scholarly journals Effect of Different DNA Demethylating Agents on In vitro Cultures of Peach Rootstock GF 677

2019 ◽  
Vol 47 (3) ◽  
Author(s):  
Miroslav BARÁNEK ◽  
Miroslav OTMAR ◽  
Marcela KREČMEROVÁ ◽  
Aleš EICHMEIER ◽  
Jana MOUDRÁ ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Crop Improvement ◽  
Digital Object Identifier ◽  
In Vitro Cultivation ◽  
Methylation State ◽  
Demethylating Agents ◽  
The Media ◽  
Somaclonal Variability ◽  
The Impact

The appearance of somaclonal variability induced by in vitro cultivation is relatively frequent and, in some cases, provides a valuable source of new phenotypes suitable for crop improvement. Numerous studies have confirmed that these changes can be explained by alterations of DNA methylation. Interestingly, a group of chemical compounds termed ‘demethylating agents’ (DMT agents) enable artificial changes to be made in the DNA methylation state. Thus, these agents are theoretically able to induce new phenotypes or more favourable properties. The objective of the present study was to verify suitable conditions for the application of different DMT agents within in vitro protocols for micropropagation using the stone fruit rootstock GF 677 as an example. The impact of these agents on the properties of plant regenerants was evaluated, and their DNA methylation state was controlled by using an AFLP protocol based on a restriction endonuclease that differed in its sensitivity to methylated cytosines. Moreover, the effect of newly synthesised derivates was compared with that of conventional compounds with a well-documented DNA-demethylating impact. Based on the results, the suitable concentration for treatment by a DMT agent was established as approximately 50 µM. Promising results were generated using a combination of DMT agents with different mechanisms of action, such as azacytidine and dihydroxypropyladenine; under these conditions, probable synergy between methyltransferase interception by the cytosine analogue and interruption of methyl group donation by dihydroxypropyladenine significantly changed the DNA methylation state of treated plants. Regarding newly synthesised compounds, the 5,6-dihydro-5-azacytosine nucleoside showed the most promising results, which can likely be explained by its higher stability in the media used for in vitro cultivation.   ********* In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 3, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue. *********

scholarly journals Cell–cell coupling and DNA methylation abnormal phenotypes in the after-hours mice

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Federico Tinarelli ◽  
Elena Ivanova ◽  
Ilaria Colombi ◽  
Erica Barini ◽  
Edoardo Balzani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Neuronal Networks ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Neuronal Cultures ◽  
Functional Impairments ◽  
After Hours ◽  
The Impact

Abstract Background DNA methylation has emerged as an important epigenetic regulator of brain processes, including circadian rhythms. However, how DNA methylation intervenes between environmental signals, such as light entrainment, and the transcriptional and translational molecular mechanisms of the cellular clock is currently unknown. Here, we studied the after-hours mice, which have a point mutation in the Fbxl3 gene and a lengthened circadian period. Methods In this study, we used a combination of in vivo, ex vivo and in vitro approaches. We measured retinal responses in Afh animals and we have run reduced representation bisulphite sequencing (RRBS), pyrosequencing and gene expression analysis in a variety of brain tissues ex vivo. In vitro, we used primary neuronal cultures combined to micro electrode array (MEA) technology and gene expression. Results We observed functional impairments in mutant neuronal networks, and a reduction in the retinal responses to light-dependent stimuli. We detected abnormalities in the expression of photoreceptive melanopsin (OPN4). Furthermore, we identified alterations in the DNA methylation pathways throughout the retinohypothalamic tract terminals and links between the transcription factor Rev-Erbα and Fbxl3. Conclusions The results of this study, primarily represent a contribution towards an understanding of electrophysiological and molecular phenotypic responses to external stimuli in the Afh model. Moreover, as DNA methylation has recently emerged as a new regulator of neuronal networks with important consequences for circadian behaviour, we discuss the impact of the Afh mutation on the epigenetic landscape of circadian biology.

scholarly journals Genome-wide DNA methylome analysis identifies methylation signatures associated with survival and drug resistance of ovarian cancers

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
David W. Chan ◽  
Wai-Yip Lam ◽  
Fushun Chen ◽  
Mingo M. H. Yung ◽  
Yau-Sang Chan ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Dna Methylation ◽  
Drug Resistance ◽  
Poor Prognosis ◽  
Ovarian Cancer Cells ◽  
High Grade ◽  
Dna Hypermethylation ◽  
Demethylating Agents ◽  
Ovarian Cancers ◽  
The Impact

