DNA targeting and interference by a bacterial Argonaute nuclease.

Members of the conserved Argonaute protein family use small RNA guides to locate their mRNA targets and regulate gene expression and suppress mobile genetic elements in eukaryotes1,2. Argonautes are also present in many bacterial and archaeal species3-5. Unlike eukaryotic proteins, several prokaryotic Argonaute proteins use small DNA guides to cleave DNA, a process known as DNA interference6-10. However, the natural functions and targets of DNA interference are poorly understood, and the mechanisms of DNA guide generation and target discrimination remain unknown. Here we analyse the activity of a bacterial Argonaute nuclease from Clostridium butyricum (CbAgo) in vivo. We show that CbAgo targets multicopy genetic elements and suppresses the propagation of plasmids and infection by phages. CbAgo induces DNA interference between homologous sequences and triggers DNA degradation at double-strand breaks in the target DNA. The loading of CbAgo with locus-specific small DNA guides depends on both its intrinsic endonuclease activity and the cellular double-strand break repair machinery. A similar interaction was reported for the acquisition of new spacers during CRISPR adaptation, and prokaryotic genomes that encode Ago nucleases are enriched in CRISPR-Cas systems. These results identify molecular mechanisms that generate guides for DNA interference and suggest that the recognition of foreign nucleic acids by prokaryotic defence systems involves common principles.

Luminescent Bacteria as Bioindicators in Screening and Selection of Enzymes Detoxifying Various Mycotoxins.

Interest in enzymes capable of neutralizing various mycotoxins is quite high. The methods used for the screening and selection of enzymes that catalyze the detoxification of mycotoxins should be sensitive and fast. However toxic compounds can be generated under the action of such enzymes. Thus, the assessment of the overall reduction in the toxic properties of reaction media towards bioluminescent bacteria seems to be the most reasonable control method allowing a quick search for the effective enzymatic biocatalysts. The influence of a wide range of mycotoxins and glucanases, which hydrolyze toxins with different chemical structures, on the analytical characteristics of luminescent photobacteria as a biosensing element has been studied. Different glucanases (ß-glucosidase and endoglucanase) were initially selected for reactions with 10 mycotoxins based on the results of molecular docking which was performed in silico with 20 mycotoxins. Finally, the biorecognizing luminescent cells were used to estimate the residual toxicity of reaction media with mycotoxins after their interaction with enzymes. The notable non-catalytic decrease in toxicity of media containing deoxynivalenol was revealed with luminous cells for both types of tested glucanases, whereas ß-glucosidase provided a significant catalytic detoxification of media with aflatoxin B2 and zearalenone at pH 6.0.

Conjugative Plasmid pPPUT-Tik1-1 from a Permafrost Pseudomonas putida Strain and Its Present-Day Counterparts Inhabiting Environments and Clinics.

A novel group of conjugative plasmids of Pseudomonas is characterized. The prototype plasmid pPPUT-Tik1-1 (153,663 bp), isolated from a permafrost strain of P. putida Tik1, carries a defective mercury transposon, Tn501, and a streptomycin resistance transposon, Tn5393. Ten plasmids and 34 contigs with backbone regions closely related to pPPUT-Tik1-1 have been found in GenBank. Two of these plasmids from clinical strains of P. putida and P. fulva are almost identical to the ancient plasmid. A characteristic feature of this group of plasmids is the presence of two genes encoding the initiators of replication (repA1 and repA2). None of these genes have high similarity with plasmid replication genes belonging to known incompatibility groups. It has been demonstrated that while pPPUT-Tik1-1-like plasmids have homologous backbone regions, they significantly differ by the molecular structure and the predicted functions of their accessory regions. Some of the pPPUT-Tik1-1-related plasmids carry determinants of antibiotic resistance and/or heavy metal salts. Some plasmids are characterized by the ability to degrade xenobiotics. Plasmids related to pPPUT-Tik1-1 are characterized by a narrow host range and are found in various species of the Pseudomonas genus. Interestingly, we also found shorter plasmid variants containing the same replication module, but lacking conjugation genes and containing other structural changes that strongly distinguish them from plasmids related to pPPUT-Tik1-1, indicating that the structure of the replication module cannot be used as the sole criterion for classifying plasmids. Overall, the results suggest that the plasmids of the novel group can be spread using conjugation in environmental and clinical strains of Pseudomonas and may play diverse adaptive functions due to the presence of various accessory regions.

