Synergistic and antibiofilm activity of DNase I and glucose oxidase loaded chitosan nanoparticles against dual-species biofilms of Listeria monocytogenes and Salmonella.

Salmonella and Listeria monocytogenes are two of the most common foodborne pathogens in the food industry. They form dual-species biofilms, which have a higher sensitivity to antimicrobial treatment and a greater microbial adhesion. In this experiment, we loaded DNase I and glucose oxidase (GOX) on chitosan nanoparticles (CSNPs) to explore their inhibitory effects on and disruption of dual-species biofilms of Salmonella enterica and L. monocytogenes. Transmission electron microscopy (TEM) showed that CSNP-DNase-GOX and CSNPs were spherical in shape. CSNP-DNase-GOX was shifted and altered compared to the infrared peaks of CSNPs. CSNPs loaded with DNase I and GOX showed an increase in the particle size and an alteration in the polydispersity index (PDI) and the zeta potential. Compared to free DNase I or GOX, DNase I and GOX loaded on CSNPs had higher stability at different temperatures. CSNP-DNase-GOX was more effective in inhibiting dual-species biofilms than CSNP-GOX. Scanning electron microscopy (SEM) and fluorescence microscopy were used to observe the structure of the biofilm, which further illustrated that CSNP-DNase-GOX disrupted the dual-species biofilms of S. enterica and L. monocytogenes.

Identification of Pulpitis-Related Potential Biomarkers Using Bioinformatics Approach.

Inflammatory reaction of pulp tissue plays a role in the pathogen elimination and tissue repair. The evaluation of severity of pulpitis can serve an instructive function in therapeutic scheme. However, there are many limitations in the traditional evaluation methods for the severity of pulpitis. Based on the Gene Expression Omnibus (GEO) database, our study discovered 843 differentially expressed genes (DEGs) related to pulpitis. Afterwards, we constructed a protein-protein interaction (PPI) network of DEGs and used MCODE plugin to determine the key functional subset. Meanwhile, genes in the key functional subset were subjected to GO and KEGG enrichment analyses. The result showed that genes were mainly enriched in inflammatory reaction-related functions. Next, we screened out intersections of PPI network nodes and pulpitis-related genes. Then, 20 genes were obtained as seed genes. In the PPI network, 50 genes that had the highest correlation with seed genes were screened out using random walk with restart (RWR). Furthermore, 4 pulpitis-related hub genes were obtained from the intersection of the top 50 genes and genes in the key functional subset. Finally, GeneMANIA was utilized to predict genes coexpressed with hub genes, and expression levels of the 4 hub genes in normal and pulpitis groups were analyzed based on GEO data. The result demonstrated that the 4 hub genes were mainly coexpressed with chemokine-related genes and were remarkably upregulated in the pulpitis group. In short, we eventually determined 4 potential biomarkers of pulpitis.

The role of long noncoding RNA THAP9-AS1 in the osteogenic differentiation of dental pulp stem cells via the miR-652-3p/VEGFA axis.

Dental pulp stem cells (DPSCs) are multipotent and may play crucial roles in dentin-pulp regeneration. Recent studies have revealed that long noncoding RNAs (lncRNAs) are implicated in the osteogenic differentiation of DPSCs. However, the specific role and potential mechanisms of the lncRNA trihydroxyacetophenone domain containing nine antisense RNA 1 (THAP9-AS1) during osteogenic differentiation of DPSCs remain unknown. In the present study, we determined that THAP9-AS1 expression was upregulated during osteogenic differentiation of DPSCs. Moreover, we investigated the biological functions of THAP9-AS1 during osteogenic differentiation of DPSCs by loss-of-function assays. THAP9-AS1 knockdown inhibited osteogenic differentiation of DPSCs by decreasing alkaline phosphatase activity, alkaline phosphatase-positive cell ratio, mineralizing matrix and mRNA, and protein levels of early osteogenic-markers. We also found that THAP9-AS1 interacted with miR-652-3p, whose downstream gene target is vascular endothelial growth factor A (VEGFA). In addition, rescue assays indicated that VEGFA rescued the effects of THAP9-AS1 knockdown during osteogenic differentiation of DPSCs. In summary, we verified that knockdown of THAP9-AS1 inhibits osteogenic differentiation of DPSCs via the miR-652-3p/VEGFA axis. Our findings may be helpful to extend research on the mechanisms underlying osteogenic differentiation of DPSCs.

