Белки CRABP – родственники или однофамильцы?

Retinoic acid being the most active metabolite of vitamin A (retinol) regulates the wide spectrum of physiological processes including embryonic development, development of immune response, hematopoiesis, glucose and lipids metabolism, etc. Retinoic acid participates in the regulation of such important aspects of life-sustaining activity as cell differentiation, proliferation and programmed cell death. This review is focused on comparison of two highly homological members of lipid-binding proteins family, CRABP1 and CRABP2. Although binding of retinoic acid is the only known function of these proteins the physiological meaning this interaction seems to be rather different. CRABBP2 binding of retinoic acid leads to the activation of RAR / RXR nuclear receptors, that act as transcription factors, and further stimulation of expression of numerous retinoic responsive genes. The meaning of CRABP1 binding of retinoic acid is less clear. Some data evidences for the similar action of CRABP1 and CRABP2 in regard to the potentiation of the retinoic acid effect, while the majority of data points on the opposite role of CRABP1, that is reduction of intracellular concentration of retinoic acid and / or decrease of retinoic acid bioavailability through the potentiation of its catabolism or sequestration in the cytosol. The most recent publications also suggest some additional functions of these proteins that could be independent of retinoic acid signalling. The data concerning the roles of these proteins in carcinogenesis and tumor progression are contradictive as well.This review covers the functions of retinoic acid as well as the molecular mechanisms mediating its activity including the different aspects of retinoic acid receptors activity. The review also comprises the comparative structural-functional analysis of CRABP proteins and probable mechanisms of their intracellular activity including those associated with retinoic acid signalling and retinoic acid-independent. A special attention is drawn to the analysis of the data on the involvement of CRABP proteins in the carcinogenesis and tumor progression. The data pointing on either oncogenic or tumor-suppressive functions are given for each protein.Ретиноевая кислота (РК) – наиболее активный метаболит витамина А (ретинола), регулирующий широкий спектр физиологических процессов в организме, в том числе эмбриональное развитие, формирование иммунного ответа, гемопоэз, метаболизм глюкозы и липидов и др. РК участвует в регуляции важнейших аспектов жизнедеятельности клеток, включая дифференцировку, пролиферацию и программируемую клеточную гибель. Обзор посвящен сравнению 2 высокогомологичных представителей семейства внутриклеточных липидсвязывающих белков CRABP1 и CRABP2, основная и единственно установленная на сегодняшний день функция которых – внутриклеточное связывание РК. Однако значение данного связывания, по-видимому, различно. Связывание СRABP2 с РК приводит к активации ядерных рецепторов (RAR / RXR), являющихся транскрипционными факторами, и последующей стимуляции экспрессии целого ряда ретиноид-респонсивных генов. Значение связывания CRABP1 с РК менее понятно. Есть свидетельства сходного действия белков CRABP1 и CRABP2 в отношении усиления эффекта РК, однако большая часть данных указывает на противоположную роль CRABP1 – снижение внутриклеточной концентрации и / или уменьшение биодоступности РК за счет усиления ее метаболизма или секвестрирования в цитоплазме. Результаты последних исследований указывают на то, что у белков СRABP могут быть функции, не связанные с проведением сигнала от РК. Также противоречивы данные о роли этих белков в канцерогенезе и опухолевой прогрессии. В обзоре рассматриваются функции РК и молекулярные механизмы, опосредующие ее активность, включая различные аспекты функционирования рецепторов РК, приводится сравнительный анализ структурно-функциональных характеристик белков CRABP и рассматриваются возможные механизмы их внутриклеточной активности, как связанные с РК, так и независимые от ретиноевого сигналинга. Особое внимание уделено анализу данных о связи белков CRABP с канцерогенезом и их участию в опухолевой прогрессии, в том числе указывающих как на опухоль-супрессорную функцию, так и на протуморогенную активность

Production and characterisation of a SARS-CoV-2 S-protein RBD homodimer with increased avidity for specific antibodies

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies.The aim of the study was to obtain and characterise a SARS-CoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies.Materials and methods: the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved mono clonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones.Results: the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD’s ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30–50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies.Conclusions: the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein

Получение и характеристика гомодимерной формы RBD S-белка SARS-CoV-2, обладающей повышенной авидностью к специфическим антителам

