dataesr/scientific-paragraphs-categorization huggingface dataset:
Complete DMD cDNA was amplified and sequenced in twenty overlapping fragments using published  [25]  and self-designed primers. Sequencing analysis were performed using Big Dye 3. 1 chemistry and ABI 35006L equipment (Applied Biosystems, Foster City, CA). Nucleotide positions were determined according to the standard DMD reference sequence (GenBank accession number NM_004006. 2), and mutation nomenclature follows the guidelines of the Human Genome Variation Society. In order to make data publicly available, mutations and associated phenotypic information were subtmited to the Leiden Open Variant Database (LOVD, www. dmd. nl), Leiden, the Neetherlands.
Analysis of splicing pathways Mutations predicted to affect splice sites or SRE motifs were analyzed by semi-quantitative QF-PCR in muscle cDNA. For each mutation, specific fluorescent labelled primers pairs encompassing the mutated exon were designed (Table  S2 ). PCR products were analyzed by capillary electrophoresis using ABI 35006L equipment and Genemapper software (Applied Biosystems, Foster City, CA). Splicing outcomes were determined by comparing fragment length with position of potential cryptic splice sites and exon length. Peak area was used to calculate the relative ratio of each transcript population. Samples were run in duplicate together with a normal control.
In both cases, most abundant inframe transcripts presented truncated protein-binding domains, actin-binding (ABD) or zinc-finger (ZZ) domains respectively. Loss of coding sequences may also have an impact on protein folding or stability. The mutations that abolished the function of natural sites presented a score reduction between 7% and 86% using HSF, and between 64% and 801% using MaxEnt (Table  2 ). The mutations that reduced the site efficiency presented a score reduction between 7% and 15% using HSF, and between 41% and 81%     To investigate the major factors determining the main alternative splicing pathway in 59 ss mutations (cryptic site activation versus exon-skipping), we analyzed several parameters: exon and intron length, density of SRE motifs, availability of cryptic sites and relative 39 ss strength.
However, the ability of these tools to predict exonskipping events in the DMD gene is limited. Analysis of the mutation entries in the Leiden database (LOVD) revealed that ESE disruption occurred in 50% of BMD nonsense mutations  [10]. Deburgrave et al. reported similar results, since 4 out of 8 mutations with confirmed mRNA exon-skipping had consequences on ESE motifs ESE motifs  [25]. In our subset of patients, we found a nonsense mutation in somatic mosaicism in a patient who presented DCM but no muscle weakness. Clinical, pathological and molecular studies in this patient are discussed in greater detail in a previous work  [49]. Exon-skipping events were found or predicted in seven BMD patients. We found that 8-mers putative splicing enhancers (PESE) and silencers (PESS) from Zhang and Chasin  [34]  are a powerful tool to predict in the DMD gene critical SRE motifs for exon recognition.
Quality control and genome-wide association analyses The analysis was conducted for autosomal SNPs with the PLINK whole genome association analysis toolset (PLINK version 1. 07 http://pngu. mgh. harvard. edu/purcell/plink/) [27]. All individuals with a call rate lower than 95% were discarded. Genotype data were obtained for 173 animals (including 89 affected piglets). Markers deviating from the Hardy-Weinberg equilibrium (p<10 -3 ) (741 SNPs), markers with a call rate lower than 95% (4944 SNPs), and markers with a minimum allele frequency (MAF) of less than 1% (8804 SNPs) were excluded from the analysis. Using these quality control criteria, 47,155 SNPs out of the initial set of 62,163 SNPs remained available for the Transmission Disequilibrium Tests. Nominal p values corresponding to genome-wide thresholds for significance or suggestive association were determined.
