Antitumor peptides are a new class of small molecules characterized by high specificity and therapeutic efficacy. One of the strategies for developing peptide-based therapeutic agents is the modification of the physicochemical properties and structural characteristics of known peptides and cytotoxic proteins. Bacillus pumilus ribonuclease, binase, possesses antitumor activity. Using bioinformatics tools, we have developed a strategy for modifying of amino acid sequence of the antitumor binase peptide to enhance its cytotoxic properties. the binase peptide. A genetic construct enabling the peptide biosynthesis in recombinant strain was engineered. The peptide preparation was purified by metal chelate affinity chromatography. Cytotoxic properties of the purified peptide were assessed according to metabolic activity of tumor cells. Intracellular localization of the peptide was determined by means of fluorescence microscopy. In the study, we have genetically modified the binase peptide in order to change its physicochemical and structural properties. It made it possible to enhance its cytotoxic potential towards human lung adenocarcinoma line A549, but specificity was decreased. Thus, that modification of structural and physicochemical properties of therapeutic peptides is an important step in the development of antitumor drugs.

Blocking the MAPK pathway is a strategy to stop cancer cells proliferation. Despite all the successes, the acquisition of drug resistance by cells, as well as the mutational status of the downstream protein KRAS, reduces the tumor response to therapy. Ribonuclease binase from Bacillus pumilus is among the agents that block this pathway through direct interaction with EGFR and RAS. The present study is aimed at the design, optimization, and characterization of a novel complex based on antitumor binase immobilized on microgranular clinoptilolite-containing rock to ensure its prolonged release in the gastrointestinal tract. A set of modern methods including transmission electron microscopy, scanning electron microscopy, and computed tomography was used to characterize the granularity, porosity and elemental composition of the carrier. The size of binase particles, measured by atomic force microscopy at 7 nm, allows enzyme penetration into meso- and macropores of the carrier. Calorimetric results confirm that binase is stable at high temperatures, even exceeding those in the body, and retains catalytic activity in the model fluids of the gastrointestinal tract. The parameters for processing a natural clinoptilolite-containing rock and the conditions for binase sorption were selected. The gradual release of the enzyme from the carrier lasts over 20 h, which provides cytotoxicity towards human adenocarcinoma cells during movement through the gastrointestinal tract. Thus, for the first time a promising long-acting complex with antitumor and detoxifying properties was successfully created.

Increasing the biological effectiveness of probiotic preparations requires the development of new stable forms in the gastrointestinal tract. Live bacteria immobilized on a sorbent belong to the latest, fourth generation of probiotics, which ensures a prolonged effect. This study is devoted to developing a new method of preparing active lactobacilli on a natural mineral carrier, a tuff containing zeolite of the clinoptilolite group, which is among the most common authigenic silicate minerals that occur in sedimentary rocks and is known as a safe ion-exchange and adsorbing detoxicant. Among the characterized lactobacilli, strains of L. plantarum, L. acidophilus, and L. crispatus possessed a high level of acid formation and stability in gastrointestinal fluids. The protective effect of the clinoptilolite-containing tuff was registered when the samples were incubated in gastric juice. The optimal technological conditions for immobilization and lyophilization were determined, and the preservation of the viability and probiotic properties of bacteria was confirmed during 8 months of storage. The release of bacteria from the carrier occurred gradually over 12 h. The data obtained show how promising the new preparation is, combining the ability to detoxify harmful intestinal metabolites and the prolonged release of probiotics.

