Poglut2/3 double knockout in mice results in neonatal lethality with reduced levels of fibrillin in lung tissues.

Fibrillin microfibrils play a critical role in the formation of elastic fibers, tissue/organ development, and cardiopulmonary function. These microfibrils not only provide structural support and flexibility to tissues, but they also regulate growth factor signaling through a plethora of microfibril-binding proteins in the extracellular space. Mutations in fibrillins are associated with human diseases affecting cardiovascular, pulmonary, skeletal, and ocular systems. Fibrillins consist of up to 47 epidermal growth factor-like repeats, of which more than half are modified by protein O-glucosyltransferase 2 (POGLUT2) and/or POGLUT3. Loss of these modifications reduces secretion of N-terminal fibrillin constructs overexpressed in vitro. Here, we investigated the role of POGLUT2 and POGLUT3 in vivo using a Poglut2/3 double knockout (DKO) mouse model. Blocking O-glucosylation caused neonatal death with skeletal, pulmonary, and eye defects reminiscent of fibrillin/elastin mutations. Proteomic analyses of DKO dermal fibroblast medium and extracellular matrix provided evidence that fibrillins were more sensitive to loss of O-glucose compared to other POGLUT2/3 substrates. This conclusion was supported by immunofluorescent analyses of late gestation DKO lungs where FBN levels were reduced and microfibrils appeared fragmented in the pulmonary arteries and veins, bronchioles, and developing saccules. Defects in fibrillin microfibrils likely contributed to impaired elastic fiber formation and histological changes observed in DKO lung blood vessels, bronchioles, and saccules. Collectively, these results highlight the importance of POGLUT2/3-mediated O-glucosylation in vivo and open the possibility that O-glucose modifications on fibrillin influence microfibril assembly and or protein interactions in the ECM environment.

Unraveling the structure, chemical composition, and conserved signaling in leech teeth.

Unlike vertebrates, the number of toothed taxa in invertebrates is very few, with leeches being the only tooth-bearing organisms in the phylum Annelida. Copious studies have been conducted regarding vertebrate teeth; however, studies regarding the structure and function of invertebrate teeth are limited. In this study, the tooth structure of leeches, specifically Hirudo nipponia and Haemadipsa rjukjuana, was revealed, which showed sharp and pointed teeth along the apex of three jaws. Understanding conserved signaling regulations among analogous organs is crucial for uncovering the underlying mechanisms during organogenesis. Therefore, to shed light on the evolutionary perspective of odontogenesis to some extent, we conducted de novo transcriptome analyses using embryonic mouse tooth germs, Hirudo teeth, and Helobdella proboscises to identify conserved signaling molecules involved in tooth development. The selection criteria were particularly based on the presence of tooth-related genes in mice, Hirudo teeth, and Helobdella proboscis, wherein 4113 genes were commonly expressed in all three specimens. Furthermore, the chemical nature of leech teeth was also examined via TEM-EDS to compare the chemical composition with vertebrate teeth. The examination of tissue-specific genetic information and chemical nature between leeches and mice revealed chemical similarities between leech and mice teeth, as well as conserved signaling molecules involved in tooth formation, including Ptpro, Prickle2, and Wnt16. Based on our findings, we propose that leech teeth express signaling molecules conserved in mice and these conserved tooth-specific signaling for dental hard tissue formation in mice would corresponds to the structural formation of the toothed jaw in leeches.

Prohibitin modulates periodontium differentiation in mice development.

