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Vol 10, No 3 (2015)


Genetic and epigenetic mechanisms of sensory maps development

Mitrukhina O., Minlebaev M., Khazipov R.


One of the central questions of neurobiology is to understand how during development billions of neurons establish synaptic connections, and what are the roles of genes and activity in the formation of specific neuronal circuits . There are two main theoretical models to describe this developmental process . The model of “tabula rasa” implies that initially there is no order in synaptic connections, and that their initial exuberant number undergoes pruning through the competition of neurons for their targets . Alternative model assumes that development of synaptic connections is strictly determined by genes, through signaling molecules that predefine highly ordered connectivity, and that the activity only plays confirmative roles . In the present review, we describe genetic and epigenetic factors involved in the development of sensory maps in barrel cortex and provide evidence that both mechanisms operate in the development of this system . While genetic mechanisms provide course topography of somatosensory map at subcolumnar precision level in its initial state, sensory-driven activity patterns, which are is expressed in barrel cortex during the critical developmental period support competition between sensory inputs (neighbor whiskers) for the cortical territories to achieve columnar level of precision in somatosensory map by the end of the critical period
Genes & Cells. 2015;10(3):6-11
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DNA of ‘minimal’ cells (mycoplasmas) in the metagenomes of Arctic permafrost

Vishnyakov I.E., Borchsenius S.N., Kayumov A.R., Shmakova L.A., Rivkina E.M.


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
Genes & Cells. 2015;10(3):12-21
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Apoptosis in regenerative histogenesis of the liver after partial hepatectomy in rats

Gazizov I.M., Gumerova A.A., Kiassov A.P.


Study of liver regeneration after partial hepatectomy in last few decades enabled to understand hourly changes of expression of different genes, levels of cytokines, determine proliferation kinetics of liver cell types. Unfortunately, processes of apoptosis, which always activates together with regeneration was not given enough attention. In a practical point of view, understanding of apoptosis is needed for development of new therapeutic approaches in liver treatment based on enhancing of anti-apoptotic signals and inducing cells into proliferation. This work was dedicated to study apoptosis during liver regeneration after partial hepatectomy in rats. We analyzed expression of PCNA, caspase 3 and 9, Bcl-2 immunohistochemically during liver regeneration on 1, 2, 3, 5, 7, 14 postoperative days. Our results have showed that caspase 9 and Bcl2 are expressed during first postoperative week, caspase-3 only in first three days. Maximal expression of caspase 9 in hepatocytes and sinusoidal cells occurs after 2 days, in cholangiocytes - after 5 days. Maximal expression of Bcl-2 in sinusoidal cells occurs after 3 days, in hepatocytes and cholangiocytes - after 5 days. So, during liver regeneration after partial hepatectomy in rats mechanisms of apoptosis and antiapoptosis are activated simultaneously. Irreversible apoptosis is possible only during first 3 postoperative days and involves only minor fraction of hepatocytes and sinusoidal cells
Genes & Cells. 2015;10(3):22-26
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Investigating the properties of membrane vesicles obtained from human cells HEK293 using cytochalasin B

Gomzikova M.O., Rizvanov A.A.


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
Genes & Cells. 2015;10(3):27-32
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Prospects of EPR spectroscopy to the intensity of free radical processes evaluation in tissue-engineering esophagus and diaphragm

Gubareva E.A., Basov A.A., Kuevda E.V., Sotnichenko A.S., Dzhimak S.S., Bolotin S.N., Gilevich I.V., Danilenko K.A., Gumenyuk I.S., Markushin V.A., Porhanov V.A., Macchiarini P.


