Определение концентрации холестерина и триглицеридов в экстраклеточных везикулах сыворотки крови с помощью коммерческих наборов

Exosomes and microvesicles, collectively referred to as small extracellular vesicles (sEV) are vesicles with an average size of about 100-150 nm. Currently, the role of sEV in various aspects of signaling in the body is being actively investigated; in addition, sEV can often serve as markers of various pathologies. The active study of the sEV composition is continuing. In this study we have demonstrated that in sEV it is possible to determine cholesterol and triglycerides concentration by using commercial kits designed for serum. The technique was tested on sEV from the blood of patients diagnosed with depression and on healthy volunteers. No differences were found in the concentration of cholesterol and triglycerides in mEV from the blood serum of depressed patients and the control group. The concentration of cholesterol and triglycerides in the samples is several times higher than the sensitivity threshold of the methods set by the manufacturer of the kits.Экзосомы и микровезикулы, совместно называемые малые экстраклеточные везикулы (мЭВ), представляют собой везикулы со средним размером около 100-150 нм. В настоящее время активно исследуется роль мЭВ в самых разных аспектах сигналинга в организме, кроме того, часто мЭВ могут служить маркерами разных патологий. Продолжается активное изучение состава мЭВ. В данной работе мы показали, что в мЭВ можно определить концентрацию холестерина и триглицеридов с помощью коммерческих наборов, предназначенных для сыворотки крови. Методика была опробована на мЭВ из крови пациентов с диагнозом депрессия и на здоровых добровольцах. Различий в концентрации холестерина и триглицеридов в мЭВ из сыворотки крови пациентов с депрессией и контрольной группы найдено не было. Концентрация холестерина и триглицеридов в образцах в несколько раз превосходит порог чувствительности методов, установленный производителем наборов

Чувствительность экстраклеточных везикул из сыворотки крови человека к различным детергентам

Blood exosomes and microvesicles, collectively known as small extracellular vesicles (sEV), are vesicles about 100-150 nm in size. Small EV are involved in various aspects of signaling in the body; in addition, they can serve as markers of various pathologies. For biochemical studies, vesicle solubilization is often required. We tested the ability of various detergents to dissolve membranes of the sEV. Small EV were isolated from the blood serum of healthy volunteers by gel filtration on Sepharose CL-2B and tried to solubilize them using the anionic detergent DOC (sodium deoxycholate), non-ionic detergent Brij 35 (polyoxyethyleneglycol dodecyl ether), zwitterionic detergent CHAPS (3 - [(3-chloramidopropyl) dimethylammonio] -1-propanesulfonate), and cationic detergent CTAB (cetyl trimethylammonium bromide). The concentration of sEV in the solution was determined by dynamic light scattering. We find DOC is the most effective for sEV solubilization.Экзосомы и микровезикулы, совместно называемые малые экстраклеточные везикулы (мЭВ), представляют собой везикулы размером около 100-150 нм. Малые ЭВ принимают участие в самых разных аспектах сигналинга в организме; кроме того, они могут служить маркерами разных патологий. Для биохимических исследований зачастую требуется солюбилизация везикул. Мы проверили способность различных детергентов растворять мембраны мЭВ. Малые ЭВ выделяли из сыворотки крови здоровых добровольцев с помощью гель-фильтрации на Sepharose CL-2B и пытались их солюбилизировать с помощью анионного детергента DOC (дезоксихолат натрия), неионного детергента Brij 35 (додециловый эфир полиэтиленгликоля), цвиттерионного детергента CHAPS (3-[(3-хлорамидопропил) диметиламмонио]-1-пропансульфонат), катионного детергента CTAB (цетил триметиламмония бромид). Концентрацию мЭВ в растворе определяли с помощью динамического светорассеяния. Самым эффективным детергентом для солюбилизации мЭВ из сыворотки крови оказался DOC

Assessment of caspase 3 activity in rabbit myocardial tissue during experimental hemodynamic overload of the left ventricle of the heart

