Issue 3 | September 2022
CRISPR/Cas9-Redacted Bacillus Subtilis Strain as the Producer of Extracellular Metalloproteinase of B. Pumilus
B. pumilus metalloproteinase was firstly isolated and characterized by Kazan Federal University scientists. Primary structure analysis showed that the novel enzyme has no analogs among prokaryotic enzymes and occupies an intermediate position between two large families of the metzinkin clan metalloproteinases – adamalysins and astacins. These families are mainly represented by eukaryotic enzymes, which play an important role in human life and health. A more detailed study of the structure and functions of novel metalloproteinase requires an efficient expression system. B. pumilus metalloproteinase gene (mprBp) was cloned into the pGP382 expression vector under a strong constitutive promoter of the degQ36 gene (PdegQ36). The resulting construct was used to transform B. subtilis Δ6 strain. This strain was constructed by CRISPR/Cas9 genome editing technology with deletion of some prophage genes of B. subtilis 168. The functional role of prophage genes is poorly understood. It is possible that prophage deletion will increase the expression of secreted enzymes. For the transformed strain we determined the dynamics of growth and accumulation of proteolytic activity by hydrolysis of azocasein. The dynamics of proteolytic activity accumulation by this strain has a different character in contrast to the protease-deficient strains carrying the gene of the investigated enzyme. The result of this work was to obtain an effective producer strain of adamalizin-like metalloproteinase of B. pumilus, which can be used in the production of the enzyme for subsequent studies.
|12 CRISPR:Cas9-REDACTED BACILLUS SUBTILIS STRAIN.pdf||636.91 KB|
Cell-Free Supernatant of Staphylococcus Aureus Culture Increases Antimicrobials Susceptibility of Pseudomonas Aeruginosa
Along with the wide spread of bacterial antibiotic resistance over the world, the treatment efficiency of infectious disease is greatly affected by the mixed biofilm formation by pathogenic bacteria. Staphylococcus aureus and Pseudomonas aeruginosa, a frequent cause of nosocomial infections, exhibit both synergistic and antagonistic interactions in co-culture, leading to various changes in the metabolic profile of bacteria, which in turn affect their sensitivity to antimicrobials. Here we show that S. aureus cell-free culture liquid exhibits bacteriostatic properties and increases the efficacy of antimicrobials against P. aeruginosa. Thus, the MICs of amikacin, gentamicin, and ciprofloxacin decreased 2-4 fold in the presence of cell-free supernatant of S. aureus 24 h culture. Furthermore, the combination of the latter with antimicrobials increased the efficacy of amikacin up to 64-fold. Thus, the combined use of cell-free culture liquid of S. aureus with broad-spectrum antibiotics can be used to increase the effectiveness of antimicrobial therapy of P. aeruginosa.
|11 CELL-FREE SUPERNATANT OF STAPHYLOCOCCUS AUREUS CULTURE.pdf||911.25 KB|
Modulating the Effect of Melatonin on Behavioral Responses of Rats in an Experimental Model of Inflammation
The problem of the influence of inflammation on the processes of higher nervous activity is still relevant. In particular, it implies an analysis of the safety and effectiveness of biological regulators’ effects with neurotropic and immunomodulatory potential. The study examined the effects of Salmonella tiphy LPS on behaviour and cognitive function in adult male Wistar rats with different levels of melatoninergic system activity, using various tests. It was found that daily injection of LPS to rats of the group А (50 μg/kg, i.p.) for 10 days leads to the formation of a special pattern of behavior with a low level of tentative research activity in the presence of increased anxiety. The rats of the group В received combined exposure to LPS (50 μg/kg, i.p.) and melatonin (5 mg/kg, orally) distinguished a higher level of various variants of motor and research activity in conditions of relatively low anxiety in contrast to the rats of the group А. The paper discusses the features and mechanisms of the formation of "painful" behavior in an experimental model of inflammation, depending on the level of activation of the components of the melatoninergic system.
|10 MODULATING THE EFFECT OF MELATONIN.pdf||637.05 KB|
Microbial production of testosterone from phytosterol is a promising alternative to the chemical synthesis from androstenedione, which is currently used. It is possible using wild-type or recombinant strains of Mycolicibacterium neoaurum. One of the key steps of biotechnology development is inoculum preparation which determines the volumetric/mass ratios of the bioreactors and affects the biotransformation rate and duration. In the present work, the conditions for the growth of testosterone-producing strain of Mycolicibacterium neoaurum recombinant strain with enhanced 17β-HSD activity were optimized. The effects of medium composition, temperature, and glucose supplements on biomass size and density were estimated. The results are of importance for the development of microbial technology for testosterone production.
