Assessment of Cre recombinase activity.

<p>Cre function was assessed by LacZ positive TE26 cells which contain a floxed terminator preventing <i>lacZ</i> expression (<b>A</b>). TE26 cells were infected for various times and MOIs and subsequently stained with X-gal for β-galactosidase (<b>B</b>) to determine the optimal infection conditions as indicated by percentage LacZ positive cells (<b>C</b>). TE26 cells were infected at an MOI of 50 for 3 hours before or after cell cycle synchronization and X-gal stained for β-galactosidase activity (<b>D</b>).</p

Protein injection into mouse embryonic stem cells (mESC).

<p>(<b>A</b>) mESC were infected with PAK-J strains at a MOI of 50 for 2.5 hours, lysed and assayed for injected ExoS-Flag by anti-Flag Western Blot. (<b>B</b>) mESC were infected with PAK-JΔ<i>STY</i>(pExoS-Flag) for 2.5 hours and subsequently fixed and immunostained with anti-Flag to illuminate translocated ExoS-Flag protein. (<b>C</b>) R26R-EYFP mESC cells contain a floxed terminator preventing downstream EYFP expression. (<b>D</b>) R26R-EYFP were infected with PAK-JΔ<i>STY</i>(pExoS54-Cre) at various MOIs for 2.5 hours. Cells were collected for FACS analysis 48 hours post infection. (<b>E</b>) EYFP-positive mESC were plated after infection to assess EYFP expression by confocal fluorescence microscopy.</p

Injection of indicated ExoS-Cre fusions.

<p>(<b>A</b>) Cre recombinase was fused in frame with various N-terminal portions of ExoS, with a nuclear localization sequence (NLS) in the fusion junction. (<b>B</b>) Detection of the five ExS54-Cre fusion proteins in PAK- JΔ<i>STY</i> harboring the corresponding fusion constructs. Bacterial cells were grown under type III inducing condition and cell lysates were subjected to Western blot using anti-Cre antibody. (<b>C</b>) TE26 cells were infected with PAK-JΔ<i>STY</i> containing the aforementioned constructs, lysed, and subject to Western Blot with anti-Cre antibody. N, no infection control; V, vector control, PAK-JΔ<i>STY</i>/pUCP20; P, T3SS mutant control, PAK-JΔ<i>popD</i>/pExoS54-Cre; Lanes1-5 are Cre fusions to ExoS17, ExoS54, ExoS96, ExoS234 and ExoS full length, respectively.</p

PCR primers used in this study.

<p>PCR primers used in this study.</p

Protein delivery by type III secretion system of <i>P. aeruginosa</i>.

<p>(<b>A</b>) Comparison of ExoS secretion by standard laboratory strains of PAO1, PAK and a hyper secreting strain PAK-J. Strains were grown under type III secretion inducing conditions and culture supernatants were subjected to Western blot analysis using anti-ExoS antibody which recognizes both ExoS and ExoT. (<b>B</b>) Immunohistochemistry of MEF cells following infection by PAK-J (60 min at MOI 20). Cells were stained with anti-ExoS antibody followed by FITC labeled secondary antibody; (i) visualization of ExoS; (ii) visualization of nuclei with Propidium Iodine stain; (iii) compilation of (i) & (ii). (<b>C</b>) Comparison of MEF viability after 3 hour infection with PAK-J or PAK-JΔ<i>STY</i> at indicated MOIs.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Transient Elastography-Based Liver Profiles in a Hospital-Based Pediatric Population in Japan

<div><p>Background & Aims</p><p>The utility of transient elastography (FibroScan) is well studied in adults but not in children. We sought to assess the feasibility of performing FibroScans and the characteristics of FibroScan-based liver profiles in Japanese obese and non-obese children.</p><p>Methods</p><p>FibroScan examinations were performed in pediatric patients (age, 1–18 yr) who visited Osaka City University Hospital. Liver steatosis measured by controlled attenuation parameter (CAP), and hepatic fibrosis evaluated as the liver stiffness measurement (LSM), were compared among obese subjects (BMI percentile ≥90%), non-obese healthy controls, and non-obese patients with liver disease.</p><p>Results</p><p>Among 214 children examined, FibroScans were performed successfully in 201 children (93.9%; median, 11.5 yr; range, 1.3–17.6 yr; 115 male). CAP values (mean±SD) were higher in the obese group (n = 52, 285±60 dB/m) compared with the liver disease (n = 40, 202±62, <i>P</i><0.001) and the control (n = 107, 179±41, <i>P</i><0.001) group. LSM values were significantly higher in the obese group (5.5±2.3 kPa) than in the control (3.9±0.9, <i>P</i><0.001), but there were no significant differences in LSM between the liver disease group (5.4±4.2) and either the obese or control group. LSM was highly correlated with CAP in the obese group (ρ = 0.511) but not in the control (ρ = 0.129) or liver disease (ρ = 0.170) groups.</p><p>Conclusions</p><p>Childhood obesity carries a high risk of hepatic steatosis associated with increased liver stiffness. FibroScan methodology provides simultaneous determination of CAP and LSM, is feasible in children of any age, and is a non-invasive and effective screening method for hepatic steatosis and liver fibrosis in Japanese obese children.</p></div

Correlation between LSM and CAP values.

<p>LSM was positively correlated with CAP in the obese group (Spearman’s ρ = 0.511) but not in the control (ρ = 0.129) or liver disease (ρ = 0.170) group.</p

Comparison of results from FibroScan, liver biopsy, and abdominal ultrasonography (AUS).

<p>a and b. Liver stiffness measurement (LSM) and controlled attenuation parameter (CAP) values from FibroScan evaluations were compared with histologic fibrosis stage and steatosis grade. Liver biopsy was performed in 8 pediatric patients, in which the underlying disease was simple obesity in 4 patients, type C hepatitis in 2 patients, type B hepatitis associated with obesity in 1 patient, and liver transplantation for treatment of congenital biliary atresia in 1 patient. Among the 5 obese patients, four patients were diagnosed with NASH, and the remaining patient was diagnosed with simple steatosis. a. Correlation between LSM value and histologic fibrosis stage. LSM was highly correlated with fibrosis stage (Spearman’s <i>ρ</i> = 0.920). b. Correlation between CAP value and histologic steatosis grade. CAP value was highly correlated with steatosis grade (<i>ρ</i> = 0.792). c. Correlation between CAP and fatty liver infiltration score calculated according to AUS findings. CAP was highly correlated with AUS fatty liver infiltration score (<i>ρ</i> = 0.713).</p

Age distribution of the patients examined by using FibroScan.

<p>A total of 214 children and adolescents (age, 1.3–17.6 years; 121 male) were examined by using FibroScan. A total of 201 patients (93.9%; median age, 11.5 years; range, 1.3–17.6 years) (black bars) were examined successfully; the evaluation was unsuccessful in the remaining 13 patients (6.1%; median age, 13.1 years; range, 2.0–17.2 years) (gray bars) due to excessive thickness of subcutaneous adipose tissue in 5 obese children (BMI percentile [mean ± 1 SD], 99.6 ± 2.2), poor cooperation in 4 young children (2.0, 3.7, 4.0, and 6.2 years), and no obvious reason in 4 non-obese and cooperative children.</p