DNA Methylation Is Dispensable for the Growth and Survival of the Extraembryonic Lineages

SummaryDNA methylation regulates development and many epigenetic processes in mammals [1], and it is required for somatic cell growth and survival [2, 3]. In contrast, embryonic stem (ES) cells can self-renew without DNA methylation [4–6]. It remains unclear whether any lineage-committed cells can survive without DNA-methylation machineries. Unlike in somatic cells, DNA methylation is dispensable for imprinting and X-inactivation in the extraembryonic lineages [7–12]. In ES cells, DNA methylation prevents differentiation into the trophectodermal fate [13]. Here, we created triple-knockout (TKO) mouse embryos deficient for the active DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b (TKO) by nuclear transfer (NT), and we examined their development. In chimeric TKO-NT and WT embryos, few TKO cells were found in the embryo proper, but they contributed to extraembryonic tissues. TKO ES cells showed increasing cell death during their differentiation into epiblast lineages, but not during differentiation into extraembryonic lineages. Furthermore, we successfully established trophoblastic stem cells (ntTS cells) from TKO-NT blastocysts. These TKO ntTS cells could self-renew, and they retained the fundamental gene expression patterns of stem cells. Our findings indicated that extraembryonic-lineage cells can survive and proliferate in the absence of DNA methyltransferases and that a cell's response to the stress of epigenomic damage is cell type dependent

Drug retention of biologics and Janus kinase inhibitors in patients with rheumatoid arthritis: the ANSWER cohort study

OBJECTIVES: This multicentre retrospective study in Japan aimed to assess the retention of biological disease-modifying antirheumatic drugs and Janus kinase inhibitors (JAKi), and to clarify the factors affecting their retention in a real-world cohort of patients with rheumatoid arthritis. METHODS: The study included 6666 treatment courses (bDMARD-naïve or JAKi-naïve cases, 55.4%; tumour necrosis factor inhibitors (TNFi) = 3577; anti-interleukin-6 receptor antibodies (aIL-6R) = 1497; cytotoxic T lymphocyte-associated antigen-4-Ig (CTLA4-Ig) = 1139; JAKi=453 cases). The reasons for discontinuation were divided into four categories (ineffectiveness, toxic adverse events, non-toxic reasons and remission); multivariate Cox proportional hazards modelling by potential confounders was used to analyse the HRs of treatment discontinuation. RESULTS: TNFi (HR=1.93, 95% CI: 1.69 to 2.19), CTLA4-Ig (HR=1.42, 95% CI: 1.20 to 1.67) and JAKi (HR=1.29, 95% CI: 1.03 to 1.63) showed a higher discontinuation rate due to ineffectiveness than aIL-6R. TNFi (HR=1.28, 95% CI: 1.05 to 1.56) and aIL-6R (HR=1.27, 95% CI: 1.03 to 1.57) showed a higher discontinuation rate due to toxic adverse events than CTLA4-Ig. Concomitant use of oral glucocorticoids (GCs) at baseline was associated with higher discontinuation rate due to ineffectiveness in TNFi (HR=1.24, 95% CI: 1.09 to 1.41), as well as toxic adverse events in JAKi (HR=2.30, 95% CI: 1.23 to 4.28) and TNFi (HR=1.29, 95%CI: 1.07 to 1.55). CONCLUSIONS: TNFi (HR=1.52, 95% CI: 1.37 to 1.68) and CTLA4-Ig (HR=1.14, 95% CI: 1.00 to 1.30) showed a higher overall drug discontinuation rate, excluding non-toxicity and remission, than aIL-6R.Ebina K., Etani Y., Maeda Y., et al. Drug retention of biologics and Janus kinase inhibitors in patients with rheumatoid arthritis: the ANSWER cohort study. RMD open 9, (2023); https://doi.org/10.1136/rmdopen-2023-003160

doi:10.1093/nar/gkn294 QUMA: quantification tool for methylation analysis

Bisulfite sequencing, a standard method for DNA methylation profile analysis, is widely used in basic and clinical studies. This method is limited, however, by the time-consuming data analysis processes required to obtain accurate DNA methylation profiles from the raw sequence output of the DNA sequencer, and by the fact that quality checking of the results can be influenced by a researcher’s bias. We have developed an interactive and easy-to-use webbased tool, QUMA (quantification tool for methylation analysis), for the bisulfite sequencing analysis of CpG methylation. QUMA includes most of the dataprocessing functions necessary for the analysis of bisulfite sequences. It also provides a platform for consistent quality control of the analysis. The QUMA web server is available a

Ozone ultrafine bubble water induces the cellular signaling involved in oxidative stress responses in human periodontal ligament fibroblasts

Periodontitis is a chronic inflammatory disease caused by oral microorganisms in the subgingival biofilm. Stable aqueous ozone ultrafine bubble water (OUFBW) has recently begun to be used as an antiseptic in the treatment of periodontitis. The effectiveness of OUFBW is thought to depend on the bactericidal actions of dissolved ozone exerted via its oxidizing effect. On the other hand, the effects of ozone on the periodontal tissues are largely unknown. In this paper we examined the cellular responses after OUFBW treatment. Human primary periodontal ligament fibroblasts (hPDLFs) or Ca9-22 human gingival epithelial cells were treated with OUFBW or UV-inactivated OUFBW. The production of reactive oxygen species (ROS), the activation of mitogen-activated protein kinase (MAPK) and the nuclear factor-kappa B (NF-κB) activation were analyzed. The transcript profiles of hPDLFs after OUFBW treatment were also analyzed by RNA sequencing (RNA-seq). Our results showed that OUFBW induces oxidative stress by generating ROS, which, in turn, activated the MAPK pathway. OUFBW triggered activation of c-Fos, a major component of the transcription factor activator protein 1 (AP-1), and also nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2), which possessed a high sensitivity to oxidative stress. The results of RNA-seq analysis revealed that the numerous genes involved in oxidative stress responses or MAPK signaling pathway were up-regulated after OUFBW treatment. Investigation of the signaling pathways activated by OUFBW highlights another aspect of the biological roles of OUFBW, in addition to its bactericidal activity, in the treatment of periodontitis