Publications by authors named "David W Greening"

86 Publications

Proteomic dissection of large extracellular vesicle surfaceome unravels interactive surface platform.

J Extracell Vesicles 2021 11;10(13):e12164

Molecular Proteomics, Baker Heart and Diabetes Institute, Melbourne, Victoria, 3004, Australia.

The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra- and extracellular signalling networks, dictates EVs' capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (L-EVs, 100-800 nm, mean 310 nm, ATP5F1A, ATP5F1B, DHX9, GOT2, HSPA5, HSPD1, MDH2, STOML2), a major EV-subtype that are distinct from small EVs (S-EVs, 30-150 nm, mean 110 nm, CD44, CD63, CD81, CD82, CD9, PDCD6IP, SDCBP, TSG101) remains limited. Using a membrane impermeant derivative of biotin to capture surface proteins coupled to mass spectrometry analysis, we show that out of 4143 proteins identified in density-gradient purified L-EVs (1.07-1.11 g/mL, from multiple cancer cell lines), 961 proteins are surface accessible. The surface molecular diversity of L-EVs include (i) bona fide plasma membrane anchored proteins (cluster of differentiation, transporters, receptors and GPI anchored proteins implicated in cell-cell and cell-ECM interactions); and (ii) membrane surface-associated proteins (that are released by divalent ion chelator EDTA) implicated in actin cytoskeleton regulation, junction organization, glycolysis and platelet activation. Ligand-receptor analysis of L-EV surfaceome (e.g., ITGAV/ITGB1) uncovered interactome spanning 172 experimentally verified cognate binding partners (e.g., ANGPTL3, PLG, and VTN) with highest tissue enrichment for liver. Assessment of biotin inaccessible L-EV proteome revealed enrichment for proteins belonging to COPI/II-coated ER/Golgi-derived vesicles and mitochondria. Additionally, despite common surface proteins identified in L-EVs and S-EVs, our data reveals surfaceome heterogeneity between the two EV-subtype. Collectively, our study provides critical insights into diverse proteins operating at the interactive platform of L-EVs and molecular leads for future studies seeking to decipher L-EV heterogeneity and function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jev2.12164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8612312PMC
November 2021

Development of Extracellular Vesicle Therapeutics: Challenges, Considerations, and Opportunities.

Front Cell Dev Biol 2021 20;9:734720. Epub 2021 Sep 20.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science (LIMS), La Trobe University, Melbourne, VIC, Australia.

Extracellular vesicles (EVs) hold great promise as therapeutic modalities due to their endogenous characteristics, however, further bioengineering refinement is required to address clinical and commercial limitations. Clinical applications of EV-based therapeutics are being trialed in immunomodulation, tissue regeneration and recovery, and as delivery vectors for combination therapies. Native/biological EVs possess diverse endogenous properties that offer stability and facilitate crossing of biological barriers for delivery of molecular cargo to cells, acting as a form of intercellular communication to regulate function and phenotype. Moreover, EVs are important components of paracrine signaling in stem/progenitor cell-based therapies, are employed as standalone therapies, and can be used as a drug delivery system. Despite remarkable utility of native/biological EVs, they can be improved using bio/engineering approaches to further therapeutic potential. EVs can be engineered to harbor specific pharmaceutical content, enhance their stability, and modify surface epitopes for improved tropism and targeting to cells and tissues . Limitations currently challenging the full realization of their therapeutic utility include scalability and standardization of generation, molecular characterization for design and regulation, therapeutic potency assessment, and targeted delivery. The fields' utilization of advanced technologies (imaging, quantitative analyses, multi-omics, labeling/live-cell reporters), and utility of biocompatible natural sources for producing EVs (plants, bacteria, milk) will play an important role in overcoming these limitations. Advancements in EV engineering methodologies and design will facilitate the development of EV-based therapeutics, revolutionizing the current pharmaceutical landscape.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcell.2021.734720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8488228PMC
September 2021

The proteomes of endometrial stromal cell-derived extracellular vesicles following a decidualizing stimulus define the cells' potential for decidualization success.

Mol Hum Reprod 2021 Sep;27(10)

Centre for Reproductive Health, Hudson Institute of Medical Research, Clayton, Victoria, Australia.

Adequate endometrial stromal cell (ESC) decidualization is vital for endometrial health. Given the importance of extracellular vesicles (EVs) in intercellular communication, we investigated how their protein landscape is reprogrammed and dysregulated during decidual response. Small EVs (sEVs) from human ESC-conditioned media at Day-2 and -14 following decidual stimuli were grouped as well- (WD) or poorly decidualized (PD) based on their prolactin secretion and subjected to mass spectrometry-based quantitative proteomics. On Day 2, in PD- versus WD-ESC-sEVs, 17 sEV- proteins were down-regulated (C5, C6; complement/coagulation cascades, and SERPING1, HRG; platelet degranulation and fibrinolysis) and 39 up-regulated (FLNA, COL1A1; focal adhesion, ENO1, PKM; glycolysis/gluconeogenesis, and RAP1B, MSN; leukocyte transendothelial migration). On Day 14, in PD- versus WD-ESC-sEVs, FLNA was down-regulated while 21 proteins were up-regulated involved in complement/coagulation cascades (C3, C6), platelet degranulation (SERPINA4, ITIH4), B-cell receptor signalling and innate immune response (immunoglobulins). Changes from Days 2 to 14 suggested a subsequent response in PD-ESC-sEVs with 89 differentially expressed proteins mostly involved in complement and coagulation cascades (C3, C6, C5), but no change in WD-ESC-sEVs ESC. Poor decidualization was also associated with loss of crucial sEV-proteins for cell adhesion and invasion (ITGA5, PFN1), glycolysis (ALDOA, PGK1) and cytoskeletal reorganization (VCL, RAC1). Overall, this study indicates varied ESC response even prior to decidualization and provides insight into sEVs-proteomes as a benchmark of well-decidualized ESC. It shows distinct variation in sEV-protein composition depending on the ESC decidual response that is critical for embryo implantation, enabling and limiting trophoblast invasion during placentation and sensing a healthy embryo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/molehr/gaab057DOI Listing
September 2021

Understanding extracellular vesicles.

