KCNJ13 Gene Deletion Impairs Cell Alignment and Phagocytosis in Retinal Pigment Epithelium Derived from Human-Induced Pluripotent Stem Cells

Purpose: The purpose of this study was to establish and analyze a cell model of Leber congenital amaurosis type 16 (LCA16), which is caused by mutations in the KCNJ13 gene encoding Kir7.1, an inward-rectifying potassium ion channel. Methods: The two guide RNAs specific to the target sites in the KCNJ13 gene were designed and KCNJ13 knock-out (KO) human-induced pluripotent stem cells (hiPSCs) were generated using the CRISPR/Cas9 system. The KCNJ13-KO hiPSCs were differentiated into retinal pigment epithelial cells (hiPSC-RPEs). The KCNJ13-KO in hiPSC-RPEs was confirmed by immunostaining. Phagocytic activity of hiPSC-RPEs was assessed using the uptake of fluorescently labeled porcine photoreceptor outer segments (POSs). Phagocytosis-related genes in RPE cells were assessed by quantitative polymerase chain reaction. Results: Most of the translated region of the KCNJ13 gene was deleted in the KCNJ13-KO hiPSCs by the CRISPR/Cas9 system, and this confirmed that the Kir7.1 protein was not present in RPE cells induced from the hiPSCs. Expression of RPE marker genes such as BEST1 and CRALBP was retained in the wild-type (WT) and in the KCNJ13-KO hiPSC-RPE cells. However, phagocytic activity and expression of phagocytosis-related genes in the KCNJ13-null hiPSC-RPE cells were significantly reduced compared to those of WT. Conclusions: We succeeded in generating an RPE model of LCA16 using hiPSCs. We suggest that Kir7.1 is required for phagocytosis of POSs by RPE cells and that impaired phagocytosis in the absence of Kir7.1 would be involved in the retinal degeneration found in LCA16

Detection of periodic patterns in microarray data reveals novel oscillating transcripts of biological rhythms in Ciona intestinalis

A circadian rhythm is a roughly 24-h cycle in biological processes and physiological phenomena such as sleep, feeding, and photosynthesis for many organisms on Earth. The circadian patterns are coordinated by rhythmical gene expression of clock genes. Time-course transcriptomic analyses involving statistical methods have shown coordination of periodic gene expression in many organisms. Here we applied the cosine fitting method COSOPT to identify novel oscillating genes in microarray data for the chordate Ciona intestinalis. This organism showed rhythmic oxygen consumption in our previous study, but there were few homologous clock genes showing rhythmic mRNA expression. To understand circadian behavior at the transcriptomic level, we analyzed the 817 of 21,938 probes showing a 23- to 25-h period by means of COSOPT. Coupling the analysis of period detection with functional annotations indicated that previously unknown rhythmic mRNA expression might exist in C. intestinalis. In addition, we are releasing our implementation of COSOPT by means of R and C. All source code and supplementary information are available from https://github.com/mhiromi/cosopt

A study on the effect of teachers' guidance for student teachers at Shinonome Elementary & Jr. High schools, based on statistical analysis of pre- & post-questionnaires (II).

本研究は昨年度の同名研究(I)に引き続き学校単位での実習指導の効果検討を目的として行った。実習を通した実習生の意識変容と実習指導の目標達成程度等から指導効果を分析するため,附属東雲小および東雲中を調査校として実習生対象に事前,事後アンケートを行った。事前アンケートでは両校とも,教員として必要な資質・能力・技能と実習で実践すべきことに関する22項目それぞれについて自信の程度を5件法,事後では事前と同じ項目について自信の獲得程度を5件法で質問した。事前,事後で回答者をペアとして対応させ個々の変容を解析したところ殆ど全ての項目で実習後に高まっていた。また,事後アンケートにおける実習指導内容項目の有効感に関する回答(4件法)および教職に対する意欲高揚に関する回答(4件法)は両校とも高いレベルであった。また実習指導の各項目の回答分布と意欲高揚に関する回答分布の関連を解析した結果,両校とも授業に直接関わる指導に有意な関連が見いだされた。特に中学校では実習生が授業の評価について自信を得ており,かつ,授業評価指導に対する有効感と教職への意欲向上との有意な関連が明らかとなった

Detection of periodic patterns in microarray data reveals novel oscillating transcripts of biological rhythms in Ciona intestinalis

A circadian rhythm is a roughly 24-h cycle in biological processes and physiological phenomena such as sleep, feeding, and photosynthesis for many organisms on Earth. The circadian patterns are coordinated by rhythmical gene expression of clock genes. Time-course transcriptomic analyses involving statistical methods have shown coordination of periodic gene expression in many organisms. Here we applied the cosine fitting method COSOPT to identify novel oscillating genes in microarray data for the chordate Ciona intestinalis. This organism showed rhythmic oxygen consumption in our previous study, but there were few homologous clock genes showing rhythmic mRNA expression. To understand circadian behavior at the transcriptomic level, we analyzed the 817 of 21,938 probes showing a 23- to 25-h period by means of COSOPT. Coupling the analysis of period detection with functional annotations indicated that previously unknown rhythmic mRNA expression might exist in C. intestinalis. In addition, we are releasing our implementation of COSOPT by means of R and C. All source code and supplementary information are available from https://github.com/mhiromi/cosopt