Effect of DMAT on the production of Ser-to-Ala and Ser-to-Asp substitution mutants of NS5A domain III of H77S.3 virus.

<p>(A) Possible Ser/Thr phospho-acceptor sites in the C-terminal region of domain III of the NS5A proteins of JFH1 and H77S virus. At the top of the panel, the H77S and JFH1 sequences are aligned: Ser residues found to be important for the NS5A-core interaction and assembly and release of infectious JFH1 virus by Masaki et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113938#pone.0113938-Masaki1" target="_blank">[35]</a> (red box), and Ser-457, identified by Tellinghuisen et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113938#pone.0113938-Tellinghuisen1" target="_blank">[11]</a> as a site of CKII phosphorylation (red arrow), are highlighted. Within the related H77S sequence, Ser-438 and Thr-442 are possible sites of CKII phosphorylation predicted by the NetPhos 1.0 server. Below are shown the C-terminal NS5A sequences of 4SA and 4SD substitution mutants. Potential Ser/Thr phospho-acceptor sites are shown in red, while Ala and Asp substitutions in the mutants are shown in bold-face type. (B) Following transfection of the RNA, cells were treated with the indicated concentration of DMAT for 48 hours. The media was then replaced with fresh medium (no drug), followed 24 hours later by harvesting of supernatant fluids for virus titration. Means ± S.E. were calculated from duplicate experiments. (C) Immunoblots for NS5A, NS2, NS3, and GAPDH from the cell lysates prepared 72 hours after transfection.</p

Enhanced H77S virus production by DMAT, a CKII inhibitor.

<p>(A) Following transfection of the JFH1 RNA, cells were treated with the indicated concentration of DMAT for 48 hours. The media was then replaced with fresh medium (no drug), followed 24 hours later by harvesting of supernatant fluids for virus titration. Means ± S.E. were calculated from duplicate experiments. (B) The effect of DMAT treatment on the production of H77S and H77S/J5Ad3 infectious particles. (C) Schematic diagram of the virus for this study. (D) Immunoblots for NS5A, NS2, NS3, and GAPDH from the cell lysates prepared 72 hours after transfection. (E) Cytotoxicity was tested by WST-1 assay. Means ± S.E. were calculated from triplicate experiments.</p

Effect of TBCA, another CKII inhibitor, on the production of H77S.3 and JFH1 virus.

<p>(A) Chemical structure of DMAT and TBCA. (B) Following transfection of the HCV RNA, cells were treated with the indicated concentration of TBCA for 48 hours. The media was then replaced with fresh medium (no drug), followed 24 hours later by harvesting of supernatant fluids for virus titration. Means ± S.E. were calculated from duplicate experiments. <i>P</i> values were determined from unpaired <i>t</i> tests.</p

Effect of DMAT on the production of 1a/2a intergenotypic HJ3-5 virus.

<p>(A) Following transfection of the HCV RNA, cells were treated with the indicated concentration of DMAT for 48 hours. The media was then replaced with fresh medium (no drug), followed 24 hours later by harvesting of supernatant fluids for virus titration. Means ± S.E. were calculated from duplicate experiments. (B) Immunoblots for NS5A, NS2, NS3, and GAPDH from the cell lysates prepared 72 hours after transfection.</p

Repeated BLI of the same mice following hydrodynamic injection.

<p>(A) Pseudocolor images of tumor growth in the liver following hydrodynamic injection of HrasG12V and shp53. Note the continuous increases in the signals from the abdominal regions of the same mice. (B) Average bioluminescence signals from the abdominal regions of the same mice at the indicated time points PHI.</p

Schematic illustration of the experimental procedure.

<p>The transposons were mixed with the plasmids encoding the <i>Sleeping Beauty</i> (SB) transposase and were then hydrodynamically delivered to the liver (see the Materials and Methods section). Once in cells, the SB transposase is expressed and binds to the IR/DRs of the transposons. The enzyme subsequently cleaves the transposons at the sites of IR/DRs and integrates them at a new location within the host genome, allowing the transgenes to be stably expressed. Mice are then subjected to repeated bioluminescence imaging over time.</p

Tumors in the livers of HrasG12V plus shp53 mice following hydrodynamic injection.

<p>(A) Gross morphology of livers harvested at 1, 2, 3, and 4 weeks PHI. Note that the tumors grew more rapidly in the right lobes. (B) H&E staining of left caudal lobes harvested at the indicated time points. Scale bar, 200 µm.</p

Assessment of the biological functions of HrasG12V, SmoM2, and shp53.

<p>(A) Activation of Ras signaling by HrasG12V was confirmed by increased phosphorylation of Akt, ERK, and MEK. (B) Increased activity of Gli transcription factors in response to expression of SmoM2 was detected using a Gli-driven luciferase reporter system. (C) UV-induced expression of p53 was suppressed by shp53 expression. Cells were transfected with either pT2/EGFP (a control) or pT2/shp53 prior to UV irradiation.</p

Tumors induced by transposon vectors encoding HrasG12V and shp53 without the <i>Sleeping Beauty</i> transposase.

<p>(A) A few large hyperplastic nodules were observed at 3 months PHI. (B) H&E staining of the tumors shown in (A). Scale bar, 200 µm.</p

Factors Affecting the Accuracy of Controlled Attenuation Parameter (CAP) in Assessing Hepatic Steatosis in Patients with Chronic Liver Disease

<div><p>Background & Aims</p><p>Controlled attenuation parameter (CAP) can measure hepatic steatosis. However, factors affecting its accuracy have not been described yet. This study investigated predictors of discordance between liver biopsy (LB) and CAP.</p><p>Methods</p><p>A total of 161 consecutive patients with chronic liver disease who underwent LB and CAP were enrolled prospectively. Histological steatosis was graded as S0 (<5%), S1 (5–33%), S2 (34–66%), and S3 (>66% of hepatocytes). Cutoff CAP values were calculated from our cohort (250, 301, and 325 dB/m for ≥S1, ≥S2, and S3). Discordance was defined as a discrepancy of at least two steatosis stages between LB and CAP.</p><p>Results</p><p>The median age (102 males and 59 females) was 49 years. Repartition of histological steatosis was as follows; S0 26.1% (n = 42), S1 49.7% (n = 80), S2 20.5% (n = 33), and S3 3.7% (n = 6). In multivariate linear regression analysis, CAP value was independently associated with steatosis grade along with body mass index (BMI) and interquartile range/median of CAP value (IQR/M_CAP) (all P<0.05). Discordance was identified in 13 (8.1%) patients. In multivariate analysis, histological S3 (odd ratio [OR], 9.573; 95% confidence interval [CI], 1.207–75.931; <i>P</i> = 0.033) and CAP value (OR, 1.020; 95% CI, 1.006–1.034; <i>P</i> = 0.006) were significantly associated with discordance, when adjusting for BMI, IQR/M_CAP, and necroinflammation, reflected by histological activity or ALT level.</p><p>Conclusions</p><p>Patients with high grade steatosis or high CAP values have a higher risk of discordance between LB and CAP. Further studies are needed to improve the accuracy of CAP interpretation, especially in patients with higher CAP values.</p></div