The protective role of curcumin in human dental pulp stem cells stimulated by lipopolysaccharide via inhibiting NF-κB p65 phosphorylation to suppress NLRP3 inflammasome activation.

OBJECTIVES: This study aims to investigate the anti-inflammatory effect of curcumin and underlying mechanisms regarding the modulation of the nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome in human dental pulp stem cells (hDPSCs). MATERIALS AND METHODS: The impact of curcumin on the viability of hDPSCs was evaluated. The effect of curcumin on the expression of IL-1ß and NLRP3 in hDPSCs stimulated by lipopolysaccharide (LPS) was assessed. Then, LPS-primed hDPSCs were pre-treated with curcumin before ATP triggering NLRP3 inflammasome activation, and NLRP3 inflammasome-related mediators were assessed. The mechanism of curcumin inactivation of LPS plus ATP-induced inflammasome associated with NF-κB pathway was explored. The NF-κB pathway related pro-inflammatory mediators at mRNA and protein levels were evaluated. The expression of NF-κB p65 and phosphorylation p65 was visualized after curcumin or NF-κB inhibitor administrating respectively in hDPSCs with an activated NLRP3 inflammasome. Statistical analysis was performed. RESULTS: While curcumin at the concentration of 0.5-5 µM showed no obvious impact on the viability of hDPSCs, it significantly decreased IL-1ß and NLRP3 mRNA expression in LPS-induced hDPSCs in a dose-dependent manner. Curcumin significantly inhibited the LPS plus ATP-primed NLRP3 inflammasome activation in hDPSCs (NLRP3, ASC, caspase-1, and IL-1ß). Curcumin evidently attenuated the LPS plus ATP-induced expression of NF-κB pathway-related pro-inflammatory mediators (IL-6, IL-8, TNF-α, and COX-2). Furthermore, curcumin effectively reduced p65 phosphorylation, which acts as an NF-κB inhibitor in hDPSCs with an activated NLRP3 inflammasome. CONCLUSIONS: Curcumin pre-treatment may exert an anti-inflammatory role via inactivation of the NLRP3 inflammasome by inhibiting NF-κB p65 phosphorylation in cultured hDPSCs. CLINICAL RELEVANCE: Curcumin may have therapeutic potential in pulp inflammation.

Odontogenesis-Associated Phosphoprotein (ODAPH) Overexpression in Ameloblasts Disrupts Enamel Formation via Inducing Abnormal Mineralization of Enamel in Secretory Stage.

Odontogenesis-associated phosphoprotein (ODAPH) is a recently discovered enamel matrix protein. Our previous study demonstrated that knockouting out Odaph in mice resulted in enamel hypomineralization. To further investigate the effect of Odaph on enamel mineralization, we constructed an Odaph overexpression mouse model, controlled by an amelogenin promoter. Our histological analysis of OdaphTg mice revealed that the enamel layer was thinner than in WT mice. An uneven, thinner enamel layer was confirmed using micro-computed tomography (uCT). It was subsequently found that the Tomes' processes lost their normal morphology, resulting in the loss of the enamel prism structure. These results indicate that Odaph overexpression in ameloblasts led to enamel dysplasia. In conjunction with this, Odaph overexpression hindered Amelx secretion, and may result in endoplasmic reticulum stress. Interestingly, uCT revealed that enamel had higher mineral density at the secretory stage; due to this, we did the histological staining for the mineralization-related proteins Alkaline phosphatase (ALPL) and Runt-related transcription factor 2 (RUNX2). It was observed that these proteins were up-regulated in OdaphTg mice versus WT mice, indicating that Odaph overexpression led to abnormal enamel mineralization. To confirm this, we transfected ameloblast-like cell line (ALC) with Odaph overexpression lentivirus in vitro and identified that both Alpl and Runx2 were strikingly upregulated in OE-mus-Odaph versus OE-NC cells. We concluded that the ectopic overexpression of Odaph in ameloblasts led to abnormal enamel mineralization. In summary, Odaph profoundly influences amelogenesis by participating in enamel mineralization.

Melanin-Like Nanoquencher on Graphitic Carbon Nitride Nanosheets for Tyrosinase Activity and Inhibitor Assay.

