[Stochastic Packaging of Cas Proteins into Exosomes].

CRISPR/Cas systems are perspective molecular tools for targeted manipulation with genetic materials, such as gene editing, regulation of gene transcription, modification of epigenome etc. While CRISPR/Cas systems proved to be highly effective for correcting genetic disorders and treating infectious diseases and cancers in experimental settings, clinical translation of these results is hampered by the lack of efficient CRISPR/Cas delivery vehicles. Modern synthetic nanovehicles based on organic and inorganic polymers have many disadvantages, including toxicity issues, the lack of targeted delivery, and complex and expensive production pipelines. In turn, exosomes are secreted biological nanoparticles that exhibit high biocompatibility, physico-chemical stability, and the ability to cross biological barriers. Early clinical trials found no toxicity associated with exosome injections. In the recent years, exosomes have been considered as perspective delivery vehicles for CRISPR/Cas systems in vivo. The aim of this study was to analyze the efficacy of CRISPR/Cas stochastic packaging into exosomes for several human cell lines. Here, we show that Cas9 protein is effectively localized into the compartment of intracellular exosome biogenesis, but stochastic packaging of Cas9 into exosomes turns to be very low (~1%). As such, stochastic packaging of Cas9 protein is very ineffective and cannot be used for gene editing purposes. Developing novel tools and technologies for loading CRISPR/Cas systems into exosomes is needed.

Methionine gamma lyase fused with S3 domain VGF forms octamers and adheres to tumor cells via binding to EGFR.

Methods for targeting enzymes exhibiting anticancer properties, such as methionine γ-lyase (MGL), have not yet been sufficiently developed. Here, we present the data describing the physico-chemical properties and cytotoxic effect of fusion protein MGL-S3 - MGL from Clostridium sporogenes translationally fused to S3 domain of the viral growth factor of smallpox. MGL-S3 has methioninase activity comparable to native MGL. In solution, MGL-S3 protein primarily forms octamers, whereas native MGL, on the contrary, usually forms tetramers. MGL-S3 binds to the surface of the neuroblastoma SH-SY5Y and epidermoid carcinoma A431 cells and, unlike native MGL, remains there and retains its cytotoxic effect after media removal. In HEK293T cells lacking EGFRs, no adhesion was recorded. Confocal fluorescence microscopy confirms the preferential adhesion of MGL-S3 to tumor cells, while it avoids getting into lysosomes. Both MGL and MGL-S3 arrest cell cycle of SH-SY5Y cells mainly in the G1 phase, while only MGL-S3 retains this ability after washing the cells.

Dynamic changes in betanin content during the growing season of table beet: their interplay with abiotic factors.

The table beet, a widespread edible root crop known for its medicinal and antioxidant properties, early maturation, good shelf life, and high contents of bioactive compounds, vitamins and minerals, is used for the production of a natural red food dye. The relevance of this study is dictated by the lack of knowledge about the dynamic changes in the content of betanin during the growing season when developing table beet cultivars with a focus on pigment extraction. The article presents the results of a study of 29 red-colored table beet accessions from the collection of the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR). Dynamic changes in the content of the pigment during the growing season were observed on two beet accessions, cvs. 'Russkaya odnosemyannaya' and 'Bordo odnosemyannaya'. Four pH versions of the buffer solution were tested, and the test results are presented. A buffer solution with pH 6.5 is recommended for research purposes. The amplitude of variability in the content of betanin in the peel (39.9-239.2 mg/100 g) and f lesh (14.4-127.5 mg/100 g) of beets was determined. It was conf irmed that the content of betanin in the peel exceeded that in the f lesh in all samples. A positive relationship between these indicators was revealed (r = 0.74, p ≤ 0.05). It was found that betanin accumulation did not occur in beet roots during the growing season. The pigment showed considerable f luctuations associated with abiotic environmental factors. Correlation analysis showed a signif icant positive relationship between air temperature and betanin content in the root f lesh (r = 0.32-0.31, p ≤ 0.05). A negative impact of environmental temperature on betanin content in the peel manifested itself on the third day (r = -0.34…-0.35, p ≤ 0.05). The negative response to precipitation was less expressed in cv. 'Bordo odnosemyannaya' due to the genotype's more active metabolism and plasticity. Structural morphological features of the photosynthetic apparatus were described for the tested accessions, and their interrelations with the studied character were specif ied. Recommendations are given concerning the choice of a planting pattern and the timing of table beet harvesting for pigment extraction.