dataesr/scientific-paragraphs-categorization huggingface dataset:
e. , data variation) and the goodness of prediction, as estimated by cross-validation. Q 2 ≥ 0. 5 can be considered as a good predictor  [14]. Cross-validation was used in each OPLS-DA model to determine the number of components and to avoid overfitting the data. A crossvalidation embedded in a Monte Carlo resampling approach  [15, 16]  was used during the construction of the model to build a confusion matrix that allowed a direct visualization of the performances of the model in terms of classification power [sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), and global accuracy]. The SIMCA P (version 11. 0; Umetrics AB) package was used for statistical data analysis. Continuous variables are expressed as mean ± SD. Statistical analysis was performed using IBM SPSS Statistics version 20 (IBM SPSS Inc, Chicago, IL).
The Spearman non-parametric test was performed to determine the correlation between metabolites. Comparisons of tumor metabolite concentrations between SDHx-related and sporadic tumors were performed using a Mann-Whitney U test. The receiver operating characteristic (ROC) curves were used to evaluate the clinical utility of metabolite quantification in the diagnosis of SDHx mutation. Areas under the ROC curve, sensitivity, and specificity were determined using MedCalc version 13. 2. 2 (MedCalc Software, Ostend, Belgium). According to the ROC curve, values exhibiting the best accuracies were chosen as the threshold for a screening test. Linear regression model was used to examine the association between metabolite levels and genetic status with adjustment for tumor location and total catecholamine levels as explanatory variables.
For multiple testings, adjusted P values were calculated using the false discovery rate (FDR) procedure with the SAS PROC MULTTEST statement  [17]. PCA was performed to visualize and analyze correlations between metabolite concentrations and SDHx status. PCA was done using the package FactoMiner (Multivariate Exploratory Data Analysis and Data Mining with R). Values of P b. 05 were considered to be statistically significant.
SDHx versus Sporadic PHEOs/PGLs Twenty-three SDHx-related PHEOs/PGLs and 48 sporadic tumors were included in the analysis. A total of 24 metabolites was recognized from the spectra obtained from all 71 tumor samples within the range of 1 to 8. 65 ppm (Supplementary Table  1 ). Besides peaks due to small watersoluble molecules, the acquired spectra display consistent broad resonances, which were attributed to three different fatty acid moieties defined as a, b, and c  [22]. A two-component OPLS-DA showed a very clear separation of the two sets of samples, a faithful representation of the Y data (R 2 Y =0. 82) and a good cumulative confidence criterion of prediction (Q 2 = 0. 7). These results are graphically represented in Figure  1. When comparing sporadic to SDHx-related tumors, the OPLS-DA model accurately classified all samples.
Histologic analysis and immunohistochemistry of mice tumor samples Tumor tissues were embedded in paraffin and 3-μm sections were cut and stained for caspase-3, Ki-67 and CD146 using respectively rabbit polyclonal anti-caspase-3 (Cell Signaling), anti-Ki67 (Dako), anti-CD146 (Abcam) antibodies. Immunodetection was performed using DAB Substrate-Chromogen (Dako) and counterstained with hematoxylin. Quantification of relative caspase-3 positive surface, Ki-67 positive cells and CD146 immunostaining in tumor tissue were evaluated by ImageJ (NIH, Bethesda, MD) softwares.
Micro-CT analysis Analysis of bone microarchitecture was performed using the high-resolution X-ray micro-CT system for small-animal imaging SkyScan-1072 (SkyScan, Kartuizersweg, Belgium) at different tumor volumes (500, 1000 and 2500 mm 3 ). All tibia/fibula were scanned using the same parameters (pixel size 18 μm, 50 kV, 0. 5-mm aluminium filter and 0. 6 degrees per rotation step). Analysis of bone parameters were performed using the CTan software (Skyscan). The bone volume (BV) of ectopic bone was quantified over the total length of tibia bearing 2500 mm 3 tumor. Trabecular structures positioned 0. 2 mm below the growth plate were quantified over a length of 1 mm at 1000 mm 3 tumor volume since at higher tumor volume trabecular bone is completely destroyed by the tumor cells. Bone volume/trabecular volume (BV/TV), trabecular thickness (Tb.
