QSAR analysis of the effect of metal ions on the peptidase activity of Bacillus thuringiensis var. israelensis IMV B-7465

Background. The catalytic activity of enzymes, which is their most important characteristic, can change significantly under the influence of effectors, for example, metal ions, and is the subject of special studies that are important for biochemistry, biotechnology, medicine, and other branches of science. Usually, the activity of enzymes in the presence of metals is assessed by the change in the rate of the enzymatic reaction. However, conducting similar experimental studies, especially for new enzymes, as in the case of peptidase Bacillus thuringiensis var. israelensis IMV B-7465, requires significant resources and extensive kinetic research. Therefore, it is advisable to use the methods of computer chemistry, the basic task of which is to search for the structure-property relationship, to build a model that can, with a high degree of probability, assess the effect of metal ions on the activity of peptidase. Objective: Objective: to develop of QSAR models to analyze and prediction the effect of metal ions on the activity of peptidase Bacillus thuringiensis var. israelensis IMV B-7465. Methods: the effect of metal ions was studied by determining the proteolytic activity of peptidase after joint incubation for 30 min in 0.0167 M Tris-HCl buffer solution (pH 7.5, 37 ° C). Final concentration of metal chlorides Li +; Na +; K +; Cs +; Cu2 +; Be2 +; Mg2 +; Ca2 +; Sr2 +; Ba2 +; Zn2 +; Cd2 +; Hg2 +; Cr3 +; Mn2 +; Co2 +; Ni2 + in the buffer solution was 4 mmol / dm3. To search for the quantitative “structure-property” relationship we used the reference data on the properties of metal ions and trend vector and random forest methods. Results: the effect of metal ions on the proteolytic activity of peptidase Bacillus thuringiensis var. israelensis IMV B-7465, some metal ions (Li +, Mn2 +, and Co2 +) activated peptidase, while others (Cu2 +, Be2 +, Cd2 +, Hg2 +, Cr3 +) inhibited the enzyme activity. Adequate statistical models without classification errors and prediction errors for the test set were constructed by nonlinear methods of trend-vector and random forest. Both models show that the most important characteristics of metal ions that affect enzyme activity are electronegativity (ENPol), first ionization potential (IP1), the entropy of ions in aqueous solution (S) and the electron affinity energy (Eae). Conclusions: methods of QSAR analysis in combination with nonlinear methods of trend vector and random forest allow to adequately describe the influence of metal ions on the activity of peptidase Bacillus thuringiensis var. israelensis IMV B-7465 due to descriptors that reflect a certain balance of their electron-donor and electron-acceptor properties (electronegativity, first ionization potential, electron affinity energy) and the degree of the hydrate shell structurization (entropy of solvation). Both statistical methods give similar values of the importance of descriptors, but only the trend vector method allows to analyze the direction of influence of specific characteristics of ions.

Antimicrobial Activity of Fomitopsis Officinalis (Vill.) Bondartsev & Singer in Pure Culture

Background. According to the World Health Organization antibiotic resistance is among the top ten threats to human health, food safety and development. Today antibiotic resistance has reached alarmingly high levels all over the world. Meanwhile, the increase in the synthetic drugs' production has led to the pathogenic mycobiota's rapid adaptation to the created chemicals, which have a narrow focus of application. That is why in modern biotechnology and pharmacology much attention is paid to natural producers of biologically active compounds, in particular – to xylotrophic fungi. It has been experimentally proven that the xylotrophic macromycete Fomitopsis officinalis or tinder fungus can be considered to be a promising producer of pharmacological substances with a broad spectrum of action. Studies of active metabolites, contained in the mycelial mass, culture fluid of the medicinal xylotrophic macromycete F. officinalis, and determination of their biological action remain relevant. Objective. The objective was to determine the antimicrobial activity of culture fluid and mycelial mass of F. officinalis different strains from the mushrooms collection (IBK Mushroom Culture Collection of the M.G. Kholodny Institute of Botany, NAS of Ukraine) against gram-negative and gram-positive bacteria species. Methods. An in vitro study of the antimicrobial activity of ethyl acetate extracts of culture fluid and aqueous-ethyl extracts of mycelial mass for F. officinalis strains IBK-5004, IBK-2497, IBK-2498 against gram-positive Staphylococcus aureus (B-918), Bacillus subtilis (В-901) and gram-negative Escherichia coli (B-906), Bacillus subtilis (B-900), Klebsiella pneumoniae (M-123) bacteria by disc-diffusion method was conducted. Results. High antimicrobial activity of tinder fungus culture fluid and mycelial mass extracts against Staphylococcus aureus was established after the 21st day of cultivation, while on the 28th day the zone of growth retardation was maximal (15–25 mm). The highest indices were recorded in F. officinalis IBK-5004 (20–25 mm) and IBK-2498 (20–24 mm) strains. Antimicrobial activity against Klebsiella pneumoniae in culture fluid extracts was manifested on the 21st and 28th days of cultivation. The highest antimicrobial activity against Klebsiella pneumoniae was observed in the culture fluid of the strain F. officinalis IBK-5004, the diameter of the growth retardation zone was 18 mm on the 28th day of cultivation. Mycelial mass's extracts showed moderate activity on the 14th day of cultivation (7-8 mm); maximal activity was recorded on the 28th day (12–22 mm). The most active strain was Fomitopsis officinalis IBK-2498. No antimicrobial activity against test organisms was detected in the following studied strains: Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis. Conclusions. It has been established that the mycelial mass and culture fluid extracts of F. officinalis IBK-5004, IBK-2497, IBK-2498 strains have high antimicrobial activity against Staphylococcus aureus and moderate antimicrobial activity against Klebsiella pneumoniae on the 21st and 28th day of cultivation.

