Kruppel-like factors (KLFs) belong to a large group of zinc finger-containing transcription factors with amino acid sequences resembling the Drosophila gap gene
. Since the first report of molecular ...cloning of the
family gene, the number of KLFs has increased rapidly. Currently, 17 murine and human KLFs are known to play crucial roles in the regulation of transcription, cell proliferation, cellular differentiation, stem cell maintenance, and tissue and organ pathogenesis. Recent evidence has shown that many KLF family molecules affect skeletal cells and regulate their differentiation and function. This review summarizes the current understanding of the unique roles of each KLF in skeletal cells during normal development and skeletal pathologies.
A zirconia oxygen sensor is an electrochemical device for rapidly measuring oxygen concentrations in molten metals at high temperatures. It is desirable to make the sensors capable of continuous, ...long-time measurements. Here, the effect on the electromotive force of a partial equilibrium state on the surface of a zirconia solid electrolyte in a zirconia oxygen sensor was investigated to reveal the factors preventing long-time measurements. The reference electrode was a mixture of W and WO2. Continuous measurements were performed in molten iron containing carbon for deoxidization. The electromotive force between the positive reference electrode and the negative sample electrode of the sensor decreased from a positive value to zero over time. This was because a metallic tungsten layer formed on the surface of the zirconia electrolyte by the reduction of the tungsten oxide via oxygen diffusion through the zirconia tube. In addition, the equilibrium in the reference electrode was disturbed during the measurements. A direct electrical current was applied to the sensor to examine the relationship between the surface and measured values. The electromotive force during the current was maintained at a higher value than that without the current because of induced re-oxidation at the zirconia surface. However, a long-time application could cause over-oxidation and dissolution of the reference electrode surface.
Zirconia oxygen sensors are used in the steel-refining process to instantly measure the oxygen concentration in molten steel. It is desirable to develop sensors that are capable of continuous and ...long-time measurements, but such measurements are difficult with conventional sensors. In this study, the factors that hinder long-time measurement by a single zirconia oxygen sensor with a mixture of Cr and Cr2O3 as the reference electrode were investigated. Continuous measurements of a molten iron containing carbon and aluminum for deoxidization were carried out with the sensor. The electromotive force between the reference electrode (positive) and sample electrode (negative) of the sensor decreased from a positive value to zero with time. This was because reduction of chromium oxide at the surface of the zirconia solid electrolyte was promoted owing to oxygen diffusion from the inside to the outside of the zirconia tube and the equilibrium in the reference electrode was disturbed during the measurements. In addition, the reference electrode at the surface partially and temporarily melted owing to diffusion of the components from the electrolyte to the reference electrode. Additionally, a test to extend the lifetime of the sensor was performed with application of a direct electrical current to the sensor. The electromotive force with application of a current was maintained at a higher value than that without application of a current owing to promotion of re-oxidation at the zirconia surface. However, it was found that a high current can cause over-oxidation and dissolution of the reference electrode at the surface.
Time-resolved serial femtosecond crystallography using an X-ray free electron laser (XFEL) in conjunction with a photosensitive caged-compound offers a crystallographic method to track enzymatic ...reactions. Here we demonstrate the application of this method using fungal NO reductase, a heme-containing enzyme, at room temperature. Twenty milliseconds after caged-NO photolysis, we identify a NO-bound form of the enzyme, which is an initial intermediate with a slightly bent Fe-N-O coordination geometry at a resolution of 2.1 Å. The NO geometry is compatible with those analyzed by XFEL-based cryo-crystallography and QM/MM calculations, indicating that we obtain an intact Fe
-NO coordination structure that is free of X-ray radiation damage. The slightly bent NO geometry is appropriate to prevent immediate NO dissociation and thus accept H
from NADH. The combination of using XFEL and a caged-compound is a powerful tool for determining functional enzyme structures during catalytic reactions at the atomic level.
In order to optimize decarburization in the RH degassing process, it is necessary to develop sensors capable of continuous and direct measurements of an oxygen concentration. However, such ...measurements are only possible for a few seconds with conventional sensors. Here, the effects that hinder long-time measurements by a single Mo/MoO2–type zirconia oxygen sensor were investigated and an approach to extend sensor lifetime was developed by the direct current (DC) voltage application. Reference electrodes containing molybdenum and molybdenum oxide were constructed in a single-closure tube of zirconia solid electrolyte. The EMF between the reference electrode (negative) and the sample electrode (positive) inserted in molten iron decreased from +300 mV owing to the formation of a metallic molybdenum layer on the inner surface of the zirconia tube. This layer was attributed to reduction of molybdenum oxide gas owing to oxygen diffusion from the inside to the outside of the tube. Because the oxygen diffusion coefficient in molybdenum is much lower than the oxygen ion diffusion coefficient in the zirconia, the layer disrupted oxygen transfer and reduced the EMF. Additionally, a DC voltage of 2.0 V was applied to the reference electrode and EMF recovered over 20 minutes. The molybdenum layer disappeared and the reference electrode was re-exposed to the electrolyte. Thus, the applied DC voltage enabled long-time measurements of oxygen content with a single Mo/MoO2–type zirconia oxygen sensor.
