CHLOROPLAST UNUSUAL POSITIONING 1 is a plant-specific actin polymerization factor regulating chloroplast movement.

Plants have unique responses to fluctuating light conditions. One such response involves chloroplast photorelocation movement, which optimizes photosynthesis under weak light by the accumulation of chloroplasts along the periclinal side of the cell, which prevents photodamage under strong light by avoiding chloroplast positioning toward the anticlinal side of the cell. This light-responsive chloroplast movement relies on the reorganization of chloroplast actin (cp-actin) filaments. Previous studies have suggested that CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) is essential for chloroplast photorelocation movement as a regulator of cp-actin filaments. In this study, we conducted comprehensive analyses to understand CHUP1 function. Functional, fluorescently tagged CHUP1 colocalized with and was coordinately reorganized with cp-actin filaments on the chloroplast outer envelope during chloroplast movement in Arabidopsis thaliana. CHUP1 distribution was reversibly regulated in a blue light- and phototropin-dependent manner. X-ray crystallography revealed that the CHUP1-C-terminal domain shares structural homology with the formin homology 2 (FH2) domain, despite lacking sequence similarity. Furthermore, the CHUP1-C-terminal domain promoted actin polymerization in the presence of profilin in vitro. Taken together, our findings indicate that CHUP1 is a plant-specific actin polymerization factor that has convergently evolved to assemble cp-actin filaments and enables chloroplast photorelocation movement.

Controlling Physical and Biochemical Parameters of Actin Nucleation Using a Patterned Model Lipid Membrane.

Self-assembly of nanoscale actin cytoskeletal proteins into filamentous networks requires organizing actin nucleation areas on the plasma membrane through recruiting actin nucleators and nucleation-promoting factors (NPFs) to the areas. To investigate impacts of the nucleation geometry on actin network assembly, we localized NPF or nucleator on defined micropatterns of laterally mobile lipid bilayers confined in a framework of a polymerized lipid bilayer. We demonstrated that actin network assembly in purified protein mixtures was confined on NPF- or nucleator-localized fluid bilayers. By controlling the shape and size of nucleation areas as well as the density and types of localized NPFs and nucleators, we showed that these parameters regulate actin network architectures. Actin network assembly in Xenopus egg extracts was also spatially controlled by patterning bilayers containing phosphatidylinositol 4,5-bisphoshate (PI(4,5)P2), an essential lipid signaling mediator. Therefore, the system provides a promising platform to investigate the physical and biochemical principles for actin network assembly.

A retrospective comparison of induction with thiopental/guaifenesin and propofol/ketamine in Thoroughbred racehorses anesthetized with sevoflurane and medetomidine during arthroscopic surgery.

This study compares clinical characteristics between induction with thiopental/guaifenesin and propofol/ketamine in Thoroughbred racehorses anesthetized with sevoflurane and medetomidine. Clinical records of 214 horses that underwent arthroscopic surgery between 2015 and 2016 were retrospectively retrieved. Horses were premedicated with medetomidine and midazolam to sedate at the adequate level for smooth induction, and then induced with either thiopental (4.0 mg/kg) and guaifenesin (100 mg/kg) in Group TG (n=91) or propofol (1.0 mg/kg) and ketamine (1.0 mg/kg) in Group PK (n=123). Anesthesia was maintained using sevoflurane with constant rate infusion of medetomidine. Quality of induction/recovery, sevoflurane requirement, cardiovascular function and recovery characteristics were evaluated. Anesthetic induction scores (median, range) for Group TG (5, 2-5) and Group PK (5, 2-5) were not significantly different. There were no significant differences in end-tidal sevoflurane concentration (mean ± standard deviation) between Group TG and Group PK (both 2.4 ± 0.2%). Dobutamine infusion rate (µg/kg/min) required for keeping mean arterial blood pressure (MAP) above 70 mmHg in Group PK (0.43, 0.10-1.40) was significantly lower than in Group TG (0.67, 0.08-1.56). Recovery score in Group PK (5, 2-5) was significantly higher than in Group TG (4, 2-5). Both propofol/ketamine and thiopental/guaifenesin provided a smooth induction of anesthesia. Moreover, induction with propofol/ketamine resulted in lower dobutamine requirements for keeping MAP above 70 mmHg during maintenance, and better quality of recovery. Induction with propofol/ketamine would be preferable to thiopental/guaifenesin in Thoroughbred racehorses anesthetized with sevoflurane and medetomidine during arthroscopic surgery.

