Proinflammatory status of oral fluid in COVID-19

At present, a search for promising ways to diagnose infection caused by SARS-CoV-2 is quite relevant. Oral fluid is not commonly used for assessment of COVID-19 risk. Its molecular profile reflects both local state of the oral cavity, and individual organs and systems, thus suggesting a reliable diagnostic platform. Systemic inflammatory response is known to play a crucial role in development of the coronavirus infection; the “cytokine storm” determines severity of the disease. The saliva-based diagnostics of clinical course in COVID-19 patients includes determination of IL-6, IL-8, C-reactive protein in oral fluid, in order to assess severity of the inflammatory process. The present study was carried out at the Department of Fundamental and Clinical Biochemistry with Laboratory Diagnostics, and Department of Pediatric Infections at the Samara State Medical University. The study involved 122 persons: 67 clinically healthy individuals comprised the control group, and the group of comparison included 55 inpatients with moderate or severe coronavirus infection (COVID-19) caused by SARS-CoV-2 virus as confirmed by PCR and/or ELISA testing. Development of the disease was accompanied by drastically increased contents of IL-6 and IL-8 in oral fluid of the patients relative to the indexes in healthy persons, i.e., several-fold for IL-6 (+ 650%) and even higher elevation of IL-8 levels (+ 26513%), as well as a 2-fold increase of C-reactive protein (+115%). When comparing the immune indexes of oral fluid in presence versus absence of respiratory insufficiency, a significant difference was found for salivary IL-6 (+173%) in the patients with grade 1-2 respiratory insufficiency as compared with patients free of respiratory disorders. Determination of these proinflammatory markers in patients with COVID-19 is of important prognostic significance when assessing development of the disease and its severity. Direct detection of their content in the oral fluid makes this method relevant, and potentially demanded for the outpatient diagnostics, being highly important during pandemics of coronavirus infection and limited medical resources. Examination of oral fluid at the pre-hospital stage is a resource-saving technology, since it does not require additional medical staff to take biomaterial, is non-invasive to the patient, and suggesting a wide range of research items, it can resolve a number of diagnostic issues, e.c., presence of specific genetic material or antibodies to SARS-CoV-2, severity of the inflammatory process and the risk of respiratory failure in the patient

Конформационные особенности лактатдегидрогеназы: влияние температурного фактора в присутствии малых молекул, математическая модель

The aim of this work was to study the conformational changes of lactate dehydrogenase under the influence of different concentrations of intermediates (pyruvate, oxaloacetate) in the temperature gradient with the  subsequent building of a mathematical model.Materials and methods. Thermolability of lactate dehydrogenase was studied using the method of differential scanning fluorimetry to determine the change in endogenous fluorescence of tryptophan and tyrosine under the conditions of stable concentration of lactate dehydrogenase and changing concentrations of pyruvate and oxaloacetate. Further, a mathematical model was developed for a more in-depth consideration of the behavior of the catalytic protein.Results. We found that pyruvate and oxaloacetate in low concentrations have a thermostabilizing effect on lactate dehydrogenase conformation; the effect of pyruvate is statistically more significant in comparison with oxaloacetate (p < 0.05). The studied ligands in high concentrations reduce the thermal stability of lactate dehydrogenase.Conclusion. Understanding the role of small molecules in the regulation of biological and catalytic processes has long remained in the background of scientific interest, but today the work in this direction is reaching a new level. The data obtained indicate the possibility of small molecules acting as ligands when interacting with enzymes.  Цель. Исследовать конформационные изменения лактатдегидрогеназы под действием различных концентраций интермедиатов (пируват, оксалоацетат) в температурном градиенте с последующим построением математической модели.Материалы и методы. Изучение термолабильности лактатдегидрогеназы проводили с  использованием метода дифференциальной сканирующей флуориметрии по изменению  эндогенной флуоресценции триптофана и тирозина в условиях стабильной концентрации лактатдегидрогеназы и изменяющихся концентраций пирувата и оксалоацетата. Далее была разработана математическая модель для более углубленного рассмотрения поведения каталитического белка.Результаты. Было выявлено, что пируват и оксалоацетат в низких концентрациях оказывают термостабилизирующее воздействие на конформацию лактатдегидрогеназы, влияние пирувата статистически более значимо в сравнении с оксалоацетатом (p < 0,05). Изучаемые лиганды в высоких концентрациях снижают термостабильность лактатдегидрогеназы.Заключение. Понимание роли малых молекул в регуляции биологических и каталитических процессов долгое время оставалось в тени научного интереса, но сегодня работа в данном направлении выходит на качественно новый уровень.  Полученные данные свидетельствуют о возможности малых молекул выступать в качестве лигандов при взаимодействии с каталитическими белками

PASS и STITCH в верификации неизвестных свойств пирувата и лактата. Обзор литературы и фрагменты собственных исследований

