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Genetic variation and epigenetic factors are thought to contribute to the development of hypersensitivity to aspirin. DNA methylation fluctuates dynamically throughout the day. To discover new CpG methylation in lymphocytes associated with aspirin-exacerbated respiratory disease (AERD), we evaluated changes in global CpG methylation profiles from before to after an oral aspirin challenge in patients with AERD and aspirin-tolerant asthma (ATA). Whole-genome CpG methylation levels of peripheral blood mononuclear cells were quantified with an Illumina 860K Infinium Methylation EPIC BeadChip array and then adjusted for inferred lymphocyte fraction (ILF) with GLINT and Tensor Composition Analysis. Among the 866,091 CpGs in the array, differentially methylated CpGs (DMCs) were found in 6 CpGs in samples from all 12 patients with asthma included in the study (AERD, n = 6; ATA, n = 6). DMCs were found in 3 CpGs in the 6 ATA samples and in 615 CpGs in the 6 AERD samples. A total of 663 DMCs in 415 genes and 214 intergenic regions differed significantly in the AERD compared with the ATA. In promoters, 126 CpG loci were predicted to bind to 38 transcription factors (TFs), many of which were factors already known to be involved in the pathogenesis of asthma and immune responses. In conclusion, we identified 615 new CpGs methylated in peripheral blood lymphocytes by oral aspirin challenge in AERD but not in ATA. These findings indicate that oral aspirin challenge induces epigenetic changes in ILFs, specifically in AERD patients, possibly via changes in TF binding, which may have epigenetic effects on the development of AERD. [ABSTRACT FROM AUTHOR]

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Purpose: Glioblastoma is the most aggressive form of brain tumors. A better understanding of the molecular mechanisms leading to its evolution is essential for the development of treatments more effective than the available modalities. Here, we aim to identify molecular drivers of glioblastoma development and recurrence by analyzing DNA CpG methylation patterns in sequential samples. Methods: DNA was isolated from 22 pairs of primary and recurrent formalin-fixed, paraffin-embedded glioblastoma specimens, and subjected to reduced representation bisulfite sequencing. Bioinformatic analyses were conducted to identify differentially methylated sites and pathways, and biostatistics was used to test correlations among clinical and pathological parameters. Results: Differentially methylated pathways likely involved in primary tumor development included those of neuronal differentiation, myelination, metabolic processes, synapse organization and endothelial cell proliferation, while pathways differentially active during glioblastoma recurrence involved those associated with cell processes and differentiation, immune response, Wnt regulation and catecholamine secretion and transport. Conclusion: DNA CpG methylation analyses in sequential clinical specimens revealed hypomethylation in certain pathways such as neuronal tissue development and angiogenesis likely involved in early tumor development and growth, while suggested altered regulation in catecholamine secretion and transport, Wnt expression and immune response contributing to glioblastoma recurrence. These pathways merit further investigations and may represent novel therapeutic targets. [ABSTRACT FROM AUTHOR]

Acetaminophen (APAP) overdose is one of the most frequent causes of acute liver failure (ALF). N-acetylcysteine (NAC) is currently being used as part of the standard care in the clinic but its usage has been limited in severe cases, in which liver transplantation becomes the only treatment option. Therefore, there still is a need for a specific and effective therapy for APAP induced ALF. In the current study, we have demonstrated that treatment with 25-Hydroxycholesterol 3-Sulfate (25HC3S) not only significantly reduced mortality but also decreased the plasma levels of liver injury markers, including LDH, AST, and ALT, in APAP overdosed mouse models. 25HC3S also decreased the expression of those genes involved in cell apoptosis, stabilized mitochondrial polarization, and significantly decreased the levels of oxidants, malondialdehyde (MDA), and reactive oxygen species (ROS). Whole genome bisulfite sequencing analysis showed that 25HC3S increased demethylation of 5mCpG in key promoter regions and thereby increased the expression of those genes involved in MAPK-ERK and PI3K-Akt signaling pathways. We concluded that 25HC3S may alleviate APAP induced liver injury via up-regulating the master signaling pathways and maintaining mitochondrial membrane polarization. The results suggest that 25HC3S treatment facilitates the recovery and significantly decreases the mortality of APAP induced acute liver injury and has a synergistic effect with NAC in propylene glycol (PG) for the injury.