Impact of COVID-19 spread on visit intervals and clinical parameters for patients with periodontitis in supportive periodontal therapy: a retrospective study.

PURPOSE: This study investigated the relationship between the number of days that hospital visits were postponed and changes in clinical parameters due to the spread of coronavirus disease 2019 (COVID-19), after the Japanese government declared a state of emergency in April 2020. METHODS: Regarding the status of postponement of appointments, we analyzed the patients who had visited the Nihon University Hospital at Matsudo for more than 1 year for supportive periodontal therapy (SPT) and classified them into low-, moderate- and high-risk subgroups according to the periodontal risk assessment (PRA). Clinical parameters for periodontal disease such as probing depth (PD), full-mouth bleeding score (FMBS), full-mouth plaque score, periodontal inflamed surface area (PISA), and periodontal epithelial surface area (PESA) were analyzed in 2 periods, from October 2019 to March 2020 and after April 2020. Correlation coefficients between days of deferral and the degree of changes in clinical parameters were calculated. RESULTS: The mean age of the 749 patients was 67.56±10.85 years, and 63.82% were female. Out of 749 patients, 33.24% deferred their SPT appointments after April 2020. The average total of postponement days was 109.49±88.84. The number of postponement days was positively correlated with changes in average PD (rs=0.474) and PESA (rs=0.443) in the high-risk subgroup of FMBS, and average PD (rs=0.293) and PESA (rs=0.253) in the high-risk subgroup of tooth number (TN). Patients belonging to the high-risk subgroups for both FMBS and TN had a positive correlation between postponement days and PISA (rs=0.56). CONCLUSIONS: The findings, the spread of COVID-19 appears to have extended the visit interval for some SPT patients. Moreover, longer visit intervals were correlated with the worsening of some clinical parameters for SPT patients with high PRA.

Comparison of early wound healing using modified papilla preservation technique between enamel matrix derivative and recombinant human fibroblast growth factor.

PURPOSE: Enamel matrix derivative (EMD) has demonstrated beneficial effects on wound healing following surgery. However, the effects of recombinant human fibroblast growth factor 2 (rhFGF-2) in periodontal regeneration therapy have not been extensively studied. This retrospective study was conducted to compare the wound healing outcomes of the modified papilla preservation technique (mPPT) between EMD and rhFGF-2 therapies. METHODS: A total of 79 sites were evaluated for early wound healing using the modified early wound healing index (mEHI), which included 6 items: incision, fibrin clotting, step, redness, swelling, and dehiscence. A numeric analog scale, along with postoperative images of the 6 mEHI items, was established and used for the evaluations. The inter-rater reliability of the mEHI was assessed via intraclass correlation coefficients (ICCs). After adjusting for factors influencing the mPPT, the differences in mEHI scores between the EMD and rhFGF-2 groups were statistically analyzed. Additionally, radiographic bone fill (RBF) was evaluated 6 months after surgery. RESULTS: The ICC of the mEHI was 0.575. The mEHI, redness score, and dehiscence scores were significantly higher in the rhFGF-2 group (n=33) than in the EMD group (n=46). Similar results were observed in the subgroup of patients aged 50 years or older, but not in those younger than 50 years. In the subgroup with non-contained bone defects, related results were noted, but not in the subgroup with contained bone defects. However, early wound healing did not correlate with RBF at 6 months after surgery. CONCLUSIONS: Within the limitations of this study, the findings suggest that early wound healing following the use of mPPT with rhFGF-2 is somewhat superior to that observed after mPPT with EMD. However, mEHI should be improved for use as a predictive tool for early wound healing and to reflect clinical outcomes after surgery.

