CRISPR/Cas9 Technique for Identification of Genes Regulating Oxaliplatin Resistance of Pancreatic Cancer Cell Line

© 2016, Springer Science+Business Media New York.Genome editing approach based on prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) system is a simple and useful way to investigate gene functions on a genome-wide scale. It is especially important for cancer research because of genetic contribution to tumor development. We applied this technique in a high-throughput screening format to find genes that could be involved in chemotherapy resistance of pancreatic cancer. We used AsPC1 cell line expressing doxycycline-inducible Cas9 to screen two sgRNA lentiviral libraries: (1) cell cycle genes (CC, 983 genes, ∼12,000 sgRNA) and (2) genome-wide (GW, ∼90,000 sgRNA). These sets of cells with different gene knockouts were treated with oxaliplatin to identify knockouts which increase sensitivity to the drug. We have performed screening both in vitro and in vivo settings. For the in vivo arm of our experiments, peritoneal carcinomatosis model in severe combined immunodeficiency (SCID) mice was created by intraperitoneal injection of AsPC1/Cas9 cells infected with sgRNA library. Genomic DNA from cells and animal tumor material was analyzed using next generation sequencing (NGS) to obtain data about representation of sgRNA. Preliminary data allowed us to identify genes potentially modulating oxaliplatin sensitivity

Autophagy induction in peripheral blood T-lymphocytes of atopic asthma patients

Autophagy is a fundamental process that ensures the regulation of T-cell homeostasis. In case of apoptosis induction disruption in the cell it could be single mechanism of the cell death. Previously was shown inhibition of lymphocyte apoptosis in patients with bronchial asthma, so the main study of this work has focused on the study development process of autophagy in T-lymphocytes of patients with bronchial asthma. The article presents the main morphological changes in cells associated with activation of autophagy (formation autophagosome). In addition to morphological changes in lymphocytes, we have shown the expression of autophagy marker protein (LC3B). We found that in T-lymphocytes of patients with severe asthma are simultaneous activation of both autophagy and apoptosis, and autophagy is a stimulus to cell death

Autophagy and LC3-associated phagocytosis: similarities and differences

Previously, autophagy was termed as a mechanism used by the cells with a lack of essential nutrients supporting homeostasis. Over the decade of studies, autophagy proved to be a more complex, ambiguous mechanism. Its activation depends on the nature of stimulus, type of immune cells and the final result. Both canonical and non-canonical autophagy, being similar in molecular events, but showing their own distinctive features, are key processes in protecting the body from penetration of intracellular pathogens, maintaining the required level of nutrients in the cell, and removing damaged organelles and cells. Canonical autophagy probably evolved as a homeostatic response to cellular stress and nutritional deficiencies, whereas non-canonical autophagy emerged as a response to suppression of inflammation. Non-canonical autophagy, hereinafter referred to as LC3-associated phagocytosis (LAP), combines the molecular mechanism of phagocytosis with an autophagy mechanism characterized by ingestion of exogenous pathogens, formation of phagosomes (laposomes) and enhanced fusion with lysosomes, followed by degradation of their contents.Significant differences were found between the processes of LAP- and canonical autophagy, which are similar in its mechanism of action. The presence of PI3K complexes in both processes, utilization and intracellular degradation of the “cargo” which is not required for the cells and organism proceeding in the lysosomes, and involvement of almost the same proteins provide similarity of their mechanisms. However, there are differences in the initiation of the processes, e.g., different types of PI3K complexes (in autophagy, PI3K III class 1 and 2 types; in LAP PI3K III, class 3 type), usage of reactive oxygen species in LAP, different types of regulatory proteins involved (ULK1, FIP200, ATG13 , Ambra1, WIPI2, ATG14 in autophagy; and Rubicon and NOX2 in LC3-associated phagocytosis), different number of layers in the membrane structure in which lysis occurs (double-membrane autophagolysosome and single-layer membrane in laposomes) clearly depict the variety of canonical and non-canonical autophagy. The two pathways are directed for different types of biological objects, i.e., intracellular pathogens, dysfunctional proteins and organelles in autophagy, and extracellular pathogens, apoptotic bodies, bacteria, utilized in LAP, thus making these mechanisms completely different in their significance.Collectively, the new data indicate that autophagy performed via both canonical and non-canonical pathways, has evolved into a host defense mechanism capable of resisting immunological and pathogenic stress and mediating immunological tolerance to both intra- and extracellular threats. The present review discusses fundamental molecular differences between these mechanisms, as well as their role in immunity, based on the latest literature data

