3 research outputs found
Smc5 depletion impedes cell cycle progression, induces DNA damage, and causes genomic instability in mouse embryonic fibroblasts
Introduction:
The structural maintenance of chromosomes 5/6 (Smc5/6) complex plays a critical role in maintaining genomic integrity. More specifically, Smc5/6 is involved in DNA replication, DNA damage repair via homologous recombination (HR), and chromosome segregation. Although its function has been extensively studied in yeast, few studies have evaluated Smc5/6 in mammalian models. Based on existing literature, we hypothesized that Smc5/6-deficient mouse embryonic fibroblasts (MEF) would accumulate DNA damage and, as a result, demonstrate abnormal mitotic progression and show evidence of replication stress.
Methods:
We used transgenic mice harboring a floxed exon 4 of the Smc5 gene (Smc5flox/flox and Smc5+/del, Ert2-Cretg/0 ) to breed and establish immortalized MEF cell lines with genotypes of Smc5flox/del, Ert2-Cretg/0 (experimental), Smc5+/flox, Ert2-Cretg/0 (control #1), and Smc5flox/del (control #2). Smc5 exon 4 was deleted by addition of 0.2µM 4-OH tamoxifen for nine days. Deletion was confirmed by PCR and protein depletion by western blot. Cells were analyzed on day 3, 6 and 9.
MEF growth characteristics and cell cycle progression were evaluated by performing cell counts and FACS analysis, respectively. We also used immunofluorescence microscopy to observe micronuclei formation and DNA bridges. Additionally, we analyzed Rad51, Sumo1, and Sumo2/3 after treating cells with hydroxyurea. Finally, we used western blot analysis to evaluate expression of the stress response marker, p53.
Results:
Smc5-depleted MEFs demonstrated several mitotic abnormalities. After six days of 4-OH tamoxifen treatment, we observed a sustained, two-fold decrease in cell proliferation compared to controls. FACS analysis showed delayed entry into S phase. DAPI staining of Smc5-depleted cells showed 12% increase in micronuclei formation and 33% increase in DNA bridges. Hydroxyurea treated cells showed an accumulation of Rad51 foci, suggesting impaired HR mechanisms. Mutation of Smc5 also resulted in a decline in Sumo1 but not Sumo2/3 foci. Lastly, western blot analysis showed significant p53 upregulation.
Conclusions:
For the first time, we have demonstrated the importance of the Smc5/6 complex in somatic mouse cells. Smc5 depletion in MEF cells compromises genomic integrity, affects cell cycle progression and leads to chromosome missegregation. We also demonstrate hypersensitivity to DNA damage agents and activation of the p53 pathway
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Brain health consequences of digital technology use
Emerging scientific evidence indicates that frequent digital technology use has a significant impact-both negative and positive-on brain function and behavior. Potential harmful effects of extensive screen time and technology use include heightened attention-deficit symptoms, impaired emotional and social intelligence, technology addiction, social isolation, impaired brain development, and disrupted sleep. However, various apps, videogames, and other online tools may benefit brain health. Functional imaging scans show that internet-naive older adults who learn to search online show significant increases in brain neural activity during simulated internet searches. Certain computer programs and videogames may improve memory, multitasking skills, fluid intelligence, and other cognitive abilities. Some apps and digital tools offer mental health interventions providing self-management, monitoring, skills training, and other interventions that may improve mood and behavior. Additional research on the positive and negative brain health effects of technology is needed to elucidate mechanisms and underlying causal relationships.

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Expression-Based Cell Lineage Analysis in Drosophila Through a Course-Based Research Experience for Early Undergraduates.
A variety of genetic techniques have been devised to determine cell lineage relationships during tissue development. Some of these systems monitor cell lineages spatially and/or temporally without regard to gene expression by the cells, whereas others correlate gene expression with the lineage under study. The GAL4 Technique for Real-time and Clonal Expression (G-TRACE) system allows for rapid, fluorescent protein-based visualization of both current and past GAL4 expression patterns and is therefore amenable to genome-wide expression-based lineage screens. Here we describe the results from such a screen, performed by undergraduate students of the University of California, Los Angeles (UCLA) Undergraduate Research Consortium for Functional Genomics (URCFG) and high school summer scholars as part of a discovery-based education program. The results of the screen, which reveal novel expression-based lineage patterns within the brain, the imaginal disc epithelia, and the hematopoietic lymph gland, have been compiled into the G-TRACE Expression Database (GED), an online resource for use by the Drosophila research community. The impact of this discovery-based research experience on student learning gains was assessed independently and shown to be greater than that of similar programs conducted elsewhere. Furthermore, students participating in the URCFG showed considerably higher STEM retention rates than UCLA STEM students that did not participate in the URCFG, as well as STEM students nationwide