Background and Purpose:

Glioblastoma (GBM), a highly aggressive form of brain tumors, has been extensively studied using OMICS methods, and the most characteristic molecular determinants have been incorporated into the histopathological diagnosis. Research data, nevertheless, only partially have been adopted in clinical practice. Here we aimed to present results of our epige­no­mic GBM profiling to better understand early and late determinants of these tumors, and to share main elements of our findings with practicing professionals.

GBM specimens were surgically obtained after first diagnosis (GBM1) and at recurrence (GBM2). DNA was extracted from 24 sequential pairs of formalin-fixed, paraffin-embedded tumor tissues. The Reduced Representation Bisulfite Sequencing kit was used for library preparation. Pooled libraries were sequenced on an Illumina NextSeq 550 instrument. Methylation controls (MC) were obtained from a publicly available database. Bioinformatic analyses were performed to identify differentially methylated pathways and their elements in cohorts of MC, GBM1 and GBM2.

Several differentially methylated pathways involved in basic intracellular and brain tissue developmental processes were identified in the GBM1 vs. MC and GBM2 vs. MC comparisons. Among differentially me­thylated pathways, those involved in immune regulation, neurotransmitter (particularly dopaminergic, noradrenergic and glutaminergic) responses and regulation of stem cell differentiation and proliferation stood out in the GBM2 vs. GBM1 comparisons.

Our study revealed biological complexity of early and late gliomagenesis encompassing mechanisms from basic intracellular through distorted neurodevelopmental processes to more specific immune and highjacked neurotransmitter pathways in the tumor microenvironment. These findings may offer considerations for therapeutic approaches.

Glioblastoma (GBM) is the most aggressive tumor of the central nervous system (CNS). The standard of care improves the overall survival of patients only by a few months. Explorations of new therapeutic targets related to molecular properties of the tumor are under way. Even though neurotransmitters and their receptors normally function as mediators of interneuronal communication, growing data suggest that these molecules are also involved in modulating the development and growth of GBM by acting on neuronal and glioblastoma stem cells. In our previous DNA CpG methylation studies, gene ontology analyses revealed the involvement of the monoamine pathway in sequential GBM. In this follow-up study, we quantitated the expression levels of four selected catecholamine pathway markers (alpha 1D adrenergic receptor—ADRA1D; adrenergic beta receptor kinase 1 or G protein-coupled receptor kinase 2—ADRBK1/GRK2; dopamine receptor D2—DRD2; and synaptic vesicle monoamine transporter—SLC18A2) by immunohistochemistry, and compared the histological scores with the methylation levels within the promoters + genes of these markers in 21 pairs of sequential GBM and in controls. Subsequently, we also determined the promoter and gene methylation levels of the same markers in an independent database cohort of sequential GBM pairs. These analyses revealed partial inverse correlations between the catecholamine protein expression and promoter + gene methylation levels, when the tumor and control samples were compared. However, we found no differences in the promoter + gene methylation levels of these markers in either our own or in the database primary–recurrent GBM pairs, despite the higher protein expression of all markers in the primary samples. This observation suggests that regulation of catecholamine expression is only partially related to CpG methylation within the promoter + gene regions, and additional mechanisms may also influence the expression of these markers in progressive GBM. These analyses underscore the involvement of certain catecholamine pathway markers in GBM development and suggest that these molecules mediating or modulating tumor growth merit further exploration.

