Study On Plasma Exosome Biomarkers Of Pregnant Women With Intrahepatic Cholestasis of Pregnancy

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Feng, Fan, Mei Jiang, Wei Liu, Di Xu, Xiao-Mei Huang, and Yong Shao. "Study On Plasma Exosome Biomarkers Of Pregnant Women With Intrahepatic Cholestasis of Pregnancy." (2021).
Background:Elevated serum total bile acid level is currently the main index for clinical diagnosis of intrahepatic cholestasis of pregnancy, but the use of TBA as a detection index has certain limitations. The early diagnosis of ICP and new treatment options still need to be further strengthened. Methods: Plasma samples were collected, and exosomes were isolated. Key differential proteins were screened by bioinformatics methods. ELISA method was used to detect the concentration of the key differential protein cluster in plasma samples, and the ROC curve was drawn to find out the best critical value. Results: There were 138 differentially expressed proteins between the ICP group and the normal group by quantitative analysis. Cluster protein was screened as a clinical validation index. The cluster protein concentration of plasma exosomes in the ICP group was significantly higher than that in the normal group (P<0.0001). ROC curve analysis showed that the best critical point for diagnosing ICP according to the plasma exosome cluster protein concentration of pregnant women was 255.28 ng/ml. In the ICP group, the best crucial point for predicting ICP with premature delivery is 286.72 ng/ml. Conclusion: the plasma exosome cluster protein concentration of ICP pregnant women is significantly higher than that of normal pregnant women. When the plasma cluster protein concentration of pregnant women is more remarkable than 255.28ng/ml, ICP can be diagnosed. When the plasma cluster protein concentration of pregnant women is higher than 286.72ng/ml, ICP pregnant women are more likely to have a premature birth.
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Caracterización de partículas coloidales en el agua del suelo mediante detección sintonizable de pulsos resistivos
The transport of colloids in soil determines the fate of pollutants, nutrients and microorganisms in the environment and the contamination of groundwater. Colloidal retention mechanisms in soils depend on complex interactions between the soil pore walls and colloids. The hypothesis of this thesis is that the interaction of the particulate colloidal pollutants with the colloids present in the soil pore water has a dramatic influence on the transport of pollutants. This is due to the fact that the filtration of colloids in the porous medium depends on the size, shape and charge of the coatings and colloidal aggregates formed between the polluting particles and the suspended soil colloids. Improving the characterization of colloidal particulate pollutants in soil water can help to explain more precisely the role of soil as a filter for pollutants. Emerging technologies in particle characterization can represent an important advance in this characterization. Specifically, the tunable resistive pulse sensing (TRPS) detection technology allows the real (non-hydrodynamic) size of individual particles to be determined with high precision in a polydisperse suspension between 40 nm and 3 micrometers, in addition to determining, also individually, their surface electrical potential. The new knowledge that this technique can provide could lead to a better understanding of the transport of particulate pollutants in the soil, which could improve the diagnosis of potential vulnerability of subsurface waters against pathogenic organisms, engineered nanoparticles and metals bound to colloids, as well as optimize the design of micro and nanopesticide formulations.
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2021
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Cardioprotection by remote ischemic conditioning is transferable by plasma and mediated by extracellular vesicles
Background Remote ischemic conditioning (RIC) by brief periods of limb ischemia and reperfusion protects against ischemia–reperfusion injury. We studied the cardioprotective role of extracellular vesicles (EV)s released into the circulation after RIC and EV accumulation in injured myocardium. Methods We used plasma from healthy human volunteers before and after RIC (pre-PLA and post-PLA) to evaluate the transferability of RIC. Pre- and post-RIC plasma samples were separated into an EV enriched fraction (pre-EV + and post-EV +) and an EV poor fraction (pre-EV- and post-EV-) by size exclusion chromatography. Small non-coding RNAs from pre-EV + and post-EV + were purified and profiled by NanoString Technology. Infarct size was compared in Sprague–Dawley rat hearts perfused with isolated plasma and fractions in a Langendorff model. In addition, fluorescently labeled EVs were used to assess homing in an in vivo rat model. (ClinicalTrials.gov, number: NCT03380663) Results Post-PLA reduced infarct size by 15% points compared with Pre-PLA (55 ± 4% (n = 7) vs 70 ± 6% (n = 8), p = 0.03). Post-EV + reduced infarct size by 16% points compared with pre-EV + (53 ± 15% (n = 13) vs 68 ± 12% (n = 14), p = 0.03). Post-EV- did not affect infarct size compared to pre-EV- (64 ± 3% (n = 15) and 68 ± 10% (n = 16), p > 0.99). Three miRNAs (miR-16-5p, miR-144-3p and miR-451a) that target the mTOR pathway were significantly up-regulated in the post-EV + group. Labelled EVs accumulated more intensely in the infarct area than in sham hearts. Conclusion Cardioprotection by RIC can be mediated by circulating EVs that accumulate in injured myocardium. The underlying mechanism involves modulation of EV miRNA that may promote cell survival during reperfusion.
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2021
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Characterisation of extracellular vesicles in the context of myocardial infarction and glucose intolerance
Introduction In response to myocardial infarction (MI), extracellular vesicles (EVs), including large (lEVs) and small (sEVs), are released within and from the heart to facilitate intercellular communication and maintain cardiac homeostasis by transporting cargo to recipient cells. Objective We investigated how glucose intolerance influences the intracardiac EV release post-MI and their content. Method B6J mice were fed chow (CD) or high-fat diet (HFD) for 3 months. MI was induced by permanent coronary artery ligation. EVs were isolated from left ventricles and quantified by tunable resistive pulse sensing. EVs were characterised by flow cytometry. EV miRNA content was determined by RNAseq and qPCR. Using cardiomyocyte specific GFP+ mice, plasma lEVs were analysed by flow cytometry to determine if cardiomyocyte EVs (CMEVs) are circulating. Labelled hypoxic cardiomyocyte cell line (HL-1) lEVs were injected in HFD/CD mice post-MI to determine target cells. Results In CD mice, EV release was significantly increased 24 h post-MI compared to sham. HFD lEV levels were significantly higher compared to sham and CD mice post-MI with no difference in sEV release between sham and MI HFD mice. Intracardiac lEVs originate from cardiomyocyte and endothelial cells in response to MI and MI + HFD respectively. qPCR analyses identified miRNA candidates that were modulated by MI and HFD. Intracardiac GFP + lEV levels were lower in HFD than in CD mice whereas levels of circulating GFP + lEVs were higher. In vivo biodistribution studies revealed a preferential uptake of hypoxic HL-1 lEVs by splenic myeloid cells in HFD spleens versus CD post-MI. Conclusion Our results show that glucose intolerance modulates intracardiac EV release post-MI and their miRNA cargo. Circulating CMEV levels as well as their uptake by splenic myeloid cells are increased. Further investigations will aim to decipher the impact of the intracardiac EV miRNA mediated transfer in the diabetic heart post-MI.
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2021
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Characterization of extracellular vesicles and synthetic nanoparticles with four orthogonal single‐particle analysis platforms
We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.
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2021
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Characterization of feces-derived bacterial membrane vesicles and the impact of their origin on the inflammatory response
The human gastrointestinal tract harbors a diverse and complex microbiome, which interacts in a variety of ways with the host. There is compelling evidence that gut microbial dysbiosis, defined as an alteration of diversity and abundance in intestinal microbes, is an etiological factor in inflammatory bowel disease (IBD). Membrane vesicles (MVs), which are nano-sized particles released by bacteria, have been found to interact with the host and modulate the development and function of the immune system. As a result MVs have been suggested to play a critical role in both health and disease. In this study we developed a method to isolate, characterize and assess the immunoreactivity of heterogeneous populations of MVs from fecal samples (fMVs) of healthy volunteers. We successfully isolated 2*109-2*1010 particles/ml from 0.5 gram of feces by using a combination of ultrafiltration and size exclusion chromatography (SEC) from 10 fecal samples. Bead-based flowcytometry in combination with tunable resistive pulse sensing (TRPS) provided a reliable method for (semi-)quantitative determination of fMVs originating from both Gram-positive and Gram-negative bacteria, while transmission electron microscopy confirmed the presence of fMVs. Real time 16s PCR on bacterial cell fractions or isolated fMVs DNA of the most common phyla (Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria) revealed differences in the relative abundance between bacteria and the fMVs. Moreover, fMVs evoke the release of TNF- by THP-1 cells in a dose-dependent matter. Also, a significant positive correlation was found between Actinobacteria/-Proteobacteria derived vesicles and the release of TNF-. It has become increasingly clear that fMVs could provide an additional layer to the definition of homeostasis or dysbiosis of the microbiota. The current study supports their potential involvement in the intestinal homeostasis or inflammatory disorders and provides putative interesting incentives for future research.
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2021
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Characterization of positively charged polyplexes by tunable resistive pulse sensing
With the approval of the first siRNA-based drugs, non-viral siRNA delivery has gained special interest in industry and academia in the last two years. For non-viral delivery, positively charged lipid and polymer formulations play a central role in research and development. However, nanoparticle size characterization, particularly of polydisperse formulations, can be very challenging. Tunable resistive pulse sensing for particle by particle measurements of size, polydispersity, zeta potential and a direct concentration promises better assessment of nanoparticle formulations. However, the current application is not optimized for positively charged particles. A supplier-provided coating solution for difficult-to-measure samples does not allow for successful measurements of positively charged nanoparticles. This article describes a new coating solution based on choline-chloride. Coating is verified by current–voltage (I-V) recordings and ultimately tested on a positively charged nanoparticle formulation comprising of siRNA and PEG-PCL-PEI polymer. This coating allows successful size, polydispersity index (PDI) and concentration measurement by tunable resistive pulse sensing of positively charged PEI-based polyplexes. This article provides the foundation for further characterization of polyplexes as well as other positively charged nanoparticle formulations based on particle by particle measurements.
