Analysis of <i>sid</i> expression levels in untreated and infected flies.

<p>Expression of <i>sid</i> was analyzed by qRT-PCR in control (not infected) and flies infected with <i>E. coli</i> flies or <i>M. luteu</i>s bacteria. <i>sid</i> expression levels were calibrated to the uninfected <i>w¹¹¹⁸</i> control level, which was assigned an arbitrary value of one. Fold expression (numbers over bars) was determined by dividing the mean value derived from the infected flies by the mean value obtained from uninfected flies (for each line; i.e., <i>W¹¹¹⁸</i> infected over uninfected <i>W¹¹¹⁸</i>). RpS15Aa expression values were very similar across all lines and not used to adjust overall expression levels.</p

Down-regulation of <i>sid</i> gene expression resulted in reduction of fly viability after infection with bacteria.

<p>Twenty five 2–3 day-old female animals were infected with <i>E. coli</i> (A) or <i>M. luteus</i> (B), from each indicated fly genotypes. All vials containing the post-infection experimental and control groups were maintained at 29°C on regular fly medium for seven days. Every day the total number of live and dead flies was recorded. Each time point represents the average of three independent trials. <i>W¹¹¹⁸</i> wild-type <i>c</i>ontrol flies were compared with experimental fly lines (<i>P</i><0.0001, Log-rank test for trend).</p

Expression profile of <i>sid</i> in the larval fat body and adult flies.

<p>(A) Expression of <i>sid</i> was analyzed by qRT<i>-</i>PCR in control flies <i>W¹¹¹⁸</i> as well as the <i>hop^Tum</i> mutant line and two transgenic fly lines, <i>UAS-sid RNAi</i> and <i>Ubiquitin-GAL4;UAS-sid RNAi</i>. Expression of <i>sid</i> in each sample was normalized to the expression of the ubiquitously expressed ribosomal RpS15Aa (CG2033) gene. Expression levels for both RpS15Aa and <i>sid</i> were calibrated to the wild type control levels, which were then assigned an arbitrary value of one, for each gene. Fold expression (numbers over bars) was determined by dividing the mean value derived from the <i>W¹¹¹⁸</i> control flies by the mean value obtained from other fly lines (ie., <i>hop^Tum sid</i> value over <i>W¹¹¹⁸ sid</i> value). Values were rounded up to the next decimal. Arrows represent fold up-regulation (up) or down-regulation (down). Each qRT-PCR experiment was performed in triplicate using RNA samples (cDNA) from the different genotypes. Bars represent the standard deviation from the mean. (C) Detection of SID protein by western blot analysis. Crude extracts from adult female flies were separated by SDS-PAGE, transferred to PVDF membranes and hybridized with rabbit anti- SID peptide antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103564#s2" target="_blank">Materials and Methods</a>.</p

Expression of SID protein in the fat body of 3^rd instar larvae.

<p>FB optical sections from control flies were exposed to anti-SID peptide antibody (A–C) and the immune complexes detected with a secondary antibody (goat anti-rabbit) labeled with Alexa Fluor 568. Only FB slices that were incubated with both primary and secondary antibodies (A–C) resulted in Alexa staining (red). Controls FBs that were only incubated with secondary antibody did not show significant staining (D–F). DAP1 (1 µg/ml) was used to localize the nuclei of FB cells (blue). The captured Z-stack images were collapsed (CZ) to visualize the full intensity of the stained FBs (A and D) and compared to a single optical section (SS; B and E). As a reference point for single sections, the section (optical slice) with the brightest nuclear staining was used. White scale bars represent a 50 µm region and shown at the bottom right of each image. Note that images containing a few cells are shown at the right at a higher magnification (C and F; yellow dashes mark region) and all were adjusted to the same level of brightness (+20) to enhance the visualization of the image. Larvae from the control fly line <i>Ubiquitin-GAL4</i> were used in these studies. Images were captured under the same experimental conditions of micro-channel plate (MCP) gain and laser power output <i>via</i> a confocal fluorescence LSM 700 microscope.</p

Analysis of survival after wounding and infection.

<p>(A) The survival rate (<b>^‡</b>) of flies after wounding with a clean needle or when injected with dead bacteria was analyzed after 7 days. The number of colony forming units (CFU) was also analyzed to determine the bacterial load over time in <i>W¹¹¹⁸</i> (blue line) and <i>Ubiquitin-GAL4;UAS-sid RNAi</i> (red line) flies infected with <i>E. coli</i> (B) or <i>M. luteus</i> (C). Significant differences in bacterial load between the two fly lines were determined <i>via</i> two-tailed paired Student’s <i>t</i>-tests with a <i>P</i> value of <0.01 (*).</p

Expression and purification of GST-SID fusion protein.

<p>(A) A GST-SID fusion protein was expressed in bacteria and partially purified with glutathione beads. The GST-SID protein was separated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103564#pone-0103564-g001" target="_blank">Figure 1</a>, and the eluted fraction was found to have few contaminants by protein staining (Coomassie R-250 blue). (B) Thrombin cleavage of the GST-SID fusion protein resulted in the release of the SID protein (arrow) as detected with anti-His antibodies (on the carboxyl-terminus of SID). (C) The thrombin-cleaved recombinant SID protein was examined for nuclease activity on DNA and RNA substrates in the presence of Cu⁺⁺ (+SID) or in the presence of thrombin alone (+T). All reactions were carried as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103564#pone-0103564-g001" target="_blank">Figure 1</a>.</p

Fruit flies deficient in <i>sid</i> expression are more susceptible to paraquat-induced oxidative stress.