Objective: Among modern gene therapy methods for combating oncology, suicidal gene therapy based on the delivery of a cytotoxic agent to target cells is of particular importance and promise. As one of such genes, the gene for ribonuclease of Bacillus pumilus 7P, binase, can be considered; the enzyme has a high antitumor potential and low immunogenicity. In addition to the choice of a transgene, another factor influencing the effectiveness of gene therapy is the method of delivering the nucleic acid to target cells. Surfactants have high functional activity and are promising means of delivering therapeutic nucleic acids. The aim of this work was to evaluate the possibility of using geminal surfactants as a means of delivering a genetic construct based on the cytotoxic binase gene into tumor cells. Methods: To optimize the transfection conditions, a reporter genetic construct carrying the binase gene fused to the gene for the green fluorescent protein TurboGFP was created, which made it possible to evaluate the delivery efficiency by the fluorescence intensity. To eliminate the toxic effect of binase on recipient cells, the RNase inhibitor gene, barstar, was introduced into the genetic construct. Results and Discussion: A high complexing ability of geminal surfactants in relation to the reporter system was shown by methods of dynamic light scattering and fluorescence spectroscopy. For surfactant 16-6-16OH, the highest transfecting activity together with a low level of cytotoxicity was found. Conclusions: Thus, the study proved the possibility of using geminal surfactants for the delivery of therapeutic nucleic acids to target cells.

Human papillomavirus type 16 (HPV16) belongs to viruses of the high-risk type and is associated by overexpression of E6 and E7 oncoproteins, which determine the oncogenic properties of the virus, such as immortalization and malignant transformation of proliferating epithelial cells. The biogenesis of redox-sensitive proteins E6 and E7 at the early stages of viral infection leads to blocking of the cell antioxidant defense system and ubiquintin-dependent degradation of p53 and Rb tumor suppressors. Maintaining high rates of tumor cell proliferation contributes to an increase in the level of reactive oxygen species (ROS) and a shift in the redox balance towards oxidative processes. Reduced glutathione (GSH) provides antioxidant protection to tumor cells through S-glutathionylation of thiol groups of redox-sensitive proteins, which leads to the appearance of multidrug-resistant forms of cancer. In this regard, drugs restoring redox balance and increasing susceptibility to antitumor therapy are of particular importance. We have established that, Bacillus pumilus RNase (binase) modulates the redox-dependent regulatory mechanisms that ensure tumor cell resistance to apoptosis in HPV-16-positive SiHa cells of cervical squamous cell carcinoma,. Binase in nontoxic concentrations initiates a number of pre-apoptogenic changes, i.e., decreases ROS and reduced glutathione (GSH) levels, suppresses the expression of the E6 oncoprotein, activates the expression of the p53 tumor suppressor, and reduces the mitochondrial potential of tumor cells. Binase-induced disruption of the integrity of the mitochondrial membrane is a signal for activation of the mitochondrial apoptosis pathway.

One of the promising directions in antitumor therapy is suicidal gene therapy based on the introduction of cytotoxic genes into tumor cells. Most often, these genes encode for enzymes of bacterial or viral origin, capable of direct or indirect killing of tumor cells. This review provides information about modern strategies for suicidal cancer gene therapy, discusses their advantages and disadvantages, and analyzes the properties of a potential candidate for creating a new highly effective suicidal system, combining the advantages of existing approaches.

Cancer is frequently coupled with the disturbance of key signaling pathways. Aberrant activation of the mitogen-activated protein kinase (MAPK) signaling cascade, occurring in over 85% of cancers, is mainly caused by the genetic alterations of its main components-oncogenes EGFR and RAS, and plays a crucial role in cell fate. The importance of EGFR and RAS proteins in a variety of tumors suggests that they would be good therapeutic targets, but at present, no effective targeted therapy against these two oncogenes has been proven. Here, we show that ribonuclease from Bacillus pumilus (binase) inhibits MAPK signaling through direct interaction with EGFR and RAS proteins. This effect contributes to the antitumor potential of binase along with its enzymatic activity. Multitargeticity of binase prevents the development of drug resistance, which is considered a major obstacle to effective anticancer treatment.

