Additional file 2: Figure S1. of Transcriptome profiling of two maize inbreds with distinct responses to Gibberella ear rot disease to identify candidate resistance genes

Comparison between ddPCR and RNA-Seq expression profiles of selected genes. The Y-axis scale corresponds to transcripts per million (TPM) for RNA-Seq data and copies/Îźl for ddPCR. Tissue samples from the 2004 and 2006 field season were used for both gene expression quantitation methods. (PDF 288 kb

Additional file 1: Table S1. of Transcriptome profiling of two maize inbreds with distinct responses to Gibberella ear rot disease to identify candidate resistance genes

Primers used for selected candidate and reference genes in droplet digital PCR validation experiment. Table S2. Hourly temperature readings during inoculation and tissue collection time periods in 2004, 2006, and 2013. Table S3. Mapping of RNA-Seq reads to the reference genome B73 V2. Table S4. List of significant differentially expressed transcripts. Table S5. Upregulated transcripts mapping within GER resistance QTL regions. (XLSX 1156 kb

Physical activity, sport and physical education in northern Ireland school children: A cross-sectional study

Internationally, insufficient physical activity (PA) is a major health concern. Children in Northern Ireland (NI) are recorded as having the lowest levels of PA in the United Kingdom (UK). To date, validated and representative data on the PA levels of NI school children are limited. The aim of this study was to provide surveillance data on self-reported PA, sport and physical education (PE) participation of school children in NI. Differences between genders and factors associated with PA were also examined. A representative sample of primary (n = 446) and post-primary (n = 1508) children was surveyed in school using validated self-report measures. Findings suggest that PA levels are low, with a minority of children (13%) meeting the PA guidelines (primary pupils 20%, post-primary pupils 11%). NI school children have lower levels of PA, PE and sports participation than UK and European peers. A trend of age-related decline across all the domains of PA was apparent. The data presented highlighted that females are less likely to achieve PA guidelines, children from lower socio-economic background participate in school and community sport less often, and that enjoyment and social support are important variables in PA adherence. Policy solutions that would support implementation e.g., mandatory minimum PE time, whole school approaches to PA promotion and targeted investment in schools, particularly in areas of deprivation and for females, are suggested

A Neurotoxic Glycerophosphocholine Impacts PtdIns-4, 5-Bisphosphate and TORC2 Signaling by Altering Ceramide Biosynthesis in Yeast

<div><p>Unbiased lipidomic approaches have identified impairments in glycerophosphocholine second messenger metabolism in patients with Alzheimer's disease. Specifically, we have shown that amyloid-β42 signals the intraneuronal accumulation of PC(<i>O</i>-16:0/2:0) which is associated with neurotoxicity. Similar to neuronal cells, intracellular accumulation of PC(<i>O</i>-16:0/2:0) is also toxic to <i>Saccharomyces cerevisiae</i>, making yeast an excellent model to decipher the pathological effects of this lipid. We previously reported that phospholipase D, a phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P₂)-binding protein, was relocalized in response to PC(<i>O</i>-16:0/2:0), suggesting that this neurotoxic lipid may remodel lipid signaling networks. Here we show that PC(<i>O</i>-16:0/2:0) regulates the distribution of the PtdIns(4)P 5-kinase Mss4 and its product PtdIns(4,5)P₂ leading to the formation of invaginations at the plasma membrane (PM). We further demonstrate that the effects of PC(<i>O</i>-16:0/2:0) on the distribution of PM PtdIns(4,5)P₂ pools are in part mediated by changes in the biosynthesis of long chain bases (LCBs) and ceramides. A combination of genetic, biochemical and cell imaging approaches revealed that PC(<i>O</i>-16:0/2:0) is also a potent inhibitor of signaling through the Target of rampamycin complex 2 (TORC2). Together, these data provide mechanistic insight into how specific disruptions in phosphocholine second messenger metabolism associated with Alzheimer's disease may trigger larger network-wide disruptions in ceramide and phosphoinositide second messenger biosynthesis and signaling which have been previously implicated in disease progression.</p></div

PC(<i>O</i>-16:0/2:0) inhibits TORC2 signaling.