Introduction: Prohibitin (PHB) is an essential scaffold protein that modulates signaling pathways controlling cell survival, metabolism, inflammation, and bone formation. However, its specific role in periodontium development remains less understood. This study aims to elucidate the expression pattern and function of PHB in periodontium development and its involvement in alveolar bone formation. Methods: Immunolocalization of PHB in the periodontium of postnatal (PN) mice were examined. Phb morpholino was micro-injected into the right-side mandible at PN5, corresponding to the position where the alveolar bone process forms in relation to the lower first molar. The micro-injection with a scramble control (PF-127) and the left-side mandibles were used as control groups. Five days post-micro-injection, immunohistochemical analysis and micro-CT evaluation were conducted to assess bone mass and morphological changes. Additionally, expression patterns of signaling molecules were examined following Phb downregulation using 24-h in vitro cultivation of developing dental mesenchyme at E14.5. Results: The immunostaining of PHB showed its localization in the periodontium at PN5, PN8, and PN10. The in vitro cultivation of dental mesenchyme resulted in alterations in Bmps, Runx2, and Wnt signalings after Phb knock-down. At 5 days post-micro-injection, Phb knocking down showed weak immunolocalizations of runt-related transcription factor (RUNX2) and osteocalcin (OCN). However, knocking down Phb led to histological alterations characterized by decreased bone mass and stronger localizations of Ki67 and PERIOSTIN in the periodontium compared 1 to control groups. The micro-CT evaluation showed decreased bone volume and increased PDL space in the Phb knock-down specimens, suggesting its regulatory role in bone formation. Discussion: The region-specific localization of PHB in the margin where alveolar bone forms suggests its involvement in alveolar bone formation and the differentiation of the periodontal ligament. Overall, our findings suggest that Phb plays a modulatory role in alveolar bone formation by harmoniously regulating bone-forming-related signaling molecules during periodontium development.

Developmental roles of glomerular epithelial protein-1 in mice molar morphogenesis.

Glomerular epithelial protein-1 (Glepp1), a R3 subtype family of receptor-type protein tyrosine phosphatases, plays important role in the activation of Src family kinases and regulates cellular processes such as cell proliferation, differentiation, and apoptosis. In this study, we firstly examined the functional evaluation of Glepp1 in tooth development and morphogenesis. The precise expression level and developmental function of Glepp1 were examined by RT-qPCR, in situ hybridization, and loss and gain of functional study using a range of in vitro organ cultivation methods. Expression of Glepp1 was detected in the developing tooth germs in cap and bell stage of tooth development. Knocking down Glepp1 at E13 for 2 days showed the altered expression levels of tooth development-related signaling molecules, including Bmps, Dspp, Fgf4, Lef1, and Shh. Moreover, transient knock down of Glepp1 revealed alterations in cellular physiology, examined by the localization patterns of Ki67 and E-cadherin. Similarly, knocking down of Glepp1 showed disrupted enamel rod and interrod formation in 3-week renal transplanted teeth. In addition, due to attrition of odontoblastic layers, the expression signals of Dspp and the localization of NESTIN were almost not detected after knock down of Glepp1; however, their expressions were increased after Glepp1 overexpression. Thus, our results suggested that Glepp1 plays modulating roles during odontogenesis by regulating the expression levels of signaling molecules and cellular events to achieve the proper structural formation of hard tissue matrices in mice molar development.

Functional modulation of lysophosphatidic acid type 2 G-protein coupled receptor facilitates alveolar bone formation.

Lipid biosynthesis is recently studied its functions in a range of cellular physiology including differentiation and regeneration. However, it still remains to be elucidated in its precise function. To reveal this, we evaluated the roles of lysophosphatidic acid (LPA) signaling in alveolar bone formation using the LPA type 2 receptor (LPAR2) antagonist AMG-35 (Amgen Compound 35) using tooth loss without periodontal disease model which would be caused by trauma and usually requires a dental implant to restore masticatory function. In this study, in vitro cell culture experiments in osteoblasts and periodontal ligament fibroblasts revealed cell type-specific responses, with AMG-35 modulating osteogenic differentiation in osteoblasts in vitro. To confirm the in vivo results, we employed a mouse model of tooth loss without periodontal disease. Five to 10 days after tooth extraction, AMG-35 facilitated bone formation in the tooth root socket as measured by immunohistochemistry for differentiation markers KI67, Osteocalcin, Periostin, RUNX2, transforming growth factor beta 1 (TGF-ß1) and SMAD2/3. The increased expression and the localization of these proteins suggest that AMG-35 elicits osteoblast differentiation through TGF-ß1 and SMAD2/3 signaling. These results indicate that LPAR2/TGF-ß1/SMAD2/3 represents a new signaling pathway in alveolar bone formation and that local application of AMG-35 in traumatic tooth loss can be used to facilitate bone regeneration and healing for further clinical treatment.