Summary: MLH1 and MSH2 are important genes for DNA mismatch repair and crossing over during meiosis and are implicated in male infertility. Therefore, the methylation patterns of the DNA mismatch repair genes MLH1 and MSH2 in oligozoospermic males were investigated. Ten oligozoospermic patients and 29 normozoospermic donors were analysed. Methylation profiles of the MLH1 and MSH2 promotors were analysed. In addition, sperm motility and seminal reactive oxygen species (ROS) were recorded. Receiver operating characteristic (ROC) analysis was conducted to determine the accuracy of the DNA methylation status of MLH1 and MSH2 to distinguish between oligozoospermic and normozoospermic men. In oligozoospermic men, MLH1 was significantly (p = .0013) more methylated compared to normozoospermic men. Additionally, there was a significant positive association (r = .384; p = .0159) between seminal ROS levels and MLH1 methylation. Contrary, no association between MSH2 methylation and oligozoospermia was found. ROC curve analysis for methylation status of MLH1 was significant (p = .0275) with an area under the curve of 61.1%, a sensitivity of 22.2% and a specificity of 100.0%. This pilot study indicates oligozoospermic patients have more methylation of MLH1 than normozoospermic patients. Whether hypermethylation of the MLH1 promoter plays a role in repairing relevant mismatches of sperm DNA strands in idiopathic oligozoospermia warrants further investigation. [ABSTRACT FROM AUTHOR]

Prenatal exposure to the antiepileptic, mood-stabilizing drug, valproic acid (VPA), increases the incidence of autism spectrum disorders (ASDs); in utero administration of VPA to pregnant rodents induces ASD-like behaviors such as repetitive, stereotyped activity, and decreased socialization. In both cases, males are more affected than females. We previously reported that VPA, administered to pregnant mice at gestational day 12.5, rapidly induces a transient, 6-fold increase in BDNF (brain-derived neurotrophic factor) protein and mRNA in the fetal brain. Here, we investigate sex differences in the induction of Bdnf expression by VPA as well as the underlying epigenetic mechanisms. We found no sex differences in the VPA stimulation of total brain Bdnf mRNA as indicated by probing for the BDNF protein coding sequence (exon 9); however, stimulation of individual transcripts containing two of the nine 5'-untranslated exons (5'UTEs) in Bdnf (exons 1 and 4) by VPA was greater in female fetal brains. These Bdnf transcripts have been associated with different cell types or subcellular compartments within neurons. Since VPA is a histone deacetylase inhibitor, covalent histone modifications at Bdnf 5'UTEs in the fetal brain were analyzed by chromatin immunoprecipitation. VPA increased the acetylation of multiple H3 and H4 lysine residues in the vicinity of exons 1, 2, 4, and 6; minimal differences between the sexes were observed. H3 lysine 4 trimethylation (H3K-4me3) at those exons was also stimulated by VPA. Moreover, the VPA-induced increase in H3K4me3 at exons 1, 4, and 6 was significantly greater in females than in males, i.e., sexually dimorphic stimulation of H3K4me3 by VPA correlated with Bdnf transcripts containing exons 1 and 4, but not 6. Neither H3K27me3 nor cytosine methylation at any of the 117 CpGs in the vicinity of the transcription start sites of exons 1, 4, and 6 was affected by VPA. Thus, of the 6 epigenetic marks analyzed, only H3K4me3 can account for the sexually dimorphic expression of Bdnf transcripts induced by VPA in the fetal brain. Preferential expression of exon 1- and exon 4- Bdnf transcripts in females may contribute to sex differences in ASDs by protecting females from the adverse effects of genetic variants or environmental factors such as VPA on the developing brain. [ABSTRACT FROM AUTHOR]

