Direct Reprogramming of Somatic Skin Cells from a Patient with Huntington's Disease into Striatal Neurons to Create Models of Pathology.

A new in vitro model of Huntington's disease (HD) was developed via a direct reprogramming of dermal fibroblasts from HD patients into striatal neurons. A reprogramming into induced pluripotent stem (iPS) cells is obviated in the case of direct reprogramming, which thus yields neurons that preserve the epigenetic information inherent in cells of a particular donor and, consequently, the age-associated disease phenotype. A main histopathological feature of HD was reproduced in the new model; i.e., aggregates of mutant huntingtin accumulated in striatal neurons derived from a patient's fibroblasts. Experiments with cultured neurons obtained via direct reprogramming make it possible to individually assess the progression of neuropathology and to implement a personalized approach to choosing the treatment strategy and drugs for therapy. The in vitro model of HD can be used in preclinical drug studies.

Motor and Cognitive Functions in Aging C57BL/6 Mice: Association with Activity of the Monoaminergic Systems in the Cerebellum and Frontal Cortex.

The activity in the open field, short- and long-term memory in the novel object recognition test, and gait features were evaluated in 6- and 12-month-old male C57BL/6 mice. The levels of norepinephrine, dopamine, serotonin, and their metabolites were determined in the cerebellum and frontal cortex. In the observed age range, a decrease in locomotion speed, impairment of gait initiation and stability, and long-term memory deficit were revealed. In the cerebral cortex, reduced levels of dopamine and its metabolites and accelerated metabolism of all neurotransmitters under study were found. In the cerebellum, the content of all studied monoamines was elevated, while dopamine metabolism was decelerated. Analysis of correlations between the neurochemical and behavioral parameters showed that the mechanisms of compensation of brain functions during the early aging may be associated with an increase in activity of the monoaminergic systems in the cerebellum.

The study of NF-κB transcription factor activation by Pseudomonas aeruginosa recombinant proteins in eukaryotic cell culture.

The transcription factor NF-κB is a key factor in the activation of immune responses; it is in turn activated by pattern recognition receptors, such as TLR and NLR receptors. The search for ligands activating innate immunity receptors is an important scientific problem due to the possibility of their use as adjuvants and immunomodulators. In this study the effect of recombinant Pseudomonas aeruginosa OprF proteins and a toxoid (a deletion atoxic form of exotoxin A) on the activation of TLR4, TLR9, NOD1, and NOD2 receptors has been investigated. The study was carried out using free and co-adsorbed on Al(OH)3 P. aeruginosa proteins and eukaryotic cells encoding these receptors and having NF-κB-dependent reporter genes. The enzymes encoded by the reported genes are able to cleave the substrate with the formation of a colored product, the concentration of which indicates the degree of receptor activation. It was found that free and adsorbed forms of the toxoid were able to activate the TLR4 surface receptor for lipopolysaccharide. OprF and the toxoid activated the intracellular NOD1 receptor, but only in the free form. This may be due to the fact that the cell lines used were not able to phagocytize aluminum hydroxide particles with protein adsorbed on them.

Preparation a Recombinant Form of Pneumolysin Protein from Streptococcus pneumoniae.

A recombinant form of pneumolysin from Streptococcus pneumoniae was obtained. By using Vector NTI Advance 11.0 bioinformatic analysis software, specific primers were designed in order to amplify the genome fragment of strain No. 3358 S. pneumoniae serotype 19F containing the nucleotide sequence encoding the full-length pneumolysin protein. A PCR product with a molecular weight corresponding to the nucleotide sequence of the S. pneumoniae genome fragment encoding the full-length pneumolysin was obtained. An expression system for recombinant pneumolysin in E. coli was constructed. Sequencing confirmed the identity of the inserted nucleotide sequence encoding the full-length recombinant pneumolysin synthesized in E. coli M15 strain. Purification of the recombinant protein was performed by affinity chromatography using Ni-Sepharose in 8 M urea buffer solution. Confirmation of the recombinant protein was performed by immunoblotting with monoclonal antibodies to pneumolysin.

AB0 and rhesus blood groups as a risk factor for ARVI COVID-19.

