Efficient Labeling of Vesicles with Lipophilic Fluorescent Dyes via the Salt-Change Method.

Fluorescent labeling allows for imaging and tracking of vesicles down to single-particle level. Among several options to introduce fluorescence, staining of lipid membranes with lipophilic dyes provides a straightforward approach without interfering with vesicle content. However, incorporating lipophilic molecules into vesicle membranes in an aqueous solution is generally not efficient because of their low water solubility. Here, we describe a simple, fast (<30 min), and highly effective procedure for fluorescent labeling of vesicles including natural extracellular vesicles. By adjusting the ionic strength of the staining buffer with NaCl, the aggregation status of DiI, a representative lipophilic tracer, can be controlled reversibly. Using cell-derived vesicles as a model system, we show that dispersion of DiI under low-salt condition improved its incorporation into vesicles by a factor of 290. In addition, increasing NaCl concentration after labeling induced free dye molecules to form aggregates, which can be filtered and thus effectively removed without ultracentrifugation. We consistently observed 6- to 85-fold increases in the labeled vesicle count across different types of dyes and vesicles. The method is expected to reduce the concern about off-target labeling resulting from the use of high concentrations of dyes.

RNA-sequencing profiling analysis of pericyte-derived extracellular vesicle-mimetic nanovesicles-regulated genes in primary cultured fibroblasts from normal and Peyronie's disease penile tunica albuginea.

BACKGROUND: Peyronie's disease (PD) is a severe fibrotic disease of the tunica albuginea that causes penis curvature and leads to penile pain, deformity, and erectile dysfunction. The role of pericytes in the pathogenesis of fibrosis has recently been determined. Extracellular vesicle (EV)-mimetic nanovesicles (NVs) have attracted attention regarding intercellular communication between cells in the field of fibrosis. However, the global gene expression of pericyte-derived EV-mimetic NVs (PC-NVs) in regulating fibrosis remains unknown. Here, we used RNA-sequencing technology to investigate the potential target genes regulated by PC-NVs in primary fibroblasts derived from human PD plaque. METHODS: Human primary fibroblasts derived from normal and PD patients was cultured and treated with cavernosum pericytes isolated extracellular vesicle (EV)-mimetic nanovesicles (NVs). A global gene expression RNA-sequencing assay was performed on normal fibroblasts, PD fibroblasts, and PD fibroblasts treated with PC-NVs. Reverse transcription polymerase chain reaction (RT-PCR) was used for sequencing data validation. RESULTS: A total of 4135 genes showed significantly differential expression in the normal fibroblasts, PD fibroblasts, and PD fibroblasts treated with PC-NVs. However, only 91 contra-regulated genes were detected among the three libraries. Furthermore, 20 contra-regulated genes were selected and 11 showed consistent changes in the RNA-sequencing assay, which were validated by RT-PCR. CONCLUSION: The gene expression profiling results suggested that these validated genes may be good targets for understanding potential mechanisms and conducting molecular studies into PD.

Pericyte-Derived Extracellular Vesicle-Mimetic Nanovesicles Restore Erectile Function by Enhancing Neurovascular Regeneration in a Mouse Model of Cavernous Nerve Injury.

