Additional file 4: of Duox mediates ultraviolet injury-induced nociceptive sensitization in Drosophila larvae

Figure S2. Confocal microscopy reveals dendrites of mdIV neurons for Ppk-td-GFP/+ (left) and Ppk-td-GFP/duox [MI11852] larvae (right). These larvae specifically express td-GFP in mdIV neurons. (PPTX 755 kb

Additional file 3: of Duox mediates ultraviolet injury-induced nociceptive sensitization in Drosophila larvae

Figure S1. A. (left) RT-PCR of 3rd instar larvae from lines Ppk-Gal4/+ (1), Ppk-Gal4 > UAS-duox-RNAi (38907) (2), and Ppk-Gal4 > UAS-Duox-RNAi (32903) (3). The primers used for duox PCR are the same as in Fig. 1. Rp49 was used as a loading control. (Right) Quantification of RT-PCR band areas by Image-J. (NIH). B. The band intensity of duox normalized to that of Rp49, and set to one for Ppk-Gal4/+. C. Larval thermal nociception assay. Rolling within 10 s of a 40 °C touch was counted as response (n = 30 per time section). Error bars denote +/− SEM. One-way ANOVA with Tukey post-test was used to analyze the differences. * and *** indicate p < 0.05 and 0.001 respectively. n.s., non-significant. (PPTX 148 kb

Effect of antidepressants (amitriptyline and trazodone) and GABAergic receptor agonists (benzodiazepine) on the viability of <i>md-TRPV1(3)</i> flies grown on capsaicin (5 mM)-containing food at 29°C.

Drugs were added to food at the concentrations indicated. Dots and vertical lines denote means and standard deviations, respectively. n = 60 for each curve; one-way ANOVA with the Tukey-Kramer method for multiple comparisons, *** P vs 1 mM amitriptyline, 0.2 mM trazodone, 5 mM benzodiazepine. All flies were 5-day-old males. md-TRPV1(3) denotes one copy of md-Gal4 and 3 copies of UAS-TRPV1.</p

Pum negatively regulates EGFR signaling on macrochaete development.

<p>Wild-type thorax and notum bears macrochaetes at specific positions (circles, A). Extra macrochaetes indicated by arrowheads are produced in transheterozygous <i>pum</i> mutants, <i>pum¹⁶⁸⁸</i>/<i>pum³</i> (B), <i>pum³</i>/<i>pum^Msc</i> (C), <i>pum¹</i>/<i>pum^Msc</i> (D), and <i>pum^Msc</i>/<i>pum¹⁶⁸⁸</i> (E). Extra macrochaetes arose by ectopic expression of EGFR in SOP (<i>sca-GAL4</i>/+; <i>UAS-EGFR</i>/+) (F). One copy reduction of <i>pum</i> greatly increased extra macrochaetes induced by enhanced EGFR signaling in SOP (<i>sca-GAL4</i>/+; <i>UAS-EGFR</i>/<i>pum¹⁶⁸⁸</i>) (G) and (<i>sca-GAL4</i>/+; <i>UAS-EGFR</i>/<i>pum^Msc</i>) (H). Ectopic expression in SOP of full-length <i>pum</i> (<i>sca-GAL4</i>/<i>UAS-pum</i>) (I) or Puf (<i>C253-GAL4</i>/UAS-<i>Pum^HD</i>) (K) eliminated macrochaetes. Concomitant expression in SOP of EGFR with either <i>pum</i> (<i>sca-GAL4</i>/<i>UAS-Pum</i>; <i>UAS-EGFR</i>) (J) or Puf (<i>C253-GAL4</i>/<i>UAS-Pum^HD</i>; <i>UAS-EGFR</i>) (L) eliminated bristles. The number of macrochaetes circled in (A) was counted (M, N). ***; P value <0.001. **; P value <0.01. The P values were obtained by student’s t-test in SigmaPlot.</p

<b>The number of macrochaetes reveals genetic interaction between Pum and EGFR.</b>

*<p>Bristles circled in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034016#pone-0034016-g003" target="_blank">Figure 3A</a> were counted. s.d., standard deviation. N, number of flies counted. P, P-value by student’s t-test.</p>**<p><i>pum</i> mutants compared to <i>w¹¹¹⁸</i>.</p

Pum binds to the potential NRE-like sequence of EGFR signaling components.