Tissue engineering can become an alternative option of treatment which is focused on restoration, replacement and regeneration of cells, tissues and failed organs. One of the tasks of regenerative medicine is creation of biological or artificial scaffolds reproducing structure of native tissue and possessing the required physical, chemical and mechanical properties for ensuring cell adhesion and formation of three-dimensional tissue as a result of recellularization with autologous cells. Biological scaffolds must be decellularized to become nonimmunogenic; however this process has to be aimed at the maximum preservation of biochemical composition, morphological structure, and maintenance of the biomechanical properties of the obtained extracellular matrix similar to the properties of native tissue One of the main issues is development of criteria of quality of the obtained scaffolds. In this work the spectroscopy EPR (electron paramagnetic resonance) method is used as one of promising biophysical methods of evaluation of decellularization EPR-spectroscopy applicability is also shown for evaluation of preservation of native organs at a cryopreservation which might already be the first stage of decellularization that is indirectly confirmed by the conducted researches
Genes & Cells. 2015;10(3):33-38
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Effect of recombinant plasmid constructs encoding combinations of dog and horse vegf and bmp2 cDNAs on mesenchymal stromal cell differentiation in vitro

Zhuravleva M.N., Zakirova E.Y., Masgutov R.F., Valiullin V.V., Deev R.V., Rizvanov A.A.


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
Genes & Cells. 2015;10(3):42-48
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Transplantation of allogeneic mesenchymal stromal cell for treating corneal ulcers in cats

Zakirova E.Y., Valeeva A.N., Faizullina R.R., Akhmetshin R.F., Nefedovskaya L.V., Rizvanov A.A.


At present, a great interest for the veterinary practice is the use of stem cells to treat diseases of the eye. This study was conducted to evaluate the therapeutic effect of allogeneic multipotent mesenchymal stromal cells derived from adipose tissue for the treatment of traumatic corneal ulcers in cats Our results indicate that the stem cells stimulate regenerative processes in the damaged cornea
Genes & Cells. 2015;10(3):49-55
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Proliferation and transdifferentiation of endocrinocytes of the rat during experimental diabetes

Kaligin M.S., Mavlikeev M.O., Titova A.A., Plushkina A.S., Titova M.A., Gumerova A.A., Kiassov A.P.


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
Genes & Cells. 2015;10(3):56-61
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Apoptosis-inducing activity of Bacillus pumilus ribonuclease and some egyptian medicinal plants extracts on human alveolar adenocarcinoma cells

Karamova N.S., Zelenikhin P.V., Miroshnik N.B., Essam A., Zakirova Y.N., Ilinskaya O.N.


Induction of apoptosis is a primary mechanism of anticancer activity of several drugs using for cancer therapy. Apoptosis-inducing activity of Bacillus pumilus ribonuclease (binase) and stem bark aqueous extracts of eight medicinal plants from Egypt in human A549 alveolar adenocarcinoma cells was studied. It was shown that binase (300 μg/ml) significantly increases the portion of apoptic cells in population after 24 h. Extracts of Albizzia lebbeck and Bauhinia variegate demonstrated a clear dose-dependent apoptosis-inducing effect. Combined treatment of A549 cells with binase and stem bark aqueous extracts of Albizzia lebbeck, Bauhinia variegate, Kigelia africana enhances induction of apoptosis in comparison with binase and extracts alone. Results obtained allow to consider combination of binase and stem bark aqueous extracts of Albizzia lebbeck, Bauhinia variegate, Kigelia africana as a source for development of low toxic, natural drugs to induce apoptosis in tumor cells
Genes & Cells. 2015;10(3):62-67
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Transplantation of cell sheets from adipose-derived mesenchymal stromal cells effectively induces angiogenesis in ischemic skeletal muscle

Makarevich P.I., Boldyreva M.A., Dergilev K.V., Gluhanyuk E.V., Gallinger J.O., Efimenko A.Y., Tkachuk V.A., Parfyonova Y.V.


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
Genes & Cells. 2015;10(3):68-77
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Regeneration of rat sciatic nerve using tubulation and allogeneic transplantation of adipose-derived mesenchymal stromal cells

Masgutov R.F., Masgutova G.A., Rogojin A.A., Zhuravleva M.N., Zakirova E.Y., Nigmetzyanova M.V., Mukhametova L.R., Shulman A.A., Mukhamedshina Y.O., Yafarova G.G., Rizvanov A.A.


