Glioblastoma (GBM) is the most malignant neoplasm with predominant astrocytic differentiation and the most frequent primary brain tumor of the adult. Here, we investigated 170 human GBM specimens deriving from 162 patients, as well as 66 healthy control tissue specimens deriving from 27 patients, and analyzed the amount of 5-hydroxymethylcytosine (5hmC) in GBMs compared to normal brain and tumor infiltration zones. Additionally, we correlated the amount of 5hmC with two different proliferation markers, Ki67 and H3S10p. Genetic characterization of GBMs enabled us to analyze the effect of isocitrate dehydrogenase 1 (IDH1) mutations, O6-methylguanin-DNA-methyltransferase (MGMT) promoter methylation, and loss of heterozygosity of chromosome 1p and 19q (LOH1p/19q) on 5hmC amount. We found that GBMs show a tremendous loss of 5hmC, and we observed that even the infiltration zones show reduced amounts of 5hmC. Interestingly, the amount of 5hmC was inversely proportional to the two investigated proliferation markers, Ki67 and H3S10p. Correlation of 5hmC amount and molecular genetic markers of GBMs showed that there are no correlations of 5hmC amount and IDH1 mutations, MGMT promoter methylation, and LOH1p/19q. Furthermore, we evaluated the intratumoral distribution of 5hmC in compact and infiltrating areas and found that the quantification of the 5hmC amount is a useful tool in evaluation of tumor infiltration. In summary, our data emphasize that GBMs show a disturbed hydroxymethylome that is disrupted by IDH1 independent pathways, and that loss of 5hmC shows astonishing intratumoral heterogeneity.

Background: Recent research indicates that long non-coding RNAs (lncRNA) represent a new family of RNAs that is of fundamental importance for controlling transcription and translation. Thereby, there is increasing evidence that lncRNAs are also important in tumourigenesis. Thereby valid expression profiling using quantitative PCR requires suitable, stably expressed normalisers to achieve reliable and reproducible data. However, no systematic analysis of suitable references in lncRNA studies in human glioma has been performed yet.Methods: In this study, we investigated 90 lncRNAs in 30 tissue specimen for the expression stability in human diffuse astrocytoma (WHO-Grade II), anaplastic astrocytoma (WHO-Grade III) and glioblastoma (WHO-Grade IV) both alone as well as in comparison with normal white matter. Our identification procedure included a rigorous bioinformatical selection process that resulted in the inclusion of only highly abundant, equally expressed lncRNAs for further analysis. Additionally, lncRNAs were classified according to their stability value using the NormFinder algorithm.Results: We identified 24 appropriate normalisers suitable for studies in diffuse astrocytoma, 22 for studies in anaplastic astrocytoma and 12 for studies in glioblastoma. Comparing all three glioma entities 7 lncRNAs showed stable expression levels. Addition of normal brain tissue resulted in only 4 suitable lncRNAs.Conclusions: Our findings indicate that 4 lncRNAs (HOXA6as, H19 upstream conserved 1 and 2, Zfhx2as and BC200) are suitable as normalisers in glioma and normal brain. These lncRNAs may thus be regarded as universal references being applicable for the accurate normalisation of lncRNA expression profiling in various glioma (WHO-Grades II-IV) alone and in combination with brain tissue. This enables to perform valid longitudinal studies, e.g. of glioma before and after malignisation to identify changes of lncRNA expressions probably driving malignant transformation.

