PROTAC: targeted drug strategy. Principles and limitations.

The PROTAC (PROteolysis TArgeting Chimera) technology is a method of targeting intracellular proteins previously considered undruggable. This technology utilizes the ubiquitin-proteasome system in cells to specifically degrade target proteins, thereby offering significant advantages over conventional small-molecule inhibitors of the enzymatic function. Preclinical and preliminary clinical trials of PROTAC-based compounds (degraders) are presented. The review considers the general principles of the design of degraders. Advances and challenges of the PROTAC technology are discussed.

[Metabolic characteristics, nutritional status and behavior factors in people with obesity, working at industrial plant].

An obesity is the topical problem worldwide, because excessive weight is the reason of metabolic changes that lead to development of serious chronic non-communicable diseases. The examination was aimed to assess clinical and metabolic health, nutritional status and behavior risk factors of obesity in people, working at the industrial plant. Material and methods. In 348 workers of the industrial plant (210 males, 138 females) examination was performed including anthropometry: body mass index (BMI), waist and hip circumferences and their ratio, blood pressure level detection, laboratory blood tests (glucose and cholesterol levels), and questionnaire on nutritional habits and physical activity. All examined were divided in three groups depending on BMI: 1st group - 151 (43.3%) persons with normal body mass, 2nd group - 125 (36%) working people with overweight and 3rd group - 72 (20.7%) people with obesity. Results. More than half of examined workers (mostly females) had overweight or obesity with predominantly abdominal type of fat distribution and I degree. Arterial hypertension was diagnosed in 41% of all working people (predominantly I degree), more frequently observed in obese (62.5%) and in males (45%). Each fourth man and woman had elevated total cholesterol level, which increased depending on BMI raise. Postprandial hyperglycemia was observed in 5.5% of cases, with the same frequency in both genders, predominantly in obesity. A significant part of responded with obesity, especially females, confirmed, that the reasons of excessive weight were stresses and somatic diseases (37%), and they preferred chocolate from confectionery. It was noted a correlation of BMI with egg consumption in males and with the consumption frequency of nuts, seeds and chocolate in females. Physical activity less than 3 times a month or never was observed in most of responded workers with overweight and obesity (73%), especially in males, as well as in females during the last month before the survey (57%). Conclusion. Preventive examination detected not only high prevalence of abdominal obesity, arterial hypertension and fat metabolism disturbances in people, working at the industrial plant, but also allowed to detect the most significant alimentary and behavioral risk factors of obesity.

[Laparoscopic management of the prostatic utricle cyst in a 4-year-old child].

The article present the clinical observation of 4 year old boy with scrotal hypospadias. Previous surgery management had unsatisfactory results due to complications of the cyst of the prostatic utricle. Laparoscopic removal of the cyst of the prostatic utricle was performed. The next step was urethral plastic. The results of the operation were satisfactory. A brief review of the literature is provided.

Structural variations of germanosilicate glasses with change in modifier cation type or Ge/Si ratio.

Raman spectroscopy was used to study glasses of three-component systems M2O-SiO2-GeO2, where M = Li, Na, K. Following the decomposition of spectra into component lines, the main structural units of both the germanate and silicate networks were determined according to peak-fitting result. With addition of germanium oxide, a change in the structure of the silicate network system was observed. In revealing highly-coordinated germanium atoms in all synthesised glasses, the study also established the dependence of their quantity on GeO2 content and modifier cation type.

Aggregation of Influenza A Virus Nuclear Export Protein.

Influenza A virus nuclear export protein (NEP) plays an important role in the viral life cycle. Recombinant NEP proteins containing (His)6-tag at either N- or C-terminus were obtained by heterologous expression in Escherichia coli cells and their high propensity for aggregation was demonstrated. Dynamic light scattering technique was used to study the kinetics and properties of NEP aggregation in solutions under different conditions (pH, ionic strength, presence of low-molecular-weight additives and organic solvents). Using atomic force microscopy, the predominance of spherical aggregates in all examined NEP preparations was shown, with some amyloid-like structures being observed in the case of NEP-C protein. A number of structure prediction programs were used to identify aggregation-prone regions in the NEP structure. All-atom molecular dynamics simulations indicate a high rate of NEP molecule aggregation and reveal the regions preferentially involved in the intermolecular contacts that are located at the edges of the rod-like protein molecule. Our results suggest that NEP aggregation is determined by different types of interactions and represents an intrinsic property of the protein that appears to be necessary for its functioning in vivo.

[Family History of Cardiovascular Disease in Patients With Early Development of Acute Coronary Syndrome].