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2021
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Characterization of systemic immunosuppression by IDH mutant glioma small extracellular vesicles
Background Gliomas are the most common primary brain tumors and are universally fatal. Mutations in the isocitrate dehydrogenase genes (IDH1 and IDH2) define a distinct glioma subtype associated with an immunosuppressive tumor microenvironment. Mechanisms underlying systemic immunosuppression in IDH mutant (mutIDH) gliomas are largely unknown. Here, we define genotype-specific local and systemic tumor immunomodulatory functions of tumor-derived glioma small extracellular vesicles (TEX). Methods TEX produced by human and murine wildtype and mutant IDH glioma cells (wtIDH and mutIDH, respectively) were isolated by size exclusion chromatography (SEC). TEX morphology, size, quantity, molecular profiles and biodistribution were characterized. TEX were injected into naive and tumor-bearing mice, and the local and systemic immune microenvironment composition was characterized. Results Using in vitro and in vivo glioma models, we show that mutIDH TEX are more numerous, possess distinct morphological features and are more immunosuppressive than wtIDH TEX. mutIDH TEX cargo mimics their parental cells, and induces systemic immune suppression in naive and tumor-bearing mice. TEX derived from mutIDH gliomas and injected into wtIDH tumor-bearing mice reduce tumor-infiltrating effector lymphocytes, dendritic cells and macrophages, and increase circulating monocytes. Astonishingly, mutIDH TEX injected into brain tumor-bearing syngeneic mice accelerate tumor growth and increase mortality compared with wtIDH TEX. Conclusions Targeting of mutIDH TEX represents a novel therapeutic approach in gliomas.
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2021
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Characterizing KRAS Membrane Structures by Data-Driven Molecular Docking
Computational Sciences and Engineering Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA, 2 NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA, 3 Department of Physics, Carnegie Mellon University, Pittsburgh, PA, USA, 4 Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, MD, USA, 5 Data Science, Argonne National Laboratory, Lemont, IL, USA, 6 Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM, USA. KRAS is a GTPase that plays an important role in cell growth and signaling pathways. of the different RAS isoforms, KRAS also has the highest prevalence of mutations related to human cancers, making it an attractive therapeutic target in these cases. Once attached to the membrane, KRAS in the active (GTP) form is capable to bind effector proteins, like RAF kinase. However, certain molecular details concerning KRAS conformation and orientational changes when interacting with the membrane and binding partners are not fully understood. To provide new insights, we used a variety of biophysical approaches to characterize KRAS structure and dynamics. Here, we focus on our results utilizing data-driven computational docking to investigate both KRAS and KRAS/ RAF1-RBD (RAS Binding Domain) complex at the membrane. with the HADDOCK program, we incorporated experimental restraints derived from our NMR paramagnetic relaxation enhancement (PRE) and neutron reflectivity (NR) measurements to dock these KRAS forms to a 70:30 POPC:POPS lipid membrane surface. Using NMR-PRE restraints alone, we performed one series of docking runs with the KRAS G-domain directly interacting with the membrane to discern membrane-proximal states. Based on our experimental evidence, and particularly from NR, a highly populated membrane-distal state also exists, where the G-domain does not directly contact the membrane but KRAS remains tethered via the C-terminal hypervariable region (HVR). Therefore, we also conducted a second series of docking runs that incorporated both NMR-PRE and NR restraints to better elucidate the conformations in this state. From these results, we were able to generate atomistic models for KRAS and KRAS/RAF1-RBD with averaged 1-D profiles closely matching the respective NR profiles. Overall, the findings should assist in elucidating the role of KRAS structural dynamics in recruiting effectors, like RAF kinase, to the membrane for activation.
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2021
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Chemical Modification of Bovine Milk Exosomes, the Biological Nanoparticles of the Future, as a Contrast Agent and Drug Delivery Vehicle
Chemically derived nanoparticles are widely used across many applications. While they showed great promise when first discovered, the main hurdles, such as clearance and targeting, have yet to be overcome. A recently discovered class of biological nanoparticles have the potential to circumvent these disadvantages. Exosomes are biological nanoparticles (30 – 150 nm) excreted from most mammalian cells. While exosomes are typically involved in cellular signaling and traditionally removed from the body to be examined for biomarkers, this work combines chemical modifications and a biological particle for diagnostics and treatment of solid tumor cancer. Exosome involvement in cancer treatment has grown over the past ten years with the encapsulation of RNA, proteins and traditional chemotherapeutics. However, this work takes these ideas and drives them into the future by using bovine milk derived exosomes as (1) an ultrasound contrasting agent and (2) a targeted and triggered chemotherapeutic drug delivery vehicle. As an ultrasound contrast agent, raw and pasteurized bovine milk exosomes were tested and found to be capable of echogenicity without altering the ability to identify key features of the exosome, including the presence of CD63 and miRNA. In the second part of this work a chemically synthesized, hypoxia responsive lipid and a tumor penetrating and targeting peptide, iRGD were integrated into the lipid bilayer of the exosome for chemotherapeutic drug delivery. These modified exosomes were characterized using a variety of techniques, including a novel adhesion assay, atomic force microscopy, and high-resolution transmission electron microscopy. The functional capacity of the modified exosomes to deliver doxorubicin to Triple Negative Breast Cancer (TNBC) cells was also evaluated using a combination of cellular internalization and cytotoxicity assays in both monolayer and 3D spheroid cultures. Overall exosomes have the iv ability to be chemically modified in a variety of ways, opening a door to a new approach to nanoparticle drug delivery and targeted imaging.
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2021
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Circulating Extracellular Vesicle Cargo as Bioinformants of 'at-risk'Carotid Artery Stenosis
Objectives Carotid artery atherosclerosis is a major cause of ischemic stroke. Managing patients with asymptomatic disease remains challenging, given the lack of reliable tests to identify the subgroup of patients prone to plaque progression and stroke. Given the functional and diagnostic roles of extracellular vesicle (EV) contents, we hypothesized that plasma EV-derived microRNA (miRNA) differs between symptomatic and asymptomatic patients. Methods EVs were isolated via serial centrifugation followed by enrichment using size exclusion chromatography (SEC) (qEVoriginal columns 70 nm; Izon Science Ltd). EV isolation was confirmed according to MISEV 2018 guidelines: Western blot analysis of common EV markers (CD63, CD81, Alix), nanoparticle tracking analysis (NTA), and cryogenic transmission electron microscopy (Cryo-TEM). Lipoprotein contamination was assessed via enzyme-linked immunosorbent assay of individual SEC fractions (R&D Systems; DAPA10, DAPB00). Next-generation sequencing was performed on EVs (HTG Molecular Diagnostics, Inc.), and differential miRNA expression evaluated using Partek Genomics Suite software (version 8.0). Results Twelve patient plasma samples were collected (n = 6 symptomatic; n = 6 asymptomatic). The average age of the cohort was 70.0 ± 5.7 years (asymptomatic, 67.0 ± 5.5 vs symptomatic, 72.5 ± 5.5 years). All patients had severe stenoses with similar peak systolic velocity (asymptomatic 403.2 ± 84.43 vs symptomatic 371.6 ± 175.25; P = .50) and internal carotid artery (ICA):common carotid artery (CCA) ratios (asymptomatic, 5.36 ± 1.07 vs symptomatic, 7.3 ± 5.00; P = .50). CD63 expression confirmed EV enrichment in fractions 7 to 10, with minimal lipoprotein contamination. EV isolation was further confirmed by CD81 and Alix expression (n = 3 patient samples per group). Cryo-TEM identified EVs as bi-layered nanoparticles with electron dense cores (Fig 1). NTA revealed no significant differences in EV concentration or size between groups (n = 3; P > .05). Principal component and heatmap analysis of miRNA sequencing data revealed symptomatic carotid plasma samples clustered together, whereas asymptomatic samples were either starkly different (n = 5) or approximated the symptomatic profiles (n = 1), suggesting a disease gradient (Fig 2). When symptomatic carotid plasma EV-miRNA profiles were compared with asymptomatic specimens, 190 miRNAs were differentially expressed, with miRNA-654-5p and miRNA-127-3p being the most upregulated, and downregulated, respectively (P < .05, fold-change −2< or >2, excluding miRNA with counts <100). Gene set enrichment identified regulation of protein metabolic processes, and negative regulation of cell communication, signaling, and signal transduction as predicted targets of differentially expressed EV-miRNA (P-value < .05). Conclusions Plasma EV-miRNA profiles may differentiate symptomatic vs asymptomatic carotid stenosis and, together with clinical characteristics, may be used in risk stratification of asymptomatic patients.
