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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Oxidative stress in high-yielding dairy goats adversely affects lactation length, milk quality, and the economics of dairy products. During the lactation period, goat mammary epithelial cells (GMECs) are often in a state of disordered metabolic homeostasis primarily caused by the overproduction of reactive oxygen species (ROS). Sulforaphane (SFN), an electrophilic compound that is enriched in broccoli, is a promising antioxidant agent for future potential clinical applications. The objective of the present study was to investigate the function of SFN on hydrogen peroxide (H2O2)-induced oxidative damage in primary GMECs and the underlying molecular mechanisms. Isolated GMECs in triplicate were pretreated with SFN (1.25, 2.5, and 5 μM) for 24 h in the absence or presence of H2O2 (400 μM) for 24 h. The results showed that SFN effectively enhanced superoxide dismutase (SOD) activity, elevated the ratio of glutathione (GSH)/glutathione oxidized (GSSG), and reduced H2O2-induced ROS and malondialdehyde (MDA) production and cell apoptosis. Mechanically, SFN-induced nuclear factor erythroid 2-related factor 2 (NRF2/NFE2L2) translocation to the nucleus through the activation of the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway coupled with inhibition of the caspase apoptotic pathway. In addition, GMECs were transfected with NFE2L2 small interfering RNA (NFE2L2 siRNA) for 48 h and/or treated with SFN (5 μM) for 24 h before being exposed to H2O2 (400 μM) for 24 h. We found that knockdown of NFE2L2 by siRNA abrogated the preventive effect of SFN on H2O2-induced ROS overproduction and apoptosis. Taken together, sulforaphane suppressed H2O2-induced oxidative stress and apoptosis via the activation of the AMPK/NFE2L2 signaling pathway in primary GMECs.