Dilated capillaries, disorganized collagen fibers and differential gene expression in periodontal ligaments of hypomorphic fibrillin-1 mice.

The periodontal ligaments (PDLs) are soft connective tissue between the cementum covering the tooth root surface and alveolar bone. PDLs are composed of collagen and elastic system fibers, blood vessels, nerves, and various types of cells. Elastic system fibers are generally formed by elastin and microfibrils, but PDLs are mainly composed of the latter. Compared with the well-known function of collagen fibers to support teeth, little is known about the role of elastic system fibers in PDLs. To clarify their role, we examined PDLs of mice under-expressing fibrillin-1 (mgR mice), which is one of the major microfibrillar proteins. The PDLs of homozygous mgR mice showed one-quarter of the elastic system fibers of wild-type (WT) mice. A close association between the elastic system fibers and the capillaries was noted in WT, homozygous and heterozygous mgR mice. Interestingly, capillaries in PDLs of homozygous mice were dilated or enlarged compared with those of WT mice. A comparable level of type I collagen, which is the major collagen in PDLs, was expressed in PDL-cells of mice with three genotypes. However, multi-oriented collagen fiber bundles with a thinner appearance were noted in homozygous mice, whereas well-organized collagen fiber bundles were seen in WT mice. Moreover, there was a marked decrease in periostin expression, which is known to regulate the fibrillogenesis and crosslinking of collagen. These observations suggest that the microfibrillar protein, fibrillin-1, is indispensable for normal tissue architecture and gene expression of PDLs.

The teeth and dentition of the filefish (Stephanolepis cirrhifer) revisited tomographically.

The upper and lower tooth-bearing jaws of the filefish (Stephanolepis cirrhifer) were scanned using a micro-CT system in order to address the existing gaps between the traditional pictures of the morphology and histology. 2D tomograms, reconstructed 3D models and virtual dissection were employed to examine and evaluate the in situ geometry of tooth implantation and the mode of tooth attachment both separately and collectively. No distinct sockets comparable to those in mammals were evident, but shallow depressions were observed in the premaxillary and the dentary. The opening of the tooth pulp cavity was not simply oriented towards the apparent tooth base in a direction opposite to the tooth apex. The opening was distorted basoposteriorly or basoanteriorly depending on the position of the tooth, and the edge of the pulp cavity opening was barely ankylosed; i.e. the sites of pleurodont ankylosis along the basoposterior or basoanterior edge of the opening appeared to closely match the contour of the shallow depression in the bone. These 3D findings appear to be very informative when considering the phylogeny of tooth attachment, suggesting that micro-CT would be a useful modality concurrent with or in advance of histological investigations.

Temporospatial distribution of osteogenic and osteoclastic cells during development of the tarsometatarsal skeleton in the chick embryo (Gallus gallus).

The morphogenesis of long bones is a multistep process that generates a variety of genetically defined forms. The tarsometatarsal (TMT) long bone morphology in birds develops through lateral fusion of three initially independent periosteal bone cylinders (BCs). Previous studies have clarified the histological details and chronology of the changes occurring during development. The present study investigated the temporospatial distribution of osteogenic and osteoclastic cells in the embryonic chicken using histochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, with particular reference to the radial growth of BCs and their subsequent fusion process. Osteogenic cells were localized preferentially in the periosteum of radially growing BCs, leaving open cancellous spaces in the BC wall. Osteoclasts observed later than embryonic day 10 were localized preferentially in the endosteal surface, and therefore the radial growth of BCs resulting from osteoblast activity was accompanied by endosteal resorption by osteoclasts, with progressive enlargement of the bone marrow spaces. During BC fusion, trabecular bridges were formed by periosteal osteogenic cells, with removal of the bone septum by endosteal osteoclasts. These findings suggest that fusion of BCs in the embryonic chicken is mediated by cellular events constituting ordinary long bone development, and not through a defined mechanism specific for fusion.

Oral and Palatal Dentition of Axolotl Arises From a Common Tooth-Competent Zone Along the Ecto-Endodermal Boundary.