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study w as t o obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Materials and methods: the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Results: the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD’s ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30–50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusions: the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Важным параметром, оцениваемым при мониторинге иммунной прослойки у населения и эффективности вакцинации населения, является уровень вируснейтрализующих антител. Разработка подхода к выявлению вируснейтрализующих антител к вирусу SARS-CoV-2 с помощью безопасного, простого и быстрого метода, не требующего использования живых вирусов, имеет большое значение для борьбы с пандемией COVID-19. Для разработки тест-систем для проведения иммуноферментного анализа (ИФА), детектирующих потенциально вируснейтрализующие антитела, необходимо получение высокоочищенного рекомбинантного рецептор-связывающего домена (RBD) S-белка, обладающего высокой авидностью к специфическим антителам. Цель работы: получение и характеристика гомодимерной формы RBD S-белка вируса SARS-CoV-2, а также клеточной линии, продуцирующей рекомбинантный RBD, для создания ИФА тест-системы для выявления потенциально вируснейтрализующих антител. Материалы и методы: дизайн генетической конструкции проводили in silico. Стабильную клеточную линию получали при помощи трансфекции клеток CHO-S, селекции на антибиотике и отбора оптимального клона. Рекомбинантный RBD очищали с использованием хроматографических методов, получали мономерную и гомодимерную формы RBD. Активность полученных форм анализировали с использованием методов Вестерн-блот, биослойной интерферометрии и непрямго ИФА. Для анализа использовали моноклональные антитела GamXRH19, GamP2C5 и h6g3, а также образцы сывороток крови добровольцев, вакцинированных препаратом Гам-КОВИД-Вак, и невакцинированных добровольцев. Результаты: получена клеточная линия CHO-S, стабильно продуцирующая рекомбинантный RBD S-белка вируса SARS-CoV-2. Показано, что при культивировании данной клеточной линии в режиме fed-batch более 7 суток рекомбинантный RBD способен образовывать гомодимеры за счет наличия неспаренных цистеинов. Количественный выход очищенного рекомбинантного RBD из культуральной жидкости составил 30–50 мг/л. Мономерная и гомодимерная формы RBD были разделены при помощи гель-фильтрации и охарактеризованы по способности взаимодействовать со специфическими моноклональными антителами, а также сыворотками крови от вакцинированных добровольцев. Продемонстрировано, что именно гомодимерная форма рекомбинантного RBD обладает повышенной авидностью к моноклональным антителам и антителам в сыворотке крови вакцинированных. Выводы: гомодимерная форма рекомбинантного RBD может являться более предпочтительной для анализа уровня антител к рецептор-связывающему домену S-белка вируса SARS-CoV-2

rAAV expressing recombinant antibody for emergency prevention and long-term prophylaxis of COVID-19

IntroductionNumerous agents for prophylaxis of SARS-CoV-2-induced diseases are currently registered for the clinical use. Formation of the immunity happens within several weeks following vaccine administration which is their key disadvantage. In contrast, drugs based on monoclonal antibodies, enable rapid passive immunization and therefore can be used for emergency pre- and post-exposure prophylaxis of COVID-19. However rapid elimination of antibody-based drugs from the circulation limits their usage for prolonged pre-exposure prophylaxis.MethodsIn current work we developed a recombinant adeno-associated viral vector (rAAV), expressing a SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibody P2C5 fused with a human IgG1 Fc fragment (P2C5-Fc) using methods of molecular biotechnology and bioprocessing.Results and discussionsA P2C5-Fc antibody expressed by a proposed rAAV (rAAV-P2C5-Fc) was shown to circulate within more than 300 days in blood of transduced mice and protect animals from lethal SARS-CoV-2 virus (B.1.1.1 and Omicron BA.5 variants) lethal dose of 105 TCID50. In addition, rAAV-P2C5-Fc demonstrated 100% protective activity as emergency prevention and long-term prophylaxis, respectively. It was also demonstrated that high titers of neutralizing antibodies to the SARS-CoV-2 virus were detected in the blood serum of animals that received rAAV-P2C5-Fc for more than 10 months from the moment of administration.Our data therefore indicate applicability of an rAAV for passive immunization and induction of a rapid long-term protection against various SARS-CoV-2 variants

SF-SRGAN: PROGRESSIVE GAN-BASED FACE HALLUCINATION

Facial hallucination is a technique that has emerged recently thanks to advances in deep learning. It can be used in various tasks such as face recognition in the wild, human identification, pedestrian re-identification, face analysis, and so on. We propose a wavelet-integrated trained face hallucination model to synthesize photorealistic face images called SF-SRGAN. The multi-stage progressive hallucination strategy is based on GAN architecture. The proposed generator consists of sequential cascade modules, each of which increases the scale by 2×. Each module has a complex structure of two branches: a progressive face hallucination branch for feature extraction and reconstruction and edge-preserving branch for high frequency detail extraction. The main difference from other progressive GAN-based face hallucination networks is that the two branches fuse followed by each cascade 2×. The model is trained and tested on popular public face datasets such as the CelebA-HQ dataset, the LFW dataset, and the Helen dataset with promising photorealistic results