Firefly luciferase is widely used in molecular biology and bioanalytical systems as a reporter molecule due to the high quantum yield of the bioluminescence, availability of stable mutant forms of the enzyme with prescribed spectral characteristics and abundance of bacterial expression systems suitable for production of recombinant proteins in limitless quantities. In this review, we described fusion proteins of luciferase with biotin-binding domain and streptavidin, with proteins A and G, antibodies, with DNA-and RNA-binding proteins, as well as fusion proteins designed for BRET systems. The firefly luciferase-based fusion proteins are represented as an effective tool for the development of different bioanalytical systems such as (1) systems in which luciferase is attached to the surface of the target and the bioluminescence signal is detected from the specific complexes formed; (2) BRET-based systems, in which the specific interaction induces changes in the bioluminescence spectrum; and (3) systems that use modified or split luciferases, in which the luciferase activity changes under the action of the analyte. All these systems have wide application in biochemical analysis of physiologically important compounds, for the detection of pathogenic bacteria and viruses, for evaluation of proteinprotein interactions, assaying of metabolites involved in cell communication and cell signaling.Abbreviations: Ab, antibody; b, biotin; bccp, biotin carboxyl carrier protein domain of acetyl-CoA carboxylase from E. coli; HRP, horseradish peroxidase; IA, immunoassay; KPBT, Klebsiella pneumoniae oxaloacetate decarboxylase; Luc, firefly luciferase; PG, pepsinogen; PSA, prostate-specific antigen; SA, streptavidin.

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The sensitive BRET system for the homogeneous immunoassay of a low-molecular weight antigen was developed using progesterone as an example. Two thermostable mutants of the Luciola mingrelica firefly luciferase (Luc)-the "red" mutant with λmax.em = 590 nm (RedLuc) and the "green" mutant with λmax.em = 550 nm (GreenLuc)-were tested as the donors. The water-soluble Alexa Fluor 610× (AF) dye was selected as the acceptor because its two absorption maxima, located at 550 and 610 nm, are close to the bioluminescence maxima of the GreenLuc and RedLuc, respectively. The methods for the synthesis of the luciferase-progesterone (Luc-Pg) conjugate and the conjugate of the dye and the polyclonal antiprogesterone antibody (AF-Ab) were developed. Both conjugates retained their functional properties, had high antigen-antibody binding activity, and demonstrated a high BRET signal. The homogeneous immunoassay system based on the BRET from the firefly luciferase to the synthetic dye was established to assay progesterone as a model antigen. Optimization of the assay conditions, the composition of the reaction mixture, and the concentrations of the donor and the acceptor made it possible to reach the minimum detectable progesterone concentration of 0.5 ng mL(-1) .

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Vitamin D deficiency is widespread globally, however available data for the Russian adult population is fragmented. This cross-sectional study used secondary data for individuals undergoing testing for vitamin D concentrations from 2013 to 2018 by InVitro laboratory. 25(OH)D serum concentration was determined using chemiluminescent microparticle immunoassay. The mean, median, and proportion with severe, deficient, insufficient and sufficient 25-hydroxyvitamin D (25(OH)D) concentrations were estimated. Splines examined the effect of latitude on 25(OH)D concentrations. Data were available for 30,040 subjects age ≥ 18 years. 24.2% of the sampled population had sufficient (30–< 150 25(OH)D ng/mL), 34% deficient (10–19.9 ng/mL) and 5.6% severely deficient (< 10 ng/mL) status. Average 25(OH)D concentrations were highest among 30–44 years and lowest amongst older adults; females had modestly higher values. Concentrations were 15% higher in fall/summer vs. winter/spring. A non-linear relationship was observed by latitude; the highest 25(OH)D concentrations were observed near 54°N, decreasing at more southern latitudes for women and more northern latitudes for both sexes. These results are comparable to other Northern European publications and limited Russian samples demonstrating low concentrations. Acknowledging that nationally-representative and randomly sampled data are needed, the present data suggest the burden may be high and identifies some population sub-groups and geographic areas with a higher potential deficiency of vitamin D.

Aim. The present article examines key methods of microbiota correction (antibiotic therapy; pro-, pre- and metabiotic therapy; faecal microbiota transplantation) used in treating inflammatory bowel disease, as well as compares the clinical trial results of these methods.Key findings. Inflammatory bowel disease (IBD) is an umbrella term used to describe a group of chronic diseases of unknown aetiology. In the past, bacteriological methods based on the isolation of a pure bacterial culture were used to determine the microbiota composition. However, such methods did not provide complete information on the microbiota composition. In recent years, preference has been given to more accurate and faster molecular genetic methods allowing a more detailed study of the key mechanisms by which microbiota affects the intestine in Crohn’s disease (CD) and ulcerative colitis (UC), as well as of the effect of microbial metabolites on their pathogenesis. The article provides an overview of main microbiota metabolites and their role in regulating the intestinal barrier function. One of the current issues consists in the development of personalised approaches to therapy and remission maintenance in IBD, including via methods for correcting the microbial composition: probiotic, prebiotic and metabiotic therapy, as well as faecal microbiota transplantation.Conclusion. The use of probiotics, prebiotics, and metabiotics can enhance the effectiveness of therapeutic regimens and significantly improve the quality of life of patients with chronic IBD. The use of antibiotics and faecal microbiota transplantation in treating IBD is the subject of extensive discussion and debate. The safety of these methods has not been confirmed so far; therefore, it is vital to continue studying their influence on the clinical condition of patients.

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A streptavidin-luciferase fusion protein comprising the thermostable mutant form of firefly luciferase Luciola mingrelica and minimal core streptavidin was constructed. The streptavidin-luciferase fusion was mainly produced in a tetrameric form with high luciferase and biotin-binding activities. It was shown that fusion has the same K values for ATP and luciferin and the bioluminescence spectra as initial luciferase. The linear dependence of the bioluminescence signal on the content of the fusion was observed within the range of 10 -10 mol per well. Successful application of obtained fusion in a biospecific bioluminescence assay based on biotin-streptavidin interactions was demonstrated by the example of a specific DNA hybridization analysis. A DNA hybridization analysis for Escherichia coli cells identification was developed using unique for these cells gadB fragment encoding glutamate decarboxylase. The amplified biotinylated GadB fragments were hybridized with the immobilized oligonucleotide probes; then, the biotin in the DNA duplexes was detected using the streptavidin-luciferase fusion protein. To reach the high sensitivity of the assay, we optimized the conditions of the assay. It was shown that the use of Pluronic for plate modification resulted in a significant reduction in the DNA detection limit which finally was 0.4 ng per well.