[Genotype-Specific Features of Cold-Induced Sweetening Process Regulation in Potato Varieties Nikulinsky, Symfonia, and Nevsky].

In this study, we performed expression analysis of genes associated with cold-induced sweetening in potato tubers: vacuolar invertase (Pain-1), sucrose synthase (SUS4), and invertase inhibitor (InvInh2). Potato varieties Nikulinsky, Symfonia, and Nevsky were used. All three varieties were found to accumulate sugars at low temperatures; the maximum accumulation of reducing sugars was observed at 4°C. It was found that the expression pattern of genes associated with cold-induced sweetening differs depending on the variety and storage duration. The increased expression of vacuolar invertase and its inhibitor is more pronounced at the beginning of storage period, whereas the increased expression of sucrose synthase is more pronounced after 3 months of storage. At early storage periods, high expression of invertase and low expression of inhibitor is observed in the Dutch variety Symfonia, and vice versa in the Russian varieties Nikulinsky and Nevsky. The involvement of the studied genes in the process of cold-induced sweetening is discussed.

[Cys-Flanked Cationic Peptides For Cell Delivery of the Herpes Simplex Virus Thymidine Kinase Gene for Suicide Gene Therapy of Uterine Leiomyoma].

Uterine leiomyoma (UL) is the most common benign tumor in women of reproductive age. Gene therapy using suicidal genes appears to be a promising approach for UL treatment. One of key factors for success of gene therapy is the right choice of genetic construct carrier. A promising group of non-viral carriers for cell delivery of expression vectors is cationic Cys-flanked peptides which form tight complexes with DNA due to electrostatic interactions and the presence of interpeptide disulfide bonds. The paper reports a comparative study of the physico-chemical, toxic, and transfectional properties of the DNA-peptide complexes obtained by matrix polymerization or oxidative polycondensation of Cys-flanked peptides using the chain growth terminator 2-amino ethanethiol. We have demonstrated the therapeutic effect of the delivery of the pPTK-1 plasmid carrying the herpes simplex virus type 1 (HSV-1) thymidine kinase gene into PANC-1, and HEK-293T cell culture as well as into primary UL cells. It has been shown that the carriers obtained by oxidative polycondensation transform primary UL cells more efficiently than those produced by matrix polymerization. Treatment with ganciclovir resulted in the death of up to 40% of UL cells transfected with the pPTK-1 plasmid. The perspectives of use of the polyR6 carrier produced by oxidative polycondensation as a tool for the development of modular peptide carriers for the purposes of UL gene therapy were discussed.

[The application of micro-cultural enzyme-linked immunosorbent assay of modified immunofluorescence technique for diagnostic of adenovirus infection.]

The particular conditions of professional activities of drafty military personnel determine wide-spread of of respiratory viruses in crew of Armed Forces. The frequent mixing of military staff conditions of carrying of infection agents , including adenoviruses. It is known, that up to 60% of acute respiratory viral disease in newly formed staff have adenovirus etiology. In these cases, the most frequently are isolated serotonins 4 and 7 of adenovirus. The diagnostic possibilities of monoclonal immunologic tests of indirect enzyme-linked immunosorbent assay and micro-cultural enzyme-linked immunosorbent assay for diagnostic of adenovirus infection are investigated. The analysis was applied to 40 clinical samples from patients with diagnosis of acute respiratory viral disease residing for treatment in military medical organizations during April-June 2014. The culture of cells A-549 infected with materials from patients was used for analysis. The evaluation of reproduction of adenovirus in infected culture of cells using both techniques was implemented by application of monoclonal antibodies to hexon of adenovirus on stage of detection. The availability of adenovirus was proved applying polymerase chain reaction in 20 samples; isolation of cell culture in 19 samples; indirect enzyme-linked immunosorbent assay - in 14 samples; micro-cultural enzyme-linked immunosorbent assay - in 14 samples. For detection of serotypes of adenovirus isolation of DNA and sequencing of 10 of analyzed samples positive for adenovirus according results of polymerase chain reaction were implemented. The results of phylogenetic analysis on site of gene string demonstrated belonging pofadenovirus out of all samples to serotype 4 (subgroup E). The sensitivity of indirect enzyme-linked immunosorbent assay and micro-cultural enzyme-linked immunosorbent assay in detection of adenovirus in cell cultures infected with materials from patients, in comparison with polymerase chain reaction, made up to 85% and 87% correspondingly. The specificity of both techniques reached 100%.