Proteomics 2021 07 17;21(13-14):e2100126. Epub 2021 Jun 17.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.202100126DOI Listing
July 2021

Impact of chemically defined culture media formulations on extracellular vesicle production by amniotic epithelial cells.

Proteomics 2021 07 22;21(13-14):e2000080. Epub 2021 Jun 22.

The Ritchie Centre, Hudson Institute of Medical Research, Clayton, Victoria, Australia.

The therapeutic properties of cell derived extracellular vesicles (EVs) make them promising cell-free alternative to regenerative medicine. However, clinical translation of this technology relies on the ability to manufacture EVs in a scalable, reproducible, and cGMP-compliant manner. To generate EVs in sufficient quantity, a critical step is the selection and development of culture media, where differences in formulation may influence the EV manufacturing process. In this study, we used human amniotic epithelial cells (hAECs) as a model system to explore the effect of different formulations of chemically defined, commercially sourced media on EV production. Here, we determined that cell viability and proliferation rate are not reliable quality indicators for EV manufacturing. The levels of tetraspanins and epitope makers of EVs were significantly impacted by culture media formulations. Mass spectrometry-based proteomic profiling revealed proteome composition of hAEC-EVs and the influence of media formulations on composition of EV proteome. This study has revealed critical aspects including cell viability and proliferation rate, EV yield, and tetraspanins, surface epitopes and proteome composition of EVs influenced by media formulations, and further insight into standardised EV production culture media that should be considered in clinical-grade scalable EV manufacture for generation of therapeutic EVs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.202000080DOI Listing
July 2021

Cancer stem cell marker DCLK1 reprograms small extracellular vesicles toward migratory phenotype in gastric cancer cells.

Proteomics 2021 07 28;21(13-14):e2000098. Epub 2021 May 28.

Cancer Inflammation Laboratory, Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia.

Doublecortin-like kinase 1 (DCLK1) is a putative cancer stem cell marker, a promising diagnostic and prognostic maker for malignant tumors and a proposed driver gene for gastric cancer (GC). DCLK1 overexpression in a majority of solid cancers correlates with lymph node metastases, advanced disease and overall poor-prognosis. In cancer cells, DCLK1 expression has been shown to promote epithelial-to-mesenchymal transition (EMT), driving disruption of cell-cell adhesion, cell migration and invasion. Here, we report that DCLK1 influences small extracellular vesicle (sEV/exosome) biogenesis in a kinase-dependent manner. sEVs isolated from DCLK1 overexpressing human GC cell line MKN1 (MKN1 -sEVs), promote the migration of parental (non-transfected) MKN1 cells (MKN1 ). Quantitative proteome analysis of MKN1 -sEVs revealed enrichment in migratory and adhesion regulators (STRAP, CORO1B, BCAM, COL3A, CCN1) in comparison to MKN1 -sEVs. Moreover, using DCLK1-IN-1, a specific small molecule inhibitor of DCLK1, we reversed the increase in sEV size and concentration in contrast to other EV subtypes, as well as kinase-dependent cargo selection of proteins involved in EV biogenesis (KTN1, CHMP1A, MYO1G) and migration and adhesion processes (STRAP, CCN1). Our findings highlight a specific role of DCLK1-kinase dependent cargo selection for sEVs and shed new light on its role as a regulator of signaling in gastric tumorigenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.202000098DOI Listing
July 2021

Proteome characterisation of extracellular vesicles isolated from heart.

Proteomics 2021 07 6;21(13-14):e2100026. Epub 2021 May 6.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science (LIMS), La Trobe University, Melbourne, Victoria, Australia.

Cardiac intercellular communication is critical for heart function and often dysregulated in cardiovascular diseases. While cardiac extracellular vesicles (cEVs) are emerging mediators of signalling, their isolation remains a technical challenge hindering our understanding of cEV protein composition. Here, we utilised Langendorff-collagenase-based enzymatic perfusion and differential centrifugation to isolate cEVs from mouse heart (yield 3-6 μg/heart). cEVs are ∼200 nm, express classical EV markers (Cd63/81/9 , Tsg101 , Pdcd6ip/Alix ), and are depleted of blood (Alb/Fga/Hba) and cardiac damage markers (Mb, Tnnt2, Ldhb). Comparison with mechanically-derived EVs revealed greater detection of EV markers and decreased cardiac damage contaminants. Mass spectrometry-based proteomic profiling revealed 1721 proteins in cEVs, implicated in proteasomal and autophagic proteostasis, glycolysis, and fatty acid metabolism; essential functions often disrupted in cardiac pathologies. There was striking enrichment of 942 proteins in cEVs compared to mouse heart tissue - implicated in EV biogenesis, antioxidant activity, and lipid transport, suggesting active cargo selection and specialised function. Interestingly, cEVs contain marker proteins for cardiomyocytes, cardiac progenitors, B-cells, T-cells, macrophages, smooth muscle cells, endothelial cells, and cardiac fibroblasts, suggesting diverse cellular origin. We present a method of cEV isolation and provide insight into potential functions, enabling future studies into EV roles in cardiac physiology and disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.202100026DOI Listing
July 2021

Proteome reprogramming of endometrial epithelial cells by human trophectodermal small extracellular vesicles reveals key insights into embryo implantation.

