Comparative Analysis of Tumor-Associated microRNAs and Tetraspanines from Exosomes of Plasma and Ascitic Fluids of Ovarian Cancer Patients.

Ovarian cancer (OC) is one of the most common and fatal types of gynecological cancer. In the early phase of OC detection, the current treatment and diagnostic methods are not efficient and sensitive enough. Therefore, it is crucial to explore the mechanisms of OC metastasis and discover valuable factors for early diagnosis of female cancers and novel therapeutic strategies for metastasis. Exosomes are known to be involved in the development, migration, and invasion of cancer cells, and their cargo could be useful for the non-invasive biopsy development. CD151- and Tspan8-positive exosomes are known to support the degradation of the extracellular matrix, and are involved in stroma remodeling, angiogenesis and cell motility, as well as the association of miR-24 and miR-101 with these processes. The objective of this study was to explore the relationship of these components of exosomal cargo, in patients with OC, to clarify the clinical significance of these markers in liquid biopsies. The levels of tetraspanins Tspan8+ and CD151+ exosomes were significantly higher in plasma exosomes of OC patients compared with healthy females (HFs). The relative levels of miR-24 and miR-101 in plasma exosomes of HFs were significantly higher than in plasma exosomes of OC patients, while the levels of these microRNAs in exosomes from plasma and ascites of ill females showed no difference. Our study revealed a strong direct correlation between the change in the ascites exosomes CD151+Tspan8+ subpopulation level and the expression levels of the ascites (R = 0.81, p < 0.05) and plasma exosomal miR-24 (R = 0.74, p < 0.05) in OC patients, which confirms the assumption that exosomal cargo act synergistically to increase cellular motility, affecting cellular processes and signaling. Bioinformatics analysis confirmed the involvement of CD151 and Tspan8 tetraspanins and genes controlled by miR-24-3p and miR-101 in signaling pathways, which are crucial for carcinogenesis, demonstrating that these tetraspanins and microRNAs are potential biomarkers for OC screening, and predictors of poor clinicopathological behavior in tumors.

Plasma Exosomes of Patients with Breast and Ovarian Tumors Contain an Inactive 20S Proteasome.

Exosomes are directly involved in governing of physiological and pathological conditions of an organism through the transfer of information from producing to receiving cells. It can be assumed that exosomes are one of the key players of tumor dissemination since they are very stable and small enough to penetrate from various tissues into biological fluids and then back, thus interacting with tissue target cells. We evaluated the enzymatic activity and the level of 20S proteasome in tissue and exosomes of healthy females (n = 39) and patients with ovarian (n = 50) and breast (n = 108) tumors to reveal the critical role of exosomal cargo in the mediation of different types of metastases. Exosomes from plasma and ascites were isolated and characterized in according to International Society for Extracellular Vesicles guidelines. The level of 20S proteasome in tissue and exosomes was determined using Western blot analysis. Chymotrypsin- and caspase-like (ChTL and CL, respectively) peptidase activities of the proteasomes were determined using fluorogenic Suc-LLVY-AMC and Cbz-LLG-AMC substrates, respectively. We observed increased levels of 20S proteasome in ovarian cancer tissue and luminal B subtype breast cancer tissue as well as in plasma exosomes from cancer patients. Moreover, the level of the 20S proteasome in plasma exosomes and ascites exosomes in patients with ovarian tumors is comparable and higher in ovarian cancer patients with low volume ascites than in patients with moderate and high-volume ascites. We also found increased ChTL and CL activities in breast cancer and ovarian cancer tissues, as well as in peritoneal metastases in ovarian cancer, while proteasomal activity in exosomes from plasma of healthy females and all patients, as well as from ascites of ovarian tumor patients were lower than detection limit of assay. Thus, regardless of the type of tumor metastasis (lymphogenous or peritoneal), the exosomes of cancer patients were characterized by an increased level of 20S proteasome, which do not exhibit enzymatic activity.

Extra Purified Exosomes from Human Placenta Contain An Unpredictable Small Number of Different Major Proteins.