Metal Nanomaterials and Hydrolytic Enzyme-Based Formulations for Improved Antifungal Activity.

Active research of metal-containing compounds and enzymes as effective antifungal agents is currently being conducted due to the growing antifungal resistance problem. Metals are attracting special attention due to the wide variety of ligands that can be used for them, including chemically synthesized and naturally obtained variants as a result of the so-called "green synthesis". The main mechanism of the antifungal action of metals is the triggering of the generation and accumulation of reactive oxygen species (ROS). Further action of ROS on various biomolecules is nonspecific. Various hydrolytic enzymes (glucanases and proteases), in turn, exhibit antifungal properties by affecting the structural elements of fungal cells (cell walls, membranes), fungal quorum sensing molecules, fungal own protective agents (mycotoxins and antibiotics), and proteins responsible for the adhesion and formation of stable, highly concentrated populations in the form of biofilms. A wide substrate range of enzymes allows the use of various mechanisms of their antifungal actions. In this review, we discuss the prospects of combining two different types of antifungal agents (metals and enzymes) against mycelial fungi and yeast cells. Special attention is paid to the possible influence of metals on the activity of the enzymes and the possible effects of proteins on the antifungal activity of metal-containing compounds.

Strategies for variable regulation of methanogenesis efficiency and velocity.

Combinations of various strategic approaches to the suppression of methanogenesis and the formation of biogas with a simultaneous decrease in the ratio of methane in its composition were investigated. Introduction of methanogenesis suppressors such as redox derivatives of humic acids, potassium persulfate (K2S2O8), possessing oxidizing and electron acceptor properties, enzyme hexahistidine-containing organophosphorus hydrolase with high lactonase activity and polypeptide antimicrobial agent bacitracin into the media with anaerobic consortia were studied. The effect of these substances was directed at various participants of the natural methanogenic consortium, as well as on the biochemical processes carried out by them. The use of K2S2O8 together with bacitracin provided maximum and almost complete suppression of CH4 production. The measured concentration of intracellular adenosine triphosphate has shown that viability of cells in the consortium remained almost the same, whereas their metabolic activity decreased. Various combinations of the above-mentioned suppressors provided different degrees of methanogenesis suppression, but redox agents played a key role in all the cases studied. Based on the accumulated data, combining suppressors in different concentrations can be used to manage the methanogenesis (efficiency and velocity of its decrease) in media with anaerobic consortia. KEY POINTS: • Various strategies for suppression of the methanogenesis were combined. • The enzyme His6-OPH was firstly used for quorum quenching in methanogenic consortium. • Velocity of methanogenesis decrease can be managed by combinations of suppressors.

Plasmids as Key Players in Acinetobacter Adaptation.

This review briefly summarizes the data on the mechanisms of development of the adaptability of Acinetobacters to various living conditions in the environment and in the clinic. A comparative analysis of the genomes of free-living and clinical strains of A. lwoffii, as well as the genomes of A. lwoffii and A. baumannii, has been carried out. It has been shown that plasmids, both large and small, play a key role in the formation of the adaptability of Acinetobacter to their living conditions. In particular, it has been demonstrated that the plasmids of various strains of Acinetobacter differ from each other in their structure and gene composition depending on the lifestyle of their host bacteria. Plasmids of modern strains are enriched with antibiotic-resistant genes, while the content of genes involved in resistance to heavy metals and arsenic is comparable to plasmids from modern and ancient strains. It is concluded that Acinetobacter plasmids may ensure the survival of host bacteria under conditions of various types of environmental and clinical stresses. A brief overview of the main mechanisms of horizontal gene transfer on plasmids inherent in Acinetobacter strains is also given.

Stem cell therapy: old challenges and new solutions.