BACKGROUND: One of the main problems of modern oncotherapy is the lack of selectivity of the antitumor drugs, leading to their systemic toxicity on the body. Targeted expression of therapeutic genes represents a new approach in cancer gene therapy. One of the ways to achieve the selectivity of action of the therapeutic genes towards tumor cells is the use of promoters that are active only in tumor cells, but not in normal cells. AIM: To evaluate the functional activity and specificity of the promoter of the human secretory leukocyte protease inhibitor hSLPI. MATERIALS AND METHODS: The promoter of the hSLPI gene was cloned into the promotorless vector pTurboGFP-PRL. The functional activity and specificity of the promoter were assessed by the expression of the mRNA of the TurboGFP reporter gene and the intensity of its luminescence in A549, MCF-7, HeLa tumor cells and WI-38 normal cells using real-time polymerase chain reaction with reverse transcription and fluorescence analysis, respectively. RESULTS: Despite the literature data, the promoter of the hSLPIa gene demonstrated high transcriptional activity only against HeLa cervical cancer tumor cells. At the same time, in the cells of lung adenocarcinoma A549 and breast cancer MCF-7, as in normal WI-38 cells, a reduced level of promoter activity was observed. CONCLUSION: The data obtained confirm the need for a preliminary assessment of the functional activity of tumor-specific promoters in relation to tumor and normal cells.

Abstract—Production of extracellular membrane vesicles plays an important role in communication in bacterial populations and in bacteria–host interactions. Vesicles as carriers of various regulatory and signaling molecules may be potentially used as disease biomarkers and promising therapeutic agents, including vaccine preparations. The composition of membrane vesicles has been deciphered for a limited number of Gram-negative and Gram-positive bacteria. In this work, for the first time, extracellular membrane vesicles of a streptomycin-resistant strain Bacillus pumilus 3-19, a producer of extracellular guanyl-preferring ribonuclease binase, are isolated, visualized, and characterized by their genome and proteome composition. It has been established that there is no genetic material in the vesicles and the spectrum of the proteins differs depending on the phosphate content in the culture medium of the strain. Vesicles from a phosphate-deficient medium carry 49 unique proteins in comparison with 101 from a medium with the high phosphate content. The two types of vesicles had 140 mutual proteins. Flagellar proteins, RNase J, which is the main enzyme of RNA degradosomes, phosphatases, peptidases, iron transporters, signal peptides, were identified in vesicles. Antibiotic resistance proteins and amyloid-like proteins whose genes are present in B. pumilus 3-19 cells are absent. Phosphate deficiency-induced binase was found only in vesicles from a phosphate-deficient medium.

Halophilic and halotolerant microorganisms have a high biotechnological potential. They are producers of biologically active substances, stress-protective agents, hydrolytic enzymes, and are used for environmental bioremediation. At the same time, the characterization of novel halotolerant bacteria and the disclosure of their salt tolerance strategy are topical fundamental problems. In the present work, a new strain MX2 was isolated from the salt well brine of the Yakshinskoe potassium-magnesium salt deposit. The isolate is represented by aerobic gram-positive non-motile bacteria that do not produce spores. The cell morphology varies from cocci to short rods that are capable of producing V-shaped forms. Colonies on the surface of agar nutrient medium were circular with an entire edge and raised center, glistening and orange. Bacteria of strain MX2 are halotolerant microorganisms capable of growing at NaCl concentrations up to 9%. Strain MX2 was sequenced. Its size was estimated at 3747717 b. p., the number of protein-coding genes — 3562. Strain MX2 was identified as belonging to the species Dietzia maris based on analysis of 16S rRNA, gyrB, rpoB, recA, ppk gene sequences and using time-of-flight mass spectrometry (MALDI-TOF-MS). D. maris MX2 has complete metabolic pathways for the synthesis of ectoine, hydroxyectoine, and trehalose, as well as transport systems for ectoine, hydroxyectoine, trehalose, glycerol, glycerol-3-phosphate, L-proline, and glycine-betaine. Thus, to ensure the osmotic balance, D. maris MX2 uses the strategy of accumulating compatible organic solutes.

— Drug delivery systems are developed to provide a necessary concentration and prolonged effect of the active substance in the organism. Orally administered protein preparations require a protection from the proteolysis in the gastrointestinal tract. Biocompatible hydrophilic polysaccharides in the composition of the matrix are especially promising, since they do not irritate the intestine and are gradually cleaved by specific glycosidases, releasing a therapeutic agent. The introduction of an insoluble porous mineral matrix into the composition of the carrier allows us to increase the concentration of the therapeutic agent in the matrix without a significant increase in the volume of the drug tablet form. In this work, a new original organomineral carrier was created based on heat-treated crushed clinoptilolite zeolite in combination with natural polysaccharides of red algae (agar–agar, agarose, and carrageenan). Granular and finely dispersed clinoptilolites in the composition of the matrix are loaded with a promising therapeutic agent ( Bacillus pumilus ribonuclease (binase)), which shows a selective cytotoxicity to tumor cells. It was established that both granular and finely dispersed zeolites in a complex with polysaccharides retain the protein better as compared with pure zeolites and provide a gradual complete release of the enzyme in 18 h; at the same time, binase retains a catalytic activity and causes apoptosis in up to 23.8% of the population of HuTu80 human duodenal adenocarcinoma cells. Data obtained substantiate the prospects of designing dosage forms based on the studied organomineral carriers.

