Mitochondrial DNA copy number changes, heteroplasmy, and mutations in plasma-derived exosomes and brain tissue of glioblastoma patients.

Glioblastoma is the most common malignant tumor of the central nervous system (CNS) in adults. Glioblastoma cells show increased glucose consumption associated with poor prognosis. Since mitochondria play a crucial role in energy metabolism, mutations and copy number changes of mitochondrial DNA may serve as biomarkers. As the brain is difficult to access, analysis of mitochondria directly from the brain tissue represents a challenge. Exosome analysis is an alternative (still poorly explored) approach to investigate molecular changes in CNS tumors. We analyzed brain tissue DNA and plasma-derived exosomal DNA (exoDNA) of 44 glioblastoma patients and 40 control individuals. Quantitative real-time PCR was performed to determine mtDNA copy numbers and the Kruskal-Wallis and Mann-Whitney U test were used for statistical analysis of data. Subsequently, sequencing libraries were prepared and sequenced on the MiSeq platform to identify mtDNA point mutations. Tissue mtDNA copy number was different among controls and patients in multiple comparisons. A similar tendency was detected in exosomes. Based on NGS analysis, several mtDNA point mutations showed slightly different frequencies between cases and controls, but the clinical relevance of these observations is difficult to assess and likely less than that of overall mtDNA copy number changes. Allele frequencies of variants were used to determine the level of heteroplasmy (found to be higher in exo-mtDNA of control individuals). Despite the suggested potential, the use of such biomarkers for the screening and/or diagnosis of glioblastomas is still limited, thus further studies are needed.

Circulating Cell-Free Nucleic Acids: Main Characteristics and Clinical Application.

Liquid biopsy recently became a very promising diagnostic method that has several advantages over conventional invasive methods. Liquid biopsy may serve as a source of several important biomarkers including cell-free nucleic acids (cf-NAs). Cf-DNA is widely used in prenatal testing in order to characterize fetal genetic disorders. Analysis of cf-DNA may provide information about the mutation profile of tumor cells, while cell-free non-coding RNAs are promising biomarker candidates in the diagnosis and prognosis of cancer. Many of these markers have the potential to help clinicians in therapy selection and in the follow-up of patients. Thus, cf-NA-based diagnostics represent a new path in personalized medicine. Although several reviews are available in the field, most of them focus on a limited number of cf-NA types. In this review, we give an overview about all known cf-NAs including cf-DNA, cf-mtDNA and cell-free non-coding RNA (miRNA, lncRNA, circRNA, piRNA, YRNA, and vtRNA) by discussing their biogenesis, biological function and potential as biomarker candidates in liquid biopsy. We also outline possible future directions in the field.

Detection of cell-free, exosomal and whole blood mitochondrial DNA copy number in plasma or whole blood of patients with serous epithelial ovarian cancer.

Ovarian tumor is one of the leading causes of cancer among women. Patients are diagnosed at an advanced stage, usually. There is a need for new specific and sensitive biomarkers. Mitochondrial DNA copy number change was observed in various cancers. Our aim was to detect mitochondrial DNA copy number in whole blood (wb-mtDNA) and in plasma (cell-free and exosome encapsulated mtDNA) in patients with serous epithelial ovarian tumor. DNA was isolated from EDTA blood and plasma obtained from 24 patients and 24 healthy controls. Exosomes were isolated from cell-free plasma, and exosome encapsulated DNA (exoDNA) was extracted. Quantitative-real-time PCR was performed with Human Mitochondrial DNA (mtDNA) Monitoring Primer Set. Kruskall­Wallis and Mann­Whitney U test were used for data analysis. Wb-mtDNA copy number was significantly different among healthy controls and patients in multiple comparison (p = 0.0090 considering FIGO stage independently, and p = 0.0048 considering early- and late-stage cancers). There was a significant decrease among early-stage, all advanced stage and all cancer patients (FIGO I: 32.5 ± 8.3, p = 0.0061; FIGO III + IV: 37.2 ± 13.7 p = 0.0139; FIGO I + III + IV: 35.6 ± 12.2, p = 0.0017) or FIGO III patients alone (32.8 ± 5.6, p = 0.00089) compared to healthy controls. We found significant increase in copy number in exosomal mtDNA in cancer patients (236.0 ± 499.0, p = 0.0155), advanced-stage cancer patients (333.0 ± 575.0, p = 0.0095), of FIGO III (362.0 ± 609.2, p = 0.0494), and FIGO IV (304.0 ± 585.0, p = 0.0393) patients alone but not in samples of FIGO I patients (10.0 ± 3.5, p = 0.3907). In multiple comparison the increase was significant considering early- and late-stage cancers (p = 0.0253). Cell-free mtDNA copy numbers were not increased significantly. We found the highest copy number of mtDNA in exosomes, followed by plasma and peripheral blood in late-stage cancer patients. We observed significant difference in wb-mtDNA copy number between healthy controls and both early- and late-stage cancer patients.

γ-Glutamyl transpeptidase (GgtA) of Aspergillus nidulans is not necessary for bulk degradation of glutathione.

Aspergillus nidulans exhibited high γ-glutamyl transpeptidase (γGT) activity in both carbon-starved and carbon-limited cultures. Glucose repressed, but casein peptone increased γGT production. Null mutation of creA did not influence γGT formation, but the functional meaB was necessary for the γGT induction. Deletion of the AN10444 gene (ggtA) completely eliminated the γGT activity, and the mRNA levels of ggtA showed strong correlation with the observed γGT activities. While ggtA does not contain a canonical signal sequence, the γGT activity was detectable both in the fermentation broth and in the hyphae. Deletion of the ggtA gene did not prevent the depletion of glutathione observed in carbon-starved and carbon-limited cultures. Addition of casein peptone to carbon-starved cultures lowered the formation of reactive species (RS). Deletion of ggtA could hinder this decrease and resulted in elevated RS formation. This effect of γGT on redox homeostasis may explain the reduced cleistothecia formation of ΔggtA strains in surface cultures.

Analysis and identification of ADP-ribosylated proteins of Streptomyces coelicolor M145.

Mono-ADP-ribosylation is the enzymatic transfer of ADP-ribose from NAD(+) to acceptor proteins catalyzed by ADP-ribosyltransferases. Using m-aminophenylboronate affinity chromatography, 2D-gel electrophoresis, in-gel digestion and MALDI-TOF analysis we have identified eight in vitro ADP-ribosylated proteins in Streptomyces coelicolor, which can be classified into three categories: (i) secreted proteins; (ii) metabolic enzymes using NAD(+)/NADH or NADP(+)/NADPH as coenzymes; and (iii) other proteins. The secreted proteins could be classified into two functional categories: SCO2008 and SC05477 encode members of the family of periplasmic extracellular solute-binding proteins, and SCO6108 and SC01968 are secreted hydrolases. Dehydrogenases are encoded by SC04824 and SC04771. The other targets are GlnA (glutamine synthetase I., SC02198) and SpaA (starvation-sensing protein encoded by SC07629). SCO2008 protein and GlnA had been identified as ADP-ribosylated proteins in previous studies. With these results we provided experimental support for a previous suggestion that ADP-ribosylation may regulate membrane transport and localization of periplasmic proteins. Since ADP-ribosylation results in inactivation of the target protein, ADP-ribosylation of dehydrogenases might modulate crucial primary metabolic pathways in Streptomyces. Several of the proteins identified here could provide a strong connection between protein ADP-ribosylation and the regulation of morphological differentiation in S. coelicolor.