Computational and Experimental Analyses for Pathogenicity Prediction of ACVRL1 Missense Variants in Hereditary Hemorrhagic Telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is a vascular disease caused by the defects of ALK1/ACVRL1 receptor signaling. In this study, we evaluated 25 recently identified ACVRL1 missense variants using multiple computational pathogenicity classifiers and experimentally characterized their signal transduction capacity. Three extracellular residue variants showed no detectable cell surface expression and impairment of bone morphogenetic protein 9 (BMP9) responsiveness of SMAD-dependent transcription in luciferase assays. Four variants with amino acid replacement in the motifs essential for the intracellular kinase function lost SMAD-dependent signaling. Most of other variations in the kinase domain also caused marked downregulation of signaling; however, two variants behaved as the wild-type ACVRL1 did, while computational classifiers predicted their functional abnormalities. Three-dimensional structure prediction using the ColabFold program supported the significance of the L45 loop and NANDOR domain of ACVRL1 for its association with SMAD1 and BMPR2, respectively, and the variations in these motifs resulted in the reduction of SMAD signaling. On the other hand, two of the GS domain variants maintained high signal transduction capacity, which did not accord with their computational pathogenicity prediction. These results affirm the requirement of a combinatory approach using computational and experimental analyses to accurately predict the pathogenicity of ACVRL1 missense variants in the HHT patients.

c-Myc/microRNA-17-92 Axis Phase-Dependently Regulates PTEN and p21 Expression via ceRNA during Reprogramming to Mouse Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) are promising cell sources for regenerative medicine and disease modeling. iPSCs are commonly established by introducing the defined reprogramming factors Oct4, Sox2, Klf4, and c-Myc. However, iPSC reprogramming efficiency remains low. Although recent studies have identified microRNAs that contribute to efficient reprogramming, the underlying molecular mechanisms are not completely understood. miR-17-92 is highly expressed in embryonic stem cells and may play an important role in regulating stem cell properties. Therefore, we examined the role of miR-17-92 in the induction of mouse iPSC production. c-Myc-mediated miR-17-92 upregulation increased reprogramming efficiency, whereas CRISPR/Cas9-based deletion of the miR-17-92 cluster decreased reprogramming efficiency. A combination of in silico and microarray analyses revealed that Pten and cyclin-dependent kinase inhibitor 1 (known as p21) are common target genes of miR-17 and miR-20a, which are transcribed from the miR-17-92 cluster. Moreover, miR-17-92 downregulated p21 in the early phase and PTEN in the mid-to-late phase of reprogramming. These downregulations were perturbed by introducing the 3' UTR of PTEN and p21, respectively, suggesting that PTEN and p21 mRNAs are competing endogenous RNAs (ceRNA) against miR-17-92. Collectively, we propose that the c-Myc-mediated expression of miR-17-92 is involved in iPSC reprogramming through the phase-dependent inhibition of PTEN and p21 in a ceRNA manner, thus elucidating an underlying mechanism of iPSC reprogramming.

Canonical Wnt signaling activation by chimeric antigen receptors for efficient cardiac differentiation from mouse embryonic stem cells.

BACKGROUND: Canonical Wnt signaling is involved in a variety of biological processes including stem cell renewal and differentiation, embryonic development, and tissue regeneration. Previous studies reported the stage-specific roles of the Wnt signaling in heart development. Canonical Wnt signal activation by recombinant Wnt3a in the early phase of differentiation enhances the efficiency of myocardial cell production from pluripotent stem cells. However, the hydrophobicity of Wnt proteins results in high cost to produce the recombinant proteins and presents an obstacle to their preparation and application for therapeutics, cell therapy, or molecular analysis of Wnt signaling. METHODS: To solve this problem, we generated an inexpensive molecule-responsive differentiation-inducing chimeric antigen receptor (designated as diCAR) that can activate Wnt3a signaling. The extracellular domains of low-density-lipoprotein receptor-related protein 6 (LRP6) and frizzeled-8 (FZD8) were replaced with single-chain Fv of anti-fluorescein (FL) antibody, which can respond to FL-conjugated bovine serum albumin (BSA-FL) as a cognate ligand. We then analyzed the effect of this diCAR on Wnt signal activation and cardiomyocyte differentiation of mouse embryonic stem cells in response to BSA-FL treatment. RESULTS: Embryonic stem cell lines stably expressing this paired diCAR, named Wnt3a-diCAR, showed TCF/ß-catenin-dependent transactivation by BSA-FL in a dose-dependent manner. Treatment with either Wnt3a recombinant protein or BSA-FL in the early phase of differentiation revealed similar changes of global gene expressions and resulted in efficient myocardial cell differentiation. Furthermore, BSA-FL-mediated signal activation was not affected by a Wnt3a antagonist, Dkk1, suggesting that the signal transduction via Wnt3a-diCAR is independent of endogenous LRP6 or FZD8. CONCLUSION: We anticipate that Wnt3a-diCAR enables target-specific signal activation, and could be an economical and powerful tool for stem cell-based regeneration therapy.

