scholarly journals Master Transcription Factors Regulate the DNA Methylation Landscape During Hepatocyte Differentiation

2020 ◽  
Author(s):  
Takahiro Suzuki ◽  
Erina Furuhata ◽  
Shiori Maeda ◽  
Mami Kishima ◽  
Yurina Miyajima ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factors ◽  
Dna Demethylation ◽  
Hepatocyte Differentiation ◽  
Endoderm Differentiation ◽  
Chromatin Activation ◽  
Specific Manner ◽  
Differentiation Stage ◽  
Differentiation Gene

BackgroundHepatocytes are the dominant cell type of the human liver, with functions in metabolism, detoxification, and in producing secreted proteins. During the process of hepatocyte differentiation, gene regulation and master transcription factors have been extensively investigated, whereas little is known about how the epigenome is regulated, particularly the dynamics of DNA methylation, and the upstream factors that have critical roles.ResultsBy examining changes in the transcriptome and the methylome duringin vitrohepatocyte differentiation, we identified putative DNA methylation-regulating transcription factors, which are likely involved in DNA demethylation and maintenance of hypo-methylation in a differentiation stage-specific manner. Of these factors, we further reveal that GATA6 induces DNA demethylation together with chromatin activation at a binding-site-specific manner during endoderm differentiation.ConclusionsThese results provide an insight into the spatiotemporal regulatory mechanisms exerted on the DNA methylation landscape by transcription factors, and uncover a new role for transcription factors in early liver development.

scholarly journals Azacitidine Blocks GATA-1-Mediated Repression of the PU.1 Gene in Human Leukemic Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5220-5220
Author(s):  
Pavel Burda ◽  
Jarmila Vargova ◽  
Nikola Curik ◽  
John Strouboulis ◽  
Giorgio Lucio Papadopoulos ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factors ◽  
Regulatory Element ◽  
Methylation Status ◽  
Erythroid Differentiation ◽  
Dna Demethylation ◽  
Leukemic Cells ◽  
Epigenetic Therapy ◽  
Myeloid Differentiation

Abstract Introduction: GATA-1 and PU.1 are two important hematopoietic transcription factors that mutually inhibit each other in progenitor cells to guide entrance into the erythroid or myeloid lineage, respectively. Expression of PU.1 is controlled by several transcription factors including PU.1 itself by binding to the distal URE enhancer (upstream regulatory element) whose deletion leads to acute myeloid leukemia (AML) (Rosenbauer F et al. 2004). Co-expression of PU.1 and GATA-1 in AML-erythroleukemia (EL) blasts prevents efficient differentiation regulated by these transcription factors. Inhibition of transcriptional activity of PU.1 protein by GATA-1 has been reported (Nerlov C et al. 2000), however it is not known whether GATA-1 can inhibit PU.1 gene in human early erythroblasts directly. We have recently found that MDS/AML erythroblasts display repressive histone modifications and DNA methylation status of PU.1 gene that respond to 5-azacitidine (AZA) leading to inhibited blast cell proliferation and stimulated myeloid differentiation (Curik N et al. 2012). We hypothesize that l eukemia blockade during early erythroid differentiation includes direct GATA-1-mediated inhibition of the PU.1 gene. Results: We herein document the GATA-1 mediated repression of the PU.1 gene in human EL cell lines (OCI-M2 and K562) together with the recruitment of DNA methyl transferase I (DNMT1) to the URE known to guide most of the PU.1 gene transcription. Repression of the PU.1 gene involves both DNA methylation at the URE and methylation/deacetylation of the histone H3 lysine-K9 residue and methylation of H3K27 at additional DNA elements and the PU.1 promoter. Inhibition of GATA-1 by siRNA as well as the AZA treatment in AML-EL led to the significant DNA-demethylation of the URE thorough the mechanism of DNMT1 depletion leading to upregulation of the PU.1 expression. Conclusions: Our data indicate that GATA-1 binds to the PU.1 gene at the URE and initiate events leading to the PU.1 gene repression in human ELs. The mechanism includes repressive epigenetic remodeling of the URE that is important for the PU.1 downregulation and leukemogenesis and that is also simultaneously sensitive to the DNA demethylation treatment with AZA. The GATA-1-mediated inhibition likely contributes to the PU.1 downregulation during progenitor cell differentiation that could be employed during leukemogenesis. Importantly, we also observed important differences between murine and human ELs and found that repression of the PU.1 gene in human ELs can become reverted by the epigenetic therapy with AZA. Our work also suggests that hypomethylating therapy using DNA methylation inhibitors in MDS/AML may become potentially effective in MDS/EL patients. We think that during early erythroid differentiation the GATA-1 binds and represses the PU.1 gene, however this is not fully completed in EL and therefore the erythroid as well as myeloid differentiation are blocked. Grants: GACR P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/1. Disclosures Off Label Use: Azacitidine, DNA demethylation agens tested in vitro in AML/MDS treatment. Stopka:Celgene: Research Funding.