Environmental Behavior of Youth and Sustainable Development

The relationship between people and nature is one of the most important current issues of human survival. This circumstance makes it necessary to educate young people who are receptive to global challenges and ready to solve the urgent problems of our time. The purpose of the article is to analyze the experience of the environmental behavior of young people in the metropolis. The authors studied articles and monographs that contain Russian and international experience in the environmental behavior of citizens. The following factors determine people’s behavior: the cognitive capabilities of people who determine the understanding and perception of nature and the value-affective component that determines the attitude towards nature. The next task of the study is surveying young people through an online survey and its analysis. The research was realized in Ekaterinburg, the administrative center of the Sverdlovsk region (Russia). The study of the current ecological situation in Ekaterinburg made it possible to conclude that the environmental problem arises not only and not simply as a problem of environmental pollution and other negative influences of human economic activity. This problem grows into transforming the spontaneous impact of society on nature into a consciously, purposefully, systematically developing harmonious interaction with it. The study results showed that, from the point of view of the youth of Ekaterinburg, the city’s ecological situation is one of the most pressing problems. Despite minor improvements over the past 3–5 years, this problem has not lost relevance, and regional authorities and city residents should be responsible for its solution. Young people know environmental practices, but they often do not apply them systematically. Ecological behavior is encouraged and discussed among friends/acquaintances. The key factors influencing the formation of environmental behavior practices are the mass media and social networks. The most popular social network for obtaining information on ecological practices among young people is Instagram, and the key persons are bloggers. This study did not reveal the influence of the socio-demographic characteristics of young people on the application of eco-behavior practices, which may indicate the need for a survey of a larger sample. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Acknowledgments: This research was supported by TPU development program

Nuclease activity associated with secreting granules by lymphocytes in patients with bronchial asthma

© 2015 Vodounon CA, et al.Background: We know, through recent studies, the existence of some morpho-biochemical peculiarities in the process of type 1 programmed death of patients' lymphocytes suffering from bronchial asthma, but little convincing data exist on the activity of enzymes involved in this physiological process. Therefore, the aim of our research was to study the enzymatic activity of secreting granules of patients' lymphocytes with bronchial asthma, according to the degree of severity. Method: The study was based on the role of granular extracts in the process of programmed death isolated lymphocytes from peripheral blood of relatively healthy individuals and asthmatic patients with different severity. The immunological characteristics of lymphocytes was done with the radial immune-diffusion method and ELISA test but the method of agarose gel electrophoresis help us to detect the catalytic activity of protein extracts of secreting granules of lymphocytes. Results: The results obtained showed that lymphocytes from asthmatic patients with severe severity are characterized by a decrease in cytotoxic T lymphocytes content balanced by an increase in T-Helper lymphocytes. We also noticed the enzymatic activity at all the groups studied but this activity was relatively high in asthmatics with severe severity. Furthermore, the study of the cationic dependence has allowed to establish an increase in enzymatic activity in all the groups studied after incubation of DNA in a medium containing Ca2+ with a pH of 7.5 unlike ions Mn2+ which seem to reduce the enzymatic activity. The expression of enzymatic activity in the presence of zinc allows us to suggest the presence of DNase acid in granules, which activity is not necessarily associated with divalent metal ions. Conclusion: Based on the above results, one might conclude that the secreting granules have a high enzymatic activity but with a strong cationic dependence. This not only allows a better understanding of the morphological changes observed during the course of apoptosis in lymphocytes of patients but also brings more to the knowledge of the enzymatic influence in the process of type 1 programmed death

Detection of macro-thyrotropinaemia in patients with Hashimotos thyroiditis and subclinical hypothyroidism

