Cell-free DNA (cfDNA) in blood is used as a source of genetic material for noninvasive pre-natal and cancer diagnostic assays in clinical practice. Recently we have started a project for new biomarker discovery with a view to developing new noninvasive diagnostic assays. While reviewing literature, it was found that exosomes may be a rich source of biomarkers, because exosomes play an important role in human health and disease. While characterizing exosomes found in human blood plasma, we observed the presence of cfDNA in plasma exosomes. Plasma was obtained from blood drawn into K 3 EDTA tubes. Exosomes were isolated from cell-free plasma using a commercially available kit. Sizing and enumeration of exosomes were done using electron microscopy and NanoSight particle counter. NanoSight and confocal microscopy was used to demonstrate the association between dsDNA and exosomes. DNA extracted from plasma and exosomes was measured by a fluorometric method and a droplet digital PCR (ddPCR) method. Size of extracellular vesicles isolated from plasma was heterogeneous and showed a mean value of 92.6 nm and a mode 39.7 nm. A large proportion of extracellular vesicles isolated from plasma were identified as exo-somes using a fluorescence probe specific for exosomes and three protein markers, Hsp70, CD9 and CD63, that are commonly used to identify exosome fraction. Fluorescence dye that stain dsDNA showed the association between exosomes and dsDNA. Plasma cfDNA concentration analysis showed more than 93% of amplifiable cfDNA in plasma is located in plasma exosomes. Storage of a blood sample showed significant increases in exosome count and exosome DNA concentration. This study provide evidence that a large proportion of plasma cfDNA is localized in exosomes. Exosome release from cells is a metabolic energy dependent process, thus suggesting active release of cfDNA from cells as a source of cfDNA in plasma. PLOS ONE | https://doi.org/10.1371/journal.pone.

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We tested whether the Steitz et al. [(1974) Proc. Natl. Acad. Sci. U.S.A., 71:593-597] model of lactose repressor (LacR) (14 x 6.0 x 4.5 nm) represented the shape of free or operator-bound LacR in solution. The model predicts a 14 nm length for bound LacR. Direct measurement, using Pt-C shadow width standards, was 9.6 +/- 0.2 nm long. Using the Steitz model, we generated a distribution of measurements and converted them into a distribution of shadow widths using gold ball standards. Direct measurement of LacR produced a narrower shadow width distribution with a larger mean size than the Steitz model predicted. Measurement along two orthogonal axes of negatively stained LacR images generated a size distribution, also converted into a shadow width distribution using the gold ball standards. Since the experimental shadow width distribution exactly matched the shadow width distribution derived theoretically from negatively stained LacR, our negative-stained images are representative of LacR's conformation in solution. Approximately 56% of negatively stained LacR had a V-shaped fold around an axis orthogonal to its length, bringing the DNA binding domains of each dimer adjacent. This open end of the V binds single operator DNA. The other 44% of the LacR tetramer is in the extended form with its DNA binding sites at opposite ends. Although the V-shaped conformation has a closed hinge with the dimers associated along a side, the extended open-hinged state remains important since LacR must bind two distant operator sites for full repression. Our measurements predict the normal presence of both conformations in nearly equal amounts, suggesting that both are equally active in repressing the lac operon.

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