[Molecular methods for diagnosing novel coronavirus infection: comparison of loop-mediated isothermal amplification and polymerase chain reaction].

INTRODUCTION: Currently, the basis for molecular diagnostics of most infections is the use of reverse transcription polymerase chain reaction (RT-PCR). Technologies based on reverse transcription isothermal loop amplification (RT-LAMP) can be used as an alternative to RT-PCR for diagnostic purposes. In this study, we compared the RTLAMP and RT-PCR methods in order to analyze both the advantages and disadvantages of the two approaches. MATERIAL AND METHODS: For the study, we used reagent kits based on RT-PCR and RT-LAMP. The biological material obtained by taking swabs from the mucous membrane of the oropharynx and nasopharynx in patients with symptoms of a new coronavirus infection was used. RESULTS: We tested 381 RNA samples of the SARS-CoV-2 virus (Coronaviridae: Coronavirinae: Betacoronavirus; Sarbecovirus) from various patients. The obtained values of the threshold cycle (Ct) for RT-PCR averaged 20.0 ± 3.7 s (1530 ± 300 s), and for RT-LAMP 12.8 ± 3.7 s (550 ± 160 s). Proceeding from the theoretical assumptions, a linear relationship between values obtained in two kits was proposed as a hypothesis; the correlation coefficient was approximately 0.827. At the same time, for samples with a low viral load (VL), the higher Ct values in RT-LAMP did not always correlated with those obtained in RT-PCR. DISCUSSION: We noted a significant gain in time for analysis using RT-LAMP compared to RT-PCR, which can be important in the context of testing a large number of samples. Being easy to use and boasting short turnaround time, RT-LAMP-based test systems can be used for mass screening in order to identify persons with medium and high VLs who pose the greatest threat of the spread of SARS-CoV-2, while RT-PCR-based diagnostic methods are also suitable for estimation of VL and its dynamics in patients with COVID-19.

[Forensic aspects of the burning of human corpses]. / Sudebno-meditsinskie aspekty sozhzheniya trupov lyudei.

Objective - to outline the basic ideas about the conditions and results of the burning of human corpses and the potential possibilities of a forensic medical examination of the burned remains. It is impossible to burn the corpse of an adult to a state of only homogeneous ash not only on the fire, but also in special crematorium chambers. After the burning of the corpse, ash and bone fragments remain in a state of black, gray and white heat. The state of the cremated remains allows, at least, establishing the number of incinerated corpses, their species and anatomical affiliation. Reconstruction of the conditions of corpse burning is possible only within the framework of a comprehensive forensic examination with the participation of specialists in the field of fire-technical affairs and forensic medical experts. The exclusive competence of specialists in the field of fire-technical affairs is the verification of the conditions of combustion (type and volume of fuel, technical features of combustion, duration of cremation, etc.).

[Rapid diagnostics of novel coronavirus infection by loop-mediated isothermal amplification].

This review presents the basic principles of application of the loop-mediated isothermal amplification (LAMP) reaction for the rapid diagnosis of coronavirus infection caused by SARS-CoV-2. The basic technical details of the method, and the most popular approaches of specific and non-specific detection of amplification products are briefly described. We also discuss the first published works on the use of the method for the detection of the nucleic acid of the SARS-CoV-2 virus, including those being developed in the Russian Federation. For commercially available and published LAMP-based assays, the main analytical characteristics of the tests are listed, which are often comparable to those based on the method of reverse transcription polymerase chain reaction (RT-PCR), and in some cases are even superior. The advantages and limitations of this promising methodology in comparison to other methods of molecular diagnostics, primarily RT-PCR, are discussed, as well as the prospects for the development of technology for the detection of other infectious agents.

[Age dynamics of secretory, absorptive, buffer ability of the mucosa inside the human nose].

