Methods for Fixing Biofilms of Staphylococcus aureus and Salmonella enterica for Microscopic Examination.

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.

[Antitumor Activity of the Combination of Topotecan and Tyrosyl-DNA-Phosphodiesterase 1 Inhibitor on Model Krebs-2 Mouse Ascite Carcinoma].

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.

[Nuclear delivery of oligonucleotides via nanocomposites based on TiO2 nanoparticles and polylysine].

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.

Synthesis of nanoscale TiO2 and study of the effect of their crystal structure on single cell response.

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.

[The morphological and karyological characteristics of MDCK and vero (B) cells cultures on plant hydrolyzate-based nutrient media].

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.

Morphological and proliferative characteristics of vero and MDCK cells during culturing in nutrient media on the basis of hydrolysates of plant proteins.

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.

[Contamination of exosome preparations, isolated from biological fluids]. / Kontaminatsiia preparatov ékzosom, vydelennykh iz biologicheskikh zhidkostei.

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.

[Characteristics of exosomes andmicroparticles discovered in human tears].

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.

Molecular-genetic and ultrastructural characteristics of 'Candidatus Ehrlichia khabarensis', a new member of the Ehrlichia genus.

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.

Fine mechanisms of ectromelia virus thymidine kinase-negative mutants avirulence.

Three independently selected spontaneous thymidine kinase-negative mutants (TK-phenotype) and a recombinant with Escherichia coli beta-galactosidase gene (LacZ+ phenotype) inserted in the viral thymidine kinase gene (tk) were derived from a plaque-cloned isolate of K-1 ectromelia virus strain (TK+ phenotype). Dramatically decreased virulence of TK- variants was observed for all routes of mouse inoculation. The kinetics of TK+ and TK- variants in various target organs indicated a significant decrease of production and dissemination of TK- mutants and recombinant in the organs of mice. In the spleen and liver of intranasally or intracerebrally infected mice TK- virus was not detected during the entire period of observation. Analysis of organs homogenates of mice intranasally infected by a mixture of recombinant with TK-LacZ+ phenotype and parental isolate with TK+LacZ- phenotype on the monolayers of TK- cells indicated that only white plaques (LacZ-) with the TK+ phenotype appeared from liver and spleen homogenates. Thus, the mouse acts as a live filter much more efficiently than any other selective systems. Ultrastructural studies showed that viral damage in animals infected by TK- variants was far less than that observed in mice, infected with wild type of ectromelia virus and pathological lessions were slight and reversible. Replication of ectromelia virus TK- variants was blocked at the viroplasma stage in cells with a high level of differentiation in contrast to TK+ variants. Most likely, such restriction of target cells assortment is the general reason of reduced virulence in the case of tk-gene inactivation.

Influence of polychemotherapy on the morphology of metastases and kidney of resistant RLS-bearing mice.

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.

Novel amphiphilic compounds effectively inactivate the vaccinia virus.

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.

Silencing of Her2, CCNB1 and PKC Genes by siRNA Results in Prolonged Retardation of Neuroblastoma Cell Division.

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.

Tumoricidal Activity of RNase A and DNase I.

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.

Use of primary human fetal chondroblast culture for xenotransplantation into rat articular cartilage defect.

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.

Study of morphofunctional peculiarities of fibroblasts isolated from human vertebral growth plates during in vitro culturing.

We studied structural and functional peculiarities of growth zone chondroblasts isolated from human fetal spines on gestation week 12 and cultured in vitro over 4 passages. The morphology of chondroblasts of different differentiation degree was described. It was found that the population of chondroblasts had certain features determined by changes in the relative content of cells of different differentiation degree. The data suggest that culturing conditions affect cell differentiation and the possibility of using primary human chondroblast culture as the experimental model of differentiating human vertebral growth plate chondroblasts in vitro.

Comparative study of pathological changes in the mouse viscera in infection caused by two orthopoxviruses.

The mechanisms of infection development in intraperitoneal inoculation of mice by ectromelia virus strain K-1 and cowpox strain EP-2 were studied. Ultrastructural parameters of virus assembly and maturation are described. Differences in the types of cells replicating the viruses and in the type of visceral injuries were detected. The studies showed a local type of strain EP-2 cowpox infection and dissemination of ectromelia strain K-1.

An analysis of features of pathogenesis in two animal models of Ebola virus infection.

Virus reproduction and the time course of changes in liver and kidney functions and in the blood clotting system were studied in the visceral organs of green monkeys and baboons infected with Ebola virus (subtype Zaire). It was shown that monocytes and macrophages were the first cells to be infected with the virus, followed by hepatocytes, adrenocorticocytes, fibroblasts, and endotheliocytes. The early and late pathologic changes in the monkey organs are described. Biochemical data on changes in blood clotting and liver and kidney functions in the course of the infection are presented. The responses of blood clotting and vascular permeability were species specific: Fibrin deposited in blood vessels in green monkeys, while hemorrhages developed in baboons. The results show that species-specific features of monkeys must be taken into account when choosing an experimental model for studying Ebola virus infection.

Venezuelan equine encephalitis virus propagation in the olfactory tract of normal and immunized mice.

The role of virus spread in the induction of damage to the central nervous system (CNS) of mice infected with Venezuelan equine encephalitis virus (VEEV) via the respiratory route was studied. The virus concentration in various organs and in the blood, the sensitivity to different doses of virus, and ultrastructural lesions in various tissues were examined. It is concluded that VEEV can enter the CNS of nonimmunized mice both by vascular and by olfactory pathways, whereas in immunized mice the olfactory pathway is the main route.