Vol 33, No 1 (2018)

One problem in the study of regulatory mechanisms in living systems is the difficulty in analysis of regulatory elements in their natural context. One promising way to solve this problem is direct in situ modification of regulatory element sequences. The new technology of gene modification based on the bacterial CRISPR/Cas system allows one to quickly and accurately modify any genomic fragment, in particular, in cells of an adult organism. This review considers principles of the CRISPR/Cas technology and its application to the study of regulatory elements, as well as the potential of using this technology in studies of regulatory systems in pancreas tumors.

To improve the safety profile and specificity in cancer gene therapy applications, а set of singleplasmid vectors for expression of suicide thymidine kinase gene of the herpes simplex virus (HSVtk) and mouse OX40L gene (m0X40L) involved in regulation of immune response was constructed. Therapeutic transgene expression was directed by a strong constitutive promoter of human cytomegalovirus (CMV), while translation occurred on an RNA template due to an encephalomyocarditis virus internal ribosome entry site (construct CMV-HSVtk-IRES-mOX40L) or 2A-peptide of Porcine Teschovirus-1 (construct CMV-HSVtk-2AmOX40L) inserted between them. The constructs obtained were functionally validated by transient transfection of C26 cells (mouse colon carcinoma). Both bicystronic vectors demonstrated strong cytotoxic activity and produced cytotoxic protein HSVtk at the similar level. To analyze efficiency of mOX40L expression, C26 transfected cells were immunofluorescence-labelled with anti-OX40L-antibodies conjugated with phycoerythrin following flow cytometric analysis. Production of mOX40L was higher in the case of CMV-HSVtk- 2A-mOX40L. Better performance of this expression vector was correlated with our data. Previously, we have demonstrated that a higher level of transgene expression was exhibited by cells transfected with the vector containing the 2A peptide sequence. Thus, transfection with the vector based on the 2A peptide sequence is optimal for achieving high efficiency of expression of both oncotherapeutic genes.

Circadian rhythms play an important role in the regulation of body metabolism, since the most important biochemical and physiological processes in the body are subjected to diurnal fluctuations. The transcription factor PERIOD3 encoded by the PER3 gene is one of the key components of the circadian negative transcription loop and, upon interaction with other proteins, affects the activity of the circadian clock. We aimed at studying genotype and allele frequencies of rs10462021 in the PER3 gene between the controls (N = 259) and different clinical types of depressive disorder, namely, depressive disorder (F32.1, 41.2) (N = 208) and recurrent depressive disorder (F 33.1) (N = 150), as well as patients with a history of suicide attempts (N = 100). No significant differences in distributions of genotype or allele frequencies between the groups were detected (p > 0.05). Therefore, there was no significant contribution of rs10462021 in the PER3 gene in the development of depressive disorder or various clinical types of depression.

Lipid-1a-phosphatase LpxE cleaves the phosphate group in the first position of lipid A of lipopolysaccharide in Gram-negative bacteria, which reduces toxicity and reactogenicity of bacterial endotoxin while maintaining its immunogenicity. Treatment with these enzymes in preparations of recombinant proteins obtained in bacterial producers, for example, in E. coli, may find application in the purification techniques of various pharmaceutical substances, primarily various protein components of vaccines. This work is devoted to the creation of an efficient method for obtaining an F. tularensis LpxE soluble protein in a heterologous expression system based on E. coli. Optimization of the bacterial production of LpxE enzyme is achieved by creating a SUMO-LpxE chimeric protein in which the SUMO polypeptide acts as a folding catalyst and an affinity target carrier for purifying the expression product. An additional increase in the level of synthesis of LpxE and reduction of its toxicity for a bacterial strain is achieved against the background of superproduction of bacterial periplasmic chaperones encoded by an auxiliary low copy plasmid. Subsequent chromatographic purification of the target recombinant protein involves the use of affinity metal chelate chromatography and a SUMO protease cleavage step of the SUMO peptide carrying the His6 N-terminal peptide with the reduction of natural primary structure of the enzyme. The developed method makes it possible to obtain a highly purified active enzyme LpxE F. tularensis with a yield of 0.26 g/L of bacterial culture without fermentation.

Cell lines derived from natural virus hosts are one of the most promising tools in virological research. The aim of this work is to establish the dynamics of RNA replication of tick-borne encephalitis virus (TBEV) in new transferable cell line of kidney of the Korean field mouse Apodemus peninsulae (ApnK) and compare it with replication in the traditional culture of fetal porcine kidney cells (SPEVs). The transplantable ApnK cell line was obtained in the laboratory of transmissive infections of the Scientific Center for Family Health and Human Reproduction Problems (Irkutsk), the identity of the culture was established by the analysis of nucleotide sequence of mitochondrial cytochrome B gene (mtCytB, GenBank access number KT983422), and the D-loop sequence of the mitochondrial genome (KT983423). Cells were inoculated with TBEV strain of the Siberian subtype 92M and the total RNA was isolated from them 0, 4, 8, 16, and 24 h after infection. In addition, RNA was also isolated 1, 4, 7, and 19 days after infection. The amount of intracellular RNA of the positive polarity (+RNA) was assessed by quantitative real-time PCR. At the initial stage of infection (0 h after infection) in ApnK cells, the intracellular +RNA concentration was significantly lower than in SPEV, 4.5 lg versus 5.5 lg of genome-equivalents/μL (p = 0.01). After 8 h, in ApnK cells there were no significant difference with points 0 and 4 (p = 0.5 and p = 0.1, respectively), whereas in SPEV at this stage the concentration of RNA was significantly higher than at points 0 and 4 (p = 0.001 and 0.01, respectively). In general, during the first day of infection, the concentration of +RNA in ApnK cells was almost ten times less than in SPEV (p = 0.001). By the fourth day after infection, ApnK cells showed maximum concentrations of +RNA, 7.8 lg of genome-equivalents/μL, with no differences with SPEV (p = 0.06). Subsequently, the amount of RNA in ApnK cells remained at the same level for 19 days, and in SPEV in late terms there was a tendency to decrease in the concentration of +RNA. The results obtained suggest that TBEV in A. peninsulae has an intracellular mechanism for controlling viral infection, which acts both at the stage of the entrance of virion into the cell and by regulating the rate of synthesis of genomic and/or replicative forms of viral RNA.

In the present study, we focus on the effect of eDNA on the initial bacterial adhesion, biofilm formation, and the mature biofilms of 17 different Salmonella serovars. We evaluated the roles of eDNA on the initial microbial adhesion, and found that some of Salmonella serotypes formed significantly more biomass in the presence of DNase I, during the early stages of biofilm formation at 28°C for 24 hours, while same strains were not produce biofilm at 37°C. We suggested that the reason for divesity among biofilm production abilities of different serovars may be due to the variability of gene expression levels at different growth temperatures. It was observed that eDNA had a notable negative effect on the initial attachment of these serovars. 45.8% of all pre-established biofilm samples of serovars were eradicated at high concentrations of DNase I (50 μg/mL). Our study highlights the serotype based role of eDNA in Salmonella strains. This report is one of the few that represents the inhibitive effect of eDNA on biofilm formation of Salmonella strains. Also, this is the fisrt evidence that eDNA has either inhibitive or stimulative effects dependind on the serovars.