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Acknowledgments This research was supported by grants from CNRS. CNJM received a studentship from CNRS and SANOFI-AVENTIS RD Exploratory Unit. JPM and GD were supported by Agence Nationale pour la Recherche (Grant number ANR-09-MNPS-022-01 ), CNRS , Université Aix-Marseille and Fondation pour la Recherche Mé...
10.1371/journal.pone.0151233
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This research was supported by grants from the Centre National pour la Recherche Scientifique (CNRS) , SANOFI-AVENTIS and Agence Nationale de la Recherche (ANR ). CNJM received a studentship from CNRS and SANOFI-AVENTIS RD Exploratory Unit. JPM and GD were supported by Agence Nationale pour la Recherche (Grant...
10.1371/journal.pone.0151233
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Describing seed size and shape using traditional and geometric morphometrics Size analysis of seeds. Four parameters representing the seed dimensions were measured using ImageJ version 1. 42 [47] (Fig 1 ). Length and width of seeds were measured on the dorsal view. Thickness was measured as the maximum width of the ...
10.1371/journal.pone.0152394
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Agricultural and Biological Sciences
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Phoenix dactylifera seed samples for morphometric analyses. When different from the country of sampling, the country of origin of the cultivar is given in parenthesis. Acc. Nb. : Accession number; Nb. seed: Number of seeds.
10.1371/journal.pone.0152394
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Agricultural and Biological Sciences
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Phoenix non dactylifera seed samples for morphometric analyses. Acc. Nb. : Accession number; Nb. seed: Number of seeds.
10.1371/journal.pone.0152394
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Agricultural and Biological Sciences
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Acknowledgments Dr. Jean-Christophe Pintaud passed away before the submission of the final version of this manuscript. This article is dedicated to our colleague and friend who died prematurely. We are immensely grateful to Jean-Christophe Pintaud. Dr. Muriel Gros-Balthazard accepts responsibility for the integrity ...
10.1371/journal.pone.0152394
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We acknowledge Susi Gomez and Michel Ferry , Phoenix station, Elche, Spain and Vincent Battesti , CNRS-MNHN, France. We are grateful to the many collaborators who gave us permission to collect seeds on their private lands. This work was supported by the Agence Nationale de la Recherche (http://www. agence-nati...
10.1371/journal.pone.0152394
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Agricultural and Biological Sciences
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This work was supported by the Agence Nationale de la Recherche (http://www. agencenationale-recherche. fr/): programme blanc , ANR-06-BLAN-0212-02 - PHOENIX project (dir. M. Tengberg; WP coordinated by J. -F. Terral ) and ANR-07-BLAN-003301 - FRUCTIMEDHIS project (dir. A. Durand; WP coordinated by J. -F. Terral...
10.1371/journal.pone.0152394
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Agricultural and Biological Sciences
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Xenopus oocyte electrophysiology. Oocytes were removed from Xenopus laevis as previously described 34, 35. After nuclear injection of cDNAs coding for each wild-type or modified AMPAR subunits (each 50-80 pg) alone or with P2X2 (10-30 pg), oocytes were incubated in Barth's solution containing 1. 8 mM CaCl 2 and gentam...
10.1038/srep31836
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Neuroscience
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47 NA objective and an iXon3 EMCCD camera (ANDOR, Belfast, UK). Imaging was performed in an extracellular solution containing a reducing and oxygen scavenging system. For direct Stochastic Optical Reconstruction Microscopy, ensemble fluorescence of Alexa647 were first converted into a dark state using a 642 nm laser 30...
10.1038/srep31836
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Quantification of western blots was performed using Image J software (National Institutes of Health), whereby phospho-specific/GluA1 or phospho-specific/actin ratios were determined. (For full length blots see Supplementary Fig. 1 ).
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Acknowledgements We are grateful to Dr. Katherine Roche at the NINDS, NIH (Bethesda) for providing us with anti-phosphoSerine567 antibodies. Microscopy experiments were done at the Bordeaux Imaging Center , a service unit of the CNRS-INSERM and Bordeaux University , member of the national infrastructure France Bi...
