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Protein kinase CK2 downregulation induces premature senescence in various human cell types via activation of the reactive oxygen species (ROS)-p53-p21Cip1/WAF1 pathway. The transcription factor “nuclear factor erythroid 2-related factor 2” (Nrf2) plays an important role in maintaining intracellular redox homeostasis. In this study, Nrf2 overexpression attenuated CK2 downregulation-induced ROS production and senescence markers including SA-β-gal staining and activation of p53-p21Cip1/WAF1 in human breast (MCF-7) and colon (HCT116) cancer cells. CK2 downregulation reduced the transcription of Nrf2 target genes, such as glutathione S-transferase, glutathione peroxidase 2, and glutathione reductase 1. Furthermore, CK2 downregulation destabilized Nrf2 protein via inhibiting autophagic degradation of Kelch-like ECH-associated protein 1 (Keap1). Finally, CK2 downregulation decreased the nuclear import of Nrf2 by deactivating AMP-activated protein kinase (AMPK). Collectively, our data suggest that both Keap1 stabilization and AMPK inactivation are associated with decreased activity of Nrf2 in CK2 downregulation-induced cellular senescence. [BMB Reports 2020; 53(5): 272-277]

Thermoanaerobacter thermocopriae-derived thermostable cycloisomaltooligosaccharide (CI)-forming enzymes catalyze the production of CIs from dextran. The primary structure of the enzyme is comprised of CI glucanotransferase (TtCITase) at the N-terminal region and long isomaltooligosaccharide-forming enzyme (TtTGase) at the C-terminal region connected by carbohydrate-binding module family 35 (CBM, TtCBM). Three truncated mutants of CI-forming enzymes were successfully produced in Corynebacterium glutamicum, a food-grade host system, and their biochemical properties were characterized. The enzymes had optimum at pH 6.0 and pH-stability (5.0-12.0). Three enzymes had optimum temperature over 55 °C and they maintained 80% activity at 55 °C for 2 h, 12 h, and 18 h, respectively. Enzymes without CBM showed weaker allosteric behavior than those of other enzymes, which suggests the important role of CBM in allosteric behavior. However, CBM bearing enzymes showed high production of CIs with various degree of polymerization. These enzymes have potential application as the encapsulating material for insoluble pharmaceutical biomaterials.

The CRISPR/Cas9 system is widely applied in genome engineering due to its simplicity and versatility. Although this has revolutionized genome-editing technology, knockin animal generation via homology directed repair (HDR) is not as efficient as nonhomologous end-joining DNA-repair-dependent knockout. Although its double-strand break activity may vary, Cas9 derived from Streptococcus pyogenens allows robust design of single-guide RNAs (sgRNAs) within the target sequence; However, prescreening for different sgRNA activities delays the process of transgenic animal generation. To overcome this limitation, multiple sets of different sgRNAs were examined for their knockin efficiency. We discovered profound advantages associated with single-stranded oligo-donor-mediated HDR processes using overlapping sgRNAs (sharing at least five base pairs of the target sites) as compared with using non-overlapping sgRNAs for knock-in mouse generation. Studies utilizing cell lines revealed shorter sequence deletions near target mutations using overlapping sgRNAs as compared with those observed using non-overlapping sgRNAs, which may favor the HDR process. Using this simple method, we successfully generated several transgenic mouse lines harboring loxP insertions or single-nucleotide substitutions with a highly efficiency of 18–38%. Our results demonstrate a simple and efficient method for generating transgenic animals harboring foreign-sequence knockins or short-nucleotide substitutions by the use of overlapping sgRNAs., Genome editing: Improving efficiency by improving DNA repair A method to improve the efficiency of repairing double-stranded DNA breaks facilitates the production of animals with edited genomes. The CRISPR/Cas9 system has been revolutionising biomedical research as it allows the insertion or deletion of any DNA sequence at specific sites by making double-strand DNA breaks. However, the insertion of precise genetic changes is limited by the relatively low efficiency of the repair mechanism, which requires a template DNA molecule to promote error-free repair of the DNA strands. A team of South Korean scientists led by Su Cheong Yeom at Seoul National University and Han Woong Lee at Yonsei University show that delivering multiple overlapping single-guide RNAs (that share at least five base pairs of the target sequence) as a DNA template significantly improves the repair and thus the production of mice with a targeted gene insertion.

