Biotechnologia Acta

28-49

Authors: O. M. Klyuchko, Z. F. Klyuchko

The purpose of the work was the examination of various databases construction as well as examination of possible errors. Peculiarities of biological objects which should be taken into account during databases design were analyzed. The methods for electronic collections with databases of biological organisms elaboration were studied. The appropriate algorithms for environmental conservation were analyzed and compared with some foreign analogs in order to study positive and negative experiences. The requirements for database with information about Noctuidae (Lepidoptera) and some Areneidae were formulated for the development of electronic information system “EcoIS”. The description of developed relational database with information about insects with analysis of selected object area were suggested taking into concideration the characteristics of biological objects and characteristics of information systems analogues. Conclusions concerning described new means with bioobjects databases and their application on the example of “EcoIS” system were done as well as some recommendations for the construction of databases with the information about living organisms basing on our experience.

57-67

Authors: Kucherenko D. Yu., Kucherenko I. S., Soldatkin O. O., Soldatkin A. P.

The aim of the work were the optimization of an amperometric glutamate-sensitive biosensor and its utilization for the determination of the glutamate concentrations in food samples. Amperometric method of measurements was used. The biosensor was based on immobilized glutamate oxidase and platinum disc electrode. The biosensor was connected to the working cell with auxiliary (platinum wire) and reference (Ag/AgCl) electrodes. The biosensor exhibited high sensitivity to glutamate, duration of one analysis was about 5 min. An influence of the ionic strength, pH, and buffer capacity on the biosensor operation was investigated. The sensitivity of biosensor to various possible interfering substances, including amino acids, was studied; high selectivity to glutamate was shown. The reproducibility of analysis of food samples and an impact of sample dilution was evaluated. Glutamate concentrations in different sauces and seasonings were measured by the developed biosensor; the results correlated well with those obtained by the spectrophotometric method (R2 = 0,988). Thus, the amperometric biosensor for glutamate determination was successfully optimized and used for measurement of glutamate concentrations in sauces and seasonings.

73-83

Authors: I. O. Nitovska, M. Yu. Vasylenko, B. V. Morgun

The aim of the work was to study the effect of introns of the rice OsAct1 and the maize hsp70 genes on the transgene expression in Nicotiana plants in order to find out of their use in the testing of vectors containing these monocot introns. Next methods were used: Agrobacterium-mediated transformation of leaves of greenhouse N. benthamiana and N. tabacum plants by vector pCB271 containing the introns of cereals, light fluorescence microscopy and fluorimetry of GFP. The presence of transgenes was detected by polymerase chain reaction. The transient GFP expression was observed in infiltrated tissue of N. benthamiana. Transgenic plants of N. tabacum resistant to kanamycin were obtained. Fluorescence of GFP in extracts of some transgenic tobacco lines was shown. The impairment of the transgene expression in some N. tabacum transformants has been observed. So, transgenes, containing introns from the hsp70 corn or from the OsAct1 rice genes downstream the promotor, are expressed in Nicotiana plants. Thus, N. benthamiana and N. Tabacum plants can be used to test vectors constructs for cereals transformation. It has been shown that the monocot introns can have the negative impact on the transgene expression in Nicotiana plants.

27-46

Authors: Manoilov K.Y.

The aim of the review was to analyze the literature data related to the application of a variety of diphtheria toxin derivatives. Although studies of the diphtheria toxin interaction with sensitive and resistant mammalian cells have been held for a relatively long time, there are still some unresolved issues concerning the molecular mechanisms of diphtheria toxin functioning. Native diphtheria toxin and parts of its molecule which preserves toxicity are used as instruments in the newest biotechnological methods of specific cell subtype ablation in multicellular organisms. New recombinant derivatives of diphtheria toxin are periodically obtained in the laboratories throughout the world. Most of these analogs of diphtheria toxin are used in biological studies as convenient tools for analysis of the functions of the natural toxin. A non-toxic analog of diphtheria toxin, protein CRM197, is used in clinical practice as a component of vaccines and as an anticancer agent. Diphtheria toxin -based targeted toxin therapy is another perspective trend for cancer therapy. Therefore, studying of diphtheria toxin derivatives is of a great relevance for biotechnology and medicine.

56-68

Authors: Vlasenko E. N., Kuznetsova O. V.

