dataesr/scientific-paragraphs-categorization huggingface dataset:
Figure1. Nine exoenzymes and interaction proteins secreted by R. pomeroyi DSS-3 when co-cultured with Synechococcus. Their annotated function was inferred by the conserved domains they contain. Gene size is represented to scale but flanking regions are not. Arrow point indicates transcription orientation. The relative abundance detected with each Synechococcus strain is indicated, n. d. meaning "not detected. " Column "Roseo BLASTp" indicates the number of sequenced Roseobacter strains that encode each protein (currently 57 in the Roseobase). NA is indicated for those proteins for which a BLASTp search did not retrieve a result in the Roseobase website. The detection of each protein in previous proteomic analyses (references [15, 17] ), where cells were pregrown in a similar way and exoproteomes were processed with the same protocol, is indicated as follows: EP, exoproteome analysis in different culture conditions with easily assimilated organic carbon (i.
This work was supported by the  FP7 Marie Curie Actions   PIEF-GA-2010-272593 ,  NERC   Independent Research Fellowship   NE/K009044/1  and the  Commissariat à l'Energie Atomique et aux Energies Alternatives. The authors acknowledge the technical support received from  Philippe Guerin  ( CEA Marcoule, France ),  Dr. Susan Slade ,  Dr. Alex Jones  ( WPH Proteomics Facility, University of Warwick ), and  Dr. Nina Kamennaya  for TEM sample preparation and image acquisition. The authors declare no conflict of interest. 5 References.
The amount of signal in the controls was usually close to the non-template control. To correct for differences in the amount of starting material, the ribosomal rpsL gene was selected as a housekeeping reference gene. Results were presented as ratios of gene expression between the target gene (target) and the reference gene (rpsL), which were obtained according to the following equation: ratio = E target CP(Control-Sample) E reference CP(Control-Sample)  (Pfaffl et al. , 2002) , where E is the real-time PCR efficiency for a given gene and CP the crossing point (CP) difference ( ) of the amplification curve with the threshold. PCR efficiencies were corrected according to LinRegPCR program  (Ruijter et al. , 2009)  and the median of individual values was calculated for each gene.
The nebulizer and the curtain gas flows were set at 50 psi using nitrogen. The Turbo V ion source was operated at 550 • C with the auxiliary gas flow (nitrogen) set at 50 psi. For peptide identification, raw data were processed in ProteinPilot v4. 0 (AB Sciex) and searched using Paragon Algorithm  (Shilov et al. , 2007)  against SwissProt Pseudomonadales database (April 2013) using the following parameters. The search effort was Rapid Identification, trypsin was specified for digestion, iodoacetamide was specified for cysteine alkylation, and the instrument specified was 5600TripleTOF. A protein score >1. 3 from software supplier (called Unused Prot Score) was used for scoring and selection of proteins. Peptides were filtered using a peptide confidence score >90. Targeted analysis was performed in SRM mode on a Nexera HPLC instrument (Shimadzu, Kyoto, Japan) hyphenated to an hybrid triple quadripole/linear ion trap mass spectrometer 5500 QTRAP (AB Sciex) equipped with an ESI Turbo V ion source.
SRM ASSAYS CONSTRUCTION SRM assays were performed directly on digests from P. aeruginosa cell pellets with a high content of proteins of interest. Antibioticresistance proteins were digested in silico using Skyline software  (Maclean et al. , 2010). All possible fully tryptic, doubly and triply charged peptides that were between 7 and 20 amino acids in length were selected. Carbamidomethyl cysteines were considered. For each peptide all singly and doubly charged product ions from ion three to the last ion were selected. The three most intense transitions were selected based on library built with previous data dependent analyses acquired on a 5600 TripleTOF (AB Sciex). Collision energy and other settings were predicted according to Skyline equations for 5500 QTRAP instrument and not optimized further.
OprD PROTEIN CASE The OprD porin presents substantial polymorphism  (Pirnay et al. , 2002) , seven representative protein sequences were retrieved from Uniprot database (Uniprot release 2013_03). Based on SRM results, four combinations of peptides (SRM profile) were observed and led to five types of protein sequences (Table  S9 ). To take into account the response factor, individual transition area values were divided by the average transition area. Averages were based on samples with transition areas above 2000 a. u. However, a different average was obtained for each SRM profile. Common peptides among SRM profiles allowed the calculation of a corrective factor (Table  S9 ).
EVALUATION OF EFFLUX SYSTEM EXPRESSION IN THE LITERATURE-BASED SET P. aeruginosa harbors 12 RND-type efflux pumps on its chromosome, however only the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY pumps have been shown to be of clinical relevance  (Poole et al. , 1993 (Poole et al. , , 1996;; Köhler et al. , 1997; Mine et al. , 1999). Among the strain test set, isolates known in the literature to have a basal level expression of efflux system were used to assess the distribution of basal level expression. Strains with expression levels statistically above this distribution were considered as efflux pump overexpressers. Using this classification scheme for both strain sets, we found that, LC-ESI-SRM outperformed RT-qPCR in the literature-based set (Table  1 ). Classification of efflux system expression levels was 100% in agreement with literature data using SRM compared to 93% using RT-qPCR.