O-fucosylation of thrombospondin type I repeats is dispensable for trafficking thrombospondin 1 to platelet secretory granules.

Thrombospondin 1 (THBS1) is a secreted extracellular matrix glycoprotein that regulates a variety of cellular and physiological processes. THBS1's diverse functions are attributed to interactions between the modular domains of THBS1 with an array of proteins found in the extracellular matrix. THBS1's three Thrombospondin type 1 repeats (TSRs) are modified with O-linked glucose-fucose disaccharide and C-mannose. It is unknown whether these modifications impact trafficking and/or function of THBS1 in vivo. The O-fucose is added by Protein O-fucosyltransferase 2 (POFUT2) and is sequentially extended to the disaccharide by ß3glucosyltransferase (B3GLCT). The C-mannose is added by one or more of four C-mannosyltransferases. O-fucosylation by POFUT2/B3GLCT in the endoplasmic reticulum has been proposed to play a role in quality control by locking TSR domains into their three-dimensional fold, allowing for proper secretion of many O-fucosylated substrates. Prior studies showed the siRNA knockdown of POFUT2 in HEK293T cells blocked secretion of TSRs 1-3 from THBS1. Here we demonstrated that secretion of THBS1 TSRs 1-3 was not reduced by CRISPR-Cas9-mediated knockout of POFUT2 in HEK293T cells and demonstrated that knockout of Pofut2 or B3glct in mice did not reduce the trafficking of endogenous THBS1 to secretory granules of platelets, a major source of THBS1. Additionally, we demonstrated that all three TSRs from platelet THBS1 were highly C-mannosylated, which has been shown to stabilize TSRs in vitro. Combined, these results suggested that POFUT2 substrates with TSRs that are also modified by C-mannose may be less susceptible to trafficking defects resulting from the loss of the glucose-fucose disaccharide.

O-fucosylation of thrombospondin type 1 repeats is essential for ECM remodeling and signaling during bone development.

Many extracellular matrix (ECM) associated proteins that influence ECM properties have Thrombospondin type 1 repeats (TSRs) which are modified with O-linked fucose. The O-fucose is added in the endoplasmic reticulum to folded TSRs by the enzyme Protein O-fucosyltransferase-2 (POFUT2) and is proposed to promote efficient trafficking of substrates. The importance of this modification for function of TSR-proteins is underscored by the early embryonic lethality of mouse embryos lacking Pofut2. To overcome early lethality and investigate the impact of the Pofut2 knockout on the secretion of POFUT2 substrates and on extracellular matrix properties in vivo, we deleted Pofut2 in the developing limb mesenchyme using Prrx1-Cre recombinase. Loss of Pofut2 in the limb mesenchyme caused significant shortening of the limbs, long bones and tendons and stiff joint resembling the musculoskeletal dysplasias in human and in mice with mutations in ADAMTS or ADAMTSL proteins. Limb shortening was evident at embryonic day 14.5 where loss of O-fucosylation led to an accumulation of fibrillin 2 (FBN2), decreased BMP and IHH signaling, and increased TGF-ß signaling. Consistent with these changes we saw a decrease in the size of the hypertrophic zone with lower levels of Collagen-X. Unexpectedly, we observed minimal effects of the Pofut2 knockout on secretion of two POFUT2 substrates, CCN2 or ADAMTS17, in the developing bone. In contrast, CCN2 and two other POFUT2 substrates important for bone development, ADAMTS6 and 10, showed a decrease in secretion from POFUT2-null HEK293T cells in vitro. These combined results suggest that the impact of the Pofut2 mutation is cell-type specific. In addition, these observations raise the possibility that the O-fucose modification on TSRs extends beyond promoting efficient trafficking of POFUT2 substrates and has the potential to influence their function in the extracellular environment.