The aim of the study was to identify the predicted spectrum of biological activity of pyruvate and lactate using modern computer modeling methods and to determine potential protein partners in intermolecular interaction.Materials and methods. The biological activity spectrum of pyruvate and lactate by the structural formula was determined using the PASS (Prediction of Activity Spectra for Substances) software. Potential protein interaction partners for small molecules were predicted using the Search Tool for Interactions Chemicals (STITCH).Results. Analyzing the obtained results in silico reveals that pyruvate and lactate exhibit diverse biological activities, molecular mechanisms, and pharmacological effects. These include regulation of lipid, protein, and carbohydrate metabolism and effects on enzyme activity and gene expression. The data on the antihypoxic, antiischemic, antitoxic, immunomodulatory, antiinflammatory, antiviral, vasoprotective, and cytoprotective effects are presented. The neuroprotective and antineurotoxic effects of pyruvate and lactate are predicted.Conclusion. The spectrum of biological activities of lactate and pyruvate were revealed by computer modeling methods, and protein interaction partners were characterized. The small molecules we studied have a coordinating role in the functioning and modulation of mediator, hormonal, receptor, immune, inflammatory, antibacterial, and antiviral responses and gene expression. The use of natural intermediates as therapeutic agents for the treatment of ischemic stroke, acute neurological disorders, and neurodegeneration is discussed, which is underlain by the stimulating effect of metabolites on neuroplasticity. These properties may be manifested through conformational rearrangement of receptors, active binding centers, expression of multiple genes, and changes in the functional manifestations of catalytic and other proteins. The obtained data will obviously expand our understanding of the role of small molecules in intermolecular metabolite – protein interactions.Цель исследования заключается в выявлении прогнозируемого спектра биологической активности пирувата и лактата с применением современных методов моделирования, определение потенциальных белковых партнеров для межмолекулярного взаимодействия.Материалы и методы. Определение спектра биологической активности пирувата и лактата по структурной формуле проводили в программном обеспечении Prediction of Activity Spectra for Substances (PASS). Прогнозирование потенциальных белковых партнеров взаимодействия для малых молекул выполняли в системе Search Tool for Interactions Chemicals (STITCH, инструмент поиска взаимодействующих химических веществ).Результаты. Анализируя полученные результаты in silico, обращает на себя внимание проявление разнообразной биологической активности молекулярных механизмов, оказываемых фармакологических эффектов пирувата и лактата. Среди них регуляция липидного, белкового, углеводного обменов, влияние на активность ферментов, экспрессию генов. Приводятся данные антигипоксического, антиишемического, антитоксического, иммуномодулирующего, противовоспалительного, противовирусного, вазопротекторного и цитопротекторного действий. Спрогнозировано нейропротекторное, антинейротоксическое действие пирувата и лактата.Заключение. Методами компьютерного моделирования раскрыт спектр биологической активности лактата и пирувата, а также охарактеризованы белки-партнеры по взаимодействию. Изучаемые нами малые молекулы выполняют координационную роль в функционировании и модуляции медиаторного, гормонального, рецепторного ответов, иммунологических, воспалительных, антибактериальных, противовирусных реакций, экспрессии генов. Обсуждается использование естественных интермедиатов в качестве терапевтических средств для лечения ишемического инсульта, острых неврологических расстройств, нейродегенерации, что имеет в своей основе стимулирующее действие метаболитов на процессы пластичности мозга. Проявление этих свойств, вероятно, реализуется через конформационную перестройку рецепторов, активных центров связывания, экспрессии множества генов, изменение функциональных проявлений каталитических и других белков. Полученные знания, очевидно, расширят наше понимание роли малых молекул в межмолекулярных взаимодействиях метаболит–белок

Conformational features of lactate dehydrogenase: Temperature effect in presence of small molecules, mathematical model

The aim. To study the conformational changes of lactate dehydrogenase under the influence of different concentrations of intermediates (pyruvate, oxaloacetate) in the temperature gradient with the subsequent building of a mathematical model. Materials and methods. Thermolability of lactate dehydrogenase was studied using the method of differential scanning fluorimetry to determine the change in endogenous fluorescence of tryptophan and tyrosine under the conditions of stable concentration of lactate dehydrogenase and changing concentrations of pyruvate and oxaloacetate. Further, a mathematical model was developed for a more in-depth consideration of the behavior of the catalytic protein. Results. We found that pyruvate and oxaloacetate in low concentrations have a thermostabilizing effect on lactate dehydrogenase conformation; the effect of pyruvate is statistically more significant in comparison with oxaloacetate (p < 0.05). The studied ligands in high concentrations reduce the thermal stability of lactate dehydrogenase. Conclusion. Understanding the role of small molecules in the regulation of biological and catalytic processes has long remained in the background of scientific interest, but today the work in this direction is reaching a new level. The data obtained indicate the possibility of small molecules acting as ligands when interacting with enzymes. © 2020 Siberian State Medical University. All rights reserved