scholarly journals Androgenic-Induced Transposable Elements Dependent Sequence Variation in Barley

2021 ◽  
Vol 22 (13) ◽  
pp. 6783
Author(s):  
Renata Orłowska ◽  
Katarzyna A. Pachota ◽  
Wioletta M. Dynkowska ◽  
Agnieszka Niedziela ◽  
Piotr T. Bednarek
Keyword(s):  
Dna Methylation ◽  
Transposable Elements ◽  
Dna Sequence ◽  
Sequence Variation ◽  
Nuclear Dna ◽  
Point Mutations ◽  
Mobile Elements ◽  
Dna Demethylation ◽  
Plant Genome

A plant genome usually encompasses different families of transposable elements (TEs) that may constitute up to 85% of nuclear DNA. Under stressful conditions, some of them may activate, leading to sequence variation. In vitro plant regeneration may induce either phenotypic or genetic and epigenetic changes. While DNA methylation alternations might be related, i.e., to the Yang cycle problems, DNA pattern changes, especially DNA demethylation, may activate TEs that could result in point mutations in DNA sequence changes. Thus, TEs have the highest input into sequence variation (SV). A set of barley regenerants were derived via in vitro anther culture. High Performance Liquid Chromatography (RP-HPLC), used to study the global DNA methylation of donor plants and their regenerants, showed that the level of DNA methylation increased in regenerants by 1.45% compared to the donors. The Methyl-Sensitive Transposon Display (MSTD) based on methylation-sensitive Amplified Fragment Length Polymorphism (metAFLP) approach demonstrated that, depending on the selected elements belonging to the TEs family analyzed, varying levels of sequence variation were evaluated. DNA sequence contexts may have a different impact on SV generated by distinct mobile elements belonged to various TE families. Based on the presented study, some of the selected mobile elements contribute differently to TE-related SV. The surrounding context of the TEs DNA sequence is possibly important here, and the study explained some part of SV related to those contexts.

Dynamic Changes Of DNA Methylation and a Functional Role For TET2 DNA Dioxygenase In Human Erythroid Differentiation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3415-3415
Author(s):  
Jie Li ◽  
Papoin Julien ◽  
Chao An ◽  
Jingpin Hu ◽  
Ari Melnick ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factors ◽  
Functional Role ◽  
Methylation Status ◽  
Erythroid Differentiation ◽  
Dna Demethylation ◽  
Parent Cell ◽  
Hematopoietic Stem ◽  
Mature Erythrocyte ◽  
Terminal Erythroid Differentiation