Biological Evaluation of Medical Devices in the Form of Suppositories for Rectal and Vaginal Use

Background. Programs of preclinical safety studies of the health care products depend on the regulatory status of the investigated products. The classification of such products, in particular suppositories for rectal and vaginal use, is a critical step of developing tactics for their biological evaluation. Adaptation of biological evaluation methods for the medical devices based on the combination of biologically active substances, as well as evaluation of the results of such studies is urgent task of biomedicine. Objective. To substantiate the regulatory status and to carry out a biological evaluation of medical devices in the form of vaginal suppositories based on octenidine dihydrochloride ("Prodexyn") and in the form of rectal suppositories based on Saw palmetto, Levisticum officinale and Calendula officinalis extracts ("Pravenor"). Methods. Biological evaluation was conducted according to the requirements of ISO 10993 standards using in vitro and in vivo biological test systems (cytotoxicity in cell culture and the MTT test, sensitizing and irritating effect in guinea pigs). Results. The cytotoxicity (СС50) of the medical device "Prodexyn" extract in Vero cell culture was 8.35 μg/ml calculated as octenidine dihydrochloride and 416.65 μg/ml calculated as dexpanthenol. "Pravenor" medical device was found to be non-toxic in Vero cell culture. According to the results of MMT assay CC50 for octenidine dihydrochloride was 1.67 μg/ml, and 83.33 μg/ml – for dexpanthenol. CC50 indicators calculated for the different active ingredients of the medical device "Pravenor" were the following: 50 mg/ml for the dwarf palm berries extract (Saw palmetto), 16.67 mg/ml for the lovage roots extract (Levisticum officinale), and 16.67 mg/ml for the calendula flowers extract (Calendula officinalis). No sensitizing or skin irritating effects were observed in guinea pigs. Conclusions. Biological evaluation of medical devices in the form of rectal suppositories "Pravenor" and vaginal suppositories "Prodexyn" performed using in vitro and in vivo biological systems. It was demonstrated an acceptable level of safety of the products. The MTT test was 5 times more sensitive than the Vero cell culture method in determination of cytotoxicity.

Human Gut Microbiome as an Indicator of Human Health

The undeniable achievement in the study of the gut microbiome as an association of different microorganisms, including viruses, that colonize various organs and systems of the body, is the establishment of the fact that some diseases that were consmicrobiotaidered as non-infectious can also be transmitted through microorganisms. This resulted in the gut microbiome being called a forgotten organ that could serve as an additional and kind of missing link for a more objective and better diagnosis and treatment of many diseases that were not considered infectious. The rapid development of gut microbiome research in recent years not only is connected with better understanding of the functioning of the microbiome by the scientific community, but also inseparable from the strategic support of each country. Global investment in researches, related to the human microbiome, has exceeded $1.7 billion over the past decade. These researches contribute to the development of new diagnostic methods and therapeutic interventions. Our review is dedicated to the analysis of the possibilities of application of the human gut microbiome for the diagnosis of diseases, and the role of the intestines in the provocation and causing of certain diseases. Significant differences in the composition and diversity of the human microbiome are shown depending on geographical location and the change of socio-economic formations towards a gradual decrease in the diversity of the gut microbiome due to three stages of human population’s existence: food production, agriculture and industrial urban life. We analyze the influence of dietary patterns, various diseases (including malignant neoplasms) and viral infections (in particular, coronavirus) on the gut microbiome. And vice versa – the influence of the gut microbiome on the drugs effect and their metabolism, which affects the host's immune response and course of the disease.

