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The proper structure of a biosafety system as a way of reducing the vulnerability of a society, economy or state in the face of a biogenic threat
стр.5-18
Gushchin VA, Manuilov VA, Makarov VV, Tkachuk AP
To understand how vulnerable are a society, an economy and a state in the face of a biohazard, one should attempt to identify any potential holes in the national biosafety system, such as the lack of important components or technologies for biological monitoring and the inadequacy of existing analytical methods used to prevent or counteract biogenic threats. In Russia, biological monitoring is quite advanced. However, the agencies that ensure proper functioning of its components lack collaboration and do not form a well-coordinated network. Each of such agencies alone cannot provide comprehensive information on the subject. In the Russian Federation, there are at least 4 state-funded programs that collect epidemiological data and are quite efficient in performing the narrow task of monitoring infections. But because there is no central database where epidemiological data can be channeled and subsequently shared, these agencies do not complete each other. This leaves the Russian society, economy and state vulnerable to biogenic threats. We need an adequately organized, modern, fully functional and effective system for monitoring biohazards that will serve as a basis for the national biosafety system and also a tool for the identification and elimination of its weaknesses.
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Multiparametric detection of bacterial contamination based on the photonic crystal surface mode detection
стр.19-24
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.
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High-performance aerosol sampler with liquid phase recirculation and pre-concentration of particles
стр.25-31
Akmalov AE, Kotkovskii GE, Stolyarov SV, Verdiev BI, Ovchinnikov PS, 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.
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Performance of the original workstation for aerosol tests under controlled conditions
стр.32-38
Kleymenov DA, Verdiev BI, Enenko AA, Gushchin VA, Tkachuk AP
Air quality monitoring is essential when it comes to protecting the urban population, especially that of big metropolises, from biohazards including biopathogens (BPs). This process is aided by different samplers and analyzers of aerosol pollutants, filters and disinfection systems. Their performance is tested using experimental aerosol formulations with a predetermined composition. Unfortunately, the majority of such systems available in Russia are only able to process a few hundred liters of air per time unit, which is too little. Big aerosol chambers (10 to 20 m) are very expensive and may not fit into a lab, necessitating an extensive overhaul. In this work we present a workstation for the detection of BP markers under controlled conditions based on the microbiological safety box MSB-III-Laminar-C-1.5 (380.150.01) that was originally designed to test the performance of samplers and analyzers of experimental aerosol formulations. Our workstation can handle the majority of BSL-1-2 BPs and, given the chamber volume that satisfies the requirements of aerosol experiments (> 4 m), can be installed in a lab with an area of >10 m.
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Comparison of fluorescence excitation modes for CdSe semi-conductor quantum dots used in medical research
стр.39-45
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.
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A study of antimicrobial activity of polyphenols derived from wood
стр.46-49
Shevelev AB, Isakova EP, Trubnikova EV, La Porta N, Martens S, Medvedeva OA, Trubnikov DV, Akbaev RM, Biryukova YK, Zylkova MV, Lebedeva AA, Smirnova MS, Deryabina YI
Due to the spreading and increasing drug resistance of pathogens, the search for novel antibiotics is becoming ever more important. Plant-derived polyphenols are a vast and promising class of compounds with a potential to fight infectious diseases. Still, they are not routinely used in clinical practice. No reports on the in vivo studies of these compounds have been presented. The aim of our work was to compare the antimicrobial activity of resveratrol (stilbene), dihydroquercetin and dihydromyricetin (flavonols) extracted from the bark and wood of conifers against the dermatophytes Staphylococcus aureus , Pseudomonas aeruginosa and Candida albicans . Using the radial diffusion assay, we established that dihydroquercetin, resveratrol and dihydromyricetin exhibit high activity against S. aureus even at the smallest possible concentrations of 0.22, 0.15, and 0.15 mM, respectively. In contrast, the highest achievable concentrations of these compounds in the solutions (21.5, 15.5 and 15.0 mM for dihydroquercetin, resveratrol and dihydromyricetin, respectively) have no effect on the growth of P. aeruginosa and C. albicans . These findings suggest that polyphenols derived from conifers could have a potential to be used as a medicine for topical application to treat bacterial infections of the skin caused by S. aureus .
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Identification of microorganisms by Fourier-transform infrared spectroscopy
стр.50-57
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 .
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Parameters of vancomycin pharmacokinetics in postoperative patients with renal dysfunction: comparing the results of a pharmacokinetic study and mathematical modeling
стр.58-64
Ramenskaya GV, Shokhin IE, Lukina MV, Andrushchishina 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 С and C were measured by high-performance liquid chromatography followed by the calculation of the area under the plasma concentration-time curve AUC. For mathematical modeling, a single-compartment model was employed; PK parameters were estimated using R 3.4.0. The values of C 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), AUC measured at the end of treatment was significantly higher than its value predicted by MM ( р = 0.011). The probability of achieving the target AUC to MIC ratio of over 400 μg•h /ml is higher in the group of patients with C = 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.