Statistical analysis All analyses were performed using GraphPad Prism 4. 0 software (GraphPad Software, La Jolla, CA, USA) or Excel. Results of in vitro experiments were analyzed with the unpaired t-test and are given as means ± SD. For in vivo experiments, results from groups treated with 1 μg/mouse/day or 5 μg/mouse/day were compared with no treated control groups using the unpaired t-test and are given as means ± SEM. Results with p < 0. 05 were considered significant.
For panels C, F, I, and N (postfusion signal), the cells were incubated at 4°C with MAb recognizing the postfusion state of MV F and stained as described in Materials and Methods, and the values on the y axis represent the relative luminescence units (RLU) of the stained cells, reflecting quantification of postfusion MAb bound. The data are averages of results for triplicate samples (with SD) from a representative experiment repeated 3 times. in the postfusion state (Fig. 7F ), as recognized by the postfusion MAb. Even at 50°C, a portion of F T461I and wt F were still present in the prefusion state; at this temperature, F L454W was all in the postfusion state, with negligible prefusion signal (Fig. 7H ). At 55°C, the prefusion MAb no longer detected any of the 3 F proteins. In fact, all three were detected by the postfusion MAb, indicating that at this temperature all the F proteins had been activated to their postfusion state.
ACKNOWLEDGMENTS This work was supported by grants from the  NIH  ( AI109050  to M. P. and  AI031971  to A. M. ) and by  INSERM  and  French Foundation for Medical Research (FRM)  awards to B. H. D. H. was supported by a  Poliomyelitis Research Foundation  award, C. M. was supported by  ANR 's  NITRODEP  project (grant  ANR-13-PDOC-0010-01 ) (http://www. agence-nationalerecherche. fr), and M. P. was supported by a  Friedman Research Scholar Award. We are grateful for the  Friedman Research Scholar Award  to M. P. , to  Lawrence Golub  for his support, to Dan and  Nancy Paduano  for support of innovative research projects in the A. M. /M. P. laboratory, and to the  Friedman Family Foundation  for support of the A. M. /M. P. Research Program. We thank  Y. Yanagi  and  S. Watanabe  for kindly providing plasmids coding for T461I and  S103I N462S N465S  mutations.
Selection criteria were (1) dendrites displayed complete Golgi impregnation, (2) the cell type was clearly identifiable, and (3) the minimum length of the dendrites was 50 m from the soma. Stacks of bright-field images with 0. 3 m spacing were acquired with a Zeiss Axioskop 50 microscope with 63ϫ oil objective (NA 1. 4; Plan-Aprochromat) coupled to a CCD camera (CoolSnap ES; Roper Scientific) operated by Metaview software (Molecular Devices). Images were analyzed with ImageJ. The number of spines of each basic type per unit dendritic length was measured. Fluorescence microscopy. Light microscopy images were acquired with a Zeiss LSM 710 inverted confocal microscope, using a 63ϫ Plan-Apochromat oil-immersion objective (NA 1. 4; Zeiss) or (for Sholl analysis) a 40ϫ oil objective (NA 1. 3; Zeiss).
To correct the radial distribution for distance-dependent increase in bin area, particle densities along the radial coordinate were divided by normalized radial distance, thus giving increased weight to particle density close to the spine center. Three-dimensional reconstruction of spine heads from serial section digital images was done using Reconstruct software (John C. Fiala, Department of Biology, Boston University, MA). For comparisons between wild-type and Chmp2b-deficient mice, acquisition of microscopy images and morphometric quantification were performed without knowledge of genotype. Purification of synaptoneurosomes and PSD fractionation. Subcellular fractionation was performed according to  Carlin et al. (1980)  with minor modifications. Samples were kept at 4°C during all procedures.