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2021
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Circulating extracellular vesicles from patients with acute chest syndrome disrupt adherens junctions between endothelial cells
Background Small cell-derived extracellular vesicles (EVs) can affect endothelial function. We previously found that patients with sickle cell disease (SCD) have greater numbers of circulating EVs than subjects without the disease, and the EVs differentially disrupt endothelial integrity in vitro. Because endothelial disruption is a critical component of acute chest syndrome (ACS), we hypothesized that EVs isolated during ACS would induce greater endothelial damage than those isolated at baseline. Methods Nine pediatric subjects had plasma isolated at baseline and during ACS from which EVs were isolated. Cultured microvascular endothelial cells were treated with EVs and then studied by immunofluorescence microscopy to localize VE-cadherin and F-actin. Results The EVs had a diameter of 95 nm. They contained CD63 and flotillin-1, which were increased in SCD patients (5–13-fold compared to control) and further increased between baseline and ACS (24–57%). The EVs contained hemoglobin, glycophorin A, and ferritin. Treatment with baseline EVs caused modest separation of endothelial cells, while ACS EVs caused substantial disruptions of the endothelial cell monolayers. EVs from subjects with ACS also caused a 50% decrease in protein levels of VE-cadherin. Conclusions These results suggest that circulating EVs can modulate endothelial integrity contributing to the development of ACS in SCD patients by altering cadherin-containing intercellular junctions. Impact - Sickle cell disease patients have circulating extracellular vesicles (EVs) that modulate endothelial integrity by altering cadherin-containing intercellular junctions. - - Disruption is more severe by EVs obtained during acute chest syndrome (ACS). - - These results expand our knowledge of the pathophysiology of acute chest syndrome and the vasculopathies of sickle cell disease.
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2021
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Circulating Serum Exosomal Long Non-Coding RNAs FOXD2-AS1, NRIR, and XLOC_009459 as Diagnostic Biomarkers for Colorectal Cancer
Background: Exosomes derived from cancer cells encapsulate various kinds of tumor-specific molecules and thus can interact with adjacent or distant cells to mediate information exchange. Long non-coding RNAs (lncRNAs) in exosomes have the potential as diagnostic and prognostic biomarkers in different types of cancers. The current study was aimed to identify circulating exosomal lncRNAs for the diagnosis of colorectal cancer (CRC). Methods: Exosomes were isolated from the serum by ultracentrifugation and verified by transmission electron microscope (TEM), qNano, and immunoblotting. Exosomal lncRNAs FOXD2-AS1, NRIR, and XLOC_009459 were selected by lncRNA microarray and validated by qPCR in 203 CRC patients and 201 healthy donors. The receiver operating characteristic curve (ROC) was used to assess the diagnostic efficiency of serum exosomal lncRNAs. Results: Exosomal FOXD2-AS1, NRIR, and XLOC_009459 (TCONS_00020073) levels were significantly upregulated in 203 CRC patients and 80 early-stage CRC patients compared to 201 healthy donors, possessing the area under the curve (AUC) of 0.728, 0.660, and 0.682 for CRC, as well as 0.743, 0.660, and 0.689 for early-stage CRC, respectively. Notably, their combination demonstrated the markedly elevated AUC of 0.736 for CRC and 0.758 for early-stage CRC, indicating their potential as diagnostic biomarkers for CRC. Conclusions: Our data suggested that exosomal lncRNAs FOXD2-AS1, NRIR, and XLOC_009459 act as the promising biomarkers for the diagnostics of CRC and early-stage CRC.
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2021
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Comparative proteome profiling in exosomes derived from porcine colostrum versus mature milk reveals distinct functional proteomes
Exosomes are membranous vesicles of endocytic origin, recently been considered as major players in cell-cell communication. Milk is highly complex, and diverse biocomponents provide adequate nutrition, transfer immunity, and promote adequate neonate development. Milk exosomes are suggested to have a key role in these processes, yet to be further explored, and the alteration of the exosomes' cargo in different stages of lactation stages is important for understanding the factors relevant in nursing and also for improving milk replacer products both for humans and animals. We isolated exosomes from porcine milk in different lactation stages and analyzed their content using a TMT-based high-resolution quantitative proteomic approach. Exosomes were isolated using ultracentrifugation coupled with size exclusion chromatography to enrich milk-derived exosomes in samples obtained at day 0, 7, and 14 after parturition, and characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. Quantitative proteomics analysis revealed different proteome profiles for colostrum exosomes and milk exosomes. The functional analysis highlighted pathways related to the regulation of homeostasis to be upregulated in colostrum exosomes, and pathways such as endothelial cell development and lipid metabolism to be upregulated in mature milk exosomes. This study endorses the importance of exosomes as active biocomponents of milk and provides knowledge for future studies exploring their role in the regulation of immunity and growth of the newborn.
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2021
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Comparative study of commercial protocols for high recovery of high-purity mesenchymal stem cell-derived extracellular vesicle isolation and their efficient labeling with fluorescent dyes
The extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) can be used as carriers for therapeutic molecules and drugs to target disordered tissues. This aimed to compare the protocols used for isolation of MSC-derived EVs by comparing EV collection conditions and three commercial purification kits. We also determined appropriate fluorescent dyes for labeling EVs. MSC-derived EVs were efficiently secreted during cell growth and highly purified by the phosphatidyl serine-based affinity kit. Although the EV membrane was more efficiently labeled with the fluorescent dye PKH67 compared to other probes, the efficiency was not enough to accurately analyze the endothelial cellular uptake of EVs. Results verified the easy protocol for isolating and fluorescently labeling EVs with commercial reagents and kits, but meanwhile, further modification of the protocol is required in order to scale up the amount of EVs derived from MSCs using fluorescent probes. Graphical Abstract The extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) can be used as carriers for therapeutic molecules and drugs. This aimed to compare the protocols used for isolation of EVs by comparing EV collection conditions and three commercial purification kits. MSC-derived EVs were efficiently secreted during cell growth and highly purified by the phosphatidyl serine-based affinity kit. Results verified the easy protocol for isolating and fluorescently labeling EVs with commercial reagents and kits, but meanwhile, further modification of the protocol is required in order to scale up the amount of EVs derived from MSCs using fluorescent probes.
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2021
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Comparison and optimization of nanoscale extracellular vesicle imaging by scanning electron microscopy for accurate size-based profiling and morphological analysis
Nanosized extracellular vesicles (EVs) have been found to play a key role in intercellular communication, offering opportunities for both disease diagnostics and therapeutics. However, lying below the diffraction limit and also being highly heterogeneous in their size, morphology and abundance, these vesicles pose significant challenges for physical characterization. Here, we present a direct visual approach for their accurate morphological and size-based profiling by using scanning electron microscopy (SEM). To achieve that, we methodically examined various process steps and developed a protocol to improve the throughput, conformity and image quality while preserving the shape of EVs. The study was performed with small EVs (sEVs) isolated from a non-small-cell lung cancer (NSCLC) cell line as well as from human serum, and the results were compared with those obtained from nanoparticle tracking analysis (NTA). While the comparison of the sEV size distributions showed good agreement between the two methods for large sEVs (diameter > 70 nm), the microscopy based approach showed a better capacity for analyses of smaller vesicles, with higher sEV counts compared to NTA. In addition, we demonstrated the possibility of identifying non-EV particles based on size and morphological features. The study also showed process steps that can generate artifacts bearing resemblance with sEVs. The results therefore present a simple way to use a widely available microscopy tool for accurate and high throughput physical characterization of EVs.
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2021
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Comparison of extracellular vesicle isolation and storage methods using high-sensitivity flow cytometry
Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major limitation for the clinical use of EVs is the lack of standardized methods for the fast and reproducible separation and subsequent detection of EV subpopulations from biofluids, as well as their storage. To advance this application area, fluorescence-based characterization technologies with single-EV resolution, such as high-sensitivity flow cytometry (HS-FCM), are powerful to allow assessment of EV fractionation methods and storage conditions. Furthermore, the use of HS-FCM and fluorescent labeling of EV subsets is expanding due to the potential of high-throughput, multiplex analysis, but requires further method development to enhance the reproducibility of measurements. In this study, we have applied HS-FCM measurements next to standard EV characterization techniques, including nanoparticle tracking analysis, to compare the yield and purity of EV fractions obtained from lipopolysaccharide-stimulated monocytic THP-1 cells by two EV isolation methods, differential centrifugation followed by ultracentrifugation and the exoEasy membrane affinity spin column purification. We observed differences in EV yield and purity. In addition, we have investigated the influence of EV storage at 4°C or -80°C for up to one month on the EV concentration and the stability of EV-associated fluorescent labels. The concentration of the in vitro cell derived EV fractions was shown to remain stable under the tested storage conditions, however, the fluorescence intensity of labeled EV stored at 4°C started to decline within one day.
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2021
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Comparison of isolation methods using commercially available kits for obtaining extracellular vesicles from cow milk
Extracellular vesicles (EV) are important for delivering biologically active substances to facilitate cell-to-cell communication. Milk-derived EV are widely known because of their potential for immune enhancement. However, procedures for isolating milk-derived EV have not been fully established. To obtain pure milk-derived EV and accurately reveal their function, such procedures must be established. The aim of the present study was to compare methods using commercially available kits for isolating milk-derived EV. Initially, we investigated procedures to remove casein, which is the major obstacle in determining milk-derived EV purity. We separated whey using centrifugation only, acetic acid precipitation, and EDTA precipitation. Then, we isolated milk-derived EV by ultracentrifugation, membrane affinity column, size exclusion chromatography (SEC), polymer-based isolation, or phosphatidylserine-affinity isolation. Using EV count per milligram of protein, which is a good indicator of purity, we determined that acetic acid precipitation was the best method for removing casein. Using nanoparticle tracking analysis, protein quantity analysis, and RNA quantity analysis, we comprehensively compared each isolation method for its purity and yield. We found that SEC-based qEV column (Izon Science) could collect purer milk-derived EV at higher quantities. Thus, a combination of acetic acid precipitation and qEV can effectively isolate high amounts of pure extracellular vesicles from bovine milk.
