[Evaluation of the effectiveness of ARVI treatment regimen including etiotropic (enisamium iodide) and symptomatic treatment].

AIM: To assess the effectiveness of the use of the antiviral drug enisamium iodide in the complex treatment of acute respiratory viral infections (ARVI) caused by various pathogens in routine clinical practice. MATERIALS AND METHODS: А prospective randomized study included 134 patients who were treated in the epidemic season of influenza and ARVI in 20182019. All patients were examined for the presence of influenza A and B viruses, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, coronaviruses, rhinoviruses, adenoviruses in nasopharyngeal swabs by PCR. Patients of the main group received enisamium iodide along with symptomatic therapy, the control group received only symptomatic therapy. The primary parameter of the effectiveness of therapy was evaluated on the scale of the general severity of the manifestations of ARVI (Total Symptom Score TSS) from the 2nd to the 4th day and by the secondary criteria of effectiveness: assessment of the duration of ARVI, the severity of fever, the proportion of patients with normal body temperature, the duration of the main clinical symptoms of acute respiratory viral infections, the proportion of patients in whom complications requiring antibiotics were noted, the dynamics of interferon status on the 6th day. To conduct a statistical analysis, depending on the efficiency parameter, the ANCOVA method with a fixed group factor and an initial score on the TSS severity scale was used as covariates, a criterion for comparing quantitative indicators in two independent groups. RESULTS: According to the results of the analysis of the primary efficacy parameter, the median (interquartile range) of the average score on the scale of the general severity of ARVI manifestations in the main group was 4.33 (3.675.83), in the comparison group 6.00 (4.677.25; p0.001). The duration of systemic and local manifestations of acute respiratory viral infections was statistically significantly less in the main group (p=0.002 and p=0.019, respectively). Prescription of additional therapy was required in 2 (2.9%) patients of the main group (patients taking enisamium iodide), compared with 8 (11.9%) patients in the control group. Serum levels of interferon  and interferon  on the last day of treatment were statistically significantly higher in patients of the main group compared with the control group (p0.001). Treatment (excellent) was evaluated by 42 (62.7%) patients, while in the control group only 17 (25.8%) patients gave similar ratings. Both patients (p0.001) and doctors (p0.002) rated therapy tolerance better in the study group. CONCLUSION: The results confirmed the safety and effectiveness of enisamium iodide as a treatment for ARVI and influenza. The antiviral, interferonogenic and anti-inflammatory properties of the drug are involved in the formation of an antiviral response and reduce the risk of complications, which makes it possible to reduce the number of symptomatic agents used.

Expression and characterization of a codon-optimized blood coagulation factor VIII.

Essentials Recombinant factor VIII (FVIII) is known to be expressed at a low level in cell culture. To increase expression, we used codon-optimization of a B-domain deleted FVIII (BDD-FVIII). This resulted in 7-fold increase of the expression level in cell culture. The biochemical properties of codon-optimized BDD-FVIII were similar to the wild-type protein. SUMMARY: Background Production of recombinant factor VIII (FVIII) is challenging because of its low expression. It was previously shown that codon-optimization of a B-domain-deleted FVIII (BDD-FVIII) cDNA resulted in increased protein expression. However, it is well recognized that synonymous mutations may affect the protein structure and function. Objectives To compare biochemical properties of a BDD-FVIII variants expressed from codon-optimized and wild-type cDNAs (CO and WT, respectively). Methods Each variant of the BDD-FVIII was expressed in several independent Chinese hamster ovary (CHO) cell lines, generated using a lentiviral platform. The proteins were purified by two-step affinity chromatography and analyzed in parallel by PAGE-western blot, mass spectrometry, circular dichroism, surface plasmon resonance, and chromogenic, clotting and thrombin generation assays. Results and conclusion The average yield of the CO was 7-fold higher than WT, whereas both proteins were identical in the amino acid sequences (99% coverage) and very similar in patterns of the molecular fragments (before and after thrombin cleavage), glycosylation and tyrosine sulfation, secondary structures and binding to von Willebrand factor and to a fragment of the low-density lipoprotein receptor-related protein 1. The CO preparations had on average 1.5-fold higher FVIII specific activity (activity normalized to protein mass) than WT preparations, which was attributed to better preservation of the CO structure as a result of considerably higher protein concentrations during the production. We concluded that the codon-optimization of the BDD-FVIII resulted in significant increase of its expression and did not affect the structure-function properties.

Proteasome inhibition and oxidative reactions disrupt cellular homeostasis during heme stress.

