Factors affecting the cleavage efficiency of the CRISPR-Cas9 system.

The CRISPR-Cas system stands out as a promising genome editing tool due to its cost-effectiveness and time efficiency compared to other methods. This system has tremendous potential for treating various diseases, including genetic disorders and cancer, and promotes therapeutic research for a wide range of genetic diseases. Additionally, the CRISPR-Cas system simplifies the generation of animal models, offering a more accessible alternative to traditional methods. The CRISPR-Cas9 system can be used to cleave target DNA strands that need to be corrected, causing double-strand breaks (DSBs). DNA with DSBs can then be recovered by the DNA repair pathway that the CRISPR-Cas9 system uses to edit target gene sequences. High cleavage efficiency of the CRISPR-Cas9 system is thus imperative for effective gene editing. Herein, we explore several factors affecting the cleavage efficiency of the CRISPR-Cas9 system. These factors include the GC content of the protospacer-adjacent motif (PAM) proximal and distal regions, single-guide RNA (sgRNA) properties, and chromatin state. These considerations contribute to the efficiency of genome editing.

Precise base editing without unintended indels in human cells and mouse primary myoblasts.

Base editors are powerful tools for making precise single-nucleotide changes in the genome. However, they can lead to unintended insertions and deletions at the target sites, which is a significant limitation for clinical applications. In this study, we aimed to eliminate unwanted indels at the target sites caused by various evolved base editors. Accordingly, we applied dead Cas9 instead of nickase Cas9 in the base editors to induce accurate substitutions without indels. Additionally, we tested the use of chromatin-modulating peptides in the base editors to improve nucleotide conversion efficiency. We found that using both dead Cas9 and chromatin-modulating peptides in base editing improved the nucleotide substitution efficiency without unintended indel mutations at the desired target sites in human cell lines and mouse primary myoblasts. Furthermore, the proposed scheme had fewer off-target effects than conventional base editors at the DNA level. These results indicate that the suggested approach is promising for the development of more accurate and safer base editing techniques for use in clinical applications.

Role of the Circadian Clock and Effect of Time-Restricted Feeding in Adenine-Induced Chronic Kidney Disease.

Most physiological functions exhibit circadian rhythmicity that is partly regulated by the molecular circadian clock. Herein, we investigated the relationship between the circadian clock and chronic kidney disease (CKD). The role of the clock gene in adenine-induced CKD and the mechanisms of interaction were investigated in mice in which Bmal1, the master regulator of the clock gene, was knocked out, and Bmal1 knockout (KO) tubule cells. We also determined whether the renoprotective effect of time-restricted feeding (TRF), a dietary strategy to enhance circadian rhythm, is clock gene-dependent. The mice with CKD showed altered expression of the core clock genes with a loss of diurnal variations in renal functions and key tubular transporter gene expression. Bmal1 KO mice developed more severe fibrosis, and transcriptome profiling followed by gene ontology analysis suggested that genes associated with the cell cycle, inflammation, and fatty acid oxidation pathways were significantly affected in the mutant mice. Tubule-specific deletion of BMAL1 in HK-2 cells by CRISPR/Cas9 led to upregulation of p21 and tumor necrosis α and exacerbated epithelial-mesenchymal transition-related gene expression upon transforming growth factor ß stimulation. Finally, TRF in the mice with CKD partially restored the disrupted oscillation of the kidney clock genes, accompanied by improved cell cycle arrest and inflammation, leading to decreased fibrosis. However, the renoprotective effect of TRF was abolished in Bmal1 KO mice, suggesting that TRF is partially dependent on the clock gene. Our data demonstrate that the molecular clock system plays an important role in CKD via cell cycle regulation and inflammation. Understanding the role of the circadian clock in kidney diseases can be a new research field for developing novel therapeutic targets.

Subcellular progression of mesenchymal transition identified by two discrete synchronous cell lines derived from the same glioblastoma.

Glioblastomas (GBM) exhibit intratumoral heterogeneity of various oncogenic evolutional processes. We have successfully isolated and established two distinct cancer cell lines with different morphological and biological characteristics that were derived from the same tissue sample of a GBM. When we compared their genomic and transcriptomic characteristics, each cell line harbored distinct mutation clusters while sharing core driver mutations. Transcriptomic analysis revealed that one cell line was undergoing a mesenchymal transition process, unlike the other cell line. Furthermore, we could identify four tumor samples containing our cell line-like clusters from the publicly available single-cell RNA-seq data, and in a set of paired longitudinal GBM samples, we could confirm three pairs where the recurrent sample was enriched in the genes specific to our cell line undergoing mesenchymal transition. The present study provides direct evidence and a valuable source for investigating the ongoing process of subcellular mesenchymal transition in GBM, which has prognostic and therapeutic implications.