Exosomes are nanovesicles (30-100 nm) containing various RNAs and different proteins. Exosomes are important in intracellular communication, immune function, etc. Exosomes from different sources including placenta were mainly obtained by different types of centrifugation and ultracentrifugations and were reported to contain from a few dozen to thousands of different proteins. First crude exosome preparations from four placentas (normal pregnancy) were obtained here using several standard centrifugations but then were additionally purified by gel filtration on Sepharose 4B. Individual preparations demonstrated different gel filtration profiles showing good or bad separation of exosome peaks from two peaks of impurity proteins and their complexes. According to electron microscopy, exosomes before gel filtration contain vesicles of different size, ring-shaped structures forming by ferritin and clusters of aggregated proteins and their complexes. After filtration through 220 nm filters and gel filtration exosomes display typically for exosome morphology and size (30-100 nm) and do not contain visible protein admixtures. Identification of exosome proteins was carried out by MS and MS/MS MALDI mass spectrometry of proteins' tryptic hydrolyzates after their SDS-PAGE and 2D electrophoresis. We have obtained unexpected results. Good, purified exosomes contained only 11-13 different proteins: CD9, CD81, CD-63, hemoglobin subunits, interleukin-1 receptor, annexin A1, annexin A2, annexin A5, cytoplasmic actin, alkaline phosphatase, serotransferin, and probably human serum albumin and immunoglobulins. We assume that a possible number of exosome proteins found previously using crude preparations may be very much overestimated. Our data may be important for study of biological functions of pure exosomes.

Non-covalent binding of nucleic acids with gold nanoparticles provides their stability and effective desorption in environment mimicking biological media.

The ability of gold nanoparticles to bind different substances has resulted in the high interest of researchers determining their usage as a promising carrier of various biological substances including nucleic acids (NAs) for therapeutic applications. Most publications report covalent binding (conjugation) of an NA to spherical AuNPs via the Au-S bond. In this work, we obtained non-covalent associates of different ssDNA, ssRNA and siRNAs with spherical gold nanoparticles (AuNPs) and examined their physico-chemical properties and stability in media mimicking intracellular space (bacterial 'cytosol') and cell culture media (10% FBS in DMEM). The 'cytosol' was obtained from E. coli and possessed nuclease activity. For the first time, we used the phosphoryl guanidine (dimethylimidazolidin-2-imine, Dmi) group for modification of 3'-ends to enhance the stability of ssRNAs and siRNAs against nuclease destruction. Trying to evaluate the material balance, we analyzed the whole nucleotide species obtained after incubation of NA-AuNPs associates in 'cytosol' and FBS and evaluated the degree of NAs destruction, a share of full-size NAs remained on the surface of the AuNPs and in the solution. Native ss- and siRNAs, both free and in composition of non-covalent associates with AuNPs, were less resistant to degrading factors than ssDNA. The introduction of two Dmi-groups into the ssDNA increased its stability in 'cytosol' three times within 2.5 h. Dmi-modified siRNAs in non-covalent associates with AuNPs were two times more stable than unmodified siRNA within 4 h. We showed that non-covalent siRNA-AuNPs associates serve as a kind of storage for full-size NAs and thereby prolong their presence in nuclease-active media. Our study showed that non-covalent binding of siRNAs with a surface of AuNPs provides desorption of both strands, which is necessary for siRNA functioning in living cells, and could be considered as an important way to construct siRNA and ssDNA delivery systems based on AuNPs.

Fine mechanisms of the interaction of silver nanoparticles with the cells of Salmonella typhimurium and Staphylococcus aureus.

Silver nanoparticles possess antibacterial effect for various bacteria; however mechanisms of the interaction between Ag-NPs and bacterial cells remain unclear. The aim of our study was to obtain direct evidence of Ag-NPs penetration into cells of Gram-negative bacterium S. typhimurium and Gram-positive bacterium S. aureus, and to study cell responses to Ag-NPs. The Ag-NPs (most 8-10 nm) were obtained by gas-jet method. S. typhimurium (7.81 × 107 CFU), or S. aureus (8.96 × 107 CFU) were treated by Ag-NPs (0.05 mg/l of silver) in orbital shaker at 190 rpm, 37 °C. Bacteria were sampled at 0.5, 1, 1.5, 2, 5 and 23 h of the incubation for transmission electron microscopy of ultrathin sections. The Ag-NPs adsorbed on outer membrane of S. typhimurium and cell wall of S. auereus; penetrated and accumulated in cells without aggregation and damaging of neighboring cytoplasm. In cells of S. aureus Ag-NPs bound with DNA fibers. Cell responses to Ag-NPs differed morphologically in S. typhimurium and S. aureus, and mainly were presented by damage of cell structures. The cytoplasm of S. aureus became amorphous, while S. typhimurium showed lumping and lysis of cytoplasm which led to formation of "empty" cells. Other difference was fast change of cell shape in S. typhimurium, and late deformation of S. aureus cells. The obtained results showed how different could be responses induced by the same NPs in relatively simple prokaryotic cells. Evidently, Ag-NPs directly interact with macromolecular structures of living cells and are exert an active influence on their metabolism.

