Precision porous templated scaffolds (PTS) are a hydrogel construct of uniformly sized interconnected spherical pores that induce a pro-healing response (reducing the foreign body reaction, FBR) ...exclusively when the pores are 30-40µm in diameter. Our previous work demonstrated the necessity of Tregs in the maintenance of PTS pore size specific differences in CD4+ T cell phenotype. Work here characterizes the role of Tregs in the responses to implanted 40µm and 100µm PTS using WT and FoxP3+ cell (Treg) depleted mice. Proteomic analyses indicate that integrin signaling, monocytes/macrophages, cytoskeletal remodeling, inflammatory cues, and vesicule endocytosis may participate in Treg activation and the CD4+ T cell equilibrium modulated by PTS resident cell-derived small extracellular vesicles (sEVs). The role of MyD88-dependent and MyD88-independent TLR4 activation in PTS cell-derived sEV-to-T cell signaling is quantified by treating WT, TLR4ko, and MyD88ko splenic T cells with PTS cell-derived sEVs. STAT3 and mTOR are identified as mechanisms for further study for pore-size dependent PTS cell-derived sEV-to-T cell signaling.
Unique cell populations colonizing only within 40µm pore size PTS generate sEVs that resolve inflammation by modifying CD4+ T cell phenotypes through TLR4 signaling.
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Small extracellular vesicles (sEVs) from mesenchymal stromal/stem cells (MSCs) are transiting rapidly towards clinical applications. However, discrepancies and controversies about the biology, ...functions, and potency of MSC-sEVs have arisen due to several factors: the diversity of MSCs and their preparation; various methods of sEV production and separation; a lack of standardized quality assurance assays; and limited reproducibility of in vitro and in vivo functional assays. To address these issues, members of four societies (SOCRATES, ISEV, ISCT and ISBT) propose specific harmonization criteria for MSC-sEVs to facilitate data sharing and comparison, which should help to advance the field towards clinical applications. Specifically, MSC-sEVs should be defined by quantifiable metrics to identify the cellular origin of the sEVs in a preparation, presence of lipid-membrane vesicles, and the degree of physical and biochemical integrity of the vesicles. For practical purposes, new MSC-sEV preparations might also be measured against a well-characterized MSC-sEV biological reference. The ultimate goal of developing these metrics is to map aspects of MSC-sEV biology and therapeutic potency onto quantifiable features of each preparation.
Small extracellular vesicles (sEVs) are largely classified into two types, plasma-membrane derived sEVs and endomembrane-derived sEVs. The latter type (referred to as exosomes herein) is originated ...from late endosomes or multivesicular bodies (MVBs). In order to release exosomes extracellularly, MVBs must fuse with the plasma membrane, not with lysosomes. In contrast to the mechanism responsible for MVB–lysosome fusion, the mechanism underlying the MVB–plasma membrane fusion is poorly understood. Here, we systematically analyze soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family proteins and identify VAMP5 as an MVB-localized SNARE protein required for exosome release. Depletion of VAMP5 in HeLa cells impairs exosome release. Mechanistically, VAMP5 mediates exosome release by interacting with SNAP47 and plasma membrane SNARE Syntaxin 1 (STX1) or STX4 to release exosomes. VAMP5 is also found to mediate asymmetric exosome release from polarized Madin-Darby canine kidney (MDCK) epithelial cells through interaction with the distinct sets of Q-SNAREs, suggesting that VAMP5 is a general exosome regulator in both polarized cells and non-polarized cells. Key words: Exosome, small extracellular vesicle (sEV), multivesicular body, SNARE, VAMP5
Extracellular vesicles (EVs), the key players in inter‐cellular communication, are produced by all cell types and are present in all body fluids. Analysis of the proteome content is an important ...approach in structural and functional studies of these vesicles. EVs circulating in human plasma are heterogeneous in size, cellular origin, and functions. This heterogeneity and the potential presence of contamination with plasma components such as lipoprotein particles and soluble plasma proteins represent a challenge in profiling the proteome of EV subsets by mass spectrometry. An immunocapture strategy prior to mass spectrometry may be used to isolate a homogeneous subpopulation of small EVs (sEV) with a specific endocytic origin from plasma or other biofluids. Immunocapture selectively separates EV subpopulations in biofluids based on the presence of a unique protein carried on the vesicle surface. The advantages and disadvantages of EV immune capture as a preparative step for mass spectrometry are discussed.