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2021
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Comparison of Syringes With Intravitreal Anti-VEGF Drugs: Particle Burden and Protein Aggregates in Brolucizumab, Aflibercept and Bevacizumab
Purpose: In a benchwork particle counting analytical evaluation, the number and type of particles in intravitreal injection formulations of three different agents against vascular endothelial growth factor were investigated. Methods: Commercially available ready-to-use aflibercept and brolucizumab glass syringes, vials containing bevacizumab (off-label use in ophthalmology), and repackaged ready-to-use plastic syringes containing bevacizumab were tested without filtration. Total visible, subvisible, and nanoparticles numbers and size distributions were quantified using light obscuration, flow imaging, resonant mass measurement (RMM), tunable resistive pulse sensing, and dynamic light scattering. Results: Repackaged bevacizumab showed overall low particle numbers, aflibercept showed high numbers of micrometer sized particles but low nanoparticle numbers, brolucizumab showed low to moderate numbers of micrometer sized particles but high nanoparticle numbers. RMM measurements identified particles in the nanometer range as either proteinaceous or silicon oil; the nature of the other particles was not further evaluated. Conclusions: Repackaged bevacizumab shows no inferior particle quality compared to ready-to-use products. It is relevant to study nanoparticle load of the products as the micrometer-sized particle numbers do not in all cases correlate to nanoparticle counts. Particularly for the high concentration product Beovu (brolucizumab), high nanoparticle numbers were found despite low numbers of micrometer sized particles. Silicone oil droplets did not account for high particle numbers as the measured numbers were low. Translational Relevance: Different side effects are registered in different frequencies with different intravitreal anti-VEGF-drugs and syringes, which are applied by injection by small 30G needles through the sclera directly to the intravitreal cavity. The study of nanoparticles and silicone oil droplets may be able to contribute to narrowing down the causes.
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2021
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Comprehensive analysis and comparison of proteins in salivary exosomes of climacteric and adolescent females
Currently, it is difficult to extract exosomes with stable physicochemical properties from saliva. Furthermore, due to inadequate availability of basic data, the application of salivary exosomes as a diagnostic material is limited. In this study, we aimed to investigate an easier method for extraction of exosomes from whole saliva and compared proteins in salivary exosomes derived from subjects of two age groups. Salivary exosomes were extracted from nine females (56.7 ± 1.17 years old; climacteric or 19.9 ± 0.20 years old; adolescent) using commercial reagents and kits and detected using western blotting with anti-exosome marker antibodies. Exosome particle size and exosome-containing proteins were identified using NanoSight® and liquid chromatography with tandem mass spectrometry, respectively. In addition, an efficient method of exosome extraction from saliva using a reagent and without the use of an ultracentrifuge was shown. Our results showed a higher total protein content and larger particle size in climacteric exosomes than in adolescent exosomes. However, adolescent exosomes showed a larger variety of proteins (780 proteins) than the climacteric exosomes (573 proteins). Altogether, 893 proteins were identified in the salivary exosomes. Although viral process-, ribosome- and structural molecule-related proteins were higher in the adolescent exosomes, the levels of major salivary proteins such as immunoglobulins and amylase, were higher in the climacteric exosomes than in the adolescent exosomes. The data presented, which show the fundamental protein composition of salivary exosomes and the changes that occur with age, are beneficial in both diagnostic and biotechnological applications.
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2021
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Dancing with Trojan horses: an interplay between the extracellular vesicles and viruses
Extracellular vesicles (EVs) are membrane-encapsulated particles released by eukaryotic and prokaryotic cells into the extracellular environment. Depending on their origin, size, and composition, EVs are grouped in several classes, with one of them being exosomes, which are small EVs (SEVs) generated within the endosomal compartment of eukaryotic cells via the unique multivesicular body pathway. Being able to deliver their content (proteins, lipids, small molecules, and nucleic acids) to other cells, exosomes/SEVs are considered as bioactive vesicles with multiple biological functions. Importantly, the composition of exosomes/SEVs depends on the cell and tissue of origin including a set of specific proteins. However, the pathological conditions may lead to the appearance of diseases-specific exosomes/SEVs containing pathology-specific cargoes utilized in the malicious cell-cell communication and spread of malady. Viruses demonstrate complex ‘dancing’ around the exosome biogenesis system, being able to hijack the host systems responsible for the exosome biogenesis. They use the exosome biogenesis system to promote packaging of their capsids, regulate virion production, and virus secretion. They also utilize a Trojan horse stratagem to place virions inside the SEVs and thereby to spread beyond their normal range of cell hosts using the normal EV uptake process. Another illustration of the virus-based utilization of Trojan horse strategy is given by the ability of human viruses to use exosomes/SEVs as carriers of their exogenous miRNA or viral proteins to the non-infected cells. Taken together, these strategies of dancing with Trojan horses can help viruses to fight with the host defense and to spread the infection.
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2021
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Deciphering the Structure and Chemical Composition of Drug Nanocarriers: From Bulk Approaches to Individual Nanoparticle Characterization
Drug nanocarriers (NCs) with sizes usually below 200 nm are gaining increasing interest in the treatment of severe diseases such as cancer and infections. Characterization methods to investigate the morphology and physicochemical properties of multifunctional NCs are key in their optimization and in the study of their in vitro and in vivo fate. Whereas a variety of methods has been developed to characterize “bulk” NCs in suspension, the scope of this review is to describe the different approaches for the NC characterization on an individual basis, for which fewer techniques are available. The accent is put on methods devoid of labelling, which could lead to artefacts. For each characterization method, the principles and approaches to analyze the data are presented in an accessible manner. Aspects related to sample preparation to avoid artefacts are indicated, and emphasis is put on examples of applications. NC characterization on an individual basis allows gaining invaluable information in terms of quality control, on: i) NC localization and fate in biological samples; ii) NC morphology and crystallinity; iii) distribution of the NC components (drugs, shells), and iv) quantification of NCs’ chemical composition. The individual characterization approaches are expected to gain increasing interest in the near future.
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2021
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Defining candidate mRNA and protein EV biomarkers to discriminate ccRCC and pRCC from non-malignant renal cells in vitro
Renal cell carcinoma (RCC) accounts for over 400,000 new cases and 175,000 deaths annually. Diagnostic RCC biomarkers may prevent overtreatment in patients with early disease. Extracellular vesicles (EVs) are a promising source of RCC biomarkers because EVs carry proteins and messenger RNA (mRNA) among other biomolecules. We aimed to identify biomarkers and assess biological functions of EV cargo from clear cell RCC (ccRCC), papillary RCC (pRCC), and benign kidney cell lines. EVs were enriched from conditioned cell media by size exclusion chromatography. The EV proteome was assessed using Tandem Mass Tag mass spectrometry (TMT-MS) and NanoString nCounter technology was used to profile 770 cancer-related mRNA present in EVs. The heterogeneity of protein and mRNA abundance and identification highlighted the heterogeneity of EV cargo, even between cell lines of a similar pathological group (e.g., ccRCC or pRCC). Overall, 1726 proteins were quantified across all EV samples, including 181 proteins that were detected in all samples. In the targeted profiling of mRNA by NanoString, 461 mRNAs were detected in EVs from at least one cell line, including 159 that were present in EVs from all cell lines. In addition to a shared EV cargo signature, pRCC, ccRCC, and/or benign renal cell lines also showed unique signatures. Using this multi-omics approach, we identified 34 protein candidate pRCC EV biomarkers and 20 protein and 8 mRNA candidate ccRCC EV biomarkers for clinical validation.
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2021
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Detection of Tumor-Associated Membrane Receptors on Extracellular Vesicles from Non-Small Cell Lung Cancer Patients via Immuno-PCR
Precision cancer medicine for non-small-cell lung cancer (NSCLC) has increased patient survival. Nevertheless, targeted agents towards tumor-associated membrane receptors only result in partial remission for a limited time, calling for approaches which allow longitudinal treatment monitoring. Rebiopsy of tumors in the lung is challenging, and metastatic lesions may have heterogeneous signaling. One way ahead is to use liquid biopsies such as circulating tumor DNA or small extracellular vesicles (sEVs) secreted by the tumor into blood or other body fluids. Herein, an immuno-PCR-based detection of the tumor-associated membrane receptors EGFR, HER2, and IGF-1R on CD9-positive sEVs from NSCLC cells and pleural effusion fluid (PE) of NSCLC patients is developed utilizing DNA conjugates of antibody mimetics and affibodies, as detection agents. Results on sEVs purified from culture media of NSCLC cells treated with anti-EGFR siRNA, showed that the reduction of EGFR expression can be detected via immuno-PCR. Protein profiling of sEVs from NSCLC patient PE samples revealed the capacity to monitor EGFR, HER2, and IGF-1R with the immuno-PCR method. We detected a significantly higher EGFR level in sEVs derived from a PE sample of a patient with an EGFR-driven NSCLC adenocarcinoma than in sEVs from PE samples of non-EGFR driven adenocarcinoma patients or in samples from patients with benign lung disease. In summary, we have developed a diagnostic method for sEVs in liquid biopsies of cancer patients which may be used for longitudinal treatment monitoring to detect emerging bypassing resistance mechanisms in a noninvasive way.
