the values were (7.66 ± 0.7) × 107 particles/mL of healthy blood and (9.4 ± 1.24) × 107 particles/mL of blood from cancer patients. Comparative analysis of exosomes using 2-D electrophoresis with subsequent analysis of 2-D proteomic maps revealed proteins missing in blood or differentially expressed in healthy females and breast cancer women. The data presented provide the possibility for identification of exosomal proteomic markers and isolation of tumor-specific exosomes, which contributes to the development of breast cancer diagnostics.

Cells of almost all tissues secrete to the extracellular environment a variety of vesicular structures. The most interesting vesicles are exosomes–microvesicles ranging from 30 to 100 nm in size. These vesicles contain various RNA, including mRNA, microRNA, as well as membrane and cytoplasmic proteins that can be transported in these particles to nearby and distantly located cells of various tissues using physiological fluids (blood, urine, saliva, etc.). Exosomes are necessary for normal functioning of the organism and their repertoire changes during the development of pathologies. This review presents the data on generation, secretion, and transport of exosomes, their structure and roles in normal conditions and in the process of the malignant tumor development. Prospects of the application of exosomal biomarkers for the development of early non-invasive cancer diagnosis are also discussed.

Exosomes represent a sort of extracellular vesicles, which transfer molecular signals in the body and contain markers of the exosome-producing cell. This study was aimed at search of exosomes in the tears of healthy humans, validation of their nature and examination of their morphological and molecular-biological characteristics. Samples of the tears individually collected from 24 healthy donors (aged 45–60 years) were centrifuged at 20000 g for 15 min to pellet cell debris. The supernatants were examined in an electron microscope using negative staining and were also used for isolation and purification of exosomes by filtration (100 nm pore-size) and double ultracentrifugation (90 min at 100000 g, 4°C). Resultant pellets were investigated by electron microscopy and immunolabeling, RNA and DNA were isolated and their sizes were determined by capillary electrophoresis, concentration and localization of nucleic acids in the isolated exosomes were studied. DNA sequencing was performed using MiSeq (Illumina, USA), data were analyzed using CLC GW 7.5 (Qiagen, USA). Sequences were mapped on human genome (hg19). Supernatants of the tears contained cell debris, spherical microparticles (20–40 nm), and vesicles; some of the vesicles had morphology and sizes corresponding to exosomes. The pellets obtained after ultracentrifugation of tears contained microparticles (17%), spherical and cup-shaped vesicles (40–100 nm, 83%), which were positive for CD63, CD9, and CD24 receptors (specific markers of exosomes). The study revealed high concentrations of exosomes in human tears; these exosomes contained both RNA (of less than 200 nucleotides in size) and DNA (of 3–9 kb in size). DNA sequencing demonstrated that about 92% of the reads was mapped to human genome.

The main components of DNA aberrant methylation in malignant cells are promoter hypermethylation of specific genes and hypomethylation of the main part of DNA, in particular, the repeated sequences of retrotransposons. The hypomethylation of retrotransposons from the LINE-1 family was revealed in the lung cancer (LC) tissue. The composition of circulating DNA (cirDNA) from the plasma and blood cell surface bound circulating DNA (csb-cirDNA) was shown earlier to be altered in the blood of cancer patients due to the accumulation of tumor-specific aberrantly methylated DNA fragments, which are currently considered to be valuable cancer markers. The present study compares LINE-1 retrotransposon methylation patterns in the cirDNA and csb-cirDNA plasma from 21 untreated LC patients and 23 healthy donors. Concentrations of methylated LINE-1 region 1 copies (LINE-1met) were assessed by quantitative real-time methylation-specific PCR. In order to normalize the LINE-1 methylation level, the LINE-1 region 2 concentration was evaluated, which was independent of the methylation status (LINE-1Ind). The LINE1met concentration in csb-cirDNA tended to decrease (by a factor of 1.4) in blood from LC patients compared to healthy donors (Mann–Whitney test, P = 0.16). The LINE-1Ind (methylation-independent) concentration in csb-cirDNA was found to be lower by a factor of three in LC patients and by a factor of four in patients with adenocarcinoma than in healthy donors. Thus, along with the expected decrease in the LINE1met concentration in csb-cirDNA from LC patients’ blood, we recorded an unexpected statistically significant increase of the LINE-1 methylation index determined as (LINE-1met/LINE-1Ind) due to the profound decrease of the total LINE-1Ind number. There were no differences between the plasma cirDNA LINE-1 methylation indexes in the LC patients and healthy donors (Mann–Whitney test, P = 0.40). The data obtained agreed with our earlier results, which showed that csb-cirDNA was highly informative material for lung cancer diagnostics.

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.

