Bulletin of RSMU

ISSN Print 2500–1094 ISSN Online 2542–1204

Bulletin of RSMU

BIOMEDICAL JOURNAL OF PIROGOV UNIVERSITY (MOSCOW, RUSSIA)

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Dimitrieva TV, Reshetov DA, Zhernovkov VE, Vlodavets DV, Zotova ED, Ermolkevich TG, Deykin AV

Genetically modified animals are an important tool for biomedical research. The CRISPR/Cas9 editing genome system is increasingly being used for production of such animals. Through microinjection, complex with guide RNA and Cas9 protein is delivered in fertilized eggs from which the animal subsequently develops with a modification in the genome. Generally, analysis of the specificity and efficiency of the system in each case is carried out after obtaining a progeny with the likely mutation. However, analysis at the preimplantation stage would allow reducing the time of the experiment, as well as understanding the reason for the birth of a small number of transgenic animals, or even lack of them in the offsprings. The paper proposes a modification of the method of preparation of total DNA from mouse blastocysts. The modification allows to easier and faster detect the results of microinjection of the CRISPR/Cas9 complex in the zygote. Having applied the method described in this paper, we successfully identified short deletions in intron 34 of dystrophin gene (DMD) in 12 out of 13 treated embryos and insertion in the break site in intron 8 of the DMD gene in 11 out of 21 samples analyzed. Using for analysis the total DNA prepared by the method proposed, you can analyze up to 20 different sites in the mouse embryo genome at the blastocyst stage without the need for full genomic amplification.

Received: 2016-06-16

Published online: 2017-01-05

DOI: 10.24075/brsmu.2016-03-02

Comments 0

VIEWS 3990

Zotova ED, Reshetov DA, Zhernovkov VE, Vlodavets DV, Dimitrieva TV, Deykin AV

Duchenne muscular dystrophy (DMD) is a common genetic disease caused by a mutation of the dystrophin gene. It leads to death in childhood. At the time of writing this paper, patients had access to supportive therapy only. However, DMD treatment methods are actively being developed. Exon skipping is a promising method. Exon skipping involves restoration of the reading frame within a gene by inducing alternative splicing. This leads to synthesis of truncated but still functional dystrophin. The paper assesses the functionality of the truncated forms of dystrophin resulting from correction of nonsense mutations and internal exon indels by exon-skipping technique. The assessment was made based on data on the phenotype of carriers of mutations in the dystrophin gene taken from the Leiden Open Variation Database (LOVD). It was revealed that the same mutation could manifest itself as a variety of phenotypes. This, perhaps, is as a result of the patients having different genetic background. For example, deletion of exon 48, for which there is 97 records in LOVD, resulted in asymptomatic diseases in 2 % of cases, Duchenne muscular dystrophy in 60 %, Becker muscular dystrophy (characterized by milder symptoms than DMD) in 12 % and intermediate phenotype in 26 % of cases. High phenotypic variability of mutations of the dystrophin gene raises the issue of limits of applying exon skipping for treatment of inherited myopathies.

Received: 2016-06-17

Published online: 2017-01-05

DOI: 10.24075/brsmu.2016-03-03

Comments 1

VIEWS 4481

Pozdniakova NV, Grigorieva EYu, Shevelev AB

This article describes a method of obtaining curcumin- and gadolinium-loaded micelles based on triblock amphiphilic polyethylene glycol and polypropylene glycol copolymers Pluronic F-127 and Pluronic P-123 (Sigma-Aldrich, USA) superficially functionalized with recombinant human -fetoprotein. The size of nanoparticles was measured using dynamic light scattering and amounted to an average of 50 to 100 nm. The micelles were stable: stored at +4 °С for 10 days, they exhibited no changes in their properties that would not fall within the standard error of measurement for the methods used for the analysis. Preliminary in vivo experiments conducted on mice showed no conspicuous toxicity of micelles with the maximum possible concentration of gadolinium, which enables their use in tumor tissue imaging in vivo.