Epigenetics has become a major field of reproductive medicine after the epigenetic regulation of gene expression was discovered. The aim of this study was to find out whether or not methylenetetrahydrofolate reductase ( MTHFR) gene promoter hypermethylation in the spermatozoa of men who were offered assisted reproduction is associated with idiopathic male infertility. Sperm DNAs from 40 idiopathic infertile men with normozoospermia and 40 controls consisting of healthy fertile men were isolated. Following the modification of DNAs by sodium bisulphite, the methylation status of the MTHFR gene promoter was quantified by pyrosequencing. No significant differences were observed between the clinical characteristics of patients and controls. The percentage of MTHFR promoter methylation in infertile men with normozoospermia (11%) was significantly higher than that in the healthy control (4.3%) group ( p = .01). A 9.5% of methylation level was determined via receiver operator characteristic ( ROC) analysis as the cut-off value. There were 21 (53%) hypermethylated men among the infertile men and 2 (5%) in the control group ( p = .0001). The intragroup analysis of the infertile group did not reveal any statistically significant differences in terms of overall clinical characteristics between hyper- and normo-methylated infertile men. Our results suggest that epigenetic silencing (hypermethylation) of MTHFR could result in an elevated risk of male infertility. [ABSTRACT FROM AUTHOR]

Bladder cancer is highly recurrent following specific transurethral resection and intravesical chemotherapy, which has prompted continuing efforts to develop novel therapeutic agents and early-stage diagnostic tools. Specific changes in protein expression can provide a diagnostic marker. In our present study, we investigated changes in protein expression during urothelial carcinogenesis. The carcinogen BBN was used to induce mouse bladder tumor formation. Mouse bladder mucosa proteins were collected and analyzed by 2D electrophoresis from 6 to 20 weeks after commencing continuous BBN treatment. By histological examination, the connective layer of the submucosa showed gradual thickening and the number of submucosal capillaries gradually increased after BBN treatment. At 12-weeks after the start of BBN treatment, the urothelia became moderately dysplastic and tumors arose after 20-weeks of treatment. These induced bladder lesions included carcinoma in situ and connective tissue invasive cancer. In protein 2D analysis, the sequentially downregulated proteins from 6 to 20 weeks included GSTM1, L-lactate dehydrogenase B chain, keratin 8, keratin 18 and major urinary proteins 2 and 11/8. In contrast, the sequentially upregulated proteins identified were GSTO1, keratin 15 and myosin light polypeptide 6. Western blotting confirmed that GSTM1 and NQO-1 were decreased, while GSTO1 and Sp1 were increased, after BBN treatment. In human bladder cancer cells, 5-aza-2′-deoxycytidine increased the GSTM1 mRNA and protein expression. These data suggest that the downregulation of GSTM1 in the urothelia is a biomarker of bladder carcinogenesis and that this may be mediated by DNA CpG methylation. [ABSTRACT FROM AUTHOR]

Abstract: Background: IL-20 is a pleiotrophic member of the IL-10 family and plays a role in skin biology and the development of haematopoietic cells. Recently, IL-20 has been demonstrated to have potential anti-angiogenic effects in non-small cell lung cancer (NSCLC) by down regulating COX-2. Methods: The expression of IL-20 and its cognate receptors (IL-20RA/B and IL-22R1) was examined in a series of resected fresh frozen NSCLC tumours. Additionally, the expression and epigenetic regulation of this family was examined in normal bronchial epithelial and NSCLC cell lines. Furthermore, the effect of IL-20 on VEGF family members was examined. Results: The expression of IL-20 and its receptors are frequently dysregulated in NSCLC. IL-20RB mRNA was significantly elevated in NSCLC tumours (p <0.01). Protein levels of the receptors, IL-20RB and IL-22R1, were significantly increased (p <0.01) in the tumours of NSCLC patients. IL-20 and its receptors were found to be epigenetically regulated through histone post-translational modifications and DNA CpG residue methylation. In addition, treatment with recombinant IL-20 resulted in decreased expression of the VEGF family members at the mRNA level. Conclusions: This family of genes are dysregulated in NSCLC and are subject to epigenetic regulation. Whilst the anti-angiogenic properties of IL-20 require further clarification, targeting this family via epigenetic means may be a viable therapeutic option in lung cancer treatment. [Copyright &y& Elsevier]

