Published online: 2018-07-13
DOI: 10.24075/brsmu.2018.026
Stem cells that penetrated deeply into the brain tissue are the main reason behind the relapses of glioblastoma multiforme after surgery. Finding new approaches to counter such relapses, including those that make use of oncolytic viruses, is a pressing issue. This study aimed to determine the sensitivity of cells of human glioblastoma multiforme to non-pathogenic enteroviruses, in vitro and in vivo (mice xenografts model). Glioblastoma tumor cells were exposed to type 1 poliovirus (Sabin vaccine strain), Coxsackie virus A7 (strain LEV8), Coxsackie virus A9 (strain LEV9) and Coxsackie virus B5 (strain LEV14). The virus reproduction intensity and cytolytic activity were assessed through infection of monolayered glioblastoma cell cultures. The ability of glialoblastoma cell cultures (enriched with tumor stem cells) to build subcutaneous tumors in immunodeficient mice after those cultures were exposed to viruses signaled the effectiveness of glioblastoma stem cells destruction. The study revealed that Coxsackie virus A7 and type 1 poliovirus possess the most pronounced oncolytic and replicative properties when tested on gliblastoma cells infected with viruses in vitro and on subcutaneous tumor xenografts in immunodeficient mice (in vivo). Type 1 poliovirus and Coxsackie virus A7 virus prevented development of tumors when glioblastoma neurospheric cell cultures were preincubated with viruses before subcutaneous implantation. Coxsackie virus B5 only managed to reduce the number of tumors developed, and Coxsackie virus A9 did not affect the tumor development at all. Thus, a number of non-pathogenic enteroviruses strains can destroy glioblastoma's stem cells, i.e. they show promise in the context of development of therapeutic agents for relapse-free treatment of glioblastomas. Ключевые
Published online: 2018-07-08
DOI: 10.24075/brsmu.2018.025
Existing therapies for glioblastoma multiforme do not ensure patient’s recovery. Oncolytic viruses (OV) represent a promising alternative as they can destroy glioblastoma-initiating stem cells, which the major cause of relapses. However, while individual OV strains are effective for some patients, they could be ineffective for others. To achieve a predictable therapeutic effect, live tumor cells of the patient need to be tested for the sensitivity to different viruses. The aim of this study was to assess how the sensitivity of tumor cells to viruses changes with passaging in cell culture. Primary glioblastoma cell cultures were prepared from excised tumors. We compared sensitivity of cells to four non-pathogenic enteroviruses (type 1 poliovirus, Coxsackie virus A7, Echoviruses 1 and 12), for freshly-explanted primary tumor cell cultures and for those that had undergone 700 divisions during the passaging. The sensitivity was assessed based on the proportion of viable cells by the MTT assay 72 hours after the cells were inoculated with serial 10-fold dilutions of virus preparations. Cells isolated from the tumors of 3 patients exhibited varying sensitivity to the used viral strains. Differences in the lowest virus dose required for the successful infection of cell cultures were as high as 105. Passaging induced sensitivity shifts, such as increased or decreased sensitivity to individual viruses. Differences in the sensitivity correlated with the ability of the infected cells to produce the virus. Based on our findings, we conclude that the sensitivity of cancer cells to viruses should be tested at very early stages of passaging, preferably in primary cultures.
Published online: 2018-07-06
DOI: 10.24075/brsmu.2018.023
Russian system of continuous medical education does not guarantee professional development of its participants: doctors do not report the number and specifics of the operations performed, self-assess their competence and compile individual professional development plans, and the professional community does not take part in these processes. Therefore, there is a need for accurate assessment of competence of plastic surgeons and objectivity of their self-assessment. We have conducted a study in the form of a single-stage questionnaire filled by the surgeons in person. The questionnaire contained two sections. The first section offered a competence self-assessment table listing 9 plastic surgery specialties; the participants used a 5-point scoring system to state their level, where 1 meant "no experience", 2 — "beginner", 3 — "specialist", 4 — "knowledgeable", 5 — "expert". The second section contained 9 test tasks (closed, univariate) used to objectively assess the level of competence of the participants. Each correct answer added 1 point to the participant's score, wrong answers added nothing. 162 people took part in the survey. The average age of the participants was 31.5 ± 6.9 years; average length of service — 4.0 ± 4.8 years. Analyzing the data, we applied the Kolmogorov-Smirnov test, Mann-Whitney test, Kruskal-Wallis test, Spearman's coefficient, used ANOVA, Levene's test, Duncan test. The values were considered statistically significant at p ˂ assessment score was 2.1 ± 0.92 points. We have discovered a statistically significant (p ˂ 0.001) correlation of the length of  0.05. The overall self-service with the level of self-assessment (rs= 0.72). The average score for the second section, the tests, was 2.6 ± 1.76 points (out of 9). The correlation between the test score and the length of service was insignificant (rs= –0.08, p = 0.3); same is true for the self-assessment (rs= –0.006, p = 0.9).
