[An effect of antipsychotic and anticholinergic treatment on cognitive function in patients with schizophrenia]. / Vliyanie antipsikhoticheskoi i antikholinergicheskoi terapii na kognitivnye funktsii u bol'nykh shizofreniei.

OBJECTIVE: To reveal the relationships between antipsychotic and anticholinergic drugs and cognitive functions in patients with schizophrenia. MATERIAL AND METHODS: The observational prospective study was conducted at the Bekhterev National Medical Center of Psychiatry and Neurology. The study involved 41 patients (22 men and 19 women) with paranoid schizophrenia, according to ICD 10 criteria, aged 30.12±8.24 years on stable antipsychotic monotherapy or in combination with anticholinergic drug (trihexiphenidyl). Cognitive functions were assessed using the «Brief Assessment of Cognitive Function in Patients with Schizophrenia¼ (BACS) scale, severity of mental state and extrapyramidal disturbances were measured using the «Positive and Negative Syndrome Scale (PANSS) and the Simpson-Angus Scale for Assessment of Extrapyramidal Side Effects (SAS). All examination procedures were performed twice at weeks 2 and 8 of therapy. Patients were divided into 2 groups according to the type of antipsychotic therapy. Twelve patients received first generation antipsychotics (FGAs) (group 1), 29 patients received second generation antipsychotics (SGAs) (group 2). RESULTS: Patients receiving SGAs had a significant decrease in the overall SAS score at week 8 of therapy compared with data at week 2, and there was an improvement in cognitive function, unlike patients receiving FGAs. There were also changes on BACS tests the digit sequencing (V=51.5, p=0.007), token motor task (V=75.5, p=0.007) and Tower of London (V=52, p=0.027) only in patients of group 2. CONCLUSION: The improved tolerance to the drug, as well as cognitive measures, was shown in patients taking SGAs by week 8. Our study confirms the importance of adhering to the minimum effective dose of antipsychotic drugs for the treatment of schizophrenia to prevent cognitive impairment, and to give preference to SGAs in the choice of treatment.

[Gyrate atrophy of the choroid and retina with ornithinemia and foveoschisis (clinical observation)]. / Atrofiya girate khorioidei i setchatki s ornitinemiei i foveoshizisom (klinicheskoe nablyudenie).

Gyrate chorioretinal atrophy (GCA) is a rare hereditary disease with certain complications; one extremely rare complication of GCA is foveoschisis. For the first time in Russian ophthalmology, a 10-year-old female child has been described to have genetically verified GCA associated with the OAT gene in combination with ornithinemia and foveoschisis. The diagnosis was made on the basis of fundus examination, perimetry data, autofluorescence, optical coherence tomography, fluorescence angiography, electroretinography, mass spectrometry with confirmation by molecular genetic research. The presented clinical case illustrates the need for an interdisciplinary approach to the diagnosis of GCA with diagnostic algorithm involving various examination methods and doctors of different specialties.

[Inherited retinal dystrophy: first results of RPE65 gene replacement therapy in Russia]. / Nasledstvennaya distrofiya setchatki: pervye rezul'taty posle RPE65-genozamestitel'noi terapii v Rossii.

PURPOSE: To present the main aspects of interdisciplinary diagnostics of patients with hereditary retinal diseases and the first results of the follow-up of patients with inherited retinal dystrophies (IRD) caused by biallelic mutations in the gene RPE65 after gene replacement therapy in Russia. MATERIAL AND METHODS: The cohort of patients consisted of six children (5-15 years old) with the diagnosis of Leber amaurosis type 2. All patients underwent a multi-disciplinary examination using conventional clinical, instrumental and molecular-genetic methods. Genetic diagnosis was established based on the results of two-stage DNA diagnostics using high-performance parallel sequencing of a custom panel and family segregation analysis by Sanger sequencing. RESULTS: In the Research Centre for Medical Genetics the first group of Russian patients with an orphan inherited retinal disease was verified, they underwent subretinal injection of the gene replacement drug Voretigene neparvovec (12 eyes) in the Helmholtz National Medical Research Center of Eye Diseases. According to the regulated terms of monitoring gene therapy patients, they were examined in the Research Centre for Medical Genetics after 1, 3, 6 and 12 months, and then once per year. Thus, the available data allows us to analyze the first results 3 months after the treatment. CONCLUSION: The presented data on inherited retinal dystrophies caused by biallelic mutations in the RPE65 gene emphasize the need to change the diagnostic algorithm in the ophthalmic practice. The use of clinical instrumental and molecular genetic diagnostic methods makes it possible to apply etiotropic treatment to patients with a disabling disease that was previously considered untreatable. The gene replacement drug Voretigene neparvovec registered in Russia showed irrefutable first positive results in all targeted patients.