<p>(A) (Upper) Schematic drawing of the yeast three-hybrid assays. An RNA containing the NRE and MS2 sequence recruits both Gal4 transcriptional activation domain (GAD)-MS2 coat protein (CP) fusion protein (GAD-MS2 CP) and lexA DNA binding domain (DBD)-Puf fusion protein (LexA-Puf). The resultant ternary complex leads to the expression of <i>lacZ</i> coding sequence through binding to <i>lexA</i> binding sequence (LexAop). (Lower) Yeast YPH500 cells harboring the <i>lex</i>A_op-<i>Lac</i>Z reporter, LexA DBD-Puf and GAD-MS2 CP were transformed with vectors that allow for expression of diverse NRE-MS2 transcripts as indicated. Liquid β-galactosidase assays were carried out for transformants. The mean ± SD values were obtained from at least three independent experiments and are presented on the Y-axis. (B) (Upper) Schematic diagram of a reporter containing <i>luciferase</i> (<i>luc</i>) coding sequence with CMV promoter (black arrow) and NREs (black box) in its 5′- and 3′-UTR, respectively. (Lower) HEK293 cells were transfected with the luciferase reporter plasmid containing NRE-like sequence as indicated, alone (−) or in combination with <i>pum</i> expression vector (+). Luciferase activities were measured and the mean ± SD values obtained from at least three independent experiments performed in triplicate (*, <i>P</i> < 0.05). The P-values were obtained by student’s t-test in SigmaPlot.</p

Pum activity in the wing disc.

<p>The 3^rd larval wing discs harboring a tub-GFP-NRE construct were visualized by GFP or redstinger fluorescence or immuno-stained by antibodies (α-GFP, α-Pum and α-HA). The <i>dpp-Gal4</i> driven expression of redstinger (A), Pum (B), Pum-IR (C), and Nos (D) were monitored, as shown in the second column. Pum activity level was monitored by GFP signals, as shown in the third column. The first column is the merged images of the second and third columns. Scale bars indicate 50 µm.</p

<b>Pum binding and repression of NRE-like sequences of EGFR and its transducers.</b>

*<p>NRE consensus sequences (<b><u>UGUA</u></b>N<b><u>AUA</u></b>).</p>**<p>Puf binding to NRE-like sequence determined by yeast three hybrid assay as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034016#pone-0034016-g005" target="_blank">Figure 5A</a>. +++, strong binding; ++, moderate binding.</p>***<p>Pum repression of a reporter containing NRE-like sequence as determined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034016#pone-0034016-g005" target="_blank">Figure 5B</a>. Y, repression; N, no repression; ND, not determined.</p

Additional file 1: of Residential radon and environmental burden of disease among Non-smokers

Table S1. Lifetime lung cancer incidence and comparison with other cause of death by the smoking status. Table S2. Ovid-MEDLINE, Ovid-EMBASE search strategy in 5th of Jan, 2016. Table S3. Documents selected for the systematic review. Table S4. Residential radon ranked by region and attributable burden of disease, 2010. (DOCX 29 kb

Accumulation of Charantin and Expression of Triterpenoid Biosynthesis Genes in Bitter Melon (<i>Momordica charantia</i>)

Charantin, a natural cucurbitane type triterpenoid, has been reported to have beneficial pharmacological functions such as anticancer, antidiabetic, and antibacterial activities. However, accumulation of charantin in bitter melon has been little studied. Here, we performed a transcriptome analysis to identify genes involved in the triterpenoid biosynthesis pathway in bitter melon seedlings. A total of 88,703 transcripts with an average length of 898 bp were identified in bitter melon seedlings. On the basis of a functional annotation, we identified 15 candidate genes encoding enzymes related to triterpenoid biosynthesis and analyzed their expression in different organs of mature plants. Most genes were highly expressed in flowers and/or fruit from the ripening stages. An HPLC analysis confirmed that the accumulation of charantin was highest in fruits from the ripening stage, followed by male flowers. The accumulation patterns of charantin coincide with the expression pattern of <i>McSE</i> and <i>McCAS1</i>, indicating that these genes play important roles in charantin biosynthesis in bitter melon. We also investigated optimum light conditions for enhancing charantin biosynthesis in bitter melon and found that red light was the most effective wavelength