Peptides were then extracted from gel pieces in three 15 min sequential extraction steps in 30 l of 50% acetonitrile; 30 l of 5% formic acid; and, finally, 30 l of 100% acetonitrile. The pooled supernatants were then dried under vacuum. For nano-LC-MS/MS analyses, the dried extracted peptides were resuspended in 5% acetonitrile and 0. 1% trifluoroacetic acid and analyzed by on-line nano-LC-MS/MS [Ultimate 3000 (Dionex) and LTQ-Orbitrap Velos pro (Thermo Fisher Scientific)]. Peptides were sampled on a 300 m ϫ 5 mm PepMap C18 precolumn and separated on a 75 m ϫ 250 mm C18 column (PepMap; Dionex). The nano-LC method consisted of a 120 min gradient at a flow rate of 300 nl/min, ranging from 5 to 37% acetronitrile in 0. 1% formic acid for 114 min before reaching 72% for the last 6 min. MS and MS/MS data were acquired using Xcalibur (Thermo Fisher Scientific).
Spray voltage and heated capillary were, respectively, set at 1. 4 kV and 200°C. Survey full-scan MS spectra (m/z ϭ 400 -1600) were acquired in the Orbitrap with a resolution of 60,000 after accumulation of 10 6 ions (maximum filling time, 500 ms). The 20 most intense ions from the preview survey scan delivered by the Orbitrap were fragmented by collision-induced dissociation (collision energy, 35%) in the LTQ after accumulation of 10 4 ions (maximum filling time, 100 ms). For bioinformatic analyses, data were processed automatically using Mascot Daemon software (version 2. 3. 2; Matrix Science). Concomitant searches against the Uniprot database (August 2012 version, Rattus taxonomy, 42,469 sequences), the classical contaminants database (67 sequences, homemade), and the corresponding reversed databases were performed using Mascot (version 2.
4). ESI-TRAP was chosen as the instrument and trypsin/P as the enzyme, and two missed cleavage were allowed. Precursor and fragment mass error tolerances were set, respectively, at 10 ppm and 0. 6 Da. Peptide modifications allowed during the search were carbamidomethyl (C, fixes) acetyl (N-ter, variable), oxidation (M, variable), and deamidation (NQ, variable). The IRMa software (version 1. 30. 4) was used to filter the results: conservation of rank 1 peptides, peptide identification False Discovery Rate Ͻ 1% (as calculated by using the reverse database strategy), and minimum of one specific peptide per identified protein group. The filtered results were uploaded into a relational mass spectrometry identification database (MSIdb), and  a homemade tool (hEIDI;  Milbradt et al. , 2014)  was used for the compilation, grouping, and comparison of the protein groups from the different samples.
hEIDI provides modules to help exploring validated search results that have been compiled into a MSIdb. The user creates contexts, which represent sets of identifications, and runs peptides and proteins grouping algorithms to cluster peptides identifying a family of proteins in these contexts. The grouping algorithm will first identify identical peptides based on their mass and sequence. Groups of proteins matching the same set of peptides are then created as is done in the IRMa toolbox for a single identification. This is typically useful to consider results at the sample level instead of the replicate level. To compare several contexts, hEIDI aligns each protein group from each context to be compared. This alignment is based on protein and peptide set similarity metrics. To be considered as specifically associated with the Chmp2b immunoprecipitate, the proteins identified by this analysis were filtered according to the following inclusion criteria: two or more unique peptides (specific spectral counts); two or more distinct protein fragments [spectral counts (SC)]; and ratio of SC in specific to control immunoprecipitate (SC 2B/SC control) Ն5.
6 EGTA, 4 Na 2 -ATP, 0. 4 Na-GTP, 10 Na-phosphocreatine, and 2. 5 MgCl 2 , adjusted to pH 7. 2 with CsOH and to 300 mOsm with Cs-acetate. Currents were recorded from a holding potential of Ϫ60 mV using an Axon 200B amplifier (Molecular Devices). Cell capacitance and series resistance compensation were applied electronically. Signals were filtered at 1 kHz, digitized at 5 kHz, and recorded using the pClamp software. Miniature EPSCs (mEPSCs) and NMDA-evoked currents were analyzed using ClampFit (Molecular Devices). Live imaging. Image acquisition and analysis were performed blind to transfected plasmids. For live imaging during chemical LTP, neurons were transferred to a perfusion chamber (Pocon), washed, and kept in an extracellular solution (125 mM NaCl, 2. 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 5 mM HEPES, 33 mM glucose, 50 M bicuculline, and 0.
