Biology subjects

Takahashi, G.

Publications and source records attributed to Takahashi, G..

3 recordsLinked to original sources

bioRxiv2021

Retinoids rescue ceruloplasmin secretion and alleviate oxidative stress in Wilson's disease-specific hepatocytes

Wilsons disease (WD) is a copper metabolic disorder, which is caused by defective ATP7B function. Here, we have generated induced pluripotent stem cells (iPSCs) from WD patients carrying compound heterozygous mutations on ATP7B. ATP7B loss- and gain-of-functions were further manifested with ATP7B-deficient iPSCs and heterozygously-corrected R778L WD patient-derived iPSCs using CRISPR-Cas9-based gene editing. Transcriptome analysis identified abnormalities of retinoid signaling pathway and lipid metabolism in WD-specific hepatocytes. Although the expression level of ATP7B protein was variable among WD-specific hepatocytes, the expression and secretion of ceruloplasmin (Cp), which is a downstream copper carrier in plasma, were consistently decreased. Cp secretion-based drug screening identified all-trans retinoic acid (ATRA) as promising candidates for rescuing Cp secretion. ATRA also alleviated reactive oxygen species (ROS) production induced by lipid accumulation in WD-specific hepatocytes. Our patient-derived iPSC-based hepatic models provide potential therapeutics for liver steatosis in WD and other fatty liver diseases.

cell biology↗

bioRxiv2021

An evolutionarily conserved coreceptor gene is essential for CLAVATA signaling in Marchantia polymorpha

Growth and development of land plants are controlled by CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) family of peptide hormones. In contrast to the genetic diversity of CLE family in flowering plants, the liverwort Marchantia polymorpha possesses a minimal set of CLE, MpCLE1(TDIF homolog) and MpCLE2 (CLV3 homolog). MpCLE1 and MpCLE2 peptides exert distinct function at the apical meristem of M. polymorpha gametophyte via specific receptors, MpTDIF RECEPTOR (MpTDR) and MpCLAVATA1 (MpCLV1), respectively, both belonging to the subclass XI of leucine-rich repeat receptor-like kinases (LRR-RLKs). Biochemical and genetic studies in Arabidopsis have shown that TDR/PXY family and CLV1/BAM family recognize the CLE peptide ligand in a heterodimeric complex with a member of subclass-II coreceptors. Here we show that three LRR-RLK genes of M. polymorpha are classified into subclass II, representing three distinct subgroups evolutionarily conserved in land plants. To address the involvement of subclass-II coreceptors in M. polymorpha CLE signaling, we performed molecular genetic analysis on one of them, MpCLAVATA3 INSENSITIVE RECEPTOR KINASE (MpCIK). Two knockout alleles for MpCIK formed narrow apical meristems marked by promMpYUC2:GUS marker, which were not expanded by MpCLE2 peptide treatment, phenocopying Mpclv1. Loss of sensitivity to MpCLE2 peptide was also observed in gemma cup formation in both Mpclv1 and Mpcik. Biochemical analysis using a Nicotiana benthamiana transient expression system revealed weak association between MpCIK and MpCLV1, as well as MpCIK and MpTDR. While MpCIK may also participate in MpCLE1 signaling, our data show that the conserved CLV3-CLV1-CIK module functions in M. polymorpha, controlling meristem activity for development and organ formation for asexual reproduction.

plant biology↗

bioRxiv2020

Rapid, precise quantification of large DNA excisions and inversions by ddPCR

The excision of genomic sequences using paired CRISPR-Cas nucleases is a powerful tool to study gene function, create disease models and holds promise for therapeutic gene editing. However, our understanding of the factors that favor efficient excision is limited by the lack of a rapid, accurate measurement of DNA excision outcomes that is free of amplification bias. Here, we introduce ddXR (droplet digital PCR eXcision Reporter), a method that enables the accurate and sensitive detection of excisions and inversions independent of length. The method can be completed in a few hours without the need for next-generation sequencing. The ddXR method uncovered unexpectedly high rates of large (>20 kb) excisions and inversions, while also revealing a surprisingly low dependence on linear distance, up to 170 kb. We further modified the method to measure precise repair of excision junctions and allele-specific excision, with important implications for disease modeling and therapeutic gene editing.

molecular biology↗