Method of preparation, visualization and ultrastructural analysis of a formulation of probiotic Bacillus subtilis KATMIRA1933 produced by solid-phase fermentation.

Probiotic preparations are used in medical treatment and in agricultural practice. They modulate numerous activities in eukaryotic hosts, such as: inhibition of pathogenic microbiota; stimulation of immunological responses; and production of antioxidants, anti-mutagens, and DNA protectors. Also, probiotic bacteria are used as a preventive measure to prevent bacterial diseases of the gastrointestinal tract. Solid-phase fermentation is reported as being used in the production of probiotic formulations where a solid substratum, such as soy and oil meal, is utilized for the growth of beneficial microorganisms. However, there are insufficient reports in the literature related to methodological approaches enabling evaluation of the final products of solid-phase fermentation. We suggest a novel method enabling evaluation of probiotic solid-state fermentation dry powders and observation of their morphology, ultrastructure, and elucidation of the quantitative distribution of probiotic microorganisms in solid substrates using electron microscopy. •The method is intended for ultrastructure microphotography of dry substances - for example, ultrastructure of solid-phase fermentation products.•The method allows preserving the ultrastructure of substrates that are damaged when soaking.•The method does not require additional equipment and reagents and can be used in all laboratories using electron microscopy.

Effect of Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 on the productivity, reproductive aging, and physiological characteristics of hens and roosters.

The study aims at investigating the effect of preparations of two bacilli strains on laying hens and roosters. Preparations were based on the strains Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895. Several groups of roosters and hens received a preparation based on either strain, or a mixture of both, from the first day to the last day of poultry in production. These preparations improved egg production, quality of sperm production, quality/hatchery of eggs, and slowed down the reproductive aging of hens. These observations were confirmed by the mathematical model proposed herein. At the molecular level, the slowing down of aging was confirmed by a decrease in the amount of mitochondrial DNA damage. Monitoring the physiological parameters of the experimental and control groups of birds showed that live weight gain in all experimental groups was higher than in the control group, and the reproductive organs of hens were more developed. There was also an improvement in the biochemical parameters of blood, the quality of the sperm of roosters, the laying of laying hens, and the morphological and biochemical parameters of the eggs. One of the most significant results is an increase in egg fertilization and a decrease in embryo death during the first 7 days of incubation.

Presence of Old Individuals in a Population Accelerates and Optimizes the Process of Selection: in silico Experiments.

One of the important components of the concept of aging-phenoptosis (programmed aging) is the notion of aging as an accelerator of evolution having the rank of subconcept. For many reasons, the main being the problematic experimental testing of evolutionary hypotheses, verification of the above-mentioned subconcept can be based primarily on analysis of the internal inconsistency of heuristic models and their correspondence to undisputedly observed facts. To illustrate the acceleration mechanism, and most importantly to structure the evolutionary process in communities that include naturally weakened individuals, V. P. Skulachev offered in 2003 a conceptual model that he later called a "fable about hares". Despite its simplicity, this model has undoubted internal logic. The natural trend in the development of conceptual models is their translation into the language of mathematics. The purpose of the present work was to create a variation of the known multi-agent model "predator-prey" that would allow us to "see" how the presence in the prey population of naturally weakened (old) members stimulates the selection of individuals with traits whose adaptive potential is not devaluated with age. The model (http://homebear.ru/PD) was developed on the Java platform, version 6, NetBeans development environment 8.2. Statistical analysis and preparation of illustrative materials were carried out using environment R, version 3.4.1. The results of numerical experiments set using our model correspond in principle to the provisions of the heuristic model of Skulachev and, consequently, confirm the absence in it of logical contradictions.

[Increase in Bacterial Resistance to Antibiotics after Cancer Therapy with Platinum-Based Drugs].

