[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.

Mechanical Transformation of Compounds Leading to Physical, Chemical, and Biological Changes in Pharmaceutical Substances.

This study demonstrates the link between the modification of the solid-phase pharmaceutical substances mechanical structure and their activity in waters with different molar ratio «deuterium-protium¼. Mechanochemical transformation of the powders of lactose monohydrate and sodium chloride as models of nutrients and components of dosage forms was investigated by the complex of physicochemical and biological methods. The solubility and kinetic activity of substances dispersed in various ways showed a positive correlation with the solvent isotope profile. Substances dissolved in heavy water were more active than solutes in natural water. Differential IR spectroscopy confirmed the modification of substituents in the sample of lactose monohydrate, demonstrating physicochemical changes during mechanical intervention. The biological activity of the compounds was determined by the method of Spirotox. The activation energy was determined by Arrhenius. Compared with the native compound, dispersed lactose monohydrate showed lower activation energy and, therefore, greater efficiency. In conclusion, proposed data confirm the statement that mechanical changes in compounds can lead to physicochemical changes that affect chemical and biological profiles.

1p/19q testing has no significance in the workup of glioblastomas.

AIMS: To determine whether testing for isolated 1p or 19q losses, or as a codeletion, has any significance in the workup of glioblastomas (GBMs). METHODS: Upfront 1p/19q testing by fluorescence in situ hybridization (FISH) and/or polymerase chain reaction (PCR)-based loss of heterozygosity (LOH) was done in 491 gliomas that were histologically diagnosed as GBMs. Outcomes were determined and measured against 1p/19q results. RESULTS: Twenty-eight showed apparent 1p/19q codeletion by either FISH and/or PCR-based LOH, but only 1/26 showed codeletion by both tests. Over 90% of tumours with apparent codeletion by either FISH or LOH also had 10q LOH and/or EGFR amplification, features inversely related to true whole-arm 1p/19q codeletion. Furthermore, only 1/28 tumours demonstrated an R132H IDH1 mutation. Neither 1p/19q codeletion by FISH nor LOH had an impact on GBM survival. Isolated losses of 1p or 19q also had no impact on survival. CONCLUSIONS: These data suggest that (i) 1p/19q testing is not useful on gliomas that are histologically GBMs; (ii) codeletion testing should be reserved only for cases with compatible morphology; and (iii) EGFR, 10q, and IDH1 testing can help act as safeguards against a false-positive 1p/19q result.

Mutant allele-specific imbalance modulates prognostic impact of KRAS mutations in colorectal adenocarcinoma and is associated with worse overall survival.

The prognostic impact of distinct KRAS mutations in colorectal carcinomas is not fully characterized. We hypothesized that the prognostic impact of KRAS mutations is modulated by KRAS mutant allele-specific imbalance (MASI). KRAS MASI was assessed by sequencing electropherograms in KRAS-mutated colorectal carcinomas (N = 394, prospectively tested). The mechanism of KRAS MASI was studied by fluorescence in situ hybridization (FISH; N = 50). FISH showed that KRAS MASI developed by chromosome 12 hyperploidy (9/18, 50%) or KRAS amplification (1/18, 5.5%). KRAS MASI was more common in tumors with KRAS codon 13 than with codon 12 mutations [24/81, 30% vs. 54/313, 17%; odds ratio (OR), 2.0, 95% confidence interval (CI), 1.2-3.5; p = 0.01]. KRAS MASI was correlated with overall survival (N = 358, median follow-up = 21 months). In a multivariate analysis, KRAS codon 13 MASI was an independent adverse prognostic factor (compared to codon 13 mutants without MASI combined with all codon 12 mutants; adjusted hazard ratio, 2.2, 95% CI: 1.2-3.9; p = 0.01). KRAS MASI arises through chromosome 12 hyperploidy or KRAS amplification and, when affects KRAS codon 13, is associated with worse overall survival.

Multicenter comparison of different real-time PCR assays for quantitative detection of Epstein-Barr virus.

