大野論文正誤表

Figure S1. DNA quality control. TapeStation profiles of gDNA isolated from FF and matching FFPE block tumor tissues from 5 lung ADC patients. In each profile, the DIN, indicative of gDNA degradation status, is also displayed (numerical assessment ranges from 10 for undamaged gDNA, to 1 for highly fragmented gDNA) (a). The Table reports the gDNA concentration (ng/ul) assessed by NanoDrop, Qubit, and TapeStation, and purity (260/280 and 260/230) (b). Additionally, AYR and DIN parameters, indicative of FFPE gDNA fragmentation status, evaluated by a multiple PCR assay and TapeStation respectively, are reported. Image of agarose gel 1 % shows the gDNA smears indicative of the different degradation status of FF and FFPE gDNAs (c). Figure S2. The workflow illustrates samples processing and WES data analysis for both exome enrichment platforms. (PDF 187 kb

Additional file 4: Table S9. of Performance comparison of two commercial human whole-exome capture systems on formalin-fixed paraffin-embedded lung adenocarcinoma samples

Coverage distribution across all the coding exons of the 623 cancer related genes in each library. For each gene, the table reports the number of coding RefSeq exons downloaded from UCSC, their presence within 21 commercial re-sequencing cancer panels and further four cancer genes databases. The coverage distribution across all coding exons was performed using the GATK DiagnoseTarget tool. For each WES capture platform we reported: the number of ‘critical’ exons (average depth of coverage < 10× for at least 20 % of the length of the interval and with insufficient median depth across all FF and FFPE libraries), the number of exon regions missed by the kit target design file, and the % of passed exons (average depth of coverage ≥ 10× for at least 20 % of the length of the interval). (XLSX 120 kb

Additional file 3: Table S7 and Table S8. of Performance comparison of two commercial human whole-exome capture systems on formalin-fixed paraffin-embedded lung adenocarcinoma samples

Table S7. Mean coverage achieved by Agilent SureSelect and Roche NimbleGen libraries within 90 PCR-capture amplicons. Mean coverage ± SD within 90 regions amplified by AmpliSeq Colon and Lung Cancer Panel v.1 (Thermo Fisher Scientific) from ‘FF’, ‘FFPE’ and ‘FF plus FFPE’ samples achieved by Agilent SureSelect and Roche NimbleGen libraries respectively. In each column, the mean coverage values are reported for each amplicon, and the heat map was created using two-color scale (lowest value is represented by dark blue and highest value by dark red). Table S8. Variant calling comparison between the two WES systems (Agilent SureSelect and Roche NimbleGen) and the AmpliSeq Colon and Lung Cancer Panel. List of FFPE and matched FF samples genetic variants called by VC v.4.2 plugin on Ion PGMTM data and GATK pipeline in both exome capture systems. All variants are annotated with gene ID, locus, reference sequence, variant allele according to the hg19 Reference Genome. The red bars show the variant allele frequency (%) detected by VC on Ion pipeline and GATK on both Agilent SureSelect and Roche NimbleGen WES (0* means variant not called but found by IGV visual inspection of BAM files). All variants are annotated for COSMIC or dbSNP (rs number) together with the codons involved and the amino acid change (AA). The 'Effect' column reports if the variant is in a coding region, discerning between nonsynonymous, synonymous and non-sense, or in an intron, downstream the gene or in a splicing region. The last four columns of the table reports the Minor Allele Frequency (MAF) reported in the 1000 Genomes Project, the prediction effect on the protein based on SIFT and Polyphen algorithms and the conservation score namely GERP. For SIFT prediction, the higher the number, the lower is the effect, whereas for Polyphen prediction is the opposite. Thus, a higher score for GERP indicates a higher conservation of the gene across 34 mammalian species. Abbreviation: - not available data. (XLSX 44 kb

Additional file 2: Table S1, Table S2, Table S3, Table S4, Table S5, and Table S6. of Performance comparison of two commercial human whole-exome capture systems on formalin-fixed paraffin-embedded lung adenocarcinoma samples

