[Evaluation of the dynamics of detection of viable SARS-CoV-2 (Coronaviridae: Betacoronavirus: Sarbecovirus) in biological samples obtained from patients with COVID-19 in a health care setting, as one of the indicators of the infectivity of the virus].

INTRODUCTION: The study of the mechanisms of transmission of the SARS-CoV-2 virus is the basis for building a strategy for anti-epidemic measures in the context of the COVID-19 pandemic. Understanding in what time frame a patient can spread SARS-CoV-2 is just as important as knowing the transmission mechanisms themselves. This information is necessary to develop effective measures to prevent infection by breaking the chains of transmission of the virus. The aim of the work is to identify the infectious SARS-CoV-2 virus in patient samples in the course of the disease and to determine the duration of virus shedding in patients with varying severity of COVID-19. MATERIALS AND METHODS: In patients included in the study, biomaterial (nasopharyngeal swabs) was subjected to analysis by quantitative RT-PCR and virological determination of infectivity of the virus. RESULTS: We have determined the timeframe of maintaining the infectivity of the virus in patients hospitalized with severe and moderate COVID-19. Based on the results of the study, we made an analysis of the relationship between the amount of detected SARS-CoV-2 RNA and the infectivity of the virus in vitro in patients with COVID-19. The median time of the infectious virus shedding was 8 days. In addition, a comparative analysis of different protocols for the detection of the viral RNA in relation to the identification of the infectious virus was carried out. CONCLUSION: The obtained data make it possible to assess the dynamics of SARS-CoV-2 detection and viral load in patients with COVID-19 and indicate the significance of these parameters for the subsequent spread of the virus and the organization of preventive measures.

Features of the Humoral Response to Infection, Vaccination, and Revaccination during COVID-19.

IgM and IgG antibodies to the SARS-CoV-2 virus are detected in subjects who have recovered from COVID-19; IgM antibodies persist in a 1/3 of infected subjects up to 12 months from the moment of the disease, while IgG antibodies are present in the vast majority of cases (97%; medium and high levels antibodies were registered in 85% of cases). By the 12th month, 40% of those who recovered still have a very high level of IgG antibodies to the S-protein (>500 BAU/ml). In the feces, urine, and blood serum of patients with long-term persistent IgM antibodies, no coronavirus antigens were detected. After vaccination with the Gam-COVID-Vac vaccine, IgG antibodies to the S-protein are detected in 100% of cases and remain at a high level for 4 months, by the 5-6th month, the level of antibodies decreases. During revaccination, the level of IgG antibodies to S-protein reaches high values earlier than during primary vaccination, and remains high for 4 months (observation period). The blood sera of recovered and vaccinated patients have a high virus-neutralizing activity (at least 1:80), while its level is somewhat higher in recovered patients.

Determination of Bactericidal Activity Spectrum of Recombinant Endolysins of ECD7, Am24, Ap22, Si3, and St11 Bacteriophages.

The bactericidal activity of recombinant endolysins LysECD7, LysAm24, LysAp22, LysSi3 and LysSt11 was assayed in multidrug resistant strains (n=120) of Salmonella enterica, E. coli, Acinetobacter baumannii, Enterobacter spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, and Campylobacter jejuni. The assay showed that the recombinant endolysins had a wide spectrum of bactericidal activity compared to endolysins of their progenitor phages. Among examined endolysins, we selected the active pharmaceutical substances with broad spectrum of bactericidal activity. Most strains were sensitive to LysECD7 (70.7%), LysAm24 (65%), and LysAp22 (58.6%), which seems to be promising causative agents for the development of finished dosage form.

Guide RNA Design for CRISPR/Cas9-Mediated Potato Genome Editing.

The activity of the pool of sgRNA molecules designed for different regions of potato coilin and phytoene desaturase genes was compared in vitro. Due to the presence of nucleotides unpaired with DNA, sgRNA is able not only to inhibit but also to stimulate the activity of the Cas9-sgRNA complex in vitro. Although the first six nucleotides located in the DNA substrate proximally to the PAM site at the 3' end are the binding sites for cas9, they had no significant effect on the activity of the Cas9-sgRNA complex.

[The evaluation of genotoxic effects in buccal epithelium under disorders of adaption status of organism.]

