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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Kodyleva TA, Kirillova AO, Tyschik EA, Makarov VV, Khromov AV, Gushchin VA, Abubakirov AN, Rebrikov DV, Sukhikh GT

The editing of the CCR5 gene in the CD4+ T cell genome is an effective way of preventing HIV-1 proliferation. Very similar strategies can be used to protect the fetus of an HIV-infected female showing a weak response to antiretroviral therapy. Inducing the “natural” CCR5delta32 mutation in a zygote may guard the fetus against HIV infection both in utero and at birth. In this study, we optimize the CRISPR-Cas9 system to induce a homozygous 32-nt deletion similar to the naturally occurring CCR5delta32 allele in the human zygote at the S-phase. Edits were done in the abnormal tripronuclear zygotes unsuitable for IVF. Sixteen tripronuclear zygotes in the S-phase obtained from WT CCR5 donors were injected with an original CRISPR-Cas9 system designed by the authors. Upon injection, the zygotes were transferred into the Blastocyst (COOK) embryo culture medium and cultured for 5 days in a CO2 incubator until blastocysts were formed (approximately 250 cells). Eight zygotes that successfully developed into blastocysts were PCR-genotyped to analyze the efficacy of genome editing. Of 16 zygotes injected with CRISPR-Cas9, only 8 reached the blastocyst stage. PCR genotyping revealed the absence of the initial WT CCR5 variant in 5 of 8 blastocysts (100% CCR5delta32 homozygous). Two had about 3% and one about 20% of WT CCR5 mosaicism. This leads us to conclude that the efficacy of the proposed CRISPR-Cas9 system for the induction of the CCR5delta32 mutation in human embryos is very high producing more than 50% of completely modified embryos.

Received: 2018-09-26

Published online: 2018-10-17

DOI: 10.24075/brsmu.2018.052

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VIEWS 15097

Ramenskaya GV, Shokhin IE, Lukina MV, Andrushishina TB, Chukina MA, Tsarev IL, Vartanova OA, Morozova TE

Mathematical modeling of pharmacokinetic (PK) and pharmacodynamic (PD) parameters essential for establishing correct dosing regimens is an alternative to pharmacokinetic studies (PKS) adopted in the clinical setting. The aim of this work was to compare the values of PK parameters for vancomycin obtained in an actual PKS and through MM in postoperative patients with kidney injury. Our prospective study included 61 patients (47 males and 14 females aged 60.59 ± 12.23 years). During PKS, drug concentrations at steady state Сtrough and Cpeak were measured by high-performance liquid chromatography followed by the calculation of the area under the plasma concentration-time curve AUC24. For mathematical modeling, a single-compartment model was employed; PK parameters were estimated using R 3.4.0. The values of Ctrough measured 48 h after the onset of antibiotic therapy during PKS were significantly lower than those predicted by MM (р = 0.004). In a group of patients with acute kidney injury (AKI), AUC24 measured at the end of treatment was significantly higher than its value predicted by MM (р = 0.011). The probability of achieving the target AUC24 to MIC ratio of over 400 μg•h /ml is higher in the group of patients with Ctrough = 10–15 μg /ml. Our findings confirm that the use of MM in postoperative patients with renal dysfunction is limited and therapeutic drug monitoring should be used instead.

Received: 2018-05-16

Published online: 2018-10-11

DOI: 10.24075/brsmu.2018.051

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VIEWS 4611

Kuzishchin YA, Martynov IL, Osipov EV, Samokhvalov PS, Chistyakov AA, Nabiev IR

Fluorescence spectroscopy is a powerful tool used in applied biological and medical research. Colloid semi-conductor quantum dots are promising fluorescent tags for simultaneous detection of different biopathogens. The techniques employing these tags can be improved by selecting the optimal modes for signal excitation and detection. The aim of the present work was to derive a mathematical expression to describe the signal-to-noise ratios in the pulsed and modulated excitation modes. Below, we compare these two modes of fluorescence excitation in ultralow quantities of quantum dots. We demonstrate that modulated excitation should be preferred for CdSe/ZnS quantum dots given that signal accumulation time is over 100 mc and the photosensor is exposed to background light of > 1 μW.

Received: 2018-07-29

Published online: 2018-10-09

DOI: 10.24075/brsmu.2018.050

Comments 0

VIEWS 5003

Akmalov AE, Kotkovskii GE, Stolyarov SV, Verdiev BI, Ovchinnikov RS, Pochtovyy AA, Tkachuk AP, Chistyakov AA

Testing the surrounding environment for the presence of biogenic aerosols is crucial in ensuring its safety for the population. It is often necessary to collect aerosol samples from large areas in short time, which demands excellent particle collection efficiency, a sufficient incoming air flow rate and a capacity to maintain the viability of the collected samples. Below we present the aerosol sampler with a high volumetric flow rate based on a two-stage particle concentration algorithm and consisting of a virtual impactor and a cyclone concentrator with a recirculating liquid phase. We provide all necessary calculations and an algorithm for modeling impactor parameters. The sampler was tested using dry and liquid formulations dispersed into the particles of 0.5 to 5 μm in diameter. We demonstrate that at volumetric flow rates over 4,000 l/min efficiency of particle collection into the liquid phase at a volume of 10 ml makes over 20% of the total aerosol mass and at volumetric flow rates over 300 l/min this value is over 60%. The proposed device maintains viability of the collected microorganisms. The sampler is portable, with flexible settings for sampling and cleaning, and can be controlled remotely over the network.