The aim of the study was to analyze clinical features of patients with premature acute coronary syndrome (ACS) in relation to family history of cardiovascular disease (CVD) and familial hypercholesterolemia (FH). MATERIALS AND METHODS: Of 2832 patients included in ORACUL 1 and ORACUL 2 multicenter observational trials 512 pts who developed premature ACS (≤55 years for men, ≤60 years for women) and had known family history and LDL level were selected for this study. Of these patients 297 had positive family history (51 with FH, 246 no FH), 215 had negative family history. RESULTS: Among patients with positive family history there were more women (31 vs 20.9 %), while among patients with negative family history there were more men (79.1 vs 69 %). The fact of regular alcohol consumption was significantly more frequently observed among patients with positive family history but without FH, compared to patients with positive family history with FH (69.6 vs 47.1 %). Women with positive family history smoked more frequently than females with negative family history (51.1 vs 31.1 %). Among patients with negative family history compared with patients with positive family history there were more people who at admission had hyperglycemia exceeding 11.1 mmol / l (10.3 vs 4.4 %). Multiple vessel disease and coronary calcinosis were present in 73.2 and 24.7 %, respectively, of patients with positive family history, and in 56.9 and 9.8 %, respectively, of those with negative family history. Among patients with positive family history multivessel disease was more frequent in the subgroup with FH, while coronary calcinosis was more frequent in the subgroup without FH. CONCLUSION: Thus, premature development of ACS might be associated not only with genetic factors but also with family history ("inheritance") of adverse habits. Herewith coronary calcinosis is more prevalent in patients with FH.

Structural study of the X-ray-induced enzymatic reduction of molecular oxygen to water by Steccherinum murashkinskyi laccase: insights into the reaction mechanism.

The laccase from Steccherinum murashkinskyi is a member of the large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates, accompanied by the reduction of dioxygen to water. The reducing properties of X-ray radiation and the high quality of the laccase crystals allow the study of the catalytic reduction of dioxygen to water directly in a crystal. A series of diffraction data sets with increasing absorbed radiation dose were collected from a single crystal of Steccherinum murashkinskyi laccase at 1.35 Šresolution. Changes in the active-site structure associated with the reduction of molecular oxygen to water on increasing the absorbed dose of ionizing radiation were detected. The structures in the series are mixtures of different states of the enzyme-substrate complex. Nevertheless, it was possible to interpret these structures as complexes of various oxygen ligands with copper ions in different oxidation states. The results allowed the mechanism of oxygen reduction catalyzed by laccases to be refined.

Heterologous Expression and Isolation of Influenza A Virus Nuclear Export Protein NEP.

Influenza A virus nuclear export protein NEP (NS2, 14.4 kDa) plays a key role in various steps of the virus life cycle. Highly purified protein preparations are required for structural and functional studies. In this study, we designed a series of Escherichia coli plasmid constructs for highly efficient expression of the NEP gene under control of the constitutive trp promoter. An efficient method for extraction of NEP from inclusion bodies based on dodecyl sulfate treatment was developed. Preparations of purified NEP with either N- or C-terminal (His)6-tag were obtained using Ni-NTA agarose affinity chromatography with yield of more than 20 mg per liter of culture. According to CD data, the secondary structure of the proteins matched that of natural NEP. A high propensity of NEP to aggregate over a wide range of conditions was observed.

Neuroprotective Effect of Carnosine on Primary Culture of Rat Cerebellar Cells under Oxidative Stress.

Dipeptide carnosine (ß-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1 mM 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20 nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2 mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20 nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2 mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1 mM AAPH and 20 nM rotenone. Using liquid chromatography-mass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2 mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells.

A hypothetical hierarchical mechanism of the self-assembly of the Escherichia coli RNA polymerase σ(70) subunit.

Diverse morphology of aggregates of amyloidogenic proteins has been attracting much attention in the last few years, and there is still no complete understanding of the relationships between various types of aggregates. In this work, we propose the model, which universally explains the formation of morphologically different (wormlike and rodlike) aggregates on the example of a σ(70) subunit of RNA polymerase, which has been recently shown to form amyloid fibrils. Aggregates were studied using AFM in solution and depolarized dynamic light scattering. The obtained results demonstrate comparably low Young's moduli of the wormlike structures (7.8-12.3 MPa) indicating less structured aggregation of monomeric proteins than that typical for ß-sheet formation. To shed light on the molecular interaction of the protein during the aggregation, early stages of fibrillization of the σ(70) subunit were modeled using all-atom molecular dynamics. Simulations have shown that the σ(70) subunit is able to form quasi-symmetric extended dimers, which may further interact with each other and grow linearly. The proposed general model explains different pathways of σ(70) subunit aggregation and may be valid for other amyloid proteins.

Biocatalytic conversion of poultry processing leftovers: Optimization of hydrolytic conditions and peptide hydrolysate characterization.

