dataesr/scientific-paragraphs-categorization huggingface dataset:
, Piscataway, NJ). Before measuring 90°l ight scattering intensity, samples were centrifuged at 20,000 ϫ g for 10 min. MARS samples in different buffers were incubated at room temperature before measurements. Particle translational diffusion coefficients were calculated from decay curves of autocorrelation of light scattering data and converted to the Rh with the Stokes-Einstein equation using Dynamics data analysis software V5 from Protein Solutions Inc. Sedimentation Velocity-Analytical ultracentrifugation (AUC) experiments were conducted in a Beckman Optima XL-A analytical ultracentrifuge using an An 60 Ti rotor and a double-sector cell of 12-mm path length. Sedimentation velocity measurements were performed at 25,000 rpm at 15 °C. Samples of MARS were extensively dialyzed against the buffers used for sedimentation analysis.
Solvent densities were calculated using SEDNTERP software. Scans were analyzed to derive the distribution of sedimentation coefficients using SEDFIT  (30). Sedimentation Equilibrium-Experiments were conducted as described previously  (31). Equilibrium was verified from the superimposition of duplicate scans recorded at 4-h intervals. The experimental sedimentation equilibrium data were fitted to a model for a single homogeneous species following the equation, c͑r͒ ϭ c͑r ref )exp{[M r (1 Ϫ ) 2 /2RT͔͑r 2 Ϫ r ref 2 ͒} (Eq. 1) where c(r) is the protein concentration at radial position r, c(r ref ) is the concentration of the protein at an arbitrary reference radial distance r ref , M r is the molecular mass, is the partial specific volume (0. 733 at 4 °C for MARS) of the solute, is the density of the solvent, is the angular velocity of the rotor, and R and T are the molar gas constant and the absolute temperature, respectively.
Small-angle X-ray Scattering-SAXS data were collected on the SWING beamline at SOLEIL Synchrotron using an Aviex CCD detector. The sample to detector distance was 1. 84 m to cover a Q-range from 0. 004 to 0. 5 Å Ϫ1 , with Q ϭ 4sin/, where 2 is the scattering angle, and ϭ 1. 03 Å is the x-ray wavelength. Accurate SAXS measurements were performed on the MARS complex eluting from an analytical size-exclusion chromatography column with 20-MDa exclusion limit (Shodex KW405-4F 4. 6 ϫ 300 mm) directly into the SAXS flowthrough capillary cell  (32)  at a flow rate of 0. 05 ml/min in a buffer containing 20 mM Hepes, pH 7. 5, 150 mM NaCl, 2 mM DTT. The volume and concentration of the MARS complex injected on the column were 0. 1 ml and 8. 5 mg/ml, respectively. The individual SAXS patterns of the frames displaying a constant radius of gyration (R g ) in the elution peak were averaged to provide a high statistics SAXS curve for the complex  (32, 33).
The individual SAXS patterns recorded before the column dead volume were also averaged to derive the buffer scattering. The data were processed and analyzed using Foxtrot, a SAXS software developed on the SWING beamline, and the program package ATSAS (v. 2. 5. 0)  (34)  or the program foXS  (35). The radius of gyration (R g ) and the intensity at the origin I(0) were derived from the distance distribution function P(r) of the particle using the program PRIMUS  (36)  as it was difficult to accurately derive the values from Guinier's approximation in the collected Q-range because of elongated shape and large dimensions of MARS particles  (34). The three-dimensional envelopes of MARS complex were determined ab initio from the scattering curve using the program DAMMIF  (37, 38) , which generates a compact beads model fitting the experimental data to minimize the discrepancy, 2 ϭ 1 n Ϫ 1 j ͫ I exp ͑Q j ͒ Ϫ cI calc ͑Q j ͒ ͑Q j ͒ ͬ 2 (Eq.