Received: 2018-07-27

Published online: 2018-10-05

DOI: 10.24075/brsmu.2018.049

Comments 0

VIEWS 5130

Korsakov AV, Hoffmann V, Pugach LI, Lagerev DG, Korolik VV, Bulatseva MB

Stillbirth rate is one of the most important indicators allowing assessment of the population's living standards and forecasting its growth rate. This study aimed to compare the frequency of stillbirths in the Bryansk region, EU and CIS countries based on the official statistical data covering the period from 1995 to 2014. It was established that male stillbirth rate is greater than female stillbirth rate both in the Bryansk region and the Russian Federation (by 14.2% and 9%, respectively), which is consistent with the worldwide trend that has the male stillbirth risk 10% higher than that for girls. Provided the dynamics remain the same, 2016 to 2021 the share of stillbirths in the Bryansk region will continue to grow and reach 28.8% by 2021, which is greater than nationwide. The gender distribution will also grow to 32.6% (male stillbirths more common than female) by 2021.

Received: 2018-06-15

Published online: 2018-10-02

DOI: 10.24075/brsmu.2018.048

Comments 0

VIEWS 5076

Petrova IO, Konopsky VN, Sukhanova AV, Nabiev IR

Conventional techniques for food and water quality control and environmental monitoring in general have a number of drawbacks. Below we propose a label-free highly accurate analytical technique for multiplex detection of biomarkers based on the analysis of propagation of Bloch waves on the surface of a photonic crystal. The technique can be used to measure molecular and cell affinity interactions in real time by recording critical and excitation angles of the surface wave on the surface of a photonic crystal. Based on the analysis of photonic crystal surface modes, we elaborated a protocol for the detection of the exotoxin A of Pseudomonas aeruginosa and the heat-labile toxin LT of Escherichia coli. The protocol exploits detection of affinity interactions between antigens pumped through a microfluidic cell and detector antibodies conjugated to the chemically activated silica chip. The proposed technique is highly sensitive, cheap and less time-consuming in comparison with surface plasmon resonance.

Received: 2018-07-28

Published online: 2018-09-29

DOI: 10.24075/brsmu.2018.047

Comments 0

VIEWS 5755

Suntsova AYu, Guliev RR, Popov DA, Vostrikova TYu, Dubodelov DV, Shchegolikhin AN, Laypanov BK, Priputnevich TV, Shevelev AB, Kurochkin IN

The need for novel techniques of rapid identification of pathogenic microorganisms arises from the massive spread of drug-resistant nosocomial strains and the emergence of centers for biohazard control. Fourier-transform infrared spectroscopy is a promising alternative to mass spectrometry as it is cost-effective, fast and suitable for field use. The aim of this work was to propose an algorithm for the identification of microorganisms in pure cultures based on the analysis of their Fourier transform infrared spectra. The algorithm is based on the automated principal component analysis of infrared spectra. Unlike its analogues described in the literature, the algorithm is capable of identifying bacteria regardless of the culture medium or growth phase. The training sample included the most prevalent causative agents of infections and sepsis in humans: Staphylococcus aureus (n = 67), Enterococcus faecalis (n = 10), Enterococcus faecium (n = 10), Klebsiella pneumoniae (n = 10), Escherichia coli (n = 10), Serratia marcescens (n = 10), Enterobacter cloacae (n = 10), Acinetobacter baumannii (n = 10), Pseudomonas aeruginosa (n = 10), and Candida albicans (n = 10). The model we built successfully passed a series of blind tests involving clinical isolates of 10 methicillin-resistant (MRSA) and 10 methicillin-sensitive (MSSA) Staphylococcus aureus strains as well as pair mixes of these cultures with clinical isolates of Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae.

Received: 2018-08-01

Published online: 2018-09-29

DOI: 10.24075/brsmu.2018.046

Comments 0

VIEWS 5102

Vaneev AN, Alova AV, Erofeev AS, Gorelkin PV, Aleksashkin AD, Beznos OV, Chesnokova NB, Kost OA, Majouga AM, Korchev Y, Klyachko NL

Reactive oxygen species (ROS) are vital metabolites in numerous biological functions. Disorders of cellular mechanisms can cause overproduction of ROS and, subsequently, oxidative damage to DNA, proteins, cells and tissues, which is associated with the pathogenesis of a number of neurodegenerative and inflammatory diseases. Development of highly sensitive, relatively simple and fast-to-implement innovative methods to detect oxidative stress requires understanding of how such disorders relate to the level of ROS. This research aimed to apply the biological fluids' ROS detection method we have developed (using the stable platinum nanoelectrode that allows assessing the level of hydrogen peroxide (H2O2) down to 1 μM) and determine the level of H2O2 in lacrimal and intraocular fluids of rabbits, as well as to investigate how the level of H2O2 changes under the influence of antioxidant therapy. The effect superoxide dismutase (SOD) nanoparticles produce on biological fluids' ROS level was shown. The level of H2O2 in lacrimal fluid increased 10 and 30 min after instillation of SOD nanoparticles. As for the intraocular fluid, H2O2 concentration starts to grow only 30 min after instillation of SOD nanoparticles, which suggests that the they penetrate the internal structures of the eye gradually. The method seems to be of value in the context of eye diseases diagnosing and treatment.

Received: 2018-06-28

Published online: 2018-09-28

DOI: 10.24075/brsmu.2018.045

Comments 0

VIEWS 4873