Only modest bleaching was observed during the experiment. Cells showing signs of damage (pearling or blebbing) were discarded. To measure changes in spine volume, image stacks were subjected to blind (adaptive) deconvolution with AutoQuant as above. The deconvolved images (scaled to 8 bit) were used as input for 3D reconstruction of dendritic morphology by the software package NeuronStudio (http://www. mssm. edu/cnic/tools. html), which allocates voxels to spines through a specific algorithm  (Rodriguez et al. , 2006 (Rodriguez et al. , , 2008)). Individual spines labeled by the software and that were clearly identifiable at both initial and final time points were manually selected. The volume of the spines before and after stimulation was estimated by counting spine voxels. The fractional increase in spine volume was calculated for each spine as follows: ⌬V V ϭ V f Ϫ V i V i , with V i the volume at 5 min before the start of the stimulation and V f the volume at 45 min after the end of the stimulation.
1C ). To define the topographical pattern of Chmp2b protein expression, we performed immunohistochemistry on mouse brain sections (Fig. 1D ). Chmp2b immunoreactivity was particularly strong in habenula, olfactory bulb, deep layers of the neocortex, amygdala, and hippocampus. This pattern generally coincided with in situ hybridization data from the Allen Brain Atlas (http://mouse. brain-map. org/gene/show/44784). In higher-magnification views, immunostaining was evident in the somatodendritic compartment of numerous neurons (e. g. , in the CA1 region of the hippocampus; Fig. 1E ). Specificity of labeling was confirmed by showing reduced staining in the brain of Chmp2b-deficient (KD) mice (Fig. 1F  ). WT and 12% for Chmp2b R19/22/26A (quantified as the percentage of Chmp2b-HApositive pixels overlapping Chmp4b-Flag puncta).
In Chmp2b-depleted neurons, the mean amplitude of mEPSCs was reduced by 43% and the mean frequency by 30% (Fig. 4 E, F  ). We also recorded excitatory currents evoked by direct activation with bath-applied NMDA. Transfection of the Chmp2b-depleting plasmid resulted in a 63% drop in NMDA receptor-mediated current density (Fig. 4G ). Cell capacitance was not significantly affected (GFP shRNA, 16. 2 Ϯ 0. 8 pF; Chmp2b shRNA, 14. 0 Ϯ 1. 0 pF; p ϭ 0. 3037, t test). Thus, Chmp2b knockdown induced a loss of functional NMDA receptors. Together, these data indicate that Chmp2b is required for maintenance of functional excitatory synapses in cultured hippocampal neurons. To confirm the specificity of the shRNA effects and to determine whether these results extended to in vivo conditions, we investigated the dendritic morphology of neurons stained by Golgi impregnation in hippocampal CA1 sections from wild-type and Chmp2b KD mice (Fig.
Actin was used as a loading control. Quantitation of the Chmp2b band in each lane was obtained by densitometry and normalized to the respective actin control. KD brain still expressed 30% of the wild-type amount. H, Western blot used for the quantitation in G. PSD-95 were used as markers of the presynaptic and postsynaptic compartments, respectively. Chmp2b was more concentrated in the membranes and in the synaptoneurosomes than in the soluble fraction (Fig. 8A ). By comparing the intensity of the Chmp2b band with recombinant protein standards, we estimated that Chmp2b represented ϳ0. 03% (w/w) of total synaptosomal protein (data not shown). The relative level of Chmp2b remained elevated in the Triton X-100-insoluble material extracted from synaptoneurosomes (PSDI fraction). Chmp4b was mostly Triton insoluble and highly enriched in the PSDI fraction.