Vertebrate dentitions arise at various places within the oropharyngeal cavity including the jaws, the palate, or the pharynx. These dentitions develop in a highly organized way, where new tooth germs are progressively added adjacent to the initiator center, the first tooth. At the same time, the places where dentitions develop house the contact zones between the outer ectoderm and the inner endoderm, and this colocalization has instigated various suggestions on the roles of germ layers for tooth initiation and development. Here, we study development of the axolotl dentition, which is a complex of five pairs of tooth fields arranged into the typically tetrapod outer and inner dental arcades. By tracking the expression patterns of odontogenic genes, we reason that teeth of both dental arcades originate from common tooth-competent zones, one present on the mouth roof and one on the mouth floor. Progressive compartmentalization of these zones and a simultaneous addition of new tooth germs distinct for each prospective tooth field subsequently control the final shape and composition of the axolotl dentition. Interestingly, by following the fate of the GFP-labeled oral ectoderm, we further show that, in three out of five tooth field pairs, the first tooth develops right at the ecto-endodermal boundary. Our results thus indicate that a single tooth-competent zone gives rise to both dental arcades of a complex tetrapod dentition. Further, we propose that the ecto-endodermal boundary running through this zone should be accounted for as a potential source of instruction factors instigating the onset of the odontogenic program.

Clinical pharmacokinetics and pharmacodynamics of intravenous alfaxalone in young Thoroughbred horses premedicated with medetomidine and midazolam.

To investigate the clinical pharmacokinetics and pharmacodynamics of intravenous alfaxalone in young Thoroughbred horses, seven Thoroughbred horses were randomly anaesthetised twice with either 1 or 2 mg/kg of intravenous alfaxalone after premedication with medetomidine (6 µg/kg intravenous) and midazolam (20 µg/kg intravenous). Blood samples were collected at predetermined time points up to two hours after administration. Plasma alfaxalone concentrations were quantified by a liquid chromatography tandem-mass spectrometry method and analysed by non-compartmental pharmacokinetic analysis. Induction and recovery qualities were good to excellent for both doses. Recovery time for the 2 mg/kg (median 90 minutes) was significantly longer than that for the 1 mg/kg (median 50 minutes). Respiratory rate for the 2 mg/kg was significantly lower than that for the 1 mg/kg, resulting in hypoxaemia. The median (range) elimination half-life, total clearance and volume of distribution were 58.2 (42.3-70.7) minutes, 11.6 (10.3-14.5) ml/minute/kg and 0.8 (0.7-0.9) l/kg for the 1 mg/kg and 59.8 (47.5-68.0) minutes, 14.7 (12.1-16.0) ml/minute/kg and 0.9 (0.9-1.2) l/kg for the 2 mg/kg, respectively. Alfaxalone is rapidly eliminated from the plasma in young Thoroughbred horses. Respiratory depression should be especially noted when alfaxalone is used in clinical practice.

Cellular origin of microfibrils explored by monensin-induced perturbation of secretory activity in embryonic primary cultures.

Fibrillin is a primary component of elastin-associated microfibrils. Since microfibrils are distributed rather ubiquitously in embryonic tissues, attention has focused on the types of cells responsible for producing fibrillin. To clarify this issue, we employed monensin-induced perturbation of secretory activity in embryonic primary cultures, as this would allow examination of both the secreted protein and the formation of extracellular fibrils in the same culture. Micromasses of avian limb bud mesoderm, its ectodermal covering and several explants from other sources were cultured in the presence and absence of monensin, and evaluated immunohistochemically using antibodies against fibrillin and cell lineage markers. The results indicated that monensin perturbation induced intracellular accumulation of fibrillin and prevented the formation of microfibrils. It was shown specifically that not only mesodermally derived fibrogenic cells and myogenic cells of skeletal and smooth muscle cell lineage, but also epithelial-type cells such as endothelial and ectodermal cells, are producers of fibrillin. This dual cellular origin of fibrillin at the ectomesenchymal interface is considered significant for understanding the formation and remodeling of microfibrils originating from the basal lamina.

Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells.

Organoid technology provides a revolutionary paradigm toward therapy but has yet to be applied in humans, mainly because of reproducibility and scalability challenges. Here, we overcome these limitations by evolving a scalable organ bud production platform entirely from human induced pluripotent stem cells (iPSC). By conducting massive "reverse" screen experiments, we identified three progenitor populations that can effectively generate liver buds in a highly reproducible manner: hepatic endoderm, endothelium, and septum mesenchyme. Furthermore, we achieved human scalability by developing an omni-well-array culture platform for mass producing homogeneous and miniaturized liver buds on a clinically relevant large scale (>108). Vascularized and functional liver tissues generated entirely from iPSCs significantly improved subsequent hepatic functionalization potentiated by stage-matched developmental progenitor interactions, enabling functional rescue against acute liver failure via transplantation. Overall, our study provides a stringent manufacturing platform for multicellular organoid supply, thus facilitating clinical and pharmaceutical applications especially for the treatment of liver diseases through multi-industrial collaborations.

Highly reproducible skeletal deformities induced by administration of ß-aminopropionitrile to developing chick embryos.

The formation of cross-linkages between and within collagen is catalyzed by lysyl oxidase, which can be inhibited by ß-aminopropionitrile (BAPN), a lathyrogen from sweet pea (Lathyrus odoratus) seeds. The quality and integrity of the collagenous template of skeletal elements depend on an appropriate concentration of collagen cross-links. In this study, chick embryos treated in ovo with BAPN on embryonic days (ED) 4-9 were found to develop multiple skeletal deformities. The most readily discernible and highly reproducible deformity was evident in the tibiotarsus, on which we focused to explore the chronology of the malformation process. Several lines of observation indicated that the bending deformity observable at ED10 in the tibiotarsus was inducible by BAPN administered on ED4-8; in other words, administration of BAPN on ED8 was sufficient to induce the deformity by ED10, whereas administration on ED9 was ineffective. Ultrastructurally, osteoclasts appeared to show enhanced activity in the medullary surface of the bone collar after BAPN administration. In addition, bone hyperplasia associated with the bending deformity was suggested to be correlated with higher osteoblast activity on the concave (or flexor) side of the tibiotarsal skeleton. These findings indicate that the bending deformity due to reduced mechanical integrity of the collagenous template is also associated with aberrant bone remodeling. (J Oral Sci 58, 255-263, 2016).

Characterization of mesenchymal progenitor cells in the crown and root pulp of primary teeth.

The existence of progenitor/mesenchymal stem cells (MSCs) was demonstrated previously in human primary/deciduous teeth. In this study, we examined dental pulp cells from root portion (root cells) of primary teeth without discernible root resorption and compared them with pulp cells from the crown portion (crown cells). Root cells and crown cells were characterized and compared to each other based on progenitor/MSC characteristics and on their generation efficiency of induced pluripotent stem (iPS) cells. Root cells and crown cells included cells manifesting typical progenitor/MSC properties such as osteogenic and adipogenic differentiation potential and clonogenicity. Interestingly, root cells showed a higher expression level of embryonic stem cell marker, KLF4, than crown cells. Moreover, the number of colony-forming unit-fibroblast and cell proliferation rate were higher for root cells than crown cells, and the efficiency of generating iPS cells from root cells was approximately four times higher than that from crown cells. Taken together, these results suggest that root cells from primary teeth show the MSC-like properties and thus could be a potent alternative source for iPS cell generation and the subsequent transplantation therapy.

Subectodermal microfibrillar bundles are organized into a distinct parallel array in the developing chick limb bud.

