Numerical analysis and measurement of glass flow in a small melting furnace

Control of glass flow in a glass tank is a key technology in the glass melting process. The flow and temperature distributions of the glass melt greatly affect the quality of glass products. However, these phenomena have not been well understood due to the difficulty involved in the measurement as a result of the high temperature of the glass melt. Α small melting furnace was developed that was heated by electrodes. The glass flow was measured and analyzed by 3-D computer simulation. The numerical results show good agreement with the experimentally measured values. It is shown that it is possible to control the glass convection using a variety of the electric boosting conditions, the heat loss through walls and the charged glass batch. The quality of glass melt was evaluated by analyzing the temperature histories of virtual particles the furnace was charged with. It is found that the temperature of the particles is high and stable near the throat, as shown by the experimental data

Role of hepcidin upregulation and proteolytic cleavage of ferroportin 1 in hepatitis C virus-induced iron accumulation.

Hepatitis C virus (HCV) is a pathogen characterized not only by its persistent infection leading to the development of cirrhosis and hepatocellular carcinoma (HCC), but also by metabolic disorders such as lipid and iron dysregulation. Elevated iron load is commonly observed in the livers of patients with chronic hepatitis C, and hepatic iron overload is a highly profibrogenic and carcinogenic factor that increases the risk of HCC. However, the underlying mechanisms of elevated iron accumulation in HCV-infected livers remain to be fully elucidated. Here, we observed iron accumulation in cells and liver tissues under HCV infection and in mice expressing viral proteins from recombinant adenoviruses. We established two molecular mechanisms that contribute to increased iron load in cells caused by HCV infection. One is the transcriptional induction of hepcidin, the key hormone for modulating iron homeostasis. The transcription factor cAMP-responsive element-binding protein hepatocyte specific (CREBH), which was activated by HCV infection, not only directly recognizes the hepcidin promoter but also induces bone morphogenetic protein 6 (BMP6) expression, resulting in an activated BMP-SMAD pathway that enhances hepcidin promoter activity. The other is post-translational regulation of the iron-exporting membrane protein ferroportin 1 (FPN1), which is cleaved between residues Cys284 and Ala285 in the intracytoplasmic loop region of the central portion mediated by HCV NS3-4A serine protease. We propose that host transcriptional activation triggered by endoplasmic reticulum stress and FPN1 cleavage by viral protease work in concert to impair iron efflux, leading to iron accumulation in HCV-infected cells

Involvement of the 3’ Untranslated Region in Encapsidation of the Hepatitis C Virus

<div><p>Although information regarding morphogenesis of the hepatitis C virus (HCV) is accumulating, the mechanism(s) by which the HCV genome encapsidated remains unknown. In the present study, in cell cultures producing HCV, the molecular ratios of 3’ end- to 5’ end-regions of the viral RNA population in the culture medium were markedly higher than those in the cells, and the ratio was highest in the virion-rich fraction. The interaction of the 3’ untranslated region (UTR) with Core <i>in vitro</i> was stronger than that of the interaction of other stable RNA structure elements across the HCV genome. A foreign gene flanked by the 3’ UTR was encapsidated by supplying both viral NS3-NS5B proteins and Core-NS2 in <i>trans</i>. Mutations within the conserved stem-loops of the 3’ UTR were observed to dramatically diminish packaging efficiency, suggesting that the conserved apical motifs of the 3´ X region are important for HCV genome packaging. This study provides evidence of selective packaging of the HCV genome into viral particles and identified that the 3’ UTR acts as a <i>cis</i>-acting element for encapsidation.</p></div

(A) Subcellular localization of NS5A (red), counter stained with LD (green in left and middle panels) or with Core (green in right panels).