Diagnostic value of postsystolic shortening of the left ventricular myocardium assessed during speckle tracking stress echocardiography on the treadmill in patients with coronary artery disease.

Aim      To evaluate the diagnostic capacity of left ventricular (LV) postsystolic shortening (PSS) values obtained by speckle-tracking stress-echocardiography (stress-EchoCG) using a treadmill test in determining the functional significance of the degree of coronary artery (CA) stenosis.Material and methods  The study included 132 patients (80 men aged 65.0±9.3 years) with suspected or previously verified diagnosis of ischemic heart disease. Stress-EchoCG with the treadmill test was performed for all patients. Strain parameters were determined by two-dimensional speckle-tracking on gray-scale images before and after the exercise. Values of LV postsystolic index (PSI) and LV mean postsystolic time (PST) were calculated. Coronary angiography was performed for all patients. Patients were divided into 3 groups based on the severity of CA stenosis according to the G. G. Gensini score.Results LV PSS values at rest did not significantly differ between the patient groups. After completion of the exercise, the mean LV PSI was significantly higher for patients with pronounced CA stenosis than in the group without CA stenosis or with moderate CA stenosis: 8.9 %  [3.8; 10.7 %] vs. 3.8 % [2.2; 6.8 %] (p=0.012) and 3.4 %  [2.2; 6.2 %] (p=0.012), respectively. The mean LV PSI after completion of the exercise indicated the presence of pronounced CA stenosis with a sensitivity of 75 % and a specificity of 61 % (area under the curve, AUC, 0.74±0.06; р<0.001). After completion of the exercise, the mean LV PST was significantly greater for patients with pronounced CA stenosis than in the group without CA stenosis or with moderate CA stenosis: 27.4 [18.7; 34.7] ms vs. 18.4 [10.8; 26.5] ms (p=0.036) and 20.9 [14.2; 29.5] ms (p=0.036), respectively. The mean LV PST after completion of the exercise exceeding 23.5 ms suggests pronounced CA stenosis with a sensitivity of 71 % and a specificity of 65 % (AUC 0.69±0.06; p=0.004). A complex evaluation of the LV PSI, the LV local contractility disorder (LCD) index, the LV PST, and LV LCD index allows enhancement of the test sensitivity in diagnozing pronounced CA stenosis.Conclusion      Determination of LV PSS in speckle-tracking stress-EchoCG may be useful for evaluating the functional significance of the degree of CA stenosis to enhance the sensitivity of stress-EchoCG in patients with pronounced CA stenosis.

Speckle Tracking Stress Echocardiography on Treadmill in Assessment of the Functional Significance of the Degree of Coronary Artery Disease.

Aim        To determine diagnostic capabilities of left ventricular (LV) global longitudinal systolic strain (GLSS) in stress echocardiography (stress-EchoCG) with a treadmill test for diagnosing the functional significance of the degree of coronary stenosis.Material and methods        The study included 121 patients (73 men aged 68.3±7.7 years) with suspected or previously diagnosed ischemic heart disease (IHD). Speckle-tracking stress-EchCG (method of tracking speckles on two-dimensional gray-scale ultrasonic images) with a treadmill test and coronarography was performed for all patients. The patients were divided into 3 groups based on the severity of coronary artery (CA) stenosis according to the Gensini scale.Results   LV GLSS at rest did not significantly differ between the study groups. After the exercise, LV GLSS was significantly lower in patients with pronounced CA stenosis than in patients without or with moderate CA stenosis (15.9±4.6 % vs. 20.6±3.7 % (p<0.001) and 19.6±3.0 % (p=0.003), respectively). Postexercise LV GLSS <16.9% suggested a pronounced CA stenosis with a sensitivity of 80% and a specificity of 70% (area under the curve, AUC, 0.76±0.06 at 95 % confidence interval, CI, 0.63-0.89; р<0.001). In the patient group without CA stenosis, LV GLSS showed a significant increase after completion of the exercise (from 19.1±3.1 to 20.6±3.7; p=0.04).Conclusion            Evaluation of LV GLSS and its dynamics in stress-EchoCG with a treadmill test may be promising in patients with IHD, since in most patients with pronounced CA stenosis, LV GLSS is reduced at baseline and further reduces in response to exercise. In patients without CA stenosis, LV GLSS increases after completing the exercise.

