Синтез цис- та транс-3-(4-гідроксифеніл)циклобутанкарбонових кислот та дослідження їх похідних як лігандів рецептора GPR-40

Aim. To synthesize cis- and trans-isomers of 3-(4-hydroxyphenyl)cyclobutanecarboxylic acid and evaluate the biological activity of their derivatives against GPR-40.Results and discussion. Cis- and trans-isomers of 3-(4-hydroxyphenyl)cyclobutanecarboxylic acid were synthesized. The derivatives of this compound were tested as GPR-40 agonists and exhibited the micromolar activity.Experimental part. The methyl ester of 3-(4-hydroxyphenyl)cyclobutanecarboxylic acid was obtained as a mixture of cis/trans-isomers in 3 steps starting from a commercially available 3-oxocyclobutanecarboxylic acid. Further transformation of this compound into isomerically pure 3-(4-hydroxyphenyl)cyclobutanecarboxylic acids was achieved in five steps based on the chromatographic separation of diastereomeric amide derivatives. New GPR-40 ligands were obtained by O-alkylation of a phenolic oxygen atom of the corresponding carboxylic acid methyl ester. The biological activity of the agonists synthesized was studied using a fluorometric bioassay and the engineered Chinese hamster ovary (CHO) stable cell line expressing the human GPR-40.Conclusions. An effective synthetic approach to 3-(4-hydroxyphenyl)cyclobutanecarboxylic acid allowing to isolate two single cis/trans-stereoisomers of this compound has been developed. In order to demonstrate the possibility for the bioisosteric replacement of the ethylene moiety in the structures of free fatty acid receptor (FFAR) agonists by the cyclobutane ring, four new GPR-40 ligands possessing the micromolar activity have been synthesized.Received: 22.08.2020 Revised: 11.10.2020 Accepted: 17.10.2020Мета. Синтезувати цис- та транс-ізомери 3-(4-гідроксифеніл)циклобутанкарбонової кислоти та виявити біологічну активність їх похідних щодо рецептора GPR-40.Результати та їх обговорення. Було синтезовано цис- та транс-ізомери 3-(4-гідроксифеніл)циклобутанкарбонової кислоти, а також їх похідні, що виявили мікромолярну активність як агоністи рецептора GPR-40.Експериментальна частина. Метиловий естер 3-(4-гідроксифеніл)циклобутанкарбонової кислоти було одержано у вигляді суміші цис- та транс-ізомерів у три стадії, виходячи з комерційно доступної 3-оксоциклобутанкарбонової кислоти. Подальше перетворення цієї речовини на ізомерно чисті 3-(4-гідроксифеніл)циклобутанкарбонові кислоти було досягнуто у п’ять стадій, що ґрунтувалося на хроматографічному розділенні діастереомерних амідних похідних. Нові ліганди рецептора GPR-40 одержано шляхом О-алкілування за фенольним атомом оксигену відповідного метилового естеру кислоти. Біологічну активність синтезованих агоністів досліджено на стабільній лінії клітин яєчників китайського хом’яка (CHO), яка експресує людський рецептор GPR-40, за допомогою флуориметричного біотесту.Висновки. Розроблено ефективний підхід до синтезу 3-(4-гідроксифеніл)циклобутанкарбонової кислоти, що дозволяє одержати її у вигляді двох індивідуальних цис/транс-стереоізомерів. Для демонстрації можливості біоізостерної заміни етиленової ланки в структурі агоністів рецепторів вільних жирних кислот (FFAR) на циклобутанове кільце було також синтезовано чотири нові ліганди GPR-40, що мали мікромолярну активність.Received: 22.08.2020 Revised: 11.10.2020 Accepted: 17.10.202

Mechanisms of autoimmune pathology in post-COVID syndrome

One of the delayed consequences of SARS-CoV-2 infection is post-acute COVID-19 – polymorphic disorders of various organ systems that affect COVID-19 convalescents and persist for more than four weeks after an acute infection. Due to the infectious nature of the COVID-19, we would like to pay special attention to complications from the immune system, especially concomitant and new-onset autoimmune pathology. This review analyzes the current state of the issue of post-acute COVID-19 complications, discusses the molecular features of the SARS-CoV-2 virus and the mechanisms underlying the impaired immune response during acute COVID-19 infection and the occurrence of autoimmune and autoinflammatory conditions during convalescence. Particular attention is paid to the molecular mimicry of antigenic determinants of the SARS-CoV-2 virus, which are structurally similar to the epitopes of human autoantigens. The current data on post-acute COVID-19 autoimmune complications from humoral immunity and the endocrine system, as well as reproductive disorders faced by male patients are presented. For the first time, we hypothesize a role of the structural homology of the human SOX13 autoantigen (HMG box factor SOX13) associated with diabetes mellitus and SARS-CoV-2 envelope (E) protein in the development of the post-acute COVID-19 autoimmune pathologies. Due to the structural similarity of the two proteins and the overlap of their immunogenic regions, we suggest that the increased risk of developing diabetes mellitus and reproductive disorders in men after suffering from COVID-19 may be associated with immunological cross-reactivity

