Infection of neutropenic mice.

<p>Partial neutropenia was induced in C57Bl/6 mice through intra-venous injection of NIMPR14 antibody on the same day as their infection with wt or ΔF2 IAV (dose of virus corresponding to the ΔF2 LD₅₀). (A) Neutropenia was checked in blood at day 4 post-injection. Results are expressed as percentages of mock-treated mice. (B) Survival curves of immunocompetent and neutropenic mice after infection. (C-D) Time-course of morbidity evolution: after infection, the weights (C) and body temperatures (D) of immunocompetent and neutropenic mice were measured every day.</p

Effects of Water and Cell Culture Media on the Physicochemical Properties of ZnMgO Nanoparticles and Their Toxicity toward Mammalian Cells

ZnMgO nanoparticles have shown potential for medical applications as an efficient antibacterial agent. In this work, we investigate the effect of water and two commonly used cell culture media on the physicochemical properties of ZnMgO nanoparticles in correlation with their cytotoxicity. In vacuum, ZnMgO nanopowder consists of MgO (nanocubes) and ZnO (nanotetrapods and nanorods) particles. Upon exposure to water or the Luria–Bertani solution, ZnO characteristic shapes were not observable while MgO nanocubes transformed into octahedral form. In addition, water caused morphological alternations in form of disordered and fragmented structures. This effect was directly reflected in UV/vis absorption properties of ZnMgO, implying that formation of new states within the band gap of ZnO and redistribution of specific sites on MgO surfaces occurs in the presence of water. In mammalian culture cell medium, ZnMgO nanoparticles were shapeless, agglomerated, and coated with surrounding proteins. Serum albumin was found to adsorb as a major but not the only protein. Adsorbed albumin mainly preserved its α-helix secondary structure. Finally, the cytotoxicity of ZnMgO was shown to strongly depend on the environment: in the presence of serum proteins ZnMgO nanopowder was found to be safe for mammalian cells while highly toxic in a serum-free medium or a medium containing only albumin. Our results demonstrate that nanostructured ZnMgO reaches living cells with modified morphology and surface structure when compared to as-synthesized particles kept in vacuum. In addition, its biocompatibility can be modulated by proteins from biological environment

PB1-F2 enhances neutrophil recruitment in the lungs.

<p>(A) Mice were infected with 1x10⁵ PFU of the wt or the ΔF2 IAV and neutrophil count was done in the BAL fluids of mock-infected (n = 5), wt-infected (n = 9) or ΔF2-infected (n = 9) mice at day 3 pi. (B-C) Mice were intranasally instilled with LPS or full-length PB1-F2 (50pmol) from WSN strain or from a highly pathogenic H5N1 avian strain. 24h later mice were euthanized, KC secretion (B) and neutrophils recruitment were quantified within BAL fluids (C).</p

Transcriptomic impact of PB1-F2 at day 4 pi.

<p>Total RNA from mock-, wt- or ΔF2-infected C57Bl/6 mice were extracted from lungs at day 4 pi and used to hybridize Agilent’s Whole Mouse Genome Microarray (4x44K; G4122F). GeneSpring software (Version 13.0; Agilent Technologies) was used to analyze differences of gene expression between mock-infected mice and wt- or ΔF2-infected mice. (A) Hierarchical clustering diagrams showing individual replicates are represented. Log-ratios (infected conditions compared to mock condition) are depicted in blue (downregulated) or red (upregulated). The magnitude of the regulation is illustrated by the intensity of the color. (B-C) “wt virus specifically regulated genes” (B) and “ΔF2 virus specifically regulated genes” (C) were uploaded on PANTHER classification System (<a href="http://pantherdb.org" target="_blank">http://pantherdb.org</a>; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165361#pone.0165361.ref034" target="_blank">34</a>]) to identify the biological processes associated with these 2 genes clusters.</p

Impact of PB1-F2 on NK cell lung recruitment.

<p>(A) Heat map of NK-specific genes differentially regulated by wt and ΔF2 viruses. Genes shown in red are up-regulated and those shown in blue are down-regulated in lungs from IAV infected mice compared to lungs from mock-infected mice. Data are expressed as fold-change <i>vs</i>. mock. (B) Histogram representing the level of expression of NK cells activating cytokines at day 4 pi in wt- and ΔF2-infected mice. Data were obtained by microarray analysis and are expressed as fold-change <i>vs</i>. mock. (C) Histogram representing the percentages of NK cells within BAL fluids of wt- or ΔF2-infected mice at day 4 pi. Data represent three independent experiments (n = 12, (*: p-value<0.05).</p

Graphical representation of the neutropenia induction timeline.

<p>Ten mice per group were used.</p

T lymphocytes recruitment is not altered by PB1-F2.

<p>Immunocompetent mice (treated with 2A3 antibody) and neutropenic mice (treated with 1A8 antibody) were infected with the wt or the ΔF2 virus. BAL fluids were collected at day 8 pi and the cellular part was characterized by FACS. (A) CD3⁺ CD11b^- CD4⁺ cell composition of the BAL fluids. (B) CD3⁺ CD11b^- CD8⁺ cell composition of the BAL fluids. (C) Viral load in lungs expressed as RNA copies normalized to the quantity of RNA used in the experiment. (D-E-F) Total RNA isolated from lungs was reverse-transcribed and used to quantify expression of several T cells markers by qPCR: IFNγ, T-Bet and GATA3. Gene expressions were normalized with the β-actin gene expression level and presented as fold increase relative to mock-treated mice. Results are the mean ± SEM values obtained from 3 animals.</p

Proposed communication path between loop 1 and the RNA groove.

<p>K113 located at the edge of β-sheet 1 strongly interacts with loop 1, in particular E73. The other extremity of the β-sheet had multiple contacts with residues of the RNA grove, in particular hydrophobic interactions between Y97 and M371, interactions between R106 and the linker backbone (residues 360–373 shown in magenta) and electrostatic interactions between K103 and E372. The linker itself was stabilized by salt bridges between E369 and R361 and E369 and R317 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030038#pone-0030038-t001" target="_blank">Table 1</a>).</p

Infection of NF-kB luciferase transgenic mice.

<p>(A) Groups of 3 immunocompetent mice were infected with 1x10⁵ PFU of the wt or the ΔF2 IAV. Four days pi, mice were anesthetized and luciferine was intra-nasally instilled (0.75 mg.kg^-1). Bioluminescence was then measured using the IVIS system. (B) Bioluminescence analysis of neutropenic mice infected with 1x10⁵ PFU of the wt or the ΔF2 IAV at day 4 pi. The scale on the right indicates the average radiance: the sum of the photons per second from each pixel inside the ROI/number of pixels (photons/sec/cm2/sr). (C) Bioluminescence activities of the 4 groups of mice were quantified using ‘Living Image’ software and represented as a diagram (***: p-value<0.001; ns: non-significant). (D) Body temperature measurement of immunocompetent mice and neutropenic mice challenged by wt or ΔF2 IAV at day 4 pi (**: p-value<0.01; ns: non-significant).</p

Influence of mutations in loop 2 of NP on RNA binding and RNA-induced oligomerization.

<p><b>A:</b> Comparison of the association to and dissociation from RNA of R361A-R204A-R208A (full triangles) and wt-R204A-R208A (open triangles); <b>B:</b> Comparison of the oligomerization kinetics of 10 µM proteins after addition of RNA (3 µM): wt NP (full squares), R361A (open squares), wt-R204A-R208A (open circles) and R361A-R204A-R208A (full triangles) (10 µM). The lines represent single exponential fits.</p