Serial measurements of body composition using bioelectrical impedance and clinical usefulness of phase angle in colorectal cancer.

BACKGROUND: Although body composition (BC) can be measured easily using bioelectrical impedance analysis (BIA), there are few studies of serial BC measurements in colorectal cancer (CRC). The purpose of the present study was to observe the serial change of BC in patients with CRC surgery from the initiation to the end of chemotherapy and to evaluate its clinical usefulness. METHODS: From July 2018 to November 2019, patients undergoing elective CRC surgery were enrolled. All clinical data were reviewed retrospectively. BIA data were collected prospectively at four time points (initial, discharge day, first chemotherapy, and 6 months later). BC was measured using a commercial BIA device. RESULTS: A total of 160 patients were enrolled, and 110 (68.8%) patients were followed. Most BC measurements, such as weight, body mass index, skeletal muscle mass, skeletal muscle index, and fat mass index, were lowest at the first chemotherapy and rebounded after 6 months. Phase angle (PhA) and the ratio of extracellular water to total body water (ECW/TBW) were "V" shaped and inverted "V" shaped, respectively, and the peaks were on discharge days. This pattern of BC showed significant difference according to sarcopenia, old age (>70 years), and advanced stage (III or IV). The change of PhA and ECW/TBW sensitively pattern differences according to clinical aspect. CONCLUSIONS: Using BIA, serial BC measurements were taken to establish a pattern based on clinical characteristics. PhA showed the most sensitive change according to the patient's clinical aspect.

Clinical effect of multimodal perioperative pain management protocol for minimally invasive colorectal cancer surgery: Propensity score matching study.

BACKGROUND: Reducing postoperative pain with less opioid is critical in postoperative care. Author developed our multimodal perioperative pain management protocol and it consists of preoperative medication, intraoperative ultrasound-guided laparoscopic transverse abdominis plane (LTAP) block and postoperative medication. This study aimed to evaluate the clinical effect of the multimodal perioperative pain management protocol for minimally invasive colorectal cancer surgery. METHODS: Of 596 colorectal surgery cases for colorectal cancer, 133 patients managed with multimodal perioperative pain protocol (group 1) and 463 patients managed without multimodal perioperative pain protocol (group 2) were enrolled in this study. To adjust for baseline differences and selection bias, operative outcomes and complications were compared after propensity score matching (PSM). RESULTS: After 1:1 propensity score matching, well-matched 133 patients in each group were evaluated. The median VAS scores on post-operative day 1 (2.1 ± 1.1 vs. 3.9 ± 1.8, p < 0.001) and day 2 (2.0 ± 1.2 vs. 3.8 ± 1.7, p < 0.001) was significantly reduced in group 1. The length of postoperative hospital stays was also significantly shorter in Group 1 (4.4 ± 3.0 vs. 5.8 ± 5.6; p = 0.014). CONCLUSION: Implementing multimodal perioperative pain protocols reduced postoperative pain and hospital stay of minimally invasive colorectal surgery.

Inhibition of MUC1 exerts cell-cycle arrest and telomerase suppression in glioblastoma cells.

Mucin 1 (MUC1) is a transmembrane glycoprotein involved in tumorigenesis of diverse cancers. However, the role of MUC1 in glioblastoma (GBM) has not yet been fully explored. In this study, the anticancer mechanism of MUC1 suppression in GBM was investigated. The expression level of MUC1 was analyzed in human glioma and paired normal brain tissues. MUC1 was overexpressed in GBM and was negatively associated with overall survival. Moreover, we silenced MUC1 to investigate its effect in GBM cell lines and found that knockdown of MUC1 inhibited cell proliferation and resulted in cell cycle arrest at G1 phase. MUC1 silencing decreased the phosphorylation of RB1 and increased the expression of CDKN1B. Gene set enrichment analysis showed that a series of genes related to cell cycle, telomere maintenance and transforming growth factor Beta (TGF-ß) signaling in epithelial mesenchymal transition (EMT) were influenced by MUC1 knockdown. Notably, the reduced TERT expression levels combined with impaired telomerase activity and the switching of telomere maintenance mechanism to alternative lengthening of telomeres (ALT) were observed after MUC1 knockdown. Our results support the role of MUC1 in oncological process in GBM which can be developed as a therapeutic target for cell cycle control and telomere maintenance mechanism.