Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers.

A novel murine plasma membrane protein has been identified in subpopulations of macrophages. It has an intracellular N-terminal domain, a transmembrane domain, and an extracellular region with a short spacer, an 89 Gly-Xaa-Yaa repeat-containing collagenous domain, and a C-terminal cysteine-rich domain. In situ hybridization and immunohistochemical staining have localized the protein to a subset of macrophages in the marginal zone of the spleen and the medullary cord of lymph nodes. No expression was observed in macrophages of liver or lung. Transfected COS cells synthesized a native trimeric plasma membrane protein that bound labeled bacteria and acetylated LDL, but not yeast or Ficoll. The results suggest that the novel protein is a macrophage-specific membrane receptor with a role in host defense, as it shows postnatal expression in macrophages, which are considered responsible for the binding of bacterial antigens and phagocytosis.

Background-The lysyl oxidases are extracellular copper enzymes that initiate the crosslinking of collagens and elastin, 5 human isoenzymes having been characterized so far. The crosslinks formed provide the tensile strength and elastic properties for various extracellular matrices, including vascular walls. We studied the role of the first described isoenzyme Lox by inactivating its gene in mice. Methods and Results-Murine Lox gene was disrupted by routine methods. Lox Ϫ/Ϫ mice died at the end of gestation or as neonates, necropsy of the live-born pups revealing large aortic aneurysms. In light microscopy, hazy and unruffled elastic lamellae in the Lox Ϫ/Ϫ aortas were observed, and electron microscopy of the aortic walls of the Lox Ϫ/Ϫ fetuses showed highly fragmented elastic fibers and discontinuity in the smooth muscle cell layers in Lox Ϫ/Ϫ fetuses. The wall of the aorta in the Lox Ϫ/Ϫ fetuses was significantly thicker, and the diameter of the aortic lumen was significantly smaller than that in the Lox ϩ/ϩ aortas. In Lox Ϫ/Ϫ fetuses, Doppler ultrasonography revealed increased impedance in the umbilical artery, descending aorta, and intracranial artery blood velocity waveforms, decreased mean velocities across cardiac inflow and outflow regions, and increased pulsatility in ductus venosus blood velocity waveforms. Conclusions-Lox

Double-stranded RNA (dsRNA) fragments are readily internalized and processed by Drosophila S2 cells, making these cells a widely used tool for the analysis of gene function by gene silencing through RNA interference (RNAi). The underlying mechanisms are insufficiently understood. To identify components of the RNAi pathway in S2 cells, we developed a screen based on rescue from RNAi-induced lethality. We identified Argonaute 2, a core component of the RNAi machinery, and three gene products previously unknown to be involved in RNAi in Drosophila: DEAD-box RNA helicase Belle, 26 S proteasome regulatory subunit 8 (Pros45), and clathrin heavy chain, a component of the endocytic machinery. Blocking endocytosis in S2 cells impaired RNAi, suggesting that dsRNA fragments are internalized by receptor-mediated endocytosis. Indeed, using a candidate gene approach, we identified two Drosophila scavenger receptors, SR-CI and Eater, which together accounted for more than 90% of the dsRNA uptake into S2 cells. When expressed in mammalian cells, SR-CI was sufficient to mediate internalization of dsRNA fragments. Our data provide insight into the mechanism of dsRNA internalization by Drosophila cells. These results have implications for dsRNA delivery into mammalian cells.Many organisms mount specific defense responses to silence invading nucleic acid sequences before these sequences integrate into the host genome and disturb cellular processes. At the core of these sequence-directed immunity mechanisms is dsRNA, 2 which becomes processed and causes gene silencing, referred to as RNA interference (RNAi) (1, 2). In addition to its defense function, RNAi guides endogenous developmental and regulatory processes and is used as a research tool to suppress the expression of cellular genes in numerous model organisms (2).Although species variations exist, the silencing mechanisms in plants, fungi, worms, insects, and mammals share common features and conserved genes. Drosophila S2 cells are widely used to carry out large scale functional screens (3-5) because dsRNA fragments (usually about 500 -700 bp) can be added directly to the cell culture medium, "soaking" the cells rather than transfecting them (6). This makes silencing in these cells easy and efficient, in contrast to mammalian cells that require small interfering RNAs (siRNAs) to be delivered by transfection. In contrast to Caenorhabditis elegans cells, which internalize dsRNA by the channel-forming transmembrane protein SID-1 (7), the mechanism of dsRNA uptake into Drosophila cells is unknown.In the cytosol, dsRNA fragments are processed into short 21-23-nucleotide dsRNA duplexes (siRNAs) by a dsRNA-specific RNase-IIItype endonuclease called Dicer-2 (8 -10). Dicer-2 is stably complexed with the dsRNA binding domain-containing protein R2D2 binding to siRNA and thereby, facilitating siRNA loading onto RNA-induced silencing complexes (RISC) (11). RISC contain a member of the Argonaute (Ago) protein family Ago-2, which has been shown to mediate siRNA-directed mRNA cleavage (12) an...