Stem cell therapy (SCT), born as therapeutic revolution to replace pharmacological treatments, remains a hope and not yet an effective solution. Accordingly, stem cells cannot be conceivable as a "canonical" drug, because of their unique biological properties. A new reorientation in this field is emerging, based on a better understanding of stem cell biology and use of cutting-edge technologies and innovative disciplines. This will permit to solve the gaps, failures, and long-term needs, such as the retention, survival and integration of stem cells, by employing pharmacology, genetic manipulation, biological or material incorporation. Consequently, the clinical applicability of SCT for chronic human diseases will be extended, as well as its effectiveness and success, leading to long-awaited medical revolution. Here, some of these aspects are summarized, reviewing and discussing recent advances in this rapidly developing research field.

Prospective Approach to the Anaerobic Bioconversion of Benzo- and Dibenzothiophene Sulfones to Sulfide.

Sulfur recovery from organic molecules such as toxic sulfones is an actual problem, and its solution through the use of environmentally friendly and nature-like processes looks attractive for research and application. For the first time, the possible bioconversion of organic sulfones (benzo-and dibenzothiophene sulfones) to inorganic sulfide under anaerobic conditions with simultaneous biogas production from glucose within a methanogenesis process is demonstrated. Biogas with a methane content of 50.7%-82.1% was obtained without H2S impurities. Methanogenesis with 99.7%-100% efficiency and 97.8%-100% conversion of benzo- and dibenzothiophene sulfones (up to 0.45 mM) to inorganic sulfide were obtained in eight days by using a combination of various anaerobic biocatalysts immobilized in a poly(vinyl alcohol) cryogel. Pure cell cultures of sulfate-reducing bacteria and/or H2-producing bacteria were tested as additives to the methanogenic activated sludge. The immobilized activated sludge "enhanced" by bacterial additives appeared to retain its properties and be usable multiple times for the conversion of sulfones under batch conditions.

Biosensitive element in the form of immobilized luminescent photobacteria for detecting ecotoxicants in aqueous flow-through systems.

We demonstrated the possibility of long-term and efficient application of a biosensitive element (BE) in the form of Photobacterium phosphoreum photobacteria immobilized in poly(vinyl alcohol) (PVA) cryogel for detecting various ecotoxicants (Zn(2) (+) , Cu(2) (+) , Hg(2) (+) , Pb(2) (+) , 2,4-dichlorophenoxyacetic acid, 2,6-dimethylphenol, pentachlorophenol, coumaphos, malathion, chlorpyrifos and methyl parathion) in flow-through media. The range of detectable concentrations of ecotoxicants was determined at 1 × 10(-8) to 1 × 10(-4) M for heavy metal ions and at 1 × 10(-8) to 1 × 10(-5) M for phenol derivatives and organophosphorus pesticides. Immobilized cells of photobacteria quantitatively reacted with these ecotoxicants; cell sensitivity exhibited no flow rate dependence in the range from 45 to 180 mL/h. At a constant concentration of ecotoxicant in the flow, the bioluminescence quenching profile of immobilized cells demonstrated an integral response. The BE could remain in a flow-through medium for at least 10 days while retaining 95% of luminescent activity in the absence of ecotoxicants. The BE tested in this work was demonstrated to have a long shelf life (> 60 weeks) at -80°C without changes in the baseline level of bioluminescence. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of novel maleimide derivatives on cell cultures with different properties.

The paper is focused on pilot study of effects of novel synthetic protein kinase inhibitors-maleimide derivatives in different concentrations on normal, transformed and multipotent cell lines. Influence on cell proliferation and morphological characteristics has been demonstrated. The chosen agents cause antiproliferative effect on transformed cells and are not cytotoxic to normal cell lines. Moreover, different maleimide derivatives' effects on multipotent cells in attached and floating states has been shown. Described results can be used for further research of the maleimide derivatives as antitumor agents.

Morphological characteristics of cultured fresh and thawed pericardium cells.

The need for selection of the optimal material for the manufacturing of cardio-patches can be resolved by the use of cryostored autologous pericardial tissue. This short communication is a concise fragment of a large-scale research and demonstrates only the efficiency of cell culturing before and after pericardial preservation in the low temperature conditions.

Integrative function of proprioceptive system in the acute effects of whole body vibration on the movement performance in young adults.