2) where n is the number of experimental points, I exp (Q j ) is the experimental intensity, I calc (Q j ) is the calculated intensity, (Q j ) is the experimental error, and c is a scaling factor. Dummy atom models from repeated calculations were superimposed and compared using the program DAMAVER  (39). EM density maps were converted into bead models in PDB-like formats using the program EM2DAM from ATSAS. Hydrodynamic parameters were calculated from bead models or crystal structures using HYDROPRO v. 10 (40).
2A ). The complex was eluted as a reasonably symmetrical peak, as assessed by the good correlation observed with the bell-shape curve obtained by fitting experimental data to a gaussian function. The deduced average partition coefficient K av corresponds to that of a globular protein of Ͼ3,000 kDa (Fig. 2A , inset). Taking into account the calculated M r of ϳ1,500 kDa, these data suggest that MARS diffuses as a non-globular molecule. We also used SEC-MALLS to determine the mass of the complex (Fig. 2B ). A mass of 1594 Ϯ 170 kDa was obtained, with a Rh of 15. 1 Ϯ 1. 1 nm, in good agreement with its theoretical mass (Table  1 ). The homogeneity of MARS was also appraised by dynamic light scattering (DLS), which gives an estimate of the size and distribution of molecules in solution. The Rh of MARS was evaluated in different buffers (HEPES/NaCl and potassium phosphate buffer) and at protein concentrations   (62).
b SDS-PAGE densitometric analysis combined with enzymatic titration. c Sum of the standard atomic volumes of each component according to Voss and Gerstein (46) (Table  I ). d Volumes were from EM images in negative stain and vitreous ice filtered to 30 and 36 Å, respectively, from Norcum and Boisset  (27). e PDB code 2AW4 (50 S); PDB codes 2AW4 ϩ 2AVY for 70 S. f Solution properties predicted from bead models or crystal structures with the program HYDROPRO  (40).
Acknowledgments-We thank Karine Madiona (Laboratoire d'Enzymologie et Biochimie Structurales, Gif-sur-Yvette) and Christophe Velours (IMAGIF, Gif-sur-Yvette) for analytical ultracentrifugation and SEC-MALLS experiments and Eric Larquet (Laboratoire d'Enzymologie et Biochimie Structurales, Gif-sur-Yvette) for fruitful discussions. We are also indebted to  Mona Norcum  ( University of Mississippi Medical Center, Jackson, MS ) and to our late colleague and friend  Nicolas Boisset  ( UniversitePierre et Marie Curie, Paris ) for kindly providing us the EM three-dimensional reconstruction maps of rabbit MARS.
NMR Spectroscopy All NMR experiments were recorded at 293K on a Varian 600 MHz spectrometer with a cryogenically cooled triple resonance 1 H/( 15 N/ 13 C) PFG probe. Proton chemical shifts were referenced to 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) as 0 ppm. 15 N and 13 C chemical shifts were referenced indirectly to DSS. The pulse sequences were taken as implemented in the Varian BioPack. NMR data were processed using NMRPipe/ NMRDraw  [22]  and spectra were analyzed using NMRViewJ and CCPNMR Analysis  [23, 24]. The HasB samples used for NMR experiments were from 0. 35 to 0. 7 mM in 50 mM sodium phosphate buffer, pH 7, 50 mM NaCl and 15% D 2 O/H 2 O (v/v). HasB mutants NMR samples were 0. 2 mM in the same buffer.
Structure Calculation HasB CTD structures were calculated using 2117 NOE-derived distance constraints, 148 dihedral angles and 23 hydrogen bonds. Distance constraints were obtained from 3D 13 C and 15 N NOESY-HSQC experiments with mixing times of 100 and 80 ms, respectively. Dihedral angle restraints were determined using TALOS+ program  [25, 26]. Hydrogen bond donors were obtained from a series of 1 H-15 N HSQC experiments of lyophilized HasB CTD dissolved in 2 H 2 O and monitored for 36 hours. The corresponding hydrogen bond acceptors were identified based on the NOE pattern from the 3D HSQC-NOESY experiments. Structure calculation was combined with automatic NOE crosspeak assignment using ARIA version 2. 2 and CNS version 1. 2  [27, 28]. Several cycles of ARIA were performed using standard protocols with spin diffusion corrections.
