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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.

Background. Extracellular vesicles (EVs) play an essential role in the communication between cells and transport of diagnostically significant molecules. A wide diversity of approaches utilizing different biochemical properties of EVs and a lack of accepted protocols make data interpretation very challenging. Scope of Review. This review consolidates the data on the classical and state-of-the-art methods for isolation of EVs, including exosomes, highlighting the advantages and disadvantages of each method. Various characteristics of individual methods, including isolation efficiency, EV yield, properties of isolated EVs, and labor consumption are compared. Major Conclusions. A mixed population of vesicles is obtained in most studies of EVs for all used isolation methods. The properties of an analyzed sample should be taken into account when planning an experiment aimed at studying and using these vesicles. The problem of adequate EVs isolation methods still remains; it might not be possible to develop a universal EV isolation method but the available protocols can be used towards solving particular types of problems. General Significance. With the wide use of EVs for diagnosis and therapy of various diseases the evaluation of existing methods for EV isolation is one of the key problems in modern biology and medicine.

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.

A series of 1,4-diazabicyclo[2.2.2]octane derivatives differing by linker moiety was evaluated for activity against several strains of both Gram-positive and Gram-negative bacteria including drug-resistant strains, one strain of fungus and influenza virus A/Puerto Rico/8/34 (H1N1). All compounds exhibited high antibacterial activity against all bacteria except Proteus vulgaris. The minimum inhibitory concentrations (MICs) of compound 1c with an o-phenylenebismethyl linker and compound 1e with a propylene aliphatic linker were found to be low and were comparable or better to the reference drug ciprofloxacin for Pseudomonas aeruginosa and Staphylococcus aureus. Additionally, a time-kill assay was performed to examine the bactericidal kinetics. Compounds 1c and 1e displayed rapid killing effects against St. aureus and Ps. aeruginosa after 2 h. Furthermore, compounds 1a–c with aromatic linkers and compound 1e showed the highest antiviral activity.

Cell-surface-bound extracellular DNA (csbDNA) is present on the outer membrane of blood cells, including both red blood cells and leukocytes. Although less well characterized than cell-free DNA (cfDNA) in plasma and serum, leukocyte and red blood cell csbDNA form a considerable fraction of the blood extracellular nucleic acids pool, with typically at least comparable amount of DNA occurring bound to the outer surface of cells as compared with circulating free DNA in plasma. The cellular origin of csbDNA is not clear; however, as with cfDNA, in patients with cancer a proportion is derived from the tumor, thus making it potentially a useful source of DNA for cancer diagnosis, prognosis, and monitoring. © 2019 IUBMB Life, 71(9):1201-1210, 2019.

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.

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.

A direct correlation between the concentration of cell-free and cell-surface-bound circulating DNA (cfDNA and csbDNA, respectively) was demonstrated. Based on an inverse correlation between blood plasma DNase activity and the cfDNA concentration, blood DNases are supposed to regulate the cfDNA concentration. However, no correlation was found between the DNase activity in blood plasma and the csbDNA concentration, indicating that blood DNases are not involved in csbDNA dissociation from the cell surface. The possibility of DNA redistribution between cfDNA and csbDNA indicates that the total pool of circulating DNA (cfDNA +csbDNA) should be used for a correct analysis of marker DNA concentrations and data standardization.

MicroRNAs (miRNAs) found in biological fluids such as blood and urine have been identified as promising biomarkers for many human disorders, including cancer, cardiopathies, and neurodegenerative diseases. However, circulating miRNAs are either encapsulated into vesicles or found in complexes with proteins and lipoproteins and, thus, require a special approach to their isolation. Acid phenol-chloroform extraction can solve this problem, but it is a labor-intensive procedure that relies heavily on the use of hazardous chemicals. Here we describe a fast and simple phenol-free protocol for miRNA isolation from biofluids. MiRNA is extracted from complexes with biopolymers by a high concentration of guanidine isothiocyanate combined with water/organic composition of solvents. Purification is finished using silica-based spin columns. Comparison of miRNA isolation from blood plasma and urine using the single-phase method and acid phenol-chloroform extraction by means of radioisotope spike-ins and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) showed similar performance of the two methods.

