Strains and Plasmids used in this study.

<p>*In this pHluorin-MotB-His₈ fusion construct, N-terminal 28 residues of MotB (Met1- Lys28) are attached to the N-terminus of pHluorin as described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019598#pone.0019598-Leake1" target="_blank">[9]</a>.</p

Pressure dependence of Poisson’s ratio of glassy Baltic amber studied by Brillouin scattering spectroscopy

Amber is a typical natural glass with very long aging time. Elastic properties of amber at high pressures have been studied using Brillouin scattering and a diamond anvil cell. Both longitudinal and transverse acoustic modes have been observed up to 12 GPa. The pressure dependences of longitudinal, shear, Young's, and bulk moduli, compressibility, and Poisson's ratio were determined. The longitudinal, shear, Young's, bulk moduli show the remarkable increase, and compressibility shows a marked decrease with increasing pressure. However, it is found that the pressure dependence of Poisson's ratio is very small. The mechanism of this small pressure dependence was discussed. The Cauchy type relation between longitudinal and shear moduli was examined. Its coefficients show the deviation from the Cauchy relation owing to the coexistence of different intermolecular interactions in amber

pH dependence of fluorescence excitation spectra and Ratio_410/470 of pHIuorin and pHluorin(M153R).

<p>(<b>A</b>) Fluorescence excitation spectra. The different colored lines refer to different pH values. red, pH 5.5; orange, pH 6.0; light green, pH 6.5; green, pH 7.0; cyan, pH 7.5; grey, pH 8.0; black, pH 8.5. (<b>B</b>) Ratio_410/470. The fluorescence excitation spectra of purified proteins were recorded on a fluorescence spectrophotometer. The measurements were done at 23°C.</p

Primers used for construction of <i>pHluorin-fliG</i> and <i>pHluorin-motB</i> strains.

<p>Primers used for construction of <i>pHluorin-fliG</i> and <i>pHluorin-motB</i> strains.</p

pH dependence of fluorescence excitation spectra and Ratio_410/470 of pHluorin(M153R)-FliG-His₆.

<p>(<b>A</b>) Fluorescence excitation spectra. The different colored lines refer to different pH values. red, pH 5.5; orange, pH 6.0; light green, pH 6.5; green, pH 7.0; cyan, pH 7.5; grey, pH 8.0; black, pH 8.5. (<b>B</b>) Ratio_410/470. The fluorescence excitation spectra of the purified protein were recorded on a fluorescence spectrophotometer. The measurements were done at 23°C.</p

Effect of the M153R mutation on Ratio_410/470.

<p>(<b>A</b>) Fluorescent intensities of SJW1103/pYC001 (pHluorin) and SJW1103/pYVM001 (pHluorin(M153R)) cells grown in T-broth at 30°C. Emission spectra with 395 nm excitation were measured by a fluorescence spectrophotometer. The measurements were done at 23°C. Inset: Immunoblotting, using polyclonal anti-GFP antibody, of whole cell proteins. (<b>B</b>) Fluorescent intensities of purified pHluorin and pHluorin(M153R).</p

Hypermethylation status and clinical outcome in patients with juvenile myelomonocytic leukemia (JMML).

<p>(A) Kaplan–Meier curves represent the probability of transplantation-free survival (TFS) in the 92 patients with JMML. TFS was defined as the probability of being alive and transplantation free. Both death and transplantation were considered events. The probability of 5-year TFS in the aberrant methylation score (AMS) 0 cohort (solid line) was significantly higher than that in the AMS 1–2 (long dashed line) and AMS 3–4 cohorts (dashed line), p < 0.001. (B) Kaplan–Meier curves represent the probability of overall survival (OS) in the 92 patients with JMML. Death was considered an event. The probability of OS in both the AMS 0 (solid line) and 1–2 cohorts (long dashed line) was significantly higher than that in the AMS 3–4 cohort (dashed line), p < 0.001.</p

Profile of genetic mutations and aberrant methylation.

<p>(A) Mutation status of RAS pathway genes and secondary genes (<i>SETBP1</i> and <i>JAK3</i>) identified as gene targets. Aberrant methylation scores (AMS) in a cohort of 92 patients with juvenile myelomonocytic leukemia are summarized. A rhombus denotes a patient with Noonan syndrome-associated myeloproliferative disorder. (B) Mutations in <i>SETBP1</i> and <i>JAK3</i> were associated with a higher AMS. The mean AMS of patients with <i>SETBP1</i> and/or <i>JAK3</i> mutations was higher than that of patients without secondary mutations (p = 0.03).</p

Summary of DNA methylation in candidate genes.

<p>(A) The dot plot represents the frequencies of methylated CpG sites for each candidate gene in the 92 patients with juvenile myelomonocytic leukemia. Aberrant hypermethylation was defined as >3 standard deviations above the mean methylation level of the healthy control population. The threshold values of each gene are shown as red broken lines. (B) Kaplan–Meier plots of the patient groups, defined by aberrant methylation of the indicated genes, are shown for <i>BMP4</i>, <i>CALCA</i>, <i>CDKN2A</i>, <i>CDKN2B</i>, <i>H19</i>, and <i>RARB</i>.</p

Patient characteristics.

<p>Patient characteristics.</p