BACKGROUND: Extracellular vesicle (EV)-mimetic nanovesicles (NVs) from embryonic stem cells have been observed to stimulate neurovascular regeneration in the streptozotocin-induced diabetic mouse. Pericytes play important roles in maintaining penile erection, yet no previous studies have explored the effects of pericyte-derived NVs (PC-NVs) in neurovascular regeneration in the context of erectile dysfunction. AIM: To investigate the potential effect of PC-NVs in neurovascular regeneration. METHODS: PC-NVs were isolated from mouse cavernous pericytes, and neurovascular regeneration was evaluated in an in vitro study. Twelve-week-old C57BL/6J mice were used to prepare cavernous nerve injury model. Erectile function evaluation, histologic examination of the penis, and Western blots were assessed 2 weeks after model creation and PC-NVs treatment. OUTCOMES: The main outcomes of this study are PC-NVs characterization, intracavernous pressure, neurovascular regeneration in the penis, and in vitro functional evaluation. RESULTS: The PC-NVs were extracted and characterized by cryotransmission electron microscopy and EV-positive (Alix, TSG101, CD81) and EV-negative (GM130) markers. In the in vivo studies, PC-NVs successfully improved erectile function in cavernous nerve injury mice (∼82% of control values). Immunofluorescence staining showed significant increases in pericytes, endothelial cell, and neuronal contents. In the in vitro studies, PC-NVs significantly increased mouse cavernous endothelial cells tube formation, Schwann cell migration, and dorsal root ganglion and major pelvic ganglion neurite sprouting. Finally, Western blot analysis revealed that PC-NVs upregulated cell survival signaling (Akt and eNOS) and induced the expression of neurotrophic factors (brain-derived neurotrophic factor, neurotrophin-3, and nerve growth factor). CLINICAL IMPLICATIONS: PC-NVs may be used as a strategy to treat erectile dysfunction after radical prostatectomy or in men with neurovascular diseases. STRENGTHS & LIMITATIONS: We evaluated the effect of PC-NVs in vitro and in a mouse nerve injury model, cavernous nerve injury. Additional studies are necessary to determine the detailed mechanisms of neurovascular improvement. Further study is needed to test whether PC-NVs are also effective when given weeks or months after nerve injury. CONCLUSION: PC-NVs significantly improved erectile function by enhancing neurovascular regeneration. Local treatment with PC-NVs may represent a promising therapeutic strategy for the treatment of neurovascular diseases. Yin GN, Park S-H, Ock J, et al. Pericyte-Derived Extracellular Vesicle-Mimetic Nanovesicles Restore Erectile Function by Enhancing Neurovascular Regeneration in a Mouse Model of Cavernous Nerve Injury. J Sex Med 2020;17:2118-2128.

BRAFV600 inhibition alters the microRNA cargo in the vesicular secretome of malignant melanoma cells.

The BRAF inhibitors vemurafenib and dabrafenib can be used to treat patients with metastatic melanomas harboring BRAFV600 mutations. Initial antitumoral responses are often seen, but drug-resistant clones with reactivation of the MEK-ERK pathway soon appear. Recently, the secretome of tumor-derived extracellular vesicles (EVs) has been ascribed important functions in cancers. To elucidate the possible functions of EVs in BRAF-mutant melanoma, we determined the RNA content of the EVs, including apoptotic bodies, microvesicles, and exosomes, released from such cancer cells after vemurafenib treatment. We found that vemurafenib significantly increased the total RNA and protein content of the released EVs and caused significant changes in the RNA profiles. RNA sequencing and quantitative PCR show that cells and EVs from vemurafenib-treated cell cultures and tumor tissues harvested from cell-derived and patient-derived xenografts harbor unique miRNAs, especially increased expression of miR-211-5p. Mechanistically, the expression of miR-211-5p as a result of BRAF inhibition was induced by increased expression of MITF that regulates the TRPM1 gene resulting in activation of the survival pathway. In addition, transfection of miR-211 in melanoma cells reduced the sensitivity to vemurafenib treatment, whereas miR-211-5p inhibition in a vemurafenib resistant cell line affected the proliferation negatively. Taken together, our results show that vemurafenib treatment induces miR-211-5p up-regulation in melanoma cells both in vitro and in vivo, as well as in subsets of EVs, suggesting that EVs may provide a tool to understand malignant melanoma progression.

Extracellular vesicle mimetics: Novel alternatives to extracellular vesicle-based theranostics, drug delivery, and vaccines.

Extracellular vesicles are nano-sized spherical bilayered proteolipids encasing various components. Cells of all domains of life actively release these vesicles to the surroundings including various biological fluids. These extracellular vesicles are known to play pivotal roles in numerous pathophysiological functions. Extracellular vesicles have distinct characteristics, like high biocompatibility, safety, and nano-sized diameters that allow efficient drug loading capacity and long blood circulation half-life. These characteristics of extracellular vesicles have engrossed many scientists to harness them as new tools for novel delivery systems. This review will highlight the current state of the arts and problems of such extracellular vesicle-based theranostics, drug delivery and vaccines, and introduce "extracellular vesicle mimetics" as the novel alternative of extracellular vesicles. We hope to provide insights into the potential of extracellular vesicle mimetics as superior substitute to the natural extracellular vesicles that can be applied to theranostics, drug delivery, and vaccines against various diseases.

Toll-Like Receptors 2 and 4 Modulate Pulmonary Inflammation and Host Factors Mediated by Outer Membrane Vesicles Derived from Acinetobacter baumannii.

