Two Main Cancer Biomarkers as Molecular Targets of Binase Antitumor Activity.

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.

Fibrin-Rhamnogalacturonan I Composite Gel for Therapeutic Enzyme Delivery to Intestinal Tumors.

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.

Structure and Functional Potential of Arctic Sea Sediment Microbiota.

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.

Bacillus pumilus proteome changes in response to 2,4,6-trinitrotoluene-induced stress.

2,4,6-Trinitrotoluene (TNT) is the most widely used nitroaromatic compound and is highly resistant to degradation. Most aerobic microorganisms reduce TNT to amino derivatives via formation of nitroso- and hydroxylamine intermediates. Although pathways of TNT degradation are well studied, proteomic analysis of TNT-degrading bacteria was done only for some individual Gram-negative strains. Here, we isolated a Gram-positive strain from TNT-contaminated soil, identified it as Bacillus pumilus using 16S rRNA sequencing, analyzed its growth, the level of TNT transformation, ROS production, and revealed for the first time the bacillary proteome changes at toxic concentration of TNT. The transformation of TNT at all studied concentrations (20-200 mg/L) followed the path of nitro groups reduction with the formation of 4-amino-2,6-dinitrotoluene. Hydrogen peroxide production was detected during TNT transformation. Comparative proteomic analysis of B. pumilus showed that TNT (200 mg/L) inhibited expression of 46 and induced expression of 24 proteins. Among TNT upregulated proteins are those which are responsible for the reductive pathway of xenobiotic transformation, removal of oxidative stress, DNA repair, degradation of RNA and cellular proteins. The production of ribosomal proteins, some important metabolic proteins and proteins involved in cell division are downregulated by this xenobiotic.

Polysiloxane Coatings Biodeterioration in Nature and Laboratory.

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.

Alginate-Based Hydrogel as Delivery System for Therapeutic Bacterial RNase.

To deliver therapeutic proteins into a living body, it is important to maintain their target activity in the gastrointestinal tract after oral administration. Secreted ribonuclease from Bacillus pumilus (binase) has antitumor and antiviral activity, which makes it a promising therapeutic agent. This globular protein of small molecular weight (12.2 kDa) is considered as a potential agent that induces apoptosis of tumor cells expressing certain oncogenes, including colorectal and duodenum cancer. The most important problem of its usage is the preservation of its structure and target activity, which could be lost during oral administration. Here, we developed alginate microspheres reinforced with divalent cations and analyzed the enzyme release from them. Using methods of scanning electron microscopy, measurements of fluorescence, enzyme catalytic activity, and determination of viability of the duodenum adenocarcinoma tumor cell line, we characterized obtained microspheres and chose calcium as a biogenic ion-strengthening microsphere structure. Among such modified additivities as beta-casein, gelatin, and carbon nanotubes introduced into microspheres, only gelatin showed a pronounced increase in their stability and provided data on the prolonged action of enzyme release from microspheres into tumor cell culture medium during 48 h in an amount of about 70% of the loaded quantity.

Structural and Functional Differences between Homologous Bacterial Ribonucleases.

Small cationic guanyl-preferring ribonucleases (RNases) produced by the Bacillus species share a similar protein tertiary structure with a high degree of amino acid sequence conservation. However, they form dimers that differ in conformation and stability. Here, we have addressed the issues (1) whether the homologous RNases also have distinctions in catalytic activity towards different RNA substrates and interactions with the inhibitor protein barstar, and (2) whether these differences correlate with structural features of the proteins. Circular dichroism and dynamic light scattering assays revealed distinctions in the structures of homologous RNases. The activity levels of the RNases towards natural RNA substrates, as measured spectrometrically by acid-soluble hydrolysis products, were similar and decreased in the row high-polymeric RNA >>> transport RNA > double-stranded RNA. However, stopped flow kinetic studies on model RNA substrates containing the guanosine residue in a hairpin stem or a loop showed that the cleavage rates of these enzymes were different. Moreover, homologous RNases were inhibited by the barstar with diverse efficiency. Therefore, minor changes in structure elements of homologous proteins have a potential to significantly effect molecule stability and functional activities, such as catalysis or ligand binding.

The composition of microbial communities in inflammatory periodontal diseases in young adults Tatars.