The purpose of the study was to analyze possible ways and intensity of synthesis of volatile flavor compounds by Pleurotus ostreatus (Jacq.:Fr.) Kumm. mushrooms in the process of intensive cultivation on sunflower husk and barley straw with the addition of vegetable oils (sunflower and corn) as a potential source of unsaturated fatty acids. Methods of sensory profile analysis and ultraviolet spectroscopy were used. Sensory profile analysis of dried samples of fruit bodies showed an increase in the intensity of mushroom, meat and grassy notes of flavor on substrates with the addition of vegetable oils in a concentration of 1% and 5% of the weight of the substrate. For the strain IBK-551 marked increase in the intensity of sweet and floral attributes of the aroma on both substrates with the addition of corn oil. UV spectroscopy of hexane extracts of dried samples of fruit bodies revealed maxima of light absorption in the range of 200−210 nm and 260−300 nm. There was a difference in intensity of light absorption of samples of different strains cultivated on substrates with the addition of vegetable oils.

78-82

Authors: O. V. Kravchenko, O. S. Panchenko

The aim of the research was the isolation from drinking water the pure cultures of iron- and manganese-oxidizing microorganisms with further assessment of their efficacy to remove these contaminants on rapid filters. To assess the effectiveness selected strains were grown on the solid nutrient medium; the suspension was prepared and was treated to zeolite loading. Ten pure cultures of iron- and manganese-oxidizing bacteria were isolated and identified as 6 genuses: Siderocapsa, Leptothrix, Sphaerotillus, Galionella, Metallogenium, Hyphomicrobium. Comparison the efficiency of genuses Leptothrix, Sphaerotillus, Metallogenium has shown that under conditions of these experiments Leptothrix more effectively removed iron and manganese at low concentrations in model solution.

23-29

Authors: T. O. Chudina, V. A. Skoryk, E. I. Legach

The aim of the study was to synthesize poly(D,L-lactide-co-glycolide) particles — PLGA and obtain their complexes with a non-toxic diphtheria toxin recombinant fragment subunit B fused with enhanced green fluorescent protein EGFP-SubB; to characterize the main physical and chemical properties of the resulting complexes. Two types of the diptheria toxoid loaded PLGA microparticles were obtained: particles with immobilized diphtheria toxoid (PLGA 1) and particles with encapsulated (PLGA) diphtheria toxoid (PLGA 2). Micropartcles obtaines were characterized for antigen loading, particle size, polydispersity index and in vitro antigen release.

40-46

Authors: М. V. Boiko, N. V. Patyka, S. М. Shulga, O. O. Tigunova, H. S. Andriiash

The aim of the research was to develop an optimal liquid medium composition for bacteria strain B. thuringiensis 87/3 which is the most favorable for the identification the potential production of biologically active components. In this work we have used new entomopathogenic bacteria strain B. thuringiensis var. thuringiensis (Bt H1) № 87/3 selected in vitro, isolated from the larvae of leaf-eating insects natural populations of Leptinotarsa decemlineata Say (L4). Nutrient medium for cultivation of Bt 87/3 was optimized on base a full-fledged xperiment, according to the Box-Behnken (33) plan. Optimumal concentrations were investigated in the medium of carbon source (15 g/l of glucose), nitrogen (10 g/l of corn extract) and phosphorus-containing inorganic salts (1.5 g/l of diammonium phosphate). When this strain grown in periodic conditions at 30 ºC, the number of viable cells reached 4.4.109 cells/ml. Nutrient medium that was optimized can be recommended for cultivation of Bacillus thuringiensis 87/3 in production conditions.

57-63

Authors: O. M. Obodovych, L. A. Sablii, V. V. Sydorenko, M. S. Korenchuk

The purpose of the study was the design of an aerator-oxidizer and optimal parameters of its work to ensure the conditions of mixing and dissolution of oxygen, in which there is no disturbance of the state of activated sludge. The testing of the work of the aeration-oxidation setup of rotor type with the use of a sludge mixture with different design of aerator-oxidizers and in different operating modes was performed. The assessment of the effect of processing on the state of the activated sludge was carried out according to standard parameters: sludge index, chemical oxygen demand. In addition, the number and state of activated sludge organisms were evaluated. The results of qualitative and quantitative analysis of activated sludge before and after processing in the setup are presented. The parameters in which the activated sludge functions in satisfactory mode are revealed.