Climate and land-use change impacts on spatiotemporal variations in groundwater recharge: A case study of the Bangkok Area, Thailand.

Groundwater contributes to the socioeconomic development of the Thai capital Bangkok and its vicinity. However, groundwater resources are under immense pressure due to population growth, rapid urbanisation, overexploitation, and climate change. Therefore, evaluating the combined impact of climate change and land-use change on groundwater recharge can be useful for developing sound groundwater management systems. In this research, the future climate is projected using three Regional Climate Models (RCMs), namely ACCESS-CSIRO-CCAM, CNRM-CM5-CSIRO-CCAM, and MPI-ESM-LR-CSIRO-CCAM for three future periods: near future (2010-2039), mid future (2040-2069), and far future (2070-2099) under two Representative Concentration Pathway (RCP) scenarios 4.5 and 8.5 as suggested in the IPCC's Fifth Assessment Report. All RCMs project the temperature to rise incessantly, although future precipitation is predicted to fluctuate (increase and decrease) among the various RCMs and RCP scenarios. A Dyna-CLUE model is employed to analyse the future land-use change scenarios (low, medium, and high urbanisation), with the aim of expanding the built-up area and creating land-use maps covering the period to 2099. A hydrological model, WetSpass, is used to estimate groundwater recharge under future climate and land-use change. The findings reveal that groundwater recharge is expected to decrease in high and medium urbanisation areas, ranging from 5.84 to 20.91 mm/yr for the RCP 4.5 scenario and 4.07 to 18.72 mm/yr for RCP 8.5. In contrast, for the low urbanisation scenario, the model projects an increase in groundwater recharge ranging from 7.9 to 16.66 mm/yr for the RCP 4.5 scenario and 5.54 to 20.04 mm/yr for RCP 8.5.

Signaling Modulation by miRNA-221-3p During Tooth Morphogenesis in Mice.

miRNAs are conserved short non-coding RNAs that play a role in the modulation of various biological pathways during tissue and organ morphogenesis. In this study, the function of miRNA-221-3p in tooth development, through its loss or gain in function was evaluated. A variety of techniques were utilized to evaluate detailed functional roles of miRNA-221-3p during odontogenesis, including in vitro tooth cultivation, renal capsule transplantation, in situ hybridization, real-time PCR, and immunohistochemistry. Two-day in vitro tooth cultivation at E13 identified altered cellular events, including cellular proliferation, apoptosis, adhesion, and cytoskeletal arrangement, with the loss and gain of miRNA-221-3p. qPCR analysis revealed alterations in gene expression of tooth-related signaling molecules, including ß-catenin, Bmp2, Bmp4, Fgf4, Ptch1, and Shh, when inhibited with miRNA-221-3p and mimic. Also, the inhibition of miRNA-221-3p demonstrated increased mesenchymal localizations of pSMAD1/5/8, alongside decreased expression patterns of Shh and Fgf4 within inner enamel epithelium (IEE) in E13 + 2 days in vitro cultivated teeth. Moreover, 1-week renal transplantation of in vitro cultivated teeth had smaller tooth size with reduced enamel and dentin matrices, along with increased cellular proliferation and Shh expression along the Hertwig epithelial root sheath (HERS), within the inhibitor group. Similarly, in 3-week renal calcified teeth, the overexpression of miRNA-221-3p did not affect tooth phenotype, while the loss of function resulted in long and slender teeth with short mesiodistal length. This study provides evidence that a suitable level of miRNA-221-3p is required for the modulation of major signaling pathways, including Wnt, Bmp, and Shh, during tooth morphogenesis.