To maintain performance of polymer composite materials (PCM) in tropical climate, it is necessary and relevant to deal with biodegradation among other factors. Increasing strength and improvement of biodegradation resistance of polymer composites simultaneously is a critical practical challenge. State-of-the-art methods of polymer composites production do not provide a possibility to address both issues at the same time. In this study, it is the first time when a method to increase strength of ED-20 epoxy-based polymer composite and improve its biodegradation resistance simultaneously is applied. In this study, the authors applied for the first time polylactide-capsulated copper oxide particles to improve biocidal and mechanical performance of ED-20 epoxy-based polymer composite. It was established that composite filled with capsulated particles has better resistance to micromycete-induced damage compared to the one filled with non-capsulated particles. Reduction of surface area affected by micromycetes isolated from samples exposed to tropical conditions was demonstrated for the composite that contained capsulated particles. The paper highlights that prevalence of Aspergillus niger is based on the high productivity of organic acids. It was found that elasticity moduli of polymer composite samples do not have significant differences. The average elasticity modulus of PCM samples was 3.4 ± 0.2 GPa before and after exposure to tropical conditions. Apparently, the thing that elasticity modulus remained the same after exposure to tropical conditions was due to the fact that only surface of the sample was subject to destruc-tion. The samples with non-capsulated particles experienced 20% decrease in ultimate strength after exposure to tropi-cal conditions while the samples with capsulated particles experienced only 10% decrease, so the material with capsu-lated particles was stronger. The fact that the elastic moduli of samples with capsulated particles remain the same after exposure to the microbial destructors indicates improved resistance of new PCM to biodegradation and confirms prom-ising practical application of the created material. Thus, this article is the first one to demonstrate that application of polylactide-capsulated copper oxide particles in combination with ED-20 epoxy-based polymer provides a possibility to obtain a new composite with improved biocidal effect

Bacillus pumilus ribonuclease (binase) exhibits cytotoxic and oncolytic properties, while causing genotoxic effects at high concentrations. Mutants that have reduced catalytic activity and preserve the antitumor properties of the native enzyme could exert lower toxic side effects. Mutant binase forms with the Lys26Ala and His101Glu single substitutions were obtained by site-directed mutagenesis. A comparative analysis of Escherichia coli- and Bacillus subtilis -based expression systems demonstrated that the latter is better to use to produce the binase mutants. The binase mutants with reduced catalytic activity were isolated and purified to homogeneity by ion exchange chromatography; the maximum yield was 25 mg/L. Catalytic activities of the mutants toward natural RNA-substrates in comparison with those for native binase were estimated at 11% and 0.02%, respectively. Like native binase, the Lys26Ala mutant was found to be cytotoxic to the A549, BT-20, and HuTu 80 tumor cell lines, but did not substantially affect normal WI-38 cells. The His101Glu mutant did not show cytotoxicity.

This study presents the results of a search for ways to prevent biodestructive processes, occurring in the natural environment of wooden architecture monuments localization. On the Sviyazhsk island since the 16th century an unique site of wooden architecture of the Volga region has been preserved. It is the Trinity Church, the only building, although rebuilt over the centuries, that has survived from the wooden medieval island-town. Climate changes, cultural tourism and living organisms activities make a significant contribution to the change in the initial state of the tree. The greatest contribution to the processes of biodamage is made by mold fungi – micromycetes. Experimental work carried out on a fragmentary sample of a structural element of the Trinity Church revealed that coating the surface of the sample with a synthetic varnish based on a mixture of linear and cyclic methylmethoxypolysiloxanes prevents the growth of microscopic fungi on the surface, among which Aspergillus niger dominates. The growth area of the sample is reduced by about 6 times compared to the untreated variant. By artificial infection of samples with spores of Aspergillus niger, Penicillus chrysogenum, Fusarium graminearum and Aspergillus puulaauensis, the growth area of varnished samples also decreases by 7 times, and the number of conidiophores, reflecting the growth of micromycetes, by 4 times. The obtained results make it possible to recommend the use of polysiloxane coatings for the protection of especially important fragments of wooden artifacts.