Abstract Erythropoiesis is a process by which multipotent hematopoietic stem cells proliferate, differentiate and eventually form mature erythrocytes. This process contains eight distinct differentiation stages including burst-forming unit-erythroid (BFU-E), colony-forming unit-erythroid (CFU-E), proerythroblast, basophilic erythroblast, polychromatic erythroblast, orthochromatic erythroblast, reticulocyte and mature erythrocyte. Unlike most cell types, an important feature of erythropoiesis is that following each of the three or four mitoses that occur during terminal erythroid differentiation, the daughter cells are distinctly different from the parent cell from which they are derived. Thus, erythropoiesis is a complex process that requires tight regulation. The most extensively studied regulators of erythroid differentiation include the EPO/EPOR system and two major transcription factors, GATA1 and KLF1. In contrast to the well-established roles of growth factors, cytokines and transcription factors in regulating erythropoiesis, the regulation of erythropoiesis by other mechanisms is much less understood. In the present study, we explore the changes in DNA methylation during human terminal erythroid differentiation and DNA methylation/demethylation in human erythropoiesis. The methylation status of DNA influences many biologic processes. It has been recently reported that global demethylation occurs during both murine and human erythropoiesis. However, the dynamics of DNA methylation changes, the underlying molecular mechanism(s), and the function of DNA demethylation in erythropoiesis are not clear. To address these issues, we performed next-generation bisulfite sequencing on highly purified human erythroblasts at distinct differentiation stages. We show that while there is a global hypomethylation as terminal erythropoiesis proceeds, stage-specific analysis revealed that a significant proportion of differential methylation includes gains of methylation. Moreover, genes that presented with DNA methylation changes could be categorized into 3 groups based on the dynamics of their methylation changes. As Ten-eleven-translocation proteins (TETs) have been implicated in DNA demethylation by converting 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (5hmc), we attempted to explore the role of TETs in DNA demethylation and terminal erythroid differentiation. We show that 5hmc is progressively increased during human terminal erythroid differentiation. Importantly, knockdown of TET2 by shRNA in human CD34+ cells impaired the production of 5hmc as well as terminal erythroid differentiation. Our findings demonstrate the complexity of DNA methylation dynamics and identify a functional role for TET2 in human erythroid differentiation. These findings provide new and novel insights into the mechanistic understanding of normal and disordered erythropoiesis. As aberrant DNA methylation underlies many hematological diseases including the dyserythropoiesis of myelodysplastic syndromes, we suggest that these finding also provide novel insights into these diseases. Disclosures: No relevant conflicts of interest to declare.

scholarly journals DNA methylation defines regional identity of human intestinal epithelial organoids and undergoes dynamic changes during development

Gut ◽  
2017 ◽  
Vol 68 (1) ◽  
pp. 49-61 ◽  
Author(s):  
Judith Kraiczy ◽  
Komal M Nayak ◽  
Kate J Howell ◽  
Alexander Ross ◽  
Jessica Forbester ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Genome Editing ◽  
Regional Identity ◽  
Dna Demethylation ◽  
Molecular Characteristics ◽  
Intestinal Epithelial ◽  
Dynamic Changes ◽  
Cellular Environment ◽  
Epigenetic Signatures

ObjectiveHuman intestinal epithelial organoids (IEOs) are increasingly being recognised as a highly promising translational research tool. However, our understanding of their epigenetic molecular characteristics and behaviour in culture remains limited.DesignWe performed genome-wide DNA methylation and transcriptomic profiling of human IEOs derived from paediatric/adult and fetal small and large bowel as well as matching purified human gut epithelium. Furthermore, organoids were subjected to in vitro differentiation and genome editing using CRISPR/Cas9 technology.ResultsWe discovered stable epigenetic signatures which define regional differences in gut epithelial function, including induction of segment-specific genes during cellular differentiation. Established DNA methylation profiles were independent of cellular environment since organoids retained their regional DNA methylation over prolonged culture periods. In contrast to paediatric and adult organoids, fetal gut-derived organoids showed distinct dynamic changes of DNA methylation and gene expression in culture, indicative of an in vitro maturation. By applying CRISPR/Cas9 genome editing to fetal organoids, we demonstrate that this process is partly regulated by TET1, an enzyme involved in the DNA demethylation process. Lastly, generating IEOs from a child diagnosed with gastric heterotopia revealed persistent and distinct disease-associated DNA methylation differences, highlighting the use of organoids as disease-specific research models.ConclusionsOur study demonstrates striking similarities of epigenetic signatures in mucosa-derived IEOs with matching primary epithelium. Moreover, these results suggest that intestinal stem cell-intrinsic DNA methylation patterns establish and maintain regional gut specification and are involved in early epithelial development and disease.

scholarly journals DNA Methylation Repels Binding of HIF Transcription Factors to Maintain Tumour Immunotolerance

