Identification of Six microRNAs as Potential Biomarkers for Pemphigus Vulgaris: From Diagnosis to Pathogenesis.

BACKGROUND: Pemphigus vulgaris (PV) is a potentially fatal autoimmune bullous disease. The role of microRNA (miRNA, miR) in the diagnosis and pathogenesis of PV remains unknown. This study aims to provide potential miRNA biomarkers for PV diagnosis and therapy options. METHODS: Serum samples were obtained from 22 PV patients, 15 mucous membrane pemphigoid (MMP) patients, and 10 normal controls (NC). Total RNA was extracted from the serum samples, and 12 selected miRNAs were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatic analyses including target gene prediction and enrichment analysis were performed. RESULTS: Twelve miRNAs were increased in the serum of the PV group compared with the NC group, in which six miRNAs had good efficacy to diagnose PV from MMP with the area under the receiver operator characteristic curves of 0.970 to 0.988. A series test for the combination of miR-584-5p and miR-155-5p reached the sensitivity and specificity of 95.5% and 100%. Bioinformatic analysis revealed target gene enrichment in the cell adhesion pathways, immune-relating pathways, and P38 mitogen-activated protein kinases signaling pathway. CONCLUSION: The study provides new insights and targets of miRNAs for the precise diagnosis and the exploration of pathogenesis for PV, which may serve as a reference for further research into autoimmune bullous diseases.

Labial Gland Mesenchymal Stem Cell Derived Exosomes-Mediated miRNA-125b Attenuates Experimental Sjogren's Syndrome by Targeting PRDM1 and Suppressing Plasma Cells.

The pathogenesis of the prototypical chronic autoimmune disorder primary Sjögren syndrome (pSS) has been thought to be B-cell-centric, based on serum autoantibodies, the increased risk of B cell lymphoma, and altered B cell subsets in patients with pSS. Over the last 10 years, therapies targeting B cells have been investigated for pSS; however, current evidence for the efficacy of B cell targeted therapies in pSS is still sparse. Mesenchymal stem cells (MSCs) might represent a promising strategy for cell therapy of autoimmune diseases via regulation of immune cells. MSC-released exosomes carry various bioactive molecules and thus have been studied in MSC-based therapy. The newly discovered labial gland MSCs (LGMSCs) have exhibited enhanced performance. Herein, we aimed to determine the effects of LGMSC-derived exosomes (LGMSC-Exos) on the symptoms of a mouse model of pSS and their regulatory effect and mechanism on B cell subsets. In vivo, treatment of the spontaneous mouse model of pSS with LGMSC-Exos resulted in reduced inflammatory infiltration and restored saliva secretion in salivary glands. In vitro, coculture of LGMSC-Exos with peripheral blood mononuclear cells of patients with pSS markedly reduced the proportions of CD19+CD20-CD27+CD38+ plasma cells among peripheral blood mononuclear cells. Further investigations provided evidence that LGMSC-Exo-derived microRNA-125b affected plasma cells of pSS by directly binding to its target gene, PRDM1 (PR domain zinc finger protein 1, also known as BLIMP1), which might be developed as a target to treat pSS. Overall, these findings provided a possible exploitable therapeutic target in pSS and provide new insights into the potential therapeutic application of exosomes in pSS and other disease mediated by B-cells.

Immobilization of Ochrobactrum sp. on Biochar/Clay Composite Particle: Optimization of Preparation and Performance for Nitrogen Removal.

The objective of this study was to prepare biochar/clay composite particle (BCCP) as carrier to immobilize Ochrobactrum sp. to degrade ammonia nitrogen (NH4 +-N), and the effects of calcined program and immobilizing material were investigated. Results reflected that the parameters were as follows: calcined temperature 400°C, heating rate 20°C min-1, and holding time 2 h, and the adsorption capacity could reach 0.492 mg g-1. Sodium alginate/polyvinyl alcohol, as embedding material, jointed with NH4 +-N adsorption process and then degraded by Ochrobactrum sp. with 79.39% degradation efficiency at 168 h. Immobilizing Ochrobactrum sp. could protect strain from high salt concentration to achieve the exceeding degradation efficiency than free bacteria, but could not block the impact of low temperature.

