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Different methods for fixing biofilms of Staphylococcus aureus and Salmonella enterica for light and electron microscopy were compared. Paraformaldehyde fixation did not preserve biofilm integrity during dehydration; Ito-Karnovsky fixation revealed cell morphology, but did not preserve the matrix. Ruthenium red combined with aldehydes allowed the matrix to be preserved and visualized. An analysis of the ultrastructure of S. aureus and S. enterica cells in biofilms and suspensions at various fixations is presented. The ultrastructure of the biofilm matrix has been described., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Bacteria of the genus Staphylococcus are significant challenge for medicine, as many species are resistant to multiple antibiotics and some are even to all of the antibiotics we use. One of the approaches to developing new therapeutics to treat staphylococcal infections is the use of bacteriophages specific to these bacteria or the lytic enzymes of such bacteriophages, which are capable of hydrolyzing the cell walls of these bacteria. In this study, a new bacteriophage vB_SepP_134 (St 134) specific to Staphylococcus epidermidis was described. This podophage, with a genome of 18,275 bp, belongs to the Andhravirus genus. St 134 was able to infect various strains of 12 of the 21 tested coagulase-negative Staphylococcus species and one clinical strain from the Staphylococcus aureus complex. The genes encoding endolysin (LysSte134_1) and tail tip lysin (LysSte134_2) were identified in the St 134 genome. Both enzymes were cloned and produced in Escherichia coli cells. The endolysin LysSte134_1 demonstrated catalytic activity against peptidoglycans isolated from S. aureus, S. epidermidis , Staphylococcus haemolyticus , and Staphylococcus warneri . LysSte134_1 was active against S. aureus and S. epidermidis planktonic cells and destroyed the biofilms formed by clinical strains of S. aureus and S. epidermidis .

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 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 (DL 4 12), ciprofloxacin and their hybrid (DL 5 Cip6); the samples were incubated for 15 and 45 min. DL 4 12 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 DL 4 12 action. Signs associated with ciprofloxacin predominated in cell damage patterns from the hybrid. We studied the effect of ciprofloxacin, DL 4 12 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.

Extracellular vesicles (EVs), carriers of molecular signals, are considered a critical link in maintaining homeostasis in mammals. Currently, there is growing interest in studying the role of EVs, including exosomes (subpopulation of EVs), in animals of other evolutionary levels, including marine invertebrates. We have studied the possibility of obtaining appropriate preparations of EVs from whole-body extract of holothuria Eupentacta fraudatrix using a standard combination of centrifugation and ultracentrifugation. However, the preparations were heavily polluted, which did not allow us to conclude that they contained vesicles. Subsequent purification by FLX gel filtration significantly reduced the pollution but did not increase vesicle concentration to a necessary level. To detect EVs presence in the body of holothurians, we used transmission electron microscopy of ultrathin sections. Late endosomes, producing the exosomes, were found in the cells of the coelom epithelium covering the gonad, digestive tube and respiratory tree, as well as in the parenchyma cells of these organs. The study of purified homogenates of these organs revealed vesicles (30-100 nm) morphologically corresponding to exosomes. Thus, we can say for sure that holothurian cells produce EVs including exosomes, which can be isolated from homogenates of visceral organs.

Exosomes are nanovesicles with a 40-150 nm diameter and are essential for communication between cells. Literature data suggest that exosomes obtained from different sources (cell cultures, blood plasma, urea, saliva, tears, spinal fluid, milk) using a series of centrifugations and ultracentrifugations contain hundreds and thousands of different protein and nucleic acid molecules. However, most of these proteins are not an intrinsic part of exosomes; instead, they co-isolate with exosomes. Using consecutive ultracentrifugation, gel filtration, and affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we isolated highly purified vesicle preparations from 18 horse milk samples. Gel filtration of the initial preparations allowed us to remove co-isolating proteins and their complexes and to obtain highly purified vesicles morphologically corresponding to exosomes. Using affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we obtained extra-purified CD9 + and CD63 + exosomes, which simultaneously contain these two tetraspanins, while the CD81 tetraspanin was presented in a minor quantity. SDS-PAGE and MALDI analysis detected several major proteins with molecular masses over 10 kDa: CD9, CD63, CD81, lactadherin, actin, butyrophilin, lactoferrin, and xanthine dehydrogenase. Analysis of extracts by trifluoroacetic acid revealed dozens of peptides with molecular masses in the range of 0.8 to 8.5 kDa. Data on the uneven distribution of tetraspanins on the surface of horse milk exosomes and the presence of peptides open new questions about the biogenesis of these extracellular vesicles.

Small interfering RNAs (siRNAs) are a powerful tool for specific suppression of protein synthesis in the cell, and this determines the attractiveness of siRNAs as a drug. Low resistance of siRNA to nucleases and inability to enter into target cells are the most crucial issues in developing siRNA-based therapy. To face this challenge, we designed multilayer nanoconstruct (MLNC) with AuNP core bearing chemically modified siRNAs. We applied chemical modifications 2'-OMe and 2'-F substitutions as well as their combinations with phosphoryl guanidine group in the internucleotide phosphate. The effect of modification on the efficiency of siRNA loading into nanocarriers was examined. The introduction of the internucleotide modifications into at least one of the strands raised the efficiency of siRNA adsorption on the surface of gold core. We also tested the stability of modified siRNA adsorbed on gold core in the presence of serum. Based on loading efficiency and stability, MLNCs with the most siRNA effective cargo were selected, and they showed an increase in biological activity compared to control MLNCs. Our study demonstrated the effect of chemical modifications of siRNA on its binding to the AuNP-based carrier, which directly affects the efficiency of target protein expression inhibition.

Extracellular vesicles (EVs) produced by various cell types are heterogeneous in size and composition. Changes in the RNA sets of EVs in biological fluids are considered the basis for the development of new approaches to minimally invasive diagnostics and the therapy of human diseases. In this study, EVs were obtained from the blood of healthy donors by centrifugation, followed by ultracentrifugation. It was shown that EVs consist of several populations including small exosome-like vesicles and larger microvesicle-like particles. The composition of EVs' RNAs was determined. A549 lung adenocarcinoma cells were incubated with EV and the NGS analysis of differentially expressed genes was performed. During the incubation of A549 cells with EVs, the levels of mRNA encoding components for the NF-kB signaling pathway increased, as well as the expression of genes controlled by the NF-kB transcription factor. Overall, our results suggest that components of EVs trigger the NF-kB signaling cascade in A549 cells, leading to the transcription of genes including cytokines, adhesion molecules, cell cycle regulators, and cell survival factors. Our data provide insight into the interaction between blood EVs and human cells and can be used for designing new tools for the diagnosis and treatment of human diseases.