Peptide hydrolysate (PH) was produced by deep controllable bioconversion of poultry processing leftovers (broiler necks), by means of a multienzyme composition, containing four commercially available enzyme preparations (Alcalase, Neutrase, Flavourzyme, Protamex). The design of multienzyme composition (MEC) was applied to yield a hydrolysate with adjusted properties, including minimized antigenicity and bitterness. The protein recovery was optimized using Box-Behnken response surface design. The individual and interactive effects of hydrolysis conditions (time, hydromodule and MEC dosage) were studied. The experimental data were analyzed by ANOVA method and a well-predictive, second order polynomial model was developed using multiple regression analysis. Optimal hydrolysis conditions were found to be: hydrolysis time 3 h, hydromodule 2.25 l/kg and dosage of MEC 0.25%. The corresponding predicted value for protein recovery was 75.34%, 2 times higher compared to traditional long-term heating hydrolysis. The PH obtained is a low allergenic product with high antioxidant capacity.

[Preparation of protoplasts of the fungus Trametes hirsuta 072 and study of the effect of antioxidants on their formation and regeneration].

The consistent application of homogenization and enzymatic treatment is required to obtain protoplasts from the basidiomycete fungus Trametes hirsuta. The maximum yield of protoplasts (∼2.5 × 107/mL) was achieved when mycelium in the exponential growth phase (60 h) was used. The maximum stability was observed in MES+ buffer during 4 h of incubation; in this case the titer reduction was 5­7%. Studies of the effect of antioxidants with different antioxidant capacities expressed in mmol equivalents of Trolox (ascorbate, 0.99; α-tocopherol, 1.0; ß-carotene, 2.14; quercetin, 3.98) indicated that the yield of protoplasts was increased in the presence of ß-carotene and quercetin by 18­24%. The studied antioxidants did not affect the protoplasts stability. The degree of regeneration of protoplasts correlated with the antioxidant capacity of the studied antioxidants and was maximal (0.4%) in the presence of ß-carotene and quercetin; it was 0.1% in the presence of MES+. The rate of protoplast growth was two times higher in the presence of ß-carotene and quercetin.

[Degradation of fluorene and fluoranthene by the basidiomycete Pleurotus ostreatus].

The dependence of the degree of fluorene and fluoranthene degradation by the fungus Pleurotus ostreatus D1 on the culture medium composition has been studied. Polycyclic aromatic hydrocarbons (PAHs) have been transformed in Kirk's medium (under conditions of laccase production) with the formation of a quinone metabolite and 9-fluorenone upon the use of fluoranthene and fluorene as substrates, respectively. More complete degradation with the formation of an intermediate metabolite, phthalic acid that has undergone subsequent utilization, has occurred in basidiomycete-rich medium (under the production of both laccase and versatile peroxidase). The formation of phthalic acid as a metabolite of fluoranthene degradation by lignolytic fungi has been revealed for the first time. The data allow the supposition that both extracellular laccase and laccase on the mycelium surface can participate in the initial stages of PAH metabolism, while versatile peroxidase is necessary for the oxidation of the formed metabolites. A scheme of fluorene metabolism by Pleurotus ostreatus D1 is suggested.

[Oil degradation by basidiomycetes in soil and peat at low temperatures].

A total of 17 basidiomycete strains causing white rot and growing on oil-contaminated substrates have been screened. Three strains with high (Steccherinum murashkinskyi), average (Trametes maxima), and low (Pleurotus ostreatus) capacities for the colonization of oil-contaminated substrates have been selected. The potential for degrading crude oil hydrocarbons has been assessed with the use of fungi grown on nonsterile soil and peat at low temperatures. Candida sp. and Rhodococcus sp. commercial strains have been used as reference organisms with oil-degrading ability. All microorganisms introduced in oil-contaminated soil have proved to be ineffective, whereas the inoculation of peat with basidiomycetes and oil-degrading microorganisms accelerated the destruction of oil hydrocarbons. The greatest degradation potential of oil-aliphatic hydrocarbons has been found in S. murashlinskyi. T. maxima turned out to be the most successful in degrading aromatic hydrocarbons. It has been suggested that aboriginal microflora contributes importantly to the effectiveness of oil-destructing microorganisms. T. maxima and S. murashkinskyi strains are promising for further study as oil-oxidizing agents during bioremediation of oil-contaminated peat soil under conditions of low temperatures.

[The screening of donor blood on antibodies to nuclear antigen of hepatitis B virus as a tool of increasing of safety of transfusion for patients with diseases of blood system.]