Changes in the Ultrastructure of Staphylococcus aureus Cells Make It Possible to Identify and Analyze the Injuring Effects of Ciprofloxacin, Polycationic Amphiphile and Their Hybrid.

The purposeful development of synthetic antibacterial compounds requires an understanding of the relationship between effects of compounds and their chemical structure. This knowledge can be obtained by studying changes in bacteria ultrastructure under the action of antibacterial compounds of a certain chemical structure. Our study was aimed at examination of ultrastructural changes in S. aureus cells caused by polycationic amphiphile based on 1,4‒diazabicyclo[2.2.2]octane (DL412), ciprofloxacin and their hybrid (DL5Cip6); the samples were incubated for 15 and 45 min. DL412 first directly interacted with bacterial cell wall, damaging it, then penetrated into the cell and disrupted cytoplasm. Ciprofloxacin penetrated into cell without visually damaging the cell wall, but altered the cell membrane and cytoplasm, and inhibited the division of bacteria. The ultrastructural characteristics of S. aureus cells damaged by the hybrid clearly differed from those under ciprofloxacin or DL412 action. Signs associated with ciprofloxacin predominated in cell damage patterns from the hybrid. We studied the effect of ciprofloxacin, DL412 and their hybrid on S. aureus biofilm morphology using paraffin sections. Clear differences in compound effects on S. aureus biofilm (45 min incubation) were observed. The results obtained allow us to recommend this simple and cheap approach for the initial assessment of antibiofilm properties of synthesized compounds.

Fixation and Visualization of Full Protein Corona on Lipid Surface of Composite Nanoconstruction.

Spontaneous sorption of proteins on the nanoparticles' surface leads to the fact that nanoparticles in biological media are always enveloped by a layer of proteins-the protein corona. Corona proteins affect the properties of nanoparticles and their behavior in a biological environment. In this regard, knowledge about the composition of the corona is a necessary element for the development of nanomedicine. Because proteins have different sorption efficacy, isolating particles with a full corona and characterizing the full corona is challenging. In this study, we propose a photo-activated cross-linker for full protein corona fixation. We believe that the application of our proposed approach will make it possible to capture and visualize the full corona on nanoparticles coated with a lipid shell.

The Composition of Small Extracellular Vesicles (sEVs) in the Blood Plasma of Colorectal Cancer Patients Reflects the Presence of Metabolic Syndrome and Correlates with Angiogenesis and the Effectiveness of Thermoradiation Therapy.

The majority of colorectal cancer patients (CRCPs) develop tumors on the background of "metabolically healthy obesity" or metabolic syndrome. The aim of the work was to study the levels of matrix metalloproteinases (MMPs) and heat shock proteins (HSPs) on the surface of blood plasma CD9-positive and FABP4-positive small extracellular vesicles (sEVs) from CRCPs depending on metabolic status and tumor angiogenesis, as well as to evaluate the sEVs markers as predictors of the effectiveness of thermoradiotherapy. In CRCPs, compared with patients with colorectal polyps (CPPs), the proportion of triple positive EVs and EVs with the MMP9+MMP2-TIMP1+ phenotype increased significantly among FABP4-positive EVs (adipocyte-derived EVs), which in general may indicate the overexpression of MMP9 and TIMP1 by adipocytes or adipose tissue macrophages in CRCPs. The results obtained have prospects for use as markers to clarify cancer risk in CPPs. One can assume that for CRCPs with metabolic syndrome or metabolically healthy obesity, it is the FABP4+MMP9+MMP2-TIMP1- population of circulating sEVs that is the most optimal biomarker reflecting tumor angiogenesis. Determining this population in the blood will be useful in monitoring patients after treatment for the early detection of tumor progression. CD9+MMP9+MMP2-TIMP1- and MMP9+MMP2-TIMP1+ subpopulations of circulating sEVs are the most promising predictors of the efficacy of thermoradiation therapy because their levels at baseline differ significantly in CRCPs with different tumor responses.