Developmental and hormonal regulation of Arabidopsis thaliana ornithine-delta-aminotransferase.

Ornithine aminotransferase (OAT) catalyzes transfer of the delta-amino group from L-ornithine to oxo-glutarate. In plants, this reaction biochemically connects urea cycle, proline cycle, and polyamine biosynthesis pathway. OAT activity is shown to be associated with biotic and abiotic stress responses and nitrogen metabolism, but its physiological role is still unclear. In our study, we decided to investigate transcriptional regulation of the OAT gene in Arabidopsis thaliana under normal conditions and in response to various growth regulators. In the present work, the reporter gene construct containing the Escherichia coli ß-glucuronidase gene (gus) under control of the A. thaliana OAT gene promoter was introduced into the genome of A. thaliana ecotype Columbia plants using the floral dip method; GUS activity was assayed in different experimental conditions including hormone treatment, low and high nitrogen and salinity. The GUS activity was analyzed histochemically. Plants were incubated with staining solution containing X-Gluc. We show that under standard growth conditions, the promoter is active during germination and in developing floral organs. OAT promoter activity specifically activates in response to different forms of auxin (IAA, NAA, and 2,4D), cytokinin (6- BAP), ethylene precursor (ACC), high nitrogen and salinity. Analysis of the OAT expression by qRT-PCR confirmed the pattern observed using the GUS reporter system. The OAT gene showed a significantly elevated expression in fourday- old seedlings and in plant roots in response to auxins and cytokinins. The analysis of the OAT promoter structure reveals cis-acting regulatory DNA elements associated with auxin regulation and abiotic stresses. The results of the study indicate that the OAT gene is involved in developmental processes and is regulated by auxin and cytokinins.

Identification of specific gene methylation patterns during motor neuron differentiation from spinal muscular atrophy patient-derived iPSC.

Spinal muscular atrophy is a progressive motor neuron disorder caused by deletions or point mutations in the SMN1 gene. It is not known why motor neurons are particularly sensitive to a decrease in SMN protein levels and what factors besides SMN2 underlie the high clinical heterogeneity of the disease. Here we studied the methylation patterns of genes on sequential stages of motor neuron differentiation from induced pluripotent stem cells derived from the patients with SMA type I and II. The genes involved in the regulation of pluripotency, neural differentiation as well as those associated with spinal muscular atrophy development were included. The results show that the PAX6, HB9, CHAT, ARHGAP22, and SMN2 genes are differently methylated in cells derived from SMA patients compared to the cells of healthy individuals. This study clarifies the specificities of the disease pathogenesis and extends the knowledge of pathways involved in the SMA progression.

Generation of a spinal muscular atrophy type III patient-specific induced pluripotent stem cell line ICGi003-A.

Spinal muscular atrophy (SMA) is a genetic disease, which characterized by the degeneration of motor neurons in the spinal cord and further striated muscle atrophy. The research of the processes in diseased neurons is complicated due to the impossibility of obtaining them safely from patients. Thus, we generated SMA type III induced pluripotent stem cell lines via using non-integrated episomal plasmid vectors. The resulting cell line expresses the major pluripotency markers and can differentiate in vitro into derivatives of three germ layers. The iPSC line can be used for further studies by providing in vitro the relevant cell types.

Generation of three Duchenne muscular dystrophy patient-derived induced pluripotent stem cell (iPSC) lines ICGi002-A, ICGi002-B and ICGi002-C.

Duchenne muscular dystrophy (DMD) is a severe and rapidly progressive hereditary muscular disease with X-linked recessive inheritance, occurring mainly in males. A complete loss of dystrophin resulted from out-of-frame deletion mutations in the DMD gene leads to Duchenne muscular dystrophy. DMD induced pluripotent stem cells (iPSCs) are a suitable cell model to study muscle development and disease mechanisms underlying muscular dystrophy and to screen novel compounds with potential therapeutic effects. We generated iPSCs from a DMD patient using non-integrating episomal plasmid vectors. The obtained iPSC lines showed ESC-like morphology, expression pluripotency markers, displayed a normal karyotype and possessed trilineage differentiation potential.