Received: 2016-06-23

Published online: 2017-01-05

DOI: 10.24075/brsmu.2016-03-04

Comments 0

VIEWS 4185

Kaplan AYa, Zhigulskaya DD, Kiriyanov DA

In this work we have tested the assumption that an individual can control a target finger of a phantom by voluntarily focusing his attention on the luminous marker located on that finger in the complex of a P300 wave–based brain-computer interface (P300 BCI) and an anthropomorphic phantom. Because each correct movement of phantom fingers indicates a sufficient mental effort aimed at this action, creating a new ideomotor training simulator of smaller movements of the hand becomes possible. Our study included 21 volunteer subjects of both sexes aged 18–25. It was shown that with P300 BCI complex the subjects learned to control phantom fingers on the first day of the experiment, the percentage of successful attempts being no less than 69 %. Failures were mainly related to the insufficient attention focus on luminous markers on the target phantom fingers. We hypothesize that P300 BCI — Hand Phantom complex can be a basis for developing a fine motor skills simulator.

Received: 2016-04-11

Published online: 2017-01-05

DOI: 10.24075/brsmu.2016-02-03

Comments 0

VIEWS 3621

Kurashenko AV, Samoilova EO, Balaeva MV, Chicherin IV, Petrov DYu, Kamenski P, Levitskii SA

Maintaining mitochondrial genome integrity is essential for the viability of the whole organism. Mitochondrial genome mutations lead to muscular dystrophies, neurodegenerative diseases, and are associated with aging. In this work a baker’s yeast (Saccharomyces cerevisiae) mitochondria model was used to investigate DNA-binding abilities of different domains of a mitochondrial Abf2p protein which participates in homologous recombination and reparation. A weak non-specific HMG1 binding to linear DNA and a specific HMG1 binding to a branched DNA with a dissociation constant of 510 nM have been discovered. The HMG2 domain itself does not bind to any DNA and either has other functions or demonstrates its DNA-binding activity in a full-length protein only.

Received: 2015-09-29

Published online: 2017-01-05

DOI: 10.24075/brsmu.2016-01-11

Comments 0

VIEWS 3794

Kharlamov VG, Kulakov VN, Lipengolts AA, Shimanovskii NL

To improve the safety of Gd3<sup>+</sup>-based contrast agents (GBCA) in clinical practice, it is recommended to use the most stable substances and to consider conditions determining their stability. The aim of this study was to compare the stability of GBCAs for magnetic resonance imaging in the presence of zinc and calcium ions and polyvinylpyrrolidone (PVP) in water, phosphate buffer solution and blood serum using proton NMR relaxometry. The study demonstrated that macrocyclic gadobutrol is more stable than all linear contrast agents. The addition of PVP (10 mg/ml) improved the stability of linear GBCAs in phosphate buffer solution and blood serum. Calcium ions have a much weaker destabilizing effect on GBCAs than zinc ions.

Received: 2015-09-29

Published online: 2017-01-05

DOI: 10.24075/brsmu.2016-01-12

Comments 0

VIEWS 3400

Frolova OP, Schukina IV, Novoselova OA, Stakhanov VA, Kazennyi AB

Despite various anti-tuberculosis measures in the setting of HIV infection, the epidemiological situation of tuberculosis in Russia is deteriorating. We have analyzed the data of statistical report form no. 61 for years 2004–2014, surveillance data on individual TB cases with HIV coinfection for years 2004–2014 (personal data) and TB care arrangements for patients with HIV in 20 regions. The main causes of the deteriorating epidemiological situation are the growing immunodeficiency in patients with TB coinfection, unseparated epidemiologically dangerous patient flows (patients with tuberculosis and HIV-infected patients) and low quality preventative measures in special care medical facilities. Chemoprophylaxis can be an effective method of controlling the spread of tuberculosis among HIV-infected patients if it is recommended by a qualified tuberculosis therapist to patients adhering to regular drug intake under supervision of medical personnel. Otherwise a large scale chemoprophylaxis can result in an increased proportion of patients with drug-resistant tuberculosis. This works suggests criteria for the evaluation of tuberculosis care effectiveness considering the pathogenesis of the disease during late stages of HIV.

Received: 2015-12-06

Published online: 2017-01-05

DOI: 10.24075/brsmu.2016-01-13

Comments 0

VIEWS 3458

Aniskevich AS, Halfin RA, Tatarinova LV

The article reviews basic methods of performance assessment of scientific medical organizations in Russia. Qualitative and quantitative effectiveness criteria are provided. International practices are described; a comparative analysis of assessment methods used in Russia and abroad is carried out. Global trends in the development of approaches to analyzing the effectiveness of scientific organizations are reviewed. Based on our analysis, a compelling rationale for developing more up-to-date criteria for evaluating the performance of scientific medical organizations is given.

Received: 2016-02-20

Published online: 2017-01-05

DOI: 10.24075/brsmu.2016-01-14

Comments 0

VIEWS 3605