Association between cheilitis granulomatosa and odontogenic infections: A case-control study.

The association between cheilitis granulomatosa and dental infections (dental caries and apical periodontitis) is still not well understood. Herein, we aimed to investigate the association in large hospital cases with cohort controls. Cheilitis granulomatosa cases (n = 181) were retrieved from Peking University Hospital of Stomatology and age- and sex-matched to controls (n = 181). The χ2 -test, Student's t-test, and Mann-Whitney U-test were used to compare the differences between groups. The χ2 -test and odds ratio were used to verify if there was an association and risk relationship. The results showed that both dental caries and apical periodontitis were associated with cheilitis granulomatosa (p < 0.001). Individuals with cheilitis granulomatosa had approximately a twofold increased frequency of dental caries than those without cheilitis granulomatosa (104/181, 57.5% vs. 53/181, 29.3%) (p < 0.001). The odds ratio of dental caries occurring in the case group compared to the control group was 3.211. The frequency of apical periodontitis in patients with cheilitis granulomatosa was significantly greater than in those without cheilitis granulomatosa (109/181, 60.2% vs. 28/181, 15.5%) (p < 0.001). The odds ratio was 8.272. Moreover, apical periodontitis was also locationally related to cheilitis granulomatosa (p < 0.001). Collectively, our study showed that the foci of dental infections are associated with cheilitis granulomatosa, suggesting that proper treatment of focal teeth may be important in the management of cheilitis granulomatosa.

Labial gland-derived mesenchymal stem cells and their exosomes ameliorate murine Sjögren's syndrome by modulating the balance of Treg and Th17 cells.

BACKGROUND: Sjögren's syndrome (SS) is a chronic, systemic autoimmune disorder characterized by sicca syndrome and/or systemic manifestations. The disease severely affects the health and life of patients, and the treatment of SS has always been a clinical challenge and essentially palliative. Mesenchymal stem cells (MSCs) have been reported to exert immunomodulatory effects and as a potential novel therapeutic strategy for SS. Labial gland-derived MSCs (LGMSCs) are a population of resident stem cells in the labial gland, first isolated by our group. Exosomes released by MSCs contain a large variety of bioactive molecules and considered to function as an extension of MSCs. METHODS: LGMSCs were isolated from patients who were needed surgery to remove the lip mucocele and LGMSCs derived exosomes (LGMSC-Exos) were isolated by ultracentrifugation. The non-obese diabetic (NOD) mice were treated with LGMSCs or LGMSC-Exos by tail vein injection. The saliva flow rate of mice was determined and salivary glands were dissected and stained with hematoxylin and eosin. In vitro, peripheral blood mononuclear cells (PBMCs) from SS patients were cocultured with LGMSCs or LGMSC-Exos. Percentage of T helper 17 (Th17) cells and regulatory T (Treg) cells were determined by flow cytometry. The serum levels of cytokines in NOD mice and in the supernatant of the co-culture system by ELISA. RESULTS: Treatment with LGMSCs or LGMSC-Exos reduced inflammatory infiltration in the salivary glands, and restored salivary gland secretory function in NOD mice. Importantly, LGMSCs or LGMSC-Exos were demonstrated to inhibit the differentiation of Th17 cells but promote the induction of Treg cells in NOD mice and PBMCs from SS patients in vitro, accompanied by reduced interleukin 17 (IL-17), interferon gamma, and IL-6 levels and enhanced transforming growth factor beta and IL-10 secretion by T cells. CONCLUSIONS: LGMSCs are potential candidates for MSCs-based therapy and LGMSC-Exos might be utilized for establishing a new cell-free therapy against SS.