Evaluation of the CORDEX regional climate models (RCMs) for simulating climate extremes in the Asian cities.

This study evaluates the ability of 21 Regional Climate Models (RCMs) from the Coordinated Regional Climate Downscaling Experiment (CORDEX) in simulating climate extremes in the fast growing Asian cities which are highly vulnerable to climate change. The three Asian cities have two different climate characteristics, namely Bangkok and its vicinity and Ho Chi Minh City in tropical climate region and Kathmandu in sub-tropical and temperate climate region. The RCMs were evaluated to simulate the six climate indices; Consecutive Dry Days (CDD), Simple Daily Intensity Index (SDII), Number of extremely heavy precipitation days (R50mm), Maximum 1-day precipitation amount (RX1day), Mean of daily maximum temperature (TX mean) and Mean of daily minimum temperature (TN mean). The performance indicators used were correlation coefficient, normalized root mean square deviation, absolute normalized root mean square deviation and average absolute relative deviation. The Entropy method was endorsed to acquire weights of these four indicators and weightage average techniques were used for ranking of 21 RCMs. The result demonstrated that the best model for one climate index is not the same best model for other climate indices. The 3 RCMs; WAS44_SMHI_RCA4_IPSL_CM5A_MR, WAS44_SMHI_RCA4_MIROC5, and WAS44_IITM_REGCM4-4_CSIRO_MK3-6-0 are the best performing RCMs for simulating future climate extremes in Bangkok and its vicinity, Ho Chi Minh city and Kathmandu valley, respectively. Therefore, they are recommended to use for climate change impact and adaptation studies in water resources management in the selected cities.

Hydrocephalus in mouse B3glct mutants is likely caused by defects in multiple B3GLCT substrates in ependymal cells and subcommissural organ.

Peters plus syndrome, characterized by defects in eye and skeletal development with isolated cases of ventriculomegaly/hydrocephalus, is caused by mutations in the ß3-glucosyltransferase (B3GLCT) gene. In the endoplasmic reticulum, B3GLCT adds glucose to O-linked fucose on properly folded thrombospondin type 1 repeats (TSRs). The resulting glucose-fucose disaccharide is proposed to stabilize the TSR fold and promote secretion of B3GLCT substrates, with some substrates more sensitive than others to loss of glucose. Mouse B3glct mutants develop hydrocephalus at high frequency. In this study, we demonstrated that B3glct mutant ependymal cells had fewer cilia basal bodies and altered translational polarity compared to controls. Localization of mRNA encoding A Disintegrin and Metalloproteinase with ThromboSpondin type 1 repeat 20 (ADAMTS20) and ADAMTS9 suggested that reduced function of these B3GLCT substrates contributed to ependymal cell abnormalities. In addition, we showed that multiple B3GLCT substrates (Adamts3, Adamts9 and Adamts20) are expressed by the subcommissural organ, that subcommissural organ-spondin ((SSPO) also known as SCO-spondin) TSRs were modified with O-linked glucose-fucose and that loss of B3GLCT reduced secretion of SSPO in cultured cells. In the B3glct mutant, intracellular levels of SSPO were reduced and BiP levels increased, suggesting a folding defect. Secreted SSPO colocalized with BiP, raising the possibility that abnormal extracellular assembly of SSPO into Reissner's fiber also contributed to impaired CSF flow in mutants. Combined, these studies underscore the complexity of the B3glct mutant hydrocephalus phenotype and demonstrate that impaired cerebrospinal fluid (CSF) flow likely stems from the collective effects of the mutation on multiple processes.

Facilitation of Bone Healing Processes Based on the Developmental Function of Meox2 in Tooth Loss Lesion.

