Токсикологическая безопасность спиртосодержащих лекарственных средств для профилактической антисептики

ЛЕКАРСТВА СОЗДАНИЕЛЕКАРСТВА КОНСТРУИРОВАНИЕЛЕКАРСТВА МОДЕЛИРОВАНИЕПРОТИВОИНФЕКЦИОННЫЕ СРЕДСТВА ЛОКАЛЬНЫЕ /АНАЛ /ТОКСИЧАНТИСЕПТИКИ /АНАЛ /ТОКСИЧАНТИСЕПТИЧЕСКИЕ СРЕДСТВА /АНАЛ /ТОКСИЧПРОТИВОИНФЕКЦИОННЫЕ СРЕДСТВА МЕСТНЫЕ /АНАЛ /ТОКСИЧЛЕКАРСТВ ТОКСИЧНОСТЬЭТАНОЛ /ТОКСИЧСПИРТ ЭТИЛОВЫЙ /ТОКСИЧЛЕКАРСТВ ФИЗИОЛОГИЧЕСКОЕ ДЕЙСТВИЕИОДХЛОРГЕКСИДИНЭКСПЕРИМЕНТЫ НА ЖИВОТНЫХКРЫСЫЦелью работы было изучение показателей токсикологической безопасности разработанных спиртосодержащих лекарственных средств "Витасепт" для профилактической антисептики. Выполнено 4 серии опытов на половозрелых белых крысах мужского пола массой 250±25 г, содержащихся в стандартных условиях. Однократное внутрижелудочное введение крысам нативных антисептических средств, содержащих спирт этиловый 72% марки "Люкс" с бриллиантовым зеленым 0,001%, с йодом кристаллическим 0,25%, с хлоргексидина биглюконатом 0,1%, а также "Этанол, раствор для наружного применения, 70%" в дозе 5300 мг/кг массы тела крыс, не вызывает гибель подопытных животных. После однократной 4-часовой аппликации в дозе 20 мг/см{2} закрытым способом и десятикратных повторных аппликаций на выстриженный участок кожи, а также хвосты крыс через 1,16 ч и в последующие 12 суток наблюдения после аппликации не вызывают гибель подопытных животных, клинические симптомы интоксикации и раздражение кожи. Все исследуемые средства имели специфический спиртовой запах, были прозрачными, подлинными, с плотностью 0,877 до 0,885 г/см{3}. Полученные результаты позволяют заключить, что исследованные инновационные лекарственные антисептические средства, содержащие экологически чистый спирт этиловый марки "Люкс" 72% с бриллиантовым зеленым 0,001%, с йодом кристаллическим 0,25%, с хлоргексидина биглюконатом 0,5% и 0,1%, а также оригинальные лекарственные антисептические средства, содержащие экологически чистый спирт этиловый марки "Люкс" 72% с бриллиантовым зеленым 0,01%, с йодом кристаллическим 0,5%, являются малоопасными, практически безвредными и не обладают кожно-раздражающим действием. Разработанные нами в настоящее время спиртосодержащие антисептики, а также производимые ОАО "Бобруйский завод биотехнологий" средства под маркой "Витасепт-СКЗ", "Витасепт-СКИ" и "Витасепт-СКО" соответствуют нормативным требованиям по токсикологическим показателям безопасности, предъявляемым к кожным антисептикам, средствам для гигиенической и хирургической обработки рук, кожи операционного и инъекционного полей.The aim of this work was to study the toxicological safety indicators of the developed alcohol-containing medicinal agents "Vitasept" for prophylactic antisepsis. 4 series of experiments were performed on adult white male rats weighing 250±25 g kept in standard conditions. A single intragastric administration to rats of native antiseptics containing 72% ethanol of the brand de luxe with diamond green 0.001%, with crystalline iodine 0.25%, with chlorhexidine bigluconate 0.1%, as well as "Ethanol, solution for external use, 70%" at a dose of 5300 mg/kg of rat body weight does not cause the death of experimental animals. After a single 4-hour application at a dose of 20 mg/cm{2} in a closed manner and ten times repeated applications on a sheared skin area, as well as rat tails after 1,16 hours and in the next 12 days of observation after application, they do not cause the death of experimental animals, clinical symptoms of intoxication and skin irritation. All studied agents had a specific alcoholic smell, were transparent, genuine, with a density of 0.877 up to 0.885 g/cm{3}. The results obtained allow us to conclude that the studied innovative medicinal antiseptic agents containing environmentally friendly 72% ethanol of the brand de luxe with brilliant green 0.001%, with crystalline iodine 0.25%, with chlorhexidine bigluconate 0.5% and 0.1%, as well as original medicinal antiseptic agents containing environmentally friendly 72% ethanol of the brand de luxe with brilliant green 0.01%, with crystalline iodine 0.5%, are slightly hazardous, practically harmless and do not produce a skin-irritating effect. Currently developed alcohol-containing antiseptics, as well as products manufactured by "Bobruisk plant of biotechnologies" under the brand names "Vitasept-SKZ", "Vitasept-SKI" and "Vitasept-SKO", comply with the regulatory requirements for toxicological safety indicators of skin antiseptics, agents for hygienic and surgical treatment of hands, skin of the operative and injection fields