Aberrant expression of selenium-containing glutathione peroxidases in clear cell renal cell carcinomas

Aim: To find putative diagnostic markers for clear cell renal cell carcinomas (ccRCC). Material and methods: Quantitative polymerase chain reaction (Q-PCR), bisulfite treatment, methylation-specific PCR, analysis on cBioPortal for Cancer Genomics. Results: We have found that expression of GPX 1, GPX3, and GPX4 genes was decreased in ccRCC. We have shown that the number of alanine (GCG) repeats at the amino terminus of the GPX1 protein is variable. It was reported earlier that an allele that possess 5 alanine repeats is associated with the increased cancer risk. According to the obtained data, the allele with the 5 alanine repeats was also present in a group of healthy donors. Moreover, the frequency of alleles with repeats was similar among ccRCC patients and healthy individuals. We found that decreased expression of GPXs genes was not associated with promoter methylation. To provide other explanation, an analysis on the gene copy number was performed. We have found the heterozygous deletions for GPX1 gene, amplification for GPX3 gene, and no change in gene copy number for GPX4. Conclusions: Our data support the hypothesis that GPX1, GPX3, and GPX4 genes may play a role in ccRCC cancerogenesis and therefore they might be considered as putative diagnostic markers for ccRCC. Key Words: clear cell renal cell carcinomas, selenium-containing GPXs, genetic and epigenetic regulation

6-gene promoter methylation assay is potentially applicable for prostate cancer clinical staging based on urine collection following prostatic massage

The detection of prostate cancer (PCa) biomarkers in bodily fluids, a process known as liquid biopsy, is a promising approach and particularly beneficial when performed in urine samples due to their maximal non‑invasiveness requirement of collection. A number of gene panels proposed for this purpose have allowed discrimination between disease‑free prostate and PCa; however, they bear no significant prognostic value. With the purpose to develop a gene panel for PCa diagnosis and prognosis, the methylation status of 17 cancer-associated genes were analyzed in urine cell‑free DNA obtained from 31 patients with PCa and 33 control individuals using methylation‑specific polymerase chain reaction (MSP). Among these, 13 genes indicated the increase in methylation frequency in patients with PCa compared with controls. No prior association has been reported between adenomatosis polyposis coli 2 (APC2), homeobox A9, Wnt family member 7A (WNT7A) and N‑Myc downstream‑regulated gene 4 protein genes with PCa. The 6‑gene panel consisting of APC2, cadherin 1, forkhead box P1, leucine rich repeat containing 3B, WNT7A and zinc family protein of the cerebellum 4 was subsequently developed providing PCa detection with 78% sensitivity and 100% specificity. The number of genes methylated (NGM) value introduced for this panel was indicated to rise monotonically from 0.27 in control individuals to 4.6 and 4.25 in patients with highly developed and metastatic T2/T3 stage cancer, respectively. Therefore, the approach of defining the NGM value may not only allow for the detection of PCa, but also provide a rough evaluation of tumor malignancy and metastatic potential by non‑invasive MSP analysis of urine samples

Scalable Production and Purification of Adeno-Associated Viral Vectors (AAV).

Here we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium in orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI) mediated transfection of a 2-plasmid system and is specified for production in milliliter to liter scales. After PEI and plasmid DNA (pDNA) complex formation the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 3-day batch process, cell cultures are further processed using different methods for lysis and recovery. Methods for the purification of viral particles are described, including iodixanol gradient purification, immunoaffinity chromatography, and ultrafiltration, as well as quantitative PCR to quantify vector titer

Isolation and whole genome sequencing of a lipophilic anaerobic bacterium, a representative of the species complex <i>Corynebacterium tuberculostearicum</i>, from a tuberculosis focus

Background. The study of the lower respiratory tract microbiome has been actively developed inrecent years with the help of whole genome sequencing (WGS) methods. Due to this, it became clear that the nature of the lungs microbiota is very different from other microbial communities inhabiting the human body. One of the important directions in the study of pathological lungs biocenosis is the study of the role of the satellite microbiota of the tuberculosis focus. The aim of the work. To isolate and characterize oxygen-tolerant anaerobes from the necrotic contents of tuberculomas. Materials and methods. Biopsy material from 5 patients with pulmonary tuberculosis was obtained during a planned surgical treatment of tuberculoma. A pure culture was isolated from one sample during anaerobic cultivation. Lipase activity of strain was determined by plating on brain heart infusion agar (HIMEDIA, India) supplemented with 0.1 % Tween-80 and 10 mM of CaCl2. Antibiotic susceptibility was determined by RAPMYCO and SLOWMYCO of TREK Diagnostic Systems (Thermo Fisher Scientific, USA). DNA from the sediment of the broth culture was isolated by the CTAB chloroform method. Whole genome sequencing was performed on a DNBSeq-G400 NGS sequencer by Genomed (Russia). Results. Based on WGS results and phylogenetic analysis, the strain was identified as Corynebacterium kefirresidentii. The strain was characterized by high lipase activity and resistance only to Isoniazid, Ethionamide and Trimethoprim/Sulfamethoxazolin. Conclusion. The isolation of a lipophilic anaerobic representative of the Corynebacterium tuberculostearicum species complex from a tuberculous focus indicates a  possible role of the non-tuberculous microbiota in the liquefaction of caseous necrosis. We assumed that in some cases, favorable conditions are created inside the tuberculous focus for the development of satellite anaerobic lipophilic microbiota

