DMP-1 expression in alveolar bone socket following Anredera cordifolia (Ten.) Steenis treatment: A histological study.

Objective: The study aimed to ascertain how Anredera cordifolia (Ten.) Steenis Gel affects the expression of protein dentin matrix protein-1 (DMP-1) in alveolar Wistar rats after tooth extraction. Materials and Methods: Rats were given A. cordifolia (Ten.) Steenis gel was in the socket after tooth extraction, and then the wound was sutured. The rats were sacrificed for 8 and 15 days following tooth extraction. The results on the 8th and 15th days demonstrate that the expression of DMP-1 in the treatment group is significantly higher than in the control group. Results: Expression of DMP-1 in the socket after tooth extraction on days 8 and 15 with a 400x magnification light microscope in both of the A. cordifolia (Ten.) Steenis gel treatment groups showed significant differences compared to the control group. Conclusion: The use of A. cordifolia (Ten.) Steenis gel can stimulate DMP-1 expression in alveolar bone after tooth extraction.

Lessons learned from the real-world diagnosis and management of hereditary hypophosphatemic rickets.

Hypophosphatemic rickets, which is often hereditary, is still under- or misdiagnosed in both children and adults, denying these individuals access to optimal management and genetic counseling. There have been recent calls to compile real-world data and share best practice on these rare conditions to guide clinical decision-making. Here we present eight clinical vignettes of patients with hypophosphatemic rickets encountered in our tertiary pediatric endocrinology practice. We describe the clinical features, genetics, and management of four cases of X-linked hypophosphatemia (PHEX mutations), one each of autosomal recessive hypophosphatemic rickets (DMP1 mutation) and autosomal recessive vitamin D-dependent rickets type 1A (CYP27B1 mutation), and two cases of distal renal tubular acidosis with FOXI1 mutation-associated hypophosphatemic rickets. Our cases prompt consideration of the (i) frequent misdiagnosis of hypophosphatemic rickets in clinical practice and the importance of comprehensive genetic testing; (ii) variable expressivity of the causative mutations; and (iii) a lack of responsiveness and/or compliance to conventional therapy and the value of burosumab in modern management, provided access is equitable. These cases highlight common real-world themes and challenges to managing patients presenting with these diverse conditions, especially the burden of disease hidden by misdiagnosis. In sharing these cases, we hope to raise awareness of these conditions, promote best practice in genetic diagnosis and management, and further advocate for reimbursement equity for the best available therapies.

Quantum Mechanics Characterization of Non-Covalent Interaction in Nucleotide Fragments.

Accurate calculation of non-covalent interaction energies in nucleotides is crucial for understanding the driving forces governing nucleic acid structure and function, as well as developing advanced molecular mechanics forcefields or machine learning potentials tailored to nucleic acids. Here, we dissect the nucleotides' structure into three main constituents: nucleobases (A, G, C, T, and U), sugar moieties (ribose and deoxyribose), and phosphate group. The interactions among these fragments and between fragments and water were analyzed. Different quantum mechanical methods were compared for their accuracy in capturing the interaction energy. The non-covalent interaction energy was decomposed into electrostatics, exchange-repulsion, dispersion, and induction using two ab initio methods: Symmetry-Adapted Perturbation Theory (SAPT) and Absolutely Localized Molecular Orbitals (ALMO). These calculations provide a benchmark for different QM methods, in addition to providing a valuable understanding of the roles of various intermolecular forces in hydrogen bonding and aromatic stacking. With SAPT, a higher theory level and/or larger basis set did not necessarily give more accuracy. It is hard to know which combination would be best for a given system. In contrast, ALMO EDA2 did not show dependence on theory level or basis set; additionally, it is faster.

Green space exposure and blood DNA methylation at birth and in childhood - A multi-cohort study.

