scRNA-seq analysis discovered suppression of immunomodulatory dependent inflammatory response in PMBCs exposed to silver nanoparticles.

The assessment of AgNPs toxicity in vitro and in vivo models are frequently conflicting and inaccurate. Nevertheless, single cell immunological responses in a heterogenous environment have received little attention. Therefore, in this study, we have performed in-depth analysis which clearly revealed cellular-metal ion association as well as specific immunological response. Our study didn't show significant population differences in PMBC between control and AgNPs group implying no toxicological response. To confirm it further, deep profiling identified differences in subsets and differentially expressed genes (DEGs) of monocytes, B cells and T cells. Notably, monocyte subsets showed significant upregulation of metallothionein (MT) gene expression such as MT1G, MT1X, MT1E, MT1A, and MT1F. On the other hand, downregulation of pro-inflammatory genes such as IL1ß and CCL3 in both CD16 + and CD16- monocyte subsets were observed. This result indicated that AgNPs association with monocyte subsets de-promoted inflammatory responsive genes suggesting no significant toxicity observed in AgNPs treated group. Other cell types such as B cells and T cells also showed negligible differences in their subsets suggesting no toxicity response. Further, AgNPs treated group showed upregulation of cell proliferation, ribosomal synthesis, downregulation of cytokine release, and T cell differentiation inhibition. Overall, our results conclude that treatment of AgNPs to PMBC cells didn't display immunological related cytotoxicity response and thus motivate researchers to use them actively for biomedical applications.

Fine-tuning of epithelial taste bud organoid to promote functional recapitulation of taste reactivity.

Taste stem/progenitor cells from posterior mouse tongues have been used to generate taste bud organoids. However, the inaccessible location of taste receptor cells is observed in conventional organoids. In this study, we established a suspension-culture method to fine-tune taste bud organoids by apicobasal polarity alteration to form the accessible localization of taste receptor cells. Compared to conventional Matrigel-embedded organoids, suspension-cultured organoids showed comparable differentiation and renewal rates to those of taste buds in vivo and exhibited functional taste receptor cells and cycling progenitor cells. Accessible taste receptor cells enabled the direct application of calcium imaging to evaluate the taste response. Moreover, suspension-cultured organoids can be genetically altered. Suspension-cultured taste bud organoids harmoniously integrated with the recipient lingual epithelium, maintaining the taste receptor cells and gustatory innervation capacity. We propose that suspension-cultured organoids may provide an efficient model for taste research, including taste bud development, regeneration, and transplantation.

Ameloblastoma modifies tumor microenvironment for enhancing invasiveness by altering collagen alignment.

Tumor progression is profoundly affected by crosstalk between cancer cells and their stroma. In the past decades, the development of bioinformatics and the establishment of organoid model systems have allowed extensive investigation of the relationship between tumor cells and the tumor microenvironment (TME). However, the interaction between tumor cells and the extracellular matrix (ECM) in odontogenic epithelial neoplasms and the ECM remodeling mechanism remain unclear. In the present study, transcriptomic comparison and histopathologic analysis revealed that TME-related genes were upregulated in ameloblastoma compared to in odontogenic keratocysts. Tumoroid analysis indicated that type I collagen is required for ameloblastoma progression. Furthermore, ameloblastoma shows the capacity to remodel the ECM independently of cancer-associated fibroblasts. In conclusion, ameloblastoma-mediated ECM remodeling contributes to the formation of an invasive collagen architecture during tumor progression.

Small-molecule binding of the axin RGS domain promotes ß-catenin and Ras degradation.

Both the Wnt/ß-catenin and Ras pathways are aberrantly activated in most human colorectal cancers (CRCs) and interact cooperatively in tumor promotion. Inhibition of these signaling may therefore be an ideal strategy for treating CRC. We identified KY1220, a compound that destabilizes both ß-catenin and Ras, via targeting the Wnt/ß-catenin pathway, and synthesized its derivative KYA1797K. KYA1797K bound directly to the regulators of G-protein signaling domain of axin, initiating ß-catenin and Ras degradation through enhancement of the ß-catenin destruction complex activating GSK3ß. KYA1797K effectively suppressed the growth of CRCs harboring APC and KRAS mutations, as shown by various in vitro studies and by in vivo studies using xenograft and transgenic mouse models of tumors induced by APC and KRAS mutations. Destabilization of both ß-catenin and Ras via targeting axin is a potential therapeutic strategy for treatment of CRC and other type cancers activated Wnt/ß-catenin and Ras pathways.

