European Cooperation in Science and Technology (COST) CM1406

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European Cooperation in Science and Technology (COST) CM1406

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Publications

MeFISH: Fluorescent detection of Target Methylated Cytosines within the Genome

Tolić, Anja; Ninković, Niccoleta Aleksandra; Rajić, Jovana; Đorđević, Miloš; Đorđević, Marija; Uskoković, Aleksandra; Grdović, Nevena; Mihailović, Mirjana; Arambašić Jovanović, Jelena; Dinić, Svetlana; Okamoto, Akimitsu; Vidaković, Melita

(Karger Publishers, 2018)

TY  - CONF
AU  - Tolić, Anja
AU  - Ninković, Niccoleta Aleksandra
AU  - Rajić, Jovana
AU  - Đorđević, Miloš
AU  - Đorđević, Marija
AU  - Uskoković, Aleksandra
AU  - Grdović, Nevena
AU  - Mihailović, Mirjana
AU  - Arambašić Jovanović, Jelena
AU  - Dinić, Svetlana
AU  - Okamoto, Akimitsu
AU  - Vidaković, Melita
PY  - 2018
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5799
AB  - Objectives: This study aim to implement a novel method, methylation-specific fluorescence in situ hybridization (MeFISH), based on microscopic visualization of DNA methylation/hydroxymethylation status at specific DNA regions in individual nuclei after pancreatic cell treatment with different compounds that possess a pronounced DNA (de)methylation capacity.
Methods: The DNA (de)methylating properties of two selected compounds caffeine (Co) and azacitidine (A) were evaluated in a Rin-5F pancreatic beta-cell line. Rin-5F cells were spin down on microscopic slides and further processed for preparing HALOs (relaxed DNA with preserved contacts with non-soluble nuclear proteins). The fluorescent visualization was achieved using ICON probe that covers region of interest in the promoter of the CXCL12 gene and target C positioned on the +26 bp, osmium for methylated cytosine (5mC)-dependent crosslinking and Tyramide Signal Amplification Systems for enhanced fluorescent staining.
Results: In control and Rin-5F cells treated with Co we were able to detect clear, single fluorescent signal that correspond to 5mC positioned on the +26 bp within the promoter region of the CXCL12 gene using MeFISH. Confirmation for the in situ hybridization specificity was achieved by omitting the crosslinking reaction with osmium. We observed a clear difference between control and Co treated samples, indicating that Co acts as pronounced DNA methylating compound. Treatment of cells with A lead to the appearance of a specific signal in a limited number of HALO preparations confirming demethylating property of A.
Conclusions: The Co acts as a pronounced DNA methylating agent in contrast to A, which demethylates CXCL12 gene and subsequently promotes higher gene expression. Higher methylation of the CXCL12 gene after cell treatment with Co leads to suppression of the gene which was observed by RT-qPCR. The analysed C, positioned on the +26 bp, may represent one of the major sites whose methylation is important for the regulation of the CXCL12 gene expression in vivo.
PB  - Karger Publishers
C3  - Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy
T1  - MeFISH: Fluorescent detection of Target Methylated Cytosines within the Genome
DO  - 10.1159/000490753
SP  - 16
ER  - 
@conference{
author = "Tolić, Anja and Ninković, Niccoleta Aleksandra and Rajić, Jovana and Đorđević, Miloš and Đorđević, Marija and Uskoković, Aleksandra and Grdović, Nevena and Mihailović, Mirjana and Arambašić Jovanović, Jelena and Dinić, Svetlana and Okamoto, Akimitsu and Vidaković, Melita",
year = "2018",
abstract = "Objectives: This study aim to implement a novel method, methylation-specific fluorescence in situ hybridization (MeFISH), based on microscopic visualization of DNA methylation/hydroxymethylation status at specific DNA regions in individual nuclei after pancreatic cell treatment with different compounds that possess a pronounced DNA (de)methylation capacity.
Methods: The DNA (de)methylating properties of two selected compounds caffeine (Co) and azacitidine (A) were evaluated in a Rin-5F pancreatic beta-cell line. Rin-5F cells were spin down on microscopic slides and further processed for preparing HALOs (relaxed DNA with preserved contacts with non-soluble nuclear proteins). The fluorescent visualization was achieved using ICON probe that covers region of interest in the promoter of the CXCL12 gene and target C positioned on the +26 bp, osmium for methylated cytosine (5mC)-dependent crosslinking and Tyramide Signal Amplification Systems for enhanced fluorescent staining.
Results: In control and Rin-5F cells treated with Co we were able to detect clear, single fluorescent signal that correspond to 5mC positioned on the +26 bp within the promoter region of the CXCL12 gene using MeFISH. Confirmation for the in situ hybridization specificity was achieved by omitting the crosslinking reaction with osmium. We observed a clear difference between control and Co treated samples, indicating that Co acts as pronounced DNA methylating compound. Treatment of cells with A lead to the appearance of a specific signal in a limited number of HALO preparations confirming demethylating property of A.
Conclusions: The Co acts as a pronounced DNA methylating agent in contrast to A, which demethylates CXCL12 gene and subsequently promotes higher gene expression. Higher methylation of the CXCL12 gene after cell treatment with Co leads to suppression of the gene which was observed by RT-qPCR. The analysed C, positioned on the +26 bp, may represent one of the major sites whose methylation is important for the regulation of the CXCL12 gene expression in vivo.",
publisher = "Karger Publishers",
journal = "Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy",
title = "MeFISH: Fluorescent detection of Target Methylated Cytosines within the Genome",
doi = "10.1159/000490753",
pages = "16"
}
Tolić, A., Ninković, N. A., Rajić, J., Đorđević, M., Đorđević, M., Uskoković, A., Grdović, N., Mihailović, M., Arambašić Jovanović, J., Dinić, S., Okamoto, A.,& Vidaković, M.. (2018). MeFISH: Fluorescent detection of Target Methylated Cytosines within the Genome. in Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy
Karger Publishers., 16.
https://doi.org/10.1159/000490753
Tolić A, Ninković NA, Rajić J, Đorđević M, Đorđević M, Uskoković A, Grdović N, Mihailović M, Arambašić Jovanović J, Dinić S, Okamoto A, Vidaković M. MeFISH: Fluorescent detection of Target Methylated Cytosines within the Genome. in Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy. 2018;:16.
doi:10.1159/000490753 .
Tolić, Anja, Ninković, Niccoleta Aleksandra, Rajić, Jovana, Đorđević, Miloš, Đorđević, Marija, Uskoković, Aleksandra, Grdović, Nevena, Mihailović, Mirjana, Arambašić Jovanović, Jelena, Dinić, Svetlana, Okamoto, Akimitsu, Vidaković, Melita, "MeFISH: Fluorescent detection of Target Methylated Cytosines within the Genome" in Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy (2018):16,
https://doi.org/10.1159/000490753 . .