Downregulation of Prolactin-Induced Protein Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells.

BACKGROUND Periodontal ligament stem cells (PDLSCs) are promising seed cells for bone tissue engineering and periodontal regeneration applications. However, the mechanism underlying the osteogenic differentiation process remains largely unknown. Previous reports showed that prolactin-induced protein (PIP) was upregulated after PDLSCs osteogenic induction. However, few studies have reported on the function of PIP in osteogenic differentiation. The purpose of the present study was to investigate the effect of PIP on osteogenic differentiation of PDLSCs. MATERIAL AND METHODS The expression pattern of PIP during PDLSCs osteogenic differentiation was detected and the effect of each component in the osteogenic induction medium on PIP was also tested by qRT-PCR. Then, the PIP knockdown cells were established using lentivirus. The knockdown efficiency was measured and the proliferation, apoptosis, and osteogenic differentiation ability were examined to determine the functional role of PIP on PDLSCs. RESULTS QRT-PCR showed that PIP was sustainedly upregulated during the osteogenic induction process and the phenomenon was mainly caused by the stimulation of dexamethasone in the induction medium. CCK-8 and flow cytometer showed that knocking down PIP had no influence on proliferation and apoptosis of PDLSCs. ALP staining and activity, Alizarin Red staining, and western blot analysis demonstrated PIP knockdown enhanced the osteogenic differentiation and mineralization of PDLSCs. CONCLUSIONS PIP was upregulated after osteogenic induction; however, PIP knockdown promoted PDLSCs osteogenic differentiation. PIP might be a by-product of osteogenic induction, and downregulating of PIP might be a new target in bone tissue engineering applications.

Nitrogen Advanced Treatment of Urban Sewage by Denitrification Deep-Bed Filter: Removal Performance and Metabolic Pathway.

This study aimed to explore the performance of denitrification deep-bed filter (DN-DBF) to treat municipal sewage for meeting a more stringent discharge standard of total nitrogen (TN) (10.0 mg L-1). A lab-scale DN-DBF was conducted to optimize operation parameters and reveal the microbiological mechanism for TN removal. The results showed that more than 12.7% TN removal was obtained by adding methanol compared with sodium acetate. The effluent TN concentration reached 6.0-7.0 mg L-1 with the optimal influent carbon and nitrogen ratio (C/N) and hydraulic retention time (HRT) (3:1 and 0.25 h). For the nitrogen removal mechanism, Blastocatellaceae_Subgroup_4 and norank_o_JG30-KF-CM45 were dominant denitrification floras with an abundance of 6-10%. Though large TN was removed at the top layer of DN-DBF, microbial richness and diversity at the middle layer were higher than both ends. However, the relative abundance of nitrite reductase enzymes (EC1.7.2.1) gradually increases as the depth increases; conversely, the relative abundance of nitrous oxide reductase gradually decreased.

Proteomics-based analysis indicating α-enolase as a potential biomarker in primary Sjögren's syndrome.

BACKGROUND: Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease. Its etiology is not well understood. Salivary glands are the main target organ in pSS, investigating the changes of salivary protein in pSS patients may not only be a valuable way of identifying new biomarkers/antigens for pSS, but also of revealing the pathogenesis underlying this autoimmune disease. In the present study, we aimed to investigate new biomarkers and explore their potential role in pSS. METHODS: In this study, α-enolase (ENO1) was found to be overexpressed in pSS by 1D gel electrophoresis/mass spectrometry. The finding was verified by Western blots, immunohistochemistry (IHC), and polymerase chain reaction (PCR) results in both saliva and labial salivary glands. The expression level of immunoglobulin G (IgG) antibody to ENO1 was then tested by enzyme-linked immunosorbent assay (ELISA). RESULTS: ENO1 autoantibody was found to be overexpressed in pSS compared with healthy controls. The effects of ENO1 overexpression on rat submandibular gland cell line SMG-C6 was investigated in vitro. The expressions of proteins related to saliva secretion and immunomodulatory were upregulated in ENO1 overexpressed SMG-C6 cells. CONCLUSIONS: Both ENO1 and anti-ENO1 autoantibody are overexpressed in pSS patients. Nevertheless, their potential role in the pathogenesis of pSS warrants further study.