Table S1. Sequencing metrics for libraries prepared with both Agilent SureSelect XT v.5 and Roche NimbleGen v.3.0  kits starting from five matched FF and FFPE tumor samples. Table S2. Variant detection comparison between matched FF-FFPE pairs. For each matched FF-FFPE pair, the number and the percentage of both SNVs and InDels common to both sample types, and unique to either FF or FFPE sample are reported. Table S3. Genotype CR and NRDR between matched FF-FFPE pairs at increasing coverage thresholds. For each matched FF-FFPE pair, the genotype CR was computed as the ratio between the sum of concordant genotypes and the sum of all genotypes called at genomic positions covered at least a certain coverage threshold (from 1 to 50×) in both samples (a). For each matched FF-FFPE pair, the NRDR was computed as the ratio between the sum of non-concordant genotypes and the sum of all non-reference genotypes called at genomic positions covered at least a certain coverage threshold (from 1 to 50×) in both samples (b). Table S4. Genotype CR and NRDR between matched FF-FFPE pairs computed for each transition type at increasing coverage thresholds. For each matched FF-FFPE pair, the genotype CR for each transition type was computed as the ratio between the sum of concordant genotypes and the sum of all genotypes called at genomic positions covered at least a certain coverage threshold (from 1 to 50×) in both samples; p-values for two-tail t-test for each comparison between two transition types are reported at the bottom of the table (a). For each matched FF-FFPE pair, the NRDR for each transition type was computed as the ratio between the sum of non-concordant genotypes and the sum of all non-reference genotypes called at genomic positions covered at least a certain coverage threshold (from 1 to 50×) in both samples; p-values for two-tail t-test for each comparison between two transition types are reported at the bottom of the table (b). Table S5. Variant detection comparison between exome libraries prepared with both Agilent SureSelect and Roche NimbleGen kit. The table reports the total number and the percentage of SNVs and InDels common to both library prep types for each sample, and unique to either Agilent SureSelect and Roche NimbleGen kit. The comparison was performed considering both the whole kit-specific target region and the 42 Mb of common target region. Table S6. Genotype CR and NRDR rates within the shared 42 Mb target region between Agilent SureSelect and Roche NimbleGen at increasing coverage thresholds. For each sample, the genotype CR was computed as the ratio between the sum of concordant genotypes and the sum of all genotypes called at genomic positions covered at least a certain coverage threshold (from 1 to 50×) in both Agilent SureSelect and Roche NimbleGen libraries (a). For each sample, the NRDR was computed as the ratio between the sum of non-concordant genotypes and the sum of all non-reference genotypes called at genomic positions covered at least a certain coverage threshold (from 1 to 50×) in both in both Agilent SureSelect and Roche NimbleGen libraries (b). (XLSX 54 kb

Easternmost Mediterranean evidence of the Zanclean flooding event and subsequent surface uplift: Adana Basin, southern Turkey

<p>According to the literature, the Adana Basin, at the easternmost part of the Mediterranean Basin in southern Turkey, records the Pliocene stage with shallow-marine to fluvial deposits. Our micropalaeontological analysis of samples from the Adana Basin reveal Late Lago–Mare biofacies with Paratethyan ostracod assemblages pertaining to the <em>Loxocorniculina djafarovi</em> zone. Grey clays rich in planktonic foraminifera lie above the Lago–Mare deposits. Within the grey clays, the continuous occurrence of the calcareous nannofossil <em>Reticulofenestra zancleana</em> and the base of the <em>Reticulofenestra pseudoumbilicus</em> paracme points to an Early Zanclean age (5.332–5.199 Ma). Both ostracod and benthic foraminifera indicate epibathyal and bathyal environments. ⁸⁷Sr/⁸⁶Sr measurements on planktonic and benthic foraminifera fall below the mean global ocean value for the Early Zanclean, indicating potentially insufficient mixing of low ⁸⁷Sr/⁸⁶Sr Mediterranean brackish ‘Lago–Mare’ water with the global ocean in the earliest Pliocene. We utilize the ages and palaeodepths of the marine sediments together with their modern elevations to determine uplift rates of the Adana Basin of 0.06 to 0.13 mm a⁻¹ since 5.2–5.3 Ma (total uplift of 350–650 m) from surface data, and 0.02–0.13 mm a⁻¹ since <em>c.</em> 1.8 Ma (total uplift of 30–230 m) from subsurface data. </p

IL-12Rβ2 expression and function in normal bronchial epithelial cells.

<p>4A. IL-12RB2 expression in human primary bronchial epithelial cells, as assessed by RT-PCR. From left to right: MW = molecular weight; NC = negative control (Raji cell line); three different NBEC cultures (NBEC #1, #2 and #3) are shown. 4B. IL-12Rβ2 surface expression in human NBEC, as assessed by flow cytometry. Open profile: IL-12Rβ2 staining; dark profile: isotype matched mAb staining. 4C. Short circuit current recordings in normal bronchial epithelial cells. The figure depicts two representative experiments from control (<i>top</i>) and IL-12 treated (<i>bottom</i>) epithelia showing responses to amiloride (10 µM, apical), forskolin (20 µM, apical and basolateral), UTP (100 µM, apical), and CFTR_inh-172 (10 µM, apical). 4D. Cytokine release by human NBEC, as assessed by Bio-Plex Assay. Pooled results from supernatants of three different bronchial epithelial cell suspensions are shown. IL-12 treatment reduced significantly the release of IL-6 (P = 0.0049), IL-8 (P = 0.0071), FGF-b (P = 0.0231), GM-CSF (P = 0.0028), IP-10 (P<0.0001), MCP-1 (P = 0.0002) and RANTES (P = 0.0108).</p

IL-12Rβ2 expression and function in human NSCLC cell lines.

