18 research outputs found

    Calibrating Focused Light-Field Cameras Using Plenoptic Disc Features

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    This paper proposes a new method for estimating calibration parameters of plenoptic cameras by minimizing the nonlinear plenoptic reprojection error. Novel plenoptic feature types are proposed as data for the calibration method. These plenoptic disc features are in a natural one-to-one correspondence with physical points in front of the camera. We exploit the intrinsic geometry of plenoptic cameras in a novel projection model that relates the plenoptic disc features to physical points. The resulting calibration quality, as quantified by mean reprojection error and 3D reconstruction error, outperforms recently published results

    Structural Characterization of a Novel Chlamydia pneumoniae Type III Secretion-Associated Protein, Cpn0803

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    Type III secretion (T3S) is an essential virulence factor used by Gram-negative pathogenic bacteria to deliver effector proteins into the host cell to establish and maintain an intracellular infection. Chlamydia is known to use T3S to facilitate invasion of host cells but many proteins in the system remain uncharacterized. The C. trachomatis protein CT584 has previously been implicated in T3S. Thus, we analyzed the CT584 ortholog in C. pneumoniae (Cpn0803) and found that it associates with known T3S proteins including the needle-filament protein (CdsF), the ATPase (CdsN), and the C-ring protein (CdsQ). Using membrane lipid strips, Cpn0803 interacted with phosphatidic acid and phosphatidylinositol, suggesting that Cpn0803 may associate with host cells. Crystallographic analysis revealed a unique structure of Cpn0803 with a hydrophobic pocket buried within the dimerization interface that may be important for binding small molecules. Also, the binding domains on Cpn0803 for CdsN, CdsQ, and CdsF were identified using Pepscan epitope mapping. Collectively, these data suggest that Cpn0803 plays a role in T3S

    Characterisation and genome sequence of the lytic Acinetobacter baumannii bacteriophage vB-AbaS-Loki

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    © 2017 Turner et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Acinetobacter baumannii has emerged as an important nosocomial pathogen in healthcare and community settings. While over 100 of Acinetobacter phages have been described in the literature, relatively few have been sequenced. This work describes the characterisation and genome annotation of a new lytic Acinetobacter siphovirus, vB-AbaS-Loki, isolated from activated sewage sludge. Sequencing revealed that Loki encapsulates a 41,308 bp genome, encoding 51 predicted open reading frames. Loki is most closely related to Acinetobacter phage IME-AB3 and more distantly related to Burkholderia phage KL1, Paracoccus phage vB-PmaS-IMEP1 and Pseudomonas phages vB-Pae-Kakheti25, vB-PaeS-SCH-Ab26 and PA73. Loki is characterised by a narrow host range, among the 40 Acinetobacter isolates tested, productive infection was only observed for the propagating host, A. baumannii ATCC 17978. Plaque formation was found to be dependent upon the presence of Ca2+ ions and adsorption to host cells was abolished upon incubation with a mutant of ATCC 17978 encoding a premature stop codon in lpxA. The complete genome sequence of vB-AbaS-Loki was deposited in the European Nucleotide Archive (ENA) under the accession number LN890663. Copyright

    Cpn0803 interacts with phosphatidylinositol and phosphatidic acid.

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    <p>His-Cpn0803 was incubated with membrane lipid strips containing purified eukaryotic membrane components and visualized by anti-his antibody and ECL reagents. Cpn0803 interacted with phosphatidylinositol and phosphatidic acid, but none of the other molecules evaluated. His-CdsL, as a negative control, did not interact with any lipid components.</p

    Pepscan mapping of the Cpn0803 binding regions shown in stereo.

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    <p>A. Pepscan epitope mapping against a Cpn0803 peptide library was performed to determine the residues of Cpn0803 responsible for mediating its interactions with CdsN, CdsF and CdsQ. Recombinant CdsN, CdsF and CdsQ was reacted against the Cpn0803 peptide library and monitored for the corresponding interacting regions. The corresponding surfaces are color-coded as follows: CdsN in blue (residues 22–26), CdsF in purple (residues 109–128), and CdsQ in orange (residues 153–161).</p

    Cpn0803 interacts with type III secretion components <i>in vitro</i>.

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    <p>A. A glutathione plate assay was applied to screen for interactions of Cpn0803 between either CdsN, CdsQ or CdsF. His-Cpn0803 was applied to GST-CdsN, -CdsQ and -CdsF immobilized on glutathione plates, washed three times with PBS, and monitored using a colorimetric assay. Data is represented on the graph as the mean ± standard deviation for each interaction. A significant interaction was considered to be two standard deviations above the negative control (GST alone against Cpn0803). As a positive control, we screened GST-Cpn0803 against His-Cpn0803, as well as GST-Lcrh-2 against His-CopN. Cpn0803 interacted significantly with CdsN, CdsQ, CdsF, and Cpn0803. while it did not interact with GST alone. GST-Lcrh-2 and His-CopN also had a significant interaction. B. We applied GST pull-down assays to corroborate the interactions found with the glutathione plate assay. GST-CdsN, -CdsQ, –CdsF or GST alone immobilized on glutathione beads were incubated with an <i>E. coli</i> lysate over-expressing His-Cpn0803 and washed with 500 mM NaCl. GST-CdsN, -CdsQ, and CdsF co-purified with Cpn0803 under 500 mM NaCl conditions while GST alone did not.</p

    Cpn0803 interacts with type III secretion components <i>in vivo</i>.

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    <p><i>C. pneumoniae</i> EB lysates were incubated with recombinant GST-CdsN, -CdsF, -CdsQ or -Cpn0803. Glutathione agarose beads were incubated with the lysates overnight, collected, and washed with 500 mM NaCl. The protein on the beads was analyzed by SDS PAGE and Western blot with anti-Cpn0803 antibody. Native Cpn0803 co-purified with GST-CdsN, -CdsF, and -CdsQ. As a positive control, Cpn0803 also co-purified with GST-Cpn0803, but not with GST alone.</p

    Stereo image of a predicted Cpn0803 hexamer colored by chain.

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    <p>We evaluated Cpn0803 for its ability to form multimers by analysis with the PISA server. Based on crystal contacts and buried surface area, the biologically active unit of Cpn0803 was predicted to be a hexamer formed by a trimer of dimers. The individual monomeric units are shown in different colours.</p

    Cpn0803 exists in a hexameric and dimeric state in solution.

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    <p>A. Gel filtration chromatography was used to analyze purified His-Cpn0803. We found two dominant peaks, one corresponding to a hexamer and one corresponding to a dimer. The elution volume of the protein standards are shown on the x-axis. B. Peak fractions in the elution volumes were collected and analyzed by anti-His Western blot. Cpn0803 was present in the peak fractions.</p
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