[Transthyretin amyloid cardiomyopathy. Features of histological diagnosis: study design].

AIM: To compare efficiency and specific features of transthyretin amyloid staining by different histological dyes and thus to assess their suitability for diagnostic purposes. MATERIALS AND METHODS: Samples of left and right heart ventricles were taken from patients over 70 years-old of both genders (n=10) with immunohistochemically verified transthyretin amyloidosis (ATTR). All samples were stained with Congo red, Alcian blue, toluidine blue and methylene violet. RESULTS: Specificity and sensitivity of Congo red staining was comparable to those of immunohistochemical staining. For verification of amyloid presence after Congo red staining one could use fluorescent microscopy instead of polarization microscopy. It allows a more accurate diagnosis of amyloidosis. Confocal microscopy with spectral unmixing improves detection sensitivity of amyloid by elimination of background fluorescence of muscle tissue and autofluorescence of lipofuscin. Alcian blue staining gives the same result as Congo red. In addition, its less labor-intensive and free of false-positive and false-negative results caused by final processing of slide preparation. Toluidine blue and methylene violet develop metachromatic staining upon binding to transthyretin fibrils, likely due to specific biochemical features of these fibrils. CONCLUSION: The most reliable method for histochemical diagnosis of ATTR is the Congo red staining with subsequent analysis using fluorescence or confocal microscopy. For diagnostic screening, the use of Sodium sulphate-Alcian blue staining method is highly promising. Metachromatic stains are less effective for ATTR diagnosis.

[Cardiac magnetic resonance imaging with contrast enhancement in treatment of atrial fibrillation].

Catheter ablation is presently the main method for interventional treatment of atrial fibrillation (AF). Despite improvements of the method and accumulation of personnel's experience, incidence of recurrent AF following catheter interventions remains high. This review addresses a possibility of using contrast-enhanced cardiac magnetic resonance imaging to increase the effectiveness of interventional treatment of arrhythmia.

A Novel Method for Amyloid Detection in Human Tissue Load Using a Fluorescent Dye - Congo Red Analogue.

The aim of the study was to develop a new technology for the detection of amyloid in human tissues based on the fluorescent dye, disodium salt of 2,7-(1-amino-4-sulfo-2-naphthylazo)fluorene (DSNAF). MATERIALS AND METHODS: Synthesis of DSNAF was performed by diazotization of 2,7-diaminofluorene in a stream of argon followed by azo coupling with naphthionic acid. Identification of DSNAF was performed using MALDI mass spectrometry. Human myocardial samples from males and females aged from 85 to 98 years (n=11) were the material for the histochemical study. Myocardial paraffin sections were stained with a 0.1% aqueous solution of Congo red or with an aqueous solution (0.1 or 0.034%) of DSNAF under the same conditions. RESULTS: It has been demonstrated for the first time that a new fluorene-based analogue of Congo red, DSNAF, can be successfully used to identify amyloid deposits in histological sections of human myocardium. In terms of the specificity and intensity of amyloid staining, DSNAF is comparable to Congo red, which is the gold standard for detecting amyloid deposits. The fluorescence intensity of DSNAF when binding to amyloid fibrils is significantly higher than the intensity of Congo red fluorescence (with a lower intensity of background fluorescence of heart muscle tissue). This is especially useful for identifying small deposits of amyloid in the human tissues which is important when using small biopsies. CONCLUSION: The advantages of using DSNAF allow us to consider the developed technology for the detection of amyloid as a new promising method of identifying amyloid deposits in human tissues.

Modeling of 3D Structure of Chimeric Constructs Based on Hemagglutinin of Influenza Virus and Immunogenic Epitopes of Streptococcus Agalactiae.

A project of an experimental recombinant vector vaccine for prevention of diseases caused by pathogenic streptococci based on ScaAB lipoprotein of Streptococcus agalactiae and a coldadapted strain of live influenza vaccine as a vector was developed. The sequence of ScaAB lipoprotein was analyzed and fragments forming immunodominant epitopes were determined. Chimeric molecules of influenza virus hemagglutinin H7 carrying insertions of bacterial origin were constructed. Based on the results of simulation, the most promising variants were selected; they represented fragments of lipoprotein ScaAB lacking N-terminal domain bound to hemagglutinin via a flexible linker. These insertions should minimally modulate the properties of the influenza strain, while retaining potential immunogenicity to a wide group of pathogenic streptococci.

