Amplification of immRNA fragments by 3´-RACE.

<p>Total RNA from <i>S</i>. <i>cerevisiae</i> CEN.PK2-1c strains carrying vectors YEPaO4 (1), YEKlO3 (2) or YEDrO5 (3) was transcribed to cDNA using a poly(A)-specific primer (+). Absence of DNA was checked by control reactions lacking the reverse transcriptase (-). ImmRNA was then amplified with one primer specific for the 5´ end of the immORF and a second primer complimentary to the poly(A)-specific anchor. <i>ERG3</i> cDNA was amplified using <i>ERG3</i> specific primers.</p

Northern blot analysis of immRNA from different genetic backgrounds.

<p>Total RNA isolated from strains expressing the respective immORFs from VLEs or nuclear vectors was probed using the respective immRNA specific DIG-labelled probes. A: <i>PaORF4</i> (1092 bp) expressed in <i>Pichia acaciae</i> from pPac1-1, pPac1-2 (1) or in <i>S</i>. <i>cerevisiae</i> 301 YEPaO4 (2). B: <i>KlORF3</i> (1287 bp) expressed in <i>K</i>. <i>lactis</i> from pGKL1, pGKL2 (1) or in <i>S</i>. <i>cerevisiae</i> CEN.PK2-1c YEKlO3 (2). C: <i>DrORF5</i> (1062 bp) expressed in <i>D</i>. <i>robertsiae</i> from pWR1A, pWR1B (1) or in <i>S</i>. <i>cerevisiae</i> CEN.PK2-1c YEDrO5 (2). D: Scheme of immORF transcription and mRNA sizes transcribed from cytoplasmic VLEs (pGKL1, pPac1-2 and pWR1B, left side) or from episomal vectors, which are located in the nucleus (right side). ImmORFs are grey shaded.</p

The codon adapted immORFs <i>PaORF4ms</i> and <i>KlORF3ms</i> confer resistance to the respective toxins PaT and zymocin.

<p>A: <i>S</i>. <i>cerevisiae</i> CEN.PK2-1c transformed with yeast episomal vectors carrying <i>ADH1pr</i> fusions of either the A/T-rich (<i>PaORF4</i> and <i>KlORF3</i>) or the A/T-decreased (<i>PaORF4ms</i> and <i>KlORF3ms</i>) immunity genes or the empty vector (control) were dropped on YPD agar, inoculated with <i>P</i>. <i>acaciae</i> (PaT) or <i>K</i>. <i>lactis</i> (zymocin) at the rim and incubated over night at 30°C. B: Microtiter plate assays. Supernatants from a <i>P</i>. <i>acaciae</i> culture were added in different concentrations (RCF) to micro cultures of the yeast strains described in A. Relative growth was determined (OD_600nm) after 24 hours at 30°C; values refer to strains grown in toxin-free medium. A relative concentration factor (RCF) of 1 relates to the toxin concentration in non diluted supernatants. Each value represents a mean of triplicates.</p

Schematic representation of the identified mRNA fragments of the immORFs <i>PaORF4</i> (A), <i>KlORF3</i> (B) and <i>DrORF5</i> (C).

<p>The cDNA fragments reached the indicated indicated nucleotide positions and were extended by a stretch of 7–66 adenyl (A) nucleotides, the length of which is indicated for each particular molecule that was obtained. Asterisks (*) mark fragments which were identified by the linker ligation method; all of the other fragments were identified by 3´-RACE. For complete gene sequences including the identified mRNA truncation sites we refer to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005005#pgen.1005005.s001" target="_blank">S1 Fig</a></p

RT-PCR analysis of immRNA.

