Far-red pentamethine cyanine dyes as fluorescent probes for the detection of serum albumins.

Benzothiazole based cyanine dyes with bridged groups in the pentamethine chain were studied as potential far-red fluorescent probes for protein detection. Spectral-luminescent properties were characterized for unbound dyes and in the presence of serum albumins (bovine (BSA), human (HSA), equine (ESA)), and globular proteins (ß-lactoglobulin, ovalbumin). We have observed that the addition of albumins leads to a significant increase in dyes fluorescence intensity. However, the fluorescent response of dyes in the presence of other globular proteins was notably lower. The value of fluorescence quantum yield for dye bearing a sulfonate group complexed with HSA amounted to 42% compared with 0.2% for the free dye. The detection limit of HSA by this dye was greater than 0.004 mg ml-1 which indicates the high sensitivity of dye to low HSA concentrations. Modelling of structure of the dyes complexes with albumin molecules was performed by molecular docking. According to these data, dyes could bind to up to five sites on the HSA molecule; the most preferable are the haemin-binding site in subdomain IB and the dye-binding site in the pocket between subdomains IA, IIA and IIIA. This work confirms that pentamethine cyanine dyes could be proposed as powerful far-red fluorescent probes applicable for highly sensitive detection of albumins.

Design, synthesis and biological evaluation of 5-amino-4-(1H-benzoimidazol-2-yl)-phenyl-1,2-dihydro-pyrrol-3-ones as inhibitors of protein kinase FGFR1.

Fibroblast growth factor receptor 1 (FGFR1) plays an important role in tumorigenesis and is therefore an attractive target for anticancer therapy. Using molecular docking approach we have identified inhibitor of FGFR1 belonging to 5-amino-4-(1H-benzoimidazol-2-yl)-phenyl-1,2-dihydro-pyrrol-3-ones with IC50 value of 3.5 µM. A series of derivatives of this chemical scaffold has been synthesized and evaluated for inhibition of FGFR1 kinase activity. It was revealed that the most promising compounds 5-amino-1-(3-hydroxy-phenyl)-4-(6-methyl-1H-benzoimidazol-2-yl)-1,2-dihydro-pyrrol-3-one and 5-amino-4-(1H-benzoimidazol-2-yl)-1-(3-hydroxy-phenyl)-1,2-dihydro-pyrrol-3-one inhibit FGFR1 with IC50 values of 0.63 and 0.32 µM, respectively, and posses antiproliferative activity against KG1 myeloma cell line with IC50 values of 5.6 and 9.3 µM. Structure-activity relationships have been studied and binding mode of this chemical class has been proposed.

[DETERMINING THE DEGREE OF DISRUPTION OF THE STRUCTURE OF THE LIVER AND THE SEVERITY OF PORTAL HYPERTENSION IN CHILDREN].

AIM: To develop a system to define the degree of liver disruption and severity of portal hypertension in children based on the International Classification of Functioning, Disability and Health (ICF). PATIENTS AND METHODS: Studied the results of laboratory and instrumental methods 382 children: 267 patients with various liver diseases, including 49 patients who underwent liver transplantation, and 115 children without liver disease. RESULTS: Based on analysis of statistical data obtained were identified 10 indicators, a set of changes which can be used to assess the degree of disruption of the structure of the liver and the severity of portal hypertension: indicators that reflect the severity of fibrosis and cirrhosis of the liver (METAVIR score on a scale at fibroelastometrii, scores are Desmet at morphological study of the liver) and indicators that reflect the severity of portal hypertension (the diameter of the portal vein, splenic vein diameter, the length of the spleen, recanalization of the umbilical vein, esophageal varices, ascites, hydropericardium, hydrothorax). Each of the indicators was assessed on a 5-point system. Number of points reflects the sum of the changes of these parameters. Decrease the number of points on 0-4% (38-40 points) is regarded as a lack of structural failure of the liver and the severity of portal hypertension by 5-24% (30-37 points)--minor violations on 25-49% (20-29 points) -moderation disorders, 50-95% (3-12 points)--severe handicaps, 96-100% (0-2 points)--absolute violation. Studied the dynamics of children with autoimmune hepatitis, Wilson's disease and chronic hepatitis C. CONCLUSION: The proposed scoring system for assessing the degree of disruption of the structure of the liver and the severity of portal hypertension can be used as an objective criterion of the severity of the pathological process, to estimate the dynamics of defeat against the background of the therapy, determining the prognosis of the disease and as a criterion of the indications for liver transplantation.