The Lysine Demethylase KDM5B Regulates Islet Function and Glucose Homeostasis

Aims. Posttranslational modifications of histones and transcription factors regulate gene expression and are implicated in beta-cell failure and diabetes. We have recently shown that preserving H3K27 and H3K4 methylation using the lysine demethylase inhibitor GSK-J4 reduces cytokine-induced destruction of beta-cells and improves beta-cell function. Here, we investigate the therapeutic potential of GSK-J4 to prevent diabetes development and examine the importance of H3K4 methylation for islet function. Materials and Methods. We used two mouse models of diabetes to investigate the therapeutic potential of GSK-J4. To clarify the importance of H3K4 methylation, we characterized a mouse strain with knockout (KO) of the H3K4 demethylase KDM5B. Results. GSK-J4 administration failed to prevent the development of experimental diabetes induced by multiple low-dose streptozotocin or adoptive transfer of splenocytes from acutely diabetic NOD to NODscid mice. KDM5B-KO mice were growth retarded with altered body composition, had low IGF-1 levels, and exhibited reduced insulin secretion. Interestingly, despite secreting less insulin, KDM5B-KO mice were able to maintain normoglycemia following oral glucose tolerance test, likely via improved insulin sensitivity, as suggested by insulin tolerance testing and phosphorylation of proteins belonging to the insulin signaling pathway. When challenged with high-fat diet, KDM5B-deficient mice displayed similar weight gain and insulin sensitivity as wild-type mice. Conclusion. Our results show a novel role of KDM5B in metabolism, as KDM5B-KO mice display growth retardation and improved insulin sensitivity.Fil: Backe, Marie Balslev. Universidad de Copenhagen; DinamarcaFil: Jin, Chunyu. Universidad de Copenhagen; DinamarcaFil: Andreone, Luz. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Sankar, Aditya. Universidad de Copenhagen; Dinamarca. The Novo Nordisk Foundation Center for Stem Cell Biology; DinamarcaFil: Agger, Karl. Universidad de Copenhagen; Dinamarca. The Novo Nordisk Foundation Center for Stem Cell Biology; DinamarcaFil: Helin, Kristian. Universidad de Copenhagen; Dinamarca. The Novo Nordisk Foundation Center for Stem Cell Biology; DinamarcaFil: Madsen, Andreas N.. Universidad de Copenhagen; DinamarcaFil: Poulsen, Steen S.. Universidad de Copenhagen; DinamarcaFil: Bysani, Madhusudhan. Lund University; SueciaFil: Bacos, Karl. Lund University; SueciaFil: Ling, Charlotte. Lund University; SueciaFil: Perone, Marcelo Javier. Universidad de Copenhagen; Dinamarca. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Holst, Birgitte. Universidad de Copenhagen; DinamarcaFil: Mandrup Poulsen, Thomas. Universidad de Copenhagen; Dinamarc

Genome-Wide Studies of Transcriptional Regulation in Human Liver Cells by High-throughput Sequencing

The human genome contains slightly more than 20 000 genes that are expressed in a tissue specific manner. Transcription factors play a key role in gene regulation. By mapping the transcription factor binding sites genome-wide we can understand their role in different biological processes. In this thesis we have mapped transcription factors and histone marks along with nucleosome positions and RNA levels. In papers I and II, we used ChIP-seq to map five liver specific transcription factors that are crucial for liver development and function. We showed that the mapped transcription factors are involved in metabolism and other cellular processes. We showed that ChIP-seq can also be used to detect protein-protein interactions and functional SNPs. Finally, we showed that the epigenetic histone mark studied in paper I is associated with transcriptional activity at promoters. In paper III, we mapped nucleosome positions before and after treatment with transforming growth factor  β (TGFβ) and found that many nucleosomes changed positions when expression changed. After treatment with TGFβ, the transcription factor HNF4α was replaced by a nucleosome in some regions. In paper IV, we mapped USF1 transcription factor and three active chromatin marks in normal liver tissue and in liver tissue of patients diagnosed with alcoholic steatohepatitis. Using gene ontology, we as expected identified many metabolism related genes as active in normal samples whereas genes in cancer pathways were active in steatohepatitis tissue. Cancer is a common complication to the disease and early signs of this were found. We also found many novel and GWAS catalogue SNPs that are candidates to be functional. In conclusion, our results have provided information on location and structure of regulatory elements which will lead to better knowledge on liver function and disease

Epigenetic alterations in blood mirror age-associated DNA methylation and gene expression changes in human liver