Subsequent solubilization of PSDI revealed that unlike PSD-95, Chmp2b and Chmp4b could be solubilized from the PSD fraction using the more stringent detergent Sarkosyl (Fig. 8B , fraction PSDIII). Solubilization of Chmp2b from synaptoneurosomes was completely ineffective with 1% digitonin, only partial with the mild nonionic detergent dodecylmaltoside, and very efficient with 1% DOC (Fig. 8C ). These data indicated that although Chmp2b and Chmp4b were associated with Triton-insoluble fractions containing postsynaptic scaffolding proteins, they did not behave as "core" proteins of the PSD, but rather as peripherally attached PSD proteins. To determine the oligomeric state of Chmp2b at synapses, gradient-purified synaptoneurosomes were lysed in DOC  (Husi and Grant, 2001). The lysates were clarified by high-speed centrifugation, and the solubilized proteins were then analyzed by gel filtration and immunoblotting of fractions.
Proteins were detected in a total of three immunoprecipitation (IP) experiments. Unique peptides (IP HMW complex) Spectral counts (IP HMW complex) Spectral counts (IP lysate) Protein name Accession number Chmp2b IgG Control IgG Chmp2b IgG Control IgG Chmp2b IgG Control IgG CHMP2B F1M8B7_RAT 2 0 2 0 2 0 CHMP2A B2RZB5_RAT 8 0 8 0 4 0 CHMP4B D4A9Z8_RAT 12 0 12 0 14 0 CHMP6 D3ZDR2_RAT 3 0 3 0 3 0 CHMP5 CHMP5_RAT 3 0 3 0 4 0 CHMP1A D4AE79_RAT 4 0 4 0 4 0 CHMP3 CHMP3_RAT 8 0 8 0 3 0 ALIX PDC6I_RAT 10 0 10 0 2 0 GLUN2B NMDE2_RAT 2 0 2 0 10 0 PSD-95 DLG4_RAT 3 0 3 0 26 7 SHANK1 SHAN1_RAT 5 0 5 0 7 0 HOMER1 HOME1_RAT 6 0 6 0 11 3.
These data suggest that the inhibitor of Dock5 acts on bone metastases via the inhibition of bone degradation by osteoclasts, thus breaking the vicious cycle between osteoclasts and metastatic cells in the bone. Dock5 inhibition preserves bone formation. Patients exposed to long-term treatment with bisphosphonates have a low bone formation that may in particular increases their susceptibility to atypical femoral fractures  23. Thus, we finally tested the effect of C21 on bone formation. We first measured bone formation parameters in the context of the above OVX curative model, which reflects the usual treatment initiation after the onset of the disease. We found that C21 treatment did not alter osteoblast numbers (Fig. 4a ) and activity, as shown by mineral apposition and bone formation rates (Fig.
5 mm aluminium filter, a rotation step of 0. 3, frame averaging of 2,  1  4 partial width and 9. 04 mm pixel size. Projection images were reconstructed using Nrecon software (Bruker-Skyscan) and further analyses and three-dimensional visualization were performed using CTan and CTvol softwares (Bruker-Skyscan). The regions of interest were defined in the trabecular zone (avoiding the cortical bone) from 0. 5 to 1. 9 mm below the femur distal growth plate and from 0. 25 to 2. 05 mm below the tibia proximal growth plate. Whole talus bones were analysed. Bone formation and histomorphometry. Healthy and OVX mice received intraperitoneal injections of 10 mg kg À 1 calcein in sodium carbonate 2% and 20 mg kg À 1 alizarin complexone, 8 and 2 days before killing, respectively. Femurs were fixed in 3.