Blood Plasma Exosomes Contain Circulating DNA in Their Crown.

It is known that circulating DNA (cirDNA) is protected from nuclease activity by proteins that form macromolecular complexes with DNA. In addition, it was previously shown that cirDNA can bind to the outer surface of exosomes. NTA analysis and real-time PCR show that exosomes from healthy females (HF) or breast cancer patients (BCP) plasma contain less than 1.4 × 10-8 pg of DNA. Thus, only a minor part of cirDNA is attached to the outer side of the exosome as part of the vesicle crown: the share of exosomal DNA does not exceed 0.025% HF plasma DNA and 0.004% BCP plasma DNA. Treatment of plasma exosomes with DNase I with subsequent dot immunoassay reveals that H2a, H2b, and H3 histones are not part of the exosomal membrane, but are part of the cirDNA-protein macromolecular complex associated with the surface of the exosome either through interaction with DNA-binding proteins or with histone-binding proteins. Using bioinformatics approaches after identification by MALDI-TOF mass spectrometry, 16 exosomal DNA-binding proteins were identified. It was shown that four proteins-AIFM1, IGHM, CHD5, and KCNIP3-are candidates for DNA binding on the outer membrane of exosomes; the crown of exosomes may include five DNA-binding proteins: H2a, H2b, H3, IGHM, and ALB. Of note, AIFM1, IGHM, and CHD5 proteins are found only in HF plasma exosomes; KCNIP3 protein is identified only in BCP plasma exosomes; and H2a, H2b, H3, and ALB are revealed in all samples of plasma exosomes. Two histone-binding proteins, CHD5 and KDM6B, have been found in exosomes from HF plasma. The data obtained indicate that cirDNA preferentially binds to the outer membrane of exosomes by association with DNA-binding proteins.

Exosomal Protease Cargo as Prognostic Biomarker in Colorectal Cancer.

OBJECTIVE: The aim of the study was to develop a model for predicting cancer risk in colorectal polyps' patients (CPPs), as well as to reveal additional prognosis factors for Stage III colorectal cancer based on differences in subpopulations of tetraspanins, tetraspanin-associated and tetraspanin-non-associated proteases in blood plasma exosomes of CPPs and colorectal cancer patients (CRCPs). METHODS: The subpopulations of CD151- and Tspan8-positive exosomes, the subpopulations of metalloproteinase at the surface of СD9-positive exosomes and the level of 20S proteasomes in plasma exosomes in 15 CPPs (tubulovillous adenomas) and 60 CRCPs were evaluated using flow cytometry and Western blotting. Logistic regression analysis was performed to predict cancer risk of CPPs. RESULTS: The levels of 20S proteasomes in exosomes, MMP9+, MMP9+/MMP2+/EMMPRIN+ in CD9-positive blood plasma exosomes are associated with the risk of malignant transformation of colorectal tubulovillous adenomas.  In patients with Stage III CRC, the levels of 20S proteasomes (less than 2 units) and MMP9+ subpopulations (more than 61%) in plasma exosomes are unfavorable prognostic factors for overall survival. The levels of 20S proteasomes and ADAM10+/ADAM17- subpopulations in CD9-positive blood plasma exosomes are the most significant values for predicting relapse-free survival. CONCLUSION: Protease cargo in CD9-positive blood plasma exosomes is prognostic biomarker for colorectal polyps and colorectal cancer.

Changes in the Ultrastructure of Staphylococcus aureus Treated with Cationic Peptides and Chlorhexidine.