Evaluation of the DNA Methylation Status of Procalcitonin Gene as a Biomarker of Local and Systemic Inflammation

Rajić, Jovana; Grdović, Nevena; Petrović Matić, Sanja; Dinić, Svetlana; Uskoković, Aleksandra; Mihailović, Mirjana; Arambašić Jovanović, Jelena; Tolić, Anja; Đorđević, Marija; Đorđević, Miloš; Poznanović, Goran; Pucar, Ana; Vidaković, Melita

(Krager Publishers, 2018)

TY  - CONF
AU  - Rajić, Jovana
AU  - Grdović, Nevena
AU  - Petrović Matić, Sanja
AU  - Dinić, Svetlana
AU  - Uskoković, Aleksandra
AU  - Mihailović, Mirjana
AU  - Arambašić Jovanović, Jelena
AU  - Tolić, Anja
AU  - Đorđević, Marija
AU  - Đorđević, Miloš
AU  - Poznanović, Goran
AU  - Pucar, Ana
AU  - Vidaković, Melita
PY  - 2018
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5800
AB  - Objectives: Procalcitonin (PCT) has recently emerged as an important biomarker for an early and accurate diagnosis of bacte­rial infection, hence suggested as biomarker of periodontal disease caused by oral pathogenic microorganisms. Altered DNA meth­ylation of PCT coding gene - calcitonin-related polypeptide a (CALCA) has been shown during systemic inflammation. The aim of this study was to evaluate the influence oflocal and/or systemic inflammation present during periodontitis and diabetes on DNA methylation status of CALCA and its potential use as epigenetic­based biomarker of these chronic inflammatory conditions. 
Methods: The study included 65 individuals divided in three groups: healthy control (n = 17), periodontitis (n = 27) and diabe­tes/periodontitis group (n = 21). Periodontitis was diagnosed us­ing International Workshop for a Classification of Periodontal Diseases and Conditions criteria (1999) while type 2 diabetes as­sessment was performed according to WHO criteria (2013). DNA methylation profile of CALCA promoter in buccal epithelial cells was analyzed by methylation specific polymerase chain reaction (MSP). 
Results: Decrease in DNA methylation of CALCA promoter was observed in periodontitis and even more pronounced in dia­betes/periodontitis compared to control group, although without statistical significance. Correlation analysis revealed statistically significant relationship between the extent of DNA methylation of the CALCA promoter and glycosylated hemoglobin. Even though it is known that life style affects DNA methylation patterns, there was no difference in DNA methylation of CALCA promoter be­tween smokers/non-smokers and normal/overweight individuals. 
Conclusions: Presented results suggest that local periodontal inflammation contributes to the change, but that only systemic inflammation significantly alters the DNA methylation profile of CALCA in buccal cells. Those results imply that DNA methylation status of CALCA reflects systemic inflammation, but additional studies are needed to estimate usefulness of this epigenetic-based biomarker for periodontal disease.
PB  - Krager Publishers
C3  - Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy
T1  - Evaluation of the DNA Methylation Status of Procalcitonin Gene as a Biomarker of Local and Systemic Inflammation
DO  - 10.1159/000490753
SP  - 12
ER  - 
@conference{
author = "Rajić, Jovana and Grdović, Nevena and Petrović Matić, Sanja and Dinić, Svetlana and Uskoković, Aleksandra and Mihailović, Mirjana and Arambašić Jovanović, Jelena and Tolić, Anja and Đorđević, Marija and Đorđević, Miloš and Poznanović, Goran and Pucar, Ana and Vidaković, Melita",
year = "2018",
abstract = "Objectives: Procalcitonin (PCT) has recently emerged as an important biomarker for an early and accurate diagnosis of bacte­rial infection, hence suggested as biomarker of periodontal disease caused by oral pathogenic microorganisms. Altered DNA meth­ylation of PCT coding gene - calcitonin-related polypeptide a (CALCA) has been shown during systemic inflammation. The aim of this study was to evaluate the influence oflocal and/or systemic inflammation present during periodontitis and diabetes on DNA methylation status of CALCA and its potential use as epigenetic­based biomarker of these chronic inflammatory conditions. 
Methods: The study included 65 individuals divided in three groups: healthy control (n = 17), periodontitis (n = 27) and diabe­tes/periodontitis group (n = 21). Periodontitis was diagnosed us­ing International Workshop for a Classification of Periodontal Diseases and Conditions criteria (1999) while type 2 diabetes as­sessment was performed according to WHO criteria (2013). DNA methylation profile of CALCA promoter in buccal epithelial cells was analyzed by methylation specific polymerase chain reaction (MSP). 
Results: Decrease in DNA methylation of CALCA promoter was observed in periodontitis and even more pronounced in dia­betes/periodontitis compared to control group, although without statistical significance. Correlation analysis revealed statistically significant relationship between the extent of DNA methylation of the CALCA promoter and glycosylated hemoglobin. Even though it is known that life style affects DNA methylation patterns, there was no difference in DNA methylation of CALCA promoter be­tween smokers/non-smokers and normal/overweight individuals. 
Conclusions: Presented results suggest that local periodontal inflammation contributes to the change, but that only systemic inflammation significantly alters the DNA methylation profile of CALCA in buccal cells. Those results imply that DNA methylation status of CALCA reflects systemic inflammation, but additional studies are needed to estimate usefulness of this epigenetic-based biomarker for periodontal disease.",
publisher = "Krager Publishers",
journal = "Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy",
title = "Evaluation of the DNA Methylation Status of Procalcitonin Gene as a Biomarker of Local and Systemic Inflammation",
doi = "10.1159/000490753",
pages = "12"
}
Rajić, J., Grdović, N., Petrović Matić, S., Dinić, S., Uskoković, A., Mihailović, M., Arambašić Jovanović, J., Tolić, A., Đorđević, M., Đorđević, M., Poznanović, G., Pucar, A.,& Vidaković, M.. (2018). Evaluation of the DNA Methylation Status of Procalcitonin Gene as a Biomarker of Local and Systemic Inflammation. in Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy
Krager Publishers., 12.
https://doi.org/10.1159/000490753
Rajić J, Grdović N, Petrović Matić S, Dinić S, Uskoković A, Mihailović M, Arambašić Jovanović J, Tolić A, Đorđević M, Đorđević M, Poznanović G, Pucar A, Vidaković M. Evaluation of the DNA Methylation Status of Procalcitonin Gene as a Biomarker of Local and Systemic Inflammation. in Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy. 2018;:12.
doi:10.1159/000490753 .
Rajić, Jovana, Grdović, Nevena, Petrović Matić, Sanja, Dinić, Svetlana, Uskoković, Aleksandra, Mihailović, Mirjana, Arambašić Jovanović, Jelena, Tolić, Anja, Đorđević, Marija, Đorđević, Miloš, Poznanović, Goran, Pucar, Ana, Vidaković, Melita, "Evaluation of the DNA Methylation Status of Procalcitonin Gene as a Biomarker of Local and Systemic Inflammation" in Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy (2018):12,
https://doi.org/10.1159/000490753 . .