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Evaluation of cardiac MRI efficacy in the diagnosis of hibernating myocardium
стр.65-69
Rustamova YK, Imanov GG, Azizov VA
The efficacy of cardiac MRI in the diagnosis of hibernating myocardium remains understudied. The existing body of evidence on this matter comes mainly from observational studies carried out in heterogenous (in terms of cardiac pathology) cohorts of patients, which complicates the interpretation of the results. The aim of our study was to evaluate the efficacy of cardiac imaging techniques in 144 patients with a history of myocardial infarction, multivessel coronary artery disease and a low ejection fraction of the left ventricle. All participants underwent stress echocardiography and cardiac MRI examinations. The following parameters were factored into: a) the number of identified segments with abnormal myocardial contractility; b) the transmurality index (scar thickness); c) the volume of the viable myocardium relative to its total mass. The study revealed that on average there were 2.72 ± 0.82 segments with contractile dysfunction per patient. Cardiac MRI was able to detect significantly more hibernating segments than stress echocardiography. On average, the difference in the number of detected segments was 36 (56; 86) at 95% CI and р < 0.01. We established that as the transmurality index increases, the number of hypokinetic segments decreases ( r = -0.78; р = 0.0314) while the number of akinetic segments ( r = -0.84; р = 0.0282) goes up. This needs to be accounted for when selecting a treatment strategy for such patients. We conclude that cardiac MRI is a more effective and sensitive diagnostic technique in patients with hibernating myocardium that allows detecting significantly more cardiac segments with contractile dysfunction than stress echocardiography. Delayed contrast enhancement is instrumental in estimating the thickness and extent of cardiac fibrosis, the parameters that should be accounted for when deciding on the treatment strategy in such patients.
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The efficacy of CRISPR-Cas9-mediated induction of the CCR5delta32 mutation in the human embryo
стр.70-74
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 CO 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.
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Levels of cell-free DNA and DNase I activity in complicated and normal pregnancies
стр.75-80
Avetisova KG, Kostyuk SV, Kostyuk EV, Ershova ES, Shmarina GV, Veiko NN, Spiridonov DS, Klimenko PA, Kurtser MA
Placental pathology is accompanied by the activation of apoptosis in the trophoblast and the subsequent increase in the concentrations of microvesicles containing placental (or fetal) DNA accumulating in the maternal blood. Fragments of fetal DNA stimulate the release of nuclear and/or mitochondrial DNA fragments by neutrophils. Therefore, one can expect that complicated pregnancies will be characterized by the dramatic elevation of total cell-free DNA (cfDNA) levels in maternal plasma. The aim of this work was to study the dynamics of plasma cfDNA concentrations and the activity of DNase I, an enzyme involved in the elimination of cfDNA from the bloodstream, in nonpregnant and pregnant women. Our study recruited 40 healthy nonpregnant women, 40 women with uncomplicated pregnancies and 35 women with the intrauterine growth restriction (IUGR) of the fetus. We did not observe the elevation of the total cfDNA concentrations in the patients with complicated pregnancies. Moreover, cfDNA concentrations in their plasma were even lower (though this difference was statistically insignificant) than in healthy pregnant and nonpregnant women. The median values of cfDNA concentrations in the group of healthy nonpregnant women were 75.5 ng/ml; in the group of healthy pregnant women, 78.0 ng/ml; and in the patients with IUGR, 42.1 ng/ml. At the same time, we observed a significant increase in DNase I activity in the plasma of women with IUGR. The median DNase I activity in the groups of healthy pregnant and nonpregnant women was 3.0 and 3.4 IU/ml, respectively. In patients with different grades IUGR of the fetus this parameter was as high as 6.3 IU/ml ( р < 0.001). Increased DNase I activity in the plasma of women with complicated pregnancies indirectly suggests a transient elevation of circulating cfDNA levels. Our study shows that the high level of activity exhibited by the cfDNA elimination system impedes the analysis of cfDNA concentrations in complicated pregnancies and skews its results. However, if cfDNA, DNase I activity and the cfDNA/DNase I ratio were all taken into account, it could be possible to develop a tool for the monitoring of cell death in the mother throughout the entire pregnancy.
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Comparative assessment of stillbirth rate in Bryansk Region, EU and CIS countries (1995-2014)
стр.81-88
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.
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Prevalence of toxocara infection in domestic dogs and cats in urban environment
стр.89-92
Kurnosova OP, Odoevskaya IM, Petkova S, Dilcheva V
Toxocariasis is the type of helminthic infection found in dogs and cats most often. It is a zoonotic disease that presents a serious threat to the national public health. Urban environment favors transmission of toxocara from animals to people; soil is the key element of such transmission. To learn the degree of toxocara invasion in domestic cats and dogs living in Moscow, we studied their feces for 7 years applying the flotation method. We found that in domestic dogs the intensity of toxocara invasion was 2.43%, but there is a big difference between puppies and adult animals: 5.53% of the former, twice as much as the latter, suffered from the invasion. The intensity of infection in adult cats was 3.97%; kittens, same as puppies, were more prone to host toxocara: 10.44% of those examined did. In general, 5.75% of cats had toxacara, which is twice as much compared to dogs; the figure applies to all ages. Stable infestation of domestic animals with this species of helminths makes them a constant source of toxocara eggs contamination in urban environments, which ups the risk of larvae toxocariasis for people.
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