Dissolving microneedles (MNs) are novel transdermal drug delivery systems that can be painlessly self-administered. This study investigated the effects of experimental conditions on the mechanical characterization of dissolving MNs for quality evaluation. Micromolding was used to fabricate polyvinyl alcohol (PVA)-based dissolving MN patches with eight different cone-shaped geometries. Axial force mechanical characterization test conditions, in terms of compression speed and the number of compression needles per test, significantly affected the needle fracture force of dissolving MNs. Characterization using selected test conditions clearly showed differences in the needle fracture force of dissolving MNs prepared under various conditions. PVA-based MNs were divided into two groups that showed buckling and unbuckling deformation, which occurred at aspect ratios (needle height/base diameter) of 2.8 and 1.8, respectively. The needle fracture force of PVA-based MNs was negatively correlated with an increase in the needle’s aspect ratio. Higher residual water or higher loading of lidocaine hydrochloride significantly decreased the needle fracture force. Therefore, setting appropriate methods and parameters for characterizing the mechanical properties of dissolving MNs should contribute to the development and supply of appropriate products.

Japanese flounder (Paralichthys olivaceus) is an important marine fish species of both fisheries and aquaculture in Northeast Asia. The commercial interest for all-female progenies due to several sex-related traits has prompted basic research on the mechanisms of sex determination in this species. By conducting a linkage analysis of the sex-determining locus, we initially identified 12 microsatellite markers linked to sex in 11 scaffolds, whose localization was restricted to a specific region of linkage group 9. Sequence analysis of this region identified 181 genes based on the UniProt database annotations. Among them, the amh gene was considered a potential candidate for sex determination because this gene is known to have taken over the role of sex determination in many teleosts. An in-depth sequence analysis of both the coding and non-coding regions of amh in XX and XY individuals detected nine SNPs linked with maleness. However, because these substitutions were synonymous, the upstream and downstream regions of amh were also investigated and a male-specific variant with deletions in the promoter region was detected. This truncated Y-specific amh variant was named amhy, and the amh shared by both sexes was named amhx. The association analysis using both females and males of the genotypic sex inferred by the presence/absence of amhy found complete association with phenotypic sex and genotype. Gene expression analysis in larvae derived from a single-pair progeny by quantitative real-time PCR detected amhy transcripts in the larval trunks between 20 and 100 days after hatching only in XY larvae. Localization of amhy by in situ hybridization was detected in presumptive Sertoli cells of XY gonads. Expression of amhx was almost undetectable in both XX and XY genotypes. Loss of Amh function by CRISPR-Cas9 induced male-to-female sex reversal, indicating that this gene was necessary for the masculinization of XY individuals. In conclusion, the complete linkage of amhy with males, its early expression in XY gonads before testicular differentiation, and the induction of sex reversal by loss-of-function mutation support the view that amhy is the sex-determining gene in this species.