Dual control of cellular heme levels by extracellular scavenger proteins and degradation by heme oxygenases is essential in diseases associated with increased heme release. During severe hemolysis or rhabdomyolysis, uncontrolled heme exposure can cause acute kidney injury and endothelial cell damage. The toxicity of heme was primarily attributed to its pro-oxidant effects; however additional mechanisms of heme toxicity have not been studied systematically. In addition to redox reactivity, heme may adversely alter cellular functions by binding to essential proteins and impairing their function. We studied inducible heme oxygenase (Hmox1)-deficient mouse embryo fibroblast cell lines as a model to systematically explore adaptive and disruptive responses that were triggered by intracellular heme levels exceeding the homeostatic range. We extensively characterized the proteome phenotype of the cellular heme stress responses by quantitative mass spectrometry of stable isotope-labeled cells that covered more than 2000 individual proteins. The most significant signals specific to heme toxicity were consistent with oxidative stress and impaired protein degradation by the proteasome. This ultimately led to an activation of the response to unfolded proteins. These observations were explained mechanistically by demonstrating binding of heme to the proteasome that was linked to impaired proteasome function. Oxidative heme reactions and proteasome inhibition could be differentiated as synergistic activities of the porphyrin. Based on the present data a novel model of cellular heme toxicity is proposed, whereby proteasome inhibition by heme sustains a cycle of oxidative stress, protein modification, accumulation of damaged proteins and cell death.

[Experience of using genfaxon for treating patients with relapsing-remitting multiple sclerosis in the Russian Federation].

UNLABELLED: Multiple sclerosis (MS) is a chronic autoimmune demyelinating disorder of the central nervous system. Nowadays some disease-modifying drugs (DMD) in the Russian Federation (RF) are biosimilars. Their full spectrum of tolerability and efficacy is to be determined. Here we present results of two retrospective-prospective studies on efficacy and safety of a biosimilar interferon beta-1a (genfaxon) in treatment of MS in the RF. AIMS: determination of safety and efficacy profile of genfaxon in a routine neurological practice in the RF. MATERIALS AND METHODS: Trials were performed in 18 MS centers in the RF. A total of 649 patients aged from 18 to 68 years with the EDSS score no more than 6.0 were treated with genfaxon for 12 months. The first group was comprised of 'naïve' patients without previous history of DMD administration. There were patients in the second group which have already received some of DMD. Statistical analysis was performed with the help of significance criteria (χ-square), t-criteria of Student for analysis of independent samplings. RESULTS: There were no serious adverse events during the period of the study. "Naïve" patients had significantly lower number of adverse events, than patients with previous history of DMD usage. Efficacy results were comparable with results published for the Rebif. CONCLUSIONS: Data, received from the studies show equal efficacy and tolerability of genfaxon compared with original DMD Rebif.

[Information theory of ageing: studying the effect of bone marrow transplantation on the life span of mice].

In this paper the method of life span extension of multicellular organisms (human) using the reservation of stem cells followed by autotransplantation has been proposed. As the efficiency of this method results from the information theory of ageing, it is important to verify it experimentally testing the basic concepts of the theory. Taking it into consideration, the experiment on the bone marrow transplantation to old mice from young closely-related donors of the inbred line was carried out. It has been shown, that transplanted animals exhibited a survival advantage, a mean life span increased by 34% as compared to the control. This result not only demonstrates the efficiency of the proposed method for life span extension of multicellular organisms, but also confirms the basis of the information theory of ageing.

[Use of fluorescence microspectral method for studying bone marrow EGFP+ cells transplantation in 5-fluorouracil-treated mice].

In this paper the experimental results of bone marrow transplantation from C57BL/6-Tg(ACTB-EGFP)1Osb/J transgenic mice into C57BL/6 mice subjected to 5-fluorouracil treatment are represented. It has been shown that EGFP+ cells engraftment in bone marrow, spleen and thymus of host mice after 5-Fu treatment significantly increased. More long-term engraftment was recorded after transplantation between closely related donors and 5-fluorouracil treatment hosts. We have also obtained data on differences in the dynamics of EGFP+ cells engraftment in host investigated organs. To assess the effect of the donor's bone marrow cells on the host immune system, functional activity of the synthetic apparatus (synthetic activity) of cells in bone marrow, spleen, thymus and blood have been investigated with fluorescence microspectral method. The results obtained allow of improving techniques for bone marrow transplantation without host irradiation in order to minimize the adverse effects.

[Comparative analysis of syngeneic and allogeneic transplantation without irradiation of the EGFP+ mice bone marrow cells with microspectral fluorescence method].

