Multiple comparisons from Evolutionary adaptation of the sensitivity of Connexin26 hemichannels to CO₂

Multiple comparisons of medians from 4 species at 3 levels of PCO2</sub

Additional file 1: of Non-compliance with randomised allocation and missing outcome data in randomised controlled trials evaluating surgical interventions: a systematic review

Search strategy

Raw data for human from Evolutionary adaptation of the sensitivity of Connexin26 hemichannels to CO₂

Pixel intensity values at 3 different levels of PCO2</sup

Raw data for mole rat from Evolutionary adaptation of the sensitivity of Connexin26 hemichannels to CO₂

Pixel intensity values at 3 different levels of PCO

Median data from Evolutionary adaptation of the sensitivity of Connexin26 hemichannels to CO₂

Median data for 4 species at 3 levels of PCO2</sub

Raw data for chicken from Evolutionary adaptation of the sensitivity of Connexin26 hemichannels to CO₂

Pixel intensity values at 3 different levels of PCO2</sub

Additional file 1: of Surgeons’ and methodologists’ perceptions of utilising an expertise-based randomised controlled trial design: a qualitative study

Topic guide – methodologists and surgeons. (DOCX 12 kb

Median data from Evolutionary adaptation of the sensitivity of Connexin26 hemichannels to CO₂

Median data for 4 species at 3 levels of PCO<sub>2</sub

The analysis of recombination junctions.

<p>A) Influence of sequence identity and local RNA structure on recombination junctions. The central panel shows the donor (dark shading) and recipient (light shading) partial genomes with protein coding regions indicated flanking a line graph of sequence identity (%) between the non-structural regions of poliovirus type 1 and type 3. Above and below are shown graphs of the mean folding energy differences (MFED; indicated as the percentage difference from 999 sequence-order randomized controls) – a measure of sequence-independent localized RNA structure – over a 250 nt. sliding window (in 30 nt. increments) in the sense and antisense genomes, indicated in solid and dotted lines respectively. The locations of precise and imprecise recombination junctions are indicated in the top and bottom panels by right angle arrows. B) The mean folding energy difference (MFED; percentage difference from 999 sequence order randomized controls) of 100 nt. regions spanning precise and imprecise junctions in donor (sub-genomic replicon) and recipient (CRE-mutant) sequences. Dark and pale shaded bars indicate the MFED of the positive- or negative-sense strand respectively. D and R indicate the donor and recipient sequences respectively. The error bar indicates the standard error. C) Precise junctions do not occur in regions of maximum sequence identity between recombination partners. Individual counts of short lengths of sequence identity between pRLucWT and pT7/SL3 (pale shading; left axis) within the 1058 nt. separating the start of the P2 coding region and the CRE and (dark shading; right axis) counts of precise recombinants exhibiting short regions of identity at the junction (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004191#ppat-1004191-g003" target="_blank">Figure 3</a>).</p

Comparison of the CRE-REP with non-replicative recombination assays and the influence of the viral polymerase on recombination.

<p>A) Recombinant virus yield in the CRE-REP and non-replicative recombination assays. Equimolar amounts of RNA generated <i>in vitro</i> from pT7/SL3 and pRLucWT or pT7/SL3Δ and ΔpRLucWT respectively – 0.5 µg total – were transfected into L929 cell monolayers. Viable progeny virus was harvested at 48 (CRE-REP) or 60 (non-replicative) hours post-transfection and quantified by plaque assay on HeLa cell monolayers. Virus yield is normalized to the yield in the CRE-REP assay, with error bars indicating standard deviation of three independent assays. B) Recombinant virus yield in the CRE-REP assay upon co-transfection of L929 cells in the presence of different concentrations of ribavirin. L929 cell monolayers were pre-treated with ribavirin at the concentrations indicated, transfected with RNA generated <i>in vitro</i> from pT7/SL3 and pRLucWT. After 48 hours in the presence of ribavirin, virus in the supernatant was quantified by plaque assay on HeLa cells. Results are presented (average of three independent assays with error bars indicating standard deviation) as the percentage with reference to transfected untreated monolayers, with statistical significance (** = <i>p</i><0.001) determined by student t tests. C) The influence of 100 mM ribavirin on the yield of viable progeny virus from a non-replicative recombination assay. L929 cell monolayers pre-treated with 100 mM ribavirin were transfected with RNA generated <i>in vitro</i> from pT7/SL3Δ and ΔpRLucWT. After 60 hours, viable recombinant virus in the supernatant was quantified by plaque assay on HeLa cells. Results are normalized to the yield of progeny recombinant virus from the same truncated templates in the absence of ribavirin, with error bars indicating the standard deviation in three independent assays. D) Comparison of recombinant virus yield in the CRE-REP assay upon co-transfection of L929 cells with RNA generated <i>in vitro</i> from pT7/SL3 and RNA from either an unmodified (wildtype; WT) poliovirus type 1 sub-genomic replicon (pRLucWT) or a derivative bearing a high-fidelity G64S substitution in the viral 3D polymerase (pRLucWT_G64S). Results are presented (average of three independent assays with error bars indicating standard deviation) as the percentage normalized to pRLucWT, with statistical significance (** = <i>p</i><0.001) determined by student t tests. E) The influence of a G64S high fidelity polymerase mutation on the yield of viable progeny in a non-replicative recombination assay. As above, L929 cell monolayers were transfected with RNA generated <i>in vitro</i> from unmodified or G64S-bearing ΔpRLucWT templates and RNA from pT7/SL3Δ. Results presented are the percentage yield normalized to the unmodified truncated templates, with error bars indicating the standard deviation of three independent assays. F) The influence of nocodazole on recombinant yield in the CRE-REP assay. As before, RNA generated <i>in vitro</i> from pT7/SL3 and pRLucWT was transfected into L929 cells previously chilled and treated with 5 µM nocodazole (+; see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004191#s4" target="_blank">Materials and Methods</a> for details). Results are normalized to the yield from the same templates in the presence of DMSO carrier alone (-), with error bars indicating the standard deviation of three independent assays (** indicates p<0.005, student t tests). G) The influence of nocodazole on non-replicative recombination. RNA was generated <i>in vitro</i> from pT7/SL3Δ and ΔpRLucWT and transfected, as above, into nocodazole (+) or DMSO carrier-treated (–) L929 cell monolayers. The results show the percentage normalized yield to controls, with error bars indicating the standard deviation of three independent assays.</p