<p>(<b>A</b>) <b>Phosphorylation of the TORC2 substrate Ypk1 is reduced following treatment.</b> TORC2-dependent Ypk1 (T662) phosphorylation status was assessed in whole cell extracts from vehicle (ethanol, EtOH), PC(<i>O</i>-16:0/2:0) (20 µM) or rapamycin (Rap, 200 ng/ml) treated wild type (YPH500) and <i>spo14</i>Δ (YKB2076) cells. Immunoblots were also probed with anti-sera for total Ypk1 to ensure equal loading. (<b>B</b>) <b><i>tor2-21</i></b><b> mutants display increased sensitivity to PC(</b><b><i>O</i></b><b>-16:0/2:0).</b> Strains expressing plasmid borne wild type <i>TOR2</i> or the temperature sensitive (ts) alleles <i>tor2-21</i> or <i>tor2-30</i> in a <i>tor1</i>Δ, <i>tor2</i>Δ or a combined <i>tor1</i>Δ <i>tor2</i>Δ background were plated in 10-fold serial dilutions on YPD plates containing vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) (3 µg/ml). Plates were incubated for 2 days at a permissive (25 C) or semi-permissive temperature (33 C). (<b>C</b>) <b>Overexpression of hyperactive Ypk2 suppresses sensitivity to PC(</b><b><i>O</i></b><b>-16:0/2:0).</b> Ypk2 wild type (Ypk2), hyperactive (D239A), kinase dead (K373A) and the double mutant (D239A and K373A) were transformed into wild type (SH100) and <i>tor2-21</i> (SH121) expressing cells. Growth was assessed following 2 days at permissive (25 C) and semi-permissive temperature (33 C) on plates containing vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) (3 µg/ml).</p

Relocalization of Slm1 by PC(<i>O</i>-16:0/2:0) does not mediate the inhibition of TORC2 signaling.

<p>(<b>A</b>) <b>PC(</b><b><i>O</i></b><b>-16:0/2:0) treatment relocalizes Slm1-GFP to foci.</b> The co localization of Slm1-GFP (YKB3035) with Lsp1-mcherry, an eisosome marker, was examined following treatment with either vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) (20 µM) for 15 min. Numbers represent the percent of Slm1-GFP foci co-localizing with Lsp1-mcherry foci. (<b>B</b>) <b>PC(</b><b><i>O</i></b><b>-16:0/2:0) treatment does not affect TORC2 interactions.</b> The indicated strains were treated with either vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) (20 µM) for 15 min. The interaction of Avo3-HA and endogenous Ypk1 with immunopurified (IP) Slm1-GFP was determined by immunoblotting with appropriate antibodies. Total levels of each protein were also examined in whole cell extracts (WCE). (<b>C</b>) <b>PC(</b><b><i>O</i></b><b>-16:0/2:0) still reduces Ypk1 phosphorylation in the presence of myriocin.</b> Wild type cells (TB50a) were pretreated with vehicle or myriocin (5 µM, 30 min) prior to adding rapamycin (Rap, 200 ng/ml) or PC(<i>O</i>-16:0/2:0) (20 µM). The ratio of TORC2-dependent Ypk1 phosphorylation to total Ypk1 was determined for each treatment condition and normalized to control. The mean is displayed below the representative blot (n = 2).</p

PC(<i>O</i>-16:0/2:0)-induced changes in PtdIns(4,5)P₂ metabolism.

<p>(<b>A</b>) <b>Mss4 is relocalized upon PC(<i>O</i>-16:0/2:0) treatment.</b> Mss4-GFP expressing cells (YKB2955) were treated with vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) (20 µM, 15 min) and localization examined. Percentage of cells with relocalized Mss4-GFP are indicated by the figure inset. (<b>B</b>) <b>Mss4 is required for PES formation.</b> Wild type (SEY6210) and <i>mss4-102</i> (AAY202) strains were grown at the indicated temperatures for one hour. Cells were subsequently treated with either vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) as previously done (20 µM, 15 min). Following treatment cells were collected into ice cold growth media and labeled with FM4-64 in ice cold growth media to visualize the PM. The percentage of cells with PES type structures for each condition are indicated by the figure inset. (<b>C</b>) <b><i>MSS4</i></b><b> and </b><b><i>STT4</i></b><b> are required for buffering against PC(<i>O</i>-16:0/2:0) toxicity.</b> The sensitivity of wild strains (SEY6210) or strains expressing a temperature sensitive alleles of either <i>STT4</i> (<i>stt4-4</i>, AAY102) or <i>MSS4</i> (<i>mss4-102</i>, AAY202) to PC(<i>O</i>-16:0/2:0) was examined by growth on plates containing vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) (3 µg/ml or 5.7 µM) for 2 days at permissive (25 C) and semi-permissive (33 C) temperatures. (<b>D</b>) <b>Overexpression of phosphatidylinositol phosphatases increase sensitivity to PC(<i>O</i>-16:0/2:0).</b> The effect of phosphatidylinositol phosphatases upon PC(<i>O</i>-16:0/2:0) sensitivity was examined by spotting 10-fold serial dilutions of wild type strain (BY4741) harboring plasmid borne, GAL-inducible <i>INP51</i>, <i>INP52</i> and <i>INP54</i> on plates containing vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) (3 µg/ml) with either dextrose or galactose as the carbon source.</p

PtdIns(4,5)P₂ is redistributed in response to PC(<i>O</i>-16:0/2:0).