The problem of age-related functional changes of organs and systems of the human body is one of the urgent problems of modern physiology and gerontology. The most important, complex system of human body is respiratory system, especially its «first line of defense¼ - the nasal cavity. Functional parameters of the nasal cavity undergo a number of specific changesat the stages of postnatal ontogenesis, the study of which has not only scientific value but also practical value for experimental physiology and clinical medicine. The article presents data on the age dynamics and adaptability of such functional parameters of the mucous membrane of the nasal cavity of the person, as secretory and suction activity, the air heater function, the buffer properties by endonasal secret. These indicators are examined at the stages of postnatal development in a comparative perspective based on the study of the age gradients.

Functioning of Yeast Pma1 H+-ATPase under Changing Charge: Role of Asp739 and Arg811 Residues.

The plasma membrane Pma1 H+-ATPase of the yeast Saccharomyces cerevisiae contains conserved residue Asp739 located at the interface of transmembrane segment M6 and the cytosol. Its replacement by Asn or Val (Petrov et al. (2000) J. Biol. Chem., 275, 15709-15716) or by Ala (Miranda et al. (2011) Biochim. Biophys. Acta, 1808, 1781-1789) caused complete blockage of biogenesis of the enzyme, which did not reach secretory vesicles. It was proposed that a strong ionic bond (salt bridge) could be formed between this residue and positively charged residue(s) in close proximity, and the replacement D739A disrupted this bond. Based on a 3D homology model of the enzyme, it was suggested that the conserved Arg811 located in close proximity to Asp739 could be such stabilizing residue. To test this suggestion, single mutants with substituted Asp739 (D739V, D739N, D739A, and D739R) and Arg811 (R811L, R811M, R811A, and R811D) as well as double mutants carrying charge-neutralizing (D739A/R811A) or charge-swapping (D739R/R811D) substitutions were used. Expression of ATPases with single substitutions R811A and R811D were 38-63%, and their activities were 29-30% of the wild type level; ATP hydrolysis and H+ transport in these enzymes were essentially uncoupled. For the other substitutions including the double mutations, the biogenesis of the enzyme was practically blocked. These data confirm the important role of Asp739 and Arg811 residues for the biogenesis and function of the enzyme, suggesting their importance for defining H+ transport determinants but ruling out, however, the existence of a strong ionic bond (salt bridge) between these two residues and/or importance of such bridge for structure-function relationships in Pma1 H+-ATPase.

[Age peculiar by endonasal homeostasis in humans.]

The state of protective functions of the nasal cavity determines its homeostasis. At the stages of postnatal development and involution of functional parameters by endonasal homeostasis undergo a number of specific changes, the study of which has not only scientific value but also practical value for experimental physiology and clinical medicine. The article presents data on the age dynamics of functional indicators by endonasal homeostasis of human as buffer properties and morphological features of nasal secret. These indicators are examined at the stages of postnatal development in a comparative perspective based on the study of the age gradients and the position of the doctrine of entropy.

[The functional state of nasal cavity in the aspect of structural-functional changes of the human organism in postnatal ontogenesis.]

Nasal cavity - «the primary line of defense¼ and a major division of the respiratory tract. A variety of functional areas and regions, structural formations of the nasal cavity allows, at the present stage of development of biology and medicine, consider it as a complex functional subsystem in the structure of the respiratory system. The age transformations of morphological and functional parameters of the nasal cavity are polymorphic and specific. An important aspect of developmental physiology of the respiratory system is a study of the relationship of these changes with morphological and functional transformations of other systems of the body. The article presents the results of the analysis of such contingency-for example, by endonasal correlations with amount of blood flow hemodynamics and functional state of the autonomic nervous system, respiratory function of the nasal cavity and respiratory system, as well as other functions of the nasal cavity and their relationship with general growth processes, critical periods of development and involution. In the process of this analysis revealed a close correlation with complex changes of the morphofunctional parameters of the systems of the human body, most pronounced during in critical periods of postnatal ontogenesis.

[CHANGES OF THE CONTENT OF DLL4 AND Jag-1 ANGIOGENESIS REGULATORS IN HUMAN DERMIS IN ONTOGENESIS].