Formation, Development, and Cross-Species Interactions in Biofilms.

Biofilms, which are essential vectors of bacterial survival, protect microbes from antibiotics and host immune attack and are one of the leading causes that maintain drug-resistant chronic infections. In nature, compared with monomicrobial biofilms, polymicrobial biofilms composed of multispecies bacteria predominate, which means that it is significant to explore the interactions between microorganisms from different kingdoms, species, and strains. Cross-microbial interactions exist during biofilm development, either synergistically or antagonistically. Although research into cross-species biofilms remains at an early stage, in this review, the important mechanisms that are involved in biofilm formation are delineated. Then, recent studies that investigated cross-species cooperation or synergy, competition or antagonism in biofilms, and various components that mediate those interactions will be elaborated. To determine approaches that minimize the harmful effects of biofilms, it is important to understand the interactions between microbial species. The knowledge gained from these investigations has the potential to guide studies into microbial sociality in natural settings and to help in the design of new medicines and therapies to treat bacterial infections.

Long non-coding RNA maternally expressed gene 3 inhibits osteogenic differentiation of human dental pulp stem cells via microRNA-543/smad ubiquitin regulatory factor 1/runt-related transcription factor 2 axis.

OBJECTIVE: The aim of the present study was to investigate the biological roles and underlying mechanism of the long non-coding RNA maternally expressed gene 3 (MEG3) on osteogenic differentiation of human dental pulp stem cells (hDPSCs). METHODS: The expression levels of MEG3, microRNA-543 (miR-543), osterix, osteopontin, osteocalcin and runt-related transcription factor 2 (RUNX2) were measured by quantitative real-time PCR (qRT-PCR). Alkaline phosphatase (ALP) activity assay and alizarin red S staining (ARS) were used to measure the impacts exerted by MEG3, miR-543 on osteogenic differentiation. Cell proliferation was measured by MTT assay. In addition, the targeted relationships between miR-543, MEG3, and Smad ubiquitin regulatory factor 1 (SMURF1) were assessed through dual luciferase reporter assay. RESULTS: During osteogenic induction, the expression of MEG3 was gradually reduced, whereas the expression of miR-543, osterix, osteopontin, osteocalcin and RUNX2 were gradually increased. Functional analysis implied that MEG3 overexpression or miR-543 inhibition reduced the cell proliferation, ALP activity, ARS levels, and decreased the expression of osteoblast-related proteins. Moreover, MEG3 promoted SMURF1 expression by directly targeting miR-543 as a competing endogenous RNA. Furthermore, overexpression of miR-543 or silencing SMURF1 could reverse the inhibitory effects of MEG3 on the osteogenic differentiation of hDPSCs. CONCLUSIONS: In conclusion, our study revealed that overexpression of MEG3 inhibited hDPSCs osteogenic differentiation via miR-543/SMURF1/RUNX2 regulatory network, which may contribute to the functional regulation and clinical applications of hDPSCs.

Removal of the root canal smear layer using Carisolv III and sodium hypochlorite.

