Overview of compounds used for training and evaluation of classifiers.

<p>The table lists all compounds along with their carcinogenicity class and CAS Registry Number. Corn oil (CO) or 0.1% carboxymethyl cellulose (CMC) was used as vehicle. Doses were selected for each compound based on the tumorigenic dose rate 50 (TD₅₀), long-term animal studies leading to liver cancer known from the literature or initial dose range-finding studies. After a dosing period of 3 or 14 days the mouse livers were subjected to gene expression analysis using an Affymetrix platform. Two treatment groups, each comprising 5–6 male and female mice, respectively, were examined for each compound, dose and point of time. Time matched control groups were included for both vehicles. Each treatment group has a unique group ID which is composed of the compound short name, the group number and the sex (Female or Male).</p

Reclassification of the compounds CPA, TAA, and WY.

<p>(<b>A</b>) Points indicate treatment groups which were originally represented by a vector containing the fold-changes of all signature genes and then transformed to its two principal components. Here, the informative genes for discriminating GC vs. NGC after 3 days of repeated dosing were used. Groups of male animals are drawn as squares and female ones as circles. The fill color of the points indicates the compound class. Polygons indicate the convex hulls of clusters corresponding to either male or female mice treated with a certain class of compounds. (<b>B</b>) Similar plot as in (A), but the multi-gene signature for GC vs. NGC classification after 14 days was used here. (<b>C</b>) The heatmap provides a graphical representation of the fold-changes of 15 selected signature genes from the 14-day signature for GC vs. NGC classification. Rows correspond to genes and columns to treatment groups. Upregulated genes are colored in red and downregulated ones in green. The colorbar on top indicates the corresponding compound classes. (<b>D</b>) Heatmaps showing confidence of predictions made by diverse C vs. NC classifiers for male (M) and female (F) mice treated with CPA, TAA and WY for 3 days (left heatmap) or 14 days (right heatmap). (<b>E</b>) Similar illustration as in (D) showing prediction outcomes of GC vs. NGC classifiers.</p

Performance comparison with signatures known from the literature.

<p>(<b>A</b>) The grouped bar plots depict the area under the ROC curves obtained for novel and known signatures for the separation of C and NC after 3 days of treatment. For this purpose, the performance of diverse classifiers was evaluated by a 3-fold cross-validation. Each bar corresponds to a certain classifier (see legend) and each group of bars refers to a certain signature. The horizontal dashed line indicates the performance that would have been achieved by random guessing. The adjacent Venn diagrams illustrate informative genes common between signatures. (<b>B</b>) Same plots as in (A), but for C vs. NC classification after 14 days of repeated dosing. (<b>C</b>) Mean ROC scores and signature overlaps for GC vs. NGC classification after 3 days of treatment. (<b>D</b>) Same plots as in (C), but for GC vs. NGC classification after 14 days of repeated dosing.</p

Classification results obtained for different signatures.

<p>(<b>A</b>) The four heatmaps show the predictions resulting from diverse binary classifiers for the discrimination of C from NC after 3 days (left two heatmaps) or 14 days (right two heatmaps) of repeated dosing. For each dosing time two heatmaps are depicted which correspond to male and female mice, respectively. The rows correspond to different classifiers and the columns to different treatment groups. The continuous prediction scores, returned from the classifiers, were transformed to confidence scores between 0 and 1, which provide an estimate of the probability of class C. The colorbar on top shows the true class annotation. The black vertical lines separate the test samples from the three folds of cross-validation. (<b>B</b>) Heatmaps illustrating the classification outcome of diverse predictors to distinguish GC from NGC based on characteristic gene expression profiles observed in male and female mouse liver samples after 3 or 14 days of administration. KNN, K-Nearest Neighbor; SVM, Support Vector Machine; PAM, Prediction Analysis for Microarrays.</p

Accuracy and stability of signatures for compound classification.

<p>(<b>A</b>) The line plots depict the mean performance of C vs. NC classification after 3 days of repeated dosing, which was achieved based on gene sets extracted with different feature selection methods. Each curve corresponds to a feature selection method and the performance was assessed depending on the number of genes selected as informative features. The prediction accuracy was assessed on the samples left out from 25 random subsamplings of the dataset (bootstraps), each containing 90% of the data, and measured in terms of area under the ROC curve. The inset bar plot depicts the ROC scores averaged across bootstraps and signature sizes. (<b>B</b>) Performance of C vs. NC classification after 14 days of treatment illustrated as in (A). (<b>C</b>) The correspondence of the extracted C vs. NC gene sets across 25 bootstraps was assessed based on the Kuncheva stability index (KI) for each of the 4 employed feature selection methods. The KI was then for each method plotted against the number of selected signature genes. (<b>D</b>) Robustness of signatures for C vs. NC classification after 14 days of treatment illustrated as in (C). (<b>E, F</b>) Prediction accuracy achieved with signatures for GC vs. NGC classification after (E) 3 days and (F) 14 days of repeated dosing, respectively, depicted as in (A). (<b>G, H</b>) Similar illustration as in (C) showing robustness of signatures for GC vs. NGC classification after (G) 3 days and (H) 14 days of administration, respectively.</p