The level of thyroid stimulating hormone is one of the diagnostic indicators of thyroid function. In subclinical hypothyroidism, its concentration in the blood serum increases, while the level of thyroid hormones remains normal. One of the reasons for this is the phenomenon of macrotyrotropinemia, in which the macro isoforms of thyrotropin (a complex of thyrotropic hormone with immunoglobulin) are present in the blood. It is assumed that the biological activity of macrotyrotropin is low, and may accumulate in the circulation, causing a falsely elevated level of thyroid-stimulating hormone in serum. The aim of this study is to identify the nature and prevalence of the macrothyrotropinemia phenomenon among patients with subclinical hypothyroidism in presence of autoimmune thyroiditis and a group of healthy donors. Materials and methods: Fifty serum samples of venous blood served as the material for the study: 30 patients with subclinical hypothyroidism in presence of autoimmune thyroiditis; 10, with manifesting hypothyroidism, 10 conditionally healthy donors without thyroid gland pathology (control group). The group was derived from results of the clinical laboratory at the Clinical Hospital at the Kazan station railway. Patients’ blood serum was screened for the presence of macrotrorotropin by polyethylene glycol precipitation method, followed by analysis by gel filtration chromatography. Results of this study were as follows: screening of blood sera was performed by gel filtration and affinity chromatography. Polyethylene glycol was shown to precipitate 50 to 100% serum thyrotropin, of which true macrotrothropin makes 56-98%. In the patients with subclinical hypothyroidism with a thyroid-stimulating hormone level of more than 10 pIU/ml, a trend towards an increase in the level of macrothyrotrophinaemia has been shown. The content of macrotyrotropin complex in patients with subclinical hypothyroidism, in whom the level of antibodies to thyroperoxidase is > 500 U/L, is significantly higher if compared to the patients with manifesting hypothyroidism. Elevated levels of antibodies to thyroperoxidase can lead to the generation of macrotyropin. Our findings have shown that the phenomenon of macrothyrotropinemia is quite common in patients with subclinical and manifesting hypothyroidism with Hashimoto thyroiditis (53.3%) and in control group (25%). Macrotyrotropin complex probably consists of thyrotropin and IgG. Patients with a thyroid-stimulating hormone level of > 10 pIU/ml are candidates for screening for the presence of the macrotyrotropin complex.The activity of the autoimmune process may correlate with the phenomenon of macrothyrotropinemia. The results can be used to develop an additional tool when choosing therapy in clinical practice

Нокаут гистоновой метилтрансферазы NSD1 приводит к снижению пролиферации и увеличению чувствительности к цисплатину клеток плоскоклеточного рака гортани