The present study investigated the effectiveness of a Carisolv III + 0.5% sodium hypochlorite (NaOCl)-based root canal irrigant for smear layer removal.Forty maxillary incisors were randomly divided into 4 groups (n = 10 per group). The canals in group A (experimental) were prepared with 0.5% NaOCl, and Carisolv III and 0.5% NaOCl was used for the final washing; groups B and C (positive controls) used 2% and 5.25% NaOCl, respectively; and group D (negative control) used phosphate-buffered saline (PBS). Ethylenediaminetetraacetic acid (EDTA) was used for all of the groups. A 5-point scoring scale and scanning electron microscopy were used to evaluate the effectiveness of the irrigants. The canals were consistently cleaner in the coronal and middle thirds than in the apical thirds (P < .05).For cleaning the root canals, 5.25% NaOCl was more effective than 2% NaOCl, 0.5% NaOCl + Carisolv III, and phosphate-buffered saline , respectively (P < .05). The 2% NaOCl solution showed similar results to 0.5% NaOCl + Carisolv III (P > .05). The combination of 5.25% NaOCl and 17% EDTA remains the most effective irrigant for removal of the root canal smear layer.A combination of Carisolv III + 0.5% NaOCl (with 17% EDTA) showed a cleaning ability similar to that of 2% NaOCl (with 17% EDTA).

Long noncoding RNA TUG1 promotes osteogenic differentiation of human periodontal ligament stem cell through sponging microRNA-222-3p to negatively regulate Smad2/7.

OBJECTIVE: To scrutinize the function of long non-coding RNA TUG1 on the osteogenic differentiation of human periodontal ligament stem cells in periodontitis and its specific mechanism. METHODS: Human periodontal ligament stem cells were extracted from human periodontium, followed by induction of osteogenic differentiation with osteogenic medium. Knockdown or overexpression of TUG1, microRNA-222-3p or Smad2/7 were performed in human periodontal ligament stem cells to observe their effect on expressions of osteogenic differentiation markers (Runx2, ALP, and OCN), and on calcium nodule formation by Alizarin red staining. Starbase software was utilized for prediction of the binding sites of TUG1 and microRNA-222-3p in addition to microRNA-222-3p and Smad2/7. Then dual-luciferase reporter gene assay was adopted to inspect the binding relationships between microRNA-222-3p and TUG1 or Smad2/7. RESULTS: Highly expressed TUG1 and lowly expressed microRNA-222-3p were found in human periodontal ligament stem cells during osteogenic differentiation. After measuring the expression of Runx2, ALP, and OCN as well as the formation of calcium nodules, we discovered that TUG1 can facilitate osteogenic differentiation of human periodontal ligament stem cells, while microRNA-222-3p had a reverse effect. Subsequently, knockdown of TUG1 and Smad2/7 or overexpression of microRNA-222-3p inhibited the osteogenic differentiation of human periodontal ligament stem cells. MicroRNA-222-3p is a target gene of TUG1, while microRNA-222-3p can negatively regulate Smad2/7. CONCLUSION: LncRNA TUG1 accelerates the osteogenic differentiation of human periodontal ligament stem cells by sponging microRNA-222-3p to regulate Smad2/7.

Root canal morphology of maxillary and mandibular first premolars analyzed using cone-beam computed tomography in a Shandong Chinese population.

The aim of this study was to explore the root morphology and root canal configuration of first premolars among Shandong Chinese residents using cone-beam computed tomography (CBCT).Randomly selected CBCT images were collected from 648 patients (44% women, 56% men). In total, 1268 maxillary and 1296 mandibular first premolars were analyzed. The number of roots and the canal configuration were recorded and identified based on Vertucci's classification.The majority of the maxillary first premolars had 1 root (67.4%), followed by 2 roots (32%). A 2-canal configuration (89%) was the most prevalent observation. For mandibular first premolars, 98.8% had 1 root and 81% presented the type I configuration. There were no statistical differences in the number of roots or morphology in terms of the left/right side or sex (P > .05).Among Chinese residents, the majority of maxillary first premolars had 1 root and 2 canals, whereas the most common anatomical configuration for mandibular first premolars was 1 root with 1 canal.

Berberine reduces inflammation of human dental pulp fibroblast via miR-21/KBTBD7 axis.

