Inside and outside of virus-like particles HBc and HBc/4M2e: A comprehensive study of the structure.

Hepatitis B virus core antigen (HBc) with the insertion of four external domains of the influenza A M2 protein (HBc/4M2e) form virus-like particles whose structure was studied using a combination of molecular modeling and cryo-electron microscopy (cryo-EM). It was also shown that self-assembling of the particles occurs inside bacterial cells, but despite the big inner volume of the core shell particle, purified HBc/4M2e contain an insignificant amount of bacterial proteins. It was shown that a fragment of the M2e corresponding to 4M2e insertion is prone to formation of amyloid-like fibrils. However, as the part of the immunodominant loop, M2e insertion does not show a tendency to intermolecular interaction. A full-atomic HBc-4M2e model with the resolution of about 3 Å (3.13 Å for particles of Т = 4 symmetry, 3.7 Å for particles of Т = 3 symmetry) was obtained by molecular modeling methods based on cryo-EM data.

Indicators of chemiluminescent and bioluminescent tests of biological liquids in the assessment of physical health.

The study includes anthropometry of 172 young male, obtained data on the length and body mass, measured the transverse diameters of the shoulders and pelvis, various body types was identified by the J.M. Tanner sexual dimorphism index (andromorphic, mesomorphic, gynecomorphic). The chemiluminescent and bioluminescent study of saliva and blood was conducted in the examined young male. We studied the indicators of the antioxidant defense system under the influence of stress. The antioxidant status of saliva was determined using the H2O2-luminol-dependent chemiluminescence method. Data on the activity of NAD (P) -dependent dehydrogenases in blood lymphocytes was obtained from a bioluminescent method of research. Young male of andromorphic body type had large overall and transverse body sizes. Indicators of antioxidant protection of saliva and blood in men of adolescence, the body type of the sexual dimorphism index J.M. Tanner was different. The persons of the andromorphic body type differed in terms of chemiluminescence in comparison with the young male of gynecomorphic body type. The results of bioluminescent blood tests suggest a violation of the catabolic and anabolic processes of carbohydrate and fat metabolism in young men of mesomorphic and gynecomorphic body types. Indicators of the system of antioxidant protection of saliva and blood reflect the sexual characteristics of the body of young male and can be used as additional criteria for diagnosing sex inversion and assessing the risk of developing socially attributed diseases.

Influence of the Linking Order of Fragments of HA2 and M2e of the influenza A Virus to Flagellin on the Properties of Recombinant Proteins.

The ectodomain of the M2 protein (M2e) and the conserved fragment of the second subunit of hemagglutinin (HA2) are promising candidates for broadly protective vaccines. In this paper, we report on the design of chimeric constructs with differing orders of linkage of four tandem copies of M2e and the conserved fragment of HA2 (76-130) from phylogenetic group II influenza A viruses to the C-terminus of flagellin. The 3D-structure of two chimeric proteins showed that interior location of the M2e tandem copies (Flg-4M2e-HA2) provides partial α-helix formation nontypical of native M2e on the virion surface. The C-terminal position of the M2e tandem copies (Flg-HA2-4M2e) largely retained its native M2e conformation. These conformational differences in the structure of the two chimeric proteins were shown to affect their immunogenic properties. Different antibody levels induced by the chimeric proteins were detected. The protein Flg-HA2-4M2e was more immunogenic as compared to Flg-4M2e-HA2, with the former offering full protection to mice against a lethal challenge. We obtained evidence suggesting that the order of linkage of target antigens in a fusion protein may influence the 3D conformation of the chimeric construct, which leads to changes in immunogenicity and protective potency.

Design of perovskite/crystalline-silicon monolithic tandem solar cells.

We present an optical model implemented in the commercial software SETFOS 4.6 for simulating perovskite/silicon monolithic tandem solar cells that exploit light scattering structures. In a first step we validate the model with experimental data of tandem solar cells that either use front- or rear-side textures and extract the internal quantum efficiency of the methyl-ammonium lead iodide (MALI) perovskite sub-cell. In a next step, the software is used to investigate the potential of different device architectures featuring a monolithic integration between the perovskite and silicon sub-cells and exploiting rear- as well as front-side textures for improved light harvesting. We find that, considering the available contact materials, the p-i-n solar cell architecture is the most promising with respect to achievable photocurrent for both flat and textured wafers. Finally, cesium-formamidinium-based perovskite materials with several bandgaps were synthetized, optically characterized and their potential in a tandem device was quantified by simulations. For the simulated layer stack and among the tested materials with bandgaps of 1.7 and 1.6 eV, the one with 1.6 eV bandgap was found to be the most promising, with a potential of reaching a power conversion efficiency of 31%. In order to achieve higher efficiencies using higher band-gap materials, parasitic absorptance in the blue spectral range should be further reduced.

