Infection of RANKL-primed BMM with <i>P. gingivalis</i> induces osteoclastogenesis in the absence of TNF-α.

<p>(A) Infection of RANKL-primed BMM with <i>P. gingivalis</i> induces osteoclastogenesis. Representative photographs are shown. (B) Effect of neutralizing antibody against mouse TNF-α on osteoclastogenesis in RANKL-primed BMM induced by TNF-α or live <i>P. gingivalis</i>. BMM were stimulated with RANKL (50 ng/ml) for 22 h and then re-stimulated by TNF-α or live <i>P. gingivalis</i> (m.o.i.  = 10) in the presence or absence of neutralizing antibody against mouse TNF-α or control IgG. At the end of culture, the culture was stained for TRAP, and TRAP-positive MNCs were counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, compared with control IgG1.</p

TLR4 is not involved in osteoclastogenesis in RANKL-primed RAW-D cells induced by infection with <i>P. gingivalis</i>.

<p>Effect of <i>E.coli</i> LPS or Pam3CSK4 (A) or <i>P. gingivalis</i> LPS (B) on osteoclastogenesis in RANKL-primed RAW-D cells. (C) Effect of heat treatment of <i>P. gingivalis</i> on osteoclastogenesis in RANKL-primed RAW-D cells. (D) Effect of polymyxin B on osteoclast formation in RANKL-primed RAW-D cells induced by <i>E. coli</i> LPS, Pam3CSK4, live <i>P. gingivalis</i>, or <i>P. gingivalis</i> LPS. RAW-D cells were primed with RANKL (50 ng/ml) for 22 h and then treated with <i>E. coli</i> LPS (100 ng/ml), Pam3CSK4 (100 ng/ml), <i>P. gingivalis</i> LPS (10 µg/ml) or live <i>P. gingivalis</i> (m.o.i.  = 10) in the presence of various concentrations of polymyxin B. After 48 h, the culture was stained for TRAP, and the number of TRAP-positive MNCs was counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01 compared to untreated controls (A, B, and C) or controls without polymyxin B (D).</p

Infection of RANKL-primed RAW-D macrophages with <i>P. gingivalis</i> induces osteoclastogenesis.

<p>(A) (B) <i>P. gingivalis</i> infection of RANKL-primed RAW-D cells induces the formation of TRAP-positive MNCs. (C) <i>P. gingivalis</i> infection of RANKL-primed RAW-D cells induces mRNA expression of the osteoclast-specific gene, cathepsin K. Total RNA was isolated, and cathepsin K expression was assessed by real-time PCR. Expression levels were normalized to GAPDH. Effect of pretreatment (D), or OPG (E) on osteoclastogenesis induced by infection with <i>P. gingivalis</i>. RAW-D cells were primed with RANKL (50 ng/ml) or TNF-α (10 ng/ml) for 22 h, then infected with <i>P. gingivalis</i>, and cultured for 24–48 h. After 24 h, RNA was extracted and analyzed for gene expression. After 48 h, the culture was stained for TRAP, and TRAP-positive MNCs were counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, *P<0.05 compared to uninfected control (B, C) or untreated control (D).</p

Expression of osteoclast signaling proteins in osteoclastogenesis in RANKL-primed RAW-D cells induced by infection.

<p>RAW-D cells were stimulated with or without RANKL (50 ng/ml) or <i>P. gingivalis</i> for 22 h. RANKL-primed RAW-D cells were then retreated with or without RANKL (50 ng/ml) or <i>P. gingivalis</i> for 24 h. Total RNA was prepared, cDNA was synthesized, and real-time PCR analysis was performed using NFATc1 (A), c-fos (B), or IFNβ (C) Taqman probes. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, *P<0.05 compared with unprimed control or RANKL-primed control.</p

RANKL but not TNF-α promotes osteoclastogenesis in RANKL-primed RAW-D cells in the presence of <i>P. gingivalis</i>.

<p>RAW-D cells were stimulated with RANKL (50 ng/ml) for 22 h and then re-stimulated with RANKL or TNF-α in the absence (A) or presence (B) of live <i>P. gingivalis.</i> The culture was stained for TRAP activity after 48 h of retreatment, and TRAP-positive MNCs were counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, compared to cultures without RANKL or TNF-α. (C) Expression of mRNAs for p55 and p75 TNF receptors, TLR2, and TLR4 in RAW-D cells treated with or without <i>P. gingivalis</i> for 22 h, or RANKL-primed RAW-D cells retreated with or without RANKL or <i>P. gingivalis</i> for 24 h. Total RNA was isolated, and mRNA expression was assessed by semi-quantitative RT-PCR using specific primers as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038500#s4" target="_blank">Materials and Methods</a></i>.</p

<i>P. gingivalis</i> induces osteoclastogenesis in RANKL-primed RAW-D cells in the absence of TNF-α.

<p>Analysis of TNF-α mRNA expression (A) or production of TNF-α protein (B) by <i>P. gingivalis</i> infected RANKL-primed RAW-D cells and unprimed cells. (C) Effect of neutralizing antibody against mouse TNF-α on osteoclast formation in RANKL-primed RAW-D cells induced by TNF-α or live <i>P. gingivalis</i>. RAW-D cells were primed with RANKL (50 ng/ml) for 22 h and then retreated with TNF-α or live <i>P. gingivalis</i> in the presence or absence of neutralizing antibody against mouse TNF-α or control IgG. After 24 h, RNA was extracted, and TNF-α mRNA expression was assessed by real-time PCR. After 48 h, cell supernatants were collected and analyzed for TNF-α by ELISA. After 48 h, the culture was stained for TRAP, and the number of TRAP-positive MNCs was counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, *P<0.05 compared with unprimed infected RAW-D or RANKL-primed uninfected control (A), unprimed control (B), or control IgG1 (C).</p

Possible role of TNF-α in osteoclastogenesis in the presence or absence of <i>P. gingivalis</i>.