10.1038/srep31836
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Integrity of the events in soybean 305423 3 40-3-2 The genetic stability of the inserted DNA over multiple generations in the single soybean events 305423 and 40-3-2 was demonstrated previously (EFSA GMO Panel, 2010b, 2012a, 2013). Integrity of these events was demonstrated by Southern analyses. 6, 7 Additional real-ti...
10.2903/j.efsa.2016.4566
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Agricultural and Biological Sciences
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Information on the expression of the inserts 8 Plants were grown in 2005 (six locations, three replicate plots) and in 2011 (10 locations, four replicate plots) under field conditions in the USA and in Canada. 9, 10 The levels of GM-HRA and CP4 EPSPS proteins in the two-event stack soybean and the two single events wer...
10.2903/j.efsa.2016.4566
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Compositional analysis 18 The seeds and forage of the soybean materials harvested from the field trials in North America in 2011 (Table 5 ) were analysed for 101 constituents (eight in forage and 93 in seeds), including the key constituents recommended for soybean by OECD (2001). Eighteen seed constituents 20 with 50%...
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Chickens were observed three times daily for clinical signs; deaths were recorded and necropsy performed on all birds found dead. Body weight and feed intake were measured every 7 days. At the end four birds per pen were taken for carcass evaluation (yield, dressing percentage, weight of thighs, breast, wings, legs, ab...
10.2903/j.efsa.2016.4566
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Agricultural and Biological Sciences
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Also, survival of soybean plants outside cultivation areas is limited mainly by a combination of low competitiveness, the absence of a dormancy phase and susceptibility to plant pathogens and cold climatic conditions. The applicant presented agronomic and phenotypic data on soybean 305423 9 40-3-2 gathered from field t...
10.2903/j.efsa.2016.4566
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Agricultural and Biological Sciences
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InnoStamp 40 ® for Multiplexed Printing of Biomolecules Using MacroStamp TM InnoStamp 40 ® (Figure 1 ) is a fully automated microcontact printer (Innopsys, Carbonne, France), which assembles different modules corresponding to every step of the microcontact printing process. It utilizes magnetic fields generated by a s...
10.3390/microarrays5040025
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Engineering
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Data Acquisition and Statistical Analysis All slides were analyzed using InnoScan 710 (Innopsys, Carbonne, France), which simultaneously scans slides at two wavelengths (PMT 532 nm and 635 nm: 100%). Images were captured using Mapix software (Innopsys, Carbonne, France). Circle grids were aligned on each spot for each ...
10.3390/microarrays5040025
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Engineering
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Acknowledgments: This work was supported by the National Research Agency (Agence Nationale pour la Recherche) , "BIOTECHNOLOGIES" program ( ANR 2010 BIOT 004 06 : Project INNODIAG to Jean Marie François and by the "pôle de compétitivité" Cancer-Bio-Santé from Midi Pyrénées, France. Julie Fredonnet was financia...
10.3390/microarrays5040025
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Engineering
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Introduction The amino acid sequence of a protein reflects the selective constraints underlying its fitness and, more generally, the evolutionary history that led to its formation [1]. A central problem is to decode this information from the sequence, and thus understand both the "architecture" of natural proteins, an...
10.1371/journal.pcbi.1004817
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Biochemistry, Genetics and Molecular Biology
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Results The SCA begins with an alignment (M sequences by L positions) representing a sampling of homologous sequences expected to share common selective pressures (Fig 1 and S1 Text, section A). Standard sequence database searching algorithms (BLAST, PSI-BLAST, etc. ) [29] together with automated alignment tools (e...
10.1371/journal.pcbi.1004817
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Biochemistry, Genetics and Molecular Biology
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In this work, we use a PFAM-based alignment of the G protein family (PFAM, PF00071, version 27. 0), and an alignment of S1A serine proteases modified from Halabi et al. [10]. After the pre-processing steps with default values for thresholds, we obtain a final G protein sequence alignment of 4978 sequences by 158 positi...
