Effects of α-amylase, endo-xylanase and exoprotease combination on dough properties and bread quality

Received: January 31st, 2021 ; Accepted: May 2nd, 2021 ; Published: May 6th, 2021 ; Correspondence: [email protected] enzymes composition is an actual alternative to chemicals to improve functional properties of flours and to generate changes in the structure of the dough and bread quality. The objective of this study was to analyze the individual and synergistic effects of enzymes preparation (α-amylase, endo-xylanase and exoprotease), newly produced in Russia, on dough properties and bread quality made from wheat flour with different amylolytic activity. Reofermentometric results revealed decreases in gas-forming capacity of dough by 10.0–13.9% when single α-amylase preparates were used. The α-amylase addition had significant effect on gas retention coefficient in flour possessed low amylolytic activity. The effect of endo-xylanase and exoprotease on hydration and amount of wheat gluten was established. The fractional composition of gluten proteins in the dough made with combination of endo-xylanase and exoprotease was established using Lowry method immediately after kneading and after fermentation. It was found that mainly water-soluble, alcohol-soluble and alkaline-soluble proteins were undergone by transformation. The bread with enzymes had a higher specific volume, porosity and aldehyde content and lower shape stability indicator than the control bread made without enzymes. Bread with enzymes was characterized by tenderer and not crumbly crumb with developed thin-walled uniform porosity compared to the control. The crusts were more brightly colored. The combined usage of α-amylase and endo-xylanase and exoprotease retarded bread staling during 5-day storage period. New enzyme composition may be a potentially strong candidate for future applications in the bread-making industry

Homobivalent Lamellarin-Like Schiff Bases: In Vitro Evaluation of Their Cancer Cell Cytotoxicity and Multitargeting Anti-Alzheimer's Disease Potential

Marine alkaloids belonging to the lamellarins family, which incorporate a 5,6-dihydro-1-phenylpyrrolo[2,1-a]isoquinoline (DHPPIQ) moiety, possess various biological activities, spanning from antiviral and antibiotic activities to cytotoxicity against tumor cells and the reversal of multidrug resistance. Expanding a series of previously reported imino adducts of DHPPIQ 2-carbaldehyde, novel aliphatic and aromatic Schiff bases were synthesized and evaluated herein for their cytotoxicity in five diverse tumor cell lines. Most of the newly synthesized compounds were found noncytotoxic in the low micromolar range (<30 μM). Based on a Multi-fingerprint Similarity Search aLgorithm (MuSSeL), mainly conceived for making protein drug target prediction, some DHPPIQ derivatives, especially bis-DHPPIQ Schiff bases linked by a phenylene bridge, were prioritized as potential hits addressing Alzheimer's disease-related target proteins, such as cholinesterases (ChEs) and monoamine oxidases (MAOs). In agreement with MuSSeL predictions, homobivalent para-phenylene DHPPIQ Schiff base 14 exhibited a noncompetitive/mixed inhibition of human acetylcholinesterase (AChE) with Ki in the low micromolar range (4.69 μM). Interestingly, besides a certain inhibition of MAO A (50% inhibition of the cell population growth (IC50) = 12 μM), the bis-DHPPIQ 14 showed a good inhibitory activity on self-induced β-amyloid (Aβ)1-40 aggregation (IC50 = 13 μM), which resulted 3.5-fold stronger than the respective mono-DHPPIQ Schiff base 9

SCLERODERMA SYSTEMATICA WITH INTERSTITIAL LUNG LESION: COMPARATIVE CLINICAL CHARACTERISTICSWITH PATIENTS WITHOUT LUNG LESION

Objective. To compare disease history data and clinical and laboratory parameters in patients with scleroderma systematica (SDS) with high-resolution computed tomography (HRCT)-verified interstitial lung lesion (ILL) versus those without lung involvement. Subjects and methods. An examination was made in 138 patients with SDS who had been consecutively admitted in 2006-2008, female/male ratio, 124 : 14; limited : diffuse : mixed forms, 78 : 40 : 20; mean age, 47±13 years; median disease duration, 6 (2.5 11) years. The history data (occupational hazards, smoking, respiratory diseases) and clinical manifestations of SDS and laboratory data were studied. The diagnosis of ILL was established on the basis of chest HRCT. Results. According to HRCT data, the signs of varying ILL were found in 82% of the patients with SDS. The duration of SDS was similar in the patients with and without lung involvement; but the latter were younger at the time of disease onset. There were no significant differences between the groups compared in history data, clinical forms of SDS, the frequency of involvement of visceral organs and systems. Crepitation was heard only in the patients with ILL. The frequency of respiratory manifestations increased with a larger number of the involved lung segments. The prevalence of ILL was found to be positively correlated with age at the onset of SDS (r=0.29;

