Influence of Helicobacter pylori culture supernatant on the ecological balance of a dual-species oral biofilm

<div><p>Abstract Dental caries is a chronic progressive disease occurring in the tooth hard tissue due to multiple factors, in which bacteria are the initial cause. Both Streptococcus mutans and Streptococcus sanguinis are main members of oral biofilm. Helicobacter pylori may also be detected in dental plaque, playing an important role in the development of dental caries. Objective The aim of this study was to investigate the effect of H. pylori culture supernatant on S. mutans and S. sanguinis dual-species biofilm and to evaluate its potential ability on affecting dental health. Material and methods The effect of H. pylori supernatant on single-species and dual-species biofilm was measured by colony forming units counting and fluorescence in situ hybridization (FISH) assay, respectively. The effect of H. pylori supernatant on S. mutans and S. sanguinis extracellular polysaccharides (EPS) production was measured by both confocal laser scanning microscopy observation and anthrone-sulfuric acid method. The effect of H. pylori supernatant on S. mutans gene expression was measured by quantitative real-time PCR (qRT-PCR) assays. Results H. pylori supernatant could inhibit both S. mutans and S. sanguinis biofilm formation and EPS production. S. sanguinis inhibition rate was significantly higher than that of S. mutans. Finally, S. mutans bacteriocin and acidogenicity related genes expression were affected by H. pylori culture supernatant. Conclusion Our results showed that H. pylori could destroy the balance between S. mutans and S. sanguinis in oral biofilm, creating an advantageous environment for S. mutans, which became the dominant bacteria, promoting the formation and development of dental caries.</p></div

DS_10.1177_0363546518756087 – Supplemental material for Anterior Cruciate Ligament Transection–Induced Cellular and Extracellular Events in Menisci: Implications for Osteoarthritis

<p>Supplemental material, DS_10.1177_0363546518756087 for Anterior Cruciate Ligament Transection–Induced Cellular and Extracellular Events in Menisci: Implications for Osteoarthritis by Jing Xie, Demao Zhang, Yunfeng Lin, Quan Yuan and Xuedong Zhou in The American Journal of Sports Medicine</p

Additional file 1 of MicroRNA-191 regulates oral squamous cell carcinoma cells growth by targeting PLCD1 via the Wnt/β-catenin signaling pathway

Additional file 1

Wnt5a promotes the adhesion but inhibits the migration of hDPCs.

<p>A: A total of 25,000 cells were seeded for the indicated times and nonadherent cells were rinsed off. Adherent cells were stained with crystal violet and were analyzed spectrophotometrically. The number of adherent cells is shown as mean±SD from three independent experiments, with the number of DMEM-treated cells set as 100%. B: Confluent monolayers of hDPCs were scratched with a pipette tip and cultured in different medium containing 5% FBS for 12 hr. The relative migration distance of the wound edge was shown as mean±SD of three independent experiments, with the migration distance of DMEM-treated cells set as 100%. Bars, 100 µm. C: HDPCs were plated on glass coverslips coated with type I collagen and cultured with different medium for the indicated times. For FACs immunostaining, anti-vinculin antibody was used, and for F-actin staining, rhodamine-phalloidin was used, arrowheads mark FACs. Bars,10 µm. D: Confluent hDPCs were incubated with Wnt5a for the indicated times and Western analyses were used to detect the expression of vinculin, phospho-paxillin, phospho-MLC, with GAPDH, total-paxillin and total-MLC as loading control. The relative protein expression at 0 min is designated 1.0. *<i>p</i> < 0.05, n=3.</p

The role of the JNK pathway in Wnt5a-dependent adhesion, migration and formation of FACs.