Acetaminophen (APAP) overdose is one of the most frequent causes of acute liver failure (ALF). N-acetylcysteine (NAC) is currently being used as part of the standard care in the clinic but its usage has been limited in severe cases, in which liver transplantation becomes the only treatment option. Therefore, there still is a need for a specific and effective therapy for APAP induced ALF. In the current study, we have demonstrated that treatment with 25-Hydroxycholesterol 3-Sulfate (25HC3S) not only significantly reduced mortality but also decreased the plasma levels of liver injury markers, including LDH, AST, and ALT, in APAP overdosed mouse models. 25HC3S also decreased the expression of those genes involved in cell apoptosis, stabilized mitochondrial polarization, and significantly decreased the levels of oxidants, malondialdehyde (MDA), and reactive oxygen species (ROS). Whole genome bisulfite sequencing analysis showed that 25HC3S increased demethylation of 5mCpG in key promoter regions and thereby increased the expression of those genes involved in MAPK-ERK and PI3K-Akt signaling pathways. We concluded that 25HC3S may alleviate APAP induced liver injury via up-regulating the master signaling pathways and maintaining mitochondrial membrane polarization. The results suggest that 25HC3S treatment facilitates the recovery and significantly decreases the mortality of APAP induced acute liver injury and has a synergistic effect with NAC in propylene glycol (PG) for the injury. [ABSTRACT FROM AUTHOR]

Mitochondria, as the center of energy production, play an important role in cell homeostasis by regulating the cellular metabolism and mediating the cellular response to stress. Epigenetic changes such as DNA and histone methylation have been increasingly recognized to play a significant role in homeostasis and stress response. The cross-talking between the metabolome and the epigenome has attracted significant attention in recent years but with a major focus on how metabolism contributes to epigenomic changes. Few studies have focused on how epigenetic modifications may alter the mitochondrial composition and activity. In this work, we designed a novel probe targeting methylated CpGs of mitochondrial DNA (mtDNA). We demonstrated the capability of our probe to reveal the spatial distribution of methylated mtDNA and capture the mtDNA methylation changes at a single-cell level. We were also able to track single-cell mtDNA and nDNA methylation simultaneously and discovered the unsynchronized dynamics of the nucleus and mitochondria. Our tool offers a unique opportunity to understand the epigenetic regulation of mtDNA and its dynamic response to the microenvironment and cellular changes.

Wnt signaling plays important roles in tumorigenesis, invasiveness and therapeutic resistance of glioblastoma (GBM). We simultaneously investigated six Wnt pathway markers (Wnt5a, Fzd-2, beta-catenin, Wnt3a, Wnt7b, Fzd-10) at epigenetic and protein levels in 21 sequential formalin-fixed paraffin-embedded GBM pairs and controls. Expression levels of Wnt5a, beta-catenin and Wnt3a proteins either moderately or significantly increased, while those of Fzd-2, Wnt7b and Fzd-10 decreased in the primary (GBM-P) and recurrent (GBM-R) tumors compared to the controls. Methylation levels within promoters and genes showed corresponding decreases for Wnt5a, beta-catenin and Wnt3a in tumors vs. controls, while that of Fzd-10 was uniformly high. Comparing the GBM-P and GBM-R pairs, proteins of Fzd-2, beta-catenin and Wnt3a were either moderately or significantly up-, while that of Wnt7b was downregulated in GBM-R, but these patterns were not accompanied by inverse methylation patterns in the corresponding promoters and genes over time. No methylation differences were noted within promoters and genes of the same markers in 112 pairs of primary and recurrent GBMs in a database, suggesting that the observed changes in protein expression levels may not be explained by CpG methylation status alone. The promoter and gene methylation rate was the highest for Fzd-10 in the database cohort too, supporting the noted low Fzd-10 protein expression. These analyses underscore the relevance of Wnt pathway molecules in the context of their methylation profiles in the development and evolution of GBM, and suggest that Wnt pathway regulation as a potential treatment target merits further studies. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is the most aggressive tumor of the central nervous system (CNS). The standard of care improves the overall survival of patients only by a few months. Explorations of new therapeutic targets related to molecular properties of the tumor are under way. Even though neurotransmitters and their receptors normally function as mediators of interneuronal communication, growing data suggest that these molecules are also involved in modulating the development and growth of GBM by acting on neuronal and glioblastoma stem cells. In our previous DNA CpG methylation studies, gene ontology analyses revealed the involvement of the monoamine pathway in sequential GBM. In this follow-up study, we quantitated the expression levels of four selected catecholamine pathway markers (alpha 1D adrenergic receptor—ADRA1D; adrenergic beta receptor kinase 1 or G protein-coupled receptor kinase 2—ADRBK1/GRK2; dopamine receptor D2—DRD2; and synaptic vesicle monoamine transporter—SLC18A2) by immunohistochemistry, and compared the histological scores with the methylation levels within the promoters + genes of these markers in 21 pairs of sequential GBM and in controls. Subsequently, we also determined the promoter and gene methylation levels of the same markers in an independent database cohort of sequential GBM pairs. These analyses revealed partial inverse correlations between the catecholamine protein expression and promoter + gene methylation levels, when the tumor and control samples were compared. However, we found no differences in the promoter + gene methylation levels of these markers in either our own or in the database primary–recurrent GBM pairs, despite the higher protein expression of all markers in the primary samples. This observation suggests that regulation of catecholamine expression is only partially related to CpG methylation within the promoter + gene regions, and additional mechanisms may also influence the expression of these markers in progressive GBM. These analyses underscore the involvement of certain catecholamine pathway markers in GBM development and suggest that these molecules mediating or modulating tumor growth merit further exploration. [ABSTRACT FROM AUTHOR]