Centaurium erythraea Methanol Extract Attenuates SNP-lnduced Oxidative Stress in Pancreatic B-Cells

Đorđević, Miloš; Mihailović, Mirjana; Arambašić Jovanović, Jelena; Grdović, Nevena; Uskoković, Aleksandra; Đorđević, Marija; Rajić, Jovana; Tolić, Anja; Poznanović, Goran; Vidaković, Melita; Dinić, Svetlana

(Krager Publishers, 2018)

TY  - CONF
AU  - Đorđević, Miloš
AU  - Mihailović, Mirjana
AU  - Arambašić Jovanović, Jelena
AU  - Grdović, Nevena
AU  - Uskoković, Aleksandra
AU  - Đorđević, Marija
AU  - Rajić, Jovana
AU  - Tolić, Anja
AU  - Poznanović, Goran
AU  - Vidaković, Melita
AU  - Dinić, Svetlana
PY  - 2018
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5801
AB  - Objectives: Centaurium erythraea ( CE) is traditionally used in Serbia for diabetes management. Since oxidative stress represents one of the major pathogenic factors that lead to diabetes and its complications, this study investigated protective effect of CE methanol extract against oxidative stress-induced pancreatic P-cell death. 
Methods: Rin-SF, rat insulinoma pancreatic P-cells, were incu­bated for 24 h with 1.25 mM sodium nitroprusside (SNP) with/ without CE extract (0.2 mg/mL) and processed immediately. Rin­SF cell viability was estimated using the MTT viability assay. Lipid peroxidation was assessed using the thiobarbituric acid-reactive substance (TBARS) assay, while the DNA damage was estimated by alkaline comet assay. Catalase (CAT) activity was determined by the rate ofH202 decomposition, whereas superoxide dismutase (SOD) activity was estimated by the epinephrine method. The ac­tivities of glutathione peroxidase (GPx) and glutathione reductase ( GR) were determined by monitoring NADPH oxidation. Relative gene expression of CAT, MnSOD, CuZnSOD, GPx, GR and insu­lin was determined by RT-qPCR. Relative protein level of antioxi­dant enzymes was estimated using immunoblot analysis. 
Results: CE methanol extract enhanced p-cell viability and in­sulin gene expression by reducing oxidative stress in SNP-treated cells. CE extract lowered DNA damage and lipid peroxidation pro­voked by SNP treatment and adjusted antioxidant enzyme activi­ties. CE treatment increased SNP-mediated attenuation of CAT, GPx and GR activities and reduced CuZnSOD and MnSOD ac­tivities that were stimulated in SNP treated cells. SNP-induced in­crease in gene expression of CAT, GPx, GR, MnSOD and CuZn­SOD was accompanied by decrease of CAT, GPx and CuZnSOD mRNA level after CE treatment. In addition, SNP treatment in­creased protein levels of CAT, GR, GPx and MnSOD and de­creased CuZnSO D protein level. CE extract reduced CAT and Mn­SOD and partially restored CuZnSOD protein level. 
Conclusions: The CE methanol extract protects pancreatic p-cells from oxidative damage by improving antioxidant defence system. Detected attenuation of oxidative stress in P-cells in vitro provide a useful platform for in vivo investigation of antioxidant and antidiabetic effect of CE extract and its potential usage as an effective supplement for diabetes treatment.
PB  - Krager Publishers
C3  - Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy
T1  - Centaurium erythraea Methanol Extract Attenuates SNP-lnduced Oxidative Stress in Pancreatic B-Cells
DO  - 10.1159/000490753
SP  - 7
ER  - 
@conference{
author = "Đorđević, Miloš and Mihailović, Mirjana and Arambašić Jovanović, Jelena and Grdović, Nevena and Uskoković, Aleksandra and Đorđević, Marija and Rajić, Jovana and Tolić, Anja and Poznanović, Goran and Vidaković, Melita and Dinić, Svetlana",
year = "2018",