Discovery of a small-molecule inhibitor of Dvl-CXXC5 interaction by computational approaches.

The Wnt/ß-catenin signaling pathway plays a significant role in the control of osteoblastogenesis and bone formation. CXXC finger protein 5 (CXXC5) has been recently identified as a negative feedback regulator of osteoblast differentiation through a specific interaction with Dishevelled (Dvl) protein. It was reported that targeting the Dvl-CXXC5 interaction could be a novel anabolic therapeutic target for osteoporosis. In this study, complex structure of Dvl PDZ domain and CXXC5 peptide was simulated with molecular dynamics (MD). Based on the structural analysis of binding modes of MD-simulated Dvl PDZ domain with CXXC5 peptide and crystal Dvl PDZ domain with synthetic peptide-ligands, we generated two different pharmacophore models and applied pharmacophore-based virtual screening to discover potent inhibitors of the Dvl-CXXC5 interaction for the anabolic therapy of osteoporosis. Analysis of 16 compounds selected by means of a virtual screening protocol yielded four compounds that effectively disrupted the Dvl-CXXC5 interaction in the fluorescence polarization assay. Potential compounds were validated by fluorescence spectroscopy and nuclear magnetic resonance. We successfully identified a highly potent inhibitor, BMD4722, which directly binds to the Dvl PDZ domain and disrupts the Dvl-CXXC5 interaction. Overall, CXXC5-Dvl PDZ domain complex based pharmacophore combined with various traditional and simple computational methods is a promising approach for the development of modulators targeting the Dvl-CXXC5 interaction, and the potent inhibitor BMD4722 could serve as a starting point to discover or design more potent and specific the Dvl-CXXC5 interaction disruptors.

CXXC5 plays a role as a transcription activator for myelin genes on oligodendrocyte differentiation.

Myelination in corpus callosum plays important role for normal brain functions by transferring neurological information between various brain regions. However, the factors controlling expression of myelin genes in myelination are poorly understood. Here, CXXC5, a recently identified protein with CXXC-type zinc finger DNA binding motif, was characterized as a transcriptional activator of major myelin genes. We identified expression of CXXC5 expression was increased by Wnt/ß-catenin signaling. CXXC5 specifically expressed in the white matter induced expression of myelin genes through the direct binding of CXXC DNA-binding motif of CXXC5 on the MBP promoter. During the differentiation of neural stem cells (NSCs) of CXXC5(-/-) mice, the expressions of myelin genes were simultaneously reduced. The CXXC5(-/-) mice exhibited severely reduction of myelin genes expression in corpus callosum as well as abnormalities in myelin structure. The disrupted structural integrity of myelin in the CXXC5(-/-) mice resulted in reduced electrical conduction amplitudes at corpus callosum. These findings indicate that the regulation of myelin genes expression by CXXC5 is important for forming myelin structure involved with axonal electrical signal transfer in the corpus callosum.

Identification of small-molecule compounds targeting the dishevelled PDZ domain by virtual screening and binding studies.

The Dishevelled (Dvl) protein, which conveys signals from receptors to the downstream effectors, is a critical constituent of the Wnt/ß-catenin signaling pathway. Because the PDZ domain of Dvl protein functions through associations with a wide range of protein partners, Dvl protein involved in the Wnt signaling pathway has been considered to be therapeutic targets in cancers. In this study, we performed structure-based pharmacophore model of the Dvl PDZ domain to discover novel small-molecule binders and identified eight compounds with micromolar affinity. The most potent compound identified, BMD4702, efficiently bound to the Dvl PDZ domain with 11.2µM affinity and had a 0.186µM KD value according to surface plasmon resonance and fluorescence spectroscopy, respectively. Combining both structural-kinetic relationship analyses and docking studies, we fourmulated that the ligand-binding site is composed of three H-bonds and three hydrophobic features. Thus, our approach led to the identification of potent binders of the Dvl PDZ domain and the findings provide novel insights into structure-based approaches to design high-affinity binders for the Dvl PDZ domain.