|9 OPTIMIZATION OF GROWTH CONDITIONS.pdf||688.63 KB|
Noroviruses with the GII.P16 polymerase gene have long been considered rare. However, since 2015, there have been reports from different geographical regions about the spread of new recombinant strains of norovirus in which GII.P16 polymerase is associated with a capsid protein of various genotypes. In the autumn of 2016, a sharp increase in the frequency of detection of noroviruses was observed in Nizhny Novgorod, which coincided with the appearance of new recombinants GII.4_Sydney and GII.2 – with GII.P16 polymerase. Based on the sequences of the RNA-dependent RNA polymerase gene present in the GenBank database, a phylogenetic analysis of noroviruses with GII.P16 specificity was performed by constructing Bayesian phylogenetic trees. Analysis of amino acid sequences showed that representatives of the novel variant of the GII.P16 polymerase gene had five substitutions compared to earlier strains. These substitutions are located near sites responsible for the enzyme activity of polymerase and could affect the transmissivity of the virus. Acquisition of a novel variant of the GII.P16 polymerase gene by noroviruses with different capsid protein genotypes probably provides certain advantages for recombinants and creates prerequisites for their wide distribution.
|8 PHYLOGENETIC ANALYSIS OF NOROVIRUSES.pdf||964.92 KB|
In this paper, we present the data on the properties of the probiotic strain Bacillus intestinalis GM2 related to adhesion, auto-, and coaggregation. GM2 exhibits a strong autoaggregation phenotype. The autoaggregation ability of GM2 did not change after trypsin treatment but was reduced under the influence of extracellular culture metabolites. Coaggregation of probiotic strains with pathogens is one of the mechanisms of displacement of pathogens from the intestinal microbiota. It was shown that the GM2 strain exhibits the ability to coaggregate with tested strains of Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, and Bacillus cereus. The studied properties of bacilli can be useful for the creation of new probiotics for poultry farming.
|7 CHARACTERIZATION OF THE ADHESIVE PROPERTIES.pdf||705.32 KB|
Morganella morganii is an important clinical pathogen with fast-paced multidrug resistance and virulence. Probiotics with potent antimicrobial activity are considered as a promising alternative to antibiotics in infection treatment. We isolated 12 lactobacilli strains of human and plant origin and characterized their beneficial properties focusing on their antagonistic activity against M. morganii. Tolerance to the hostile gastrointestinal environment, surface properties (hydrophobicity and autoaggregation), and acidification rate values varied considerably between strains and were strain-specific. Most Lactobacillus strains showed antibiotic resistance profiles typical for lactobacilli. Lactobacilli demonstrated inhibitory activity towards the growth of M. morganii in the agar-overlay assay, produced bacteriocins and coaggregated with M. morganii cells, but did not affect the growth of the pathogen during co-culturing in the mixed-species biofilms. Lactiplantibacillus plantarum strain FCa3L was selected as the candidate strain with potential probiotic properties for further investigation.
|6 ANTAGONISTIC ACTIVITY OF NEWLY ISOLATED LACTOBACILLUS.pdf||699.64 KB|
Nowadays, manned cosmonautics is faced with the task of carrying out a long-term space flight beyond the limits of low Earth orbit. Under the conditions of an orbital space flight, a person is exposed to a number of adverse effects on the body, among which microgravity is especially distinguished. Prolonged exposure to microgravity can lead to severe immune impairment. At present, immunological studies of cosmonauts can be conducted only after they return to Earth at the end of a space flight, as a result of which the use of ground-based models that imitate specific space factors seems to be an advanced direction. A well-studied microgravity model is bed rest, during which volunteers are kept in strict rest in bed. Granulocytes, as representatives of innate immunity, are the first among immune cells to respond to an altered state of the body; therefore, researches of the influence of bed rest on the granulocyte phenotypic characteristics can provide important information for the development of prophylaxis measures to the immune disorders’ development when exposed to microgravity. The work used data obtained from six subjects. The impact of bed rest was determined at the end of the model, after 21 days. The following granulocyte clusters of differentiation (CD) were studied by flow cytometry: CD25, CD64, CD23, CD14, CD16, CD36, CD11b, CD18, CD286. Tendencies to a decrease in the percentage of CD64+ subpopulation and an increase in the percentage of CD23+, as well as CD25+ subpopulation of granulocytes after 21 days of bed rest were revealed.