Quantification of Epstein-Barr virus (EBV) in peripheral blood is important for the diagnosis and management of serious EBV diseases, including posttransplant lymphoproliferative disorder. A variety of PCR-based methods are currently in use; however, there is little information on their comparability. This study assessed the relative performance of different quantitative assays. A multicenter comparative study was performed at eight sites using three panels consisting of serial dilutions of quantified EBV DNA and extracts from a total of 19 whole-blood specimens. Samples were distributed and tested blindly. Instrumentation, probe chemistries, amplification targets, and other test-related aspects varied considerably between laboratories. Each laboratory's calibration curve indicated strong evidence of a consistent log-linear relationship between viral load and cycle threshold, suggesting that intralaboratory tracking of a given patient would yield similar relative quantitative trends among the participating test sites. There was strong concordance among laboratories with respect to qualitative test results; however, marked quantitative discordance was seen. For most samples, the across-laboratory interquartile range of the reported viral load (in copies/microl) was roughly 0.6 log-units, and for one sample the overall range was approximately 4.2 log-units. While intralaboratory tracking of patients may yield similar results, these data indicate a need for caution when attempting to compare clinical results obtained at different institutions and suggest the potential value to be gained by more standardized testing methodology.

Solid variant of papillary thyroid carcinoma: incidence, clinical-pathologic characteristics, molecular analysis, and biologic behavior.

Solid variant is a rare and poorly characterized variant of papillary thyroid carcinoma. In this study we analyzed 20 primary cases of the solid variant of papillary carcinoma found in a series of 756 papillary carcinomas operated at the Mayo Clinic between 1962 and 1989. The criteria for classification included predominantly (>70%) solid growth pattern of primary tumor, retention of cytologic features typical of papillary carcinoma, and absence of tumor necrosis. For each case of the solid variant, a control case of classical papillary carcinoma matched by age, sex, tumor size, and length of follow-up was selected. The follow-up ranged from 6 to 32 years. Two patients with the solid variant of papillary carcinoma (10%) died from disease 7 and 10 years after initial surgery, while another two patients (10%) are alive with lung metastases. In contrast, the control group had no cases with distant metastases or death from disease. Molecular analyses showed a similar prevalence of RET /PTC rearrangements in both groups. In conclusion, the solid variant of papillary carcinoma is associated with a slightly higher frequency of distant metastases and less favorable prognosis than classical papillary carcinoma. However, it should be distinguished from poorly differentiated thyroid carcinoma, which has a reported lower survival rate compared with the solid variant of papillary carcinoma.

On target cell numbers in radiation-induced H4-RET mediated papillary thyroid cancer.

Radiation-induced human papillary thyroid cancer (PTC) is associated with chromosomal inversions that involve the genetic loci H4 and RET on chromosome 10. Recently, experimental data has shown that these loci lie in very close spatial proximity in a high proportion of adult human thyroid cells. Applying the generalized formulation of dual radiation action to this H4-to-RET geometric distance data, we predict here the radiation dose-response of H4-RET induction. The predicted H4-RET dose-response has a linear-to-quadratic transition dose of approximately 7 Gy, suggesting the validity of linear risk extrapolations to very low doses for H4-RET mediated radiation-induced PTC. In conjunction with A-bomb survivor data, the predicted H4-RET dose-response yields estimates of the number of PTC target cells that are of the order of approximately 10(6) to approximately 10(7) cells, i.e. considerably less than the total number of follicular cells in the thyroid gland.

Proximity of chromosomal loci that participate in radiation-induced rearrangements in human cells.

Rearrangements involving the RET gene are common in radiation-associated papillary thyroid cancer (PTC). The RET/PTC1 type of rearrangement is an inversion of chromosome 10 mediated by illegitimate recombination between the RET and the H4 genes, which are 30 megabases apart. Here we ask whether despite the great linear distance between them, RET and H4 recombination might be promoted by their proximity in the nucleus. We used two-color fluorescence in situ hybridization and three-dimensional microscopy to map the positions of the RET and H4 loci within interphase nuclei. At least one pair of RET and H4 was juxtaposed in 35% of normal human thyroid cells and in 21% of peripheral blood lymphocytes, but only in 6% of normal mammary epithelial cells. Spatial contiguity of RET and H4 may provide a structural basis for generation of RET/PTC1 rearrangement by allowing a single radiation track to produce a double-strand break in each gene at the same site in the nucleus.

Chromosomal breakpoint positions suggest a direct role for radiation in inducing illegitimate recombination between the ELE1 and RET genes in radiation-induced thyroid carcinomas.