There is an urgent need to develop systems for nucleic acid delivery, especially for the creation of effective therapeutics against various diseases. We have previously shown the feasibility of efficient delivery of small interfering RNA by means of gold nanoparticle-based multilayer nanoconstructs (MLNCs) for suppressing reporter protein synthesis. The present work is aimed at improving the quality of preparations of desired MLNCs, and for this purpose, optimal conditions for their multistep fabrication were found. All steps of this process and MLNC purification were verified using dynamic light scattering, transmission electron microscopy, and UV-Vis spectroscopy. Factors influencing the efficiency of nanocomposite assembly, colloidal stability, and purification quality were identified. These data made it possible to optimize the fabrication of target MLNCs bearing small interfering RNA and to substantially improve end product quality via an increase in its homogeneity and a decrease in the amount of incomplete nanoconstructs. We believe that the proposed approaches and methods will be useful for researchers working with lipid nanoconstructs.

Small interfering RNA (siRNA) is the most important tool for the manipulation of mRNA expression and needs protection from intracellular nucleases when delivered into the cell. In this work, we examined the effects of siRNA modification with the phosphoryl guanidine (PG) group, which, as shown earlier, makes oligodeoxynucleotides resistant to snake venom phosphodiesterase. We obtained a set of siRNAs containing combined modifications PG/2'-O-methyl (2'-OMe) or PG/2'-fluoro (2'-F); biophysical and biochemical properties were characterized for each duplex. We used the UV-melting approach to estimate the thermostability of the duplexes and RNAse A degradation assays to determine their stability. The ability to induce silencing was tested in cultured cells stably expressing green fluorescent protein. The introduction of the PG group as a rule decreased the thermodynamic stability of siRNA. At the same time, the siRNAs carrying PG groups showed increased resistance to RNase A. A gene silencing experiment indicated that the PG-modified siRNA retained its activity if the modifications were introduced into the passenger strand.

Fluorophore (FD) labeling is widely used for detection and quantification of various compounds bound to nanocarriers. The systems, composed of gold nanoparticles (GNPs) and oligonucleotides (ONs) labeled with FDs, have wide applications. Our work was aimed at a systemic study of how FD structure (in composition of ON-FDs) influenced the efficiency of their non-covalent associates' formation with GNPs (ON-FD/GNPs). We examined ONs of different length and nucleotide composition, and corresponding ON-FDs (FDs from a series of xanthene, polymethine dyes; dyes based on polycyclic aromatic hydrocarbons). Methods: fluorometry, dynamic light scattering, high performance liquid chromatography, gel electrophoresis, molecular modeling and methods of thermodynamic and statistical analysis. We observed significant, differing several times, changes in surface density and Langmuir constant values of ON-FDs vs. ONs, evidence for the critical significance of FD nature for binding of ON-FDs with GNPs. Surface density of ON-FD/GNPs; hydrophobicity and total charge of ON or ON-FD; and charge and surface area of FDs were revealed as key factors determining affinity (Langmuir constant) of ON or ON-FDs for GNPs. These factors compose a specific set, which makes possible the highly reliable prediction of efficiency of ONs and ON-FDs binding with GNPs. The principal possibility of creating an algorithm for predictive calculation of efficiency of ONs and GNPs interaction was demonstrated. We proposed a hypothetical model that described the mechanism of contact interaction between negatively charged nano-objects, such as citrate-stabilized GNPs, and ONs or ON-FDs.

Topotecan is a cytostatic drug from the camptothecin group, it acts by inhibiting topoisomerase 1 (TOP1). Tyrosyl-DNA phosphodiesterase 1 (TDP1) is capable of interfering with the action of TOP1 inhibitors, reducing their therapeutic efficacy. Suppression of TDP1 activity may enhance the effects of topotecan. In this work, we investigated the effect of the antitumor drug topotecan alone and in combination with a TDP1 inhibitor, a hydrazinothiazole derivative of usnic acid, on Krebs-2 mouse ascites tumors. We have previously shown that this derivative efficiently inhibits TDP1. In the present work, we show that both topotecan and the TDP1 inhibitor have an antitumor effect when evaluated separately. The combination of topotecan and the TDP1 inhibitor additively reduces both the weight of the ascites tumor and the number of cells in ascites. In mice, the TDP1 inhibitor alone or in combination with topotecan eliminated the tumor cells. After the combined intraperitoneal administration of these two compounds, we observed cells in which lipid droplets occupied almost the entire cytoplasm and the accumulation of cell detritus, which was absent in the samples collected from mice treated with each compound separately.

Multifunctional gold nanoparticles (AuNPs) may serve as a scaffold to integrate diagnostic and therapeutic functions into one theranostic system, thereby simultaneously facilitating diagnosis and therapy and monitoring therapeutic responses. Herein, albumin-AuNP theranostic agents have been obtained by conjugation of an anticancer nucleotide trifluorothymidine (TFT) or a boron-neutron capture therapy drug undecahydro- closo -dodecaborate (B 12 H 12 ) to bimodal human serum albumin (HSA) followed by reacting of the albumin conjugates with AuNPs. In vitro studies have revealed a stronger cytotoxicity by the AuNPs decorated with the TFT-tagged bimodal HSA than by the boronated albumin conjugates. Despite long circulation time, lack of the significant accumulation in the tumor was observed for the AuNP theranostic conjugates. Our unique labelling strategy allows for monitoring of spatial distribution of the AuNPs theranostic in vivo in real time with high sensitivity, thus reducing the number of animals required for testing and optimizing new nanosystems as chemotherapeutic agents and boron-neutron capture therapy drug candidates.