It is well known that chronic overload of the cardiac left ventricle is accompanied by an increase in the cardiomyocyte apoptosis rate. However, direction and extent of changes in programmed cell death under an acute overload of the left ventricle still requires detailed investigation (as its pathogenesis significantly differs from chronic overload). Caspase-3 activity has been investigated in left ventricle myocardium of rabbits on days 1, 3, and 5 after modeling of left ventricle hemodynamic overload caused by experimental stenosis of the ascending aorta. Control group included intact animals. It was found that caspase-3 activity significantly increased in both ventricles on day 1; it increased more than twofold above control values on day 3 and decreased up to nearly control values on day 5. Based on these data it was concluded that the acute hemodynamic overload of the left ventricle may be a cause of increased apoptosis in the myocardial tissue of both cardiac ventricles during first days of the pathological process. © Pleiades Publishing, Ltd., 2011

Assessment of caspase 3 activity in rabbit myocardial tissue during experimental hemodynamic overload of the left ventricle of the heart

It is well known that chronic overload of the cardiac left ventricle is accompanied by an increase in the cardiomyocyte apoptosis rate. However, direction and extent of changes in programmed cell death under an acute overload of the left ventricle still requires detailed investigation (as its pathogenesis significantly differs from chronic overload). Caspase-3 activity has been investigated in left ventricle myocardium of rabbits on days 1, 3, and 5 after modeling of left ventricle hemodynamic overload caused by experimental stenosis of the ascending aorta. Control group included intact animals. It was found that caspase-3 activity significantly increased in both ventricles on day 1; it increased more than twofold above control values on day 3 and decreased up to nearly control values on day 5. Based on these data it was concluded that the acute hemodynamic overload of the left ventricle may be a cause of increased apoptosis in the myocardial tissue of both cardiac ventricles during first days of the pathological process. © Pleiades Publishing, Ltd., 2011

The Missing Link: How Exosomes and miRNAs can Help in Bridging Psychiatry and Molecular Biology in the Context of Depression, Bipolar Disorder and Schizophrenia

MicroRNAs (miRNAs) only recently have been recognized as promising molecules for both fundamental and clinical neuroscience. We provide a literature review of miRNA biomarker studies in three most prominent psychiatric disorders (depression, bipolar disorder and schizophrenia) with the particular focus on depression due to its social and healthcare importance. Our search resulted in 191 unique miRNAs across 35 human studies measuring miRNA levels in blood, serum or plasma. 30 miRNAs replicated in more than one study. Most miRNAs targeted neuroplasticity and neurodevelopment pathways. Various limitations do not allow us to make firm conclusions on clinical potential of studied miRNAs. Based on our results we discuss the rationale for future research investigations of exosomal mechanisms to overcome methodological caveats both in studying etiology and pathogenesis, and providing an objective back-up for clinical decisions. © 2019, Springer Science+Business Media, LLC, part of Springer Nature

Оптимизация иммуноферментного метода для определения цилиарного нейротрофического фактора в слезной жидкости человека

