The 3′ UTR landscape of the <i>C</i>. <i>albicans</i> transcriptome.

<p>(A) Comparison of 3′ UTRs as determined by our study and Bruno <i>et al</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.ref042" target="_blank">42</a>]. Of the 3′ UTR that are called by both technologies, 84.5% are within 100 bases (r = 0.4684 (<i>p</i> much < 0.001, n = 2697. Of note, the correlation is highly significant because of the high numbers and would be classed as of moderate strength). Where there is a difference, it is due to filtering differences: we have used the most abundant 3′ UTR, whereas Bruno <i>et al</i> used the longest 3′ UTR for which there was evidence, including minor alternative 3′ UTR isoforms. (B) Graph showing 3′ UTRs are overall shorter in <i>C</i>. <i>albicans</i> than <i>S</i>. <i>cerevisiae</i>. The global positions of polyadenylation in the forward direction and, where it exists, the position of any anti-parallel overlapping adenylated RNA running in the reverse direction. Note any effect of filtering is avoided by this approach as all adenylation sites are utilized in this comparison (370997 and 201547 sites in <i>C</i>. <i>albicans</i> and <i>S</i>. <i>cerevisiae</i> respectively). (C) Comparison between the 3′ UTR lengths of the 3552 orthologous genes between <i>C</i>. <i>albicans</i> and <i>S</i>. <i>cerevisiae</i>. Genes annotated to GO “Mitochondrion” are labeled in red. (D) Comparison of the distance of the Puf3 binding site in putative mRNA targets conserved between <i>S</i>. <i>cerevisiae</i> and <i>C</i>. <i>albicans</i> relative to the closest coding sequence (CDS). (E) Comparison of the distance of the Puf3 binding site in putative mRNA targets conserved between <i>S</i>. <i>cerevisiae</i> and <i>C</i>. <i>albicans</i> relative to the polyadenylation site (PA).</p

The <i>C</i>. <i>albicans</i> Puf3 regulon and mitochondrial roles.

<p>(A) Venn diagram showing the number of mRNAs with a Puf3 binding motif in the 3′ UTRs in <i>S</i>. <i>cerevisiae</i> and <i>C</i>. <i>albicans</i> (only genes which contain orthologs in both species are depicted here). (B) Functional groupings of the Puf3 regulon in <i>C</i>. <i>albicans</i>. The data used to produce this chart is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.s014" target="_blank">S4 Dataset</a>. (C) Growth of <i>C</i>. <i>albicans</i> wild type and <i>puf3Δ/Δ</i> mutant on plates supplied with glucose, glycerol or lactate. Ten-fold serial dilutions were made starting from OD₆₀₀ = 0.5, and plates were photographed after 2 days of growth. (D) Mitochondria in the indicated strains were stained with MitoTracker and imaged as described in Materials and Methods. (E) <i>C</i>. <i>albicans</i> growth on plates was tested as in (C), in the presence or absence of CCCP. Growth was observed on glycerol plates where mitochondrial function is essential.</p

Effects of <i>CCR4</i> on the extracellular matrix and biofilm stability.

<p>(A) Quantification of total extracellular matrix (ECM) in <i>C</i>. <i>albicans</i> biofilms. Biofilms were grown in Spider medium and collected after 48 h. The proportion of ECM was calculated relative to the total biofilm biomass (cells + ECM), as determined by dry weight measurement (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#sec010" target="_blank">Materials and Methods</a>). Results were calculated from three biological repeats in technical triplicates. Shown are the averages and the standard errors. <i>P</i> value of the difference between WT and <i>ccr4Δ/Δ</i> is shown as * <0.05. (B) Biofilms were grown in 96-well microtiter plates in RPMI-MOPS for 24 h and then exposed to zymolyase (20T) prepared in RPMI-MOPS + 0.9% NaCl (1:1 ratio) for 24 h. The remaining biomass was quantified by crystal violet staining. Results were calculated from three biological repeats in technical duplicates. Shown are the averages and the standard errors. <i>P</i> value of the difference between WT and <i>ccr4Δ/Δ</i> is shown as * < 0.05. (C) Matrix 1,3 β-glucan was quantified from biofilms grown in Spider medium as described in Materials and Methods. The yield of 1,3 β-glucan was calculated as the weight of 1,3 β-glucan (pg) per 1 μg of biofilm cells. Results were calculated from 6 biological repeats. Shown are the averages and the standard errors. Differences: WT versus <i>ccr4Δ/Δ</i>, <i>p</i> = 0.067; <i>ccr4Δ/Δ</i> + <i>CCR4</i> versus <i>ccr4Δ/Δ</i>, <i>p</i> = 0.062. (D) qPCR analysis for the indicated transcripts was performed on biofilms grown for 48 h in Spider medium and data normalized to <i>SCR1</i> RNA. Error bars are ± standard errors of the average of 3 biological replicates. <i>P</i> values are as follows: ** <0.01 or * <0.05.</p

Uncoupling of mitochondrial oxidative phosphorylation stimulates biofilm matrix production.