<p>Huh7.5.1 cells were co-transfected with pCAG/NS3-5B, pRluc (R) or pRluc-3’UTR (R3), together with pCAG/C-NS2 (C-NS2) (middle and right panels) or an empty vector (left panel), and were immunostained for NS5A, Core and LD at 48 hr post-transfection. Scale bars represent 20 μm. Co-localization between NS5A and LD (<b>B</b>) or NS5A and Core (<b>C</b>) was assessed by Pearson’s correlation coefficient (PCC) and intensity correlation quotient (ICQ) analyses. For each group, co-localization was analyzed in 30 cells. N: pCAG/NS3-5B, R: pRluc, encoding Rluc; R3: pRluc-3’UTR, encoding Rluc followed with 3’ UTR of JFH-1. Data were present as mean ± SEM, n = 30. * <i>P</i><0.05, ** <i>P</i><0.01, Student’s <i>t</i> test.</p

Characteristics of HCV RNAs in infected cells and culture supernatant.

<p>(<b>A</b>) Normalized 3’:5’-end ratios of HCV RNA from cells (whole cell), supernatants (whole sup) and fractions with the highest infectivity (Top Inf. fraction) of cultures infected with HCVcc JFH-1 or J6/JFH-1. The ratio values calculated from NS5B (3’ end) and 5’ UTR (5’ end) qRT-PCR were normalized by the reference ratio (0.459; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005441#ppat.1005441.s002" target="_blank">S1D Fig</a>). The reference ratio was arbitrarily set to 1 (Rr) and the normalized 3’:5’-end ratios were shown. (<b>B</b>) Distribution of HCV RNA (5’ end, 3’ end) in fractions from culture supernatants and lysates of cells infected with HCVcc JFH-1, and the infectivity of each fraction. The y-axis indicates number of HCV RNA copies/ml (left) and infectivity in terms of viral RNA copies per μg of total RNA (right) from cells inoculated with equal aliquots of each fraction. Infectivity was measured by quantification of HCV RNA in the infected cells, 2 days post-infection. Blue, red and black lines represent quantity of 5’ end, 3’ end and infectivity, respectively. (<b>C</b>) Correlation of 3’:5’-end ratios with infectivity of the fractions obtained from supernatant and cells following HCVcc (JFH-1) infection as shown in (<b>B</b>). Correlations were estimated by way of linear regression and statistical significance was set at <i>P</i> = 0.01. (<b>D</b>) Comparison of 3’:5’-end ratios of high infectious (HI) and low infectious (LI) fractions. The median value of the infectivity of the fractions was used to split the fractions into HI and LI groups. Fractions derived from JFH-1, as shown in Fig 1B were used. Values are the mean ± SEM (n = 4 for whole cell and whole sup; n = 2 for Top Inf. fraction, n = 5 for supernatant HI and LI fraction groups and n = 7 for intracellular HI and LI fraction groups); ** <i>P</i><0.01, Student’s <i>t</i> test.</p

Effect of mutations in 3’ UTR on Core-binding and HCVtcp production.

<p>(<b>A</b>) Schematic representation of designed mutants in replication-defective subgenomic replicon SGR-JFH1/Gluc/GND. Colored shadows and dashed lines were used to depict the deletion boundaries. (<b>B</b>) Production of HCVtcp by using the mutant subgenomic replicons. HCVtcp production (upper) and expression of subgenome (Gluc) and Core in the producer cells (middle and lower) were shown. (<b>C</b>) Predicted structures of SLI and II of 3’ UTR are depicted along with the substitution mutations introduced. The resultant mutants were named STIM, STIIM, LIM, LIIM and LI&IIM. (<b>D</b>) The interactions of Core with 3’ UTR mutants shown in (<b>C</b>). (<b>E</b>) Production of HCVtcp by replication-defective subgenomic replicons with 3’ UTR mutations shown in (<b>C</b>). HCVtcp production (upper), expression of subgenome and Core in the producer cells (middle and lower) are shown. HCVtcp production was determined as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005441#ppat.1005441.g002" target="_blank">Fig 2</a>. Results shown represent the mean of three independent experiments ± SEM. HCV RNA copies are indicated as numbers per μg of total RNA for each assay, and Gluc activities are indicated as RLU per μl. VSL: variable region and poly (U/UC) tract, 3’X: the 3’ X tail, ×: GND mutants in NS5B.</p

Entire 3’ UTR is required for Core-binding to produce HCVtcp.

