Cumulative frequency (percent) distribution of raw DEFB-CN determined from DNA obtained from blood and saliva samples.

<p>(Panel A) We observed a slightly lower mean DEFB-CN among gDNA templates isolated from whole blood samples (—_█—) relative to DEFB-CN estimated from gDNA extracted from saliva samples (^•••▴^•••) (<i>p</i><0.05, t-test). However, when stratified by sample type, there were no significant differences found between OM prone (—_█—) and control (^•••▴^•••) DEFB-CN derived from either whole blood samples (Panel B) (<i>p</i> = 0.55, t-test) or saliva samples (Panel C) (<i>p</i> = 0.37, t-test).</p

Subject Demographics.

<p>Subject Demographics.</p

Univariate analyses of subject-level characteristics.

<p>Univariate analyses of subject-level characteristics.</p

Cumulative DEFB-CN frequency (percent) distribution among OM prone and control subjects.

<p>The overall distribution of DEFB-CN did not differ significantly among OM prone (—_█—) or control (<b>^•••</b>▴<b>^•••</b>) study subjects (p>0.05).</p

Nasopharyngeal colonization pattern among study subjects.

<p>*Indicates a significant difference (<i>p</i>-value <0.05) between OM prone and healthy controls.</p

High-density <i>S</i>. Typhimurium (1e8 CFU/ml) in buffer alone (tris-maleate pH 6.4), related to supplemental Fig 1.

High-density S. Typhimurium (1e8 CFU/ml) in buffer alone (tris-maleate pH 6.4), related to supplemental Fig 1.</p

Nucleotide sequence of feCath gene and flanking region.

<p>(A) Gene and mRNA schematic of feCath showing four exons and interspersed intronic sequences. The mRNA sequence includes for the 5′ and 3′ untranslated regions, a coding region for signal sequence, cathelin-like domain, mature peptide, and a polyadenylated tail. (B) Nucleotide sequence of <i>feCath</i> gene and flanking regions. Numbering begins arbitrarily at the 5′ most nucleotide. Lowercase letters indicate intronic and flanking sequence, uppercase letters indicate exonic sequence with deduced amino acid sequence of coding region below in three-letter code. Polyadenylation signal is underlined. The cDNA sequence was deposited in GenBank (Accession #: HQ221766).</p

3′ RACE Analysis and Identification of feCath.

<p>(A) Schematic of 3′ RACE strategy targeting the signal sequence region and the propeptide (cathelin) domain with sense primers and using antisense adaptor primer, AP1. Feline bone marrow RNA was reverse transcribed with oligo-dT conjugated to adaptor primer 1/2 (AP1/2). Sense primers (sequences found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018756#pone-0018756-t001" target="_blank">Table 1</a>) were used to amplify cathelicidin related sequences in the pool of bone marrow cDNA. (B) Agarose gel electrophoresis analysis of 3′ RACE PCR products from (A). HaeIII digested Phi-X174 phage DNA used as the marker.</p

Purification and Structure of feCath.

<p>(A) Reverse phase HPLC purification of synthetic feCath using a gradient of acetonitrile. Dotted line and inset indicate elution gradient. Arrow indicates positive fraction for feCath. AU-PAGE of synthetic LL-37 and feCath showing peptide homogeneity and equal peptide concentrations by Simply Blue staining (inset). (B) CD spectra of feCath (50 µM) in water (gray line) and 10 mM SDS (black line).</p

Gel electrophoresis of DNA binding assay.

<p>A DNA ladder (HaeIII digested Phi-X174, 200 ng) was incubated with increasing concentrations of peptide (100 ng, 300 ng, 1000 ng) for 5 minutes in 0.05% acetic acid at room temperature. The reaction mixture was resolved using agarose gel electrophoresis (2% w/v). Arrow indicates retarded migration of Phi-X174 DNA into the gel.</p