Pneumonia due to Gram-negative bacteria is associated with high mortality. Acinetobacter baumannii is a Gram-negative bacterium that is associated with hospital-acquired and ventilator-associated pneumonia. Bacteria have been described to release outer membrane vesicles (OMVs) that are capable of mediating systemic inflammation. The mechanism by which A. baumannii OMVs mediate inflammation is not fully defined. We sought to investigate the roles that Toll-like receptors (TLRs) play in A. baumannii OMV-mediated pulmonary inflammation. We isolated OMVs from A. baumannii cultures and intranasally introduced the OMVs into mice. Intranasal introduction of A. baumannii OMVs mediated pulmonary inflammation, which is associated with neutrophil recruitment and weight loss. In addition, A. baumannii OMVs increased the release of several chemokines and cytokines in the mouse lungs. The proinflammatory responses were partially inhibited in TLR2- and TLR4-deficient mice compared to those of wild-type mice. This study highlights the important roles of TLRs in A. baumannii OMV-induced pulmonary inflammation in vivo.

Gram-negative and Gram-positive bacterial extracellular vesicles.

Like mammalian cells, Gram-negative and Gram-positive bacteria release nano-sized membrane vesicles into the extracellular environment either in a constitutive manner or in a regulated manner. These bacterial extracellular vesicles are spherical bilayered proteolipids enriched with bioactive proteins, lipids, nucleic acids, and virulence factors. Recent progress in this field supports the critical pathophysiological functions of these vesicles in both bacteria-bacteria and bacteria-host interactions. This review provides an overview of the current understanding on Gram-negative and Gram-positive bacterial extracellular vesicles, especially regarding the biogenesis, components, and functions in poly-species communities. We hope that this review will stimulate additional research in this emerging field of bacterial extracellular vesicles and contribute to the development of extracellular vesicle-based diagnostic tools and effective vaccines against pathogenic Gram-negative and Gram-positive bacteria.

EVpedia: A community web resource for prokaryotic and eukaryotic extracellular vesicles research.

For cell-to-cell communication, all living cells including archaea, bacteria, and eukaryotes secrete nano-sized membrane vesicles into the extracellular space. These extracellular vesicles harbor specific subsets of proteins, mRNAs, miRNAs, lipids, and metabolites that represent their cellular status. These vesicle-specific cargos are considered as novel diagnostic biomarkers as well as therapeutic targets. With the advancement in high-throughput technologies on multiomics studies and improvements in bioinformatics approaches, a huge number of vesicular proteins, mRNAs, miRNAs, lipids, and metabolites have been identified, and our understanding of these complex extracellular organelles has considerably increased during these past years. In this review, we highlight EVpedia (http://evpedia.info), a community web portal for systematic analyses of prokaryotic and eukaryotic extracellular vesicles research.

Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing.

Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions.

Extracellular vesicles derived from Gram-negative bacteria, such as Escherichia coli, induce emphysema mainly via IL-17A-mediated neutrophilic inflammation.

Recent evidence indicates that Gram-negative bacteria-derived extracellular vesicles (EVs) in indoor dust can evoke neutrophilic pulmonary inflammation, which is a key pathology of chronic obstructive pulmonary disease (COPD). Escherichia coli is a ubiquitous bacterium present in indoor dust and secretes nanometer-sized vesicles into the extracellular milieu. In the current study, we evaluated the role of E. coli-derived EVs on the development of COPD, such as emphysema. E. coli EVs were prepared by sequential ultrafiltration and ultracentrifugation. COPD phenotypes and immune responses were evaluated in C57BL/6 wild-type (WT), IFN-γ-deficient, or IL-17A-deficient mice after airway exposure to E. coli EVs. The present study showed that indoor dust from a bed mattress harbors E. coli EVs. Airway exposure to E. coli EVs increased the production of proinflammatory cytokines, such as TNF-α and IL-6. In addition, the repeated inhalation of E. coli EVs for 4 wk induced neutrophilic inflammation and emphysema, which are associated with enhanced elastase activity. Emphysema and elastase activity enhanced by E. coli EVs were reversed by the absence of IFN-γ or IL-17A genes. In addition, during the early period, lung inflammation is dependent on IL-17A and TNF-α, but not on IFN-γ, and also on TLR4. Moreover, the production of IFN-γ is eliminated by the absence of IL-17A, whereas IL-17A production is not abolished by IFN-γ absence. Taken together, the present data suggest that E. coli-derived EVs induce IL-17A-dependent neutrophilic inflammation and thereby emphysema, possibly via upregulation of elastase activity.