Host susceptibility and environmental factors are important for the development of gingivitis and periodontitis, but bacterial biofilms attached to the teeth and gingival tissues play a crucial role. We have analyzed and compared the subgingival microbial communities between subjects with dental plaque biofilm-induced generalized chronic gingivitis (CG), localized initial (Stage I) periodontitis (IP) and healthy controls (HC) of young people aged 18-19 years permanently residing in the city of Kazan (Tatarstan, Russia). The results showed that the α-diversity in groups with CG and IP was higher than in the healthy group. In a course of periodontal disease, a decrease in the relative abundance of dominates genera Rothia and Streptococcus was observed along with increase of class TM7-3 (Candidatus Saccharibacteria phylum) representatives. Also, the increase of red complex representatives Porphyromonadeceae, Treponema and Tannerella was detected together with statistically significant increase of Filifactor, Parvimonas, Peptostreptococcaceae, Veillonellaceae, Tissierelaceae and Mogibacteriaceae. Analysis of our data suggests that transition from HC to IP may be accompanied by a decrease in microbial diversity and a reduction in the abundance of family Rs-045 (Candidatus Saccharibacteria phylum), Desulfovibrionaceae Corynebacterium, Campylobacter and Selenomonas in young adults Kazan Tatars.

The Cytotoxicity of RNase-Derived Peptides.

Bacterial ribonuclease binase exhibits a cytotoxic effect on tumor cells possessing certain oncogenes. The aim of this study was to identify the structural parts of the binase molecule that exert cytotoxicity. Out of five designed peptides, the peptides representing the binase regions 21-50 and 74-94 have the highest cytotoxic potential toward human cervical HeLa and breast BT-20 and MCF-7 cancer cells. The peptides B21-50 and B74-94 were not able to enter human lung adenocarcinoma A549 cells, unlike BT-20 cells, explaining their failure to inhibit A549 cell proliferation. The peptide B74-94 shares similarities with epidermal growth factor (EGF), suggesting the peptide's specificity for EGF receptor overexpressed in BT-20 cells. Thus, the binase-derived peptides have the potential of being further developed as tumor-targeting peptides.

Anti-Influenza Activity of the Ribonuclease Binase: Cellular Targets Detected by Quantitative Proteomics.

Unpredictable influenza pandemics, annual epidemics, and sporadic poultry-to-human avian influenza virus infections with high morbidity and mortality rates dictate a need to develop new antiviral approaches. Targeting cellular pathways and processes is a promising antiviral strategy shown to be effective regardless of viral subtypes or viral evolution of drug-resistant variants. Proteomics-based searches provide a tool to reveal the druggable stages of the virus life cycle and to understand the putative antiviral mode of action of the drug(s). Ribonucleases (RNases) of different origins not only demonstrate antiviral effects that are mediated by the direct RNase action on viral and cellular RNAs but can also exert their impact by signal transduction modulation. To our knowledge, studies of the RNase-affected cell proteome have not yet been performed. To reveal cellular targets and explain the mechanisms underlying the antiviral effect employed by the small extra-cellular ribonuclease of Bacillus pumilus (binase) both in vitro and in vivo, qualitative shotgun and quantitative targeted proteomic analyses of the influenza A virus (IAV) H1N1pdm09-infected A549 cells upon binase treatment were performed. We compared proteomes of mock-treated, binase-treated, virus-infected, and virus-infected binase-treated cells to determine the proteins affected by IAV and/or binase. In general, IAV demonstrated a downregulating strategy towards cellular proteins, while binase had an upregulating effect. With the help of bioinformatics approaches, coregulated cellular protein sets were defined and assigned to their biological function; a possible interconnection with the progression of viral infection was conferred. Most of the proteins downregulated by IAV (e.g., AKR1B1, AKR1C1, CCL5, PFN1, RAN, S100A4, etc.) belong to the processes of cellular metabolism, response to stimulus, biological regulation, and cellular localization. Upregulated proteins upon the binase treatment (e.g., AKR1B10, CAP1, HNRNPA2B1, PFN1, PPIA, YWHAB, etc.) are united by the processes of biological regulation, cellular localization, and immune and metabolic processes. The antiviral activity of binase against IAV was expressed by the inversion of virus-induced proteomic changes, resulting in the inhibition of virus-associated processes, including nuclear ribonucleoprotein export (NCL, NPM1, Nup205, and Bax proteins involved) and cytoskeleton remodeling (RDX, PFN1, and TUBB) induced by IAV at the middle stage of single-cycle infection in A549 cells. Modulation of the immune response could be involved as well. Overall, it seems possible that binase exerts its antiviral effects in multiple ways.

