Allelochemicals of the phenoxazinone class act at physiologically relevant concentrations

<p>Plants compete with their neighbors via the release of chemical compounds into the rhizosphere. These phytotoxins originate from a series of secondary metabolites and can be processed further by soil-living microorganisms before exerting their activity on the target plant. To determine the molecular mode of action and the physiological relevance of potential phytotoxins, it is important to simulate true-to-life conditions in laboratory experiments, for example by applying physiologically relevant concentrations. Here, we report on an improved experimental setting to study the function of allelochemicals of the benzoxazolinone class. By adjusting the solvent and the application of the chemicals, we reduced by more than 2fold the concentration that is necessary to induce growth defects in the model plant Arabidopsis thaliana.</p

Individual courses of HCV viral load over time.

<p>Numbers indicate patient numbers as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080528#pone-0080528-t001" target="_blank">Table 1</a>. </p

Attenuated SeV variants in primary human hepatocytes (PHH).

<p>PHH from three different donors were infected with SeV D52 and all six recombinant SeV variants (MOI of 0.1). <b>(A)</b> Exemplarily chosen pictures of PHH 72 hpi for seven different recombinant viruses (detection of GFP by fluorescence microscopy). Bar represents 200 µm. For donor 1, the analysis of the SeV FmutC/Y_termV_ko variant was not done (n.d.). <b>(B)</b> 72 h after the inoculation with the different viruses, infected cells were counted in three randomly chosen areas and calculated as a ratio of all cells in the same area. Data are shown in mean and SD of three independent experiments in triplicates.</p

Growth kinetics and spreading of newly generated SeV variants in different cell types.

<p><b>(A+B)</b> Growth kinetics of six different recombinant SeV viruses over a 96×10⁷ TCID₅₀ is depicted. <b>(A)</b> Malignant human hepatoma cells PLC/PRF/5, Hep3B and HuH7. <b>(B)</b> Non-malignant MRC-5 fibroblasts and primary human hepatocytes (PHH) from three different donors. <b>(C+D)</b> Detection of EGFP reporter protein expression over a 72 h observation period by fluorescence microscopy as a surrogate marker for viral replication and spread to neighboring cells. Size bar: 200 µm. <b>(C)</b> Infection of PLC/PRF/5 hepatoma cells. <b>(D)</b> Infection of MRC-5 human fibroblast cells.</p

Comparison of attainable peak SeV titers during replication in different cell types.

<p>The mean peak titer (sum of the infectious virus particles determined via TCID₅₀ for super natant and lysate) over a period of 96 h was determined including all experiments with the investigated three hepatoma cell lines (HuH7, Hep3B, PLC/PRF/5) and compared to the achievable peak titer in non-malignant cells (MRC-5 or PHH). Numbers over the columns display fold-changes between the mean of all hepatoma cells compared to either MRC-5 or PHH. The dotted line shows the amount of inoculated viral particles during the initial infection (5×10³ TCID₅₀). Data represent mean and SEM.</p

The amount of infectious virus particles in tumor tissue was quantified with the TCID₅₀ method.

<p>48₅₀ titration (mean and standard deviation of two (D52) or three analyzed tumors of each group).</p

Growth kinetics of newly generated SeV variants in Vero producer cells.

<p>Growth kinetics of six different recombinant SeV viruses over a 96×10⁷ TCID₅₀ was inserted for better orientation.</p

Generation of recombinant Sendai virus variants.

<p><b>(A)</b> Schematic representation of SeV genomes and the protein sequence of the SeV-F protein cleavage-site of newly generated SeV virus variants. At the 3′-end all variants encode a reporter gene for enhanced green fluorescent protein (EGFP). In contrast to the P and F gene wild-type variant (D52), in all SeV Fmut variants the wild-type F protein cleavage-site (VPQSR) was replaced by the oligobasic cleavage-site of Newcastle disease virus F protein (RRQKR). The seven wild-type P gene encoded proteins are a result of multiple open reading frames (ORF; C′, C, Y1, Y2) and RNA editing, respectively, leading to a frame shift and thus to the V or W ORF's. For attenuation in non-malignant cell types, different mutations were introduced in C (ORFs; C′-, C-, Y1-, Y2) or V/W-ORFs. For the V_ko variants (no V and no W proteins), mutations of the editing-site within the P frame were introduced without changing the amino acid sequence of the P protein. Thus, P protein but no truncated P protein variants (V and W proteins) can be synthesized. For the C protein deficient variants (C_ko: no C′ and C proteins; Y_ko: no Y1 and Y2 proteins), inserted point mutations are marked by numbers from 1-5 (1: M1T, 2: L5stop, 3: L11stop, 4: M24T, 5: M30T; numbers refer to amino acid position in C/Y-frame whereas 1 refers to the initiator methionine of the C protein). <b>(B)</b> Schematic overview of functional ORFs from the P gene of different SeV variants. The SeV P-gene wild-type expression pattern is shown for D52 and Fmut (1.+2.).</p

Peak titers in different cell types.

<p>Mean peak titers of resulting progeny virus of the six different SeV variants in Vero cells, three hepatoma cell lines (Hep3B, HuH7, PLC/PRF/5) and two non-malignant cells (MRC-5 and PHH). Numbers represent the mean of three independent experiments and are given as TCID₅₀/2 ml.</p

Quantification of cellular viability in a dose and time-dependent manner.

<p>Infection experiments with Vero and PLC/PRF/5 (at MOIs of 1 and 0.1) cells were performed with all variants of the newly generated Sendai viruses as well as the wild type variant SeV D52. Cellular viability was investigated via CytoTox-Glo™ assay at different time points post infection (0 h, 24 hpi, 48 hpi, 72 hpi, 96 hpi). The assay was performed in triplicates and repeated three times; data are shown as mean and SEM. SeV 1 (green line): SeV D52, SeV 2: SeV Fmut, SeV 3: SeV FmutV_ko, SeV 4: SeV FmutC_ko, 5: SeV FmutY_ko, 6: SeV FmutC_koV_ko, 7: SeV FmutC/Y_termV_ko, TX: Triton X-100 (positive control for the induction of a maximum grade, chemically-mediated destruction of test cells).</p