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2021
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Development of a new methodology to determine size differences of nanoparticles with nanoparticle tracking analysis
The current frontiers in Biology thus in Medicine and Pharmacy are at the nanoscale. Indeed, this is the relevant scale for extracting or synthetizing, visualizing, counting, characterizing and/or modifying nanoparticles. Nanoparticles are highly diverse including: extracellular vesicles (e.g.: exosomes), proteins, viruses and nanovectors or drug delivery systems for instance. To quantify the concentration of nano-sized objects, a growing number of size-tracking instruments is being developed. However, to date, the generated data is only used to provide a concentration measurement. The objective of this study was to determine which sizes of nanoparticles are statistically significant between 2 groups of samples. Using different samples (in silico data; calibrated beads; various biological samples), an approach that statistically compares 2 groups of samples was developed and validated. The proof of concept of the proposed approach was illustrated with applications in the field of Biology, Medicine and Pharmacy using liposomes and extracellular vesicles. For the first time to our knowledge, our results suggest that the presented approach enables comparing different groups of biological samples. It may be extended to situations such as batch 1 versus batch 2; healthy versus disease or non-treated versus treated.
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2021
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Development of fast, reliable and automated isolation and fractionation methods for nanosized subpopulations of human biomacromolecules
This doctoral thesis describes the development of fast, reliable and automated isolation and fractionation methods for nanosized subpopulations of human biomacromolecules. The focus of the study was on subpopulations of lipoproteins and extracellular vesicles (EVs) that are important in the detection of different diseases, such as atherosclerotic cardiovascular diseases and cancer, and may even possess therapeutic potential. In the thesis, immunoaffinity chromatography (IAC) with selective antibodies immobilized on the monolithic disk columns were utilized for the selective isolation of biomacromolecules from human plasma, while asymmetrical flow field-flow fractionation (AsFlFFF or AF4) was able to fractionate relevant subpopulations of biomacromolecules (e.g., small dense low-density lipoproteins, exomeres, and exosomes) from the isolates. Continuous flow quartz crystal microbalance (QCM) and partial filling affinity capillary electrophoresis (PF-ACE) were employed to study the affinity of the interactions between the antibody and lipoproteins. The first step was to develop a method to study interactions between antibody and lipoproteins to select a high affinity antibody useful for the isolation of lipoprotein subpopulations by IAC. The interaction data obtained with PF-ACE was analyzed to determine the heterogeneity of the interactions with adsorption energy distribution calculations, while the QCM data was processed with interaction maps. The affinity constants obtained with QCM and PF-ACE agreed well with each other. Next, the IAC methods were developed to capture EVs of different cellular origins from human plasma using anti-CD9 monoclonal antibody (mAb), while anti-CD61 mAb was exploited to capture platelet-derived EVs. The anti-apolipoprotein B-100 (anti-apoB-100) mAb was exploited to immunocapture apoB-100 containing lipoproteins. The anti-apoB-100 mAb was also characterized by the PF-ACE and QCM studies. Appropriate elution conditions were found for the IAC methods, which has often been an issue with magnetic beads-based immunoaffinity methods. Since IAC allowed selective isolation of EVs and lipoproteins, a size-based separation to their subpopulations with AsFlFFF was introduced as a successive step. This enabled additional characterization of subpopulations by nanoparticle tracking analysis, western blotting, electron microscopy, capillary electrophoresis coupled with laser-induced fluorescent detection, zeta potential measurements, as well as free amino acids and glucose analysis with hydrophilic interaction liquid chromatography-tandem mass spectrometry. Finally, IAC was successfully on-line coupled to AsFlFFF, resulting in quick and automated isolation and fractionation of the subpopulations of EVs and lipoproteins. The constructed IAC-AsFlFFF system was able to process reliably 18–38 samples in 24 h with only minor operator involvement, resulting in highly reproducible and gentle fractionation of EV subpopulations in the size range of exomeres and exosomes. Polymeric monolithic disk columns were utilized for the first time for the IAC-based isolation of EVs and their subpopulations from human plasma, and for the detection of exomeres in CD9+ EVs and CD61+ platelet-derived EVs from human plasma samples. The results demonstrated that CD61+ EVs are potentially taking part in gluconeogenesis based on free amino acids and glucose present as cargo.
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2021
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Diagnostic potential of extracellular vesicle‑associated microRNA‑10b and tumor markers for lung adenocarcinoma
MicroRNAs (miRNAs/miRs) in extracellular vesicles (EVs) are potential diagnostic markers. The purpose of the present study was to investigate potential EV miRNA biomarkers for lung adenocarcinoma (LUAD). Potential miRNAs were identified by searching public databases and verified by examining clinical samples. The diagnostic value of EV‑associated miR‑10b, plasma miR‑10b and tumor markers (TMs), including α‑fetoprotein (AFP), neuron‑specific enolase, carcinoembryonic antigen (CEA), cytokeratin 19 fragment 21‑1 (CYFRA211), pro‑gastrin‑releasing‑peptide, carbohydrate antigen (CA)125, CA153, CA199 and CA724, was evaluated via receiver operating characteristic curve analysis. By searching the Gene Expression Omnibus and The Cancer Genome Atlas databases, miR‑10b was identified as a potential biomarker. The analysis of clinical samples suggested that EV‑associated miR‑10b from plasma was significantly differentially expressed between LUAD and control samples. EV‑associated miR‑10b could function as a diagnostic marker for LUAD, with an AUC of 0.998, which was higher than the AUCs for TMs such as AFP, CEA, CYFRA211, CA125, CA153, CA199, CA724, pro‑gastrin‑releasing‑peptide and neuron‑specific enolase. In conclusion, EV‑associated miR‑10b may be a potential diagnostic biomarker for LUAD that is superior to plasma miR‑10b and TMs.
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2021
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Dually targeted bioinspired nanovesicle delays advanced prostate cancer tumour growth in vivo
Prostate cancer (PC) is second-leading cancer in men, with limited treatment options available for men with advanced and metastatic PC. Prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) have been exploited as therapeutic targets in PC due to their upregulation in the advanced stages of the disease. To date, several PSA- and PSMA-activatable prodrugs have been developed to reduce the systemic toxicity of existing chemotherapeutics. Bioinspired nanovesicles have been exploited in drug delivery, offering prolonged drug blood circulation and higher tumour accumulation. For the first time, this study describes the engineering of dually targeted PSA/PSMA nanovesicles for advanced PC. PSMA-targeted bioinspired hybrids were prepared by hydrating a lipid film with anti-PSMA-U937 cell membranes and DOX-PSA prodrug, followed by extrusion. The bioinspired hybrids were characterised using dynamic light scattering, transmission electron microscopy, Dot blot, flow cytometry and Western blot. Cellular binding and toxicity studies in PC cancer cell lines were carried out using flow cytometry, confocal microscopy, and resazurin assay. Finally, tumour targeting and therapeutic efficacy studies were performed in solid and metastatic C4-2B-tumor-bearing mice. Interestingly, our PSMA-targeted hybrids demonstrated high cell uptake in PSMA-expressing cells with significant accumulation in solid and metastatic C4-2B tumour tissues following intravenous administration. More promisingly, our dually targeted PSA/PSMA hybrid significantly slowed down the C4-2B tumour growth in vivo, compared to free DOX-PSA and non-targeted PSA-hybrid. Our PSA/PSMA bioinspired hybrid could offer a highly selective treatment for advanced PC with lower side effects. Statement of significance This study investigates a new approach to treat prostate cancer using dually targeted bioinspired nanovesicle . Our bioinspired vesicles are made mainly of a human blood cell membrane with a ligand recognising a specific marker (PSMA) on the surface of the prostate cancer cells. The present work describes the successful loading of a doxorubicin prodrug linked to a PSA- activatable peptide into these targeted bioinspired nanovesicle , where the active PSA enzyme presents in these cells converts the drug to its active form. Our dually targeted PSA/PSMA hybrid vesicles has successfully improved site-specific prodrug delivery to tackle advanced prostate cancer, offering a novel and effective prostate cancer treatment.
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2021
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Effects of endurance racing on horse plasma extracellular particle miRNA
Background Physical exercise is an essential factor in preventing and treating metabolic diseases by promoting systemic benefits throughout the body. The molecular factors involved in this process are poorly understood. Micro RNAs (miRNAs) are small non‐coding RNAs that inhibit mRNA transcription. MiRNAs, which can participate in the benefits of exercise to health, circulate in plasma in extracellular particles (EP). Horses that undergo endurance racing are an excellent model to study the impact of long‐duration/low intensity exercise in plasma EP miRNAs. Objectives To evaluate the effects of 160 km endurance racing on horse plasma extracellular particles and their miRNA population. Study design Cohort study. Methods We collected plasma from 5 Arabian horses during five time‐points of an endurance ride. Extracellular particles were purified from plasma and characterised by electron microscopy, resistive pulse sensing (qNano), and western blotting. Small RNAs were purified from horse plasma EP, and sequencing was performed. Results Endurance racing increased EP concentration and average diameter compared to before the race. Western blotting showed a high concentration of extracellular vesicles proteins 2 h after the race, which returned to baseline 15 h after the race. MicroRNA differential expression analysis revealed increasing levels of eca‐miR‐486‐5p during and after the race, and decreasing levels of eca‐miR‐9083 after the end. Conclusions This study adds new data about the variation in plasma EP concentrations after long‐distance exercise and brings new insights about the roles of exercise‐derived EP miRNAs during low‐intensity endurance exercise.