The frequency of AB0 and Rhesus blood groups was studied in 12120 patients with COVID-19, 5180 convalescent plasma donors and 118801 healthy donors from Moscow, Smolensk, Yakutsk, Minsk and Gomel. In infected individuals, the frequency of blood group A was significantly higher than in uninfected individuals (41,54 % and 34,39 % respectively, p<0,05), and the frequency of blood group 0, on the contrary, was significantly lower (27,69 % and 36,71 %, p<0,05). The frequency of blood group A was particularly high among patients who died from ARVI COVID-19 - 45,51 % vs. 34,39 %, p=0,008. In some groups of patients, there was a decrease in the frequency of Rh-negative individuals (2,23 % vs. 8,30 %, p<0,001).

Pathomorphological Changes in the Brain in Dynamics of Long-Term Dust Exposure.

Histological and morphometric studies of brain autopsy material showed that the development of hypoxic changes in miners starts at the early stages of working in the dusty atmosphere. Edema of the pericellular and perivascular zones and the pia mater, degenerative changes in some nerve cells and even their loss and formation of gliosis foci were identified. The revealed changes in neurons progressed with increasing the duration of working under hazardous conditions.

[Five year experience in ibrutinib therapy for relapsed and refractory mantle cell lymphoma in real world Russian clinical practice].

BACKGROUND: Mantle cell lymphoma (MCL) is a rare and clinically aggressive lymphoma subtype. Current approaches have greatly improved patients outcomes, but relapse is inevitable. In phase IIIII clinical trials, ibrutinib has shown significant activity in patients with relapsed or refractory (R/R) MCL. AIM: To assess efficacy and toxicity of ibrutinib monotherapy in patients with R/R MCL in routine practice outside of clinical trials. MATERIALS AND METHODS: The study enrolled patients with confirmed R/R MCL who had received at least one line of previous chemotherapy. ECOG 24, cytopenia, infectious complications, hemorrhagic syndrome were not exclusion criteria. Patients received daily oral ibrutinib 560 mg until progression or unacceptable toxicity. RESULTS: From May 2015 to September 2020 ibrutinib therapy was started in 106 patients with R/R MCL in 16 regions of Russia. The median age was 66 years; ECOG2 18%, blastoid variant (or Ki6740% or WBC50109/l) 43%. The median number of previous treatment lines was 2 (111). The ORR was 78.4% (CRR 27.4%). The median PFS was 13.6 months and OS 23.2 months. In the blastoid group the median PFS was 4.4 months vs 36.5 months in the alternative group (p0.001), the median OS 9.0 vs 41.0 (p=0.001). The median OS of patients after progression on ibrutinib was 3.2 months. The common complications are hemorrhages (63%), diarrhea (62%), myalgia and muscle cramps (60%), infections (31%), skin and nail toxicity 15%, arrhythmia 8%. None of recipients had to completely discontinue ibrutinib therapy due to complications. CONCLUSION: Ibrutinib is effective and well tolerated in routine practice of R/R MCL treatment and our results are consistent with international clinical trials. The favorable toxicity profile and the high response rate made it possible to prescribe ibrutinib in severe somatic status, cytopenia, and even in the presence of infectious complications.

[The value of a low-protein diet and ketoanalogues of essential amino acids in the сontrol of protein carbamylation and toxic effects of urea in chronic kidney disease].

Chronic kidney disease (CKD) is characterized by high mortality from cardiovascular diseases, the development of which is facilitated by traditional risk factors (typical for the general population) and by nontraditional ones (specific to patients with CKD) as well. These factors include also uremic toxins, for which a causal relationship has been established with specific pathological processes in patients with CKD, comprising the development of vascular dysfunction and accelerated progression of atherosclerosis. Urea has long been considered not as a uremic toxin, but as a marker of metabolic imbalance or dialysis efficiency (Kt/V) in CKD patients. In recent years, more and more publications have appeared on the study of the toxic effects of urea with the development of toxic-uremic complications and the phenotype of premature aging, common in CKD. It was found that an increase in urea levels in uremic syndrome causes damage to the intestinal epithelial barrier with translocation of bacterial toxins into the bloodstream and the development of systemic inflammation, provokes apoptosis of vascular smooth muscle cells, as well as endothelial dysfunction, which directly contributes to the development of cardiovascular complications. The indirect effects of increased urea levels are associated with carbamylation reactions, when isocyanic acid (a product of urea catabolism) changes the structure and function of proteins in the body. Carbamylation of proteins in CKD patients is associated with the development of renal fibrosis, atherosclerosis and anemia. Thus, urea is now regarded as an important negative agent in the pathogenesis of complications in CKD. Studies on a low-protein diet with using ketoanalogues of essential amino acids to minimize the accumulation of urea and other uremic toxins demonstrate the clinical benefit of such an intervention in slowing the progression of CKD and the development of cardiovascular complications.

