Experimental Measurement of Ignition Delay Times of Thermally Cracked <i>n</i>‑Decane in a Shock Tube

The ignition characteristics of endothermic hydrocarbon fuels (with different pyrolysis degrees) were investigated in a shock tube using <i>n</i>-decane as model compound. Six component surrogates (CH₄/C₂H₄/C₂H₆/C₃H₆/C₃H₈/<i>n</i>-C₁₀H₂₂, marked as cracked <i>n</i>-decane) for thermally cracked <i>n</i>-decane were proposed based on the chemical compositions from the thermal stressing of <i>n</i>-decane on electrically heated tube under 5 MPa. Ignition delay times were measured behind reflected shock waves over a temperature range of 1296–1915 K, a pressure of 0.1–0.2 MPa, and equivalence ratios of 0.5–2.0. <i>n</i>-Decane showed a shorter ignition delay time than cracking gas at 0.1 MPa, demonstrating higher reactivity. For cracked <i>n</i>-decane, it was found that thermal cracking could improve the ignitability under certain conditions to a limited degree, i.e., at <i>T</i> > 1480 K for <i>x</i> = 37.97% and <i>x</i> = 17.61% and at <i>T</i> < 1480 K for <i>x</i> = 62.15% (<i>x</i> represents the conversion of thermal cracking of <i>n</i>-decane) in this work. Unimolecular decomposition reactions of <i>n</i>-decane producing active radicals and H atom would help chain initiation via H-abstraction reaction for unreacted fuels. This initial stage might accelerate ignition by activating cracking gas at these conditions. The empirical correlations for the ignition delay time of cracking gas and <i>n</i>-decane were also analyzed and compared with previous works. Two models were also used to simulate the experimental data of <i>n</i>-decane and cracking gas and showed good agreement with experimental results, and a combined model was utilized to predict results of cracked <i>n</i>-decane

DV1 infection of HUH-7 and shRIG-I cells.

<p>(A) Whole cell lysate from DV1-infected cells were subjected to Western blot analysis and probed for the antibodies indicated and visualized by enhanced chemiluminescence. The nitrocellulose membranes were then reprobed for β-actin (loading control). (B) Qualitative RT-PCR for MDA5 mRNA level in DV1-infected cells from different time points. Total RNA was isolated and subjected to RT-PCR analysis. GAPDH was used as a control for equal RNA templates. (C) Uninfected shRIG-I and HUH-7 cells were stimulated with synthetic polyI:C. Cell lysates were assessed after 24 h of stimulation by Western blot for MDA5. β-actin was used as a control for equal loading of cell lysates. (D) Real-time RT-PCR analysis of MDA5 expression in mock and DV1-infected cells. Total RNA was isolated, used for cDNA preparation and subjected to real-time RT-PCR analysis. Bar histograms show the average difference in gene expression between mock and DV1-infected cells based on at least two independent experiments. (E) siRNA silencing technique was used to silence RIG-I gene. In HUH-7 cells, and not in A549 cells, RIG-I silencing co-induced silencing of MDA5 gene. (F) A549 cells were transfected with siRNA for EGFP, RIG-I and/or MDA5. Whole cell lysate from mock and DV1-infected A549 cells were subjected to immunoblotting (IB) and RT-PCR analysis. Results show that DV1 infection is significantly higher in cell line deficient for both RIG-I and MDA5. (G) Whole cell lysate from HUH-7 and shRIG-I cells infected with either wild type or UV-treated DV1 virus were subjected to immunoblotting (IB) and RT-PCR analysis. DV1 was UV-inactivated by exposing the virus to a UV-lamp (wavelength, 254 nm) at a distance of 5 cm for 1 hour. Results show that UV-treated DV1 infection did not activate RIG-I and MDA5. (H) Specificity of monoclonal NS3 antibody. Myc-tagged DV1 NS1 and NS3 constructs were transfected into HUH-7 cells. Cell lysates were harvested 24 h later and used to test specificity of monoclonal NS3 antibody. DV1-infected HUH-7 cell lysate was used as a control. Anti-NS3 antibody was found to be specific for DV1 NS3 protein.</p

Over-expression of RIG-I and MDA5 in wild type HUH-7 cells.

