Biohimiâ

This review examines modern approaches to studying double-strand break (DSB) DNA repair in mammalian cells, employing the CRISPR/Cas9 system. Due to its flexibility and efficacy, the Cas9 nuclease is used in numerous genetic reporters. We discuss various fluorescence-based genetic reporters used to monitor the repair process. Among the innovative Cas9-based methods, special attention is given to the techniques that examine both single and multiple DSBs, including approaches such as DSB-TRIP and ddXR. These methods open new possibilities for investigating structural rearrangements or analyzing random genomic sites. Additionally, the review considers how DSBs induced by Cas9 differ from those made by other nucleases and how these peculiarities could impact DNA repair mechanisms. Understanding these differences is crucial for planning experiments aimed at studying DSB repair.

Th2 cytokines (IL-4, IL-5, and IL-13) play an important role in the development of allergies, including allergic rhinitis (AR). IL-13 promotes mucus hypersecretion in the airways and IL-5 recruits eosinophils to the nasal mucosa, leading to increased inflammation and tissue damage. Drugs based on monoclonal antibodies that block the activity of these cytokines are being developed for the treatment of allergic diseases. However, studies of drugs that target IL-13 alone (such as Tralokinumab and Lebrikizumab) were not successful. Given that IL-5 and IL-13 have different roles in AR, simultaneous inhibition of both cytokines may be a promising approach. New methods of regulating gene activity, such as RNA interference (RNAi), offer new perspectives for the development of drugs. This study describes a complex consisting of siRNAs that inhibit the activity of Il5 or Il13 genes and a currier peptide LTP. The effects of this complex on the allergic inflammation in a mouse model of AR was studied. Suppression of Il5 expression decreased nasal hyperreactivity and reduced the number of goblet cells in the respiratory epithelium of AR-induced mice. Inhibiting the Il13 gene had a more beneficial effect than suppression Il5 alone, further contributing to reducing the number of cells infiltration the nasal cavity. When both Il5 and Il13 were suppressed simultaneously, the result was similar to that of Il13 inhibition alone. Likely, IL-13 plays a more significant role in the development of AR than IL-5. As a result, the possibility of using RNAi for anti-cytokine therapy for AR has been demonstrated. However, dual inactivation of IL-5 and IL-13 by siRNAs does not provide any advantages over inactivating IL-13 alone in the current mouse model of AR. However, the lack of success of anti-IL-13 therapy in clinical practice indicates the promise of an approach based on the dual blocking of IL-5 and IL-13.

The evolution of proteins from restriction–modification systems containing an endonuclease domain of the RE_AlwI family and either two DNA methyltransferases, each with a domain of the MethyltransfD12 family, or one DNA methyltransferase with two domains of this family was studied. It was found that all such systems recognize one of three DNA sequences, namely GGATC, GATGC or GATGG, and the restriction endonucleases of these systems are divided by sequence similarity into three clades that unambiguously correspond to specificities. The DNA methyltransferase domains of these systems are divided into two groups based on sequence similarity, with two domains of each system belonging to different groups. Within each group, the domains are divided into three clades according to their specificity. Evidence of multiple interspecific horizontal transfers of systems as a whole is found, as well as evidence of gene transfer between systems, including transfer of one of the DNA methyltransferases with a change in specificity. Evolutionary relationships of DNA methyltransferases from such systems with other DNA methyltransferases, including orphan DNA methyltransferases, were revealed.

The effect of salt stress was studied in mesophyll (M) and bundle sheath (BS) chloroplasts of maize plants treated with NaCl for 5 days. Pigment content, chlorophyll fluorescence at 77 K, activity of photosystems (PS) I and II, polypeptide compositions and ultrastructure of the thylakoid membranes were determined in plants grown under different salinity conditions (0, 100, 200, and 250 mM NaCl). The salt treatment caused a decrease in fluorescence, photochemical activity of PSII and PSI, as well as the protein content of the thylakoids. At high salt concentrations, the F735/F686 nm fluorescence ratio in M chloroplasts was reduced, while it was stimulated in BS chloroplasts. The photochemical activity of PSII was reduced in both chloroplasts, while there was no statistically significant difference in the activity of PSI compared to the control. According to the analysis of the protein content of the thylakoid membranes of M and BS chloroplasts, polypeptides belonging to the core antenna of PSII (47 kDa and 43 kDa) and LHCII (28-24 kDa) were present in both types of membranes, but their intensity was weak in BS thylakoids. The synthesis of the 68 kDa apoprotein belonging to the core of PSI was inhibited in the M membranes. There was no noticeable change in the membrane system of BS thylakoids. Salt stress had a greater impact on the ultrastructure of M chloroplasts than on BS ones and caused the formation of granal stacking in BS chloroplast. These results may indicate different responses of the two chloroplast types to salt stress.