The use of platinum-based anticancer drugs is limited by both their side effects and their effect on normal microflora's metagenome. Drugs that possess mutagenic and genotoxic properties may cause mutations in microbial genomes that contribute to the emergence of resistance to antimicrobial preparations and the development of complications after chemotherapy. The effects of cisplatin and oxaliplatin on microorganisms were studied using bacterial biosensors - E. coli strains MG1655 pKatG-lux, which reacts to the generation of hydrogen peroxide; MG1655 pSoxS-lux, which reacts to the superoxide anion radical; and the MG1655 pColD-lux strain, which detects DNA damage. The biosensor tests demonstrated high levels of genotoxicity for both drugs and some differences in the spectrum of reactive oxygen species generated. Ascor-bate reduced genotoxicity of cisplatin by 41%. Nonlethal doses of cisplatin induced a three- to sevenfold increase in the frequency of the mutations that confer the resistance of E. coli to rifampicin and ciprof loxacin. Ascorbate also reduced frequency of the mutations by 65%. Thus, the effect of these drugs was probably associated with the generation of reactive oxygen species and induction of SOS response. The risk of secondary antibiotic-resistant infections may be decreased by applying antioxidants and antimutagens. At the same time, these increases may also decrease the anti-tumoral action of these compounds.

Methods to Assess the Antioxidative Properties of Probiotics.

Probiotics prove useful in correcting and preventing numerous health conditions, including those having severe impact on society, e.g., obesity and cancer. Notably, these capabilities of probiotics appear to be associated with their antioxidant properties. The mechanisms of antioxidant action of probiotics range from immediate biochemical scavenging of reactive substances to induction of signaling events leading to increased capacity of the host's cytoprotective systems. Since the antioxidant effects of probiotics significantly vary in types and details, a broad selection of methods of assessment of these properties is required in order to identify, characterize, and develop novel probiotics for medical purposes, as well as to explain the mechanisms of action of probiotics already in use in healthcare. This review revises the versatile toolbox, which can be used to assess the antioxidant properties of probiotics.

Mitochondria as a Signaling Hub and Target for Phenoptosis Shutdown.

Mitochondria have long been studied as the main energy source and one of the most important generators of reactive oxygen species in the eukaryotic cell. Yet, new data suggest mitochondria serve as a powerful cellular regulator, pathway trigger, and signal hub. Some of these crucial mitochondrial functions appear to be associated with RNP-granules. Deep and versatile involvement of mitochondria in general cellular regulation may be the legacy of parasitic behavior of the ancestors of mitochondria in the host cells. In this regard, we also discuss here the perspectives of using mitochondria-targeted compounds for systemic correction of phenoptotic shifts.

[The role of a lysine residue in the antioxidant and dna-protective activity of oligopeptides].

Oligopeptides present in the living cell were found to have antioxidative activity and to be involved in the regulation of antioxidant balance by interaction with the redox-dependent cellular signaling cascades. Experiments on animal models have shown that the introduction of oligopeptides causes geroprotective and adaptogenic effects. In the present work, we investigate the biological action of a number of synthetic oligopeptides using bacterial biosensors. This approach allows us to precisely estimate the antioxidant properties of the compounds without affecting their participation in regulatory cascades typical to eukaryotic cells. It has been shown that the ability of oligopeptides to protect cells from action of physical prooxidant factors (UV irradiation) is related to the presence of a lysine residue in the molecule. For chemical pro-oxidants (dioxidine), we have observed a similar, though less strict pattern. This effect also correlates with DNA-protective activity of the investigated oligopeptides.

DNA-protection and antioxidant properties of fermentates from Bacillus amyloliquefaciens B-1895 and Bacillus subtilis KATMIRA1933.