CROSS-PROTECTIVE PROPERTIES OF AN INFLUENZA VACCINE BASED ON HBC4M2E RECOMBINANT PROTEIN.

One of the main problems in the area of influenza prophylaxis and pandemic prevention is the development of cross-reactive vaccines, i.e. vaccines directed against all subtypes of human influenza viruses. Such vaccines are being developed in many countries for more than 10 years. A number of vaccines are presently undergoing clinical trials. We created Uniflu candidate vaccine based on recombinant HBc4M2e protein consisting of 4 tandem-connected copies of the highly conserved ectodomain of M2 protein of the influenza A virus. These 4 copies were genetically fused to the carrier protein, namely hepatitis B core antigen. Commercially available Derinat was used as adjuvant in the candidate vaccine. Preclinical studies on laboratory animals (mice, ferrets) demonstrated that immunization with Uniflu leads to significantly higher level of specific immunoglobulins in the blood and bronchoalveolar lavages. Moreover, it produces immunoglobulins belonging to subtype IgG2a that is the most important mediator of antibody-dependent cytotoxicity. The vaccine under review stimulates the proliferation of T-lymphocytes, as well as the formation of CD4+ and CD8+ T-cells synthesizing ɣ-IFN. When infected with the lethal doses (5 LD50) of influenza A viruses of the subtypes H1N1, H2N2, H3N2, and H1N1pdm09, immunized animals typically developed mild form of illness. This kept them alive in 90-100% of cases, which demonstrated almost complete protection from death. Replication of the virus in the lungs of immunized mice was reduced by 1.8-4.8 log10. High immunogenicity of the vaccine, and reduced clinical symptoms following experimental infection, were demonstrated in ferrets as well. The developed recombinant vaccine Uniflu has high specific activity and cross-protection. Uniflu can be proposed as pre-pandemic vaccine, provided that it passes clinical trials.

A Fusion Protein Based on the Second Subunit of Hemagglutinin of Influenza A/H2N2 Viruses Provides Cross Immunity.

Conserved fragments of the second subunit of hemagglutinin (HA2) are of great interest for the design of vaccine constructs that can provide protective immunity against influenza A viruses of different subtypes. A recombinant fusion protein, FlgMH, was constructed on the basis of flagellin and a highly conserved HA2 fragment (35-107) of influenza viruses of the subtype A/H2N2, containing B cell, CD4+ T cell, and CD8+ T cell epitopes. The native conformation of the HA2 fragment was partially preserved upon its attachment to the C-terminus of flagellin within the recombinant fusion protein FlgMH. FlgMH was shown to stimulate a mixed Th1/Th2 response of cross-reactive antibodies, which bind to influenza viruses of the first phylogenetic group (H1, H2, H5), to the target sequence as well as the induction of specific cytotoxic T cells (CD3+CD8+IFNγ+). Immunization with the recombinant protein protected animals from a lethal influenza infection. The developed FlgMH protein is a promising agent that may be included in an influenza vaccine with a wide spectrum of action which will be able to stimulate the T and B cell immune responses.

[THE ENERGY FUNCTION OF RAT CARDIAC MITOCHONDRIA UNDER ARTIFICIAL HYPOBIOSIS].

We investigated the energy activity of mitochondria from rat cardiomyocytes under the artificial carbon dioxide hypobiosis, which led to physiological changes in the organism (the decrease of body temperature, the reduction of heart rate, etc.). The respiratory and phosphorylation activities in mitochondria of cardiomyocytes is reduced when using two oxidation substrates (succinate and malate), which characterize the rate of the oxygen consumption by the mitochondria. The partial uncoupling of the oxidation and phosphorylation processes when using the malate unlike succinate was established. The activity of NADH-KoQ-oxidoreductase (complex I of the respiratory chain) is inhibited, but the activities of succinate dehydrogenase and cytochrome oxidase don't change. Probably, the priority of the succinate use under the artificial hypobiosis provides the support of the mitochondria functional activity on a sufficient energy level. It is evidenced by the ATP-synthetase activity. The modifications of the structural and functional state of the inner mitochondria membrane of the cardiomyocytes are directed to the adaptation under the artificial carbon dioxide hypobiosis.

[Ebola hemorrhagic fever: Properties of the pathogen and development of vaccines and chemotherapeutic agents].