The cyto-morphologic analysis of buccal epithelium is one of technique of evaluation of adaptation status of organism and xenogeneic intoxication. The analysis of reactivity of cells of buccal epithelium is used to obtain information concerning genetic alterations in human cells that is extremely important for proper decision making related to issues of population health protection. The smoking results in manifestation of tension of adaptation mechanisms and significant increasing of risk of development cytogenetic disorders in cells of buccal epithelium of oral cavity. The study was carried out concerning morphology of buccal epithelial cells of smoking youths. The cytogram demonstrated a reliable decreasing of number of normal cells and increasing of percentage of cells with various cytogenetic alterations. The cells with micro-nuclei are observed twice more often that can be considered as an integral indicator of genetic disorders in inter phase. In smokers, among cells with signs of completion of nucleus destruction the cells with karyorrhexis are registered reliably more often. The results of micro-nucleus test are an unfavorable prognostic indication testifying high degree of genotoxicity of tobacco smoke and meaning a disorder of cytogenetic homeostasis and decreasing of adaptation reserve of organism.

Multi-subunit BCG booster vaccine GamTBvac: Assessment of immunogenicity and protective efficacy in murine and guinea pig TB models.

New innovative vaccines are highly needed to combat the global threat posed by tuberculosis. Efficient components-antigens and adjuvants-are crucial for development of modern recombinant TB vaccines. This study describes a new vaccine (GamTBvac) consisting of two mycobacterial antigen fusions (Ag85A and ESAT6-CFP10)-with dextran-binding domain immobilized on dextran and mixed with an adjuvant consisting of DEAE-dextran core, and with CpG oligodeoxynucleotides (TLR9 agonists). GamTBvac and its components were assessed for immunogenicity and protective efficacy in GamTBvac-prime/boost and BCG-prime/ GamTBvac-boost in murine and guinea pig TB models. Results show that in both infectious models, GamTBvac has a strong immunogenicity and significant protective effect against Mycobacterium tuberculosis strain H37Rv under aerosol and intravenous challenges. GamTBvac showed a particularly strong protective effect as a BCG booster vaccine.

Searching for active mobile genetic elements in dsRNA fraction of Pinus sylvestris having witches broom abnormalities.

The most common type of coniferous mobile genetic elements are retrotransposons. Despite of their early positive impact on evolution of modern coniferous species they can have a significant negative impact for Forestry and breeding. Breaking genomic structural integrity mobile elements can cause phenotypic defects of plants. In this regard, the study of the diversity of coniferous mobile genetic elements is particularly interesting. In the present paper, we describe mobile genetic elements in dsRNA fraction of Pinus sylvestris having witches broom abnormalities. In result of assembled contigs analysis by RepeatMasker 70 mobile genetic elements were identified. A 68 of that were retroelements. Most of elements represented by Gypsy (16 contigs) and Copia (48 contigs). In 4 cases retroelements specific to Pinus taeda were identified. In most cases fragments of integrase (24), reverse transcriptase (22) and RNaseH (15) were identified. Results of the study may be of interest for coniferous breeding and genetic specialists. The raw data of these experiments have been deposited at NCBI under the accession number SAMN06185845.

A conserved region in the Closterovirus 1a polyprotein drives extensive remodeling of endoplasmic reticulum membranes and induces motile globules in Nicotiana benthamiana cells.

In infected plant cells, closterovirus replicative polyproteins 1a and 1ab drive membrane remodeling and formation of multivesicular replication platforms. Polyprotein 1a contains a variable Central Region (CR) between the methyltransferase and helicase domains. In a previous study, we have found that transient expression of the Beet yellows virus CR-2 segment (aa 1305-1494) in Nicotiana benthamiana induces the formation of ~1µm mobile globules originating from the ER membranes. In the present study, sequence analysis has shown that a part of the CR named the "Zemlya region" (overlapping the CR-2), is conserved in all members of the Closterovirus genus and contains a predicted amphipathic helix (aa 1368-1385). By deletion analysis, the CR-2 region responsible for the induction of 1-µm globules has been mapped to aa 1368-1432. We suggest that the conserved membrane-modifying region of the BYV 1a may be involved in the biogenesis of closterovirus replication platforms.