Comparative analysis of lncRNA and mRNA expression profiles between periodontal ligament stem cells and gingival mesenchymal stem cells.

Oral tissue-derived mesenchymal stem cells, such as periodontal ligament stem cells (PDLSCs) and gingival mesenchymal stem cells (GMSCs), possess different biological characteristics, but the molecular mechanism remains unclear, which restricts their application in tissue engineering. Long noncoding RNAs (lncRNAs) are known to be significant regulators of gene expression, but our knowledge about their roles in the regulation of stem cell biological properties is still limited. This study compared the lncRNA and mRNA expression profiles between PDLSCs and GMSCs through microarray analysis, and applied bioinformatics methods to analyze and predict the function and connection of differentially expressed genes, aiming to screen potential key regulators of diverse biological characteristics in PDLSCs and GMSCs. Microarray analysis showed that 2162 lncRNAs and 1347 mRNAs were significantly differentially expressed between PDLSCs and GMSCs. Gene ontology (GO) analysis and pathway analysis indicated that these differentially expressed genes were involved in diverse biological processes and signaling pathways. The gene signal network and pathway relation network predicted some potentially important regulators. The coding-noncoding gene coexpression network (CNC network) revealed many potential lncRNA-mRNA connection pairs that participated in the regulation of biological behaviors. These results stressed the roles of lncRNAs in controlling stem cell biological behaviors and provided guides for molecular mechanistic study of different biological characteristics in PDLSCs and GMSCs.

High-fat and high-glucose microenvironment decreases Runx2 and TAZ expression and inhibits bone regeneration in the mouse.

BACKGROUND: Type 2 diabetes mellitus (T2DM) and hyperlipidemia are negatively related to bone regeneration. The aim of this study was to evaluate the effect of high-fat and high-glucose microenvironment on bone regeneration and to detect the expression of runt-related transcription factor 2 (Runx2) and transcriptional co-activator with PDZ-binding domain (TAZ) during this process. METHODS: After establishing a high-fat and high-glucose mouse model, a 1 mm × 1.5 mm bone defect was developed in the mandible. On days 7, 14, and 28 after operation, bone regeneration was evaluated by hematoxylin-eosin staining, Masson staining, TRAP staining, and immunohistochemistry, while Runx2 and TAZ expression were detected by immunohistochemistry, RT-PCR, and Western blot analysis. RESULTS: Our results showed that the inhibition of bone regeneration in high-fat and high-glucose group was the highest among the four groups. In addition, the expression of Runx2 in high-fat, high-glucose, and high-fat and high-glucose groups was weaker than that in the control group, but the expression of TAZ only showed a decreasing trend in the high-fat and high-glucose group during bone regeneration. CONCLUSIONS: In conclusion, these results suggest that high-fat and high-glucose microenvironment inhibits bone regeneration, which may be related to the inhibition of Runx2 and TAZ expression.

Effect of 1α, 25-dihydroxyvitamin D3 on the osteogenic differentiation of human periodontal ligament stem cells and the underlying regulatory mechanism.