72-78

Authors: L. N. Churkina, V. V. Klochko, S. .D Zagorodnya, L. V. Yaroshenko, O. B. Luitko

The aim of the work was the research of antibiotic batumin action on the biofilm formed by staphylococci and on the process of the biofilm formation by strain-producer Pseudomonas batumici. The minimal inhibiting concentration (MIC) of batumin was studied according to The Clinical and Laboratory Standards Institute standards — CLSI. Biofilm formation was studied using photometric O’Toole method with flatbed photometer at wavelength 540 nm. The batumin penetrated into biofilms of staphylococci and reduced the biomass of the formed biofilm of Staphylococcus aureus and S. epidermidis at the antibiotic concentration of 0.5 μg/ml, which was only twice higher than MIC of batumin for planktonic cells. The degree of biofilm changes in the presence of batumin differed for different staphylococcus strains. However, these differences did not depend on strains sensitivity or their resistance to the antibiotic. The ability of batumin strain-producer to form the biofilm and the ability of antibiotic to stimulate this process in concentrations of 1 and 10 μg/ml was established for the first time. In addition, it was found that in the Pseudomonas batumici B-321 planktonic culture in the presence of 10 μg/ml of batumin, the increase in length of producer cells on average by 15% was noted. The obtained data shed the light on batumin antimicrobic action on the staphylococci biofilm. It can be argued that is a perspective for treatment of staphylococcal infections. It enabled to consider batumin as a promising drug for the staphylococcal infections treatment.

25-38

Authors: O. Yu. Galkin, A. G. Komar, M. O. Pys’menna

The aim of this research was to analyze recent scientific literature, as well as national and international legislature on manifacturing process validation of biopharmaceutical production, in particular devices for serological diagnostics. Technology validation in the field of medical devices for serological diagnostics is most influenced by the Technical Regulation for Medical Devices for in vitro Diagnostics State Standards of Ukraine – SSU EN ISO 13485:2015 “Medical devices. Quality management system. Requirements for regulation”, SSU EN ISO 14971:2015 “Medical devices. Instructions for risk management”, Instruction ST-N of the Ministry of Healthcare of Ukraine 42-4.0:2014 “Medications. Suitable industrial practice”, State Pharmacopoeia of Ukraine and Instruction ICH Q9 on risk management. Current recommendations for validations of drugs manufacturing process, including biotechnological manufacturing, can not be directly applied to medical devices for in vitro diagnostics. It was shown that the specifics of application and raw materials require individual validation parameters and process validations for serological diagnostics devices. Critical parameters to consider in validation plans were provided for every typical stage of production of in vitro diagnostics devices on the example of immunoassay kits, such as obtaining protein antigens, including recombinant ones, preparations of mono- and polyclonal antibodies, immunoenzyme conjugates and immunosorbents, chemical reagents etc. The bottlenecks of technologies for in vitro diagnostics devices were analyzed from the bioethical and biosafety points of view.

58-63

Authors: Yu. P. Rud, M. I. Maistrenko, L. P. Buchatskiy

The aim of the research was to investigate diseased fish species Labeo (Labeo bicolor) and Sack-gill Catfish (Heteropneustes fossilis) by testing several diagnostic systems targeting fish herpesviruses in purpose to determine the etiology of the outbreak infection in aquarium fishes at the Kуiv Zoo during summer 2017. During a fish health inspection of aquariums in Kyiv Zoo, an Cyprinid herpesvirus 3 − CyHV-3 was detected in Labeo and Sack-gill Catfish. Preliminary examination of infected fish revealed a range of lesions particularly in internal organs and tissues. The gills of diseased fish were characterized by hyperaemia and necrosis. The infringement of liver color and structure, kidney swelling and gallbladder necrosis were observed in both fish species. The virus did not grow in fish cell lines of RTG-2, FHM and EPC, that was evident by absence of any morphological changes in appropriate cell lines. Also initially fish were tested for parasitic and bacterial infections and they were determined to be non-infected. Our results demonstrated that specific oligonucleotide primers for thymidine kinase gene of CyHV-3 were successfully amplified the specific DNA fragments. The length of polymerase chain reaction product, as expected, was 264 nucleotide pairs. The amplified specific fragments were identical to the area of thymidine kinase gene CyHV-3, as was shown by sequence analysis. The identity of nucleotide sequences composed 97‒99%. The same positive results were obtained using primers that recommended by the International Epizootic Bureau, fragments in size of 409 and 292 were also obtained. In our opinion, CyHV-3 was brought to Ukraine by import of aquarium fish avoiding sanitary control of transboundary transportation. Therefore, the uncontrolled import of aquarium and cultured fish species is a serious problem because imported fish could be as a source of highly pathogenic infections for industrial aquaculture.