Exosomes in the nose induce immune cell trafficking and harbour an altered protein cargo in chronic airway inflammation.

BACKGROUND: Exosomes are nano-sized extracellular vesicles participating in cell-to-cell communication both in health and disease. However, the knowledge about the functions and molecular composition of exosomes in the upper airways is limited. The aim of the current study was therefore to determine whether nasal exosomes can influence inflammatory cells and to establish the proteome of nasal lavage fluid-derived exosomes in healthy subjects, as well as its alterations in individuals with chronic airway inflammatory diseases [asthma and chronic rhinosinusitis (CRS)]. METHODS: Nasal lavage fluid was collected from 14 healthy subjects, 15 subjects with asthma and 13 subjects with asthma/CRS. Exosomes were isolated with differential centrifugation and the proteome was analysed by LC-MS/MS with the application of two exclusion lists as well as using quantitative proteomics. Ingenuity Pathways Analysis and GO Term finder was used to predict the functions associated with the exosomal proteome and a migration assay was used to analyse the effect on immune cells by nasal exosomes. RESULTS: Firstly, we demonstrate that nasal exosomes can induce migration of several immune cells, such as monocytes, neutrophils and NK cells in vitro. Secondly, a mass spectrometry approach, with the application of exclusion lists, was utilised to generate a comprehensive protein inventory of the exosomes from healthy subjects. The use of exclusion lists resulted in the identification of ~15 % additional proteins, and increased the confidence in ~20 % of identified proteins. In total, 604 proteins were identified in nasal exosomes and the nasal exosomal proteome showed strong associations with immune-related functions, such as immune cell trafficking. Thirdly, a quantitative proteomics approach was used to determine alterations in the exosome proteome as a result of airway inflammatory disease. Serum-associated proteins and mucins were more abundant in the exosomes from subjects with respiratory diseases compared to healthy controls while proteins with antimicrobial functions and barrier-related proteins had decreased expression. CONCLUSIONS: Nasal exosomes were shown to induce the migration of innate immune cells, which may be important as the airway epithelium is the first line of defence against pathogens and allergens. The decreased expression in barrier and antimicrobial exosomal proteins in subjects with airway diseases, could possibly contribute to an increased susceptibility to infections, which have important clinical implications in disease progression.

Proteomics of extracellular vesicles: Exosomes and ectosomes.

Almost all bacteria, archaea, and eukaryotic cells shed extracellular vesicles either constitutively or in a regulated manner. These nanosized membrane vesicles are spherical, bilayered proteolipids that harbor specific subsets of proteins, DNAs, RNAs, and lipids. Recent research has facilitated conceptual advancements in this emerging field that indicate that extracellular vesicles act as intercellular communicasomes by transferring signals to their target cell via surface ligands and delivering receptors and functional molecules. Recent progress in mass spectrometry-based proteomic analyses of mammalian extracellular vesicles derived from diverse cell types and body fluids has resulted in the identification of several thousand vesicular proteins that provide us with essential clues to the molecular mechanisms involved in vesicle cargo sorting and biogenesis. Furthermore, cell-type- or disease-specific vesicular proteins help us to understand the pathophysiological functions of extracellular vesicles and contribute to the discovery of diagnostic and therapeutic target proteins. This review focuses on the high-throughput mass spectrometry-based proteomic analyses of mammalian extracellular vesicles (i.e., exosomes and ectosomes), EVpedia (a free web-based integrated database of high-throughput data for systematic analyses of extracellular vesicles; http://evpedia.info), and the intravesicular protein-protein interaction network analyses of mammalian extracellular vesicles. The goal of this article is to encourage further studies to construct a comprehensive proteome database for extracellular vesicles that will help us to not only decode the biogenesis and cargo-sorting mechanisms during vesicle formation but also elucidate the pathophysiological roles of these complex extracellular organelles.

Bacterial protoplast-derived nanovesicles as vaccine delivery system against bacterial infection.

The notion that widespread infectious diseases could be best managed by developing potent, adjuvant-free vaccines has resulted in the use of various biological immune-stimulating components as new vaccine candidates. Recently, extracellular vesicles, also known as exosomes and microvesicles in mammalian cells and outer membrane vesicles in Gram-negative bacteria, have gained attention for the next generation vaccine. However, the more invasive and effective the vaccine is in delivery, the more risk it holds for severe immune toxicity. Here, in optimizing the current vaccine delivery system, we designed bacterial protoplast-derived nanovesicles (PDNVs), depleted of toxic outer membrane components to generate a universal adjuvant-free vaccine delivery system. These PDNVs exhibited significantly higher productivity and safety than the currently used vaccine delivery vehicles and induced strong antigen-specific humoral and cellular immune responses. Moreover, immunization with PDNVs loaded with bacterial antigens conferred effective protection against bacterial sepsis in mice. These nonliving nanovesicles derived from bacterial protoplast open up a new avenue for the creation of next generation, adjuvant-free, less toxic vaccines to be used to prevent infectious diseases.