Antitumour Activity of the Ribonuclease Binase from Bacillus pumilus in the RLS40 Tumour Model Is Associated with the Reorganisation of the miRNA Network and Reversion of Cancer-Related Cascades to Normal Functioning.

The important role of miRNA in cell proliferation and differentiation has raised interest in exogenous ribonucleases (RNases) as tools to control tumour-associated intracellular and extracellular miRNAs. In this work, we evaluated the effects of the RNase binase from Bacillus pumilus on small non-coding regulatory RNAs in the context of mouse RLS40 lymphosarcoma inhibition. In vitro binase exhibited cytotoxicity towards RLS40 cells via apoptosis induction through caspase-3/caspase-7 activation and decreased the levels of miR-21a, let-7g, miR-31 and miR-155. Intraperitoneal injections of binase in RLS40-bearing mice resulted in the retardation of primary tumour growth by up to 60% and inhibition of metastasis in the liver by up to 86%, with a decrease in reactive inflammatory infiltration and mitosis in tumour tissue. In the blood serum of binase-treated mice, decreases in the levels of most studied miRNAs were observed, excluding let-7g, while in tumour tissue, the levels of oncomirs miR-21, miR-10b, miR-31 and miR-155, and the oncosuppressor let-7g, were upregulated. Analysis of binase-susceptible miRNAs and their regulatory networks showed that the main modulated events were transcription and translation control, the cell cycle, cell proliferation, adhesion and invasion, apoptosis and autophagy, as well as some other tumour-related cascades, with an impact on the observed antitumour effects.

Bacillus pumilus ribonuclease rescues mice infected by double-stranded RNA-containing reovirus serotype 1.

Reoviruses (respiratory enteric orphan viruses) are nonenveloped viruses with segmented dsRNA genome. Viruses in the family Reoviridae are quite stable in the environment. Recently, they have been identified with various pathologies and physiologic dysfunctions in a wide range of organs and tissues, including the hepatobiliary system, the myocardium, lungs, and endocrine tissues. Although most cases of reovirus infection are mild or subclinical diseases, the prevention measures are currently needed, especially for young children suffering from dehydrating gastroenteritis. To inhibit viral replication, different RNases targeting viral RNA are proposed. Here, we first have shown that RNase from Bacillus pumilus (binase) acts as an antiviral agent at the level of the whole animal organism infected by Mammalian orthoreovirus 1 strain Lang (TL1). The results obtained on the mice model infected with 10 LD50 and 20 LD50 doses of reovirus indicate the restoration of mice physiological parameters under binase treatment at the dose of 50 µg/mouse. Thus, our research supports the relevance of binase as a promising antiviral agent that affects viral RNA.

Zeolites as Carriers of Antitumor Ribonuclease Binase.

Natural and synthetic zeolites have many applications in biomedicine and nutrition. Due to its properties, zeolites can absorb therapeutically active proteins and release them under physiological conditions. In this study we tested the clinoptilolite, chabazite, and natrolite ability to be loaded by antitumor ribonuclease binase and the cytotoxicity of the obtained complexes. We found the optimal conditions for binase loading into zeolites and established the dynamic of its release. Cytotoxic effects of zeolite-binase complexes toward colorectal cancer Caco2 cells were characterized after 24 and 48 h of incubation with cells using MTT-test. Zeolites were toxic by itselfs and reduced cells viability by 30% (clinoptilolite), 40% (chabazite), and 70% (natrolite) after 48 h of incubation. Binase complexes with clinoptilolite as well as chabazite always demonstrated enhanced toxicity (up to 57 and 60% for clinoptilolite and chabazite, respectively) in comparison with binase and zeolites separately. Our results contribute to the perspective development of binase-based complexes for therapy of colorectal cancer for or the treatment of malignant skin neoplasms where the complexes can be used in pasty form.

The Native Monomer of Bacillus Pumilus Ribonuclease Does Not Exist Extracellularly.