The concentration of ciliary neurotrophic factor (CNTF) was measured in lacrimal fluid (LF) using Human CNTF Quantikine ELISA kit (“R&D Systems”, USA) on a ChemWell 2910 automatic analyzer (“Awareness Technology Inc.”, USA). We initially attempted to use commercial kits, designed for serum and plasma CNTF detection, to quantify lacrimal CNTF. The results, however, were rarely above the minimum detection level of the kits, most likely due to matrix complexity and low concentrations of CNTF in diluted LF (LF had to be diluted because of the small volume of collected samples). The optimal sensitivity and the lowest background for the best minimum quantifiable value were determined empirically. Phosphate buffer solution containing 1% bovine serum albumin was selected as an optimal diluent for CNTF measurements in small fluid samples. A standard curve was produced using the calibrating solutions 0-250 pg/ml. Acid treatment of LF samples before the analysis allowed to increase the detectable concentration of the CNTF two-fold. The 1:3 dilution was selected based on the available volume of collected LF and a reasonable variation coefficient. The described protocol allowed to develop a sandwich ELISA optimized for lacrimal CNTF.Определение цилиарного нейротрофического фактора – ciliary neurotrophic factor (CNTF) в слёзной жидкости (СЖ) человека рутинными лабораторными методами затруднено из-за его низкой концентрации в структурах глаза и недостаточного количества материала для анализа. Были подобраны условия для определения CNTF в СЖ твердофазным методом иммуноферментного анализа (ИФА) на основе набора Human CNTF Quantikine ELISA Kit (“R&D Systems”, США) с использованием автоматического иммуноферментного анализатора ChemWell 2910 Combi (“Awareness Technology Inc.”, США). Предварительная кислотная обработка проб СЖ позволила вдвое повысить концентрацию CNTF. Полностью автоматизированная система проведения анализа и выбор разбавителя с низкой оптической плотностью бланка (0.011) обеспечили линейную зависимость калибровочного графика от 0 до 250 пг/мл с возможностью его использования для расчета концентрации CNTF в области низких значений. Рабочие характеристики оптимизированной тест-системы ИФА (линейность метода, предел обнаружения, воспроизводимость, интерференция матрицы) в указанном диапазоне концентраций соответствовали критериям приемлемости для используемого метода определения. Разведение образцов СЖ в четыре раза, совмещённое с кислотной обработкой проб, оказалось оптимальным для получения стабильных результатов при проведении анализа. В этом случае концентрация CNTF в пробе составляла в среднем 10.4±0.4 пг/мл с коэффициентом вариации 4.2% и близостью определяемых значений к ожидаемым величинам на уровне 104%

Elevated Levels of Serum Exosomes in Patients with Major Depressive Disorder

Exosomes are small (30-100 nm) vesicles of the endocytic membrane that may be found in the endosome system of multivesicular organs and are released after fusion of multivesicular bodies with the plasma membrane. Exosomes function as carriers for specific loads and are stable in biological fluids; hence, exosomes are considered as potential biomarkers of different diseases. The degree of involvement and functional significance of exosomes in pathogenesis of depression have been poorly studied. The development of diagnostic methods for depressive disorders based on the analysis of circulating exosomes is very promising, despite the methodological complications related to their isolation and identification. The purpose of this study was to isolate exosomes from the blood serum of patients with major depressive disorder (MDD) and compare them with healthy volunteers using different methods of evaluation. The concentration of serum exosomes assessed using the methods of dynamic light scattering, nanoparticle tracking analysis, and the enzyme-linked immunoassay was statistically elevated in patients with MDD compared to healthy volunteers. The possible connection between the increased level of serum exosomes with the cell-mediated immune activation that is seen in depression is discussed

Properties and Prospects of Application of the Whey Protein Lactoferrin in Medicine and Veterinary Medicine (Review)

Introduction. Lactoferrin is a biologically active substance, a natural glycoprotein from the group of iron-containing proteins, transferrin. This article is devoted to a review of the literature data on the properties and prospects for the use of lactoferrin in medicine and veterinary medicine. Text. The problem of widespread use of lactoferrin is the need to use a large volume of raw materials, the difficulty of extracting it from agricultural raw materials and subsequent purification. In this view, at present, it is very promising to use transgenic animals to obtain such a valuable compound in the required volume. Discovered in 1939 in cow's milk, lactoferrin exhibits a wide spectrum of biological activity, new properties of this protein are constantly being established. The most studied are the immunomodulatory effects, anti-inflammatory, antimicrobial, antiviral, antifungal activities of lactoferrin, and the search for the possibility of using this compound for the treatment and prevention of the new coronavirus infection SARS-CoV-2, as well as for the prevention of postCOVID complications, has begun. In recent years, the antitumor activity of lactoferrin has been actively studied, as well as the prospects for its use as a biomarker for early diagnosis of neurodegenerative diseases. Lactoferrin can play a special role in the delivery of drugs to target organs, as well as in the manufacture of functional food products and various drugs for both humans and animals. Conclusion. Thus, the performed theoretical study and the data obtained on a wide spectrum of biological activity of lactoferrin confirm the prospects and expediency of the development of formulations and technology of drugs, functional food products, veterinary drugs and animal products based on lactoferrin. © 2022, Center of Pharmaceutical Analytics. All rights reserved