AFM-Based High-Throughput Nanomechanical Screening of Single Extracellular Vesicles.

The mechanical properties of extracellular vesicles (EVs) are known to influence their biological function, in terms of, e.g., cellular adhesion, endo/exocytosis, cellular uptake, and mechanosensing. EVs have a characteristic nanomechanical response which can be probed via force spectroscopy (FS) and exploited to single them out from nonvesicular contaminants or to discriminate between subtypes. However, measuring the nanomechanical characteristics of individual EVs via FS is a labor-intensive and time-consuming task, usually limiting this approach to specialists. Herein, we describe a simple atomic force microscopy based experimental procedure for the simultaneous nanomechanical and morphological analysis of several hundred individual nanosized EVs within the hour time scale, using basic AFM equipment and skills and only needing freely available software for data analysis. This procedure yields a "nanomechanical snapshot" of an EV sample which can be used to discriminate between subpopulations of vesicular and nonvesicular objects in the same sample and between populations of vesicles with similar sizes but different mechanical characteristics. We demonstrate the applicability of the proposed approach to EVs obtained from three very different sources (human colorectal carcinoma cell culture, raw bovine milk, and Ascaris suum nematode excretions), recovering size and stiffness distributions of individual vesicles in a sample. EV stiffness values measured with our high-throughput method are in very good quantitative accord with values obtained by FS techniques which measure EVs one at a time. We show how our procedure can detect EV samples contamination by nonvesicular aggregates and how it can quickly attest the presence of EVs even in samples for which no established assays and/or commercial kits are available (e.g., Ascaris EVs), thus making it a valuable tool for the rapid assessment of EV samples during the development of isolation/enrichment protocols by EV researchers. As a side observation, we show that all measured EVs have a strikingly similar stiffness, further reinforcing the hypothesis that their mechanical characteristics could have a functional role.

Intersectin goes nuclear: secret life of an endocytic protein.

Intersectin 1-short (ITSN1-s) is a 1220 amino acid ubiquitously expressed scaffold protein presenting a multidomain structure that allows to spatiotemporally regulate the functional interaction of a plethora of proteins. Besides its well-established role in endocytosis, ITSN1-s is involved in the regulation of cell signaling and is implicated in tumorigenesis processes, although the signaling pathways involved are still poorly understood. Here, we identify ITSN1-s as a nucleocytoplasmic trafficking protein. We show that, by binding to importin (IMP)α, a small fraction of ITSN1-s localizes in the cell nucleus at the steady state, where it preferentially associates with the nuclear envelope and interacts with lamin A/C. However, upon pharmacological ablation of chromosome region maintenance 1 (CRM-1)-dependent nuclear export pathway, the protein accumulates into the nucleus, thus revealing its moonlighting nature. Analysis of deletion mutants revealed that the coiled coil (CC) and Src homology (SH3) regions play the major role in its nucleocytoplasmic shuttling. While no evidence of nuclear localization signal (NLS) was detected in the CC region, a functional bipartite NLS was identified within the SH3D region of ITSN1-s (RKKNPGGWWEGELQARGKKRQIGW-1127), capable of conferring energy-dependent nuclear accumulation to reporter proteins and whose mutational ablation affects nuclear import of the whole SH3 region. Thus, ITSN1-s is an endocytic protein, which shuttles between the nucleus and the cytoplasm in a CRM-1- and IMPα-dependent fashion.

Exosomes Secreted by HeLa Cells Shuttle on Their Surface the Plasma Membrane-Associated Sialidase NEU3.

Sialidases are glycohydrolases that remove terminal sialic acid residues from oligosaccharides, glycolipids, and glycoproteins. The plasma membrane-associated sialidase NEU3 is involved in the fine-tuning of sialic acid-containing glycans directly on the cell surface and plays relevant roles in important biological phenomena such as cell differentiation, molecular recognition, and cancer transformation. Extracellular vesicles are membranous structures with a diameter of 0.03-1 µm released by cells and can be detected in blood, urine, and culture media. Among extracellular vesicles, exosomes play roles in intercellular communication and maintenance of several physiological and pathological conditions, including cancer, and could represent a useful diagnostic tool for personalized nanomedicine approaches. Using inducible expression of the murine form of NEU3 in HeLa cells, a study of the association of the enzyme with exosomes released in the culture media has been performed. Briefly, NEU3 is associated with highly purified exosomes and localizes on the external leaflet of these nanovesicles, as demonstrated by enzyme activity measurements, Western blot analysis, and dot blot analysis using specific protein markers. On the basis of these results, it is plausible that NEU3 activity on exosome glycans enhances the dynamic biological behavior of these small extracellular vesicles by modifying the negative charge and steric hindrance of their glycocalyx. The presence of NEU3 on the exosomal surface could represent a useful marker for the detection of these nanovesicles and a tool for improving our understanding of the biology of these important extracellular carriers in physiological and pathological conditions.