Proteomic analysis of extracellular vesicles derived from Mycobacterium tuberculosis.

The release of extracellular vesicles, also known as outer membrane vesicles, membrane vesicles, exosomes, and microvesicles, is an evolutionarily conserved phenomenon from bacteria to eukaryotes. It has been reported that Mycobacterium tuberculosis releases extracellular vesicles harboring immunologically active molecules, and these extracellular vesicles have been suggested to be applicable in vaccine development and biomarker discovery. However, the comprehensive proteomic analysis has not been performed for M. tuberculosis extracellular vesicles. In this study, we identified a total of 287 vesicular proteins by four LC-MS/MS analyses with high confidence. In addition, we identified several vesicular proteins associated with the virulence of M. tuberculosis. This comprehensive proteome profile will help elucidate the pathogenic mechanism of M. tuberculosis. The data have been deposited to the ProteomeXchange with identifier PXD001160 (http://proteomecentral.proteomexchange.org/dataset/PXD001160).

In vivo kinetic biodistribution of nano-sized outer membrane vesicles derived from bacteria.

Evaluation of kinetic distribution and behaviors of nanoparticles in vivo provides crucial clues into their roles in living organisms. Extracellular vesicles are evolutionary conserved nanoparticles, known to play important biological functions in intercellular, inter-species, and inter-kingdom communication. In this study, the first kinetic analysis of the biodistribution of outer membrane vesicles (OMVs)-bacterial extracellular vesicles-with immune-modulatory functions is performed. OMVs, injected intraperitoneally, spread to the whole mouse body and accumulate in the liver, lung, spleen, and kidney within 3 h of administration. As an early systemic inflammation response, increased levels of TNF-α and IL-6 are observed in serum and bronchoalveolar lavage fluid. In addition, the number of leukocytes and platelets in the blood is decreased. OMVs and cytokine concentrations, as well as body temperature are gradually decreased 6 h after OMV injection, in concomitance with the formation of eye exudates, and of an increase in ICAM-1 levels in the lung. Following OMV elimination, most of the inflammatory signs are reverted, 12 h post-injection. However, leukocytes in bronchoalveolar lavage fluid are increased as a late reaction. Taken together, these results suggest that OMVs are effective mediators of long distance communication in vivo.

Comparison of confocal microscopy and two-photon microscopy in mouse cornea in vivo.

High-resolution imaging of the cornea is important for studying corneal diseases at cellular levels. Confocal microscopy (CM) has been widely used in the clinic, and two-photon microscopy (TPM) has recently been introduced in various pre-clinical studies. We compared the performance of CM and TPM in normal mouse corneas and neovascularized mouse corneas induced by suturing. Balb/C mice and C57BL/6 mice expressing green fluorescent protein (GFP) were used to compare modalities based on intrinsic contrast and extrinsic fluorescence contrast. CM based on reflection (CMR), CM based on fluorescence (CMF), and TPM based on intrinsic/extrinsic fluorescence and second harmonic generation (SHG) were compared by imaging the same sections of mouse corneas sequentially in vivo. In normal mouse corneas, CMR visualized corneal cell morphologies with some background noise, and CMF visualized GFP expressing corneal cells clearly. TPM visualized corneal cells and collagen in the stroma based on fluorescence and SHG, respectively. However, in neovascularized mouse corneas, CMR could not resolve cells deep inside the cornea due to high background noise from the effects of increased structural irregularity induced by suturing. CMF and TPM visualized cells and induced vasculature better than CMR because both collect signals from fluorescent cells only. Both CMF and TPM had signal decays with depth due to the structural irregularity, with CMF having faster signal decay than TPM. CMR, CMF, and TPM showed different degrees of image degradation in neovascularized mouse corneas.

Vesiclepedia: a compendium for extracellular vesicles with continuous community annotation.

Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.

Small RNA deep sequencing discriminates subsets of extracellular vesicles released by melanoma cells--Evidence of unique microRNA cargos.