Identification of CNV's PennCNV software  [29]  was used to detect pig CNVs surrounding the SOX9 gene on SSC12. Analysis was performed with all the SNP of the chip (62,163) but only the results obtained on SSC12 were considered. PennCNV software integrates, in a joint-calling algorithm, a Hidden Markov Model (HMM) including the pedigree information when available, the total signal intensity (Log R ratio -LRR) and allelic intensity ratio (B allele frequency -BAF) at each SNP marker, the distance between neighboring SNPs and the population frequency of allele B (PFB). Both LRR and BAF were exported from GenomeStudio (Illumina San Diego, USA) and the PFB file was calculated based on the BAF of each marker. CNV calling was performed using the default parameters of the HMM model with 0. 01 as UF factor.
5U of Shrimp Alkaline Phosphatase (Promega) and 0. 8 U of Exonuclease I (New England Biolabs). Sequencing was performed on a 3730 ABI capillary DNA sequencer using a Big Dye terminator V3. 1 cycle sequencing kit (Applied Biosystems, Inc. ). Sequences were analyzed using the CodonCode Aligner software v4. 0. 4 (http://www. codoncode. com/aligner/), and any polymorphisms were detected by comparison with the reference sequence. Multiple alignments of the core region of TESCO was performed with multalin software  [30]. In total, five distinct sequences (alleles) were obtained for SOX9 (Genbank Accession numbers KF422597, KF422598, KF422599, KF422600, KF422601), and 3 for the TESCO region (Genbank Accession numbers KF422602, KF422603, KF422604).
Detection of CNV To investigate whether a chromosomal rearrangement (deletion or duplication) surrounding the SOX9 gene could be causal, genotyping data obtained with the 60K SNP chip were analyzed with PennCNV. The analyses of SNP arrays provide normalized total signal intensity and allelic intensity ratios which represent overall copy numbers and allelic contrasts. To detect CNV, we jointly analyzed the 60KSNP genotypes of the 89 affected piglets. Successive windows of 3 markers, corresponding to 150kb windows on average (given the density of the chip) were analyzed. Among all the affected individuals of our design, no structural variations were identified in the 3. 14 Mb segment encompassing SOX9.
Positions were defined from position 9033579 (=1) on the pig draft sequence for SOX9 and 9047123 (=1) for TESCO. Five different alleles were detected for SOX9 (Genbank Accession numbers: KF422597, KF422598, KF422599, KF422600, KF422601), and 3 for the TESCO (KF422602, KF422603, KF422604). doi: 10. 1371/journal. pone. 0079882. g004 testicular tissue) and of true hermaphrodites for which testislike gonads and ovarian-like gonads had been studied separately. Results had shown that the level of SOX9 increases in testis-like gonads of XX males and of true hermaphrodites, whereas it remains at a normal female level in the ovarian-like tissues of the latter. However, whereas these results alone were not proof for genetic causality, a SOX9 genetic determinism has now stronger support with the association signal obtained here.
Acknowledgements We gratefully acknowledge BIOPORC gathering the French pig breeding companies (ADN,  NUCLEUS, Gene+ and Pen Ar Lan ) for their contribution in the constitution of the collection. We also thank  LABOGENA  for its technical assistance with the microarray genotyping. We thank  J. Demars ,  H. Gilbert  and P. mulsant for helpful discussion and revision of the manuscript. The authors also thank  Byron C Jones  for its precious help for the English revision of the manuscript.
The project was supported by the  French National Research Agency (Agence Nationale de la Recherche ,  ANR-08-GENM-040   SwAn  project) and by  BIOPORC. SR PhD is funded by LABOGENA and the  Research Ministry  ( CIFFRE grant ). The  ANR  and  LABOGENA had no role in study design , data collection and analysis, decision to publish, or preparation of the manuscript. BIOPORC has contributed to the construction of the collection used for the design; BIOPORC had no role in the analysis and the preparation of the manuscript.