This study is devoted to interactions of oligonucleotides containing specific DNA motifs with and transport into human endothelial cells. Those motifs are overrepresented in a pool of short DNA firmly bound to the surfaces of these cells. The data obtained by various methods suggest that the binding of oligonucleotides to the cell membrane and their accumulation in endothelial cells depend on both the presence of specific DNA motifs (CATGCAT, GATCCA, or TACGT) and their combination in the oligonucleotides under study. The oligonucleotides penetrate into endothelial cells via receptor-mediated endocytosis, which is confirmed by flow cytometry and fluorescence microscopy in the presence of potential inhibitors and competitors of intracellular transport.

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.

In the current study we have investigated the protein content of blood plasma deoxyribonucleoprotein complexes. The complexes were isolated using affinity chromatography with immobilized polyclonal anti-histone antibodies. Proteins were separated by SDS PAAGE and identified by MALDI-TOF mass-spectrometry. 111 and 56 proteins (excluding histones), respectively, were identified with a good score in deoxyribonucleoprotein complexes of healthy females and breast cancer patients. However, only four of these proteins were found in 30 % of all samples. Fourteen proteins previously described as tumor specific proteins were found in cancer patients whereas not one of them was found in healthy individuals. The data obtained demonstrate the involvement of different cellular and extracellular proteins in circulating cell-free DNA.

Extracellular nucleic acids are found in human blood and cell culture medium as cell-free or being adsorbed at cell surface. In the last years, the circulating extracellular nucleic acids in blood were shown to be associated with certain diseases. Attempts are made to develop non-invasive methods of early tumor diagnostics based on analysis of circulating DNA and RNA.This article reviews accumulating data regarding cell-free and cell-surface-bound extracellular nucleic acid nature and generation mechanisms. Their existence as a constituent of the naturally occurring complexes with proteins or membrane-bearing particles is discussed with regard to their homeostatic concentration and distribution in healthy donor blood which are significantly altered in cancer patients. Gene-target and whole-genome studies reveal significant differences in gene representation between extracellular DNA and genome DNA. Overrepresentation of regions with high transcription activity has led to proposal that extracellular DNA generation is strongly dependent on the parent genome functionality, which is associated with chromosome packaging and DNA methylation levels.Recent studies provide evidence of the circulating nucleome organization complexity indicating that discovery of extracellular DNA generation and circulation patterns in healthy condition and cancer is essential to enable the development of proper approaches for the selection of valid diagnostic markers.

MicroRNAs (miRNAs) have been identified as promising biomarkers in cancer and other diseases. Packaging of miRNAs into vesicles and complexes with proteins ensures their stability in biological fluids but also complicates their isolation. Conventional protocols used to isolate cell-free RNA are generally successful in overcoming these difficulties; however, they are costly, labor-intensive, or heavily reliant on the use of hazardous chemicals. Here we describe a protocol that is suitable for isolating miRNAs from biofluids, including blood plasma and urine. The protocol is based on precipitation of proteins, denaturation of miRNA-containing complexes with octanoic acid and guanidine isothiocyanate, and subsequent purification of miRNA on spin columns. The efficacy of miRNA extraction by phenol-chloroform extraction, miRCURY RNA isolation kit--biofluids (Exiqon), and the proposed protocol was compared by quantitative reverse-transcription PCR of miR-16 and miR-126. The proposed protocol was slightly more effective for isolating miRNA from plasma and significantly superior to the other two methods for miRNA isolation from urine. Spectrophotometry and SDS-PAGE data suggest that the disparity in performance between miRCURY Biofluids and the proposed protocol can be attributed to differences in precipitation mechanisms, as confirmed by the retention of different proteins in the supernatant.

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.

Since the association of circulating DNA level changes with tumor growth was discovered many attempts have been made to develop the sensitive and robust blood-based tests for early tumor diagnostics. Both genomic as well as mitochondrial DNA quantification in the circulation have been extensively evaluated as a diagnostic and prognostic tool to monitor cancer therapy. Cell-free DNA bearing the same genetic and epigenetic changes as the tumor tissues were shown to be detectable in plasma / serum of cancer patients indicating the principal possibility to create the minimally invasive diagnostic tests based on tumor-specific DNA markers. Apart from circulating DNA, tumor-derived RNA in plasma / serum was found to be a promising approach for the development of cancer markers. Results of the last two years establish the quantification of the tumor-derived microRNAs in plasma / serum as an extremely promising approach for cancer diagnostics. The aim of this publication was to review the recently reported studies on the circulating DNA and RNA in cancer patients and to estimate their impact on making the ongoing research closer to clinical application.