Graphitic C3N4 (g-C3N4) nanosheets are a type of emerging graphene-like carbon-based nanomaterials with high fluorescence and large specific surface areas that hold great potential for biosensor applications. However, current g-C3N4 based biosensors have prevailingly been limited to coordination with metal ions, and it is of great significance to develop new designs for g-C3N4 nanosheets based biosensors toward biomarkers of general interest. We report the development of a novel g-C3N4 nanosheet-based nanosensor strategy for highly sensitive, single-step and label-free detection of tyrosinase (TYR) activity and its inhibitor. This strategy relies on the catalytic oxidation of tyrosine by TYR into melanin-like polymers, which form a nanoassembly on the g-C3N4 nanosheets and quench their fluorescence. This strategy was demonstrated to provide excellent selectivity and superior sensitivity and to enable rapid screening for TYR inhibitors. Therefore, the developed approach might create a useful platform for diagnostics and drugs screening for TYR-based diseases including melanoma cancer.

Transforming growth factor-ß1 regulates expression of the matrix metalloproteinase 20 (Mmp20) gene through a mechanism involving the transcription factor, myocyte enhancer factor-2C, in ameloblast lineage cells.

Matrix metalloproteinase-20 (Mmp20) plays an essential role in amelogenesis during tooth development and is regulated by transforming growth factor-ß1 (TGF-ß1) in mouse ameloblast lineage cells (ALCs). The objective of this study was to explore the role of myocyte enhancer factor-2C (MEF2C), a key transcription factor in craniofacial development, in TGF-ß1-induced Mmp20 gene expression. We investigated Mmp20 expression in ALCs over-expressing MEF2C and in ALCs with MEF2C knocked down. We also analyzed activity of the Mmp20 promoter using a transient reporter gene-expression assay in cultured ALCs. Putative transcription factor-binding sites for MEF2C and TGF-ß1 on the Mmp20 promoter were analyzed with bioinformatics tools and examined using an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). The expression of Mmp20 was induced, in a dose-dependent manner, by MEF2C over-expression, and TGF-ß1-induced Mmp20 expression was blocked by MEF2C knockdown in ALCs. There was a TGF-ß1/MEF2C-responsive region, including a putative MEF2-binding site, between base pairs -356 and -73 of the Mmp20 promoter. Mutation of the putative MEF2-binding site significantly reduced Mmp20 promoter activity upon activation with MEF2C or TGF-ß1. In conclusion, TGF-ß1-induced Mmp20 expression in ALCs was regulated through the MEF2-binding site on the Mmp20 promoter and thus mediated by the MEF2C signaling pathway.

Remarkable Enhancement of Antioxidant Activity of the Ovalbumin-EGCG Conjugate through a Novel Preceding Selective Protection Grafting Strategy.

Conventional radical grafting of proteins with catechins consumed the most antioxidant-active hydroxyls during grafting, thus failing to effectively retain antioxidant activity in conjugates. In this study, a novel strategy of selective protection of the most reactive hydroxyls before grafting was developed to preserve the most reactive hydroxyls and effectively retain antioxidant activity in conjugates. Selective protection of the most reactive hydroxyls of (-)-epigallocatechin-3-gallate (EGCG) was successfully realized in a yield of 87% applying trimethyl orthopropionate and catalytic calcium triflate at 40 °C. The novel ovalbumin (OVA)-EGCG conjugate with 93% grafting ratio was prepared by radical grafting with the selectively protected EGCG and subsequent deprotection. Substantially enhanced antioxidant performance of the novel OVA-EGCG conjugate in liposomes was unveiled with notably reduced curcumin degradation and leakage. The strategy and approaches developed in this study will be valuable to effectively improve the antioxidant activities of protein-catechin grafting conjugates.

Metformin alleviates junctional epithelium senescence via the AMPK/SIRT1/autophagy pathway in periodontitis induced by hyperglycemia.

