EAST might act as a salt sensor that modulates gene expression in response to changing ion (Na⁺/K⁺) concentrations.

<p>A change in EAST dosage may lead to the sensing of incorrect ion levels, which may result in inappropriate physiological responses.</p

EAST-GFP accumulates in chromosome regions of low transcriptional activity.

<p>(A) EAST-GFP (green) is excluded from highly transcribed chromosome regions (arrow) in detergent treated larval salivary glands cells. Run-on transcription was visualized using BrUTP (red). TOPRO3 (white) was used to label DNA. (B) In <i>east</i>^hop7 mutants, like in wildtype, regions of high BrUTP incorporation (arrow) are found in interbands. Note that the banding pattern shows less contrast compared to EAST-GFP overexpressing or wildtype (not shown) cells. Bars correspond to 10 µm in all panels.</p

east corresponds to <i>suppressor of white-spotted.</i>

<p>All flies shown are homozygous for <i>w</i>^sp1. (A) In male pharates dissected out of pupal cases, the mutations <i>east</i>^hop7 and <i>su(w</i>^sp<i>)</i>¹ suppress the loss of eye pigmentation resulting from the white mutation <i>w</i>^sp1. (B) The mutations <i>east</i>^hop7 and <i>su(w</i>^sp<i>)</i>¹ show complementation. Like <i>su(w</i>^sp<i>)</i>¹/<i>su(w</i>^sp<i>)</i>¹, the combination <i>east</i>^hop7/<i>su(w</i>^sp<i>)</i>¹ restores eye pigmentation to almost wildtype levels. Heterozygous flies of each mutation display intermediate eye pigmentation.</p

Characterization of EAST-GFP localization and mobility in larval salivary glands.

<p>(A) Varying the salt concentration can modulate localization of EAST-GFP. At 100 mM, the distribution is mostly chromosomal, at 150 mM chromosomal and nucleoplasmic and at 200 mM no chromosomal-like pattern is detectable. (B-D) The mobility of EAST-GFP was assessed by FRAP. (B, C) Increasing the salt concentration lowers the affinity to chromatin. An increase in salt from 50 mM (B) to 100 mM (C) leads to a faster recovery of fluorescence after bleaching chromosome regions bound by EAST-GFP. (D) Removing the C-terminal residues 1535-2301 of EAST leads to an increase in mobility. At a salt concentration of 50 mM, the truncated version of EAST associates with a lower affinity than the full-length version. (E) The diagram shows the recovery in seconds after bleaching the indicated nuclear regions for 4 seconds at a laser intensity of 100%. The two different variants of EAST-GFP were expressed using the <i>ftz-GAL4</i> driver. Cells were permeabilized in buffers containing 50 mM NaCl supplemented with varying amounts of KCl to reach the indicated salt concentrations. The recovery of the non detergent treated cell (non-perm) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000412#pone-0000412-g001" target="_blank">Figure 1A</a> is indicated for comparison. The Bar in A represents 10 µm and applies to all panels.</p

Change of localization of EAST-GFP during apoptosis.

<p>(A) In a 13 h APF old pupa, EAST-GFP (green) shows mainly extrachromosomal localization. The nuclear lamina (red) is still intact. DNA (blue) was labeled with TOPRO-3. (B) Co-localization of EAST-GFP with intact polytene chromosomes can be observed in a 19 hour APF old pupa. The destruction of the lamina indicates that part of the apoptotic program is executed. Note the barely detectable anti-lamin staining in salivary glands compared to neighboring diploid cells (arrow). The Bar in A represents 10 µm and also applies to B.</p

Additional file 1: Table S1. of Live imaging of muscle histolysis in Drosophila metamorphosis

List of gene perturbations tested for muscle defects in pilot screen. (PDF 120 kb

Additional file 3: Figure S2. of Live imaging of muscle histolysis in Drosophila metamorphosis

Two different Cp1-shRNAs silence expression of a Cp1-mKO2 reporter during prepupal (A) and pupal (B) stages. The construct HMS00725 (second row) was used in most experiments. (PDF 6018 kb

