The vertebrate skeletal system has various functions, including support, movement, protection, and the production of blood cells. The development of cartilage and bones, the core components of the skeletal system, is mediated by systematic inter- and intracellular communication among multiple signaling pathways in differentiating progenitors and the surrounding tissues. Recently, Pannexin (Panx) 3 has been shown to play important roles in bone development in vitro by mediating multiple signaling pathways, although its roles in vivo have not been explored. In this study, we generated and analyzed Panx3 knockout mice and examined the skeletal phenotypes of panx3 morphant zebrafish. Panx3(-/-) embryos exhibited delays in hypertrophic chondrocyte differentiation and osteoblast differentiation as well as the initiation of mineralization, resulting in shortened long bones in adulthood. The abnormal progression of hypertrophic chondrogenesis appeared to be associated with the sustained proliferation of chondrocytes, which resulted from increased intracellular cAMP levels. Similarly, osteoblast differentiation and mineralization were delayed in panx3 morphant zebrafish. Taken together, our results provide evidence of the crucial roles of Panx3 in vertebrate skeletal development in vivo.

Objective-Mutations in the GJB2, GJB6 and SLC26A4 genes are a frequent cause of hearing loss in a number of populations. However, little is known about the genetic causes of hearing loss in the Korean population.Methods-We sequenced the GJB2 and GJB6 genes to examine the role of mutations in these genes in twenty-two hearing loss patients. We also sequenced the SLC26A4 gene in seven patients with inner ear malformations, including enlarged vestibular aqueduct (EVA) revealed by computer tomography.Results-Coding sequence mutations in GJB2 were identified in 13.6% of the patients screened. Two different mutations, 235delC and T86R were found in three unrelated patients. The 235delC was the most prevalent mutation with an allele frequency of 6.9% in our patient group. No mutations, including 342 kb deletion, were found in GJB6 gene. Three different variants of SLC26A4 were identified in the EVA patients, including one novel mutation. Four EVA patients carried two mutant alleles of SLC26A4, and at least one allele in all patients was the H723R mutation, which accounted for 75% of all mutant alleles.Conclusions-Our results suggest that GJB2 and SLC26A4 mutations together make up a major cause of congenital hearing loss in the Korean population. Further studies may be able to identify other common variants that account for a significant fraction of hearing loss in the Korean population.

Extracellular vesicles (EVs) carrying tumor cell-derived programmed death-ligand 1 (PD-L1) interact with programmed death 1 (PD-1)-producing T cells, thus significantly lowering a patient's response to immune checkpoint blockade drugs. No drug that reinvigorates CD8+ T cells by suppressing EV PD-L1 has been approved for clinical usage. Here we have identified macitentan (MAC), an FDA-approved oral drug, as a robust booster of antitumor responses in CD8+ T cells by suppressing tumor cell-derived EV PD-L1. Methods: EV was analyzed by the data from nanoparticle tracking, immunoblotting analyses, and nano-flow cytometry. Antitumor immunity was evaluated by luciferase assay and immune phenotyping using flow cytometry. Clinical relevance was analyzed using the cancer genome atlas database. Results: MAC inhibited secretion of tumor-derived EV PD-L1 by targeting the endothelin receptor A (ETA) in breast cancer cells and xenograft models. MAC enhanced CD8+ T cell-mediated tumor killing by decreasing the binding of PD-1 to the EV PD-L1 and thus synergizing the effects of the anti-PD-L1 antibody. MAC also showed an anticancer effect in triple-negative breast cancer (TNBC)-bearing immunocompetent mice but not in nude mice. The combination therapy of MAC and anti-PD-L1 antibody significantly improved antitumor efficacy by increasing CD8+ T cell number and activity with decreasing Treg number in the tumors and draining lymph nodes in TNBC, colon, and lung syngeneic tumor models. The antitumor effect of MAC was reversed by injecting exogenous EV PD-L1. Notably, ETA level was strongly associated with the innate anti-PD-1 resistance gene signature and the low response to the PD-1/PD-L1 blockade. Conclusion: These findings strongly demonstrate that MAC, already approved for clinical applications, can be used to improve and/or overcome the inadequate response to PD-1/PD-L1 blockade therapy.