The goal of this study was to examine the contents of D114 and Jag-1 angiogenesis regulators in human dermis at different age periods. D114 and Jag-1 were demonstrated by indirect immunohistochemistry in skin sections of fetuses of 20-40 gestational weeks and in persons aged from birth to 85 years. D114 was studied in 150 skin samples of 72 females and 78 males, while Jag-1 was examined in 120 samples of 58 females and 62 males. It is found that the immunoreactivity was mainly expressed by the endothelial cells. Vessels, which gave a positive reaction to D114 and Jag-1, were found throughout the entire thickness of the dermis, both in fetuses, and people of all age groups. Expression of D114 in the vessels of dermal microvasculature was shown to increase from 20 weeks of gestation to 20 years. With the further age increase, the intensity of the reaction of blood vessels for D114 was decreased. Expression of Jag-1 in dermal microvessels was enhanced from 20 weeks of gestation to 85 years. The results are discussed in connection with the role of D114 and Jag-1 in angiogenesis in human dermis during ontogeny.

[Age features of mucociliary system the mucous membrane of the nasal cavity of the human].

The mucociliary system of the mucosa of the nasal cavity has a complex structural-functional organization and a wide range of functionality. Essential components of this system are the motor activity of ciliated epithelium and mucociliary transport time. With age, the mucous membrane of the nasal cavity undergoes specific changes, detection of which is of practical importance for gerontology and medicine. This article presents the results of studying the main components of the mucociliary apparatus of the nasal cavity of a person in mature, elderly and senile age, we investigated the relationship of functional changes of the mucociliary system with age-related structural reorganization of the epithelial layer of the nasal mucosa. Identified critical periods functional mucociliary system of nasal cavity of man: old age (out of definitivno state) and aging (involution of the mucociliary apparatus).

[Age-related changes of hemodynamic potential of the nasal cavity of a human].

Vascular system of the nasal cavity has a complex structural organization and a wide range of functionality. However, with age, the hemodynamics of the nasal cavity undergoes specific changes, detection of which is of practical importance for gerontology and medicine. This paper presents the results of the study of hemodynamics of the nasal cavity in Mature, elderly and senile age by the method of rheorinography . Identified critical periods of functional changes of blood flow of the nasal cavity: the elderly (exit definitivno state) and old age (involution hemodynamic function and the reduction of its compensatory capacity).

Ion-beam-assisted spatial modulation of inhomogeneous broadening of a quantum well resonance: excitonic diffraction grating.

We propose a method of spatial modulation of inhomogeneous broadening of a quantum-well excitonic resonance based on local generation of defects produced by a focused ion beam. The method is applied to fabrication of excitonic diffraction grating in a single quantum-well InGaAs/GaAs structure by irradiating the sample with a beam of 35-keV He+ ions of exposure doses <1012 cm-2. The spectrum of resonant diffraction on such a structure is narrower than that of reflectivity and decreases much faster with increasing temperature. A proposed model of formation of the diffractive response based on the single scattering approximation well describes the results of the spectral and temperature measurements.

Role of loop L5-6 connecting transmembrane segments M5 and M6 in biogenesis and functioning of yeast Pma1 H+-ATPase.

The L5-6 loop is a short extracytoplasmic stretch (714-DNSLDID) connecting transmembrane segments M5 and M6 and forming along with segments M4 and M8 the core through which cations are transported by H+-, Ca2+-, K+,Na+-, H+,K+-, and other P2-ATPases. To study structure-function relationships within this loop of the yeast plasma membrane Pma1 H+-ATPase, alanine- and cysteine-scanning mutagenesis has been employed. Ala and Cys substitutions for the most conserved residue (Leu717) led to complete block in biogenesis preventing the enzyme from reaching secretory vesicles. The Ala replacement at Asp714 led to five-fold decrease in the mutant expression and loss of its activity, while the Cys substitution blocked biogenesis completely. Replacements of other residues did not lead to loss of enzymatic activity. Additional replacements were made for Asp714 and Asp720 (Asp®Asn/Glu). Of the substitutions made at Asp714, only D714N partially restored the mutant enzyme biogenesis and functioning. However, all mutant enzymes with substituted Asp720 were active. The expressed mutants (34-95% of the wild-type level) showed activity high enough (35-108%) to be analyzed in detail. One of the mutants (I719A) had three-fold reduced coupling ratio between ATP hydrolysis and H+ transport; however, the I719C mutation was rather indistinguishable from the wild-type enzyme. Thus, substitutions at two of the seven positions seriously affected biogenesis and/or functioning of the enzyme. Taken together, these results suggest that the M5-M6 loop residues play an important role in protein stability and function, and they are probably responsible for proper arrangement of transmembrane segments M5 and M6 and other domains of the enzyme. This might also be important for the regulation of the enzyme.