In this study, a unique fiber system in the subectodermal mesenchyme of the chick limb bud was visualized immunohistochemically with the use of a novel monoclonal antibody termed "FB1." This antibody stained a subset of extracellular fibers in the embryonic mesenchyme. Among the fibers visualized, those running perpendicularly to the limb bud ectoderm became progressively prominent in their thickness and length, and organized into a parallel array in the subectodermal region. This fiber system was distinct from that of major collagens, fibronectin, or tenascin. A molecule immunoprecipitated with FB1 comigrated with JB3 antigen, or chicken fibrillin-2. The fibers visualized immunohistochemically by FB1 and JB3 were indistinguishable from each other, and ultrastructurally appeared to be bundles composed of tubular-like microfibrils that originated directly from the ectodermal basal lamina. They lacked the amorphous deposits that are characteristic of elastin. A similar array of subectodermal fibers was also found in the developing axilla and some truncal regions, again well before the development of a definitive dermis. These findings suggest that a parallel array of subectodermal FB1-positive fibers constitutes a precocious fiber system in the presumptive dermis prior to the substantial formation of collagenous fibers. These fibers could be developmentally linked to oxytalan fibers, which are known to be present in the papillary dermis in mature cutaneous tissue.

Progressive bundling of fibrillin microfibrils into oxytalan fibers in the chick presumptive dermis.

Dorsoventral fibers in the presumptive dermis of the chick limb bud reported first by Hurle's group in 1989 are now revealed as bundles of fibrillin microfibrils (Isokawa et al., 2004). The bundles, which could be called oxytalan fibers at the light microscopic level, are aligned perpendicularly to the overlying ectoderm and form a unique fiber array, originating directly from the basal lamina. This well-oriented organization is beneficial in examining the process of in vivo bundling of microfibrils into oxytalan fibers. In this study, sections through the presumptive limb dermis were preferentially prepared from chick embryos at Days 4-6 (ED4-6). Immunohistochemically, fibrillin-positive dots representing cross-sectioned surfaces of individual fibers, increased in size from ED4 to 6, but their number per unit area remained constant. Ultrastructurally, a single oxytalan fiber at ED4 consisted of ∼15 microfibrils; the latter number increased fourfold from ED4 to 5 and threefold from ED5 to 6. Oxytalan fibers were all closely associated with mesenchymal cell; notably, the fibers at ED5 and 6 were held in a shallow ditch on the cell body or by lamellipodial cytoplasmic protrusion. In the sites of cell-fiber adhesion, microfibrils in the periphery of an oxytalan fiber appeared to adhere directly or by means of short flocculent strands to a nearby cell membrane; the latter showed a thickening of plasmalemma and its undercoat, indicating the presence of adhesive membrane specification. These findings suggest that the bundling of microfibrils is a progressive and closely cell-associated process.

Bilateral, asymmetric anomalies of the anterior bellies of digastric muscles.

Bilateral, asymmetric anomalies of the anterior bellies of digastric muscles were observed during dissection of the submental region. Specifically, four extra muscle bundles were found between the anterior bellies of the digastric muscle. Although anomalies of the anterior bellies of digastric muscles are often observed, this complicated pattern of digastric anomalies has not been previously reported. Our findings and previous observations illustrate the morphogenetic complexity of the anterior belly of the digastric muscle derived from the first pharyngeal arch, which gives rise to jaw musculature such as the mylohyoid muscle.

Development of the tarsometatarsal skeleton by the lateral fusion of three cylindrical periosteal bones in the chick embryo (Gallus gallus).

An avian tarsometatarsal (TMT) skeleton spanning from the base of toes to the intertarsal joint is a compound bone developed by elongation and lateral fusion of three cylindrical periosteal bones. Ontogenetic development of the TMT skeleton is likely to recapitulate the changes occurred during evolution but so far has received less attention. In this study, its development has been examined morphologically and histologically in the chick, Gallus gallus. Three metatarsal cartilage rods radiating distally earlier in development became aligned parallel to each other by embryonic day 8 (ED8). Calcification initiated at ED8 in the midshaft of cartilage propagated cylindrically along its surface. Coordinated radial growth by fabricating bony struts and trabeculae resulted in the formation of three independent bone cylinders, which further became closely apposed with each other by ED13 when the periosteum began to fuse in a back-to-back orientation. Bone microstructure, especially orientation of intertrabecular channels in which blood vasculature resides, appeared related to the observed rapid longitudinal growth. Differential radial growth was considered to delineate eventual surface configurations of a compound TMT bone, but its morphogenesis preceded the fusion of bone cylinders. Bony trabeculae connecting adjacent cylinders emerged first at ED17 in the dorsal and ventral quarters of intervening tissue at the mid-diaphyseal level. Posthatch TMT skeleton had a seemingly uniform mid-diaphysis, although the septa persisted between original marrow cavities. These findings provide morphological and histological bases for further cellular and molecular studies on this developmental process.