Cytokine profile and expression of FYN, ZAP-70 and LAT during concanavalin a stimulation in patients with resistant bronchial asthma

Background: Bronchial asthma (BA) is one of the most spreading chronic lung pathology in the world. The disease is characterized by high heterogeneity of clinical phenotypes including resistant forms which provoke significant clinical problem. Immune shift from Th2 to alternative immunological response is considered to be a mechanism of drug-resistance in BA treatment but this issue is not considerably studied yet. Aims: Detection of distinctive patterns in cytokine secretion and genetic expression (ZAP-70, FYN and LAT) of naïve and concanavalin A stimulated lymphocytes in patients with resistant BA. Materials and methods: The study enrolled ten patients in each group: subjects with treatment resistant BA, severe BA, and controls (30 in total). During the experiment, all patients with BA received treatment according to the condition. For each participant lymphocytes isolation from venous blood was performed. Cells were cultured with concanavalin A and without stimulation. Concentrations of cytokines IL-2, IL-12, TNF-α, IL-4, IL-5, and IL-6 in supernatants were measured with ELISA. Reverse transcription polymerase chain reaction was used to detect the mRNA expression of LAT, ZAP-70, and FYN genes. Results: Significant disease contribution to the lymphocyte secretion profile was established without concanavalin A stimulation: increased levels of IL-2 and IL-4 was observed in lymphocytes of patients with resistant BA if compared to the results of gorup with severe BA. Patients with resistant BA were characterized by weak cytokine response to the stimulation: only TNF-α and IL-5 levels were significantly increased whereas in group with severe BA all cytokines concentrations increased except IL-12, in controls - except IL-12 and IL-2. Significant FYN upregulation was identified in resistant BA group if compared with other groups, and in severe BA patients if compared with controls. The concanavalin A-stimulated cells showed increased expression of ZAP-70 in cells of patients with resistant BA compared to control group. Conclusions: Lymphocytes from patients with resistant BA are characterized by lack of cytokine response to concanavalin A stimulation, alteration of cytokine secretion, and genetic expression profile similar to cells with low sensitivity to apoptosis. The FYN gene is a perspective target for finding approaches to overcome resistance to steroid drugs in bronchial asthma. © 2018 Izdatel'stvo Meditsina. All rights reserved

Дифференциация и субпопуляционный состав VEGFR2+ моноцитов крови и костного мозга при ишемической кардиомиопатии