<p>The wild type stain DAY185 was used for these experiments. (A) qPCR analysis of the expression of the indicated mitochondrial biogenesis genes in 48 h grown biofilm samples upon treatment with CCCP. Error bars are ± standard errors of the average of three biological replicates. <i>P</i> values are as follows: ** <0.01, * < 0.05. (B) SEM of biofilms in the presence or absence of 20 μM CCCP. The ECM is indicated with black arrows. Scale bar = 10 μm. (C) The susceptibility to zymolyase of CCCP-treated biofilms grown in RPMI-MOPS was determined by crystal violet staining. Results were calculated from three biological repeats in technical duplicates. Shown are the averages and the standard errors. <i>P</i> value is * < 0.05.</p

Biofilm mRNA targets of the Pumilio RNA binding protein Puf3.

<p>(A) Analysis of the gene expression data from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.ref014" target="_blank">14</a>] revealed that 162 genes down-regulated in biofilms are annotated to GO “Mitochondrion”, and 36/162 contain a Puf3 recognition element in their 3’ UTR. The putative biofilm-regulated Puf3 targets include several important mitochondrial biogenesis factors, such as mitochondrial ribosomal subunits, proteins required for respiratory chain function and assembly, and proteins that belong to the mitochondrial protein import machinery. Gene Ontology analysis was performed using the tools in the Candida Genome Database. (B) Cartoon of the Puf3 RNA binding domain from <i>S</i>. <i>cerevisiae</i> showing binding to the core recognition element and the interaction with the -2 cytosine (based on the structure reported in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.ref039" target="_blank">39</a>]. (C) Alignment of the PUM domains of the <i>C</i>. <i>albicans</i> and <i>S</i>. <i>cerevisiae</i> Puf3 proteins containing the 8–8’ repeat. The LAS motif of the Puf3 binding pocket that interacts with the -2 cytosine is boxed in blue, and this binding pocket is conserved in the <i>C</i>. <i>albicans</i> protein (see also [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.ref066" target="_blank">66</a>]).</p

Model for the role of posttranscriptional gene regulation and mitochondrial activity in biofilm matrix production and stress protection.

<p>As the biofilms mature, environmental changes, such as hypoxia and nutrients, lead to lowering of mitochondrial activity. Lower mitochondrial activity might be sensed as a stress signal and drive the production of protective extracellular matrix. Cell wall integrity pathways are known to function in matrix production [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.ref015" target="_blank">15</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.ref016" target="_blank">16</a>], and mitochondrial function has been linked to pathways of fungal cell wall integrity (reviewed in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.ref027" target="_blank">27</a>]), thus providing a plausible mechanism of mitochondrial control over biofilm matrix production. Mitochondrial dysfunction could also lead to weaker cell walls and cell lysis, further contributing to extracellular matrix deposition. Posttranscriptional regulators, such as the Ccr4-NOT mRNA deadenylase and Puf3, coordinate biofilm maturation pathways by responding to nutrients and hypoxia to adjust mitochondrial biogenesis, as well as the expression of genes needed for biofilm matrix production.</p

<i>C</i>. <i>albicans</i> Puf3 is a repressor of mRNA stability in glucose and lactate growth conditions.