Supported by crystallography studies, secreted ribonuclease of Bacillus pumilus (binase) has long been considered to be monomeric in form. Recent evidence obtained using native polyacrylamide gel electrophoresis and size-exclusion chromatography suggests that binase is in fact dimeric. To eliminate ambiguity and contradictions in the data we have measured conformational changes, hypochromic effect, and hydrodynamic radius of binase. The immutability of binase secondary structure upon transition from low to high protein concentration was registered, suggesting the binase dimerization immediately after translocation through the cell membrane and leading to detection of binase dimers only in the culture fluid regardless of ribonuclease concentration. Our results made it necessary to take a fresh look at the binase stability and cytotoxicity towards virus-infected or tumor cells.

Bacterial ribonuclease binase exerts an intra-cellular anti-viral mode of action targeting viral RNAs in influenza a virus-infected MDCK-II cells.

BACKGROUND: Influenza is a severe contagious disease especially in children, elderly and immunocompromised patients. Beside vaccination, the discovery of new anti-viral agents represents an important strategy to encounter seasonal and pandemic influenza A virus (IAV) strains. The bacterial extra-cellular ribonuclease binase is a well-studied RNase from Bacillus pumilus. Treatment with binase was shown to improve survival of laboratory animals infected with different RNA viruses. Although binase reduced IAV titer in vitro and in vivo, the mode of action (MOA) of binase against IAV at the molecular level has yet not been studied in depth and remains elusive. METHODS: To analyze whether binase impairs virus replication by direct interaction with the viral particle we applied a hemagglutination inhibition assay and monitored the integrity of the viral RNA within the virus particle by RT-PCR. Furthermore, we used Western blot and confocal microscopy analysis to study whether binase can internalize into MDCK-II cells. By primer extension we examined the effect of binase on the integrity of viral RNAs within the cells and using a mini-genome system we explored the effect of binase on the viral expression. RESULTS: We show that (i) binase does not to attack IAV particle-protected viral RNA, (ii) internalized binase could be detected within the cytosol of MDCK-II cells and that (iii) binase impairs IAV replication by specifically degrading viral RNA species within the infected MDCK-II cells without obvious effect on cellular mRNAs. CONCLUSION: Our data provide novel evidence suggesting that binase is a potential anti-viral agent with specific intra-cellular MOA.

Metabolic Activity of Micromycetes Affecting Urban Concrete Constructions.

Concrete resistance to the destructive action of microorganisms is considered as a measure of its durability and is increasingly being raised as an important issue. We focused our study on the biodeterioration of concrete specimens widely used as a building material of urban houses by micromycetes isolated from the inner wall surface of the former military hospital in Kazan city, Tatarstan, Russia. Fungal community consists of 9 Penicillium isolates, 6 Aspergillus, 2 Trichoderma, and 1 isolate of Alternaria. First, we have identified two dominant isolates, Aspergillus fumigatus and Penicillium brevicompactum, and characterized their destructive properties according to the radial growth rate, antagonistic activity towards bacterial habitants of concrete, and production of organic acids. Then, we have demonstrated that five tested brands of high-strength concrete differ in bioreceptivity. The alterations in concrete resistances to compression and flexure after fungal attack were recorded at the trend level, mainly due to a short exposure time of concrete to fungal destructors in tests recommended by national Russian standard. Finally, using scanning electron microscopy we have shown that colonization of concrete by the dominant fungi includes their penetration into the thickness of concrete and germination in cracks. Elementary analysis revealed the decrease of calcium content on about 41% after fungal growth on the concrete in liquid phase and on 32% by superficial growth in comparison with the samples without fungal treatment.

Secretome of Intestinal Bacilli: A Natural Guard against Pathologies.

Current studies of human gut microbiome usually do not consider the special functional role of transient microbiota, although some of its members remain in the host for a long time and produce broad spectrum of biologically active substances. Getting into the gastrointestinal tract (GIT) with food, water and probiotic preparations, two representatives of Bacilli class, genera Bacillus and Lactobacillus, colonize epithelium blurring the boundaries between resident and transient microbiota. Despite their minor proportion in the microbiome composition, these bacteria can significantly affect both the intestinal microbiota and the entire body thanks to a wide range of secreted compounds. Recently, insufficiency and limitations of pure genome-based analysis of gut microbiota became known. Thus, the need for intense functional studies is evident. This review aims to characterize the Bacillus and Lactobacillus in GIT, as well as the functional roles of the components released by these members of microbial intestinal community. Complex of their secreted compounds is referred by us as the "bacillary secretome." The composition of the bacillary secretome, its biological effects in GIT and role in counteraction to infectious diseases and oncological pathologies in human organism is the subject of the review.

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.