10.1371/journal.pcbi.1004817
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Biochemistry, Genetics and Molecular Biology
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5 × M 0 ). Structure of evolutionary conservation: For a large and diverse alignment (M 0 > 100, minimally), the evolutionary conservation of each amino acid a at position i taken independently of other positions is measured by the statistical quantity D a i , the Kullback-Leibler relative entropy of f a i given q a , ...
10.1371/journal.pcbi.1004817
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Biochemistry, Genetics and Molecular Biology
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Discussion A fundamental goal in biology is to understand the architectural principles of proteins-the pattern of constraints on and between amino acids that underlies folding, biochemical activities, and adaptation. An emerging approach is to leverage the growing databases of protein sequences to statistically infer t...
10.1371/journal.pcbi.1004817
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Biochemistry, Genetics and Molecular Biology
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Materials and Methods Multiple sequence alignments were obtained from previous work [10] or from the PFAM database (release 27. 0, accession codes PF00071 (G proteins), PF00186 (DHFR), and PF13354 (class A β-lactamases)), and were subject to pre-processing with default parameter values as described in Box 1. Referenc...
10.1371/journal.pcbi.1004817
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All calculations were carried out using a new python implementation of the statistical coupling analysis (pySCA v6. 2), following the algorithms described in Box 1 and in the main text. Step-by-step tutorials for executing the analysis for the four protein families are provided in the S3 Text and accompany the toolbox ...
10.1371/journal.pcbi.1004817
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Supporting Information S1 Text. Statistical Coupling Analysis: supplementary methods and codes. We provide a more detailed description of the SCA method. The pySCA toolbox is available for download through GitHub (https://github. com/reynoldsk/pySCA), and with online instructions at http:// reynoldsk. github. io/pySCA....
10.1371/journal.pcbi.1004817
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Acknowledgments We thank J. Bloom , A. Murray , and members of the Ranganathan Lab for critical review of the manuscript and I. Junier and S. Leibler for valuable discussions.
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This work was supported by Agence Nationale de la Recherche ( ANR-10-PDOC-004-01 , O. R. ), the Gordon and Betty Moore Foundation (KAR) , the National Institutes of Health ( RO1EY018720-05 , R. R. ), the Robert A. Welch Foundation ( I-1366 , R. R. ), and the Green Center for Systems Biology. The funders had no ...
10.1371/journal.pcbi.1004817
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All data and materials described in this paper are available from the GitHub software repository: https://github. com/reynoldsk/ pySCA and http://reynoldsk. github. io/pySCA.
10.1371/journal.pcbi.1004817
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We assessed the feasibility of both SNP datasets, UNEAK and transcriptome mapping, to investigate the diversity of 91 samples of wild pearl millet sampled across its distribution area. Both approaches produced several tens of thousands of single nucleotide variants, but differed in the way the variants were identified,...
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These steps depend on several parameters, such as minimum coverage, for a read to be considered as a TAG, or the number of mismatches between two TAGs to be considered as alternative copies of one locus or different loci. The TASSEL "no reference genome" pipeline is implemented in the UNEAK (Universal Network Enabled A...
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UNEAK Pipeline Raw sequences were processed with a modification of the TASSEL-GBS pipeline (Glaubitz et al. , 2014) : the UNEAK pipeline (Lu et al. , 2013). With the UNEAK pipeline, the alignment of TAGs to a reference genome is replaced by the creation of a pair of TAGs and network filtering to enable SNP discovery ...
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Biochemistry, Genetics and Molecular Biology
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4. 7 (DePristo et al. , 2011) to handle indels. SNPs and genotypes were called using UnifiedGenotyper. A total of 236,897 SNPs were then filtered for no more than three mismatches per 10 bp window, a HARD_TO_VALIDATE mapping quality (MQ) filter was applied [MQ0 ≥ 4 && ((MQ0/(1. 0 * DP)) > 0. 1], and filtering was per...
