Quantitative and functional characterisation of extracellular vesicles after passive loading with hydrophobic or cholesterol-tagged small molecules.

Extracellular vesicles (EVs) are nanosized intercellular messengers that bear enormous application potential as biological drug delivery vehicles. Much progress has been made for loading or decorating EVs with proteins, peptides or RNAs using genetically engineered donor cells, but post-isolation loading with synthetic drugs and using EVs from natural sources remains challenging. In particular, quantitative and unambiguous data assessing whether and how small molecules associate with EVs versus other components in the samples are still lacking.

High efficiency preparation of monodisperse plasma membrane derived extracellular vesicles for therapeutic applications.

Extracellular vesicles (EVs) are highly interesting for the design of next-generation therapeutics. However, their preparation methods face challenges in standardization, yield, and reproducibility. Here, we describe a highly efficient and reproducible EV preparation method for monodisperse nano plasma membrane vesicles (nPMVs), which yields 10 to 100 times more particles per cell and hour than conventional EV preparation methods.

Surface display of functional moieties on extracellular vesicles using lipid anchors.

Extracellular vesicles (EVs) are efficient natural vehicles for intercellular communication and are under extensive investigation for the delivery of diverse therapeutics including small molecule drugs, nucleic acids, and proteins. To understand the mechanisms behind the biological activities of EVs and develop EV therapeutics, it's fundamental to track EVs and engineer EVs in a customized manner. In this study, we identified, using single-vesicle flow cytometry and microscopy, the lipid DOPE (dioleoyl phosphatidyl ethanolamine) as an efficient anchor for isolated EVs.

EVAnalyzer: High content imaging for rigorous characterisation of single extracellular vesicles using standard laboratory equipment and a new open-source ImageJ/Fiji plugin.

Extracellular vesicle (EV) research increasingly demands for quantitative characterisation at the single vesicle level to address heterogeneity and complexity of EV subpopulations. Emerging, commercialised technologies for single EV analysis based on, for example, imaging flow cytometry or imaging after capture on chips generally require dedicated instrumentation and proprietary software not readily accessible to every lab. This limits their implementation for routine EV characterisation in the rapidly growing EV field.

Large-scale production of extracellular vesicles: Report on the "massivEVs" ISEV workshop.

Extracellular vesicles (EVs) large-scale production is a crucial point for the translation of EVs from discovery to application of EV-based products. In October 2021, the International Society for Extracellular Vesicles (ISEV), along with support by the FET-OPEN projects, "The Extracellular Vesicle Foundry" (evFOUNDRY) and "Extracellular vesicles from a natural source for tailor-made nanomaterials" (VES4US), organized a workshop entitled "massivEVs" to discuss the potential challenges for translation of EV-based products. This report gives an overview of the topics discussed during "massivEVs", the most important points raised, and the points of consensus reached after discussion among academia and industry representatives.

Nanomaterial Exposure, Extracellular Vesicle Biogenesis and Adverse Cellular Outcomes: A Scoping Review.

The progressively increasing use of nanomaterials (NMs) has awakened issues related to nanosafety and its potential toxic effects on human health. Emerging studies suggest that NMs alter cell communication by reshaping and altering the secretion of extracellular vesicles (EVs), leading to dysfunction in recipient cells. However, there is limited understanding of how the physicochemical characteristics of NMs alter the EV content and their consequent physiological functions.

Synergy of Human Platelet-Derived Extracellular Vesicles with Secretome Proteins Promotes Regenerative Functions.

Platelet-rich plasma is a promising regenerative therapeutic with controversial efficacy. We and others have previously demonstrated regenerative functions of human platelet lysate (HPL) as an alternative platelet-derived product. Here we separated extracellular vesicles (EVs) from soluble factors of HPL to understand the mode of action during skin-organoid formation and immune modulation as model systems for tissue regeneration.

-Derived Outer Membrane Vesicles (OMVs): Role in Bacterial Pathogenesis?