[The Relationship of the Degree of Impairment of the Structure and Function of the Liver with its Chronic Illnesses in Children].

OBJECTIVE: Our aim was to on the basis of determining the degree of violation of the structure and function of the liver establish their relationships and to assess the dynamics of liver disease in its chronic illnesses in children. METHODS: With the help of the developed scoring systems were used to assess the degree of liver dysfunction and the degree of disruption of the structure of the liver and the severity of portal hypertension. RESULTS: The results of the diagnostic methods 252 children aged 1 to 17 years (mean age of 11.8±3,5) with Wilson disease (WD), autoimmune hepatitis (AIH), chronic hepatitis C (CHC) were analyzed; 48 patients underwent liver transplantation. In children with WD, AIHand CHC liverfunction reduced by 41.3±12.9% to 28.8±12.5% and 19.1±7.8% respectively. Structure of the liver in children with WD, AIH and CHC was disturbed by 25.0±8.1% to 20.4±9.2% and 6.8±4.4% respectively. Thefunction and structure violations of the liver more pronounced in liver cirrhosis. The use of the developed scoring systems to monitor the severity of liver damage in the dynamics and evaluation of the effectiveness of the therapy is demonstrated. The degree of liver dysfunction is directly dependent on the degree of its structure. Abnormal liverfunction ≥40% and ≥40% of its structure with treatment failure can be used as a criterion of indicationsfor elective liver transplantation with its chronic diseases in children. CONCLUSION: Developed a point system to determine liver function and a point system to determine disruption of the structure of the liver and the severity of portal hypertension in children can serve as an objective criterion for assessing the severity of liver disease, monitoring their changes in the dynamics with the assessment of the effectiveness of the therapy and making decisions about the need for routine liver transplantation in its chronic illnesses in children.

[OPTICAL BIOPSY FOR DIAGNOSIS OF ESOPHAGEAL MUCOSAL CHANGES IN CHILDREN].

AIM: To analyze the value of confocal laser endomicroscopy in diagnostics of upper gastrointestinal tract mucosa changes in children. PATIENTS AND METHODS: In the current study a total of 116 children aged from 3 to 18 years old undergo conventional endoscopy with confocal laser endomicroscopy supplemented with mucosal biopsy followed by traditional histology in the period from 2011 until 2014. To determine the prognostic value of the of probe based CLE in the evaluation of normal and pathological changes of the esophageal mucosa a comparison of results of optical biopsy with the data obtained during the standard histological examination were performed. RESULTS: After results of probe-based CLE and traditional histology were comprised optical biopsy showed 88.8% sensitivity and 88.3% specificity to esophagitis with Spearmen correlation 0.79 (p = 0.001); 92.3% sensitivity and 95.3% specificity to metaplastic changes of esophageal mucosa with Spearmen correlation 0.85 (p = 0.001); 92.4% sensitivity and 95.2% specificity in differential diagnosis of esophageal polyps with Spearmen correlation 0.95 (p = 0.001). CONCLUSION: Confocal endomicroscopy may become one of the leading methods in pediatric gastroenterology since it allows the endoscopists to inspect the mucosa at the cellular level during the endoscopic procedure and can help in establishing the diagnosis.

[DETERMINING THE DEGREE OF LIVER DYSFUNCTION IN CHILDREN WITH POSITIONS OF THE INTERNATIONAL CLASSIFICATION OF FUNCTIONING, DISABILITY AND HEALTH (ICF)].

