Development of a rapamycin-inducible protein-knockdown system in the unicellular red alga Cyanidioschyzon merolae.

An inducible protein-knockdown system is highly effective for investigating the functions of proteins and mechanisms essential for the survival and growth of organisms. However, this technique is not available in photosynthetic eukaryotes. The unicellular red alga Cyanidioschyzon merolae possesses a very simple cellular and genomic architecture and is genetically tractable but lacks RNA interference machinery. In this study, we developed a protein-knockdown system in this alga. The constitutive system utilizes the destabilizing activity of the FRB domain of human target of rapamycin (TOR) kinase or its derivatives to knock down target proteins. In the inducible system, rapamycin treatment induces the heterodimerization of the human FKBP12-rapamycin binding (FRB) domain fused to the target proteins with the human FK506-binding protein 12 (FKBP) fused to S-phase kinase associated protein 1 (SKP1) or Cullin 1 (CUL1), subunits of the SCF E3 ubiquitin ligase. This results in the rapid degradation of the target proteins through the ubiquitin-proteasome pathway. With this system, we successfully degraded endogenous essential proteins such as the chloroplast division protein Dynamin related protein 5B (DRP5B) and E2 transcription factor (E2F), a regulator of the G1/S transition, within 2-3 hours after rapamycin administration, enabling the assessment of resulting phenotypes. This rapamycin-inducible protein-knockdown system contributes to the functional analysis of genes whose disruption leads to lethality.

Post-discharge sedentary behavior and light-intensity physical activity-associated stroke recurrence in patients with minor ischemic stroke: A preliminary retrospective observational study.

BACKGROUND AND PURPOSE: Evidence regarding whether reducing sedentary behavior and increasing physical activity levels to prevent stroke recurrence is insufficient. Therefore, this study preliminarily investigated whether post-discharge sedentary behavior and physical activity levels in patients with minor ischemic stroke were associated with stroke recurrence. METHODS: This retrospective observational study included 73 patients (aged 72.0 years) with minor ischemic stroke from a previous study. The outcome was recurrent stroke 2 years after stroke onset, assessed using medical records. Exposure factors including sedentary behavior and physical activity levels 6 months post-discharge were measured using accelerometers; patients were classified into the recurrence or non-recurrence groups. Logistic regression analyses were then conducted to determine whether sedentary behavior and physical activity 6 months after discharge were associated with stroke recurrence. RESULTS: Six patients experienced stroke recurrence (recurrence rate, 8.2%). The recurrence group showed greater sedentary behavior (recurrence group 68.0%, non-recurrence group 52.0%, p = 0.007) and less light-intensity physical activity (LPA) (recurrence group 21.0%, non-recurrence group 37.0%, p = 0.002) than in the non-recurrence group. Logistic regression analysis showed that sedentary behavior (odds ratio = 1.083, 95% confidence interval = 1.007-1.165, p = 0.032) and LPA (odds ratio = 0.874, 95% confidence interval = 0.785-0.975, p = 0.015) were independent factors for recurrence of stroke. DISCUSSION: Post-discharge sedentary behavior and LPA in patients with minor ischemic stroke were associated with stroke recurrence. Results suggest that reducing post-discharge sedentary behavior and increasing LPA may be crucial for reducing the risk of stroke recurrence in patients with minor ischemic stroke.

Selenoprotein P expression in glioblastoma as a regulator of ferroptosis sensitivity: preservation of GPX4 via the cycling-selenium storage.

Glioblastoma (GBM) is one of the most aggressive and deadly brain tumors; however, its current therapeutic strategies are limited. Selenoprotein P (SeP; SELENOP, encoded by the SELENOP gene) is a unique selenium-containing protein that exhibits high expression levels in astroglia. SeP is thought to be associated with ferroptosis sensitivity through the induction of glutathione peroxidase 4 (GPX4) via selenium supplementation. In this study, to elucidate the role of SeP in GBM, we analyzed its expression in GBM patients and found that SeP expression levels were significantly higher when compared to healthy subjects. Knock down of SeP in cultured GBM cells resulted in a decrease in GPX1 and GPX4 protein levels. Under the same conditions, cell death caused by RSL3, a ferroptosis inducer, was enhanced, however this enhancement was canceled by supplementation of selenite. These results indicate that SeP expression contributes to preserving GPX and selenium levels in an autocrine/paracrine manner, i.e., SeP regulates a dynamic cycling-selenium storage system in GBM. We also confirmed the role of SeP expression in ferroptosis sensitivity using patient-derived primary GBM cells. These findings indicate that expression of SeP in GBM can be a significant therapeutic target to overcome anticancer drug resistance.