Proteomics 2021 07 6;21(13-14):e2000210. Epub 2021 May 6.

Baker Heart and Diabetes Institute, Molecular Proteomics, Melbourne, Victoria, Australia.

Embryo implantation into the receptive endometrium is critical in pregnancy establishment, initially requiring reciprocal signalling between outer layer of the blastocyst (trophectoderm cells) and endometrial epithelium; however, factors regulating this crosstalk remain poorly understood. Although endometrial extracellular vesicles (EVs) are known to signal to the embryo during implantation, the role of embryo-derived EVs remains largely unknown. Here, we provide a comprehensive proteomic characterisation of a major class of EVs, termed small EVs (sEVs), released by human trophectoderm cells (Tsc-sEVs) and their capacity to reprogram protein landscape of endometrial epithelium in vitro. Highly purified Tsc-sEVs (30-200 nm, ALIX , TSG101 , CD9/63/81 ) were enriched in known players of implantation (LIFR, ICAM1, TAGLN2, WNT5A, FZD7, ROR2, PRICKLE2), antioxidant activity (SOD1, PRDX1/4/6), tissue integrity (EZR, RAC1, RHOA, TNC), and focal adhesions (FAK, ITGA2/V, ITGB1/3). Functionally, Tsc-sEVs were taken up by endometrial cells, altered transepithelial electrical resistance, and upregulated proteins implicated in embryo attachment (ITGA2/V, ITGB1/3), immune regulation (CD59, CD276, LGALS3), and antioxidant activity (GPX1/3/4, PRDX1/2/4/5/6): processes that are critical for successful implantation. Collectively, we provide critical insights into Tsc-sEV-mediated regulation of endometrial function that contributes to our understanding of the molecular basis of implantation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.202000210DOI Listing
July 2021

Secreted midbody remnants are a class of extracellular vesicles molecularly distinct from exosomes and microparticles.

Commun Biol 2021 03 25;4(1):400. Epub 2021 Mar 25.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science (LIMS), La Trobe University, Melbourne, VIC, 3086, Australia.

During the final stages of cell division, newly-formed daughter cells remain connected by a thin intercellular bridge containing the midbody (MB), a microtubule-rich organelle responsible for cytokinetic abscission. Following cell division the MB is asymmetrically inherited by one daughter cell where it persists as a midbody remnant (MB-R). Accumulating evidence shows MB-Rs are secreted (sMB-Rs) into the extracellular medium and engulfed by neighbouring non-sister cells. While much is known about intracellular MB-Rs, sMB-Rs are poorly understood. Here, we report the large-scale purification and biochemical characterisation of sMB-Rs released from colon cancer cells, including profiling of their proteome using mass spectrometry. We show sMB-Rs are an abundant class of membrane-encapsulated extracellular vesicle (200-600 nm) enriched in core cytokinetic proteins and molecularly distinct from exosomes and microparticles. Functional dissection of sMB-Rs demonstrated that they are engulfed by, and accumulate in, quiescent fibroblasts where they promote cellular transformation and an invasive phenotype.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42003-021-01882-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7994562PMC
March 2021

Correction: Recent advances in bioanalytical methods to measure proteome stability in cells.

Analyst 2021 Apr 11;146(7):2400. Epub 2021 Mar 11.

Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia.

Correction for 'Recent advances in bioanalytical methods to measure proteome stability in cells' by Shouxiang Zhang et al., Analyst, 2021, DOI: .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d1an90022fDOI Listing
April 2021

Transglutaminase-2, RNA-binding proteins and mitochondrial proteins selectively traffic to MDCK cell-derived microvesicles following H-Ras-induced epithelial-mesenchymal transition.

Proteomics 2021 07 19;21(13-14):e2000221. Epub 2021 Mar 19.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.

Epithelial-mesenchymal transition (EMT) describes an evolutionary conserved morphogenic process defined by loss of epithelial characteristics and acquisition of mesenchymal phenotype, and altered patterns of intercellular communication, leading to functional changes in cell migration and invasion. In this regard, we have previously reported that oncogenic H-Ras induced EMT in Madin-Darby Canine Kidney (MDCK) cells (21D1 cells) trigger changes in the protein distribution pattern in cells, exosomes, and soluble protein factors (secretome) which modulate the tumor microenvironment. Here, we report that shed microvesicles (also termed microparticles/ectosomes) secreted from MDCK cells following oncogenic H-Ras-induced EMT (21D1-sMVs) are biochemically distinct from exosomes and parental MDCK-sMVs. The protein spectra of RNA-binding proteins and mitochondrial proteins in 21D1-sMVs differ profoundly compared to those of exosomes, likewise proteins associated with suppression of anoikis. We show that 21D1-sMVs promote cell migration, confer anchorage-independent growth, and induce EMT in parental MDCK cells. An unexpected and novel finding was the selective sorting of tissue transglutaminase-2 (TGM2) into 21D1-sMVs; there was no evidence of TGM2 in MDCK-sMVs. Prior treatment of 21D1-sMVs with neutralizing anti-TGM2 or anti-FN1 antibodies attenuates the invasive capability of fibroblasts. These finding suggest that microvesicle-associated TGM2 may play an important contributory role in the EMT process and warrants further investigation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.202000221DOI Listing
July 2021

Recent advances in bioanalytical methods to measure proteome stability in cells.

Analyst 2021 Apr 26;146(7):2097-2109. Epub 2021 Feb 26.

Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia.

