Additional file 1: of Altered lung biology of healthy never smokers following acute inhalation of E-cigarettes

Supplemental Methods. Inclusion / exclusion criteria for heathy never smokers. Table S1. E-cigarette Effects Scale. Table S2A. Initiation of E-cigarette Vital Signs. Table S2B. Time point Differences of E-cigarette Vital Signs. Table S3. Urine Nicotine Metabolites Pre- and Post-Exposure to E-Cigarette Aerosols. Table S4. Small Airway Epithelium Differentially Expressed Genes Following Acute Inhalation of E-cigarettes with Nicotine. Table S5. Small Airway Epithelium Differentially Expressed Genes Following Acute Inhalation of E-cigarettes without Nicotine. Table S6. Alveolar Macrophage Differentially Expressed Genes Following Acute In-halation of E-cigarettes with Nicotine. Table S7. Alveolar Macrophage Differentially Expressed Genes Following Acute Inhalation of E-cigarettes without Nicotine. (PDF 199 kb

Role of OSGIN1 in mediating smoking-induced autophagy in the human airway epithelium

<p>Enhanced macroautophagy/autophagy is recognized as a component of the pathogenesis of smoking-induced airway disease. Based on the knowledge that enhanced autophagy is linked to oxidative stress and the DNA damage response, both of which are linked to smoking, we used microarray analysis of the airway epithelium to identify smoking upregulated genes known to respond to oxidative stress and the DNA damage response. This analysis identified <i>OSGIN1</i> (oxidative stress induced growth inhibitor 1) as significantly upregulated by smoking, in both the large and small airway epithelium, an observation confirmed by an independent small airway microarray cohort, TaqMan PCR of large and small airway samples and RNA-Seq of small airway samples. High and low <i>OSGIN1</i> expressors have different autophagy gene expression patterns in vivo. Genome-wide correlation of RNAseq analysis of airway basal/progenitor cells showed a direct correlation of <i>OSGIN1</i> mRNA levels to multiple classic autophagy genes. In vitro cigarette smoke extract exposure of primary airway basal/progenitor cells was accompanied by a dose-dependent upregulation of OSGIN1 and autophagy induction. Lentivirus-mediated expression of OSGIN1 in human primary basal/progenitor cells induced puncta-like staining of MAP1LC3B and upregulation of <i>MAP1LC3B</i> mRNA and protein and <i>SQSTM1</i> mRNA expression level in a dose and time-dependent manner. OSGIN1-induction of autophagosome, amphisome and autolysosome formation was confirmed by colocalization of MAP1LC3B with SQSTM1 or CD63 (endosome marker) and LAMP1 (lysosome marker). Both <i>OSGIN1</i> overexpression and knockdown enhanced the smoking-evoked autophagic response. Together, these observations support the concept that smoking-induced upregulation of OSGIN1 is one link between smoking-induced stress and enhanced-autophagy in the human airway epithelium.</p

Lung function and chest high resolution computed tomography (HRCT) scans.

<p><b>A.</b> Forced expiratory volume in 1 sec (FEV1), forced vital capacity (FVC), and diffusing capacity (DLCO), all as % predicted. <b>B.</b> Ratio of FEV1/FVC as % observed. <b>C.</b> HRCT quantification of emphysema by −950 Hounsfield Units (HU) divided into top (upper lung zones) and bottom (lower lung zones) quartiles by lung volume.</p

High Correlation of the Response of Upper and Lower Lobe Small Airway Epithelium to Smoking

<div><p>The distribution of lung disease induced by inhaled cigarette smoke is complex, depending on many factors. With the knowledge that the small airway epithelium (SAE) is the earliest site of smoking-induced lung disease, and that the SAE gene expression is likely sensitive to inhaled cigarette smoke, we compared upper <i>vs.</i> lower lobe gene expression in the SAE within the same cigarette smokers to determine if the gene expression patterns were similar or different. Active smokers (n = 11) with early evidence of smoking-induced lung disease (normal spirometry but low diffusing capacity) underwent bronchoscopy and brushing of the upper and lower lobe SAE in order to compare upper <i>vs</i> lower lobe genome-wide and smoking-responsive gene expression by microarray. Cluster and principal component analysis demonstrated that, for each individual, the expression of the known SAE smoking-responsive genes were highly correlated in upper and lower lobe pairs, although, as expected, there were differences in the smoking-induced changes in gene expression from individual to individual. These observations support the concept that the heterogeneity observed among smokers in the anatomic distribution of smoking-induced disease are not secondary to the topographic differences in the effects of cigarette smoke on the airway epithelium.</p></div

