Applied Biochemistry and Microbiology最新文献

Isocitric acid (ICA) is an isomer of citric acid (CA), which is widely used in the food industry, household chemicals, and other fields. ICA is a constituent in some fruit juices and differs from CA due to a number of valuable properties, particularly those related to its high antioxidant activity. ICA is used in sports nutrition to enhance blood oxygenation and is promising for use in medical preparations, particularly for the treatment of iron deficiency anemia and preventing vascular thrombosis. The limited scale of ICA use is largely determined by its high production cost. Previously known biotechnological methods of its production, based on Yarrowia lipolytica yeast platforms using glycerol or ethanol as carbon sources, allowed to obtain ICA mixed with CA. Recent results on the study of genetic control over mitochondrial transport of ICA in Y. lipolytica cells and the development of efficient genetic engineering methodologies for this yeast open up opportunities for a significant reduction in the cost and expansion of the scale of biotechnological production of this acid using as a raw material glucose molasses obtained by deep processing of grain. This could significantly reduce the cost of IСA and expand the scope and scale of its use.

Adenoviral vectors represent a safe and highly immunogenic platform for vaccine delivery. Non-replicating viral vectors are widely used in vaccinology due to properties such as high replicative activity, ease of manipulation, safety and immunogenicity, as well as scalability of their production and resistance to heat treatment procedures. In addition, vaccines based on adenoviral vectors induce sustained antigen-specific cellular and humoral immune responses. To elucidate the conditions for cultivating adenovirus vector serotype 26 (Ad26) in HEK293 cells, the effects of multiplicity of infection (MOI) on the cell maximum density, viability and productivity were investigated. As a result, optimal parameters for Ad26 cultivating were selected. The use of Ad26 at a dose in the range of (3.20‒4.16) × 10⁹ hexon gene DNA copies to infect 1 L of HEK293 cell suspension allowed us to obtain viral material with a high yield of virus particles (about 2 × 10¹²/L). In addition, it was demonstrated that replacing glutamine in the nutrient medium with the more degradation-resistant dipeptide GlutaMAX™ (Thermo Fisher Scientific, USA) resulted in a decrease in cell doubling time to 22‒24 h while maintaining cell viability at 92‒98% during the exponential growth phase. Results obtained were used in the manufacture of the Ad26-component of the Gam-COVID-Vac vaccine, which made it possible to satisfy the high demand for the vaccine during the mass immunization campaign against COVID-19 in the Russian Federation.

This review contains up-to-date information about existing methods of gene therapy (GT) and GT-products approved for use in the world and Russian Federation, as well as those currently at the stage of development and clinical trials. The stages of GT-drug development as well as methods for targeted delivery of genetic material into cells and tissues are considered. Particular attention is paid to the characteristics of viral vectors and some methods of their design and targeting. Approaches for modifying the sensitivity of viruses to endogenous (pH, redox potential, enzyme content) and exogenous (light, magnetic field, temperature) stimuli are discussed, as are various methods used for this purpose, such as pseudotyping, incorporation of a targeting ligand into the viral genome, and physical exposure. The various adapters are also described in detail. All of these modification methods are based on rational design or directed evolution approaches. Fundamental research in this field of science is aimed at developing new strategies for the treatment of various diseases, including those still considered incurable.

Inflammatory bowel diseases (IBD) are a group of chronic gastrointestinal tract diseases. The main causes of IBD are believed to be a disruption in the balance of microbiota and function of the intestinal epithelium. The treatment for IBD includes a combination of drugs and dietary nutrition. Until now, there have been no in vitro studies on the functional state and transcriptome of cultured intestinal epithelial cells depending on alimentary factors such as products of dietary therapeutic nutrition and biologically active additives used to correct the functional state of the intestinal barrier. We analyzed the transcriptome of differentiated Caco-2 cells, which modeled the intestinal barrier, and showed that Saccharomyces cerevisiae hydrolysate had a pronounced positive effect on the intestinal epithelium. Thus, this product can be recommended for correction of the microbiome and restoration of functional activity of the intestine in IBD, as well as for prevention of diseases of the gastrointestinal tract.

A technology for sugar beet genotyping has been developed (Beta vulgaris) at ten microsatellite loci: FDSB1001, FDSB1033, SB04, SB09, SB15, Unigene16898, Unigene17623B, Unigene17923, Unigene26753, and Unigene27833. The technology can be used for efficient, accurate, and rapid identification of sugar beet lines and hybrids. The proposed approach includes multiplex PCR for all loci in one test tube followed by electrophoretic analysis of the obtained DNA fragments in one capillary of a genetic analyzer. One of the key elements of the technology, determining the accuracy and reproducibility of the analysis, is the use at the electrophoresis stage of an additional length standard, an allelic ladder consisting of DNA fragments of the analyzed microsatellite loci. The advantages of the proposed technology include the possibility of standardization and automation of the method in a 96-well plate format, which allows for the establishment of a mass analysis process. As a result of this study, the DNA profiles of six modern sugar beet hybrids (Azimut, Visit, Korvet, Rubin, Fregat, and Uspekh) and their component lines were obtained for the first time. The developed technology can be used effectively for genetic identification of sugar beet lines, assessment of the homogeneity of plant material, and quality control of hybridization at all stages of the breeding process and can become a reliable laboratory tool for supporting industrial seed production of this crop.

