Applied Biochemistry and Microbiology最新文献

The parameters of lyophilization of substances of organ-tissue and cultural origin containing a fixed rabies virus were determined. The objects of this research were the “Moscow 3253,” СVS, and “Saratov” strains of the rabies virus used in the production of commercial and experimental batches of rabies immunoglobulin from horse blood serum. For substances containing “Moscow 3253,” CVS, and “Saratov” strains, the effects of both freezing and eutectic temperatures were identical. The use of the developed lyophilization protocol allowed us to obtain samples as well-formed porous tablets. The infectivity of the freeze-dried Moscow 3253, CVS, and Saratov strains was 6.2, 7.1, and 6.8 logID₅₀/mL, respectively. Thus, the proposed freeze-drying regime is acceptable for obtaining samples of rabies virus strains used for the production of rabies immunoglobulin.

Genetic polymorphism of a collection of accessions of the genus Lupinus was studied using SSR marking technology. The average level of intraspecies DNA polymorphism for L. albus, L. angustifolius, and L. luteus was found to be 25.8, 21.6, and 34.7%, respectively. The informative markers for genotyping and identification of Russian lupine varieties and breeding samples were determined. As a result of assessment of the genetic distance, genetically divergent accessions were identified that can serve as promising material for use in breeding programs for white (L. albus) and yellow (L. luteus) lupine, namely, SN 8-12, SN 96-15 DT1, KCI-22 d-15, KCI 22 d-17, 19-PIO-18-3, and SN 12-11-02-2-4-1. Based on the data obtained, molecular formulas of the studied samples were developed and reference genetic passports were compiled for the lupine varieties Belorozovyi 144, Nadyezhnyi, Antei, and Kurshavel. The practical results of this research and new knowledge on the structure of the genomes of Russian lupine varieties can be used in variety testing, seed production, and as a system of information support for the processes of selection and preservation of the national gene pool of lupine, a valuable agricultural crop.

Qualitative and quantitative characteristics of the aqueous phase obtained as a result of hydrothermal liquefaction of biomass of excessive activated sludge from municipal and domestic sewage treatment plants in Kaliningrad have been investigated experimentally. The content of organic compounds averaged 11 592 ± 571 mg O/dm³, and the biochemical index was 0.57. Aromatic compounds, carboxylic acids, and heterocyclic nitrogen-containing compounds were found in the composition of the aqueous phase. From activated sludge of treatment facilities of PJSC Nizhnekamskneftekhim, Republic of Tatarstan, 11 isolates–destructors of components of the aqueous phase were isolated. Their cultural and morphological characteristics were evaluated, and the necessary dilution of the aqueous phase for their cultivation was determined. It was noted that, in the process of cultivation of isolates, S2, S7, and S11 are able to neutralize components of the aqueous phase effectively in comparison with other isolated cultures: the efficiency of degradation by COD was 69, 73, and 75%, respectively. Based on 16S rRNA gene sequencing, isolate S2 had the highest similarity to Pseudomonas glycinae MS586 (99.52%), S7 with Paenarthhobacter nicotinovorans DSM 420 (99.8%), and isolate S11 with Comamonas testosteroni KS 0043 (99.6%). The most active isolates can be used for the treatment of highly concentrated wastewater before preliminary discharge into the sewerage network and natural water bodies.

The yeast microflora of brined Ossetia cheeses made from raw milk by small farms in various regions of North and South Ossetia has been studied for the first time. A total of 17 yeast species belonging to ten genera were identified. It is shown that the composition of the yeast microflora of Ossetia cheeses depends on the place of production, the microflora of the raw milk and starter culture, and the type of milk. Most of the Ossetian cheeses studied were dominated by lactose-utilizing yeasts Kluyveromyces lactis and Debaryomyces hansenii, as well as by lactose-negative yeasts Saccharomyces cerevisiae, Pichia kudriavzevii, and P. fermentans. The composition of the microflora of artisanal Ossetia cheeses is more diverse than that of commercial cheeses produced from pasteurized milk.

Traditional two-dimensional (2D) cell cultures have limited capabilities for modeling complex interactions between viruses and host cells, which reduces the efficiency of the studies when trying to transfer data obtained to in vivo models. Three-dimensional (3D) cell cultures are the most physiologically relevant models, without the limitations inherent to 2D cultures. The use of 3D models opens up new possibilities when studying viral infections and developing new antiviral drugs and preventive measures. We constructed a biological model based on 3D spheroids from the human embryonic lung cell line (HELC-3) and tested it on the example of the analysis of replicative activity of herpes simplex virus type 1, strain L₂ (HSV-1/L₂). The spheroids were obtained when cultivating the cells in hydrogel microwells. The replicative activity of HSV-1 in 3D culture of HELC-3 cells was estimated by a cytopathic effect of the culture liquid of virus-infected spheroids on a monolayer culture of the same cells (2D format). It was demonstrated that HSV-1 replicates efficiently in spheroids. Thus, the model based on 3D spheroids from HELC-3 cells can be considered as a promising tool for studying the HSV-1 replication cycle in vitro, molecular mechanisms of its pathogenesis and persistence, as well as the analysis of potential antiviral drugs and vaccines.