<p>(A) RIG-I and MDA5 constructs were over-expressed in HUH-7 cells and lysates harvested 48 hours post infection. Immunoblotting for antibodies stated on the left shows a decrease in viral antigen in cells double transfected with both plasmids. Real-time RT-PCR analysis of gene expression in mock and RIG-I and/or MDA5 transfected cells. Total RNA was isolated, used for cDNA preparation and subjected to real-time RT-PCR analysis. Rows below figure show negative strand viral RNA. (B) IFN-β production was assayed using ELISA. Symbols indicate significantly different from vector transfected and infected cells (*P<0.05).</p

siRNA silencing of TLR3 in HUH7 and shRIG-I cell lines.

<p>siTLR3 was transfected in HUH7 and shRIG-I cells and total RNA was tested for DV1 negative strand RNA by semi-quantitative RT-PCR. Rows below figure shows quantitative analysis of DV1 negative strand RNA, TLR3 and IFN-β production in infected HUH7 and shRIG-I cells. Symbols indicate significantly different from vector transfected and infected cells (**P<0.01).</p

DV1 replication and propagation in HUH-7 and shRIG-I cells.

<p>(A) Flow cytometric analysis showed enhanced DV1 infection in shRIG-I cells as compared to HUH-7 cells. Cells were stained with anti-Dengue E or IBV S (an isotype control) antibodies and analyzed by flow cytometry. (B) Tissue Culture Infectious Dose 50 (TCID50) assay. Virus titers in 50% tissue culture infectious doses (TCID₅₀)/ml were determined according to Reed and Muench <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000926#pntd.0000926-Reed1" target="_blank">[45]</a>. Symbols indicate significantly different from infected HUH-7 (**P<0.01). (C) Qualitative RT-PCR detection of IFN-related gene expression. Cells grown in 6-well plates were infected with DV1 for up to 72 h. Total RNA was extracted, used for cDNA synthesis and subjected to RT-PCR analysis. GAPDH was used as a control for equal RNA templates. (D) Real-time RT-PCR analysis of gene expression in mock and DV1-infected cells. Total RNA was isolated, used for cDNA preparation and subjected to real-time RT-PCR analysis. Bar histograms show the average difference in gene expression between mock and DV1-infected cells based on at least two independent experiments. IFN-β production was assayed using ELISA (lower panel). Symbols indicate significantly different from infected HUH-7 cells (**P<0.01). (E) Total cellular protein was extracted and analyzed by immunoblot. IRF3 dimerization was assessed by native PAGE with anti-IRF3 antibody as a probe. Tubulin protein level was assessed by SDS PAGE to ensure equal loading of cell lysate. Arrows indicate IRF3 dimer and monomer. Row below figure shows ratio of dimer: monomer analysed by densitometry.</p

Endoplasmic reticulum (ER) stress-induced expression of XBP1 mRNA and calreticulin protein expression.

<p>(A, upper panel) XBP1 splicing in DV1-infected cells. Total RNA samples were prepared and RT-PCR analysis was performed. sXBP1, spliced form of XBP1; uXBP1, unspliced form of XBP1. (A, lower panel) Analysis of calreticulin expression in DV1-infected cells. Whole cell lysate from DV1-infected cells were subjected to Western blot analysis and probed for the calreticulin and visualized by enhanced chemiluminescence. The nitrocellulose membranes were then reprobed for β-actin (loading control). Note increase in calreticulin expression in DV1-infected shRIG-I cells. (B, upper panel) <i>In situ</i> DNA fragmentation analysis (TUNEL) of mock and DV1-infected cells was carried out by flow cytometry. (B, lower panel) Flow cytometric analysis of DNA content by PI staining: DV1-infected HUH-7 and shRIG-I cells were analyzed after 72 hpi. The PI uptake rate was determined by flow cytometry and analyzed using WinMDI 2.7 software. Percentage of cells in sub-G1 (M1 region), indicative of cell death, in each sample is shown. DV1-infected shRIG-I cells showed significant increase in sub-G1 content.</p