Profiling surface proteins on individual exosomes using a proximity barcoding assay

Exosomes have been implicated in numerous biological processes, and they may serve as important disease markers. Surface proteins on exosomes carry information about their tissues of origin. Because of the heterogeneity of exosomes it is desirable to investigate them individually, but this has so far remained impractical. Here, we demonstrate a proximity-dependent barcoding assay to profile surface proteins of individual exosomes using antibody-DNA conjugates and next-generation sequencing. We first validate the method using artificial streptavidin-oligonucleotide complexes, followed by analysis of the variable composition of surface proteins on individual exosomes, derived from human body fluids or cell culture media. Exosomes from different sources are characterized by the presence of specific combinations of surface proteins and their abundance, allowing exosomes to be separately quantified in mixed samples to serve as markers for tissue-specific engagement in disease

Human erythrocyte-derived nanovesicles can readily be loaded with doxorubicin and act as anticancer agents

Purpose: In future therapeutics new formulas are needed that assure lower doses, fewer side effects, targeted administration and protection of the drug from degradation. In a first step to fulfil the requirements defined above, we carried out an in vitro study by developing a new procedure to encapsulate drugs using native vesicles first from prostasomes and then from erythrocyte membranes known to be well tolerated. The new method for production of drug delivery vesicles utilized osmotic loading of detergent resistant membranes (DRMs). Materials and methods: DRMs of prostasomes and prepared human erythrocyte membranes were extracted and separated in a sucrose gradient at a density of 1.10 g/mL containing 1% Triton X-100. These DRMs were characterized by electron microscopy (transmission and scanning EM) and loaded with low and high molecular compounds. PC3 prostate cancer cells were treated with doxorubicin loaded DRMs in triplicate. DAPI (nuclear fluorescent stain) was included and fluorescence microscopic pictures were taken before the cells were trypsinized and counted after 48h. Results: The content of the well separated band was observed ultrastructurally as small spherical, double layered membrane vesicles, (DRM vesicles) which harbored hyperosmolar sucrose of the gradient. Encapsulated hyperosmolar sucrose induced a transient osmotic lysis of the DRM vesicles when suspended in isotonic buffer containing loading molecules allowing vesicular inclusion. After this proof of concept, the method was finally employed for doxorubicin loading of DRM vesicles from human erythrocytes. When incubating such vesicles with PC3 cells a complete arrest of growth was observed in sharp contrast to PC3 cells incubated with plain doxorubicin in similar conditions. Conclusion: The present results open up new possibilities for using DRM vesicles as drug delivery vesicles.Louise Dubois and Liza Löf contributed equally to this work.</p

Human erythrocyte-derived nanovesicles can readily be loaded with doxorubicin and act as anticancer agents

Purpose: In future therapeutics new formulas are needed that assure lower doses, fewer side effects, targeted administration and protection of the drug from degradation. In a first step to fulfil the requirements defined above, we carried out an in vitro study by developing a new procedure to encapsulate drugs using native vesicles first from prostasomes and then from erythrocyte membranes known to be well tolerated. The new method for production of drug delivery vesicles utilized osmotic loading of detergent resistant membranes (DRMs). Materials and methods: DRMs of prostasomes and prepared human erythrocyte membranes were extracted and separated in a sucrose gradient at a density of 1.10 g/mL containing 1% Triton X-100. These DRMs were characterized by electron microscopy (transmission and scanning EM) and loaded with low and high molecular compounds. PC3 prostate cancer cells were treated with doxorubicin loaded DRMs in triplicate. DAPI (nuclear fluorescent stain) was included and fluorescence microscopic pictures were taken before the cells were trypsinized and counted after 48h. Results: The content of the well separated band was observed ultrastructurally as small spherical, double layered membrane vesicles, (DRM vesicles) which harbored hyperosmolar sucrose of the gradient. Encapsulated hyperosmolar sucrose induced a transient osmotic lysis of the DRM vesicles when suspended in isotonic buffer containing loading molecules allowing vesicular inclusion. After this proof of concept, the method was finally employed for doxorubicin loading of DRM vesicles from human erythrocytes. When incubating such vesicles with PC3 cells a complete arrest of growth was observed in sharp contrast to PC3 cells incubated with plain doxorubicin in similar conditions. Conclusion: The present results open up new possibilities for using DRM vesicles as drug delivery vesicles.<p>Louise Dubois and Liza Löf contributed equally to this work.</p

Detecting individual extracellular vesicles using a multicolor in situ proximity ligation assay with flow cytometric readout

Flow cytometry is a powerful method for quantitative and qualitative analysis of individual cells. However, flow cytometric analysis of extracellular vesicles (EVs), and the proteins present on their surfaces has been hampered by the small size of the EVs - in particular for the smallest EVs, which can be as little as 40 nm in diameter, the limited number of antigens present, and their low refractive index. We addressed these limitations for detection and characterization of EV by flow cytometry through the use of multiplex and multicolor in situ proximity ligation assays (in situ PLA), allowing each detected EV to be easily recorded over background noise using a conventional flow cytometer. By targeting sets of proteins on the surface that are specific for distinct classes of EVs, the method allows for selective recognition of populations of EVs in samples containing more than one type of EVs. The method presented herein opens up for analyses of EVs using flow cytometry for their characterization and quantification

Diagnostics for a troubled backbone: testing topologicalhypotheses of trapelioid lichenized fungi in a large-scalephylogeny of Ostropomycetidae (Lecanoromycetes)

Trapelioid fungi constitute a widespread groupof mostly crust-forming lichen mycobionts that are key tounderstanding the early evolutionary splits in theOstropomycetidae, the second-most species-rich subclassof lichenized Ascomycota. The uncertain phylogeneticresolution of the approximately 170 species referred tothis group contributes to a poorly resolved backbone forthe entire subclass. Based on a data set including 657newly generated sequences from four ribosomal and fourprotein-coding gene loci, we tested a series of a priori andnew evolutionary hypotheses regarding the relationshipsof trapelioid clades within Ostropomycetidae. We foundstrong support for a monophyletic group of nine coretrapelioid genera but no statistical support to reject thelong-standing hypothesis that trapelioid genera are sisterto Baeomycetaceae or Hymeneliaceae. However, we canreject a sister group relationship to Ostropales with highconfidence. Our data also shed light on several longstandingquestions, recovering Anamylopsoraceae nestedwithin Baeomycetaceae, elucidating two major monophyleticgroups within trapelioids (recognized here asTrapeliaceae and Xylographaceae), and rejecting themonophyly of the genus Rimularia. We transfer elevenspecies of the latter genus to Lambiella and describe thegenus Parainoa to accommodate a previously misunderstoodspecies of Trapeliopsis. Past phylogenetic studies inOstropomycetidae have invoked Bdivergence order^ fordrawing taxonomic conclusions on higher level taxa.Our data show that if backbone support is lacking, contrastingsolutions may be recovered with different oradded data. We accordingly urge caution in concludingevolutionary relationships from unresolved phylogenies