[Echocardiography in the Assessment of Postsystolic Shortening of the Left Ventricle Myocardium of the Heart].

Echocardiography allows evaluating left ventricular (LV) myocardial contractility; however, the visual assessment of contractility is subjective and requires considerable experience. Modern technologies for assessment of LV myocardial contractility, such as tissue Doppler and speckle-tracking echocardiography, provide quantitative estimation of various parameters of myocardial strain, including the LV postsystolic shortening. Several studies have demonstrated the value of postsystolic shortening for evaluation of myocardial ischemia and "ischemic memory" in patients with cardiovascular diseases. This review analyzes experimental and clinical studies that addressed LV postsystolic shortening.

Mutational Spectrum of Spast (Spg4) and Atl1 (Spg3a) Genes In Russian Patients With Hereditary Spastic Paraplegia.

Hereditary spastic paraplegia (HSP) comprises a heterogeneous group of neurodegenerative disorders, it share common symptom - of progressive lower spastic paraparesis. The most common autosomal dominant (AD) forms of HSP are SPG4 (SPAST gene) and SPG3 (ATL1 gene). In the current research we investigated for the first time the distribution of pathogenic mutations in SPAST and ATL1 genes within a large cohort of Russian HSP patients (122 probands; 69 famillial cases). We determined the frequencies of genetic abnormalities using Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and Next Generation Sequencing (NGS) of targeted gene panels. As a result, SPG4 was diagnosed in 30.3% (37/122) of HSP cases, where the familial cases represented 37.7% (26/69) of SPG4. In total 31 pathogenic and likely pathogenic variants were detected in SPAST, with 14 new mutations. Among all detected SPAST variants, 29% were gross deletions and duplications. The proportion of SPG3 variants in Russian cohort was 8.2% (10/122) that were all familial cases. All 10 detected ATL1 mutations were missense substitutions, most of which were in the mutational hot spots of 4, 7, 8, 12 exons, with 2 novel mutations. This work will be helpful for the populational genetics of HSP understanding.

[TITLE].

The purpose of study is to establish features of autoimmune reaction of children with Crohn's disease. The sampling included 62 patients aged from 2 to 17 years with diagnosis of Crohn's disease. The evaluation was carried out concerning concentration in blood serum of immunoglobulins IgA, IgM, IgG, IgЕ, antibodies to Saccharomyces cerevisiae (ASCA) classes IgA, IgG и IgЕ, antibodies to Candida albicans classes IgA, IgM, IgG и IgЕ, anti-neutrophilic cytoplasmic antibodies (ANCA) to myeloperoxidase (MPO), to proteinase 3 (PR3), anti-nuclear antibodies (ANA), antibodies to DNAds, DNAss (to double-helical and single-stranded DNA), antibodies to antigens of small and large intestines, pancreas, circulating immune complexes. The hyperimmunoglobulinemia was diagnosed in 47 (75.8%) out of 62 patients with Crohn's disease. The increased level of IgM in blood was detected in 29 patients (46.8%). The hyperimmunoglobulinemia У was established in 19 (30.6%) out of 62 children. The hypoimmunoglobulinemia was detected in 22 (35.5%) of patients and in 17 (77.3%) out of them the disimmunoglobulinemia type IV (isolated decreasing of concentration of IgA). The evaluation of rate of occurrence of specifc antibodies in blood serum demonstrated that in patients most frequently was detected presence of specifc IgE to Saccharomyces cerevisiae (70.9%). The increased level of ASCA (IgA, IgG) was detected in 22 (35.5%) patients. The concentration of antibodies to DNAds, DNAss in blood exceeded standard value in 4.8% and 16.1% patients correspondingly. The increased level of circulating immune complex was established in 20 (32.3%) patients. The concentration of ANA corresponded to standard values in all 62 (100%) patients. The evaluation of results of correlation analysis established a strong positive correlation of concentration in blood of antibodies to antigens of small and large intestines; average positive correlation of level of antibodies to antigens of small intestine and IgM, ANCA PR3, ASCA IgE, antibodies to Candida albicans classes IgM, IgG, IgE, antibodies to antigens of pancreas; average degree of positive correlation between concentration of antibodies to antigens of large intestine and IgA, IgM, circulating immune complex, ANCA PR3, DNAss, ASCA IgE, antibodies to antigens of pancreas; strong positive correlation between concentrations of IgA to Candida albicans and ANA. The detection of auto-to antibodies Saccharomyces cerevisiae, Candida albicans, ANCA, antigens of small and large intestines, pancreas and expressed degree of correlation of many indices of autoimmune reaction indicate to intensity of immune pathological process under Crohn's disease. Under Crohn's disease, the formation of antibodies to ASCA is a prognostically unfavorable sign. The immune diagnostic under Crohn's disease is necessary for evaluating severity of course of disease, differential diagnostic, establishment of prognosis and selection of individual immune correcting therapy.