Background: The proprioceptive system coordinates locomotion, but its role in short-term integration and recovery of motor activity in imbalance of motor patterns and body remains debated. The aim of this study is investigating the functional role of proprioceptive system in motor patterns and body balance in healthy young adults. Methods: 70 participants (aged 20.1 ± 0.3) were divided into experimental groups EG1 (n = 30), EG2 (n = 30), control group (CG, n = 10). EG1 performed single WBV session on Power Plate (7 exercises adapted to Functional Movement Screen (FMS). EG2 performed single session of FMS Exercises (FMSE). CG didn't perform any physical activity. All participants performed pre- and post-session of FMS and stabilometric measurements. Results: FMS total score in EG1 increased by 2.0 ± 0.2 (p0 < 0.001), this was significantly differed (p0 < 0.001) from EG2 and CG. Acute effects of WBV and FMSE on rate of change and standard deviation (SD) of pressure center (COP) were shown in all groups during Static Test (p0 < 0.01). SD increased (p0 < 0.01) in Given Setting Test in EG1 and EG2, and in Romberg Test (p0 < 0.001) in EG1. Length, width and area (p0 < 0.01) of confidence ellipse, containing 95% of the statokinesiogram points, decreased in Static Test in EG1; width and area (p0 < 0.01) decreased in EG2 group. Significant (p0 < 0.01) decrease in Given Setting Test was in EG1, and significant (p0 < 0.01) increase was in Romberg Test (open eyes) in CG. Maximum amplitude of COP oscillations: significantly (p0 < 0.01) decreasing along X and Y axes in EG1 and EG2, and along Y axis in CG during Static Test; along Y axis (p0 < 0.01) in all groups during Given Setting Test. Significant differences were identified (p0 < 0.01) in calculated energy consumption for COP moving during all stabilometric tests. However, inter-group differences in COP after acute WBV and FMSE sessions have not been identified. Conclusions: Acute WBV session eliminates the deficits in motor patterns which is not the case after acute FMSE session, which, according to our integrative movement tuning hypothesis, is due to high activation of integrative function of proprioceptive system. Efficacy of WBV and FMSE on COP performance indicates a high sensitivity of postural control to different levels of proprioceptive system activity.

Using Fungi in Artificial Microbial Consortia to Solve Bioremediation Problems.

There is currently growing interest in the creation of artificial microbial consortia, especially in the field of developing and applying various bioremediation processes. Heavy metals, dyes, synthetic polymers (microplastics), pesticides, polycyclic aromatic hydrocarbons and pharmaceutical agents are among the pollutants that have been mainly targeted by bioremediation based on various consortia containing fungi (mycelial types and yeasts). Such consortia can be designed both for the treatment of soil and water. This review is aimed at analyzing the recent achievements in the research of the artificial microbial consortia that are useful for environmental and bioremediation technologies, where various fungal cells are applied. The main tendencies in the formation of certain microbial combinations, and preferences in their forms for usage (suspended or immobilized), are evaluated using current publications, and the place of genetically modified cells in artificial consortia with fungi is assessed. The effect of multicomponence of the artificial consortia containing various fungal cells is estimated, as well as the influence of this factor on the functioning efficiency of the consortia and the pollutant removal efficacy. The conclusions of the review can be useful for the development of new mixed microbial biocatalysts and eco-compatible remediation processes that implement fungal cells.

"Stop, Little Pot" as the Motto of Suppressive Management of Various Microbial Consortia.

The unresolved challenges in the development of highly efficient, stable and controlled synthetic microbial consortia, as well as the use of natural consortia, are very attractive for science and technology. However, the consortia management should be done with the knowledge of how not only to accelerate but also stop the action of such "little pots". Moreover, there are a lot of microbial consortia, the activity of which should be suppressively controlled. The processes, catalyzed by various microorganisms being in complex consortia which should be slowed down or completely cancelled, are typical for the environment (biocorrosion, landfill gas accumulation, biodegradation of building materials, water sources deterioration etc.), industry (food and biotechnological production), medical practice (vaginitis, cystitis, intestinal dysbiosis, etc.). The search for ways to suppress the functioning of heterogeneous consortia in each of these areas is relevant. The purpose of this review is to summarize the general trends in these studies regarding the targets and new means of influence used. The analysis of the features of the applied approaches to solving the main problem confirms the possibility of obtaining a combined effect, as well as selective influence on individual components of the consortia. Of particular interest is the role of viruses in suppressing the functioning of microbial consortia of different compositions.

