Chemical modification to suppress metal ions elution of dental orthodontic wire surface

<p>In this study, we investigated on suppression of metal ions elution on dental orthodontic wire. To suppress metal ion elution from stainless wire, two types of surface chemical modifications were carried out. The obtained wire were immersed into several aqueous solution then the eluted ions (Cr and Ni, which are well known to cause a metal allergy) were determined using coupled plasma mass spectrometry (ICP-MS). By both electropolymerization and self-assembly monolayer formation, the ion elution was effectively reduced. These results suggested that these surface modifications succeeded to suppress the metal ions.</p

<i>P. gingivalis</i> LPS-induced Wnt5a expression was inhibited by wedelolactone or dnIκBα.

<p>(A, B) THP-1 cells were pre-treated with the IKK inhibitor wedelolactone or the IKK inhibitor VII for 1 hr. The expression of Wnt5a mRNA was detected by real-time PCR. Relative expression levels of Wnt5a are presented. DMSO is a solvent control for both inhibitors. (C) THP-1 cells were transfected with a dominant-negative form of IκBα (dnIκBα) or the parent plasmid (pUSE) for 12 hrs. Fold induction of Wnt5a mRNA expression is shown. *p<0.05.</p

Characteristics of the Study Subjects.

*<p>Mann-Whitney U test for continuous data, and Fisher's exact test for binary data.</p>**<p>Measured at the sampling sites.</p

Wnt5a was specifically up-regulated in THP-1 cells by <i>P. gingivalis</i> LPS.

<p>(A) HGF-1 and THP-1 cells were stimulated with <i>A. actinomycetemcomitans</i> sonicated extract, <i>P. gingivalis</i> sonicated extract, <i>P. gingivalis</i> LPS, and TNF-α for 4 hrs, and the expression of Wnt5a mRNA was detected by RT-PCR. PCR products were electrophoresed on a 1.5% agarose gel and visualized by UV illumination. GAPDH served as the internal control. (B) THP-1 cells were stimulated with 1 µg/ml of <i>P. gingivalis</i> LPS for 0.5, 2, 4, 12, or 24 hrs, and the expression of Wnt5a mRNA was detected by real-time PCR. Relative expression levels of Wnt5a are shown. (C) THP-1 cells were stimulated with 0.01–10 µg/ml of <i>P. gingivalis</i> LPS (black bars) or <i>E. coli</i> LPS (gray bars) for 4 hrs, and then the expression of Wnt5a mRNA was detected by real-time PCR. Relative expression levels of Wnt5a are presented. (D) THP-1 cells were stimulated with <i>E. coli</i> 055:B5 LPS (middle and right upper panels) or <i>P. gingivalis</i> LPS (middle and right lower panels) for 30 min or 4 hrs, and then the expression of surface TLR2 and TLR4 protein was determined by flow cytometry. Left upper panel shows no-staining condition, and left lower panel shows un-stimulated condition with staining. (E, F, G, H) The expression of Wnt5a mRNA was detected by real-time PCR. Relative expression levels of Wnt5a mRNA are shown. (E) THP-1 cells were stimulated with 10⁴–10⁷ cells/ml of live <i>P. gingivalis</i> for 4 hrs. (F, G) Primary human gingival fibroblasts (HGF) and human monocytes were stimulated with 1 µg/ml of <i>P. gingivalis</i> LPS for 4 hrs. Monocytes were pretreated with the NF-κB inhibitor MG132 for 1 hr. Here we describe typical dates of three samples. (H) THP-1 cells were stimulated with <i>P. gingivalis</i> LPS for 4 hrs after being transfected with TLR2 siRNA, TLR4 siRNA or control siRNA for 72 hrs. *p<0.05.</p

Induction of Wnt5a expression is partly JAK/STAT dependent.

<p>(A–D) The expression of Wnt5a mRNA was detected by real-time PCR. Relative expression levels of Wnt5a are presented. (A, B) THP-1 cells were pre-treated with AG490 (A) or fludarabine (B) for 1 hr and then stimulated with <i>P. gingivalis</i> LPS for 4 hrs. DMSO is a solvent control for both inhibitors. (C) THP-1 cells were stimulated with <i>P. gingivalis</i> LPS for 4 hrs after transfection with STAT1 siRNA or control siRNA for 18 hrs. (D) THP-1 cells were pre-treated with STA21 for 1 hr and stimulated with <i>P. gingivalis</i> LPS for 4 hrs. *p<0.05.</p

Induction of Wnt5a expression is NF-κB dependent.

<p>(A) THP-1 cells were stimulated with <i>E. coli</i> LPS or <i>P. gingivalis</i> LPS for 30 mins or 4 hrs. Whole cell extracts were prepared and analyzed by Western blot by using antibodies against IκBα. β-actin served as the protein loading control. (B) THP-1 cells were stimulated with <i>P. gingivalis</i> LPS for 4 hrs after being transfected with NF-κB reporter plasmid for 12 hrs, and the transcription activity was assessed by luminometer. The activity is represented by the relative luciferase activity. (C) Nuclear extracts were prepared and EMSA was performed with γ³²P-labeled oligonucleotides representing the NF-κB consensus sequence as a probe. Anti-p65 antibody was used for supershift assays. The lower arrow shows the DNA-protein complex, and the upper arrow shows the supershifted band. *p<0.05.</p

The levels of Wnt5a mRNA were significantly up-regulated in chronic periodontitis tissues.