Green space exposure has been associated with improved mental, physical and general health. However, the underlying biological mechanisms remain largely unknown. The aim of this study was to investigate the association between green space exposure and cord and child blood DNA methylation. Data from eight European birth cohorts with a total of 2,988 newborns and 1,849 children were used. Two indicators of residential green space exposure were assessed: (i) surrounding greenness (satellite-based Normalized Difference Vegetation Index (NDVI) in buffers of 100 m and 300 m) and (ii) proximity to green space (having a green space ≥ 5,000 m2 within a distance of 300 m). For these indicators we assessed two exposure windows: (i) pregnancy, and (ii) the period from pregnancy to child blood DNA methylation assessment, named as cumulative exposure. DNA methylation was measured with the Illumina 450K or EPIC arrays. To identify differentially methylated positions (DMPs) we fitted robust linear regression models between pregnancy green space exposure and cord blood DNA methylation and between cumulative green space exposure and child blood DNA methylation. Two sensitivity analyses were conducted: (i) without adjusting for cellular composition, and (ii) adjusting for air pollution. Cohort results were combined through fixed-effect inverse variance weighted meta-analyses. Differentially methylated regions (DMRs) were identified from meta-analysed results using the Enmix-combp and DMRcate methods. There was no statistical evidence of pregnancy or cumulative exposures associating with any DMP (False Discovery Rate, FDR, p-value < 0.05). However, surrounding greenness exposure was inversely associated with four DMRs (three in cord blood and one in child blood) annotated to ADAMTS2, KCNQ1DN, SLC6A12 and SDK1 genes. Results did not change substantially in the sensitivity analyses. Overall, we found little evidence of the association between green space exposure and blood DNA methylation. Although we identified associations between surrounding greenness exposure with four DMRs, these findings require replication.

Study on the mechanism and degradation behavior of Encifer adhaerens DNM-S1 capturing dimethyl phthalate.

The global concern surrounding pollution caused by phthalates is escalating, with dimethyl phthalate (DMP) emerging as one of the most prevalent contaminants within the phthalates (PAEs) category. Although the biodegradation of DMP is considered both safe and efficient, its underlying degradation mechanism is not yet fully elucidated, and the degradation performance can be somewhat inconsistent. To address this issue, our study isolated a DMP-degrading bacterium (DNM-S1) from a vegetable greenhouse. The resulting data revealed that DNM-S1 exhibited a remarkable degradation performance, successfully degrading 84.98% of a 2000 mg L-1 DMP solution within 72 h. Remarkably, it achieved complete degradation of a 50 mg L-1 DMP solution within just 3 h. DMP degradation by DNM-S1 was also found to be efficient even under low-temperature conditions (10 °C). Our research further indicates that DNM-S1 is capable of capturing DMP through the ester bond in the bacterium's cell wall fatty acids, forming hydrogen bonds through hydrophobic interactions. The DMP was then transported into the DNM-S1 protoplasm using an active transport mechanism. Interestingly, the secondary metabolites of DNM-S1 contained natural carotenoids, which could potentially counteract the damaging effects of PAEs on cell membrane permeability. In summary, these findings highlight the potential of DNM-S1 in addressing PAEs pollution and provide new insights into the metabolic mechanism of PAEs degradation.

Genome-Wide Identification and Expression Analysis of the DMP and MTL Genes in Sweetpotato (Ipomoea batatas L.).

Sweetpotato (Ipomoea batatas L.) is a strategic crop with both economic and energy value. However, improving sweetpotato varieties through traditional breeding approaches can be a time-consuming and labor-intensive process due to the complex genetic nature of sweetpotato as a hexaploid species (2n = 6x = 90). Double haploid (DH) breeding, based on in vivo haploid induction, provides a new approach for rapid breeding of crops. The success of haploid induction can be achieved by manipulating specific genes. Two of the most critical genes, DMP (DUF679 membrane proteins) and MTL (MATRILINEAL), have been shown to induce haploid production in several species. Here, we identified and characterized DMP and MTL genes in sweetpotato using gene family analysis. In this study, we identified 5 IbDMPs and 25 IbpPLAs. IbDMP5 and IbPLAIIs (IbPLAIIκ, IbPLAIIλ, and IbPLAIIµ) were identified as potential haploid induction (HI) genes in sweetpotato. These results provide valuable information for the identification and potential function of HI genes in sweetpotato and provide ideas for the breeding of DH lines.

Metaplastic regeneration in the mouse stomach requires a reactive oxygen species pathway.