Participation of Free-Radical Processes in Structural and Metabolic Disturbances in the Lung Tissues Caused by Exposure to Coal-Rock Dust and their Adaptogenic Correction.

Adaptive correction of structural and metabolic disturbances in the lungs caused by longterm exposure to coal-rock dust were studied in experiments on rats. It was shown that the complex antioxidant preparation containing dihydroquercetin compensated disturbances in the redox balance in the lung tissue, prevented the formation of dust granulomas, and reduced the severity of degenerative changes in the bronchopulmonary system.

Immunization with Recombinant Pneumolysin Induces the Production of Antibodies and Protects Mice in a Model of Systemic Infection Caused by Streptococcus pneumoniae.

Immunogenic and protective activity of recombinant pneumolysin was studied in experiments on male BALB/c mice. The mice were immunized intraperitoneally with recombinant pneumolysin sorbed on Al(OH)3 (200 µg per mouse). In 2 weeks after immunization, the isotypes of antibodies to recombinant pneumolysin in the serum of immunized mice were determined by ELISA. The animals were infected with Streptococcus pneumoniae serotype 3. Immunization with recombinant pneumolysin induced the production of anti-pneumolysin antibodies, mainly of IgG1 subisotype. On day 21 after intraperitoneal infection with S. pneumoniae serotype 3 in a dose of 106 microbial cells, the survival rate of animals immunized with recombinant pneumolysin in a dose of 25 µg/mouse was 67% vs. 0% in the control (p<0.001). Recombinant pneumolysin could be considered as a promising protective antigen for inclusion in the serotype-independent vaccine against S. pneumoniae.

The Molecular Mechanisms of Gametic Incompatibility in Invertebrates.

Fertilization (gamete fusion followed by zygote formation) is a multistage process. Each stage is mediated by ligand-receptor recognition of gamete interaction molecules. This recognition includes the movement of sperm in the gradient of egg chemoattractants, destruction of the egg envelope by acrosomal proteins, etc. Gametic incompatibility is one of the mechanisms of reproductive isolation. It is based on species-specific molecular interactions that prevent heterospecific fertilization. Although gametic incompatibility may occur in any sexually reproducing organism, it has been studied only in a few model species. Gamete interactions in different taxa involve generally similar processes, but they often employ non-homologous molecules. Gamete recognition proteins evolve rapidly, like immunity proteins, and include many taxon-specific families. In fact, recently appeared proteins particularly contribute to reproductive isolation via gametic incompatibility. Thus, we can assume a multiple, independent origin of this type of reproductive isolation throughout animal evolution. Gametic incompatibility can be achieved at any fertilization stage and entails different consequences at different taxonomic levels and ranges, from complete incompatibility between closely related species to partial incompatibility between distantly related taxa.

Study of the Osteoindictive Properties of Protein-Modified Polylactide Scaffolds.

Bone marrow mesenchymal stromal cells are multipotent and can differentiate into cells of various tissues, which determines their high importance for clinical application. We performed an in vitro study of the osteogenic potential of mesenchymal stromal cells cultured on intact polylactide scaffolds or scaffolds modified with collagen I or fibrin. Scanning electron microscopy showed that the cells formed osteogenic nodules or osteogenic nodules on both intact and fibrin-modified polylactide scaffolds. Spectrophotometric detection of alkaline phosphatase activity on days 7 and 11 showed that mesenchymal stromal cell grown on intact polylactide scaffolds and on scaffolds modified with collagen type I or fibrin more intensively synthesized alkaline phosphatase than in the control (culture plastic). This dependence increases in the presence of osteogenic differentiation factors in the medium. After long-term culturing (4 weeks), the presence of calcium deposits detected by alizarin red staining confirmed the osteoinductive properties of intact and protein-modified polylactide scaffolds. These findings suggest that polylactide scaffolds and collagen I increase the osteogenic differentiation potential of mesenchymal stromal cells.

