Changes in haemodynamic parameters for the control; hypovolaemia and endotoxaemia rabbits.

<p>The central bar is the mean, the box represents the inter-quartile range and the whiskers the range. Significant differences between the control and experimental groups of p<0.05 are noted by an ‘*’ and p<0.01 by an ‘¤’.</p

Gant chart showing schema of blood sampling and electrical stimulation.

<p>Control rabbits and the experimental groups all received the same number of stimulations and blood samples. Endotoxaemic rabbits had five stimulations and three blood samples that were not analysed, as according to protocol, so as to ensure identical experimental conditions for all groups.</p

Changes in biochemistry, catecholamines and haematocrit in the hypovolaemic rabbits.

<p>Interquartile ranges [IQR] and standard deviations are shown (SD) according to the distribution of the variables.</p>*<p>p<0.05,</p>¤<p>p<0.01.</p

Changes in biochemistry, catecholamines and haematocrit in the endotoxin group.

<p>Interquartile ranges [IQR] and standard deviations are shown (SD) according to the distribution of the variables.</p>*<p>p<0.05,</p>¤<p>p<0.01.</p

<i>PTPLA</i> mutation in the initial confirmation panel of CNM dogs.

<p>(A) Wild-type (wt = 610 bp) and <i>PTPLA^cnm</i> (<i>cnm</i> = wt+238 bp) alleles in a healthy carrier (FR-1) and an affected (FR-2) Labrador from the French experimental pedigree. (B) Segregation of the <i>PTPLA^cnm</i> allele in a four-generation pedigree of a client-owned US proband female (arrow). (C) Genotypes of client-owned Labradors from several countries, diagnosed with CNM-related myopathies (asterisks; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046408#pone.0046408.s004" target="_blank">Table S1</a>). US-6 is a champion known to have produced CNM pups; DE-5 is a control affected by a neuropathy and FR-2 was reloaded for size comparison.</p

A 3.8-Mb haplotype is highly associated with CNM.

<p>The acrocentric region of the <i>PTPLA</i> locus within canine chromosome 2 (CFA2) is depicted. Positions of genotyped SNPs are indicated. The short and long haplotypes associated with CNM are shown in green and red, respectively. For each SNP, the allele detected in the CNM associated haplotype is indicated and represented as a grey box. The alternative allele is represented as a white box. For each SNP, the minor allele frequency (MAF) in the healthy population of Labradors is given. The <i>PTPLA^cnm</i> allele is represented by a black dot (•) and the wild-type <i>PTPLA⁺</i> allele by a “+”. Frequencies of long 3.8-Mb haplotypes in each population of CNM or healthy dogs are given below each haplotype. For haplotypes with frequencies >10%, width of haplotypes is proportional to its frequency. Haplotypes with frequencies below 3% have been omitted and are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046408#pone.0046408.s003" target="_blank">Figure S3</a>.</p

Number of genotyped dogs used in this study.

<p>The whole panel includes all dogs for which samples were received for testing purposes. The initial confirmation panel includes dogs with an early diagnosis of HLMR or phenotypically similar myopathies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046408#pone.0046408.s004" target="_blank">Table S1</a>).</p

Client-owned US Labradors share similar morphological and histopathological features with French CNM dogs from the experimental pedigree.

<p>French CNM (A–C) and US HMLR (D–F) affected dogs have atrophic skeletal muscles, the most affected being those of pelvic limbs (e.g. <i>biceps femoris</i> muscle, arrows in A,D). B,C,E,F are Hematoxylin-Eosin-stained transverse sections of the <i>biceps femoris</i> muscle from 6-month-old (B, FR-4; E, US-18) or 10-year-old (C,F) affected Labradors. Early signs include groups of atrophic fibers, surrounded by endomysial (e) and perimysial (p) fibrosis. In older dogs, increased internalized or centralized nuclei (asterisks) and fatty infiltration (f) are observed. Scale bar = 50 µm.</p

Subcellular localization of Bmcc1s in primary neurons.

<p>(A–C) Confocal section images of primary neurons after 7 days of culture immunostained for endogenous Bmcc1s (green) and α-tubulin or neurofilament subunit M (NF-M) (red). Merge images showed that Bmcc1s colocalizes with α-tubulin (A) and NF-M (C) immunoreactivity signal. Boxed regions in A and C indicate the fields enlarged in each image. B. In nocodazole-treated primary neurons (10 µM, 1 h), Bmcc1s followed the disrupted α-tubulin microtubular staining. (D) Immunogold labeling and electron microscopy analysis of primary neurons showed that Bmcc1s localized on cytoskeleton-type structures compatible with microtubules (left) and intermediate filaments (right). Bars: 10 µm (A–C); 100 nm (D).</p

Morphological changes induced by Bmcc1s overexpression requires MAP6.

<p>Confocal microscopy image projections of cells transfected with a Bmcc1s-V5 or GFP expressing plasmid, and stained for V5 (green) and F-actin (detected with TRITC-conjugated phalloidin in red). Cells were fixed 24 h after transfection. (A) primary astrocytes; (B) primary neurons. The morphology of GFP-expressing cells (green) was unchanged compared to untransfected cells. In contrast, Bmcc1s-V5-expressing astrocytes and neurons developed numerous membrane protrusions (white arrowheads). Images in B illustrate representative confocal projections of the effect of Bmcc1s-V5 on neuritic growth and number in wild-type neurons. The whole Bmcc1s-V5 transfected neuron is shown in the insert. Histograms present means ± sd of the length of the longest neurite and of the number of neurites. *** p-value<0.0001 ** p-value<0.001. ns, not significant for 3 independent experiments using the two sample independent t-test. In neurons, length of the longest neurite, and number of neurites (or cell extensions starting from the soma) were significantly increased by Bmcc1s-V5 transfection, but not in MAP6-deleted neurons. Bars: 10 µm.</p