A Complex of Pyrosequencing-Based Methods for Detection of Somatic Mutations in Codons 600 and 601 of the BRAF gene.

The aim of the study is to develop methods for the differentiation of mutations in the BRAF codon 600 and to increase the sensitivity of the K601E mutation detection. Materials and Methods: The nucleotide sequence of the BRAF codons 592-602 was identified using the PyroMark Q24 genetic analysis system. The mutations search in codon 600 was conducted using the 600-S primer in line with the following order of adding nucleotides: GCTGTCАTCTGCTAGCTAGAC (corresponding to nucleotides 1799-1786). The K601E mutation was detected using the 601-S primer in line with the following order of nucleotide addition: GCTACTCACTGTAG (corresponding to nucleotides 1801-1793). The analytical characteristics of the proposed methods for somatic mutations' detection were determined using dilutions of plasmid DNA samples containing the BRAF gene region without mutations or with one of the following mutations: V600E, V600R, V600K, V600M, and K601E. Validation was performed on 132 samples of biological material obtained from the thyroid nodules. Results: The developed methods allow to determine 2% of the V600E or V600M mutations, 1% of the V600K and V600R mutations, and 3% of the K601E mutations in samples with high DNA concentration; it is also possible to confidently detect at least 5% of the mutant allele for all mutations in low concentration samples (less than 500 copies/PCR). During biological material testing, 53 samples with the V600E mutation were detected; the proportion of the mutant allele was 4.9-50.0%. Conclusion: A complex of methods for determination of the nucleotide sequence of the BRAF codons 592-601 and the algorithm for testing samples and analyzing mutations in the BRAF codons 600-601 was developed. The method provides sufficient sensitivity to detect frequent mutations in codons 600 and 601 and allows them to be precisely differentiated.

Differences in Fatty Acid Composition between Orb-Weaver Spiders Inhabiting a Riparian Zone and a Steppe Are Associated with the Consumption of Different Chironomid Taxa.

Amphibiotic insects, chironomids of the genera Glyptotendipes and Chironomus, which emerged from saline Lake Shira, differed in composition and content of fatty acids, including the essential eicosapentaenoic acid (20:5n-3, EPA), and upon flying out they were concentrated in different territories, the riparian zone and remote arid steppe zone, respectively. Potential consumers of chironomids adults, the orb-weaver spiders Larinoides suspicax, which inhabited both zones, also differed in fatty acid composition. The main difference in their biochemical composition was a significantly higher level of EPA in spiders from the riparian zone that likely to be explained by consumption of the Glyptotendipes adults enriched in this fatty acid and concentrated only within this zone. The higher level of EPA, which is deficient in terrestrial ecosystems, in orb-weaver spiders from the riparian zone of the saline lake may potentially promote a successful survival of the consumers in the arid landscape.

[Mechanisms for the Development of Blood Pressure Variability and the Potential of Antihypertensive Drugs in Their Correction].

Blood pressure variability (BPV) is the fluctuations of blood pressure over a certain period of time under the influence of various factors. The issue of increased BPV is of particular clinical importance due to high predictive value of this parameter as a risk factor for fatal and non-fatal cardiovascular, cerebrovascular and renal events. It is proved that in the BPV increasing, the key role is played by impairments in arterial baroreflexes, which, in turn, are mediated by increased vascular stiffness, impact of angiotensin II and the sympathetic nervous system, endothelial dysfunction, nitric oxide deficiency and aging, including the vascular aging. Antihypertensive drugs that targeting largest amount of pathophysiological mechanisms in BPV increasing have a most advantages in correcting excessive pressure fluctuations. In this regard such drugs are perindopril and amlodipine, which can eliminate almost the entire spectrum of increased BPV causes and, therefore, optimally reduce the cardiovascular risk.

[Single Pill Indapamide and Perindopril Arginine Combination: Optimal Coupling of Vascular Protection and Hemodynamic Risk Factors Reduction in Patients with Essential Arterial Hypertension].