<p>mRNA of <i>PaORF4</i>/<i>PaORF4ms</i> and <i>KlORF3</i>/<i>KlORF3ms</i> or the control gene <i>ERG3</i> was analysed by reverse transcription (+RT) using total RNA as the template. The absence of contaminating DNA was checked by control reactions lacking the reverse transcriptase (-RT). A: mRNA of the original A/T-rich immORFs <i>PaORF4</i> and <i>KlORF3</i> was analysed using total RNA isolated from <i>S</i>. <i>cerevisiae</i> CEN.PK2-1c strains expressing <i>ADH1pr</i>-<i>PaORF4</i> (1) or <i>ADH1pr</i>-<i>KlORF3</i> (2) from nuclear single copy vectors (nuc sc) or nuclear high copy vectors (nuc hc) or from <i>S</i>. <i>cerevisiae</i> F102.2 MS1607 expressing <i>PaORF4</i> from a cytoplasmic VLE (cyt). PC: as a positive control for checking primer binding vector DNA carrying the immunity genes <i>PaORF4</i> or <i>KlORF3</i> was used. B: Reverse transcription of mRNA of the codon adjusted, A/T-decreased immunity genes <i>PaORF4ms</i> and <i>KlORF3ms</i> using as the template total RNA isolated from strains expressing <i>ADH1pr</i>-<i>PaORF4ms</i> (1) or <i>ADH1pr</i>-<i>KlORF3ms</i> (2) from high copy vectors. C: Schematic representation of RT-PCR analyses for the detection of immRNAs. Primers, depicted as small arrows, are denoted as such (for sequences see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005005#pgen.1005005.s006" target="_blank">S3 Table</a>). wt denotes the original immunity gene sequences (grey shaded large arrows), ms denotes the modified (G/C rich) sequences (white large arrows). The expected sizes of RT-PCR products refer to the size of the structural genes and are given in base pairs (bp).</p

The zymocin immORF confers full immunity to exo zymocin and intracellularly expressed ACNase subunit γ-toxin.

<p>(A) Exo-zymocin resistance of isogenic WT, <i>elp3</i>∆, and WT strains carrying YEKlO3ms (<i>ADHpr-KlORF3ms</i>) analyzed by microtiter plate assay as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005005#pgen.1005005.g001" target="_blank">Fig 1</a>. (B) Intracellular coexpression of zymocin immORF and γ-toxin. Tenfold serial dilutions of cultures were spotted on YPD media (no zymocin, γ-toxin OFF and zymocin), YPD media supplemented with zymocin (zymocin) or YNB media with galactose as the sole carbon source (γ-toxin ON). All strains analyzed in (B) carry pRK57 for GAL driven, intracellular expression of γ-toxin and additionally carry YEKlO3 (<i>ADH1pr-KlORF3</i>) or YEKlO3ms (<i>ADH1pr-KlORF3ms</i>). WT and <i>elp3</i>∆ indicate the additional presence or absence of a deletion in <i>ELP3</i> (<i>elp3</i>∆).</p

Descriptive analysis of post-operative data.

<p>Descriptive analysis of post-operative data.</p

Influence of factor XIII activity on post-operative transfusion in congenital cardiac surgery—A retrospective analysis

<div><p>Objectives</p><p>Coagulation factor XIII (FXIII) plays a key role in fibrin clot stabilization—an essential process for wound healing following cardiothoracic surgery. However, FXIII deficiency as a risk for post-operative bleeding in pediatric cardiac surgery involving cardiopulmonary bypass (CPB) for congenital heart disease (CHD) is controversially discussed. Thus, as primary outcome measures, we analyzed the association of pre-operative FXIII activity and post-operative chest tube drainage (CTD) loss with transfusion requirements post-operatively. Secondary outcomes included the influence of cyanosis and sex on transfusion.</p><p>Methods</p><p>Our retrospective analysis (2009–2010) encompassed a single center series of 76 cardio-surgical cases with CPB (0–17 years, mean age 5.61 years) that were post-operatively admitted to our pediatric intensive care unit (PICU). The observational period was 48 hours after cardiac surgery. Blood cell counts and coagulation status, including FXIII activity were routinely performed pre- and post-operatively. The administered amount of blood products and volume expanders was recorded electronically, along with the amount of CTD loss. Uni- and multivariate logistic regression analysis was performed to calculate the associations (odds ratios) of variables with post-operative transfusion needs.</p><p>Results</p><p>FXIII activities remained stable following CPB surgery. There was no association of pre- and post-operative FXIII activities and transfusion of blood products or volume expanders in the first 48 hours after surgery. Similarly, FXIII showed no association with CTD loss. Cyanosis and female sex were associated with transfusion rates.</p><p>Conclusions</p><p>Although essentially involved in wound healing and clotting after surgery, FXIII activity does not serve as a valid predictor of post-operative transfusion need.</p></div

Analysis of the associations between intra-operative volume management and post-operative blood tests / transfusion requirements.

<p>Analysis of the associations between intra-operative volume management and post-operative blood tests / transfusion requirements.</p

Multivariate logistic regression analysis of post-operative transfusion requirement.

<p>Multivariate logistic regression analysis of post-operative transfusion requirement.</p