Methylated flavones of the hairy root culture Scutellaria baicalensis.

Perennial plants in northern Dauria (Zabaikalsky region) grow in low temperatures in winter and in a dry hot summer. The prairies of northern Dauria are rich in a variety of medicinal herbs, including S. baicalensis, which has roots that are in demand for traditional Chinese medicine. In addition to two monomethylated flavones (wogonin and oroxylin A), determining the pharmacological significance of the root, there is also a minority of their polymethylated congeners. Little is known about their role in the plant or their connection with the conditions of growth and cultivation of their hairy root culture (HRC). Therefore, the purpose of this study was to determine whether and to what extent the biosynthesis of the latter is retained in the hairy root culture established from wild plants of Dauria. The composition of the main methylated flavones of HRC was established using LC-MS and a previously unknown pentamethylated flavone was found in the roots. This study showed a more significant accumulation of polymethylated flavones in the root of the wild plant than in HRC.

[Peculiarities of the Mechanism of Interactions of Catalytic Antibodies with Organophosphorus Substrates].

Catalytic antibodies are a promising model for creating highly specific biocatalysts with predetermined activity. However, in order to realize the directed change or improve their properties, it is necessary to understand the basics of catalysis and the specificity of interactions with substrates. In the present work, a structural and functional study of the Fab fragment of antibody A5 and a comparative analysis of its properties with antibody A17 have been carried out. These antibodies were previously selected for their ability to interact with organophosphorus compounds via covalent catalysis. It has been established that antibody A5 has exceptional specificity for phosphonate X with bimolecular reaction rate constants of 510 ± 20 and 390 ± 20 min^(-1)М^(-1) for kappa and lambda variants, respectively. 3D-Modeling of antibody A5 structure made it possible to establish that the reaction residue L-Y33 is located on the surface of the active site, in contrast to the A17 antibody, in which the reaction residue L-Y37 is located at the bottom of a deep hydrophobic pocket. To investigate a detailed mechanism of the reaction, A5 antibody mutants with replacements L-R51W and H-F100W were created, which made it possible to perform stopped-flow kinetics. Tryptophan mutants were obtained as Fab fragments in the expression system of the methylotrophic yeast species Pichia pastoris. It has been established that the effectiveness of their interaction with phosphonate X is comparable to the wild-type antibody. Using the data of the stopped-flow kinetics method, significant conformational changes were established in the phosphonate modification process. The reaction was found to proceed using the induced-fit mechanism; the kinetic parameters of the elementary stages of the process have been calculated. The results present the prospects for the further improvement of antibody-based biocatalysts.

Change in the Content of Immunoproteasomes and Macrophages in Rat Liver At the Induction of Donor-Specific Tolerance.