Vertebrates form their skeletal tissues from three distinct origins (the neural crest, paraxial mesoderm, and lateral plate mesoderm) through two distinct modes of ossification (intramembranous and ...endochondral ossification). Since the paraxial mesoderm generates both intramembranous and endochondral bones, it is thought to give rise to both osteoprogenitors and osteo-chondroprogenitors. However, it remains unclear what directs the paraxial mesoderm-derived cells toward these different fates in distinct skeletal elements during human skeletal development. To answer this question, we need experimental systems that recapitulate paraxial mesoderm-mediated intramembranous and endochondral ossification processes. In this study, we aimed to develop a human pluripotent stem cell (hPSC)-based system that models the human intramembranous ossification process. We found that spheroid culture of the hPSC-derived paraxial mesoderm derivatives generates osteoprogenitors or osteo-chondroprogenitors depending on stimuli. The former induced intramembranous ossification, and the latter endochondral ossification, in mouse renal capsules. Transcriptional profiling supported the notion that bone signatures were enriched in the intramembranous bone-like tissues. Thus, we developed a system that recapitulates intramembranous ossification, and that enables the induction of two distinct modes of ossification by controlling the cell fate of the hPSC-derived paraxial mesoderm derivatives.
•We modeled the intramembranous ossification process using human stem cells.•Skeletal progenitors were generated from stem cell-derived mesoderm derivatives.•The progenitors induced intramembranous and endochondral ossification in vivo.•RNA-sequencing supported enrichment of bone signatures in the induced tissues.
The pregnane X receptor (PXR, NR1I2) belongs to the nuclear receptor family and functions as a xenobiotic and endobiotic sensor by binding to various molecules through its relatively flexible ...ligand-binding domain. In addition to these well-known canonical roles, we previously reported that PXR represses osteoblast differentiation. However, the mechanisms underlying the PXR-mediated repression of osteoblast differentiation remains unknown. In this study, we analyzed the changes in global gene expression profiles induced by PXR in calvarial osteoblasts cultured in standard fetal bovine serum (in which PXR induces repression of differentiation), and in those cultured in charcoal-stripped fetal bovine serum (in which PXR does not induce repression of differentiation). The comparison revealed that PXR attenuated the Hedgehog-mediated signaling in culture conditions that induced PXR-mediated repression of differentiation. Real-time PCR analysis showed that PXR repressed the Hedgehog signaling-induced genes such as Gli1 and Hhip, and conversely induced the Hedgehog signaling-repressed genes such as Cdon, Boc, and Gas1. Activation of Smo-mediated signaling in osteoblasts following treatment with a Smo agonist (SAG) significantly restored Gli-mediated transcriptional activity and osteoblast differentiation. Our results demonstrate the osteoblast-autonomous effects of PXR and identify a novel regulation of Hedgehog signaling by nuclear receptors.
Abstract Trichorhinophalangeal syndrome (TRPS) is a genetic disorder caused by point mutations or deletions in the gene-encoding transcription factor TRPS1. TRPS patients display a range of skeletal ...dysplasias, including reduced jaw size, short stature, and a cone-shaped digit epiphysis. Certain TRPS patients experience early onset coxarthrosis that leads to a devastating drop in their daily activities. The etiologies of congenital skeletal abnormalities of TRPS were revealed through the analysis of Trps1 mutant mouse strains. However, early postnatal lethality in Trps1 knockout mice has hampered the study of postnatal TRPS pathology. Here, through epigenomic analysis we identified two previously uncharacterized candidate gene regulatory regions in the first intron of Trps1. We deleted these regions, either individually or simultaneously, and examined their effects on skeletal morphogenesis. Animals that were deleted individually for either region displayed only modest phenotypes. In contrast, the Trps1Δint/Δint mouse strain with simultaneous deletion of both genomic regions exhibit postnatal growth retardation. This strain displayed delayed secondary ossification center formation in the long bones and misshaped hip joint development that resulted in acetabular dysplasia. Reducing one allele of the Trps1 gene in Trps1Δint mice resulted in medial patellar dislocation that has been observed in some patients with TRPS. Our novel Trps1 hypomorphic strain recapitulates many postnatal pathologies observed in human TRPS patients, thus positioning this strain as a useful animal model to study postnatal TRPS pathogenesis. Our observations also suggest that Trps1 gene expression is regulated through several regulatory elements, thus guaranteeing robust expression maintenance in skeletal cells.
•SAMP8 mice show different cusp size in specific region of lower molar.•The distal cusp size of SAMP8 mice display exaggerated distal reduction in disto-lingual entoconid.•The aged SAMP8 mice show ...severe regional occlusal wear at entoconid of lower molar.
The position and size of the major cusps in mammalian molars are arranged in a characteristic pattern that depends on taxonomy. In humans, the cusp which locates distally within each molar is smaller than the mesially located cusp, which is referred to as “distal reduction”. Although this concept has been well-recognized, it is still unclear how this reduction occurs. Current study examined whether senescence-accelerating mouse prone 8 (SAMP8) mice could be a possible animal model for studying how the mammalian molar cusp size is determined.
SAMP8 mice were compared with parental control (SAMR1) mice. Microcomputed tomography images of young and aged mice were captured to observe molar cusp morphologies. Cusp height from cement-enamel junction and mesio-distal length of molars were measured. The statistical comparison of the measurements was performed by Mann-Whitney U test.
SAMP8 mice showed reduced development of the disto-lingual cusp (entoconid) of lower second molar when compared with SAMR1 mice. The enamel thickness and structure was disturbed at entoconid, and aged SAMP8 mice displayed severe wear of the entoconid in lower second molar. These phenotypes were observed on both sides of the lower second molar.
In addition to the general senescence phenotype observed in SAMP8 mice, this strain may genetically possess molar cusp phenotypes which is determined prenatally. Further, SAMP8 mice would be a potential model strain to study the genetic causes of the distal reduction of molar cusp size.
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