Pavel Zelenikhin

Antifungal activity of oligochitosans (short chain chitosans) against some Candida species and clinical isolates of Candida albicans: molecular weight-activity relationship.

A series of oligochitosans (short chain chitosans) prepared by acidic hydrolysis of chitosan and characterized by their molecular weight, polydispersity and degree of deacetylation were used to determine their anticandidal activities. This study has demonstrated that oligochitosans show a high fungistatic activity (MIC 8-512 μg/ml) against Candida species and clinical isolates of Candida albicans, which are resistant to a series of classic antibiotics. Flow cytometry analysis showed that oligochitosan possessed a high fungicidal activity as well. For the first time it was shown that even sub-MIC oligochitosan concentration suppressed the formation of C. albicans hyphal structures, cause severe cell wall alterations, and altered internal cell structure. These results indicate that oligochitosan should be considered as a possible alternative/additive to known anti-yeast agents in pharmaceutical compositions.

Oncogenic c-kit transcript is a target for binase.

Mutational activation of c-Kit receptor tyrosine kinase is common in acute myelogenous leukemia (AML). One such activating point mutation is the N822K replacement in the c-Kit protein. Here we investigate the selective cytotoxic effect of binase - RNase from Bacillus intermedius - on FDC-P1-N822K cells. These cells were derived from myeloid progenitor FDC-P1 cells, in which ectopic expression of N822K c-kit gene induces interleukin-3 independent growth. In order to determine whether the sensitivity of these cells to binase is caused by the expression of c-kit oncogene, the cytotoxicity of the RNase was studied in the presence of selective inhibitor of mutated c-Kit imatinib (Gleevec). Inhibition of mutated c-Kit protein leads to the loss of cell sensitivity to the apoptotic effect of binase, while the latter still decreases the amount of cellular RNA. Using green fluorescent protein as an expression marker for the c-Kit oncoprotein, we demonstrate that the elimination of c-Kit is the key factor in selective cytotoxicity of binase. Quantitative RT-PCR with RNA samples isolated from the binase-treated FDC-P1-N822K cells shows that binase treatment results in 41% reduction in the amount of с-kit mRNA. This indicates that the transcript of the activated mutant c-kit is the target for toxic action of binase. Thus, the combination of inhibition of oncogenic protein with the destruction of its mRNA is a promising approach to eliminating malignant cells.

Binase cleaves cellular noncoding RNAs and affects coding mRNAs

Bacterial RNases are promising tools for the development of anticancer drugs. Neoplastic transformation leads to enhanced accumulation of rRNA and tRNA, and altered expression of regulatory noncoding RNAs. Cleavage of RNA in cancer cells is the main reason for the cytotoxic effects of exogenic RNases. We have shown that binase, a cytotoxic ribonuclease from Bacillus intermedius, affects the total amount of intracellular RNA and the expression of proapoptotic and antiapoptotic mRNAs. For four cell lines, we visualized cellular RNA by fluorescence microscopy, and determined RNA levels, viability and apoptosis by flow cytometry. We found that the level of cellular RNA was decreased in cells that were sensitive to the cytotoxic effects of binase. The RNA level was lowered by 44% in HEK cells transfected with the hSK4 gene of the Ca2+‐activated potassium channels (HEKhSK4) and by 20% in kit‐transformed myeloid progenitor FDC‐P1iR1171 cells. The most significant decrease in RNA levels was registered in the subpopulations of apoptotic cells. However, the binase‐induced RNA decrease did not correlate with apoptosis. Kit‐transformed cells with binase‐induced RNA decrease retained viability if the interleukin‐dependent proliferation pathway was activated. Using quantitative RT‐PCR with RNA samples isolated from the binase‐treated HEKhSK4 cells, we found that the amount of mRNA of the antiapoptotic bcl‐2 gene in vivo was reduced about two‐fold. In contrast, expression of the proapoptotic genes p53 and hSK4 was increased 1.5‐fold and 4.3‐fold, respectively. These results show that binase is a regulator of RNA‐dependent processes of cell proliferation and apoptosis.