2020 ◽  
Author(s):  
Flora D’anna ◽  
Laurien Van Dyck ◽  
Jieyi Xiong ◽  
Hui Zhao ◽  
Rebecca V. Berrens ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factors ◽  
Immune Checkpoint ◽  
Binding Sites ◽  
Dna Demethylation ◽  
Transcript Expression ◽  
Dependent Manner ◽  
Cell Type ◽  
Dna Hypomethylation ◽  
Cell Type Specific

AbstractBackgroundHypoxia is pervasive in cancer and other diseases. Cells sense and adapt to hypoxia by activating hypoxia-inducible transcription factors (HIFs), but it is still an outstanding question why cell types differ in their transcriptional response to hypoxia.ResultsHere, we report that HIFs fail to bind CpG dinucleotides that are methylated in their consensus binding sequence, both in in vitro biochemical binding assays and in vivo studies of differentially methylated isogenic cell lines. Based on in silico structural modelling, we show that 5-methylcytosine indeed causes steric hindrance in the HIF binding pocket. A model wherein cell-type-specific methylation landscapes, as laid-down by the differential expression and binding of other transcription factors under normoxia control cell-type-specific hypoxia responses is observed. We also discover ectopic HIF binding sites in repeat regions which are normally methylated. Genetic and pharmacological DNA demethylation, but also cancer-associated DNA hypomethylation, expose these binding sites, inducing HIF-dependent expression of cryptic transcripts. In line with such cryptic transcripts being more prone to cause double-stranded RNA and viral mimicry, we observe low DNA methylation and high cryptic transcript expression in tumours with high immune checkpoint expression, but not in tumours with low immune checkpoint expression, where they would compromise tumour immunotolerance. In a low-immunogenic tumour model, DNA demethylation upregulates cryptic transcript expression in a HIF-dependent manner, causing immune activation and reducing tumour growth.ConclusionsOur data elucidate the mechanism underlying cell-type specific responses to hypoxia, and suggest DNA methylation and hypoxia to underlie tumour immunotolerance.

scholarly journals Modulation of DNA Methylation Influences Cartilage Formation in Murine Chondrogenic Models

2021 ◽  
Author(s):  
Judit Vágó ◽  
Katalin Kiss ◽  
Edina Karanyicz ◽  
Roland Takács ◽  
Csaba Matta ◽  
...  
Keyword(s):  
Dna Methylation ◽  
In Situ Hybridization ◽  
Dna Methyltransferase ◽  
Mouse Cell ◽  
Dna Demethylation ◽  
Specific Gene ◽  
Cartilage Formation

The aim of this study was to investigate the role of DNA methylation in the regulation of in vitro and in vivo cartilage formation. Based on the data of an RNA chip-assay performed on chondrifying BMP2-overexpressing C3H10T1/2 cells, the relative expression of Tet1 (tet methylcytosine dioxygenase 1), Dnmt3a (DNA methyltransferase 3) and Ogt (O-linked N-acetylglucosamine transferase) genes was examined with RT-qPCR in mouse cell-line based and primary micromass cultures. RNA probes for in situ hybridization were used on frozen sections of 15-day-old mouse embryos. DNA methylation was inhibited with 5-azacytidine during culturing. We found very strong but gradually decreasing expression of Tet1 throughout the entire course of in vitro cartilage differentiation along with strong signals in the cartilaginous embryonic skeleton. Dnmt3a and Ogt expressions did not show significant changes with RT-qPCR and gave weak in situ hybridization signals. Inhibition of DNA methylation applied during early stages of differentiation reduced cartilage-specific gene expression and cartilage formation. In contrast, it had stimulatory effect when added to differentiated chondrocytes. Our results indicate that the DNA demethylation-inducing Tet1 is a significant epigenetic factor of chondrogenesis, and inhibition of DNA methylation exerts distinct effects in different phases of in vitro cartilage formation.

scholarly journals lncRNA DIGIT and BRD3 protein form phase-separated condensates to regulate endoderm differentiation

