Modulatory Effects of the Recombinant Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Spike S1 Subunit Protein on the Phenotype of Camel Monocyte-Derived MacrophagesHussen, Al-Mubarak, Shawaf
et alBiology (Basel) (2025) 14 (3)
Abstract: Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an emerging zoonotic pathogen with different pathogenesis in humans and camels. The mechanisms behind the higher tolerance of camels to MERS-CoV infection are still unknown. Monocytes are innate myeloid cells that are able, depending on the local stimulation in their microenvironment, to differentiate into different functional subtypes of macrophages with an impact on the adaptive immune response. Several in vitro protocols have been used to induce the differentiation of monocyte-derived macrophages (MDMs) in human and several veterinary species. Such protocols are not available for camel species. In the present study, monocytes were separated from camel blood and differentiated in vitro in the presence of different stimuli into MDM. Camel MDMs generated in the presence of a combined stimulation of monocytes with LPS and GM-CSF resulted in the development of an M1 macrophages phenotype with increased abundance of the antigen-presentation receptor MHCII molecules and a decreased expression of the scavenger receptor CD163. The expression pattern of the cell markers CD163, CD14, CD172a, CD44, and CD9 on MDM generated in the presence of the MERS-CoV S1 protein revealed similarity with M-CSF-induced MDM, suggesting the potential of the MERS-CoV S1 protein to induce an M2 macrophages phenotype. Similarly to the effect of M-CSF, MERS-CoV-S protein-induced MDMs showed enhanced phagocytosis activity compared to non-polarized or LPS/GM-CSF-polarized MDMs. Collectively, our study represents the first report on the in vitro generation of monocyte-derived macrophages (MDMs) in camels and the characterization of some phenotypic and functional properties of camel MDM under the effect of M1 and M2 polarizing stimuli. In addition, the results suggest a polarizing effect of the MERS-CoV S1 protein on camel MDMs, developing an M2-like phenotype with enhanced phagocytosis activity. To understand the clinical relevance of these in vitro findings on disease pathogenesis and camel immune response toward MERS-CoV infection, further studies are required.
Tetraspanins affect membrane structures and the trafficking of molecular partners: what impact on extracellular vesicles?Rubinstein, Théry, Zimmermann
Biochem Soc Trans (2025)
Abstract: Tetraspanins are a family of 33 proteins in mammals believed to play a crucial role in the compartmentalization of various associated proteins within cells and membranes. Recent studies have elucidated the structure of several tetraspanin members, revealing that while the four transmembrane domains typically adopt a cone-shaped configuration in crystals, other conformations are also possible. This cone-shaped structure may explain why tetraspanins are often enriched in curved and tubular cellular structures, such as microvilli, tunneling nanotubes, retraction fibers, or at the site of virus budding, and may contribute to the formation or maintenance of these structures. Tetraspanins have also been detected on midbody remnants and migrasomes, as well as on extracellular vesicles (EVs), for which CD9, CD81, and CD63 are widely used as markers. Although their impact on certain membrane structures and their ability to regulate the function and trafficking of associated proteins would suggest a potential role of tetraspanins either in EV formation or in regulating their protein composition, or both, efforts to characterize these roles have been complicated by conflicting results. In line with the interaction of certain tetraspanins with cholesterol, two recent studies have suggested that the presence or organization of oxysterols and cholesterol in EVs may be regulated by Tspan6 and CD63, respectively, paving the way for further research on the influence of tetraspanins on the lipid composition of EVs.© 2025 The Author(s).
Next Generation Aqueous Two-Phase System for Gentle, Effective, and Timely Extracellular Vesicle Isolation and Transcriptomic AnalysisSu, Jeyhani, Thillainadesan
et alJ Extracell Vesicles (2025) 14 (3), e70058
Abstract: The isolation of extracellular vesicles (EVs) using currently available methods frequently compromises purity and yield to prioritize speed. Here, we present a next-generation aqueous two-phase system (next-gen ATPS) for the isolation of EVs regardless of scale and volume that is superior to conventional methods such as ultracentrifugation (UC) and commercial kits. This is made possible by the two aqueous phases, one rich in polyethylene glycol (PEG) and the other rich in dextran (DEX), whereby fully encapsulated lipid vesicles preferentially migrate to the DEX-rich phase to achieve a local energy minimum for the EVs. Isolated EVs as found in the DEX-rich phase are more amenable to biomarker analysis such as nanoscale flow cytometry (nFC) when using various pre-conjugated antibodies specific for CD9, CD63 and CD81. TRIzol RNA isolation is further enabled by the addition of dextranase, a critical component of this next-gen ATPS method. RNA yield of next-gen ATPS-isolated EVs is superior to UC and other commercial kits. This negates the use of specialized EV RNA extraction kits. The use of dextranase also enables more accurate immunoreactivity of pre-conjugated antibodies for the detection of EVs by nFC. Transcriptomic analysis of EVs isolated using the next-gen ATPS revealed a strong overlap in microRNA (miRNA), circular RNA (circRNA) and small nucleolar RNA (snoRNA) profiles with EV donor cells, as well as EVs isolated by UC and the exoRNeasy kit, while detecting a superior number of circRNAs compared to the kit in human samples. Overall, this next-gen ATPS method stands out as a rapid and highly effective approach to isolate high-quality EVs in high yield, ensuring optimal extraction and analysis of EV-encapsulated nucleic acids.© 2025 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.
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