Biosafety Analysis of Metabolites of Streptomyces tauricus Strain 19/97 M, Promising for the Production of Biological Products.

A biosafety study was carried out concerning the metabolites of Streptomyces tauricus strain 19/97 M. This strain is a promising producer of biological preparations and shows antagonistic properties against Fusarium fungi, which cause Fusarium wilt disease. The strain has a pronounced biological activity against conifers, cereals and legumes. The treatment of planting material reduces infections, increases germination and furthers plant productivity. Using metabolites, we understood the culture liquid separated by filtration after the cultivation of the strain. Animals of different taxonomic affiliations were used as test objects: (CBA × C57BI/6) F1 hybrid mice (Mus musculus) (warm-blooded organisms), Daphnia magna Straus (planktonic crustaceans) and the unicellular alga Chlorella vulgaris Beijer. In the study, we were guided by the test standards for acute oral toxicity and irritation to the skin, mucous membranes of the eyes and inhalation toxicity. The research results showed that the metabolites of the strain are not acutely toxic to organisms of different taxonomic levels. The metabolites of the strain do not have an irritating effect on the skin and mucous membranes of warm-blooded animals. Based on the studies carried out, metabolites can be used for creating a fungicidal biological preparation.

Cellulose-based scaffolds for fluorescence lifetime imaging-assisted tissue engineering.

Quantitative measurement of pH and metabolite gradients by microscopy is one of the challenges in the production of scaffold-grown organoids and multicellular aggregates. Herein, we used the cellulose-binding domain (CBD) of the Cellulomonas fimi CenA protein for designing biosensor scaffolds that allow measurement of pH and Ca2+ gradients by fluorescence intensity and lifetime imaging (FLIM) detection modes. By fusing CBD with pH-sensitive enhanced cyan fluorescent protein (CBD-ECFP), we achieved efficient labeling of cellulose-based scaffolds based on nanofibrillar, bacterial cellulose, and decellularized plant materials. CBD-ECFP bound to the cellulose matrices demonstrated pH sensitivity comparable to untagged ECFP (1.9-2.3 ns for pH 6-8), thus making it compatible with FLIM-based analysis of extracellular pH. By using 3D culture of human colon cancer cells (HCT116) and adult stem cell-derived mouse intestinal organoids, we evaluated the utility of the produced biosensor scaffold. CBD-ECFP was sensitive to increases in extracellular acidification: the results showed a decline in 0.2-0.4 pH units in response to membrane depolarization by the protonophore FCCP. With the intestinal organoid model, we demonstrated multiparametric imaging by combining extracellular acidification (FLIM) with phosphorescent probe-based monitoring of cell oxygenation. The described labeling strategy allows for the design of extracellular pH-sensitive scaffolds for multiparametric FLIM assays and their use in engineered live cancer and stem cell-derived tissues. Collectively, this research can help in achieving the controlled biofabrication of 3D tissue models with known metabolic characteristics. STATEMENT OF SIGNIFICANCE: We designed biosensors consisting of a cellulose-binding domain (CBD) and pH- and Ca2+-sensitive fluorescent proteins. CBD-tagged biosensors efficiently label various types of cellulose matrices including nanofibrillar cellulose and decellularized plant materials. Hybrid biosensing cellulose scaffolds designed in this study were successfully tested by multiparameter FLIM microscopy in 3D cultures of cancer cells and mouse intestinal organoids.