Acoustic purification of extracellular microvesicles.

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are released by most cell types into the extracellular space and represent the pathophysiological condition of their source cells. Recent studies demonstrate that EVs derived from infected cells and tumors contribute to disease pathogenesis. However, very few studies have rigorously characterized exosomes and microvesicles in infectious diseases. In this study, we focused on subpopulations of EVs during the human enterovirus infection and explored the distinct traits and functions of EVs. We construct an effective immunomagnetic method to isolate exosomes and MVs from enterovirus‐infected cells excluding virion. The morphology and sizes of exosomes and MVs have no significant alteration after enterovirus infection. Meanwhile, our study observed that the enterovirus infection could induce exosome secretion but not MVs. In vivo study showed that there was differential biodistribution between exosomes and MVs. Using deep RNA sequencing, we found that the cargo information in MVs rather than in exosomes could accurately reflect pathological condition of original cells. Our study demonstrated that it should be considered to use MVs as clinical diagnostics during in enterovirus infection because their composition is reflective of pathological changes.

Advances and challenges in sample preparation and analytical methods for water-soluble vitamins: Application in food, clinical, pharmaceutical samples.

Extracellular vesicles play a crucial role in feto-maternal communication and provide an important paracrine signaling mechanism in pregnancy. We hypothesized that fetal cells-derived exosomes and microvesicles (MVs) under oxidative stress (OS) carry unique cargo and traffic through feto-maternal interface, which cause inflammation in uterine cells associated with parturition. Exosomes and MVs, from primary amnion epithelial cell (AEC) culture media under normal or OS-induced conditions, were isolated by optimized differential centrifugation method followed by characterization for size (nanoparticle tracking analyzer), shape (transmission electron microscopy), and protein markers (western blot and immunofluorescence). Cargo and canonical pathways were identified by mass spectroscopy and ingenuity pathway analysis. Myometrial, decidual, and cervical cells were treated with 1 × 107 control/OS-derived exosomes/MVs. Pro-inflammatory cytokines were measured using a Luminex assay. Statistical significance was determined by paired T-test (P< 0.05). AEC produced cup-shaped exosomes of 90-150nm and circular MVs of 160-400nm. CD9, heat shock protein 70, and Nanog were detected in exosomes, whereas OCT-4, human leukocyte antigen G, and calnexin were found in MVs. MVs, but not exosomes, were stained for phosphatidylserine. The protein profiles for control versus OS-derived exosomes and MVs were significantly different. Several inflammatory pathways related to OS were upregulated that were distinct between exosomes and MVs. Both OS-derived exosomes and MVs significantly increased pro-inflammatory cytokines (granulocyte-macrophage colony-stimulating factor, interleukin 6 (IL-6), and IL-8) in maternal cells compared with control (P< 0.05). Our findings suggest that fetal-derived exosomes and MVs under OS exhibited distinct characteristics and a synergistic inflammatory role in uterine cells associated with the initiation of parturition.

Cell-derived microvesicles (MVs), which are biogenic nanosized membrane-bound vesicles that convey bioactive molecules between cells, have recently received attention for use as natural therapeutic platforms. However, the medical applications of MV-based delivery platforms are limited by the lack of effective methods for the efficient isolation of MVs and the convenient tuning of their targeting properties. Herein, we report the development of magnetic and folate (FA)-modified MVs based on a donor cell-assisted membrane modification strategy. MVs inherit the membrane properties of their donor cells, which allows them to be modified with the biotin and FA on their own membrane. By conjugating with streptavidin-modified iron oxide nanoparticles (SA-IONPs), the MVs can be conveniently, efficiently, and rapidly isolated from the supernatant of their donor cells using magnetic activated sorting. Moreover, the conjugated magnetic nanoparticles and FA confer magnetic and ligand targeting activities on the MVs. Then, the MVs were transformed into antitumor delivery platforms by directly loading doxorubicin via electroporation. The modified MVs exhibited significantly enhanced antitumor efficacy both in vitro and in vivo. Taken together, this study provides an efficient and convenient strategy for the simultaneous isolation of cell-derived MVs and transformation into targeted drug delivery nanovectors, thus facilitating the development of natural therapeutic nanoplatforms.