Persistent infections with the human pathogen () have been closely associated with the induction and progression of a wide range of gastric disorders, including acute and chronic gastritis, ulceration in the stomach and duodenum, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric adenocarcinoma. The pathogenesis of is determined by a complicated network of manifold mechanisms of pathogen-host interactions, which involves a coordinated interplay of pathogenicity and virulence factors with host cells. While these molecular and cellular mechanisms have been intensively investigated to date, the knowledge about outer membrane vesicles (OMVs) derived from and their implication in bacterial pathogenesis is not well developed.

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.

Molecular basis for AU-rich element recognition and dimerization by the HuR C-terminal RRM.

Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate-rich elements (AU-rich elements; AREs). These are a major class of elements within 3' untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3.

Structural basis of siRNA recognition by TRBP double-stranded RNA binding domains.

The accurate cleavage of pre-micro(mi)RNAs by Dicer and mi/siRNA guide strand selection are important steps in forming the RNA-induced silencing complex (RISC). The role of Dicer binding partner TRBP in these processes remains poorly understood. Here, we solved the solution structure of the two N-terminal dsRNA binding domains (dsRBDs) of TRBP in complex with a functionally asymmetric siRNA using NMR, EPR, and single-molecule spectroscopy.

Posttranscriptional Regulation of mRNA by HuR Facilitates DNA Repair and Resistance to PARP Inhibitors.

The majority of pancreatic ductal adenocarcinomas (PDAC) rely on the mRNA stability factor HuR (ELAV-L1) to drive cancer growth and progression. Here, we show that CRISPR-Cas9-mediated silencing of the HuR locus increases the relative sensitivity of PDAC cells to PARP inhibitors (PARPi). PDAC cells treated with PARPi stimulated translocation of HuR from the nucleus to the cytoplasm, specifically promoting stabilization of a new target, poly (ADP-ribose) glycohydrolase () mRNA, by binding a unique sequence embedded in its 3' untranslated region.

Impact of HuR inhibition by the small molecule MS-444 on colorectal cancer cell tumorigenesis.

Colorectal cancer (CRC) is the third most common cancer and a leading cause of cancer-related mortality. Observed during CRC tumorigenesis is loss of post-transcriptional regulation of tumor-promoting genes such as COX-2, TNFα and VEGF. Overexpression of the RNA-binding protein HuR (ELAVL1) occurs during colon tumorigenesis and is abnormally present within the cytoplasm, where it post-transcriptionally regulates genes through its interaction with 3'UTR AU-rich elements (AREs).

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.

HuR Contributes to TRAIL Resistance by Restricting Death Receptor 4 Expression in Pancreatic Cancer Cells.

Unlabelled: Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal cancers, in part, due to resistance to both conventional and targeted therapeutics. TRAIL directly induces apoptosis through engagement of cell surface Death Receptors (DR4 and DR5), and has been explored as a molecular target for cancer treatment. Clinical trials with recombinant TRAIL and DR-targeting agents, however, have failed to show overall positive outcomes.

Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties.

Unlabelled: Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC).

The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing.

Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid-transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA-induced silencing complexes), encounter of the target mRNA, and Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA- and siRNA-loaded Ago2 populations co-sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon-induced protein kinase (PACT).

The mRNA stability factor HuR inhibits microRNA-16 targeting of COX-2.

Commonly observed in colorectal cancer is the elevated expression of the prostaglandin (PG) synthase COX-2. In normal intestinal epithelium, the COX-2 mRNA is targeted for rapid decay through the 3'-untranslated region (3'-UTR) adenylate- and uridylate (AU)-rich element (ARE), whereas in tumors ARE-mediated decay is compromised. Here we show that the COX-2 ARE can mediate degradation through microRNA (miRNA)-mediated regulation.

mRNA stability alterations mediated by HuR are necessary to sustain the fast growth of glioma cells.

Regulation of mRNA decay is an important mechanism controlling gene expression. Steady state levels of mRNAs can be markedly altered by changes in the decay rate. The control of mRNA stability depends on sequences in the transcript itself and on RNA-binding proteins that dynamically bind to these sequences.