Quantification of ß region IgA paraproteins - should we include immunochemical "heavy/light chain" measurements? Counterpoint.

Serum protein electrophoresis (SPE), serum immunofixation (s-IFE), free light chain measurement (FLC) and nephelometric measurements of total immunoglobulin in serum (IgTot) are some of the laboratory tests required for the management of plasma cell proliferative disorders. The monoclonal protein is usually visible on SPE as a spike (M-spike) in the γ region and the derived densitogram is used to quantify it relative to serum total protein concentration. IgA M-protein, however, often migrates in the ß region on SPE and its quantification can be masked by other serum proteins that migrate in this region. The immunoassay Hevylite™ (heavy/light chain, HLC) seems to solve this problem: it quantifies the involved/uninvolved isotype, calculating the ratio IgAκ/IgAλ, considered indicative of clonal proliferation. However, this test seems redundant in the case of artifacts on SPE such as obvious hemolysis or lipemia, or if the IgA M-spike is clearly visible in the ß region. In conclusion whereas the IgA HLC assay does not represent an alternative to SPE and s-IFE in the diagnostic patient workup, it may prove to be an alternative to SPE, s-IFE and total IgA quantification in risk stratification and evaluation of response to therapy in patients affected by MM and other monoclonal plasma proliferative disorders.

Colorimetric nanoplasmonic assay to determine purity and titrate extracellular vesicles.

Extracellular Vesicles (EVs) - cell secreted vesicles that carry rich molecular information of the parental cell and constitute an important mode of intercellular communication - are becoming a primary topic in translational medicine. EVs (that comprise exosomes and microvesicles/microparticles) have a size ranging from 40 nm to 1 µm and share several physicochemical proprieties, including size, density, surface charge, and light interaction, with other nano-objects present in body fluids, such as single and aggregated proteins. This makes separation, titration, and characterization of EVs challenging and time-consuming. Here we present a cost-effective and fast colorimetric assay for probing by eye protein contaminants and determine the concentration of EV preparations, which exploits the synergy between colloidal gold nanoplasmonics, nanoparticle-protein corona, and nanoparticle-membrane interaction. The assay hits a limit of detection of protein contaminants of 5 ng/µL and has a dynamic range of EV concentration ranging from 35 fM to 35 pM, which matches the typical range of EV concentration in body fluids. This work provides the first example of the exploitation of the nanoparticle-protein corona in analytical chemistry.

USP44 overexpression drives a MYC-like gene expression program in neuroblastoma through epigenetic reprogramming.

Neuroblastoma is an embryonic cancer that contributes disproportionately to death in young children. Sequencing data have uncovered few recurrently mutated genes in this cancer, although epigenetic pathways have been implicated in disease pathogenesis. We used an expression-based computational screen that examined the impact of deubiquitinating enzymes on patient survival to identify potential new targets. We identified the histone H2B deubiquitinating enzyme USP44 as the enzyme with the greatest impact on survival in patients with neuroblastoma. High levels of USP44 significantly correlate with metastatic disease, unfavorable histology, advanced patient age, and MYCN-amplification. The subset of patients with tumors expressing high levels of USP44 had a significantly worse survival, including those with tumors lacking MYCN amplification. We showed experimentally that USP44 regulates neuroblastoma cell proliferation, migration, invasion, and neuronal development. Depletion of the histone H2B ubiquitin ligase subunit RNF20 resulted in similar findings, strongly implicating this histone mark as the target of USP44 activity in this disease. Integration of transcriptome and epigenome in analyses demonstrates a distinct set of genes that is regulated by USP44, including those in Hallmark MYC target genes in both murine embryonic fibroblasts and the SH-SY5Y neuroblastoma cell line. We conclude that USP44 is a novel epigenetic regulator that promotes aggressive features and may be a novel target in neuroblastoma. Implications: This study identifies a new genetic marker of aggressive neuroblastoma and identifies the mechanisms by which its overactivity contributes to pathophysiology in this disease.