Proteome stability constitutes an essential aspect of protein homeostasis (proteostasis). Proteostasis networks maintain proteins and their interactors in a defined conformation for their activity, localisation, and function. However, endogenous or exogenous stressors can perturb proteostasis integrity and deplete folding capacity, generating destabilized folding intermediates and deleterious aggregated species. Over the years, protein unfolding, misfolding and aggregation have been reported to be associated with aging and many diseases such as neurodegenerative diseases, diabetes, cardiac disease and toxicity, and cancers. Therefore, monitoring proteome stability is central to understanding underlying biological processes and mechanisms of disease progression. Herein, we review the recent bioanalytical methods to measure protein stability in cells on a proteome-wide scale.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d0an01547dDOI Listing
April 2021

Proteomic profiling of human uterine extracellular vesicles reveal dynamic regulation of key players of embryo implantation and fertility during menstrual cycle.

Proteomics 2021 07 19;21(13-14):e2000211. Epub 2021 Mar 19.

Baker Heart and Diabetes Institute, Molecular Proteomics, Melbourne, Victoria, Australia.

Endometrial extracellular vesicles (EVs) are emerging as important players in reproductive biology. However, how their proteome is regulated throughout the menstrual cycle is not known. Such information can provide novel insights into biological processes critical for embryo development, implantation, and successful pregnancy. Using mass spectrometry-based quantitative proteomics, we show that small EVs (sEVs) isolated from uterine lavage of fertile women (UL-sEV), compared to infertile women, are laden with proteins implicated in antioxidant activity (SOD1, GSTO1, MPO, CAT). Functionally, sEVs derived from endometrial cells enhance antioxidant function in trophectoderm cells. Moreover, there was striking enrichment of invasion-related proteins (LGALS1/3, S100A4/11) in fertile UL-sEVs in the secretory (estrogen plus progesterone-driven, EP) versus proliferative (estrogen-driven, E) phase, with several players downregulated in infertile UL-sEVs. Consistent with this, sEVs from EP- versus E-primed endometrial epithelial cells promote invasion of trophectoderm cells. Interestingly, UL-sEVs from fertile versus infertile women carry known players/predictors of embryo implantation (PRDX2, IDHC), endometrial receptivity (S100A4, FGB, SERPING1, CLU, ANXA2), and implantation success (CAT, YWHAE, PPIA), highlighting their potential to inform regarding endometrial status/pregnancy outcomes. Thus, this study provides novel insights into proteome reprograming of sEVs and soluble secretome in uterine fluid, with potential to enhance embryo implantation and hence fertility.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.202000211DOI Listing
July 2021

Human myeloma cell- and plasma-derived extracellular vesicles contribute to functional regulation of stromal cells.

Proteomics 2021 07 5;21(13-14):e2000119. Epub 2021 Mar 5.

Myeloma Research Group, Australian Centre for Blood Diseases, Monash University/Alfred Health, Melbourne, Victoria, Australia.

Circulating small extracellular vesicles (sEV) represent promising non-invasive biomarkers that may aid in the diagnosis and risk-stratification of multiple myeloma (MM), an incurable blood cancer. Here, we comprehensively isolated and characterized sEV from human MM cell lines (HMCL) and patient-derived plasma (psEV) by specific EV-marker enrichment and morphology. Importantly, we demonstrate that HMCL-sEV are readily internalised by stromal cells to functionally modulate proliferation. psEV were isolated using various commercial approaches and pre-analytical conditions (collection tube types, storage conditions) assessed for sEV yield and marker enrichment. Functionally, MM-psEV was shown to regulate stromal cell proliferation and migration. In turn, pre-educated stromal cells favour HMCL adhesion. psEV isolated from patients with both pre-malignant plasma cell disorders (monoclonal gammopathy of undetermined significance [MGUS]; smouldering MM [SMM]) and MM have a similar ability to promote cell migration and adhesion, suggesting a role for both malignant and pre-malignant sEV in disease progression. Proteomic profiling of MM-psEV (305 proteins) revealed enrichment of oncogenic factors implicated in cell migration and adhesion, in comparison to non-disease psEV. This study describes a protocol to generate morphologically-intact and biologically functional sEV capable of mediating the regulation of stromal cells, and a model for the characterization of tumour-stromal cross-talk by sEV in MM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.202000119DOI Listing
July 2021

Human Plasma Extracellular Vesicle Isolation and Proteomic Characterization for the Optimization of Liquid Biopsy in Multiple Myeloma.

Methods Mol Biol 2021 ;2261:151-191

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia.

Cancer cells secrete membranous extracellular vesicles (EVs) which contain specific oncogenic molecular cargo (including oncoproteins, oncopeptides, and RNA) into their microenvironment and the circulation. As such, EVs including exosomes (small EVs) and microvesicles (large EVs) represent important circulating biomarkers for various diseases, including cancer and its progression. These circulating biomarkers offer a potentially minimally invasive and repeatable targets for analysis (liquid biopsy) that could aid in the diagnosis, risk stratification, and monitoring of cancer. Although their potential as cancer biomarkers has been promising, the identification and quantification of EVs in clinical samples remain challenging. Like EVs, other types of circulating biomarkers (including cell-free nucleic acids, cf-NAs; or circulating tumor cells, CTCs) may represent a complementary or alternative approach to cancer diagnosis. In the context of multiple myeloma (MM), a systemic cancer type that causes cancer cells to accumulate in the bone marrow, the specific role for EVs as biomarkers for diagnosis and monitoring remains undefined. Tumor heterogeneity along with the various subtypes of MM (such as non-secretory MM) that cannot be monitored using conventional testing (e.g. sequential serological testing and bone marrow biopsies) render liquid biopsy and circulating tumor-derived EVs a promising approach. In this protocol, we describe the isolation and purification of EVs from peripheral blood plasma (PBPL) collected from healthy donors and patients with MM for a biomarker discovery strategy. Our results demonstrate detection of circulating EVs from as little as 1 mL of MM patients' PBPL. High-resolution mass spectrometry (MS)-based proteomics promises to provide new avenues in identifying novel markers for detection, monitoring, and therapeutic intervention of disease. We describe biophysical characterization and quantitative proteomic profiling of disease-specific circulating EVs which may provide important implications for the development of cancer diagnostics in MM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-0716-1186-9_10DOI Listing
March 2021

A Protocol for Isolation, Purification, Characterization, and Functional Dissection of Exosomes.