ETS-dependent enhancers for endothelial-specific expression of serum/glucocorticoid-regulated kinase 1 during mouse embryo development.

Serum/glucocorticoid-regulated kinase 1 (SGK1) is predominantly expressed in endothelial cells of mouse embryos, and Sgk1 null mice show embryonic lethality due to impaired vascular formation. However, how the SGK1 expression is controlled in developing vasculature remains unknown. In this study, we first identified a proximal endothelial enhancer through lacZ reporter mouse analyses. The mouse Sgk1 proximal enhancer was narrowed down to the 5' region of the major transcription initiation site, while a human corresponding region possessed relatively weak activity. We then searched for distal enhancer candidates using in silico analyses of publicly available databases for DNase accessibility, RNA polymerase association and chromatin modification. A region approximately 500 kb distant from the human SGK1 gene was conserved in the mouse, and the mouse and human genomic fragments drove transcription restricted to embryonic endothelial cells. Minimal fragments of both proximal and distal enhancers had consensus binding elements for the ETS transcription factors, which were essential for the responsiveness to ERG, FLI1 and ETS1 proteins in luciferase assays and the endothelial lacZ reporter expression in mouse embryos. These results suggest that endothelial SGK1 expression in embryonic vasculature is maintained through at least two ETS-regulated enhancers located in the proximal and distal regions.

Tmem100-BAC-EGFP mice to selectively mark and purify embryonic endothelial cells of large caliber arteries in mid-gestational vascular formation.

Embryonic vascular development is achieved through the complex arrays of differentiation, proliferation, migration and mutual interaction of different cell types, and visualization as well as purification of unique cell populations are fundamental in studying its detailed mechanisms using in vivo experimental models. We previously demonstrated that Tmem100 was a novel endothelial gene encoding a small transmembrane protein, and that Tmem100 null mice showed embryonic lethality due to severe impairment of vascular formation. In the present study, we generated an EGFP reporter mouse line using a 216 kb genomic region containing mouse Tmem100 gene. A novel line designated as Tmem100-BAC-EGFP mice precisely recapitulated the Tmem100 expression profile at the mid-gestational stage, which was highly enriched in endothelial cells of large caliber arteries in mouse embryos. FACS experiments demonstrated that Tmem100-BAC-EGFP mice served to selectively purify a specific population of arterial endothelial cells, indicating their usefulness not only for the research concerning Tmem100 expression and function but also for comparative analysis of multiple endothelial cell subgroups in embryonic vascular development.

Metabolic remodeling during somatic cell reprogramming to induced pluripotent stem cells: involvement of hypoxia-inducible factor 1.

Induced pluripotent stem cells (iPSCs) were first established from differentiated somatic cells by gene introduction of key transcription factors, OCT4, SOX2, KLF4, and c-MYC, over a decade ago. Although iPSCs can be applicable for regenerative medicine, disease modeling and drug screening, several issues associated with the utilization of iPSCs such as low reprogramming efficiency and the risk of tumorigenesis, still need to be resolved. In addition, the molecular mechanisms involved in the somatic cell reprogramming to pluripotency are yet to be elucidated. Compared with their somatic counterparts, pluripotent stem cells, including embryonic stem cells and iPSCs, exhibit a high rate of glycolysis akin to aerobic glycolysis in cancer cells. This is known as the Warburg effect and is essential for maintaining stem cell properties. This unique glycolytic metabolism in iPSCs can provide energy and drive the pentose phosphate pathway, which is the preferred pathway for rapid cell proliferation. During reprogramming, somatic cells undergo a metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis trigged by a transient OXPHOS burst, resulting in the initiation and progression of reprogramming to iPSCs. Metabolic intermediates and mitochondrial functions are also involved in the epigenetic modification necessary for the process of iPSC reprogramming. Among the key regulatory molecules that have been reported to be involved in metabolic shift so far, hypoxia-inducible factor 1 (HIF1) controls the transcription of many target genes to initiate metabolic changes in the early stage and maintains glycolytic metabolism in the later phase of reprogramming. This review summarizes the current understanding of the unique metabolism of pluripotent stem cells and the metabolic shift during reprogramming, and details the relevance of HIF1 in the metabolic shift.

Chimeric G-CSF Receptor-Mediated STAT3 Activation Contributes to Efficient Induction of Cardiomyocytes from Mouse Induced Pluripotent Stem Cells.