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.

We describe a simple and efficient method to detect antibodies against native epitopes following immunization with denatured proteins and peptides. With this method, soluble antigens genetically fused with placental alkaline phosphatase (AP) are used as probes to detect antibodies immobilized on nitrocellulose membranes. The AP-tagged proteins can be produced in sufficient amounts using transient transfection of eukaryotic cells with an appropriate cDNA fragment in a commercial AP-tag vector. The intrinsic thermo-stable phosphatase activity of a tagged protein obviates the need for its purification. To evaluate the method, three recently identified proteins of the FcR family, FCRLA, FCRL1, and FCRL4, were fused with AP and tested in a reaction with various polyclonal and monoclonal antibodies raised by immunization with bacterially produced antigens and peptide conjugates. All the three probes demonstrated high specificity in analysis of immune sera and hybridoma supernatants. Sensitivity of the assay varied depending on antibody tested and, in some cases, was in the subnanogram range. The results obtained show that AP-tagged proteins are useful tools for discrimination of antibodies against native epitopes when production of antigen in its native conformation is laborious and expensive.

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.

Extracellular nucleic acids are found in different biological fluids in the organism and in the environment: DNA is a ubiquitous component of the organic matter pool in the soil and in all marine and freshwater habitats. Data from recent studies strongly suggest that extracellular DNA and RNA play important biological roles in microbial communities and in higher organisms. DNA is an important component of bacterial biofilms and is involved in horizontal gene transfer. In recent years, the circulating extracellular nucleic acids were shown to be associated with some diseases. Attempts are being made to develop noninvasive methods of early tumor diagnostics based on analysis of circulating DNA and RNA. Recent observations demonstrated the possibility of nucleic acids exchange between eukaryotic cells and extracellular space suggesting their participation in so far unidentified biological processes.

The concentration of extracellular DNA and RNA in blood plasma of healthy donors, trauma patients, patients with breast and lung cancer, nonmalignant breast tumors and nonmalignant lung diseases were estimated. Significant amounts of extracellular RNA were found in plasma of trauma patients. The concentration of DNA and RNA in plasma of trauma patients correlates with the extent of posttraumatic organ failure. Extracellular RNA was not found in the plasma of breast cancer patients and patients with nonmalignant breast tumors, whereas a very high concentration of extracellular RNA was found in patients with malignant and nonmalignant diseases of lung.

Extracellular nucleic acids in cultures of A431 and HeLa cells were investigated. The data obtained demonstrate the presence of high weight DNA and RNA in the extracellular medium. Temporal changes of extracellular nucleic acids levels in growth medium were investigated.

Affinity modified with Flu-DAP-p(N)16degU oligonucleotide-binding proteins were isolated by affinity chromatography using Ultrogel A2-anti fluorescein antibodies. After separation by SDS-PAGE the proteins with molecular masses about 68 kDa were MS/MS sequenced and identified as keratin K1, keratin K10, keratin K2e and albumin.

Concentration of circulating DNA probes is required to increase the amount of DNA involved in subsequent study (by polymerase chain reaction, sequencing, and microarray). This work was dedicated to the comparison of five different methods used for concentration of DNA circulating in blood. Precipitation of circulating DNA with acetone in the presence of triethylamine provides minimal DNA loss, high reproducibility, and at least three times higher DNA yield in comparison with the standard ethanol protocol.

The subcellular localisation of oligodeoxynucleotides (ODN) is a major limitation for their use against nuclear targets. In this study we demonstrate that an antisense ODN directed against cytosolic phospholipase A(2) (cPLA2) mRNA is efficiently taken up and accumulates in the nuclei of endothelial cells (HUVEC), human monocytes and HeLa cells. Gel shift experiments and incubation of cells with oligonucleotide derivatives show that the anti-cPLA2 oligo binds a 37 kDa protein in nuclear extracts. The TAAAT sequence was identified as the major binding motif for the nuclear protein in competition experiments with mutated ODNs. Modification of the AAA triplet resulted in an ODN which failed to localise in the nucleus. Moreover, inserting a TAAAT motif into an ODN localising in the cytosol did not modify its localisation. The 37 kDa protein was purified and identified after peptide sequencing as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It was shown by confocal microscopy that GAPDH co-localises with anti-cPLA2 ODN in the nucleus and commercial GAPDH effectively binds the oligo. Competition experiments with increasing concentration of NAD(+) co-factor indicate that the GAPDH Rossmann fold is a docking site for antisense oligonucleotides containing a TAAAT motif.