In pyloric metaplasia, mature gastric chief cells reprogram via an evolutionarily conserved process termed paligenosis to re-enter the cell cycle and become spasmolytic polypeptide-expressing metaplasia (SPEM) cells. Here, we use single-cell RNA sequencing (scRNA-seq) following injury to the murine stomach to analyze mechanisms governing paligenosis at high resolution. Injury causes induced reactive oxygen species (ROS) with coordinated changes in mitochondrial activity and cellular metabolism, requiring the transcriptional mitochondrial regulator Ppargc1a (Pgc1α) and ROS regulator Nf2el2 (Nrf2). Loss of the ROS and mitochondrial control in Ppargc1a-/- mice causes the death of paligenotic cells through ferroptosis. Blocking the cystine transporter SLC7A11(xCT), which is critical in lipid radical detoxification through glutathione peroxidase 4 (GPX4), also increases ferroptosis. Finally, we show that PGC1α-mediated ROS and mitochondrial changes also underlie the paligenosis of pancreatic acinar cells. Altogether, the results detail how metabolic and mitochondrial changes are necessary for injury response, regeneration, and metaplasia in the stomach.

[General practitioners' perspectives on the perceived effectiveness of the disease management programs for type 2 diabetes and coronary heart disease: Results of a focus group study]. / Die wahrgenommene Effektivität der Disease Management Programme für Diabetes mellitus Typ 2 und Koronare Herzkrankheit aus Sicht von Hausärzt*innen ­ Ergebnisse einer Fokusgruppenstudie.

BACKGROUND: The majority of patients in disease management programs (DMPs) for type 2 diabetes (T2DM) and coronary heart disease (CHD) in Germany are enrolled by their general practitioner (GP). The aim of this study was, in the context of upcoming DMP expansions, to elicit GPs' current experiences and opinions regarding the perceived effectiveness and acceptance of the DMPs T2DM and CHD, as well as to determine beneficial and hindering aspects of the implementation of these programs from a GP's perspective. METHODS: In August and September 2020, 20 GPs of teaching practices of the University Hospital Cologne with experiences in DMPs were interviewed in semi-structured focus group discussions. Their expectations, attitudes and opinions regarding the DMPs T2DM and CHD were evaluated and analyzed according to the content-structuring qualitative content analysis by Kuckartz. RESULTS: The DMP T2DM was rated as generally positive by the respondents due to the structured treatment including regular foot and eye examinations, close patient contacts and perceptions of improved health outcomes. The DMP CHD was rated more negatively by the respondents because of a high and partly unnecessary documentation workload and limited therapeutic freedom, leading to a perceived ineffectiveness for patients' health outcomes. Thus, there was a discrepancy in the perceived effectiveness of the examined DMPs, causing a lower acceptance of the DMP CHD. Therefore, some of the respondents tended to enroll fewer patients into the DMP CHD or to drop out of the DMP CHD. DISCUSSION: In order to increase the acceptance and sustainability of DMPs some elements of the DMP CHD as well as the remuneration and the documentation need to be reconsidered. Additionally, future studies on the acceptance of DMPs should differentiate between different DMPs in order to generate valid results.

IgA nephropathy in a boy with frequently relapsing nephrotic syndrome.

A Japanese boy developed nephrotic syndrome (NS) and had microscopic hematuria at 8 years old. Renal biopsy was performed. Light microscopy study revealed mesangial proliferation and all immunofluorescent stains (including IgA) were negative, so he was diagnosed with non-IgA diffuse mesangial proliferation (DMP). Complete remission was achieved at 13 days after the initiation of oral prednisolone, and hematuria also disappeared 3 days later, but the patient developed frequently relapsing nephrotic syndrome. Cyclosporine A (CyA) was introduced at 10 years old, and there were no relapses between then and when it was discontinued at 12 years old. A second renal biopsy revealed minimal change without CyA nephrotoxicity. However, there was repeated relapse of NS after discontinuation, so CyA was reintroduced 8 months later, and NS remained in remission thereafter. Microscopic hematuria appeared at 13 years old, however, with gross hematuria appearing at the time of infection. A third renal biopsy revealed mesangial proliferation with IgA-dominant deposition, so the patient was diagnosed with IgA nephropathy. Currently (14 years old), CyA treatment has been discontinued and the patient is undergoing lisinopril therapy for IgA nephropathy, but there are still relapses of NS. To the best of our knowledge, there have been no previous reports of a patient with non-IgA DMP at the onset of NS who had later development of IgA nephropathy. The patient showed non-IgA DMP at the onset, suggesting that NS with non-IgA DMP and IgA nephropathy has some common pathophysiology. Treatment for NS, such as PSL and/or CyA treatment, may suppress the clinical manifestation of late IgA nephropathy.

On-demand chlorine dioxide solution enhances odontoblast differentiation through desulfation of cell surface heparan sulfate proteoglycan and subsequent activation of canonical Wnt signaling.