Extracted proteins were then dissolved in rehydratation buffer  [24]  in a final volume of 350 mL. The protein concentration was determined on an aliquot by Bradford assay. Proteins were separated on 12% w/v polyacrylamide 1D gel SDS-PAGE and on 2D gels using pH 4 to 7 non-linear IEF strips and 12% w/v polyacrylamide gel  [24]. The second dimension separation was run at 50 mA/gel for 4 h. Gel images were captured using a GS-800 densitometer (Bio-Rad) and analyzed using the Bio-rad PDQuest 2D analysis software. Bands or spots of interest were dissected, submitted to in-gel trypsin digestion  [24] , and analysed by MALDI-TOF using an AutoFlex III mass spectrometer (Bruker Daltonics) and a FlexControl software Version 3. 3. The spectrometer was used in a positive/reflector mode using peptide calibration standards (Bruker Daltonics) as references.
Samples were spotted onto MTP 384 ground steel targets using freshly prepared matrix solution composed of 2,5-dihydroxybenzoic acid (20 mg. mL 21 in trifluoroacetic acid and acetonitrile). Each spectrum was established over an average of 500-1000 laser shots and 2,5-dihydroxybenzoic acid as matrix. The MS peak list was submitted for fingerprinting using Biotools (Version 3. 2). The NCBI data base was searched using MASCOT (http://www. matrixscience. com/cgi/nph-mascot. exe).
B. Cereus Surface Hydrophobicity and Biofilm Formation Studies The polarity of control and SP treated B. cereus was studied using the MATS technique  [25]  and two solvent couples: chloroform/ hexadecane and ethyl acetate/n-decane. The effect of SP on B. cereus biofilms was investigated using a LSM 710 CLSM (Zeiss). GFP-transformed bacteria grown for 13 h in the presence of SP or SPrev were poured in dishes containing sterile glass slides. After 2 h a slide was heat fixed to observe the initial adhesion. The other slides were covered by LB medium containing SP or SPrev and incubated for 5 or 24 h without agitation. In order to control their structure, the biofilms were scanned every micrometer in depth at 3 random positions. Three-dimensional (3D) images and ortho cuts (3D/z) were reconstructed using ZenH 2009 software (Zeiss).
Statistical Analysis Cytotoxicity (LDH), caspase 1 and MATS experiments were conducted independently at least three times at different days. The results are expressed as Means 6 SEM and statistical differences were determined using the Student's t-test. Significant differences were noted as w, ww and www for p-values ,0. 05, ,0. 01 and ,0. 001, respectively. For each biofilm, the thickness was calculated from a minimum of 20 measures in different fields using the ZenH 2009 software (Zeiss). The mean biofilm thickness was quantified over three different experiments and the Student ttest was used to compare the means. SDS-PAGE electrophoresis, bidimensional electrophoresis, MALDI-TOF and immunoprecip-itation figures are representative of three independent experiments.
Acknowledgments We thank  S. Leblanc  ( BioGalenys SAS ) for the HPLC analysis of cereulide and  Dr Sastalla  ( NIH ) for providing the pTeTON-GFP plasmid. Figure 9. Inhibition of the effects of Substance P (SP) on the cytotoxic activity of Bacillus cereus, Staphylococcus aureus and Staphylococcus epidermidis. (A) Before infection, bacteria were exposed to SP in the presence of thermal water from  Uriage-les-Bains (UTW) or (B) TefloseH (TF)  (w = p,0. 05,ww = p,0. 01,www = p, 0. 001 ). doi:10. 1371/journal. pone. 0078773. g009.
Functional analysis of the RPS7 mutations The high degree of evolutionary conservation of both mutated amino acids (p. V156G and p. Y177S) suggests that these alterations may disrupt normal function. The consequences of the RPS7 Mtu and RPS7 Zma mutant proteins were first assessed using the computational analyses PANTHER and SIFT. Results from the PANTHER coding SNP analysis tool  [18]  suggested that both mutations are likely to be deleterious (PANTHER subPSEC score of 25. 00 and 25. 06 for RPS7 Mtu and RPS7 Zma , respectively). Similarly, the Sorting Tolerant From Intolerant (SIFT) algorithm  [19]  predicted both alleles to affect protein function, however, the high degree of conservation in the 72 database sequences at each position resulted in a low confidence level for the prediction (SIFT prediction score 0.