Binase penetration into alveolar epithelial cells does not induce cell death

Microbial ribonucleases possess a broad spectrum of biological activities, which demonstrate stimulating properties at low concentrations and cytotoxicity and genotoxicity at high concentrations. Mechanisms of their penetration into the cells still remain unclear. In this study penetration of Bacillus intermedius RNase (binase) in alveolar lung epithelial cells, type II (ATII) pneumocytes, has been investigated. Using immunofluorescence analysis we have shown for the first time internalization of binase by primary non-differentiated pneumocytes ATII. The enzyme did not penetrate in MLE-12 (Murine Lung Epithelial-12 cells). However, binase was cytotoxic towards tumor MLE-12 cells, but not ATII cells. These results clearly indicate higher sensitivity of tumor cells to binase compared to normal cells; they also demonstrate that penetration of the enzyme into alveolar epithelial cells is not directly associated with their death.

Evaluation of a method for the determination of antibacterial activity of chitosan

A method for the determination of the antimicrobial activity of chitosan with the use of organic salts for the production of pH in the range of 5.5–8.2 was studied. The double-dilution method demonstrated the effectiveness of the determination of the antimicrobial activity of chitosan samples with different molecular weights and solubilities. It was found that the antibacterial activity increased at low pH values with increasing molecular weight, but chitosans with a molecular weight of 5–6 kDa showed higher activity at neutral and slightly alkaline pH levels. Determination of the antimicrobial activity of various chitosan samples at different pH values allowed a more reliable assessment of the potential biological activity of chitosan.

Binase Immobilized on Halloysite Nanotubes Exerts Enhanced Cytotoxicity toward Human Colon Adenocarcinoma Cells

Many ribonucleases (RNases) are considered as promising tools for antitumor therapy because of their selective cytotoxicity toward cancer cells. Binase, the RNase from Bacillus pumilus, triggers apoptotic response in cancer cells expressing RAS oncogene which is mutated in a large percentage of prevalent and deadly malignancies including colorectal cancer. The specific antitumor effect of binase toward RAS-transformed cells is due to its direct binding of RAS protein and inhibition of downstream signaling. However, the delivery of proteins to the intestine is complicated by their degradation in the digestive tract and subsequent loss of therapeutic activity. Therefore, the search of new systems for effective delivery of therapeutic proteins is an actual task. This study is aimed to the investigation of antitumor effect of binase immobilized on natural halloysite nanotubes (HNTs). Here, we have developed the method of binase immobilization on HNTs and optimized the conditions for the enzyme loading and release (i); we have found the non-toxic concentration of pure HNTs which allows to distinguish HNTs- and binase-induced cytotoxic effects (ii); using dark-field and fluorescent microscopy we have proved the absorption of binase-loaded HNTs on the cell surface (iii) and demonstrated that binase-halloysite nanoformulations possessed twice enhanced cytotoxicity toward tumor colon cells as compared to the cytotoxicity of binase itself (iv). The enhanced antitumor activity of biocompatible binase-HNTs complex confirms the advisability of its future development for clinical practice.

Antitumor Macrophage Response to Bacillus pumilus Ribonuclease (Binase)

Extracellular bacterial ribonucleases such as binase from Bacillus pumilus possess cytotoxic activity against tumor cells with a potential for clinical application. Moreover, they may induce activation of tumor-derived macrophages either into the M1-phenotype with well-documented functions in the regulation of the antitumor immune response or into M2-macrophages that may stimulate tumor growth, metastasis, and angiogenesis. In this study, binase or endogenous RNase1 (but not RNA or short oligonucleotides) stimulated the expression of activated NF-κB p65 subunit in macrophages. Since no changes in MyD88 and TRIF adaptor protein expression were observed, toll-like receptors may not be involved in RNase-related NF-κB pathway activation. In addition, short exposure (0.5 hr) to binase induced the release of cytokines such as IL-6, МСР-1, or TNF-α (but not IL-4 and IL-10), indicative for the polarization into antitumor M1-macrophages. Thus, we revealed increased expression of activated NF-κB p65 subunit in macrophages upon stimulation by binase and RNase1, but not RNA or short oligonucleotides.