The molecular mechanisms leading to brain tumors still remain unclear. Nevertheless, there is increasing evidence that epigenetic effects play crucial roles in tumor development and progression. Thereby, 5-hydroxymethylcytosine (5hmC) represents a further base modification of cytosine besides 5-methylcytosine (5mC). In addition to the role of 5hmC as an intermediate in demethylation, 5hmC is of reasonable importance for cellular control. Previous studies showed that loss of 5hmC is a hallmark of human malignancies, e.g. in glioma, melanoma, and myeloid tumors. In myeloid malignancies studies showed that loss of 5hmC was due to mutations within ten-eleven-translocation (TET) genes, enzymes being responsible for conversion of 5mC to 5hmC. Nevertheless, till date there are no genetic characterization data of TET enzymes available for glioma.In this study, we genetically characterized TET2 and TET3 alterations in 50 human gliomas (WHO-Grade II-IV) and in 19 healthy brain samples. We identified 7 genetic alterations within TET2 (p.V218M, p.G355N, p.P363L, p.L1721W, p.P1723S, p.I1762V, p.H1778R). Additionally, we performed quantification of 5hmC amount and added functional prediction analysis of identified TET alterations to evaluate the biological impact of these alterations on the hydroxymethylome. An analysis of TET3 showed no non-synonymous alterations.In summary, we did not find correlations of TET alterations with 5hmC amount. Thus, our data emphasize that, in contrast to leukemia, loss of 5hmC in glioma is not caused by TET gene alterations. Moreover, other disturbances, such as disrupted gene expressions or functional inhibitions of TET proteins may be responsible for the aberrant epigenome of human glioma.

The epigenome is of fundamental importance for development and ageing. The discovery of 5-hydroxymethylcytosine (5hmC), a further base modification of cytosine beyond 5-methylcytosine, might be of high relevance in understanding the complexity of the human brain, as 5hmC is found in great extent in brain tissue. The aim of this study was to investigate the quantity of 5hmC containing nuclei by immunohistochemistry in human and murine brains at several developmental stages. We performed immunohistochemical stainings on frontal cortex, white matter and cerebellar cortex of 15 healthy controls. Three cases each were assigned to five age groups (foetus, adolescent, adult, elderly, aged). Additionally, cortex and cerebellum of 15 mice sacrificed between day 0 and 120 after birth were investigated. We found marked alterations of 5hmC amount during ageing. In human cortex there was an increase of 5hmC of 50%, in white matter we found an increase of even 200% during ageing. In the cerebellum both internal granular cell layer and molecular cell layer showed a significant increase of 5hmC till adulthood. Purkinje cell nuclei showed constantly positive signals for 5hmC. These data were paralleled in murine brains. Co-labelling of 5hmC and markers for mature and immature cells in murine cerebellar cortex at the age of 7 days revealed that 5hmC was found in mature but not in immature cells. In conclusion, the findings described in this study emphasise the importance of 5hmC in brain development and ageing and will help to better understand the complexity and plasticity of the brain.

Cidaroid sea urchins are the sister clade to all other extant echinoids and have numerous unique features, including unusual primary spines. These lack an epidermis when mature, exposing their high‐magnesium calcite skeleton to seawater and allowing the settlement of numerous epibionts. Cidaroid spines are made of an inner core of classical monocrystalline skeleton and an outer layer of polycrystalline magnesium calcite. Interestingly, cidaroids survived the Permian‐Triassic crisis, which was characterized by severe acidification of the ocean. Currently, numerous members of this group inhabit the deep ocean, below the saturation horizon for their magnesium calcite skeleton. This suggests that members of this taxon may have characteristics that may allow them to resist ongoing ocean acidification linked to global change. We compared the effect of acidified seawater (pH 7.2, 7.6, or 8.2) on mature spines with a fully developed cortex to that on young, growing spines, in which only the stereom core was developed. The cortex of mature spines was much more resistant to etching than the stereom of young spines. We then examined the properties of the cortex that might be responsible for its resistance compared to the underlying stereomic layers, namely morphology, intramineral organic material, magnesium concentration, intrinsic solubility of the mineral, and density. Our results indicate that the acidification resistance of the cortex is probably due to its lower magnesium concentration and higher density, the latter reducing the amount of surface area in contact with acidified seawater. The biofilm and epibionts covering the cortex of mature spines may also reduce its exposure to seawater.