The epsilon hinge-ear region regulates membrane localization of the AP-4 complex.

Adaptor protein (AP) complexes are key factors for the spatial and temporal regulation of intracellular trafficking events. Four complexes (AP-1, -2, -3, -4) are known, among which AP-4 is only poorly characterized. Recent work suggests a role for AP-4 in the intracellular trafficking of the ß-amyloid precursor protein and molecular genetics showed that the loss of functional AP-4 is associated with congenital neuronal disorders of severe cognitive dysfunction. To unravel the molecular mechanisms controlling AP-4 functions, we established the intracellular expression of recombinant AP-4 complex. This approach combined with the analysis of mutant complexes allowed us to discover that the epsilon adaptin hinge-ear region has a function in membrane recruitment of AP-4. We further show that this process is phosphorylation dependent and involves PP2A-like protein phosphatases and a staurosporine-sensitive kinase. Deletion of the residues 839-871 in the carboxy-terminal region of the hinge of epsilon adaptin abrogated the membrane/cytosol recycling of AP-4. As targets of phosphorylation, we identified three serine residues: S847, S868 and S871. We conclude that the terminal hinge region and the appendage of the AP-4 epsilon subunit are involved in membrane association in a process that is controlled by phosphorylation and dephosphorylation events.

Surface functionalization of extracellular vesicle nanoparticles with antibodies: a first study on the protein corona "variable".

To be profitably exploited in medicine, nanosized systems must be endowed with biocompatibility, targeting capability, the ability to evade the immune system, and resistance to clearance. Currently, biogenic nanoparticles, such as extracellular vesicles (EVs), are intensively investigated as the platform that naturally recapitulates these highly needed characteristics. EV native targeting properties and pharmacokinetics can be further augmented by decorating the EV surface with specific target ligands as antibodies. However, to date, studies dealing with the functionalization of the EV surface with proteins have never considered the protein corona "variable", namely the fact that extrinsic proteins may spontaneously adsorb on the EV surface, contributing to determine the surface, and in turn the biological identity of the EV. In this work, we explore and compare the two edge cases of EVs modified with the antibody Cetuximab (CTX) by chemisorption of CTX (through covalent binding via biorthogonal click-chemistry) and by formation of a physisorbed CTX corona. The results indicate that (i) no differences exist between the two formulations in terms of binding affinity imparted by molecular recognition of CTX versus its natural binding partner (epidermal growth factor receptor, EGFR), but (ii) significant differences emerge at the cellular level, where CTX-EVs prepared by click chemistry display superior binding and uptake toward target cells, very likely due to the higher robustness of the CTX anchorage.

Symptomatic Uncomplicated Diverticular Disease (SUDD): Practical Guidance and Challenges for Clinical Management.

Symptomatic Uncomplicated Diverticular Disease (SUDD) is a syndrome within the diverticular disease spectrum, characterized by local abdominal pain with bowel movement changes but without systemic inflammation. This narrative review reports current knowledge, delivers practical guidance, and reveals challenges for the clinical management of SUDD. A broad and common consensus on the definition of SUDD is still needed. However, it is mainly considered a chronic condition that impairs quality of life (QoL) and is characterized by persistent left lower quadrant abdominal pain with bowel movement changes (eg, diarrhea) and low-grade inflammation (eg, elevated calprotectin) but without systemic inflammation. Age, genetic predisposition, obesity, physical inactivity, low-fiber diet, and smoking are considered risk factors. The pathogenesis of SUDD is not entirely clarified. It seems to result from an interaction between fecal microbiota alterations, neuro-immune enteric interactions, and muscular system dysfunction associated with a low-grade and local inflammatory state. At diagnosis, it is essential to assess baseline clinical and Quality of Life (QoL) scores to evaluate treatment efficacy and, ideally, to enroll patients in cohort studies, clinical trials, or registries. SUDD treatments aim to improve symptoms and QoL, prevent recurrence, and avoid disease progression and complications. An overall healthy lifestyle - physical activity and a high-fiber diet, with a focus on whole grains, fruits, and vegetables - is encouraged. Probiotics could effectively reduce symptoms in patients with SUDD, but their utility is missing adequate evidence. Using Rifaximin plus fiber and Mesalazine offers potential in controlling symptoms in patients with SUDD and might prevent acute diverticulitis. Surgery could be considered in patients with medical treatment failure and persistently impaired QoL. Still, studies with well-defined diagnostic criteria for SUDD that evaluate the safety, QoL, effectiveness, and cost-effectiveness of these interventions using standard scores and comparable outcomes are needed.

