The mean, standard deviation and coefficient of variation for all repetitions (N = 7) of the dynamic phantom.

<p>Lac and Pyr refer to the total volume under the spectral and temporal curves for each tracer; <i>k_PL</i> is the forward reaction rate (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071274#pone.0071274.e006" target="_blank">Equations 6</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071274#pone.0071274.e010" target="_blank">8</a>).</p

A schematic view of the dynamic chemical phantom structure.

<p>The injection and exhaust ports were fitted with catheters to facilitate rapid mixture of reagents at isocenter. A thin acrylic sheet was attached to the top to seal the fill cavity. This top could be removed to allow cleaning after injection. The phantom rested on a sled that allowed convenient removal and insertion of the phantom and included warm circulating water to maintain constant temperature.</p

Dynamic signal evolution across (N = 7) injections.

<p>The mean signal for lactate and pyruvate, normalized to peak carbon signal for each injection, are displayed with error bars that indicate the minimum and maximum values at each time over all injections. Total HP ¹³C was estimated by summing signal from HP ¹³C Lactate and HP ¹³C Pyruvate. The average linewidth for pyruvate and lactate peaks were 19±5 Hz and 17±5 Hz, respectively.</p

Spectroscopic images of the reaction carried out in a standard imaging phantom.

<p>Proton imaging (top left) shows the phantom structure in high resolution. Spectroscopic imaging data acquired using a radial EPSI sequence allows metabolite-specific visualization of tracer distribution (bottom row). Spectroscopic data can be intrinsically registered to high-resolution proton images (top center and right).</p

Construction of subgenomic replicons and HCV protein expression in transfected Huh-7.5 cells.

<p>(A) Schematic representation of subgenomic (SG) replicons and their replication efficiency. Huh-7.5 cells were transfected with the indicated constructs and plated at decreasing cell number concentrations under neomycin selection to determine transduction efficiency of each construct, with GDD⁻ serving as a negative control. NS3_1629–1637 epitopes are listed by month(s) first detected in an in vivo chimpanzee CH503 infection model. (B) Replication of SG HCV RNA inside Huh-7.5 cells. Huh-7.5 cells were transfected with subgenomic replicons as in (A), and assayed for RNA replication 6 d post-transfection using a real time qRT-PCR Taqman assay as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000143#s2" target="_blank">Materials and Methods</a>. The minimum sensitivity of detection was 1,000 HCV copies/µg RNA). (C) Western blot of replicon-transfected Huh-7.5 cell lysates. The expression of HCV proteins NS3 and NS5A were detected post-transfection using anti-NS3/anti-NS5A monoclonal Abs. Lysates from Huh-7.5 cells transfected with replicons containing the BB7 epitope served as a positive control, untransfected Huh-7.5 cells were used as a negative control, and β-actin served as a positive control for input protein.</p

NS3_1629–1637 epitope evolution during in vitro viral infection.

<p>At indicated times post-infection, cells were harvested and the nucleotide sequence of the viral NS3_1629–1637 epitope was cloned and examined. Input sequence is listed above each timepoint, and “# of clones” denotes the number of individual colonies with the displayed sequence. Dashes represent no amino acid change from the listed input sequence.</p

Expression and recognition of the chimpanzee Patr-B1701 molecule on the surface of Huh-7.5 cells.

<p>(A) Surface expression of MHC class I on Huh-7.5 cells transfected with a plasmid containing the Patr-B1701 molecule and zeocin selection marker. No difference in surface expression was observed when compared to untransfected Huh-7.5 cells. Isotype control is depicted in grey. (B) CTL lysis of transfected Huh-7.5 cells. Huh-7.5 cells expressing the Patr-B1701 (Huh-7.5/B1701) molecule were pulsed with wild-type peptide and incubated with increasing amounts of CTL clone 4A specific for the NS3_1629–1637 wild-type epitope. Cells presenting this peptide on the Patr-B1701 molecule are lysed by CTLs as efficiently as EBV-transformed autologous B cells presenting peptide (B1701T). Untransfected Huh-7.5 cells served as a negative control. (C) CD8+ T cell clone IFNγ response to Huh-7.5/B1701 cells presenting exogenous peptide. Huh-7.5/B1701 cells were loaded with parent HCV1/910 NS3_1629–1637 or mutant NS3_1629–1637 peptide as in (B) and cocultured with a CD8+ T cell clone targeting the NS3_1629–1637 epitope. Huh-7.5/B1701 cells presenting parent HCV1/910 NS3_1629–1637 but not mutant peptide at concentrations of 0.5 µg/ml and lower could elicit an IFNγ response from the CD8+ T clone. Cocultures were stimulated with PHA as a positive control, and unpulsed Huh-7.5/B1701 cells or Huh-7.5 cells pulsed with parent HCV1/910 NS3_1629–1637 peptide served as negative controls. Plots depicted are gated on CD3+ T cells.</p

IR cytotoxicity in ATC is driven by changes in ROS levels.

<p>A) ATC (U-HTH83) intra-cellular ROS levels can be manipulated through the addition of exogenous ROS sources (H₂O₂) or ROS scavenging NAC. B) IR induces a dose dependent increase in intra-cellular ROS levels, which is neutralized by the addition of NAC. C) IR cytoxicity as measured using surviving fraction can be potentiated by the addition of H₂O₂ or reversed by NAC. Data are presented as averages with error bars representing standard deviation. Each experiment was performed at least in duplicate. *indicates p-value <0.05 compared to corresponding control condition unless otherwise indicated as in panel C. All experiments were conducted using the U-HTH83 cell line. (CNT = control, DCFDA = 2′,7′-dichlorodihydrofluorescein diacetate).</p

ATC tumors display significant metabolic heterogeneity.

<p>A) Control and irradiated tumors (IR) were serially sectioned and H&E staining was used to evaluate overall tumor architecture. Control tumors were substantially larger, but the majority of the tumor core consisted of non viable tissue (NVT). In contrast the majority of the irradiated tumor volume consisted of apparently viable tissue (VT). Irradiated tumors exhibited a high degree of aberrant cellular morphology as illustrated in the right lower panel inset. B) ATC tumor imaged at 2 weeks following tumor cell injection. Single snapshot imaging was performed 20 seconds after injection of labeled pyruvate. Spatial heat maps were generated from raw data and superimposed onto T2 weighted anatomic images for both pyruvate and lactate. Chemical spectra obtained in two separate voxels demonstrating differential conversion of pyruvate into lactate are shown.</p

Perturbations in oxidative stress and reducing potential are reflected in altered lactate generation.

<p>A) Endogenous reactive oxygen species (ROS) are generated by multiple cellular processes in both the mitochondria and cytoplasm. Exogenous ROS can increase the free radical burden inside the cell. Reducing equivalents in the form of NAD and NADP moieties are generated through multiple metabolic pathways and can cycle rapidly throughout various cellular compartments. Reducing equivalents are utilized by the cell to neutralize ROS. The conversion of pyruvate into lactate requires the presence of NADH; the conversion rate of labeled pyruvate into lactate therefore is an indirect measure of global cellular reducing potential and ROS stress. B) ATC cells were exposed to varying dose of radiation. Cells were harvested at indicated time points and lactate production was assayed biochemically. Data are presented as means, with error bars indicating standard deviation. *indicates p-value compared to control time point <0.05 using a two-tailed Student’s t test.</p