Additional file 2: Figure S1. of Live imaging of muscle histolysis in Drosophila metamorphosis

Expression of the 24B-GAL4 driver in metamorphosis. In (A) prepupae and (B) pupae, the mesodermal 24B-Gal4 driver is expressed in multi-nucleated muscles (outlined in black) and more apically located mono-nucleated cells (white arrow). Cells were labelled with the mitochondrial marker UAS-Mito-GFP (green) and UAS-histone-mKO (magenta). (B) During pupation, mono-nucleated cells (white arrow) undergo PCD and show nuclear fragmentation, while nuclei (black arrow) in muscles undergoing histolysis, like the DIOM2s (outlined in black), condense without showing fragmentation (see Fig. 2). (PDF 218 kb

Oral Administration and Selective Uptake of Polymeric Nanoparticles in <i>Drosophila</i> Larvae as an <i>in Vivo</i> Model

In this article, <i>Drosophila</i> larvae are applied as an <i>in vivo</i> model to investigate the transport and uptake of polymeric nanoparticles in the larval digestive tract after oral administration. After feeding the larvae with food containing bare and chitosan-coated Poly­(d,l-lactic-<i>co</i>-glycolic acid) (PLGA) nanoparticles encapsulated with BODIPY, time-lapse imaging of live larvae is used to monitor the movement of fluorescent nanoparticles in the anterior, middle, and posterior midgut of the digestive tract. Also, the dissection of the digestive tract enables the analysis of cellular uptake in the midgut. Bare PLGA nanoparticles travel through the whole midgut smoothly while chitosan-coated PLGA nanoparticles have a long retention time in the posterior midgut. We identify that this retention occurs in the posterior segment of the posterior midgut, and it is termed as the retention segment. During transport in the midgut, chitosan-coated nanoparticles pass through the near-neutral anterior midgut and become highly positively charged when entering into the highly acidic middle midgut. After traveling through the near-neutral anterior segment of the posterior midgut, chitosan-coated nanoparticles have a long retention time of ∼10 h in the retention segment, indicating that the chitosan coating greatly enhances mucoadhesive ability and promotes cellular uptake in this part of the midgut. The dynamic behavior of orally administered nanoparticles in <i>Drosophila</i> larvae is in agreement with studies in other animal models. A <i>Drosophila</i> larva has the potential to evolve into a low-cost drug screening model through real time imaging, which will accelerate the development of improved nanoparticle formulations for oral drug delivery

Wounding increased lipid peroxidation and nitrative damage but not protein carbonylation.

<p>Levels of F₂-isoprostanes levels in skin and wounds were compared by normalizing against arachidonic acid (A) or tissue weight (B). Results shown are mean ± S.E.M, n = 5. Wounds were compared to skin using 1-way ANOVA with Dunnett’s post-hoc test. Asterisks denote level of significance when compared to skin. Control and H₂O₂ wounds were also compared against each other using 2-way ANOVA but the differences was not statistically significant. (C) Levels of arachidonic acid in skin and wound tissues. Results shown are mean ± S.E.M, n = 5. Wounds were compared to skin using 1-way ANOVA with Dunnett’s post-hoc test. Asterisks denote level of significance when compared to skin. Control and H₂O₂ wounds were also compared against each other using 2-way ANOVA but the differences was not statistically significant. (D) Levels of protein carbonyls in wounds were compared to intact skin and expressed as fold change. The results shown are the mean fold change ± S.E.M. No difference in the levels of protein carbonyl was observed in control wounds and 166 mM H₂O₂ treated wounds. (E) Comparison of 3-nitrotyrosine level in skin and wounds. Results shown are mean ± S.E.M., n = 5. The 3-nitrotyrosine levels of skin were compared to control wounds or H₂O₂ treated wounds and analyzed with 1-way ANOVA followed by Dunnett’s post-hoc test. Levels of 3-nitrotyrosine were significantly higher at day 6 after wounding. Levels of 3-nitrotyrosine in control and 166 mM H₂O₂ treated wounds were also compared using 2-way ANOVA and the differences were not statistically significant. *p<0.05, ** p<0.01, ***p<0.001.</p