Chromosome instability in neuroblastoma: A pathway to aggressive disease.

For over 100-years, genomic instability has been investigated as a central player in the pathogenesis of human cancer. Conceptually, genomic instability includes an array of alterations from small deletions/insertions to whole chromosome alterations, referred to as chromosome instability. Chromosome instability has a paradoxical impact in cancer. In most instances, the introduction of chromosome instability has a negative impact on cellular fitness whereas in cancer it is usually associated with a worse prognosis. One exception is the case of neuroblastoma, the most common solid tumor outside of the brain in children. Neuroblastoma tumors have two distinct patterns of genome instability: whole-chromosome aneuploidy, which is associated with a better prognosis, or segmental chromosomal alterations, which is a potent negative prognostic factor. Through a computational screen, we found that low levels of the de- ubiquitinating enzyme USP24 have a highly significant negative impact on survival in neuroblastoma. At the molecular level, USP24 loss leads to destabilization of the microtubule assembly factor CRMP2 - producing mitotic errors and leading to chromosome missegregation and whole-chromosome aneuploidy. This apparent paradox may be reconciled through a model in which whole chromosome aneuploidy leads to the subsequent development of segmental chromosome alterations. Here we review the mechanisms behind chromosome instability and the evidence for the progressive development of segmental alterations from existing numerical aneuploidy in support of a multi-step model of neuroblastoma progression.

Comparison of separation methods for immunomodulatory extracellular vesicles from helminths.

Helminths survive within their host by secreting immunomodulatory compounds, which hold therapeutic potential for inflammatory conditions. Helminth-derived extracellular vesicles (EVs) are one such component proposed to possess immunomodulatory activities. Due to the recent discovery of helminth EVs, standardised protocols for EV separation are lacking. Excretory/secretory products of the porcine helminth, Ascaris suum, were used to compare three EV separation methods: Size exclusion chromatography (SEC), ultracentrifugation (UC) and a combination of the two. Their performance was evaluated by EV yield, sample purity and the ability of EVs to suppress lipopolysaccharide (LPS)-induced inflammation in vitro. We found that all three separation methods successfully separated helminth EVs with a similar EV yield. Functional studies showed that EVs from all three methods reduced LPS-induced levels of tumour necrosis factor (TNF-α) in a dose-dependent manner. Overall, the three separation methods showed similar performance, however, the combination of UC+SEC presented with slightly higher purity than either method alone.

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. Overall, the review of the existing EV manufacturing, upscaling challenges and directions for their resolution highlighted in the workshop painted an optimistic future for the expanding EV field.

Cigarette Smoke Exposure, Pediatric Lung Disease, and COVID-19.