The effectiveness of application of biodegradable conduit (NeuraGen; INTEGRA, USA) in combination with adipose derived multipotent mesenchymal stem cells and fibrin sealant (Tissucol-Kit; Baxter AG, Austria) on posttraumatic peripheral nerve regeneration was investigated using a rat model of sciatic nerve injury. We demonstrate that our proposed cell therapy confers a neuroprotective effect under our experimental design
Genes & Cells. 2015;10(3):78-82
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Conduit based on poly(ε-caprolactone) filled with fibrin-based hydrogel with mesenchymal stromal cells for peripheral nerve defect reconstitution

Mukhamedshina Y.O., Masgutov R.F., Masgutova G.A., Zuravleva M.N., Shulman A.P., Galieva L.R., Rogozin A.F., Chelyshev Y.A., Rizvanov A.A.


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
Genes & Cells. 2015;10(3):83-87
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Tryptophanyl-tRNA synthase gene expression as genetic marker of the athletes’ overtraining

Nurbekov M.K., Elov A.A., Il'in A.B., Ibragimova M.Y., Zhdanov R.I.


In connection with a necessity the development of effective biomarkers of sportsman physical and overtraining (distress) states, the study of a role of triptophanyl-tRNA synthase, TRSase, and corresponding gene in process is of special interest. Preliminary estimate of specificity and level of mRNA expression of TRSase gene is carried out using method 1: scanning of gels with an image of separation of polymerase chain reaction PCR products to detect quantitatively the content of the mRNA and/or cDNA copies compare, in parallel, to DNA quantity markers to compose a calibrating curve. Final estimation of TRSase gene expression is carries out by real time PCR (method 2) using calibrating curve (technique 1) and relative quantitative estimation of a number of specific mRNA copies with reference gene involved (technique 2). The level of TRSase gene expression represents a criterium of organism response, which is adequate to a stress strength (overtraining) It is proposed to detect the overtraining state at sportsmen using registrating an increased TRSase gene expression. In our pilot project, the distress overtraining state is determined as increased level of specific TRSase gene expression in 1,45 fold higher at the samples under the study compare to control samples. The TRSase mRNA expression before training is increased by 1 2-1 6 fold compare to one after training
Genes & Cells. 2015;10(3):88-93
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Comparative analysis of secretory profile of human mesenchymal stromal cells differentiated in myogenic direction

Samchuk D.P., Pulin A.A., Eremin I.I., Gilmutdinova I.R., Korsakov I.N., Zorin V.L., Zorina A.I., Grinakovskaya O.S., Lazareva N.L., Eremin P.S., Petrikina A.P., Gomzyakov A.E., Deev R.V., Timashkov D.A., Vit'ko N.K., Kotenko K.V., Kopnin P.B.


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
Genes & Cells. 2015;10(3):94-105
pages 94-105 views

Soluble and immobilized papain and trypsin as destroyers of bacterial biofilms

Trizna E.Y., Baydamshina D.R., Kholyavka M.G., Sharafutdinov I.S., Hairutdinova A.R., Khafizova F.A., Zakirova E.Y., Hafizov R.G., Bogachev M.I., Kayumov A.R.


The proteolytic enzymes are widely used in medicine as a wound healing agents, removing necrotic tissues and serving as an alternative to surgery. The ability of soluble and immobilized papain and trypsin to destroy bacterial biofilm was investigated. We show that treatment with papain leads to disruption of biofilms formed by Pseudomonas aerugenosa, Escherichia coli, Micrococcus luteus, and in a lesser extent of Staphylococcus aureus and Staphylococcus epidermidis. It is shown that none of the investigated enzymes has mutagenicity and cytotoxicity, and causes no increase in the amount of necrotic cells in culture in vitro
Genes & Cells. 2015;10(3):106-112
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Impact of histological material fixation on the results of immunohistochemistry

Vaganova A.N.


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
Genes & Cells. 2015;10(3):113-119
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Analysis of the draft law «On Biomedical cellular products»

Solonitsyn L.A., Sazonov S.V.
Genes & Cells. 2015;10(3):120-121
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Genes & Cells. 2015;10(3):122-124
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