RNAs play many essential roles in gene expression and are involved in various human diseases. Although genome editing technologies have been established, the engineering of sequence-specific RNA-binding proteins that manipulate particular cellular RNA molecules is immature, in contrast to nucleotide-based RNA manipulation technology, such as siRNA- and RNA-targeting CRISPR/Cas. Here, we demonstrate a versatile RNA manipulation technology using pentatricopeptide-repeat (PPR)-motif-containing proteins. First, we developed a rapid construction and evaluation method for PPR-based designer sequence-specific RNA-binding proteins. This system has enabled the steady construction of dozens of functional designer PPR proteins targeting long 18 nt RNA, which targets a single specific RNA in the mammalian transcriptome. Furthermore, the cellular functionality of the designer PPR proteins was first demonstrated by the control of alternative splicing of either a reporter gene or an endogenous CHK1 mRNA. Our results present a versatile protein-based RNA manipulation technology using PPR proteins that facilitates the understanding of unknown RNA functions and the creation of gene circuits and has potential for use in future therapeutics.

Laparoscopic cholecystectomy using an ultrasound surgical aspirator has been performed in our department since March 1991. The horn cover was altered in order to be inserted through a trocar 10 mm in diameter. The main purpose of this device is to explore Calot's triangle by fragmentation and aspiration of the fatty tissue without damaging the nerves, vessels, and cystic duct. First the serosa of the Calot's triangle is cut via electrocautery with the sharp-angle hook dissector we designed. Then the cystic duct and cystic artery are efficiently exposed by the ultrasound surgical aspirator. This procedure is perfectly adapted for laparoscopic cholecystectomy. We obtained favorable results with the ultrasound surgical aspirator in 135 cases including 40 cases with a negative gallbladder, as evaluated by endoscopic retrograde cholangiography. In conclusion, the ultrasound surgical aspirator is suitable for skeletonizing the cystic duct and cystic artery, and the procedure is perfectly safe.

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