Antimicrobial peptides, including synthetic ones, are becoming increasingly important as a promising tool to fight multidrug-resistant bacteria. We examined the effect of cationic peptides H2N-Arg9-Phe2-C(O)NH2 and H2N-(Lys-Phe-Phe)3-Lys-C(O)NH2 on Staphylococcus aureus, which remains one of the most harmful pathogens. Antiseptic chlorhexidine served as reference preparation. We studied viability of S. aureus and examined its ultrastructure under treatment with 100 µM of R9F2 or (KFF)3K peptides or chlorhexidine using transmission electron microscopy of ultrathin sections. Bacterial cells were sampled as kinetic series starting from 1 min up to 4 h of treatment with preparations. Both peptides caused clearly visible damage of bacteria cell membrane within 1 min. Incubation of S. aureus with R9F2 or (KFF)3K peptides led to cell wall thinning, loss of cytoplasm structure, formation of mesosome-derived multimembrane structures and "decorated fibers" derived from DNA chains. The effect of R9F2 peptides on S. aureus was more severe than the effect of (KFF)3K peptides. Chlorhexidine heavily damaged the bacteria cell wall, in particular in areas of septa formation, while cytoplasm kept its structure within the observation time. Our study showed that cell membrane damage is critical for S. aureus viability; however, we believe that cell wall disorders should also be taken into account when analyzing the effects of the mechanisms of action of antimicrobial peptides (AMPs).

Amphiphilic "Like-a-Brush" Oligonucleotide Conjugates with Three Dodecyl Chains: Self-Assembly Features of Novel Scaffold Compounds for Nucleic Acids Delivery.

The conjugation of lipophilic groups to oligonucleotides is a promising approach for improving nucleic acid-based therapeutics' intracellular delivery. Lipid oligonucleotide conjugates can self-aggregate in aqueous solution, which gains much attention due to the formation of micellar particles suitable for cell endocytosis. Here, we describe self-association features of novel "like-a-brush" oligonucleotide conjugates bearing three dodecyl chains. The self-assembly of the conjugates into 30-170 nm micellar particles with a high tendency to aggregate was shown using dynamic light scattering (DLS), atomic force (AFM), and transmission electron (TEM) microscopies. Fluorescently labeled conjugates demonstrated significant quenching of fluorescence intensity (up to 90%) under micelle formation conditions. The conjugates possess increased binding affinity to serum albumin as compared with free oligonucleotides. The dodecyl oligonucleotide conjugate and its duplex efficiently internalized and accumulated into HepG2 cells' cytoplasm without any transfection agent. It was shown that the addition of serum albumin or fetal bovine serum to the medium decreased oligonucleotide uptake efficacy (by 22.5-36%) but did not completely inhibit cell penetration. The obtained results allow considering dodecyl-containing oligonucleotides as scaffold compounds for engineering nucleic acid delivery vehicles.

Changes in the Ultrastructure of Candida albicans Treated with Cationic Peptides.

Candida albicans is becoming increasingly harmful for humans, which determines the need for new effective antifungal preparations. Currently, when testing antifungals, various morphological methods are used, among which transmission electron microscopy (TEM) is not the leading one. In this work, we used TEM to study the submicroscopic changes in C. albicans cells induced by cationic peptides R9F2 and (KFF)3K. Studies were performed on C. albicans-34 strain from the Collection of EMTC of ICBFM SB RAS in logarithmic phase. R9F2 and (KFF)3K showed an antifungal effect (MIC 10 and 20 µM) and suppressed fungal hyphal growth. Semithin and ultrathin sections of fungal suspensions incubated with 10 µM of peptides were studied at regular intervals from 15 min to 24 h. The first target of both peptides was plasmalemma, and its "alignment" was the only common morphological manifestation of their effect. Other changes in the plasmalemma and alteration of the vacuole and cell wall ultrastructure distinctly differed in cells treated with R9F2 and (KFF)3K peptides. In general, our work has shown pronounced differences of the temporal and morphologic characteristics of the effect of peptides, evidently related to their physicochemical properties. The benefit of TEM studies of ultrathin sections for understanding the mechanisms of action of antifungal drugs is shown.

Proteomic Analysis of Blood Exosomes from Healthy Females and Breast Cancer Patients Reveals an Association between Different Exosomal Bioactivity on Non-tumorigenic Epithelial Cell and Breast Cancer Cell Migration in Vitro.