Heparan sulfate proteoglycans (HSPGs) surround the surface of odontoblasts, and their modification affects their affinity for Wnt ligands. This study proposes applying Matching Transformation System® (MA-T), a novel chlorinated oxidant, to enhance dentinogenesis. MA-T treatment in odontoblasts decreased sulfation of HSPG and upregulated the expression of dentin sialophosphoprotein (Dspp) and Dentin Matrix Protein 1 (Dmp1) via activation of canonical Wnt signaling in vitro. Ex vivo application of MA-T also enhanced dentin matrix formation in developing tooth explants. Reanalysis of a public single-cell RNA-seq dataset revealed significant Wnt activity in the odontoblast population, with enrichment for Wnt10a and Wnt6. Silencing assays showed that Wnt10a and Wnt6 were redundant in inducing Dspp and Dmp1 mRNA expression. These Wnt ligands' expression was upregulated by MA-T treatment, and TCF/LEF binding sites are present in their promoters. Furthermore, the Wnt inhibitors Notum and Dkk1 were enriched in odontoblasts, and their expression was also upregulated by MA-T treatment, together suggesting autonomous maintenance of Wnt signaling in odontoblasts. This study provides evidence that MA-T activates dentinogenesis by modifying HSPG and through subsequent activation of Wnt signaling.

A Nanozymatic-Mediated Smartphone Colorimetric Sensing Platform for the Detection of Dimethyl Phthalate (DMP) and Dibutyl Phthalate (DBP).

Plasticizers are a type of toxic substance that may remain in food, posing significant health risks including carcinogenic, teratogenic, mutagenic, and other adverse effects. In this study, a novel strategy was employed by combining Pt@Au nanozymes with high catalytic properties to created two catalytic signal probes, designated as Pt@Au@Ab1 and Pt@Au@Ab2, specifically designed for the detection of dimethyl phthalate (DMP) and dibutyl phthalate (DBP). These catalytic signal probes served as the foundation for the development of a colorimetric immunoassay, enabling the simultaneous detection of both DMP and DBP. The colorimetric immunoassay is capable of detecting DMP in the range of 0.5-100 µg/L with a limit of detection as low as 0.1 µg/L and DBP in the range of 1-32 µg/L with a low limit of detection of 0.5 µg/L. The developed immunoassay can be used for the determination of the DMP and DBP in baijiu and plastic bottled drinks. The recovery rate is in the range of 96.4% and 100.5% and the coefficient of variation is between 1.0% and 7.2%. This innovative colorimetric immunoassay offers a robust tool for the simultaneous quantification of DMP and DBP in real samples.

Commercial beers: A source of phthalates and di-ethylhexyl adipate.

Beer is one of the most consumed beverages worldwide. Different materials used along its production and packaging can result in human exposure to phthalates and adipates. The aim of this study was to assess simultaneously the levels of phthalates and di-ethylhexyl adipate (DEHA) in commercial beer samples (n = 66) with a method based on DLLME and detection with GC-MS/MS, and further evaluate human exposure. Six out of seven compounds studied were found in the beers analysed, with levels ranging from 1.77 to 205.40 µg/L. The most prevalent was DEHA at 205.40 µg/L, while dimethyl phthalate (DMP) was not present in any sample. Samples with 5-6 % alcohol, packed in aluminium cans and produced in an industrial environment presented the highest level of these contaminants. Despite low-risk exposure to phthalates and adipate with beer, it is important to remember the ubiquitous nature of these compounds, which can lead to cumulative exposure.

Loss of Bmp2 impairs odontogenesis via dysregulating pAkt/pErk/GCN5/Dlx3/Sp7.

