Developmental impairments of craniofacial bone and cartilage in transgenic mice expressing FGF10.

Mutations in a common extracellular domain of fibroblast growth factor receptor (FGFR)-2 isoforms (type IIIb and IIIc) cause craniosynostosis syndrome and chondrodysplasia syndrome. FGF10, a major ligand for FGFR2-IIIb and FGFR1-IIIb, is a key participant in the epithelial-mesenchymal interactions required for morphogenetic events. FGF10 also regulates preadipocyte differentiation and early chondrogenesis in vitro, suggesting that FGF10-FGFR signaling may be involved in craniofacial skeletogenesis in vivo. To test this hypothesis, we used a tet-on doxycycline-inducible transgenic mouse model (FGF10 Tg) to overexpress Fgf10 from embryonic day 12.5. Fgf10 expression was 73.3-fold higher in FGF10 Tg than in wild-type mice. FGF10 Tg mice exhibited craniofacial anomalies, such as a short rostrum and mandible, an underdeveloped (cleft) palate, and no tympanic ring. Opposite effects on chondrogenesis in different anatomical regions were seen, e.g., hyperplasia in the nasal septum and hypoplasia in the mandibular condyle. We found an alternative splicing variant of Fgfr2-IIIb with a predicted translation product lacking the transmembrane domain, and suggesting a soluble form of FGFR2-IIIb (sFGFR2-IIIb), differentially expressed in some of the craniofacial bones and cartilages. Thus, excessive FGF10 may perturb signal transduction of the FGF-FGFR, leading to craniofacial skeletal abnormalities in FGF10 Tg mice.

Five-Year Follow-Up of a Child with Non-Syndromic Oligodontia from before the Primary Dentition Stage: A Case Report.

BACKGROUND: Congenital absence of teeth is a major dental abnormality in pediatric dentistry and the absence of six or more teeth is defined as oligodontia. Few reports of patients with non-syndromic oligodontia without systemic disease have continued dental follow-up from an early age. METHODS: We performed the five-year follow-up from before the eruption of the primary dentition of a Japanese child with non-syndromic oligodontia and analyzed changes in dental arch growth. RESULTS: At the oral examination at the age of 1 year and 2 months, eight primary incisors were congenitally absent. Therefore, we made dentures for the patient at the age of 3 years and 4 months. From the age of 5 years and 1 month, the child received articulation training for dysarthria from a speech therapist to improve the function and appearance of the oral cavity. Measurement of the patient's dental models revealed a particularly narrow dental arch, especially between the primary canines. CONCLUSIONS: Our findings highlight the importance of treatment for patients with non-syndromic oligodontia from an early age by multiple medical professionals, recognizing that the missing teeth affect the growth of the maxillofacial region.

Single-Cell RNA-Sequencing Reveals the Breadth of Osteoblast Heterogeneity.

The current paradigm of osteoblast fate is that the majority undergo apoptosis, while some further differentiate into osteocytes and others flatten and cover bone surfaces as bone lining cells. Osteoblasts have been described to exhibit heterogeneous expression of a variety of osteoblast markers at both transcriptional and protein levels. To explore further this heterogeneity and its biological significance, Venus-positive (Venus+) cells expressing the fluorescent protein Venus under the control of the 2.3-kb Col1a1 promoter were isolated from newborn mouse calvariae and subjected to single-cell RNA sequencing. Functional annotation of the genes expressed in 272 Venus+ single cells indicated that Venus+ cells are osteoblasts that can be categorized into four clusters. Of these, three clusters (clusters 1 to 3) exhibited similarities in their expression of osteoblast markers, while one (cluster 4) was distinctly different. We identified a total of 1920 cluster-specific genes and pseudotime ordering analyses based on established concepts and known markers showed that clusters 1 to 3 captured osteoblasts at different maturational stages. Analysis of gene co-expression networks showed that genes involved in protein synthesis and protein trafficking between endoplasmic reticulum (ER) and Golgi are active in these clusters. However, the cells in these clusters were also defined by extensive heterogeneity of gene expression, independently of maturational stage. Cells of cluster 4 expressed Cd34 and Cxcl12 with relatively lower levels of osteoblast markers, suggesting that this cell type differs from actively bone-forming osteoblasts and retain or reacquire progenitor properties. Based on expression and machine learning analyses of the transcriptomes of individual osteoblasts, we also identified genes that may be useful as new markers of osteoblast maturational stages. Taken together, our data show much more extensive heterogeneity of osteoblasts than previously documented, with gene profiles supporting diversity of osteoblast functional activities and developmental fates. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

One-Pot Production of L-threo-3-Hydroxyaspartic Acid Using Asparaginase-Deficient Escherichia coli Expressing Asparagine Hydroxylase of Streptomyces coelicolor A3(2).

