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.

Strategies Shaping the Transcription of Carbohydrate-Active Enzyme Genes in Aspergillus nidulans.

Understanding the coordinated regulation of the hundreds of carbohydrate-active enzyme (CAZyme) genes occurring in the genomes of fungi has great practical importance. We recorded genome-wide transcriptional changes of Aspergillus nidulans cultivated on glucose, lactose, or arabinogalactan, as well as under carbon-starved conditions. We determined both carbon-stress-specific changes (weak or no carbon source vs. glucose) and carbon-source-specific changes (one type of culture vs. all other cultures). Many CAZyme genes showed carbon-stress-specific and/or carbon-source-specific upregulation on arabinogalactan (138 and 62 genes, respectively). Besides galactosidase and arabinan-degrading enzyme genes, enrichment of cellulolytic, pectinolytic, mannan, and xylan-degrading enzyme genes was observed. Fewer upregulated genes, 81 and 107 carbon stress specific, and 6 and 16 carbon source specific, were found on lactose and in carbon-starved cultures, respectively. They were enriched only in galactosidase and xylosidase genes on lactose and rhamnogalacturonanase genes in both cultures. Some CAZyme genes (29 genes) showed carbon-source-specific upregulation on glucose, and they were enriched in ß-1,4-glucanase genes. The behavioral ecological background of these characteristics was evaluated to comprehensively organize our knowledge on CAZyme production, which can lead to developing new strategies to produce enzymes for plant cell wall saccharification.

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.

Disruption of SCO5461 gene coding for a mono-ADP-ribosyltransferase enzyme produces a conditional pleiotropic phenotype affecting morphological differentiation and antibiotic production in Streptomyces coelicolor.

The SCO5461 gene of Streptomyces coelicolor A3(2) codes for an ADP-ribosyltransferase enzyme that is predicted to be a transmembrane protein with an extracellular catalytic domain. PCR-targeted disruption of the gene resulted in a mutant that differentiated normally on complex SFM medium; however, morphological differentiation in minimal medium was significantly delayed and this phenotype was even more pronounced on osmotically enhanced minimal medium. The mutant did not sporulate when it was grown on R5 medium, however the normal morphological differentiation was restored when the strain was cultivated beside the wild-type S. coelicolor M145 strain. Comparison of the pattern of ADP-ribosylated proteins showed a difference between the mutant and the wild type, fewer modified proteins were present in the cellular crude extract of the mutant strain. These results support our previous suggestions that protein ADP-ribosylation is involved in the regulation of differentiation and antibiotic production and secretion in Streptomyces.

Identification of ß-lactamases in human and bovine isolates of Staphylococcus aureus strains having borderline resistance to penicillinase-resistant penicillins (PRPs) with proteomic methods.

Methicillin and oxacillin-hydrolyzing enzymes of 6 borderline methicillin-resistant and 1 methicillin-resistant Staphylococcus aureus strains isolated from human clinical samples and 4 borderline methicillin-resistant S. aureus strains isolated from bovine mastitis were investigated. As previous studies suggested the involvement of an additional enzyme besides the penicillinase BlaZ in the determination of borderline resistance, we analyzed the expressed extracellular and membrane-bound ß-lactamases with 2-D gel electrophoresis and mass spectrometry. Our analysis showed that the penicillin-hydrolyzing BlaZ alone was responsible for the hydrolysis of both methicillin and oxacillin. All supernatant and membrane fractions contained the same enzyme with slight sequence variations. The size and pI of the proteins were also variable, probably due to spontaneous hydrolysis and/or posttranslational modifications. Interestingly, we found also cytotoxins and other virulence factors in some nitrocefin-hydrolyzing dots, suggesting that those proteins might have a role in the reduction of local antibiotic concentration.

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.

Lack of A-factor production induces the expression of nutrient scavenging and stress-related proteins in Streptomyces griseus.

The small gamma-butyrolactone A-factor is an important autoregulatory signaling molecule for the soil-inhabiting streptomycetes. Starvation is a major trigger for development, and nutrients are provided by degradation of the vegetative mycelium via a process of programmed cell death, reusing proteins, nucleic acids, and cell wall material. The A-factor regulon includes many extracellular hydrolases. Here we show via proteomics analysis that many nutrient-scavenging and stress-related proteins were overexpressed in an A-factor non-producing mutant of Streptomyces griseus B-2682. Transcript analysis showed that this is primarily due to differential transcription of the target genes during early development. The targets include proteins relating to nutrient stress and environmental stress and an orthologue of the Bacillus sporulation control protein Spo0M. The enhanced expression of these proteins underlines the stress that is generated by the absence of A-factor. Wild-type developmental gene expression was restored to the A-factor non-producing mutant by the signaling protein Factor C in line with our earlier observation that Factor C triggers A-factor production.

Investigation of beta-Lactamases in clinical isolates of Staphylococcus aureus for further explanation of borderline methicillin resistance.

BACKGROUND: Borderline methicillin resistance in Staphylococcus aureus is due to beta-lactamase overproduction and/or specific methicillinases. METHODS: beta-Lactamase activity in culture supernatants and in cytoplasmic membrane fractions was estimated by bioassay and by SDS-PAGE combined with nitrocefin assay. RESULTS: During the investigation of borderline methicillin-resistant Staphylococcus aureus (BORSA) strains VU94 and 822 two beta-lactamases were detected in the membranes, with molecular weights of 13 and 30 kDa. The latter could be found in the culture supernatants, too. In the presence of globomycin, this enzyme disappeared from the membrane, and the oxacillin-hydrolyzing activity of the membrane decreased to the level of susceptible strains. Both beta-lactamases were detected in the methicillin-resistant Staphylococcus aureus strain studied, but the susceptible strains possessed only the first enzyme. CONCLUSIONS: The 30-kDa beta-lactamase proved to be a methicillinase, and it can be one of the main causes of the borderline phenotype of BORSA strains. The other enzyme is one of the smallest beta-lactamases published to date.

Staphylococci isolated from animals and food with phenotypically reduced susceptibility to beta-lactamase-resistant beta-lactam antibiotics.

The antibiotic resistance pattern of 1921 Staphylococcus strains isolated from animals and food within the last two years were examined using diffusion tests. Among them there were only 35 strains of S. aureus having an inhibition zone diameter of 15 mm or less, and 4 strains of coagulase-negative staphylococci (CNS) having a zone diameter of 18 mm or less to 1-microg oxacillin disk. These 39 strains were examined also by E-test to oxacillin and for the detection of the mecA gene by PCR in order to determine whether they might be real methicillin-resistant staphylococci. Among the 39 strains there were only two that were susceptible to penicillin by disk diffusion method; however, further examination by the penicillinase test showed that they produced beta-lactamase. While 19 (15 S. aureus, 4 CNS) strains were resistant and 7 strains were intermediate to oxacillin in disk diffusion test, the E-test gave 8 resistant and 5 intermediate results. Six out of the 8 oxacillin-resistant strains examined by disk diffusion and E-test harboured the mecA gene. Thus only 6 out of the examined 1921 strains proved to be mecA positive. These methicillin-resistant, mecA-positive strains (5 of the S. aureus strains and 1 of the S. epidermidis) originated from two dairy herds. The results prove that methicillin-resistant S. aureus (MRSA) strains in animals are really rare in Hungary. Eighteen strains were chosen and screened for minimal inhibitory concentration (MIC) of oxacillin with or without clavulanic acid or sulbactam, and three of them produced methicillinase enzyme.