The review summarizes literature data on modern instrumental, microscopic, and molecular (metabolomics, proteomics, genetics and epigenetics) methods currently used for early breast cancer diagnostics. Special attention is paid to analytical capacities and perspectives of their application in clinical practice.

Extracellular nucleic acids (exNAs) were originally described in blood of healthy and sick people in 1948; however, little attention was paid to them until 1960th. exNAs are intensively studied now, especially during the last five years. The major attention is paid to exNAs as the source of a diagnostic material, whereas mechanisms of their generation, as well as mechanisms responsible for their long-term circulation in blood-stream still need better understanding. Some authors believe that the processes of apoptosis and necrosis represent the main source of blood circulating deoxyribonucleic acids (cirDNA), while others suggest nucleic acid secretion by healthy and tumor cells. Circulating DNA were found to be stable in blood for a long time, escaping from the action of DNA hydrolyzing enzymes, possibly, due to formation of various supramolecular complexes. This review summarizes literature data which support all the theories describing appearance of cirDNA; certain attention is paid to features of circulation and structure of the cirDNA and factors affecting time of DNA circulation in blood.

To study DNA transport in isolated mitochondria from potato tubers (Solanum tuberosum), we developed an approach permitting the analysis of internalized DNA by quantitative PCR. It was shown that mitochondrial transport of recombinant plasmid pBluescript IIKS (+) of the size of 3050 bp and DNA fragment of 89 bp amplified from human LINE1 repeat did take place, apparently through different membrane translocation mechanisms. Regardless of the length of the polynucleotide chain, DNA transport into mitochondria was a reversible process.

The review considers the main approaches to the identification of lung cancer (LC) markers, including genetic, epigenetic, protein, transcriptomic, proteomic, metabolic, and miRNA markers. Emphasis is placed on epigenetic markers, which are the most promising because epigenetic changes are among the earliest events in malignant transformation. Special attention is given to circulating tumor markers, which can be detected in easily accessible biological fluids with minimally invasive methods and may be useful for screening the risk groups for LC, diagnosing cancer before its clinical manifestation, monitoring the tumor in remission after therapy, and verifying the diagnosis based on standard clinical and instrumental methods of diagnostics. Extracellular nucleic acids (circulating in blood, circNA) are highlighted as a potential source of material for early diagnosis of LC, prediction of the efficiency of antitumor treatment, posttreatment monitoring, and disease prognosis.

Intensive studies of molecular mechanisms responsible for tumor transformation results in identification of new proteins and their genes involved into tumor development. These proteins may be used as markers of tumor transformation of cells and the level of their expression may be evaluated by means of modern highly sensitive and technological methods of analysis. This review summarized literature data on currently used immunohistochemical and molecular genetic markers of gastric cancer. It highlights genetic and epigenetic changes detected in nucleic acids of tumor tissue cells in malignant and benign gastric diseases as well as in the level of DNA circulating in blood of patients with gastric cancer.

Extracellular DNA and RNA were extracted from blood plasma and cell surface-bound fractions of healthy women and patients with fibroadenoma and breast cancer. Frequency of methylation of RASSF1A, Cyclin D2, and RARβ2 genes was detected in the extracellular DNA using methylation-specific PCR. Methylation of at least one of these genes was found in plasma of 13% patients with nonmalignant breast fibroadenoma and in 60% of breast cancer patients. Employment cell-surface bound DNA as the substrate for PCR increased the detection frequency of gene methylation up to 87% in patients with fibroadenoma and 95% in breast cancer patients. In clinically healthy women the methylation markers have not been found in extracellular DNA. GAPDH, RASSF8, Ki-67 mRNAs, and 18S rRNA copies were quantified using RT-qPCR of extracellular RNA circulating in blood of patients with breast tumors and healthy controls. The major part of blood extracellular RNA is associated with cell surface. ROC analysis has shown that differences in concentrations 18S RNA, RASSF8, and Ki-67 mRNAs in blood plasma are highly sensitive and specific in discrimination of benign and malignant breast tumors. Thus, analysis of methylated forms of tumor suppressor genes in blood extracellular and quantification of specific extracellular RNA circulating in blood plasma may detect mammary gland tumors and discriminate malignant and benign neoplasms.

Circulating nucleic acids were discovered more than 30 years ago, but did not attract much attention until the past decade. This review summarizes the data on the sources of extracellular DNA circulating in the blood, features of its circulation, and pathways of its removal. The possibility of using circulating DNA in medical diagnosis is discussed.

The data about deoxyribonuclease activity in blood of healthy donors and patients with malignant and autoimmune diseases are described. Methodological aspects of the analysis of DNase activity and factors which interfere with DNase activity in blood are discussed.