<p>Macrophages respond to infection with <i>P. gingivalis</i> by producing TNF-α, which stimulates osteoclastogenesis in osteoclast precursor cells in the absence of <i>P. gingivalis</i> (A). However, osteoclast precursor cells primed with RANKL do not produce TNF-α and respond differentially to various stimuli. (B) Cells that are not stimulated do not differentiate into osteoclasts. (C) Cells that are continuously re-stimulated with RANKL differentiate into osteoclasts in an NFATc1- and NF-κB-dependent manner in the presence of <i>P. gingivalis</i>. (D) Cells that are infected with <i>P. gingivalis</i> differentiate into osteoclasts in an NFATc1-dependent and NF-κB-independent manner. TNF-α does not stimulate osteoclastogenesis in osteoclast precursor cells in the presence of <i>P. gingivalis</i>, whereas RANKL stimulates osteoclastogenesis in the presence or absence of <i>P. gingivalis</i>.</p

Photoinduced Deformation of Rigid Azobenzene-Containing Polymer Networks

Photoresponsive poly­(amide acid) (PAA) gels containing multiple azobenzene units in a rigid aromatic backbone were synthesized. A centimeter-long cantilever made up of the photoresponsive PAA gel exhibited reversible bending motions upon blue (442 nm) and visible light (>490 nm) irradiation. The network structure in the PAA gels during alternating photoirradiation of blue and visible light was characterized using <i>in situ</i> scanning microscopic dynamic light scattering (SMILS), which revealed reversible mesh-size changes synchronized with the photoisomerization of azobenzene moieties. The photomechanical responses of the PAA gel were likely due to photoinduced contracting and stretching motions of the polymer backbone. A numerical calculation of photon absorptions revealed that photoisomerization in a very thin layer of the surface (∼40 μm) generated large macroscopic motion and large strain in the gel cantilever. The photoresponsive capability is, however, reduced or eliminated when the PAA gels are transformed to the corresponding polyimide (PI) gels, due to the large shrinkage caused by poor solubility of the backbone in the polyimide state

Image_1_Starch Synthase IIa-Deficient Mutant Rice Line Produces Endosperm Starch With Lower Gelatinization Temperature Than Japonica Rice Cultivars.TIF

<p>The gelatinization temperature of endosperm starch in most japonica rice cultivars is significantly lower than that in most indica rice cultivars. This is because three single nucleotide polymorphisms in the Starch synthase (SS) IIa gene in japonica rice cultivars (SSIIa^J) significantly reduce SSIIa activity, resulting in an increase in amylopectin short chains with degree of polymerization (DP) ≤ 12 compared to indica rice cultivars (SSIIa^I). SSIIa forms a trimeric complex with SSI and starch branching enzyme (BE) IIb in maize and japonica rice, which is likely important for the biosynthesis of short and intermediate amylopectin chains (DP ≤ 24) within the amylopectin cluster. It was unknown whether the complete absence of SSIIa further increases amylopectin short chains and reduces gelatinization temperature and/or forms altered protein complexes due to the lack of a suitable mutant. Here, we identify the SSIIa-deficient mutant rice line EM204 (ss2a) from a screen of ca. 1,500 plants of the rice cultivar Kinmaze (japonica) that were subjected to N-methyl-N-nitrosourea mutagenesis. The SSIIa gene in EM204 was mutated at the boundary between intron 5 and exon 6, which generated a guanine to adenine mutation and resulted in deletion of exon 6 in the mRNA transcript. SSIIa activity and SSIIa protein in developing endosperm of EM204 were not detected by native-PAGE/SS activity staining and native-PAGE/immunoblotting, respectively. SSIIa protein was completely absent in mature seeds. Gel filtration chromatography of soluble protein extracted from developing seeds showed that the SSI elution pattern in EM204 was altered and more SSI was eluted around 300 kDa, which corresponds with the molecular weight of trimeric complexes in wild type. The apparent amylose content of EM204 rice grains was higher than that in its parent Kinmaze. EM204 also had higher content of amylopectin short chains (DP ≤ 12) than Kinmaze, which reduced the gelatinization temperature of EM204 starch by 5.6°C compared to Kinmaze. These results indicate that EM204 starch will be suitable for making foods and food additives that easily gelatinize and slowly retrograde.</p

Ultrasonography predicts achievement of Boolean remission after DAS28-based clinical remission of rheumatoid arthritis

<p><i>Objectives.</i> To determine whether ultrasonography (US) predicts Boolean remission in rheumatoid arthritis (RA) patients who had achieved disease activity score in 28 joints (DAS28)-based remission criteria.</p> <p><i>Methods.</i> Thirty-one RA patients in DAS28-based clinical remission were recruited. US semiquantitatively determined Gray scale (GS) and power Doppler (PD) signal scores in the bilateral wrists and all metacarpophalangeals and proximal interphalangeals. Total GS score and total PD score were calculated as the sum of individual scores for each joint.</p> <p><i>Results.</i> Among 22 RA patients, who maintained DAS28 remission for 2 years, 16 met Boolean remission criteria at the end of study. Both total GS and total PD scores at baseline were significantly lower in Boolean remission group than non-remission group. There was no significant difference in other baseline parameters, including duration of disease, duration of remission, mTSS, and disease activity composite parameters between the two groups. Among the factors for Boolean remission criteria at 2 years, patient global assessment score was associated with total GS score at the entry, while swollen joint count was related to total PD score.</p> <p><i>Conclusions.</i> Null or low grade of GS and PD findings in US are associated with achieving Boolean remission. Thus, US is essential for assessment and prediction of “deeper remission” of RA.</p