Methods Mol Biol 2021 ;2261:105-149

Baker Heart and Diabetes Institute, Melbourne, VIC, Australia.

Extracellular vesicles (EVs) are membrane-enclosed vesicles released by cells. They carry proteins, nucleic acids, and metabolites which can be transferred to a recipient cell, locally or at a distance, to elicit a functional response. Since their discovery over 30 years ago, the functional repertoire of EVs in both physiological (e.g., organ morphogenesis, embryo implantation) and pathological (e.g., cancer, neurodegeneration) conditions has cemented their crucial role in intercellular communication. Moreover, because the cargo encapsulated within circulating EVs remains protected from degradation, their diagnostic as well as therapeutic (such as drug delivery tool) applications have garnered vested interest. Global efforts have been made to purify EV subtypes from biological fluids and in vitro cell culture media using a variety of strategies and techniques, with a major focus on EVs of endocytic origin called exosomes (30-150 nm in size). Given that the secretome comprises of soluble secreted proteins, protein aggregates, RNA granules, and EV subtypes (such as exosomes, shed microvesicles, apoptotic bodies), it is imperative to purify exosomes to homogeneity if we are to perform biochemical and biophysical characterization and, importantly, functional dissection. Besides understanding the composition of EV subtypes, defining molecular bias of how they reprogram target cells also remains of paramount importance in this area of active research. Here, we outline a systematic "how to" protocol (along with useful insights/tips) to obtain highly purified exosomes and perform their biophysical and biochemical characterization. This protocol employs a mass spectrometry-based proteomics approach to characterize the protein composition of exosomes. We also provide insights on different isolation strategies and their usefulness in various downstream applications. We outline protocols for lipophilic labeling of exosomes to study uptake by a recipient cell, investigating cellular reprogramming using proteomics and studying functional response to exosomes in the Transwell-Matrigel™ Invasion assay.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-0716-1186-9_9DOI Listing
March 2021

Analysis of Annotated and Unannotated Long Noncoding RNAs from Exosome Subtypes Using Next-Generation RNA Sequencing.

Methods Mol Biol 2021 ;2254:195-218

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science (LIMS), La Trobe University, Melbourne, VIC, Australia.

Long noncoding RNAs (lncRNAs) contain >200 nucleotides and act as regulatory molecules in transcription and translation processes in both normal and pathological conditions. LncRNAs have been reported to localize in nuclei, cytoplasm, and, more recently, extracellular vesicles such as exosomes. Exosomal lncRNAs have gained much attention as exosomes secreted from one cell type can transfer their cargo (e.g., protein, RNA species, and lipids) to recipient cells and mediate phenotypic changes in the recipient cell. In recent years, many exosomal lncRNAs have been discovered and annotated and are attracting much attention as potential markers for disease diagnosis and prognosis. It is expected that many exosomal lncRNAs are yet to be identified. However, characterization of unannotated exosomal RNAs with non-protein-coding sequences from massive RNA sequencing data is technically challenging. Here, we describe a method for the discovery of annotated and unannotated exosomal lncRNA. This method includes a large-scale isolation and purification strategy for exosome subtypes, using the human colorectal cancer cell line (LIM1863) as a model. The method inputs RNA sequencing clean reads and performs transcript assembly to identify annotated and unannotated exosomal lncRNAs. Cutoffs (length, number of exon, classification code, and human protein-coding probability) are used to identify potentially novel exosomal lncRNAs. Raw read count calculation and differential expression analysis are also introduced for downstream analysis and candidate selection. Exosomal lncRNA candidates are validated using RT-qPCR. This method provides a template for exosomal lncRNA discovery and analysis from next-generation RNA sequencing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-0716-1158-6_12DOI Listing
March 2021

Exosomes Derived from the Human Primary Colorectal Cancer Cell Line SW480 Orchestrate Fibroblast-Led Cancer Invasion.

Proteomics 2020 07;20(14):e2000016

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, 3086, Australia.

In localized tumors, basement membrane (BM) prevents invasive outgrowth of tumor cells into surrounding tissues. When carcinomas become invasive, cancer cells either degrade BM or reprogram stromal fibroblasts to breach BM barrier and lead invasion of cancer cells into surrounding tissues in a process called fibroblast-led invasion. However, tumor-derived factors orchestrating fibroblast-led invasion remain poorly understood. Here it is shown that although early-stage primary colorectal adenocarcinoma (SW480) cells are themselves unable to invade Matrigel matrix, they secrete exosomes that reprogram normal fibroblasts to acquire de novo capacity to invade matrix and lead invasion of SW480 cells. Strikingly, cancer cells follow leading fibroblasts as collective epithelial-clusters, thereby circumventing need for epithelial to mesenchymal transition, a key event associated with invasion. Moreover, acquisition of pro-invasive phenotype by fibroblasts treated with SW480-derived exosomes relied on exosome-mediated MAPK pathway activation. Mass spectrometry-based protein profiling reveals that cancer exosomes upregulate fibroblasts proteins implicated in focal adhesion (ITGA2/A6/AV, ITGB1/B4/B5, EGFR, CRK), regulators of actin cytoskeleton (RAC1, ARF1, ARPC3, CYFIP1, NCKAP1, ICAM1, ERM complex), and signalling pathways (MAPK, Rap1, RAC1, Ras) important in pro-invasive remodeling of extracellular matrix. Blocking tumor exosome-mediated signaling to fibroblasts therefore represents an attractive therapeutic strategy in restraining tumors by perturbing stroma-driven invasive outgrowth.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.202000016DOI Listing
July 2020

Sustained subcutaneous delivery of secretome of human cardiac stem cells promotes cardiac repair following myocardial infarction.