Abstract Background In contrast to stable genetic events, epigenetic changes are highly plastic and play crucial roles in tumor evolution and development. Epithelial ovarian cancer (EOC) is a highly heterogeneous disease that is generally associated with poor prognosis and treatment failure. Profiling epigenome-wide DNA methylation status is therefore essential to better characterize the impact of epigenetic alterations on the heterogeneity of EOC. Methods An epigenome-wide association study was conducted to evaluate global DNA methylation in a retrospective cohort of 80 mixed subtypes of primary ovarian cancers and 30 patients with high-grade serous ovarian carcinoma (HGSOC). Three demethylating agents, azacytidine, decitabine, and thioguanine, were tested their anti-cancer and anti-chemoresistant effects on HGSOC cells. Results Global DNA hypermethylation was significantly associated with high-grade tumors, platinum resistance, and poor prognosis. We determined that 9313 differentially methylated probes (DMPs) were enriched in their relative gene regions of 4938 genes involved in small GTPases and were significantly correlated with the PI3K-AKT, MAPK, RAS, and WNT oncogenic pathways. On the other hand, global DNA hypermethylation was preferentially associated with recurrent HGSOC. A total of 2969 DMPs corresponding to 1471 genes were involved in olfactory transduction, and calcium and cAMP signaling. Co-treatment with demethylating agents showed significant growth retardation in ovarian cancer cells through differential inductions, such as cell apoptosis by azacytidine or G2/M cell cycle arrest by decitabine and thioguanine. Notably, azacytidine and decitabine, though not thioguanine, synergistically enhanced cisplatin-mediated cytotoxicity in HGSOC cells. Conclusions This study demonstrates the significant association of global hypermethylation with poor prognosis and drug resistance in high-grade EOC and highlights the potential of demethylating agents in cancer treatment. Graphic abstract

scholarly journals Methylmercury Epigenetics

Toxics ◽  
2019 ◽  
Vol 7 (4) ◽  
pp. 56 ◽  
Author(s):  
Megan Culbreth ◽  
Michael Aschner
Keyword(s):  
Dna Methylation ◽  
Mirna Expression ◽  
Epigenetic Modifications ◽  
Nervous System Development ◽  
Gene Promoters ◽  
Toxic Response ◽  
The Impact ◽  
The Brain

Methylmercury (MeHg) has conventionally been investigated for effects on nervous system development. As such, epigenetic modifications have become an attractive mechanistic target, and research on MeHg and epigenetics has rapidly expanded in the past decade. Although, these inquiries are a recent advance in the field, much has been learned in regards to MeHg-induced epigenetic modifications, particularly in the brain. In vitro and in vivo controlled exposure studies illustrate that MeHg effects microRNA (miRNA) expression, histone modifications, and DNA methylation both globally and at individual genes. Moreover, some effects are transgenerationally inherited, as organisms not directly exposed to MeHg exhibited biological and behavioral alterations. miRNA expression generally appears to be downregulated consequent to exposure. Further, global histone acetylation also seems to be reduced, persist at distinct gene promoters, and is contemporaneous with enhanced histone methylation. Moreover, global DNA methylation appears to decrease in brain-derived tissues, but not in the liver; however, selected individual genes in the brain are hypermethylated. Human epidemiological studies have also identified hypo- or hypermethylated individual genes, which correlated with MeHg exposure in distinct populations. Intriguingly, several observed epigenetic modifications can be correlated with known mechanisms of MeHg toxicity. Despite this knowledge, however, the functional consequences of these modifications are not entirely evident. Additional research will be necessary to fully comprehend MeHg-induced epigenetic modifications and the impact on the toxic response.

scholarly journals SUN-715 IIM May Influence Matured Oocytes’ DNA Methylation of PCOS Patients

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Congru Li ◽  
Yang Yu
Keyword(s):  
Dna Methylation ◽  
In Vitro Fertilization ◽  
Embryonic Development ◽  
Embryo Transfer ◽  
Cpg Island ◽  
Reproductive Technologies ◽  
Childbearing Age ◽  
Vitro Fertilization ◽  
The Impact

