Embedding of the phylogeny of the TIR domain of the adaptor molecules into the phylogeny of the TIR domain of the TLRs for mouse and human shows that early gene duplication gave rise to extant adaptor families and paralleled the diversification of TLRs.

<p>Open circles indicate cospeciation events. Closed circles indicate duplication events. Wavy lines indicate failure to diverge and dashed lines indicate loss. Lines with arrows denote "host" switching- the co-option of an adaptor by a different TLR. These data are consistent with the vertebrate MyD88 begin recruited from an earlier invertebrate-like MyD88 that was likely associated with TLR9-like TLRs.</p

Maximum likelihood phylogeny of the TIR domain of the TLR family and TLR adaptor molecules shows strong support for individual branches corresponding to vertebrate TLR3, TLR4, TLR5, and invertebrate TLR9.

<p>Additionally, the invertebrate TLR9 taxa cluster with vertebrate TLRs outside of the main invertebrate TLR branch. A single cluster contains both vertebrate and invertebrate MyD88, but other TLR adaptor molecules form three groups: TIRAP, SARM; and a single branch for both TRIF and TRAM. The tree is rooted by the outgroup TIR domain Homo sapiens interleukin 1 receptor. The numbers indicate boot-strap support out of 1000. Only values above 500 are indicated.</p

Maximum likelihood phylogeny of the TIR domain of the TLR adaptor molecules alone is consistent with the topology observed in <b>Figure 1</b>.

<p>The tree is rooted by the outgroup TIR domain of Homo sapiens TLR5. The numbers indicate boot-strap support out of 1000. Only values above 500 are indicated.</p

RNAi of four <i>D. melanogaster de novo</i> genes causes arrest at the pharate stage.

<p>We knocked down expression of four <i>de novo</i> genes using phiC31 UAS-RNAi lines (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003860#s4" target="_blank">methods</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003860#pgen.1003860.s002" target="_blank">Figure S1</a>) and found that adult RNAi flies did not eclose. (A) By using a GFP marked <i>Actin</i>-Gal4 driver, we found that RNAi (red, diamond) and control (blue, square) flies had similar death rates before the adult stage (wandering larvae were sorted for GFP status and subsequently allowed to develop in separate vials). At the time of pupation, survival rates were not significantly different, but prior to the time of eclosion all RNAi individuals had died (A). By observing developing pupae each day, we found that RNAi pupae but not control pupae arrested at the pharate adult stage, just prior to eclosion (<i>CG34434</i> (B) and <i>CG32582</i> (C) are shown, other crosses similar) with a number of fully pigmented adult features visible (e.g., eyes, wings, legs). A single <i>CG32582</i>-RNAi pupa is shown with a scale for reference (D). The raw number of animals of each genotype are shown as numbers on the plot. As observed with the <i>Actin</i>-Gal4 cross, control but not RNAi adults were produced for all of the crosses.</p

Neutrality index and direction of selection estimates for four <i>de novo</i> genes.

<p>Neutrality index and direction of selection estimates for four <i>de novo</i> genes.</p

Testes biased expression of <i>de novo</i> genes is conserved across species.

<p>We compared the expression of sequences or genes that were collinear to <i>D. melanogaster de novo</i> genes across a number of tissues in the five species of the <i>melanogaster</i> subgroup. In <i>D. sechellia</i> (B) and <i>D. erecta</i>, (D) we dissected male reproductive tracts from flies, and compared expression across the reproductive tracts (Testes “t”, blue), the remainder of the male (Carcass “c”, red), and whole females (Females “f”, green). In <i>D. yakuba</i> (C) and <i>D. simulans</i>, (A) we further dissected male reproductive tracts into testes and accessory glands (“ag”, purple). When available, two biological replicates are shown. Expression shown is relative to the same set of <i>Actin5c</i> primers across all 5 species. In those cases where the gene was expressed at a moderate level in any tissue (shown with a *), expression was always higher in the testes than in female-derived tissues suggesting preservation of testes-bias in expression. For <i>CG31909</i>, which is almost entirely deleted in <i>D. yakuba</i> and <i>D. erecta</i>, primers were designed to the closest alignable sequence to the <i>D. melanogaster</i> gene region, and expression was not detected. <i>CG32582</i> was deleted in <i>D. yakuba</i> and expression was not detected in <i>D, erecta</i>. Despite not containing an open reading frame in <i>D. simulans</i> and <i>D. sechellia</i>, however, <i>CG32582</i> was expressed in a testes-biased manner in these species. Likewise, <i>CG32690</i> was expressed stably in both <i>D. yakuba</i> and <i>D. erecta</i> despite the presence of no ORF in these species. Finally, although the band is not visible for CG31406 in <i>D. yakuba</i> on this gel, the ct values for the testes samples (but not other samples) indicated expression similar to the <i>D. simulans</i> testes samples.</p