UNLABELLED: DNA protective and antioxidant activity of Bacillus amyloliquefaciens B-1895 and Bacillus subtilis KATMIRA1933 were evaluated by Escherichia coli-based Lux biosensors. Two biosensor strains of E. coli, MG1655 (pColD-lux) and MG1655 (pSoxS-lux), which react on DNA damage and superoxide-anion radical activity, were used. SOS-response and Sox-response were stimulated by addition of dioxidine (2,3-Quinoxalinedimethanol,1,4-dioxide) and paraquat (N,N'-dimethyl-4,4'-bipyridinium dichloride) respectively. Preparations of both Bacillus fermentates demonstrated DNA protective and antioxidant (superoxide scavenging) activity (up to 60·19%). The strain К1933 is, in general, characterized by higher DNA protective activity (28·85%), with parameters of antioxidant activity of both bacilli strains being statistically not significantly different. Sporogenous potential probiotic micro-organisms with antioxidant and DNA protective activities can become an effective tool for compensation of various negative oxidative stress processes in humans. SIGNIFICANCE AND IMPACT OF THE STUDY: In humans, oxidative stress is a cause or an important component of many serious diseases, as well as being one of the age influencing factors. Environmental stresses lead to the increase in levels of reactive oxygen species (ROS). Oxidative DNA damage is a side effect of nonspecific inflammation. These human health challenging factors trigger the search for health-promoting bacteria capable of production of antioxidants and DNA-protectors. In this study, two Bacillus strains of interest were shown to produce noticeable DNA protective and antioxidant activities.

Aging Saves Populations from Extinction under Lack of Resources: in silico Experiments.

By admitting the programmed organism death (phenoptosis) concept, it inevitably raises a question as to what advantages it gives to communities containing elderly and naturally weakened individuals. We believe that the broadest prevalence of the aging phenomenon is accounted for, particularly, by the fact that in certain situations occurrence of elderly individuals may guarantee not only evolution, but also the mere existence of populations. The goal of our study was to create a mathematical model illustrating the occurrence of situations when existence of elderly individuals accounts for population survival, whereas an ageless population would be completely extinguished. A logic basis for such model is as follows: 1) natural populations exist under conditions of uneven renewal of resources; 2) if resources are renewed at a high level and rapidly compensated by their restoration, then a population strives to achieve a maximum size, which is limited not by resource availability, but specific biological mechanisms; 3) rate of resource influx may decline down to zero very rapidly (e.g. during drought); 4) a capacity, at least, for some individuals to survive during resource shortage is crucial for survival of the entire population; 5) rapid extinction of the weaker elderly individuals saves resources for survival of the younger and stronger ones. A multi-agent simulation is used as a mathematical basis for the proposed model (http://www.winmobile.biz). In silico experiments confirmed the lack of fatal contradictions in our logical construction. The presence of the aged individuals once the aging program has been turned at the age of 25-30 years results in a 24-26% increase in lifetime of the population.

Possible mechanisms of fullerene C60 antioxidant action.

Novel mechanism of antioxidant activity of buckminsterfullerene C60 based on protons absorbing and mild uncoupling of mitochondrial respiration and phosphorylation was postulated. In the present study we confirm this hypothesis using computer modeling based on Density Functional Theory. Fullerene's geroprotective activity is sufficiently higher than those of the most powerful reactive oxygen species scavengers. We propose here that C60 has an ability to acquire positive charge by absorbing inside several protons and this complex could penetrate into mitochondria. Such a process allows for mild uncoupling of respiration and phosphorylation. This, in turn, leads to the decrease in ROS production.

Effect of plastoquinone derivative 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SkQ1) on estrous cycle and 17ß-estradiol level in rats.

Administration of the plastoquinone derivative 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) to female Wistar rats with regular estrous cycle once a day for two weeks at doses of 25 nmol/kg (but not 250 nmol/kg) leads to increase in proestrus duration by reducing the phase of diestrus and metestrus. Neither dose caused significant changes in serum 17ß-estradiol level for any stage of the cycle. However, relative elongation of the proestrus stage leads to an increase in average per cycle estradiol levels by 20%.

Superoxide scavenging activity of plastoquinone derivative 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1).

The plastoquinone derivative 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) has the ability to scavenge superoxide anion radical. This ability is manifested both in vitro and in vivo in experiments with the bacterium Escherichia coli. The protective effect of SkQ1 in vivo significantly exceeds that of ascorbic acid.

Cellularity loss and Dilman's problem: an in silico study.