abstract = "Objectives: Centaurium erythraea ( CE) is traditionally used in Serbia for diabetes management. Since oxidative stress represents one of the major pathogenic factors that lead to diabetes and its complications, this study investigated protective effect of CE methanol extract against oxidative stress-induced pancreatic P-cell death. 
Methods: Rin-SF, rat insulinoma pancreatic P-cells, were incu­bated for 24 h with 1.25 mM sodium nitroprusside (SNP) with/ without CE extract (0.2 mg/mL) and processed immediately. Rin­SF cell viability was estimated using the MTT viability assay. Lipid peroxidation was assessed using the thiobarbituric acid-reactive substance (TBARS) assay, while the DNA damage was estimated by alkaline comet assay. Catalase (CAT) activity was determined by the rate ofH202 decomposition, whereas superoxide dismutase (SOD) activity was estimated by the epinephrine method. The ac­tivities of glutathione peroxidase (GPx) and glutathione reductase ( GR) were determined by monitoring NADPH oxidation. Relative gene expression of CAT, MnSOD, CuZnSOD, GPx, GR and insu­lin was determined by RT-qPCR. Relative protein level of antioxi­dant enzymes was estimated using immunoblot analysis. 
Results: CE methanol extract enhanced p-cell viability and in­sulin gene expression by reducing oxidative stress in SNP-treated cells. CE extract lowered DNA damage and lipid peroxidation pro­voked by SNP treatment and adjusted antioxidant enzyme activi­ties. CE treatment increased SNP-mediated attenuation of CAT, GPx and GR activities and reduced CuZnSOD and MnSOD ac­tivities that were stimulated in SNP treated cells. SNP-induced in­crease in gene expression of CAT, GPx, GR, MnSOD and CuZn­SOD was accompanied by decrease of CAT, GPx and CuZnSOD mRNA level after CE treatment. In addition, SNP treatment in­creased protein levels of CAT, GR, GPx and MnSOD and de­creased CuZnSO D protein level. CE extract reduced CAT and Mn­SOD and partially restored CuZnSOD protein level. 
Conclusions: The CE methanol extract protects pancreatic p-cells from oxidative damage by improving antioxidant defence system. Detected attenuation of oxidative stress in P-cells in vitro provide a useful platform for in vivo investigation of antioxidant and antidiabetic effect of CE extract and its potential usage as an effective supplement for diabetes treatment.",
publisher = "Krager Publishers",
journal = "Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy",
title = "Centaurium erythraea Methanol Extract Attenuates SNP-lnduced Oxidative Stress in Pancreatic B-Cells",
doi = "10.1159/000490753",
pages = "7"
}
Đorđević, M., Mihailović, M., Arambašić Jovanović, J., Grdović, N., Uskoković, A., Đorđević, M., Rajić, J., Tolić, A., Poznanović, G., Vidaković, M.,& Dinić, S.. (2018). Centaurium erythraea Methanol Extract Attenuates SNP-lnduced Oxidative Stress in Pancreatic B-Cells. in Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy
Krager Publishers., 7.
https://doi.org/10.1159/000490753
Đorđević M, Mihailović M, Arambašić Jovanović J, Grdović N, Uskoković A, Đorđević M, Rajić J, Tolić A, Poznanović G, Vidaković M, Dinić S. Centaurium erythraea Methanol Extract Attenuates SNP-lnduced Oxidative Stress in Pancreatic B-Cells. in Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy. 2018;:7.
doi:10.1159/000490753 .
Đorđević, Miloš, Mihailović, Mirjana, Arambašić Jovanović, Jelena, Grdović, Nevena, Uskoković, Aleksandra, Đorđević, Marija, Rajić, Jovana, Tolić, Anja, Poznanović, Goran, Vidaković, Melita, Dinić, Svetlana, "Centaurium erythraea Methanol Extract Attenuates SNP-lnduced Oxidative Stress in Pancreatic B-Cells" in Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy (2018):7,
https://doi.org/10.1159/000490753 . .

