The positional and numerical effect of N 6-methyladenosine in tracrRNA on the DNA cleavage activity of Cas9.

Post-transcriptional modifications on the guide RNAs utilized in the Cas9 system may have the potential to impact the activity of Cas9. In this study, we synthesized a series of tracrRNAs containing N 6-methyadenosine (m6A), a prevalent post-transcriptional modification, at various positions. We evaluated the effect of these modifications on the DNA cleavage activity of Cas9. Our results show that multiple m6As in the anti-repeat region of tracrRNA reduce the DNA cleavage activity of Cas9. This suggests that the m6A-modified tracrRNA can be used for Cas9 only when the number and the position of the modified residue are properly chosen in tracrRNA.

Inhibition of glutathione S-transferase omega 1-catalyzed protein deglutathionylation suppresses adipocyte differentiation.

Glutathione S-transferase omega 1 (GstO1) is closely associated with various human diseases, including obesity and diabetes, but its functional mechanism is not fully understood. In the present study, we found that the GstO1-specific inhibitor C1-27 effectively suppressed the adipocyte differentiation of 3T3-L1 preadipocytes. GstO1 expression was immediately upregulated upon the induction of adipocyte differentiation, and barely altered by C1-27. However, C1-27 significantly decreased the stability of GstO1. Moreover, GstO1 catalyzed the deglutathionylation of cellular proteins during the early phase of adipocyte differentiation, and C1-27 inhibited this activity. These results demonstrate that GstO1 is involved in adipocyte differentiation by catalyzing the deglutathionylation of proteins critical for the early phase of adipocyte differentiation. [BMB Reports 2023; 56(8): 457-462].

Split-tracrRNA as an efficient tracrRNA system with an improved potential of scalability.

Due to the relatively long sequence, tracrRNAs are chemically less synthesizable than crRNAs, leading to limited scalability of RNA guides for CRISPR-Cas9 systems. To develop shortened versions of RNA guides with improved cost-effectiveness, we have developed a split-tracrRNA system by nicking the 67-mer tracrRNA (tracrRNA(67)). Cellular gene editing assays and in vitro DNA cleavage assays revealed that the position of the nick is critical for maintaining the activity of tracrRNA(67). TracrRNA(41 + 23), produced by nicking in stem loop 2, showed gene editing efficiency and specificity comparable to those of tracrRNA(67). Removal of the loop of stem loop 2 was further possible without compromising the efficiency and specificity when the stem duplex was stabilized via a high GC content. Binding assays and single-molecule experiments suggested that efficient split-tracrRNAs could be engineered as long as their binding affinity to Cas9 and their reaction kinetics are similar to those of tracrRNA(67).

Adipogenic function of tetranectin mediated by enhancing mitotic clonal expansion via ERK signaling.

Tetranectin (TN), an adipogenic serum protein, enhances adipocyte differentiation, however, its functional mechanism has yet to be elucidated. In the present study, we investigated the adipogenic function of TN by using medium containing TN-depleted fetal bovine serum (TN-del-FBS) and recombinant mouse TN (mTN). The adipocyte differentiation of 3T3-L1 cells was significantly enhanced by mTN supplementation essentially at differentiation induction, which indicated a potential role of the protein in the early differentiation phase. The adipogenic effect of mTN was more significant with insulin in the differentiation induction cocktail, implicating their close functional relationship. mTN enhanced not only the proliferation of growing cells, but also mitotic clonal expansion (MCE) that is a prerequisite for adipocyte differentiation in the early phase. Consistently, mTN increased the phosphorylation of ERK in the early phase of adipocyte differentiation. Results of this study demonstrate that the adipogenic function of mTN is mediated by enhancing MCE via ERK signaling. [BMB Reports 2021; 54(7): 374-379].

Polysaccharide from Codium fragile Induces Anti-Cancer Immunity by Activating Natural Killer Cells.

Natural polysaccharides exhibit beneficial immune modulatory effects, including immune stimulatory and anti-cancer activities. In this study, we examined the effect of Codium fragile polysaccharide (CFP) on natural killer (NK) cell activation, and its effect on tumor-bearing mice. Intravenous CFP treatment of C57BL/6 mice resulted in the upregulation of CD69, which is a marker associated with NK cell activation. In addition, intracellular levels of interferon (IFN)-γ and the cytotoxic mediators perforin and granzyme B were markedly increased in response to the CFP treatment of splenic NK cells. IFN-γ production by NK cells was directly induced by CFP, whereas the upregulation of CD69 and cytotoxic mediators required IL-12. Finally, intraperitoneal treatment with CFP prevented CT-26 (murine carcinoma) tumor cell infiltration in the lungs, without significantly reducing the body weight. In addition, treatment with CFP prevented B16 melanoma cell infiltration in the lung of C57BL/6 mice. Moreover, the anti-tumor effect was diminished by the depletion of NK cells. Therefore, these data suggest that CFP may be used as an NK cell stimulator to produce a phenomenon that contributes to anti-cancer immunity.

Human Peripheral Blood Dendritic Cell and T Cell Activation by Codium fragile Polysaccharide.

Natural polysaccharides exhibit an immunostimulatory effect with low toxicity in humans and animals. It has shown that polysaccharide extracted from Codium fragile (CFP) induces anti-cancer immunity by dendritic cell (DC) activation, while the effect of CFP has not examined in the human immune cells. In this study, we found that CFP promoted the upregulation of CD80, CD83 and CD86 and major histocompatibility complex (MHC) class I and II in human monocyte-derived dendritic cells (MDDCs). In addition, CFP induced the production of proinflammatory cytokines in MDDCs. Moreover, CFP directly induced the activation of Blood Dendritic Cell Antigen (BDCA)1+ and BDCA3+ subsets of human peripheral blood DCs (PBDCs). The CFP-stimulated BDCA1+ PBDCs further promoted activation and proliferation of syngeneic CD4 T cells. The CFP-activated BDCA3+ PBDCs activated syngeneic CD8 T cells, which produced cytotoxic mediators, namely, cytotoxic T lymphocytes. These results suggest that CFP may be a candidate molecule for enhancing immune activation in humans.

Highly sensitive and straightforward methods for the detection of cyanide using profluorescent glutathionylcobalamin.

The extreme toxicity of cyanide and its continued use in various industries have raised concerns over environmental contamination and, therefore, considerable attention has given to develop facile and sensitive methods of cyanide detection. In this study, we developed highly sensitive and straightforward methods of cyanide detection using eosin-labeled glutathionylcobalamin (E-GSCbl) containing fluorescent eosin-labeled glutathione (E-GSH) as the upper axial ligand to the cobalt. E-GSH fluorescence was strongly quenched in E-GSCbl. The E-GSH ligand of E-GSCbl was replaced specifically by cyanide, showing recovery of the E-GSH fluorescence. This profluorescent property of E-GSCbl enabled detection of cyanide in aqueous solutions, yielding a lower detection limit of 10 nM (0.26 µg L-1). Moreover E-GSH exhibited strong luminescence under UV-light that was quenched in E-GSCbl, and this allowed naked-eye detection of cyanide at concentrations as low as 100 nM. This study demonstrates that profluorescent E-GSCbl is a highly sensitive cyanide chemosensor that can detect nanomolar concentrations of cyanide.