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Overlap between the Two SNP Datasets We aligned the Hapmap file of TAG sequences on the transcriptome using BWA version 0. 7. 5 (Li and Durbin, 2009) with -n 3, allowing for a maximum number of three alignments to output. We only report TAGs that had a unique hit. In order to identify SNPs shared by the two datasets,...
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Biochemistry, Genetics and Molecular Biology
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Diversity Statistics and Population Genetics Structure We performed most analyses in the R environment (R Core Team, 2015 3 ). We performed a principal component analysis (PCA) using SMARTPCA (Patterson et al. , 2006; Price et al. , 2006) as implemented in the R package SNPRelate (Zheng et al. , 2012). We used the R...
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Mapping and SNP Discovery Both pipelines produced a similarly high number of SNPs. With the UNEAK pipeline, we were able to identify 262,928 biallelic SNPs. After filtering for depth (DP ≤ 51, 50. 5% filtered) and missing data (NA ≥ 0. 1, 41. 2% filtered), we obtained 21,913 good quality SNPs. With the TM approach, a t...
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1) removed 25 and 13. 5%, respectively (Figure 2 ). The final sets of SNPs revealed that the quality of the two approaches was equivalent. The UNEAK final set of 21,913 SNPs had a mean DP per site and per sample of 7. 24 ± 3. 63 sd and an average missing rate per sample of 0. 04 ± 0. 04 sd. The TM final set of 22,262 ...
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A total of 21,410 TAG loci (8%) mapped on 13,177 transcriptome contigs (26%). The mapping was relatively good since 94% of the mapped TAGs had a unique hit, among which 96% had a perfect 64 bp match. The mean MQ of these unique hits was 34 ± 9 sd. Among the 21,943 good quality TAGs, we found 3,146 TAGS (14%) that had g...
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Genetic Structure and Genetic Diversity The two datasets showed very similar inference of genetic structure. We identified K = 3 grouping populations geographically in a Western, Center, and Eastern clusters with both datasets (Figure 3 ). Correlations between admixture values from both approaches within each cluster ...
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More generally, the regression of PCA coordinates between the two pipelines showed that most individuals qualified as slight outliers had three times more missing data in the UNEAK pipeline than in the TM pipeline. However, overall, we observed very good individual quality and a very strong congruent inference of popul...
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On the other hand, TM SFS appeared to overestimate their number. As a result, π was 2. 2 times higher with the UNEAK dataset and Tajima's D-values consequently differed considerably with a positive Tajima's D-value of 2. 74 for UNEAK and negative value of -0. 65 for TM dataset.
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DISCUSSION In this study, we compared two bioinformatics pipelines and their impact on population genetics statistics. Investigating genomic diversity is still challenging for non-model organisms with large genomes. RRL sequencing approaches, such as RNA-seq and GBS approaches, have been proposed to reduce genome compl...
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GBS Reads Biased Toward Coding Regions The quality of our two final datasets is as good as the datasets used in other population genetic studies with final coverage ranging from 5 to 10 and missing values rates below 0. 3. Many RRL datasets may have low coverage in studies whose design aims for more individuals or loci...
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Effect of Pipelines on SNPs Identified Pipeline specifics influence the number of SNPs discovered and their distribution properties. There are major differences in how SNPs are called between pipelines, because pipelines deal somewhat differently with sequencing errors, base quality values, SNP calling and genotype cal...
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Both final datasets had similar coverage and similar missing rates. Thus, it that sense, it would have little effect on number of SNPs discovered and population genetics estimates between datasets. Another possible bias comes from repetitive regions in the genome, such as paralogs, and is not always easy to identify wi...
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, 2013; Davey et al. , 2013; Gautier et al. , 2013). By construction, in RRL based on restriction enzymes, reads will start at the same mapping position which is the RE site, therefore applying PCR duplicates filter will not be possible unless a paired-end sequencing approach and random sheering is used (Davey et al. ...
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Within the TM pipeline, we applied a SNP clustering filter with no more than three SNPs per 10 bp. Nevertheless, it allowed quite a number of SNPs in a 100 bp read. For instance in the TM datasets, 56% of SNPs were less than 64 pb away. Since the UNEAK approach only allows 1 SNP per 64 pb, more than 50% of TM SNPs woul...
