miRNAs originate from primary transcripts (pri-miRNAs) with characteristic stem-loop structures. Accurate processing of pri-miRNAs is required for functional miRNAs. Here, using pri-miR166 family as a paradigm, we report the decisive role of pri-miRNA terminal loops in miRNA biogenesis. We found that multi-branched terminal loops in pri-miR166s substantially suppressed miR166 expression in vivo. Unlike canonical processing of pri-miRNAs, terminal-loop-branched (TLBed) pri-miRNAs can be processed by Dicer-like1 (DCL1) complexes bi-directionally: from base to loop and from loop to base, resulting in productive and abortive processing of miRNAs, respectively. In either case, DCL1 complexes canonically cut pri-miRNAs at a distance of 16-17 base pairs (bp) from a reference single-stranded loop region. DCL1 also adjusts processing sites toward an internal loop through its helicase domain. Thus, these results provide new insight into the poorly understood processing mechanism of pri-miRNAs with complicated secondary structures.

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Background and objective:Oxidative stress has been suggested as an important pathogenic factor contributing to chronic periodontitis with diabetes mellitus (CPDM). Previous studies have revealed the potential therapeutic properties of baicalein (BCI) in oxidative stress-related diseases; however, the antioxidant effects of BCI on therapy for individual with CPDM remain largely unexplored. Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a critical role in cellular defence against oxidative stress. In this study, we aim to determine whether BCI prevents diabetesrelated periodontal tissue destruction by regulating Nrf2 signaling pathway. Material and methods: Human gingival epithelial cells (hGECs) were challenged with high glucose (HG, 25 mmol/L) and/or lipopolysaccharide (LPS, 20 µg/mL). Reactive oxygen species (ROS) were detected by fluorescence-activated cell sorting. The changes of antioxidant-related genes, including Nrf2, catalase (Cat), glutamatecysteine ligase catalytic subunit (Gclc), superoxide dismutase 1 (Sod1), and superoxide dismutase 2 (Sod2), were quantified by real-time PCR. The localization of phospho-Nrf2 (pNrf2, S40) in the nucleus was detected by immunofluorescence staining and laser scanning confocal microscope (LSCM). PNrf2 and total form of Nrf2 were determined using western blot. The above indicators together with mitochondrial membrane potential (MMP) were further investigated in hGECs pre-treated with different concentrations of BCI (0.01, 0.1, or 0.5 µg/mL) before stimulated with HG plus LPS (GP). Finally, the role of BCI in activating Nrf2 signaling pathway and relieving the alveolar bone absorption was examined in the CPDM model of Sprague Dawley rats.CPDM rats were oral gavaged with BCI (50, 100, or 200 mg/kg daily). The pNrf2 was detected by immunohistochemistry, and the alveolar bone absorption was examined by microcomputed tomography. Results:Our results showed that ROS were significantly increased in both groups of HG and LPS, with the strongest generation in the GP group. In terms of ROSrelated gene expression, we found that the mRNA levels of Nrf2, Cat, Gclc, Sod1, and Sod2 were significantly decreased in HG and LPS groups. In consistent with the strongest induction of ROS in GP group, the gene expression in GP group was further

Background Human periodontal ligament fibroblasts (HPDLFs) represent the first line of defense against pathogens in the periodontal tissue. Porphyromonas gingivialis (P. gingivalis) has been known to be most strongly associated with periodontitis. MicroRNA (miR)‐146a is involved in the inflammatory regulation of periodontitis. However, the regulatory mechanism of miR‐146a on in P. gingivalis‐induced inflammation response in HPDLFs was still unclear. The aim of this study was to investigate whether miR‐146a plays a key role in P. gingvalis‐induced inflammation responses through regulation of TRAF6 in HPDLFs. Methods MiR‐146a expression was measured by real‐time polymerase chain reaction (PCR) in HPDLFs stimulated with P. gingivalis and its lipopolysaccharide (LPS). IL‐1ß, IL‐6, and IL‐8 were determined by enzyme‐linked immunosorbent assay (ELISA) in the culture supernatants of HPDLFs after transfected with miR‐146a mimic or inhibitor. Meanwhile, the expression of TRAF6 was measured by real‐time PCR and Western blot. Then, we used luciferase reporter assay to detect whether miR‐146a binds to the 3′‐UTR of TRAF6. By using small interfering RNA (siRNA) of TRAF6, the phosphorylation of p38 mitogen‐activated protein kinase (MAPK) was measured by Western blot. Finally, after inhibition of TRAF6 and p38 in HPDLFs, we analyzed the expression of miR‐146a upon P. gingivalis challenge. Results P. gingivalis and its LPS significantly induced miR‐146a expression in HPDLFs. Overexpression of miR‐146a significantly suppressed the IL‐1ß, IL‐6 and IL‐8 secretion, TRAF6 expression, and p38 phosphorylation. In contrast, the levels of these indexes significantly increased by inhibition of miR‐146a. Furthermore, MiR‐146a directly binds to the 3′‐UTR of TRAF6 in P. gingivalis‐induced HPDLFs, but not in P. gingivalis LPS stimulation. Suppression of TRAF6 could inhibit the phosphorylation of p38. Finally, inhibition of TRAF6 and p38 significantly abolished P. gingivalis‐induced miR‐146a upregulation in HPDLFs. Conclusions MiR‐146a contribute to negative regulation of P. gingivalis‐induced proinflammatory cytokines secretion in HPDLFs though TRAF6/p38 MAPK pathway. Maintaining miR‐146a homeostasis plays a key role in controlling inflammatory response in periodontal tissues.