<p>Wild type (WT) cells (YPH500) expressing (<b>A</b>) GFP-2×PH^PLCδ (PtdIns(4,5)P₂) (<b>B</b>) GFP-PH^Fapp (PtdIns(4)P) or (<b>C</b>) GFP-FYVE^EEA1 (PtdIns(3)P) were treated with either vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) (20 µM, 15 min) and localization of the GFP probe quantified. The percentage of cells displaying a redistribution of the fluorescent reporter is reported in the inset of the figure.</p

PC(<i>O</i>-16:0/2:0) disrupts sphingolipid metabolism leading to changes in Mss4-GFP localization.

<p>(<b>A</b>) <b>PC(</b><b><i>O</i></b><b>-16:0/2:0) treatment disrupts sphingolipid metabolism.</b> Wild type (BY4741) cells were treated with vehicle or PC(<i>O</i>-16:0/2:0) (20 µM) for the indicated times (min). Lipids were extracted and sphingolipid levels were quantified and expressed as a log₂ fold change of PC(<i>O</i>-16:0/2:0) treated from vehicle treated control. LCB, long chain base; IPC, inositol phosphorylceramide; MIPC, mannosyl phosphorylceramide; DHS(-P), dihydrosphingosine (1-phosphate); PHS(-P), phytohydrosphingosine; LC, long chain (acyl chain is equal to or less than 22 carbons); VLC, very long chain (more than 22 carbons). (<b>B</b>) <b>Treatment with ceramide promotes PES formation and inhibits actin cytoskeleton polarization.</b> Wild type (BY4741) cells expressing GFP-2×PH^PLCδ were grown in YPD in the presence of vehicle (EtOH), PC(<i>O</i>-16:0/2:0), Cer(d18:1/2:0) or Cer(d18:0/2:0) (20 µM, 15 min) prior to imaging live or fixing and staining for acting cytoskeleton polarization as described in methods. The percentage of cells displaying a redistribution of the fluorescent reporter or proper actin polarization is reported in the inset of the respective figure. (<b>C</b>) <b>Inhibition of sphingolipid metabolism prevents the relocalization of Mss4.</b> Mss4-GFP (YKB2955) expressing cells were pretreated with vehicle or myriocin (5 µM) for 30 min and subsequently treated with vehicle or PC(<i>O</i>-16:0/2:0) (20 µM, 15 min) as previously done. Pretreatment with myriocin inhibited PC(<i>O</i>-16:0/2:0)-dependent changes in PES formation.</p

Characterization of PM changes in PC(<i>O</i>-16:0/2:0)-treated cells.

<p><b>Large PM invaginations are present in PC(</b><b><i>O</i></b><b>-16:0/2:0)-treated cells.</b> Wild type cells (BY4742) exposed (<b>B, C, D, E and F</b>) or not (<b>A</b>) to PC(<i>O</i>-16:0/2:0) for 15 min were processed for EM as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004010#pgen.1004010-Griffith1" target="_blank">[55]</a>. Panel (<b>C</b>) is an inset of panel (<b>B</b>). Panels (<b>D</b>), (<b>E</b>) <b>and</b> (<b>F</b>) show magnifications of the large PM invaginations induced by PC(<i>O</i>-16:0/2:0), which very likely represent the PES. The asterisks indicate the peripheral ER that is associated with the PM. CW, cell wall; ER, endoplasmic reticulum; M, mitochondrion; V, vacuole. Bars in panels (<b>A</b>) and (<b>B</b>), 500 µm; bars in panels (<b>C</b>), (<b>D</b>), (<b>E</b>) <b>and</b> (<b>F</b>), 100 µm. <b>PES formation still occurs in the presence of depolymerised actin.</b> (<b>G</b>) Wild type cells (YPH500) expressing GFP-2×PH^PLCδ were treated with Latrunculin A (5 µM, 30 min) to induce depolymerization of the actin cytoskeleton prior to treatment with PC(<i>O</i>-16:0/2:0) (20 µM, 15 min) and imaged live. The percentage of cells displaying a redistribution of the fluorescent reporter is reported in the inset of the figure. (<b>H</b>) An aliquot of cells was also fixed following treatment for imaging of the actin cytoskeleton by staining with Rhodamine-conjugated phalloidin. The percentage of small budded cells displaying a polarized actin cytoskeleton is reported in the inset of the figure.</p