|5 IMPACT OF 21-DAY BED REST ON THE PHENOTYPIC FEATURES.pdf||306.12 KB|
The Transcription Factor Satb2 Regulates the Development of the Neuronal Network of the Cerebral Cortex through the Protein Kinases Signaling Cascade
Using fluorescence microscopy, morphological analysis of cell growth, and PCR analysis, we have shown that deletion of the Satb2 transcription factor in mouse cortical neurons results in impaired neuronal network development in vitro. It was found that primary cell cultures of the cerebral cortex obtained from Satb2-null mice are not able to form a developed network of neurites during 5 days of cultivation, while cells from control mice are characterized at this time by a fully developed network of neuronal processes. Analysis of protein kinases expression involved in the processes of neuron differentiation and neurites growth showed that deletion of Satb2 leads to suppression of the expression of genes encoding protein kinase C (PKC), protein kinase B (Akt/PKB), mitogen-activated protein kinase 8 (MAPK8) and Ca2+/ calmodulin-dependent protein kinase II (CaMKII), while not affecting the expression of phosphatidylinositol 3-kinase (PI3K). Activation of neurites outgrowth and differentiation of Satb2-null neurons was achieved by the application of exogenous activators of Akt/PKB, CaMKII and PI3K, but not PKC, the expression and activity of which is probably completely suppressed by the deletion of Satb2.
|4 THE TRANSCRIPTION FACTOR SATB2 REGULATES THE DEVELOPMENT.pdf||1.34 MB|
The Effect of Ionizing Radiation in Combination with Chemotherapy Drug Doxorubicin on A431 Human Epidermoid Carcinoma Cell Treatment
Radiotherapy is one of the most effective and most commonly used methods of cancer treatment. However, as a result of irradiation, there are side effects that occur as a result of ionizing radiation on healthy tissues. The use of a combined approach with the use of low doses of radiation and antitumor drugs that have a radiosensitizing effect may be one of the ways to reduce side effects and overcome the resistance of malignant cells. This study was undertaken to evaluate the combined effects of radiotherapy and the chemotherapeutic agent Doxorubicin on A431 human epidermoid carcinoma cell line. The cells were incubated with the antitumor antibiotic Doxorubicin and then exposed to high-energy electron ionizing radiation. The cell viability was examined using the MTT assay. The results showed that Doxorubicin acts as a radiosensitizer. Moreover, the combined effect of Doxorubicin and high-energy ionizing radiation of electrons is additive. According to the obtained results, combination therapy used in the treatment of oncological diseases can significantly reduce the radiation dose and minimize the side effects that occur during high doses of irradiation.
|3 THE EFFECT OF IONIZING RADIATION.pdf||395.29 KB|
Therapy of Pressure Sores via Activation of Regenerative Processed in Tissues by Low-Temperature Glow-Type Plasma Discharges of Glow Type
Pressure sores remain an important clinical problem with significant socioeconomic implications. The pressure sores treatment via reparative processes activation in tissues by low-temperature plasma discharges was studied in the present work. Low-temperature plasma discharges were initiated by high-frequency 0.11, 2.64, 6.78, and 13.56 MHz current. It is shown that the optimal current frequency for the generation of the cold plasma is 6.78 MHz. This current frequency was used in clinical studies of pressure sores treatment with cold plasma discharges of the glow type. The efficiency of treatment was evaluated by analysis of histological samples, histochemical and bacteriological methods. Low-temperature plasma discharge treatment improved the dynamic of pressure sore healing, activated reparative processes in injured tissues, and decreased bacteria numbers in a wound. The most pronounced effect was observed after 14-21 days. The low-temperature plasma discharges accelerated pressure sores healing from 14 to 16% compared with non-treated by cold plasma wound. The effect depended on the pressure sores etiology. Low-temperature plasma discharges of glow type may be considered as an effective approach to pressure sores therapy.
|2 THERAPY OF PRESSURE SORES VIA ACTIVATION.pdf||950.22 KB|
Photodynamic therapy (PDT) has been successfully used to treat many types of tumors. However, the widespread use of PDT is limited by a number of factors, including low selectivity of photosensitizer (PS) accumulation in tumor tissue. We have synthesized the novel third-generation photosensitizer, conjugate of zinc complex of chlorine e6 with maltose and biotin (Chl-Mal-B7). The introduction of maltose and biotin is intended to provide high selectivity to tumor cells often characterized by high-level expression of receptors for these molecules. It was shown that Chl-Mal-B7 intensively absorbs light and fluoresces in a far-red spectral region with a quantum yield of about 10%. Chl-Mal-B7 demonstrated photoinduced toxicity in submicromolar concentrations against cancer cells that is several times more effective compared to nonmalignant cells.
|1 NOVEL CHLORINE E6 CONJUGATE WITH DUAL TARGETING TO CANCER CELLS.pdf||747.88 KB|