Abstract Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of childbearing age and is the main cause of anovulatory infertility. To increase the number of oocytes obtained, controlled ovarian stimulation (COS) has become a routine choice for in vitro fertilization-embryo transfer (IVF-ET), which is one of the common assisted reproductive technologies for PCOS patients. However, for these patients, there is a high risk of ovarian hyperstimulation syndrome (OHSS). Obtaining in vitro maturation (IVM) of immature oocytes, and then in vitro fertilization and embryo transfer of mature oocytes provides a possible way for people to solve the above problems. Since the IVM technology will expose oocytes to in vitro conditions for a longer period of time, theoretically increasing the risk of the oocytes being affected by the culture environment, further research and explorations are needed for study in gene programming, epigenetics, etc. Therefore, to explore the impact of IVM operation on embryonic development is of great significance for further clarifying assisted reproductive safety and improving IVM operation conditions. Here we focused on DNA methylation reprogramming process which was essential for embryonic development. We tested the DNA methylation of sperm, IVM oocytes and IVM generated early stage embryos including pronucleus, 4cell, 8cell, morula, inner cell mass, trophoectoderm (TE) as well as six-week embryos by Nimble Gen Human DNA Methylation 3x729K CpG Island Plus RefSeq Promoter Array and compared the data with our published genome-wide DNA methylomes of human gametes and early embryos generated from in vivo maturation oocytes. We showed that IVM embryos show abnormal DNA methylation reprogramming pattern. By analyzing the abnormally reprogrammed promoters, we further found that IVM may affect the functions of demethylation related genes. Oocytes from IVM manipulation were tested with higher DNA methylation levels, and their abnormal methylated promoters mainly enriched in immune and metabolism pathways. Furthermore, we investigated the DNA methylation of TE, which was directly related with implantation process and revealed the abnormal methylated promoters were related with metabolism pathway too. Our data support that IVM may influence the DNA methylome of oocytes, which in turn affects the methylome of their embryos. However, due to the limited number of samples and the inability of the chip to cover all CpG sites, the results of this study require further research and validation.

DNMT3B Expression Delays Leukemogenesis,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3446-3446
Author(s):  
Petra Tschanter ◽  
Isabell Schulze ◽  
Nicole Bäumer ◽  
Beate Surmann ◽  
Konstantin Agelopoulos ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Malignant Disease ◽  
Bone Marrow Cells ◽  
Dna Methyltransferases ◽  
Leukemic Cells ◽  
Epigenetic Changes ◽  
Leukemia Development ◽  
The Impact ◽  
Dnmt3b Expression

Abstract Abstract 3446 Acute myeloid leukaemia (AML) is a malignant disease with poor prognosis, which is, among other biological features, characterized by epigenetic changes including alterations in DNA methylation. DNA methyltransferases (DNMT) play an important role in regulation of DNA methylation and mutations of DNMT3A are frequently found in AML. We analyzed the effects of DNMT overexpression on leukemogenesis using an inducible DNMT3B mouse model (Linhart et al., 2007). To analyse the impact of DNMT3B overexpression on leukemia we retrovirally co-transduced lineage-negative bone marrow cells of wildtype and DNMT3Btg mice with a MSCV-cMyc-bcl2 and a MSCV-tTA-GFP containing vector. Under these conditions, doxycycline suppressed DNMT3B expression whereas absence of doxycycline led to overexpression of DNMT3B on the mRNA and protein level. DNMT3B overexpression was not toxic since colony formation in vitro did not differ between DNMT3B expressing and physiologically expressing cells. To analyze leukemogenesis, 5 × 104 sorted GFP-positive cells were transplanted into sublethally irradiated wildtype recipients. Both recipients of transduced wildtype cells and recipients of transduced DNMT3Btg cells developed leukemia with a tendency of delayed leukemogenesis in DNMT3B overexpressing mice. GFP positive leukemic cells were sorted and doxycycline regulated DNMT3B expression was verified by Western blot analysis in vitro. To determine the repopulation capacity of the leukemic cells we performed transplantation of GFP-positive primary leukemia cells into secondary wildtype recipients. Leukemia of both, wildtype and DNMT3B-overexpressing donors was transplantable and lethal. However, DNMT3Btg leukemic cells were severely impaired in leukemia development in secondary recipients. Secondary recipients of leukemic DNMT3Btg cells died significantly later (p= 0.02). Taken together, these findings demonstrate that DNMT3B expression impairs leukemia maintenance. Loss of DNMT activity might contribute to the pool size of leukemia initiating cells. Disclosures: Krug: Boehringer Ingelheim: Research Funding.