CXXC5 is a transcriptional activator of Flk-1 and mediates bone morphogenic protein-induced endothelial cell differentiation and vessel formation.

CXXC5 is a member of a small subset of proteins containing CXXC-type zinc-finger domain. Here, we show that CXXC5 is a transcription factor activating Flk-1, a receptor for vascular endothelial growth factor. CXXC5 and Flk-1 were accumulated in nucli and membrane of mouse embryonic stem cells (mESCs), respectively, during their endothelial differentiation. CXXC5 overexpression induced Flk-1 transcription in both endothelium-differentiated mESCs and human umbilical vein endothelial cells (HUVECs). In vitro DNA binding assay showed direct interaction of CXXC5 on the Flk-1 promoter region, and mutation on its DNA-binding motif abolished transcriptional activity. We showed that bone morphorgenetic protein 4 (BMP4) induced CXXC5 transcription in the cells, and inhibitors of BMP signaling suppressed the CXXC5 induction and the consequent Flk-1 induction by BMP4 treatment. CXXC5 knockdown resulted in suppression of BMP4-induced stress fiber formation (56.8 ± 1.3% decrease, P<0.05) and migration (54.6 ± 1.9% decrease, P<0.05) in HUVECs. The in vivo roles of CXXC5 in BMP-signaling-specific vascular development and angiogenesis were shown by specific defect of caudal vein plex vessel formation (57.9 ± 11.8% decrease, P<0.05) in cxxc5 morpholino-injected zebrafish embryos and by suppression of BMP4-induced angiogenesis in subcutaneously injected Matrigel plugs in CXXC5(-/-) mice. Overall, CXXC5 is a transcriptional activator for Flk-1, mediating BMP signaling for differentiation and migration of endothelial cell and vessel formation.

The novel linkage between Fuz and Gpr161 genes regulates sonic hedgehog signaling during mouse embryonic development.

Sonic hedgehog (Shh) signaling regulates embryonic morphogenesis utilizing primary cilia, the cell antenna acting as a signaling hub. Fuz, an effector of planar cell polarity (PCP) signaling, involves Shh signaling via cilia formation, while the G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling. The range of phenotypic malformations observed in mice bearing mutations in either of these two genes is similar; however, their functional relations have not been previously explored. This study identified the genetic and biochemical link between Fuz and Gpr161 in mouse embryonic development. Fuz was genetically epistatic to Gpr161 via Shh signaling during mouse embryonic development. The FUZ biochemically interacted with GPR161, and Fuz regulated Gpr161 ciliary trafficking via ß-arrestin2. Our study suggested the novel Gpr161-Fuz axis that regulates Shh signaling during mouse embryonic development.

MAST4 regulates stem cell maintenance with DLX3 for epithelial development and amelogenesis.

The asymmetric division of stem cells permits the maintenance of the cell population and differentiation for harmonious progress. Developing mouse incisors allows inspection of the role of the stem cell niche to provide specific insights into essential developmental phases. Microtubule-associated serine/threonine kinase family member 4 (Mast4) knockout (KO) mice showed abnormal incisor development with low hardness, as the size of the apical bud was decreased and preameloblasts were shifted to the apical side, resulting in amelogenesis imperfecta. In addition, Mast4 KO incisors showed abnormal enamel maturation, and stem cell maintenance was inhibited as amelogenesis was accelerated with Wnt signal downregulation. Distal-Less Homeobox 3 (DLX3), a critical factor in tooth amelogenesis, is considered to be responsible for the development of amelogenesis imperfecta in humans. MAST4 directly binds to DLX3 and induces phosphorylation at three residues within the nuclear localization site (NLS) that promotes the nuclear translocation of DLX3. MAST4-mediated phosphorylation of DLX3 ultimately controls the transcription of DLX3 target genes, which are carbonic anhydrase and ion transporter genes involved in the pH regulation process during ameloblast maturation. Taken together, our data reveal a novel role for MAST4 as a critical regulator of the entire amelogenesis process through its control of Wnt signaling and DLX3 transcriptional activity.

PIBF1 regulates trophoblast syncytialization and promotes cardiovascular development.