NMR Backbone Dynamics, Data Acquisition and Analysis One set of T 1 and T 2 experiments and two sets of steadystate 1 H-15 N NOE were recorded on a Varian Inova 500 MHz spectrometer equipped with a triple resonance z-pulse field gradient probe using standard Varian BioPack pulse sequences. For T 1 experiments, relaxation delays were 0. 02, 0. 06, 0. 26, 0. 50, 1. 01, 1. 20, 1. 50 and 2. 00 s, and for T 2 experiments, 0. 01, 0. 03, 0. 05, 0. 07, 0. 09, 0. 13, 0. 15 and 0. 17 s. The analysis was done using the Rate Analysis package in NMRViewJ. The R 1 and R 2 relaxation rates were determined by fitting the T 1 and T 2 peak intensities to a single-exponential decay. NOE values are the ratios between the intensities of corresponding peaks in the spectra recorded with and without pre-saturation of the amide protons.
Docking of the Complex Structure A model of the complex of HasB CTD with the 21-mer peptide was built using the standard protocols of program HADDOCK, version 2. 0  [34]. Initial coordinates of HasB CTD in the free form were taken from our PDB entry 2M2K whereas the free peptide was defined in extended conformation. Twenty-three ambiguous interaction restraints, defined on the basis of the CSP and surface accessibility analysis were used as the docking driving information together with three intermolecular NOEs, which also assured the correct orientation of the peptide relative to HasR. Two hundred complex structures were generated and refined in water. Twenty structures with lowest energy values were then analyzed. The lowest energy structure of the complex, representative of this family of structure, was chosen as the model of the complex shown here.
Isothermal Titration Calorimetry (ITC) ITC was performed at 25uC using a MicroCal VP titration calorimeter (MicroCal-GE Healthcare) under the same conditions as described previously  [20]. The buffer was 20 mM sodium phosphate, pH 7, 50 mM NaCl for all the titrations, except for experiments carried out in the presence of HasR where 20 mM sodium phosphate, pH 7, 0. 08% ZW 3-14 was used. The heat of dilution of peptide/protein injections was determined either by injecting the ligand into the buffer alone or by injecting the ligand into the cell after the saturation of the protein binding site. The value obtained was subtracted from the heat of reaction to give the effective heat of binding. The resulting titration data were analyzed using Microcal-ORIGIN software package. The molar binding stoichiometry (N), association constant (K a ; K d = 1/K a ) and enthalpy changes (DH) of binding were determined by fitting the binding isotherm to a model with one set of sites.
One approach to answer this question consists in combining the NMR shift mapping with a few distance constraints obtained from intermolecular NOEs. In our case, some of HasB CTD residues in the interface region were in the intermediate chemical exchange regime and thus were not seen in the spectra. Consequently, the number of distance constraints between HasB CTD and the peptide was very limited. Three distance constraints were used as selection filter in the docking program HADDOCK  [34]  in combination with twenty-three NMR defined ambiguous interaction restraints to calculate the HasB CTD -TonB box complex model. In the model of the complex HasB CTD -TonB box peptide, HasB CTD conserves its overall structure. The peptide is bound parallel to the strand b3 of HasB CTD. The N-terminal part of the peptide between positions 95 and 100 (G 95 ALALD 100 ) forms an additional strand b4, facing the stretch of b3 residues 117-120, hence contributing to the formation of an intermolecular fourstranded b-sheet.
portion of the protein is flexible and unstructured. On the other hand, the values of R1, R2 and the NOEs for the residues N36 to F127 are representative of a well-structured region with low flexibility and slow internal motions. The atomic coordinates of the ensemble of 20 lowest energy conformers of HasB CTD have been deposited in RCSB Protein Data Bank (http://www. rcsb. org) under the accession code 2M2K.