The detrimental effects of tobacco exposure on children's health are well known. Nonetheless, the prevalence of secondhand or direct cigarette smoke exposure (CSE) in the pediatric population has not significantly decreased over time. On the contrary, the rapid incline in use of e-cigarettes among adolescents has evoked public health concerns since increasing cases of vaping-induced acute lung injury have highlighted the potential harm of these new "smoking" devices. Two pediatric populations are especially vulnerable to the detrimental effects of cigarette smoke. The first group is former premature infants whose risk is elevated both due to their prematurity as well as other risk factors such as oxygen and mechanical ventilation to which they are disproportionately exposed. The second group is children and adolescents with chronic respiratory diseases, in particular asthma and other wheezing disorders. Coronavirus disease 2019 (COVID-19) is a spectrum of diseases caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has spread worldwide over the last year. Here, respiratory symptoms ranging from mild to acute respiratory distress syndrome (ARDS) are at the forefront of COVID-19 cases among adults, and cigarette smoking is associated with worse outcomes in this population, and cigarette smoking is associated with worse outcomes in this population. Interestingly, SARS-CoV-2 infection affects children differently in regard to infection susceptibility, disease manifestations, and complications. Although children carry and transmit the virus, the likelihood of symptomatic infection is low, and the rates of hospitalization and death are even lower when compared to the adult population. However, multisystem inflammatory syndrome is recognized as a serious consequence of SARS-CoV-2 infection in the pediatric population. In addition, recent data demonstrate specific clinical patterns in children infected with SARS-CoV-2 who develop multisystem inflammatory syndrome vs. severe COVID-19. In this review, we highlight the pulmonary effects of CSE in vulnerable pediatric populations in the context of the ongoing SARS-CoV-2 pandemic.

A plasmon-based nanoruler to probe the mechanical properties of synthetic and biogenic nanosized lipid vesicles.

Nanosized lipid vesicles are ubiquitous in living systems (e.g. cellular compartments or extracellular vesicles, EVs) and in formulations for nanomedicine (e.g. liposomes for RNA vaccine formulations). The mechanical properties of such vesicles are crucial in several physicochemical and biological processes, ranging from cellular uptake to stability in aerosols. However, their accurate determination remains challenging and requires sophisticated instruments and data analysis. Here we report the first evidence that the surface plasmon resonance (SPR) of citrated gold nanoparticles (AuNPs) adsorbed on synthetic vesicles is finely sensitive to the vesicles' mechanical properties. We then leverage this finding to show that the SPR tracking provides quantitative access to the stiffness of vesicles of synthetic and natural origin, such as EVs. The demonstration of this plasmon-based "stiffness nanoruler" paves the way for developing a facile, cost-effective and high-throughput method to assay the mechanical properties of dispersions of vesicles of nanometric size and unknown composition at a collective level.

MicroRNA­34a­5p expression in the plasma and in its extracellular vesicle fractions in subjects with Parkinson's disease: An exploratory study.

Parkinson's disease (PD) is an important disabling age­related disorder and is the second most common neurodegenerative disease. Currently, no established molecular biomarkers exist for the early diagnosis of PD. Circulating microRNAs (miRNAs), either vesicle­free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood­based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR­34a­5p because of its well­documented involvement in neurobiology. To explore a differential profile of circulating miR­34a­5p in PD, PD patients and age­matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). Characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. Circulating miR­34a­5p levels were evaluated using qPCR in plasma and in each EV type. miR­34a­5p was significantly up­regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from PD patients and ROC analysis indicated a good diagnostic performance in discriminating patients from controls (AUC=0.74, P<0.05). Moreover, miR­34a­5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck Depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR­34a­5p in PD via the use of pure SEVs.

Corrigendum: Augmented COlorimetric NANoplasmonic (CONAN) Method for Grading Purity and Determine Concentration of EV Microliter Volume Solutions.

[This corrects the article DOI: 10.3389/fbioe.2019.00452.].

"Percorso Giacomo": An Italian Innovative Service of Perinatal Palliative Care.

The perspective proposed by this article will focus on perinatal palliative care as a strategy for improving the quality of life of neonates with life-limiting conditions when extending the patient's life is no longer the goal of care. This manuscript reports the creation of an innovative program of perinatal palliative care called "Percorso Giacomo" (Giacomo's Pathway) at Sant'Orsola Hospital in Bologna, Italy in 2013. Key features include interdisciplinary collaboration between professionals from obstetrics, neonatology and other specialties aiming to reach the most detailed fetal and neonatal diagnosis and prognosis; communication and engagement with the family to discuss goals of care and prepare a birthing plan that follows the family's desires and expectations; and personalized care to achieve comfort for each newborn and support for families according to their social, cultural, and religious backgrounds.

Fourier-transform Infrared (FT-IR) spectroscopy fingerprints subpopulations of extracellular vesicles of different sizes and cellular origin.

