[Isolation of extracellular micro-vesicles from cell culture medium: comparative evaluation of methods]. / Sravnitel'nyi analiz metodov vydeleniia vnekletochnykh mikrovezikul iz kul'tural'noi sredy.

Extracellular vesicles (EV) are secreted by cells of multicellular organisms. EV mediate specific mode of intercellular communication by "horizontal" exchange of substances and information. This phenomenon seems to have an essential biological significance and became a subject of intensive research. Biogenesis, structural and functional features of the EV is being commonly studies in in vitro condition. Several methods of EV isolation from cell culture medium are established, however selection of method might influence on obtained results. The choice of the optimal method depends usually from the amount of medium and the aims of the research while is still challenging issue. We performed a comparative analysis of four different methods of EV isolation from cell culture medium: differential ultracentrifugation, ultracentrifugation with a 30% sucrose/D2O "cushion", precipitation with plant proteins and immune-affinity capturing. EV isolated by different approaches were compared in terms of following parameters: size, concentration, morphology of EV, contamination by non-vesicular particles, content of exosomal tetraspanins on the EV surface, content of total proteins, RNA, and several glioma-associated miRNAs. Applied methods included nano-patricle tracking analysis (NTA), dynamic light scattering (DLS), cryo-electron microscopy, flow cytometry and RT-qPCR. On the base of obtained results, we developed practical recommendations that may help researchers to make a best choice of EV isolation method.

[Comparative analysis of RT-qPCR based methodologies for microRNA detection.]

Many pathological states are accompanied by characteristic changes in the cellular profile of microRNAs - small molecules that regulate gene expression at the posttranscriptional level. This allows us to consider miRNA as a promising class of biological markers. In the work, a direct comparison of three RT-qPCR methodologies (s-Loop, u-Elong and 2-Tail) for miRNA analysis was performed. A synthetic miRNA-451 analog was used to determine the efficiency of miRNA molecule detection and analysis of the miRNA-29b, miRNA-375 and miRNA-451 profiles in OAW42 and HT29 cell lines was carried out. By the methods of 2-Tail and s-Loop, seven different miRNA were also analyzed in 13 clinical specimens. The results of the study show that in the 2-Tail and s-Loop approaches, RT-qPCR demonstrated high reproducibility in results of miRNA analysis, and a linear dependence of the mimic миРНК-451detection efficiency in the range of 107 to 103 molecules per reaction was registered. On a number of significant criteria, the two technologies turned out to be relatively equivalent, i.e. any of them can be used as a basis for the method of clinical diagnostics.

AGGREGATION BY LECTIN-METHODICAL APPROACH FOR EFFECTIVE ISOLATION OF EXOSOMES FROM CELL CULTURE SUPERNATANT FOR PROTEOME PROFILING.

Exosomes are small membrane vesicles secreted by most cell types in vivo and in vitro. Exosomes are found in cell culture media, blood, urine, amniotic fluid, malignant ascite fluids and contain distinct subsets of microRNAs and proteins depending upon the tissue from which they are secreted. Thus exosomes constitute potential biomarkers of human diseases, such as cancer. A major bottleneck in the development of exosome-based diagnostic assays is the challenging purification of these vesicles; this requires time-consuming and instrument-based procedures. Isolation of exosomes can be a tedious, non-specific, and difficult process. Here, we provide a preparative technique for isolation of exosomes based on their ability to aggregate in the presence of lectins. The new method for lectin-based isolation of exosomes from cell culture media was developed as a sample preparation step for exosome-based protein biomarker research.

Possibilities of estimating the prognosis for development of preinvasive cervical neoplasia by the latest molecular biology methods.

Secondary prevention of cervical cancer is the identification and treatment for preinvasive forms of the disease, which include cervical intraepithelial neoplasia (CIN). The traditional method of identification of CIN is cytological however the sensitivity and specificity of this method is limited. The efficacy of a test for human papillomavirus as well as new molecular-biological methods for assessing the prognosis of development of dysplasia and choice of appropriate treatment tactics are still in the process of discussion. The article contains information about different possibilities of using molecular-biological methods for assessing the prognosis of development of CIN and cervical cancer.

[Oncoendocrinology: interim results and new challenges].

Over the few past years there have been passed many significant and positive changes in various fields of oncology due to both the use of achievements, stimulated by previous generations, and the progress of modern technology. This largely concerns endocrinology of malignant tumors, which is reflected in this article on the basis of the experience of the N.N.Petrov Research Institute of Oncology gained during recent times. Above all it is about the features of tumors of hormone-dependent tissues, hormonal and metabolic shifts, associated with them, and the ways of their correction based on the principles of personalized medicine.

[Exosomal intercellular communication system and its role in the process of metastatic dissemination].

Metabolism and information between cells is the basis of the existence of any multicellular organism. Malfunction of the intercellular communication play an important and sometimes decisive role in the pathogenesis of the most diseases, including cancer. According to traditional views, functional integration of individual cells in tissues, organs and organ systems is mediated by the efficient work of regulatory systems: nervous, immune, endocrine. Over the past few years the attention of scientists is attracted the ability of cells to "communicate" with the help of nanoscale vesicular formations, or so-called exosomes. There are accumulated data that the cells of the most tissues secrete exosomes into the intercellular environment, after which, by means of stream of blood or lymph, exosomes transferred to anatomically distant sites where they are accepted by the other cells. It is showed that the content of exosomes are not random and that vesicular transport may be targeted and to play a significant physiological and even "pathophysiological" role. The aim of this review is the analysis and integration of modern scientific data on the role of exosomes in the process of tumor progression and presentation of possible ways and methods of using these data in the practice of oncology.