[Mast cells of the human pineal gland.]

The purpose of this study was to assess the possibilities of identifying mast cells using different histochemical and immunohistochemical methods and elucidating the features of their localization in the human pineal gland. The undertaken study showed that mast cells are an essential component of the human pineal gland, regardless of age. The data obtained indicate an increase in the number of mast cells in the pineal gland with age. Mast cells are mostly located in the pineal stroma and their preferred location has not been related to concrements, cysts or melanin accumulations. Mast cells in the pineal gland are predominantly non-degranulating, which indicates their inactive state. The detectability of mast cells in the pineal gland depended significantly on the applied method of staining of the preparations. The largest number of mast cells was revealed by tryptase immunohistochemistry, which should be used to accurately determine the population of mast cells of the pineal gland.

[The new 2017 European society of cardiology (ESC) guidelines: important changes for introduction into clinical practice]. / Novye rekomendatsii Evropeiskogo obshchestva kardiologov (ESC) 2017 g.: vazhnye izmeneniia dlia vnedreniia v klinicheskuiu praktiku.

The paper gives an overview of the European Society of Cardiology (ESC) guidelines updated in 2017. The revised and amended guidelines for areas, such as dual antiplatelet therapy (DAT), treatment of patients with ST-segment elevation myocardial infarction (STEMI), and management of patients with valvular heart disease and peripheral artery disease, were presented in late summer of this year. The authors of this paper present an independent analysis and discussion of new data on the key issues of diagnosis and treatment in patients in the above areas. The recommendations on DAT pay special attention to the timing of the therapy and to the choice of its drugs. The updated data on the treatment of patients with STEMI accurately determine the time to percutaneous coronary interventions, approaches to revascularization; the updates touch upon fibrinolytic therapy and new approaches to lipid-lowering therapy too. Recommendations for the management of patients with peripheral artery atherosclerosis propose for the first time a section devoted to the choice of antiplatelet therapy (an antiaggregant and/or an anticoagulant) depending on the clinical situation.

Analysis of Immune Epitopes of Respiratory Syncytial Virus for Designing of Vectored Vaccines Based on Influenza Virus Platform.

The immunoepitope database was used for analysis of experimentally detected epitopes of the respiratory syncytial virus (RSV) proteins and for selection of the epitope combinations for subsequent designing of recombinant vectored anti-RSV vaccines based on attenuated influenza viruses. Three cassettes containing the most promising B- and T-cell RSV epitopes were selected: peptide F (243-294) supporting the formation of humoral immunity in animals; fragment M2-1 (70-101+114-146) containing two MHC I epitopes (82-90 and 127-135); and MHC II-epitope (51-66). The selected constructions contained no neoepitopes causing undesirable effects of vaccination, such as immunotolerance or autoimmunity.

[MORPHOLOGICAL BASES OF REORGANIZATION OF THE RAT CEREBELLAR CORTEX IN SENESCENCE].

A histological and immunohistochemical study of the cerebellar cortex of senescent Wistar rats (36-month-old) was performed in comparison to young rats (36-month-old).The cerebellar cortex of senescent animals typically showed degeneration of the Purkinje cells accompanied by immunochemically determined loss of the calcium-binding protein calbindin. These results suggest that presence of calbindin in the Purkinje cells is a criterion of functional activity of these neurons. The Purkinje cells degeneration is accompanied by lesion of the synaptophysin-containing basket cell networks, which is an indication of impaired function of inhibitory (GABAergic) axo-axonal synapses. During senescence significant rear-rangement is observed in the glomeruli of the granular layer of the cerebellum, which are the structures where primary afferent animals disintegrate suggesting of alteration in the transmission of information from the cerebrum to the cerebellum.

[Method for simultaneous visualization of mast cells and nerve terminals in the rodent thymus].

The purpose of this study was to develop the method for the simultaneous visualization of mast cells (MCs) and nerve terminals, based on generally accepted techniques of histochemical identification of MCs with alcian blue and immunohistochemical detection of synaptophysin. The protocol presented allows simultaneous identification of mast cells and nerve terminals in the sections of paraffin-embedded thymus of laboratory mammals with high selectivity and good reproducibility. The method can be used for both visualization of spatial relationship between MCs and nerve terminals and independent research of the innervation of mammalian internal organs. Zinc-ethanol-formaldehyde is recommended as an optimal fixative.