[Age-related changes of the content of angiomatin and endostatin in human skin].

Human skin structures stained positively for angiomotin or endostatin were studied by indirect immunohistochemical method. Skin specimens from frontal surface of the lower part of the neck (from upper corner of standard autopsy skin incision) from human fetuses died antenatally from 20 to 40 weeks of pregnancy, humans who died from different causes from 1 day to 85 years of life were obtained at autopsy. Positive staining for angiomotin or endostatin in the skin was found in epidermal cells, fibroblasts, sweat and sebaceous glands, blood vessels of the dermis. Blood vessels stained positively for angiomotin were detected in skin samples in all ages. Age-dependent decrease in the content of angiomotin in blood vessels of the dermis was detected. Most prominent decrease in angiomotin content in dermal blood vessels was found in 61-85 years age-group. Endostatin positive blood vessels were also detected in skin samples of all ages. However, the intensity of staining for endostatin in dermal blood vessels was increased during aging. It can be proposed that changes in the content of angiomotin and endostatin yield a negative impact on angiogenesis in human skin during aging.

[Blood vessels in human dermis during aging].

A factor that potentially influences on skin aging is blood supply which determines global conditions for an organ or a tissue functioning, including skin. Scientific data on conditions of blood supply in the skin during aging are insufficient and contradictory. Therefore, this work was aimed to the study of age-related changes in the number of blood vessels in the human dermis. Blood vessels were visualized with immunohistochemical technique to two endothelial markers, as von Willebrand factor and antigen CD31. The results showed that von Willebrand factor and antigen CD31 are present in endothelial cells of blood vessels of dermis in all examined age periods, from 20 weeks of pregnancy to 85 yeas. Intensity of immunohistochemical staining to von Willebrand factor is enhanced during age. Intensity of staining to CD31 is not changed with age. The number of blood vessels positively stained either to von Willebrand factor or to CD31 in dermis was decreased gradually with age. A total number of fibroblasts in dermis decreased with age. The number of PCNA+ fibroblasts in dermis showing their proliferative activity was decreased with the progression of age. The decrease in the number of blood vessels is statistically associated with that in the general number of fibroblasts and proliferating fibroblasts. Hence, a factor that leads to aged decrease in the number of dermal fibroblasts is diminished blood supply, and actions targeted to enhancement of blood supply are to be in the basis of clinical approaches to prophylaxis and treatment aging changes of the skin.

[Effect of point substitutions of Asp-714 and Asp-720 residues on the structure and function of the H+ -ATPase of the yeast plasma membrane].

Membrane-spanning M5 and M6 segments, which play a role in the formation of cation transport sites in H(+)-, Ca2(+)-, K(+)-, Na(+)-, and other P2-ATPases, are connected by a short extracytoplasmic loop. In the yeast plasma membrane H(+)-ATPase, which belongs to a family of P2-ATPases, the loop is connected to M5 and M6 through the Asp-714 and Asp-720 residues. In this work, the effect of point amino, acidreplacements of Asp-714 and Asp-720 by Ala, Val, Asn, and Glu residues on the function of the enzyme was studied. The Asp714Asn point mutant possessed activities similar to those of the wild-type enzyme, whereas the replacement of Asp-714 by other amino acid residues disrupted biogenesis and led to a loss of activity. All mutants with substitution of Asp-720 were expressed and possessed relatively high activity. The D720V mutant displayed significantly reduced expression levels, activity, H+ transport, and ATP hydrolyzing activity. Thus, substitutions of Asp-714, except for the D714N mutant, led to significant defects in biogenesis and/or function of the enzyme. The results indicate the important role for the Asp-714 residue in biogenesis, structure stability, and enzyme function.