The RET/PTC3 rearrangement is formed by fusion of the ELE1 and RET genes, and is highly prevalent in radiation-induced post-Chernobyl papillary thyroid carcinomas. We characterized the breakpoints in the ELE1 and RET genes in 12 post-Chernobyl pediatric papillary carcinomas with known RET/PTC3 rearrangement. We found that the breakpoints within each intron were distributed in a relatively random fashion, except for clustering in the Alu regions of ELE1. None of the breakpoints occurred at the same base or within a similar sequence. There was also no evidence of preferential cleavage in AT-rich regions or other target DNA sites implicated in illegitimate recombination in mammalian cells. Modification of sequences at the cleavage sites was minimal, typically involving a 1-3 nucleotide deletion and/or duplication. Surprisingly, the alignment of ELE1 and RET introns in opposite orientation revealed that in each tumor the position of the break in one gene corresponded to the position of the break in the other gene. This tendency suggests that the two genes may lie next to each other but point in opposite directions in the nucleus. Such a structure would facilitate formation of RET/PTC3 rearrangements because a single radiation track could produce concerted breaks in both genes, leading to inversion due to reciprocal exchange via end-joining.

Frequent loss of heterozygosity at chromosome 3p14.2-3p21 in human pancreatic islet cell tumours.

OBJECTIVE: Pancreatic islet betacell tumours occur either sporadically or as part of inherited neoplastic syndromes, most commonly multiple endocrine neoplasia (MEN) type 1. Recently, a transgenic mouse model has been established in which the expression of the SV40 large T antigen was targeted to betacells by the rat insulin promoter, leading to the development of multiple pancreatic betacell tumours. In the advanced stages of tumour evolution, these tumours exhibited a high prevalence of loss of heterozygosity (LOH) on mouse chromosomes 9 and 16, at regions syntenic with regions 3q, 3p21, 6q12, 15q24 and 22q of the human genome. DESIGN: Loss of heterozygosity in human islet cell tumours was analysed in a PCR based approach at regions of the human genome syntenic with the mouse loci linked to pancreatic betacell tumours as well as the MEN1 gene on chromosome 11q13. These included 35 microsatellite markers in the human chromosomal regions 3q, 3p21, 6q12, 11q13, 15q24 and 22q. PATIENTS: 21 patients diagnosed with insulinoma were analysed. Histologically, 16 tumours were benign, while 5 were malignant insulinomas. RESULTS: Thirteen of 21 (62%) tumours were found to have loss of genetic material on chromosome 3. The shortest region of overlap implicated a deletion at 3p14.2-3p21 region, corresponding to the marker D3S1295. We did not detect a substantial frequency of LOH in the other syntenic regions, except for the region of MEN 1 gene on 11q13 found to be deleted in 6 (29%) cases, including 3 of 4 tumours from MEN 1 families. Deletions of 3p14. 2-3p21 were observed in 8 of 15 (53%) benign tumours, and in 5 of 6 (83%) malignant neoplasms. CONCLUSIONS: These results indicate the high frequency of 3p14.2-3p21 deletions in human pancreatic betacell neoplasms. These finding suggest the presence of a tumour suppressor gene in this region, that may be important in the microevolution of these tumours towards malignancy.

Prevalence of minisatellite and microsatellite instability in radiation-induced post-Chernobyl pediatric thyroid carcinomas.

Exposure to ionizing radiation induces different forms of genomic instability in cultured cells and experimental animals. A higher rate of germline mutations at human hypervariable minisatellite loci was reported in children born from parents exposed to radiation after Chernobyl, implicating genome destabilization as a possible mechanism responsible for late radiation effects in humans. To test if radiation-induced carcinogenesis in the thyroid gland may be associated with somatic minisatellite instability or microsatellite instability, we utilized a PCR-based approach to study normal and tumor DNA from 17 pediatric post-Chernobyl papillary thyroid carcinomas for mutations at three different minisatellite loci (D1S80, D17S30, ApoB), and 27 microsatellite loci of di-, tri-, or tetranucleotide repeats. Minisatellite instability was found in three (18%) tumors, with one of them exhibiting mutations in all three minisatellite loci, whereas two others showed mutations in one of two informative markers. By contrast, none of 20 sporadic thyroid cancers from patients with no history of radiation exposure was positive for minisatellite instability. Microsatellite analysis of post-Chernobyl tumors revealed a mutation in one (6%) tumor only at the locus of D10S1412, whereas all other 26 microsatellite markers showed identical patterns in each normal/tumor pair. Our results suggest that somatic cell microsatellite instability does not contribute to radiation-induced thyroid carcinogenesis. However, somatic minisatellite mutation events are present in a subset of radiation-induced, but not sporadic, thyroid cancers, suggesting that this type of genomic instability may play a role in radiation-induced tumorigenesis in the thyroid gland.