Ebola hemorrhagic fever (EHF) epidemic currently ongoing in West Africa is not the first among numerous epidemics in the continent. Yet it seems to be the worst EHF epidemic outbreak caused by Ebola virus Zaire since 1976 as regards its extremely large scale and rapid spread in the population. Experiments to study the agent have continued for more than 20 years. The EHF virus has a relatively simple genome with seven genes and additional reading frame resulting from RNA editing. While being of a relatively low genetic capacity, the virus can be ranked as a standard for pathogenicity with the ability to evade the host immune response in uttermost perfection. The EHF virus has similarities with retroviruses, but belongs to (-)RNA viruses of a nonretroviral origin. Genetic elements of the virus, NIRV, were detected in animal and human genomes. EHF virus glycoprotein (GP) is a class I fusion protein and shows more similarities than distinctions in tertiary structure with SIV and HIV gp41 proteins and even influenza virus hemagglutinin. EHF is an unusual infectious disease, and studying the molecular basis of its pathogenesis may contribute to new findings in therapy of severe conditions leading to a fatal outcome.

Ebola hemorrhagic fever: Properties of the pathogen and development of vaccines and chemotherapeutic agents.

Ebola hemorrhagic fever (EHF) epidemic currently ongoing in West Africa is not the first among numerous epidemics in the continent. Yet it seems to be the worst EHF epidemic outbreak caused by Ebola virus Zaire since 1976 as regards its extremely large scale and rapid spread in the population. Experiments to study the agent have continued for more than 20 years. The EHF virus has a relatively simple genome with seven genes and additional reading frame resulting from RNA editing. While being of a relatively low genetic capacity, the virus can be ranked as a standard for pathogenicity with the ability to evade the host immune response in uttermost perfection. The EHF virus has similarities with retroviruses, but belongs to (-)RNA viruses of a nonretroviral origin. Genetic elements of the virus, NIRV, were detected in animal and human genomes. EHF virus glycoprotein (GP) is a class I fusion protein and shows more similarities than distinctions in tertiary structure with SIV and HIV gp41 proteins and even influenza virus hemagglutinin. EHF is an unusual infectious disease, and studying the molecular basis of its pathogenesis may contribute to new findings in therapy of severe conditions leading to a fatal outcome.

Oxidative phosphorylation in mitochondria of small-intestinal enterocytes at chronic and single exposure to low power ionizing radiation. / Okysne fosforyljuvannja v mitohondrijah enterocytiv tonkoi' kyshky za hronichnoi' ta razovoi' dii' ionizujuchoi' radiacii' nyz''koi' potuzhnosti.

Objective - to investigate the intensity of oxidation and phosphorylation processes in the small intestine enterocytes mitochondria of the rats under chronic and single exposure to ionizing radiation of low power. Materials and methods. The single irradiation of the rats with X-rays was performed using the RUM-17 equipment (at the dose of 0.055 Gy/min) for absorbed doses of 0.1; 0.5 and 1.0 Gy. The functional state of mitochondria was evaluated in 1 h and 24 h after irradiation. The chronic external γ-irradiation (at the dose rate of 0.72 cGy/min to achieve a total dose of 0.3; 0.6 and 1.0 Gy was performed using the "Etalon" equipment which contained 60Co. The intensity of the oxidation and phosphorylation activity of the small intestine enterocytes mitochondria was assessed by the polarographic method. Results. The use of malate as exogenous substrate allowed to assess the functioning of all areas of the electron transport chain of mitochondria in experimental conditions. The increase in the intensity of mitochondrial respiration was found in all conditions and terms of research in response to irradiation. In chronic ionizing radiation appears partial separation of interface processes of oxidation and phosphorylation, as evidenced by the decrease in value of the index control and ADP / O and reducing efficiency phosphorylation (decrease of value Vf). For single exposure disconnection of coupling processes oxidation and phosphorylation were not accompanied by changes in the index of oxidative phosphorylation, however, was characterized by reduced ratio V4S/V4АТP. Conclusions. Single and chronic exposure to low power of ionizing radiation leads to an increase in intensity of respiration and disruption of oxidative phosphorylation as a result of separation of interface processes of oxidation and phosphorylation. In chronic exposure received changes were accompanied by inhibition of ATP from ADP and FN because no change of ATP hydrolase activity of mitochondria was noticed. The identified functional disruption of enterocytes mitochondria by ionizing radiation may be associated with structural and functional changes of the respiratory chain, activity both enzymes of electron transport chain and H+ -ATPase. Effects of ionizing radiation of low power can lead to disruption of mitochondrial bioenergetic function - one of the earliest manifestations of cellular dysfunction.

