Sequestration of membrane cholesterol by cholesterol-binding proteins inhibits SARS-CoV-2 entry into Vero E6 cells.

Membrane lipids and proteins form dynamic domains crucial for physiological and pathophysiological processes, including viral infection. Many plasma membrane proteins, residing within membrane domains enriched with cholesterol (CHOL) and sphingomyelin (SM), serve as receptors for attachment and entry of viruses into the host cell. Among these, human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use proteins associated with membrane domains for initial binding and internalization. We hypothesized that the interaction of lipid-binding proteins with CHOL in plasma membrane could sequestrate lipids and thus affect the efficiency of virus entry into host cells, preventing the initial steps of viral infection. We have prepared CHOL-binding proteins with high affinities for lipids in the plasma membrane of mammalian cells. Binding of the perfringolysin O domain four (D4) and its variant D4E458L to membrane CHOL impaired the internalization of the receptor-binding domain of the SARS-CoV-2 spike protein and the pseudovirus complemented with the SARS-CoV-2 spike protein. SARS-CoV-2 replication in Vero E6 cells was also decreased. Overall, our results demonstrate that the integrity of CHOL-rich membrane domains and the accessibility of CHOL in the membrane play an essential role in SARS-CoV-2 cell entry.

Characterization of Nanohybridosomes from Lipids and Spruce Homogenate Containing Extracellular Vesicles.

Introduction: Lipid nanovesicles associated with bioactive phytochemicals from spruce needle homogenate (here called nano-sized hybridosomes or nanohybridosomes, NSHs) were considered. Methods: We formed NSHs by mixing appropriate amounts of lecithin, glycerol and supernatant of isolation of extracellular vesicles from spruce needle homogenate. We visualized NSHs by light microscopy and cryogenic transmission electron microscopy and assessed them by flow cytometry, dynamic light scattering, ultraviolet-visual spectroscopy, interferometric light microscopy and liquid chromatography-mass spectrometry. Results: We found that the particles consisted of a bilayer membrane and a fluid-like interior. Flow cytometry and interferometric light microscopy measurements showed that the majority of the particles were nano-sized. Dynamic light scattering and interferometric light microscopy measurements agreed well on the average hydrodynamic radius of the particles Rh (between 140 and 180 nm), while the concentrations of the particles were in the range between 1013 and 1014/mL indicating that NSHs present a considerable (more than 25%) of the sample which is much more than the yield of natural extracellular vesicles (EVs) from spruce needle homogenate (estimated less than 1%). Spruce specific lipids and proteins were found in hybridosomes. Discussion: Simple and low-cost preparation method, non-demanding saving process and efficient formation procedure suggest that large-scale production of NSHs from lipids and spruce needle homogenate is feasible.

Small Cellular Particles from European Spruce Needle Homogenate.

Small cellular particles (SCPs) are being considered for their role in cell-to-cell communication. We harvested and characterized SCPs from spruce needle homogenate. SCPs were isolated by differential ultracentrifugation. They were imaged by scanning electron microscope (SEM) and cryogenic transmission electron microscope (cryo TEM), assessed for their number density and hydrodynamic diameter by interferometric light microscopy (ILM) and flow cytometry (FCM), total phenolic content (TPC) by UV-vis spectroscopy, and terpene content by gas chromatography-mass spectrometry (GC-MS). The supernatant after ultracentrifugation at 50,000× g contained bilayer-enclosed vesicles whereas in the isolate we observed small particles of other types and only a few vesicles. The number density of cell-sized particles (CSPs) (larger than 2 µm) and meso-sized particles (MSPs) (cca 400 nm-2 µm) was about four orders of magnitude lower than the number density of SCPs (sized below 500 nm). The average hydrodynamic diameter of SCPs measured in 10,029 SCPs was 161 ± 133 nm. TCP decreased considerably due to 5-day aging. Volatile terpenoid content was found in the pellet after 300× g. The above results indicate that spruce needle homogenate is a source of vesicles to be explored for potential delivery use.

Autologous Platelet and Extracellular Vesicle-Rich Plasma as Therapeutic Fluid: A Review.

The preparation of autologous platelet and extracellular vesicle-rich plasma (PVRP) has been explored in many medical fields with the aim to benefit from its healing potential. In parallel, efforts are being invested to understand the function and dynamics of PVRP that is complex in its composition and interactions. Some clinical evidence reveals beneficial effects of PVRP, while some report that there were no effects. To optimize the preparation methods, functions and mechanisms of PVRP, its constituents should be better understood. With the intention to promote further studies of autologous therapeutic PVRP, we performed a review on some topics regarding PVRP composition, harvesting, assessment and preservation, and also on clinical experience following PVRP application in humans and animals. Besides the acknowledged actions of platelets, leukocytes and different molecules, we focus on extracellular vesicles that were found abundant in PVRP.

