Additional file 1: Figure S1. of Conditional knock-out of lipoic acid protein ligase 1 reveals redundancy pathway for lipoic acid metabolism in Plasmodium berghei malaria parasite

Genotype and phenotype analysis of LplA1-cKO transgenic parasite clones. a PCR verification of genotypes of the LplA1-cKO parasite clones. b Regulation of GFP expression by ATc in cloned LplA1-cKO parasites. c Mean GFP fluorescence intensity of 9 LplA1-cKO clones. Each clone was analysed by flow cytometry in triplicate, and the mean + SD was reported. Table S1. Nucleotide sequences of primers and the 2A peptide used in the study. (DOCX 678 kb

Structure of Al-CPI and structural comparison of Al-CPI with CEW cystatin and human cystatin C.

<p>(A) Monomer structure of Al-CPI. (B) Structural comparison of Al-CPI (green), CEW cystatin (cyan), and cystatin C (magenta) around the α-helix core. (C) Structural comparison of Al-CPI (green), CEW cystatin (cyan) and cystatin C (magenta) around the active site segment. (D) Sequence alignment of five type-2 cystatins from <i>A. lumbricoides</i>, chicken egg white (CEW) and soft tick salivary gland (T.S. cystatin) and human cystatin C and D. The amino acid residues partially conserved among the five cystatins are highlighted in blue, and fully conserved are highlighted in red. The amino acid residues indexed with red arrow were selected for distance measurement as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096069#pone-0096069-t002" target="_blank">Table 2</a>.</p

Structural basis of differential inhibition activity of Al-CPI to cathepsin L (Cat. L) and cathepsin B (Cat. B).

<p>(A) Molecular docking was performed for Al-CPI and Cat. L. (B) Docking of AL-CPI against Cat. B suggested the insert segment of Cat. B would crash with the L2 of Al-CPI. (C) The sequence alignment of four cathepsins showing the unique insert segment of Cat. B.</p

The docking energy between Al-CPI and various enzymes.

a)<p>E_RDock, the RDOCK score is defined as: E_elec2+beta×E_sol. E_elec2: the electrostatic energy of the protein complex after the first and second CHARMm minimizations. E_sol: the desolvation energy of the protein complex calculated by the ACE method.</p

Comparison of Al-CPI with other monomeric type-2 cystatins^a).

a)<p>The Cα of conserved amino acid residues were chosen for all the distance measurements.</p

Protease inhibition of wild-type Al-CPI and the mutants.

<p>IC₅₀ values of wild type Al-CPI and five mutated forms to cathepsin B, C, L and S were shown. The inhibition activities of Al-CPI and its mutants were analyzed as described in Materials and.</p

Inhibition activities against cathepsin B, C, L and S by recombinant cysteine protease inhibitor <i>Ascaris lumbricoides</i> (Al-CPI).

<p>Cathepsins were incubated with the fluorogenic substrates in the absence or presence of serially diluted Al-CPI in appropriate buffer for 15 min. and reaction was stopped with stopping buffer. The amount of product was measured fluorometrically with excitation at 360 nm and emission at 460 nm. The inhibitory activity of Al-CPI was expressed as a percentage of the total activity detected in reactions without Al-CPI. The half maximal inhibitory concentration (IC₅₀) values of Al-CPI based on initial reaction velocities were determined by nonlinear regression analysis and are shown on each plot. Data shown are from one of three experiments.</p

Docking analysis of Al-CPI with cathepsin L.

<p>(A) Docking between Al-CPI and cathepsin L. (B) 90° rotation around the x-axis from the view of (A). (C–E) The detailed analysis of interactions between cathepsin L and Al-CPI N-terminal (C), loop 1 (D) and loop 2 (E). Inhibitor (Al-CPI) residues are indicated with an “i” after the sequence number to distinguish them from those of the enzyme (cathepsin).</p

Data collection and refinement statistics.

<p>The values in parentheses refer to statistics in the highest bin.</p>a)<p>R_merge = ∑_hkl∑_i|I_i(hkl)- | / ∑_hkl∑_iI_i(hkl), where I_i(hkl) is the intensity of an observation and is the mean value for its unique reflection; summations are over all reflections.</p>b)<p>R-factor  = ∑_h|Fo(h)-Fc(h)|/∑_hFo(h), where Fo and Fc are the observed and calculated structure-factor amplitudes, respectively.</p>c)<p>R_free was calculated with 5% of the data excluded from the refinement.</p>d)<p>Root-mean square-deviation from ideal values.</p>e)<p>Categories were defined by MolProbity.</p

Five-year outcomes from a prospective trial of image-guided accelerated hypofractionated proton therapy for prostate cancer

<p><b>Purpose:</b> To report 5-year outcomes of a prospective trial of image-guided accelerated hypofractionated proton therapy (AHPT) for prostate cancer.</p> <p><b>Patients and methods:</b> 215 prostate cancer patients accrued to a prospective institutional review board-approved trial of 70Gy(RBE) in 28 fractions for low-risk disease (<i>n</i> = 120) and 72.5Gy(RBE) in 29 fractions for intermediate-risk disease (<i>n</i> = 95). This trial excluded patients with prostate volumes of ≥60 cm³ or International Prostate Symptom Scores (IPSS) of ≥15, patients on anticoagulants or alpha-blockers, and patients in whom dose-constraint goals for organs at risk (OAR) could not be met. Toxicities were graded prospectively according to Common Terminology Criteria for Adverse Events (CTCAE), version 3.0. This trial can be found on ClinicalTrials.gov (NCT00693238).</p> <p><b>Results:</b> Median follow-up was 5.2 years. Five-year rates of freedom from biochemical and clinical disease progression were 95.9%, 98.3%, and 92.7% in the overall group and the low- and intermediate-risk subsets, respectively. Actuarial 5-year rates of late radiation-related CTCAE v3.0 grade 3 or higher gastrointestinal and urologic toxicities were 0.5% and 1.7%, respectively. Median IPSS before treatment and at 4+ years after treatment were 6 and 5 for low-risk patients and 4 and 6 for intermediate-risk patients.</p> <p><b>Conclusions:</b> Image-guided AHPT 5-year outcomes show high efficacy and minimal physician-assessed toxicity in selected patients. These results are comparable to the 5-year results of our prospective trials of standard fractionated proton therapy for patients with low-risk and intermediate-risk prostate cancer. Longer follow-up and a larger cohort are necessary to confirm these findings.</p