00, median conservation 3. 59 for both RPS7 Mtu and RPS7 Zma ). Collectively, the computational analysis was consistent with the Rps7 Mtu and Rps7 Zma alleles both having functional consequences. The potential effects of p. V156G and p. Y177S on RPS7 secondary structure (Figure  1D , Figure  S2 ) were also examined. There is no three-dimensional protein structure currently available for metazoan RPS7, therefore we used the available structures from Tetrahymena thermophila (PDB ID 2XZM) and Saccharomyces cerevisiae (PDB ID 3U5C), whose RPS7 ortholog sequences share 37% and 55% residue identity with mouse RPS7, respectively  [20, 21]. RPS7 structural elements appear to be highly conserved across species: the S. cerevisiae and T. thermophila proteins themselves share only 34% amino acid identity, yet their solved threedimensional structures superimpose well, and are consistent with secondary structural predictions of mouse RPS7 generated using PSSpred (http://zhanglab.
Postnatally, we observed that melanocytes were present in the tail skin of Rps7 Zma /+ mice (Figure  S7E, S7F ), yet no dark skin phenotype occurred in the tails or footpads of adult Rps7 Zma /+ or Rps7 Mtu /+mice (Figure  S7C-S7F ). This indicates that these Rps7 mutations do not cause epidermal melanocytosis, as was previously observed for mutation of Rps19 and Rps20  [5]. Collectively, these data show that mutation of Rps7 causes a severe, early reduction in melanoblasts that is sufficient to account for the white belly spotting observed in viable heterozygotes. Enlarged ventricles, cortical thinning, and deficits in working memory. We examined the neuroanatomical features of the Rps7 mutants, employing histological techniques and magnetic resonance microscopy (MRM). Gross observations and histological staining revealed marked cortical thinning accompanied by enlarged ventricles in adult Rps7 Mtu /+ (Figure  7A-7B ) and perinatal Rps7 Zma /+ mice (Figure  7C , and data not shown).
Genetic mapping and sequencing DNA was extracted from mice using standard techniques. For Mtu, the mutation was mapped using 11 affected animals and 51 polymorphic MIT makers that were amplified by PCR and visualized on a 4% agarose gel stained with ethidium bromide. Fine mapping was performed with 13 additional SNPs that distinguished between BALB/c and 101. Seventeen known and predicted genes were identified in the Mtu critical interval from the Ensembl database (ENSMUSG00000020633, ENSMUSG00 000066544, ENSMUSG00000020636, ENSMUSG00000036655, ENSMUSG00000061477, ENSMUSG00000020630, ENSMU-SG00000020629, ENSMUSG00000020628, ENSMUSG0000 0036613, ENSMUSG00000061911, ENSMUSG00000020674, ENSMUSG00000020673, ENSMUSG00000020672, ENSMU-SG00000043061, ENSMUSG00000044573, ENSMUSG00000 020669, ENSMUSG00000036136).
Ribosomal analysis For Northern blots for rRNA processing, RNA was extracted from mouse tissues with Trizol (Invitrogen) according to the manufacturer's protocol. Total RNA was fractionated on formaldehyde-agarose gels and transferred to Gene Screen Plus membrane (NEN). To detect rRNA precursor transcripts, ITS1 probe was prepared by 59 end-labeling of a 28-mer oligonucleotide (GCTCCTCCACAGTCTCCCGTTAATGATC) with 25 mCi of c [32P]-ATP and T4 polynucleotide kinase according to standard protocols. Hybridization was carried out overnight at 42uC in 66 SSPE, 1% SDS, 0. 25 mg/ml ssDNA and 56 Denhardt's solution. After hybridization, the blot was washed twice with 1% SDS in 26 SSPE for 30 min at 37uC. Quantitation of signals was obtained by phosphor screen scanning with a STORM PhosphorImager and ImageQuant software analysis (Molecular Dynamics).