Proper placental development in early pregnancy ensures a positive outcome later on. The developmental relationship between the placenta and embryonic organs, such as the heart, is crucial for a normal pregnancy. However, the mechanism through which the placenta influences the development of embryonic organs remains unclear. Trophoblasts fuse to form multinucleated syncytiotrophoblasts (SynT), which primarily make up the placental materno-fetal interface. We discovered that endogenous progesterone immunomodulatory binding factor 1 (PIBF1) is vital for trophoblast differentiation and fusion into SynT in humans and mice. PIBF1 facilitates communication between SynT and adjacent vascular cells, promoting vascular network development in the primary placenta. This process affected the early development of the embryonic cardiovascular system in mice. Moreover, in vitro experiments showed that PIBF1 promotes the development of cardiovascular characteristics in heart organoids. Our findings show how SynTs organize the barrier and imply their possible roles in supporting embryogenesis, including cardiovascular development. SynT-derived factors and SynT within the placenta may play critical roles in ensuring proper organogenesis of other organs in the embryo.

Fabrication of functional ameloblasts from hiPSCs for dental application.

Tooth formation relies on two types of dental cell populations, namely, the dental epithelium and dental mesenchyme, and the interactions between these cell populations are important during tooth development. Although human-induced pluripotent stem cells (hiPSCs) can differentiate into dental epithelial and mesenchymal cells, organoid research on tooth development has not been established yet. This study focused on the hiPSC-derived human ameloblast organoid (hAO) using a three-dimensional (3D) culture system. hAOs had similar properties to ameloblasts, forming enamel in response to calcium and mineralization by interaction with the dental mesenchyme. hAOs simultaneously had osteogenic and odontogenic differentiation potential. Furthermore, hAOs demonstrated tooth regenerative potential upon interaction with the mouse dental mesenchyme. Our findings provide new insights into a suitable hiPSC-derived dental source and demonstrate that hAOs can be beneficial not only for tooth regeneration but also for the study of various dental diseases for which treatment has not been developed yet.

Harnessing the dental cells derived from human induced pluripotent stem cells for hard tissue engineering.

INTRODUCTION: Most mineralized tissues in our body are present in bones and teeth. Human induced pluripotent stem cells (hiPSCs) are promising candidates for cell therapy to help regenerate bone defects and teeth loss. The extracellular matrix (ECM) is a non-cellular structure secreted by cells. Studies on the dynamic microenvironment of ECM are necessary for stem cell-based therapies. OBJECTIVES: We aim to optimize an effective protocol for hiPSC differentiation into dental cells without utilizing animal-derived factors or cell feeders that can be applied to humans and to mineralize differentiated dental cells into hard tissues. METHODS: For the differentiation of both dental epithelial cells (DECs) and dental mesenchymal cells (DMCs) from hiPSCs, an embryoid body (EB) was formed from hiPSCs. hiPSC were differentiated into neural crest cells with an induction medium utilized in our previous study, and hiPSC-derived DECs were differentiated with a BMP-modulated customized medium. hiPSC-dental cells were then characterized, analyzed, and validated with transcriptomic analysis, western blotting, and RT-qPCR. To form mineralized tissues, hiPSC-derived DECs were recombined with hiPSC-derived DMCs encapsulated in various biomaterials, including gelatin methacryloyl (GelMA), collagen, and agar matrix. RESULTS: These hiPSC-derived dental cells are highly osteogenic and chondro-osteogenic in photocrosslinkable GelMA hydrogel and collagen type I microenvironments. Furthermore, hiPSC-derived dental cells in agar gel matrix induced the formation of a bioengineered tooth. CONCLUSION: Our study provides an approach for applying hiPSCs for hard tissue regeneration, including tooth and bone. This study has immense potential to provide a novel technology for bioengineering organs for various regenerative therapies.

