AR-A014418, a glycogen synthase kinase-3ß inhibitor, mitigates lipopolysaccharide-induced inflammation in rat dental pulp stem cells via NLR family pyrin domain containing 3 inflammasome impairment.

Background/purpose: Cell pyroptosis and gingival inflammation have been implicated in periodontitis progression. Our previous study revealed that AR-A014418, a pharmacological inhibitor of glycogen synthase kinase-3ß (GSK-3ß), can enhance the migratory and osteogenic differentiation abilities of rat dental pulp stem cells (rDPSCs). The present study aimed to explore the effect of AR on the inflammation of rDPSCs. Materials and methods: The primary rDPSCs were isolated and identified by flow cytometry, as well as Oil red O and Alizarin Red S staining. The rDPSCs were cultured and exposed to lipopolysaccharide (LPS) before treating them with different concentrations of AR-A014418. The cell viability was detected using the CCK-8 assay. The generation and secretion of pro-inflammatory cytokines (IL-18, TNF-α, L-1ß, and IL-6) were examined by qPCR and ELISA, respectively. To investigate the activation of the NLRP3 inflammasome, the expression levels of pro-caspase 1, cleaved caspase 1, as well as NLRP3 were analyzed by western blotting and immunofluorescence, respectively. Results: In the rDPSCs, LPS prohibited cell viability and enhanced the generation and secretion of pro-inflammatory cytokines. LPS upregulated NLRP3 and cleaved caspase-1 protein levels and promoted ASC speck formation in the rDPSCs. AR-A014418 administration effectively blocked the LPS-induced inflammation of the rDPSCs in a dose-dependent way. Mechanistically, AR-A014418 significantly restrained the up-regulation of NLRP3 and cleaved caspase-1 in LPS-treated rDPSCs. Conclusion: Collectively, our findings suggest that AR-A014418 significantly mitigates LPS-induced inflammation of rDPSCs by blocking the activation of the NLRP3 inflammasome.

Glycogen synthase kinase-3ß inhibitor promotes the migration and osteogenic differentiation of rat dental pulp stem cells via the ß-catenin/PI3K/Akt signaling pathway.

Background/purpose: Glycogen synthase kinase-3ß (GSK3ß) inhibitor enhances bone formation, while dental pulp stem cells (DPSC) are potentially used to repair bone defects. The present study aimed to investigate the effect of AR-A014418 (AR, a specific glycogen synthase kinase-3ß inhibitor) on the migration and osteogenic differentiation of rat-derived dental pulp stem cells (rDPSCs), and further explore the underlying mechanism. Materials and methods: rDPSCs were isolated from rats, and then cultured with different concentrations of AR with or without LY294002 (a PI3K inhibitor). Then, cell viability, migration, osteogenic differentiation, and the involvement of PI3K pathway were detected by CCK-8 assay, Transwell assay, Alizarin Red S Staining, Alkaline phosphatase (ALP) assay, Western blot, and RT-PCR, respectively. Results: Our present study demonstrated that AR of various concentrations (1 µM, 2.5 µM, and 5 µM) not only promoted the rDPSC proliferation and migration, but also increased calcium deposition, the activity of alkaline phosphatase (ALP), and levels of osteogenic markers (RUNX2, OPN, OCN, and OSX) in rDPSCs. It was also found that the administration of AR resulted in an increase in the expression level of p-GSK3ß (Ser), ß-catenin, p-PI3K, and p-Akt, and a reduction in p-GSK3ß (Tyr216). Furthermore, PI3K inhibitor LY294002 abrogated the enhanced cell migration and osteogenic differentiation of rDPSCs induced by AR. Conclusion: Our results provide evidence that AR significantly promotes migration and osteogenic differentiation of rDPSCs by activating ß-catenin/PI3K/Akt signaling pathway.

[Effect of chlorhexidine-loaded halloysite nanotube on human dentin bond strength].

PURPOSE: To evaluate the effect of chlorhexidine-loaded halloysite aluminosilicate clay nanotube (HNT) incorporation into a two-step etch-and-rinse (ONE-STEP) and a self-etch (XENO Ⅴ) adhesive on human dentin tensile shear strength (TSS). METHODS: Ten groups (n=12) were prepared according to the adhesive system and amount of CHX/HNT incorporated (5%, 10%, 15%) as follows: XE control(A1), XE experimental control (A2), 5% CHX/HNT-XE (A3), 10%CHX/HNT-XE (A4), 15% CHX/HNT-XE (A5), OS control (B1), OS experimental control (B2), 5% CHX/HNT-OS (B3), 10% CHX/HNT-OS (B4), 15% CHX/HNT-OS (B5). TSS testing was performed after 24 h. Scanning electron microscopy of the resin-dentin interface of selected specimens was carried out. One-way ANOVA was used to evaluate the TSS date in XE groups and OS groups. Two-way ANOVA was used to evaluate the effect of the adhesive system and the CHX/HNT content on dentin TSS with SPSS 19.0 software package. RESULTS: The TSS date of 5% CHX/HNT-XE was significantly higher than the other groups. The TSS date of 10% CHX/HNT-OS was significantly higher than the other groups (P<0.05). No significant difference was detected between adhesive systems and dentin TSS. significant difference was found between CHX/HNT content and dentin TSS. CONCLUSIONS: CHX/HNT addition into dentin adhesive increased bond strength to dentin.

MIST: a Multilocus Identification System for Trichoderma.

Due to the rapid expansion in microbial taxonomy, precise identification of common industrially and agriculturally relevant fungi such as Trichoderma species is challenging. In this study, we introduce the online multilocus identification system (MIST) for automated detection of 349 Trichoderma species based on a set of three DNA barcodes. MIST is based on the reference databases of validated sequences of three commonly used phylogenetic markers collected from public databases. The databases consist of 414 complete sequences of the nuclear rRNA internal transcribed spacers (ITS) 1 and 2, 583 sequence fragments of the gene encoding translation elongation factor 1-alpha (tef1), and 534 sequence fragments of the gene encoding RNA polymerase subunit 2 (rpb2). Through MIST, information from different DNA barcodes can be combined and the identification of Trichoderma species can be achieved based on the integrated parametric sequence similarity search (blastn) performed in the manner of a decision tree classifier. In the verification process, MIST provided correct identification for 44 Trichoderma species based on DNA barcodes consisting of tef1 and rpb2 markers. Thus, MIST can be used to obtain an automated species identification as well as to retrieve sequences required for manual identification by means of phylogenetic analysis.IMPORTANCE The genus Trichoderma is important to humankind, with a wide range of applications in industry, agriculture, and bioremediation. Thus, quick and accurate identification of Trichoderma species is paramount, since it is usually the first step in Trichoderma-based research. However, it frequently becomes a limitation, especially for researchers who lack taxonomic knowledge of fungi. Moreover, as the number of Trichoderma-based studies has increased, a growing number of unidentified sequences have been stored in public databases, which has made the species identification more ambiguous. In this study, we provide an easy-to-use tool, MIST, for automated species identification, a list of Trichoderma species, and corresponding sequences of reference DNA barcodes. Therefore, this study will facilitate the research on the biodiversity and applications of the genus Trichoderma.