Additional file 1 of Ex-vivo RNA expression analysis of vaccine candidate genes in COPD sputum samples

Additional file 1: Table S1. PCR primer sequences for NTHi and Mcat genes. Figure S1. NTHi gene RNA concentrations in NTHi positive samples did not differ between stable visits (ST) and exacerbation visits (EX). Figure S2. Mcat gene RNA concentrations in Mcat positive samples did not differ between stable visits (ST) and exacerbation visits (EX). Additional methods

Schematic representation of the contribution of SslE to <i>E.coli</i> pathogenesis.

<p>Gut mucus forms two layers, an inner firm mucus layer devoid of bacteria, and an outer layer that is not sterile and is a major habitat for commensal bacteria. <i>E. coli</i> can penetrate this barrier through the SslE-mediated enzymatic degradation of the mucus, targeting epithelial cells. This interaction will eventually lead to IL-8 release and neutrophils recruitment.</p

Stable iron isotopes and microbial mediation in red pigmentation of the Rosso Ammonitico (mid-late Jurassic, Verona area, Italy).

The iron (Fe) isotopic composition of 17 Jurassic limestones from the Rosso Ammonitico of Verona (Italy) have been analyzed by Multiple-Collector Inductively Coupled Plasma Mass Spectrometry (MC-ICP-MS). Such analysis allowed for the recognition of a clear iron isotopic fractionation (mean -0.8 per thousand, ranging between -1.52 to -0.06 per thousand) on a millimeter-centimeter scale between the red and grey facies of the studied formation. After gentle acid leaching, measurements of the Fe isotopic compositions gave delta(56)Fe values that were systematically lower in the red facies residues (median: -0.84 per thousand, range: -1.46 to +0.26 per thousand) compared to the grey facies residues (median: -0.08 per thousand, range: -0.34 to +0.23 per thousand). In addition, the red facies residues were characterized by a lighter delta(56)Fe signal relative to their corresponding leachates. These Fe isotopic fractionations could be a sensitive fingerprint of a biotic process; systematic isotopic differences between the red and grey facies residues, which consist of hematite and X-ray amorphous iron hydroxides, respectively, are hypothesized to have resulted from the oxidizing activity of iron bacteria and fungi in the red facies. The grey Fe isotopic data match the Fe isotopic signature of the terrestrial baseline established for igneous rocks and low-C(org) clastic sedimentary rocks. The Fe isotopic compositions of the grey laminations are consistent with the influx of detrital iron minerals and lack of microbial redox processes at the water-interface during deposition. Total Fe concentration measurements were performed by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) (confirmed by concentration estimations obtained by MC-ICP-MS analyses of microdrilled samples) on five samples, and resultant values range between 0.30% (mean) in the grey facies and 1.31% (mean) in the red facies. No correlation was observed between bulk Fe content and pigmentation or between bulk Fe content and Fe isotopic compositions. The rapid transformation of the original iron oxyhydroxides to hematite could have preserved the original isotopic composition if it had occurred at about the same temperature. This paper supports the use of Fe isotopes as sensitive tracers of biological activities recorded in old sedimentary sequences that contain microfossils of iron bacteria and fungi. However, a careful interpretation of the iron isotopic fractionation in terms of biotic versus abiotic processes requires supporting data or direct observations to characterize the biological, (geo)chemical, or physical context in relation to the geologic setting. This will become even more pertinent when Fe isotopic studies are expanded to the interplanetary realm.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

<i>E. coli</i> growth rate in association with HT29-MTX cells.

<p>Using the transwell system, three sets of non-differentiated cells (A) and three sets of differentiated-mucus—producing- cells (B) were infected with WT and <i>sslE</i> KO bacteria for 2 hrs. Medium was removed to eliminate non-adhering bacteria and two sets of wells were used to do a total association assay, while the other wells were further incubated for 24 and 48 hrs at air liquid interface. At the end of the incubation period a total association assay was performed. The data presented are means ± standard deviations for 3 replicate experiments (n = 9).*P≤0.05. Error bars, SD.</p

Growth curves of strain IHE3034 in the presence of mucin.

<p>Growth curves of (A) IHE3034 and IHE3034Δ<i>sslE</i> strains (B) IHE3034Δ<i>sslE</i>::<i>sslE</i>_WT and IHE3034Δ<i>sslE</i>::<i>sslE</i>_mut, are shown in M9 minimal medium with and without the addition of mucin harvested from HT29-MTX cells after 6 h of incubation (Dash line). Measurements were performed in triplicate.</p

Kinetics of transepithelial electrical resistance in HT29-MTX cells over a 21 day period of differentiation.

<p>(A) TEER values were measured at different time points throughout a 21 day period of differentiation. (B) RT-PCR analysis of MUC mRNAs. After 21 days of differentiation, HT29-MTX mRNA was isolated, and cDNA was used to compare the level of MUC gene expression. Data are represented as relative fold increase of MUC mRNA in differentiated mucus-producing cells <i>versus</i> non-differentiated (control) cells. Control cells were assigned a value of 1.0. Levels of the different transcripts were normalized to β-actin, used as a house keeping gene. Error bars represent the SD.</p

SslE contributes to the capacity of IHE3034 strain to reach the surface of mucus-producing epithelial cells.