[Peculiarity of reassortment of current wild type influenza viruses with master donor viruses for live influenza vaccine].

The live attenuated influenza vaccine (LAIV) currently licensed in Russia consists of the reassortant viruses with hemagglutinin (HA) and neuraminidase (NA) gene segments from the circulating wild-type viruses and the six internal protein-encoding gene segments from cold-adapted master donor viruses (MDV) A/Leningrad/134/17/57 (H2N2) or B/USSR/60/69. Presently, only classical reassortment technique is approved for the generation of Russian LAIV strains. In this work, we describe the obstacles to the development of LAIV 6:2 vaccine strains depending on the phenotypic properties of the wild-type viruses used for reassortment. It was demonstrated that the highest percentage of 6:2 vaccine reassortants could be achieved when wild-type parental viruses were resistant to non-specific gamma-inhibitors. It was shown that it was impossible to generate 6:2 vaccine reassortants possessing six internal genes of the AILeningrad113417/57 (H2N2) master donor virus and avian HA and NA genes from H5N1-PR8 viruses using classical reassortment technique. It was suggested that strong constellation effects between the gene segments of the parental viruses could affect the virus gene reassortment. A strong interaction between the genome segments encoding neuraminidase of avian origin and PB2 gene of PR8 virus was observed. When the PB2 gene was inherited from cold-adapted master donor virus, the neuraminidase was also found to be of MDV origin.

Isolation and properties of recombinant inulinases from Aspergillus sp.

The genes inuA and inu1, encoding two inulinases (32nd glycosyl hydrolase family) from filamentous fungi Aspergillus niger and A. awamori, were cloned into Penicillium canescens recombinant strain. Using chromatographic techniques, endoinulinase InuA (56 kDa, pI 3) and exoinulinase Inu1 (60 kDa, pI 4.3) were purified to homogeneity from the enzymatic complexes of P. canescens new transformants. The properties, such as substrate specificity, pH- and T-optima of activity, stability at different temperatures, influence of cations and anions on the catalytic activity, etc., of both recombinant inulinases were studied.

Isolation and properties of xyloglucanases of Penicillium sp.

Using chromatographic technique, xyloglucanase (XG) A (25 kDa, pI 3.5, 12th glycosyl hydrolase family) was isolated from the enzyme complex secreted by the mycelial fungus Penicillium canescens, and xyloglucanases XG 25 (25 kDa, pI 4.1, 12th glycosyl hydrolase family) and XG 70 (70 kDa, pI 3.5, 74th glycosyl hydrolase family) were isolated from the enzyme complex of Penicillium verruculosum. Properties of the isolated enzymes (substrate specificity, optimal ranges of pH and temperature for enzyme activity and stability, effect of metal ions on catalytic activity) were compared with the properties of xyloglucanases XG 32 of Aspergillus japonicus, XG 78 of Chrysosporium lucknowense, and XG of Trichoderma reesei. The gene xegA encoding XG A of P. canescens was isolated, and the amino acid sequence of the corresponding protein was determined.

[Analysis of morphological manifestations of the inflammatory reaction in wistar rat spinal cord in experimental model].

Morphological changes in the spinal cord of rats with different intensity of pathological symptoms were studied at the peak of the experimental encephalomyelitis development. Light-microscopical and immunohistochemical methods were used. Distribution of proliferating cell nuclear antigen (PCNA), astrocyte marker - glial fibrillar acidic protein (GFAP), and microglia and macrophage marker Iba-1, was studied. Heterogeneity in morphological manifestations of the experimental allergic encephalomyelitis was shown. Four typical patterns of morphological manifestations of the disease were demonstrated depending on the preferential involvement of pia mater, vessels, spinal cell nuclei or conductive tracts in the pathological process.

Regulatory activity of heterologous gene-activator xlnR of Aspergillus niger in Penicillium canescens.

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, beta-xylosidase, alpha-L-arabinofuranosidase, alpha-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while beta-galactosidase, beta-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-beta-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous beta-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.

Morphofunctional characteristics of the lumbar enlargement of the spinal cord in rats.