Identification of exosomal microRNA panel as diagnostic and prognostic biomarker for small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) has an exceptionally poor prognosis; as most of the cases are initially diagnosed as extensive disease with hematogenous metastasis. Therefore, the early diagnosis of SCLC is very important and may improve its prognosis. METHODS: To investigate the feasibility of early diagnosis of SCLC, we examined exosomal microRNAs (miRNAs) present in serum obtained from patients with SCLC. First, exosomes were isolated in serum from patients with SCLC and healthy individuals and were characterized using particle size and protein markers. Additionally, miRNA array was performed to define SCLC-specific exosomal miRNAs. Second, the obtained miRNAs were further validated employing a large cohort. Finally, the ability to diagnose SCLC was estimated by area under the curve (AUC), and intracellular mRNA change patterns were verified through validated miRNAs. RESULTS: From the miRNA array results, we selected 51-miRNAs based on p-values and top 10 differentially expressed genes, and 25-miRNAs were validated using quantitative reverse transcription-polymerase chain reaction. The 25-miRNAs were further validated employing a large cohort. Among them, 7-miRNAs showed significant differences. Furthermore, 6-miRNAs (miR-3565, miR-3124-5p, miR-200b-3p, miR-6515, miR-3126-3p and miR-9-5p) were up-regulated and 1-miRNA (miR-92b-5p) was down-regulated. The AUC value of each miRNA sets between 0.64 and 0.76, however the combined application of 3-miRNAs (miR-200b-3p, miR-3124-5p and miR-92b-5p) remarkably improved the diagnostic value (AUC = 0.93). Gene ontology analysis revealed that the 3-miRNA panel is linked to various oncogene pathways and nervous system development. When the 3-miRNAs were introduced to cells, the resulting changes in total mRNA expression strongly indicated the presence of lung diseases, including lung cancer. In addition, the 3-miRNA panel was significantly associated with a poorer prognosis, although individual miRNAs have not been validated as prognostic markers. CONCLUSION: Our study identified SCLC-specific exosomal miRNAs, and the 3-miRNAs panel (miR-200b-3p, miR-3124-5p and miR-92b-5p) may serve as a diagnostic and prognostic marker for SCLC.

Development of a screening platform to discover natural products active against SARS-CoV-2 infection using lung organoid models.

BACKGROUND: Natural products can serve as one of the alternatives, exhibiting high potential for the treatment and prevention of COVID-19, caused by SARS-CoV-2. Herein, we report a screening platform to test the antiviral efficacy of a natural product library against SARS-CoV-2 and verify their activity using lung organoids. METHODS: Since SARS-CoV-2 is classified as a risk group 3 pathogen, the drug screening assay must be performed in a biosafety level 3 (BSL-3) laboratory. To circumvent this limitation, pseudotyped viruses (PVs) have been developed as replacements for the live SARS-CoV-2. We developed PVs containing spikes from Delta and Omicron variants of SARS-CoV-2 and improved the infection in an angiotensin-converting enzyme 2 (ACE2)-dependent manner. Human induced pluripotent stem cells (hiPSCs) derived lung organoids were generated to test the SARS-CoV-2 therapeutic efficacy of natural products. RESULTS: Flavonoids from our natural product library had strong antiviral activity against the Delta- or Omicron-spike-containing PVs without affecting cell viability. We aimed to develop strategies to discover the dual function of either inhibiting infection at the beginning of the infection cycle or reducing spike stability following SARS-CoV-2 infection. When lung cells are already infected with the virus, the active flavonoids induced the degradation of the spike protein and exerted anti-inflammatory effects. Further experiments confirmed that the active flavonoids had strong antiviral activity in lung organoid models. CONCLUSION: This screening platform will open new paths by providing a promising standard system for discovering novel drug leads against SARS-CoV-2 and help develop promising candidates for clinical investigation as potential therapeutics for COVID-19.

Adult dental epithelial stem cell-derived organoids deposit hydroxylapatite biomineral.

Ameloblasts are specialized cells derived from the dental epithelium that produce enamel, a hierarchically structured tissue comprised of highly elongated hydroxylapatite (OHAp) crystallites. The unique function of the epithelial cells synthesizing crystallites and assembling them in a mechanically robust structure is not fully elucidated yet, partly due to limitations with in vitro experimental models. Herein, we demonstrate the ability to generate mineralizing dental epithelial organoids (DEOs) from adult dental epithelial stem cells (aDESCs) isolated from mouse incisor tissues. DEOs expressed ameloblast markers, could be maintained for more than five months (11 passages) in vitro in media containing modulators of Wnt, Egf, Bmp, Fgf and Notch signaling pathways, and were amenable to cryostorage. When transplanted underneath murine kidney capsules, organoids produced OHAp crystallites similar in composition, size, and shape to mineralized dental tissues, including some enamel-like elongated crystals. DEOs are thus a powerful in vitro model to study mineralization process by dental epithelium, which can pave the way to understanding amelogenesis and developing regenerative therapy of enamel.

