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1. Human adenovirus type 3 restores pharmacologically inhibited exosomal cargo in lung carcinoma cells

   https://doi.org/10.3389/fphar.2024.1339862  

   Ipinmoroti, Ayodeji O; Pandit, Rachana; Crenshaw, Brennetta J; Sims, Brian; Matthews, Qiana L (February 2024, Frontiers in Pharmacology)

   Kalesh, Karunakaran (Ed.)

   Introduction:Drug repurposing is fast growing and becoming an attractive approach for identifying novel targets, such as exosomes for cancer and antiviral therapy. Exosomes are a specialized class of extracellular vesicles that serve as functional mediators in intercellular communication and signaling that are important in normal physiological functions. A continuously growing body of evidence has established a correlation between the abnormal release of exosomes with various viral disease pathologies including cancer. Cells that are virus-infected release exosomes known to influence the process via the loading and transfer of viral components, such as miRNA, small (s) RNA, DNA, and proteins. Inhibition of exosome release may abate the spread and severity of viral infection, thus making exosomes an attractive target for antiviral therapies. We previously demonstrated the pharmacological inhibition of exosomes. Methods:Herein, we used a cell-based assay to determine the effect of Human adenovirus type 3 (HAdV3) on the exosome inhibition process by azole and Heparin derivatives. HAdV3-infected cells were treated with two concentrations of each inhibitor at different time points. Results:HAdV3 activities led to increased total sRNA, DNA, and exosome particle concentrations via particle tracking in the presence of Climbazole and Heparin relative to uninfected exosomes. In addition, there was an increased expression of classical markers such as ALG-2 interacting protein X (ALIX), and tetraspanin (CD63), (p< 0.05) and upregulated transcription factor interferon regulatory factor (IRF) 8 in the presence of HAdV3 after 24 hours (h) of treatment. Whereas higher concentrations of Climbazole and Heparin sodium salt were found to inhibit total exosome protein (p< 0.001) and exo-RNA (p< 0.01) content even in the presence of HAdV3 relative to infected exosomes only. Activities of HAdV3 in the presence of selected inhibitors resulted in the positive regulation of exosome related DNA damage/repair signaling proteins. Blocking exosome secretion partially obstructed viral entry. Immunological studies revealed that HAdV3 fiber protein levels in A549 cells were reduced at all concentrations of Climbazole and Heparin and both multiplicities of infections (p< 0.001). Discussion:Our findings suggest that while HAdV may bolster inhibited exosome content and release when modulating certain activities of the endosomal pathway mediators, HAdV entry might be constrained by the activities of these pharmacological agents. 

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2. Isolation of Extracellular Vesicles from Arabidopsis

   https://doi.org/10.1002/cpz1.352  

   Chen, Angela; He, Baoye; Jin, Hailing (January 2022, Current Protocols)

   Abstract Extracellular vesicles (EVs) in plants have emerged as key players in cell‐to‐cell communication and cross‐kingdom RNAi between plants and pathogens by facilitating the exchange of RNA, proteins, and other molecules. In addition to their role in intercellular communication, plant EVs also show promise as potential therapeutics and indicators of plant health. However, plant EVs exhibit significant heterogeneity in their protein markers, size, and biogenesis pathways, strongly influencing their composition and functionality. While mammalian EVs can be generally classified as exosomes that are derived from multivesicular bodies (MVBs), microvesicles that are shed from the plasma membrane, or as apoptotic bodies that originate from cells undergoing apoptosis, plant EVs remain poorly studied in comparison. At least three subclasses of EVs have been identified inArabidopsisleaves to date, including Tetraspanin‐positive exosomes derived from MVBs, Penetration 1 (PEN1)‐positive EVs, and EVs derived from exocyst‐positive organelles (EXPO). Differences in the plant starting material and isolation techniques have resulted in different purities, quality, and compositions of the resulting EVs, complicating efforts to better understand the role of these EVs in plants. We performed a comparative analysis on commonly used plant EV isolation methods and have identified an effective protocol for extracting clean apoplastic washing fluid (AWF) and isolating high‐quality intact and pure EVs ofArabidopsis thaliana. These EVs can then be used for various applications or studied to assess their cargos and functionality in plants. Furthermore, this process can be easily adapted to other plant species of interest. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Isolation of EVs from the apoplastic fluid ofArabidopsis thaliana Basic Protocol 2: Density gradient fractionation of EVs Basic Protocol 3: Immuno‐isolation of EVs usingArabidopsistetraspanin 8 (TET8) antibody 

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3. Therapeutic Cell Repopulation of the Liver: From Fetal Rat Cells to Synthetic Human Tissues

   https://doi.org/10.3390/cells12040529  

   Shafritz, David A; Ebrahimkhani, Mo R; Oertel, Michael (February 2023, Cells)