The experimental results on syngeneic and allogeneic transplantation of whole fraction of mice bone marrow cells without irradiation have been presented. Data on the dynamics of the donor cell colonization of bone marrow, spleen, thymus and blood of the recipient mice were obtained. The degree of immunogenicity of donor cells with syngeneic and allogeneic bone marrow transplantation based on the microspectral fluorescence method was evaluated. Within the framework of the experiment a low degree of immunogenicity of donor cells with syngeneic and allogeneic transplantation is shown. Importantly, allogeneic bone marrow transplantation did not cause any reduction in the mean life span of mice. These data and the results of our previous studies, demonstrating the mean life span increased by 34% with syngeneic transplantation in line C57BL/6 EGFP+, allow for developing different methods of cell therapy with no risk of fatal consequences of the immunological incompatibility between donor and recipient.

[On the possibility of determination of stereological characteristics of interactions of biological micro objects with nanoparticles based on analysis of 2-dimensional images of cytological and histological specimens].

In this article the method for studying 3-dimensional (stereological) characteristics of interactions of biological micro objects with nanoparticles on a basis of morphodensitometry analysis of standard 2-dimensional images of cytological and histological specimens is proposed. The performance of the task of determining the distance from registered nanoparticle to the center of the nucleus of the cell is described in detail. It is shown that using specific nanoparticles the results obtained may find application in science and diagnostics. Furthermore, it is possible to employ these results in nanotoxicology, in particular for determination of quantity characteristics of translocation of nanoparticles of different types in biological structures.

[Informational hypothesis of aging: how does the germ line "avoid" the aging?].

An informational hypothesis of aging has been formulated, and an imitational model of the survival of a population of multicellular organisms under conditions of informational degradation of the cell genetic material (accumulation of random errors in the genome) has been constructed. It is assumed that the basic mechanism of "rejuvenation" (decrease in the number of errors) of the genetic material during its transition from parents to their progeny is the phenomenon of crossing-over during gametogenesis and the competitive selection of gametes participating in the formation of the progenitor genetic material. Within the framework of the imitational model, it is shown that this mechanism, which exists in most eukaryotes, provides the stability of the population gene pool in a large number of generations, whereas in a single organism, the amount of genome errors increases with aging.

[Application of theoretical group methods in the modeling of complex systems].

It has been shown that theoretical group methods can be applied in studies of complex physical and biological systems in which the phenomenon of self-organization takes place. The problem of calculating the parameters of climatic sensitivity with allowance for the cyclone-anticyclone structure of the atmosphere optically dense in the infrared region is considered as an example.

Recombinant human alpha-1 proteinase inhibitor: towards therapeutic use.

Human alpha-1-proteinase inhibitor is a well-characterized protease inhibitor with a wide spectrum of anti-protease activity. Its major physiological role is inhibition of neutrophil elastase in the lungs, and its deficiency is associated with progressive ultimately fatal emphysema. Currently in the US, only plasma-derived human alpha-1-proteinase inhibitor is available for augmentation therapy, which appears to be insufficient to meet the anticipated clinical demand. Moreover, despite effective viral clearance steps in the manufacturing process, the potential risk of contamination with new and unknown pathogens still exists. In response, multiple efforts to develop recombinant versions of human alpha-1-proteinase inhibitor, as an alternative to the plasma-derived protein, have been reported. Over the last two decades, various systems have been used to express the human gene for alpha-1-proteinase inhibitor. This paper reviews the recombinant versions of human alpha-1-proteinase inhibitor produced in various hosts, considers current major safety and efficacy issues regarding recombinant glycoproteins as potential therapeutics, and the factors that are impeding progress in this area(1).

Circular dichroism and cross-linking studies of bacteriorhodopsin mutants.

Circular dichroism (CD) spectroscopy was employed for native (wild type, WT) bacteriorhodopsin (bR) and several mutant derivatives: R134K, R134H, R82Q, S35C, L66C, and R134C/E194C. Comparative analysis of the CD spectra in visible range shows that only R134C/E194C exhibits biphasic CD, typical for native bR, the other mutants demonstrate CD spectra with significantly smaller or absent negative band. Since the biphasic CD is a feature of hexagonal lattice structure composed by bR trimers in the purple membrane, these mutants and WT were examined by cross-linking studies, which confirmed the same trend towards trimeric organization. Therefore, a single amino acid substitution may lead to drastically different CD spectra without disruption of bR trimeric organization. Thus, although disruption of bR trimeric crystalline lattice structure (e.g., solubilization with detergents) directly results in the disappearance of characteristic bilobe in visible CD, the lack of the bilobe in the CD alone does not predict the absence of trimers.

[Consideration of a priori information and type of an experimental error using the method of system identification based on the minimal quadratic discrepancy criterion].

