Per- and poly fluoroalkyl substances (PFAS) pose significant global health risks. Although the use of classical PFAS such as perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) is regulated, the toxicological effects of alternative PFASs remain unknown. Cleft palate is a congenital condition influenced by both environmental and genetic factors. Although PFOS has been linked to cleft palate, the effects of other PFAS compounds remain unexplored. The aim of this study was to clarify the involvement of classical and alternative PFAS (PFHxA and PFHxS) in human embryonic palatal mesenchymal cell (HEPM) proliferation. Following PFAS treatment for 48 h, cell viability, apoptosis, and expression of cell cycle-related proteins were tested. In addition, miRNA levels and predicted target genes were measured, and a rescue experiment against PFHxS was conducted using an miR-374a-5p inhibitor. Among the four PFASs, PFHxS decreased the number of cells showing cyclin- and cyclin-dependent kinase reduction. In addition, PFHxS treatment upregulated miR-374a-5p and downregulated its downstream genes. Furthermore, miR- 374a-5p inhibitor alleviated the PFHxS-induced reduction in cell proliferation. These findings therefore indicate that miR-374a-5p plays a key role in the development of PFHxS-induced cleft palate and that alternative PFAS may have a highly toxic effect on HEPM cells.

We previously showed that tenascin-XB (TNXB) contributes to tumor suppressor function. The present study aimed to assess the tumor-suppressive mechanism of TNXB by focusing on immune cell infiltration into the tumor microenvironment (TME). We revealed that B16-OVA melanoma cells (MO5)-bearing TNXB-deficient (Tnxb−/−) mice exhibited significant tumor progression and a poor survival rate. Allogeneic mixed lymphocyte reaction showed reduced numbers and increased activation of both CD4+ and CD8+ T cells from Tnxb−/− spleens. Moreover, T cell activation assay further proved that CD4+ and CD8+ T cells from Tnxb−/− mice were more activated than those from WT mice. RT-qPCR analysis showed that expression of T cell activation-related cytokines and chemokines was significantly decreased in tumor tissues from Tnxb−/− mice. Flow cytometry analysis revealed a reduced infiltration level of CD8+ T cells in both naïve spleens and tumor tissues in Tnxb−/− mice. Ultimately, total activation of CD8+ T cells was decreased in tumor tissues in Tnxb−/− mice. In conclusion, we found that although Tnxb−/− CD4+ and CD8+ T cells tend to be activated more than WT CD4+ and CD8+ T cells, CD8+ T cell infiltration and activation level were attenuated in tumor sites of Tnxb−/− mice.

Serial section scanning electron microscopy (SEM) is useful for revealing the three-dimensional (3D) architecture of organelles by acquiring backscattered electron images of ultrathin serial sections of resin-embedded tissues on solid substrates. However, comprehensive analyses of organelle function require a combination of ultrastructural and molecular localization data. In the present study, we developed a novel 3D immuno-electron microscopy (immuno-EM) approach that combines Tokuyasu cryosectioning with serial section SEM to elucidate the spatial distribution of organelle-associated proteins. Thick cryosections of tissues were immunolabeled with primary antibodies and FluoroNanogold-conjugated secondary antibodies, followed by gold enhancement, resin embedding, and serial sectioning and SEM. Serial tomographic images of organelles were aligned and segmented to generate 3D reconstructions. To demonstrate the effectiveness of the method, we visualized the localization of GM130, a representative cis-Golgi matrix protein, in a 3D model of the Golgi apparatus in rat pituitary gonadotropes. The 3D model revealed a spherical Golgi apparatus composed of five cisternae arranged in cis-trans order, with GM130 localized on the outer cisternae, consistent with previous findings. Our 3D immuno-EM technique enables the detailed 3D visualization of the Golgi apparatus and other organelles as well as analyses of the spatial distribution of target proteins in their 3D reconstructions.

Ghrelin-producing cells (ghrelin cells) in rat fundic glands were analyzed three-dimensionally by combining serial section scanning electron microscopy with immunogold labeling to elucidate their ultrastructural characteristics. This approach enabled unambiguous identification of ghrelin cells and the three-dimensional (3D) reconstruction of their organelles with special reference to primary cilium. Rat ghrelin cells were morphologically classified into two types: ciliated ghrelin cells possessing primary cilia and non-ciliated ghrelin cells. In ciliated cells, primary cilia protruded from basal bodies located near the Golgi apparatus and were largely or entirely enclosed within ciliary pockets. Ghrelin-positive secretory granules were electron-dense and spherical. Ciliated and non-ciliated cells contained both large (250–350 nm) and small (100–200 nm) ghrelin-positive granules. In ciliated cells, the granules were densely accumulated in a localized region of the cytoplasm, being opposite the Golgi apparatus across the nucleus; in non-ciliated cells they were densely distributed throughout the cytoplasm. The ultrastructure of the basement membrane and overall 3D configuration of the Golgi apparatus also differed between the two cell types. Our results provide novel insights into the morphological organization of ghrelin cells, whose 3D ultrastructural features have been unclear in conventional two-dimensional transmission electron microscopy using single ultrathin sections.