Senescent cells accumulate in various tissues over time, and contribute to tissue dysfunction and aging-associated phenotypes. Accumulating evidence suggests that cellular senescence can be inhibited through pharmacological intervention, as well as through treatment with soluble factors derived from embryonic stem cells (ESCs). In an attempt to investigate the anti-senescence factors secreted by ESCs, we analyzed mouse ESC-derived extracellular miRNAs in conditioned medium (CM) via miRNA array analysis. We selected mmu-miR-291a-3p as a putative anti-senescence factor via bioinformatics analysis. We validated its inhibitory effects on replicative, adriamycin-induced, and ionizing radiation-induced senescence in human dermal fibroblasts. Treatment of senescent cells with mmu-miR-291a-3p decreased senescence-associated-β-galactosidase activity, enhanced proliferative potential, and reduced mRNA and protein expression of TGFBR2, p53, and p21. Mmu-miR-291a-3p in CM was enclosed in ESC-derived exosomes and exosomes purified from ESC-CM inhibited cellular senescence. The inhibitory effects of mmu-miR-291a-3p were mediated through the TGFBR2 signaling pathway. Hsa-miR-371a-3p and hsa-miR-520e, the human homologs of mmu-miR-291a-3p, showed similar anti-senescence activity. Furthermore, mmu-miR-291a-3p accelerated the excisional skin wound healing process in aged mice. Our results indicate that the ESC-derived mmu-miR-291a-3p is a novel candidate agent that can be utilized for cell-free therapeutic intervention against aging and aging-related diseases.

The etch characteristics of HfO 2 by atomic-layer etching ͑ALET͒ were investigated using a BCl 3 /Ar neutral beam. The effect of ALET on surface modification and etch-depth control was also examined. Self-limited etching of HfO 2 could be obtained using BCl 3 ALET. This was attributed to the absorption of BCl 3 by the Langmuir isotherm during the absorption stage and the vaporization of hafnium-chlorides/boron oxychlorides formed on the surface during the desorption stage. In addition, the surface composition of HfO 2 was not altered by etching during ALET.The downscaling of metal-oxide-semiconductor field-effect transistors ͑MOSFETs͒ has created the need for high-dielectric-constant ͑k͒ materials to replace SiO 2 for reducing the gate-leakage current while maintaining the gate-dielectric capacitance. Among the various high-k materials, hafnium oxide ͑HfO 2 ͒ is a potential material in next-generation MOSFETs due to its high-dielectric constant ͑25-30͒, wide bandgap, low leakage current, and superior thermal stability. 1,2 In order to apply HfO 2 to next-generation MOSFET devices, HfO 2 films should be patterned by dry etching, because the wet etching of HfO 2 has several disadvantages such as difficulty in controlling the etch rate, severe undercut, etc. Therefore, there have been many studies on the dry etching of HfO 2 using halogen-based plasma etching for applications to MOSFET devices. A precise etch rate is required in the plasma etching for HfO 2 instead of a high etch rate due to the low thickness of the material. Moreover, an extremely high etch selectivity over the underlayer material is required. In addition, the damage on the etched surface is intolerable. However, these conventional plasma-etching processes tend to physically damage the surface of the devices by creating surface defects, including structural disruption, an intermixing layer, or stoichiometry modification, and increasing surface roughness, due to use of energetic reactive ions to achieve vertical etch profiles. 3,4 In addition, these halogen-based plasma etchings showed finite etch selectivity between HfO 2 and the underlying materials. 5,6 These problems decrease the device performance.Atomic-layer etching ͑ALET͒ may be the most suitable method for etching HfO 2 in next-generation MOSFET devices because it may etch HfO 2 with no physical damage and with atomic-scale etch controllability. Generally, ALET is a cyclic process that consists of four sequential steps: ͑i͒ adsorption of reactant molecules on the surface, ͑ii͒ evacuation of the remaining reactant molecules, ͑iii͒ Ar + ion-beam irradiation to the reactant adsorbed surface for the desorption of chemisorbed species, and ͑iv͒ evacuation of the etch products. 7,8 However, during step ͑iii͒ with Ar + beam irradiation, the device can be damaged electrically due to the charged particles, such as positive ions generated in the plasma.In this article, ALET of HfO 2 was carried out using BCl 3 adsorption, followed by Ar neutral-beam irradiation instead of Ar + ionbeam irradiation to a...

