Binding of recombinant RFX1 to wild-type and G1613A mutant NRE.

<p>(<b>A</b>) Sequence alignment of the consensus binding sequence of RFX1 and the NREγ region. The consensus sequence of RFX1 was aligned with the sequence of the NREγ region (nt. 1605–1617). The underlined ‘G’ corresponds to the nt.1613 position in our study. (<b>B</b>) <i>In vitro</i> expression of RFX1 using radioactive ³⁵S methionine. 1–5 µg of RFX1-expression construct was used for each reaction. The <i>in vitro</i> synthesized RFX1 protein was revealed using 10% SDS-PAGE and the signals were detected by autoradiography for 2 days. RFX1 of apparent size of 130 kDa was successfully expressed. The smear observed may be due to the degradation of proteins of large size. No band was shown in the empty vector control. The 62 kDa protein in the control correspond to the luciferase protein expressed from a control plasmid provided by the kit. (<b>C</b>) Electrophoretic mobility shift assay of Wt and Mut radioactive labeled oligos with recombinant RFX1 protein. The complexes were analyzed by native polyacrylamide gel electrophoresis. Non-specific oligos and <i>in vitro</i> transcribed luciferase protein were used as negative control. The labeling is depicted as previously described.</p

Analysis of 803 NRE sequences of HBV genotype C.

a<p>The nucleotide 1613 is the only hot spot mutation within nt. 1611–1619. The HBV NRE sequences were obtained from National Center for Biotechnology Information (GenBank) database (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>).</p

The prevalence of G1613A mutation in HBV carriers and HCC patients.

<p>*The G1613A mutation was found significantly higher prevalent in HCC patients in subgenotype Cs.</p>#<p>R represents purines, A or G.</p>##<p>P value was determined using chi's square analysis in SPSS software. A <i>P</i>-value of .05 or less is regarded as statistically significant.</p

The effect of G1613A mutation on the HBV DNA level.

<p>The relative intracellular and extracellular HBV DNA levels were measured by quantitative real-time PCR (<b>A</b> and <b>B</b>) and Southern blot analysis (<b>C</b> and <b>D</b>) respectively after transfection with 1.3× HBV genomes into HuH7 cells. Except the indicated mutations, all the constructs have the same genetic background. Results in real-time PCR were normalized to the wild-type and shown in mean values ± S.D. from 3 independent experiments. The increases of extracellular HBV DNA level with statistical significance are indicated by asterisks. In Southern blot analysis, the amount of HBV DNA extracted from both intracellular and extracellular cores particles from each plate of transfected cells was normalized using the SEAP activity as an internal control. The relative signals were quantified by densitometer and shown below each graphs for comparison. GenA: Huh7 cells transfected with a replicative competent 1.5× HBV plasmid (Genotype A) as positive control. ND: not determined.</p

The relationship between the prevalence of G1613A mutation and viral load in HBV chronic carriers of subgenotype Cs.

<p>*The G1613A mutation is associated with higher serum viral load in female carriers.</p>#<p>P value was determined using chi's square analysis in SPSS software. A <i>P</i>-value of .05 or less is regarded as statistically significant.</p>##<p>Harzard Ratio is an estimation of relative risk.</p

Schematic diagrams of HBV genome and core promoter.

<p>(<b>A</b>) The genome organization of 1.3× HBV genome used in this study (nt. 980–2000). The numbering of the nucleotide start at the unique <i>EcoR</i>I site as ‘1’ indicated in the middle of the genome. The two ends contains repeated parts from nt. 980–1821 and nt. 1825–2000 (dark grey colour) including the polyadenylation signal located downstream of the core promoter. The whole genome was cloned into pUC18 vector. The genes in the genome are partially overlapping. ORF: open reading frame; pro: promoter; X: region coding for X protein; PreC/C: precore/core region coding the precore and core protein; PreS/S: region coding for large, middle and small surface proteins; P: region coding for viral polymerase. (<b>B</b>) The major elements in HBV core promoter. The core promoter overlaps with 5′ end of X ORF and enhancer II (ENII). The activity of the basal core promoter (BCP) is regulated by the upper regulatory region (URR), which is further divided into negative regulatory element (NRE) and the core upstream regulatory sequence (CURS).</p

Northern blot analysis of HBV RNA.

<p>HuH7 were transfected with 1.3× HBV genomes with indicated mutations. All the constructs have the same genetic background except the indicated mutations. Total RNA was extracted at 5-day posttransfection.10 µg of total RNA were loaded in each lane. X, C and S indicate the 3.9-kb X gene transcript, the C gene transcripts and the S gene transcripts, respectively. The relative signals of the total core RNA (C) were quantified by densitometer and showed below for comparison. The 18S rRNA showed at the bottom is the internal control of RNA loading. ND: not determined.</p

The effect of G1613A mutations on the levels of HBsAg and HBeAg.

<p>(<b>A</b>) Intracellular level of HBsAg, (<b>B</b>) extracellular level of HBsAg, (<b>C</b>) intracellular level of HBeAg and (<b>D</b>) extracellular level of HBeAg were measured after transfection with 1.3× HBV genomes with indicated mutations into HuH7 cells by ELISA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021856#s2" target="_blank">materials and methods</a>. All the constructs have the same genetic backgrounds except the indicated mutations. The mutations were introduced into the HBV genome by site directed mutagenesis. The levels of the antigens were normalized to the transfection efficiency measured as the co-transfected SEAP activities. The decrease with statistical significance in the HBeAg in the extracellular media is indicated by asterisk(s). Data were presented as normalized values in 4 independent experiments (mean ± S. D.).</p

Score plots of PLS-DA distinguishing between the microbial community data of controls and NASH patients.

<p>(A and B) OTU level, (C and D) genus level.</p

Firmicutes phylogeny and principal component analysis (PCA) plot based on Unifrac distances between the Firmicutes sequences in control and NASH subjects.

<p>(A) The Firmicutes phylogeny was reconstructed from the OTU representative sequences in the control and NASH samples, and their relative abundance was indicated by gradient color from red to blue. (B) PCA plot of controls and NASH patients. The percentage of variation explained by each principal component was indicated in the parenthesis.</p