The junctional epithelium (JE) serves a crucial protective role in the periodontium. High glucose-related aging results in accelerated barrier dysfunction of the gingival epithelium, which may be associated with diabetic periodontitis. Metformin, an oral hypoglycemic therapeutic, has been proposed as a anti-aging agent. This study aimed to clarify the effect of metformin on diabetic periodontitis and explore its mechanism in ameliorating senescence of JE during hyperglycemia. The db/db mice was used as a diabetic model mice and alterations in the periodontium were observed by hematoxylin-eosin staining and immunohistochemistry. An ameloblast-like cell line (ALC) was cultured with high glucose to induce senescence. Cellular senescence and oxidative stress were evaluated by SA-ß-gal staining and Intracellular reactive oxygen species (ROS) levels. Senescence biomarkers, P21 and P53, and autophagy markers, LC3-II/LC3-I, were measured by western blotting and quantitative real-time PCR. To construct a stable SIRT1 (Sirtuin 1) overexpression cell line, we transfected ALCs with lentiviral vectors overexpressing the mouse SIRT1 gene. Cellular senescence was increased in the JE of db/db mice and the periodontium was destroyed, which could be alleviated by metformin. Moreover, oxidative stress and cellular senescence in a high glucose environment were reduced by metformin in in-vitro assays. The autophagy inhibitor 3-MA and SIRT1 inhibitor EX-527 could dampen the effects of metformin. Overexpression of SIRT1 resulted in increased autophagy and decreased oxidative stress and cellular senescence. Meanwhile, AMPK (AMP-activated protein kinase) inhibition reversed the anti-senescence effects of metformin. Overall, these results suggest that metformin alleviates periodontal damage in db/db mice and cellular senescence in ALCs under high glucose conditions via the AMPK/SIRT1/autophagy pathway.

Recent Advances in Engineering Carriers for siRNA Delivery.

RNA interference (RNAi) technology has been a promising treatment strategy for combating intractable diseases. However, the applications of RNAi in clinical are hampered by extracellular and intracellular barriers. To overcome these barriers, various siRNA delivery systems have been developed in the past two decades. The first approved RNAi therapeutic, Patisiran (ONPATTRO) using lipids as the carrier, for the treatment of amyloidosis is one of the most important milestones. This has greatly encouraged researchers to work on creating new functional siRNA carriers. In this review, the recent advances in siRNA carriers consisting of lipids, polymers, and polymer-modified inorganic particles for cancer therapy are summarized. Representative examples are presented to show the structural design of the carriers in order to overcome the delivery hurdles associated with RNAi therapies. Finally, the existing challenges and future perspective for developing RNAi as a clinical modality will be discussed and proposed. It is believed that the addressed contributions in this review will promote the development of siRNA delivery systems for future clinical applications.

Longitudinal assessment of Indiana dentists' participation in Medicaid before and after expansion.

BACKGROUND: Although Medicaid expansion aims to eliminate financial barriers to health care for low-income people in the United States, health care accessibility cannot be guaranteed without clinicians who provide health care to Medicaid recipients. This study examined the characteristics of Indiana dentists that are associated with the likelihood of participating in Medicaid after expansion in 2015. METHODS: This study included Indiana-licensed dentists who renewed their licenses in 2018 and provided supplemental data elements related to demographics, education and training, and professional characteristics. Dentists' Medicaid engagement behavior was categorized on the basis of when claims were submitted from 2014 through 2017. Statistical analyses included the χ2 test and generalized multinomial logit model. RESULTS: Overall, 2,037 Indiana-licensed dentists were included in the study. Of these, 802 (39.4%) were continually active in Medicaid during the study period, and 116 (5.7%) became active after expansion. Dentists had a greater likelihood of engaging in Medicaid after expansion if they were female, specialized in oral and maxillofacial surgery, practiced in a group practice, and were located in a rural county. CONCLUSIONS: This study shows that dentists with certain demographic and practice characteristics had a greater likelihood of participation in Indiana Medicaid after expansion in 2015. Several findings from this study are consistent with previous research regarding the emerging trends in workforce diversity and show the impact of expansion policies on the dental safety net. PRACTICAL IMPLICATIONS: This study presents an effective framework for the use of administrative and regulatory data sources for state-level analysis of the Medicaid safety net.

Dual-mode of electrochemical-colorimetric imprinted sensing strategy based on self-sacrifice beacon for diversified determination of cardiac troponin I in serum.

Diversified determination of cardiac troponin I (cTnI) in serum is of great significance for the clinical diagnose and daily monitoring of acute myocardial infarction. Herein, an electrochemical-colorimetric dual-mode imprinted sensing strategy was proposed. Firstly, the aptamer-functionalized Fe3+-polydopamine (Apt@Fe3+-PDA) was constructed as the self-sacrifice beacon. Furthermore, an epitope magnetic molecularly imprinted polymer was prepared to recognize cTnI. Once the cTnI was captured, the beacon was then bound to it through the aptamer, which formed a sandwich-like complex. Under acidic condition, the Apt@Fe3+-PDA was disintegrated to release large amount of Fe3+, and further converted to Prussian blue (PB). On one hand, the electrochemical response of the PB was sensitive to the cTnI amount. On the other hand, the addition of the yellow K3[Fe(CN)6] into different amounts of the produced PB was able to result in different colors, which enabled the visual detection of cTnI. Under the optimal condition, the sensing strategy exhibited a linear range from 1.0 × 10-2 to 1.0 × 103 ng mL-1, and the detection limits were 3.2 and 7.4 pg mL-1 for the electrochemical and colorimetric modes, respectively. Besides, It is demonstrated that the dual-mode strategy shows favorable selectivity and stability in the assays, indicating a diversified application prospect in both clinic analysis and daily care.