The variants of the identification method were considered that take the a priori information about the evolution of a system under study and the type of experimental errors of the dynamic parameters of the system into account. An example of using this method for the identification of a biochemical reaction is given where the error in measuring the dynamic parameters (concentration of substances) has both an absolute and a relative components.

[A new method of identifying a systems based on the minimal quadratic discrepancy criterion for biophysics problems]. / Novyi metod identifikatsii sistem na osnove kriteriia minimal'noi kvadratichnoi neviazki dlia zadach biofiziki.

A wide range of biophysical systems are described by nonlinear dynamic models mathematically presented as a set of ordinary differential equations in the Cauchy explicit form: [formula: see text] Fij(X1(t),..,XN(t),t), (i = 1,...,N, j = 1,...,M), where Fij (X1(t), ..., XN(t), t) is a set of basis functions satisfying the Lipschitz condition. We investigate the problem of evaluation of model constants aij (the system identification) using experimental data about the time dependence of the dynamic parameters of the system Xi(t). A new method of system identification for the class of similar nonlinear dynamic models is proposed. It is shown that the problem of identifying an initial nonlinear model can be reduced to the solution of a system of linear equations for the matrix of the dynamic model constants [aj]i. It is proposed to determine the set of dynamic model constants aij using the criterion of minimal quadratic discrepancy for the time dependence of the set of dynamic parameters Xi(t). An important special case of the nonlinear model, the quadratic model, is considered. Test problems of identification using this method are presented for two nonlinear systems: the Van der Pol type multiparametric nonlinear oscillator and the strange attractor of Ressler, a widely known example of dynamic systems showing the stochastic behavior.

Application of a new method of nonlinear dynamical system identification to biochemical problems.

The system identification method for a variety of nonlinear dynamic models is elaborated. The problem of identification of an original nonlinear model presented as a system of ordinary differential equations in the Cauchy explicit form with a polynomial right part reduces to the solution of the system of linear equations for the constants of the dynamical model. In other words, to construct an integral model of the complex system (phenomenon), it is enough to collect some data array characterizing the time-course of dynamical parameters of the system. Collection of such a data array has always been a problem. However difficulties emerging are, as a rule, not principal and may be overcome almost without exception. The potentialities of the method under discussion are demonstrated by the example of the test problem of multiparametric nonlinear oscillator identification. The identification method proposed may be applied to the study of different biological systems and in particular the enzyme kinetics of complex biochemical reactions.

Oral administration of homocysteine leads to increased plasma triglycerides and homocysteic acid-additional mechanisms in homocysteine induced endothelial damage?

Increased plasma homocyst(e)ine is strongly correlated with occlusive arterial diseases. A series of different hypotheses have been reported including involvement of free oxygen radicals and therefore oxidative stress. We determined plasma homocyst(e)ine and homocysteic acid levels after oral low dose homocysteine thiolactone administration to rats for a period of six weeks. Plasma levels of homocyst(e)ine and triglycerides were significantly elevated in the group fed homocysteine thiolactone. GC/MS determination of ketone body formation showed that the underlying mechanism for the increase of triglycerides seems to be inhibition of fatty acid oxidation. Homocysteic acid was detected in the experimental group exclusively. The present study showing a homocyst(e)ine correlated increase of plasma triglycerides by the inhibition of fatty acid oxidation may well propose an additional role of triglycerides for vascular pathology. The presence of homocysteic acid in the experimental group only would support the free oxygen radical hypothesis for the development of vascular changes but homocysteic acid as a potent neurotransmitter could play an independent role in the pathogenesis.

(2)H- and(13)C-labeled amino acids generated by obligate methylotrophs Biosynthesis and MS monitoring.

Biosynthetic preparation of(2)H- and(13)C- labeled amino acids was studied using a leucine-producing mutant of the obligate methylotroph,Methylobacillus flagellatum. The strain was cultivated in various media containing(13)C- or(2)H-analogs of methanol. The total protein from each experiment was subjected to acid hydrolysis and converted into a mixture of dansyl amino acid methyl esters. The samples of excreted leucine were converted into methyl esters of dansyl and benzyloxycarbonyl derivatives. Electron impact mass spectrometry was performed to detect stable isotope enrichment of the amino acids. According to the mass spectrometric analysis it is feasible to use methylotrophic microorganisms for the preparation of(2)H- and(13)C- analogs of amino acids by labeled methanol bioconversion; the excreted amino acids can be convenient for express analysis as an indicator of isotopic enrichment of the total protein. The data obtained testified to a high efficiency of dansyl derivatization for mass spectrometric analysis of complex amino acid mixtures.