Rationale: Inadequate expression of suppressor of cytokine signaling 3 (SOCS3) with subsequent activation of its target, the transcription factor STAT3, has been implicated in tumorigenesis and cancer progression in the lung and other organs. Our lab has recently reported the novel capability of alveolar macrophages (AMs) to secrete SOCS3 within microvesicles (MVs). While AM delivery of MV-encapsulated SOCS3 was shown to suppress inflammatory signaling in recipient lung epithelial cells, the potential significance of this process in restraining the development of lung cancer has not been studied. Methods: A KRAS G12D mutant mouse model was utilized to determine dysfunction of AM SOCS3 secretion in lung cancer. Mice were administered adenoviral Cre recombinase via intratracheal instillation, resulting in formation of lung tumors after 16 weeks. Bronchoalveolar lavage (BAL) fluid and AMs were isolated from the lungs of KRAS and wild-type (WT) mice, and analysis of SOCS3 secretion in BAL or AM cell culture medium was done via ELISA after sonication to disrupt vesicles. In vitro experiments utilized human adenocarcinoma cells (A549) or KRAS mutant rat lung epithelial cells (RLE-G12V). Proliferation, apoptosis and transformation were assessed by Cyquant assay, Annexin V staining, and soft agar assay, respectively. For SOCS3 provision studies, natural AM-derived MVs (isolated by ultracentrifugation) or synthetic liposomes containing recombinant SOCS3 were utilized. Results: Levels of secreted SOCS3 were ~50% lower in KRAS mice BAL than in WT BAL fluid. Additionally, although AMs isolated from KRAS mice contained similar amounts of intracellular SOCS3 and released similar numbers of MVs as those from WT mice, their ex vivo capacity for SOCS3 secretion was significantly lower than that of WT AMs. To determine whether provision of exogenous SOCS3 could inhibit tumorigenesis, synthetic SOCS3 liposomes were administered to RLE-G12V cells prior to chemical transformation with N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG). Addition of exogenous SOCS3 had the capacity to significantly inhibit colony formation in soft agar. To investigate effects of exogenous SOCS3 on established tumor cell function, A549 cells were exposed to both natural AM-derived MVs and SOCS3-containing liposomes and effects on proliferation and apoptosis were measured. Both liposomes and natural MVs significantly induced apoptosis and inhibited proliferation. Finally, the reduction in secreted SOCS3 observed in the mouse model was confirmed in BAL samples of a cohort of NSCLC lung cancer patients compared to healthy volunteers. Conclusion: We report a novel dysregulation of immune surveillance in the form of decreased SOCS3 secretion by AMs that is elicited by the tumor microenvironment, and that may promote tumorigenesis via sustained STAT3 activation. Future studies will focus on the mechanism underlying this defect and whether rescuing SOCS3 secretion can inhibit cancer progression in vivo. Citation Format: Jennifer M. Speth, Loka R. Penke, Joseph Bazzill, Daniel J. Schneider, Douglas A. Arenberg, James J. Moon, Venkateshwar G. Keshamouni, Marc Peters-Golden. Vesicular secretion of suppressor of cytokine signaling 3 by alveolar macrophages is dysregulated in NSCLC and its provision inhibits tumor cell function [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A35.

Microvesicles (MVs) are extracellular vesicles secreted by various cell types that are involved in intercellular communication. We hypothesized that in human periodontal disease, the pocket epithelium releases MVs, which then modulate gene expression in the underlying fibroblasts to control periodontal inflammation. MVs were isolated from culture medium of gingival epithelial cells (GECs) treated with oral bacterial biofilm extract or left untreated. Biofilm treatment significantly increased MV release from the GECs. Mass spectrometry of GEC-MVs identified a total of 2,173 proteins, of which about 80% were detected in MVs from both control and biofilm-treated GECs. Among 80 signature genes of human gingival fibroblasts, 20 were significantly regulated (P < 0.05) by MVs from control and biofilm-treated GECs in a similar manner. Matrix metalloproteinase 1 and 3 and interleukin 6 and 8 showed the strongest regulation at the mRNA and protein levels. Several cellular signaling pathways were activated by GEC-MVs in human gingival fibroblasts, including Smad and mitogen-activated protein kinase–associated pathways ERK1/2, JNK, and p38. However, ERK1/2 signaling dominated in the MV-induced gene expression changes. The results demonstrate that GEC-MVs have a strong regulatory effect on the expression of fibroblast genes associated with inflammation and matrix degradation and that bacterial biofilm stimulates the generation of GEC-MVs. This suggests that bacterial biofilms can contribute to the initiation and progression of periodontal disease by promoting a tissue-destructive phenotype in gingival fibroblasts via the enhanced secretion of epithelial MVs.