Cardiovasc Res 2021 02;117(3):918-929

Departments of Medicine and Surgery, University of Melbourne, Melbourne, VIC, Australia.

Aims: To establish pre-clinical proof of concept that sustained subcutaneous delivery of the secretome of human cardiac stem cells (CSCs) can be achieved in vivo to produce significant cardioreparative outcomes in the setting of myocardial infarction.

Methods And Results: Rats were subjected to permanent ligation of left anterior descending coronary artery and randomized to receive subcutaneous implantation of TheraCyte devices containing either culture media as control or 1 × 106 human W8B2+ CSCs, immediately following myocardial ischaemia. At 4 weeks following myocardial infarction, rats treated with W8B2+ CSCs encapsulated within the TheraCyte device showed preserved left ventricular ejection fraction. The preservation of cardiac function was accompanied by reduced fibrotic scar tissue, interstitial fibrosis, cardiomyocyte hypertrophy, as well as increased myocardial vascular density. Histological analysis of the TheraCyte devices harvested at 4 weeks post-implantation demonstrated survival of human W8B2+ CSCs within the devices, and the outer membrane was highly vascularized by host blood vessels. Using CSCs expressing plasma membrane reporters, extracellular vesicles of W8B2+ CSCs were found to be transferred to the heart and other organs at 4 weeks post-implantation. Furthermore, mass spectrometry-based proteomic profiling of extracellular vesicles of W8B2+ CSCs identified proteins implicated in inflammation, immunoregulation, cell survival, angiogenesis, as well as tissue remodelling and fibrosis that could mediate the cardioreparative effects of secretome of human W8B2+ CSCs.

Conclusions: Subcutaneous implantation of TheraCyte devices encapsulating human W8B2+ CSCs attenuated adverse cardiac remodelling and preserved cardiac function following myocardial infarction. The TheraCyte device can be employed to deliver stem cells in a minimally invasive manner for effective secretome-based cardiac therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/cvr/cvaa088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898942PMC
February 2021

Fat Therapeutics: The Clinical Capacity of Adipose-Derived Stem Cells and Exosomes for Human Disease and Tissue Regeneration.

Front Pharmacol 2020 3;11:158. Epub 2020 Mar 3.

O'Brien Institute Department, St Vincent's Institute for Medical Research, Fitzroy, VIC, Australia.

Fat grafting is a well-established surgical technique used in plastic surgery to restore deficient tissue, and more recently, for its putative regenerative properties. Despite more frequent use of fat grafting, however, a scientific understanding of the mechanisms underlying either survival or remedial benefits of grafted fat remain lacking. Clinical use of fat grafts for breast reconstruction in tissues damaged by radiotherapy first provided clues regarding the clinical potential of stem cells to drive tissue regeneration. Healthy fat introduced into irradiated tissues appeared to reverse radiation injury (fibrosis, scarring, contracture and pain) clinically; a phenomenon since validated in several animal studies. In the quest to explain and enhance these therapeutic effects, adipose-derived stem cells (ADSCs) were suggested as playing a key role and techniques to enrich ADSCs in fat, in turn, followed. Stem cells - the body's rapid response 'road repair crew' - are on standby to combat tissue insults. ADSCs may exert influences either by releasing paracrine-signalling factors alone or as cell-free extracellular vesicles (EVs, exosomes). Alternatively, ADSCs may augment vital immune/inflammatory processes; or themselves differentiate into mature adipose cells to provide the 'building-blocks' for engineered tissue. Regardless, adipose tissue constitutes an ideal source for mesenchymal stem cells for therapeutic application, due to ease of harvest and processing; and a relative abundance of adipose tissue in most patients. Here, we review the clinical applications of fat grafting, ADSC-enhanced fat graft, fat stem cell therapy; and the latest evolution of EVs and nanoparticles in healing, cancer and neurodegenerative and multiorgan disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fphar.2020.00158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062679PMC
March 2020

Proteomic Insights into Endometrial Receptivity and Embryo-Endometrial Epithelium Interaction for Implantation Reveal Critical Determinants of Fertility.

Proteomics 2020 01;20(1):e1900250

Baker Heart and Diabetes Institute, Molecular Proteomics, Melbourne, Victoria, 3004, Australia.