Cluster analysis of upper <i>vs</i> lower lobe gene expression to assess if, for each individual, the expression of known smoking-responsive genes is highly correlated in the upper <i>vs</i> lower lobes.

<p>Shown is a cluster analysis of small airway epithelium 529 known smoking-responsive probe sets corresponding to 372 genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072669#pone.0072669-StruloviciBarel1" target="_blank">[25]</a>. Note that the smoking-responsive gene expression of the upper <i>vs</i> lower lobes for each of the 11 subjects clusters together.</p

Sampling of small airway epithelium of upper <i>vs</i> lower lobes.

<p><b>A, B.</b> Fluoroscopy of the upper and lower lobes showing the position of the brush used for sampling (arrows). <b>C.</b> Epithelial cell types in the upper <i>vs</i> lower lobes. Shown is data (mean ± standard error) based on counting 500 cells in cytocentrifuge preparations of upper <i>vs</i> lower lobe small airway epithelial samples. p values for paired t-test between upper and lower lobes are shown.</p

Demographics of the Study Population and Airway Epithelial Samples.¹

1<p>Data are presented as mean ± standard deviation.</p>2<p>B = Black, W = White, O = Other.</p>3<p>Pulmonary function testing parameters are given as % of predicted value with the exception of FEV1/FVC, which is reported as % observed; FVC - forced vital capacity; FEV1 - forced expiratory volume in 1 sec; TLC - total lung capacity; DLCO - diffusing capacity.</p>4<p>Small airway epithelium.</p>5<p>As a % of small airway epithelium recovered.</p

Principal component and multivariate vector length analysis of upper <i>vs</i> lower lobe gene expression of known smoking-responsive genes [25].

<p><b>A.</b> Principal component analysis (PCA) of the 529 probe sets of the smoking responsive list. Red = upper lobe samples; blue = lower lobe samples. The line connecting the red and blue data points connects the upper and lower lobe samples from the same individual. <b>B.</b> Plot of the distribution of the median distance between the upper and lower lobe samples for each individual in the space of 529 probe sets in the smoking- responsive list as measured by vector length (red circle) compared to the distribution of median distances between upper and lower lobe samples for 100 sets of 529 probe sets selected at random from the genome-wide list of probe sets present in at least 20% of the samples, minus the 529 smoking-responsive list (boxplot: middle bar is overall median, box contains the inner quartiles, and the whiskers, the 0.95 quartiles).</p

Lung function and chest high resolution computed tomography (HRCT) scans.

<p><b>A.</b> Forced expiratory volume in 1 sec (FEV1), forced vital capacity (FVC), and diffusing capacity (DLCO), all as % predicted. <b>B.</b> Ratio of FEV1/FVC as % observed. <b>C.</b> HRCT quantification of emphysema by −950 Hounsfield Units (HU) divided into top (upper lung zones) and bottom (lower lung zones) quartiles by lung volume.</p

Exome Sequencing of Only Seven Qataris Identifies Potentially Deleterious Variants in the Qatari Population

<div><p>The Qatari population, located at the Arabian migration crossroads of African and Eurasia, is comprised of Bedouin, Persian and African genetic subgroups. By deep exome sequencing of only 7 Qataris, including individuals in each subgroup, we identified 2,750 nonsynonymous SNPs predicted to be deleterious, many of which are linked to human health, or are in genes linked to human health. Many of these SNPs were at significantly elevated deleterious allele frequency in Qataris compared to other populations worldwide. Despite the small sample size, SNP allele frequency was highly correlated with a larger Qatari sample. Together, the data demonstrate that exome sequencing of only a small number of individuals can reveal genetic variations with potential health consequences in understudied populations.</p> </div