BMP2 signaling plays a pivotal role in odontoblast differentiation and maturation during odontogenesis. Teeth lacking Bmp2 exhibit a morphology reminiscent of dentinogenesis imperfecta (DGI), associated with mutations in dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) genes. Mechanisms by which BMP2 signaling influences expressions of DSPP and DMP1 and contributes to DGI remain elusive. To study the roles of BMP2 in dentin development, we generated Bmp2 conditional knockout (cKO) mice. Through a comprehensive approach involving RNA-seq, immunohistochemistry, promoter activity, ChIP, and Re-ChIP, we investigated downstream targets of Bmp2. Notably, the absence of Bmp2 in cKO mice led to dentin insufficiency akin to DGI. Disrupted Bmp2 signaling was linked to decreased expression of Dspp and Dmp1, as well as alterations in intracellular translocation of transcription factors Dlx3 and Sp7. Intriguingly, upregulation of Dlx3, Dmp1, Dspp, and Sp7, driven by BMP2, fostered differentiation of dental mesenchymal cells and biomineralization. Mechanistically, BMP2 induced phosphorylation of Dlx3, Sp7, and histone acetyltransferase GCN5 at Thr and Tyr residues, mediated by Akt and Erk42/44 kinases. This phosphorylation facilitated protein nuclear translocation, promoting interactions between Sp7 and Dlx3, as well as with GCN5 on Dspp and Dmp1 promoters. The synergy between Dlx3 and Sp7 bolstered transcription of Dspp and Dmp1. Notably, BMP2-driven GCN5 acetylated Sp7 and histone H3, while also recruiting RNA polymerase II to Dmp1 and Dspp chromatins, enhancing their transcriptions. Intriguingly, BMP2 suppressed the expression of histone deacetylases. we unveil hitherto uncharted involvement of BMP2 in dental cell differentiation and dentine development through pAkt/pErk42/44/Dlx3/Sp7/GCN5/Dspp/Dmp1.

Regulation of Osteoblast to Osteocyte Differentiation by Cyclin-Dependent Kinase-1.

Osteocytes have recently been identified as a new regulator of bone remodeling, but the detailed mechanism of their differentiation from osteoblasts remains unclear. The purpose of this study is to identify cell cycle regulators involved in the differentiation of osteoblasts into osteocytes and determine their physiological significance. The study uses IDG-SW3 cells as a model for the differentiation from osteoblasts to osteocytes. Among the major cyclin-dependent kinases (Cdks), Cdk1 is most abundantly expressed in IDG-SW3 cells, and its expression is down-regulated during differentiation into osteocytes. Inhibition of CDK1 activity reduces IDG-SW3 cell proliferation and differentiation into osteocytes. Osteocyte and Osteoblast-specific Cdk1 knockout in mice (Dmp1-Cdk1KO ) results in trabecular bone loss. Pthlh expression increases during differentiation, but inhibiting CDK1 activity reduces Pthlh expression. Parathyroid hormone-related protein concentration is reduced in the bone marrow of Dmp1-Cdk1KO mice. Four weeks of Parathyroid hormone administration partially recovers the trabecular bone loss in Dmp1-Cdk1KO mice. These results demonstrate that Cdk1 plays an essential role in the differentiation from osteoblast to osteocyte and the acquisition and maintenance of bone mass. The findings contribute to a better understanding of the mechanisms of bone mass regulation and can help develop efficient therapeutic strategies for osteoporosis treatment.

Chromatographic reversed HPLC and TLC-densitometry methods for simultaneous determination of serdexmethylphenidate and dexmethylphenidate in presence of their degradation products-with computational assessment.

Two Chromatographic methods have been established and optimized for simultaneous determination of serdexmethylphenidate (SER.DMP) and dexmethylphenidate (DMP) in the presence of their degradation products. The first method is a reversed phase high performance liquid chromatography with diode array detection (HPLC-DAD). Isocratic separation was carried out on Waters X-bridge Shield RP18 column (150×3.9×5 µm particle size) using a mixture of 5 mM phosphate buffer (pH 5.5): acetonitrile (40:60, v/v) as a mobile phase, flow rate 1 mL/min and detection at 220 nm. The second method is a thin-layer chromatography (TLC)-densitometry method using methanol: chloroform (70:30, v/v) as a mobile phase and UV scanning at 220 nm. In HPLC method, the linearity range of SER.DMP was (2.5-25 µg/mL); with LOD (0.051 µg/mL) and LOQ (0.165 µg/mL) while for DMP was (2.5-25 µg/mL); with LOD and LOQ of (0.098 µg/mL) and (0.186 µg/mL), respectively. For TLC method the sensitivity range of SER.DMP was (5-25 µg/mL), LOD was (0.184 µg/spot), while LOQ was (0.202 µg/ spot) whereas for DMP the sensitivity range was (5-25 µg/mL) with LOD of (0.115 µg/ spot) and LOQ of (0.237 µg/ spot), respectively. SER.DMP was found to be equally labile to acidic and alkaline hydrolysis, whereas DMP was sensitive to acidic hydrolysis only. Both drugs were successfully determined in presence of acidic and basic degradants by the two developed methods (stability indicating assay method). Chromatographic separation of the degradation products was carried out on TLC aluminum silica plates 60 F254, as a stationary phase, using methanol: dichloroethane: acetonitrile (60:20:20 v/v), as a mobile phase. The degradation pathway was confirmed using TLC, IR, 1H-NMR and mass spectroscopy; moreover, the separation power was correlated to the computational results by applying molecular dynamic simulation. The developed methods were validated according to the International Conference on Harmonization (ICH) guidelines demonstrating good accuracy and precision. They were successfully applied for quantitation of SER.DMP and DMP in pure and capsule forms. The results were statistically compared with those obtained by the reported method in terms of accuracy, precision and robustness, and no significant difference was found.