Small extracellular vesicles (sEVs) are important mediators of cell-cell communication with respect to diverse physiological processes. To further understand their physiological roles, understanding ...blood sEV homoeostasis in a quantitative manner is desired. In this study, we propose novel kinetic approaches to estimate the secretion and clearance of mouse plasma-derived sEVs (MP-sEVs) based on the hypothesis that blood sEV concentrations are determined by a balance between the secretion and clearance of sEVs. Using our specific and sensitive sEV labelling technology, we succeeded in analysing MP-sEV clearance from the blood after intravenous administration into mice. This revealed the rapid disappearance of MP-sEVs with a half-life of approximately 7 min. Moreover, the plasma sEV secretion rate, which is presently impossible to directly evaluate, was calculated as 18 μg/min in mice based on pharmacokinetic (PK) analysis. Next, macrophage-depleted mice were prepared as a model of disrupted sEV homoeostasis with retarded sEV clearance. MP-sEV concentrations were increased in macrophage-depleted mice, which probably reflected a shift in the balance of secretion and clearance. Moreover, the increased MP-sEV concentration in macrophage-depleted mice was successfully simulated using calculated clearance rate constant, secretion rate constant and volume of distribution, suggesting the validity of our PK approaches. These results demonstrate that blood sEV concentration homoeostasis can be explained by the dynamics of rapid secretion/clearance.
•This is the first time that multiplexed sEV-circRNAs analysis combined with machine learning for GC detection.•This platform for multiplexed sEV-circRNAs detection offering a non-invasive, highly ...sensitive approach for early GC diagnosis.•A sensitive and cost-effective electrochemical platform was developed using TDA tag coupled with DT-CHA.•MLP was chosen from five machine learning algorithms to analyze clinical electrochemical data for GC precise staging.
Small extracellular vesicle associated circular RNAs (sEV-circRNAs) are emerging as promising biomarkers for gastric cancer diagnosis. Current research predominantly focuses on identifying these biomarkers through high-throughput sequencing. However, there has been insufficient exploration into the practical application of sEV-circRNAs for early gastric cancer diagnosis. In this study, we developed a sensitive electrochemical platform that leverages tetrahedron-Dox-AuNPs (TDA) tag and DNA tetrahedron-enhanced catalytic hairpin assembly (DT-CHA) to detect sEV-circRNAs. Based on the dual signal amplification of the TDA tag and DT-CHA, the platform can achieve low-concentration detection of the target, with a detection limit of 153.1 aM and a linear range from 1 fM to 1 nM. By profiling four sEV-circRNAs (circNRIP1, circRANGAP1, circCORO1C, and circSHKBP1) in a gastric cancer cohort and combining suitable ML diagnostic model, this platform performed well in distinguishing healthy donors from early GC patients. Thus, this confluence of a multi-biomarker approach with machine learning analysis, applied to plasma sEV-circRNAs, emerges as an important strategy for cancer screening.
Loss of functionality during aging of cells and organisms is caused and accompanied by altered cell-to-cell communication and signalling. One factor thereby is the chronic accumulation of senescent ...cells and the concomitant senescence-associated secretory phenotype (SASP) that contributes to microenvironment remodelling and a pro-inflammatory status. While protein based SASP factors have been well characterized, little is known about small extracellular vesicles (sEVs) and their miRNA cargo. Therefore, we analysed secretion of sEVs from senescent human dermal fibroblasts and catalogued the therein contained miRNAs. We observed a four-fold increase of sEVs, with a concomitant increase of >80% of all cargo miRNAs. The most abundantly secreted miRNAs were predicted to collectively target mRNAs of pro-apoptotic proteins, and indeed, senescent cell derived sEVs exerted anti-apoptotic activity. In addition, we identified senescence-specific differences in miRNA composition of sEVs, with an increase of miR-23a-5p and miR-137 and a decrease of miR-625-3p, miR-766-3p, miR-199b-5p, miR-381-3p, miR-17-3p. By correlating intracellular and sEV-miRNAs, we identified miRNAs selectively retained in senescent cells (miR-21-3p and miR-17-3p) or packaged specifically into senescent cell derived sEVs (miR-15b-5p and miR-30a-3p). Therefore, we suggest sEVs and their miRNA cargo to be novel, members of the SASP that are selectively secreted or retained in cellular senescence.
