Vol 10, No 3 (2015)

During the long geological time enzymes, nucleic acids, viruses and viable microorganisms can be kept in permafrost. It is difficult to get a holistic view of the microbial community of permafrost using only classical microbiological methods. The analysis of metagenomes of permafrost allowed us to identify the genetic material of ancient mycoplasmas - pathogens of humans, animals and plants Sampling, isolation of total DNA from soil, sequencing (Illumina), metagenomic data processing (MG-RAST, M5nr, UniProt, Krona). Mycoplasma species composition in permafrost soil samples of different origin, but of comparable age (31-32 thousand years), was predicted A comparative analysis of short polypeptides encoded by fragments of ancient DNA with corresponding parts of proteins of modern mycoplasmas was done We discuss the phylogenetic history of Mollicutes, the plasticity of mycoplasma genomes, and the pathogenic potential of the permafrost

The preparation method of membrane vesicles from human cells using cytochalasin B allows to overcome the limitations of human cells natural microvesicles, associated with the complex procedure of isolation and limited output. Membrane vesicles (MV) prepared from human cells are a promising vector for delivering of various bioactive substances. We performed the preparation of MV from human cells HEK293 using cytochalasin B and size determination of the MV. Then we studied the influence of applied MV concentration and intravesicular substance concentration on the substance delivery effectiveness to recipient cells. It was found that MV ranging in size from 164.2 nm to 3580 nm, but the most of MV sized from 164.2 nm to 712,4 nm (84. 6%). MV are able to enclose the cytoplasmic contents of the parent cells and deliver it to recipient cells, the amount of delivered substance (CFDA SE) to the recipient cells is proportional to the loaded substances into MV

Gene therapy is one of the most promising fields in modern regenerative medicine, though today there is no approved veterinary gene therapy drugs on the market. We have created species-specific gene-engineering plasmid constructs based on plasmid DNA encoding genes of dog and horse vascular endothelial growth factor and bone morphogenetic protein 2, which can be potentially used in treatment of domestic animals traumas and locomotor system disorders In vitro studies of these constructs have shown their effect on stimulation of osteogenic, chondrogenic differentiation and angiogenesis in mesenchymal stem cells in vitro

Some studies have found an increase number of α-cells in experimental diabetes, which may cause rising of blood glucose levels, along with the lack of insulin. But the mechanism of increasing the amount of glucagon-positive cells is still unknown. The aim of the study was to investigate the proliferative activity and the possibility of differentiation of α- and β-cells of the islets of Langerhans of pancreas during experimental diabetes in rats The work was performed on 33 white mongrel male rats. After alloxan injection, blood glucose levels were measured by glucose oxidase method and the expression of insulin, glucagon, and proliferating cell nuclear antigen was studied. Isolated proliferating glucagon-positive cells were found only on day 14 of the experiment. At the same time of the experiment bigormonal cells were found that synthesize insulin and glucagon. The results of the double staining for PCNA and glucagon showed that the increasing number of glucagon-positive cells in early stages of experimental diabetes is not related to their proliferation Probably it is due to differentiation of the progenitor cells of the islets in pancreas

Delivery of cells is a promising approach to induce blood vessel formation for treatment of ischemia. Still, efficacy of these methods has been shown to be below expectations due to the fact that injection procedures used to transplant cells can diminish their survival rate. To circumvent this problem a technique known as “cell sheets” can be utilized. Cell sheets are minimal tissue-engineered constructs that comprise of cells along with their extracellular matrix proteins Present study investigates application of cell sheets from adipose-derived mesenchymal stromal cells (AD-MSC) to stimulate angiogenesis. In a mouse model of limb ischemia we demonstrate that subcutaneous implantation of a cell sheet from 1 mln AD-MSC effectively stimulates angiogenesis and restores perfusion of ischemic muscle compared to untreated animals with limb ischemia. Histology also indicates that cell sheet transplantation results in decreased necrosis of skeletal muscle and retain of AD-MSC at Day 14 with certain prevalence of proliferating and minimal amount of apoptotic cells within cell sheet Furthermore, comparison of cell sheet-treated animals vs. injection of the same dose of AD-MSC shows that cell sheet delivery was superior to routine injection-based delivery in terms of limb perfusion and tissue protection Obtained results indicate that local application of AD-MSC cell sheets to promote angiogenesis and protect skeletal muscle from ischemia can be a promising approach for therapeutic use

In this study we used promising therapeutic strategy for stimulation of peripheral nerve regeneration - implantation of poly(ε-caprolactone) nerve conduit filled with fibrin hydrogel Tissucol in combination with mesenchymal stem cells. In vitro studies showed that the coating by Tissucol of poly(ε-caprolactone) substrate promotes cell proliferation. In vivo results of peripheral nerve defect reconstitution in rats using the conduit based on poly (ε-caprolactone) filled with fibrin hydrogel with mesenchymal stem cells confirmed the effectiveness of the proposed approach

Up to this day there are lots of data accumulated about the role of cytokines in regulation of different tissues homeostasis independently of inflammation framework. Skeletal muscles produce a wide range of biologically active molecules both in a normal condition and after injuries of different etiologies. Moreover, cultures of cells isolated from muscle tissue show same properties. In this regard identification of cytokines profile secreted by cells with myogenic potential is of particular importance as it will help to choose optimal cell types and their sources for clinical application Our research group previously demonstrated the possibility of obtainment of myogenic cells from gingival mucosa derived multipotent mesenchymal stromal cells (MMSC) However, secretory profile of this myogenic cells is not thoroughly investigated to this day The study was conducted on cultures of skin fibroblasts, MMSc derived from the attached and alveolar parts of the gingival mucosa and gingival mucosa MMSc, differentiated in a myogenic direction cells were isolated from skin and gingival mucosa biopsy specimens of 15 healthy volunteers. ELISA assay was performed for evaluation of 48 proinflammatory and anti-inflammatory cytokines, chemokines and growth factors Our data demonstrates tendency of most investigated proteins secretion gradual increase in the following sequence: skin fibroblasts - attached gingival mucosa MMSC - alveolar gingival mucosa MMSC - differentiated myoblasts, including factors directly involved in myogenesis, skeletal muscle homeostasis and regeneration Thus, alveolar gingival mucosa MMSC both before and after induction of myogenic differentiation potentially could facilitate skeletal muscle regeneration Our results indicate that subpopulation of MMSC derived from alveolar gingival mucosa are perspective candidates for clinical usage in patients with skeletal muscle disorders

A common method of tissue fixation is the fixation in formalin. This fixing method is related to the chemical transformations of molecules and may influence the stability of their epitopes. The extent of this impact is aggravated by prolonged tissue stay in the fixative. To standardize the result of research, it should thus be standardized length of tissue fixation. The allowable fixation delay after the separation of tissue from a circulatory system also must be limited. However, in the modern time, the new requirements for the time to result and preservation of the molecular composition in the tissue leads to introducing of new technologies and fixing reagents into the practice There is a trend to move to coagulating fixatives, based on the various alcohols. The impact of such reactives on biomolecules in general is gentler. The feature of the protein antigens as an object of study is a high diversity of chemical structure that dictates the necessity for an individualized approach to development of immunohistochemical staining protocol including factors that the tissue meets before the staining procedure. Such optimization procedure is carried out mainly empirical. Existi ng immunohistochemical tests are adapted for the formalin-fixed tissue, and their use after the fixation in other conditions requires preliminary studies for protocol adaptation and optimization