<p>(A) Schematic representation of the experimental procedure. (B), Percentage of mucus trapped (left panel) and cell associated (right panel) <b>I</b>HE3034 (WT), IHE3034Δ<i>sslE</i> (KO), IHE3034Δ<i>sslE</i>::<i>sslE</i>_WT (COMPL) and IHE3034Δ<i>sslE</i>::<i>sslE</i>_mut (MUT) bacteria after 4hrs of infection. <i>n</i> = 4, **P≤0.01; Error bars, SD; percentages were calculated on recovered CFU respect to the starting inoculum. (C) Co-infection experiments: percentage of mucus trapped (left panel) and cell associated (right panel) <b>I</b>HE3034 (WT), IHE3034Δ<i>sslE</i> (KO) or IHE3034Δ<i>sslE</i> plus IHE3034 (KO_COINF) bacteria. Strains were plated on both non-selective and selective plates to differentiate WT and Δ<i>sslE</i> for CFU counts. <i>n</i> = 4, **P≤0.01; Error bars, SD;</p

SslE induces IL-8 secretion and stimulates neutrophil chemotaxis.

<p>(A) IL-8 levels in supernatants from the apical compartments of polarized HT29-MTX cells infected with IHE3034 WT and IHE3034Δ<i>SslE</i> KO bacteria. Data show mean chemokine concentrations in culture supernatants representative of three independent experiments *P≤0.05. Error bars, SD. (B) To measure neutrophil chemotaxis, bottom chambers of transwell supports were filled with supernatants deriving from HT29-MTX infected cells. Neutrophils were added to the upper chambers. After 1 h at 37°C, cells that had migrated toward the lower compartments were quantified by flow cytometry. DMEM has been used as a negative control and recombinant IL-8 as a positive control. The graph represents a typical experiment out of three performed with similar results. **** P ≤ 0.0001. Error bars, SD.</p

Modulation of SslE gene expression upon interaction with intestinal epithelial cells.

<p>(A) SslE transcription level in bacteria adhering to differentiated cells and in planktonic organisms. (B) SslE transcription level upon the interaction with differentiated or not differentiated HT29-MTX cells. Starting mRNA copy number of the unknown samples was determined using the comparative ΔΔ_{<b><i>CT</i></b>} method, and levels of the different transcripts were normalized to 16S rRNA, used as a housekeeping gene. Error bars represent the SD. **, P ≤ 0.01. n = 3.</p

cAMP production and PKA activation in T cells treated with high and low concentrations of CyaA and ET.

<p>(A) Time course analysis of cAMP production in purified peripheral blood T lymphocytes treated with high (CyaA hi, 45 nM ; ET hi, 110 nM) (<i>top left</i>) or low (CyaA lo, 0.28 nM; ET lo, 0.11 nM) (<i>top right</i>) concentrations of CyaA or ET, or activated by TCR/CD3 cross-linking (<i>bottom right</i>). The histogram on the bottom left panel also includes the quantification of cAMP in lysates of T cells treated with the adenylase cyclase deficient CyaA and ET mutants (45 nM CyaA-E5, 110 nM EL1) for 2 h or 6 h, respectively. The results, which show the levels of cAMP measured in T cell lysates, are expressed as fmoles/10⁶ cells. Representative experiments, each carried out on duplicate samples from individual healthy donors, are shown (<i>n</i>≥4). (B) <i>Top</i>, Immunoblot analysis of the phosphorylation state of PKA substrates in post-nuclear supernatants of T cells treated with 45 nM CyaA (CyaA hi) or 0.28 nM CyaA (CyaA lo), or 110 nM ET (ET hi) or 0.11 nM ET (ET lo), for 2 h (CyaA) or 6 h (ET), and then lysed as such or after stimulation for 1 min with anti-CD3 mAb (CD3). A sample stimulated with anti-CD3 mAb alone was also included. The immunoblot was carried out using an antibody which recognizes a phosphorylated PKA consensus sequence (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000325#s4" target="_blank">Materials and Methods</a>). The stripped filter was reprobed with a phosphospecific antibody which recognizes the active form of CREB <i>(middle)</i>. The fold activation of CREB in CyaA/ET treated samples <i>vs</i> untreated control in the experiment shown was the following: CyaA low, 8.3; CyaA high, 19.0; ET low, 8.7; ET high, 79.9. The levels of phospho-CREB in the samples treated with CyaA or ET in combination with anti-CD3 mAb <i>vs</i> samples treated with anti-CD3 mAb alone (taken as 100%) were the following: CyaA low+CD3, 98.1±4.8%; CyaA high+CD3, 103.4±8.7%; ET low+CD3, 102.1±3.2%; ET high+CD3, 112.1±8.1% (<i>n</i> = 3). A control anti-actin blot is shown below. None of the treatments modified the expression levels of CREB (data not shown). Representative experiments are presented (<i>n</i>≥3). The migration of molecular mass markers is indicated. (C) Quantification of CREB phosphorylation in post-nuclear supernatants of T cells treated with 45 nM CyaA (CyaA hi)/CyaA-E5 or 0.28 nM CyaA (CyaA lo), or 110 nM ET (ET hi) /EL1 or 0.11 nM ET (ET lo), for 2 h (CyaA) or 6 h (ET). Where indicated, cells were pretreated for 1 h with 20 µM H89. A sample stimulated for 30 min with 100 µM 8-CPT was included as positive control. The data were obtained by laser densitometry of anti-phospho-CREB immunoblots. The results are expressed as relative CREB phosphorylation (fold activation <i>vs</i> untreated controls) (<i>n</i> = 2). (D) Quantification of cAMP in lysates of T cells treated as in B. The results, which show the levels of cAMP measured in T cell lysates, are expressed as fmoles/10⁶ cells. A representative experiment, carried out on duplicate samples from an individual healthy donor, is shown (<i>n</i> = 3). ***<i>P</i>≤0.001; **<i>P</i>≤0.01; *<i>P</i>≤0.05. Error bars, SD.</p