Identification of extracellular vesicle (EV) subpopulations remains an open challenge. To date, the common strategy is based on searching and probing set of molecular components and physical properties intended to be univocally characteristics of the target subpopulation. Pitfalls include the risk to opt for an unsuitable marker set - which may either not represent the subpopulation or also cover other unintended subpopulations - and the need to use different characterization techniques and equipment. This approach focused on specific markers may result inadequate to routinely deal with EV subpopulations that have an intrinsic high level of heterogeneity. In this paper, we show that Fourier-transform Infrared (FT-IR) spectroscopy can provide a collective fingerprint of EV subpopulations in one single experiment. FT-IR measurements were performed on large (LEVs, ~600 nm), medium (MEVs, ~200 nm) and small (SEVs ~60 nm) EVs enriched from two different cell lines medium: murine prostate cancer (TRAMP-C2) and skin melanoma (B16). Spectral regions between 3100-2800 cm-1 and 1880-900 cm-1, corresponding to functional groups mainly ascribed to lipid and protein contributions, were acquired and processed by Principal Component Analysis (PCA). LEVs, MEVs and SEVs were separately grouped for both the considered cell lines. Moreover, subpopulations of the same size but from different sources were assigned (with different degrees of accuracy) to two different groups. These findings demonstrate that FT-IR has the potential to quickly fingerprint EV subpopulations as a whole, suggesting an appealing complement/alternative for their characterization and grading, extendable to healthy and pathological EVs and fully artificial nanovesicles.

Biogenic supported lipid bilayers as a tool to investigate nano-bio interfaces.

HYPOTHESIS: Extracellular Vesicles (EVs) are natural nanosized lipid vesicles involved in most intercellular communication pathways. Given their nature, they represent natural cell membrane models, with intermediate complexity between real and synthetic lipid membranes. Here we compare EVs-derived (EVSLB) and synthetic Supported Lipid Bilayers (SLBs) in the interaction with cationic superparamagnetic iron oxide nanoparticles (SPIONs). The aim is twofold: (i) exploit SPIONs as nanometric probes to investigate the features of EVSLBs as novel biogenic platforms; (ii) contribute at improving the knowledge on the behavior of SPIONs with biological interfaces. EXPERIMENTS: Quartz Crystal Microbalance, X-ray Reflectivity, Grazing-incidence Small-angle X-ray Scattering, Atomic Force Microscopy, Confocal Microscopy data on SPIONs-EVSLB were systematically compared to those on SPIONs challenging synthetic SLBs, taken as references. FINDINGS: The ensemble of experimental results highlights the much stronger interaction of SPIONs with EVSLBs with respect to synthetic SLBs. This evidence strongly supports the hypotheses on the peculiar structure of EVSLBs, with cushioned non-flat areas and extended exposed surface; in addition, it suggests that these features are relevant in the response of biogenic membranes to nano-objects. These findings contribute to the fundamental knowledge on EVSLBs, key for their development both as biomimetic membranes, or as platforms for biomedical applications.

Augmented COlorimetric NANoplasmonic (CONAN) Method for Grading Purity and Determine Concentration of EV Microliter Volume Solutions.

This protocol paper describes how to assign a purity grade and to subsequently titrate extracellular vesicle (EV) solutions of a few microliters in volume by microplate COlorimetric NANoplasmonic (CONAN) assay. The CONAN assay consists of a solution of gold nanoparticles (AuNPs) into which the EV preparation is added. The solution turns blue if the EV preparation is pure, whereas it stays red if soluble exogenous single and aggregated proteins (SAPs; often referred to as protein contaminants) are present. The color change is visible by the naked eye or can be quantified by UV-Vis spectroscopy, providing an index of purity (a unique peculiarity to date). The assay specifically targets SAPs, and not the EV-related proteins, with a detection limit <50 ng/µl (an order of magnitude higher resolution than that of the Bradford protein assay). For pure solutions, the assay also allows for determining the EV number, as the color shift is linearly dependent on the AuNP/EV molar ratio. Instead, it automatically reports if the solution bears SAP contaminants, thus avoiding counting artifacts. The CONAN assay proves to be robust and reliable and displays very interesting performances in terms of cost (inexpensive reagents, run by standard microplate readers), working volumes (1-2 µl of sample required), and time (full procedure takes <1 h). The assay is applicable to all classes of natural and artificial lipid microvesicles and nanovesicles.