Environmental DNA (eDNA) is genetic material obtained directly from samples of various natural substrates, rather than directly from a living organism. The use of eDNA simplifies monitoring of secretive and low-abundance species that are difficult to detect using classical environmental survey methods. Understanding the capabilities and limitations of eDNA-based methods requires information about the influence of various factors on the rate of degradation of nucleic acids in the environment. We studied the rate of DNA degradation at a temperature of 14°C in water from Svirskaya Bay of Lake Ladoga with pH 7.3 in the absence of light. For this purpose, highly specific primers and a TaqMan probe were developed and tested for the D-loop fragment of mitochondrial DNA of the domestic chicken (Gallus gallus domesticus). The choice of this particular genetic material as exogenous DNA was due to the minimal risk of contamination of natural water in the sampling area. At the start of the experiment, chicken DNA (galDNA) was added to the water samples to a concentration of 50 μg/L and detected by real-time PCR after 6 h and 1, 2, 3, 8, and 14 days. Using the developed protocol, we identified target D-loop sequences in natural water samples during the first three days of the experiment, with trace amounts of galDNA detected up to eight days.

The production of the most studied, active and widespread groups of brassinosteroids by salt-tolerant bacteria Priestia megaterium Ср-1, Rhodococcus jostii CA-6 and Pseudomonas koreensis FP2/1 isolated from saline soil samples was studied. It was shown that the studied bacterial strains are capable of producing steroidal phytohormones of the brassinolide, 24-epibrassinolide and 28-homobrassinolide groups, as well as brassinosteroid B-ketones, B-lactones and 6-deoxo derivatives. This circumstance may be the cause of the growth-stimulating activity of these bacteria and their effect on plant adaptation to stress factors.

The effects of exogenous abscisic acid (ABA) on early germination processes in seeds of two cereal crops, wheat and triticale (a hybrid of wheat and rye), are investigated. A clear relationship was established between growth processes and both the concentration of ABA and the imbibition stage at which ABA was applied. At moderate to high concentrations (10^–6–10^–4 M), ABA acted as an inhibitor of physiological processes. Early stages of seed germination, specifically those involving physical swelling and radicle emergence, were found to be highly sensitive to ABA. Furthermore, a high concentration of exogenous ABA (10^–4 M) inhibited the mobilization of protein reserves in the wheat embryo, which may contribute to the overall inhibition of germination.

Upon immunochemical determination of low-molecular-weight compounds capable of binding only to one antibody molecule (haptens), competitive interactions of the hapten in the sample and the hapten-carrier conjugate (usually, a hapten-protein conjugate) with the antibodies are conducted. The characteristics of such analytical systems largely depend on the properties of the conjugates used. However, the relationships between the conjugate composition and the achieved limit of detection (LOD) are described fragmentarily and are not systematized. In this study, a panel of nine conjugates of bisphenol A, a relevant toxic environmental pollutant, with soybean trypsin inhibitor (STI) and bovine serum albumin is considered. The composition of the conjugates synthesized by the carbodiimide-succinimide method was determined using fluorescamine. Concentration dependences of binding between antibodies and conjugates immobilized in microplates and competitive interaction with free bisphenol A were studied. The enzyme-linked immunoassay (ELISA) implemented using various conjugates was characterized by LODs and working ranges. It was demonstrated that the minimum LOD of 1.9 ng/mL was achieved using the bisphenol A–STI conjugate with a 5 : 1 composition. The developed ELISA was successfully tested for the detection of bisphenol A in natural and drinking water.

A highly efficient expression system for recombinant Escherichia coli type II L-asparaginase (EC 3.5.1.1) has been developed based on a synthetic gene optimized for the energy of secondary structure hairpins formation in the 5'-region of mRNA. A producer strain, E. coli BL21[DE3]/pET28a-AsnSYN was created, providing an accumulation of up to 291 ± 9 mg/L of enzymatically active protein (44.5 ± 2.6 mg/(L AU)) when cultivated in stirred flasks, which is 50% higher than the control strain with a natural gene at an induction time of 3 hours. Optimization of the codon composition of the gene made it possible to increase the energy of secondary mRNA structure formation in the 5'-region from –70 to –47 kcal/mol, which, as we assume, contributed to improved translation efficiency. For the developed producer strain, asparaginase that meets pharmacopoeial purity requirements can be obtained with a total yield of ≥25% and a specific activity of >250 IU/mg. The preparation contains no visible impurities according to electrophoresis data and less than 3% multimeric forms according to size exclusion chromatography data. The results obtained demonstrate the promise of using synthetic genes with optimized DNA structure for the industrial production of therapeutic enzymes.