1α, 25­dihydroxyvitamin D3 (1,25­D3), an active vitamin D metabolite, is a well­known regulator of osteogenic differentiation. However, how 1,25­D3 regulates osteogenic differentiation in human periodontal ligament stem cells (hPDLSCs) remains to be fully elucidated. The present study aimed to clarify this issue through well­controlled in vitro experiments. After hPDLSCs were treated with 1,25­D3, immunofluorescence and western blotting were used to detect the expression of vitamin D receptor; Cell Counting Kit­8 and western blotting were used to assay the cell proliferation ability. Alkaline phosphatase staining, Alizarin Red staining and western blotting were used to detect the osteogenic differentiation. It was found that treating hPDLSCs with 1,25­D3: i) Inhibited cell proliferation; ii) promoted osteogenic differentiation; iii) upregulated the expression of transcriptional coactivator with PDZ­binding motif (TAZ), an important downstream effector of Hippo signaling that has been demonstrated to be involved in the osteogenic differentiation of stem/progenitor cells; and iv) that co­treatment of TAZ­overexpressing hPDLSCs with 1,25­D3 synergistically stimulated the expression of osteogenic markers. These results suggested that the induction of osteogenic differentiation promoted by 1,25­D3 in hPDLSCs involves, at least in part, the action of TAZ.

A comprehensive study on donor-matched comparisons of three types of mesenchymal stem cells-containing cells from human dental tissue.

BACKGROUND AND OBJECTIVE: Mesenchymal stem cells (MSCs) have been widely used in tissue engineering, such as for regenerating the supporting structures of teeth destroyed by periodontal diseases. In recent decades, dental tissue-derived MSCs have drawn much attention owing to their accessibility, plasticity and applicability. Dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs) and gingival MSCs (GMSCs) are the most readily available MSCs among all types of dental MSCs. The purpose of this study was to comprehensively compare the characteristics of MSCs from dental pulp (DP), periodontal ligament (PDL) and gingiva (G) in vitro and thus provide insight into optimizing the performance of cells and seed cell selection strategies for tissue regeneration. MATERIALS AND METHODS: In this study, patient-matched (n = 5) cells derived from DP, PDL and G which, respectively, contained DPSCs, PDLSCs and GMSCs were evaluated using multiple methods in terms of their proliferation, senescence, apoptosis, multilineage differentiation and stemness maintenance after long-term passage. RESULTS: Mesenchymal stem cells-containing cells from G (MSCs/GCs) showed superior proliferation capability, whereas patient-matched MSCs-containing cells from PDL (MSCs/PDLCs) exhibited excellent osteogenic and adipogenic differentiation ability; MSCs-containing cells from DP (MSCs/DPCs) achieved mediocre results in both aspects. In addition, MSCs/GCs were the least susceptible to senescence, while MSCs/PDLCs were the most prone to ageing. Furthermore, the biological properties of these three types of cells were all affected after long-term in vitro culture. CONCLUSION: These three types of dental MSCs showed different biological characteristics. MSCs/PDLCs are the best candidate cells for bone regeneration, but the application of MSCs/PDLCs might be limited to certain number of passages. Improving the differentiation of MSCs/GCs remains the key issue regarding their application in tissue engineering.

Lipopolysaccharide from Escherichia coli stimulates osteogenic differentiation of human periodontal ligament stem cells through Wnt/ß-catenin-induced TAZ elevation.

Human periodontal ligament stem cells (PDLSCs), a type of dental tissue-derived mesenchymal stem cells (MSCs), can be clinically applied in periodontal tissue regeneration to treat periodontitis, which is initiated and sustained by bacteria. Lipopolysaccharide (LPS), the major component of the outer membrane of gram-negative bacteria, is a pertinent deleterious factor in the oral microenvironment. The aim of this study was to investigate the effect of LPS on the proliferation and osteogenic differentiation of PDLSCs, as well as the mechanisms involved. Proliferation and osteogenic differentiation of PDLSCs were detected under the stimulation of Escherichia coli-derived LPS. The data showed that E. coli-derived LPS did not affect the proliferation, viability, and cell cycle of PDLSCs. Furthermore, it promoted osteogenic differentiation with the activation of TAZ. Lentivirus-mediated depletion of TAZ (transcriptional activator with a PDZ motif) was used to determine the role of TAZ on LPS-induced enhancement of osteogenesis. PDLSCs cultured in osteogenic media with or without LPS and DKK1 (Wnt/ß-catenin pathway inhibitor) were used to determine the regulatory effect of Wnt signaling. We found that TAZ depletion offset LPS-induced enhancement of osteogenesis. Moreover, treatment with DKK1 offset LPS-induced TAZ elevation and osteogenic promotion. In conclusion, E. coli-derived LPS promoted osteogenic differentiation of PDLSCs by fortifying TAZ activity. The elevation and activation of TAZ were mostly mediated by the Wnt/ß-catenin pathway. PDLSC-governed alveolar bone tissue regeneration was not necessarily reduced under bacterial conditions and could be modulated by Wnt and TAZ.