70-75

Authors: T. Y. Trokhymchuk, L. A. Ganova, N. Ya. Spivak

The purpose of the work was to study the ability of test systems DIA-HIV ½ and DIA-HIV-Ag/Ab produced by PJSC “SPC “Diaproph-Med” to detect HIV infection. Reference blood serum of HIV-positive patients was tested using immunoassay analysis. It was shown that early detection of HIV infection is more effective using test kits of the fourth generation, which should be used for primary screening for HIV, and third-generation test systems should be used to confirm the positive results of primary analysis.

18-27

Authors: A. P. Kharkova, Y. A. Garmash, O. S. Hnatiuk, O. Y. Luzina, S. V. Danilovskyi, A.Y. Kuznetsova, O. H. Minchenko

To study effect of glutamine deprivation on the expression of genes encoding the key proliferation associated factors on a relation to inhibition of inositol requiring enzyme-1 IRE1 in U87 glioma cells was the aim of the research. It was shown that glutamine withdrawal down-regulated the expression of DEK, BRCA1, LIF, and COL6A1 genes in control glioma cells (transfected by empty vector), up-regulated ADGRE5 gene expression, and did not significantly change the expression of TPD52 and GNPDA. Inhibition of ІRE1 signaling enzyme activity modified the effect of glutamine deprivation on the expression of TPD52, BRCA1, LIF, DEK, ADGRE5, and COL6A1 genes: introduces the effect of glutamine deprivation on TPD52 and GNPDA1, reduced – on COL6A1, and enhanced – on ADGRE5, DEK, and BRCA1 in U87 glioma cells. Therefore, glutamine deprivation affect the expression level of most studied genes in U87 glioma cells in relation to the functional activity of IRE1 signaling enzyme, which is responsible for control of cell proliferation and glioma growth.

35-44

Authors: V. M. Minarchenko, R. V. Kutsyk, N. P. Kovalska, B. O. Movchan, O. V. Hornostai, O. M. Strumensk, L. M. Makhynia

The aim of our research was to investigate the influence of silver nanoparticles on the physical and chemical features of plant oils of dogrose, flax, cedar, amaranth and watermelon and their antimicrobial activity. Plant oils were saturated with silver nanoparticles using electron-beam technology for depositing a molecular stream of metal in a vacuum. To characterize the rancidity of plant oils, the acid, iodine, peroxide, ester and saponification values were determined. A sharp drop in the iodine number and an increase in the peroxide number in oils saturated with silver nanoparticles were observed, as compared to pure oils, indicating a decrease in the number of unsaturated bonds in fatty acids and the formation of peroxides in oils. All pure plant oils and a separate sample of silver nanoparticles suppressed the growth of only E. faecalis colonies. Plant oils that were saturated with silver nanoparticles delayed the growth of S. aureus, S. epidermidis, E. faecalis, E. coli, P. aeruginosa, and C. albicans; the greatest delay in the growth of colonies was caused by flaxseed oil. Thus, the features of the plant oils under study essentially changed after they are aturated with silver nanoparticles. It can be assumed that the metal acted as a catalyst for peroxide oxidation of lipids in the investigated plant oil samples, the products of which caused toxic effects on cultures of bacteria and fungi in the experiment.

53-60

Authors: N. Ya. Levchyck, A. V. Liubinska, N. V. Skrypchenko, O. I. Dziuba, N. M. Kachalova, O. L. Mironov

The aim of the research was to detect the biological activity of aqueous solutions of various samples of amber from the Trancedniestria province, and to characterize their effect on the physiological processes of higher plants. The allelopathic and cytostatic effects of amber solutions were studied using biotesting methods in open soil conditions and in culture in vitro. The test-object was the seedlings of cucumber Cucumis sativus L. It was shown that the activity and nature of the action of the samples depend on the region of origin of the amber, the degree of shredding and concentration of the solution. According to the results, fine fractions of amber show biostimulating effects on plants. Thus, amber and its aqueous solutions can be effectively used for cultivating agricultural plants in vivo and in vitro conditions. For stimulation of the increasing of the plant stem mass it is recommended to use amber B1, to increase the mass of roots - amber B4, and amber B2 - to stimulate stem growth in length.