20-Hydroxyecdysone Protects Pancreatic Islets and Liver in Streptozotocin-lnduced Diabetic Rats

Đorđević, Marija; Arambašić Jovanović, Jelena; Mihailović, Mirjana; Uskoković, Aleksandra; Grdović, Nevena; Dinić, Svetlana; Đorđević, Miloš; Tolić, Anja; Rajić, Jovana; Hunyadi, Attila; Vidaković, Melita

(Krager Publishers, 2018)

TY  - CONF
AU  - Đorđević, Marija
AU  - Arambašić Jovanović, Jelena
AU  - Mihailović, Mirjana
AU  - Uskoković, Aleksandra
AU  - Grdović, Nevena
AU  - Dinić, Svetlana
AU  - Đorđević, Miloš
AU  - Tolić, Anja
AU  - Rajić, Jovana
AU  - Hunyadi, Attila
AU  - Vidaković, Melita
PY  - 2018
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5802
AB  - Objective: 20-hydroxyecdysone (20HE), a steroid hormone that modulates molting response in insects exerts many pharma­cological effects in mammals, most of which appear beneficial. The aim of this study was to investigate whether 20HE is able to reduce the destruction of beta-cells of the islets of Langerhans and ame­liorate hyperglycemia induced changes in liver tissue in strepto­zotocin (STZ)-induced rat model of diabetes. 
Methods: An experimental model of diabetes was induced in rats by the administration of 35 mg/kg STZ intraperitoneally for 4 consecutive days. 20HE was administered orally at a dose of20 mg/ kg body weight for four weeks, starting from the last day of STZ administration. Pancreas tissue sections were analyzed by hema­toxylin and eosin staining and immunohistochemical staining with insulin. Estimation of oxidative damage of DNA and lipids in the liver were detected by comet assay and thiobarbituric acid-re­active substance assay, respectively. Liver sections were analyzed by hematoxylin/eosin and Masson's trichrome staining. 
Results: Diabetic rats treated with the 20HE displayed several improved biochemical parameters in the circulation: reduced hy­perglycemia, lower triglyceride concentration and reduced glycat­ed hemoglobin. The administration of 20HE to diabetic rats also led to positive histological changes of pancreatic islets and increase in the number of insulin-positive cells in the islets which was ac­companied by increased serum insulin level. These results show that 20HE administration to diabetic rats restrained islet destruc­tion and partially restored the number of insulin-positive cells. In addition, treatment of 20HE attenuated diabetes-induced liver damage in rats according to lower level of DNA damage and reduc­tion of oxidative damage oflipids. This result is in accordance with observed improvement of liver architecture in 20HE treated dia­betic rats. Staining of collagen and increase in E-cadherin and de­creases in a-smooth muscle actin revealed that administration of 20HE to diabetic rats attenuated fibrotic process in the liver. 
Conclusions: 20HE administration seems to be beneficial for improving the hyperglycemia by increasing beta-cell mass and preventing diabetic complications in liver by attenuating fibrotic process.
PB  - Krager Publishers
C3  - Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy
T1  - 20-Hydroxyecdysone Protects Pancreatic Islets and Liver in Streptozotocin-lnduced Diabetic Rats
DO  - 10.1159/000490753
SP  - 14
ER  - 
@conference{
author = "Đorđević, Marija and Arambašić Jovanović, Jelena and Mihailović, Mirjana and Uskoković, Aleksandra and Grdović, Nevena and Dinić, Svetlana and Đorđević, Miloš and Tolić, Anja and Rajić, Jovana and Hunyadi, Attila and Vidaković, Melita",
year = "2018",