Lung cancer is a complex disease that often manifests at the point when treatment is not effective. Introduction of blood-based complementary diagnostics using molecular markers may enhance early detection of this disease and help reduce the burden of lung cancer. Here we evaluated the diagnostic potential of seven plasma miRNA biomarkers (miR-21, -19b, -126, -25, -205, -183, -125b) by quantitative reverse transcription PCR. Influence clinical and demographical characteristics, including age, tumor stage and cancer subtype on miRNA levels was investigated. Four miRNAs were significantly dysregulated (miR-19b, -21, -25, -183) in lung cancer patients. Combination of miR-19b and miR-183 provided detection of lung cancer with 94.7% sensitivity and 95.2% specificity (AUC = 0.990). Thus, miRNAs have shown the potential to discriminate histological subtypes of lung cancer and reliably distinguish lung cancer patients from healthy individuals.

The protein-binding properties of dodecathymidine derivatives (prooligos) bearing either methyl- or tert-butyl-S-acylthioethyl (Me- or tBuSATE) protecting groups were evaluated. The dissociation constants (Kd) were estimated for complexes of prooligos with serum blood proteins and lactoferrin using prooligos to compete the binding of the radiolabeled, alkylating probe oligonucleotide CIRp(T)12 with the proteins. tBuSATE and MeSATE prooligos have decreased affinity of binding with serum proteins and lactoferrin compared with their parent oligos. These data suggest that prooligos should cause less side effects which combined with their stability to nucleases and enhanced permeability into cells make them potentially useful for design of therapeutics.

The effect of polyethylenimine on the mitogenic stimulation of murine splenocytes by DNA and oligonucleotides has been investigated. It was found that polyethylenimine masks the mitogenic stimulation effect of the compounds.

The analysis of circulating nucleic acids (cirNAs) in blood provides helpful information for medical diagnostics (1). Thus, there has been intensive investigation of the factors influencing nucleic acid concentrations in plasma and serum, such as changes in DNA concentrations in serum and plasma during storage of blood samples (2) and the influence of blood processing on cell-free DNA quantification (3)(4)(5). Recent observations demonstrated that cirNAs are found not only free in blood plasma but also bound to the cell surface (6). Physiologic factors determining cirNA concentrations in blood remain to be elucidated. In the present study, we compared the concentration of cell-surface–bound and cell-free cirNAs in blood samples from 15 healthy men and 20 healthy women. Venous blood (8 mL) was collected into 13-mL tubes (cat. no. 55.459; Sarstedt) containing 2 mL of sterile phosphate-buffered saline (PBS) containing 50 mmol/L EDTA. The samples were stored at 4 °C and treated within 4 h after blood collection. Plasma was collected after low-speed centrifugation …

Recent studies suggest that extracellular vesicles may be the key to timely diagnosis and monitoring of genito-urological malignancies. In this study we investigated the composition and content of extracellular vesicles found in the urine of healthy donors and prostate cancer patients. Urine of 14 PCa patients and 20 healthy volunteers was clarified by low-speed centrifugation and total extracellular vesicles fraction was obtain by high-speed centrifugation. The exosome-enriched fraction was obtained by filtration of total extracellular vesicles through a 0.1 μm pore filter. Transmission electron microscopy showed that cell-free urine in both groups contained vesicles from 20 to 230 nm. Immunogold staining after ultrafiltration demonstrated that 95% and 90% of extracellular vesicles in healthy individuals and cancer patients, respectively, were exosomes. Protein, DNA and RNA concentrations as well as size distribution of extracellular vesicles in both fractions were analyzed. Only 75% of the total protein content of extracellular vesicles was associated with exosomes which amounted to 90-95% of all vesicles. Median DNA concentrations in total extracellular vesicles and exosome-enriched fractions were 18 pg/ml and 2.6 pg/ml urine, correspondingly. Urine extracellular vesicles carried a population of RNA molecules 25 nt to 200 nt in concentration of no more than 290 pg/ml of urine. Additionally, concentrations of miR-19b, miR-25, miR-125b, and miR-205 were quantified by qRT-PCR. MiRNAs were shown to be differently distributed between different fractions of extracellular vesicles. Detection of miR-19b versus miR-16 in total vesicles and exosome-enriched fractions achieved 100%/93% and 95%/79% specificity/sensitivity in distinguishing cancer patients from healthy individuals, respectively, demonstrating the diagnostic value of urine extracellular vesicles.