Aim. To identify disturbances of differentiation and subpopulation composition of VEGFR2+ cells in the blood and bone marrow associated with the features of the cytokine profile in the blood and bone marrow in patients with coronary artery disease (CAD) with and without ischemic cardiomyopathy (ICM).Materials and methods. The study included 74 patients with СAD with and without ICM (30 and 44 people, respectively) and 18 healthy donors. In all patients with СAD, peripheral blood sampling was performed immediately before coronary artery bypass grafting, and bone marrow samples were taken during the surgery via a sternal incision. In the healthy donors, only peripheral blood sampling was performed. In the bone marrow and blood samples, the number of VEGFR2+ cells (CD14+VEGFR2+ cells) and their immunophenotypes CD14++CD16-VEGFR2+, CD14++CD16+VEGFR2+, CD14+CD16++VEGFR2+, and CD14+CD16-VEGFR2+ was determined by flow cytometry. Using enzyme-linked immunosorbent assay, the levels of VЕGF-А, TNFα, M-CSF, and IL-13, as well as the content of MCP-1 (only in the blood) and the M-CSF / IL-13 ratio (only in the bone marrow) were determined.Results. The content of CD14+VEGFR2+ cells in the blood of CAD patients with and without ICM was higher than normal values due to the greater number of CD14++CD16-VEGFR2+, CD14++CD16+VEGFR2+, and CD14+CD16++VEGFR2+. In the bone marrow of the patients with ICM, the content of CD14++CD16-VEGFR2+, CD14+CD16++VEGFR2+, and CD14+CD16-VEGFR2+ was lower than in patients with CAD without ICM, and the number of CD14++CD16+VEGFR2+ cells corresponded to that in the controls. Regardless of the presence of ICM in CAD, a high concentration of TNFα and normal levels of VEGF-A and IL-13 were observed in the blood. In CAD without ICM, an excess of MCP-1 and deficiency of M-CSF were revealed in the blood. In the bone marrow, the levels of VEGF-A, TNFα, M-CSF, and IL-13 were comparable between the groups of patients against the background of a decrease in the M-CSF / IL-13 ratio in the patients with ICM.Conclusion. Unlike CAD without cardiomyopathy, in ICM, no excess of VEGFR2+ cells and MCP-1 in the blood is observed, which hinders active migration of CD14+CD16++VEGFR2+ cells from the myeloid tissue, and a decrease in the M-CSF / IL-13 ratio in the bone marrow disrupts differentiation of other forms of VEGFR2+ cells, preventing vascular repair.Цель: установить нарушения дифференцировки и субпопуляционного состава VEGFR2+ моноцитов в крови и костном мозге во взаимосвязи с особенностями цитокинового профиля крови и костного мозга у больных ишемической болезнью сердца (ИБС), страдающих и не страдающих ишемической кардиомиопатией (ИКМП).Материалы и методы. В исследование вошли 74 больных ИБС, страдающих и не страдающих ИКМП (30 и 44 человека соответственно), и 18 здоровых доноров. У всех больных ИБС забор периферической крови производился непосредственно перед операцией коронарного шунтирования, а костного мозга – из разреза грудины во время операции. У здоровых доноров забирали только периферическую кровь.  В костном мозге и крови методом проточной цитофлуориметрии определяли численность VEGFR2+ моноцитов (CD14+VЕGFR2+ клеток) и их иммунофенотипов CD14++CD16-VEGFR2+, CD14++CD16+VEGFR2+, CD14+CD16++VEGFR2+, CD14+CD16-VEGFR2+, методом иммуноферментного анализа регистрировали концентрацию VЕGF-А, TNFα, M-CSF, IL-13, а также содержание MCP-1 (только в крови) и соотношение M-CSF/IL-13 (только в костном мозге).Результаты. Содержание CD14+VEGFR2+ клеток в крови у больных ИБС без кардиомиопатии и с ИКМП было выше нормы из-за большей численности CD14++CD16-VEGFR2+, CD14++CD16+VEGFR2+ и CD14+CD16++VEGFR2+ форм. В костном мозге у больных ИКМП содержание CD14++CD16-VEGFR2+, CD14+CD16++VEGFR2+ и CD14+CD16-VEGFR2+ форм было ниже, чем у больных ИБС без кардиомиопатии, а количество CD14++CD16+VEGFR2+ клеток соответствовало их числу в группе сравнения. Вне зависимости от наличия ИКМП при ИБС в крови отмечалась высокая концентрация TNFα, нормальный уровень VEGF-А и IL-13; при ИБС без кардиомиопатии – избыток МСР-1 и дефицит M-CSF в крови. В костном мозге концентрация VЕGF-А, TNFα, M-CSF, IL-13 была сопоставимой между группами больных на фоне снижения M-CSF/IL-13 у пациентов с ИКМП.Заключение. В отличие от ИБС без кардиомиопатии при ИКМП не формируется избыток VEGFR2+ моноцитов и МСР-1 в крови, что затрудняет активную миграцию CD14+CD16++VEGFR2+ клеток из миелоидной ткани, а снижение M-CSF/IL-13 в костном мозге нарушает дифференцировку остальных форм VEGFR2+ моноцитов, препятствуя репарации сосудов

Аллергия на металлы

Allergic reactions associated with sensitisation to metals are a common but underexplored problem. Due to the frequent use of metals and their alloys there has been an increase in the number of registered cases of allergic reactions. Recently there have been cases when allergic reactions were induced by metals that were previously considered absolutely inert and non-allergenic, such as gold, palladium and others. The aim of this work was to summarise scientific data on allergic reactions to metals and their diagnosis in humans. In medicine, alloys of nickel, palladium and gold are used in the manufacture of both surgical instruments and various implants used in orthopedics, endovascular surgery, gynecology and dentistry. Allergic reactions to these metals may lead to failure of artificial joints, thrombosis of endovascular stents, stomatitis, gingivitis, and dermatitis. The most frequent allergic reaction to metals is contact dermatitis which is most frequently caused by nickel. Metal allergies are diagnosed by skin tests. There are no Russian-made diagnostic systems for detecting metal allergies. The diagnosis of allergic contact dermatitis is performed with the help of AllerTest test kit («TRUE Test», Denmark). Therefore, elaboration of a domestic diagnostic test for timely detection of allergies to metals is still relevant.Аллергические реакции, связанные с сенсибилизацией к металлам, являются распространенной, но недостаточно изученной проблемой. В связи с частым использованием металлов и их сплавов регистрируют все больше случаев аллергических реакций, вызванных их применением. В последнее время стали появляться случаи аллергических реакций даже на те металлы, которые ранее считались абсолютно инертными и не аллергенными, такими как золото, палладий и другие. Целью данной работы являлось обобщение научной информации о возникновении аллергических реакций на металлы и проблемах их диагностики у человека. В медицине сплавы на основе никеля, палладия и золота используют как для изготовления хирургических инструментов, так и для производства различных имплантов, применяемых в ортопедии, эндоваскулярной хирургии, гинекологии и стоматологии. Аллергические реакции на металлы могут приводить к нарушениям функции искусственного сустава, тромбозу эндоваскулярных стентов, стоматитам, гингивитам. Наиболее частым проявлением аллергической реакции на металлы является контактный дерматит. Лидирующую позицию в этиологии контактного дерматита занимает никель. Диагностика аллергии на металлы заключается в постановке кожных тестов. В Российской Федерации отсутствуют отечественные диагностические системы, позволяющие выявить аллергическую реакцию на металлы. В этих целях применяется набор для диагностики аллергического контактного дерматита – тест-система «АллерТест» («TRUE Test», Дания). В связи с этим, является актуальной разработка отечественного диагностического теста для своевременного выявления аллергических реакций на металлы