The cooperative thermomechanical properties of plant-derived polymers have been studied insufficiently, although this feedstock has a very high potential. In the present paper, we analyzed the changes in the structure and physicochemical properties of lignin-rich biomass induced by thermomechanical pretreatment. Low-temperature treatment allows one to retain the original supramolecular structure of the cell walls, while an appreciably high disintegration degree is reached. This increases the reactivity of the material in the subsequent heterogeneous reactions. Mechanical pretreatment at medium temperatures (10 °C), when almost all cell wall polymers except for low-molecular-weight lignin are in the glassy state, enhances the mobility of cell wall polymers and causes sufficient cellulose disordering, while the specific surface area is not significantly increased. High-temperature pretreatment of reed biomass is accompanied by pore formation and lignin release from the cell wall structure, which opens up new prospects for using this biomass as a matrix to produce core-shell-structured sorbents of heavy metals. The energy consumed by mechanochemical equipment for the activation of reed biomass was determined., Competing Interests: The authors declare no conflicts of interest.

Gold nanoparticles (AuNPs) are a platform for the creation of nanoconstructions that can have a variety of functions, including the delivery of therapeutic nucleic acids. We previously designed a AuNP/small interfering RNA (siRNA) nanoconstruction consisting of siRNA noncovalently bound on the AuNP surface and showed that this construction, when coated with a lipid shell, was an efficient vehicle for the delivery of siRNA into cells. The goal of the present work was to study the possibility of scaling up the synthesis of AuNP-siRNA and its long-term storage without loss of physicochemical characteristics and siRNA duplex integrity as well as siRNA surface density. Dynamic light scattering, transmission electron microscopy, UV-vis spectroscopy, and electrophoresis were used to study the effect of scaling up the AuNP-siRNA synthesis and long term storage of its suspension on physicochemical properties of the samples and integrity of the siRNA duplex. It was shown that a ten-fold increase in the volume of the reaction mixture decreased the surface density of siRNA by about 10%, which influenced the corresponding physicochemical characteristics of the AuNP-siRNA suspension. The storage of the AuNP-siRNA suspension at 4 °C for different times resulted in the formation of particle clusters of high colloidal stability as demonstrated by conventional methods. These clusters completely disintegrated when albumin was added, indicating that they are agglomerates (and not aggregates) of AuNP-siRNA. The AuNPs-siRNA nanoconstruction demonstrated integrity of the siRNA duplex and high stability of the siRNA surface density during storage for seven months at 4 °C. Thus, it can be concluded that it is possible to scale-up the synthesis of noncovalent AuNP-siRNA and to obtain a nanoconstruction possessing high stability in terms of physicochemical characteristics and siRNA surface density for a long period., (Copyright © 2019, Epanchintseva et al.; licensee Beilstein-Institut.)

Nanoconstructions composed of lipid vesicles and inorganic units (nanoparticles, metal complexes) arouse much interest across materials science and nanotechnology as hybrid materials combining useful functionalities from both parts. Ideally, these units are to be embedded into the bilayer to keep the biophysical performance of lipid vesicles having inorganic moieties screened from the environment. This can be achieved by doping a lipid bilayer with cluster complexes of transition metals. In this work, we report the preparation of nanoparticles from trinuclear W 3 S 4 cluster complexes and egg phosphatidylcholine. A systematic study of their properties was performed by the differential scanning calorimetry, NMR spectroscopy, dynamic light scattering, and transmission electron microscopy. Phospholipids and clusters have been found to spontaneously self-assemble into novel cluster-lipid hybrid materials. The behavior of clusters in the hydrophobic lipid environment is determined by the structure of the ligands and cluster-to-lipid ratio. Intact cluster complexes bearing compact hydrophobic ligands are embedded into the hydrophobic midplane of a lipid bilayer, whereas cluster complexes bearing larger ligands drive the aggregation of lipids and cluster complexes. Considering these differences, it could be possible to obtain different self-assembled associates such as cluster-doped liposomes or lipid-covered crystals. These cluster-lipid hybrids can be a platform for the design of new materials for nanotechnology.

A novel lytic Raoultella phage, RP180, was isolated and characterized. The RP180 genome has 44,851 base pairs and contains 65 putative genes, 35 of them encoding proteins whose functions were predicted based on sequence similarity to known proteins. The RP180 genome possesses a gene synteny typical of members of the subfamily Guernseyvirinae. Phylogenetic analysis of the RP180 genome and similar phage genomes revealed that phage RP180 is the first member of the genus Kagunavirus, subfamily Guernseyvirinae, that is specific for Raoultella sp. The genome of RP180 encodes a putative protein with similarity to CRISPR-like Cas4 nucleases, which belong to the pfam12705/PDDEXK_1 family. Cas4-like proteins of this family have been shown to interfere with the bacterial host type II-C CRISPR-Cas system.

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.

Exosomes are nanovesicles (40-100 nm) containing various RNAs and different proteins. Exosomes are involved in intracellular communication and immune system function. Exosomes from different sources are usually isolated using standard methods-centrifugation and ultracentrifugations. Exosomes isolated by these procedures were reported to contain from a few dozen to thousands of different proteins. Here crude vesicle preparations from five placentas (normal pregnancy) were first obtained using standard centrifugation procedures. According to electron-microscopic studies, these preparations contained vesicles of different size (30-225 nm), particles of round shape of average electron density ("nonvesicles" 20-40 nm) (A), structured clusters of associated proteins and shapeless aggregations (B), as well as ring-shaped 10-14 nm structures formed by ferritin (C). After additional purification of the vesicle preparations by gel filtration on Sepharose 4B, the main part of protein structures was removed; however, the preparations still contained small admixtures of components A-C. Further purification of the preparations by affinity chromatography on Sepharose bearing immobilized antibodies against exosome surface protein CD81 led to isolation of highly purified exosomes (40-100 nm). These exosomes according to electron microscopy data contained tetraspanin embedded in the membrane, which was stained with antibodies against CD81 conjugated with 10-12 nm gold nanoparticles. SDS-PAGE and MALDI MS and MS/MS mass spectrometry of tryptic hydrolysates of proteins contained in these exosomes revealed eleven major proteins (>10 kDa): hemoglobin subunits, CD81, interleukin-1 receptor, annexin A5, cytoplasmic actin, alpha-actin-4, alkaline phosphatase, human serum albumin, serotransferrin, and lactotrasferrin. Using MALDI mass analysis of the highly purified exosomes, we for the first time found that in addition to the large proteins (>10 kDa), exosomes having affinity to CD81 contain more than 27 different peptides and small proteins of 2-10 kDa. This finding can be useful for revealing biological functions of pure exosomes. © 2018 IUBMB Life, 70(11):1144-1155, 2018., (© 2018 International Union of Biochemistry and Molecular Biology.)