MLH1 and MSH2 are important genes for DNA mismatch repair and crossing over during meiosis and are implicated in male infertility. Therefore, the methylation patterns of the DNA mismatch repair genes MLH1 and MSH2 in oligozoospermic males were investigated. Ten oligozoospermic patients and 29 normozoospermic donors were analysed. Methylation profiles of the MLH1 and MSH2 promotors were analysed. In addition, sperm motility and seminal reactive oxygen species (ROS) were recorded. Receiver operating characteristic (ROC) analysis was conducted to determine the accuracy of the DNA methylation status of MLH1 and MSH2 to distinguish between oligozoospermic and normozoospermic men. In oligozoospermic men, MLH1 was significantly (p = .0013) more methylated compared to normozoospermic men. Additionally, there was a significant positive association (r = .384; p = .0159) between seminal ROS levels and MLH1 methylation. Contrary, no association between MSH2 methylation and oligozoospermia was found. ROC curve analysis for methylation status of MLH1 was significant (p = .0275) with an area under the curve of 61.1%, a sensitivity of 22.2% and a specificity of 100.0%. This pilot study indicates oligozoospermic patients have more methylation of MLH1 than normozoospermic patients. Whether hypermethylation of the MLH1 promoter plays a role in repairing relevant mismatches of sperm DNA strands in idiopathic oligozoospermia warrants further investigation., (© 2017 Blackwell Verlag GmbH.)

Pyrosequencing is a technique that uses a sequencing-by-synthesis system which is designed to quantify single-nucleotide polymorphisms (SNPs). Artificial C/T SNP creation via bisulfite modification permits measurement of DNA methylation locally and globally in real time. Alteration in DNA methylation has been implicated in aging, as well as aging-related conditions such as cancer, as well as cardiovascular, neurodegenerative, and autoimmune diseases. Considering its ubiquitous presence in divergent clinical pathologies, quantitative analysis of DNA CpG methylation both globally and at individual genes helps to elucidate the regulation of genes involved in pathophysiological conditions. The ability to detect and quantify the methylation pattern of DNA has the potential to serve as an early detection marker and potential drug target for several diseases. Here, we provide a detailed technical protocol for pyrosequencing supplemented by critical information about assay design and nuances of the system that provides a strong foundation for beginners in the field.