   Progenitor cells isolated from the fetal liver can provide a unique cell source to generate new healthy tissue mass. Almost 20 years ago, it was demonstrated that rat fetal liver cells repopulate the normal host liver environment via a mechanism akin to cell competition. Activin A, which is produced by hepatocytes, was identified as an important player during cell competition. Because of reduced activin receptor expression, highly proliferative fetal liver stem/progenitor cells are resistant to activin A and therefore exhibit a growth advantage compared to hepatocytes. As a result, transplanted fetal liver cells are capable of repopulating normal livers. Important for cell-based therapies, hepatic stem/progenitor cells containing repopulation potential can be separated from fetal hematopoietic cells using the cell surface marker δ-like 1 (Dlk-1). In livers with advanced fibrosis, fetal epithelial stem/progenitor cells differentiate into functional hepatic cells and out-compete injured endogenous hepatocytes, which cause anti-fibrotic effects. Although fetal liver cells efficiently repopulate the liver, they will likely not be used for human cell transplantation. Thus, utilizing the underlying mechanism of repopulation and developed methods to produce similar growth-advantaged cells in vitro, e.g., human induced pluripotent stem cells (iPSCs), this approach has great potential for developing novel cell-based therapies in patients with liver disease. The present review gives a brief overview of the classic cell transplantation models and various cell sources studied as donor cell candidates. The advantages of fetal liver-derived stem/progenitor cells are discussed, as well as the mechanism of liver repopulation. Moreover, this article reviews the potential of in vitro developed synthetic human fetal livers from iPSCs and their therapeutic benefits. 

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4. Immunofluorescence Microscopy

   https://doi.org/10.1002/cpz1.842  

   Galati, Domenico F.; Asai, David J. (August 2023, Current Protocols)

   Abstract Visualizing fluorescence‐tagged molecules is a powerful strategy that can reveal the complex dynamics of the cell. One robust and broadly applicable method is immunofluorescence microscopy, in which a fluorescence‐labeled antibody binds the molecule of interest and then the location of the antibody is determined by fluorescence microscopy. The effective application of this technique includes several considerations, such as the nature of the antigen, specificity of the antibody, permeabilization and fixation of the specimen, and fluorescence imaging of the cell. Although each protocol will require fine‐tuning depending on the cell type, antibody, and antigen, there are steps common to nearly all applications. This article provides protocols for staining the cytoskeleton and organelles in two very different kinds of cells: flat, adherent fibroblasts and thick, free‐swimmingTetrahymenacells. Additional protocols enable visualization with widefield, laser scanning confocal, and eSRRF super‐resolution fluorescence microscopy. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescence staining of adherent cells such as fibroblasts Basic Protocol 2: Immunofluorescence of suspension cells such asTetrahymena Basic Protocol 3: Visualizing samples with a widefield fluorescence microscope Alternate Protocol 1: Staining suspension cells adhered to poly‐l‐lysine‐coated coverslips Alternate Protocol 2: Visualizing samples with a laser scanning confocal microscope Alternate Protocol 3: Generating super‐resolution images with SRRF microscopy 

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5. Evaluating PC12 Cell Differentiation Following Exosome Treatments

   Justin McGee, Cyntanna Hawkins (October 2019, Proceedings of The National Conference On Undergraduate Research (NCUR) 2019 Kennesaw State University Kennesaw, Georgia April 11-13, 2019)

   Exosomes are extracellular vesicles ranging in size from 40 to 100nm that are used for extracellular communication to control the cell niche environment with regards to differentiation. Initial studies in our lab have shown that exosomes isolated from differentiating neurites can cause differentiation in cells absent typical neuronal hormones. However, these studies only used exosomes isolated after complete differentiation. To further clarify the role of exosomes in cell differentiation, we isolated exosomes from NGF-treated PC12 cells after two, four and six days. The exosomes were taken at the specific times due to a slight majority of cells showing differentiation at day three, with an overwhelming majority showing differentiation at day six. Day two, four and six were thus good markers in the development of cell differentiation. Each set of exosomes was added to a different well of untreated cells, which were then observed for differentiation daily over a six-day period. The results showed a considerably larger amount of percent differentiation in cells treated with the day six exosomes compared to cells treated by the day two and day four exosomes. The change in number of differentiated cells in different exosome treatments indicate that exosomal contents change over time. In an effort to make sure that no NGF contamination occurred in our treatments, an NGF-ELISA was completed testing all the treatment exosomes with results showing no NGF. Additionally, exosomes were isolated from NGF spiked media to show that no NGF was carried over in our exosome isolation process. Both tests further evidence that our exosomes did not contain NGF. Our overall results indicate that the isolated exosomes changed contents over time and that their contents caused greater percent differentiation over time. Funding provided by the Cell Biology Education Consortium (CBEC) and through the AR-EPSCoR Center for Advanced Surface Engineering. 

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