In this work, we report the assemblage of hydrogels from phosphorus dendrimers in the presence of biocompatible additives and the study of their interactions with nucleic acids. As precursors for hydrogels, phosphorus dendrimers of generations 1⁻3 based on the cyclotriphosphazene core and bearing ammonium or pyridinium acetohydrazones (Girard reagents) on the periphery have been synthesized. The gelation was done by the incubation of dendrimer solutions in water or phosphate-buffered saline in the presence of biocompatible additives (glucose, glycine or polyethylene glycol) to form physical gels. Physical properties of gels have been shown to depend on the gelation conditions. Transmission electron microscopy revealed structural units and well-developed network structures of the hydrogels. The hydrogels were shown to bind nucleic acids efficiently. In summary, hydrogels of phosphorus dendrimers represent a useful tool for biomedical applications.

The nuclear delivery of nucleic acid derivatives is an essential prerequisite for successful antisense therapy. Using laser confocal and electron microscopy, we have studied the uptake of fluorescently labeled oligonucleotides in the form of nanocomposites with polylysine and TiO2 nanoparticles into Caco2, MDCK, and HeLa cells. In all three cell lines, bright fluorescence has been detected after 30 min in the nuclei (excluding the nucleoli) of the interphase cells; no substantial increase in the intensity of the signal was observed for next 24 hours. In all cells undergoing mitosis, the signal was localized in the cytoplasm with zones of higher intensity around chromatin. In some cells, at the beginning of interphase (G-1 phase), fluorescence was not detected at all. The latter may be explained by the brief moment in the cell cycle when oligonucleotides delivered in the nanocomposite cannot be taken up by cells. The studied nanocomposites are prone to aggregation. The degree of aggregation increases with the storage time up to complete loss of the ability of the nanocomposites to penetrate the cells.

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.

Gold nanoparticles (GNPs) are used in complicated nanoconstructions, and their preparation implies careful analysis of the intermediate and resulting products, including visualisation of the NPs. Visualisation of protein and/or organic polymer covers on GNPs using electron microscopy (EM) was a goal of this study. We covered GNPs with human serum albumin or PEG, and then added a second layer of branched or linear polyethyleneimine. EM studies were supplemented with dynamic light scattering, spectrophotometry and gel electrophoresis, which confirmed the presence and integrity of a cover on GNPs in mixtures with uranylacetate (UA) or phosphotungstic acid (PTA). Covered GNPs were contrasted 'on a drop' or in suspension with UA (pH 4.5) or PTA (pH 0.5, 3.0, 5.0 and 7.0), and studied by transmission EM. A cover on GNPs becomes visible as the result of direct interaction of UA or PTA with the components of a layer. The same NPs could look 'naked' or demonstrate a distinct cover of average electron density. The most distinct images of the layers were obtained using PTA at pH 0.5. Thus, visualisation of protein and/or polymeric layers covering the GNPs by EM depends on the type of contrasting reagent and contrasting conditions, but does not depend on surface charge of the NPs and the chemical nature of a cover., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aim of our study was to attract the attention of researchers at the problem of contamination of exosome preparations. Using a transmission electron microscope JEM-1400 ("JEOL", Japan) we have examined exosome preparations, isolated according to the conventional scheme of sequential centrifugation from different biological fluids: plasma and urine of healthy persons and patients with oncologic diseases, bovine serum, and culture fluid (MDCK, MDA-MB и MCF-7 cells). All exosome preparations (over 200) contained exosomes, which were identified by immuno-electron microscopy using antibodies to tetraspanins CD63 or CD9. Besides exosomes, all the studied preparations contained contaminating structures: distinct particles of low electron density without limiting membrane ("non-vesicles"). Two main kinds of the "non-vesicles" species were found in exosome preparations: 20-40 nm in size, representing 10-40% of all structures in the preparations; and 40-100 nm in size (identical to exosomes by size). Morphology of the "non-vesicles" allowed to identify them as lipoproteins of intermediate and low density (20-40 nm), and very low density (40-100 nm). The highest level of the contamination was detected in exosome preparations, isolated from blood samples. The results of our study indicate the need to control the composition of exosome preparation by electron microscopy and take into account the presence of contaminating structures in analysis of experimental data.

Human peripheral blood contains RNA in cells and in extracellular membrane vesicles, microvesicles and exosomes, as well as in cell-free ribonucleoproteins. Circulating mRNAs and noncoding RNAs, being internalized, possess the ability to modulate vital processes in recipient cells. In this study, with SOLiD sequencing technology, we performed identification, classification, and quantification of RNAs from blood fractions: cells, plasma, plasma vesicles pelleted at 16,000 g and 160,000 g , and vesicle-depleted plasma supernatant of healthy donors and non-small cell lung cancer (NSCLC) patients. It was determined that 16,000 g blood plasma vesicles were enriched with cell-free mitochondria and with a set of mitochondrial RNAs. The variable RNA set of blood plasma 160,000 g pellets reflected the prominent contribution of U1, U5, and U6 small nuclear RNAs' fragments and at the same time was characterized by a remarkable depletion of small nucleolar RNAs. Besides microRNAs, the variety of fragments of mRNAs and snoRNAs dominated in the set of circulating RNAs differentially expressed in blood fractions of NSCLC patients. Taken together, our data emphasize that not only extracellular microRNAs but also circulating fragments of messenger and small nuclear/nucleolar RNAs represent prominent classes of circulating regulatory ncRNAs as well as promising circulating biomarkers for the development of disease diagnostic approaches., Competing Interests: The authors declare that there are no competing interests.

