Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disorder caused by mutations in the Dmd gene encoding Dystrophin12. DMD model animals, such as mdx mice and canine X-linked muscular dystrophy dogs, have been widely utilized in the development of a treatment for DMD3. Here, we demonstrate the generation of Dmd-mutated rats using a clustered interspaced short palindromic repeats (CRISPR)/Cas system, an RNA-based genome engineering technique that is also adaptive to rats. We simultaneously targeted two exons in the rat Dmd gene, which resulted in the absence of Dystrophin expression in the F0 generation. Dmd-mutated rats exhibited a decline in muscle strength, and the emergence of degenerative/regenerative phenotypes in the skeletal muscle, heart, and diaphragm. These mutations were heritable by the next generation, and F1 male rats exhibited similar phenotypes in their skeletal muscles. These model rats should prove to be useful for developing therapeutic methods to treat DMD.

Halogen bonding catalysis has recently gained increasing attention as a powerful tool to activate organic molecules. However, the variety of the catalyst structure has been quite limited so far. Herein, we report the first example of the use of an iodoalkyne as a halogen bond donor catalyst. By using an iodoalkyne bearing a pentafluorophenyl group as a catalyst, thioamides were efficiently activated and reacted with 2-aminophenol to generate benzoxazoles in good yield. Mechanistic studies, including C NMR spectroscopic analysis and several control experiments, provided concrete evidence that this catalytic activation is based on halogen bonding. Thus, the results obtained in this study demonstrate that iodoalkynes can serve as a new scaffold for future development of halogen bonding catalysis.

Ammonium influx into plant roots via the high-affinity transport system (HATS) is down-modulated under elevated external ammonium, preventing ammonium toxicity. In ammonium-fed Arabidopsis, ammonium transporter 1 (AMT1) trimers responsible for HATS activity are allosterically inactivated in a dose-dependent manner via phosphorylation of the conserved threonine at the carboxyl-tail by the calcineurin B-like protein 1-calcineurin B-like protein-interacting protein kinase 23 complex and other yet unidentified protein kinases. Using transcriptome and reverse genetics in ammonium-preferring rice, we revealed the role of the serine/threonine/tyrosine protein kinase gene OsACTPK1 in down-modulation of HATS under sufficient ammonium. In wild-type roots, ACTPK1 mRNA and protein accumulated dose-dependently under sufficient ammonium. To determine the function of ACTPK1, two independent mutants lacking ACTPK1 were produced by retrotransposon Tos17 insertion. Compared with segregants lacking insertions, the two mutants showed decreased root growth and increased shoot growth under 1 mm ammonium due to enhanced ammonium acquisition, via aberrantly high HATS activity, and use. Furthermore, introduction of OsACTPK1 cDNA fused to the synthetic green fluorescence protein under its own promoter complemented growth and the HATS influx, and suggested plasma membrane localization. Root cellular expression of OsACTPK1 also overlapped with that of ammonium-induced OsAMT1;1 and OsAMT1;2. Meanwhile, threonine-phosphorylated AMT1 levels were substantially decreased in roots of ACTPK1-deficient mutants grown under sufficient ammonium. Bimolecular fluorescence complementation assay further confirmed interaction between ACTPK1 and AMT1;2 at the cell plasma membrane. Overall, these findings suggest that ACTPK1 directly phosphorylates and inactivates AMT1;2 in rice seedling roots under sufficient ammonium.

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