Dentin Matrix Protein 1 Regulates Mineralization of MC3T3-E1 Cells via the TNAP-ANK-ENPP1 Axis.

BACKGROUND: Dentin matrix protein 1 (DMP1) is central to matrix mineralization. Clarification of the function of DMP1 is crucial to understanding normal bone formation and pathological calcification. The tissue-nonspecific alkaline phosphatase (TNAP) -progressive ankylosing enzyme (ANK) -extracellular nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) axis induces deposition of hydroxyapatite (HA) and pyrophosphate dehydrate (CPPD) by regulating pyrophosphate (PPi). Here, we investigated the mechanism by which DMP1 and the TNAP-ANK-ENPP1 axis participate in mineralization. METHODS: Expression of DMP1, TNAP, NPP1, and ANK genes in MC3T3-E1 cells was detected by RT-qPCR before and after treatment with DMP1 siRNA. An enzyme-linked immunosorbent assay was used to determine expression of DMP1 protein, TNAP activity was detected by SIGMAFAST p-nitrophenyl phosphate tablets, and mineralization of osteoblasts was determined by alizarin red staining. PPi levels were determined radiometrically and equalized for cell DNA. Levels of calcium, inorganic phosphate, zinc, and magnesium were assessed by standard laboratory techniques. RESULTS: After DMP1 gene silencing, expressions of TNAP, ENPP1, and ANK were correspondingly reduced. DMP1 altered extravesicular and intravesicular ion levels through the TNAP-ENPP1-ANK axis in MC3T3-E1 cells. CONCLUSIONS: DMP1 regulated mineralization of MC3T3-E1 cells via the TNAP-ANK-ENPP1 axis and affected TNAP activity by two processes-rapid regulation of the Zn2+ transporter (ZnT) and transcriptional regulation of hysteresis. However, DMP1 may affect expression of ENPP1 and ANK only via hysteresis transcriptional regulation. DMP1, as a calcium trap or catalytic enzyme, appears to have a role in collagen mineralization.

LPS-induced inflammation potentiates dental pulp stem cell odontogenic differentiation through C5aR and p38.

AIM: Inflammation is a complex host response to harmful infection or injury, and it seems to play a crucial role in tissue regeneration both positively and negatively. We have previously demonstrated that the activation of the complement C5a pathway affects dentin-pulp regeneration. However, limited information is available to understand the role of the complement C5a system related to inflammation-mediated dentinogenesis. The aim of this study was to determine the role of complement C5a receptor (C5aR) in regulating lipopolysaccharide (LPS)-induced odontogenic differentiation of dental pulp stem cells (DPSCs). MATERIAL AND METHODS: Human DPSCs were subjected to LPS-stimulated odontogenic differentiation in dentinogenic media treated with the C5aR agonist and antagonist. A putative downstream pathway of the C5aR was examined using a p38 mitogen-activated protein kinase (p38) inhibitor (SB203580). RESULTS: Our data demonstrated that inflammation induced by the LPS treatment potentiated DPSC odontogenic differentiation and that this is C5aR dependent. C5aR signaling controlled the LPS-stimulated dentinogenesis by regulating the expression of odontogenic lineage markers like dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP-1). Moreover, the LPS treatment increased the total p38, and the active form of p38 expression, and treatment with SB203580 abolished the LPS-induced DSPP and DMP-1 increase. CONCLUSIONS: These data suggest a significant role of C5aR and its putative downstream molecule p38 in the LPS-induced odontogenic DPSCs differentiation. This study highlights the regulatory pathway of complement C5aR/p38 and a possible therapeutic approach for improving the efficiency of dentin regeneration during inflammation.