Artificial ribonucleases (aRNases) are small compounds catalysing RNA cleavage. Recently we demonstrated that aRNases readily inactivate various viruses in vitro. Here, for three series of aRNases (1,4-diazabicyclo [2.2.2]octane-based and peptide-like compounds) we show that apart from ribonuclease activity the aRNases display chaotropic-like and membranolytic activities. The levels of membranolytic and chaotropic-like activities correlate well with the efficiency of various viruses inactivation (enveloped, non-enveloped, RNA-, DNA-containing). We evaluated the impact of these activities on the efficiency of virus inactivation and found: i) the synergism between membranolytic and chaotropic-like activities is sufficient for the inactivation of enveloped viruses (influenza A, encephalitis, vaccinia viruses) for 1,4-diazabicyclo [2.2.2]octane based aRNases, ii) the inactivation of non-enveloped viruses (encephalomyocarditis, acute bee paralysis viruses) is totally dependent on the synergism of chaotropic-like and ribonuclease activities, iii) ribonuclease activity plays a leading role in the inactivation of RNA viruses by aRNases Dp12F6, Dtr12 and K-D-1, iv) peptide-like aRNases (L2-3, K-2) being effective virus killers have a more specific mode of action. Obtained results clearly demonstrate that aRNases represent a new class of broad-spectrum virus-inactivating agents., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Recent studies suggest that extracellular vesicles may be the key to timely diagnosis and monitoring of genito-urological malignancies. In this study we investigated the composition and content of extracellular vesicles found in the urine of healthy donors and prostate cancer patients. Urine of 14 PCa patients and 20 healthy volunteers was clarified by low-speed centrifugation and total extracellular vesicles fraction was obtain by high-speed centrifugation. The exosome-enriched fraction was obtained by filtration of total extracellular vesicles through a 0.1 μm pore filter. Transmission electron microscopy showed that cell-free urine in both groups contained vesicles from 20 to 230 nm. Immunogold staining after ultrafiltration demonstrated that 95% and 90% of extracellular vesicles in healthy individuals and cancer patients, respectively, were exosomes. Protein, DNA and RNA concentrations as well as size distribution of extracellular vesicles in both fractions were analyzed. Only 75% of the total protein content of extracellular vesicles was associated with exosomes which amounted to 90-95% of all vesicles. Median DNA concentrations in total extracellular vesicles and exosome-enriched fractions were 18 pg/ml and 2.6 pg/ml urine, correspondingly. Urine extracellular vesicles carried a population of RNA molecules 25 nt to 200 nt in concentration of no more than 290 pg/ml of urine. Additionally, concentrations of miR-19b, miR-25, miR-125b, and miR-205 were quantified by qRT-PCR. MiRNAs were shown to be differently distributed between different fractions of extracellular vesicles. Detection of miR-19b versus miR-16 in total vesicles and exosome-enriched fractions achieved 100%/93% and 95%/79% specificity/sensitivity in distinguishing cancer patients from healthy individuals, respectively, demonstrating the diagnostic value of urine extracellular vesicles.

Exosomes represent a sort of extracellular vesicles, which transfer molecular signals in organism and possess markers of producing cells. Our study was aimed at search of exosomes in the tears of healthy humans, confirmation of their nature and examination of exosome morphological and molecular-biological characteristics. The tears (110-340 ml) were collected from 24 healthy donors (aged 46-60 years); individual probes were centrifuged at 20000 g for 15 min to pellet cell debris. The supernatants were examined in electron microscope using negative staining; and they were also used for isolation and purification of the exosomes by filtration (100 nm pore-size) and double ultracentrifugation (90 min at 100000 g, 4°C). The "pellets" were subjected to electron microscopy, immunolabeling. The RNA and DNA were isolated from the samples, and their sizes were evaluated by capillary electrophoresis, the concentration and localization of nucleic acids were determined. Sequencing of DNA was performed using MiSeq ("Illumina", USA), data were analyzed using CLC GW 7.5 ("Qiagen", USA). Sequences were mapped on human genome (hg19). Electron microscopy revealed in supernatants of the tears cell debris, spherical microparticles (20-40 nm), membrane vesicles and macromolecular aggregates. The "pellets" obtained after ultracentrifugation, contained microparticles (17%), spherical and cup-shaped EVs (40-100 nm, 83%), which were positive for CD63, CD9 and CD24 receptors (specific markers of exosomes). Our study showed presence of high amount of exosomes in human tears, and relation of the exosomes with RNA (size less than 200 nt) and DNA (size was 3-9 kb). Sequencing of the DNA showed that about 92% of the reads mapped to human genome.

Recently, a new Ehrlichia genetic variant, Ehrlichia sp. Khabarovsk, was identified in tissue samples of small mammals captured in the Russian Far East. To further characterize Ehrlichia sp. Khabarovsk, tissue homogenate from a naturally infected gray red-backed vole (Myodes rufocanus) was passaged three times in newborn laboratory mice. Using nested PCR Ehrlichia sp. Khabarovsk DNA was detected in tissue samples from infected mice at 1-4 weeks post inoculation. Electron microscopic examination revealed morulae containing gram-negative bacterial cells in monocytes of mouse spleen and liver. The size and ultrastructure of these cells corresponded to those described previously and allowed us to identify the bacteria as Ehrlichia sp. The comparison of ehrlichial 16S rRNA, groEL and gltA genes and putative GroEL and GltA amino acid sequences has demonstrated that Ehrlichia sp. Khabarovsk, like Ehrlichia ruminantium, is more distant from all other Ehrlichia species than these species are between themselves. Phylogenetic analysis has shown that Ehrlichia sp. Khabarovsk belongs to the clade formed by Ehrlichia spp. but clusters separately from other Ehrlichia species and genetic variants. These data indicate that Ehrlichia sp. Khabarovsk can be considered as a new candidate species. We propose to designate it as 'Candidatus Ehrlichia khabarensis' according to the territory where this species was found., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Background: Antioxidant supplements have been suggested as a strategy to decrease the risk of age-related cataract, but there is no evidence that antioxidants can reduce the signs of the disease. Recently, we showed that the mitochondrial antioxidant SkQ1 can partially reverse cataract signs in senescence-accelerated OXYS rats. The aim of the present study was the histomorphological examination of the influence of SkQ1 eye drops on the cataract development in OXYS rats., Methods: OXYS rats received SkQ1 eye drops (250 nM) from 9 to 12 months of age. Ophthalmoscopic examination was carried out before and after treatment. Light and electron microscopy were used for histomorphological examination. Expression of the Cryaa and Cryab genes was determined using real-time PCR. αB-crystallin expression was detected using Western blotting., Results: SkQ1 completely prevented the cataract development in OXYS rats, and in some of the animals diminished the signs of the disease. Light and electron microscopy showed that SkQ1 attenuated the (typical for cataract) alterations in the lens capsule and epithelial cells, ameliorated disturbances of the hexagonal packing geometry of lens fibers, and improved ultrastructure of the epithelial cells. The levels of mRNA of α-crystallins genes which encode small heat shock proteins αA- and αB-crystallin that play a central role in maintaining lens transparency were significantly lower in the OXYS rats' lenses than in Wistar rats (control). SkQ1 normalized the level of mRNA of Cryaa, and significantly increased the level of Cryab mRNA as well as αB-crystallin protein in the lens of OXYS rats to the level of the control Wistar rats., Conclusion: SkQ1 eye drops hold promise as a treatment of cataract.

Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with limited therapeutic options. Since HCC has been shown to be immunogenic, immunotherapy is considered a promising therapeutic approach. Small interfering RNAs (siRNAs), depending on their structure and sequence, can trigger the innate immune system, which can potentially enhance the adaptive anticancer immune response in the tumor-bearing subjects. Immunostimulatory properties of nucleic acids can be applied to develop adjuvants for HCC treatment., Methods: The transplantable HCC G-29 tumor in male CBA/LacSto (CBA) mice was used to study the effects of immunostimulatory RNA on tumor growth. Tumor size, metastases area in different organs of mice and mouse survival rate were analyzed. Furthermore the mouse serum IFN-α levels were measured using ELISA., Results: In the present study, we found that a 19-bp RNA duplex (ImmunoStimulattory RNA or isRNA) with 3-nt overhangs at the 3'-ends of specific sequence displays immunostimulatory, antitumor, and antimetastatic activities in mice bearing HCC G-29. Our results demonstrate that isRNA strongly increases the level of interferon-α (IFN-α) by up to 25-fold relative to the level in mice injected with Lipofectamine alone (Mock), and to a lesser extent increases the level of proinflammatory cytokine interleukin-6 (IL-6) (by up to 5.5-fold relative to the Mock level), in mice blood serum. We showed that isRNA reliably (P < 0.05) inhibits primary tumor growth in mice compared to the mock group. Furthermore, injections of isRNA significantly enhanced necrotic processes in the center of the primary tumor, and decreased by twofold the width of the undifferentiated peripheral zone and the number of mitotic cells in this zone. The results showed that isRNA efficiently reduces the area of metastases in the liver, kidneys, and heart of CBA/LacSto mice with HCC., Conclusions: The obtained results clearly demonstrate immunostimulatory and antimetastatic properties of the isRNAs in mice with HCC. Consequently, this short double-stranded RNA can be considered as a potential adjuvant for the therapy of HCC.

Specific alterations (mutations, deletions, insertions) of virus genomes are crucial for the functional characterization of their regulatory elements and their expression products, as well as a prerequisite for the creation of attenuated viruses that could serve as vaccine candidates. Virus genome tailoring can be performed either by using traditionally cloned genomes as starting materials, followed by site-directed mutagenesis, or by de novo synthesis of modified virus genomes or parts thereof. A systematic nomenclature for such recombinant viruses is necessary to set them apart from wild-type and laboratory-adapted viruses, and to improve communication and collaborations among researchers who may want to use recombinant viruses or create novel viruses based on them. A large group of filovirus experts has recently proposed nomenclatures for natural and laboratory animal-adapted filoviruses that aim to simplify the retrieval of sequence data from electronic databases. Here, this work is extended to include nomenclature for filoviruses obtained in the laboratory via reverse genetics systems. The previously developed template for natural filovirus genetic variant naming, (/)///-, is retained, but we propose to adapt the type of information added to each field for cDNA clone-derived filoviruses. For instance, the full-length designation of an Ebola virus Kikwit variant rescued from a plasmid developed at the US Centers for Disease Control and Prevention could be akin to "Ebola virus H.sapiens-rec/COD/1995/Kikwit-abc1" (with the suffix "rec" identifying the recombinant nature of the virus and "abc1" being a placeholder for any meaningful isolate designator). Such a full-length designation should be used in databases and the methods section of publications. Shortened designations (such as "EBOV H.sap/COD/95/Kik-abc1") and abbreviations (such as "EBOV/Kik-abc1") could be used in the remainder of the text, depending on how critical it is to convey information contained in the full-length name. "EBOV" would suffice if only one EBOV strain/variant/isolate is addressed.

Novel hybrids of fluorescein-labeled poly(ethylene glycol)-modified single-walled carbon nanotubes (SWCNTs) with nucleic acids were prepared. 5'-Pyrene conjugates of oligodeoxyribonucleotides were used to construct the noncovalent hybrids, with the pyrene residues acting as anchor groups, immobilizing an oligonucleotide on the SWCNT surface. The hybrid formation characteristics were studied using ζ-potential measurements and adsorption isotherm plots. Transmission electron microscopy (TEM) of the samples stained with contrast agents proved that the pyrene conjugates of oligonucleotides were adsorbed onto the surfaces of the functionalized SWCNTs. On the basis of the MTT assay, the functionalized SWCNTs and their hybrids with oligonucleotides exhibited low toxicity toward HeLa, KB-3-1, and KB-8-5 cells. A TEM study of ultrathin sections of cells treated with SWCNTs revealed that the nanotubes directly interacted with the cellular surface.

Gold nanorods (GNRs) are considered one of the most promising forms of nanoparticles for nanobiotechnology; however, the problem of their toxicity is currently not resolved. We synthesised GNRs, modified with linear polyethyleneimine (PEI-GNRs), and examined their physicochemical and some biological properties in comparison with GNRs modified with BSA and spherical gold nanoparticles (sGNPs) modified with the same agents. The influence of the buffer, cell culture media, and serum on hydrodynamic diameter and zeta potential of all GNPs was studied. Simultaneously, the size, shape, and formation of a corona were examined by transmission electron microscopy (TEM). PEI-GNRs and GNPs were nontoxic for BHK-21 and HeLa cells (MTT test). Penetration of all GNPs into BHK-21, melanoma B16, and HeLa cells was examined after 30 min, 3 h, and 24 h of incubation using TEM ultrathin sections. PEI-GNRs and PEI-sGNPs demonstrated fast and active penetration into cells by caveolin-dependent and lipid raft-mediated endocytosis and accumulated in endosomes and lysosomes. BSA-modified GNPs showed prolonged flotation and a significant delay in cell penetration. The results show that the charge of initial NPs determines penetration into cells. Thus, the designed PEI-GNRs were nontoxic and stable in cell culture media and could efficiently penetrate cells.

