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In this study, we present a model for visualization of the exosome transfer of p53-GFP protein between cultured mammalian cells. The temporal parameters of the accumulation of fluorescent labeled protein in recipient cells have been analyzed. We employed HEK293 cells transfected with p53∆Y126-GFP plasmid (a GFP clone) as donor of the exosomes, while the original HEK293 cell line was used as a recipient. Our results provide evidence that the transfer of the GFP protein from cell to cell is carried out via exosomes. It has been shown that the accumulation of this protein by the recipient cells in cocultures of these cells takes a prolonged time. The temporal parameters of this transfer differ between the cells within the same population.

Clinical and radiological features of tuberculosis and sarcoidosis are quite overlapping, and therefore, a diagnostic dilemma often persists. There are no commonly accepted criteria for the diagnosis of sarcoidosis due to the lack of data on the etiology of the disease. The exclusion of tuberculosis in every patient with suspected sarcoidosis is a mandatory stage of diagnosis, especially in countries with a high burden of tuberculosis. A prospective study was conducted with two groups of patients: group I (n = 50)-patients with pulmonary sarcoidosis established according to standard criteria; group II (n = 28)-patients with pulmonary tuberculosis with bacterial excretion. The control group (n = 24) was presented by healthy subjects. The examination complex included x-ray, bacteriological, immunological (Mantoux test with 2 TE, TB.SPOT test), and histological methods. All patients and healthy subjects were assessed for immune complexes with the use of the dynamic light scattering (DLS) method and adding of "healthy lung tissue extract" antigens and specific tuberculosis antigens ESAT-6 and SFP-10 in vitro. Significant differences were found in determining specific immune complexes in patients with pulmonary sarcoidosis and pulmonary tuberculosis. Registration of specific immune complex formation with "healthy lung tissue extract" in 100% cases may indicate the autoimmune nature of sarcoidosis. The absence of the immune complex formation in response to ESAT-6/SFP-10 antigens can be used for the differential diagnosis of two diseases. The diagnostic significance of the DLS method was 100% for sarcoidosis and 92.2% for tuberculosis. The data obtained in the study allows not only understanding the etiology of sarcoidosis, but also obtaining new criteria for the differential diagnosis of tuberculosis and pulmonary sarcoidosis.

Plant-derived extracellular vesicles (EVs) gain more and more attention as promising carriers of exogenous bioactive molecules to the human cells. Derived from various edible sources, these EVs are remarkably biocompatible, biodegradable and highly abundant from plants. In this work, EVs from grapefruit juice were isolated by differential centrifugation followed by characterization of their size, quantity and morphology by nanoparticle tracking analysis, dynamic light scattering, atomic force microscopy and cryo-electron microscopy (Cryo-EM). In Cryo-EM experiments, we visualized grapefruit EVs with the average size of 41 ± 13 nm, confirmed their round-shaped morphology and estimated the thickness of their lipid bilayer as 5.3 ± 0.8 nm. Further, using cell culture models, we have successfully demonstrated that native grapefruit-derived extracellular vesicles (GF-EVs) are highly efficient carriers for the delivery of the exogenous Alexa Fluor 647 labeled bovine serum albumin (BSA) and heat shock protein 70 (HSP70) into both human peripheral blood mononuclear cells and colon cancer cells. Interestingly, loading to plant EVs significantly ameliorated the uptake of exogenous proteins by human cells compared to the same proteins without EVs. Most importantly, we have confirmed the functional activity of human recombinant HSP70 in the colon cancer cell culture upon delivery by GF-EVs. Analysis of the biodistribution of GF-EVs loaded with 125I-labeled BSA in mice demonstrated a significant uptake of the grapefruit-derived extracellular vesicles by the majority of organs. The results of our study indicate that native plant EVs might be safe and effective carriers of exogenous proteins into human cells.

Simple Summary Exosomes are nanoscale membrane extracellular vesicles that are involved in intercellular communication and signaling, and are a promising tool in biomedicine for drug delivery. Despite the progress in practical application and morphological characterization, information about their biomechanical properties is still scarce. The presence of non-membrane particles called exomeres with similar functions has recently been reported. We applied the atomic force microscopy technique to study the biomechanical properties of both types of particles in air and in liquid. We found a correlation between the biomechanical properties of the vesicles, their size, structure, and function. Our data provide useful information for a better understanding of the biomechanical characteristics of extracellular vesicles and non-membrane extracellular particles and their AFM detection. Abstract While extracellular vesicles (EVs) are extensively studied by various practical applications in biomedicine, there is still little information on their biomechanical properties due to their nanoscale size. We identified isolated blood plasma vesicles that carried on biomarkers associated with exosomes and exomeres and applied atomic force microscopy (AFM) to study them at single particle level in air and in liquid. Air measurements of exosomes revealed a mechanically indented internal cavity in which highly adhesive sites were located. In contrast, the highly adhesive sites of exomeres were located at the periphery and the observed diameter of the particles was ~35 nm. In liquid, the reversible deformation of the internal cavity of exosomes was observed and a slightly deformed lipid bi-layer was identified. In contrast, exomeres were not deformed and their observed diameter was ~16 nm. The difference in diameters might be associated with a higher sorption of water film in air. The parameters we revealed correlated with the well-known structure and function for exosomes and were observed for exomeres for the first time. Our data provide a new insight into the biomechanical properties of nanoparticles and positioned AFM as an exclusive source of in situ information about their biophysical characteristics.