Alopecia areata (AA) is an autoimmune disease that results in spot baldness in humans. Adequate animal models for AA are currently lacking. The objective of this study was to elucidate the mechanism of autoimmune-like alopecia (ALA) in C57BL/6.CD80CD86-deficient (B6.CD80CD86-/- ) mice. Incidence and severity of alopecia were analysed in 58 B6.CD80CD86-/- mice using histological examination, flow cytometry, multiplex enzyme-linked immunosorbent assay, quantitative RT-PCR and CD25 inhibition test. Both male and female B6.CD80CD86-/- mice showed almost 100% incidence of hair loss at 40 weeks of age. Moreover, CD4+FoxP3+Treg (Treg) cell population in B6.CD80CD86-/- mice was significantly lower than in B6 mice, which presumably underlined autoimmune reaction. Histologically, B6.CD80CD86-/- mice showed CD4+ and CD8+ T-cell infiltration around terminal follicle region and exhibited hair follicle destruction in the anagen or catagen stage. Negative correlation between the number of CD4+FoxP3+ Tregs and ALA was confirmed by the CD25 depletion test in B6 mice, as follicle destruction was similar to that observed in B6.CD80CD86-/- animals. CD80CD86 deficiency disrupted CD4+FoxP3+ Treg homoeostasis and prompted the development of ALA. We demonstrated that B6.CD80CD86-/- mice might have several advantages as an ALA model, because they exhibited high incidence of disease phenotype and epipathogenesis similar to that observed in human AA.

Anagen effluvium develops because of disturbances in the hair follicle cycle, leading to acute and severe hair loss in humans. The objective of this study was to establish a mouse model of anagen effluvium by 5-bromo-2'-deoxyuridine (BrdU) treatment, and evaluate the pathological changes and underlying mechanisms. We treated 9-10-day-old pups and 3-7-week-old C57BL/6 mice with BrdU. After successfully inducing hair loss in the neonatal pups, microscopic, immunohistochemical and flow cytometry analyses were conducted. BrdU induced early onset alopecia in neonates and caused epidermal thickening and hair shaft breakage. BrdU appeared to incorporate the CD326-positive keratinocyte layer and induced p53-related apoptosis. Keratinocyte apoptosis caused immune cell infiltration in the dermal region; M2 macrophages and neutrophils were dominant. The BrdU-induced hair loss was dose-dependent, and alopecia was visible at a dose range of 25-200 μg/g bodyweight. The BrdU-induced anagen effluvium mouse model is novel and easily established by administrating four simple BrdU injections to pups; these mice showed synchronized onset of alopecia symptoms with little individual variation. Moreover, this model showed an alopecia phenotype similar to that of human anagen effluvium with acute, severe and widespread hair loss.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder involving changes in normal bowel movements. The pathophysiology of IBS is not clearly understood owing to the lack of identifiable pathological abnormalities and reliable biomarkers. The aim of this study was to discover the novel and reliable biomarker for IBS. In this study, neonatal maternal separation (NMS) stress model was used for the IBS mouse model. Further assessment was conducted with whole gastrointestinal transit test, quantitative RT-PCR, histological examination, and western blot. Male pups developed symptoms similar to those of human IBS with diarrhea (IBS-D), such as low-grade inflammation, stool irregularity, and increased bowel motility. NMS stress influenced to the interstitial cells of Cajal (ICC) and induced altered bowel motility, resulting in IBS-D-like symptoms. In addition, we found neuronal nitric oxide synthase (nNOS) to be a novel biomarker for ICC under NMS stress. nNOS expression was only observed in the ICC of the submucosal plexus of IBS-D mice, and the inhibition of nNOS changed the phenotype from IBS-D to IBS with constipation. Our study demonstrates that early-life stress can influence to ICC and modulate bowel activity and that nNOS might be used as a biomarker for ICC stimulation in IBS.

Some G-protein-coupled receptors have been reported to require accessory proteins with specificity for proper functional expression. In this study, we found that CXCR1 interacted with REEP5 and REEP6, but CXCR2 did not. Overexpression of REEP5 and REEP6 enhanced IL-8-stimulated cellular responses through CXCR1, whereas depletion of the proteins led to the downregulation of the responses. Although REEPs enhanced the expression of a subset of GPCRs, in the absence of REEP5 and REEP6, CXCR1 was expressed in the plasma membrane, but receptor internalization and intracellular clustering of β-arrestin2 following IL-8 treatment were impaired, suggesting that REEP5 and REEP6 might be involved in the ligand-stimulated endocytosis of CXCR1 rather than membrane expression, which resulted in strong cellular responses. In A549 lung cancer cells, which endogenously express CXCR1, the depletion of REEP5 and REEP6 significantly reduced growth and invasion by downregulating IL-8-stimulated ERK phosphorylation, actin polymerization and the expression of genes related to metastasis. Furthermore, an in vivo xenograft model showed that proliferation and metastasis of A549 cells lacking REEP5 and REEP6 were markedly decreased compared to the control group. Thus, REEP5 and REEP6 could be novel regulators of G-protein-coupled receptor signaling whose functional mechanisms differ from other accessory proteins.