Aim: Polychemotherapy (PCT), widely used for the antitumor treatment has a pronounced toxic effect on the organism, and its cytostatic effect sometimes is canceled by multidrug resistance of a neoplasia. Comprehension of the nature and development of pathological changes caused by the PCT during the treatment of cancer is very important to improve the efficiency of the therapy and to clarify the mechanisms of tumor-host interactions. This study was aimed to examine PCT impact on kidney cells and tissues in mice with transplanted resistant lymphosacroma (RLS) and to analyze morphology of metastases of the tumor in kidney during PCT., Materials and Methods: Male mice CBA/LacSto (55 animals) were intramuscularly implanted in the right hind paw by 105 cells/ml of tumor RLS (a diffuse large B-cell lymphosarcoma) with multi-drug resistance (MDR) phenotype. Mice received combination of cyclophosphamide (50 mg/kg), oncovin (0.1 mg/kg), hydroxydaunorubicin (4 mg/kg), and prednisone (5 mg/kg) accordingly to CHOP scheme each 7 days after inoculation of the tumor. The kidneys were sampled on days 1, 3 and 7 after each series of injection of PCT preparations and processed for light and electron microscopy, immunohistochemical analysis of Ki-67 and Apaf-1 proteins also was performed., Results: Tumor RLS produced metastases comprised of small cells in the kidneys of mice after 8 days post inoculation. Application of PCT resulted in destruction of small-cell metastases and development of many large-cell metastases in kidney. Application of PCT induced the development of prominent damage of nephron cells, primarily in S3 segments of proximal tubules. Even one series of PCT caused reduction of basal plasma folds in these cells and alteration of mitochondria. Damage of proximal tubules and involvement of distal tubules, renal bodies and interstitial tissue in the pathologic process, increased during the experiment. This work presents the description of morphological changes in kidney as well as of the tumor metastases under PCT influence., Conclusion: The obtained data should be considered while designing of remedies for recovery of internal organs functions after antitumor PCT.

Human bocavirus (HBoV) is associated with acute gastroenteritis in humans, occurring mostly in young children and elderly people. Four bocavirus genotypes (HBoV1-HBoV4) have been found so far. Since there were no data on the contribution of HBoV to gastroenteritis in Russia, 1781 fecal samples collected from infants hospitalized with acute gastroenteritis in Novosibirsk, Russia during one year were tested for the presence of nucleic acids from HBoV and three major gastrointestinal viruses (rotavirus A, norovirus II, and astrovirus). HBoV was detected only in 1.9% of the samples: HBoV1 was detected in 0.6% and HBoV2, in 1.3%. Complete genome sequencing of three Novosibirsk isolates was performed. An evolutionary analysis of these sequences and the available sequences of human and great apes bocaviruses demonstrated that the current HBoV genotypes diverged comparatively recently, about 60-300years ago. The independent evolution of bocaviruses from chimpanzees and gorillas commenced at the same time period. This suggests that these isolates of great apes bocaviruses belong to separate genotypes within the species of human bocavirus, which is actually the primate bocavirus. The rate of mutation accumulation in the genome of primate bocaviruses has been estimated as approximately 9×10(-4)substitutions/site/year. It has been demonstrated that HBoV1 diverged from the ancestor common with chimpanzee bocavirus approximately 60-80years ago, while HBoV4 separated from great apes bocaviruses about 200-300years ago. The hypothesis postulating independent evolution of HBoV1 and HBoV4 genotypes from primate bocaviruses has been proposed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Recent studies demonstrated the ability of artificial ribonucleases (aRNases, small organic RNA cleaving compounds) to inactivate RNA-viruses via the synergetic effect of viral RNA cleavage and disruption of viral envelope [1,2]. Herein, we describe the antiviral activity of aRNases against DNA-containing vaccinia virus: screening of aRNases of various structures revealed that amphiphilic compounds built of positively charged 1,4-diazabicyclo[2.2.2] octane substituted at the bridge nitrogen atoms with aliphatic residues efficiently inactivate this virus. The first stage was the destruction of viral membrane and structure of surface proteins (electron microscopy data). Thus, 1,4-diazabicyclo[2.2.2] octane-based aRNases are novel universal agents inactivating both RNA- and DNA-containing viruses., (Copyright © 2012. Published by Elsevier B.V.)

To study the effect of nanoscale titanium dioxide (TiO(2)) on cell responses, we synthesized four modifications of the TiO(2) (amorphous, anatase, brookite, and rutile) capable of keeping their physicochemical characteristics in a cell culture medium. The modifications of nanoscale TiO(2) were obtained by hydrolysis of TiCl(4) and Ti(i-OC(3)H(7))(4) (TIP) upon variation of the synthesis conditions; their textural, morphological, structural, and dispersion characteristics were examined by a set of physicochemical methods: XRD, BET, SAXS, DLS, AFM, SEM, and HR-TEM. The effect of synthesis conditions (nature of precursor, pH, temperature, and addition of a complexing agent) on the structural-dispersion properties of TiO(2) nanoparticles was studied. The hydrolysis methods providing the preparation of amorphous, anatase, brookite, and rutile modifications of TiO(2) nanoparticles 3-5 nm in size were selected. Examination of different forms of TiO(2) nanoparticles interaction with MDCK cells by transmission electron microscopy of ultrathin sections revealed different cell responses after treatment with different crystalline modifications and amorphous form of TiO(2). The obtained results allowed us to conclude that direct contact of the nanoparticles with cell plasma membrane is the primary and critical step of their interaction and defines a subsequent response of the cell.