<p>2A. IL-12RB2 expression in NSCLC cell lines, as assessed by RT-PCR. From left to right: MW = molecular weight; NC = negative control (Raji cell line); PC = positive control (total tonsil B cells); different NSCLC cell lines (Colo699, Calu6, Calu1, A549, SK-MES-1, GLC82 and Calu6/β2 cells) are shown. 2B. Left panel. IL-12Rβ2 protein expression in Calu6/β2 cells, as assessed by flow cytometry. Open profile: IL-12Rβ2 staining; dark profile: isotype matched antibody staining. Right panel. IL-6 intracellular staining in Calu6/β2 cells cultured with medium or hrIL-12 for 48 h, as assessed by flow cytometry. Open profile: IL-6 staining in cells cultured with medium; dark profile: isotype matched antibody staining, dashed line: IL-6 staining in cells cultured with IL-12. 2C. Angiogenic activity of supernatants from Calu6/β2 cells cultured with medium alone or hrIL12. CAM treated with sponges loaded with supernatant from the untreated cells were surrounded by allantoic vessels developing radially towards the implant in a ‘spoked-wheel’ pattern (upper left panel). When supernatants from hrIL-12treated Calu6/β2 cells was tested, a significant reduction (P = 0.001) of the angiogenic response was appreciable (upper right panel). Lower panels show the angiogenic activity of Calu6/β2 cells in the presence of an anti-IL-6 mAb (left panel) or of an anti-VEGF-C mAb (right panel). These experiments were repeated three times. Original magnification: ×50.</p

Anti-tumor activity of IL-12 on NSCLC <i>in vivo.</i>

<p>3A. Volume of tumors grown after Calu6/β2 cell inoculation orthotopically (left panel) or subcutaneously (right panel) in PBS and hrIL-12 treated animals. Animals injected orthotopically were sacrificed after 23 days, those injected subcutaneously after 14 days. Volume of tumors grown after inoculation orthotopically (left panel) or subcutaneously (right panel) of Calu6 cell transfected with the empty vector hrIL-12 treated animals was also shown (empty vector+IL12). The differences in size between tumors removed from PBS and hrIL-12 treated mice were evaluated by Mann-Whitney U test. Boxes indicate values between the 25^th and 75^th percentiles, whisker lines represent highest and lowest values for each group. Horizontal lines represent median values. 3B. Tumors (developed after s.c injection) injected subcutaneously with Calu6/β2 cells in PBS-treated SCID/NOD mice are mostly formed of nests of undifferentiated, pleomorphic and proliferating cells (mitotic (figures) features indicated by arrows) rapidly infiltrating the underlying muscle layers (arrowheads) (a), and supplied by a distinct microvessel network, as assessed by laminin staining (b). In hrIL-12 treated mice, tumor histology is altered by the appearance of large areas of ischemic-hemorrhagic necrosis (N) (c) associated with defective microvascular architecture (d) (×400). Orthotopical injection of Calu6/β2 cells gave rise, in PBS-treated mice, to tumors with istopathological features (e) similar to those of subcutaneously developed tumors (a) and supplied by a well developed microvascular network (f). As observed in subcutaneous tumors, in orthotopic tumors as well hrIL-12 treatment induced wide necrosis (g) and severe microvascular alterations (h) (×400). 3C. Human Angiogenesis PCR Array on tumors explanted from hrIL-12 <i>vs</i> PBS treated animals 23 days after orthotopic inoculation of Calu6/β2 cells. Histogram shows fold expression changes of genes in tumors from hrIL-12 <i>vs</i> PBC treated mice. 3D. Tumors from PBS-treated mice express VEGF-C (a) and IL-6 (c). Expression of VEGF-C and IL-6 is strongly reduced (b and d, respectively) in tumors from hrIL-12 treated mice. (×400).</p

IL-12Rβ2 expression and function in human lung adenocarcinoma.

<p>1A. Histological features and IL-12Rβ2 expression in human bronchioloalveolar lung carcinomas. Non-mucinous bronchioloalveolar carcinoma typically shows columnar neoplastic cells growing along the alveolar septa (a). In 41.4% of adenocarcinomas, neoplastic cells lack IL12Rβ2 expression (b), though a few may sometimes retain it (inset in b). By contrast, alveoli unaffected by malignant process express IL-12Rβ2 (c), as observed in the remaining tumors (d). (×400). 1B. Angiogenic activity of supernatants from one representative lung ADC sample cultured in the presence or absence of hrIL12. CAM treated with sponges loaded with supernatant from the untreated cells were surrounded by allantoic vessels developing radially towards the implant in a ‘spoked-wheel’ pattern (left panel). When supernatants from hrIL-12 treated lung ADC sample was tested, a significant reduction (P = 0.001) of the angiogenic response was appreciable (right panel). These experiments were repeated three times. Original magnification: ×50. 1C. Pooled results from human angiogenesis PCR array performed in three lung ADC samples cultured in the presence or absence of hrIL-12 are shown. Histogram shows fold expression changes of genes in primary samples treated with hrIL-12 vs medium.</p