FGF4 and FGF9 have synergistic effects on odontoblast differentiation.

The purpose of this study was to investigate whether fibroblast growth factor 4 (FGF4) and FGF9 are active in dentin differentiation. Dentin matrix protein 1 (Dmp1) -2A-Cre transgenic mice, which express the Cre-recombinase in Dmp1-expressing cells, were crossed with CAG-tdTomato mice as reporter mouse. The cell proliferation and tdTomato expressions were observed. The mesenchymal cell separated from neonatal molar tooth germ were cultured with or without FGF4, FGF9, and with or without their inhibitors ferulic acid and infigratinib (BGJ398) for 21 days. Their phenotypes were evaluated by cell count, flow cytometry, and real-time PCR. Immunohistochemistry for FGFR1, 2, and 3 expression and the expression of DMP1 were performed. FGF4 treatment of mesenchymal cells obtained promoted the expression of all odontoblast markers. FGF9 failed to enhance dentin sialophosphoprotein (Dspp) expression levels. Runt-related transcription factor 2 (Runx2) was upregulated until day 14 but was downregulated on day 21. Compared to Dmp1-negative cells, Dmp1-positive cells expressed higher levels of all odontoblast markers, except for Runx2. Simultaneous treatment with FGF4 and FGF9 had a synergistic effect on odontoblast differentiation, suggesting that they may play a role in odontoblast maturation.

Generation of bicistronic Dmp1-Cre knock-in mice using a self-cleaving 2A peptide.

INTRODUCTION: The conditional manipulation of genes using the Cre recombinase-locus of crossover in P1 (Cre/loxP) system is an important tool for revealing gene functions and cell lineages in vivo. The outcome of this method is dependent on the performance of Cre-driver mouse strains. In most cases, Cre knock-in mice show better specificity than randomly inserted Cre transgenic mice. However, following knock-in, the expression of the original gene replaced by Cre is lost. MATERIALS AND METHODS: We generated a new differentiated osteoblast- and osteocyte-specific Cre knock-in mouse line that carries the viral T2A sequence encoding a 2A self-cleaving peptide at the end of the coding region of the dentin matrix protein 1 (Dmp1) gene accompanied by the Cre gene. RESULTS: We confirmed that Dmp1-T2A-Cre mice showed high Cre expression in osteoblasts, osteocytes, odontoblasts, and periodontal ligament cells and that the 2A self-cleaving peptide efficiently produced both Dmp1 and Cre proteins. Furthermore, unlike the Dmp1 knockout mice, homozygous Dmp1-T2A-Cre mice showed no skeletal abnormalities. Analysis using the Cre reporter strain confirmed differentiated osteoblast- and osteocyte-specific Cre-mediated recombination in the skeleton. Furthermore, recombination was also detected in some nuclei of skeletal muscle cells, spermatocytes, and intestinal cells. CONCLUSION: 2A-Cre functions effectively in vivo, and Dmp1-T2A-Cre knock-in mice are a useful tool for studying the functioning of various genes in hard tissues.

Involvement of Dmp1 in the Precise Regulation of Hair Bundle Formation in the Developing Cochlea.

Dentin matrix protein 1 (Dmp1) is a highly phosphorylated, extracellular matrix protein that is extensively expressed in bone and teeth but also found in soft tissues, including brain and muscle. However, the functions of Dmp1 in the mice cochlea are unknown. Our study showed that Dmp1 was expressed in auditory hair cells (HCs), with the role of Dmp1 in those cells identified using Dmp1 cKD mice. Immunostaining and scanning electron microscopy of the cochlea at P1 revealed that Dmp1 deficiency in mice resulted in an abnormal stereociliary bundle morphology and the mispositioning of the kinocilium. The following experiments further demonstrated that the cell-intrinsic polarity of HCs was affected without apparent effect on the tissue planer polarity, based on the observation that the asymmetric distribution of Vangl2 was unchanged whereas the Gαi3 expression domain was enlarged and Par6b expression was slightly altered. Then, the possible molecular mechanisms of Dmp1 involvement in inner ear development were explored via RNA-seq analysis. The study suggested that the Fgf23-Klotho endocrine axis may play a novel role in the inner ear and Dmp1 may regulate the kinocilium-stereocilia interaction via Fgf23-Klotho signaling. Together, our results proved the critical role of Dmp1 in the precise regulation of hair bundle morphogenesis in the early development of HCs.