Distribution of energetic particles and secondary radiation according to orbital station "MIR" data obtained in 1991.

A set of instruments for measuring energetic particle fluxes, containing two neutron detectors under different plexiglas shielding thicknesses, a scintillation detector, measuring energy release >0.1 MeV and 0.5 MeV and a Geiger counter were launched onboard OS 'MIR'. The latitude dependencies of the cosmic ray measurements were obtained and studied. The distributions of primary particle fluxes (protons and elections) as well as secondary particle fluxes (bremsstrahlung gamma-rays and neutrons) produced in interactions of radiation belt particles with the station materials were obtained. The electron belt, generated during the storm of March 24 1991, is studied.

Prevalence of mutations of ras and p53 in benign and malignant thyroid tumors from children exposed to radiation after the Chernobyl nuclear accident.

Starting 4 years after the Chernobyl accident, a dramatic increase in incidence of thyroid carcinoma was noticed in children from contaminated areas. The incidence of benign thyroid lesions in the exposed population was also increased. To study the possible role of ras and p53 genes in radiation-induced thyroid tumorigenesis, 33 papillary carcinomas, one follicular carcinoma and 22 benign lesions removed from children aged 5-19 were screened for point mutations of H-, K-, and N-ras, as well as of p53 (exons 5-8) using single strand conformation polymorphism (SSCP) analysis. Ras point mutations were detected in 1/1 case of follicular carcinoma (N-ras codon 61 CAAgln-->AAAlys), and in 3/7 follicular adenomas (N-ras codon 61 CAAgln-->CGAarg x 2, CAAgln-->AAAlys). None of the cases of papillary thyroid carcinoma was positive for ras oncogene abnormalities. The lack of K-ras mutations was confirmed by allele-specific oligonucleotide hybridization (ASOH), and by sequencing in five cases. Somatic point mutations in p53 were found by SSCP in 2/33 papillary thyroid carcinomas, with one missense mutation (exon 5, codon 160 ATGmet-->GTGval) and another silent mutation (codon 182, TGCcys-->TGTcys). Immunohisto-chemically, focally positive p53 staining was found in four papillary carcinomas being primarily confined to solid and poorly-differentiated areas in tumors. These data demonstrate that as opposed to the few reports on tumors arising after therapeutic external irradiation, ras mutations are not primary events in the development of post-Chernobyl thyroid papillary carcinomas. p53 mutations do not appear to be important in the development of these tumors, but may in some cases have a role in progression to a more aggressive phenotype that has not yet fully manifested in these pediatric neoplasms.

[Extraction and freeze-drying of herpes virus C3-1 isolated from turkeys]. / Ekstrahirane i liofilizirane na kherpesen virus shcham C3-1, izoliran ot puiki.

Possibilities for producing dried preparations of turkey herpes virus C3-1 by use of various ultra-sound desintegrator-systems and different ways of processing the cell suspensions containing the virus were studied. It was established that cell-less virus of a 1.5 to 2 log smaller quantity than the initial virus containing cell suspension can be produced by ultrasound generator with an indirect action -- a vibrating membrane, which transmits the impulses through a liquid medium and the container's walls on the object. The use of a generator with direct action -- titanic probe placed into the suspension results in an insignificant loss and in some cases even leads to higher titer. In the process of freeze drying the protective media SPGA, 5% pepton and 5% horse serum produce almost equal results. Highest dried virus yield was produced by lyophilization of entire (without centrifugal separation) ultrasounded cell suspension. In such cases the titer of the freeze-drying virus was by 0.2-0.4 log higher than the initial cell virus determined after the microplate method. The authors are of the opinion that titration by this method does not give an exact criterion for comparison of virus content in frozen cell and freeze-dried preparations. A proper protection against Marek's disease was established in laboratory and terrain experiments with chickens following immunization by freze-dried preparations of the strain C3-1 (losses were reduced 3-4 times). An immunization dose of 2000 POU lyophilized virus is recommended for practical use.