OBJECTIVE: This study is to investigate the effect and underlying mechanisms of berberine (BBR) on the proliferation and inflammatory levels of lipopolysaccharide induced human dental pulp fibroblast (LPS-HDPF). METHODS: Different concentrations of LPS were used to induce inflammatory response of HDPF. Cell proliferation was observed in BBR treated HDPF after transfection of miR-21 mimic, miR-21 inhibitor or sh-kBTBD7 plasmids and their negative controls. ELISA was employed to detect the expressions of inflammation related cytokines. Real-time quantitative RT-PCR was applied to estimate the expressions of miR-21 and KBTBD7. KBTBD7 expression was detected by Immunocytochemistry. Western blot analysis showed the protein levels of KBTBD7, Phospho-IKKα/ß, IKKß, Phospho-NF-κB p65, NF-κB p65, and IκBα. Luciferase reporter gene assay was used to determine the interaction between miR-21 and KBTBD7. RESULTS: LPS could promote inflammatory response in HDPF. Down-regulated miR-21 and up-regulated KBTBD7 were found in LPS-HDPF. BBR (25 uM) treatment in LPS-HDPF could ameliorate cell inflammatory response, presented by reduced expressions of IL-1ß, IL-6 and TNF-α, as well as enhanced cell proliferation and miR-21 expression. Moreover, miR-21 negatively targets KBTBD7. Over-expression of miR-21 or silencing of KBTBD7 could enhance the protective role of BBR on LPS-HDPF by inhibiting inflammatory response and promoting cell proliferation. Transfection of miR-21 overexpression or KBTBD7 silencing in BBR treated LPS-HDPF could inhibit activation of NF-κB signal pathway. CONCLUSION: Evidence in this study suggested that BBR mediates LPS induced inflammation in HDPF via miR-21/KBTBD7 axis to regulate NF-κB signal pathway, which may provide theoretical basis for BBR in prevention of pulpitis.

Berberine Promotes Osteogenic Differentiation of Human Dental Pulp Stem Cells Through Activating EGFR-MAPK-Runx2 Pathways.

Similar to the mesenchymal stem cells (MSCs), dental pulp stem cells (DPSCs) also have pluripotent differentiation characteristic and may be more ideal for tissue regeneration, especially in tooth regeneration engineering. However, bacterial infection may be a powerful obstacle. Berberine (BBR), known with antibacterial effects, was recently found to play functions in bone formation through promoting osteogenic differentiation from pluripotent stem cells. However, whether BBR also function in DPSCs osteogenic differentiation has not yet been reported. Primary DPSCs were isolated from dental pulp tissues extracted from human impacted mandibular third molars, and identified by flow cytometry for cell surface antigen molecules. A dexamethasone osteogenic medium was used to induce DPSCs osteogenic differentiation. BBR (1 µM and 5 µM) was pre-added to into medium, and then cell proliferation, spheroid formation and osteogenic differentiation capacities of DPSCs were analyzed, as well as the underlying molecules modulation mechanism. Flow cytometry identified that CD44, CD90, CD81 and CD105 positively expressed in the isolated hDPSCs, with CD34 and CD45 negetively expressed. BBR enhanced the cell proliferation of hDPSCs in a dose-dependent pattern, and promoted dexamethasone-induced osteogenic differentiation via enhancing Runx2 transcription factor activity followed by upregulating osteogenesis markers expression, whereas the adipogenic differentiation of hDPSCs was suppressed dramatically by BBR. The EGFR and MAPK pathways were activated by BBR, and inhibitors for these pathways significantly suppressed the osteogenic differentiation promotion of BBR. These results have revealed a novel mechanism that berberine might promote hDPSCs osteogenic differentiation through activating EGFR-MAPK-Runx2 signaling pathways.