Acknowledgments We thank the  Plate-forme de Biophysique of the Institut Pasteur  for providing access to the microcalorimeter and  Emmanuel Frachon  from the  Plate-forme 5-Production de Proteines Recombinantes et d'Anticorps of the Institut Pasteur  for expert assistance in bacterial cell production in fermentors. We are grateful to  Hilde De Reuse  for helpful discussion about HP TonB and  Fatoumata Waggeh  for assistance with production of mutant proteins.
This work was funded by the  Institut Pasteur , the  CNRS  and a grant from the  ANR (Agence Nationale de la Recherche ,  08-BLAN-HEMETRANS ). GCA was funded by postdoctoral fellowships from the  Mairie de Paris  and the  ANR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Acknowledgements We thank the  European Research Council  under the European Community's  Seventh Framework Program  (FP7/2007-2013)/  ERC Starting Grant  agreement no. 257099. We also thank the  Agence Nationale pour la Recherche  ( ANR-09-BLAN-034-02 ) and a  Marie Curie Career Integration Grant  for postdoctoral fellowships (E. B. and Y. R. ). We acknowledge the  University of Strasbourg (UdS) , the  Centre National de la Recherche Scienti-que (CNRS) , the international center for Frontier Research in Chemistry (icFRC), the  Laboratory of Excellence for Complex Systems Chemistry (LabEx CSC) , and the  Institut Universitaire de France (IUF).
pneumophila strain Paris (CR628336) and its plasmid (CR628338) as described previously  (43). Hybridizations were performed following the manufacturer's recommendations (Corning) using 250 pmol of Cy3-and Cy5labeled cDNA. Slides were scanned on a GenePix 4000A scanner (Axon Instruments), and the laser power and photomultiplier tubes (PMTs) were adjusted to balance the two channels. The resulting files were analyzed using Genepix Pro 5. 0 software. Spots were excluded from analysis if they contained high background fluorescence, slide abnormalities, or weak intensity. To obtain statistical data for the gene expression profiles, all microarrays were performed in duplicate with a dye swap. Data and statistical analysis for microarrays. Data normalization and differential analysis were conducted using R software (http://www.
R-project. org). No background subtraction was performed, but a careful graphical examination of all the slides was performed to ensure a homogeneous, low-level background in both channels. A loess normalization  (44)  was performed on a slide-by-slide basis (BioConductor package marray; http://bioconductor. org/packages/2. 2/bioc/html/marray. html). Differential analysis was carried out separately for each comparison between the two time points, using the VarMixt package  (45)  together with the P value adjustment method pioneered by Benjamini and Yekutieli  (46). Only genes with a 1. 5-fold or greater change in two or more independent experiments were considered further. Empty and flagged spots were excluded from the data set, and only genes without missing values for the comparison of interest were analyzed.
BMD Analysis Bone morphological analysis was performed using an eXplore CT 120 scanner (GE Healthcare, Little Chalfont, United Kingdom). After removing soft tissues, left femurs were placed in PBS buffer with 10% formaldehyde at 4uC for one week and scanned. Acquisition consisted of 360 views acquired in 1u increments collected in one full gantry rotation, with a 20 ms exposure/view and X-ray tube settings being 100 kV and 50 mA. CT images were reconstructed using a modified conebeam algorithm with an isotropic voxel of 0. 045 6 0. 045 6 0. 045 mm3. CT scans were analyzed using MicroViewH version 2. 3 software (GE Healthcare). A hydroxyapatite calibration phantom (SB3, Gamex RMI, WI) was used to convert gray-scale levels to HA density values. Trabecular bone of the distal femur was selected for bone mineral density and bone volume fraction (BVF = Bone Volume/Total volume) analyses by fitting a cylindrical region (r = 0.
Bone Micro-architecture Analysis To perform a measurement, the specimen was mounted on a turntable that could be shifted automatically in the axial direction (angular step: 0. 675u/reconstruction angular range: 186. 30u). Aluminum filter (0. 5 mm thick) was placed between X-rays source and sample. Micro-architecture (secondary spongiosa) was analyzed using X-ray radiation micro-CT SkyScan 1072 (BRUKER-MICROCT, Kontich, Belgium). Pictures of 1024*1024 pixels were obtained using 37 kV and 215 mA. According to camera settings final pixels measured 5. 664 mm leading to a voxel of 1. 817*10 27 mm 3. Calculation of histomorphometric parameters was performed using CTAn H and NreconH softwares (version 1. 11. and 1. 6. 1. 7 respectively).