Evaluation of Physicochemical Characteristics in Drinking Water Sources Emphasized on Fluoride: A Case Study of Yancheng, China.

In this study, the concentration of fluoride and the associated health risks for infants, children, and adults were analyzed and compared for three drinking water sources in Yancheng City, Jiangsu Province, China. To analyze the relationship between the water quality parameters of pH, fluoride (F-), sulfate (SO42-), chloride (Cl-), total dissolved solids (TDS), total alkalinity (TAlk), sodium (Na⁺), and potassium (K⁺), statistical analyses including correlation analysis, R-mode cluster analysis and factor analysis were performed based on monthly data from the year 2010 to 2015. The results indicated: (1) Fluoride concentrations in the drinking water sources ranged from 0.38 to 1.00 mg L-1 (mean = 0.57 mg L-1) following the order of Tongyu River > Yanlong Lake > Mangshe River; (2) fluoride concentrations in 22.93% of the collected samples were lower than 0.5 mg L-1, which has the risk of tooth cavities, especially for the Mangshe River; (3) the fluoride exposure levels of infants were higher than children and adults, and 3.2% of the fluoride exposure levels of infants were higher than the recommended toxicity reference value of 122 µg kg-1 d-1 as referenced by Health Canada, which might cause dental fluorosis issues; (4) the physico-chemical characteristics are classified the into four groups reflecting F-- TAlk, Na⁺-K⁺, SO42--Cl-, and pH-TDS, respectively, indicating that fluoride solubility in drinking water is TAlk dependent, which is also verified by R-mode cluster analysis and factor analysis. The results obtained supply useful information for the health department in Yancheng City, encouraging them to pay more attention to fluoride concentration and TAlk in drinking water sources.

Dental safety net capacity: An innovative use of existing data to measure dentists' clinical engagement in state Medicaid programs.

BACKGROUND: The demand for dentists available for state Medicaid populations has long outpaced the supply of such providers. To help understand the workforce dynamics, this study sought to develop a novel approach to measuring dentists' relative contribution to the dental safety net and, using this new measurement, identify demographic and practice characteristics predictive of dentists' willingness to participate in Indiana's Medicaid program. METHODS: We examined Medicaid claims data for 1,023 Indiana dentists. We fit generalized ordered logistic regression models to measure dentists' level of clinical engagement with Medicaid. Using a partial proportional odds specification model, we estimated proportional adjusted odds ratios for covariates and separate estimates for each contrast of nonproportional covariates. RESULTS: Though 75% of Medicaid-enrolled dentists were active providers, only 27% of them had 800 or more claims during fiscal year 2015. As has been shown in previous studies, our findings from the proportional odds model reinforced certain demographic and practice characteristics to be predictive of dentists' participation in state Medicaid programs. CONCLUSIONS: In addition to confirming predictive factors for Medicaid enrollment, this study validated the clinical engagement measure as a reliable method to assess the level of Medicaid participation. Prior studies have been limited by self-reported data and variations in Medicaid claims reporting. PRACTICAL IMPLICATIONS: Our findings have implications for state Medicaid policymakers by enabling access to data regarding dental providers' level of participation in Medicaid in addition to identifying factors predictive of such participation. This information will inform Medicaid program plans and provider recruitment efforts.

Conjugated polymer nanoparticles-based fluorescent biosensor for ultrasensitive detection of hydroquinone.

This work describes a simple and sensitive fluorescent method for detection of hydroquinone utilizing conjugated polymer nanoparticles (CPNs). The CPNs serve both as a catalyst to accelerate the conversion of hydroquinone to benzoquinone and a fluorescent probe. In the presence of hydroquinone, the fluorescence of CPNs can be effectively quenched by benzoquinone. The detection limit of hydroquinone was down to 5 nM and excellent selectivity toward possible interferences was obtained. This method was successfully applied for hydroquinone detection in lake water and satisfactory results were achieved.

Combination of Runx2 and Cbfß upregulates Amelotin gene expression in ameloblasts by directly interacting with cis­enhancers during amelogenesis.