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, 2014). With some pipelines, the error rate estimate is considered to be constant across the genome, while other pipelines estimate an error rate for each base (Hohenlohe et al. , 2011). Error rate estimates can also account for dependency between sequencing errors, (or not; Han et al. , 2014). In GATK software, it ...
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This way of handling the error sequencing rate might be the main reason why UNEAK SFS underestimates low frequency SNPs compared to the expected distribution with a population at equilibrium. It would also explain why so few SNPs are shared, since only frequent SNPs can be found by both datasets, which was confirmed by...
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, 2014). This could also have an effect on the identification of population structure (Harvey et al. , 2015; Rodríguez-Ezpeleta et al. , 2016). With the UNEAK pipeline only allowing 1 bp mismatch, it is the maximum stringency level for an RRL pipeline. Yet, we saw no effect on population structure and we observed very...
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On the other hand, several statistics using low frequency variants differed considerably depending on the dataset used. Tajima's D test (Tajima, 1989 ) is based on the SFS pattern, where an excess of rare variants is the sign of a population expansion or positive selection and inversely, a reduction in rare variants i...
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A number of F ST outlier tests have been developed and extensively used for the discovery of candidate genes (Beaumont and Nichols, 1996; Vitalis et al. , 2001; Beaumont and Balding, 2004; Foll and Gaggiotti, 2008; Bonhomme et al. , 2010; Günther and Coop, 2013; Duforet-Frebourg et al. , 2014) and are based on the ex...
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Moreover, since few SNPs are shared, both datasets could be combined, thereby significantly increasing the SNPs used.
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FUNDING This work was supported by grant N • ANR-12-PDOC-009-01 from the Agence National de la Recherche to CB-S, YV is supported by a grant N • ANR-13-BSV7-0017-01.
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Images Acquisition and Quantification of c-Fos + Nuclei Quantification was performed by identifying spot positions. c-Fos + were counted with ICY software (http://icy. bioimageanalysis. org/) after acquired images using a digital camera (Nikon DXM 1200) of an Olympus BX600 microscope coupled to a software (Mercator Pro...
10.3389/fpsyt.2016.00171
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Biochemistry, Genetics and Molecular Biology
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Experiment II When considering all animals (i. e. , before subgroup separation), we used ANOVAs usingVAR3 statistical software (42) with an alpha level of 0. 05. In order to test global differences from chance level (50%) we use Wilcoxon rank sum test, paired version (Z of the Wilcoxon test is displayed in Statistica...
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Biochemistry, Genetics and Molecular Biology
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Experiment III Non-parametric analyses were performed using R software (version 2. 13. 2 (2011-09-30) copyright (c) 2011 the R foundation for Statistical computing with Rcmdr-package), as some of the scored behavior would not follow a Gaussian distribution. We used Wilcoxon rank sum test for two samples, the 43 ) wa...
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Metabolomics Analysis. Metabolomics analyses have been performed by Criblage Biologique Marseille (CRIBIOM) platform. For the analysis of CSF, protein of 50 L was removed by methanol precipitation using 200 L of cold methanol (-20 ∘ C) followed by 5 min centrifugation at 14000 rpm. The supernatant was recovered and fil...
10.1155/2016/9740353
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Health Professions
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This result demonstrates for the first time that NU has an effect on metabolism of CSF. These metabolites are important for the proper physiological functioning of the brain. For instance, the two first belong to the metabolic pathway of arginine and proline and N-methylsalsolinol is implicated on the balance impairmen...
10.1155/2016/9740353
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Health Professions
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Results Molecular Dynamic simulation of the XRCC4/ Lig4 C-ter complex. To get quantitative insights into the dynamics of critical residues involved in the interaction between XRCC4 and the Lig 4 C-ter domain (residues 654-911), we performed a 10 ns Molecular Dynamics (MD) simulation on this complex. From this run, we a...