Background: Plant-derived extracellular vesicles increasingly gain attention as promising carriers of exogenous bioactive molecules to the human cells. Due to their various edible sources they are distinctly biocompatible, biodegradable and easily available in significant amounts. Methods: In present work, extracellular vesicles from grapefruit juice were isolated by differential centrifugation and characterized in terms of size, quantity, and morphology by nanoparticle tracking analysis, dynamic light scattering, atomic force microscopy, and cryo-electron microscopy (Cryo-EM). In Cryo-EM experiments we have visualized grapefruit vesicles with average size of 41 ± 13 nm, confirmed their round-shaped morphology and estimated the thickness of their lipid bilayer as 5.3 ± 0.8 nm. Results: Using in vitro cell culture models, we have shown that grapefruit-derived extracellular vesicles (GF-EVs) are highly efficient carriers for the delivery of the exogenous Alexa flour 647 labelled BSA and HSP70 proteins into human colon cancer HCT-116 and DLD1 cells. Both proteins when loaded to plant vesicles were captured by human intestinal cells much more efficiently compare to their free state. Additionally, the functional activity of human recombinant HSP70 delivered by GF-EVs in the tissue culture cells has been confirmed. Conclusions: The results clearly indicate the high potential of native plant vesicles for the safe delivery of therapeutic proteins into human cells. Here, we reported the first demonstration of effective loading of natural plant-derived extracellular nanovesicles with exogenous proteins and their successful delivery into human cells.

Background: Extracellular vesicles (EVs) are small membrane vesicles released from different types of cells. EVs are found in many human biological fluids. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies with the plasma membrane. This type of vesicles is characterized by specific exosomal markers. Exosomes extracted from peripheral body liquids could have specific properties associated with different physiological conditions as well as human disorders, including neurodegenerative diseases. Gaucher disease (GD) – is the most common form of lysosomal storage disorders caused by mutations in the glucocerebrosidase (GBA) gene. Lysosome functionality is critical for the regulation of extracellular vesicle secretion and content. In model animals, the inhibition of glucocerebrosidase has been shown to increase the secretion of extracellular vesicles in brain tissues. Amount evaluation of EVs and their size in the biological fluids of patients with GD has not been early performed; therefore, it is unknown whether lysosomal dysfunction found in GD patients influences the plasma pool of EVs. The aim of this study was to evaluate the amount of blood plasma EVs in patients with GD and their characterization for morphology and size. Methods: EVs were isolated from the blood plasma of 8 GD patients and 8 controls by ultracentrifugation, and were characterized using cryo-electron microscopy (cryo-EM), nanoparticle tracking analysis (NTA), and dynamic light scattering (DLS). Also, the presence of exosomal markers CD9, CD63, CD81, and HSP70 was analyzed by flow cytometry and western blot. Results: Here, it was first shown an increased proportion of exosome fraction in EVs from plasma of GD patients compared to controls by DLS and cryo-EM (p

Exosomes, a special type of microparticles produced by cells, are currently of considerable interest for researchers. The term “exosomes” denotes extracellular vesicles of less than 120 nm in size derived from intracellular multivesicular bodies. Multiple studies that address the distinctive features of exosome structure and biochemical composition in various pathological states imply the possibility of development of novel diagnostic techniques based on the detection of changes in the pool of proteins and nucleic acids transported by exosomes. However, methods for isolation and investigation of exosomes are rather difficult to develop because of a small size of these vesicles. A novel approach for preparative-scale isolation of exosomes based on the phenomenon of binding and aggregation of these particles in the presence of lectins has been put forward in the present study. The method developed is relatively cost-effective, allows for the isolation of exosomes from various biological fluids, and has been validated for the subsequent analysis of the protein composition of the exosomes in view of the possible clinical applications. The validation showed that the sedimentation of lectin-aggregated exosomes is a suitable approach for the isolation of these microvesicles from the complete conditioned culture medium in a research-laboratory setup.

A mirror-symmetry motif was discovered in the N-terminus of the influenza virus PB1 protein. Structure of peptide comprised of the corresponding part of PB1 (amino acid residues 6-25) was investigated by circular dichroism and in silico modeling. We found that peptide PB1 (6-25) in solution assumes beta-hairpin conformation. A truncated peptide PB1 (6-13), containing only half of the mirror-symmetry motif, appeared to stabilize the beta-structure of the original peptide and, at high concentrations, was capable of reacting with peptide to form insoluble aggregates in vitro. Ability of PB1 (6-13) peptide to interact with the N-terminal domain of PB1 protein makes it a potential antiviral agent that inhibits PA-PB1 complex formation by affecting PB1 N-terminus structure.

Summary form only given. The pulsed electric discharges (PED) in water attract more and more attention of researchers. There are at least three reasons: their bactericidal action to pathogenic bacteria and spores of fungi, which are opportunistic to the human beings; destroying ability concerning some of organic compounds, including toxic ones; high productivity of oxide nanoparticles of various metals. The erosion of electrodes at electric discharges in water takes place. As a result of the erosion the metal nanoparticles are in water. Hence some part of the oxide nanoparticles transforms to the ions, the action of the water treated by the PED to the microorganisms must be than more intensively, than the ions are more toxic. The ions of metals on a degree of their toxicity to microorganisms are located as follows; Ag > Cu > Cd. Zn. Pb > Mn, Fe > Mg, Ca. The purpose of this investigation was to determine quantitative relations between parameters of electric pulses, such as their duration, current rise rate, the energy expenditure and specific erosion of electrodes metal, and also characteristics of produced nanoparticles. in particular, function of distribution of nanoparticles sub fractions in the dimension. It was found that at duration of the discharge pulse from 5 to 10 mus there is optimum ratio of those two above parameters, i.e. high specific erosion of electrodes of 5 mg/C at dominant output of nanoparticles with dimension of about 20 nm. which most effectively influence onto bacteria and spores. Besides increase of energy expenditure results in displacement of maxima of distribution of mass of subfractions to greater dimension of nanoparticles and clusters formed by them.