Highly proliferating WI-38T (WI-38T^HP-1/2) exhibit pre-malignant phenotypes.

<p>(<b>A</b>) The growth curve of WI-38T and WI-38T^HP-1/2 cells over 10 days is depicted. (<b>B</b>) The colony formation capability of WI-38T and WI-38T^HP-1/2 is depicted.</p

Mapping of the der(X)t(X;17) breakpoint.

<p>(<b>A</b>) A scheme of the bacterial artificial chromosome (BAC) probes used for the FISH analysis in order to localize the translocation. Filled squares represent probes detecting 17q material on both 17q and Xq, empty squares represent probes detecting 17q material only on 17q and not on Xq. A magnification of the breakpoint region is illustrated on the left-hand side of the panel. (<b>B</b>) The breakpoint region is localized in the 17q24.3 region. Fluorescence <i>in situ</i> hybridization analysis along the 17q arm of WI-38T^HP-1 reveals the breakpoint region in the interval between two probes: RP11-387O17 (left-hand side – red) which is detected on chrX in addition to its normal position on chr17. In contrast, probe RP11-304I14 (right-hand side – red) is detected only on the two normal copies of chr17 and not on chrX. ChrX material is marked in green. (<b>C</b>) No aberrations in chromosome Xq material are detected. Fluorescence <i>in situ</i> hybridization along Xq arm. The two probes detecting the most distant area of the Xq arm (RP11-304H and RP11-26A - red) are visible only on the two copies of chrX and not on chr17. The chr17 centromeric region is marked in green.</p

The der(X)t(X;17) translocation is selected in tumors-derived from WI-38T^HP-1 cells harboring the GSE56 and H-Ras^V12 constructs.

<p>(<b>A</b>) The WI-38T^HP-1 cells that express the GSE56 and oncogenic H-Ras^V12 exhibit genomic instability. Spectral karyotyping (SKY) analysis of WI-38T^HP-1 cells which were introduced with GSE56 and oncogenic H-Ras^V12 reveals genomic instability manifested by polysomy and several genomic aberrations. (<b>B</b>) A SKY analysis of WI-38T^HP-1 tumor-derived cells is shown, the der(X)t(X;17) translocation is circled. (<b>C</b>) A FISH analysis on WI-38T^HP-1 tumor-derived cells that harbor a shRNA against p53 (shp53) and oncogenic H-Ras^V12. The probe detecting chr17q24.3 (RP11-387O17 - red) is visible on two copies of chr17 and on one copy of chrX. A probe detecting the most telomeric X chromosomal region (RP11-26A4) is marked in green.</p

Gain of the BPTF genomic locus and mRNA levels in human tumors.

<p>(<b>A</b>) Representative pictures of the various chromosomal rearrangements in the BPTF locus in six human tumor types. FISH analysis using probe RP11-387O17 (detecting 17q24.3 region in the BPTF locus) and a probe detecting chr17 centromeric material to distinguish between polysomy and gain of the BPTF locus. The nuclei presented are of lung adenocarcinoma cells and are representative of the different probe patterns detected in the other tumors (colon, neuroblastomas and leukimias). a) normal ploidy. b) chr17 polysomy. c) partial trisomy of 17q24.3. d) gain of 17q24.3. (<b>B</b>) A graph depicts the percentage of samples containing gain of 17q24.3 (a sample in which over 30% of cells exhibit gain was considered a positive sample) as assessed by FISH of a paraffin tissue array containing 143 tumor samples. (<b>C</b>) Expression of BPTF in several human tumors. Depicted is the relative expression of BPTF in several human cancers compared to normal tissues as obtained from the ONCOMINE database.</p

Localization of the breakpoint region within the fourth intron of BPTF gene.

<p>(<b>A</b>) DNA samples from primary WI-38 and WI-38T^HP-1 were labeled and hybridized to Agilent custom arrays covering the 0.5-Mb region (the distance between probe RP11-387O17 and RP11-304I14) on chromosome 17 with an average probe spacing of 0.5 kb. (<b>B</b>) Quantitative Real-Time PCR analysis on genomic DNA from WI-38T^HP-1/2 in the BPTF gene revealed a 1.5 fold increase in BPTF genomic dosage compared to WI-38T cells. Two sets of primers were used to detect the gene dosage of BPTF. One set annealing to the 5-prime end of the BPTF gene (BPTF-exon 2) and the second set annealing to the 3-prime end of it (BPTF-exon 30).</p

A Novel Host-Proteome Signature for Distinguishing between Acute Bacterial and Viral Infections