2019 ◽  
Author(s):  
Kaveh Daneshvar ◽  
M. Behfar Ardehali ◽  
Isaac A. Klein ◽  
Arcadia J. Kratkiewicz ◽  
Chan Zhou ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transcription Factors ◽  
Histone H3 ◽  
Domain Family ◽  
Endoderm Differentiation ◽  
Family Protein ◽  
Cellular Components ◽  
Specific Sequences

AbstractGene programs that control differentiation are regulated through the interplay between DNA, RNA, and protein. Cooperation among these fundamental cellular components can occur through highly structured interactions connecting domains formed by specific sequences of nucleotides, ribonucleotides, and/or amino acids and also through the assembly of biomolecular condensates. Here, we show that endoderm differentiation is regulated through the interaction of the long noncoding (lnc) RNA DIGIT and the bromodomain and extra-terminal (BET) domain family protein BRD3. BRD3 forms phase-separated condensates that contain DIGIT, occupies enhancers of endoderm transcription factors, and is required for endoderm differentiation. Purified BRD3 binds to acetylated histone H3 lysine 18 (H3K18ac) in vitro and occupies regions of the genome enriched in H3K18ac during endoderm differentiation, including the key transcription factors that regulate endoderm differentiation. DIGIT is also enriched in regions of H3K18ac, and depletion of DIGIT results in decreased recruitment of BRD3 to these regions. Our findings support a model where cooperation between DIGIT and BRD3 at regions of H3K18ac regulates the transcription factors that drive endoderm differentiation and suggest a broader role for protein-lncRNA phase-separated condensates as regulators of transcription in development.

scholarly journals TET3 overexpression facilitates DNA reprogramming and early development of bovine SCNT embryos

Reproduction ◽  
2020 ◽  
Vol 160 (3) ◽  
pp. 379-391
Author(s):  
Jian Zhang ◽  
Linlin Hao ◽  
Qian Wei ◽  
Sheng Zhang ◽  
Hui Cheng ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Specific Activity ◽  
Mammalian Species ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Dna Demethylation ◽  
Cell Nuclei ◽  
Active Dna Demethylation ◽  
Pluripotency Genes

Somatic cell nuclear transfer (SCNT) has been successfully used for cloning in a variety of mammalian species. However, SCNT reprogramming efficiency is relatively low, in part, due to incomplete DNA methylation reprogramming of donor cell nuclei. We previously showed that ten-eleven translocation 3 (TET3) is responsible for active DNA demethylation during preimplantation embryonic development in bovines. In this study, we constructed TET3-overexpressing cell lines in vitro and observed that the use of these fibroblasts as donor cells increased the blastocyst rate by approximately 18 percentage points compared to SCNT. The overexpression of TET3 in bovine SCNT embryos caused a decrease in the global DNA methylation level of the pluripotency genes Nanog and Oct-4, ultimately resulting in an increase in the transcriptional activity of these pluripotency genes. Moreover, the quality of bovine TET3-NT embryos at the blastocyst stage was significantly improved, and bovine TET3-NT blastocysts possessed more total number of cells and fewer apoptotic cells than the SCNT blastocysts, similar to in vitro fertilization (IVF) embryos. Nevertheless, DNA methylation of the imprinting control region (ICR) for the imprinted genes H19-IGF2 in SCNT embryos remained unaffected by TET3 overexpression, maintaining parent-specific activity for further development. Thus, the results of our study provide a promising approach to rectify incomplete epigenetic reprogramming and achieve higher cloning efficiency.

42 EFFECT OF S-ADENOSYLHOMOCYSTEINE, A NON-TOXIC EPIGENETIC MODIFYING REAGENT, ON PORCINE FEMALE DONOR CELLS AND CLONED EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 169 ◽  
Author(s):  
J. T. Kang ◽  
J. Y. Choi ◽  
S. J. Park ◽  
S. J. Kim ◽  
J. H. Moon ◽  
...  
Keyword(s):  
Dna Methylation ◽  
X Chromosome ◽  
Dna Demethylation ◽  
Control Group ◽  
Time Interval ◽  
Normal Donor ◽  
In Vitro Development ◽  
Donor Cells ◽  
Vitro Development