OBJECTIVE: to investigate the impact of indapamide / perindopril single-pill combination (I / P SPC) on arterial stiffness parameters, blood pressure (BP) level and BP variability (BPV) in middle-aged patients with stage II grade 1-2 essential arterial hypertension (EAH). Materials and methods. We retrospectively formed a group of patients with stage II grade 1-2 EAH who had not previously received regular antihypertensive therapy (AHT) (n=52, mean age 52.9±6.0 years). All patients were treated with I / P SPC and all of them achieved target office BP level (less than 140 / 90 mm Hg). After 12 weeks of follow-up (from the time of reaching the target BP) assessment of AHT effectiveness (general clinical data, ambulatory blood pressure monitoring [ABPM], volume sphygmography, echocardiography), and vascular stiffness evaluation were performed. RESULTS: At the end of follow-up office systolic BP (SBP), diastolic BP (DBP), pulse BP, day-time, night-time and 24­hour SBP and DBP significantly (p<0.001 for all) decreased. According to the ABPM data day-time, nighttime, and 24­hour systolic BPV significantly decreased (p=0.029, p=0.006 and p<0.001, respectively); day-time and 24­hour diastolic BPV also significantly decreased (p=0.001 and p<0.001, respectively). Day-night standard deviation (SDdn) significantly decreased too (p=0.002 and p<0.001, respectively). Volumetric sphygmography showed significant decrease of right cardio-ankle vascular index (CAVI) (from 8.20±1.29 to 7.58±1.44, p=0.001) and of left CAVI (from 8.13±1.40 to 7.46±1.43, p<0.001), as well as reduction of the number o f patients with a right- and / or left-CAVI >9.0 (from 32.7 to 11.5 %, p=0.018). According to assessment of arterial stiffness using the Vasotens24 software package, the arterial stiffness index (ASI) significantly (p<0.001) decreased from 153.5±29.9 to 138.3±20.0 (by -9.2±13.1 %). Transthoracic echocardiography data demonstrated significant decrease (p<0.001) in effective arterial elastance (from 1.82±0.43 to 1.58±0.36 mm Hg; by -11.85±16.29 %) and significant (p<0.001) increase in the arterial compliance - from 1.27±0.34 to 1.54±0.38 mm Hg / ml (+26.95±38.06 %). CONCLUSION: In AHT naive patients 40-65 years old with stage II grade 1-2 EAH therapy with I / P SPC provided effective 24­hour BP control, reduced BPV and improved arterial stiffness parameters.

[Cognitive functions, emotional status, MRI measurements in treatment-naïve middle-aged patients with uncomplicated essential arterial hypertension]. / Kognitivnye funktsii, émotsional'nyi status i pokazateli magnitno-rezonansnoi tomografii u nelechenykh patsientov srednego vozrasta s neoslozhnennoi arterial'noi gipertenziei.

AIM: To study cognitive functions, anxiety and depression levels, 24-hour blood pressure (BP) profile, cerebral blood flow (CBF) perfusion in treatment-naive middle-aged patients with uncomplicated essential arterial hypertension (EAH) depending on the white matter hyperintensities (WMH) burden. MATERIAL AND METHODS: Forty-one hypertensive patients (mean age 46.2±4.6 years) and 41 healthy volunteers (mean age 50.3±6.7 years) were enrolled to the study. All subjects underwent brain MRI (MAGNETOM Skyra 3.0T, T1, T2 FSE, T2 FLAIR, T1 MPRAGE, ASL), Montreal cognitive assessment (MoCa), 10-word learning task, verbal fluency test, trail making test, Stroop color and word test, anxiety and depression assessment with Hamilton rating scales, 24-hour blood pressure monitoring (ABPM). RESULTS: WMH were found in 22 (53.7%) hypertensive patients and in 3 (7.3%) healthy volunteers (p=0.0002). Hypertensive patients had the significantly lower CBF compared to controls (p<0.001). CONCLUSION: WMH were identified in treatment-naive middle-aged patients with uncomplicated mild to moderate EAH. There was an association between WMH and lower CBF in the cortical plate of frontal lobes, SBP variability and worse cognition. Cerebral hypoperfusion can cause cognitive impairment even in the earliest stages of EAH, which increases due to emotional disorders.

[Day-by Day Blood Pressure Variability: Methodological Aspects, Prognostic Value, Effects of Antihypertensive Therapy].