We developed a novel process for efficient synthesis of L-threo-3-hydroxyaspartic acid (L-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693-D246N) were adaptable to the direct hydroxylation of L-aspartic acid; however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesize L-THA. By using these recombinant enzymes, L-THA was obtained by L-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube. L-THA was obtained in a small amount with a molar yield of 0.076% by using intact Escherichia coli expressing the asnO gene, and thus, two asparaginase-deficient mutants of E. coli were investigated. A remarkably increased L-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of the asnO gene was enhanced by using the T7 promoter in E. coli instead of the lac promoter, the L-THA yield was significantly increased to 92%. By using a combination of the E. coli asparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently, L-THA was successfully obtained from L-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production of L-THA.

Efficient asymmetric synthesis of abeo-abietane-type diterpenoids by using the intramolecular Heck reaction.

The synthesis of the abeo-abietane-type diterpenoids, i.e., (-)-dichroanal B, (-)-dichroanone, and taiwaniaquinone H, was achieved by using the intramolecular asymmetric Heck reaction. Our synthetic routes required fewer steps and gave a much higher overall yield and ee within shorter steps than those for racemic and antipodal forms reported to date (10, 12, and 13 steps with an overall yield of 50%, 40%, and 39%, and 94%, 98%, and 98% ee, respectively).

Inorganic iodine incorporation into soil organic matter: evidence from iodine K-edge X-ray absorption near-edge structure.

The transformation of inorganic iodine (I(-) and IO(3)(-)) incubated in soils with varying amounts of organic matter (Andosols from the surface layer of an upland field and forest, as well as Acrisols from surface and subsurface layers of an upland field) was investigated by using the iodine K-edge X-ray absorption near-edge structure (XANES). After 60d of reaction, both I(-) and IO(3)(-) were transformed into organoiodine in surface soils containing sufficient amounts of organic matter, whereas IO(3)(-) remained unchanged in the subsurface soil of Acrisols with low organic matter contents. Transformation of IO(3)(-) into organoiodine was not retarded when the microbial activity in soil was reduced by gamma-ray irradiation, suggesting that microbial activity was not essential for the transformation of inorganic iodine into organoiodine. Soil organic matter has the ability to transform inorganic iodine into organoiodine.

Interaction between cyclooxygenase (COX)-1- and COX-2-products modulates COX-2 expression in the late phase of acute inflammation.

Prostanoid production depends on the activity of two cyclooxygenase (COX) isoforms. It is appreciated that COX-1 plays a role in physiological processes, whereas COX-2 acts in pathological conditions. However their roles, particularly roles of COX-1, have not yet been fully established in inflammation. Here, we examined the effects of COX inhibitors, having differential isoform selectivity, on the late phase of rat carrageenin-induced pleurisy to elucidate the role of COX-2 expressed in the draining lymph nodes and found substantial contribution of COX-1-product(s). Protein and mRNA of COX-2 were detectable with Western blotting analysis and reverse-transcription polymerase chain reaction (RT-PCR) analysis in parathymic lymph nodes, peaking at 48 h after induction of pleurisy. Microsomal prostaglandin E synthase (mPGES)-1 was detectable by immunohistochemical analysis in cells with dendritic processes, a morphological characteristic similar to that of COX-2 expressing cells. Although aspirin, indomethacin and a COX-1 inhibitor, ketorolac, significantly decreased the volume of pleural exudate, they did not affect the levels of COX-2 and mPGES-1 in the lymph node 24 h after induction of pleurisy. In contrast, COX-2 inhibitors, nimesulide and NS-398, had no effect on the exudate volume, but they increased the number of COX-2- and mPGES-1-expressing cells and extension of their dendritic processes with significant increase in the COX-2 level, which were antagonised by ketorolac. These results suggest that COX-2-expressing cells may negatively self-regulate their functions by producing PGE2 via mPGES-1: migration into the draining lymph node and their differentiation. Moreover, COX-1- and COX-2-derived prostanoids may play differential or sometimes antagonistic roles in the late phase of acute inflammation.