The effects of pH on DNA methylation state: In vitro and post-mortem brain studies

2008 ◽  
Vol 174 (1) ◽  
pp. 123-125 ◽  
Author(s):  
Carl Ernst ◽  
Patrick O. McGowan ◽  
Vesselina Deleva ◽  
Michael J. Meaney ◽  
Moshe Szyf ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Post Mortem ◽  
Methylation State ◽  
Post Mortem Brain ◽  
Effects Of Ph

scholarly journals Culture Medium and Sex Drive Epigenetic Reprogramming in Preimplantation Bovine Embryos

2021 ◽  
Vol 22 (12) ◽  
pp. 6426
Author(s):  
Sebastian Canovas ◽  
Elena Ivanova ◽  
Meriem Hamdi ◽  
Fernando Perez-Sanz ◽  
Dimitrios Rizos ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Assisted Reproductive Technologies ◽  
Developmental Process ◽  
Reproductive Technologies ◽  
Sexually Dimorphic ◽  
Element Analysis ◽  
Global Methylation ◽  
The Impact

Assisted reproductive technologies impact transcriptome and epigenome of embryos and can result in long-term phenotypic consequences. Whole-genome DNA methylation profiles from individual bovine blastocysts in vivo- and in vitro-derived (using three sources of protein: reproductive fluids, blood serum and bovine serum albumin) were generated. The impact of in vitro culture on DNA methylation was analyzed, and sex-specific methylation differences at blastocyst stage were uncovered. In vivo embryos showed the highest levels of methylation (29.5%), close to those produced in vitro with serum, whilst embryos produced in vitro with reproductive fluids or albumin showed less global methylation (25–25.4%). During repetitive element analysis, the serum group was the most affected. DNA methylation differences between in vivo and in vitro groups were more frequent in the first intron than in CpGi in promoters. Moreover, hierarchical cluster analysis showed that sex produced a stronger bias in the results than embryo origin. For each group, distance between male and female embryos varied, with in vivo blastocyst showing a lesser distance. Between the sexually dimorphic methylated tiles, which were biased to X-chromosome, critical factors for reproduction, developmental process, cell proliferation and DNA methylation machinery were included. These results support the idea that blastocysts show sexually-dimorphic DNA methylation patterns, and the known picture about the blastocyst methylome should be reconsidered.

The impact of distinct culture media inLeishmania infantumbiology and infectivity

Parasitology ◽  
2013 ◽  
Vol 141 (2) ◽  
pp. 192-205 ◽  
Author(s):  
NUNO SANTARÉM ◽  
JOANA CUNHA ◽  
RICARDO SILVESTRE ◽  
CÁTIA SILVA ◽  
DIANA MOREIRA ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Culture Media ◽  
Rational Approach ◽  
Cycle Progression ◽  
The Media ◽  
Parasite Biology ◽  
Basic Characteristics ◽  
The Impact

SUMMARYAn ideal culture medium forLeishmaniapromastigotes should retain the basic characteristics of promastigotes found in sandflies (morphology and infectivity). Furthermore, the media should not create a bias in experimental settings, thus enabling the proper extrapolation of results. To assess this we studied several established media for promastigote growth. We analysed morphology, viability, cell cycle progression, metacyclic profile, capacity to differentiate into axenic amastigotes and infectivity. Furthermore, using a rational approach from the evaluated media we developed a simple serum-free medium (cRPMI). We report that parasites growing in different media present different biological characteristics and distinctin vitroandin vivoinfectivities. The developed medium, cRPMI, proved to be a less expensive substitute for traditional serum-supplemented media for thein vitromaintenance of promastigotes. In fact, cRPMI is ideal for the maintenance of parasites in the laboratory, diminishing the expected loss of virulence over time typical of the parasite cultivation. Ultimately this report is a clear warning that the normalization of culture media should be a real concern in the field as media-specific phenomena are sufficient to induce biological bias with consequences in infectivity and general parasite biology.

scholarly journals The impact of in vitro cultivation on the natural life cycle of the tick-borne relapsing fever spirochete Borrelia turicatae

PLoS ONE ◽  
2020 ◽  
Vol 15 (10) ◽  
pp. e0239089
Author(s):  
Aparna Krishnavajhala ◽  
Brittany A. Armstrong ◽  
Job E. Lopez
Keyword(s):  
Life Cycle ◽  
In Vitro Cultivation ◽  
Relapsing Fever ◽  
The Impact ◽  
Natural Life
Sign in / Sign up

Export Citation Format

Share Document