In this article we present problems of prognostic value of day-by-day blood pressure (BP) variability, its role in development and progression of damage of target organs in arterial hypertension, and impact on risk of cardiovascular, cerebral-vascular complications, and mortality. We also discuss methodological aspects of assessment of day-by-day BP variability. The article contains literature data on effects of antihypertensive therapy on variability of home BP, and consideration of evidence base of possibilities of the fixed amlodipine/perindopril combination for lowering day-by-day BP variability.

[Blood Pressure Variability. Visit-to-Visit Blood Pressure Variability].

The article is devoted to the modern state of the problem of blood pressure variability (BPV). Along with discussion of classification and methods of diagnosis it contains data on prognostic value of visit-to-visit BPV. We have also reviewed effect on BPV of various regimens of antihypertensive therapy and presented evidence base supporting ability of amlodipine/perindopril fixed dose combination to lower visit-to-visit BPV.

[24­Hour Arterial Pressure Variability: Prognostic Significance, Methods of Evaluation, Effect of Antihypertensive Therapy].

We discuss in this article problems of prognostic significance of 24­hour arterial pressure variability (24hAPV), as well as the role of 24hAPV in development and progression of various target organs damage by arterial hypertension. We also present literature data on impact of various regimens of antihypertensive therapy on 24hAPV, and on ability of fixed-dose perindopril/amlodipine to lower 24­hAPV.

[A change in adrenal responsiveness in sotalol-treated patients with paroxysmal atrial fibrillation depending on autonomic nervous system tone].

AIM: To evaluate the efficacy of sotalol depending on the magnitude of changes in adrenal responsiveness and autonomic nervous system tone in patients with paroxysmal atrial fibrillation (AF). SUBJECTS AND METHODS: Twenty-six patients with paroxysmal AF in the presence of coronary heart disease (CHD) and hypertension were examined. Sinus rhythm variability and sympathicotonic and vagotonic disorders were studied in patients with paroxysmal AF before and during sotalol treatment. A commercial Beta-ARM-Agat kit was used to estimate the adrenal responsiveness of erythrocyte membranes (ß-APM), which can judge the body's individual sensitivity to ß-adrenoblockers. RESULTS: Sotalol used in the average therapeutic doses of 160-240 mg did not reduce ejection fraction or increase atrioventricular conduction up to abnormal values. In patients with borderline and mild hypertension, the drug lowered blood pressure statistically significantly (p=0.01) and was well tolerated. The drug increased the sensitivity of ß-adrenoblockers in patients with adrenergic AF. CONCLUSION: The effect of sotalol on the autonomic nervous system manifested in the higher power of a high-frequency spectral component of heart rate variability than in that of a low-frequency one. Long-term sotalol administration significantly reduced ß-APM, increasing the sensitivity of adrenoceptors.

FOR EXTRAINTESTINAL MANIFESTATIONS OF INFLAMMATORY BOWEL DISEASE IN CHILDREN.

Inflammatory bowel disease (IBD) are systemic diseases with different intestinal and extra-intestinal manifestations (ElM). This fact can determin an examination plan and the disease course and prognosis. Up to 8.4% of children with IBD have ELM like the first symptom of the disease. The range of ElM of iBD in children is differ from that one of adult patients: rarer arthropathy and arthritis, skin and liver diseases, an extremely rare eye disease. The physical development disorder is a typical children's manifestation of the IBD, the "failure of the weight curve" is an early symptom of the IBD debut (82% of examined children), especially ulcerative colitis. The article discusses the clinical features and therapy of the most frequent extraintestinal manifestations of IBD in children: skin, mucous, eye diseases, axial and peripheral arthropathies, primary scierosing cholangitis, physical, sexual development disorders, decreased bone mineral density. Most often, we observed patients with arthritis and primary scierosing cholangitis - up to 12.5% of patients with active disease. A growth failure was observed in 20% of children with IBD debut, and remained in 9.7% children despite adequate anti-inflammatory therapy. Decreased bone mineral density in children with IBD observed in 30% cases (ISCD-recommendations). Extraintestinal manifestations are very important to make the early diagnosis of IBD without any intestinal symptoms. The pediatrician can suspect a systemic diseases course. ElMs adversely impact upon patientsquality of life and some can be life-threatening.