Despite application of decreed modes of laboratory analysis of components of donors' blood, the risk of infection of recipients with hepatitis B virus continues to be actual. The isolated identification of HBsAg provides no control of all categories of persons infected with hepatitis B virus. The analysis of presence of antibodies to nuclear antigen of hepatitis B virus that are the first out of antiviral ones and are preserved for life, is an expedient technique of screening testing of donor's blood that permits implementing an additional selection of donors. During March 2014 - March 2015, cohort of regular anti-hepatitis B virus negative donors of blood and its components. The testing of blood samples for anti-hepatitis B virus can be recommended as a routine test increasing viral safety of blood transfusions for patients with diseases of blood system.

[Investigation of the Dependence of Escherichia coli RNA Polymerase σ70-Subunit Structure on Ionic Strength by Molecular Dynamics Simulation Method].

The σ70-subunit of E. coli RNA polymerase (a small protein, being a part of RNA holoenzyme, and responsible for initiation of transcription of constitutive genes) is modeled at different ionic strengths. Two variants of the location of C-end domain 4 are obtained. At low ionic strength domain 4 interacts with the region of high negative charge 190-210 AK within NCR domain. At high ionic strength this region was screened and domain 4 was free and set away from domain NCR. We suppose that this leads to the increase in polymerization rate. Simulation data do not confirm any hypothesis about a self-inhibition mechanism.

Comparative proteomic study of the basidiomycete Trametes hirsuta grown on different substrates.

Protein profiles of the basidiomycete Trametes hirsuta grown on standard medium without laccase as an inducer and on medium supplemented with CuSO4 were analyzed using a differential proteomics approach. Protocols developed for isolation and purification of extracellular and intracellular proteins of the mycelium allowed us to show extensive extraction of protein components. Simultaneously, components hampering two-dimensional electrophoresis (pigments, low molecular mass metabolites) were removed from the samples, and high-resolution protein maps were obtained. Analysis of the basidiomycete secretomes revealed qualitative changes in the protein profile: the addition of CuSO4 as an inducer resulted in increase in the produced laccase isoforms and/or isozymes from 7 to 11, whereas its pI range change exceeded 2 units. The number of separated intracellular protein components was 552 and 502 for the control medium and medium with the inducer, respectively. Comparative analysis of the protein maps revealed five regions with the most pronounced differences in the protein profiles. The proteins of interest were identified using MALDI-TOF/TOF mass spectrometry with subsequent peptide fingerprinting. Some intracellular proteins (ß-subunits of ATP synthase, molecular chaperones, chaperone activator) upregulated during the growth on the inducer-containing medium were identified. These proteins are supposed to be involved in the regulation of laccase biosynthesis during folding and secretion of the enzyme.

[Transformation of humic substances of highly oxidized brown coal by the basidiomycetes fungi Trametes hirsuta and Trametes maxima].

The ability of the white rot basidiomycetes Trametes hirsuta and Trametes maxima to transform coal humic substances (HS's) under the conditions of solid phase cultivation in the presence or absence of an easily available source of corbon (glucose) has been studied. It was shown that during the growth of the fungal strains used in media, containing HS's, destructive and condensation processes of HS transformation proceeded simultaneously. Based on a comparative physicochemical analysis of the initial HS's and HS's transformed by the fungi, it was established that, despite the introduction of glucose may favor a deeper transformation of HS's by basidiomycetes, the general direction of their modification is dominant reduction or oxidation and is determined by the physiological biochemical peculiarities of the strain used.

Investigation of activity of recombinant mengovirus RNA-dependent RNA polymerase and its mutants.

The activities of wild-type mengovirus RNA polymerase (RdRP) and of its three mutants with C-terminal tryptophan residue replaced by residues of alanine (W460A), phenylalanine (W460F), or tyrosine (W460Y) were studied. The proteins were expressed in E. coli and purified by affinity chromatography with the IMPACT system. The isolated recombinant proteins were studied using a cell-free replication system on elongation of oligo(U) primer on RNA template corresponding to the 3'-terminal 366-meric fragment of the mengovirus RNA. The activities of the mutant polymerases were comparable to that of the wild-type enzyme.

[Comparative analysis of the ligninolytic potential of basidiomycetes belonging to different taxonomic and ecological groups].

Screening of the ligninolytic activity of basidiomycetes from the Culture Collection of the Komarov Botanical Institute, Russian Academy of Sciences, belonging to diterent taxonomic and ecological groups was performed. The patterns of the position of taxa of active producers of ligninolytic enzymes in the modern system of fungi were identified. Cluster analysis showed that the group of fungi with the greatest lign- inolytic and degradation potential includes representatives of the families Pleurotaceae, Polyporaceae, and Phanerochaetaceae, which perform the first stages of wood decomposition. As a result, species of interest for the further study of their oxidative potential and use in biotechnology were selected.