In the present study, we examined the bone healing capacity of Meox2, a homeobox gene that plays essential roles in the differentiation of a range of developing tissues, and identified its putative function in palatogenesis. We applied the knocking down of Meox2 in human periodontal ligament fibroblasts to examine the osteogenic potential of Meox2. Additionally, we applied in vivo periodontitis induced experiment to reveal the possible application of Meox2 knockdown for 1 and 2 weeks in bone healing processes. We examined the detailed histomorphological changes using Masson's trichrome staining and micro-computed tomography evaluation. Moreover, we observed the localization patterns of various signaling molecules, including α-SMA, CK14, IL-1ß, and MPO to examine the altered bone healing processes. Furthermore, we investigated the process of bone formation using immunohistochemistry of Osteocalcin and Runx2. On the basis of the results, we suggest that the knocking down of Meox2 via the activation of osteoblast and modulation of inflammation would be a plausible answer for bone regeneration as a gene therapy. Additionally, we propose that the purpose-dependent selection and application of developmental regulation genes are important for the functional regeneration of specific tissues and organs, where the pathological condition of tooth loss lesion would be.

Developmental Roles of FUSE Binding Protein 1 (Fubp1) in Tooth Morphogenesis.

FUSE binding protein 1 (Fubp1), a regulator of the c-Myc transcription factor and a DNA/RNA-binding protein, plays important roles in the regulation of gene transcription and cellular physiology. In this study, to reveal the precise developmental function of Fubp1, we examined the detailed expression pattern and developmental function of Fubp1 during tooth morphogenesis by RT-qPCR, in situ hybridization, and knock-down study using in vitro organ cultivation methods. In embryogenesis, Fubp1 is obviously expressed in the enamel organ and condensed mesenchyme, known to be important for proper tooth formation. Knocking down Fubp1 at E14 for two days, showed the altered expression patterns of tooth development related signalling molecules, including Bmps and Fgf4. In addition, transient knock-down of Fubp1 at E14 revealed changes in the localization patterns of c-Myc and cell proliferation in epithelium and mesenchyme, related with altered tooth morphogenesis. These results also showed the decreased amelogenin and dentin sialophosphoprotein expressions and disrupted enamel rod and interrod formation in one- and three-week renal transplanted teeth respectively. Thus, our results suggested that Fubp1 plays a modulating role during dentinogenesis and amelogenesis by regulating the expression pattern of signalling molecules to achieve the proper structural formation of hard tissue matrices and crown morphogenesis in mice molar development.

Signaling Modulations of miR-206-3p in Tooth Morphogenesis.

MicroRNAs (miRNAs) are a class of naturally occurring small non-coding RNAs that post-transcriptionally regulate gene expression in organisms. Most mammalian miRNAs influence biological processes, including developmental changes, tissue morphogenesis and the maintenance of tissue identity, cell growth, differentiation, apoptosis, and metabolism. The miR-206-3p has been correlated with cancer; however, developmental roles of this miRNA are unclear. In this study, we examined the expression pattern and evaluated the developmental regulation of miR-206-3p during tooth morphogenesis using ex-vivo culture method. The expression pattern of miR-206-3p was examined in the epithelium and mesenchyme of developing tooth germ with stage-specific manners. Perturbation of the expression of miR-206-3p clearly altered expression patterns of dental-development-related signaling molecules, including Axin2, Bmp2, Fgf4, Lef1 and Shh. The gene expression complemented with change in cellular events including, apoptosis and proliferation which caused altered crown and pulp morphogenesis in renal-capsule-calcified teeth. Especially, mislocalization of ß-Catenin and SMAD1/5/8 were observed alongside dramatic alterations in the expression patterns of Fgf4 and Shh. Overall, our data suggest that the miR-206-3p regulate the cellular physiology during tooth morphogenesis through modulation of the Wnt, Bmp, Fgf, and Shh signaling pathways to form proper tooth pulp and crown.

Mapping groundwater resiliency under climate change scenarios: A case study of Kathmandu Valley, Nepal.