Multiscale, Converging Defects of Macro-Porosity, Microstructure and Matrix Mineralization Impact Long Bone Fragility in NF1

<div><p>Bone fragility due to osteopenia, osteoporosis or debilitating focal skeletal dysplasias is a frequent observation in the Mendelian disease Neurofibromatosis type 1 (NF1). To determine the mechanisms underlying bone fragility in NF1 we analyzed two conditional mouse models, Nf1Prx1 (limb knock-out) and Nf1Col1 (osteoblast specific knock-out), as well as cortical bone samples from individuals with NF1. We examined mouse bone tissue with micro-computed tomography, qualitative and quantitative histology, mechanical tensile analysis, small-angle X-ray scattering (SAXS), energy dispersive X-ray spectroscopy (EDX), and scanning acoustic microscopy (SAM). In cortical bone of Nf1Prx1 mice we detected ectopic blood vessels that were associated with diaphyseal mineralization defects. Defective mineral binding in the proximity of blood vessels was most likely due to impaired bone collagen formation, as these areas were completely devoid of acidic matrix proteins and contained thin collagen fibers. Additionally, we found significantly reduced mechanical strength of the bone material, which was partially caused by increased osteocyte volume. Consistent with these observations, bone samples from individuals with NF1 and tibial dysplasia showed increased osteocyte lacuna volume. Reduced mechanical properties were associated with diminished matrix stiffness, as determined by SAM. In line with these observations, bone tissue from individuals with NF1 and tibial dysplasia showed heterogeneous mineralization and reduced collagen fiber thickness and packaging. Collectively, the data indicate that bone fragility in NF1 tibial dysplasia is partly due to an increased osteocyte-related micro-porosity, hypomineralization, a generalized defect of organic matrix formation, exacerbated in the regions of tensional and bending force integration, and finally persistence of ectopic blood vessels associated with localized macro-porotic bone lesions.</p></div

Diminished organic matrix properties in Nf1Prx1 mice.

<p>(<b>A</b>) Picrosirius red stained bone sections imaged with polarized light. Homogenous red staining in controls indicates highly packed and thick collagen. Heterogeneous red-yellow-green staining of Nf1Prx1 bone sections is indicative of diminished packaging and thickness of bone collagen. Note, there is no picrosirius red staining within non-mineralized bone tissue (osteoid) surrounding blood vessels. Scale bar shows 20 µm. (<b>B</b>) Silver staining (AgNOR) detects the osteocytic network and other accumulations of acidic matrix proteins. Note the large unstained area around blood vessels in Nf1Prx1 humerus. Scale bar shows 50 µm. (<b>C</b>) Sections of humerus cortex stained with Toluidin and Safranin O. Toluidin stained osteocyte (Ot.) appear blue with dark blue nuclei (black arrowhead). Presence of nuclei suggests vitality of cells in the bone cortex. Osteoid (dotted line) near to the blood vessel was stained light blue (Toluidin) or light red (Safranin O). Light blue or red staining indicates that bone lesions are not composed of cartilaginous matrix, which with these methods stains purple (Toluidin) or dark red (Safranin O) (not shown). Ot. in non-mineralized areas are viable (nuclei marked with grey arrow head). Scale bar represents 50 µm. Abbreviations: blood vessels (bv), bone (b), bone marrow (bm), osteoid (o).</p

Diminished bone stiffness, bone mineralization and organic matrix formation in Nf1Prx1 mice.

<p>(<b>A</b>) Scanning acoustic microscopy (SAM) was applied to show elastic properties of the bone matrix. SAM measurements were performed within six ROIs E1a–E3b on anterior (a) and posterior (b) bone cortex. Images illustrate impedance values (Z in MRayl) according to the color scale (left). Nf1Prx1 mutants show diminished impedance values within all analyzed ROIs. (<b>B</b>) Quantitative evaluation of SAM measurements reveals decreased Z values in Nf1Prx1 humerus in all analyzed ROIs (control n = 4, Nf1Prx1 n = 4). Nf1Col1 humeri did not demonstrate diminished Z values (control n = 5, Nf1Col1 n = 4). (<b>C</b>) The cross sections of humerus at the level E2 with color coded BMD values measured by phantom calibrated microCT. The yellow and green coloring indicates low and high BMD values, respectively. Scale bar - 600 µm. (<b>D</b>) Cortical bone mineral density (BMD) was assessed by microCT within region E1–E3 in humeri of Nf1Prx1 (control n = 5, Nf1Prx1 n = 5) mice. BMD was reduced in the Nf1Prx1 model in all ROIs (for definition see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086115#pone.0086115.s001" target="_blank">Fig.S1</a>). Statistical significance - t-test, * p≤0.05 and ** p≤0.01.</p

Nf1Prx1 bone tissue shows diminished mechanical strength and increased micro-porosity due to increased osteocyte lacuna size.