Aim: To study the impact of aging on DNA methylation and mRNA expression in human liver. Experimental procedures: We analysed genome-wide DNA methylation and gene expression in human liver samples using Illumina 450K and HumanHT12 expression BeadChip arrays. Results: DNA methylation analysis of ∼455,000 CpG sites in human liver revealed that age was significantly associated with altered DNA methylation of 20,396 CpG sites. Comparison of liver methylation data with published methylation data in other tissues showed that vast majority of the age-associated significant CpG sites overlapped between liver and blood, whereas a smaller overlap was found between liver and pancreatic islets or adipose tissue, respectively. We identified 151 genes whose liver expression also correlated with age. Conclusions: We identified age-associated DNA methylation and expression changes in human liver that are partly reflected by epigenetic alterations in blood

ATAC-seq reveals alterations in open chromatin in pancreatic islets from subjects with type 2 diabetes

Impaired insulin secretion from pancreatic islets is a hallmark of type 2 diabetes (T2D). Altered chromatin structure may contribute to the disease. We therefore studied the impact of T2D on open chromatin in human pancreatic islets. We used assay for transposase-accessible chromatin using sequencing (ATAC-seq) to profile open chromatin in islets from T2D and non-diabetic donors. We identified 57,105 and 53,284 ATAC-seq peaks representing open chromatin regions in islets of nondiabetic and diabetic donors, respectively. The majority of ATAC-seq peaks mapped near transcription start sites. Additionally, peaks were enriched in enhancer regions and in regions where islet-specific transcription factors (TFs), e.g. FOXA2, MAFB, NKX2.2, NKX6.1 and PDX1, bind. Islet ATAC-seq peaks overlap with 13 SNPs associated with T2D (e.g. rs7903146, rs2237897, rs757209, rs11708067 and rs878521 near TCF7L2, KCNQ1, HNF1B, ADCY5 and GCK, respectively) and with additional 67 SNPs in LD with known T2D SNPs (e.g. SNPs annotated to GIPR, KCNJ11, GLIS3, IGF2BP2, FTO and PPARG). There was enrichment of open chromatin regions near highly expressed genes in human islets. Moreover, 1,078 open chromatin peaks, annotated to 898 genes, differed in prevalence between diabetic and non-diabetic islet donors. Some of these peaks are annotated to candidate genes for T2D and islet dysfunction (e.g. HHEX, HMGA2, GLIS3, MTNR1B and PARK2) and some overlap with SNPs associated with T2D (e.g. rs3821943 near WFS1 and rs508419 near ANK1). Enhancer regions and motifs specific to key TFs including BACH2, FOXO1, FOXA2, NEUROD1, MAFA and PDX1 were enriched in differential islet ATAC-seq peaks of T2D versus non-diabetic donors. Our study provides new understanding into how T2D alters the chromatin landscape, and thereby accessibility for TFs and gene expression, in human pancreatic islets

Additional file 3. of lobChIP: from cells to sequencing ready ChIP libraries in a single day

EVOware script for running lobChIP on the Tecan EVO pipetting robot

HDAC7 is overexpressed in human diabetic islets and impairs insulin secretion in rat islets and clonal beta cells

Aims/hypothesis: Pancreatic beta cell dysfunction is a prerequisite for the development of type 2 diabetes. Histone deacetylases (HDACs) may affect pancreatic endocrine function and glucose homeostasis through alterations in gene regulation. Our aim was to investigate the role of HDAC7 in human and rat pancreatic islets and clonal INS-1 beta cells (INS-1 832/13). Methods: To explore the role of HDAC7 in pancreatic islets and clonal beta cells, we used RNA sequencing, mitochondrial functional analyses, microarray techniques, and HDAC inhibitors MC1568 and trichostatin A. Results: Using RNA sequencing, we found increased HDAC7 expression in human pancreatic islets from type 2 diabetic compared with non-diabetic donors. HDAC7 expression correlated negatively with insulin secretion in human islets. To mimic the situation in type 2 diabetic islets, we overexpressed Hdac7 in rat islets and clonal beta cells. In both, Hdac7 overexpression resulted in impaired glucose-stimulated insulin secretion. Furthermore, it reduced insulin content, mitochondrial respiration and cellular ATP levels in clonal beta cells. Overexpression of Hdac7 also led to changes in the genome-wide gene expression pattern, including increased expression of Tcf7l2 and decreased expression of gene sets regulating DNA replication and repair as well as nucleotide metabolism. In accordance, Hdac7 overexpression reduced the number of beta cells owing to enhanced apoptosis. Finally, we found that inhibiting HDAC7 activity with pharmacological inhibitors or small interfering RNA-mediated knockdown restored glucose-stimulated insulin secretion in beta cells that were overexpressing Hdac7. Conclusions/interpretation: Taken together, these results indicate that increased HDAC7 levels caused beta cell dysfunction and may thereby contribute to defects seen in type 2 diabetic islets. Our study supports HDAC7 inhibitors as a therapeutic option for the treatment of type 2 diabetes