Groundwater resources of Kathmandu Valley in Nepal are under immense pressure from multiple stresses, including climate change. Due to over-extraction, groundwater resources are depleting, leading to social, environmental and economic problems. Climate change might add additional pressure by altering groundwater recharge rates and availability of groundwater. Mapping groundwater resilience to climate change can aid in understanding the dynamics of groundwater systems, facilitating the development of strategies for sustainable groundwater management. Therefore, this study aims to analyse the impact of climate change on groundwater resources and mapping the groundwater resiliency of Kathmandu Valley under different climate change scenarios. The future climate projected using the climate data of RCM's namely ACCESS-CSIRO-CCAM, CNRM-CM5-CSIRO-CCAM and MPI-ESM-LR-CSIRO-CCAM for three future periods: near future (2010-2039), mid future (2040-2069) and far future (2070-2099) under RCP 4.5 and RCP 8.5 scenarios were bias corrected and fed into the Soil and Water Assessment Tool (SWAT), a hydrological model, to estimate future groundwater recharge. The results showed a decrease in groundwater recharge in future ranging from 3.3 to 50.7 mm/yr under RCP 4.5 and 19-102.1 mm/yr under RCP 8.5 scenario. The GMS-MODFLOW model was employed to estimate the future groundwater level of Kathmandu Valley. The model revealed that the groundwater level is expected to decrease in future. Based on the results, a groundwater resiliency map of Kathmandu Valley was developed. The results suggest that groundwater in the northern and southern area of the valley are highly resilient to climate change compared to the central area. The results will be very useful in the formulation and implementation of adaptation strategies to offset the negative impacts of climate change on the groundwater resources of Kathmandu Valley.

Effects of resveratrol on bone-healing capacity in the mouse tooth extraction socket.

BACKGROUND AND OBJECTIVE: After tooth extraction, the extraction socket undergoes several steps of soft and hard tissue healing. The healing process of the extraction socket is modulated by a range of signaling factors and biochemical agents. It has been reported that resveratrol, a polyphenolic compound, exhibits various biological effects, including anti-inflammatory, anti-carcinogenic, antioxidant, and anti-aging effects, and protects cardiovascular and bone tissues. In this study, we examined the cellular effects of resveratrol on human periodontal ligament (hPDL) cells and osteoblast-like (MC3T3-E1) cells and evaluated the bone-healing capacity of tooth extraction sockets in mice. MATERIAL AND METHODS: Resveratrol was applied to hPDL and MC3T3-E1 cells to detect cell proliferation and alkaline phosphatase (ALP) activity, and qPCR was employed to understand the gene expression level in vitro. For in vivo experiment, six-week-old C57BL/6 male mice were randomly divided into control (n = 15) and experimental (n = 15) groups and maxillary first molars were extracted by surgery. Experimental groups received 50-µM resveratrol on extraction sockets and analyzed the degree of new bone formation. RESULTS: Treatment of hPDL and MC3T3-E1 cells with resveratrol increased the cell proliferation and ALP activity and enhanced the expression of ALP, BMP-2, BMP-4, and OC genes. Resveratrol enhanced new bone formation in the lingual extraction socket in mice. CONCLUSION: These results suggest that resveratrol increases the cellular physiology of PDL and osteoblast including their proliferation and differentiation and may play an important role in bone-healing capacity after tooth extraction.

Cyclosporine A eyedrops with self-nanoemulsifying drug delivery systems have improved physicochemical properties and efficacy against dry eye disease in a murine dry eye model.

