Analysis of the regenerative capacity of human serum exosomes after a simple multistep separation from lipoproteins.

Due to the abundance of lipoproteins in blood, it is challenging to characterize the biological functions and components of blood-derived extracellular vesicles. The aim of this study was to develop a multiple-step purification protocol to separate serum exosomes from serum proteins and lipoproteins and assess their regenerative potential. Exosomes were isolated by concentrating them in human serum using ultracentrifugation (UC), followed sequentially by density gradient (DG) UC and size exclusion chromatography (SEC). Purity and characterization were assessed by western blots, Lipoprint®, enzyme-linked immunosorbent assay, electron microscopy, mass spectrometry, and nanoparticle tracking analysis. Functionality was assessed by cell proliferation analysis and with an in vivo subcutaneous angiogenesis model. SEC alone isolated nano-sized vesicles possessing vesicle markers TSG101 and CD9, but there was a substantial presence of apolipoprotein B, predominantly derived from very-low- and intermediate-density lipoprotein particles. This was reduced to an undetectable level using the combined UC DG SEC approach. Mass spectrometry identified 224 proteins in UC DG SEC isolates relative to the 135 from SEC, with considerable increases in exosome-related proteins and reductions in lipoproteins. A consistent but limited increase in human dermal fibroblast proliferation and evidence of neovascularization enhancement were observed after exposure to UC DG SEC exosomes. An UC DG SEC purification protocol considerably improved the removal of lipoproteins during isolation of serum exosomes. The purified exosomes stimulated cell proliferation and potentially increased an in vivo angiogenic response. This multistep purification allows for more accurate identification of serum exosome functional activity and composition.

A case report: delayed right ventricular pericardial fistula and aneurysm following penetrating traumatic injury-a controversial aetiology.

BACKGROUND: Right ventricular aneurysms (RVAs) are rare. We present a case with a combined RVA and right ventricular pericardial fistula resulting in a pericardial effusion and cardiac tamponade. The RVA was detected 47 days after the patient suffered a gunshot wound. This report adds to the body of scarce literature on RVA aetiology, diagnoses, and treatment. CASE SUMMARY: A 30-year-old male patient presented with worsening respiratory distress over a 7-day period with clinical signs of cardiac tamponade following a history of a gunshot (with associated liver laceration, pulmonary embolism, right nephrectomy, and sepsis) 47 days prior. Transthoracic echocardiography showed a large circumferential pericardial effusion and an RVA. The patient was emergently taken for surgical repair of the RVA. DISCUSSION: Our case presents a delayed presentation of a gunshot heart and an aetiology with indications of and against a true aneurysm. It brings attention to possible complications of penetrating precordial injuries, with the need for consideration and possible evaluation at follow-up. The literature on the operative excision of RVA is reviewed and various aetiological factors and consequences are discussed.

DNA methylation analysis on purified neurons and glia dissects age and Alzheimer's disease-specific changes in the human cortex.

BACKGROUND: Epigenome-wide association studies (EWAS) based on human brain samples allow a deep and direct understanding of epigenetic dysregulation in Alzheimer's disease (AD). However, strong variation of cell-type proportions across brain tissue samples represents a significant source of data noise. Here, we report the first EWAS based on sorted neuronal and non-neuronal (mostly glia) nuclei from postmortem human brain tissues. RESULTS: We show that cell sorting strongly enhances the robust detection of disease-related DNA methylation changes even in a relatively small cohort. We identify numerous genes with cell-type-specific methylation signatures and document differential methylation dynamics associated with aging specifically in neurons such as CLU, SYNJ2 and NCOR2 or in glia RAI1,CXXC5 and INPP5A. Further, we found neuron or glia-specific associations with AD Braak stage progression at genes such as MCF2L, ANK1, MAP2, LRRC8B, STK32C and S100B. A comparison of our study with previous tissue-based EWAS validates multiple AD-associated DNA methylation signals and additionally specifies their origin to neuron, e.g., HOXA3 or glia (ANK1). In a meta-analysis, we reveal two novel previously unrecognized methylation changes at the key AD risk genes APP and ADAM17. CONCLUSIONS: Our data highlight the complex interplay between disease, age and cell-type-specific methylation changes in AD risk genes thus offering new perspectives for the validation and interpretation of large EWAS results.

Altered Long Noncoding RNA Expression Precedes the Course of Parkinson's Disease-a Preliminary Report.

Parkinson's disease (PD) is a slowly progressing neurodegenerative disorder that affects approximately seven million patients worldwide. Despite intensive research, the molecular mechanisms initiating and promoting PD are still unknown. However, it is assumed that environmental factors trigger PD. Recent research demonstrated that long noncoding RNAs (lncRNA) interfere in transcriptional and translational processes modulating gene expression reflecting environmental influences. Nevertheless, there is no systematic analysis available that investigates the impact of lncRNAs on PD. In the current study, we performed a comprehensive analysis on expression levels of 90 well-annotated lncRNAs in 30 brain specimens deriving from 20 PD patients and 10 controls as a preliminary report on the significance of lncRNAs in PD. Expression profiling of lncRNAs revealed that five lncRNAs are significantly differentially expressed in PD. While H19 upstream conserved 1 and 2 is significantly downregulated in PD, lincRNA-p21, Malat1, SNHG1, and TncRNA are significantly upregulated. An analysis on expression levels and PD stages revealed that the identified dysregulated lncRNA are altered already in early disease stage and that they precede the course of PD. In summary, this is the first comprehensive analysis on lncRNAs in PD revealing significantly altered lncRNAs. Additionally, we found that lncRNA dysregulations precede the course of the disease. Thus, the five newly identified lncRNAs may serve as potential new biomarkers appropriate even in early PD. They may be used in monitoring disease progression and they may serve as potential new targets for novel therapeutic approaches.

Profiling of methylation and demethylation pathways during brain development and ageing.

Numerous signal pathways are epigenetically controlled during brain development and ageing. Thereby, both 5-methylcytosine (5mC) and the newly described 5-hydroxymethylcytosine (5hmC) are highly exhibited in the brain. As there is an uneven distribution of 5hmC in the brain depending on age and region, there is the need to investigate the underlying mechanisms being responsible for 5hmC generation and decline. The aim of this study was to quantify expression levels of genes that are associated with DNA methylation/demethylation in different brain regions and at different ages. Therefore, we investigated frontal cortex and cerebellum of 40 mice (strain C57BL/6), each eight mice sacrificed at day 0, 7, 15, 30 and 120 after birth. We performed expression profiling of methylation/demethylation genes depending on age and brain region. Interestingly, we see significant expression differences of genes being responsible for methylation/demethylation with a significant reduction of expression levels during ageing. Validating selected expression data on protein level using immunohistochemistry verified the expression data. In conclusion, our findings demonstrate that the regulation of methylation/demethylation pathways is highly controlled depending on brain region and age. Thus our data will help to better understand the complexity and plasticity of the brain epigenome.

Genetic Characterization of Ten-Eleven-Translocation Methylcytosine Dioxygenase Alterations in Human Glioma.

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.