10.1038/srep22878
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Biochemistry, Genetics and Molecular Biology
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Therefore we needed to establish the structure of the Lig4 clamp domain separated from XRCC4. Since the C-terminal structure of the Lig4 was only available with its XRCC4 partner (PDB entry 3II6), we first virtually assessed its intrinsic flexibility when present alone by running a 15ns MD simulation in a periodic box ...
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Biochemistry, Genetics and Molecular Biology
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2A, B ). We set the cluster radius to 2 Å and extracted eleven major main chain conformers for subsequent docking. These clamp structures thus likely represented the most probable conformations of Lig4 in solution when not in complex with XRCC4 (Fig. 2A ). This conformationally sampled Lig4 clamp receptor was then used...
10.1038/srep22878
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Biochemistry, Genetics and Molecular Biology
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Purified XRCC4 was incubated with increasing amounts of purified Lig4 C-ter domain and the reaction mixture was run on a native gel. At equimolar concentrations, both components were fully engaged in complex formation, thus confirming their functionality (Fig. 5A ). In the interaction buffer used, molecules #231, #3101...
10.1038/srep22878
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Biochemistry, Genetics and Molecular Biology
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We used AutoDock Vina 34 as docking program and the Relaxed Complex Scheme (RCS) method 28 to account for protein flexibility during the screening of potential inhibitors. RCS relies on molecules docking on several protein conformations computationally extracted by MD simulation. RCS has previously proven successfu...
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Biochemistry, Genetics and Molecular Biology
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54 (2H, t, J = 7. 5 Hz, Ar), 7. 68 (1H, bs, Ar), 8. 15 (2H, d, J = 7. 2 Hz, Ar). 13CNMR (DMSO-d6, 75 MHz) δ ppm: 168. 99, 162. 25, 158. 86, 155. 12, 142. 54, 137. 32, 133. 90, 130. 31, 128. 79, 128. 61, 128. 24, 128. 02, 127. 56, 126. 19, 71. 75, 70. 26, 70. 07, 68. 90, 65. 76, 59. 39, 55. 39, 26. 51. ES-HRMS (positi...
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Samples were heated from 20 to 89 °C. Each fluorescence measurement was taken every 0. 3 °C/tps. Data were analyzed using the CFX Manager Software (Bio-Rad Laboratories, USA) and the Tm value estimated from the transition midpoint of the fluorescence curve, which corresponds to the temperature at which half of the prot...
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A spacing of 1 Angstrom was used with the center of the grid placed at the center of gravity of the protein. During the docking, the side chains of the clamp were kept as rigid. An ensemble of ZINC database 32 and in-house molecules was used. Raltegravir, a potent selective orally bioavailable HIV-integrase inhibitor...
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Reactive compounds (covalent binders), warheads (non-covalent binders) as well as the PAINS compounds have been eliminated [55] [56] [57] , and the rest of the library has been filtered for druglikeness 58, 59. The initial part of the work was done using the Screening Assistant v. 60. The final filtered database con...
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MD simulations of 10ns for each 29 C-ter Lig4/molecule complexes were performed to determine the stability over time (RMSD) of the interaction, persistent contacts for each molecule inside the clamp domain as well as details on the molecule binding mode and energy. The binding energy was calculated from the last 4ns of...
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Biochemistry, Genetics and Molecular Biology
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2 NMR software (Bruker). For TR-NOESY spectra, protein buffer was the same as for STD-NMR experiments. Samples were prepared with 165 μL in 3 mm capillaries containing 1 mM of ligands from 100% DMSO d 6 stock solutions and with or without 14 μM of C-ter Lig4 protein. Experiments were measured with 8 scans and a mixing ...
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Then, 10 pmol purified recombinant XRCC4 protein was added in the mix with the reaction buffer (20 mM Tris pH 8. 0, 50 mM KCl, 0. 1 mM DTT and 5% glycerol) in 10 μl final reaction volume (the final concentration of each protein was 10 μM). The reaction was incubated during 15 min at room temperature. Reactions were loa...