[The use of cluster analysis and logistic regression for assessing the risk of Alzheimer's disease in patients with mild cognitive impairment, amnestic type]. / Ispol'zovanie klasternogo analiza i logisticheskoi regressii dlia otsenki riska bolezni Al'tsgeimera u patsientov s sindromom miagkogo kognitivnogo snizheniia amnesticheskogo tipa.

AIM: The evaluation of the risk of Alzheimer disease (AD) in patients with cognitive impairment, amnestic type (aMCI) on the basis of cluster analysis and logistic regression with the use of such markers of inflammation as enzymatic activity of leukocyte elastase (LE) and the functional activity of α1-proteinase inhibitor (α1-PI). MATERIAL AND METHODS: The study object of statistical analysis was the database, including the results of LE activity and functional α1-PI activity in blood plasma of 78 outpatients with aMCI (25 men and 53 women, aged 44 to 89 years (69.1±9.95). RESULTS AND CONCLUSION: Clustering by k-means and classification by logistic regression indicate a high probability of AD in patients with aMCI depending on the activity of LE and α1-PI in blood plasma. The total coincidence of objects included in the clusters and in the AD risk group was 94%. The high coincidence of two different methods of grouping confirms the previously stated notion of the possibility of identifying patients with the high risk of AD among patients with aMCI by the activity of LE and α1-PI in the blood.

[MICROBIOCENOSIS, IMMUNE SYSTEM AND HEREDITY].

The formation of pro-/eukaryotic systems is the general biological mechanism of formation and variability of the phenotype of plants, animals, human beings under the influence of external wednesday, i.e. formation of adaptive potency conditions to external wednesday that increases the <> prokaryotic structures in sustaining body health. Prominent role in the formation of the phenotype of micro media, immunological tolerance (immunological programming), as a basis for the formation of individual pro-/eukaryotic interactions in perinatal age, the dominant role of maternal influence in this process on the one hand, micro-variability due to external stress impact on the other, makes it possible to consider pro-/eukaryotic interaction as a possible mechanism of perinatal programming and epigenetics inheritance and therefore, as one possible approach for correction of chronic and congenital pathology This points to the need to improve monitoring of the formation microbiocenosis of children, improve the methods of assessment and correction.

Complete Genome Sequence of Alkaliphilus metalliredigens Strain QYMF, an Alkaliphilic and Metal-Reducing Bacterium Isolated from Borax-Contaminated Leachate Ponds.

Alkaliphilus metalliredigens strain QYMF is an anaerobic, alkaliphilic, and metal-reducing bacterium associated with phylum Firmicutes QYMF was isolated from alkaline borax leachate ponds. The genome sequence will help elucidate the role of metal-reducing microorganisms under alkaline environments, a capability that is not commonly observed in metal respiring-microorganisms.

[NOVEL APPROACH TO COMPOSITION OF, BACTERIOPHAGE MIXTURES FOR ANTIBACTERIAL THERAPY].