The innovation of NO2 gas sensors is highly desirable in environmental monitoring and human safety. Herein, a macroporous SnO2/MoS2 inverse opal hierarchitecture has been constructed with substantial interface charge transfer,...

The scaffolds for stem cell-based bone tissue engineering should hold the ability to guide stem cells osteo-differentiating. Otherwise, stem cells will differentiate into unwanted cell types or will form tumors in vivo. Alginate, a natural polysaccharide with great biocompatibility, was widely used in biomedical applications. However, the limited bioactivity and poor osteogenesis capability of pristine alginate hampered its further application in tissue engineering. In this work, a bone forming peptide-1 (BFP-1), derived from bone morphogenetic protein-7, was grafted to alginate polymer chains to prepare peptide-decorated alginate porous scaffolds (pep-APS) for promoting osteo-differentiation of human mesenchymal stem cells (hMSCs). SEM images of pep-APS exhibited porous structure with about 90% porosity (pore size 100-300 μm), which was appropriate for hMSCs ingrowth. The adhesion, proliferation and aggregation of hMSCs grown on pep-APS were enhanced in vitro. Moreover, pep-APS promoted the alkaline phosphatase (ALP) activity of hMSCs, and the osteo-related genes expression was obviously up-regulated. The immunochemical staining and western blot analysis results showed high expression level of OCN and Col1a1 in the hMSCs grown on pep-APS. This work provided a facile and valid strategy to endow the alginate polymers themselves with specific bioactivity and prepare osteopromoting scaffold with enhanced osteogenesis ability, possessing potential applications in stem cell therapy and regenerative medicine.

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Background Oxidative stress mediated by hyperglycemia damages cell-reparative processes such as mitophagy. Down-regulation of mitophagy is considered to be a susceptible factor for diabetes mellitus (DM) and its complications. However, the role of mitophagy in DM-associated periodontitis has not been fully elucidated. Apoptosis of human gingival epithelial cells (hGECs) is one of the representative events of DM-associated periodontitis. Thus, this study aimed to investigate PTEN-induced putative kinase 1 (PINK1)-mediated mitophagy activated in the process of high glucose (HG)-induced hGECs apoptosis. Methods For dose–response studies, hGECs were incubated in different concentrations of glucose (5.5, 15, 25, and 50 mmol/L) for 48 h. Then, hGECs were challenged with 25 mmol/L glucose for 12 h and 48 h, respectively. Apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL), caspase 9 and mitochondrial membrane potential (MMP). Subsequently, autophagy was evaluated by estimating P62, LC3 II mRNA levels, LC3 fluorescent puncta and LC3-II/I ratio. Meanwhile, the involvement of PINK1-mediated mitophagy was assessed by qRT-PCR, western blotting and immunofluorescence. Finally, hGECs were transfected with shPINK1 and analyzed by MMP, caspase 9 and annexin V-FITC apoptosis. Results The number of TUNEL-positive cells and caspase 9 protein were significantly increased in cells challenged with HG (25 mmol/L) for 48 h (HG 48 h). MMP was impaired both at HG 12 h and HG 48 h, but the degree of depolarization was more serious at HG 48 h. The autophagy improved as the amount of LC3 II increased and p62 decreased in HG 12 h. During this process, HG 12 h treatment induced PINK1-mediated mitophagy. PINK1 silencing with HG 12 h resulted in MMP depolarization and cell apoptosis. Conclusions These results suggested that loss of the PINK1 gene may cause mitochondrial dysfunction and increase sensitivity to HG-induced apoptosis of hGECs at the early stage. PINK1 mediated mitophagy attenuates early apoptosis of gingival epithelial cells induced by high glucose.