Induction of donor specific tolerance (DST) by the introduction of donor cells into a recipient's portal vein is one of the approaches used to solve the problem of transplant engraftment. However, the mechanism of DST development remains unclear to this moment. In the present work, we first studied the change in the content of immunoproteasomes and macrophages of the liver at early stages of the development of allospecific portal tolerance in rats by Western blotting and flow cytofluorimetry. On the basis of the data obtained, we can conclude that the induction of DST is an active process characterized by two phases during which the level of the proteasome immune subunits LMP2 and LMP7 in liver mononuclear cells, including Kupffer cells, and the number of Kupffer cells change. The first phase lasts up to 5 days after the beginning of DST induction; the second phase - from 5 to 14 days. In both phases, the level of the subunits LMP2 and LMP7 in the total pool of mononuclear cells and Kupffer cells increases, with maximum values on days 1 and 7. In addition, the total number of Kupffer cells increases in both phases with a shift in several days. The most noticeable changes take place in the second phase. The third day is characterized by a lower content of mononuclear cells expressing immunoproteasomes compared to the control value in native animals. Presumably, at this time point a "window of opportunity" appears for subsequent filling of an empty niche with cells of different subpopulations and, depending on this fact, the development of tolerance or rejection. The results obtained raise the new tasks of finding ways to influence the cellular composition in the liver and the expression of immunoproteasomes on the third day after the beginning of DST induction to block the development of rejection.

Evolution of catalytic centers of antibodies by virtual screening of broad repertoire of mutants using supercomputer.

It is proposed to perform quantum mechanical/molecular dynamics calculations of chemical reactions that are planned to be catalyzed by antibodies and then conduct a virtual screening of the library of potential antibody mutants to select an optimal biocatalyst. We tested the effectiveness of this approach by the example of hydrolysis of organophosphorus toxicant paraoxon using kinetic approaches and X-ray analysis of the antibody biocatalyst designed de novo.

A Study of the Protective Properties of an Antibody-Based Antidote Metabolizing Organophosphorus Pesticide Paraoxon.

A catalytic antibody A17 and its mutants highly efficiently interact with organophosphorus pesticide paraoxon. In this work, we studied the protective properties of antibody A17-K47 in paraoxon poisoning using a mouse model. The optimal paraoxon dose simulating the acute toxic effect of organophosphorus compounds was 550 µg/kg. The pharmacokinetic parameters of A17-K47 antibody were t1/2distr =7.2±1.4 min, t1/2el =330±20 min. The antibody did not cause toxic effects when administered at a ten-fold calculated therapeutic dose (610 mg/kg). The drug did not reduce mortality from acute paraoxon poisoning; however, the absence of drug toxicity opens up prospects for its use in symptomatic treatment of chronic paraoxon poisoning.

[Investigation of the surface layer of 3D-matrices for tissue engineering]. / Issledovanie poverkhnostnogo sloia 3D-matriksov dlia tkanevoi inzhenerii.

Electrospinning is a convenient and promising manufacturing method a variety of materials for tissue engineering. 3D matrices fabricated by electrospinning from solutions of polycaprolactone with human serum albumin or gelatin in 1,1,1,3,3,3-hexafluoroisopropanol were studied. The microstructure of the 3D matrices and surface of the fibers were investigated using scanning electron microscopy. Protein distribution in the surface layer was studied by modification of protein amino groups with N-(2-hydroxyethyl)phenazine and X-ray photoelectron spectroscopy. It was shown, that concentration of the proteins in the surface layer of fibers exceeded their concentration in the initial electrospun solution up to 12 times and the surface layer was enriched in the protein inversely to the concentration of the protein in solution. The minor part of the proteins was released from fibers during first 30-60 min after swelling in water. Treatment of matrices with proteinase K hydrolyzed about 1/3 of the surface exposed human serum albumin. Thus, both methods can be used to study the surface content of the materials produced by electrospinning from blends of synthetic and natural polymers, however X-ray photoelectron spectroscopy appears to be more convenient and informative.

Increased Eps15 homology domain 1 and RAB11FIP3 expression regulate breast cancer progression via promoting epithelial growth factor receptor recycling.