[Treatment of surgical stress response by neuroleptics, leu-enkephalin, glycyl-proline dipeptide, and electroanesthesia in experimental animals].

We have studied the effects of chlorpromazine, acepromazine, droperidol, and transcranial electroanesthesia upon evacuation function of the stomach in piglets and the effects of leu-enkephalin and glycyl-proline upon secretory activity of the stomach in dogs and rats during surgical stress to optimize anesthetic dosage. All pharmaceutical and nonpharmaceutical methods of anesthesia used in the experiments were implemented by actiovating stress-limiting systems in post-operational period. Leu-enkephalin, apart from stimulating the stress-limiting factors of neurohumoral systems of the organism, shifted the vector ratio of aggressive and protective factors of gastric mucosa toward strengthening of the latter. The opposite trends of changes in the secretory activity of the stomach of rats in response to surgical stress can be leveled using glycyl-proline as a component of anesthesia.

Optimization of parameters of physiological systems in animals under conditions of experimental stress exposure.

Physiological and biomedical experiments were performed. We estimated the parameters of tree main levels of physiological systems as a response of an organism to stress. Mechanisms of physiological defense of an organism against stress should be considered systemically. The response of physiological systems of animal organism was modeled by the activation of latent functional reserves by various exposures to stress (abdominal surgery, cranial electrotherapy stimulation, cranial electrotherapy stimulation with simultaneous administration of droperidol, aminazine, vetranquil, and carbacholine).

[The role of adenosine Al receptors and mitochondrial K+ATP channels in the mechanism of increasing the resistance to acute hypoxia in the combined effects of hypoxia and hypercapnia].

We studied the role of the role of mitoK+ATp channels and Al-adenosine receptor in the mechanism of increasing the resistance to acute hypoxia after hypoxic, hypercapnic and hypercapnic-hypoxic preconditioning. It is shown that mitochondrial ATP-sensitive potassium channels and Al-adenosine receptors, an important mechanism of preconditioning have a high value to increase the resistance to acute hypoxia/ischemia in the combined effect of hypoxia and hypercapnia. However, with regard to the adenosine receptor, this mechanism is realized without the participation hypercapnic component, which apparently starts neuroprotection without activation of the adenosine Al receptors.

Functional and dynamic state of inner mitochondrial membrane of sarcoma 37 in mice under administration of sodium dichloroacetate.

The activity of enzymes of the respiratory chain and structural-dynamic properties of the inner mitochondrial membrane (IMM) of sarcoma 37 (S37) in mice under sodium dichloroacetate (SDA) administration in a daily dose of 86 mg/kg of body weight starting from the 2nd day after tumor transplantation were investigated. The dynamic and structural state of the IMM components was determined using the fluorescent probes. With S37 growth the intensification of glycolytic metabolism occurred on the background of suppressed functional capacity of mitochondrial respiratory chain enzymes. The changes of conformational properties of protein molecules and the increase of IMM lipid phase microviscosity were shown. The administration of SDA promotes the decrease of lactate content and the increase of pyruvate dehydrogenase activity in S37. This was accompanied by further suppression of the functional activity of the respiratory chain complexes and H+-ATPase coupled with conformational modification ofprotein molecules and changes of the structural orderliness of the IMM lipid phase, possibly due to intensification of reactive oxygen species generation.

[Immunogenicity of recombinant proteins including ectodomain of M2 influenza virus A].

Two recombinant proteins with three copies of the ectodomain of the conserved influenza protein M2 (M2e) of influenza viruses were developed: A (H1N1)pdm09, A/Kurgan/05/05 (H5N1), and M2e consensus sequence of the human influenza A virus (H1N1, H2N2, H3N2) based on flagellin and core antigen of hepatitis B (HBc). The first recombinant protein comprised flagellin fused to three tandem copies of M2e, the second preparation was based on non-covalent interaction between M2e peptides and HBc. The immunogenicity of two preparations was comparatively tested. A covalent linkage of flagellin with M2e significant increased the immunogenicity of the target antigen compared with non-covalent interaction M2e and HBc. Flagellin as a protein carrier of M2e induced mainly IgG1 subclass, whereas HBc stimulated more balanced Th1/Th2 response. Our study showed a decrease in the viral titers in lung tissues of immunized mice after lethal challenge of A/PR/8/34 (H1N1). The study revealed a possibility to obtain a vaccine preparation with equal immunogenicity both against human influenza viruses and highly pathogenic avian influenza viruses.

[The functional state of the mitochondrial respiratory chain of the small intestine enterocytes of the rats under the low dose rate X-ray total external exposure].