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Acknowledgements We thank Pascal Demange and Pascal Ramos for assistance in the NMR experiments. The authors thank Dr. Neil Johnson for the careful reading of the manuscript. This work was supported by grants from the « Institut National du Cancer » ( PLBio2010 XXL-screen program ) and the « Ligue Nationale Co...
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Thus, they can be implemented without a priori knowledge on the nature of adaptive traits (Luikart et al. 2003; Stinchcombe and Hoekstra 2008). Combined with the use of large molecular marker datasets, these so-called genome scan methods have accelerated the discovery of putatively adaptive variants in natural populat...
10.1093/cz/zow088
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Biochemistry, Genetics and Molecular Biology
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Population Genomics Methods to Detect Polygenic Selection The advent of next-generation sequencing (Shendure and Ji 2008) has made possible the generation of large datasets consisting of dense arrays of markers, typically single nucleotide polymorphisms (SNPs), covering the whole genome of a species. This in turn has...
10.1093/cz/zow088
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Biochemistry, Genetics and Molecular Biology
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This type of information is unfortunately not available in large populations of unrelated individuals, which is a quite common situation in marine populations. A powerful alternative to detect QTL in populations that do not contain closely related individuals is to use a GWAS approach (Goddard and Hayes 2009). GWAS me...
10.1093/cz/zow088
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2011; Robinson et al. 2013). Under the polygenic inheritance model, the proportion of phenotypic variance explained by each chromosome is proportional to its length or gene content (Figure 3B ). This expectation provides a direct way to assess the hypothesis of polygenicity by evaluating the strength of the correlatio...
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Acknowledgments Cynthia Riginos and two anonymous reviewers provided very helpful comments that allowed us to improve the original manuscript.
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Approximately 1 × 10 6 cells in 1. 2 ml ice-cold PBS were fixed by slowly adding 3 ml cold 100% ethanol while vortexing to obtain a 70% final concentration. The incubation procedure with primary and secondary antibodies was as described for immunofluorescence, with centrifugation at 2,200 rpm for 10 min at each step. F...
10.1083/jcb.201602057
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Incubation procedures with primary (NeuN) and secondary antibodies were as described earlier. Cells were resuspended in 500 µl PI/RNase (550825; BD) solution for 15 min in the dark. Acquisition was performed on a FAC SCalibur (BD) with 2 × 10 5 cells analyzed with CellQuestPro software (BD). Debris and doublets were ex...
10.1083/jcb.201602057
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Biochemistry, Genetics and Molecular Biology
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Synchronization and stimulation were performed as described earlier. Acquisition was performed with the Zen software (ZEI SS) calibrated as follows: seven fields per well, one frame every 10 min for 12 h, 15 z-stacks of 2 µm. Other microscope acquisitions were performed on either an SP5 DM600B confocal microscope (40×/...
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Database search and data analysis Peak lists extraction from Xcalibur raw files were automatically performed using Proteome Discoverer software (version 1. 4. 0. 288; Thermo Fisher Scientific). The following parameters were set for creation of the peak lists: parent ions in the mass range 300-5,000 and no grouping of M...
10.1083/jcb.201602057
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Statistical analysis For experiments involving a single pair of conditions, statistical significance between the two sets of data were analyzed with a two-tailed, unpaired Student's t test with Prism5 (GraphPad software). For datasets containing more than two samples, one-way analysis of variance with a classical Bonfe...
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Online supplemental material Fig. S1 shows validation of siRNAs. Fig. S2 shows still images from Incucyte recordings. Fig. S3 shows immunofluorescence staining for Anillin and MgcRacGAP. Fig. S4 shows the effect of activation of other RTKs on abscission and phosphorylation of CitK. Fig. S5 shows cytometry paramete...
10.1083/jcb.201602057
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Acknowledgments The EphB2-GFP, EphB2-3YF-GFP, HA-EphB2 WT , and HA-EphB2 K661R constructs were gifts from Dr. Tony Pawson. The CitK cDNA was provided by Dr. Ferdinando Di Cunto. We are grateful to Dr. Elena Pasquale and Dr. Andreas Merdes for sharing plasmids. FACS analyses were performed at the Centre de Biolog...