Exosomes are important intercellular communication vehicles, secreted into body fluids by multiple cell types, including tumor cells. They contribute to the metastatic progression of tumor cells through paracrine signalling. It has been recently discovered that blood circulating exosomes contain distinguishable fractions of free and cell-surface-associated vesicles. We evaluated the influence of protein cargoes from exosomes from plasma, and exosomes from the total blood of healthy females (HFs) and breast cancer patients (BCPs), on cell motility. We conducted a mass spectrometric analysis of exosomal contents isolated from samples using ultrafiltration and ultracentrifugation approaches and verified their nature using transmission electron microscopy, nanoparticle tracking analysis and flow cytometry. We observed that malignant neoplasm-associated proteins in exosomes from BCP total blood were detected more often than in plasma (66% vs. 59%). FunRich analysis to assess Gene Ontology (GO) enrichment revealed that proteins with catalytic activities, transporter functions and protein metabolism activities were increased in exosomes from BCP blood. Finally, GO analysis revealed that proteomic profiles of exosomes from HF total blood were enriched with proteins inhibiting cell migration and invasion, which explains the low stimulating activity of exosomes from HF total blood on SKBR-3 cancer cell migration velocity. This allows exosomes to act as intermediaries providing intercellular communications through horizontal transfer of RNA and functionally active proteins, potentially affecting the development of both primary neoplasms and distant metastases.

Blood Circulating Exosomes Contain Distinguishable Fractions of Free and Cell-Surface-Associated Vesicles.

BACKGROUND: Considering exosomes as intercellular transporters, inevitably interacting with the plasma membrane and the large available surface of blood cells, we wonder if a fraction of circulating exosomes is associated with the surface of blood cells. OBJECTIVE: The aim of this study was to develop an efficient protocol for isolating exosomes associated with the surface of blood cells and to further investigate the characteristics of this fraction in a healthy state and during the development of breast cancer, as well as its possible implication for use in diagnostic applications. METHODS: Blood samples were collected from Healthy Females (HFs) and breast cancer patients (BCPs). Exosomes extracted from blood plasma and eluted from the surface of blood cells were isolated by ultrafiltration with subsequent ultracentrifugation. RESULTS: Transmission Electron Microscopy (TEM), along with immunogold labeling, demonstrated the presence of exosomes among membrane-wrapped extracellular vesicles (EVs) isolated from both plasma and blood cell eluates. TEM, nanoparticle tracking analysis, and NanoOrange protein quantitation data showed that cell-associated exosomes constituted no less than 2/3 of total blood exosome number. Exosomes, ranging from 50-70 nm in size, prevailed in the blood of breast cancer patients, whereas smaller exosomes (30-50 nm) were mostly observed in the blood of healthy women. Analysis of specific proteins and RNAs in exosomes circulating in blood demonstrated the significant differences in the packing density of the polymers in exosomes of HFs and BCPs. Preliminary data indicated that detection of cancer-specific miRNA (miR-103, miR-191, miR-195) in exosomes associated with the fraction of red blood cells allowed to discriminate HFs and BCPs more precisely compared to cell-free exosomes circulating in plasma. CONCLUSION: Our data provide the basis for using blood cell-associated exosomes for diagnostic applications.

What information can be obtained from the tears of a patient with primary open angle glaucoma?

Since tears are a biological fluid, they have a potential diagnostic value for ophthalmic diseases. The aim of this study was to compare tear supernatants and pellets obtained from patients suffering from primary open angle glaucoma (POAG) and healthy persons (HPs) using transmission electron microscopy (TEM) and molecular biological methods. Tear supernatants and pellets were prepared using ultrafiltration and ultracentrifugation and were examined by negative staining and immunogold labelling TEM. DNA of the pellets was isolated, quantified and sequenced using a MiSeq (Illumina, USA) genomic sequencer with the Reagent Kit v3 (600 cycles, Illumina, USA). MicroRNA was isolated and quantified from the pellets; miR-146b, miR-16 and miR-126 were detected using TaqMan MicroRNA Assays (Applied Biosystems, USA). TEM of tear supernatants from both POAG patients and HPs revealed identical constituents: spherical or cup-shaped vesicles, "non-vesicles", cell debris and macromolecular aggregates. Pellets of POAG patients and HPs contained small extracellular vesicles (sEVs) non-labelled vesicles and "non-vesicles"; pellets of sick persons also contained sEVs with "a capsule". POAG-patient tear pellets showed elevated concentrations of genomic ds-DNA and SINE-repeats, and different expressions of miR-146b, miR-16 and miR-126 and a different set of bacterial DNA in comparison with pellets obtained from the tears of HPs. The data obtained indicate that the tears of HPs and POAG patients could serve as an object for TEM studies and as a source of sEV-containing preparations (pellets), which, in turn, could be used for the isolation and study of genomic ds-DNA and RNA. Our data provide the basis for using tears for diagnostic applications.