Late deposition of elastin to vertical microfibrillar fibers in the presumptive dermis of the chick embryonic tarsometatarsus.

Fibrillin microfibrils are integral components of elastic fibers and serve as a scaffold for elastin deposition. However, microfibrillar fibers (MFs) are not necessarily committed to develop into so-called elastic fibers. In dermis, elastin-free oxytalan MFs originating from the dermoepidermal junction are continuous to elaunin-type MFs (with a small amount of elastin) in the deeper papillary dermis, whereas the reticular dermis contains elastic fibers, or MFs embedded largely in elastin. In this study, we have investigated temporospatial patterns of elastin deposition on the MFs in tarsometatarsal presumptive dermis. While the earliest expression of elastin was demonstrated immunohistochemically as early as embryonic day 4 (ED4) in the wall of cardiac outflow and pharyngeal arch arteries, its deposition in the tarsometatarsus was first detected at ED6 in the deeper mesenchyme and at ED13 in the subectodermal mesenchyme. In the latter tissue, MFs had been organized perpendicularly to the covering ectoderm by ED4, well before an overt accumulation of collagenous matrix. Elastin deposition was observed initially in a punctate manner at ED13 and afterward became continuous along MFs. However, a characteristic spaced array of subectodermal vertical MFs was disorganized by ED17. These findings suggest that elastin deposition in the subectodermal MFs is not deployed by continuous, orderly propagation from elastic fibers in the deeper mesenchyme but occurs de novo in multiple foci along vertical MFs. Moreover, the present chronology of elastin deposition indicates that subectodermal, elastin-free MFs function as a transient, but primary fibrous structure in the presumptive dermis before the accumulation of collagenous matrix.

Potent platelet-derived growth factor-beta receptor (PDGF-betaR) inhibitors: Synthesis and structure-activity relationships of 7-[3-(cyclohexylmethyl)ureido]-3-{1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl}quinoxalin-2(1H)-one derivatives.

We found previously that 7-[3-(cyclohexylmethyl)ureido]-3-{1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl}quinoxalin-2(1H)-one (7d-6) has considerable potency as a PDGF inhibitor. This compound showed potent inhibitory activity in a PDGF-induced CPA (Cell Proliferation Assay) and APA (Auto-Phosphorylation Assay) (IC50 = 0.05 micromol/l in CPA, 0.03 micromol/l in APA). Therefore, we tried to develop a novel and effective PDGF-betaR inhibitor by optimizing a series of its derivatives. We found that trifluoroacetic acid (TFA)-catalyzed coupling of pyrrolo[2,3-b]pyridines with quinoxalin-2-ones proceeded efficiently under mild oxidation condition with manganese(IV) oxide (MnO2) in situ, so this method was applied to prepare a series of derivatives. Results of in vitro screening of newly synthesized derivatives identified compound 7d-9 as having potent (IC50 = 0.014 micromol/l in CPA, 0.007 micromol/l in APA) and selective [IC50 values against vascular endothelial growth factor receptor 2 (VEGFR2, kinase domain region, KDR), epidermal growth factor receptor (EGFR), c-Met (hepatocyte growth factor receptor) and insulin growth factor I receptor (IGF-IR)/IC50 against PDGFR were each >1000] inhibitory activity. Moreover, in this series of derivatives, 7b-2 showed potent inhibitory activity toward both PDGF- and VEGF-induced signaling (PDGFR: IC50 = 0.004 micromol/l in CPA, 0.0008 micromol/l in APA, KDR: IC50 = 0.008 micromol/l in APA). Herein we report a new and convenient synthetic method for this series of derivatives and its SAR study.