<p><b>(</b>A) A cartoon depicting the location of primers used for specific amplification of the <i>MET3p</i>-driven <i>COX23</i> and <i>MRPL25</i> genes. Detection of this allele was specific, as demonstrated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.s003" target="_blank">S3 Fig</a>. (B) qPCR showing time dependent decay of <i>COX23</i> and <i>MRPL25</i> genes following transcriptional shutdown of <i>MET3p</i> in glucose. Fold expression was represented as ratio of expression levels for each time point after dividing with the expression levels at time 0. Decay curves were obtained with the nonlinear regression (curve fit) method using the exponential, one phase decay equation in the GraphPad “Prism 6” software. The half-life (T1/2) was also calculated using this equation by plotting the decay curve of 3–4 biological replicates separately, and is shown as the average ± standard error. (C) The experiments were performed as described in (B), but with lactate as the carbon source. Data represent the average and standard error from 4 biological replicates. (D) The cartoon depicts mutations in the core and -2C positions of the Puf3 binding motif in the <i>COX23</i> 3′ UTR. Decay curves are from wild type <i>C</i>. <i>albicans</i> strains expressing either a <i>COX23</i> construct with either the wild type or the mutant Puf3 recognition element. The experiments were performed as in (B). Strains were grown in lactate media and shown are results of 2 biological replicates. (E) The experiment was performed as in (C), but concomitant with addition of methionine and cysteine to inhibit transcription, the temperature was raised to 37°C. Shown are results of 3 biological repeats.</p

The mRNA deadenylase Ccr4 regulates extracellular matrix production in biofilms.

<p>(A) qPCR analysis of the expression of mitochondrial biogenesis genes in biofilms grown for 48 h in Spider medium. <i>SCR1</i> RNA was used for normalization. Error bars are ± standard errors of the average of 3 biological replicates. <i>P</i> values are as follows: *** <0.001, ** <0.01, * <0.05. Additional genes are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.s007" target="_blank">S7 Fig</a>. (B) Scanning electron microscopy of biofilms formed by <i>C</i>. <i>albicans</i> wild type (DAY185), <i>ccr4Δ/Δ</i> and <i>pop2Δ/Δ</i> mutants and their respective complemented strains. Mature biofilms (48 h) grown in Spider medium were assessed. Experiments were repeated at least twice and inset boxes are regional amplifications. Scale bar = 10 μm. Similar results were obtained when biofilms were grown in RPMI-MOPS or YNB (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.s009" target="_blank">S9 Fig</a>). (C) The data for RPMI-MOPS are from the control samples in the experiments performed to assess biofilm susceptibility to zymolyase (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.g007" target="_blank">Fig 7B</a>). Biofilms were grown for 48 h in the indicated media. Total biofilm biomass was determined by staining with crystal violet. Bars represent averages ± standard errors from three biological replicates. <i>P</i> value of the difference between WT versus <i>ccr4Δ/Δ</i> is shown as ** <0.01 in RPMI-MOPS and *<0.05 in Spider media. (D) Metabolic activity of <i>C</i>. <i>albicans</i> biofilms was determined using the XTT reduction assay. Results were calculated from three independently grown biofilms for each of the strains assayed in technical duplicates. The error bar represents standard errors. <i>P</i> value of the difference between WT and <i>ccr4Δ/Δ</i> is shown as * <0.05.</p

Posttranscriptional regulation of the mitochondrial ribosome and the <i>COX</i> genes by carbon source and Puf3 in <i>S</i>. <i>cerevisiae</i>.

<p>Decay of the indicated mRNAs was measured in wild type and <i>puf3Δ</i> strains grown in glucose (A) or lactate (B) following transcriptional repression at 37°C. The decay curves and half-life (T1/2) were calculated as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005590#pgen.1005590.g004" target="_blank">Fig 4</a>. The data are shown as the average and standard error of 2–4 biological replicates.</p

Prolonging the circulatory retention of SPIONs using dextran sulfate : in vivo tracking achieved by functionalisation with near-infrared dyes

<p>Intraperitoneal (Panels <b>A</b> and <b>C</b>) and oral (Panel <b>B</b>) glucose tolerance tests were performed on 6 hr fasted conscious C57BL/6J WT (closed symbols) and <i>Slc30a8</i> KO (open symbols) male mice as described in Materials and Methods. The IPGTT results in Panel <b>A</b> show the mean glucose concentrations ± S.E.M. in WT (n = 11; mean age ∼20 weeks) and <i>Slc30a8</i> KO (n = 9; mean age ∼20 weeks) animals. The OGTT results in Panel <b>B</b> show the mean glucose concentrations ± S.E.M. in WT (n = 16; mean age ∼22 weeks) and <i>Slc30a8</i> KO (n = 9; mean age ∼22 weeks) animals. The IPGTT results in Panel <b>C</b> show the mean glucose concentrations ± S.E.M. in WT (n = 17; mean age ∼4 weeks) and <i>Slc30a8</i> KO (n = 19; mean age ∼4 weeks) animals. *p<0.05 <i>versus</i> WT.</p