Fast and simple tool for the quantification of biofilm-embedded cells sub-populations from fluorescent microscopic images

Fluorescent staining is a common tool for both quantitative and qualitative assessment of pro- and eukaryotic cells sub-population fractions by using microscopy and flow cytometry. However, direct cell counting by flow cytometry is often limited, for example when working with cells rigidly adhered either to each other or to external surfaces like bacterial biofilms or adherent cell lines and tissue samples. An alternative approach is provided by using fluorescent microscopy and confocal laser scanning microscopy (CLSM), which enables the evaluation of fractions of cells subpopulations in a given sample. For the quantitative assessment of cell fractions in microphotographs, we suggest a simple two-step algorithm that combines single cells selection and the statistical analysis. To facilitate the first step, we suggest a simple procedure that supports finding the balance between the detection threshold and the typical size of single cells based on objective cell size distribution analysis. Based on a series of experimental measurements performed on bacterial and eukaryotic cells under various conditions, we show explicitly that the suggested approach effectively accounts for the fractions of different cell sub-populations (like the live/dead staining in our samples) in all studied cases that are in good agreement with manual cell counting on microphotographs and flow cytometry data. This algorithm is implemented as a simple software tool that includes an intuitive and user-friendly graphical interface for the initial adjustment of algorithm parameters to the microphotographs analysis as well as for the sequential analysis of homogeneous series of similar microscopic images without further user intervention. The software tool entitled BioFilmAnalyzer is freely available online at https://bitbucket.org/rogex/biofilmanalyzer/downloads/.

Binase-Induced Changes of Tumor Cell Membranes

Exogenous ribonucleases of Bacilli can selectively induce apoptosis of malignant cells. The ability of Bacillus pumilus ribonuclease, binase, to induce processes leading to a dynamic disruption of the integrity of A549 human pulmonary adenocarcinoma cell membranes was analyzed. The influence of different enzyme concentrations on the state of the cytoplasmic membrane of cells and mitochondrial membranes was characterized. Using the methods of flow cytofluorometry and fluorescence microscopy, it has been established that binase leads to disruption in normal functioning of both types of membranes, with mitochondrial membranes affected first. The study made it possible to identify and visualize the effects of binase on the membrane structures of target cells and to confirm that bacterial RNase induces apoptosis of target cells mainly through the “internal” (mitochondrial) pathway.

Coprecipitation Method of Synthesis, Characterization, and Cytotoxicity of Pr3+:LaF3 (CPr = 3, 7, 12, 20, 30%) Nanoparticles

The Pr3+:LaF3 (CPr = 3, 7, 12, 20, 30%) nanoparticles were characterized by means of high-resolution transmission electron microscopy, X-ray diffraction, optical spectroscopy, energy dispersive X-ray spectroscopy, dynamic light scattering, and MTT assay. It was revealed that the average diameter of all the NPs is around 14–18 nm. The hydrodynamic radius of the Pr3+:LaF3 (CPr = 7%) nanoparticles strongly depends on the medium. It was revealed that hydrodynamic radii of the Pr3+:LaF3 (CPr = 7%) nanoparticles in water, DMEM, and RPMI-1640 biological mediums were 18 ± 5, 41 ± 6, and 186 ± 8 nm, respectively. The Pr3+:LaF3 (CPr = 7%) nanoparticles were nontoxic at micromolar concentrations toward COLO-320 cell line. The lifetime curves were fitted biexponentially, and for the Pr3+:LaF3 (CPr = 7%) NPs, the luminescence lifetimes of Pr3+ ions were 480 ± 2 and 53 ± 5 nanosec.