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Diabetes is characterized by hyperglycaemia resulting from a deficiency in insulin secretion and/or action leading to severe diabetic complications. Despite numerous efforts, recovery and maintenance of functional beta-cells is still an unresolved task in diabetes therapy. Considering anti-diabetic properties of medicinal herb Centaurium erythraea Rafn (CE), this study aimed to analyze protective effects of the CE extract on Rin-5F beta-cell line exposed to diabetogenic agent streptozotocin (STZ). Cytoprotective concentration of CE extract (0.25 mg/mL) and IC50 dose of STZ (12mM) were determined using cell viability assay (MTT). The level of insulin mRNA and the concentration of insulin released from beta-cells in a culture medium were analyzed by RT-qPCR and ELISA, respectively. Activity of Akt, ERK and p38 kinases, as well as nuclear levels of islet-enriched Pdx1 and MafA proteins were assessed by Western blot analysis. In comparison to STZ-treated cells, CE extract/STZ co-treatment increased viability of Rin- 5F cells for 12%. STZ-treated beta-cells displayed reduced mRNA level of insulin to 63% and reduced insulin secretion to 76% in comparison to controls, while application of CE extract improved insulin mRNA level to 77% and insulin secretion to 90% of the control level. Improved viability and functionality of beta-cells could be ascribed to a CE extractmediated modulation of the activities of pro-survival Akt, ERK and p38 kinases and Pdx- 1 and MafA factors involved in regulation of beta-cell proliferation and insulin expression/secretion. The results of this study suggest that CE extract promotes proliferative and pro-survival pathways in beta-cells and improves their functional properties.

In light of increasing prevalence of diabetes over the past few decades and increasing lifespan of human population, there is a need to better monitor the quality of life of diabetic patients. Epidemiological evidence shows protective effect of regular consumption of diets rich in plant polyphenols against development of diabetes. Centaurium erythraea Rafn (CE) is traditionally used in Serbia for diabetes treatment. Previous phytochemical studies with aerial parts of CE showed it can substantially alleviate oxidative stress, one of the major pathogenic factors that lead to diabetes and its complications. Chronic hyperglycemia, the hallmark of diabetes, leads to increased production of free radicals which affect red blood cells (RBCs) structure and function. Considering RBCs role as oxygen transporters and consequences of impaired oxygen delivery in diabetes, the main goal of this research was to evaluate the protective effect of the methanol extract of aerial parts of CE against oxidative challenge in RBCs of rats with streptozotocin (STZ)-induced diabetes. The CE extract (100 mg/kg) was given daily and orally two weeks before, during diabetes induction (i.p. injection of STZ (40 mg/kg) for five consecutive days), and for four weeks after the STZ injections (animals designated as pre-treated group), or for four weeks after diabetes induction (post-treated group). Daily application of CE extract to STZ-induced diabetic rats improved the redox status of RBCs, observed as reduced lipid peroxidation and alleviated oxidative damage due to improved glutathione system and antioxidant enzyme activity, such as catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR). Ameliorated RBCs’ redox status was accompanied with improvement of major biochemical indicators of diabetes. CE extract increased serum insulin level, reduced blood glucose and glycated hemoglobin concentrations and improved lipid profile of diabetic rats. Furthermore, the CE extract reduced non-enzymatic glycation and enzymatic glycosylation and improved parameters which correlate with RBC aggregation and deformability. The protective effect of CE extract was more pronounced in pre-treated diabetic group. According to these results, Centaurium erythraea methanol extract has a great potential for use as dietary supplement in diabetes management. Since plenty of bioactive compounds, including polyphenols were determined in CE extract, identification of active metabolites from CE and their tissue distribution after intake could provide a useful source of potential novel antidiabetic pharmaceutical entities.

Introduction: Trachoma is the most common cause of infectious blindness worldwide, initiated by repeated infection of the conjunctiva with Chlamydia trachomatis (Ct). The resulting chronic inflammation and formation of fibrotic tissue eventually lead to corneal damage. Based on the facts that epithelial to mesenchymal transition (EMT) plays an important role in the development of fibrosis and that EMT is epigenetically regulated process, the aims of this study were to reveal the capacity of Ct to induce EMT in vitro and to unveil potential underlying epigenetic mechanisms. Methods: Human conjunctival epithelial (HCjE) cells were infected with 107 IFU of Ct for 72 h. EMT-inducing signaling pathways, as well as mRNA and protein expression of EMT markers (E-cadherin, fibronectin and α-SMA) were evaluated by RT-qPCR, Immunoblotting and Immunocytochemistry. DNA methylation patterns of selected regions of E-cadherin, fibronectin and α-SMA genes were examined by Methylation-Specific PCR, High Resolution Melting analysis and Bisulfite Sequencing. Results: Infection with Ct was accompanied with the activation of EMT-inducing signaling pathways, downregulation of epithelial marker E-cadherin and upregulation of mesenchymal markers fibronectin and α-SMA. While DNA methylation status of E-cadherin gene promoter correlated with its expression, methylation status of fibronectin and α-SMA genes couldn’t be related to their expression levels. Conclusion: Ct infection of HCjE cells triggers EMT that goes along with changes in the methylation profile of the E-cadherin promoter. Sequence of events described herein could contribute to scarring process in trachoma and open up possibilities for development of new therapeutic strategies in trachoma treatment. Brajušković G, Đorđević A, editors. CoMBoS. 1st Congress of Molecular Biologists of Serbia; 2017 Sep 20-22; Belgrade, Serbia. Belgrade: University of Belgrade, Faculty of Biology; 2017. p. 70.

Introduction: Since diabetes is characterized by impaired ability of pancreatic betacells to respond and/or produce insulin, new approaches for renewal and replacement of deficient beta-cells are indispensable. The aim of this study is direct pancreatic alpha- to beta-cells transdifferentiation by using a new synthetic epigenetic tool, Epi-CRISPR system. Using Epi-CRISPR system we aim to introduce targeted DNA methylation and subsequent repression of genes responsible for maintaining alpha-cell identity. Methods: AlphaTC1-6 cells (α-cells) were transiently transfected with dCas9-Dnmt3a- Dnmt3L constructs and one or four different vectors containing guide RNA components for specific targeting the promoter region of aristaless-related homeobox gene (Arx). The success of α-cells transdifferentiation into insulinproducing cells was evaluated by measuring Arx and insulin mRNA level, amount of secreted insulin and by immunostaining of insulin/glucagon in the cells. Results: We observed Arx transcriptional repression in α-cell transfected with Epi- CRISPR construct that targets the Arx gene promoter inducing subsequent methylation. At fifth day post-transfection the expression of Arx was decreased in α- cells followed by consequent increase in insulin (mRNA and protein level). At the same time, the glucagon levels remained unchanged. At twelfth day posttransfection the transfected cells start to lose glucagon while still secreting insulin. Conclusion: This study is near to confirm Epi-CRISPR system functionality and to verify the concept of cell transdifferentiation through silencing of genes responsible for maintaining cell phenotype. The obtained results will be valuable for later Epi- CRISPRs use in mouse in vivo models of diabetes and eventually as a future therapy for diabetes attenuation in humans. Brajušković G, Đorđević A, editors. CoMBoS. 1st Congress of Molecular Biologists of Serbia; 2017 Sep 20-21; Belgrade, Serbia. Belgrade: University of Belgrade, Faculty of Biology; 2017. p. 73.