In vitro fertilization has overcome infertility issues for many couples. However, achieving implantation of a viable embryo into the maternal endometrium remains a limiting step in optimizing pregnancy success. The molecular mechanisms which characterize the transient state of endometrial receptivity, critical in enabling embryo-endometrial interactions, and proteins which underpin adhesion at the implantation interface, are limited in humans despite these temporally regulated processes fundamental to life. Hence, failure of implantation remains the "final frontier" in infertility. A human coculture model is utilized utilizing spheroids of a trophectoderm (trophoblast stem) cell line, derived from pre-implantation human embryos, and primary human endometrial epithelial cells, to functionally identify "fertile" versus "infertile" endometrial epithelium based on adhesion between these cell types. Quantitative proteomics identified proteins associated with human endometrial epithelial receptivity ("epithelial receptome") and trophectoderm adhesion ("adhesome"). As validation, key "epithelial receptome" proteins (MAGT-1/CDA/LGMN/KYNU/PC4) localized to the epithelium of receptive phase (mid-secretory) endometrium obtained from fertile, normally cycling women but is largely absent from non-receptive (proliferative) phase tissues. Factors involved in embryo-epithelium interaction in successive temporal stages of endometrial receptivity and implantation are demonstrated and potential targets for improving fertility are provided, enhancing potential to become pregnant either naturally or in a clinical setting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.201900250DOI Listing
January 2020

Chronic methamphetamine interacts with BDNF Val66Met to remodel psychosis pathways in the mesocorticolimbic proteome.

Mol Psychiatry 2021 Aug 10;26(8):4431-4447. Epub 2019 Dec 10.

School of Psychology and Public Health, La Trobe University, Melbourne, VIC, Australia.

Methamphetamine (Meth) abuse has reached epidemic proportions in many countries and can induce psychotic episodes mimicking the clinical profile of schizophrenia. Brain-derived neurotrophic factor (BDNF) is implicated in both Meth effects and schizophrenia. We therefore studied the long-term effects of chronic Meth exposure in transgenic mice engineered to harbor the human BDNF polymorphism expressed via endogenous mouse promoters. These mice were chronically treated with an escalating Meth regime during late adolescence. At least 4 weeks later, all hBDNF Meth-treated mice exhibited sensitization confirming persistent behavioral effects of Meth. We used high-resolution quantitative mass spectrometry-based proteomics to biochemically map the long-term effects of Meth within the brain, resulting in the unbiased detection of 4808 proteins across the mesocorticolimbic circuitry. Meth differentially altered dopamine signaling markers (e.g., Dat, Comt, and Th) between hBDNF and hBDNF mice, implicating involvement of BDNF in Meth-induced reprogramming of the mesolimbic proteome. Targeted analysis of 336 schizophrenia-risk genes, as well as 82 growth factor cascade markers, similarly revealed that hBDNF genotype gated the recruitment of these factors by Meth in a region-specific manner. Cumulatively, these data represent the first comprehensive analysis of the long-term effects of chronic Meth exposure within the mesocorticolimbic circuitry. In addition, these data reveal that long-term Meth-induced brain changes are strongly dependent upon BDNF genetic variation, illustrating how drug-induced psychosis may be modulated at the molecular level by a single genetic locus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41380-019-0617-8DOI Listing
August 2021

Human Endometrial Extracellular Vesicles Functionally Prepare Human Trophectoderm Model for Implantation: Understanding Bidirectional Maternal-Embryo Communication.

Proteomics 2019 12 30;19(23):e1800423. Epub 2019 Oct 30.

Molecular Proteomics, Baker Heart and Diabetes Institute, Melbourne, VIC, 3004, Australia.

Embryo implantation into maternal endometrium is critical for initiation and establishment of pregnancy, requiring developmental synchrony between endometrium and blastocyst. However, factors regulating human endometrial-embryo cross talk and facilitate implantation remain largely unknown. Extracellular vesicles (EVs) are emerging as important mediators of this process. Here, a trophectoderm spheroid-based in vitro model mimicking the pre-implantation human embryo is used to recapitulate important functional aspects of blastocyst implantation. Functionally, human endometrial EVs, derived from hormonally treated cells synchronous with implantation, are readily internalized by trophectoderm cells, regulating adhesive and invasive capacity of human trophectoderm spheroids. To gain molecular insights into mechanisms underpinning endometrial EV-mediated enhancement of implantation, quantitative proteomics reveal critical alterations in trophectoderm cellular adhesion networks (cell adhesion molecule binding, cell-cell adhesion mediator activity, and cell adherens junctions) and metabolic and gene expression networks, and the soluble secretome from human trophectodermal spheroids. Importantly, transfer of endometrial EV cargo proteins to trophectoderm to mediate changes in trophectoderm function is demonstrated. This is highlighted by correlation among endometrial EVs, the trophectodermal proteome following EV uptake, and EV-mediated trophectodermal cellular proteome, important for implantation. This work provides an understanding into molecular mechanisms of endometrial EV-mediated regulation of human trophectoderm functions-fundamental in understanding human endometrium-embryo signaling during implantation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.201800423DOI Listing
December 2019

Part II: Special Issue on Extracellular Vesicles and Exosomes.

Proteomics 2019 04;19(8):e1900121

Head, Molecular Proteomics, Baker Heart and Diabetes Institute, La Trobe Institute for Molecular Science, La Trobe University, Australia.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.201900121DOI Listing
April 2019

Oncogenic and Non-Malignant Pancreatic Exosome Cargo Reveal Distinct Expression of Oncogenic and Prognostic Factors Involved in Tumor Invasion and Metastasis.

Proteomics 2019 04 9;19(8):e1800158. Epub 2019 Apr 9.

Metabolic Signaling, School of Pharmacy and Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, 6102, Perth, Western Australia, Australia.