While confocal fluorescence microscopy has provided valuable insights into chromatin organization in plant nuclei, its diffraction-limited resolution constrains the investigation of chromatin architecture, motivating the use of super-resolution techniques such asSingle-Molecule Localization Microscopy (SMLM). Among these approaches, direct stochastic optical reconstruction microscopy (dSTORM) provides nanoscale resolution in individual cells, enabling precise visualization of chromatin domains, histone modifications, and nuclear organization. While such methods are increasingly applied in mammalian systems, their use in plant biology remains limited, largely due to technical challenges in sample preparation. Here, we present a streamlined and reproducible workflow for SMLM imaging of nuclei isolated from Arabidopsis thaliana. This protocol starts with seedling fixation to preserve nuclear morphology, followed by gentle tissue chopping and centrifugation to enrich intact nuclei. Isolated nuclei are then fluorophore-labeled in liquid medium and immobilized on low-melting agarose pads, a strategy that enhances stability during prolonged single-molecule imaging sessions. These steps collectively minimize background fluorescence, improve labeling consistency, and increase reproducibility across biological replicates. The resulting preparations provide enhanced clarity for visualizing chromatin modifications and nuclear architecture in plants. By lowering the technical barriers to implement SMLM imaging in Arabidopsis, this protocol provides a versatile means to investigate epigenetic regulation, chromatin organization, and nuclear topological variations at the nanoscale. This work establishes a methodological foundation for applying SMLM to plants, bridging the gap with mammalian cell biology and opening new opportunities to study how nuclear architecture contributes to genome regulation in response to developmental and environmental cues in plant systems.

Intercellular communications between lung epithelial cells and alveolar macrophages play an essential role in host defense against acute lung injury. Hyperoxia-induced oxidative stress is an established model to mimic human lung injury. We show that after hyperoxia-associated oxidative stress, a large amount of extracellular vesicles (EVs) are detectable in bronchoalveolar lavage fluid (BALF) and culture medium of lung epithelial cells. Microvesicles (MVs), but not exosomes (Exos) or apoptotic bodies (Abs), are the main type of EVs found in the early stages after hyperoxia. Among all the MV compositions, small RNAs are altered the most significantly after hyperoxia-associated oxidative stress. We further confirmed that hyperoxia up-regulates the levels of certain specific miRNAs in the epithelial cell-derived MVs, such as the miR-320a and miR-221. Functionally, the hyperoxia-induced epithelial MVs promote macrophage activation in vitro and facilitate the recruitment of immunomodulatory cells in vivo detected in BALF. Using MV as a cargo, delivery of the specific miRNA-enriched epithelial MVs (miR-221 and/or miR-320a) also triggers macrophage-mediated pro-inflammatory effects. Collectively, epithelial cell-derived MVs promote macrophage-regulated lung inflammatory responses via MV-shuttling miRNAs.

Pre-analytical issues in the measurement of circulating microparticles: current recommendations and pending questions

Recent advances of boronate affinity materials in sample preparation

Wood-based materials such as composites or laminates play an important role in today's furniture industry, especially in manufacturing high-quality kitchen and dining room furniture. One important aspect after fabrication is the investigation of these materials to derive quality metrics such as surface stain and scuff resistance. Current sample preparation methods are mostly straightforward and rely on cutting and grinding the materials under test, including sensitive wood substrates. To investigate cross-sections and different layer topologies, characterisation techniques such as atomic force microscopy (AFM) and infrared (IR) microscopy are of potential interest. However, a huge limitation is that current sample preparation methods lead to smeared coatings on the sample cross-sections and high surface roughness. Hence, these methods are not applicable for the sample preparation in measuring AFM and IR microscopy. Therefore, new preparation techniques need to be developed accordingly. This article presents a new approach towards coated wood-based sample preparation including embedding processes to use those samples for AFM and IR microscopy technologies. The proposed method has been evaluated by obtaining AFM, IR and microscopy measurements of more than four different wood-based samples such as (i) raw paper, (ii) impregnated paper, (iii) melamine-coated chipboards and (iv) medium-density fibreboards. The investigation results showed a significant improvement in sample preparation, as well as clear chemical and physical characterisation over whole sample construction, including coating layers, for wood-based materials.

Microvesicles in the brain: Biomarker, messenger or mediator?

Biological and pharmaceutical samples represent formidable challenges in sample preparation that hold important consequences for bioanalysis and genotoxic impurity quantification. This Feature will emphasize significant advances toward the development of rapid, sensitive, and selective sample preparation methods.

Current trends and challenges in sample preparation for metallic nanoparticles analysis in daily products and environmental samples: A review

New challenges in sample preparation: Miniaturized stir bar sorptive dispersive microextraction as a high-throughput and feasible approach for low-availability sample preparation