Introduction. The histone methylation regulates gene expression and plays a role in genomic stability participating in DNA repair. Dimethylation of histone 3 lysine 36 (H3K36me2) is an important histone modification which is responsible for gene expression activation. H3K36me2 is a product of methyltransferase activity of NSD1, NSD2, NSD3, and ASH1L proteins. NSD1 mutations are known to often occur in head and neck squamous carcinoma. The presence of NSD1 mutations highly correlates with increased survival, especially for patients with laryngeal cancer. The aim of this study was an in vitro investigation of the role of NSD1 in the cell proliferation of laryngeal squamous cell cancer and non-small lung cancer cells, as well as a study of the effect of disruption of the NSD1 gene expression on cisplatin treatment response.Material and Methods. Using TCGA, correlation analysis was performed to compare NSD1 wild type and mutant patient survival. NSD1 knockout cell lines models of laryngeal and non-small cell lung cancer were developed using the CRISPR/ Cas9 system. The effect of NSD1 knockout on H3K36me2 level was evaluated by western blot. Proliferation and IC50 of cisplatin in control and knockout cells were studied as well.Results. It was demonstrated that NSD1 knockout decreased the H3K36me2 level and cell proliferation in laryngeal squamous cell cancer cells and increased the sensitivity of head and neck cancer cells to cisplatin treatment, while there was no effect of NSD1 knockout in a non-small cell lung cancer cell line.Conclusion. Based on the data obtained, it can be concluded that the NSD1 protein is a potential target for inhibitor development following in vitro and in vivo testing in head-neck squamous cell carcinoma models. More studies are needed for better understanding of the regulation of tumor cell growth by NSD1. Введение. Метилирование гистонов является одним из механизмов, участвующих в регуляции экспрессии генов и поддерживающих стабильность генома, участвуя в репарации ДНК. Диметилирование лизина 36 на гистоне 3 (H3K36me2) является одной из важнейших гистоновых модификаций, которая характеризуется как эпигенетическая метка, ответственная за активацию экспрессии генов. H3K36me2 – продукт ферментной активности белков NSD1, NSD2, NSD3 и ASH1L. Известно, что мутации гистоновой метилтрансферазы NSD1 часто встречаются при плоскоклеточном раке головы и шеи, и наличие мутаций NSD1 положительно коррелирует с повышенной выживаемостью пациентов. Особенно эта тенденция выражена у пациентов с раком гортани.Целью исследования стали изучение роли NSD1 в росте клеток плоскоклеточного рака гортани и немелкоклеточного рака легкого и оценка влияния нарушения экспрессии NSD1 на чувствительность клеток к цисплатину.Материалы и методы. С использованием базы данных TCGA был проведен корреляционный анализ между наличием мутаций в гене NSD1 и выживаемостью пациентов. С помощью системы геномного редактирования CRISPR/Cas9 были созданы клеточные линии рака гортани и немелкоклеточного рака легкого с нокаутом гена NSD1. Методом иммуноблоттинга был оценен эффект нокаута NSD1 на уровень H3K36me2 в контрольных и нокаутных клетках. Также были проведены тест на пролиферацию клеток и тест на определение жизнеспособности клеток под действием цисплатина.Результаты. Нокаут NSD1 эффективно снижает уровень H3K36me2 в клеточных линиях рака гортани и рака легкого. Также на созданных клеточных моделях было продемонстрировано, что нокаут NSD1 значительно понижает пролиферативную активность клеток рака гортани и повышает эффективность лечения цисплатином, в то время как в клетках немелкоклеточного рака легкого такого эффекта обнаружено не было.Заключение. На основе полученных данных можно сделать вывод о том, что белок NSD1 является потенциальной мишенью для разработки ингибиторов с последующим тестированием in vitro и in vivo на моделях плоскоклеточного рака головы и шеи, особенно рака гортани. Для более детального понимания того, как NSD1 регулирует рост опухолевых клеток, нужны дополнительные исследования

Излечение саркоидоза по данным диспансерного наблюдения 1992— 1999 гг.

Sarcoidosis coruse was analyzed in 103 patients with long-term loss of sarcoidosis activity and 25 recovered ones. Favourable factors included well-timed diagnosing of the disease, initial moderate inhibition of immune system, an absence of pronounced bronchial obstruction. An acute course with fever, accelerated erythrocytes settling rate and lymphocytosis of broncho-alveolar lavage fluid are not considered to be signs of poor outcome.Анализировалось течение саркоидоза у 103 больных с длительной потерей активности процесса и у 25 человек с излечением. К числу благоприятных факторов течения отнесены: своевременное выявление заболевания, умеренно выраженное исходное снижение иммунитета, отсутствие выраженных обструктивных изменений бронхов. Острое течение с лихорадкой, высокой СОЭ, УЭ и лимфоцитозом БАС не являются признаками плохого прогноза

Rugged Single Domain Antibody Detection Elements for Bacillus anthracis Spores and Vegetative Cells

Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs) were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors

Эффективность и безопасность эрибулина при различных подтипах рака молочной железы: данные из реальной клинической практики в России

The article presents a pooled experience of the use of eribulin in the real clinical practice of treatment of metastatic breast cancer in Russian oncological institutions. The effectiveness of the drug in monotherapy with HER2‑negative breast cancer was analyzed, groups of patients with most effective use of eribulin were identified depending on the localization of metastases, the most effective lines of therapy. The effectiveness of the drug in combination with trastuzumab in HER2‑positive breast cancer is described, as well as toxic reactions. В статье представлен обобщенный опыт применения эрибулина в реальной клинической практике онкологических учреждений РФ при метастатическом раке молочной железы. Проанализирована эффективность препарата в монотерапии при HER2-отрицательном раке молочных желез, выделены группы больных в зависимости от локализации метастазов, линии терапии, в которых препарат оказался максимально эффективным. Описана эффективность препарата в комбинации с трастузумабом при HER2-положительном раке молочной железы, а также токсические реакции.