Acknowledgments First we would like to thank in the friendliest way  Marie-Jeanne Davicco  ,  Patrice Lebecque  and  Sylvie Mercier  for their every day science support and their major contribution in the data collection. We thank  C. Pique ,  Jiake Xu ,  Zhenlin Li  for valuable material,  Paul Pilet  for bone microarchitecture analysis, Aurelie  Moulinoux-Wauquier  and  Fanny Furlan  for their technical assistance.
ACKNOWLEDGMENTS We thank  Dr. Soraya Giuliana Victoria  for critical reading of the manuscript and the lab members for useful discussion. TNT work in Chiara Zurzolo lab is supported by the  European Union   FP7 (Priority , Grant  222887 ) and by  ANR  ( ANR-09-BLAN-0122 ,  ANR-09-NEUR-002-03 ). Elise Delage is supported by the  Pasteur-Roux Fellowship Program.
3B , see accession no. GSE48540 in Material and methods). Because the CD49a molecule is up-regulated in the VSMC commitment program  [20, 21] , we focalized on VSMC-specific molecules in CD146 À/Low and CD146 High cells such as CNN1, which was significantly up-regulated in CD146 High clonal MSCs (Fig. 3B b ). To confirm these data, we performed transcriptome analysis by qRT-PCR of CNN1 and found that CNN1 was significantly up-regulated in CD146 High cells (Fig. 3C ). We evaluated other VSMC-specific transcripts and found a tendency for up-regulation of early VSMC markers, smooth muscle protein 2a (SM22a) and elastin (ELN) in CD146 High cells (Fig. S2B ). After gene array and transcriptome analysis, we studied CNN1 protein expression in CD146 À/low and CD146 High populations. Most.
Acknowledgements This work was supported by research grants from the  Institut National du Cancer. (INCa, France). We thank the  "Plateau technique de cytom etrie du Centre de Physiopathologie de Toulouse-Purpan ,"  Inserm UMR1043-CNRS UMR5282-Universit e Toulouse III, France , and technical assistance by  Fatima Ezzahra L'Faqihi-Olive  and  Val erie Duplan-Eche. We thank  Na€ ıs Prade  for her help in microarray analysis.
Toxicity Assays Rice weevils (Sitophilus oryzae, Coleoptera) were reared on wheat seeds at 27. 5uC and 70% RH. Tests and survival analysis were performed on adults feeding on food pellets (composed of wheat flour and water) incorporating PA1b (extracted from pea seeds for control tests or produced in tobacco) at 400 mg. g 21 , as described in detail in  [10]. LT 50 values were calculated using the SIMFIT software (http://www. simfit. man. ac. uk). The PA1b resistant strain, ISOR3, was reared in an identical way except that the weevils were fed on pea seeds rather than on wheat seeds. Spodoptera frugiperda Sf9 cells were grown and used for viability assays, according to  [23].
Construction of the PA1 and PA1 Truncated Variants Expression Plasmid The sequences of all the primers used in this paper are given in the Table  S1  and were synthesized by Eurogentec (France). The cDNA used for PCR corresponds to the pea gene with accession number AJ574795. The entire PA1 cDNA codes for a peptide with a signal sequence (SS), the PA1b peptide, an initial propeptide (PP), the PA1a peptide, and a second propeptide (PP). The entire PA1 cDNA (SS-PA1b-PP-PA1a-PP) was amplified, by PCR, with two specific primers (Sac1PA1F and BamH1PA1R) to incorporate the enzyme restriction sites Sac1 and BamH1. In the same way, the SS-PA1b-PP-PA1a, the SS-PA1b-PP, the SS-PA1b, and the PA1b (with inclusion of a start codon) cDNAs were amplified with, respectively, the specific primers Sac1PA1F and BamH1PA1R, BamH1PA1R and Sac1PA1-P3F; BamH1PA1R and Sac1PA1b+P2F; BamH1PA1R and Sac1PA1bF; and BamH1PA1-P1 and Sac1PA1bF.