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Davy ( ANR-15-CE13-0010-01 ), and in part by the French Ministry of Research with the Investissement d'Avenir Infrastructures Nationales en Biologie et Santé program ( Proteomics French Infrastructure project, ANR-10-INBS-08 ). A. Besson is supported by grants from the Fondation ARC pour la Recherche sur le Ca...
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approaches offer a promising alternative to these methods. They permit sequencing of pre-selected regions of the genome (see the review from ref. 9) that have been determined a priori to be informative for the research question and are generally effective across a range of sample types and qualities. This allows the ge...
10.1038/srep33753
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This separation of time scales holds true for several metapopulation models, including range expansions 19. Contrasting the shape of the genealogy at different sampling levels (e. g. "single deme" vs. "scatter", where each lineage comes from a different deme) indirectly provides information on the long term Nm 18. It...
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Results Genetic diversity and data summary. The demographic history of the metapopulation of C. melanopterus was examined using two datasets: the first based on estimates from a "single deme" (SID) in Northern Australia, the second based on a collection of samples taken from various locations in the Indo-Pacific, which...
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1. Overall, 2605 and 1946 high quality single nucleotide polymorphisms (SNPs) were called for the scatter and single deme dataset, respectively. As expected, we detected a higher number of SNPs from SCD (considering samples from different locations) than SID, and SNP density is higher in introns than in exons for both ...
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S2, panel b and d ). Overall, this confirms that the signal in our dataset is incompatible with panmixia and thus a simple demographic model of a single isolated population is not appropriate to fully describe the demographic history of these samples. Demographic inferences: indirect approach. First, simple population...
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Therefore, a model with two demographic changes (model CHG2; Fig. 2, panel c ) was also tested in order to account for possible more recent demographic events (e. g. post-glacial expansion), as described for other marine species 30, 31. Posterior probabilities of model CHG1 and model CHG2 are similar for both datasets...
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2, panel d ) with 100 demes to both datasets. The choice of an island model is justified by the absence of panmixia in the PCA and by the near-uniform Gst matrix (Supplementary Table S5 ), suggesting similar genetic distances across all samples except Oman. Model FIM is defined by three parameters, namely Nm, the time...
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Importantly, the two datasets produced similar estimates of both Nm and T i (Table 3 ). We further confirmed this result by applying FIM to the complete dataset of 18 samples (SCD + SID without shared samples) (Table 3 ). With this value of Nm ≈ 40 in the metapopulation, the signature of the ancestral expansion at T ...
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Detection of a recent bottleneck. We used ABC to test whether the absence of a bottleneck in our dataset reflects the real demographic history of C. melanopterus, or a lack of power to detect it. To this end, we simulated pseudo observed data sets (pods) under a modified FIM model (FIM-BOTT) characterised by two additi...
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5 , Supplementary Fig. S7 ). SCD does not show any significant reduction in Nm est until 500 generations ago, while SID starts showing a decrease almost immediately, after just 10 generations. The decline of Nm est in SID is faster when I bott = 1,000 than for I bott = 100. In both cases, the distributions of Nm est fo...
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When we fit the FIM model independently to both SCD and SID, we found a metapopulation characterised by a value of Nm ~40 (95% CI: 20-110), with an onset of formation around ~59,000 (95% CI: 6,000-165,000) generations ago. This level of connectivity is consistent with philopatry in this species, as previously described...
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Indeed, we simulated from the posterior distribution of the FIM the expected Fst between two demes at 14 independent microsatellites loci with the same mutation rate as in Vignaud et al. 29. We found the Fst distribution to be compatible with the value they found between East and West Australia, which are more distant ...
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5 and Supplementary Fig. S7 ), suggesting that the comparison of the two datasets may help detecting recent changes in effective population size. These results (both the ABC-skyline models and the Nm estimates) can be interpreted from a coalescent point of view: a recent bottleneck will produce a burst of coalescent e...
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