Systematic characterization of extracellular vesicle sorting domains and quantification at the single molecule – single vesicle level by fluorescence correlation spectroscopy and single particle imaging

Extracellular vesicles (EV) convey biological information by transmitting macromolecules between cells and tissues and are of great promise as pharmaceutical nanocarriers, and as therapeutic per se. Strategies for customizing the EV surface and cargo are being developed to enable their tracking, visualization, loading with pharmaceutical agents and decoration of the surface with tissue targeting ligands. While much progress has been made in the engineering of EVs, an exhaustive comparative analysis of the most commonly exploited EV-associated proteins, as well as a quantification at the molecular level are lacking. Here, we selected 12 EV-related proteins based on MS-proteomics data for comparative quantification of their EV engineering potential. All proteins were expressed with fluorescent protein (FP) tags in EV-producing cells; both parent cells as well as the recovered vesicles were characterized biochemically and biophysically. Using Fluorescence Correlation Spectroscopy (FCS) we quantified the number of FP-tagged molecules per vesicle. We observed different loading efficiencies and specificities for the different proteins into EVs. For the candidates showing the highest loading efficiency in terms of engineering, the molecular levels in the vesicles did not exceed ca 40–60 fluorescent proteins per vesicle upon transient overexpression in the cells. Some of the GFP-tagged EV reporters showed quenched fluorescence and were either non-vesicular, despite co-purification with EVs, or comprised a significant fraction of truncated GFP. The co-expression of each target protein with CD63 was further quantified by widefield and confocal imaging of single vesicles after double transfection of parent cells. In summary, we provide a quantitative comparison for the most commonly used sorting proteins for bioengineering of EVs and introduce a set of biophysical techniques for straightforward quantitative and qualitative characterization of fluorescent EVs to link single vesicle analysis with single molecule quantification

RNA delivery by extracellular vesicles in mammalian cells and its applications.

The term 'extracellular vesicles' refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell-cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle-associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo. Further progress in unravelling the basic mechanisms of extracellular vesicle biogenesis, transport, and cargo delivery and function is needed for successful clinical implementation. This Review focuses on the current state of knowledge pertaining to packaging, transport and function of RNAs in extracellular vesicles and outlines the progress made thus far towards their clinical applications

Genetically determined variation of constitutive major histocompatibility complex class II antigen expression in various rat strains and cell types

No abstract availabl

A Wide-Field Fluorescence Microscope Extension for Ultrafast Screening of One-Bead One-Compound Libraries Using a Spectral Image Subtraction Approach

The increasing involvement of academic institutions and biotech companies in drug discovery calls for cost-effective methods to identify new bioactive molecules. Affinity-based on-bead screening of combinatorial one-bead one-compound libraries combines a split-mix synthesis design with a simple protein binding assay operating directly at the bead matrix. However, one bottleneck for academic scale on-bead screening is the unavailability of a cheap, automated, and robust screening platform that still provides a quantitative signal related to the amount of target protein binding to individual beads for hit bead ranking. Wide-field fluorescence microscopy has long been considered unsuitable due to significant broad spectrum autofluorescence of the library beads in conjunction with low detection sensitivity. Herein, we demonstrate how such a standard microscope equipped with LED-based excitation and a modern CMOS camera can be successfully used for selecting hit beads. We show that the autofluorescence issue can be overcome by an optical image subtraction approach that yields excellent signal-to-noise ratios for the detection of bead-associated target proteins. A polymer capillary attached to a semiautomated bead-picking device allows the operator to efficiently isolate individual hit beads in less than 20 s. The system can be used for ultrafast screening of >200,000 bead-bound compounds in 1.5 h, thereby making high-throughput screening accessible to a wider group within the scientific community

Geotechnische Untersuchungen im Rahmen des Demonstrationsversuchs 'Thermische Simulation der Streckenlagerung' Schlussbericht (9. Bericht)

The ultimate disposal of radioactive wastes by means of emplacement in drifts of a salt mine has to be based on information about the thermomechanical performance of compact salt rock as well as salt rock grit used as a backfilling material. The work performed in the reporting period encompasses experimental and theoretical geotechnical analyses of the stresses affecting the rock and their modifications induced by drifting and heat generated by the waste forms. The measured changes in stress have been interpreted by numerical model calculations and deliverd input data for further development of geotechnical measuring methods. (DG)SIGLEAvailable from TIB Hannover: F97B950+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman

Geotechnische und grossnumerische Untersuchungen zur direkten Endlagerung von Brennelementen. Teilprojekt 1: Thermische Simulation der Streckenlagerung Schlussbericht (8. Bericht)

hort-term tension measurements show that a reduced rock tension level exists in the Thermal Storage Simulation field (TSS). This tension level is markedly lower than the lithostatic rock pressure to be expected in the salt rock. The long-term tension measurements show that tension maxima will develop until early in 1993 in the close-in area of the experimental fields, for instance in the pillar between the galleries A and B. Creep tests with salt rock specimens from the TSS field show that the creep performance at room temperature of the Na2#beta# rock salt to be considered in the experimental fields can be sufficiently accurately described by the reference creep law for stationary creep used by the BGR (Bundesanstalt f. Geowissenschaften und Rohstoffe).The experimental in-situ results on the permeability of the salt rock in the experimental fields indicate i.a. very low permeabilities, except for a restricted, local area within the loosening zone below gallery B. This exhibits relatively high permeabilities. The experimental in-situ results on the permeability of the backfilling in the experimental galleries show that during the experimental measuring period, an only small change in permeability due to compaction of the backfilling material was observed. This result agrees with results of compaction tests performed by the GSF. The measured results show a difference in permeabilities between backfilling material subject to heating up, and colder material. (orig.)Wesentliche Ergebnisse: 1. Die Kurzzeitspannungsmessungen belegen, dass im TSS-Versuchsfeld ein reduziertes Gebirgsspannungsniveau vorliegt, das deutlich unter dem im unverritzten Salzgebirge zu erwartenden lithostatischen Gebirgsdruck liegt. 2. Die Langzeitspannungsmessungen zeigen, dass im Nahbereich der Versuchsstrecken, z.B. im Pfeiler zwischen Strecke A und B, bis ca. Anfang 1993 Spannungsmaxima durchlaufen werden. 3. Kriechversuche an Salzpruefkoerpern aus dem TSS-Feld belegen, dass das Kriechverhalten des im Bereich der Versuchsstrecken anstehenden Na2#beta#-Steinsalzes fuer Raumtemperatur ausreichend genau mit dem Referenzkriechgesetz der BGR fuer stationaeres Kriechen beschrieben werden kann. 4. Die experimentellen In-situ-Untersuchungen zur Permeabilitaet des Salzgebirges im Bereich der Versuchsstrecken weisen i.a. auf sehr geringe Durchlaessigkeiten hin. Davon ausgenommen ist ein lokal begrenzter Untersuchungsbereich in der Auflockerungszone unterhalb der Strecke B. Hier treten vergleichsweise hohe Permeabilitaeten auf. 5. Die experimentellen In-situ-Untersuchungen zur Permeabilitaet des Versatzes in den Strecken belegen, dass im Versuchszeitraum nur eine geringe Veraenderung der Durchlaessigkeit infolge Kompaktion des Versatzes aufgetreten ist. Dieser Befund stimmt mit Ergebnissen der GSF zum Kompaktionsverhalten ueberein. Unterschiede in der Durchlaessigkeit des Versatzes im erwaermten und im kaelteren Streckenbereich lassen sich aufgrund der Messergebnisse nachweisen. (orig.)Available from TIB Hannover: FR 5540(8)+8 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman

Geotechnisches Verhalten verschiedener Salzgesteine. Teilprojekt 3 In-situ-Messtechnik im Salz. Schlussbericht

TIB Hannover: FR 3623+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

In-situ-Messtechnik im Salz : Ermittlung des Spannungs-Deformationsverhaltens von Salzgebirge durch Messungen in der Umgebung von Grubenhohlraeumen und Bestimmung des sekundaeren und des primaeren Spannungszustandes Schlussbericht

Contains photosSIGLETIB: FR 785 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman

Exosomes surf on filopodia to enter cells at endocytic hot spots, traffic within endosomes, and are targeted to the ER

Exosomes are nanovesicles released by virtually all cells, which act as intercellular messengers by transfer of protein, lipid, and RNA cargo. Their quantitative efficiency, routes of cell uptake, and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake, which saturates with dose and time and reaches near 100% transduction efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. After internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction, and suggest unanticipated routes of subcellular cargo delivery