Exosomes are small extracellular membrane vesicles important in intercellular communication, with their oncogenic cargo attributed to tumor progression and pre-metastatic niche formation. To gain an insight into key differences in oncogenic composition of exosomes, human non-malignant epithelial and pancreatic cancer cell models and purified and characterized resultant exosome populations are utilized. Proteomic analysis reveals the selective enrichment of known exosome markers and signaling proteins in comparison to parental cells. Importantly, valuable insights into oncogenic exosomes (362 unique proteins in comparison to non-malignant exosomes) of key metastatic regulatory factors and signaling molecules fundamental to pancreatic cancer progression (KRAS, CD44, EGFR) are provided. It is reported that oncogenic exosomes contain factors known to regulate the pre-metastatic niche (S100A4, F3, ITGβ5, ANXA1), clinically-relevant proteins which correlate with poor prognosis (CLDN1, MUC1) as well as protein networks involved in various cancer hallmarks including proliferation (CLU, CAV1), invasion (PODXL, ITGA3), metastasis (LAMP1, ST14) and immune surveillance escape (B2M). The presence of these factors in oncogenic exosomes offers an understanding of select differences in exosome composition during tumorigenesis, potential components as prognostic and diagnostic biomarkers in pancreatic cancer, and highlights the role of exosomes in mediating crosstalk between tumor and stromal cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.201800158DOI Listing
April 2019

Surfaceome of Exosomes Secreted from the Colorectal Cancer Cell Line SW480: Peripheral and Integral Membrane Proteins Analyzed by Proteolysis and TX114.

Proteomics 2019 04;19(8):e1700453

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science (LIMS), La Trobe University, Melbourne, Victoria, 3086, Australia.

Exosomes are important bidirectional cell-cell communicators in normal and pathological physiology. Although exosomal surface membrane proteins (surfaceome) enable target cell recognition and are an attractive source of disease marker, they are poorly understood. Here, a comprehensive surfaceome analysis of exosomes secreted by the colorectal cancer cell line SW480 is described. Sodium carbonate extraction/Triton X-114 phase separation and mild proteolysis (proteinase K, PK) of intact exosomes is used in combination with label-free quantitative mass spectrometry to identify 1025 exosomal proteins of which 208 are predicted to be integral membrane proteins (IMPs) according to TOPCONS and GRAVY scores. Interrogation of UniProt database-annotated proteins reveals 124 predicted peripherally-associated membrane proteins (PMPs). Surprisingly, 108 RNA-binding proteins (RBPs)/RNA nucleoproteins (RNPs) are found in the carbonate/Triton X-114 insoluble fraction. Mild PK treatment of SW480-GFP labeled exosomes reveal 58 proteolytically cleaved IMPs and 14 exoplasmic PMPs (e.g., CLU/GANAB/LGALS3BP). Interestingly, 18 RBPs/RNPs (e.g., EIF3L/RPL6) appear bound to the outer exosome surface since they are sensitive to PK proteolysis. The finding that outer surface-localized miRNA Let-7a-5p is RNase A-resistant, but degraded by a combination of RNase A/PK treatment suggests exosomal miRNA species also reside on the outer surface of exosomes bound to RBPs/RNPs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.201700453DOI Listing
April 2019

Proteomic and Post-Translational Modification Profiling of Exosome-Mimetic Nanovesicles Compared to Exosomes.

Proteomics 2019 04 3;19(8):e1800161. Epub 2019 Apr 3.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Victoria, 3086, Australia.

Issues associated with upscaling exosome production for therapeutic use may be overcome through utilizing artificial exosomes. Cell-derived mimetic nanovesicles (M-NVs) are a potentially promising alternative to exosomes for clinical applicability, demonstrating higher yield without incumbent production and isolation issues. Although several studies have shown that M-NVs have similar morphology, size and therapeutic potential compared to exosomes, comprehensive characterization and to what extent M-NVs components mimic exosomes remain elusive. M-NVs were generated through the extrusion of cells and proteomic profiling demonstrated an enrichment of proteins associated with membrane and cytosolic components. The proteomic data herein reveal a subset of proteins that are highly abundant in M-NVs in comparison to exosomes. M-NVs contain proteins that largely represent the parental cell proteome, whereas the profile of exosomal proteins highlight their endosomally derived origin. This advantage of M-NVs alleviates the necessity of endosomal sorting of endogenous therapeutic proteins or RNA into exosomes. This study also highlights differences in protein post-translational modifications among M-NVs, as distinct from exosomes. Overall this study provides key insights into defining the proteome composition of M-NVs as a distinct from exosomes, and the potential advantage of M-NVs as an alternative nanocarrier when spontaneous endosomal sorting of therapeutics are limited.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.201800161DOI Listing
April 2019

Somatic proteome of Haemonchus contortus.

Int J Parasitol 2019 03 14;49(3-4):311-320. Epub 2019 Feb 14.

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

Currently, there is a dearth of proteomic data to underpin fundamental investigations of parasites and parasitism at the molecular level. Here, using a high throughput LC-MS/MS-based approach, we undertook the first reported comprehensive, large-scale proteomic investigation of the barber's pole worm (Haemonchus contortus) - one of the most important parasitic nematodes of livestock animals worldwide. In total, 2487 unique H. contortus proteins representing different developmental stages/sexes (i.e. eggs, L3s and L4s, female (Af) and male (Am) adults) were identified and quantified with high confidence. Bioinformatic analyses of this proteome revealed substantial alterations in protein profiles during the life cycle, particularly in the transition from the free-living to the parasitic phase, and key groups of proteins involved specifically in feeding, digestion, metabolism, development, parasite-host interactions (including immunomodulation), structural remodelling of the body wall and adaptive processes during parasitism. This proteomic data set will facilitate future molecular, biochemical and physiological investigations of H. contortus and related nematodes, and the discovery of novel intervention targets against haemonchosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijpara.2018.12.003DOI Listing
March 2019

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.

J Extracell Vesicles 2018 23;7(1):1535750. Epub 2018 Nov 23.

Institute of Biomedicine and Molecular Immunology (IBIM), National Research Council (CNR) of Italy, Palermo, Italy.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/20013078.2018.1535750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322352PMC
November 2018
-->