Producing a sufficient number of cardiomyocytes from pluripotent stem cells has been of great demand for cardiac regeneration therapy. However, it remains challenging to efficiently differentiate cardiomyocytes with low costs. Reportedly, granulocyte colony-stimulating factor (G-CSF) receptor (GCSFR) signaling activates signal transducers and activators of transcription (STAT) signaling and enhances cardiac differentiation from embryonic stem cells or induced pluripotent stem cells (iPSCs). To economically and efficiently produce cardiomyocytes from iPSCs through GCSFR/STAT axis activation, we constructed antibody/receptor chimeras that can respond to an inexpensive small molecule. Single-chain Fv of anti-fluorescein (FL) antibody was ligated to transmembrane/cytoplasmic domains of GCSFRs, enabling transduction of GCSFR signaling in response to FL-conjugated bovine serum albumin (BSA-FL) as an alternative ligand. Mouse iPSC lines constitutively expressing these chimeric receptors exhibited increased BSA-FL-induced STAT3 phosphorylation in a dose-dependent manner, which was abolished by an inhibitor of Janus tyrosine kinase (JAK). In addition, BSA-FL stimulation also increased the incidence of beating embryoid bodies and upregulated cardiac-specific gene expressions after differentiation in these iPSC lines. Therefore, the chimeric GCSFRs activated endogenous GCSFR signaling at least via the JAK/STAT3 pathway, thereby enhancing cardiac differentiation from iPSCs. This approach, as an economical strategy, could contribute to stem cell-based cardiac regeneration therapy.

Improving laser cooling efficiencies of Yb-doped yttrium aluminum garnet by utilizing non-resonant anti-Stokes emission at high temperatures.

Laser cooling in solids has been demonstrated, and cooling from room temperature to cryogenic temperatures has so far been the main focus of research. However, operation at high temperatures can be advantageous. We propose that laser cooling of Yb-doped yttrium aluminum garnet ceramic discs is enhanced above 300 K. Photoluminescence (PL) spectra is evidence that the phonon-assisted energy transfer from resonant states, which exhibit a narrow PL peak, to inhomogeneously distributed energy states becomes stronger at higher temperatures. This results in an enhanced non-resonant anti-Stokes PL at high temperatures. The ideal cooling efficiency determined from the PL spectrum at 470 K is 1.7 times higher than that at 300 K.

Assessing the Nature of the Distribution of Localised States in Bulk GaAsBi.

A comprehensive assessment of the nature of the distribution of sub band-gap energy states in bulk GaAsBi is presented using power and temperature dependent photoluminescence spectroscopy. The observation of a characteristic red-blue-red shift in the peak luminescence energy indicates the presence of short-range alloy disorder in the material. A decrease in the carrier localisation energy demonstrates the strong excitation power dependence of localised state behaviour and is attributed to the filling of energy states furthest from the valence band edge. Analysis of the photoluminescence lineshape at low temperature presents strong evidence for a Gaussian distribution of localised states that extends from the valence band edge. Furthermore, a rate model is employed to understand the non-uniform thermal quenching of the photoluminescence and indicates the presence of two Gaussian-like distributions making up the density of localised states. These components are attributed to the presence of microscopic fluctuations in Bi content, due to short-range alloy disorder across the GaAsBi layer, and the formation of Bi related point defects, resulting from low temperature growth.

Serum/glucocorticoid-regulated kinase 1 as a novel transcriptional target of bone morphogenetic protein-ALK1 receptor signaling in vascular endothelial cells.

Bone morphogenetic protein 9 (BMP9)/BMP10-ALK1 receptor signaling is essential for endothelial differentiation and vascular morphogenesis. Mutations in ALK1/ACVRL1 and other signal-related genes are implicated in human vascular diseases, and the Alk1/Acvrl1 deletion in mice causes severe impairment of vascular formation and embryonic lethality. In the microarray screen to search for novel downstream genes of ALK1 signaling, we found that the mRNA and protein expression of serum/glucocorticoid-regulated kinase 1 (SGK1) was rapidly up-regulated by the BMP9 stimulation of cultured human endothelial cells. The increase in SGK1 mRNA was completely blocked by the transcriptional inhibitor actinomycin D and significantly suppressed by the siRNA treatment against the co-SMAD transcription factor SMAD4. Upon the BMP9 treatment of endothelial cells, phosphorylated SMAD1/5/9 bound to a consensus site upstream of the SGK1 gene, which was necessary for BMP9-dependent increment of the luciferase reporter activity driven by the SGK1 proximal enhancer. The Sgk1 mRNA expression in mouse embryos was enriched in vascular endothelial cells at embryonic day 9.0-9.5, at which Sgk1 null mice showed embryonic lethality due to abnormal vascular formation, and its mRNA as well as protein expression was clearly reduced in Alk1/Acvrl1 null embryos. These results indicate that SGK1 is a novel target gene of BMP9/BMP10-ALK1 signaling in endothelial cells and further suggest a possibility that down-regulation of the Sgk1 expression may be involved in the mechanisms of vascular defects by the ALK1 signaling deficiency.

Increasing conversion efficiency of two-step photon up-conversion solar cell with a voltage booster hetero-interface.

Development of high-efficiency solar cells is one of the attractive challenges in renewable energy technologies. Photon up-conversion can reduce the transmission loss and is one of the promising concepts which improve conversion efficiency. Here we present an analysis of the conversion efficiency, which can be increased by up-conversion in a single-junction solar cell with a hetero-interface that boosts the output voltage. We confirm that an increase in the quasi-Fermi gap and substantial photocurrent generation result in a high conversion efficiency.