<p>Upper panel; Total RNA was extracted from periodontitis tissues, and the expression of Wnt5a mRNA was detected by RT-PCR. PCR products were electrophoresed on a 1.5% agarose gel and visualized by UV illumination. β-actin served as the internal control. Results are representative of five patients (right panel). Lower panel; The relative mRNA levels of Wnt5a. The horizontal line within each box represents the median expression level in each group.</p

TAS-116 effects on cell viability and RAS-RAF-MEK-ERK signaling in <i>RAS</i>-mutated MM cell lines.

<p>(A) NCI-H929, INA6, MM.1S, and RPMI-8226 MM cell lines were cultured with TAS-116 (0–5 μM) for 24, 48, or 72 h. In each case, cell viability was assessed with the MTT assay of triplicate cultures and expressed as the percentage of the untreated control. Data are the mean ± SD. (B) NCI-H929 and RPMI-8226 cells were treated with the indicated doses of TAS-116 for 24 h. Whole-cell lysates were subjected to western blotting using p-B-Raf, B-Raf, p-C-Raf, C-Raf, p-MEK1/2, MEK1/2, p-ERK, ERK, p-Akt (S473), Akt, PARP, and β-actin Abs. FL, full-length; CF, cleaved form.</p

Downregulation of RAS inhibits growth and enhances cytotoxicity of doxorubicin and bortezomib in <i>RAS</i>-mutated MM cell lines.

<p>(A) INA6 cells were transiently transfected with non-targeting or <i>NRAS</i> siRNA, and RPMI-8226 and RPMI-8226 DOX40 cells were transiently transfected with non-targeting or <i>KRAS</i> siRNA. The cell number and viability 48 h later were assessed with trypan blue exclusion. Whole-cell lysates were subjected to western blotting to confirm the downregulation of NRAS and KRAS expression using NRAS, KRAS, HRAS, and β-actin Abs. Data are the mean ± SD of triplicate wells. (B) INA6 and NCI-H929 cells were transiently transfected with non-targeting or <i>NRAS</i> siRNA, and RPMI-8226 and RPMI-8226 DOX40 cells were transiently transfected with non-targeting or <i>KRAS</i> siRNA. After 48 h, whole-cell lysates were subjected to western blotting using p27, cyclin D1, NRAS and β-actin Abs. (C) INA6 cells were transiently transfected with non-targeting or <i>NRAS</i> siRNA and then treated with or without bortezomib (5 nM) for 48 h. RPMI-8226 cells were transiently transfected with non-targeting or <i>KRAS</i> siRNA and then treated with or without doxorubicin (0.1 μM) for 48 h. In each case, cell viability was assessed with the MTT assay of triplicate cultures and expressed as the percentage of the untreated control. Data are the mean ± SD. (D) RPMI-8226 DOX40 cells were transiently transfected with non-targeting or <i>KRAS</i> siRNA and then treated with or without doxorubicin (1 μM) for 24 h. Cell viability was assessed with the MTT assay of triplicate cultures and expressed as the percentage of the untreated control. Data are the mean ± SD.</p

Combination of TAS-116 plus tipifarnib, dabrafenib, or AZD6244 blocks the growth stimulatory effect of the bone marrow microenvironment.

<p>(A) NCI-H929, INA6, MM.1S, and RPMI-8226 cells were treated with TAS-116 (1 μM) either alone or in combination with tipifarnib (NCI-H929: 2 μM, INA6: 0.5 μM, MM.1S: 2 μM, RPMI-8226: 2 μM), dabrafenib (NCI-H929: 10 μM, INA6: 2 μM, MM.1S: 10 μM, RPMI-8226: 10 μM), or AZD6244 (NCI-H929: 10 μM, INA6: 2 μM, MM.1S: 20 μM, RPMI-8226: 20 μM) for 48 h. Apoptotic cells were analyzed with flow cytometry using annexin V/PI staining. Each treatment was tested in triplicate wells, and apoptosis was assessed as the percentage of annexin V-positive cells. TAS, TAS-116; Tipi, tipifarnib; Dabra, dabrafenib; AZD, AZD6244. (B) MM.1S and NCI-H929 cells were cultured with TAS-116 (2 μM), AZD6244 (20 μM), or TAS-116 plus AZD6244 for 24 h in the presence or absence of BMSC supernatant. Whole-cell lysates were subjected to western blotting using PARP, caspase 3, and β-actin Abs. FL, full-length; CF, cleaved form; SN, supernatant. (C) MM.1S cells were cultured with TAS-116 (1 μM), AZD6244 (20 μM), or TAS-116 plus AZD6244; and NCI-H929 cells were cultured with TAS-116 (1 μM), AZD6244 (10 μM), or TAS-116 plus AZD6244 for 48 h in the presence or absence of BMSC supernatant. Apoptotic cells were analyzed with flow cytometry using annexin V/PI staining. Each treatment was tested in triplicate wells, and apoptosis was assessed as the percentage of annexin V-positive cells. TAS, TAS-116; AZD, AZD6244; SN, supernatant (*: <i>P</i> < 0.05; **: <i>P</i> < 0.01).</p