Assessment of Small Cellular Particles from Four Different Natural Sources and Liposomes by Interferometric Light Microscopy.

Small particles in natural sources are a subject of interest for their potential role in intercellular, inter-organism, and inter-species interactions, but their harvesting and assessment present a challenge due to their small size and transient identity. We applied a recently developed interferometric light microscopy (ILM) to assess the number density and hydrodynamic radius (Rh) of isolated small cellular particles (SCPs) from blood preparations (plasma and washed erythrocytes) (B), spruce needle homogenate (S), suspension of flagellae of microalgae Tetraselmis chuii (T), conditioned culture media of microalgae Phaeodactylum tricornutum (P), and liposomes (L). The aliquots were also assessed by flow cytometry (FCM), dynamic light scattering (DLS), ultraviolet-visible spectrometry (UV-vis), and imaging by cryogenic transmission electron microscopy (cryo-TEM). In Rh, ILM showed agreement with DLS within the measurement error in 10 out of 13 samples and was the only method used here that yielded particle density. Cryo-TEM revealed that representative SCPs from Tetraselmis chuii flagella (T) did not have a globular shape, so the interpretation by Rh of the batch methods was biased. Cryo-TEM showed the presence of thin filaments in isolates from Phaeodactylum tricornutum conditioned culture media (P), which provides an explanation for the considerably larger Rh obtained by batch methods than the sizes of particles observed by cryo-TEM images. ILM proved convenient for assessment of number density and Rh of SCPs in blood preparations (e.g., plasma); therefore, its use in population and clinical studies is indicated.

A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications.

Tumor growth and metastasis strongly rely on cell-cell communication. One of the mechanisms by which tumor cells communicate involves the release and uptake of lipid membrane encapsulated particles full of bioactive molecules, called extracellular vesicles (EVs). EV exchange between cancer cells may induce phenotype changes in the recipient cells. Our work investigated the effect of EVs released by teratocarcinoma cells on glioblastoma (GBM) cells. EVs were isolated by differential centrifugation and analyzed through Western blot, nanoparticle tracking analysis, and electron microscopy. The effect of large EVs on GBM cells was tested through cell migration, proliferation, and drug-sensitivity assays, and resulted in a specific impairment in cell migration with no effects on proliferation and drug-sensitivity. Noticeably, we found the presence of the EGF-CFC founder member CRIPTO on both small and large EVs, in the latter case implicated in the EV-mediated negative regulation of GBM cell migration. Our data let us propose a novel route and function for CRIPTO during tumorigenesis, highlighting a complex scenario regulating its effect, and paving the way to novel strategies to control cell migration, to ultimately improve the prognosis and quality of life of GBM patients.

Interaction between Microalgae P. tricornutum and Bacteria Thalassospira sp. for Removal of Bisphenols from Conditioned Media.

We studied the efficiency of three culture series of the microalgae Phaeodactylum tricornutum (P. tricornutum) and bacteria Thalassospira sp. (axenic microalgae, bacterial culture and co-culture of the two) in removing bisphenols (BPs) from their growth medium. Bacteria were identified by 16S ribosomal RNA polymerase chain reaction (16S rRNA PCR). The microorganism growth rate was determined by flow cytometry. Cultures and isolates of their small cellular particles (SCPs) were imaged by scanning electron microscopy (SEM) and cryogenic transmission electron microscopy (Cryo-TEM). BPs were analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). Our results indicate that some organisms may have the ability to remove a specific pollutant with high efficiency. P. tricornutum in axenic culture and in mixed culture removed almost all (more than 99%) of BPC2. Notable differences in the removal of 8 out of 18 BPs between the axenic, mixed and bacterial cultures were found. The overall removals of BPs in axenic P. tricornutum, mixed and bacterial cultures were 11%, 18% and 10%, respectively. Finding the respective organisms and creating microbe societies seems to be key for the improvement of wastewater treatment. As a possible mediating factor, numerous small cellular particles from all three cultures were detected by electron microscopy. Further research on the mechanisms of interspecies communication is needed to advance the understanding of microbial communities at the nano-level.

Stability of Erythrocyte-Derived Nanovesicles Assessed by Light Scattering and Electron Microscopy.