The topography of the lumbar enlargement of the spinal cord in rats was studied; an immunohistochemical method was used to determine the distribution of synaptophysin--a membrane protein of synaptic vesicles. Synaptophysin-immunoreactive structures were detected in the gray matter of all Rexed laminae, around most neurons and in the neuropil. Previously undescribed subpial synaptic contacts were detected immunohistochemically in the white matter and confirmed by electron microscopy. A non-myelinated component of the corticospinal tract, including axonal varicosities and synaptic contacts, was observed in the dorsal part of the white matter of the lumbar enlargement of the spinal cord.

Isolation and characterization of extracellular alpha-galactosidases from Penicillium canescens.

Two alpha-galactosidases were purified to homogeneity from the enzymatic complex of the mycelial fungus Penicillium canescens using chromatography on different sorbents. Substrate specificity, pH- and temperature optima of activity, stability under different pH and temperature conditions, and the influence of effectors on the catalytic properties of both enzymes were investigated. Genes aglA and aglC encoding alpha-galactosidases from P. canescens were isolated, and amino acid sequences of the proteins were predicted. In vitro feed testing (with soybean meal and soybean byproducts enriched with galactooligosaccharides as substrates) demonstrated that both alpha-galactosidases from P. canescens could be successfully used as feed additives. alpha-Galactosidase A belonging to the 27th glycosyl hydrolase family hydrolyzed galactopolysaccharides (galactomannans) and alpha-galactosidase C belonging to the 36th glycosyl hydrolase family hydrolyzed galactooligosaccharides (stachyose, raffinose, etc.) of soybean with good efficiency, thus improving the digestibility of fodder.

[Morphofunctional characteristic of the rat distal spinal cord after its complete experimental section with subsequent treadbun training of the animal].

Motor activity of rats has been studied after complete experimental section of spinal cord at the lower thoracic level. A treadbun training performed one day after the operation has been shown to lead to the appearance of movement of hindlimbs and to restoration of function of support of the body weight. In our opinion, the key moment in initiation of locomotor movements is stimulation of foot. Morphoimmunohistochemical study (detection of nuclear protein of proliferation cells, synaptophysin, and glial fibrillary acid protein) of the lumbar enlargement has allowed revealing reorganization of motoneurons, interneurons, and afferent chain in the distal part of the sectioned spinal cord. In trained animals there are observed the normal structure of motoneurons and the appearance of aggregates of synaptophysin-immunoreactive structures lost after the operation.

Isolation and characterization of extracellular pectin lyase from Penicillium canescens.

Pectin lyase A (molecular weight 38 kD by SDS-PAGE, pI 6.7) was purified to homogeneity from culture broth of the mycelial fungus Penicillium canescens using chromatographic techniques. During genomic library screening, the gene encoding pectin lyase A from P. canescens (pelA) was isolated and sequenced, and the amino acid sequence was generated by applying the multiple alignment procedure (360 residues). A theoretical model for the three dimensional structure of the protein molecule was also proposed. Different properties of pectin lyase A were investigated: substrate specificity, pH- and temperature optimum of activity, stability under different pH and temperature conditions, and the effect of Ca2+ on enzyme activity. In the course of the laboratory trials, it was demonstrated that pectin lyase A from P. canescens could be successfully applied to production and clarification of juice.

[Morpho-functional characterictic of rat lumbar spinal cord enlargement].

Topography and the distribution of synaptophysin-immunoreactive structures were studied in the rat lumbar spinal cord enlargement. Synaptophysin (synaptic vesicle marker) was found in the gray matter of all Rexed laminae around most neurons and in neuropil. Subpial synaptic contacts, that were not described previously, were found in the white matter by immunohistochemistry and their presence was confirmed by electron microscopy. They were localized directly beneath the astrocyte processes, that cover the spinal cord externally. In the dorsal portion of the white matter within the lumbar spinal cord enlargement, the unmyelinated synaptophysin-immunoreactive component of pyramidal tract was detected which included axonal varicosities and synaptic contacts.

[Use of a preparation from fungal pectin lyase in the food industry].

A new enzyme preparation of fungal pectin lyase (EC 4.2.2.10) was shown to be useful for the production of cranberry juice and clarification of apple juice in the food industry. A comparative study showed that the preparation of pectin lyase is competitive with commercial pectinase products. The molecular weight of homogeneous pectin lyase was 38 kDa. Properties of the homogeneous enzyme were studied. This enzyme was most efficient in removing highly esterified pectin.