The repair protein O6-methylguanine-DNA methyltransferase (MGMT) is regulated epigenetically, mainly by the methylation of the MGMT promoter. MGMT promoter methylation status has emerged as a prognostic and predictive biomarker for patients with newly diagnosed glioblastoma (GBM). However, a strong negative correlation between MGMT promoter methylation and MGMT protein expression cannot be applied as a rule for all GBM patients. In order to investigate if the DNA methylation status of MGMT enhancers is associated with MGMT promoter methylation, MGMT expression, and the overall survival (OS) of GBM patients, we established assays based on high-resolution melting analysis and pyrosequencing for one intragenic and three intergenic MGMT enhancers. For CpGs in an enhancer located 560 kb upstream of the MGMT promoter, we found a significant negative correlation between the methylation status and MGMT protein levels of GBM samples expressing MGMT. The methylation status of CpGs in the intragenic enhancer (hs696) was strongly negatively correlated with MGMT promoter methylation and was significantly higher in MGMT-expressing GBM samples than in MGMT-non-expressing GBM samples. Moreover, low methylation of CpGs 01–03 and CpGs 09–13 was associated with the longer OS of the GBM patients. Our findings indicate an association between MGMT enhancer methylation and MGMT promoter methylation, MGMT protein expression, and/or OS.

Seeds of “sweet lupins” have been playing an increasing role in the food industry. Lupin proteins may be used for producing a variety of foods, including pasta, bread, cookies, dairy products, and coffee substitutes. In a small percentage of the population, lupin consumption may elicit allergic reactions, either due to primary sensitization to lupin or due to cross-allergy with other legumes. Thus, lupin has to be declared on commercial food products according to EU food regulations. In this study, we investigated the influence of roasting seeds of the L. angustifolius cultivar “Boregine” on the detectability of lupin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), ELISAs, LC-MS/MS, and real-time PCR. Seeds were roasted by fluidized bed roasting, and samples were drawn at seed surface temperatures ranging from 98 °C to 242 °C. With increasing roasting temperature, the extractability of proteins and DNA decreased. In addition, roasting resulted in lower detectability of lupin proteins by ELISAs and LC-MS/MS and lower detectability of DNA by real-time PCR. Our results suggest reduced allergenicity of roasted lupin seeds used for the production of “lupin coffee”; however, this has to be confirmed in in vivo studies.

The consumption of edible insects is a promising approach to meet the increasing global demand for food. Commercialization of edible insects in the EU is regulated by the Novel Food regulation. To date, the yellow mealworm (Tenebrio molitor larva), the migratory locust (Locusta migratoria), the house cricket (Acheta domesticus), and the buffalo worm (Alphitobius diaperinus larva) have been authorized in the EU for human consumption. We aimed to develop a method based on DNA barcoding and high-resolution melting (HRM) analysis for the identification and differentiation of these four EU-authorized edible insect species in food. A primer pair previously designed for DNA metabarcoding, targeting a ~200 bp sequence of mitochondrial 16S rDNA, allowed discrimination between the four insect species in highly processed food. However, house cricket and migratory locust could not unambiguously be differentiated from tropical house cricket, desert locust, superworm, cowpea weevil, and sago worm, respectively. This problem could be solved by designing primers specific for house cricket and migratory locust. By combining these primers with the insect primers, additional polymerase chain reaction (PCR) products for house cricket and migratory locust were obtained, resulting in more complex melt curves compared to the unauthorized insect species. The optimized PCR-HRM assay is a very cost-efficient screening tool for authentication of EU-authorized edible insect species in food.

The response of glioblastoma (GBM) patients to the alkylating agent temozolomide (TMZ) vitally depends on the expression level of the repair protein O6-methylguanine-DNA methyltransferase (MGMT). Since MGMT is strongly regulated by promoter methylation, the methylation status of the MGMT promoter has emerged as a prognostic and predictive biomarker for GBM patients. By determining the methylation levels of the four enhancers located within or close to the MGMT gene, we recently found that enhancer methylation contributes to MGMT regulation. In this study, we investigated if methylation of the four enhancers is associated with SNP rs16906252, TERT promoter mutations C228T and C250T, TERT SNP rs2853669, proliferation index Ki-67, overall survival (OS), age, and sex of the patients. In general, associations with genetic variants, clinical parameters, and demographic characteristics were caused by a complex interplay of multiple CpGs in the MGMT promoter and of multiple CpGs in enhancer regions. The observed associations for intragenic enhancer 4, located in intron 2 of MGMT, differed from associations observed for the three intergenic enhancers. Some findings were restricted to subgroups of samples with either methylated or unmethylated MGMT promoters, underpinning the relevance of the MGMT promoter status in GBMs.

The methylation status of the MGMT gene promoter is recognized as a key predictive biomarker for glioblastoma patients, influencing treatment decisions and outcomes. Emerging evidence suggests that enhancer methylation may also play a role in gene regulation and is associated with various clinical parameters, genetic variants, and demographic factors. This study aimed to assess DNA methylation levels in intergenic and intragenic MGMT enhancers to investigate their relationship with MGMT promoter methylation, MGMT protein expression, and clinical and demographic characteristics in glioblastoma. We developed 18 pyrosequencing assays to analyze 54 CpGs, including 34 in intergenic and 20 in intragenic enhancers. The assays were applied to tumor cells derived from 38 glioma patients. Intragenic enhancer CpGs showed significantly higher methylation than intergenic enhancer CpGs. Intragenic enhancer methylation showed a strong negative correlation with MGMT promoter methylation. For several CpGs in intragenic enhancers, an inverse L-shaped relationship between methylation levels and MGMT expression was observed. We identified distinct associations between enhancer methylation and clinical and demographic parameters. Intergenic enhancer methylation was primarily linked to the TERT SNP rs2853669 genotype, Ki-67 expression, age, and sex, whereas intragenic enhancer methylation was associated with MGMT promoter methylation, MGMT expression, overall survival, and progression-free survival. Further studies with larger patient cohorts are needed to validate the clinical relevance of intergenic and intragenic MGMT enhancer methylation in glioblastoma.

Many cancer-associated deaths result from metastases rather than primary tumors. Growing evidence suggests that DNA methylation alterations are crucial for inducing a plastic phenotype that allows cancer cells to adapt to the metastatic microenvironment. Brain metastases of melanoma (MBM) and glioblastoma (GB) share a neuroectodermal origin and the brain as tissue of residence, but their epigenetic regulation is poorly understood. Aiming at elucidating shared and tumor-distinct features, we analyzed the methylation of MGMT regulatory elements. We focused on MGMT because MGMT promoter methylation is used as a predictive marker for temozolomide response in GB, but its role in MBM has been discussed controversially. By targeting 12 CpG dinucleotides (CpGs) in the promoter, 68 CpGs in intergenic enhancers, and 31 CpGs in intragenic enhancers, we identified shared features, including an L-shaped relationship between promoter methylation and MGMT protein expression and an inverse L-shaped relationship between intragenic enhancer methylation and MGMT protein expression. GB exhibited higher methylation, particularly in promoter and intergenic enhancers, and stronger associations between methylation and overall survival than MBM. These results highlight both conserved and tumor-specific MGMT regulation, reflecting the complexity of epigenetic control in brain malignancies and emphasizing divergent evolution between MBM and GB.