X-irradiation chronosensitivity and circadian rhythmic proliferation in healthy and sarcoma-carrying rats' bone marrow.

Mitotic activity of bone marrow and tumor of sarcoma-bearing rats and of bone marrow of healthy controls is circadian periodic. Whereas the circadian rhythm in mitotic activity of bone marrow is similar for intact and tumor-bearing rats, with a peak occurring shortly after the onset of the dark (activity) span, the circadian rhythm in mitotic activity of tumor has a much smaller amplitude and a different acrophase occurring late in the dark span. This difference in acrophase of mitotic activity of bone marrow and tumor has important implications for scheduling the administration of oncotherapy. For the tumor model examined herein, it suggests the possibility to achieve concomitantly near maximal efficacy and near minimal myelotoxicity, a result awaiting further investigation in humans with different kinds of malignancies.

[Individual variability in the number of stem cells in mammalian tissues]. / Individual'naia variabel'nost' chisla stvolovykh kletok nekotorykh tkanei mlekopitaiushchikh.

The distribution of animal groups (3--4 control or gamma-irradiated mice per group) were obtained when analysing published data on the mean number of the CFUs in the femoral bone marrow or stem cells of small intestinal crypt. Almost 50% of such groups have the mean number less than the geometric mean. The mean value for the group of animals deviation from the expected geometric mean for all animal population for more than 2.5 times, both for the larger values and for the smaller, was about 5% in such groups for the marrow CFUs and 20% for intestinal stem cells. The percentage of mice, that died from acute radiation sickness after acute irradiation, was related with the parameter D(0), that characterized the CFUs survivability on the exponential part of the survivability curve. The negative correlation (r = -0.83) was determined between the mean value of the small intestine crypt stem cells and the ability of intestinal stem cells to the radiation damage reparation, evaluated through the parameter D(q) of the stem enterocyte survival curve for this animal groups.

[Monotonous and wave-form changes in the myelogram of rats over a 24-hour period]. / Monotonnye i volnoobraznye izmeneniia mielogrammy krys v techenie sutok.

Circadian changes of the mean value of each component of rat myelogram and of the proliferative pool of bone marrow cells were obtained. These changes were imposed on a monotonous decrease in the case of subpopulations of granulocytes or on a monotonous increase in the case of subpopulations of the erythroid cells and lymphocytes (from 12.00 to 09.00 next day). This process was not influenced by the elimination of the lymphocytes from the individual myelograms. These myelogram changes appeared to be due to the circadian mature cell production rhythm and flows of the lymphocytes and of mature granulocytes between the bone marrow and the peripheral blood. These changes also suggest the antiphase monotonous correction of the erythrocytic and granulocytic precursors production during several days.

[The mechanism of the formation of a circadian rhythm of bone marrow proliferation in rats]. / O mekhanizme formirovaniia sutochnogo ritma proliferatsii kostnogo mozga u krys.

Flow cytofluorimetry and statmokinetic method were used to study the circadian rhythm of bone marrow proliferation in Pliss' lymphosarcoma-bearing and intact rats. These data were compared to those obtained in the study of the mitotic activity of the bone marrow in cancer patients. It was found that, already at early stage, tumor affected the circadian rhythm of bone marrow proliferation, reducing the amplitude of oscillations. A model simulating formation of the circadian rhythm of the bone marrow was suggested basing on the possibility to arrest cells at the end of G1 phase. The rate of transition of G1 cells to S phase was determined not only by endogenous "set-points" of the rhythm which formed the basic wave of proliferation but also by conditions of animal upkeep.

[A model of the interclonal interactions in chronic myeloleukemia]. / Model' mezhklonal'nykh vzaimodeistvii pri khronicheskom mieloleikoze.

A new model of chronic myelogenous leukemia (CML) pathogenesis based on the idea of some cellular clones dominance over the others at the stem cells level is described. The model gives good explanations to all basic clinical variants of CML as well as to all well-known cytological phenomena to be found recently in this type of leukemia. The most probable mechanisms which might be responsible for the above-mentioned phenomenon of dominance between different cellular clones are discussed.

[The rate of tumor growth and cell loss in cervical cancer]. / Skorost' rosta opukholi i kletochnaia poteria pri rake sheiki matki.