STUDY OF THE EFFICIENCY AND SAFETY OF MYCOPHENOLATE MOFETIL THERAPY IN PATIENTSWITH SYSTEMIC SCLERODERMA

Interstitial lung disease (ILD) is one of the major causes of death in systemic scleroderma (SSD). Treatment of these patients remains difficult and controversial. Mycophenolate mofetil (MPM) has been in vitro shown to inhibit overproduction of type I collagen and hence may be effective against SSD. Objective: to study the efficiency and safety of MPM therapy in patients with SSD and clinically relevant ILD in an open-label prospective study. Subjects and methods. Ten patients with SSD (7 and 3 with its diffuse and limited forms, respectively) and ILD were given MPM in combination with glucocorticoids (mean daily dose was 10+4 mg). The mean MPM therapy duration was 11.4+1.3 months. The Rodnan total skin thickness score, flexion index, forced vital capacity (FVC), diffusing capacity of the lung for carbon monoxide (DLCO), and European Scleroderma Study Group (EScSG) activity index were estimated and a 6-minute walk test (6MWT) was carried out before and after MPM therapy. Results. After therapy, the whole group showed a significant reduction in skin scores from 12.9+9.8 to 5.6+3.2 (p=0.036) and EScSG from 3.9+1.4 to 2.25+1.03 (p=0.015) and an increase in exercise tolerance from 446+155 to 535+78 m (p=0.03) as evidenced by 6MWT. The degree of flexion contractures decreased from 15+21 to 3.7+11.3 mm (p>0.05). FVC (77.8+18.7% versus 73.8+11.3%) and DLCO (45+14.4% versus 42+16.4%) were significantly unchanged. A 10% or more clinically significant fall was noted in FVC and DLCO in 3 and 1 patients, respectively. In the remaining patients, the lung functional test results remained stable. MPM tolerability was satisfactory. All the patients completed their course of treatment. Conclusion. Stabilization of lung function with higher exercise tolerance and significantly reduced skin density allow therapy with MPM in combination with low-dose glucocorticoids to be regarded as an effective and well-tolerated treatment in patients with ILD in the presence of SS

Применение иммуноанализа для решения актуальных проблем стандартизации препаратов аллергенов

The present article describes principal problematic issues that refer to the standardization of allergen preparations. It is shown that at present domestic allergen preparations are being standardized by protein content in allergenic material, however, the mentioned characteristic does not reflect true allergic activity of a drug. There is a need in harmonization of domestic standardization technology for allergen preparations with the approaches of EMA (European Medicines Agency) and FDA (Food and Drug Administration), regulating their standardization in allergenic activity units, as well as the use of modern immunoassay methods for quantitative evaluation such as radioallergosorbent test, chemiluminescence analysis, immunoallergological test, enzyme-linked immunosorbent assay with inhibition phase etc.Иммунохимические методы анализа основаны на связывании исследуемого белка специфичными антителами, с последующим выявлением образовавшегося комплекса «антиген-антитело» с помощью метки, которая легко детектируется с использованием высокочувствительных устройств типа счетчиков радиоактивных частиц, спектрофотометров, флуориметров и т.п. Радиоиммунологический анализ (РИА) был разработан в 50-х годах прошлого столетия R.S. Yalow и S.A. Berson, которые в качестве метки использовали изотоп125I [1]. Применение твердых носителей (нитроцеллюлозы, полистирольных плат) для сорбции антител (АТ) или антигенов (АГ) положило начало развитию гетерогенного (твердофаз ного) анализа. Иммобилизация антигена на твердом носителе позволила предотвратить агрегацию в растворе и осуществить с помощью отмывки физическое разделение иммунокомплексов от свободных компонентов. В середине 1960-х годов появились более безопасные варианты иммуноанализа, не требующие специальных условий работы - ИФА, имму-нохемилюминесцентный анализ. В них используются специфичные АТ, меченные молекулами ферментов или специальными красителями, такими как люминол, люцегенин, флюо-ресцеина ацетат, флуоресцеина изотиоцианат. В настоящее время иммуноанализ широко применяется в научных исследованиях, в различных областях медицины, Приведены основные проблемные вопросы в стандартизации препаратов аллергенов. Показано, что в настоящее время отечественные препараты аллергенов стандартизуются по содержанию белка в аллергенном материале -показателю, не отражающему истинную аллергенную активность препарата. Назрела необходимость гармонизации отечественной технологии стандартизации препаратов аллергенов с учетом подходов ЕМА (European Medicines Agency) и FDA (Food and Drug Administration), регламентирующих стандартизацию препаратов в единицах аллергенной активности и применение для количественной оценки данного показателя современных методов иммуноанализа - радиоаллергосорбентный тест (РАСТ), хемолюминесцентный анализ, иммуноаллергосорбентный тест, иммуноферментный анализ (ИФА) с этапом ингибирования и другие