Extracellular vesicles (EVs) are gaining increasing amounts of attention due to their potential use in diagnostics and therapy, but the poor reproducibility of the studies that have been conducted on these structures hinders their breakthrough into routine practice. We believe that a better understanding of EVs stability and methods to control their integrity are the key to resolving this issue. In this work, erythrocyte EVs (hbEVs) were isolated by centrifugation from suspensions of human erythrocytes that had been aged in vitro. The isolate was characterised by scanning (SEM) and cryo-transmission electron microscopy (cryo-TEM), flow cytometry (FCM), dynamic/static light scattering (LS), protein electrophoresis, and UV-V spectrometry. The hbEVs were exposed to various conditions (pH (4-10), osmolarity (50-1000 mOsm/L), temperature (15-60 °C), and surfactant Triton X-100 (10-500 µM)). Their stability was evaluated by LS by considering the hydrodynamic radius (Rh), intensity of scattered light (I), and the shape parameter (ρ). The morphology of the hbEVs that had been stored in phosphate-buffered saline with citrate (PBS-citrate) at 4 °C remained consistent for more than 6 months. A change in the media properties (50-1000 mOsm/L, pH 4-10) had no significant effect on the Rh (=100-130 nm). At pH values below 6 and above 8, at temperatures above 45 °C, and in the presence of Triton X-100, hbEVs degradation was indicated by a decrease in I of more than 20%. Due to the simple preparation, homogeneous morphology, and stability of hbEVs under a wide range of conditions, they are considered to be a suitable option for EV reference material.

Cytotoxic Activity of LLO Y406A Is Targeted to the Plasma Membrane of Cancer Urothelial Cells.

Identification of novel agents for bladder cancer treatment is highly desirable due to the high incidence of tumor recurrence and the risk of progression to muscle-invasive disease. The key feature of the cholesterol-dependent toxin listeriolysin O mutant (LLO Y406A) is its preferential activity at pH 5.7, which could be exploited either directly for selective targeting of cancer cells or the release of accumulated therapeutics from acidic endosomes. Therefore, our goal was to compare the cytotoxic effect of LLO Y406A on cancer cells (RT4) and normal urothelial cells (NPU), and to identify which cell membranes are the primary target of LLO Y406A by viability assays, life-cell imaging, fluorescence, and electron microscopy. LLO Y406A decreased viability, altered cell morphology, provoked membrane blebbing, and induced apoptosis in RT4 cells, while it did not affect NPU cells. LLO Y406A did not cause endosomal escape in RT4 cells, while the plasma membrane of RT4 cells was revealed as the primary target of LLO Y406A. It has been concluded that LLO Y406A has the ability to selectively eliminate cancer urothelial cells through pore-forming activity at the plasma membrane, without cytotoxic effects on normal urothelial cells. This promising selective activity merits further testing as an anti-cancer agent.

Decrease in Cellular Nanovesicles Concentration in Blood of Athletes More Than 15 Hours After Marathon.

INTRODUCTION: Cellular nanovesicles (CNVs), that are shed from cells, have been recognized as promising indicators of health status. We analyzed the effect of long-distance running on concentration of CNVs, along with some standard blood parameters, in 27 athletes two days before and >15 hours after physical effort. METHODS: CNVs were isolated by repetitive centrifugation and washing of samples, and assessed by flow cytometry. Cholinesterase (ChE) and glutathione S-transferase (GST) activity were measured spectrophotometrically. Interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) concentrations were measured using enzyme-linked immunosorbent assay (ELISA). C-reactive protein (CRP) was measured with immunoturbidimetric determination and lipidogram parameters were measured by enzymatic colorimetric assay. Flow cytometry was used for blood cell count and mean platelet volume (MPV) measurement. RESULTS: More than 15 hours after physical effort a decrease was found in CNVs' concentration in isolates from blood (46%; p<0.05), in ChE activity in whole blood (47%; p<0.001), in plasma (34%; p<0.01), and in erythrocyte suspension (54%; p<0.001), as well as in GST activity in erythrocyte suspension (16%; p<0.01) and in IL-6 concentration in plasma (63%; p<0.05). We found no change in GST activity in plasma and in TNF-α concentration in plasma. Correlations (>0.8; p<0.001) between CNVs' concentration and ChE activity, and GST activity, respectively, in erythrocyte suspension were found. CONCLUSION: We found that >15 hours post-physical effort, CNVs' concentration was below the initial value, concomitant with other measured parameters: ChE and GST activity as well as IL-6 concentration, indicating a favorable effect of physical effort on health status. CNVs' concentration and ChE activity in isolates from peripheral blood proved to have potential as indicators of the response of the human body to inflammation after physical effort. Physical activity should be considered as an important factor in preparation of subjects for blood sampling in procedures focusing on CNV-containing diagnostic and therapeutic compounds.