Recent research indicates that the C-terminal Eps15 homology domain 1 is associated with epithelial growth factor receptor-mediated endocytosis recycling in non-small-cell lung cancer. The aim of this study was to determine the clinical significance of Eps15 homology domain 1 gene expression in relation to phosphorylation of epithelial growth factor receptor expression in patients with breast cancer. Primary breast cancer samples from 306 patients were analyzed for Eps15 homology domain 1, RAB11FIP3, and phosphorylation of epithelial growth factor receptor expression via immunohistochemistry. The clinical significance was assessed via a multivariate Cox regression analysis, Kaplan-Meier curves, and the log-rank test. Eps15 homology domain 1 and phosphorylation of epithelial growth factor receptor were upregulated in 60.46% (185/306) and 53.92% (165/306) of tumor tissues, respectively, as assessed by immunohistochemistry. The statistical correlation analysis indicated that Eps15 homology domain 1 overexpression was positively correlated with the increases in phosphorylation of epithelial growth factor receptor ( r = 0.242, p < 0.001) and RAB11FIP3 ( r = 0.165, p = 0.005) expression. The multivariate Cox proportional hazard model analysis demonstrated that the expression of Eps15 homology domain 1 alone is a significant prognostic marker of breast cancer for the overall survival in the total, chemotherapy, and human epidermal growth factor receptor 2 (-) groups. However, the use of combined expression of Eps15 homology domain 1 and phosphorylation of epithelial growth factor receptor markers is more effective for the disease-free survival in the overall population, chemotherapy, and human epidermal growth factor receptor 2 (-) groups. Moreover, the combined markers are also significant prognostic markers of breast cancer in the human epidermal growth factor receptor 2 (+), estrogen receptor (+), and estrogen receptor (-) groups. Eps15 homology domain 1 has a tumor suppressor function, and the combined marker of Eps15 homology domain 1/phosphorylation of epithelial growth factor receptor expression was identified as a better prognostic marker in breast cancer diagnosis. Furthermore, RAB11FIP3 combines with Eps15 homology domain 1 to promote the endocytosis recycling of phosphorylation of epithelial growth factor receptor.

Native structure of rat liver immune proteasomes.

Native structure of active forms of rat liver immune proteasomes has been studied by two-dimensional electrophoresis method modified for analysis of unpurified protein fractions. The developed method allowed revealing the proteasome immune subunits LMP7 and LMP2 in 20S subparticles and in the structures bound to one or two PA28αß activators, but not to the PA700 activator, which is involved in the hydrolysis of ubiquitinated proteins. The results obtained indicate the participation of the immune proteasomes in delicate regulatory mechanisms based on the production of biologically active peptides and exclude their participation in processes of crude degradation of "rotated" ubiquitinated proteins.

[Expression Of DNA-Encoded Antidote to Organophosphorus Toxins in the Methylotrophic Yeast Pichia Pastoris].

A platform for the cloning and expression of active human butyrylcholinesterase (BuChE) in the yeast Pichia pastoris is first presented. Genetic constructs for BuChE gene expression, separately and in conjunction with a proline-rich peptide called proline-rich attachment domain (PRAD), are based on the vector pPICZαA. It is shown that the highest level of production is achieved in the expression of a BuChE gene without PRAD pPICZαA. It is found that one can obtain up to 125 mg of active enzyme from 1 L of culture medium at an optimal pH environment (pH 7.6), an optical seed culture density of 3 o.u., and an optimum methanol addition mode of (0.5% methanol in the first day and 0.2% thereafter from the second day).

New Genetic Constructs for Generation of Stable Therapeutic Antibodies to Organophosphorus Toxins in Methylotrophic Yeasts Pichia Pastoris.

We propose a new method of obtaining of stable Fab-fragments of antibodies in Pichia pastoris expression system. Recently, we obtained Fab-fragments of antibodies neutralizing organophosphorus toxins. However, high yield of the target products was not attained because of high level of proteolytic degradation. In the present study, we identified sites of proteolytic degradation in Fab-fragments and endogenous proteases performing degradation, which allowed obtaining optimized genetic constructs for expression of antibody heavy chains (IgGγ1) and kappa and lambda isotypes of light chains. Co-transformation of these vectors allowed obtaining Fab-fragments of antibodies to organophosphorus toxins without proteolytic degradation of the product.