Carbon-Rich Plasma-Deposited Silicon Oxycarbonitride Films Derived from 4-(Trimethylsilyl)morpholine as a Novel Single-Source Precursor.

4-(trimethylsilyl)morpholine O(CH2CH2)2NSi(CH3)3 (TMSM) was investigated as a single-source precursor for SiCNO films synthesis. Optical emission spectroscopy of plasma generated from TMSM/He, TMSM/H2, and TMSM/NH3 gas mixtures revealed the presence of N2, CH, H, CN, and CO species. The last two are suggested to be responsible for the lowering of carbon concentration in the films in comparison with the precursor. The refractive index ranged from 1.5 to 2.0, and bandgap varied from 2.0 to 4.6 eV, which pointed that some of the films can be used as antireflective coatings in silicon photovoltaic cell technologies and dielectric layers in electronic devices.

Combination of Enzymes with Materials to Give Them Antimicrobial Features: Modern Trends and Perspectives.

Multidrug-resistant bacteria form serious problems in many areas, including medicine and the food industry. At the same time, great interest is shown in the transfer or enhancement of antimicrobial properties to various materials by modifying them with enzymes. The use of enzymes in biomaterials with antimicrobial properties is important because enzymes can be used as the main active components providing antimicrobial properties of functionalized composite biomaterials, or can serve as enhancers of the antimicrobial action of certain substances (antibiotics, antimicrobial peptides, metal nanoparticles, etc.) against cells of various microorganisms. Enzymes can simultaneously widen the spectrum of antimicrobial activity of biomaterials. This review presents the most promising enzymes recently used for the production of antibacterial materials, namely hydrolases and oxidoreductases. Computer modeling plays an important role in finding the most effective combinations between enzymes and antimicrobial compounds, revealing their possible interactions. The range of materials that can be functionalized using enzymes looks diverse. The physicochemical characteristics and functionalization methods of the materials have a significant impact on the activity of enzymes. In this context, fibrous materials are of particular interest. The purpose of this review is to analyze the current state of the art in this area.

Carrier Variety Used in Immobilization of His6-OPH Extends Its Application Areas.

Organophosphorus hydrolase, containing a genetically introduced hexahistidine sequence (His6-OPH), attracts the attention of researchers by its promiscuous activity in hydrolytic reactions with various substrates, such as organophosphorus pesticides and chemical warfare agents, mycotoxins, and N-acyl homoserine lactones. The application of various carrier materials (metal-organic frameworks, polypeptides, bacterial cellulose, polyhydroxybutyrate, succinylated gelatin, etc.) for the immobilization and stabilization of His6-OPH by various methods, enables creation of biocatalysts with various properties and potential uses, in particular, as antidotes, recognition elements of biosensors, in fibers with chemical and biological protection, dressings with antimicrobial properties, highly porous sorbents for the degradation of toxicants, including in flow systems, etc. The use of computer modeling methods in the development of immobilized His6-OPH samples provides in silico prediction of emerging interactions between the enzyme and immobilizing polymer, which may have negative effects on the catalytic properties of the enzyme, and selection of the best options for experiments in vitro and in vivo. This review is aimed at analysis of known developments with immobilized His6-OPH, which allows to recognize existing recent trends in this field of research, as well as to identify the reasons limiting the use of a number of polymer molecules for the immobilization of this enzyme.

Destruction of Mycotoxins in Poultry Waste under Anaerobic Conditions within Methanogenesis Catalyzed by Artificial Microbial Consortia.

To reduce the toxicity of modern feeds polluted by mycotoxins, various sorbents are added to them when feeding animals. A part of the mycotoxins is excreted from the body of animals with these sorbents and remains in the manure. As a result, bulk animal wastes containing mixtures of mycotoxins are formed. It is known that it is partially possible to decrease the initial concentration of mycotoxins in the process of anaerobic digestion (AD) of contaminated methanogenic substrates. The aim of this review was to analyze the recent results in destruction of mycotoxins under the action of enzymes present in cells of anaerobic consortia catalyzing methanogenesis of wastes. The possible improvement of the functioning of the anaerobic artificial consortia during detoxification of mycotoxins in the bird droppings is discussed. Particular attention was paid to the possibility of effective functioning of microbial enzymes that catalyze the detoxification of mycotoxins, both at the stage of preparation of poultry manure for methanogenesis and directly in the anaerobic process itself. The sorbents with mycotoxins which appeared in the poultry wastes composed one of the topics of interest in this review. The preliminary alkaline treatment of poultry excreta before processing in AD was considered from the standpoint of effectively reducing the concentrations of mycotoxins in the waste.