<p>In a typical viability experiment, twenty 2–3 day-old female flies were placed in a vial with solid medium containing 5 mM and 10 mM paraquat and maintained at room temperature for four days. The fly lines used in this study are shown on the right side of the graph. Every day the total number of live and dead flies was tallied. Three replica vials were used for each fly strain with the same indicated genotypes. Each bar represents the standard error of the mean of three independent experiments. <i>W¹¹¹⁸</i> wild-type <i>c</i>ontrol flies were compared with experimental fly lines (<i>P</i><0.0001, Log-rank test for trend).</p

Amino acid sequence alignment of conserved elements among active sites of different nonspecific nucleases.

<p>Sequence alignment of the region containing the catalytic-residues of the SmNuc (<i>S.mercescens</i>), SID (<i>Drosophila</i>), two human mitochondrial endonucleases (Endo-G and Endo-GL), and the mosquito CuQu Endo nuclease. The compared nucleases have the conserved RGH motif called the NUC domain that is present in all active family members. The Histidine (*) within the RGH is the general based residue required for catalytic activity while the conserved Asparagine (**) is required for cation binding. Arrows on top of the aligned sequences indicate the two beta sheets and the grey line indicates the position of the alpha helix.</p

Searching in Mother Nature for Anti-Cancer Activity: Anti-Proliferative and Pro-Apoptotic Effect Elicited by Green Barley on Leukemia/Lymphoma Cells

<div><p>Green barley extract (GB) was investigated for possible anti-cancer activity by examining its anti-proliferative and pro-apoptotic properties on human leukemia/lymphoma cell lines. Our results indicate that GB exhibits selective anti-proliferative activity on a panel of leukemia/lymphoma cells in comparison to non-cancerous cells. Specifically, GB disrupted the cell-cycle progression within BJAB cells, as manifested by G2/M phase arrest and DNA fragmentation, and induced apoptosis, as evidenced by phosphatidylserine (PS) translocation to the outer cytoplasmic membrane in two B-lineage leukemia/lymphoma cell lines. The pro-apoptotic effect of GB was found to be independent of mitochondrial depolarization, thus implicating extrinsic cell death pathways to exert its cytotoxicity. Indeed, GB elicited an increase of TNF-α production, caspase-8 and caspase-3 activation, and PARP-1 cleavage within pre-B acute lymphoblastic leukemia Nalm-6 cells. Moreover, caspase-8 and caspase-3 activation and PARP-1 cleavage were strongly inhibited/blocked by the addition of the specific caspase inhibitors Z-VAD-FMK and Ac-DEVD-CHO. Furthermore, intracellular signaling analyses determined that GB treatment enhanced constitutive activation of Lck and Src tyrosine kinases in Nalm-6 cells. Taken together, these findings indicate that GB induced preferential anti-proliferative and pro-apoptotic signals within B-lineage leukemia/lymphoma cells, as determined by the following biochemical hallmarks of apoptosis: PS externalization, enhanced release of TNF-α, caspase-8 and caspase-3 activation, PARP-1 cleavage and DNA fragmentation Our observations reveal that GB has potential as an anti-leukemia/lymphoma agent alone or in combination with standard cancer therapies and thus warrants further evaluation <i>in vivo</i> to support these findings.</p> </div

Dose-dependent activation of caspase-8 pathway by GB in Nalm-6 cells monitored <i>via</i> flow cytometry.

<p>(A) After 4 h of GB treatment, the percentage of caspase-8-positive cells exhibiting green fluorescence signal is indicated on the <i>y</i>-axis, whereas the different cell treatments are indicated on the <i>x</i>-axis. Each bar represents the average of three independent measurements, and the error bars are their corresponding standard deviations. Representative flow cytometric dot plots (B–G) that were used to determine the distribution of caspase-8-positive cells are depicted. The diverse dot (event) color in each plot, designates just a density gradient; low-density region blue and high-density red. The flow cytometer acquisition settings were as following: FL1 and FL2 detectors were plotted at <i>x</i>-axis versus <i>y</i>-axis, respectively. Cells were exposed to 10 µl (B) and 50 µl (C) of GB for 4h and then, stained with FITC-IETD-FMK, as detailed in Materials and methods; this bar is imperceptible due a low value (<0.1%). (D) A set of GB-treated cells was concurrently exposed to 50 µl of GB and 20 µM of Z-VAD-FMK cell-permeant pan-caspase inhibitor (VAD). (E) PBS solvent control, where an error bar 0.11% is not noticeable and (G) untreated cells were included. (F) As a positive control for induction of caspase-8 activation, 2 mM of H₂O₂ was utilized. Approximately, 1x10⁴ events were acquired and analyzed per sample using CXP software. GB 10 µl = 0.3 ± 0.009 mg/ml, and GB 50 µl = 1.5 ±0.048 mg/ml lyophilized powder. The significance of the differences between 10 µl GB-treated cells as compared to 50 µl PBS-treated cells, and also, with untreated cells, is of <i>P</i> = 0.00039 (*) and <i>P</i> = 0.00203 (‡), respectively; whereas 50 µl GB-treated cells as compared with 50 µl PBS-treated cells was consistently <i>P</i><0.0001(**) in both data sets. GB 10 µl = 0.3 ± 0.009 mg/ml, and GB 50 µl = 1.5 ± 0.048 mg/ml lyophilized powder.</p