7% paraformaldehyde and embedded in methyl methacrylate plastic after serial dehydration with a graded ethanol series to xylene. Five-mm sections were directly imaged using a Nanozoomer run by NDPI software (Hamamatsu). Bone histomorphometry was performed with Bioquant Osteo II version 12. 1. 6 (Bioquant Image Analysis, Nashville, TN). The region of interest was defined as 1. 2 mm long starting 100 mm from the distal femur growth plate. We assessed mineral apposition rate and bone formation rate by calcein and alizarin complexone double staining, osteoblast number by Goldner's trichrome staining and osteoclast number by TRAP/Von Kossa/alcian blue staining. For bone metastasis experiment, tibias were embedded in paraffin, sectioned, stained with TRAP/haematoxylin-eosin staining and analysed for osteoclast numbers in the region of interest defined as 1.
Beads were washed three times in lysis buffer before addition of Laemmli sample buffer. Rac was revealed by western blot using ECL western reagent (PerkinElmer, Villebonsur-Yvette, France) with a monoclonal anti-antibody used 1:10,000 (Millipore, Saint-Quentin en Yvelines, France) followed by a secondary antibody to mouse coupled to horseradish peroxidase used 1:10,000 (GE Healthcare Life Science, Velizy Villacoublay, France) (Supplementary Fig. 6 ). Quantification of activation was performed by densitometric analysis using ImageJ software. The relative amount of active Rac was determined relative to the total amount of the protein in whole-cell lysates. Immunofluorescence staining. Osteoclasts were differentiated on glass coverslips in 24-well plates and treated with 100 mM C21 or vehicle for 1, 2 or 4 h, followed or not by overnight recovery.
All experiments were carried out at least in triplicate. Statistical analysis. We assessed statistical significance with the non-parametric Mann-Whitney test or using the Kruskall-Wallis test followed by Dunn's post-test, in case of more than two samples. For comparisons including time courses, we used two-way analysis of variance. Paired t-test was used to determine whether there is a significant difference between the tumour areas measured on four different sections of the same tibia. All analyses were performed using GraphPad Prism (GraphPad Software, Inc. , La Jolla, CA). P values o0. 05 were considered statistically significant. All data are presented as the mean±s. e. m. Cauchy's equation was used to determine the confidence intervals of a quotient.
Acknowledgements We thank  P. Balaguer  ( INSERM U896 ,  Institut de Recherche en Cancerologie de Montpellier, France ) for luciferase-expressing B16BL6 cells;  R. Lebrun  ( Montpellier RIO Imaging platform and LabEx CeMEB m-CT facility ),  G. Valette  and  G. Cazals  ( Mass spectrometry platform, Institut des Biomolecules Max Mousseron, Montpellier, France ),  K. Chebli  ( Institut de Genetique Moleculaire de Montpellier, France ),  I. Ait-Arsa  (IRCM animal facility, Montpellier, France),  M. Plays  and  S. Mennechet  ( CNRS animal facilities, Montpellier, France ) for the technical help with animals;  F. Apparailly ,  P. Plence ,  D. Matyre  and  J. Quentin  ( INSERM U844 ,  Institut des Neurosciences de Montpellier, France ) for CIA procedure training; and  Anne Morel  and  Clement Kerneur  ( CNRS UMR 5237 ,  CRBM, Montpellier, France ) for the technical assistance.
Imaging was performed with the equipment of the Montpellier RIO Imaging platform (www. mri. cnrs. fr)and the  University of Saint-Etienne PLEXAN facility. We also thank  Lionel Duarte  ( LEBS, Gifsur-Yvette, France ) for protein purifications,  Christophe Velours  ( IMAGIF platform, Centre de Recherche de Gif, France ) for size-exclusion chromatography coupled to multi-angle light scattering analysis and  Raphae ¨l Rodriguez  and  Angelica Mariani  ( Institut de Chimie de Substances Naturelles, CNRS, Gif-sur-Yvette ) for complementary LC-MS and NMR analysis of C21. This work was supported by research grants from the  Fondation pour la Recherche Medicale  (FRM grant #  DVO20081013473  to A. B. ), the  Institut National du Cancer  (grant #  INCa-4361  to A. B. ), the  Agence Nationale de la Recherche  ( ANR  grant #  ANR-2011-BLAN-006  to A.