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2021
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Effects of exercise on exosome release and cargo in in vivo and ex vivo models: A systematic review
Exercise-released exosomes have been identified as novel players to mediate cell-to-cell communication in promoting systemic beneficial effects. This review aimed to systematically investigate the effects of exercise on exosome release and cargo, as well as provide an overview of their physiological implications. Among the 436 articles obtained in the database search (WOS, Scopus, and PubMed), 19 articles were included based on eligibility criteria. Results indicate that exercise promotes the release of exosomes without modification of its vesicle size. The literature has primarily shown an exercise-driven increase in exosome markers (Alix, CD63, CD81, and Flot-1), along with other exosome-carried proteins, into circulation. However, exosome isolation, characterization, and phenotyping methodology, as well as timing of sample recovery following exercise can influence the analysis and interpretation of findings. Moreover, a large number of exosome-carried microRNAs (miRNAs), including miR-1, miR-133a, miR-133b, miR-206, and miR-486, in response to exercise are involved in the modulation of proliferation and differentiation of skeletal muscle tissue, although antigen-presenting cells, leukocytes, endothelial cells, and platelets are the main sources of exosome release into the circulation. Collectively, with the physiological implications as evidenced by the ex vivo trials, the release of exercise-promoted exosomes and their cargo could provide the potential therapeutic applications via the role of intercellular communication.
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2021
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Effects of exosomes derived from human umbilical vein endothelial cells on apoptosis of pre-chondrogenic cells stimulated by inflammatory factors
·To investigate the effect of exosomes derived from umbilical vein endothelial cells (HUVECs) on apoptosis of murine pre-chondrogenic cell line ATDC5 cells under inflammatory stimulation. ·The exosomes derived from HUVECs were isolated by using an exosome isolation kit. Western blotting was used to detect the exosome marker proteins, including tumor susceptibility gene 101 (Tsg101), cluster differentiation 9 (CD9) and apoptosis linked gene-2-interacting protein X (Alix). The morphology of exosomes was observed by transmission electron microscope, and the size of exosomes was identified by particle size detection. Fluorescence microscope was used to observe the ATDC5 cell uptake of exosomes and the production of reactive oxygen species (ROS). TUNEL staining and flow cytometry were used to examine the effect of exosomes on ATDC5 cell apoptosis stimulated by interleukin-1β (IL-1β). Western blotting was used to detect the effect of exosomes on the expression levels of ATDC5 apoptosis-related proteins such as B-cell lymphoma/leukemia 2 (Bcl-2), Bcl-2 associated X protein (Bax), cleaved caspase-3 (c-caspase-3) and anti-oxidative stress-related proteins such as nuclear factor E2 related factor 2 (Nrf-2), Kelch-like ECH-associated protein 1 (Keap-1), heme oxygenase 1 (HO-1) and NADPH quinone oxidoreductase-like protein 1 (NQO-1) under IL-1β stimulation. ·Under the transmission electron microscope, the HUVEC-derived exosomes were oval, hollow, double-layered, and positively expressed exosome markers CD9, Alix and Tsg101. Compared with the ATDC5 cells stimulated by IL-1β, ATDC5 cells stimulated by IL-1β incubated with exosomes had higher level of ROS (P=0.000) and higher apoptosis rate (P=0.000). The expression of Bax, c-caspase-3 and Keap-1 increased, and the expression of Bcl-2, Nrf-2, HO-1 and NQO-1 decreased in ATDC5 cells exposed to IL-1β and exosomes compared to ATDC5 cells only exposed to IL-1β. ·HUVEC-derived exosomes may promote ATDC5 cells apoptosis under the stimulation of IL-1β by inhibiting the ability of ATDC5 cell to resist oxidative stress.
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2021
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Effects of Solidification Conditions on Grain Refinement Capacity of TiC in Directionally Solidified Ti6Al4V Alloy
In this study, the effects of solidification conditions on the grain refinement capacity of heterogeneous nuclei TiC in directionally solidified Ti6Al4V alloy were investigated using experimental and numerical approaches. Ti6Al4V powder with and without TiC particles in a Ti6Al4V sheath was melted and directionally solidified at various solidification rates via the floating zone melting method. In addition, by using the phase field method, the microstructural evolution of directionally solidified Ti6Al4V was simulated by varying the temperature gradient G and solidification rate V. As the solidification rate increased, the increment of the prior β grain number by TiC addition also increased. There are two reasons for this: first, the amount of residual potent heterogeneous nuclei TiC is larger. Second, the amount of TiC particles that can nucleate becomes larger. This is because increasing the constitutional undercooling ΔTc leads to the activation of a smaller radius of heterogeneous nuclei and a higher nucleation probability from each radius. At a cooling rate R higher than that in the floating zone melting experiment (R = 3 to 1000 K/s), the maximum degree of constitutional undercooling ΔTc,Max has a peak value, which suggests that constitutional undercooling ΔTc has a smaller contribution at higher cooling rates, such as those that occur during electron beam melting (EBM), including laser powder bed fusion (LPBF).
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2021
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Embryonic stem cell-derived exosomes attenuate transverse aortic constriction induced heart failure by increasing angiogenesis
Background: Although there are concerns regarding their clinical use, embryonic stem cells (ESCs) hold a great promise for cardiac repair. Exosomes deriving from ESCs constitute a promising alternative for heart restoration. However, their effects in hypertension-induced heart failure are still unknown. Objective and Methods: To investigate the effects of ESCs-derived exosomes on hypertension-induced heart failure and the underlying mechanisms, sustained transverse aortic constriction (TAC) was performed on 8-week-old C57BL/6 male mice. After 1 months, ESCs-derived exosomes were isolated and injected intravenously once a week for 6 weeks. Echocardiography, wheat germ agglutinin (WGA), Masson staining, immunohistochemistry, and tube formation assays were all involved in our study. Results: Proteomics analyses revealed that ESC-derived exosomes contain FGF2 protein. Tube formation induced by these exosomes could be inhibited by FGF2R siRNA interference. ESCs-derived exosomes evidently attenuated TAC-induced heart failure, improving cardiac function and promoting myocardial angiogenesis which can be attenuated by selective FGF2 inhibitor AZD4547. Conclusions: ESC-derived exosomes attenuate TAC-induced heart failure mostly by promoting myocardial angiogenesis. FGF2 signaling plays a vital role in the myocardial angiogenesis induced by ESC-derived exosomes. Keywords: embryonic stem cells, exosomes, angiogenesis, transverse aortic constriction, heart failure
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2021
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Endothelial Progenitor Cell-Derived Extracellular Vesicles: Potential Therapeutic Application in Tissue Repair and Regeneration
Recently, many studies investigated the role of a specific type of stem cell named the endothelial progenitor cell (EPC) in tissue regeneration and repair. EPCs represent a heterogeneous population of mononuclear cells resident in the adult bone marrow. EPCs can migrate and differentiate in injured sites or act in a paracrine way. Among the EPCs’ secretome, extracellular vesicles (EVs) gained relevance due to their possible use for cell-free biological therapy. They are more biocompatible, less immunogenic, and present a lower oncological risk compared to cell-based options. EVs can efficiently pass the pulmonary filter and deliver to target tissues different molecules, such as micro-RNA, growth factors, cytokines, chemokines, and non-coding RNAs. Their effects are often analogous to their cellular counterparts, and EPC-derived EVs have been tested in vitro and on animal models to treat several medical conditions, including ischemic stroke, myocardial infarction, diabetes, and acute kidney injury. EPC-derived EVs have also been studied for bone, brain, and lung regeneration and as carriers for drug delivery. This review will discuss the pre-clinical evidence regarding EPC-derived EVs in the different disease models and regenerative settings. Moreover, we will discuss the translation of their use into clinical practice and the possible limitations of this process.
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2021
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Enhancing the Stabilization Potential of Lyophilization for Extracellular Vesicles
Extracellular vesicles (EV) are an emerging technology as immune therapeutics and drug delivery vehicles. However, EVs are usually stored at −80 °C which limits potential clinical applicability. Freeze-drying of EVs striving for long-term stable formulations is therefore studied. The most appropriate formulation parameters are identified in freeze-thawing studies with two different EV types. After a freeze-drying feasibility study, four lyophilized EV formulations are tested for storage stability for up to 6 months. Freeze-thawing studies revealed improved colloidal EV stability in presence of sucrose or potassium phosphate buffer instead of sodium phosphate buffer or phosphate-buffered saline. Less aggregation and/or vesicle fusion occurred at neutral pH compared to slightly acidic or alkaline pH. EVs colloidal stability can be most effectively preserved by addition of low amounts of poloxamer 188. Polyvinyl pyrrolidone failed to preserve EVs upon freeze-drying. Particle size and concentration of EVs are retained over 6 months at 40 °C in lyophilizates containing 10 mm K- or Na-phosphate buffer, 0.02% poloxamer 188, and 5% sucrose. The biological activity of associated beta-glucuronidase is maintained for 1 month, but decreased after 6 months. Here optimized parameters for lyophilization of EVs that contribute to generate long-term stable EV formulations are presented.
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2021
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Enzymatically active apurinic/apyrimidinic endodeoxyribonuclease 1 is released by mammalian cells through exosomes
The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1), the main AP-endonuclease of the DNA base excision repair pathway, is a key molecule of interest to researchers due to its unsuspected roles in different nonrepair activities, such as: i) adaptive cell response to genotoxic stress, ii) regulation of gene expression, and iii) processing of microRNAs, which make it an excellent drug target for cancer treatment. We and others recently demonstrated that APE1 can be secreted in the extracellular environment and that serum APE1 may represent a novel prognostic biomarker in hepatocellular and non-small-cell lung cancers. However, the mechanism by which APE1 is released extracellularly was not described before. Here, using three different approaches for exosomes isolation: commercial kit, nickel-based isolation, and ultracentrifugation methods and various mammalian cell lines, we elucidated the mechanisms responsible for APE1 secretion. We demonstrated that APE1 p37 and p33 forms are actively secreted through extracellular vesicles (EVs), including exosomes from different mammalian cell lines. We then observed that APE1 p33 form is generated by proteasomal-mediated degradation and is enzymatically active in EVs. Finally, we revealed that the p33 form of APE1 accumulates in EVs upon genotoxic treatment by cisplatin and doxorubicin, compounds commonly found in chemotherapy pharmacological treatments. Taken together, these findings provide for the first time evidence that a functional Base Excision Repair protein is delivered through exosomes in response to genotoxic stresses, shedding new light into the complex noncanonical biological functions of APE1 and opening new intriguing perspectives on its role in cancer biology.