Оптимизация метода исследования маммосферообразования для оценки il-6 индуцированной стволовой пластичности дифференцированных клеток рака молочной железы

The aim of the study was mammosphere assay optimization for quantifcation of IL6-induced stemness in differentiated (СD44– ) T47D breast cancer cells.Material and Methods. The effect of three commonly used cell-detaching methods (TrypLE, accutase, cell scrapper) at various confuence (40–50 % and 70–80 %) on cell viability, phenotypic profle and mammosphere formation was tested. The cell viability was examined using AnnexinV/propidium iodide assay. The phenotypic profle was analyzed by fow cytometry with fuorescent markers CD24 and CD44.Results. Detachment of the cells using scrapper led to substantial increase in early apoptotic and late apoptotic cells in comparison with TrypLE and accutase. Dissociation with TrypLE reduced the percentage of detected CD44+ positive cells, whereas accutase saved the surface marker. The number of mammosphere and their diameter did not differ between groups. Incubation of differentiated (CD44– CD24+) T47D cells with IL-6 for 24 hours resulted in an appearance of CD44+CD24+ and CD44+CD24–/low subpopulation. Furthermore, the differentiated cells after 24 hours of IL6 exposure formed 3 times more mammospheres compared to the control.Conclusion. Usage of cells with confuence of no more than 80 % and accutase for detachment of cells is recommended for mammosphere assay. Incubation of CD44– CD24+ T47D cells with IL6 for 24 hours is suffcient for stimulation of stemness plasticity. Цель исследования – оптимизация метода оценки образования маммосфер после индуцированной интерлейкином 6 (IL6) стволовой пластичности у дифференцированных (CD44– ) опухолевых клеток линии рака молочной железы человека T47D.Материал и методы. Проведена оценка жизнеспособности, цитофлуориметрического профиля клеток и их способности к маммосферообразованию при различной исходной конфлюентности (40–50 % и 70–80 %) и методах снятия с поверхности пластика (использование TrypLE, аккутазы, скребка для клеток). Жизнеспособность клеток оценивали методом проточной цитометрии с использованием флуоресцентно-меченного АннексинV и йодистого пропидия. Динамику изменения экспрессии поверхностных маркеров клеток оценивали методом проточной цитометрии с флуоресцентно-меченными антителами к CD24 и CD44.Результаты. Применение скребка для снятия клеток приводило к существенному повышению количества клеток с цитофлуориметрическими признаками раннего и позднего апоптоза по сравнению с использованием TrypLE и аккутазы. Использование TrypLE приводило к снижению количества CD44+ клеток, тогда как аккутаза не влияла на представленность данного маркера. Количество маммосфер и их средний диаметр не отличались между исследуемыми группами. Стимуляция дифференцированных (CD44– CD24+) клеток линии T47D IL6 уже через 24 ч приводила к появлению популяций CD44+CD24+ прогениторных и CD44+CD24–/low опухолевых стволовых клеток. Дифференцированные клетки после стимуляции IL6 в течение 24 ч образовывали в 3 раза больше маммосфер по сравнению с контролем.Заключение. Для оценки маммосферообразования целесообразно использовать клетки с конфлюентностью до 80 % и применять аккутазу для снятия с поверхности пластика. Для стимуляции стволовой пластичности у дифференцированных клеток линии T47D достаточной является их инкубация с IL6 в течение 24 ч.