In situ hybridization For whole mount, Pmel17-containing plasmid (Riken cDNA clone G370069C13; GenBank Acc: BB766987) was digested with Kpn1 and transcribed with T3 polymerase to generate DIGlabeled in situ probes. Hybridizations were performed using published protocols  [61]  with the following modifications. After probe hybridization, Ribonuclease A digestion was omitted, and Tris-buffered saline was used in place of PBS. BM-purple substrate (Roche, Molecular Biochemicals) was used in place of 5-bromo-4chloro-3-indolyl phosphate/nitroblue tetrazolium. Embryos were photographed using a Zeiss SteREO Discovery V12 microscope with a Zeiss AxioCam camera. Probes to detect Rps7 by in situ hybridization were amplified by PCR and cloned into the pCRII-TOPO vector (Invitrogen) using the following primer pairs: R7_IN_F AAGGAAATCGAAGTTGGTG and R7_IN_ R AATTAACATCCTTGCCTGTG.
D1 microscope. For apoptosis and proliferation experiments, transverse cryo-sections collected at the level of the forelimb from 3 embryos of each genotype, 6 sections per embryo, were counted for CC3+ and PH3+ cells. InStat software (GraphPad Software, Inc. ) was used for one-way ANOVA statistical analysis. No significant difference among the three genotypes was observed in PH3+ cell counts (p = 0. 902). CC3 counts were significantly different among genotypes (p = 0. 0005) and a Tukey-Kramer multiple comparison post test was used for pairwise comparison among all genotypes with the following results: Rps7+/+ versus Rps7 Zma /+ (p,0. 001), Rps7+/+ versus Rps7 Zma /+; Trp53 KO /+ (P. 0. 05, no significant difference), Rps7 Zma /+ versus Rps7 Zma /+; Trp53 KO /+ (p,0. 01).
MRM imaging Late gestation (E18. 5) embryos were collected, fixed in 4% paraformaldehyde overnight, washed extensively in PBS, and stored in 70% ethanol. Prior to imaging, embryos were rehydrated in a solution of PBS and 0. 5% (v/v) Magnevist (Bayer Healthcare Pharmaceuticals, Wayne, NJ), a contrast agent. MRM was performed on specimens using a Bruker 14. 1T MR imaging spectrometer (Bruker Biospin, Billerica, MA) using a multi-echo RARE technique  [63]  with TR/TE = 200/6. 9 ms, 8-echoes, and 4 signal averages. The resulting 3D images were acquired in 53 min with an acquisition resolution of 50 microns isotropic. Manual segmentation of the whole brain and 10 major anatomical regions was performed using Amira visualization software (v5. 2. 2, Visage Imaging Inc. , Andover MA, USA) with guidance from an anatomical reference atlas  [64].
The three-dimensional structures of RPS7 orthologs from S. cerevisiae (PDB ID 3U5C_H, red) and T. thermophila (PDB ID 2XZM_3, gray) are superimposed. The locations of the residues homologous to mouse p. V156 (*) and p. Y177 (arrow) are shown in green (S. cerevisiae) and in yellow (T. thermophila). Image generated with UCSF Chimera. doi:10. 1371/journal. pgen. 1003094. g001.
Acknowledgments We thank  Arturo Incao  for assistance with colony maintenance and all members of the  Pavan and Flint laboratories  for helpful discussions. Additional thanks go to  David Bodine  and  Brian Brooks  for discussions,  Brenda Klaunberg  and the  NIH Mouse Imaging Facility  for MRM imaging assistance,  Mark Bryant  for pathology analysis,  Stephen Wincovitch  for microscopy and imaging assistance,  Stacie Anderson  for FACS assistance,  Andy Baxevanis  for assistance with protein structure analysis, and the  NHGRI ES  and  Tg Mouse Core  for assistance in maintaining the ENU screen.