Therapeutic muds (peloids), which are widely used for body healing, improve metabolism and have antibacterial, anti-inflammatory and analgesic effects due to enrichment with necessary microelements and biological active substances. However, the microbiological component of these effects is not well studied.OBJECTIVE:To characterize the microbiome of therapeutic muds, used in the Tatarstan Republic, by identifying spectrum of cultivated microorganisms, using molecular analysis of bacterial communities, and by determining their biodiversity and functional potential based on revealed genetic determinants.MATERIAL AND METHODS:The study design of 5 peloids samples (local sapropels and peat deposits of swamp; 3 samples of Crimean sulfide muds) included three main techniques: isolation and taxonomic determination of cultivated microorganisms by time-of-flight mass-spectrometry; molecular analysis of peloids bacterial communities by 16S RNA high-throughput sequencing; identification of functional profiles of communities by their genetic determinant using Global Mapper tool on iVikodak platform.RESULTS:Experimental studies have confirmed the safety of examined peloids, where non-pathogenic cultivated bacteria belonging mainly to Bacillus and Rhodococcus genera were dominant. Metagenomic analysis showed that Firmicutes, Proteobacteria and Actinobacteria were predominant in all samples in different ratios. It has been established, that there is both the internal biodiversity of each sample and difference between them. The functional profile of microbial communities was determined based on the identification of bacterial genes. It has been revealed that all communities have an ability to synthesize antibiotics, as well as to decompose dangerous xenobiotics - polyaromatic hydrocarbons, cyclic compounds, and dioxins.CONCLUSION:Various microbial communities, which were identified in the therapeutic muds, contribute both to the clearance of toxicants in the peloids and to the antibacterial properties of the latter. The obtained priority results create a fundamental basis for the subsequent study of the role of peloids' microbiome of different origin in their healing action.

Objective: Recent studies have revealed the biodiversity of both cultivated and uncultivated microbiomes in extreme environments. It has been shown that terrestrial subsurface ecosystems contain vast metabolic potential. Heterotrophic bacteria living in karst caves with an organic substrate deficit represent a special reserve for the isolation of metabolite producers. Here, we cultivated a bacterial community collected from biofilms in Kapova Cave (Shulgan–Tash Nature Reserve, Bashkortostan), and assessed its ability to synthesize secreted hydrolytic enzymes including RNases, proteases, and amylases.Materials and Methods: Isolated bacteria were identified by V3-V4 16S rRNA region sequencing. Enzymatic activities were assessed by measuring transparency zones around colonies grown on the appropriate substrate (RNA, casein, starch). Functional profiles of the communitieswere predicted using the Global Mapper module on iVikodak. Taxonomic, structural, and compositional diversity were calculated using Shannon–Wiener and Bray–Curtis indices.Results: Eighty-nine percent of 102 bacterial isolates were Proteobacteria, whereas other isolates were divided into three other phyla, Actinobacteria, Firmicutes, and Bacteroidetes that comprised 5%, 4%, and 2% of the isolates, respectively. Genus Pseudomonas was predominant with 42 isolates. Six isolates showed no extracellular enzymatic activity at all, 73 isolates expressed protease, 57 isolates expressed amylase, and 71 isolates had RNase activity. All three extracellular enzymes were expressed by 39isolates.Conclusion: The biodiversity of cultivated microbiota from Kapova Cave was characterized. Bacteria that produce large amounts of protease, RNase and amylase were identified as Stenotrophomonas rhizophila, Lysinibacillus fusiformis, and Pseudomonas stutzeri, respectively.