Melanoma cells release different types of extracellular vesicles (EVs) into the extracellular milieu that are involved with communication and signaling in the tumor microenvironment. Subsets of EVs include exosomes, microvesicles, and apoptotic bodies that carry protein and genetic (RNA) cargos. To define the contribution of the RNA cargo of melanoma cell derived EVs we performed small RNA sequencing to identify different small RNAs in the EV subsets. Using validated centrifugation protocols, we separated these EV subsets released by the melanoma cell line MML-1, and performed RNA sequencing with the Ion Torrent platform. Various, but different, non-coding RNAs were detected in the EV subsets, including microRNA, mitochondrial associated tRNA, small nucleolar RNA, small nuclear RNA, Ro associated Y-RNA, vault RNA and Y-RNA. We identified in total 1041 miRNAs in cells and EV subsets. Hierarchical clustering showed enrichment of specific miRNAs in exosomes, including hsa-miR-214-3p, hsa-miR-199a-3p and hsa-miR-155-5p, all being associated with melanoma progression. Comparison of exosomal miRNAs with miRNAs in clinical melanoma samples indicate that multiple miRNAs in exosomes also are expressed specifically in melanoma tissues, but not in benign naevi. This study shows for the first time the presence of distinct small RNAs in subsets of EVs released by melanoma cells, with significant similarities to clinical melanoma tissue, and provides unique insights into the contribution of EV associated extracellular RNA in cancer.

Immunization with Escherichia coli outer membrane vesicles protects bacteria-induced lethality via Th1 and Th17 cell responses.

Outer membrane vesicles (OMVs), secreted from Gram-negative bacteria, are spherical nanometer-sized proteolipids enriched with outer membrane proteins. OMVs, also known as extracellular vesicles, have gained interests for use as nonliving complex vaccines and have been examined for immune-stimulating effects. However, the detailed mechanism on how OMVs elicit the vaccination effect has not been studied extensively. In this study, we investigated the immunological mechanism governing the protective immune response of OMV vaccines. Immunization with Escherichia coli-derived OMVs prevented bacteria-induced lethality and OMV-induced systemic inflammatory response syndrome. As verified by adoptive transfer and gene-knockout studies, the protective effect of OMV immunization was found to be primarily by the stimulation of T cell immunity rather than B cell immunity, especially by the OMV-Ag-specific production of IFN-γ and IL-17 from T cells. By testing the bacteria-killing ability of macrophages, we also demonstrated that IFN-γ and IL-17 production is the main factor promoting bacterial clearances. Our findings reveal that E. coli-derived OMV immunization effectively protects bacteria-induced lethality and OMV-induced systemic inflammatory response syndrome primarily via Th1 and Th17 cell responses. This study therefore provides a new perspective on the immunological detail regarding OMV vaccination.

Comparative interactomes of SIRT6 and SIRT7: Implication of functional links to aging.

Sirtuins are NAD(+) -dependent deacetylases that regulate a range of cellular processes. Although diverse functions of sirtuins have been proposed, those functions of SIRT6 and SIRT7 that are mediated by their interacting proteins remain elusive. In the present study, we identified SIRT6- and SIRT7-interacting proteins, and compared their interactomes to investigate functional links. Our interactomes revealed 136 interacting proteins for SIRT6 and 233 for SIRT7 while confirming seven and 111 proteins identified previously for SIRT6 and SIRT7, respectively. Comparison of SIRT6 and SIRT7 interactomes under the same experimental conditions disclosed 111 shared proteins, implying related functional links. The interaction networks of interactomes indicated biological processes associated with DNA repair, chromatin assembly, and aging. Interactions of two highly acetylated proteins, nucleophosmin (NPM1) and nucleolin, with SIRT6 and SIRT7 were confirmed by co-immunoprecipitation. NPM1 was found to be deacetylated by both SIRT6 and SIRT7. In senescent cells, the acetylation level of NPM1 was increased in conjunction with decreased levels of SIRT6 and SIRT7, suggesting that the acetylation of NPM1 could be regulated by SIRT6 and SIRT7 in the aging process. Our comparative interactomic study of SIRT6 and SIRT7 implies important functional links to aging by their associations with interacting proteins. All MS data have been deposited in the ProteomeXchange with identifiers PXD000159 and PXD000850 (http://proteomecentral.proteomexchange.org/dataset/PXD000159, http://proteomecentral.proteomexchange.org/dataset/PXD000850).