[Manifestations of polymorphism of ß1-adrenoreceptors in patients with newly diagnosed cardiac rhythm disorders].

Individual peculiarities of the receptor apparatus of cardiomyocytes may determine pathological features of heart activity and susceptibility to pharmaceuticals. The possible role of beta-adrenoreceptor polymorphism in the development of cardiac rhythm disturbances is assessed by PCR. Special attention is given to A145G polymorphism of the ADRB1 gene in 127 patients with primary cardiac rhythm disorders. It was shown that AJ45G polymorphism (Ser49Gly) at DNA sites encoding for the amino acid sequence of beta-1 adrenoreceptors can influence the development of sex-specific cardiac rhythm disorders.

[Symptomatic epilepsy in inflammatory demyelinating diseases].

Based on the own observations, we have summarized the features of symptomatic epilepsy in multiple sclerosis and conducted a clinical analysis of the most diagnostically difficult cases of inflammatory and demyelinating diseases in which epilepsy was the major clinical syndrome. The cases of post-infectious acute disseminated encephalomyelitis, idiopathic cerebral angiitis, Rasmussen's encephalitis are reviewed. Peculiarities of MRI structural brain lesions in these patients are discussed. The use of additional laboratory studies, including a study of cerebrospinal fluid, is considered.

[The role of unconscious emotional-motivational stimuli in heroin addiction].

Eighty patients with heroin addiction, aged from 15 to 20 years (most of them over the age of 16), were examined. The duration of illness was from 6 months to 3 years. The examination was conducted 2-3 weeks after arresting of withdrawal syndrome. In that period, patients had different mental disorders: depressive-dysphoric, neurosis-like, anxiety-hypochondriac, hysteriform and others. The aim of the examination was to find peculiarities of functioning of different cortical regions in the process of appraisal of conscious and unconscious stimuli using the method of visual evoked potentials with P300 recording. Stimuli were words highlighted on the screen--indifferent and potentially significant for a patient (e.g. heroin). The stimulus related to the dominating motivation caused in patients the higher diffuse cortical activation on the unconscious level compared to non-targeted stimuli.

[Metabolic activity of peripheral blood neutrophils during treatment of children with acute lymphoblastic leukemia].

Neutrophilic metabolic activity was studied in 21 children with lymphoblastic leukemia during chemotherapy. After one-year chemotherapy, reduced cell activities of catalase and glutathione reductase were observed in children with septic complications during a follow-up in the presence of lower neutrophilic lactate levels. There was a direct correlation between lactate levels and catalase activity. Since the reduction in the levels of lactate in the neutrophils is attended by their apoptosis, this suggests that there is an association of delayed cell death with a fall in the phagocytic antioxidative defense potential.

[Use of venofer for iron deficiency correction in patients undergoing programmed hemodialysis]. / Primenenie preparata "Venofer" dlia korrektsii defitsita zheleza u patsientov, nakhodiashchikhsia na programmnom gemodialize.

AIM: To study iron metabolism in patients on programmed hemodialysis (PH) in oral and intravenous administration of iron drugs; to compare clinical and financial results of using such drugs. MATERIAL AND METHODS: A two-stage trial studied iron metabolism in 158 PH patients on replacement therapy with erythropoetin. They received correction of iron deficiency with oral drugs (stage I) and venofer (stage II). RESULTS: The study of iron metabolism has found its deficiency in 2/3 patients receiving oral iron: absolute (48%) and relative (20%). Administration of venofer led to a 2-fold increase in the number of patients with normal iron metabolism. The target Hb and Ht were achieved in 2.5 times more patients than before venofer treatment. The dose of erythpoetin in such cases was reduced by 40%. Side effects were not observed. The week cost of venofer treatment per patient was lower by 22.5$ than the cost of treatment with oral iron drugs. CONCLUSION: Venofer correction of iron deficiency in PH patients is more effective both clinically and financially than use of oral iron preparations.

[Immunosuppressive components of extracellular lipopolysaccharide highly virulent strain Salmonella typhimurium 1468]. / Immunosupressivnye komponenty ékstratselliuliarnogo lipopolisakharida vysokovirulentnogo shtamma Salmonella typhimurium 1468.