<div><p>Bacterial and viral infections are often clinically indistinguishable, leading to inappropriate patient management and antibiotic misuse. Bacterial-induced host proteins such as procalcitonin, C-reactive protein (CRP), and Interleukin-6, are routinely used to support diagnosis of infection. However, their performance is negatively affected by inter-patient variability, including time from symptom onset, clinical syndrome, and pathogens. Our aim was to identify novel viral-induced host proteins that can complement bacterial-induced proteins to increase diagnostic accuracy. Initially, we conducted a bioinformatic screen to identify putative circulating host immune response proteins. The resulting 600 candidates were then quantitatively screened for diagnostic potential using blood samples from 1002 prospectively recruited patients with suspected acute infectious disease and controls with no apparent infection. For each patient, three independent physicians assigned a diagnosis based on comprehensive clinical and laboratory investigation including PCR for 21 pathogens yielding 319 bacterial, 334 viral, 112 control and 98 indeterminate diagnoses; 139 patients were excluded based on predetermined criteria. The best performing host-protein was TNF-related apoptosis-inducing ligand (TRAIL) (area under the curve [AUC] of 0.89; 95% confidence interval [CI], 0.86 to 0.91), which was consistently up-regulated in viral infected patients. We further developed a multi-protein signature using logistic-regression on half of the patients and validated it on the remaining half. The signature with the highest precision included both viral- and bacterial-induced proteins: TRAIL, Interferon gamma-induced protein-10, and CRP (AUC of 0.94; 95% CI, 0.92 to 0.96). The signature was superior to any of the individual proteins (P<0.001), as well as routinely used clinical parameters and their combinations (P<0.001). It remained robust across different physiological systems, times from symptom onset, and pathogens (AUCs 0.87-1.0). The accurate differential diagnosis provided by this novel combination of viral- and bacterial-induced proteins has the potential to improve management of patients with acute infections and reduce antibiotic misuse.</p></div

Baseline characteristics of the study cohort patients.

<p>Values are presented as total numbers, followed by the corresponding percentages in brackets. Only microorganisms that were detected in more than five patients are presented. CNS- central nervous system, GI—gastroenteritis, LRTI—lower respiratory tract infection, UTRI—upper respiratory tract infection, UTI—urinary tract infection, N/A—healthy controls or patients in which data was not obtained. Influenza A subgroup included H1N1 strains. The atypical bacteria subgroup included <i>Chlamydophila pneumoniae</i>, <i>Mycoplasma pneumonia</i> and <i>Legionella pneumophila</i>. The Enteric viruses subgroup included Rota virus, Astrovirus, Enteric Adenovirus and Norovirus G I/II. In the clinical syndrome analysis the LRTI group included pneumonia, bronchiolitis, acute bronchitis, and laryngitis; the URTI group included pharyngitis, acute otitis media, acute sinusitis and acute tonsillitis.</p><p>Baseline characteristics of the study cohort patients.</p

Signature measures of accuracy for diagnosing bacterial vs viral infections.

<p>Left: Performance estimates and their 95% CIs were obtained using a leave-10%-out cross-validation on all patients in the study cohort (n_Bacterial = 319, n_Viral = 334), Unanimous sub-cohort (n_Bacterial = 256, n_Viral = 271), and Microbiologically confirmed sub-cohort (n_Bacterial = 68, n_Viral = 173). Right: The analysis was repeated after filtering out patients with an equivocal immune response (study cohort [n_Bacterial = 290, n_Viral = 277, n_equivocal = 86], Unanimous [n_Bacterial = 233, n_Viral = 232, n_equivocal = 62] and Microbiologically confirmed [n_Bacterial = 64, n_Viral = 160, n_equivocal = 17]), which resembles the way clinicians are likely to use the signature. Additional measures of accuracy, including positive predictive value and negative predictive value, and their dependency on bacterial prevalence are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120012#pone.0120012.s005" target="_blank">S5 Data</a>.</p><p>Signature measures of accuracy for diagnosing bacterial vs viral infections.</p

Signature performance is robust across different patient subgroups and outperforms lab parameters and protein biomarkers.

<p>(A) Signature AUCs in subgroups of the study cohort (bacterial and viral) are depicted. Square size is proportional to number of patients and error bars represent 95% CI. In the Pathogens analysis, each virus was compared to bacteria affecting the same physiological system, indicated in brackets. R-respiratory, C-central nervous system, G-gastrointestinal, U-urinary, K-skin, S-systemic (i.e. non-localized). Only pathogens detected in more than 5 patients are presented. PED—pediatric emergency departments, ED—emergency departments. For subgroup definitions see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120012#pone.0120012.t001" target="_blank">Table 1</a> legend. (B) Performance of clinical and lab parameters as well as the best performing pair (ANC and Lym %), triplet (ANC, Lym % and Pulse), and quadruplets (ANC, Lym %, Pulse, Mono %) of parameters, the values of which were combined using a logistic regression. Comparison was done on the entire study cohort (n = 653), apart from pulse (recorded in 292 bacterial and 326 viral patients), and respiratory rate (recorded in 292 bacterial and 326 viral patients). The signature performed significantly better (<i>P</i><10^–15) than the optimal quadruplet. (C) The signature performed significantly better (<i>P</i><10^–8) than biomarkers with a well-established role in the host response to infections. For each of the select biomarkers, analysis was performed in a subgroup of the study cohort (43≤n≤154 for each analysis, a convenience sample, n depended on the strength of the signal). Error bars represent 95% CI.</p

The proteins TRAIL, IP-10 and CRP are differentially expressed in bacterial, viral and non-infectious patients.

<p>Box plots for TRAIL (A), IP-10 (B), and CRP (C), measured over the entire study cohort (n = 765) are presented. Red line and circle correspond to group median and average respectively; t-test p-values between bacterial and viral groups and between infectious (bacterial and viral) vs non-infectious (including healthy subjects) are depicted.</p