The aim of this study was to create biodegradable scaffolds based on natural silk fabrics for regenerative medicine. Fabrics with different densities gas-chiffon (15 g/m²) and double-sided satin (155 g/m²) were used as matrices. The samples were preliminarily treated with a water–alcohol solution of calcium chloride for different periods of time to obtain the samples with different degrees of degradation. Morphological studies using light microscopy confirmed structural changes in the tissues after this treatment. Degradation of the obtained scaffolds was studied in vitro using Fenton’s reagent with different duration of incubation (15, 30, and 45 days). The rate of scaffold destruction depended on the type of fabric and time of preliminarily treatment with calcium chloride. The highest degradation rate was observed for gas-chiffon, while satin demonstrated slower destruction. The results obtained can be used when optimizing the properties of silk fabrics for their use in regenerative medicine and development of efficient methods to control their biodegradation.

At present, biosensor tools for analyzing intracellular energy processes are actively being developed. In this work, a set of plasmid constructs with luciferase genes from Luciola mingrelica and Photorhabdus luminescens was proposed for obtaining biosensor cells based on bacteria and eukaryotes. The possibility of targeted expression of luciferase both in the cytoplasm and in the mitochondrial matrix was realized due to the use of signal sequences. It was demonstrated that the resulting biosensors are suitable for analyzing the cell pool of macromolecular compounds and reducing equivalents, such as ATP, NADH, and flavin mononucleotide (FMN). We hope that this tool will be in demand for the analysis of the activity of intracellular chaperones, as well as toxicological and other investigations.

The enzymes that are least represented but most in demand in biotechnology are highly specific proteases that can hydrolyze difficult-to-degrade substrates. Micromycetes from the genus Aspergillus have been identified as producers of these proteases, which have diverse biochemical properties. For Aspergillus ochraceus VKM F-4104D, which has been previously studied as a producer of a protease that activates protein C and human factor X, its ability to secrete proteolytic enzymes that act on globular and fibrillar proteins has been investigated when cultured in nine different nutrient media with varying nitrogen sources. Maximum collagenolytic and fibrinolytic activity was observed when using a medium containing bovine collagen as an inducer. During preparative isoelectric focusing and subsequent zymography, two proteases with isoelectric points of 5.6 and 6.2 and molecular weights of 74 and 33 kilodaltons, respectively, were identified. As a result of genomic analysis of A. ochraceus VKM F-4104D, 121 potential secreted proteases have been predicted. Transcriptomic analysis revealed that when grown on a medium containing bovine collagen with high proteolytic activity, the microorganism expresses 117 extracellular proteases. Of these, only two were identified using traditional enzymological methods. Thus, the use of more advanced techniques allows for the detection of previously unidentified proteases with different activities among organisms for which information on physiology has been gathered or individual enzymes have been described previously, whereas traditional methods for identifying new enzymes often yield sparse or unrepresentative results.

The conditions of recombinant ɑ-amylase production in an induction-free cultivation system based on Bacillus mojavensis are described. Fermentation was carried out in pilot fermenters under deep cultivation, which ensured the production of experimental batches in the quantities necessary for comprehensive studies of the samples obtained. Gradual accumulation of the recombinant enzyme in the bioreactor reaches a maximum after 31 hours. Some biochemical parameters of the recombinant enzyme were studied. Its thermostability was established, and the pH optimum of activity manifestation was determined—the maximum efficiency of substrate hydrolysis was observed when the sample was preheated for 30 min at a temperature of 55°C and pH equal to 7.2. An inversely proportional relationship between the change in the level of amylolytic activity and the amount of calcium chloride added during cultivation was revealed. The possibility of creating strains producing recombinant ɑ-amylase on the basis of B. mojavensis strain BDV-1 was demonstrated.

Using cryostructuring techniques, a new biocompatible, wide-porous gelatin-hyaluronate material was prepared, which was successfully tested as a carrier of a protein-peptide bioregulator. The following physicochemical parameters of the synthesized carrier were determined: the total water-holding capacity (51.2 ± 5.8 g H₂O/g polymer), the degree of swelling (7.03 ± 0.65 g H₂O/g polymer) of the polymer inside the macropore walls of the spongy material and the compressive modulus of elasticity (12.3 ± 1.5 kPa). The presence of a system of interconnected macropores with a cross section from 20 to 210 μm in the material is shown. Next, the resulting carrier was loaded with a protein-peptide bioregulator isolated from bovine sclera and used in the experiments on organ cultivation of a biological model: the posterior section of the newt eye. As a result, a significant protective effect of the scleral bioregulator included in the sponge carrier on the viability of tissues and cells of a biological model was revealed and the prospects of a new biocompatible cryogenically structured material for dosed release of the bioregulator into the culture medium was shown.