Early Detection of Male-Predominant Phenotypes in the Pattern of Ultrasonic Vocalizations Emitted by Autism Spectrum Disorder Model (Crmp4-Knockout) Mice

Male predominance is a known feature of autism spectrum disorder (ASD). Although ASD mouse models can be useful for elucidating mechanisms underlying abnormal behaviors relevant to human ASD, suitable models to analyze sex differences in ASD pathogenesis remain insufficient. Herein, we used collapsin response mediator protein 4 (Crmp4)-knockout (KO) mice exhibiting ASD-like phenotypes in a male-predominant manner and analyzed ultrasonic vocalizations (USVs) to detect potential differences between genotypes and sexes during the early postnatal period. We recorded isolation-induced USVs emitted from wild-type (WT) and Crmp4-KO littermates and compared the total number of USVs between genotypes and sexes. We classified USVs into 10 types based on internal pitch changes, lengths, and shapes and compared the number of USVs in each type by genotypes and sex. Male Crmp4-KO mice exhibited a reduction in the total number of USVs. Crmp4-KO decreased the number of USVs in 7 out of 10 USV types, and male KO mice exhibited a greater reduction than females in 3 of the 7 types. This study offers a suitable ASD animal model and tool for assessing sex-based communication deficits during the early postnatal period, both of which would be valuable for elucidating the underlying mechanism

An Easy-to-Use Polystyrene Microchip-based Cell Culture System

In this study, we developed an integrated, low-cost microfluidic cell culture system that is easy to use. This system consists of a disposable polystyrene microchip, a polytetrafluoroethylene valve, an air bubble trap, and an indium tin oxide temperature controller. Valve pressure resistance was validated with a manometer to be 3 MPa. The trap protected against bubble contamination. The temperature controller enabled the culture of Macaca mulatta RF/6A 135 vascular endothelial cells, which are difficult to culture in glass microchips, without a CO2 incubator. We determined the optimal coating conditions for these cells and were able to achieve stable, confluent culture within 1 week. This practical system is suitable for low-cost screening and has potential applications as circulatory cell culture systems and research platforms in cell biology

Structure and developmental expression of hatching enzyme genes of the Japanese eel Anguilla japonica: an aspect of the evolution of fish hatching enzyme gene

We isolated seven cDNA clones from embryos of the Japanese eel Anguilla japonica. Each deduced amino acid sequence consisted of a signal peptide, a propeptide and a mature enzyme portion belonging to the astacin protease family. A phylogenetic analysis showed that the eel enzymes resembled the high choriolytic enzyme (HCE) of medaka Oryzias latipes, and the hatching enzymes of the zebra fish Danio rerio and masu salmon Oncorhynchus masou. Hatching enzymes of these teleosts belonged to the group of the medaka HCE, and not the medaka low choriolytic enzyme (LCE), another hatching enzyme of medaka. Southern blot analysis showed that the genes of the eel hatching enzymes were multicopy genes like the medaka HCE genes. However, one of the eel hatching enzyme genes comprised eight exons and seven introns, and the exon-intron organization was similar to the medaka LCE gene, which is a single-copy gene. The molecular evolution of the fish hatching enzyme genes is discussed. In addition, whole-mount in situ hybridization and immunocytochemistry showed that the eel hatching enzyme was first expressed in the pillow anterior to the forebrain of early neurula, and finally in the cell mass on the yolk sac of later stage embryos. The early differentiation profile of eel hatching gland cells was similar to that of medaka, masu salmon and zebrafish, whereas the final location of the gland cells was different among fishes