abstract = "Objective: 20-hydroxyecdysone (20HE), a steroid hormone that modulates molting response in insects exerts many pharma­cological effects in mammals, most of which appear beneficial. The aim of this study was to investigate whether 20HE is able to reduce the destruction of beta-cells of the islets of Langerhans and ame­liorate hyperglycemia induced changes in liver tissue in strepto­zotocin (STZ)-induced rat model of diabetes. 
Methods: An experimental model of diabetes was induced in rats by the administration of 35 mg/kg STZ intraperitoneally for 4 consecutive days. 20HE was administered orally at a dose of20 mg/ kg body weight for four weeks, starting from the last day of STZ administration. Pancreas tissue sections were analyzed by hema­toxylin and eosin staining and immunohistochemical staining with insulin. Estimation of oxidative damage of DNA and lipids in the liver were detected by comet assay and thiobarbituric acid-re­active substance assay, respectively. Liver sections were analyzed by hematoxylin/eosin and Masson's trichrome staining. 
Results: Diabetic rats treated with the 20HE displayed several improved biochemical parameters in the circulation: reduced hy­perglycemia, lower triglyceride concentration and reduced glycat­ed hemoglobin. The administration of 20HE to diabetic rats also led to positive histological changes of pancreatic islets and increase in the number of insulin-positive cells in the islets which was ac­companied by increased serum insulin level. These results show that 20HE administration to diabetic rats restrained islet destruc­tion and partially restored the number of insulin-positive cells. In addition, treatment of 20HE attenuated diabetes-induced liver damage in rats according to lower level of DNA damage and reduc­tion of oxidative damage oflipids. This result is in accordance with observed improvement of liver architecture in 20HE treated dia­betic rats. Staining of collagen and increase in E-cadherin and de­creases in a-smooth muscle actin revealed that administration of 20HE to diabetic rats attenuated fibrotic process in the liver. 
Conclusions: 20HE administration seems to be beneficial for improving the hyperglycemia by increasing beta-cell mass and preventing diabetic complications in liver by attenuating fibrotic process.",
publisher = "Krager Publishers",
journal = "Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy",
title = "20-Hydroxyecdysone Protects Pancreatic Islets and Liver in Streptozotocin-lnduced Diabetic Rats",
doi = "10.1159/000490753",
pages = "14"
}
Đorđević, M., Arambašić Jovanović, J., Mihailović, M., Uskoković, A., Grdović, N., Dinić, S., Đorđević, M., Tolić, A., Rajić, J., Hunyadi, A.,& Vidaković, M.. (2018). 20-Hydroxyecdysone Protects Pancreatic Islets and Liver in Streptozotocin-lnduced Diabetic Rats. in Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy
Krager Publishers., 14.
https://doi.org/10.1159/000490753
Đorđević M, Arambašić Jovanović J, Mihailović M, Uskoković A, Grdović N, Dinić S, Đorđević M, Tolić A, Rajić J, Hunyadi A, Vidaković M. 20-Hydroxyecdysone Protects Pancreatic Islets and Liver in Streptozotocin-lnduced Diabetic Rats. in Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy. 2018;:14.
doi:10.1159/000490753 .
Đorđević, Marija, Arambašić Jovanović, Jelena, Mihailović, Mirjana, Uskoković, Aleksandra, Grdović, Nevena, Dinić, Svetlana, Đorđević, Miloš, Tolić, Anja, Rajić, Jovana, Hunyadi, Attila, Vidaković, Melita, "20-Hydroxyecdysone Protects Pancreatic Islets and Liver in Streptozotocin-lnduced Diabetic Rats" in Selected Abstracts from the 3rd European Summer School on Nutrigenomics; 2018 Jun 25-29; Jesi, italy (2018):14,
https://doi.org/10.1159/000490753 . .