Protease Cargo in Circulating Exosomes of Breast Cancer and Ovarian Cancer Patients

Background: As is known, exosomes play an important role in promoting progression of cancers by increasing its invasive potential. The aim of this study was to evaluate the levels of tetraspanine-associated (ADAM-10) and tetraspanine-nonassociated proteases (20S proteasomes) in exosomes from culture medium, plasma exosomes of patients with breast tumors and plasma and ascites of ovarian tumor patients. Methods: MCF-7 and SVO-3 culture mediums and blood samples from healthy females (n = 30, HFs), patients with diffuse dyshormonal dysplasia of the breast (n=28, BBTPs), breast cancer patients (n=32, BCPs), borderline ovarian tumor patients (n=20, BOTPs) and blood and ascites samples ovarian cancer patients (n=35, OCPs) were included in the study. Exosomes from plasma, ascites and culture mediums were isolated and characterized in according to Extracellular Vesicles Society. The expression levels of 20S proteasome and ADAM-10 in exosomes were determined using flow cytometry and western blot analysis, correspondingly. Results: The subpopulation composition of the exosomes from MCF-7 culture medium and from blood plasma of HFs and breast diseases patients is similar, however CD9/CD24 subpopulation significantly increased at cell supernatant. The similar results was obtained for exosomes from SVO-3 medium and blood plasma and ascites of ovary tumor patients, but CD9/CD24 subpopulation significantly decreased at cells and illness samples, however CD63/CD24 exosomes increased significantly from cell supernatant. 20S proteasome level is significantly increased in exosomes from MCF-7 and SVO-3 culture medium, breast tumor patients and OCPs plasma in comparison to HUVEC culture medium and HFs plasma samples. At CD9-positive exosomes from BCPs plasma and MCF-7 was reveal a high expression of ADAM-10 and low expression is from BBDPs plasma and ovarian tumor patients plasma/ ascites samples. Exosomes from ascites OCP had high expression of ADAM-10 in the CD24-positive subpopulation. Conclusion: Breast and ovarian cancer development is connected with functioning of immune proteasome forms in plasma and ascites exosomes, while increased ADAM10 expression at CD9-positive exosome was associated with breast cancer and at CD24-positive subpopulation ­ with ovarian cancer. Obtained data confirm role of exosomal proteases in tumor progression.

Relation between Tetraspanin- Associated and Tetraspanin- Non- Associated Exosomal Proteases and Metabolic Syndrome in Colorectal Cancer Patients

Purpose: Exosomal proteases are important in regulation of molecular signaling from growth factor receptors and adhesion molecules and also the regulation of cell motility and protein folding. The aim of this study was to evaluate the level of ADAM10, ADAM17 and 20S proteasomes in exosomes isolated from colorectal cancer patients (CRCPs) in relation with clinical and histopathological parameters. Methods: Blood plasma exosomes of 60 CRCPs at stage T2-4N0-2M0-1 and 10 control subjects (CSs) with colorectal polyps were isolated using ultrafiltration in combination with ultracentrifugation. The level of tetraspanin-associated (ADAM20 and ADAM17) and tetraspanin-non-associated (20S proteasome) proteases were evaluated by flow cytometry and western blot analysis. Results: The ADAM10-/ ADAM17- population predominated in plasma exosomes of CRCPs and the level of ADAM10+ exosomes was significantly higher in exosomes of CSs compared with CRCPs. No difference was found between subpopulations of ADAM10/ADAM17 exosomes and level of exosomal 20S proteasomes in terms of sex, age and tumor grade. Simultaneous decrease of ADAM10+/ADAM17-subpopulation of exosomes and level of exosomal 20S proteasomes in patients with metastatic CRC was observed compared with patients with non-metastatic CRC. The level of ADAM17+ exosomes significantly reduced in exosomes of CRCPs with metabolic syndrome compared to CRCPs without metabolic syndrome( 3.97±0.71 (%) vs. 13.04±1.34 (%), respectively (p<0.05). A decrease in the 20S proteasomes level in plasma exosomes was revealed in CRCPs with metabolic syndrome compared with CRCPs without metabolic disorders ( 1.90±0.25 (r.u.) vs. 2.92±0.42 (r.u.) respectively( (p<0.05). Conclusion: According to findings of this study, it seems that exosomal proteases can be promising molecular predictors of hematogenous metastasis in patients with non-metastatic CRC. Further studies on subpopulation composition of exosomes CRCPs are need for elucidating the role of tetraspanin-associated and tetraspanin-non-associated exosomal proteases in CRC development and progression.