Online service for interpretation of the resistance prediction results to bedaquiline by the molecular data

Background. Bedaquiline is a new and promising anti-tuberculosis drug, but longterm use requires resistance. This is due to mutations in the atpE and mmpR genes in M. tuberculosis (MBT).The aim of the research was to test a system for automated interpretation of results for predicting resistance to bedaquiline by the molecular data.Materials and methods. DNA was isolated from strains of M. tuberculosis in the Irkutsk region and Yakutia. The total quantity of DNA samples was 27 strains from Yakutia and 21 strains from the Irkutsk region. The study of MBT genomes was carried out on the DNA previously obtained by the authors in the territories of the Irkutsk region (n = 5), Yakutia (n = 4), Buryatia (n = 3), Zabaykalskiy kray (n = 4) and the Far East (n = 8). We used the BSATool program to detect bedaquiline resistance based on  Sanger and genomic data. Sanger sequencing analyzed the atpE and  mmpR genes, and whole genome sequencing examined mutations in the same sequences, as well as additionally in mmpL5, mmpS5, Rv0678, Rv1979c, and pepQ.Results. Complete agreement between the phenotypic and genotypic analysis of resistance to bedaquiline was found for three strains from Yakutia. One genome with significant mutations to bedaquiline was identified. A conclusion was made about the importance of molecular analysis of target genes with subsequent detection of resistance to bedaquiline in silico

Body weight, metabolism and clock genes

Biological rhythms are present in the lives of almost all organisms ranging from plants to more evolved creatures. These oscillations allow the anticipation of many physiological and behavioral mechanisms thus enabling coordination of rhythms in a timely manner, adaption to environmental changes and more efficient organization of the cellular processes responsible for survival of both the individual and the species. Many components of energy homeostasis exhibit circadian rhythms, which are regulated by central (suprachiasmatic nucleus) and peripheral (located in other tissues) circadian clocks. Adipocyte plays an important role in the regulation of energy homeostasis, the signaling of satiety and cellular differentiation and proliferation. Also, the adipocyte circadian clock is probably involved in the control of many of these functions. Thus, circadian clocks are implicated in the control of energy balance, feeding behavior and consequently in the regulation of body weight. In this regard, alterations in clock genes and rhythms can interfere with the complex mechanism of metabolic and hormonal anticipation, contributing to multifactorial diseases such as obesity and diabetes. The aim of this review was to define circadian clocks by describing their functioning and role in the whole body and in adipocyte metabolism, as well as their influence on body weight control and the development of obesity

Генетический анализ вируса гриппа А / Н1N1 "пандемический" в условиях эпидемии

Genetic analysis of pandemic influenza A / H1N1 virus during epidemia.Генетический анализ вируса гриппа А / Н1N1 "пандемический" в условиях эпидемии

підручник

Кримінальне право України. Загальна частина : підручник / [Харченко В. Б., Житний О. О., Орлов Ю. В. та ін.] ; за заг. ред. О. М. Литвинова; МВС України, Харків. нац. ун-т внутр. справ. – Харків, 2020. – 428 с. – ISBN 978-966-610-097-2.Lytvynov O.M. (ed.), 2020. Criminal Law of Ukraine. General Part [Kryminalne pravo Ukrainy. Zahalna chactyna]. Kharkiv: Kharkivskyi natsionalnyi universytet vnutrishnikh sprav.У підручнику розглянуто основні питання навчальної дисципліни «Кримінальне право України. Загальна частина». У матеріалах курсу під час розгляду основних інститутів і вчень Загальної частини кримінального права України використано актуальні нормативні акти й судову практику. Для курсантів і студентів вищих навчальних закладів юридичного профілю, викладачів, аспірантів та ад’юнктів, науковців і практичних працівників, а також широкого кола читачів, які цікавляться проблематикою протидії злочинності.The textbook examines the main issues of the discipline "Criminal law of Ukraine". In the course materials, when considering the main institutions and teachings of the General Part of the Criminal Law of Ukraine, the current regulations and judicial practice were used. For cadets and students of higher educational institutions of a legal profile, teachers, graduate students and adjuncts, scientific and practical workers, as well as a wide range of readers interested in the problems of combating crime.В учебнике рассмотрены основные вопросы учебной дисциплины «Уголовное право Украины. Общая часть ». В материалах курса при рассмотрении основных институтов и учений Общей части уголовного права Украины использованы актуальные нормативные акты и судебная практика. Для курсантов и студентов высших учебных заведений юридического профиля, преподавателей, аспирантов и адъюнктов, научных и практических работников, а также широкого круга читателей, интересующихся проблематикой противодействия преступности