Therapeutic genome methylation for cell reprogramming editing: use of Epi-CRISPR-induced targeted DNA

Vidaković, Melita; Đorđević, Marija; Arambašić Jovanović, Jelena; Tolić, Anja; Đorđević, Miloš; Mihailović, Mirjana; Grdović, Nevena; Uskoković, Aleksandra; Rajić, Jovana; Poznanović, Goran; Dinić, Svetlana; Jurkowski, Tomasz

(Belgrade: University of Belgrade, Faculty of Biology, 2017)

TY  - CONF
AU  - Vidaković, Melita
AU  - Đorđević, Marija
AU  - Arambašić Jovanović, Jelena
AU  - Tolić, Anja
AU  - Đorđević, Miloš
AU  - Mihailović, Mirjana
AU  - Grdović, Nevena
AU  - Uskoković, Aleksandra
AU  - Rajić, Jovana
AU  - Poznanović, Goran
AU  - Dinić, Svetlana
AU  - Jurkowski, Tomasz
PY  - 2017
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5816
AB  - Introduction and aim: Diabetes is the perfect candidate for cell replacement therapy since it is caused by either an absolute (type 1 diabetes) or relative (type 2 diabetes) defect of insulin-producing pancreatic beta cells (b-cells}. We focused on applying a novel synthetic epigenetic tool (Epi-CRISPRs) for a straightforward, one­-step transdifferentiation of mouse pancreatic alpha (a-cells) to b-cell by targeted DNA methylation and suppression of genes essential for maintaining pancreatic cell identity (homeobox Arx gene (Arx)). 
Methods: The a-cells were transiently transfected with four different Epi-CRISPR constructs and co-transfected with a single guided RNA (gRNA} or with a mix of different gRNAs all targeting different promoter regions of Arx. After 5, 8 and 12 days post-transfection, DNA and RNA were isolated and the cells were immunostained. The transdifferentiated cells were analysed for key features of bona fide cells, using qPCR to assess Arx expression, and immunostaining of insulin/glucagon and ELISA for measuring secreted insulin. 
Results: We succeeded to transiently transfect a-cells with Epi-CRISPR constructs and 275 gRNA/mix gRNA. The suppression of Arx in a-cells was confirmed on days 5 and 8 post-transfection. The reduction of glucagon synthesis and beginning of insulin production in transfected a-cell was confirmed and visualised by immunostaining. Whether DNA methylation-mediated suppression of Arx in a-cells lead to their transdifferentiation to insulin-producing cells, will be confirmed by bisulfite sequencing. 
Conclusion: We are on the right course of developing a clear-cut technology capable of providing a perfect delivery system for increasing the number of insulin­producing cells in vitro.
PB  - Belgrade: University of Belgrade, Faculty of Biology
C3  - Book of Abstracts: 1st Congress of Molecular Biologists of Serbia: CoMBoS; 2017 Sep 20-21; Belgrade, Serbia.
T1  - Therapeutic genome methylation for cell reprogramming editing: use of Epi-CRISPR-induced targeted DNA
SP  - 10
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5816
ER  - 
@conference{
author = "Vidaković, Melita and Đorđević, Marija and Arambašić Jovanović, Jelena and Tolić, Anja and Đorđević, Miloš and Mihailović, Mirjana and Grdović, Nevena and Uskoković, Aleksandra and Rajić, Jovana and Poznanović, Goran and Dinić, Svetlana and Jurkowski, Tomasz",
year = "2017",
