Additional file 1: Table S1. of Aesthetic discomfort in hand osteoarthritis: results from the LIège Hand Osteoarthritis Cohort (LIHOC)

Radiological severity and impact of hand osteoarthritis on functional disability, health-related quality of life, and psychological status. (DOC 38 kb

Comparison of genes affected after PTH or VitD3 treatment between 5–6dpf.

<p>(A) List of common genes and their respective log2(fold change) in the two conditions. (B) Comparison of the number of genes affected by PTH or VitD3 treatment. The number of probes resulting in different hybridization signals is given, with the numbers in parenthesis and the graph showing the numbers of IPA-annotated genes. (C) Network constructed using the common genes and extended using the genes affected in one of the two conditions. The color overlay indicates the fold change after VitD3 (left) or PTH (right) treatment. Genes up-regulated (red), down-regulated (green), (*) indicates that the gene is represented by two or more probes on the microarray.</p

Network of genes affected in "relative microgravity" experiments.

<p>A network was constructed using the genes common to all three experiments, or the genes common only to 3g>1g and 3g>axe. Color overlay indicates the fold change relative to the 3g sample taken as control. Genes up-regulated (red), down-regulated (green), (*) indicates that the gene is represented by two or more probes on the microarray.</p

Morphometric analysis results of bone matrix staining after 5 days chemical treatments.

<p>The distances are measured in pixels. Mean ± SD and t-test analysis were calculated for each measure on at least 20 individuals. * <i>p <</i> 0.05, ** <i>p <</i> 0.01 and ***<i>p <</i> 0.001. (A) Distances after VitD3 treatment. (B) Distances after PTH treatment. (C) Area of the parasphenoid bone results after 5 days PTH or VitD3 treatment. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126928#pone.0126928.g001" target="_blank">Fig 1</a>. <b>A)</b> Analysis of the bone skeleton after VitD3 treatment revealed a significant increase of the distance between maxillae (m), consistent with a broader jaw as already observed by cartilage morphometry. The length of the head skeleton is also increased upon VitD3 treatment with a longer distance between the anterior part of the head (an) and the notochord (n), and between an and the parasphenoid (p) bone. Other measures are not significantly modified (A, C). B) PTH treatment caused an increase of the distance between the anterior part of the head and the summit “a” of the parasphenoid, mainly due to a significant decrease of the size of the parasphenoid (p) (C). Some structures are missing, such as the anguloarticular (aa), branchiostegal ray2 (br2), ceratohyal (ch) and/or maxilla (m). However, a significant broadening of the posterior head skeleton is revealed by the increased distance between left and right ("up" and "down") branchiostegal rays1 (br1), entopterygoids (en) and also the opercula (o) (B).</p

(A-D) Cartilage and bone elements of the head skeleton in 10dpf zebrafish.

<p>(A) Alcian blue staining of head cartilage representing the landmarks used for morphometry. (B) Schematic representation of the different head cartilage elements. anterior limit (an), articulation (ar), ceratobranchial pairs 1 to 4 (cb1-4), ceratohyal (ch), ethmoid plate (et), hyosymplectic (h), Meckel's cartilage (mk), palatoquadrate (pq), posterior limit (po). (C) Alizarin red staining of cranial bones representing the landmarks used for morphometry. (D) Schematic representation of the different cranial bone elements with 29 landmarks used for chemicals treatments and 15 landmarks for the 3g and the relative-hypergravity. The 15 landmarks are anguloarticular (aa), anterior (an), branchiostegal ray1 (br1), entopterygoid (en), maxilla (m), notochord (n), opercle (o), parasphenoid (p). Note that the parasphenoid is a triangular bone defined by its anterior summit (a) and two posterior summits (b,c). The 29 landmarks include the 15 named before with branchiostegal ray2 (br2), cleithrum (c), ceratobranchial 5 (cb), ceratohyal (ch), dentary (d), hyomandibular (hm). (<b>E-J</b>) <b>10dpf zebrafish larvae after 5 days chemical treatments.</b> (E-G) Alcian blue staining of cartilage. (H-J) Alizarin red staining of bone. (E,H) Controls in DMSO. (F,G) no significant effect of, respectively VitD3 and PTH on cartilage development, nor on chondrocyte shape or size (inlays showing close-up). I: increase of bone development after VitD3 treatment. (J) decrease of bone development after PTH treatment. Ventral views, anterior to the left, (E-J) scale bar = 250μm.</p

Number of genes affected in the various hypergravity experiments.

<p>The absolute number of probes resulting in a statistically significant hybridization signal is given for each condition. In parentheses, the corresponding number of genes with an annotation in IPA is given, while the Venn diagrams represent the number of genes unique to each condition and genes common to two or three conditions.</p

Extent of bone formation in 10dpf larvae after 5days chemical treatments.

<p>Bone development is classified for each element into different categories: Absent (no structure present; red), early ossification (beginning of the bone ossification; yellow), advanced ossification (the structure is present and already developed as the control; green) and over ossification (the structure is more developed compared to the control; purple). Cumulated frequencies in % are represented for each element. As no significant difference was observed for paired structures between left and right (up and down), their scores have been combined. Statistical analysis was performed by X² of Pearson and a logistic regression. (A) Cumulated frequency after 5days VitD3 treatment. To obtain this, values were attributed to each element according to its category and added up for each larva: 0 for absent, 1 for early, 2 for advanced, and 4 for over ossification (B) Cumulated frequency after 5days PTH treatment.</p

Biological functions associated to "relative microgravity"-affected genes.

<p>Ingenuity Pathway Analysis of the lists of genes affected at 6dpf after 6 days at 1g (1g), or after 5 days at 3g and returned to 1g on the centrifuge axis (3g>axe) or outside of the centrifuge room (3g>1g), each time compared to 3g hypergravity treatment for 6 days (3g). Columns indicate respectively the function, the range of p-values (significance) associated to various sub-functions, and the number of genes concerned (N).</p><p>Biological functions associated to "relative microgravity"-affected genes.</p

Schematic overview of the different hypergravity experiments.

<p>(A) larvae are placed at hypergravity at 5dpf until 9dpf (3g), while (control) larvae are kept at normal gravity for 9 days. Total mRNA was extracted at 6dpf and batches of larvae were fixed at 9dpf for Alizarin red staining of bone matrix. (B) Experiment in which the control larvae were placed at 3g and kept at 3g until 6dpf (3g), or returned at 5dpf to 1g outside (3g>1g) or on the axis of the centrifuge (3g>axe) for one day. An additional batch of larvae was kept at normal gravity until 6dpf (1g). RNA extraction and Alizarin red staining are performed at 6dpf. For abbreviations see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126928#pone.0126928.g001" target="_blank">Fig 1</a>.</p

Summary graphs comparing the bone formation scores for each structure in the different experiments.

<p>Statistical analysis was performed by X² of Pearson and a logistic regression. In red, the scores are significantly increased. In green, the scores are significantly decreased. (A) PTH. (B) VitD3. (C) 3g hypergravity between 5–6dpf (D) "relative microgravity". For abbreviations see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126928#pone.0126928.g001" target="_blank">Fig 1</a>.</p