(D) The predicted structural locations of mutated amino acids in Rps7 Mtu and Rps7 Zma alleles. The three-dimensional structures of RPS7 orthologs from S. cerevisiae (PDB ID 3U5C_H, red) and T. thermophila (PDB ID 2XZM_3, gray) are superimposed. The locations of the residues homologous to mouse p. V156 (*) and p. Y177 (arrow) are shown in green (S. cerevisiae) and in yellow (T. thermophila). Image generated with UCSF Chimera. doi:10. 1371/journal. pgen. 1003094. g001.
A major goal of bio-inspired artificial intelligence is to design artificial neural networks with abilities that resemble those of animal nervous systems. It is commonly believed that two keys for evolving nature-like artificial neural networks are (1) the developmental process that links genes to nervous systems, which enables the evolution of large, regular neural networks, and (2) synaptic plasticity, which allows neural networks to change during their lifetime. So far, these two topics have been mainly studied separately. The present paper shows that they are actually deeply connected. Using a simple operant conditioning task and a classic evolutionary algorithm, we compare three ways to encode plastic neural networks: a direct encoding, a developmental encoding inspired by computational neuroscience models, and a developmental encoding inspired by morphogen gradients (similar to HyperNEAT).
This encoding produces very regular neural networks because it has to treat each neuron in a map in the exact same way as the other neurons of the same map. The second encoding is a simplified version HyperNEAT  [12] , called HNN, for Hyper Neural Network (Methods). HyperNEAT-like encodings are developmental encodings in which morphogen gradients are described as feed-forward networks of mathematical functions that operate in a Cartesian space. This indirect approach allows them to encode large networks with Nature-like connection patterns (symmetry, symmetry with variations, repetition, etc. ). The last encoding is a classic direct encoding in which evolution directly acts on the structure and the parameters of the ANN (Methods). This encoding has no bias to produce regular networks. To understand the relationship between encodings, regularity and learning abilities, we have to assess the regularity of evolved ANNs.
This work was funded by the  Agence Nationale de la Recherche  (http://www. agence-nationale-recherche. fr/) by the grants  Creadapt  ( ANR-12-JS03-0009 ) and  EvoNeuro  ( ANR-09-EMER-005 ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Statistical analysis The allelic and genotypic frequencies were estimated with the aid of the Genepop software version 4. 0. 10  [37] , using the exact test with Levene's correction to calculate the number of expected homozygotes or heterozygotes. The exact test of population differentiation based on allelic and genotypic frequencies between the French and Brazilian populations was also carried out using the Genepop program  [37]. Adherences of genotypic proportions to expectations under Hardy-Weinberg equilibrium were tested by the exact test of Guo and Thompson  [38]  using Genepop 4. 0. 10  [36]. Linkage disequilibrium (LD) between 3'UTR polymorphic sites was evaluated for each group as well as for the entire population using the Arlequin program version v3. 5. 1. 2 [39]. Given the positive LD between SNPs, but unknown gametic phase, the most likely haplotypes for each sample were determined by two independent computational methods, without taking into account any prior information: (i) the expectation-maximization (EM) algorithm  [40]  implemented with the PL-EM software  [41]  and (ii) a coalescence-based method implemented with the PHASE v2 software  [42].
Concordant haplotypes according to both methods were submitted to the exact test of population differentiation based on haplotype frequencies between French and Brazilian populations using the Arlequin program. For the analysis of sHLA-G levels and their respective associations with the 3'UTR genotypes, haplotypes and diplotypes we used nonparametric methods for comparing two sample groups (Mann-Whitney) or three sample groups [Kruskal-Wallis (P KW ) followed by the Dunn's posttest]. Correlations between sHLA-G levels and age and gender were determined using the Spearman rank correlation test. These analyses were performed using SPSS Statistics (17. 0. 2) (SPSS Software) and GraphPad InStat 3. 06 (GraphPad Software). For all instances, P values < 0. 05 were considered to be significant.