Aspects of Lyophilization of Cardiac Bioimplant

The use of implants of biological origin in clinical practice has led to the search for methods of long-term storage of tissues without damaging their functional and structural characteristics. Xenografts (extracted from pericardium of pigs, horses, bulls) are drawing more and more interest. The bovine pericardium is exposed to chemical and physical factors providing complete purification of tissue from cells and their components. Such scaffolds are protein (collagen) complexes that fully replicate the microstructure of the pericardial tissue. Lyophilisation ensures long-term preservation of the extracellular matrix properties. The principle of the method is in drying pre-frozen tissue, in which water is sublimated. The method is intended for storage, transportation, and the subsequent use of the bioimplant in clinical practice. However, the lyophilization process may be accompanied by various undesirable factors that can lead to denaturation of the matrix protein or loss of its functionality and structure. To preserve the natural microstructure, stabilizers or various modifications (slow/fast freezing, reducing the degree of supercooling, etc.) of the lyophilization process are applied to biological prostheses. In this review, the main processes of lyophilization of biological tissue are described, which can affect the operation of a cardiac implant. A deep understanding of the parameters of the lyophilization process is crucial for creation of stable tissue grafts and their subsequent long-term storage.

Efficiency of Combined Use of Fullerene C60 and Bovine Serum Albumin for Rehabilitation of Vitrified Fragments of Rat Immature Seminiferous Tubules

Background. Today, cryopreserved reproductive tissues are used to treat some forms of male infertility. However, after long-term preservation of fragments of seminiferous tubules of testes (FSTT) in a low-temperature bank (-196 °С) their morphological and functional characteristics decrease reversibly. To solve this problem after freezing-thawing, the use of rehabilitation media with special additives is promising. Due to the fact that serum albumin and fullerene C60 have powerful protective and antioxidant properties, their use allows to stabilize the plasma membrane, osmotic pressure, and reduce free radicals that make them promising candidates to use in the development of rehabilitation media for biological objects after cryopreservation. Objective. The efficacy of fullerene C60, bovine serum albumin (BSA), and their combination as components of rehabilitation medium of vitrified FSTT of immature rats was studied. Methods. Vitrified-thawed samples of FSTT were incubated (22 °C) for 30 minutes in Leibovitz's medium with addition of 15 mg/mL C60, 5 g/L BSA or their combination. Control samples were incubated in the medium without C60 or BSA addition. Metabolic activity (MTT test), histomorphological data, total antioxidant status (TAS), reactive oxygen species (ROS) production, activity of g-glutamyltransferase (gGGT), and glucose-6-phosphate dehydrogenase were determined in the samples after rehabilitation in the investigated media. Results. The use of C60 led to the increase of metabolic (by 1.26 times) and TAS (by 1.74 times) activities, to the decrease in the number of ROS+ cells (by 1.35 times) and to the improvement of the spermatogenic epithelium binding to the basement membrane versus control sample. Application of BSA did not significantly affect the studied biochemical indices but decreased the number of tubules with desquamation of spermatogenic epithelium in histological sections. The combined use of BSA and C60 had the best effect among investigated rehabilitation media that led to the increase of metabolic activity (by 1.51 times), TAS activity (by 1.78 times), gGGT activity (by 1.59 times), histostructure restoration and the decrease in the number of ROS+ cells (by 1.45 times) compared to the control samples. Conclusions. The use of C60 and BSA combination increases the metabolic and antioxidant activity of vitrified FSTT and also has a positive effect on their histostructural characteristics compared to control samples. It should be noted that the effect of С60 and BSA addition to rehabilitation medium exceeds the results of using the investigated additives separately (by the metabolic and gGGT activity as well as architectonics of vitrified FSTT). These data relate to reproductive medicine and can be used to develop an effective rehabilitation protocol for vitrified FSTT.

The Effect of Magnetite Nanoparticles on the Growth and Development of Nicotiana Tabacum Plants in Vivo and in Vitro Culture