METHODS: Based on a retrospective analysis of biochemical blood parameters which characterize the role of liver function in the metabolism of proteins, fats and carbohydrates (considered indicators of ALT, AST, De Ritis coefficient, bilirubin, albumin, fibrinogen, prothrombin, transferrin, ceruloplasmin, cholesterol, urea, ammonia, glucose, lactate) in 95 children without liver pathology, 15 children who died of liver failure, 295 patients with various liver diseases who were treated in the SCCH, a scale system was developed as a support tool to assess liver dysfunction. RESULTS: Each biochemical indicator was assessed on a five-point scale. The level of a biochemical indicator, which corresponded to the absence of disorders, was estimated as 4 points, corresponding to "insignificant disorders"--as 3 points, "moderate disorders"--as 2 points, "severe disorders"--as 1 point, "absolute disorders"--as 0 points. The total score is the estimate of the degree of liver dysfunction. According to the recommendations of the International Classification of Functioning, Limitations of vital activities and Health, the decrease of the number of points on 0-4% (54-56 points) corresponds to the absence of the liver dysfunction, on 5-24% (43-53 points)--insignificant disorders of liver function, on 25-49% (29-42 points)--moderate hepatic impairment, on 50-95% (3-28 points)--severe disturbances of liver function, on 96-100% (0-2 points)--absolute dysfunction of the liver. CONCLUSIONS: A scoring system of assessing liver dysfunction can be applied at any stage of the examination and treatment of children of any age, as used in biochemical parameters do not depend on the age of the patient. It is an objective criterion for assessing the degree of liver dysfunction and can be used to assess the severity of the pathological process in the dynamics determining the prognosis of the disease and can be the criterion of the indications for liver transplantation, and also used during the of medico-social expert examination.

[Chronic hepatitis C in children: problems and prospects].

The analysis of a problem state of chronic hepatitis C in children was conducted. Data on primary incidence of chronic hepatitis C at the children's population as on the territory of the Russian Federation, and abroad are submitted. Problems of diagnosis of a HCV infection are studied. The survey analysis of risk of contamination is carried out, possible ways of transfer are highlighted, features of a course of a HCV infection in children are shown, synchronising frequency of a process is displayed. The problem is analysed now, and further prospects in treatment of chronic viral hepatitis C in children are estimated.

[Identification of low-molecular inhibitors of proteinase ASK1].

Protein kinase ASK1 (Apoptosis signal-regulating kinase 1) plays a key role in cell differentiation, aging and apoptosis. High activity of the kinase is associated with several pathologies. The ASK1 inhibitors might be therapeutic for patients with neurodegenerative, cardiovascular diseases and fibrous histiocytoma. In this work the identification of ASK1 inhibitors was performed by the methods of computer modeling and biochemical testing in vitro. The virtual screening experiments were carried out targeting the ATP binding site of ASK1 by browsing the database which contained 164 840 compounds of diverse chemical classes. The best-scored 300 ligands have been taken for the kinase assay analysis. In vitro tests revealed that derivatives of 2-thioxo-thiazolidin-4-one exhibited inhibitory activity against ASK1. The most active compound was 5-bromo-3-(4-oxo-2-thioxo-thiazolidin-5-ylidene)-1,3-dihydro-indol-2-one (IC50 = 2 microM). Binding mode for inhibitors of this class with ASK1 ATP-binding site was proposed. Our results can be used for further optimization and developing more potent and selective inhibitors of ASK1.

Docking and fusion of synaptic vesicles in cell-free model system of exocytosis.

The present study involves the testing and characterization of synaptic vesicle (SV) docking and fusion as the steps of exocytosis using two different approaches in vitro. The interaction of SVs was determined by the changing of particles size in suspensions by the method of dynamic light scattering (DLS). Fluorescence assay is represented for studying the mechanism of SV membrane fusion. The sizes of membrane particles were shown to increase in the medium containing cytoplasmic proteins of synaptosomes. Therefore, the cytosolic proteins are suggested to promote the SVs into close proximity where they may become stably bound or docked. The specific effect of synaptosomal cytosolic proteins on the interaction of SVs in the cell-free system was demonstrated. The incubation of SVs with liver cytosol proteins or in the bovine serum albumin solution did not lead to the enlargement of the particles size. The fusion reaction of the SVs membranes occurred within the micromolar range of Ca(2+) concentrations. Our studies have shown that in vitro process of exocytosis can be divided into Ca(2+)-independent step, termed docking and followed by fusion step that is triggered by Ca(2+). The role of cytosolic proteins of synaptosomes in docking and fusion of SVs in cell-free system was further confirmed.