Protein enhancer of sevenless 2B, E(sev)2B, is a key adapter protein in the Ras/MAPK signaling pathway which has been reported to be involved in innate immunity. In this study, the gene that encodes ...AsE(sev)2B was isolated from A. sinica. It was found to contain a 636 bp open reading frame encoding 211 amino acids with a calculated molecular mass of 24.357 kDa and a predicted isoelectric point of 5.39. The predicted protein contains a N-terminal Src homology 3 domain (SH3), a central Src homology 2 domain (SH2), and a C-terminal Src homology 3 domain (SH3). Homology analysis revealed that AsE(sev)2B shares 49%–95% identity with E(sev)2B homologs from other species. In this study, the expression pattern and location of AsE(sev)2B during different stages of embryonic development and bacterial challenge were investigated by means of real-time qPCR, Western blotting and immunohistochemistry. Results showed that the highest expression level of AsE(sev)2B was at 0 h. After challenged by Gram-positive bacteria and Gram-negative bacteria, AsE(sev)2B was remarkably upregulated at 106 cellsL−1 bacterial concentrations. These results suggested that AsE(sev)2B plays a vital role during early embryonic development and in immune responses against bacterial challenge.
•Open reading frame of E(sev)2B was isolated and characterized from Artemia sinica.•AsE(sev)2B was expressed at all developmental stages using RT-qPCR and Western blotting.•The signals of AsE(sev)2B were mainly detected in the cephalothorax by immunohistochemistry.•AsE(sev)2B was remarkably upregulated under stimulated by Gram-positive and Gram-negative bacteria.
Extracellular vesicles (EVs) are particles released from most cell types delimited by a lipid bilayer. Small EVs (sEVs) are nanosized (<200 nm) and include exosomes. Brain-derived sEVs may provide a ...source for new biomarkers of brain status. CD9, CD63, and CD81 are major members of the tetraspanin family frequently used as sEV markers. However, according to a recent report, tetraspanins were not equally expressed in all sEVs, but rather show heterogeneity that reflects the expression levels in their secretory cells. We therefore investigated tetraspanin heterogeneity of sEVs in biofluids commonly used for clinical laboratory tests, and those in the brain. Expression levels and distributions of CD9, CD63 and CD81 on sEVs were determined in serum, plasma, and cerebrospinal fluid (CSF) samples collected from each healthy donor, and in post-mortem brain tissue samples. We found heterogeneous mixes of sEVs with various tetraspanin combinations among sEVs, and the predominant types and heterogeneous patterns of tetraspanins were specific to sample type. Hierarchical clustering revealed that brain sEVs were similar to those in the CSF, but different from those in peripheral blood. Our findings both provide basic information and contribute to the development of biomarkers for neurological and psychiatric disorders.
•Expression and distribution of tetraspanins in biofluids and brain sEV were examined.•Heterogeneous patterns and predominant tetraspanins were specific for sample types.•Tetraspanin heterogeneity suggest close relationship between brain and CSF.
Studies revealed that the levels of small extracellular vesicle (sEV) programmed cell death-ligand 1 (PD-L1) may serve as a predictive biomarker for the clinical responses to immunotherapy. While, ...the currently available enzyme-linked immunosorbent assay (ELISA)-based quantitative analysis of sEV PD-L1 is tedious and time-consuming and have low sensitivity. Additionally, to avoid the interference signal from free forms of PD-L1, sEVs were isolated from plasma by ultracentrifugation with low recovery and rigorous steps. Since the mass and volume of PD-L1-positive sEVs were significantly larger than free forms of PD-L1, we developed a free-separation anisotropy probe (FSAP) for sensitive and rapid quantitative detection of sEV PD-L1 in human plasma samples. It could avoid the interference of free forms of PD-L1 and shorten the whole process to 30 min. The level of PD-L1-positive sEVs was quantified by FSAP with high sensitivity and low detection limit (375 sEVs/µL). We studied plasma samples from 15 oral squamous cell carcinoma patients (OSCCP) and 15 healthy donors (HD), we found that cancer patients have higher levels of sEVs than healthy donors. Most importantly, FSAP can detect amounts of PD-L1-positive sEVs in plasma samples that diluted more than 70 times and distinguish OSCC patients from healthy donors well.
•A biosensor free-separation anisotropy probe (FSAP) was constructed for the detection of sEV PD-L1 with separation-free.•The method avoids the interference of free forms of PD-L1, its sensitivity is higher than other traditional methods.•It has a low detection limit of 375 sEVs/µL and the testing process takes only 30 min.•Healthy donors also could be distinguished from patients through the plasma samples which were diluted 70 times.•sEV: small extracellular vesicle; PD-L1: programmed cell death-ligand 1
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