AIM: Evaluate antibacterial activity of an experimental mixture of phages, belonging to several well-studied species. MATERIALS AND METHODS: The study was carried out using a group of 55 clinical Pseudomonas aeruginosa strains of various origins,- 4 mono-species mixtures of 32 virulent bacteriophages (species phiKZ-, phiKMV-, phiPBl-, PaP3-like phages) and 2 novel phages, phiMK (species PaK-P2) and phiPerm5. Activity of preparations from mono-species mixtures of bacteriophages ofvarious species were compared with activity of 3 commercial mixtures. Standard methods of study of bacteriophages were used: determination of lytic activity by seeding onto bacterial lawns of P. aeruginosa, restriction analysis of phage DNA for confirmation of their be- longing to certain species. RESULTS: Cumulative activity of 6 mono-species mixtures of virulent phages was shown to be similar to lytic activity of commercial therapeutic mixtures used against P. aeruginosa infections. 54 of 55 strains of clinical isolates of P: aeruginosa showed sensitivity to experimental mixtures composed of mono-species mixtures of bacteriophages. 53 strains were lysed by commercial preparations. Wherein the possibility of accidental inclusion of moderate -bacteriophages in the experimental mixture is excluded. CONCLUSION: A possibility of creation of highly active therapeutic antibacterial preparations against P. aeruginosa using mono-species mixtures of 6 species of lytic bacteriophages is shown Use of such a mixture in therapy of lung infections reduces the risk of emergence of bacterial strains with increased virulence and patho- genicity during prolonged administration.

Specificity of morphologic changes in target organs, associated with exposure to coal rock dust and fluorine compounds.

The studies revealed specificity of morphologic changes in target organs, depending on acting occupational hazard. Evidences are that inhalation of coal rock dust causes irreversible sclerotic and degenerative changes mostly in lungs and bronchi even on 6th week of the experiment. In liver, changes in parenchyma and stroma are controlled by reparative processes by 9th week. Accumulation of sodium fluoride in the body causes irreversible necrotic changes mostly in liver, on 6th week of the intoxication. With that, morphologic changes in lungs and bronchi are minor, characterized by immune inflammation with degenerative changes only after the 9th week. Irrespective of the acting hazard, vascular changes are characterized by media and intima hypertrophy with endothelial dystrophy and hyalinosis since the 6th week of the experiment.

Experimental studies of intracellular liver protective mechanisms in development of chronic fluorine intoxication.

The authors studied intracellular liver protective mechanisms in development of chronic fluorine intoxication. Findings are that synthesis of protective proteins HIF-1α, HOx-1, HOx-2 and HSP72, restricting free radical oxidation in hepatocytes increased in liver at early stages (1-3 weeks) of exposure to fluorine. At late terms of chronic fluorine intoxication (6-12 weeks), damaging effects of fluorine result from its genotixicity - ability to suppress synthesis of intracellular protective proteins and enzymes of main metabolic cycles in liver.

Experimental Study of the Mechanisms of Intracellular Defense in Cardiomyocytes Associated with Stages of Anthracosilicosis Development.

Mechanisms of intracellular defense of rat cardiomyocytes were studied in dynamics of anthracosilicosis development induced by long-term inhalation of coal and rock dust. It was shown that synthesis of transcription factor HIF-1α and protective proteins increased in the heart at the early stages of coal and rock dust inhalation (1-3 weeks), and these changes limited the development of free radical oxidation and activated metabolism of glucose and fatty acids. Exposure to coal and rock dust for 6-12 weeks activated free radical oxidation and decreased basal metabolism in cardiomyocytes.

[PROTEOMIC ANALYSIS OF ADAPTIVE MECHANISMS TO SALINITY STRESS IN MARINE GASTROPODS LITTORINA SAXATILIS].

Salinity is one of the most important abiotic environmental factors affecting marine animals. If salinity deviate from optimum, adaptive mechanisms switch on to maintain organism's physiological activity. In this study, the reaction of the snails Littorina saxatilis from natural habitats and in response to experimental salinity decreasing was analyzed on proteomic level. The isolation of all snails inside their shells and gradually declining mortality was observed under acute experimental salinity decrease (down to 10 per hundred). Proteomic changes were evaluated in the surviving experimental mollusks compared to control individual using differential 2D gel-electrophoresis (DIGE) and subsequent LC-MS/MS-identification of proteins. Approximately 10% of analyzed proteins underwent up- or down regulation during the experiment. Proteins of folding, antioxidant response, intercellular matrix, cell adhesion, cell signaling and metabolic enzymes were identified among them. Proteome changes observed in experimental hypoosmotic stress partially reproduced in the proteomes of mollusks that live in conditions of natural freshening (estuaries). Possible mechanisms involved in the adaptation process of L. saxatilis individuals to hypo-osmotic stress are discussed.