Immunosuppressive activity of culture liquid substrate (CFS) of highly virulent strain Salmonella typhimurium has been studied. A model of delayed hypersensitivity (DHS) to nonbacterial antigen in mice, a method of gel-filtration through the sephadex column G-200, immunosorbents were used. Three components with immunosuppressive activity: thermolabile component and thermostable one with direct immunosuppressive action and the third thermolabile component which manifested inductive immunosuppressive activity only after redox treatment have been revealed in the strain CFS. O-specific and lipid parts were found in the composition of all the components. This allowed them to be related to lipopolysaccharide.

[Computed tomography in the evaluation of the bronchopulmonary system in late periods after childhood partial resection of the lung]. / KT v otsenke sostoianiia bronkho-legochnoi sistemy v otdalennye sroki posle perenesennoi v detskom vozraste rezektsii chasti legkogo.

The authors used computed tomography (CT) as a basis to analyze the anatomic status of the bronchopulmonary system in 28 patients undergone resection of a part of the lung in childhood for chronic nonspecific lung inflammations and malformations. The findings confirmed the high diagnostic value of CT in the evaluation of the lung in different postoperative periods and showed a certain range of changes varying from significant in the lung operated on to progression of the primary disease to the collateral lung.

[Immunosuppressive effects of pathogenic gram-negative bacteria]. / Immunosupressivnoe deistvie patogennykh gramotritsatel'nykh bakterii.

The review deals with the Immunosuppressive effects of virulent gram-negative bacteria (Salmonella, Shigela, Pseudomonas aeruginosa), with the importance of these effects for the bacteria to survive in the infected body. The above bacteria affect the immunity system in a different way, yet have common features. They are characterized by the occurrence of endotoxin shock, by the suppression of the phagocytic system and cell-mediated immunity. A significant role in suppressing a cellular immune response is played by the lipopolysaccharide of virulent bacteria that greatly differs from that of nonvirulent strains. The immunosuppressive activity of the bacteria and their lipopolysaccharide is closely related to their virulent properties.

[Dependence of immunosuppressive action of lipopolysaccharide on degree of Salmonella pathogenicity]. / Zavisimost' immunosupessivnogo deistviia lipopolisakharida ot stepeni patogennosti sal'monell.

Immunosuppressive activity of Salmonella typhimurium extracellular lipopolysaccharide (LPS) was studied. In this study isogenic S. typhimurium strains with different degree of virulence were used. The attenuation of these strains was linked with mutations on their chromosome (altered synthesis of RNA polymerase or gyrase DNA) or their own virulence plasmid (the insertion of transposon Tn-5). To obtain LPS fraction with different molecular weights, the filtrate of bacterial culture was subjected to gel filtration through a column packed with Sephadex G-200. The immunosuppressive action of LPS fractions was determined on the model of delayed-type hypersensitivity to nonbacterial antigen in experiments on BALB/c mice. The study revealed that transposon-mediated mutation on plasmid, accompanied by the attenuation of salmonellae, led to the loss of immunosuppressive activity of the high-molecular heat-sensitive component of LPS; only the second heat-resistant component with medium molecular weight retained its activity. The presence of two chromosomal attenuating mutations (rifr nalr) was accompanied by the loss of immunosuppressive activity in both components of LPS.

[Lipopolysaccharides of Shigella sonnei]. / Lipopolisakharidy Shigella sonnei.

Immunobiological properties of native lipopolysaccharides (LPS) from virulent and avirulent strains of Shigella sonnei bacteria (LPS-V and LPS-A, respectively) were studied. In avirulent bacteria, LPS-V induced immunosuppressive activity specific of the virulent strain. LPS of the avirulent strain, whereas LPS-A lacked this property. Native LPS-V with immunosuppressive activity were isolated from the virulent strain by and immune affinity method. Treatment of LPS-V with phenol or TCA abolished its activity and converted it into the LPS-A form. The data showed that LPS-A can be converted back to the LPS-V form by redox treatment. This approach seems to be promising for activating LPS extracted from cells with TCA or a water-phenol mixture.