Sur8/Shoc2 involves both inhibition of differentiation and maintenance of self-renewal of neural progenitor cells via modulation of extracellular signal-regulated kinase signaling.

Sur8/Shoc2 is a scaffold protein that regulates the Ras-extracellular signal-regulated kinase (ERK) pathway. However, the roles of Sur8 in cellular physiologies are poorly understood. In this study, Sur8 was severely repressed in the course of neural progenitor cell (NPC) differentiation in the cerebral cortex of developing rat embryos. Similarly, Sur8 was also critically reduced in cultured NPCs, which were induced differentiation by removal of basic fibroblast growth factor (bFGF). Sur8 regulation occurs at the protein level rather than at the mRNA level as revealed by both in situ hybridization and reverse transcriptase polymerase chain reaction analyses. The role of Sur8 in NPC differentiation was confirmed by lentivirus-mediated Sur8 knockdown, which resulted in increased differentiation, whereas exogenous expression of Sur8 inhibited differentiation. Contrastingly, NPC proliferation was promoted by overexpression, but was suppressed by Sur8 knockdown. The role of Sur8 as an antidifferentiation factor in the developing rat brain was confirmed by an ex vivo embryo culture system combined with the lentivirus-mediated Sur8 knockdown. The numbers and sizes of neurospheres were reduced, but neuronal outgrowth was enhanced by the Sur8 knockdown. The Ras-ERK pathway is involved in Sur8-mediated regulations of differentiation, as the treatment of ERK kinase (MEK) inhibitors blocks the effects of Sur8. The regulations of NPCs' differentiation and proliferation by the Ras-ERK pathway were also shown by the rescues of the effects of bFGF depletion, neuronal differentiation, and antiproliferation by epidermal growth factor. In summary, Sur8 is an antidifferentiation factor that stimulates proliferation for maintenance of self-renewal in NPCs via modulation of the Ras-ERK pathway.

Transcriptomic Comparison Analysis between Ameloblastoma and AM-1 Cell Line.

Cancer initiation and progression are profoundly along with the crosstalk between cancer cells and the surrounding stroma. Accumulating evidence has shown that the therapy targeting the extracellular matrix (ECM) would regress tumor growth and invasion in the most common carcinomas. However, it remains largely unexplored in several rare tumors like odontogenic tumors. Ameloblastoma (AM) is the representative odontogenic epithelial tumor in the jawbone, and it usually infiltrates into adjacent bone marrow and has unlimited growth capacity and a high potential for recurrence. This study aims to investigate the role of collagen-rich ECM during the invasion of AM. Transcriptomic analysis revealed that ECM- and epithelial-to-mesenchymal transition (EMT)-related genes were up-regulated in AM compared to ameloblastoma cell line, AM-1. Tumoroid forming analysis showed that Collagen-rich ECM is indispensable for AM progression, especially for aggressive growth patterns and collective invasion.

Inhibition of L-type voltage-gated calcium channel-mediated Ca2+ influx suppresses the collective migration and invasion of ameloblastoma.

OBJECTIVES: Ameloblastoma (AM) has been known as a benign but locally invasive tumour with high recurrence rates. Invasive behaviour of the AM results in destruction of the adjacent jawbone and the non-detectable remnants during surgery, interrupting the complete elimination of cancer cells. METHODS: To explore novel targets for the tumour cell invasion, a transcriptomic analysis between AM and odontogenic keratocyst were performed through next-generation sequencing in detail. RESULTS: Enrichment of CACNA1C gene (encoding Cav1.2) in AM, a subunit of the L-type voltage-gated calcium channel (VGCC) was observed for the first time. The expression and channel activity of Cav1.2 was confirmed by immunostaining and calcium imaging in the patient samples or primary cells. Verapamil, L-type VGCC blocker revealed suppression of the Ca2+ -induced cell aggregation and collective invasion of AM cells in vitro. Furthermore, the effect of verapamil in suppressing AM invasion into the adjacent bone was confirmed through orthotopic xenograft model specifically. CONCLUSION: Taken together, Cav1.2 maybe considered to be a therapeutic candidate to decrease the collective migration and invasion of AM.