The influence of the low-rate ionizing radiation (0.055 Gy/min) at the doses of 0.1; 0.5 and 1.0 Gy on the functional state of the mitochondria respiratory chain of the rat small intestine enterocytes was investigated. The dysfunction of the electron transport chain enzymes and changes in the content of cytochromes b, c, a in the mitochondrial inner membrane were revealed 1, 12 and 24 hours after exposure to radiation. The revealed disorders indicate early membrane sensitivity to the radiation effect. The inhibition of the H+ -ATPase activity in the studied dose range indicates the decrease of the mitochondrial energy capacity.

[Oxidative and hydrolytic deterioration of palm oil and fat products based on it under various conditions of storage and transportation].

Studies have been conducted on the effect of storage conditions for refined deodorized palm oil on the quality and safety: in containers made of ferrous metals (mild steel) at unregulated temperature, in sealed plastic bags at the temperature -20 degrees C in stainless steel under stratification of nitrogen at the temperature of 40+/-1 degrees C. The choice of the objects of study determined by the normative documents of the Russian Federation governing the transportation and storage of vegetable oils and fat products based on them. All samples of palm oil with peroxide value of 1,0 to 1,5 meq O2/kg indicated the presence of a weak foreign taste, is not peculiar impersonalfat, the samples with peroxide value above 1,5 meq O2/kg were observed pronounced off-flavors and odors characteristic of stale oil. Rancidity was observed in samples having peroxide value of 2,0 meq O2/kg or more. Free acid value and anizidin value for the studied period changed to a lesser extent, from 0,06 to 0,1 mg KOH/g and from 1,2 to 1,4 respectively. It is proved that, transportation/storage of palm oil at the temperature above 50 degrees C without stratification of nitrogen greatly accelerates the process of oxidative damage. Based on these data we can recommend transportation/storage and management process with the least possible time of contact of melted palm oil with oxygen to produce high-quality final product (within 2-3 hours from the time of melting).

Plant-produced recombinant influenza vaccine based on virus-like HBc particles carrying an extracellular domain of M2 protein.

Conventional influenza vaccines are based on a virus obtained in chicken embryos or its components. The high variability of the surface proteins of influenza virus, hemagglutinin and neuraminidase, requires strain-specific vaccines matching the antigenic specificity of newly emerging virus strains to be developed. A recombinant vaccine based on a highly conservative influenza virus protein M2 fused to a nanosized carrier particle can be an attractive alternative to traditional vaccines. We have constructed a recombinant viral vector based on potato X virus that provides for expression in the Nicotiana benthamiana plants of a hybrid protein M2eHBc consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen (HBc). This vector was introduced into plant cells by infiltrating leaves with agrobacteria carrying the viral vector. The hybrid protein M2eHBc was synthesized in the infected N. benthamiana plants in an amount reaching 1-2% of the total soluble protein and formed virus-like particles with the M2e peptide presented on the surface. Methods of isolation and purification of M2eHBc particles from plant producers were elaborated. Experiments on mice have shown a high immunogenicity of the plant-produced M2eHBc particles and their protective effect against lethal influenza challenge. The developed transient expression system can be used for production of M2e-based candidate influenza vaccine in plants.

[A new method of producing biologically active nanocomplexes by non-covalent conjugation of proteins with viral particles].

Currently, a range of biologically active molecules have been attached to plant and bacterial viras nanoscaffolds, yielding stable nanoparticles that display multiple copies of the desired molecule. In this paper we propose a new method of non-covalent attachment of peptides to the surface of virios. We have demonstrated that this method is efficient in a model system that includes tobacco mosaic virus particles, synthetic polycation (quaternized poly(4-vinylpyridine) carrying ethyl ethyl pendant radicals) and polypeptide of interest. This principle of step-by-step binding to the surface of virions was used for electrostatic association with hydrophilic fragment of influenza virus haemagglutinin.

[Functioning of the antioxidant system in epithelial cells of small intestine under the influence of ionizing radiation of low dose rate].

The investigations of the functional state of the antioxidant system in epithelial cells from rat small intestine in dynamics after X-ray irradiation (0.1; 0.5 and 1.0 Gy) at a low absorbed dose rate (55 mGy/min) were performed. The obtained results point out the ambiguity of the antioxidant system reaction to the activation of oxidative processes at different doses of irradiation. The multidirectional changes of these antioxidant enzymatic activities whose functioning is characterized by the early post-irradiation recovery depending on the value of the absorbed dose were observed. The sensitivity of superoxide dismutase, glutathione transferase and glutathione peroxidase in mitochondria of epithelial cells to the irradiation exposure in the investigated range of absorbed doses was shown.