An In Vitro Comparative Study of Multisource Derived Human Mesenchymal Stem Cells for Bone Tissue Engineering.

Mesenchymal stem cells (MSCs) have been considered promising tools for tissue engineering and regenerative medicine. However, the optimal cell source for bone regeneration remains controversial. To better identify seed cells for bone tissue engineering, we compared MSCs from seven different tissues, including four from dental origins, dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), gingival MSCs (GMSCs), and dental follicle stem cells (DFSCs); two from somatic origins, bone marrow-derived MSCs (BM-MSCs) and adipose-derived stem cells (ADSCs); and one from birth-associated perinatal tissue umbilical cord (UCMSCs). We cultured the cells under a standardized culture condition and studied their biological characteristics. According to our results, these cells exhibited similar immunophenotype and had potential for multilineage differentiation. MSCs from dental and perinatal tissues proliferated more rapidly than those from somatic origins. Simultaneously, DPSCs and PDLSCs owned stronger antiapoptotic ability under the microenvironment of oxidative stress combined with serum deprivation. In respect to osteogenic differentiation, the two somatic MSCs, BM-MSCs and ADSCs, demonstrated the strongest ability for osteogenesis compared to PDLSCs and DFSCs, which were just a little bit weaker than the formers. However, GMSCs and UCMSCs were the most pertinacious ones to differentiate to osteoblasts. We also revealed that the canonical intracellular protein kinase-based cascade signaling pathways, including PI3K/AKT, MAPK/ERK, and p38 MAPK, possessed different levels of activation in different MSCs after osteoblast induction. Our conclusions suggest that PDLSCs might be a good potential alternative to BM-MSCs for bone tissue engineering.

Antitumor activity of the plant extract morin in tongue squamous cell carcinoma cells.

Morin is a naturally occurring bioflavonoid originally isolated from members of the Moraceae family of flowering plants and it possesses antitumor activity in various human cancer cells. The present study explored the antitumor effects of morin in tongue squamous cell carcinoma (TSCC) cells in vitro and investigated the underlying molecular events. A TSCC cell line was treated with different doses of morin for up to 48 h. Analyses of cell viability, using Cell Counting Kit­8 (CCK­8), EdU incorporation, colony formation, flow cytometric analysis of cell cycle distribution and apoptosis, wound healing assay, western blot analysis and qRT­PCR assays, were then performed. The data revealed that morin treatment reduced Cal27 cell proliferation and reduced the migration capacity of tumor cells in a dose­dependent manner. Morin treatment also significantly upregulated mammalian sterile 20­like 1 (MST1) and MOB kinase activator 1 (MOB1) phosphorylation in CAL27 cells, but suppressed nuclear translocation of yes­associated protein (YAP) through the induction of YAP phosphorylation in Cal27 cells. Moreover, the expression of YAP­targeting genes, such as CTGF, CYR61 and ANKRD, was downregulated in morin­treated TSCC cells, indicating that morin was able to activate the Hippo signaling pathway to inhibit YAP nuclear translocation and YAP­related transcriptional activity in TSCC cells. In conclusion, the data from the present study demonstrated that morin produces anti­TSCC activity in vitro through activation of the Hippo signaling pathway and the downstream suppression of YAP activity in TSCC cells. Future studies should assess the clinical antitumor effects of morin.