Background. Nanomaterials are easily modified and have unique characteristics associated with a large reactive surface Due to these properties, nanomaterials are used in various branches of sciences and technology, such as pharmaceuticals, biotechnology, chemical technology, etc. Recently, the effect of magnetite nanoparticles on the morphological properties of plants has been actively studied for their further use as nanoadditives to increase yields and improve the properties of agricultural plants. Tobacco (Nicotiana tabacum) is a model object of plant biotechnology, it is used to study the effect of various factors on dicotyledonous plants, so it was chosen to study the effect of magnetite on the growth, development, and mass accumulation by plants. Objective. We are aimed to study the effect of magnetite nanoparticles on the growth and development of Nicotiana tabacum in vivo and in vitro. Methods. The ability of tobacco to produce biogenic magnetic nanoparticles by searching for mammal proteins homologues in theNicotiana tabacum proteome using the Blast NCBI program was studied using comparative genomics methods. The plants were divided into groups (control, magnetite nanoparticle concentration 0.1 mg/cm3, magnetite nanoparticle concentration 1 mg/cm3) for both in vivo and in vitro experiments. Analysis of plant parameters was performed every 14 days to study the dynamics of the effects of magnetite nanoparticles. Results. It was determined that magnetite nanoparticles at a concentration of 0.1 mg/cm3 in culture in vitro and in vivo significantly affect the growth of the root system and sprouts of Nicotiana tabacum. On the 56th day of plant cultivation in vitro on a salivary medium supplemented with magnetite nanoparticles at a concentration of 0.1 mg/cm3, an increase in the shoot length by 13.3%, root length by 31.7%, and the mass of absolutely dry substances by 18.75% was observed compared to the control. Treatment of magnetite nanoparticles with a suspension at a concentration of 0.1 mg/cm3 led to more pronounced results when growing tobacco in vivo. So, on the56th day, the root length increased by 23.3%, the length of the shoot – by 19.2%, and the mass of absolutely dry substances – by2 times, the first leaves appeared 2 days earlier compared to the control. The addition of magnetite nanoparticles to the substrate on which the plants were grown in vivo at a concentration of 1 mg/cm3 inhibits the growth of tobacco. Conclusions. Studies have shown the expediency of using magnetic nanoparticles at a concentration of 0.1 mg/cm3 as nanofertilizers in tobacco cultivation.

The Role of Cord Blood in the Regulation of the Cellular and Humoral Link of Immunity in Experimental Atopic Dermatitis

Background. Atopic dermatitis (AD) as one of the most common diseases of autoimmune genesis in the structure of dermatological practice, is characterized by itching, dryness, thickening of the skin, characteristic rashes. The drugs of choice in the treatment of AD are steroidal anti-inflammatory drugs. However, the development of unwanted side effects is a serious problem attributed to using hormone therapy. The search for effective methods of treating AD is an urgent task of medicine and in particular dermatology. At the same time, there is an obvious need for the participation in the solution of this problem also of specialists-immunologists working in the field of application of cell therapy drugs, acting on various pathogenetic links of the disease. The development of new or optimization of existing methods of treating AD is the urgent task facing them. Objective. Evaluation of the immunocorrective effect of lyophilized (lHCBL) and cryopreserved human cord blood leucoconcetrate (cHCBL) on a AD model. Methods. The experiments were carried out on 6-month-old Wistar rats. Upon induction of AD, the inflammation focus was formed on the rat's back (9–10 cm2) by daily rubbing in a 5% alcohol-acetone solution of dinitrochlorobenzene (DNCB) for 21 days. cHCBL and lHCBL were injected intraperitoneally, 0.5 ml at a dose of 5´106 cells in one day after the final DNCB treatment. The adhesive and phagocytic activity of the cells of the peritoneal cavity, the level of circulating immune complexes, the population and subpopulation of lymphocytes (CD3+, CD4+, CD8+, CD16+, CD4+CD25+), the immunoregulatory index of lymphocytes, the concentration of immunoglobulins in the blood serum were determined. Results. For AD induced by DNCB, systemic changes in the immune status are characteristic, which is expressed by changes in the parameters of cellular and humoral immunity. The most fundamental changes in cell subpopulations in spleen of rats with AD were revealed: a decrease in the number of total T-lymphocytes and their two main subpopulations (CD4+ and CD8+ cells). Against this background, changes were noted in the monocytic-phagocytic and humoral systems of immunity. The paper shows the effectiveness of the use of cHCBL and lHCBL in the correction of pathological manifestations of experimental AD. On the background of treatment, the features of the immunocorrective effect of each of the drugs were noted. Thus, when assessing intergroup values, a more pronounced increase in T-reg was revealed in rats of the 5th group – 3.9 [3.8; 4.0] versus 3.2 [3.0; 3.3] in the 4th group (P < 0.01); IgA level – 1.6 [1.5; 1.7] versus 1.3 [1.2; 1.4] (P < 0.01). Conclusions. Thus, lHCBL exhibits immunocorrective activity in the treatment of experimental AD, surpassing in some parameters the activity of сHCBL, which is promising for its use in clinical practice.