RNA-containing viruses represent a global threat to the health and wellbeing of humans and animals. Hence, the discovery of new approaches for the design of novel vaccines and antiviral compounds attains high attention. Here we describe the potential of artificial ribonucleases (aRNases), low molecular weight compounds capable to cleave phosphodiester bonds in RNA under mild conditions, to act as antiviral compounds via destroying the genome of non-enveloped RNA viruses, and the potential of utilizing honey bee larvae and adult bees (Apis mellifera) as a novel experimental system for the screening of new antiviral compounds. Pre-incubation of an Acute bee paralysis virus (ABPV) suspension with aRNases D3-12, K-D-1 or Dp12F6 in a concentration-dependent manner increased the survival rate of bee larvae and adult bees subsequently infected with these preparations, whereas incubation of the virus with aRNases ABL3C3 or L2-3 had no effect at all. The results of RT-PCR analysis of viral RNA isolated from aRNase-treated virus particles confirmed that virus inactivation occurs via degradation of viral genomic RNA: dose-dependent inactivation of ABPV correlates well with the cleavage of viral RNA. Electron microscopy analysis revealed that the morphology of ABPV particles inactivated by aRNases remains unaffected as compared to control virus preparations. Altogether the obtained results clearly demonstrate the potential of aRNases as a new virus inactivation agents and bee larvae/ABPV as a new in vivo system for the screening of antiviral compounds., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Deregulation of the expression of the genes that are involved in the control of the cell cycle impairs cellular differentiation and leads to cell death. This process can result in uncontrollable cell proliferation and, subsequently, cancer development. In this study, we examined the effect of the silencing of cancer-related genes by small interfering RNAs (siRNA) targeted at mRNAof Her2, cyclin B1 (CCNB1), and protein kinase C(PKC) on the proliferation of human cancer cells of different origins. Maximum silencing ofCCNB1,Her2(in KB-3-1, SK-N-MC, MCF-7 cells), andPKC(in MCF-7 cells) was achieved 72 h after transfection of the corresponding siRNAs, and 12 days after the transfection, the initial levels of the target mRNAs were fully recovered. Silencing ofHer2,CCNB1,andPKCdifferently effected the proliferation of the cell lines under study. The most pronounced antiproliferative action of the investigated siRNAs was observed in neuroblastoma SK-N-MC cells (3 - 10-fold reduction in the proliferation rate) even after the recovery of the initial levels of expression ofthe Her2,CCNB1, andPKС genes. The obtained data indicate that theCCNB1 andPKCgenes can be used as targets in the development of drugs for neuroblastoma treatment.

MDCK and Vero (B) cell cultures were propagated during 10 passages in the experimental nutrient media containing the soybean powder hydrolyzate prepared using trypsin and bromelain enzymes and the rice powder hydrolysate prepared with trypsin and in the control DMEM and SFM4 MegaVir media. The karyological, morphological, and proliferative characteristics of continuous cultures were examined and compared. The experimental media supplied with 3% fetal bovine serum (FBS) (Gibco, U.S.A.) showed high growth-enhancing properties and failed to affect their morphology. After propagated during 10 passages in the experimental media preserved a stable karyotype. MDCK cell cultures in the nutrient media based on rice and soybean powder hydrolyzates low (2%) in FBS caused no substantial changes in the proliferation indices and morphological and karyological characteristics of cell cultures.

Cholesterol amphiphiles containing positively charged groups (pyridinium, N-methylimidazolium, N-methylmorpholinium, and N-methylpiperidinium) linked via β-glucosyl spacer were prepared by alkylation of the corresponding bases with 6-О-mesyl-β-D-cholesteryl glucopyranoside. IC(50) values were in the range 20-35μM for the series of compounds and liposomal formulations with DOPE (1:1) were significantly less toxic. The liposomal formulations provided the accumulation of FITC-labeled oligonucleotide in cells, and the efficiency of this process was comparable to that of Lipofectamine 2000. Cationic liposomes were able to deliver siRNA into the cells, and the liposomal formulation 7d/DOPE provided the most pronounced down-regulation of EGFP expression both in the presence and in the absence of serum (up to 30%)., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

In our work the antitumor and antimetastatic activities of RNase A and DNase I were studied using two murine models of pulmonary (Lewis lung carcinoma) and liver (hepatoma A-1) metastases. We found that intramuscular administration of RNase A at the dose range of 0.1-50 µ g/kg retarded the primary tumor growth by 20-40%, and this effect disappeared with the increase in RNase A dose over 0.5 mg/kg. DNase I showed no effect on the primary tumor growth. The intramuscular administration of RNase A (0.35-7 µ g/kg) or DNase I (0.02-2.3 mg/kg) resulted in a considerable decrease in the metastasis number into the lungs of animals with Lewis lung carcinoma and a decrease of the hepatic index of animals with hepatoma 1A. A histological analysis of the organs occupied by metastases revealed that the administration of RNase A and DNase I induced metastasis pathomorphism as manifested by the destruction of oncocytes, an increase in necrosis and apoptosis foci in metastases, and mononuclear infiltration. Our data indicated that RNase A and DNase I are highly promising as supplementary therapeutics for the treatment of metastasizing tumors.

Morphological and proliferative characteristics of cultured Vero and MDCK cells were compared after growth in nutrient media of the basis of enzymatic hydrolysates of rice flour proteins and soybean flour proteins or control media (DMEM and Axcevir-MDCK). These media had a strong stimulatory effect on the growth of cultured Vero cells (addition of 3% fetal bovine serum, Gibco), but did not modulate the morphology of this culture. Culturing of MDCK cells in nutrient media on the basis of enzymatic hydrolysates of rice and soybean flour proteins with low content of fetal bovine serum (2%, Gibco) was accompanied by insignificant changes in the index of proliferation and morphological characteristics of cultured cells.

We report the isolation and detailed characterization of the novel strain, Partizansk/2006, of Powassan virus (POWV) from a human case of infection, which occurred in Primorsky krai, Russia, in 2006. Comparative complete genome sequence analysis of the Far Eastern strains Spassk-9 (1975), Nadezdinsk-1991 and Partizansk/2006 of POWV revealed that these strains are 99.8% similar to the LB strain, which was isolated in Canada in 1958. Phylogenetic analysis of 5' UTR sequences of five other strains of POWV isolated from 1972 to 1986 in Primorsky krai produced similar results. Presumably, Far Eastern POWV has common putative ancestor with LB strain POWV from North America, and the time of divergence of these POWVs is relatively short. We conclude that POWV has become endemic in Far Eastern Russia.

The possibility of xenotransplantation of human fetal chondroblasts was studied. Filling of the rat articular cartilage defect with a tissue-engineering construction based on primary culture of human fetal chondroblasts and chitosan gel caused no immune rejection over 60 days and provided the formation of organotypical regenerate due to proliferation and differentiation of donor fetal chondroblasts and their integration in the recipient cartilage tissue.