An immunomodulatory activity of circulating DNA (cirDNA) is implemented via the interactions of cirDNA with the targets exposed on the cell membrane and/or intracellular targets. The goal of this work was to identify the cellular targets of immunoinhibiting cell-surface-bound cirDNA (csbDNA) using its oligodeoxyribonucleotide (ODN) analogs containing the nucleotide motifs frequently found in csbDNA and displaying the same effects.The binding of [(32)P]-labeled single- and double-stranded ODNs (ss- and ds-ODNs) with membrane-cytosolic (MC) extracts and living human umbilical vein endothelial cells (HUVEC) was studied by electromobility shift assay (EMSA). Complexes of biotinylated ODNs with target proteins were affinity isolated using streptavidin Sepharose with subsequent SDS-PAGE and identified by MALDI-TOF mass spectrometry.Both ss- and ds-ODNs form strong ODN-protein complexes with similar electrophoretic mobilities after incubation with the MC extracts of HUVEC either when added extracellularly or lipofected into cells. The ODN-binding proteins were identified as the DNA-binding components of DNA-dependent protein kinase (DNA-PK), namely, Ku70 and Ku80 proteins. Diverse cellular localizations and functions of the Ku proteins demand further clarification of Ku70/80 role as a mediator of the csbDNA immunoinhibiting effects.

Plasmid pUC19 DNA was shown to stimulate in vitro proliferation of CBA mouse splenocytes in a dose-dependent manner. Simultaneous treatment of the cells with the plasmid DNA and Con A or LPS produced an additive effect, while PMA acted synergistically with DNA. Monovalent Fab fragments of rabbit anti-mouse Ig (RAMIg) antibodies significantly inhibited plasmid DNA-induced polyclonal lymphocyte activation suggesting the involvement of Ig receptors in this process. Affinity modification of lymphocytes membrane-cytosole proteins with a 32P-labeled alkylating oligonucleotide derivative resulted in labeling of 67–82 and 23 kDa polypeptides corresponding to IgD and IgM heavy and light chains respectively. The immunoglobulin nature of the 82 and 23 kDa oligonucleotide-binding polypeptides was confirmed by immunoprecipitation with RAMIg antibodies.

The presence of various genomic sequences in the pool of extracellular DNA was studied by cloning and sequencing the DNA eluted from the surface of HeLa cells. Sequences of 19 genes, 10 pseudogenes, and 41 repeated elements were found in 103 clones. Sequences of LINE repeats were found in 17% of the clones; consequently real-time PCR assay for quantification of LINE repeats was chosen to study the influence of protein transport inhibitors on the concentration of free and cell-surface-bound DNA in the cultures of HUVEC and HeLa cells. Treatment of both cell lines with the inhibitors did not interfere with the concentration of extracellular DNA in the growth medium, except for chloroquine, which doubled the concentration of extracellular DNA in HUVEC culture. The treatment of HUVECs with monensin, glyburide, or methylamine decreases the cell-surface-bound DNA concentration by 30, 35, and 19%, respectively. The incubation of HeLa cells with monensin reduces the concentration of cell-surface-bound DNA by 15%; however, the treatment with glyburide increases cell-surface-bound DNA concentration by 50%. The data obtained demonstrate the involvement of vesicular transport in generation of extracellular DNA.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a well-studied glycolytic protein with energy production as its implied occupation. It has established itself lately as a multifunctional protein. Recent studies have found GAPDH to be involved in a variety of nuclear and cytosolic pathways ranging from its role in apoptosis and regulation of gene expression to its involvement in regulation of Ca2+ influx from endoplasmic reticulum. Numerous studies also indicate that GAPDH interacts with microtubules and participates in cell membrane fusion. This review is focused on the cytosolic functions of the protein related to vesicular transport. Suggestions for future directions as well as the model of protein polymer structure and possible post-translational modifications as a basis for its multifunctional activities in the early secretory pathway are given.

