Measurement of the Lifetime Difference in D0 Meson Decays

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Immigration Policies in the Western Europe and the Radicalised Youths under its Crevasses

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Aft1 localization is affected in the absence of Yap5.

<p>(A) Wild-type (WT) <i>yap5</i> (<i>yap5</i>) <i>grx4</i> (<i>grx4</i>) mutant strains were transformed with a high-copy <i>AFT1-GFP</i> plasmid. Exponentially growing cells in SD medium were untreated (SD) or treated with 2 mM of FeSO₄ for 30 min. GFP-Aft1 fusion protein was visualized by fluorescence microscopy. (B) Cells with nuclear fluorescence were counted as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037434#s4" target="_blank"><i>Materials and Methods</i></a>. Approximately 100 cells were analyzed per condition. Values are the mean of two biological duplicates (200 cells) ± s.d. (C) Exponentially growing cells from wild-type (WT) and <i>yap5</i> mutant (<i>yap5</i>) strains were upshifted to high-Fe medium, by supplementation of SC medium with 2 mM of Fe SO4, and harvested at the indicated time-points. RNAs were isolated and the expression of <i>FET3</i> was assessed by qRT-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037434#s4" target="_blank"><i>Materials and Methods</i></a>. Values are the mean of biological triplicates ± s.d. (D) WT and <i>yap5</i> strains were transformed with the plasmid pCM64-<i>CTH2</i>-FeRE-<i>CYC1</i>-LacZ (<i>CTH2:LacZ</i>). Cells were grown exponentially in SD medium or in Sd medium supplemented with 2 mM of FeSO₄, for 30 min (+FeSO₄) and ß-galactosidase activity was assayed as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037434#s4" target="_blank"><i>Materials and Methods</i></a>. Values are the mean of biological decaplicates ± s.d. * P<0.01 **P<0.05.</p

Functional categories of genes differentially expressed in <i>yap5</i> mutant cells.

<p>BY4742 wild-type and <i>yap5</i> mutant strains were grown to exponential phase in SC medium and challenged with 2 mM of FeSO₄ for 60 min. RNA of Fe-treated cells was isolated and analyzed with DNA microarrays as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037434#s4" target="_blank"><i>Materials and Methods</i></a>. Genes up- or downregulated were sorted into functional categories according to MIPS database. Dubious open reading frame unlikely to encode a protein (100) were not considered. A list of all the genes included in each functional category is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037434#pone.0037434.s003" target="_blank">Table S3</a>.</p

<i>GRX4</i> gene expression is dependent on Yap5.

<p>(A) Exponentially growing cells from wild-type (WT) and <i>yap5</i> mutant (<i>yap5</i>) strains were upshifted to high-Fe medium, by supplementation of SC medium with the indicated FeSO₄ concentrations, and harvested at the indicated time-points. The expression of <i>GRX4</i> was assessed by qRT-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037434#s4" target="_blank"><i>Materials and Methods</i></a>. Values are the mean of biological triplicates ± s.d. (B) WT and <i>yap5</i> strains were transformed with a plasmid carrying <i>GRX4</i>-HA, treated with 5 and 15 mM of FeSO₄ for 20 min and analyzed by Western blot with an anti-HA antibody. Pgk1 protein levels were used as loading control. (C) Representation of the <i>GRX4</i> constructs without (a) or with (b,c,d) mutations in the YREs. (D) Mutant <i>grx4</i> cells expressing the constructs depicted in (C) were grown under Fe-adequate or Fe overload (5 mM FeSO₄) conditions, and the expression of <i>GRX4</i> was assessed by qRT-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037434#s4" target="_blank"><i>Materials and Methods</i></a>. Values are the mean of biological triplicates ± s.d. (E) <i>yap5</i> cells were transformed with HA-tagged Yap5 and grown in SD medium not supplemented (−Fe) or supplemented (+Fe) for 15 min with 2 mM of FeSO₄, before being processed for ChIP. ChIP analysis was performed using probes specific for <i>GRX4</i>. (F) ChIP analyses combined with qRT-PCR, were used to determine the fold enrichment of <i>GRX4</i>, <i>SCR1</i> and <i>ARN2</i>. The sequence enrichment in the ChIP (i.e. IP/IN) was normalized using the <i>ACT</i> gene as a reference.</p

Genes involved in iron homeostasis whose expression is altered in the <i>yap5</i> mutant.

*<p>FC-Fold Change.</p

Genes involved in iron homeostasis with expression altered in Fe excess.

<p>Genes involved in iron homeostasis with expression altered in Fe excess.</p

Yap5 is not the only regulator of <i>CCC1</i> gene.

<p>(A) Exponentially growing cells from wild-type (WT), <i>yap5</i> mutant (<i>yap5</i>) and <i>ccc1</i> mutant (<i>ccc1</i>) strains were harvested, serially diluted and spotted onto control SC plates or SC plates containing the designated FeSO₄ concentrations. (B) WT and <i>yap5</i> strains were upshifted to high-Fe medium, by supplementation of SC medium with 2 mM of FeSO4, and harvested at the indicated time-points. The expression of <i>CCC1</i> was assessed by qRT-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037434#s4" target="_blank"><i>Materials and Methods</i></a>. Values are the mean of biological triplicates ± s.d. (C) Schematic representation of the truncated promoter version of <i>CCC1</i> gene (sp<i>CCC1</i>). (D) <i>ccc1</i> strain was transformed with a plasmid harboring sp<i>CCC1</i> gene (<i>ccc1</i>CCC1>), fpCCC1 gene (<i>ccc1</i>CCC1>) or the plasmid alone (<i>ccc1</i><<i>vector</i>>). Wild-type strain was transformed with the empty plasmid (WT<<i>vector</i>>). Exponentially growing cells were harvested, serially diluted and spotted onto control SD plates or SD plates containing 9 mM of FeSO₄.</p

Transfer RNAs used in this study.

<p>In order to reconstruct the early stages of the CUG identity alteration in <i>C. albicans</i>, <i>S. cerevisiae</i> leucine tRNAs containing the anticodons UAG or CAG were expressed in C. <i>albicans</i>. A) The respective tRNA genes were cloned into plasmid pUA12, which is based on the <i>C. albicans</i> pRM1 vector. B) A leucine tRNA gene containing the near-cognate anticodon (5′-UAG-3′) for the CUG codon was used as a low decoding efficiency tRNA (pUA13). C) Two tRNA_CAG^Leu genes, containing anticodons cognate for the CUG codon were used for higher CUG decoding efficiency, one contained G₃₃ (pUA14; medium decoding efficiency) and the other contained U₃₃ (pUA15; high decoding efficiency), in the anticodon-loop. D) The <i>S. cerevisiae</i> tRNA_AGA^Ser gene was used as negative control (pUA16).</p

Summary of gene expression changes at 7 h and 24 h after OGD.

<p>(<b>A</b>) Student's <i>t</i>-test analysis was applied to the microarray data to identify all genes whose expression was significantly different between conditions (<i>p</i><0.05). Up-regulated and down-regulated genes include those whose expression levels had a fold change ≥2.0. (<b>B and C</b>) Number of up-regulated (<b>B</b>) or down-regulated (<b>C</b>) transcripts at 7 h and 24 h after 2 h OGD. The intersection represents the number of transcripts whose transcription was changed in both recovery periods. The transcripts used in this analysis were considered differentially expressed after using two cut-off criteria: a <i>p</i>-value <0.05 and a fold change of 2.0. The VENNY informatic tool was used to compare the lists of transcripts to obtain the Venn Diagrams.</p