Comparative study of quality of life 9 months post-COVID-19 infection with SARS-CoV-2 of varying degrees of severity: impact of hospitalization vs. outpatient treatment.

Purpose: This experimental study was conducted during the post-COVID-19 period to investigate the relationship between the quality of life 9 months after and the severity of the SARS-CoV-2 infection in two scenarios: hospitalization (with/without medical oxygen) and outpatient treatment. Methods: We employed the EQ-5D-5L Quality of Life tests and the PSQI as a survey to evaluate respondents' quality of life 9 months after a previous SARS-CoV-2 infection of varying severity. Results: We identified a clear difference in the quality of life of respondents, as measured on the 100-point scale of the EQ-5D-5L test, which was significantly lower 9 months after a previous SARS-CoV-2 infection for Group 1 (n = 14), respondents who had received medical attention for SARS-CoV-2 infection in a hospital with oxygen treatment, compared to those with the SARS-CoV-2 infection who were treated without oxygen treatment (Group 2) (n = 12) and those who were treated on an outpatient basis (Group 3) (n = 13) (H = 7.08 p = 0.029). There were no intergroup differences in quality of life indicators between hospitalized patients (Group 2) and groups 1 and 3. PSQI survey results showed that "mobility," "self-care," "daily activities," "pain/discomfort," and "anxiety/ depression" did not differ significantly between the groups, indicating that these factors were not associated with the severity of the SARS-CoV-2 infection. On the contrary, the respondents demonstrated significant inter-group differences (H = 7.51 p = 0.023) and the interdependence of respiratory difficulties with the severity of clinically diagnosed SARS-CoV-2 infection. This study also demonstrated significant differences in the values of sleep duration, sleep disorders, and daytime sleepiness indicators between the three groups of respondents, which indicate the influence of the severity of the infection. The PSQI test results revealed significant differences in "bedtime" (H = 6.00 p = 0.050) and "wake-up time" (H = 11.17 p = 0.004) between Groups 1 and 3 of respondents. At 9 months after COVID-19, respondents in Group 1 went to bed at a later time (pp = 0.02727) and woke up later (p = 0.003) than the respondents in Group 3. Conclusion: This study is the first of its kind in the current literature to report on the quality of life of respondents 9 months after being diagnosed with COVID-19 and to draw comparisons between cohorts of hospitalized patients who were treated with medical oxygen vs. the cohorts of outpatient patients. The study's findings regarding post-COVID-19 quality of life indicators and their correlation with the severity of the SARS-CoV-2 infection can be used to categorize patients for targeted post-COVID-19 rehabilitation programs.

Quorum Sensing as a Trigger That Improves Characteristics of Microbial Biocatalysts.

Quorum sensing (QS) of various microorganisms (bacteria, fungi, microalgae) today attracts the attention of researchers mainly from the point of view of clarifying the biochemical basics of this general biological phenomenon, establishing chemical compounds that regulate it, and studying the mechanisms of its realization. Such information is primarily aimed at its use in solving environmental problems and the development of effective antimicrobial agents. This review is oriented on other aspects of the application of such knowledge; in particular, it discusses the role of QS in the elaboration of various prospective biocatalytic systems for different biotechnological processes carried out under aerobic and anaerobic conditions (synthesis of enzymes, polysaccharides, organic acids, etc.). Particular attention is paid to the biotechnological aspects of QS application and the use of biocatalysts, which have a heterogeneous microbial composition. The priorities of how to trigger a quorum response in immobilized cells to maintain their long-term productive and stable metabolic functioning are also discussed. There are several approaches that can be realized: increase in cell concentration, introduction of inductors for synthesis of QS-molecules, addition of QS-molecules, and provoking competition between the participants of heterogeneous biocatalysts, etc.).