Arctic ecosystems are affected by negative influence of climate change, pollution, and overexploitation of resources. Microorganisms playing a key role in preserving extreme econiches are poorly studied and require the use of modern methods for studying both their biodiversity and physiological activity. We applied Illumina MiSeq to the high-throughput 16S rRNA sequencing study of four Laptev Sea sediments from 64 - 185 m depth, using next generation sequencing enables rapid analysis of composition and diversity of prokaryotic communities. Although the dominant phylum in all samples was Proteobacteria, only the deepest sample contained a high number of archaeal organisms (19%) with the predominance of Methanosarcinaceace family in comparison with less 1% in the other three samples. This deepest sample had the lowest biodiversity and richness indices. Comparison of functional profiles of communities using Global Mapper tool revealed similar average abundance of infectiousness, drug resistance and environmental adaptation determinants in all samples, and high functional abundance for xenobiotic degradation in two samples. Among cultivated bacteria which could be promising producers of secreted RNase the representatives of Bacillus and Lysinibacillus genera were found. Our results contribute to improve our understanding of richness and ecological role of Laptev Sea microbiota.

Therapy of colorectal cancer with protein drugs, including targeted therapy using monoclonal antibodies, requires the preservation of the drug’s structure and activity in the gastrointestinal tract or bloodstream. Here, we confirmed experimentally the fundamental possibility of creating composite protein–polysaccharide hydrogels based on non-degrading rhamnogalacturonan I (RG) and fibrin as a delivery vehicle for antitumor RNase binase. The method is based on enzymatic polymerization of fibrin in the presence of RG with the inclusion of liposomes, containing an encapsulated enzyme drug, into the gel network. The proposed method for fabricating a gel matrix does not require the use of cytotoxic chemical cross-linking agents and divalent cations, and contains completely biocompatible and biodegradable components. The process proceeds under physiological conditions, excluding the effect of high temperatures, organic solvents and ultrasound on protein components. Immobilization of therapeutic enzyme binase in the carrier matrix by encapsulating it in liposomes made from uncharged lipid made it possible to achieve its prolonged release with preservation of activity for a long time. The release time of binase from the composite carrier can be regulated by variation of the fibrin and RG concentration.

— Probiotic preparations are popular means for restoring the intestinal microbiota; their effectiveness requires long-term preservation of bacteria viability, induction of their proliferation, and manifestation of their functional properties in the body. Carriers of diverse nature are designed to ensure a prolonged release of the probiotic during their transit through the gastrointestinal tract. In the present work, a natural zeolite-containing rock with 25% clinoptilolite, which has sorption, detoxifying, and ion-exchange properties, was used as a carrier. Using transmission microscopy methods, the structure of the carrier was characterized, and the distribution of pores depending on their equivalent diameter was determined. It was shown that the size of the cavities was sufficient for adsorption of bacteria in the macropores and on the surface of the particles. Three Lactobacillus strains loaded onto a carrier and freeze-dried remained viable for 10 months.

Objects and structures made of organic glass require protection from damage caused by external factors. Light, humidity, temperature, dust pollution and, undoubtedly, microorganisms lead to the deterioration of optical and mechanical properties. Polysiloxane-based protective coatings, consisting of silicon–oxygen backbones linked together with organic side groups attached to the silicon atoms, are widely used. However, the polysiloxane coatings themselves also cannot avoid deterioration during operation that implies the constant development of new protective materials. Here, we created a new cross-linked polysiloxane that covers organic glasses to enhance their resistance to aggressive external factors, and investigated its own resistance to damage induced by micromycetes in natural tropical conditions and in the laboratory. It has been established that the surface of coatings in the tropics is prone to fouling with micromycetes, mainly of the genera Aspergillus and Penicillium, which produce oxalic, malic, lactic, and citric acids contributing to the biodeterioration of polysiloxane. The testing of monolithic polycarbonate, polymethyl methacrylate, and triplex coated with polysiloxane showed that they retained significant resistance to abrasion and transparency at a level of more than 90% under aggressive natural conditions. Under artificial laboratory conditions, the infection of samples with micromycete spores also revealed their growth on surfaces and a similar trend of damage.