A case of maxillary second premolar with 3 teeth and 4 root canals and a literature review / 口腔疾病防治

Objective @#To explore the discovery and treatment of multiple root canals in the maxillary second premolar to provide a reference for root canal therapy.@*Methods @#A case of maxillary second premolar occlusal discomfort accompanied by cold and heat stimulation pain and spontaneous pain was analyzed retrospectively.@*Results@# In this case, the number of roots and root canals in the tooth was determined by CBCT to be three roots with four canals. Root canal therapy was performed with Ni-Ti endodontic files and thermoplastic gutta-percha via root optical microscopy. The clinical symptoms disappeared after the operation.@*Conclusion@#The maxillary second premolar root canal system is complex, and additional root canals can be found and located with CBCT and optical microscopy. High-quality root canal therapy was completed.

[Mandibular first molar with C-shaped root canal system: a two-case report].

The root and canal anatomy of the mandibular first molar is highly complicated. A mandibular first molar with a C-shaped root canal system is rare. This article describes two different cases of this system. Case 1 has a complete C-shaped fused root. The root canal is characterized by a mesial lingual root canal and a C-shaped fused root canal. Case 2 has a C-shaped fused root in the buccal region and a distal lingual root. The root canal is characterized by a type Ⅱ-Ⅰroot canal in the buccal region and a distal lingual root canal.

[4i, a novel PPARγ ligand, inhibits the production of inflammatory cytokines in murine macrophages by blocking NF-κB and MAPK pathway].

Objective To investigate the anti-inflammatory activity and mechanism of peroxisome proliferator activated receptor gamma (PPARγ) ligand 4i. Methods Mouse macrophages RAW264.7 at the logarithmic phase were induced by 100 ng/mL lipopolysaccharide (LPS). The effect of 4i (10 µmol/L) on the production of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) was investigated by ELISA. The effect of 4i on the protein levels of nuclear factor κB (NF-κBp65), IκBα, JNK, ERK1/2, p38MAPK were detected by Western blot analysis. Furthermore, the role of PPARγ in the regulation of inflammatory-related NF-κB and MAPK signaling pathways was analyzed using GW9662 (5 µmol/L) which was a highly irreversible PPARγ antagonist. And the docking analysis was carried out using SYBYL 8.1 software to investigate the binding interaction of 4i with PPARγ ligand. Results The 4i significantly decreased the production of TNF-α and IL-6 in a time-dependent manner. It also inhibited the phosphorylation of NF-κBp65, IκBα, JNK, ERK1/2 and p38MAPK in varying degrees, and the suppressive effect of 4i could be reversed by GW9662. The 4i exhibited a strong binding ability with PPARγ. Conclusion The anti-inflammation effect of 4i was due to the interaction with PPARγ, thereby suppressing the activation of NF-κB and MAPK cascades and resulting in the decreased levels of TNF-α and IL-6.

miR-140-5p-mediated regulation of the proliferation and differentiation of human dental pulp stem cells occurs through the lipopolysaccharide/toll-like receptor 4 signaling pathway.

Human dental pulp stem cells (DPSCs) are oral mesenchymal stem cells with potential to differentiate into various cell types. Recent studies of DPSCs have focused on microRNAs (miRNAs), a class of small noncoding RNAs that play crucial roles in regulating DPSC phenotypes. In the current study, the expression of miR-140-5p was significantly decreased during lipopolysaccharide (LPS)-mediated differentiation of DPSCs in vitro. Overexpression of miR-140-5p enhanced proliferation of DPSCs and inhibited DPSC differentiation, whereas suppression of miR-140-5p produced the opposite effect. Moreover, the expression of toll-like receptor 4 (TLR-4), a critical regulator of DPSCs, was negatively correlated with the levels of miR-140-5p. A luciferase reporter analysis confirmed that miR-140-5p could regulate TLR-4 by directly binding to the 3'-untranslated region (3'-UTR) of the TLR4 mRNA. Additionally, we suppressed TLR-4 expression by treating cells with a TLR-4 inhibitor, CLI-095, and demonstrated that the effect of the miR-140-5p inhibitor on DPSC proliferation and differentiation could be partially reversed by blocking TLR-4. Taken together, our data suggest that miR-140-5p is a novel miRNA that regulates DPSC proliferation and differentiation.