Sequence Alignments and Comparative Modeling Sequence alignments were performed using CLUSTAL OME-GA  [29]. Comparative modeling was performed using the ORCHESTRAR homology modeling program in the SYBYL-X 2. 0 software package (TRIPOS Inc. , St Louis, MO). The PA1b NMR structure (PDB code: 1P8b) was used as a template to build the 3D structure model of the PA1b isoforms. Lipophilic potentials were calculated and represented using the MOLCAD option of the SYBYL-X 2. 0 software package.
Identification of a Mutation in the Sec7 Domain of GBF1 in R1 and R2 Cells The lack of BFA inhibition of secretion in R1 and R2 cells could result from a mutation that reduces the interaction of BFA with its target, the GBF1-ArfNGDP complex. To investigate this hypothesis, we sequenced the coding sequence of GBF1 and of the four known BFA-sensitive human Arfs (Arf1, Arf3, Arf4 and Arf5). Three isoforms of GBF1 have been reported  [42]. We only detected the expression of GBF1 isoform 3 (Genbank accession number NM_001199379. 1) in Huh-7 and Huh-7-derived cells. The complete GBF1 coding sequence was determined. Four point mutations were found, as compared to the reference sequence. These mutations led to amino-acid substitutions at positions 547, 1396, 1725, and 1833 of the coding sequence of GBF1 isoform 3 (table 2).
In contrast, we observed in R1 and R2 cells a mutation that was absent from GBF1 of Huh-7 and cells of group 2. It is a substitution of a thymine by an adenine at position 2788 of the sequence of reference. This mutation leads to the change of the methionine 832 ATG codon into a leucine TTG codon (figure  5A ). This mutation affects a residue of the Sec7 domain, which is the domain interacting with Arf and with BFA. In both cell lines, the mutation was observed together with the wild type sequence, suggesting that only one copy of the GBF1 gene is mutated in R1 and R2 cells (figure  5A ). We also sequenced the Sec7 domains of the two other BFAsensitive Arf-GEFs (BIG1 and BIG2) and the coding sequences of the 4 human Arfs known to be sensitive to BFA inhibition. No additional mutation was observed in any of the resistant cell lines (data not shown).
For androgen-depletion versus androgen-stimulation cells were cultured in phenol-red free RPMI media containing 10 % charcoal-stripped serum (CSS) (Gibco; Invitrogen) +/-treatment with 10 nM DHT for 24 hours. Enzalutamide (Selleck Chemicals) was used at 10 μM for 24 hours  (18). Cells were lysed in Passive lysis buffer and luciferase activity measured using the Dual luciferase reporter kit (Promega). All luciferase data was normalized for transfection efficiency by correcting for constitutive Renilla luciferase activity and is presented relative to the relevant control cells.
Immunofluorescence staining of cells Cells were fixed  (11)  and incubated with primary antibodies, for 1 hour; FHL2 (1:500 mouse or 1: 200 rabbit), filamin (1:1000; N-or C-terminal), paxillin (1:500) and AR-V7 (1:500). Following washing with PBS, cells were incubated with Alexa Fluor-conjugated secondary antibodies (1:600) for 1 hour. Alexa Fluor-594 phalloidin (1:500) staining for 1 hour was used to detect actin. To detect nuclei, propidium iodide or TO-PRO®-3 iodide staining was performed. Image J 1. 43u software (National Institutes of Health, USA) was used to quantify the nuclear:cytoplasmic ratio of FHL2 staining. The mean nuclear:cytoplasmic ratio was calculated from three independent experiments. For each experiment 10 cells were analyzed, and for each cell the fluorescence intensity of 10 randomly selected points in the nucleus and cytoplasm were measured and the average nuclear:cytoplasmic ratio determined.