Hearing loss, which is genetically heterogeneous, can be caused by mutations in the mitochondrial DNA (mtDNA). The A1555G mutation of the 12S ribosomal RNA (rRNA) gene in the mtDNA has been associated with both aminoglycoside-induced and non-syndromic hearing loss in many ethnic populations. Here, we report for the first time the clinical and genetic characterization of nine Korean pedigrees with aminoglycoside-induced and non-syndromic hearing loss. These Korean families carry in the A1555G mutation of 12S rRNA gene and exhibit variable penetrance and expressivity of hearing loss. Specifically, the penetrance of hearing loss in these families ranged between 28.6% and 75%, with an average of 60.8%. These results were higher than the 29.8% penetrance that was previously reported in a Chinese population but similar to the 65.4% and 54.1% penetrance observed in a large Arab-Israeli population and nineteen Spanish pedigrees, respectively. The mutational analysis of the complete mtDNA genome in these families showed that the haplogroups of the Korean population, which belongs to the eastern Asian population, were similar to those of the Chinese population but different from the Spanish population, which belongs to the European-Caucasian population. The mtDNA variants that may act as modifier factors were also found to be similar to the Chinese population. Although the mtDNA haplogroups and variants were similar to the eastern Asian population, we did find some differing phenotypes, although some subjects had the same variants. This result suggests that both the ethnic background and environmental factors lead to a variable phenotype of the A1555G mutation.

According to clinical studies, statins improve the efficacy of programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) blockade therapy for breast cancer; however, the underlying mechanisms are unclear. Herein, we showed that atorvastatin (ATO) decreased the content of PD-L1 in extracellular vesicles (EVs) by reducing cellular PD-L1 expression and inhibiting EV secretion in breast cancer cells, thereby enhancing the efficacy of anti-PD-L1 therapy. ATO reduced EV secretion by regulating the Rab proteins involved in EV biogenesis and secretion. ATO-mediated inhibition of the Ras-activated MAPK signaling pathway downregulated PD-L1 expression. In addition, ATO strongly promoted antitumor efficacy by inducing T cell-mediated tumor destruction when combined with an anti-PD-L1 antibody. Moreover, suppression of EV PD-L1 by ATO improved the reactivity of anti-PD-L1 therapy by enhancing T-cell activity in draining lymph nodes of EMT6-bearing immunocompetent mice. Therefore, ATO is a potential therapeutic drug that improves antitumor immunity by inhibiting EV PD-L1, particularly in response to immune escape during cancer.

Despite their potent antitumor activity, clinical application of immune checkpoint inhibitors has been significantly limited by their poor response rates (<30%) in cancer patients, primarily due to immunosuppressive tumor microenvironments. As a representative immune escape mechanism, cancer-derived exosomes have recently been demonstrated to exhaust CD8 + cytotoxic T cells. Here, it is reported that sulfisoxazole, a sulfonamide antibacterial, significantly decreases the exosomal PD-L1 level in blood when orally administered to the tumor-bearing mice. Consequently, sulfisoxazole effectively reinvigorates exhausted T cells, thereby eliciting robust antitumor effects in combination with anti-PD-1 antibody. Overall, sulfisoxazole regulates immunosuppression through the inhibition of exosomal PD-L1, implying its potential to improve the response rate of anti-PD-1 antibodies.