Cell-free nucleic acids (NA) from human urine were investigated. Concentrations of DNA and RNA in the urine of healthy people were independent of gender and were in the range of 6 ng/mL to 50 ng/mL and 24 ng/mL to 140 ng/mL, respectively. DNA fragments of 150-400 bp represent the main part of cell-free DNA, along with DNA fragments up to 1,300 bp, which were found in male urine, and DNA fragments up to 19 kbp, which were found in female urine. Analysis of circulating DNA, isolated from blood of breast cancer patients and cell-free DNA isolated from their urine by methylation-specific PCR, demonstrates that the presence of methylated promoters of RASSF1A and RARbeta2 genes in plasma was accompanied by the detection of the same methylated markers in urine. The data obtained demonstrate applicability of cell-free urine DNA in cancer diagnostics.

The study of mechanisms of nucleic acid transport across the cell membrane is valuable both for understanding the biological function of extracellular nucleic acids and the practical use of nucleic acids in gene therapy. It has been clearly demonstrated that cell surface proteins are necessary for transport of nucleic acids into cells. A large amount of data has now been accumulated about the proteins that participate in nucleic acid transport. The methods for revealing and identification of these proteins, possible mechanisms of protein-mediated transport of nucleic acids, and cellular functions of these proteins are described.

Using affinity columns with immobilized poly(A), poly(G), poly(U), poly(C), and poly(A).poly(U) and poly(G) x poly(C) duplexes several polyribonucleotide-binding blood plasma proteins have been captured. Albumin and keratins K1 and K2e have been detected to bind polypurine tracts. The in vitro glycated albumin binds poly(A) and poly(G) more efficiently than the unmodified protein. The major polypyrimidine-binding blood plasma protein (28 kDa) can catalyze the hydrolysis of poly(U).

The procedure based on binding of nucleic acids with glass surface in presence of chaotropic salts was adapted for efficient isolation of 100–10000 b.p. DNA fragments and 50–10,000 b. RNA fragments. The method provide 90% and 85% efficacy of isolation of 100 b.p. DNA and 100 b. RNA fragments respectively. High molecular weight nucleic acids are isolated with 98% efficacy. Isolated nucleic acids are free from contaminations, influencing nucleic acids modifying enzymes and fluorochromes. The method is rapid, simple and cost‐effective.

The frequency of APC, RASSF1A, RARbeta, CDH1 and CDH13 gene promoter methylation in samples of DNA isolated from breast and lung patient plasma was studied in order to develop the noninvasive tumor-specific DNA detection method. Methylation of at least one of genes was detected in extracellular DNA from most of the cancer blood specimens. The results obtained indicate that promoter hypermethylation of a number of marker genes represents a promising serum marker for early breast and lung cancer detection.

Concentrations of extracellular DNA and RNA in the blood of healthy donors and patients with malignant and nonmalignant breast tumors were investigated. Cell-surface-bound extracellular DNA and RNA were detached by PBS-EDTA treatment or mild trypsin treatment of erythrocytes and leukocytes. In healthy donors, almost all extracellular nucleic acids (98%) are bound at the surface of blood cells. In the blood of cancer patients, extracellular nucleic acids were found in plasma and not at the cell surface. In patients with nonmalignant breast tumors, extracellular nucleic acids were found both at the surface of blood cells and in plasma. In healthy donors, the cell-surface-bound DNA is represented by 20-kbp DNA fragments and smaller fragments that varied in amounts in different fractions.

Investigation of the kinetics of nucleic acid release by HeLa (human cervical carcinoma cell line) and A431 (human squamous carcinoma cell line) cells is presented. The released DNA and RNA were shown to accumulate in culture medium and at the cell surface. A portion of cell surface bound RNA can be eluted with PBS/EDTA. Mild trypsin treatment is required for complete detachment of cell surface bound RNA and cell surface bound DNA. Electrophoretic analysis reveals characteristic patterns of cell-associated and free RNA and DNA molecules.