We assume that prolonged trends of increasing concentration of hormones could be a consequence of deterioration of functioning of glands producing inhibitors of their synthesis. Such deterioration would result from loss of cellularity of the glands. Experiments in silico carried out using the model at http://www.winmobile.biz/monstr/ show that, in principle, the diversity of hormonal effects that accompany phenoptosis of multicellular organisms can be provided with a simple "software mechanism". This mechanism is based on the gradual loss of cellularity as a result of continuous run of apoptosis in some cells of the glands due to natural fluctuations in levels of intracellular inducers of apoptosis. The main practical sense of our work lies in the illustration of the fact that substances inhibiting cellularity loss can theoretically be effective suppressors of hormonal changes characteristic for aging.

Mitochondrial-targeted plastoquinone derivatives. Effect on senescence and acute age-related pathologies.

Plastoquinone, a very effective electron carrier and antioxidant of chloroplasts, was conjugated with decyltriphenylphosphonium to obtain a cation easily penetrating through membranes. This cation, called SkQ1, is specifically targeted to mitochondria by electrophoresis in the electric field formed by the mitochondrial respiratory chain. The respiratory chain also regenerates reduced SkQ1H(2) from its oxidized form that appears as a result of the antioxidant activity of SkQ1H(2). SkQ1H(2) prevents oxidation of cardiolipin, a mitochondrial phospholipid that is especially sensitive to attack by reactive oxygen species (ROS). In cell cultures, SkQ1 and its analog plastoquinonyl decylrhodamine 19 (SkQR1) arrest H(2)O(2)-induced apoptosis. When tested in vivo, SkQs (i) prolong the lifespan of fungi, crustaceans, insects, fish, and mice, (ii) suppress appearance of a large number of traits typical for age-related senescence (cataract, retinopathies, achromotrichia, osteoporosis, lordokyphosis, decline of the immune system, myeloid shift of blood cells, activation of apoptosis, induction of ß-galactosidase, phosphorylation of H2AX histones, etc.) and (iii) lower tissue damage and save the lives of young animals after treatments resulting in kidney ischemia, rhabdomyolysis, heart attack, arrhythmia, and stroke. We suggest that the SkQs reduce mitochondrial ROS and, as a consequence, inhibit mitochondria-mediated apoptosis, an obligatory step of execution of programs responsible for both senescence and fast "biochemical suicide" of an organism after a severe metabolic crisis.

Antimutagenic activity of mitochondria-targeted plastoquinone derivative.

The ability of cationic plastoquinone derivative 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SkQ1) to modify processes of spontaneous and induced mutagenesis was studied. It is shown that daily introduction of this compound into male Wistar rats in doses of 25 and 250 nmol/kg during two weeks decreases spontaneous level of chromosome aberrations in anaphase in the eye cornea from 0.39 +/- 0.09 to 0.13 +/- 0.08 and 0.14 +/- 0.05, respectively. The level of 8-hydroxy-2'-deoxyguanosine in blood serum of the investigated animals decreases from 32.12 +/- 1.55 to 25.90 +/- 2.26 and 25.76 +/- 1.50 ng/ml, respectively. These facts indicate that the decrease in spontaneous clastogenesis is caused by decreased level of DNA damage by endogenous reactive oxygen species. A higher dose of SkQ1 also decreases to control level chromosome aberrations caused by oxygen under pressure of 0.5 MPa for 60 min. It is also shown in experiments with bacterial biosensors that SkQ1 is able to efficiently protect cells against genotoxic effect of UV radiation at 300-400 nm.

Effect of plastoquinone derivative 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) on contents of steroid hormones and NO level in rats.

Introduction of the plastoquinone derivative 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) into male Wistar rats once a day for two weeks in doses of 25 and 250 nmol/kg led to elevation of 17ß-estradiol level in blood serum by 33 and 41%, respectively. At the same time, nitrate and nitrite contents in the rat blood serum increased by 49 and 34%, respectively. ESR spectroscopy with diethyldithiocarbamateiron complex as a spin trap showed more than twofold increase in NO production in lungs, but not in blood, liver, and intestines, following the SkQ1 daily introduction at a dose of 25 nmol/kg.