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Development of biofluid-based molecular diagnostic tests for cancer is an important step towards tumor characterization and real-time monitoring in a minimally invasive fashion. Extracellular vesicles (EVs) are released from tumor cells into body fluids and can provide a powerful platform for tumor biomarkers because they carry tumor proteins and nucleic acids. Detecting rare point mutations in the background of wild-type sequences in biofluids such as blood and cerebrospinal fluid (CSF) remains a major challenge. Techniques such as BEAMing (beads, emulsion, amplification, magnetics) PCR and droplet digital PCR (ddPCR) are substantially more sensitive than many other assays for mutant sequence detection. Here, we describe a novel approach that combines biofluid EV RNA and BEAMing RT-PCR (EV-BEAMing), as well droplet digital PCR to interrogate mutations from glioma tumors. EVs from CSF of patients with glioma were shown to contain mutant IDH1 transcripts, and we were able to reliably detect and quantify mutant and wild-type IDH1 RNA transcripts in CSF of patients with gliomas. EV-BEAMing and EV-ddPCR represent a valuable new strategy for cancer diagnostics, which can be applied to a variety of biofluids and neoplasms.

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BackgroundRNA from exosomes and other microvesicles contain transcripts of tumour origin. In this study we sought to identify biomarkers of glioblastoma multiforme in microvesicle RNA from serum of affected patients.MethodsMicrovesicle RNA from serum from patients with de-novo primary glioblastoma multiforme (N = 9) and normal controls (N = 7) were analyzed by microarray analysis. Samples were collected according to protocols approved by the Institutional Review Board. Differential expressions were validated by qRT-PCR in a separate set of samples (N = 10 in both groups).ResultsExpression profiles of microvesicle RNA correctly separated individuals in two groups by unsupervised clustering. The most significant differences pertained to down-regulated genes (121 genes > 2-fold down) in the glioblastoma multiforme patient microvesicle RNA, validated by qRT-PCR on several genes. Overall, yields of microvesicle RNA from patients was higher than from normal controls, but the additional RNA was primarily of size < 500 nt. Gene ontology of the down-regulated genes indicated these are coding for ribosomal proteins and genes related to ribosome production.ConclusionsSerum microvesicle RNA from patients with glioblastoma multiforme has significantly down-regulated levels of RNAs coding for ribosome production, compared to normal healthy controls, but a large overabundance of RNA of unknown origin with size < 500 nt.

Oral streptococci are able to produce growth-inhibiting amounts of hydrogen peroxide (H2O2) as byproduct of aerobic metabolism. Several recent studies showed that the produced H2O2is not a simple byproduct of metabolism but functions in several aspects of oral bacterial biofilm ecology. First, the release of DNA from cells is closely associated to the production of H2O2inStreptococcus sanguinisandStreptococcus gordonii. Extracellular DNA is crucial for biofilm development and stabilization and can also serve as source for horizontal gene transfer between oral streptococci. Second, due to the growth inhibiting nature of H2O2, H2O2compatible species associate with the producers. H2O2production therefore might help in structuring the initial biofilm development. On the other hand, the oral environment harbors salivary peroxidases that are potent in H2O2scavenging. Therefore, the effects of biofilm intrinsic H2O2production might be locally confined. However, taking into account that 80% of initial oral biofilm constituents are streptococci, the influence of H2O2on biofilm development and environmental adaptation might be under appreciated in current research.

SummaryMicrocystis is a cosmopolitan genus of cyanobacteria and occurs in many different forms. Large surface blooms of the cyanobacterium are well known in eutrophic lakes throughout the globe. We evaluated the role of microcystins (MCs) in promoting and maintaining bloom-forming cell aggregates at environmentally relevant MC concentrations (0.25-10 mg l

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