PURPOSE: We aimed to compare the physicochemical properties and in vivo efficacy of commercially available nanoemulsion cyclosporine A (CsA) eyedrops in benzalkonium chloride (BAC)-induced dry eye disease (DED). METHODS: Particle size analysis was performed on conventional 0.05% CsA (Restasis, C-CsA) and two new types of 0.05% CsA eyedrops based on a self-nanoemulsifying drug delivery system (SNEDDS, SNEDDS-N and -T). Turbidometry, pH measurements and instability indices of each CsA solution were measured. DED was induced with BAC, and animals were treated with vehicle or CsA preparations. Tear volume and fluorescein staining scores were evaluated on days 7 and 14. Eyes were enucleated and subjected to IHC, TUNEL staining, periodic acid-Schiff (PAS) staining, real-time PCR and western blotting. RESULTS: Both SNEDDSs had lower and more uniform particle size distribution than C-CsA, and a similar optical density to phosphate-buffered saline and stable pH, in contrast to the high turbidity and unstable pH of C-CsA. Aqueous tear volume and fluorescein staining scores were improved in C-CsA- and SNEDDS-treated mice. Numbers of PAS-positive goblet cells and levels of inflammatory mediators were decreased by both C-CsA and SNEDDS, although SNEDDS resolved inflammation more effectively than C-CsA. CONCLUSIONS: Cyclosporine A eyedrops with SNEDDS have improved physicochemical properties and treatment efficacy in BAC-induced DED.

ADAMTS9 and ADAMTS20 are differentially affected by loss of B3GLCT in mouse model of Peters plus syndrome.

Peters plus syndrome (MIM #261540 PTRPLS), characterized by defects in eye development, prominent forehead, hypertelorism, short stature and brachydactyly, is caused by mutations in the ß3-glucosyltransferase (B3GLCT) gene. Protein O-fucosyltransferase 2 (POFUT2) and B3GLCT work sequentially to add an O-linked glucose ß1-3fucose disaccharide to properly folded thrombospondin type 1 repeats (TSRs). Forty-nine proteins are predicted to be modified by POFUT2, and nearly half are members of the ADAMTS superfamily. Previous studies suggested that O-linked fucose is essential for folding and secretion of POFUT2-modified proteins and that B3GLCT-mediated extension to the disaccharide is essential for only a subset of targets. To test this hypothesis and gain insight into the origin of PTRPLS developmental defects, we developed and characterized two mouse B3glct knockout alleles. Using these models, we tested the role of B3GLCT in enabling function of ADAMTS9 and ADAMTS20, two highly conserved targets whose functions are well characterized in mouse development. The mouse B3glct mutants developed craniofacial and skeletal abnormalities comparable to PTRPLS. In addition, we observed highly penetrant hydrocephalus, white spotting and soft tissue syndactyly. We provide strong genetic and biochemical evidence that hydrocephalus and white spotting in B3glct mutants resulted from loss of ADAMTS20, eye abnormalities from partial reduction of ADAMTS9 and cleft palate from loss of ADAMTS20 and partially reduced ADAMTS9 function. Combined, these results provide compelling evidence that ADAMTS9 and ADAMTS20 were differentially sensitive to B3GLCT inactivation and suggest that the developmental defects in PTRPLS result from disruption of a subset of highly sensitive POFUT2/B3GLCT targets such as ADAMTS20.

An endoplasmic reticulum stress regulator, Tmbim6, modulates secretory stage of mice molar.

To understand the role of endoplasmic reticulum (ER)-stress in mice molar development, we studied Tmbim6 that antagonizes the unfolded protein response, using Tmbim6 knockout (KO) mice and in vitro organ cultivation with knocking down using small interfering RNA. During molar development, Tmbim6 is expressed in developing tooth at E14-E16, postnatal0 (PN0), and PN6. Mineral content in Tmbim6 KO enamel was reduced while dentin was slightly increased revealing ultrastructural changes in pattern formation of both enamel and dentin. Moreover, odontoblast differentiation was altered with increased Dspp expression at PN0 followed by altered AMELX localizations at PN5. These results were confirmed by in vitro organ cultivation and showed altered Bmp signaling, proliferation, and actin rearrangement in the presumptive ameloblast and odontoblasts that followed the altered expression of differentiation and ER stress-related signaling molecules at E16.5. Overall, ER stress modulated by Tmbim6 would play important roles in patterned dental hard tissue formation in mice molar within a limited period of development.