Replicate B FASTQ quality-filtered sequencing files from the manuscript 'Alterations in airway microbiota in patients with acute lung injury after burn and inhalation injury'

The second set (B) of duplicate FASTQ sequencing files for 16S rRNA gene sequencing done on airway samples from victims of burn and inhalation injury. Quality filtered using Illumina CASAVA software. Duplicate barcoded paired-end reads were sequenced on the Illumina MiSeq using the molecule tagging method described by Lundberg et. al. (Nature Methods, 2013, DOI: 10.1038/nmeth.2634) and the OTU table was generated using the MTToolbox pipeline (Yourstone, 2015, BMC Bioinformatics, DOI: 10.1168/1471-2105-15-284). The OTU table was imported into the program Explicet (www.explicet.org; Robertson et. al., Bioinformatics, 2013, DOI: 10.1093/bioinformatics/btt526), OTUs identified as the same species were condensed, and this table was exported and used for analysis in R

Stepwise gene model evolution of six <i>D. melanogaster de novo</i> genes.

<p>We used BLASTZ alignments as well as our own MAUVE alignments to infer the evolution of six <i>D. melanogaster de novo</i> genes – <i>CG31909</i> (A), <i>CG33235</i> (B), <i>CG31406</i> (C), <i>CG34434</i> (D), <i>CG32582</i> (E) and <i>CG32690</i> (F). The current <i>D. melanogaster</i> gene model is shown on top, and blocks of sequence that are collinear and align to parts of the <i>D. melanogaster</i> gene (by BLASTZ) are shown below. Blue blocks represent putative protein coding sequence, grey blocks non-coding sequence. <i>D. simulans</i>, <i>D. yakuba</i>, and <i>D. ananassae</i> collinear blocks are shown as appropriate, with the size of the block indicating the relative length of the alignment. The proportion of <i>D. melanogaster</i> bases aligned and the sequence similarity of aligned bases are shown on each block (proportion/similarity). Large scale deletions are shown using vertical lines. The inferred gene model at the nodes is also shown as faded blocks. Finally, expression was measured (using RT-PCR) in each species where collinear sequence could be found. Species where expression was detected are bolded on the phylogeny and the green dot on the phylogeny indicates the inferred start of transcription. A red dot indicates cases where transcription was lost or the gene was lost in that lineage as described.</p

Alterations in airway microbiota in patients with PaO₂/FiO₂ ratio ≤ 300 after burn and inhalation injury

<div><p>Background</p><p>Injury to the airways after smoke inhalation is a major mortality risk factor in victims of burn injuries, resulting in a 15–45% increase in patient deaths. Damage to the airways by smoke may induce acute respiratory distress syndrome (ARDS), which is partly characterized by hypoxemia in the airways. While ARDS has been associated with bacterial infection, the impact of hypoxemia on airway microbiota is unknown. Our objective was to identify differences in microbiota within the airways of burn patients who develop hypoxemia early after inhalation injury and those that do not using next-generation sequencing of bacterial 16S rRNA genes.</p><p>Results</p><p>DNA was extracted from therapeutic bronchial washings of 48 patients performed within 72 hours of hospitalization for burn and inhalation injury at the North Carolina Jaycee Burn Center. DNA was prepared for sequencing using a novel molecule tagging method and sequenced on the Illumina MiSeq platform. Bacterial species were identified using the MTToolbox pipeline. Patients with hypoxemia, as indicated by a PaO₂/FiO₂ ratio ≤ 300, had a 30% increase in abundance of <i>Streptococcaceae</i> and <i>Enterobacteriaceae</i> and 84% increase in <i>Staphylococcaceae</i> as compared to patients with a PaO₂/FiO₂ ratio > 300. Wilcoxon rank-sum test identified significant enrichment in abundance of OTUs identified as <i>Prevotella melaninogenica (p</i> = 0.042), <i>Corynebacterium</i> (<i>p</i> = 0.037) and <i>Mogibacterium</i> (<i>p</i> = 0.048). Linear discriminant effect size analysis (LefSe) confirmed significant enrichment of <i>Prevotella melaninognica</i> among patients with a PaO₂/FiO₂ ratio ≤ 300 (<i>p</i><0.05). These results could not be explained by differences in antibiotic treatment.</p><p>Conclusions</p><p>The airway microbiota following burn and inhalation injury is altered in patients with a PaO₂/FiO₂ ratio ≤ 300 early after injury. Enrichment of specific taxa in patients with a PaO₂/FiO₂ ratio ≤ 300 may indicate airway environment and patient changes that favor these microbes. Longitudinal studies are necessary to identify stably colonizing taxa that play roles in hypoxemia and ARDS pathogenesis.</p></div

Effects of RNAi using “GD” lines targeting <i>de novo</i> genes.

*<p>Males/total offspring.</p