abstract = "Introduction and aim: Diabetes is the perfect candidate for cell replacement therapy since it is caused by either an absolute (type 1 diabetes) or relative (type 2 diabetes) defect of insulin-producing pancreatic beta cells (b-cells}. We focused on applying a novel synthetic epigenetic tool (Epi-CRISPRs) for a straightforward, one­-step transdifferentiation of mouse pancreatic alpha (a-cells) to b-cell by targeted DNA methylation and suppression of genes essential for maintaining pancreatic cell identity (homeobox Arx gene (Arx)). 
Methods: The a-cells were transiently transfected with four different Epi-CRISPR constructs and co-transfected with a single guided RNA (gRNA} or with a mix of different gRNAs all targeting different promoter regions of Arx. After 5, 8 and 12 days post-transfection, DNA and RNA were isolated and the cells were immunostained. The transdifferentiated cells were analysed for key features of bona fide cells, using qPCR to assess Arx expression, and immunostaining of insulin/glucagon and ELISA for measuring secreted insulin. 
Results: We succeeded to transiently transfect a-cells with Epi-CRISPR constructs and 275 gRNA/mix gRNA. The suppression of Arx in a-cells was confirmed on days 5 and 8 post-transfection. The reduction of glucagon synthesis and beginning of insulin production in transfected a-cell was confirmed and visualised by immunostaining. Whether DNA methylation-mediated suppression of Arx in a-cells lead to their transdifferentiation to insulin-producing cells, will be confirmed by bisulfite sequencing. 
Conclusion: We are on the right course of developing a clear-cut technology capable of providing a perfect delivery system for increasing the number of insulin­producing cells in vitro.",
publisher = "Belgrade: University of Belgrade, Faculty of Biology",
journal = "Book of Abstracts: 1st Congress of Molecular Biologists of Serbia: CoMBoS; 2017 Sep 20-21; Belgrade, Serbia.",
title = "Therapeutic genome methylation for cell reprogramming editing: use of Epi-CRISPR-induced targeted DNA",
pages = "10",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5816"
}
Vidaković, M., Đorđević, M., Arambašić Jovanović, J., Tolić, A., Đorđević, M., Mihailović, M., Grdović, N., Uskoković, A., Rajić, J., Poznanović, G., Dinić, S.,& Jurkowski, T.. (2017). Therapeutic genome methylation for cell reprogramming editing: use of Epi-CRISPR-induced targeted DNA. in Book of Abstracts: 1st Congress of Molecular Biologists of Serbia: CoMBoS; 2017 Sep 20-21; Belgrade, Serbia.
Belgrade: University of Belgrade, Faculty of Biology., 10.
https://hdl.handle.net/21.15107/rcub_ibiss_5816
Vidaković M, Đorđević M, Arambašić Jovanović J, Tolić A, Đorđević M, Mihailović M, Grdović N, Uskoković A, Rajić J, Poznanović G, Dinić S, Jurkowski T. Therapeutic genome methylation for cell reprogramming editing: use of Epi-CRISPR-induced targeted DNA. in Book of Abstracts: 1st Congress of Molecular Biologists of Serbia: CoMBoS; 2017 Sep 20-21; Belgrade, Serbia.. 2017;:10.
https://hdl.handle.net/21.15107/rcub_ibiss_5816 .
Vidaković, Melita, Đorđević, Marija, Arambašić Jovanović, Jelena, Tolić, Anja, Đorđević, Miloš, Mihailović, Mirjana, Grdović, Nevena, Uskoković, Aleksandra, Rajić, Jovana, Poznanović, Goran, Dinić, Svetlana, Jurkowski, Tomasz, "Therapeutic genome methylation for cell reprogramming editing: use of Epi-CRISPR-induced targeted DNA" in Book of Abstracts: 1st Congress of Molecular Biologists of Serbia: CoMBoS; 2017 Sep 20-21; Belgrade, Serbia. (2017):10,
https://hdl.handle.net/21.15107/rcub_ibiss_5816 .