<p>(A) Diagram of the analytical setup used for tensile experiment. Measurements were done on laser dissected cortical bone slices from P90 mice. (B) A typical tensile stress-strain pattern in controls showed a linear elastic modulus (E-modulus) phase that is followed by the yield point plateau and the ultimate stress point. Note, increased elasticity and diminished ultimate strength in bone slices of Nf1Prx1 humerus compared to controls. (C) Diminished E-modulus (slope of tensile curve) and (D) ultimate stress point (point of maximal load before tissue failure) in bone tissue slices from 3 months old Nf1Prx1 (n = 14) and control (n = 15) mice. (E) Quantitative microCT analysis of the total (summed) lacunae porosity (Lc.V/BV) within the lacunae range from 100-4000 µm³. Note the significant increase of Lc.V/BV in Nf1Col1 and Nf1Prx1 mice. (F) The summed volume of Ot. lacunae (Lc.V) within the size range 100–4000 µm³ with an increment of 100 µm³ (histogram). Note the shift of summed Lc.V fractions towards higher volume range in Nf1Col1 and Nf1Prx1 mutants. Insert (a) - Nf1Col1 mice demonstrated reduced summed Lc. volume in the intervals 200–300 and 300–400 µm³ and (b) - summed Lc.V in fractions 800–900 and 900–1000 µm³. Insert (c) - Nf1Prx1 mice showed reduced summed Lc. volume in the intervals 100–200 and 200–300 µm³ and (d) - summed Lc.V in fractions 700–800 and 800–900 µm³. (G) The total number of Ot. lacunae (Lc.N) was unaffected in both mouse models. (H) Ot. lacunae morphology (range from 100–4000 µm³) assessed by measuring the x, y and z axis length in a selected volume of ROI E2. Note, lacunae size is increased in all dimensions in Nf1Col1 and Nf1Prx1 mice. The number of analyzed bone samples: Nf1Prx1 n = 5, controls n = 5; Nf1Col1 n = 3, controls n = 3. Statistical analysis was performed with unpaired Student t-test, * p≤0.05 ** p≤0.01.</p

Cortical bone in NF1 tibial dysplasia is characterized by heterogeneous mineralization, diminished collagen thickness and increased micro-porosity.

<p>(A) MicroCT analysis of human cortical bone samples (tibia). Bone mineral density was calibrated according to Houndsfield units (HU) and color coded (see HU scale). HU values measured in ROI1 (cortical bone without blood vessels) and ROI2 (cortical bone near blood vessel) were in control specimen similar. However, in the NF1 bone sample HU values were in proximity of large vessels decreased (ROI2). The analyzed bone sample was obtained pre-fracture from an individual affected by NF1 with tibial bowing. (B) Two further mineralized bone samples were obtained from cortical bone of NF1 individuals with pseudarthrosis. Samples were analyzed by picrosirius red histology and imaged in polarized light (large image) and bright light (inset). Collagen fibers follow osteonal organization in control specimens. Note that in the NF1 cortical bone sample collagen fiber organization appears less orderly with abundant thin (green) collagen fibers. (C) Ot. morphology was visualised with AgNOR staining. Ot. are spindle shaped (inset) and regularly distributed in control cortical bone. In contrast, Ot. are round and irregularly distributed in NF1 cortical bone. (D) Histomorphometry of AgNOR stained bone sections showing: relative Ot. area (Ot.Ar/B.Ar), Ot. number per bone area (Ot.N/B.Ar), individual Ot. area (Ot.Ar) and individual Ot. circumference (Ot.Cir). Presented data are mean values with standard deviations (control n = 3, NF1 n = 2). (E) Volumetric microCT analysis showing increased specific lacunae (Ot.) volume (Lc.Vol) and surface (Lc.Sur) in NF1 tibial dysplasia cortical bone as compared to controls. Statistical analysis was performed with unpaired t-test, ** p≤0.01. Abbreviations: blood vessels (bv) and bone (b). All scale bars represent 50 µm.</p