Metalloproteinases at the surface of small extrcellular vesicles in advanced ovarian cancer: Relationships with ascites volume and peritoneal canceromatosis index.

Metalloproteinases and their extracellular matrix metalloproteinase inducer (EMMPRIN) play an essential role in the regulation of signaling from growth factors receptors and adhesion molecules, cell motility and extracellular matrix degradation. The aim of the study was to evaluate the relationship between the levels of small extracellular vesicles (sEVs) metalloproteinases, such as ADAM10, ADAM17, MMP2, MMP9 and EMMPRIN and ascites volume and peritoneal canceromatosis index in advanced ovarian cancer patients (OCPs). The subpopulations of metalloproteinases at the surface of sEVs of borderline ovarian tumor patients (BOTPs) (n = 20, 36.5 ±â€¯2.5 years) and previously untreated advanced OCPs (n = 35, 56.5 ±â€¯2.5 years) were evaluated using flow cytometry. The metalloproteinase subpopulations of CD9-positive sEVs isolated from plasma of BOTPs and OCPs appeared to be quite similar. However, a significant difference in the expression of ADAM-metalloproteinases in ascites sEVs was found between BOTPs and OCPs. The level of sEVs metalloproteinases in OCPs significantly depended on the ascites volume. A statistically significant relationship between the level of ADAM10+/ADAM17- subpopulation in plasma sEVs and the peritoneal canceromatosis index was found (R = 0.66, p < .05). The levels of metalloproteinases and EMMPRIN in circulating sEVs, as well as the assessment of individual subpopulations may be promising approaches to OCPs managing.

Purified horse milk exosomes contain an unpredictable small number of major proteins.

Exosomes are 40-100 nm nanovesicles containing RNA and different proteins. Exosomes containing proteins, lipids, mRNAs, and microRNAs are important in intracellular communication and immune function. Exosomes from different sources are usually obtained by combination of centrifugation and ultracentrifugation and according to published data can contain from a few dozens to thousands of different proteins. Crude exosome preparations from milk of eighteen horses were obtained for the first time using several standard centrifugations. Exosome preparations were additionally purified by FPLC gel filtration. Individual preparations demonstrated different profiles of gel filtration showing well or bad separation of exosome peaks and one or two peaks of co-isolating proteins and their complexes. According to the electron microscopy, well purified exosomes displayed a typical exosome-like size (30-100 nm) and morphology. It was shown that exosomes may have several different biological functions, but detection of their biological functions may vary significantly depending on the presence of exosome contaminating proteins and proteins directly into exosomes. Exosome proteins were identified before and after gel filtration by MALDI MS and MS/MS spectrometry of protein tryptic hydrolyzates derived by SDS PAGE and 2D electrophoresis. The results of protein identification were unexpected: one or two peaks co-isolating proteins after gel-filtration mainly contained kappa-, beta-, alpha-S1-caseins and its precursors, but these proteins were not found in well-purified exosomes. Well-purified exosomes contained from five to eight different major proteins: CD81, CD63 receptors, beta-lactoglobulin and lactadherin were common to all preparations, while actin, butyrophilin, lactoferrin, and xanthine dehydrogenase were found only in some of them. The article describes the morphology and the protein content of major horse milk exosomes for the first time. Our results on the decrease of major protein number identified in exosomal preparations after gel filtration may be important to the studies of biological functions of pure exosomes.