Amelotin (Amtn) is a recently identified enamel protein secreted by ameloblasts at late stage of enamel development. Runt­related transcription factor 2 (Runx2) in combination with the coactivator core­binding factor ß (Cbfß) regulates the early stages of tooth development. The aim of the present study was to investigate the role of Runx2 in the regulation of Amtn gene expression in ameloblasts. Immunohistochemistry was performed and the results revealed that Runx2 protein was predominantly expressed in the nuclei of ameloblasts during the transition stage and the maturation stage of enamel development, whereas Cbfß was expressed in ameloblasts from the secretory stage to the maturation stage. Reverse transcription­quantitative polymerase chain reaction results demonstrated that Runx2 knockdown decreased Amtn expression in ameloblast­lineage cells and co­expression of Runx2 and Cbfß in ameloblast lineage cells induced an upregulation in Amtn gene expression. Two putative Runx2­binding sites within the Amtn promoter were identified using bioinformatics analysis. Results of an electrophoretic mobility shift assay and chromatin immunoprecipitation indicated that Runx2/Cbfß bound to specific DNA sequences. Site­directed mutagenesis of the Runx2 binding sites within the Amtn promoter resulted in decreased basal promoter activity and did not affect the overexpressed Runx2/Cbfß. The results of the present study suggest that Runx2 upregulates Amtn gene expression via binding directly to Runx2 sites within the Amtn promoter during amelogenesis.

Ablation of Runx2 in Ameloblasts Suppresses Enamel Maturation in Tooth Development.

Runt-related transcription factor 2 (Runx2) is involved in the early stage of tooth development. However, only few studies have reported the role of Runx2 in enamel development, which may be attributed to that Runx2 full knockout mice cannot survive after birth. In the present study, we successfully established a Runx2-deficient mouse model using a conditional knockout (cKO) method. We observed a significant reduction in the degree of mineralization and the decreased size of enamel rods in cKO mice. Histological analysis showed the retained enamel proteins in enamel layer at maturation stage in cKO molars. Further analysis by qRT-PCR revealed that the expressions of genes encoding enamel structure proteins, such as amelogenin (AMELX), ameloblastin (AMBN) and enamelin (ENAM), were increased in cKO enamel organs. On the other hand, the expression of kallikrein-related peptidase-4 (KLK4) at the mRNA and protein levels was dramatically decreased from late secretory stage to maturation stage in cKO enamel organs, while the expression of matrix metalloproteinase-20 (MMP-20) was not significantly altered. Finally, immunohistochemistry indicated that the uptake of amelogenins by ameloblasts was significantly decreased in cKO mice. Taken together, Runx2 played critical roles in controlling enamel maturation by increasing synthesis of KLK4 and decreasing synthesis of AMELX, AMBN and ENAM.

Radiographic, Histologic, and Biomechanical Evaluation of Combined Application of Platelet-rich Fibrin with Blood Clot in Regenerative Endodontics.

INTRODUCTION: No in vivo study has been reported on the mechanical reinforcement of a tooth after regenerative endodontic treatment (RET). The present work aimed to evaluate the concurrent use of platelet-rich fibrin (PRF) with a blood clot (BC) in RET concerning periapical healing, root development, and tooth structural reinforcement. METHODS: In our study, 24 premolars from 3 beagle dogs were assigned into control, BC, and PRF + BC groups. Periapical healing was monitored with quantitative measurements of root elongation and thickening radiographically. Tooth biomechanical integrity was assessed with the fracture resistance test. Histologic evaluation was conducted. RESULTS: There was a significant difference among the periapical radiolucency decreasing rate of the control (43.75%) and the BC (100%) and PRF + BC (100%) groups (P < .05). The increase of root length and thickness in both the BC and PRF + BC groups was significantly greater than that in the control group (P < .05). No significant difference was detected between the 2 experimental groups regarding periapical healing or root development (P > .05). Teeth in the BC (249.3 ± 64.1 N) and PRF + BC (281.7 ± 37.8 N) groups had significantly higher fracture resistance than those in the control group (108.5 ± 23.3 N) (P < .05). No significant difference was revealed between the BC and PRF + BC groups (P > .05). Histologic evidence of cementumlike tissue deposition along the canal wall with scattered bonelike tissue in the canal was observed. CONCLUSIONS: Either a combination of PRF with BC or BC alone could improve periapical healing, induce root development, and reinforce tooth structure. No additional benefit of PRF to BC in RET was found.