<p>(<b>A</b>) Schematic representation of HCV genome and the regions used for the Core-RNA interaction assay. Stem-loops and colored lines depict <i>in vitro</i> synthesized and folded RNA fragments used. (<b>B</b>) <i>In vitro</i> interactions of RNA fragments with Core determined by AlphaScreen. Results for comparison among structure clusters across HCV genome (left) and among 3’ UTR and the fragments within the region (right) were obtained from two independent assays. (<b>C</b>) <i>Trans</i>-packaging of EmGFP RNA into HCV particles indicated by transduced RNA level in the inoculated cells (upper). Transduction of EmGFP RNA was determined at 12 hr post-inoculation. EmGFP RNA level in the co-transfected producer cells (lower) is shown. N: pCAG/NS3-5B, G: p/EmGFP, G3H: p/EmGFP-H3’UTR, encoding EmGFP followed with 3’ UTR of H77. G3J: p/EmGFP-J3’UTR, encoding EmGFP followed with 3’ UTR of JFH-1. cont; control with cells co-transfected with a pCAG-Neo empty vector, p/EmGFP-J3’UTR and pCAG/NS3-5B. (<b>D</b>) Entry of HCVtcp was blocked by anti-CD81 antibody (α-CD81), carried out as described in Fig 3C. Data were present as mean ± SEM, n = 4. (<b>E</b>) Comparison of <i>tran</i>-packaging of EmGFP RNAs, directed by 3’- or 5’ UTR. 5G3J: p/5’UTR-EmGFP-J3’UTR, encoding EmGFP flanked by 5’ UTR and 3’ UTR of JFH-1. 5G: p/5’UTR-EmGFP, addition of 5’ UTR at upstream of EmGFP. (<b>C, D, E</b>) Results shown represent the means of three independent experiments ± SEM. RNA copies are indicated as numbers per μg of total RNA.</p

<i>Trans</i>-packaging system based on a replication-defective subgenomic replicon.

<p>(<b>A</b>) Schematic representation of the HCV <i>trans</i>-packaging system. (<b>B</b>) Production of HCVtcp from replication-competent or replication-defective subgenomic replicon (WT or GND, respectively). Transfection with an empty vector pCAG-Neo and pHHSGR-JFH1/Gluc/GND was used to determine the background control (nc), unless described elsewhere. HCVtcp production was determined by quantification of the viral RNA in the transduced cells at 12 hr post-inoculation. The lower panel showed Gaussia luciferase (Gluc) activity released from producer cells. Core expression in producer cells was assessed by immunoblotting. (C) Blocking of HCVtcp entry by anti-CD81 antibody (left), and inoculation of Huh7-25 cells (right). Huh7.5.1 cells were pre-incubated with 20 μg/ml of anti-CD81 antibody (α-CD81) or mouse IgG (IgG) for 1 hr, followed by inoculation with GND HCVtcp. HCV RNA levels in the transduced cells (upper) and Gluc activity released from producer cells (lower) are shown. (<b>D</b>) Deletion of UTR impaired production of HCVtcp. Upper graph: Production of HCVtcp from GND and UTR deletion mutants. HCV RNA level in the transduced cells (upper graph) and producer cells (middle graph) were determined by qRT-PCR targeting NS5B region as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005441#ppat.1005441.s002" target="_blank">S1 Fig</a>. Northern blot analysis of HCV RNA in Huh7.5.1 cells transfected with GND, Δ5’ UTR or Δ3’ UTR constructs (lower graph), Huh 7.5.1 cells were transfected with the mutant constructs and subjected to RNA extraction 72 hr post-transfection. 10 μg of total RNA was loaded to formaldehyde denaturing agarose gel electrophoresis and followed by Northern hybridization; a DIG-labeled RNA probe targeting to NS5B was used. 28S rRNA was used to demonstrate equal loading. Comparable Core expression in the producer cells was determined by western blotting. (<b>B, C, D</b>) Results of HCV RNA in transduced and producer cells, and reporter activity in producer cells represent the means of three independent experiments ± SEM. HCV RNA copies are indicated as numbers per μg of total RNA for each assay, and Gluc activities are indicated as RLU per μl.</p