Despite great advances in the field of cloning techniques, the efficiency of production of cloning animals is very low. Maybe the poor outcome of somatic cell nuclear transfer (SCNT) is thought to be a consequence of incomplete reprogramming of the donor cell or cloned embryos. The objective of this study was to investigate the effects of treatment with S-adenosylhomocysteine (SAH), the reversible nontoxic inhibitor of DNA methyltransferases (DNMT), on porcine female fibroblast donor cells and in vitro development of cloned embryos. We hypothesized that SAH targeting DNA methylation could alter chromatin configuration and turn it more amenable to reprogramming. Thus, the female fibroblast donor cells were cultured in media containing respective concentrations of SAH [0 (control), 0.1, 0.5, and 1 mM) for 2 passages. One-way ANOVA was used to determine significant differences in the data and a Tukey test was done to determine statistical differences among groups. Compared with nontreated controls, the cells treated with SAH, especially 1 mM, revealed significantly (P < 0.05) reduced global DNA methylation, proved by commercial kit and immunocytochemistry analysis, and elevation of transcript levels for X chromosome-linked genes (XIST and HPRT), estimated by real-time PCR analysis compared with the control group. It was suggested that treatment with SAH in female cells could make cells into more valuable donor cells for cloning. In another trial, cloned embryos using normal donor cells were cultured in media containing 1 mM SAH for 0 (control), 12, and 24 h after activation on different time interval of DNMT inhibition, transferred to PZM5 media, and subsequently cultured for 7 days. Treatment with SAH for 12 h resulted in 13.0 ± 1.9% blastocyst production, which was significantly greater than cloned embryos treated with SAH for 24 h (11.2 ± 2.1%) and control cloned embryos (9.1 ± 1.2%). It was suggested that the appropriate DNMT inhibition might have an important role in in vitro development of porcine SCNT, and improving effects on developmental competency of cloned embryos. We concluded that SAH induced global DNA demethylation that partially reactivated the X chromosome and that a hypomethylated genome may facilitate the nuclear reprogramming process. This study was supported by IPET (no. 311011-05-1-SB010), MKE (no. 10033839-2012-21), Institute for Veterinary Science, the BK21 program, and TS Corporation.

Altered chromatin condensation of heat-stressed spermatozoa perturbs the dynamics of DNA methylation reprogramming in the paternal genome after in vitro fertilisation in cattle

2014 ◽  
Vol 26 (8) ◽  
pp. 1107 ◽  
Author(s):  
Mohammad Bozlur Rahman ◽  
Md. Mostofa Kamal ◽  
Tom Rijsselaere ◽  
Leen Vandaele ◽  
Mohammed Shamsuddin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Chromatin Condensation ◽  
Dna Demethylation ◽  
In Vitro Fertilisation ◽  
Paternal Genome ◽  
Time Points ◽  
Fertilisation Rate ◽  
De Novo Methylation

Shortly after penetration of the oocyte, sperm DNA is actively demethylated, which is required for totipotent zygotic development. Aberrant DNA methylation is thought to be associated with altered chromatin condensation of spermatozoa. The objectives of this study were to investigate the dynamics of DNA methylation reprogramming in the paternal pronucleus and subsequent fertilisation potential of heat-stressed bull spermatozoa having altered chromatin condensation. Hence, bovine zygotes (n = 1239) were collected at three different time points (12, 18 and 24 h post insemination, hpi), and stained with an antibody against 5-methylcytosine. Fluorescence intensities of paternal and maternal pronuclei were measured by ImageJ. DNA methylation patterns in paternal pronuclei derived from heat-stressed spermatozoa did not differ between time points (P > 0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24 hpi, respectively. Moreover, heat-stressed spermatozoa showed a highly reduced (P < 0.01) fertilisation rate compared with non-heat-stressed or normal control spermatozoa (53.7% vs 70.2% or 81.5%, respectively). Our data show that the normal pattern of active DNA demethylation followed by de novo methylation in the paternal pronucleus is perturbed when oocytes are fertilised with heat-stressed spermatozoa, which may be responsible for decreased fertilisation potential.

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