Published online: 2018-07-04
DOI: 10.24075/brsmu.2018.022
Cystic fibrosis is a severe autosomal recessive disease caused by mutations in the CFTR gene. The most common CFTR mutation occurring in the European population is F508del. Advances in the management of patients with cystic fibrosis aimed at blocking disease progression have considerably improved the prognosis, but gene therapy has turned to be less effective than expected. Capable of correcting mutations direct in the cells, genome editing, and specifically the CRISPR-Cas9 technology, raises hope of causal treatment for patients with cystic fibrosis. The aim of this work was to compare and improve the efficacy of F508del editing using different combinations of guide RNAs and Cas9. The study was carried out in HEK293T cells. The efficacy of editing was assessed for both plasmid and genomic sites by T7E1 analysis. The best effect was demonstrated by a combination of SaCas9 and sgRNA targeting F508del: 29% of alleles were successfully edited. A combination of SpCas9 and a similar sgRNA showed low efficacy due to the low expression of this guide RNA. All attempts to improve its expression failed. SgRNA stabilization by introducing a G-quadruplex into the sgRNA sequence and adding GG to the 5′-region also did not work. Perhaps, low performance of this guide RNA is determined by its nucleotide sequence, limiting its use.
Published online: 2018-07-01
DOI: 10.24075/brsmu.2018.021
The use of CRIPSR-Cas systems in genome editing has recently become one of the major research areas. Meanwhile, CAS proteins can be employed to develop novel techniques for molecular diagnostics. Traditional approaches to the identification of microorganisms have a few drawbacks: they are time-consuming (microbiological methods), insufficiently sensitive (immunoassays), expensive or labor-intensive (PCR, sequencing). The aim of this work was to obtain a functionally active Cas13a protein that could be used as a diagnostic tool and study its behavior under different conditions and at various target concentrations. We constructed an expression vector with the cas13a gene of Leptotrichia wadei under the control of T7 promoter. We obtained a functionally active Cas13a RNAse with pre-programmed activity, guide RNA, and a fragment of influenza B RNA sequence serving as a target. The functional activity of Cas13 RNAse was assessed by fluorescence in the reaction mix containing guide RNA, target RNA, and a molecular RNA beacon. The obtained protein Cas13a was able to specifically recognize the target and did not exhibit any non-specific RNAse activity. This study can become a basis for developing a novel, rapid, specific and sensitive method for pathogen detection.
Published online: 2018-06-27
DOI: 10.24075/brsmu.2018.019
CRISPR-Cas is an immune system of prokaryotes that protects them against alien replicons, mainly viruses and plasmids. Short sequences (spacers) complementary to the regions of a viral or plasmid genome are inserted into a CRISPR array conferring resistance to reinfection. Infections caused by Escherichia coli still present a serious challenge for clinical medicine. The aim of this study was to scan the genome of Escherichia coli HS for CRISPR-Cas components. The search was conducted using MacSyFinder (Macromolecular System Finder, ver. 1.0.2.), a program for bioinformatic modelling. Sequence homology searches were done using makeblastdb (ver. 2.2.28) and HMMER (ver. 3.0) tools. Bioinformatics-based methods allowed us to detect one CRISPR-Cas system in the studied genome of Escherichia coli HS and read the spacer sequences of its CRIPSR array. The protospacer regions complementary to the spacer sequences of the detected CRISPR array are typical for a few types of phages. Based on these findings, one can assess the degree of bacterial resistance to alien genetic elements.