Time of tumor volume doubling, mitotic index and share of pathologic mitoses were identified in 61 patients with squamous-cell cervical tumors and the importance of cell loss evaluated. Tumor doubling time ranged 58-1500 days (average 314 +/- 37); mitotic index--0.1-3.7% (average 1.07 +/- 0.07%); percentage of pathologic mitoses 1-89% (average 28.9 +/- 1.6%); cell loss--92.0-99.9% (average 98.2 +/- 0.2%). Cell loss was found to be significantly different in groups of patients characterized by different rates of tumor growth and was directly proportional to tumor doubling time. There were no significant differences in mitotic index or pathologic mitosis share in groups with different rates of tumor growth. It is suggested that cell loss is determined by the tumor-bearer's antitumor systems to a considerable degree.

[Calculation of the duration of mitosis for a population with an exponential growth of the cell number]. / Raschet dlitel'nosti mitoza dlia populiatsii s éksponentsial'nym rostom chisla kletok.

A formula was received for the mean mitotic duration of a cell population being at the phase of exponential growth of the cell number with the cell loss: tM = nM.tD.(1-phi)/ln 2, where nM is the mitotic index, tD is the doubling time of the cell number, and phi is the Steel cell loss factor. In the case when after irradiation of such a population a 100% radiation G2-block arises, the method of calculation of the tM according to the curve of the relative mitotic index decrease was shown to be independent on the value of parameter phi and to coincide with the same method to be used in the case when the cell population is at the steady state before irradiation. As the result of analysis of literary experimental data the following values were received: tM = 20 min and tM = 37-42 min for two cell subpopulations of the Ehrlich ascite tumour, resp., tM = 39 min for epithelial cells of the mouse cornea, and tM = 29 min for enterocytes of the mouse jejunum. It has been also shown that the values of the dose of cell irradiation and of the mean duration tG2" of the subphase G2" localized at the end of phase G2 and preceded by the G2-block satisfy the next formula: Ig D = -a . Ig tG2" + b.

[Calculation of Steel's cell loss factor and of the cellular kinetic parameters for a population with an exponential growth of the cell number and an equal probability of cell death in all phases of the life cycle]. / Raschet faktora kletochnykh poter' Stila i nekotorykh parametrov kletochnoi kinetiki dlia populiatsii s éksponentsial'nym rostom chisla kletok i ravnoveroiatnostnoi gibel'iu kletok vo vsekh fazakh zhiznennogo tsikla.

A method of calculation of parameter m (equal to a doubled probability of birth of cells which did not pass into phase G0 from the mitotic cycle) and of the proliferative pool was shown to be the same for the exponential growth phase of cell population with the equal probability of cell death (lambda = const) at all phases of the life cycle (the Steel model of type E), and for the case "lambda = 0" (the Steel model of type B). The method is proposed for the calculation of parameter lambda and of the Steel cell lose factor phi = lambda/[lambda + [ln 2)/tD)]. It is noted that the value of parameter phi for the type E model can be estimated by the Steel formula: phi congruent to 1-tDpot/tD--where tD is the doubling time of the cell number, and tDpot = tcln 2/ln m. Here tc is the mean mitotic cycle duration.

[Calculation of Steel's cell loss factor and cell kinetic parameters for a population with exponential growth of cell number and cell death in the G1 and/or G0 phase with probabilities less than 1]. / Raschet faktora kletochnykh poter' Stila i nekotorykh parametrov kletochnoi kinetiki dlia populiatsii s éksponentsial'nym rostom chisla kletok i gibel'iu kletok v faze G1 i (ili) v faze G0 s veroiatnostiami, men'shimi 1.

The method of calculation of the proliferative pool and of the Steel's loss factor was shown to be the same for the exponential growth state with cell death at the G1-phase and/or at the G0-phase with propabilities less than one (the model of F type), and for the exponential growth state model of C type. A method is proposed for calculation of the number of the cell kinetics parameters on the data of percentage of labeled cells cultivated with 3H-thymidine. An unsatisfactory agreement between the exponential growth state model of F type with the interpretation of the experimental of Drewinco e. a. (1978) was shown. A more satisfactory explanation of these data has been offered.