An Experimental Study of the Cryopreserved Placenta Extract Effect On the Sodium Diclofenac Anti-Inflammatory Activity

Background. As a means of correcting the ulcerogenic effect of nonsteroidal anti-inflammatory drugs, our attention was attracted by a cryopreserved extract of the human placenta, which has a multivector spectrum of biological activity. To date, there is no information about its effect on the specific activity of this class of medicines (anti-inflammatory, analgesic, etc.) in published sources. Objective. We are aimed to characterize the effect of cryopreserved placenta extract on the anti-inflammatory activity of diclofenac sodium when administered separately in a model of acute exudative inflammation. Methods. Experimental studies in vivo were conducted on 28 nonlinear male laboratory rats. The model of acute exudative inflammation was reproduced by subplantar injection of 0.1 ml of 1.0% aqueous solution of λ-karagenin into the right hind limb of rats. Cryopreserved placenta extract was administered intramuscularly at a dose of 0.16 ml/kg 60 minutes before diclofenac sodium (8 mg/kg). Results. Preventive administration of diclofenac sodium caused an antiexudative effect as early as 30 minutes after administration of λ-karagenin – its anti-inflammatory activity was 11.0%, which is 4.6 times higher than similar indicators at the same time in rats injected with placental cryoextract. At 60 minutes of observation, diclofenac sodium was comparable in anti-inflammatory activity with cryopreserved placenta extract: 28.6% and 22.2%, respectively, but at 120 and 180 minutes, diclofenac sodium exceeded the studied cryoextract in antiphlogistic effect by 1.6 times in both periods of observation. The anti-inflammatory effect of the combined separate administration of placenta cryoextract and diclofenac sodium before λ-karagenin for 30 and 60 minutes was 12.7% and 32.3%, respectively, which is comparable with analogous indicators against the background of diclofenac sodium monotherapy. However, at 120 minutes of observation, the group of combined use of placenta cryoextract and diclofenac sodium showed the greatest anti-inflammatory effect among rats of all the studied groups – 52.6%, which was 2.2 times higher than the indicators of the placenta cryoextract monotherapy group and 1.4 times lower than the indicators of the rats of the diclofenac sodium monotherapy group. Conclusions. 4 hours after administration, placental cryoextract had a suppressive effect on kinins like diclofenac sodium, and in the prostaglandin period of caragenin-induced inflammation against the background of combined use of the studied cryoextract and diclofenac sodium, the anti-inflammatory activity was 46.4 %. This suggests a suppressive effect on the production of prostaglandins as a possible mechanism of anti-exudative action of cryopreserved placenta extract.

Comparative Analysis of Classification Algorithms in the Analysis of Medical Images From Speckle Tracking Echocardiography Video Data

Background. Machine learning allows applying various intelligent algorithms to produce diagnostic and/or prognostic models. Such models can be used to determine the functional state of the heart, which is diagnosed by speckle-tracking echocardiography. To determine the patient's heart condition in detail, a classification approach is used in machine learning. Each of the classification algorithms has a different performance when applied to certain situations. Therefore, the actual task is to determine the most efficient algorithm for solving a specific task of classifying the patient's heart condition when applying the same speckle-tracking echocardiography data set. Objective. We are aimed to evaluate the effectiveness of the application of prognostic models of logistic regression, the group method of data handling (GMDH), random forest, and adaptive boosting (AdaBoost) in the construction of algorithms to support medical decision-making on the diagnosis of coronary heart disease. Methods. Video data from speckle-tracking echocardiography of 40 patients with coronary heart disease and 16 patients without cardiac pathology were used for the study. Echocardiography was recorded in B-mode in three positions: long axis, 4-chamber, and 2-chamber. Echocardiography frames that reflect the systole and diastole of the heart (308 samples in total) were taken as objects for classification. To obtain informative features of the selected objects, the genetic GMDH approach was applied to identify the best structure of harmonic textural features. We compared the efficiency of the following classification algorithms: logistic regression method, GMDH classifier, random forest method, and AdaBoost method. Results. Four classification models were constructed for each of the three B-mode echocardiography positions. For this purpose, the data samples were divided into 3: training sample (60%), validation sample (20%), and test sample (20%). Objective evaluation of the models on the test sample showed that the best classification method was random forest (90.3% accuracy on the 4-chamber echocardiography position, 74.2% on the 2-chamber, and 77.4% on the long axis). This was also confirmed by ROC analysis, wherein in all cases, the random forest was the most effective in classifying cardiac conditions. Conclusions. The best classification algorithm for cardiac diagnostics by speckle-tracking echocardiography was determined. It turned out to be a random forest, which can be explained by the ensemble approach of begging, which is inherent in this classification method. It will be the mainstay of further research, which is planned to be performed to develop a full-fledged decision support system for cardiac diagnostics.