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2021
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Estradiol driven metabolism in transwomen associates with reduced circulating extracellular vesicle microRNA-224/452
Objective Sex steroid hormones like estrogens have a key role in the regulation of energy homeostasis and metabolism. In transwomen, gender-affirming hormone therapy like estradiol (in combination with antiandrogenic compounds) could affect metabolism as well. Given that the underlying pathophysiological mechanisms are not fully understood, this study assessed circulating estradiol-driven microRNAs (miRs) in transwomen and their regulation of genes involved in metabolism in mice. Methods Following plasma miR-sequencing (seq) in a transwomen discovery (n = 20) and validation cohort (n = 30), we identified miR-224 and miR-452. Subsequent systemic silencing of these miRs in male C57Bl/6 J mice (n = 10) was followed by RNA-seq-based gene expression analysis of brown and white adipose tissue in conjunction with mechanistic studies in cultured adipocytes. Results Estradiol in transwomen lowered plasma miR-224 and -452 carried in extracellular vesicles (EVs) while their systemic silencing in mice and cultured adipocytes increased lipogenesis (white adipose) but reduced glucose uptake and mitochondrial respiration (brown adipose). In white and brown adipose tissue, differentially expressed (miR target) genes are associated with lipogenesis (white adipose) and mitochondrial respiration and glucose uptake (brown adipose). Conclusion This study identified an estradiol-drive post-transcriptional network that could potentially offer a mechanistic understanding of metabolism following gender-affirming estradiol therapy.
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2021
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Evaluating comparative β-glucan production aptitude of Saccharomyces cerevisiae, Aspergillus oryzae, Xanthomonas campestris, and Bacillus natto
β-glucan is a natural polysaccharide derivative composed of a group of glucose monomers with β-glycoside bonds that can be synthesized intra- or extra-cellular by various microorganisms such as yeasts, bacteria, and moulds. The study aimed to discover the potential of various microorganisms such as Saccharomyces cerevisiae, Aspergillus oryzae, Xanthomonas campestris, and Bacillus natto in producing β-glucan. The experimental method used and the data were analyzed descriptively. The four microorganisms above were cultured under a submerged state in Yeast glucose (YG) broth for 120 h at 30 °C with 200 rpm agitation. During the growth, several parameters were examined including total population by optical density, the pH, and glucose contents of growth media. β-glucan was extracted using acid-alkaline methods from the growth media then the weight was measured. The results showed that S. cerevisiae, A. oryzae X. campestris, and B. natto were prospective for β-glucans production in submerged fermentation up to 120 h. The highest β-glucans yield was shown by B. natto (20.38%) with the β-glucans mass of 1.345 ± 0.08 mg and globular diameter of 600 μm. The highest β-glucan mass was achieved by A. oryzae of 82.5 ± 0.03 mg with the total population in optical density of 0.1246, a final glucose level of 769 ppm, the pH of 6.67, and yield of 13.97% with a globular diameter of 1400 μm.
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2021
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Evidence of Immune Modulators in the Secretome of the Equine Tapeworm Anoplocephala perfoliata
Anoplocephala perfoliata is a neglected gastro-intestinal tapeworm, commonly infecting horses worldwide. Molecular investigation of A. perfoliata is hampered by a lack of tools to better understand the host–parasite interface. This interface is likely influenced by parasite derived immune modulators released in the secretome as free proteins or components of extracellular vesicles (EVs). Therefore, adult RNA was sequenced and de novo assembled to generate the first A. perfoliata transcriptome. In addition, excretory secretory products (ESP) from adult A. perfoliata were collected and EVs isolated using size exclusion chromatography, prior to proteomic analysis of the EVs, the EV surface and EV depleted ESP. Transcriptome analysis revealed 454 sequences homologous to known helminth immune modulators including two novel Sigma class GSTs, five α-HSP90s, and three α-enolases with isoforms of all three observed within the proteomic analysis of the secretome. Furthermore, secretome proteomics identified common helminth proteins across each sample with known EV markers, such as annexins and tetraspanins, observed in EV fractions. Importantly, 49 of the 454 putative immune modulators were identified across the secretome proteomics contained within and on the surface of EVs in addition to those identified in free ESP. This work provides the molecular tools for A. perfoliata to reveal key players in the host–parasite interaction within the horse host.
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2021
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Evidence of Immune Modulators in the Secretome of the Equine Tapeworm Anoplocephala perfoliata. Pathogens 2021, 10, 912
Anoplocephala perfoliata is a neglected gastro-intestinal tapeworm, commonly infecting horses worldwide. Molecular investigation of A. perfoliata is hampered by a lack of tools to better understand the host–parasite interface. This interface is likely influenced by parasite derived immune modulators released in the secretome as free proteins or components of extracellular vesicles (EVs). Therefore, adult RNA was sequenced and de novo assembled to generate the first A. perfoliata transcriptome. In addition, excretory secretory products (ESP) from adult A. perfoliata were collected and EVs isolated using size exclusion chromatography, prior to proteomic analysis of the EVs, the EV surface and EV depleted ESP. Transcriptome analysis revealed 454 sequences homologous to known helminth immune modulators including two novel Sigma class GSTs, five α-HSP90s, and three α-enolases with isoforms of all three observed within the proteomic analysis of the secretome. Furthermore, secretome proteomics identified common helminth proteins across each sample with known EV markers, such as annexins and tetraspanins, observed in EV fractions. Importantly, 49 of the 454 putative immune modulators were identified across the secretome proteomics contained within and on the surface of EVs in addition to those identified in free ESP. This work provides the molecular tools for A. perfoliata to reveal key players in the host–parasite interaction within the horse host.
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2021
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Exosomal CD47 plays an essential role in immune evasion in ovarian cancer
Ovarian cancer is largely diagnosed at advanced stages upon detection of multiple peritoneal dissemination, resulting in poor outcomes. CD47 is overexpressed in tumors, facilitates tumor immune evasion, and is located on exosomes. We aimed to investigate the role of exosomal CD47 in ovarian cancer progression. Prognostic significance of CD47 expression in ovarian cancer was examined using a public database including 1,435 patients and validated with 26 patients at our institution. CD47 expression was associated with poor progression-free survival and inversely correlated with macrophage infiltration in ovarian cancer tissues. Exosomes were collected from ovarian cancer cell lines, and CD47 expression on exosomes was confirmed via flow cytometry. Inhibition of exosome secretion with GW4869 and exosome uptake with 5-(N-ethyl-N-isopropyl)-amiloride inhibited the surface CD47 expression on ovarian cancer cells and promoted phagocytosis by macrophages. RAB27A (a key regulator of exosome release) knockdown inhibited exosome secretion and led to CD47 downregulation in ovarian cancer cells. In a xenograft mouse model, suppression of the release of tumor-derived exosomes by GW4869 or RAB27A knockdown suppressed tumor progression and enhanced M1 macrophage phagocytosis in cancer tissues. Collectively, CD47 expression was correlated with poor prognoses in patients with ovarian cancer, suggesting the importance of immune evasion. CD47 was expressed on exosomes and the inhibition of exosome secretion and/or uptake enhanced cancer cell phagocytosis by macrophages, and thus, suppressed peritoneal dissemination. This suggests the potential of a novel immune checkpoint therapeutic agent that focuses on exosomes. Implications: Mechanistic insight from the current study suggests that exosomal CD47 may be an advantageous therapeutic target in ovarian cancer.