<p>A: HDPCs were pretreated with 10 µM or 30 µM SP600125 for 30 min and the levels of phospho-JNK and total JNK were measured by Western blot analysis. The relative protein expression without SP600125 treatment is designated 1.0. B ,C: Cell adhesion and wound healing assays were performed as in Figure 1, but the cells were hDPCs which were pretreated with 30 µm SP600125 for 30 min, and the observed time in the wound healing assay was 16 hr. Bars, 100 µm. D: Vinculin immunostaining and phalloidin staining were performed at 15min as in Figure 1C, but the hDPCs were pretreated with 30µM SP600125 for 30 min before being seeded onto glass slides. Arrowheads mark FACs. The number of FACs and the relative fluorescence were analyzed as in Figure 1C. Bars,10 µm. E: Pretreatment with 30 µM SP600125 for 30 min, hDPCs were incubated with Wnt5a CM for the indicated times, the cell lysates were collected and immunoblotted with antibodies to phospho-MLC, phospho-paxillin, total-paxillin, total-MLC and vinculin. The promotion of phospho-paxillin by Wnt5a CM was delayed until after 60 min, but no changes were seen in the expression of phospho-MLC. *<i>p</i> < 0.05, n = 3.</p

<i>Sirt6</i> deletion does not affect the development of the tooth germs.

<p>(A) Micro-Computed Tomography testing revealed that the KO group differed from the WT group in several ways. The size of the tooth and mesial and distal roots were reduced in the KO group, and the diameter of the apical foramen of their distal root canal was wider. (B) H&E staining confirmed that there was no significant difference between the two groups with respect to <i>in utero</i> tooth germ morphogenesis. However, at or after DPN14, the first mandibular molar of the <i>Sirt6</i> KO group suffered an obvious growth retardation and developmental delay when compared to the WT group (E13.5-2W) (n = 6). Bars, as shown at the corner of the images.</p

Wnt5a has no effect on β-catenin expression or translocation in hDPCs.

<p>A: Western analyses of β-catenin in cytosolic or nuclear fractions of cells cultured in Wnt5a CM for indicated times. Cytosolic and nuclear signals were normalized to GAPDH and H3, respectively. The relative expression of β-catenin at 0min is designated 1.0. B: Immunofluorescence microscopy of hDPCs following culture with rhWnt5a or Wnt5a CM for 1 hr. β-catenin signal is in green. Bars,30 µm. C: Wnt5a up-regulates the expression of GTP-RhoA and phospho-JNK. RhoA activity stimulated by Wnt5a was detected by GST-Pull down assay at the indicated times, the expression of total RhoA, phospho-JNK, and total-JNK was detected by Western blot analysis. The relative protein expression at 0min is designated 1.0. *<i>p</i> < 0.05, n=3.</p

Wnt5a activated JNK signaling is dependent and independent of RhoA signaling.

<p>A: Lysates from hDPCs were obtained following transfection with adenoviral vectors encoding GFP, RhoA WT, RhoA T19N and RhoA Q63L for 48 hr and the levels of phospho-JNK and total-JNK were measured by Western blot analysis. The normalized amount of phospho-JNK in GFP adenovirus infected hDPCs is designated 1.0. B: HDPCs infected with adenoviruses encoding the RhoA mutant for 48 hr were cultured with Wnt5a CM and the cell lysates were obtained at the indicated times, with the level of phospho-JNK and total-JNK measured by Western analyses. The relative amount of phospho-JNK is normalized to the total amount, at 0min, is designated 1.0. *<i>p</i> < 0.05, n=3.</p

Additional file 4: of Cell cycle control, DNA damage repair, and apoptosis-related pathways control pre-ameloblasts differentiation during tooth development

Scatterplots between module membership measure (x-axis) and gene significance of GO terms in the brown module (A-E) and blue module (F-J). The grey dashed line in the plot is the threshold for choosing significantly expressed genes, and the threshold value is –log10(0.05). (TIFF 1712 kb

<i>Sirt6 r</i>egulates the mRNA levels of the osteogenic/ adipogenic -related transcription factor in the differentiation of DMCs.

<p>qPCR revealed that the <i>Sirt6</i> KO group experienced a substantial down-regulation in the mRNA levels of the osteogenic-related transcription factor, whereas the levels of the adipogenic-related transcription factor were increased significantly. The gene expression level was normalized to GAPDH (n = 3 to 6), *<i>p</i><0.05; **<i>p</i><0.01versus CO; CO = control.</p