Bulletin of Russian State Medical University

CRISPR-Сas systems are widespread in bacteria and archaea. They provide adaptive immunity against bacterial phages and plasmids and exert a few important functions like regulation of gene expression, DNA repair or virulence formation. We have analyzed the CRISPR-Cas systems of 7 M. tuberculosis lineages with fully sequenced genomes, namely Beijing, B0/W-148, EAI, Haarlem, Ural, LAM, and S. The CRISPR-Cas systems present in the analyzed genomes belong to type III-A. M. tuberculosis lineages differ in their CRISPR-Cas structure; in the Beijing lineage a part of the system is reduced. We have conducted a search for the functionally related partners and compensatory mechanisms of cas-genes using a method of phylogenetic profiling. The obtained phylogenetic profiles show that some genes have undergone similar evolutionary events. The reduction of the system’s part in the Beijing lineage was accompanied by at least two evolutionary losses and one acquisition of genome regions. Exploration of alternative CRISPR-Cas functions in M. tuberculosis and their possible associations with other gene systems remains an exciting challenge.

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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.

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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.

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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.

Multidrug resistance pumps (MDR pumps) of bacteria confer protection against aggressive environmental factors. The genes coding for MDR pumps are thought to be variable. They belong to the group of the so-called contingency genes, i.e are necessary for bacterial adaptation to the changing environment. The aim of the present work was to establish how conserved are the sequences of genes coding for MDR pumps. We analyzed the sequences of AcrA, AcrB and TolC proteins of different Escherichia coli strains. Using sequence alignment tools, we demonstrated that strains originating in different countries and cultured in the labs for a long time are amazingly conserved in terms of AcrAB-TolC sequences. They resemble housekeeping genes, suggesting the involvement of the AcrAB-TolC pump into permanent “cleaning” of various biotic and abiotic agents.

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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, 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 their sensitivity to different viruses. The aim of this study was to assess how sensitivity of tumor cells to viruses changes with passaging in the cell culture. Primary glioblastoma cell cultures were prepared from excised tumors. We compared the sensitivity of the cells to four non-pathogenic enteroviruses (type 1 poliovirus, Coxsackievirus A7, Echoviruses 1 and 12) for freshly explanted primary tumor cell cultures and for those that had undergone 700 divisions during passaging. Cell sensitivity was assessed by the MTT assay based on the proportion of viable cells 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 the 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.

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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.

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There are a few major factors determining the success of in vitro fertilization (IVF), including the potential of embryos to implant and the receptivity of the endometrium, which, in turn, are directly dependent on the ovarian reserve, i.e. the quality and quantity of oocytes. Diminished ovarian reserve can be inferred from reduced blood flow to the uterus on Doppler sonography. Based on the results of 3D power Doppler imaging of uterine blood flow on the day of ET and Virtual Organ Computer-aided AnaLysis (VOCAL), we attempted to predict the outcomes of IVF and embryo transfer (ET) in 56 female patients with different ovarian reserves. Blood flow was measured in the uterus, subendometrial region and endometrium, and IVF cycle outcomes were subsequently assessed. We have established an association between the characteristics of uterine and subendometrial blood flow and the outcomes of IVF cycles and ET. No such association has been established for the endometrium, though. Therefore, a 3D power Doppler examination on the day of embryo transfer provides valuable information on the endometrium receptivity and can be used as a prognostic marker of IVF success.

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Traditionally, anti-age therapies employ ultraviolet radiation and exposure to ozone, nitric oxide and electromagnetic fields. Non-thermal atmospheric-pressure plasma (NTAPP) combines the effects of all those techniques. The aim of our study was to assess the feasibility of low-dose NTAPP application in anti-age facial skin therapy. Ten female patients aged 50 to 55 years were examined and three facial zones were chosen for the experiment: the T-zone (the central part of the forehead) and the “crow’s feet” areas on the right and left sides of the face. Ultrasonography was performed on the DUB SkinScaner before the treatment course and 24 hours after the last treatment. Cleansed skin was exposed to a low-energy NTAPP helium jet generated by the HELIOS system (Plasma Research and Production, Russia). Exposure time was 5 min per zone. Each participant received 10 NTAPP procedures on alternate days. Before therapy, the skin condition in all participants fitted into morphotype 3. Ultrasonography of the studied zones revealed a considerable deformation of the skin surface, a thickening of the epidermis with a distinct border between the epidermis and the dermis, a slight thinning of the dermis, its relatively homogenous echogenicity, and a blurred border between the dermis and the hypodermis. After the course was completed, all patients demonstrated an evener skin surface, reduced epidermal thickness and reduced acoustic density of the epidermis and the dermis; the dermis tended to have above average thickness. The most significant changes were observed for the wrinkles: they became less pronounced in the “crow’s feet” area. Exposure to NTAPP caused the epidermal corneum to diminish in thickness; it also stimulated microcirculation and improved the condition of the hydrolipidic film, all of which ultimately led to the effacement of wrinkles. Treatment produced no adverse effects on the skin or its appendages.

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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 ˂ 0.05. The overall selfassessment score was 2.1 ± 0.92 points. We have discovered a statistically significant (p ˂ 0.001) correlation of the length of service with the level of self-assessment (r = 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 (r = -0.08, p = 0.3); same is true for the self-assessment (r = -0.006, p = 0.9).

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The health of the cervical spine (CS) and the functional state of the pectoral girdle are interdependent. Injuries to the pectoral girdle can be an underlying cause of CS pain, including cervicalgia. The aim of this study was to evaluate the condition of the cervical spine in patients with cervicalgia developed after a pectoral girdle injury using radiographic and physical examinations. The study included 400 patients complaining of cervicalgia. Pain intensity was evaluated on the visual analog scale (VAS); the impact of the condition on patients’ lives was assessed using the Neck Disability Index (Russian language). During physical examinations, the general health of the spine was evaluated and abnormalities in the cervical spine were noted. All participants underwent a radiographic scan of the cervical spine in the lateral and anterior-posterior projections; 49.5% of patients underwent postural digital radiography to evaluate their CS sagittal profile. All patients received an MRI scan. Based on the results, we identified certain functional changes in the cervical spine which possibly caused cervicalgia. Structurally and functionally, the changes were divided into static and dynamic. We conclude that cervical spinal pain is a common problem among patients with previous pectoral girdle injury.

Knee osteochondritis desiccans, or Koenig's disease, is commonly found in active young people engaged in manual labor, sports etc., i.e. socially active population. Today, we have a good number of surgical method to treatment of this disorder; however, there is still much controversy about their application, and no single approach is considered to be the optimal one. Plus, high-quality biomaterials required for the intervention are sometimes unavailable. The analysis of the results of treatment of patients (spanning several years) proved urgency of the problem and highlighted the necessity to solve a number of related issues. This paper presents the algorithm of surgical treatment of knee osteochondritis desiccans. We have provided surgery validation criteria, suggested optimal methods of correction of local cartilage defects (depending on the degree of damage and patient's age) and outlined some recommendations based on our practical experience.

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