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2021
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Exosome-Depleted Excretory-Secretory Products of the Fourth-Stage Larval Angiostrongylus cantonensis Promotes Alternative Activation of Macrophages Through Metabolic Reprogramming by the PI3K-Akt Pathway
Angiostrongylus cantonensis (AC), which parasitizes in the brain of the non-permissive host, such as mouse and human, is an etiologic agent of eosinophilic meningitis. Excretory-secretory (ES) products play an important role in the interaction between parasites and hosts’ immune responses. Inflammatory macrophages are responsible for eosinophilic meningitis induced by AC, and the soluble antigens of Angiostrongylus cantonensis fourth stage larva (AC L4), a mimic of dead AC L4, aggravate eosinophilic meningitis in AC-infected mice model via promoting alternative activation of macrophages. In this study, we investigated the key molecules in the ES products of AC L4 on macrophages and observed the relationship between metabolic reprogramming and the PI3K-Akt pathway. First, a co-culture system of macrophage and AC L4 was established to define the role of AC L4 ES products on macrophage polarization. Then, AC L4 exosome and exosome-depleted excretory-secretory products (exofree) were separated from AC L4 ES products using differential centrifugation, and their distinct roles on macrophage polarization were confirmed using qPCR and ELISA experiments. Moreover, AC L4 exofree induced alternative activation of macrophages, which is partially associated with metabolic reprogramming by the PI3K-Akt pathway. Next, lectin blot and deglycosylation assay were done, suggesting the key role of N-linked glycoproteins in exofree. Then, glycoproteomic analysis of exofree and RNA-seq analysis of exofree-treated macrophage were performed. Bi-layer PPI network analysis based on these results identified macrophage-related protein Hexa as a key molecule in inducing alternative activation of macrophages. Our results indicate a great value for research of helminth-derived immunoregulatory molecules, which might contribute to drug development for immune-related diseases. Keywords: Angiostrongylus cantonensis, exosome-depleted excretory-secretory products, N-linked glycoproteins, macrophage polarization, mechanism
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2021
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Exosome-mediated mRNA delivery for SARS-CoV-2 vaccination
Background In less than a year from its zoonotic entry into the human population, SARS-CoV-2 has infected more than 45 million people, caused 1.2 million deaths, and induced widespread societal disruption. Leading SARS-CoV-2 vaccine candidates immunize with the viral spike protein delivered on viral vectors, encoded by injected mRNAs, or as purified protein. Here we describe a different approach to SARS-CoV-2 vaccine development that uses exosomes to deliver mRNAs that encode antigens from multiple SARS-CoV-2 structural proteins. Approach Exosomes were purified and loaded with mRNAs designed to express (i) an artificial fusion protein, LSNME, that contains portions of the viral spike, nucleocapsid, membrane, and envelope proteins, and (ii) a functional form of spike. The resulting combinatorial vaccine, LSNME/SW1, was injected into thirteen weeks-old, male C57BL/6J mice, followed by interrogation of humoral and cellular immune responses to the SARS-CoV-2 nucleocapsid and spike proteins, as well as hematological and histological analysis to interrogate animals for possible adverse effects. Results Immunized mice developed CD4+, and CD8+ T-cell reactivities that respond to both the SARS-CoV-2 nucelocapsid protein and the SARS-CoV-2 spike protein. These responses were apparent nearly two months after the conclusion of vaccination, as expected for a durable response to vaccination. In addition, the spike-reactive CD4+ T-cells response was associated with elevated expression of interferon gamma, indicative of a Th1 response, and a lesser induction of interleukin 4, a Th2-associated cytokine. Vaccinated mice showed no sign of altered growth, injection-site hypersensitivity, change in white blood cell profiles, or alterations in organ morphology. Consistent with these results, we also detected moderate but sustained anti-nucleocapsid and anti-spike antibodies in the plasma of vaccinated animals. Conclusion Taken together, these results validate the use of exosomes for delivering functional mRNAs into target cells in vitro and in vivo, and more specifically, establish that the LSNME/SW1 vaccine induced broad immunity to multiple SARS-CoV-2 proteins. Competing Interest Statement S.J.G is a paid consultant for Capricor, holds equity in Capricor, and is co-inventor of intellectual property licensed by Capricor. S.J.T. is co-inventor of intellectual property licensed by Capricor. C.G. is co-inventor of intellectual property licensed by Capricor. N.A. is an employee of Capricor.
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2021
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Exosomes for Wound Treatment: Purification Optimization, Bioactive Components Identification and Drug Loading
The application of exosomes as therapeutic agents and drug delivery systems has gained increasing popularity over the last decades owing to their natural functions in intercellular communication processes. Exosomes are nanosized membrane vesicles of endosomal origin, which are constitutively released by cells into the extracellular space. They consist of functional proteins, nucleic acids and lipids, which enable them to imitate the biological functions of their producing parent cells. While proteins and nucleic acids have been identified as key players in the biological activity of exosomes, potential contributions of constitutional lipids to these effects remain largely unknown. Moreover, the purification process of exosomes continues to be a critical issue in exosome research since the definition of standardized exosome purification conditions is still pending. Several isolation methods are currently available, yet their potential impact on the exosome functionality has been rarely assessed in sufficient detail. Finally, when investigated as drug delivery platforms, mostly hydrophobic drugs have been loaded into exosomes, therefore leaving the loading capacity of current processes for hydrophilic classical drugs largely unaddressed. Furthermore, the impact of the loading methods on the exosome integrity and intrinsic bioactivity remains incompletely understood as the vesicle characterization is often restricted to analyzing their basic physicochemical properties and cellular uptake as well as monitoring the drug response. This thesis is aimed at addressing central questions related to the characterization of stem cell-derived exosomes as therapeutic entities and drug carriers, mainly in the context of wound healing. The major objectives specifically encompass 1) the optimization of exosome production and purification parameters, and an investigation of their effect on the exosome properties, 2) the identification of the role of selected exosomal components in processes required for wound healing, and 3) a comprehensive appraisal of the impact of several drug loading methods on the exosome integrity and functionality. Chapter 1 introduces the research field of exosomes and presents currently available purification methods. Moreover, the therapeutic applications of stem cell-derived exosomes are portrayed, focusing on their potential use in wound healing. Chapter 2 presents a synopsis of currently available synthetic carriers and exosomes as drug delivery platforms. In addition, drug encapsulation techniques for exosomes are presented and discussed. In Chapter 3, a standardized exosome preparation protocol is described. Special attention was paid to the interplay between production/purification conditions and exosome 2 characteristics to ultimately establish the optimal conditions delivering a high yield of bioactive exosomes. Subsequently, the activity of stem cell-derived exosomes in skin wound healing was assessed both in vitro and in vivo. The potential involvement of the transmembrane enzyme CD73 and exosomal lipids in the wound healing-promoting effects of stem cell exosomes was reported. It was found that the extent of the different exosome components’ activities was dependent on the target cell type. Specifically, CD73 contributed significantly to the in vitro migratory/mitogenic activity of stem cell exosomes on keratinocytes, but had no effect on endothelial cells. Exosomal lipids, on the other hand, were involved in the in vitro and in vivo activity of stem cell exosomes in blood vessel formation and maturation, but did not promote proliferation/migration of keratinocytes or fibroblasts in vitro. Chapter 4 explores processes for the encapsulation of non-membrane permeable hydrophilic low molecular weight compounds (i.e. pyranine and pentoxifylline) into exosomes. The loading efficiency of several methods was compared, and the osmotic shock procedure was identified as the most efficient one. Subsequently, the potential impact of the loading processes on the functionality of stem cell-derived exosomes was assessed using physicochemical characterization and biological activity methods. Only two out of five tested encapsulation processes (i.e. freeze-thawing and osmotic shock) preserved the structural and biological integrity of stem cell exosomes. In Chapter 5, the main findings of the current work are recapitulated and discussed. In addition, an outlook on yet unsolved challenges in the exosome research area is provided.
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2021
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Exosomes released by imatinib‑resistant K562 cells contain specific membrane markers, IFITM3, CD146 and CD36 and increase the survival of imatinib‑sensitive cells in the presence of imatinib
Chronic myeloid leukemia (CML) is a malignant hematopoietic disorder distinguished by the presence of a BCR‑ABL1 fused oncogene with constitutive kinase activity. Targeted CML therapy by specific tyrosine kinase inhibitors (TKIs) leads to a marked improvement in the survival of the patients and their quality of life. However, the development of resistance to TKIs remains a critical issue for a subset of patients. The most common cause of resistance are numerous point mutations in the BCR‑ABL1 gene, followed by less common mutations and multiple mutation-independent mechanisms. Recently, exosomes, which are extracellular vesicles excreted from normal and tumor cells, have been associated with drug resistance and cancer progression. The aim of the present study was to characterize the exosomes released by imatinib‑resistant K562 (K562IR) cells. The K562IR‑derived exosomes were internalized by imatinib‑sensitive K562 cells, which thereby increased their survival in the presence of 2 µM imatinib. The exosomal cargo was subsequently analyzed to identify resistance‑associated markers using a deep label‑free quantification proteomic analysis. There were >3,000 exosomal proteins identified of which, 35 were found to be differentially expressed. From this, a total of 3, namely the membrane proteins, interferon‑induced transmembrane protein 3, CD146 and CD36, were markedly upregulated in the exosomes derived from the K562IR cells, and exhibited surface localization. The upregulation of these proteins was verified in the K562IR exosomes, and also in the K562IR cells. Using flow cytometric analysis, it was possible to further demonstrate the potential of CD146 as a cell surface marker associated with imatinib resistance in K562 cells. Taken together, these results suggested that exosomes and their respective candidate surface proteins could be potential diagnostic markers of TKI drug resistance in CML therapy.
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2021
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Exosomes‐mediated transfer of long noncoding RNA LINC01133 represses bladder cancer progression via regulating the Wnt signaling pathway
Bladder cancer (BC), as one of the most common malignant cancers of the urinary system, has a high incidence and mortality rates. Recently, increasing studies have indicated that exosomes can mediate cellular communication in assorted cancers, including BC. Long noncoding RNAs (lncRNAs) have also been confirmed to take part in the regulation of many cancers. Long intergenic non-protein coding RNA 1133 (LINC01133) is an lncRNA and its roles in several cancers have been revealed. However, the functions of exosomes and LINC01133 in BC are still not elucidated. In our research, functional assays were conducted to evaluate the function of LINC01133, as well as the influence of exosomes and LINC01133 on BC cells. Western blot assay, immunofluorescence assay, electron microscope, and nanoparticle tracking analysis were applied for detecting the characteristics of exosomes. Bioinformatics tools and quantitative reverse-transcription polymerase chain reaction were performed to test the expression of LINC01133 in BC cells and exosomes of the immortalized human uroepithelial cell line (SV-HUC-1). Luciferase reporter assay was performed to measure the activity of the Wnt pathway. We discovered that LINC01133 expression was high in exosomes of SV-HUC-1 and low in that of BC cells. Additionally, exosomes restrained cell viability, proliferation, migration, and invasion. Similarly, LINC01133 exerted the same function on BC cells. In addition, the Wnt signaling pathway could be inactivated by LINC01133. Finally, in vivo experiments demonstrated that cell growth could be suppressed by overexpressed LINC01133. In short, exosomes-mediated transfer of lncRNA LINC01133 repressed BC progression via regulating the Wnt signaling pathway.
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2021
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