[Age-related changes in mast cells and eosinophils of human dermis].

In this study, quantitative analysis of inflammatory effectors--mast cells and eosinophils--in derma of people of different ages is performed. The study shows that mast cell quantity in derma increases with age. Eosinophils are rarely observed in human dermis. There are no age-correlated changes of dermal eosinophils quantity observed. Age-correlated dermal fibroblast quantity is established. PCNA+ fibroblast percentage demonstrating their proliferative pool also reliably decreases with age. Results of correlation analysis show that age-correlated increase in mast cells' quantity is reliably correlated with decrease in total number and percentage of PCNA+ fibroblasts in derma. Consequently, age-correlated increase in dermal mast cell may be proposed to be one of the inflammatory and aging mechanisms. Mast cells, whose number increases with aging, may influence dermal fibroblast number with aging.

[Age-related changes in the number of CD45+ cells in human dermis].

The aim of this work was the study of age-related changes in the number of CD45+ cells in human dermis. The results showed that the number of CD45+ cells in dermis increased gradually with age. At the same time, a total number of fibroblasts in dermis decreased with age. The number of PCNA+ fibroblasts in dermis showing their proliferative activity was observed to be decreased with the progression of age. The results of the correlation analysis show that the age-related increase of CD45+ cells number is statistically associated with the decrease in the total number of fibroblasts and with the quantity of PCNA+ fibroblasts in dermis through age. Therefore, accumulation of CD45+ cells in human dermis with age may be regarded as a mechanism which brings about inflammatory reaction and emergence of signs of aging. It is also possible that the increased number of bone marrow derived cells has an impact on the age-related decline in the number of fibroblasts in dermis with age.

[Age-related changes in the number and proliferation of fibroblasts in the human skin].

Skin aging is important medical and social problem in modern world. A goal of present work was to estimate changes in the number of fibroblasts and PCNA-positive fibroblasts in human dermis at different ages. Total number of fibroblasts per unit area was counted in slides stained with haematoxylin and eosin. PCNA which is present in cells being in the cell cycle was revealed immunohistochemically. Results showed a decrease in a total number of fibroblasts in dermis through aging. The number of PCNA-positive fibroblasts in dermis is also decreased with progression of age. It may be concluded that age-related decrease in dermal fibroblasts number is associated with diminished proliferation of these cells. Causes which lead to a decrease in proliferation are not clear, and new researches are needed to reveal them.

[Tension-free plasty in the surgery of abdominal wall hernias].

The article presents an experience with application of tension-free plasty of the anterior abdominal wall using reticular endoprostheses. New endoprostheses of Reperen are developed and applied, a method of sutureless fixation of polypropylene net in the abdominal wall tissues is proposed when performing plasty for great ventral hernias. Advantages of new methods are shown compared with analogs both in selective and in emergency surgery, in different age groups of patients. The direct and long-term postoperative results and parameters of quality of life are investigated.

Point mutations in Pma1 H+-ATPase of Saccharomyces cerevisiae: influence on its expression and activity.

Yeast Pma1 H+-ATPase is a key enzyme of cell metabolism generating electrochemical proton gradient across the plasma membrane, thus playing an important role in the maintenance of ion homeostasis in the cell. Using site-directed mutagenesis, we have previously replaced all 21 amino acid residues in the transmembrane segment M8 with Ala (Guerra et al. (2007) Biochim. Biophys. Acta, 1768, 2383-2392). In this work, we present new data on the role of these amino acid residues in the structure-function relationship in the enzyme and cell tolerance to heat shock. Mutations Q798A and I799A are lethal for cells regardless of expression of the enzyme in secretory vesicles or plasma membrane. The F796A mutation causes enzyme and cell sensitivity to heat shock when expressed in secretory vesicles. The I794A mutation increases temperature sensitivity of cells when the enzyme is expressed either in secretory vesicles or, to a lesser extent, in plasma membrane. The E803A mutation has no significant influence on the ATPase and cell sensitivity to heat shock; however, it causes a shift in the equilibrium between E1 and E2 conformations of the enzyme towards E1.