[Hyperbranched polylysines modified with histidine and arginine: the optimization of their DNA compacting and endosomolytic properties].

The DNA compacting and transfection properties of hyperbranched polylysines whose N-terminal amino groups were modified with histidine and arginine were studied. The histidine-modified hyperbranched polylysines were shown to provide higher efficacy of binding and transfection in comparison with unmodified or hyperbranched arginine-containing polylysines. This fact was explained by the intrinsic endosomolytic activity of the histidine-modified polymers. The dependence between the quantity of the amino acids that modified the terminal lysine residues in the hyperbranched polylysines, the efficacy of their DNA binding, and the transfection activity of the DNA complexes with the corresponding carriers was found. The possibility to increase the transfection activity of the DNA complexes with the hyperbranched polylysines by glycerin or the JTS-1 amphipathic nonapeptide was studied. At the same time, their simultaneous use was found to result in a transfection decrease.

[Detection of somatic mosaicism in humans using polymerase chain reaction with random primers].

Using the method PCR amplification with random primers, DNA samples from human embryonic organs and tissues were examined. Among 27 oligonucleotide primers tested, 10 primers, producing stable, well-reproducible profiles of amplification products, were chosen for further analysis. With the help of two primers (447 and R45), the differences in RAPD PCR profiles obtained from the tissues of one embryo, were revealed. These differences were associated with the change of mobility, or with the fragment gain/loss in the RAPD profile, and could be caused either by genomic rearrangements, or mutations involving the regions of the DNA-primer pairing. Different epigenetic factors, like methylation, can also play the role in this process.

[Lysine dendrimers as vectors for delivering genetic constructs to eukaryotic cells].

Asymmetrical lysine dendrimers are promising as vectors for delivering gene expression constructs into mammalian cells. The condensing, protective, and transfection properties were studied for pentaspherical lysine dendrimer D5 and its analog D5C10, modified with capric acid residues at the outer sphere; in addition, the transfection activity was assayed for complexes DNA-dendrimer-endosomolytic peptide JTS-1. Fatty acid residues incorporated in lysine dendrimers proved to improve their ability to bind DNA, to protect DNA from nuclease degradation, and to ensure its transfer into the nucleus. Peptide JTS-1 introduced in DNA-dendrimer complexes significantly increased their transfection activity. The potentiating effect of JTS-1 was especially high with the DNA-D5C10 complex. An excess of JTS-1 changed the structure of the complexes and reduced their transfection activity. It was assumed that dendrimers D5 and D5C10 are promising vectors for delivering DNA to eukaryotic cells and provide a basis for constructing more refined nonvirus module carriers.

GENETIC DIVERSITY OF ADENOVIRUSES CIRCULATING AMONG THE MILITARY IN THE NORTH-WEST REGION.

The contribution of adenovirus (AV) infections to the overall structure of acute viral respiratory infections among young people of draft age can reach as high as 64.6%. Wide dissemination, the incidence of AV-associated pneumonias and lethal outcomes in the case of some complicated infections illustrate the urgency of studying the antigenic diversity of AVs circulating among the military. 991 nasopharyngeal swabs from patients hospitalized in military health facilities with symptoms of acute respiratory infections from 2014 to 2017 were detected by real-time PCR. Sanger sequencing was performed using forward and reverse primers matching the fiber gene. AVs were detected in 326 samples. In 80 of those, AVs were present in combination with other respiratory viruses, as follows: 26 with respiratory syncytial viruses (RSV), 49 with rhinoviruses, 2 with bocaviruses, 1 with RSV and rhinovirus, 1 with parainfluenza virus, and 1 with metapneumovirus. 31 samples were sequenced. Thirty AVs belonged to group E (serotype 4), and 1 AV belonged to group B (serotype 7).

[Digestive enzyme activity in growing rats consuming nitrates against a background og protein insufficiency]. / Aktivnost' pishchevaritel'nykh fermentov u rastushchikh krys pri potreblenii imi nitratov na fone belkovoi nedostatochnosti.

The effects of sodium nitrate on the activity of digestive enzymes in mucosal and submucosal layers of the small intestine as well as in liver and kidney were studied in the growing rats maintained on either standard or protein-free diets. The enzyme systems were more sensitive in the growing rats to protein deprivation than in adult animals. However, the digestive enzymes were more resistant against nitrate intoxication in the growing rats.