STRENGTHS AND WEAKNESSES OF LABEL-FREE SRM vs. OTHERS MASS SPECTROMETRY-BASED TECHNIQUES Several mass spectrometry-based approaches have been used to perform quantitative proteomics and have been reviewed recently, for instance, to study carbapenem resistance in Acinetobacter baumannii  (Tiwari and Tiwari, 2014). Here, we deliberately chose a bottom-up approach and more precisely a peptide-centric strategy because separation, solubilization, ionization, and fragmentation of peptides was easier and more effective than for the parent proteins. A targeted approach was rationally preferred to a shotgun approach. Reproducibility is lacking for shotgun approaches  (Tabb et al. , 2010)  and identification is limited to database exhaustiveness  (Duncan et al. , 2010). SRM is the dedicated targeted approach and was therefore selected.
Preparation and recombinant expression of the site-directed mutant E449G The pPICZ-abf containing the cDNA encoding PoAbf (EMBL Data Library accession number HE565356) was used for recombinant expression in P. pastoris as previously reported  (Amore et al. 2012). Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and the pPICZ-abf as template. The mutagenic oligonucleotides adopted as primers are ACCTTC-TACGAGGGAGGATACGCCGCTATTAG (Fw) and CTAATAGCGGCGTATCCTCCCTCGTAGAAGGT (Rev), with the mutated nucleotides underlined and bold. The mutated gene was sequenced to confirm that only the desired mutations were inserted. The wild type and mutated proteins were overexpressed, purified and assayed as previously described  (Amore et al.
Bioinformatic analysis and homology modelling The sequence of PoAbf belongs to the GH51 enzyme family, identified in the Cazy (carbohydrate-active enzymes) database  (Lombard et al. 2014 ). However, the analysis of the sequence of PoAbf revealed that it shows very low sequence identity (below 14 %) with many sequences from characterized GH51 enzymes, like Thermotogs maritima and Thermobacillus xylanilyticus. The sequence of PoAbf shows high sequence identity with 50 GH51 sequences from the public database, which together probably form a subfamily of GH51 enzymes distantly related to the main GH51 enzyme family. In order to localise the catalytic residues on PoAbf sequence, 50 GH51 sequences homologous to PoAbf were aligned and revealed the strict conservation of E293, E371, E449 E480 and E488.
The Glu nucleophile conserved among the GH51 enzyme sequences corresponding to E281 from Tm-AFase and E298 from Tx-Abf is shown encased in orange with an orange dot, and does not align with the conserved Glu nucleophile of Pleurotus ostreatus as it was not superimposing with the nucleophile from the GH51 characterized enzymes. Therefore, in order to support the hypothesis that E371 is the acid/base catalyst and to localise the nucleophile residue, a homology model of PoAbf was generated using the Tm-AFase (PDB: 2VRQ) and Tx-Abf (PDB: 3UG3) structures as templates. The superimposition of PoAbf model on the structure of Tm-AFase and Tx-Abf confirmed the putative role of acid/base catalyst for E371 as it superimposes well with E172 and E176 from Tm-AFase and Tx-Abf, respectively. From this result, we could also determine the putative nucleophile residue, namely E449, as it superimposes with E281 from Tm-AFase and E298 from Tx-Abf (Fig.
Acknowledgements This work was supported by Grant from the  Ministero dell'Università e della Ricerca Scientifica-Industrial Research  Project "Integrated agro-industrial chains with high energy efficiency for the development of eco-compatible processes of energy and biochemicals production from renewable sources and for the land valorization (EnerbioChem)"  PON01_01966 , funded in the frame of Operative National Programme Research and Competitiveness 2007-2013 D. D. Prot. n. 01/Ric. 18. 1. 2010. The authors thank  Dr. Régis Fauré  of  LISBP-INSA de Toulouse INSA/CNRS   5504-UMR INSA/INRA 792 ,  135 avenue de Rangueil, 31077 Toulouse  for the useful suggestions.
Acknowledgments The EM observations and analyses were performed by AP and CdC in the  PiCSL EM core facility (Institut de Biologie du D eveloppement/Aix Marseille Universit e, Marseille) , a member of the  France BioImaging French research infrastructure. We thank the members of this EM facility for expert technical assistance and  Lionel Spinelli  for advices in statistics. Funding AP, CD and AG held fellowships from  Aix Marseille Universit  e. This work was supported by the  Center National de la Recherche Scientifique , the  Institut National de la Sant e et de la Recherche M edicale , and  Aix-Marseille Universit  e. This work was also supported by a grant from the  Fondation pour la Recherche M edicale (FRM SC).
Elo calculations were executed in R (custom code developed in R Development Core Team 2004, www. R-project. org). Red dots = 'alpha males that controlled female harems over the season; blue dots = 'beta males that reliably held harem flanking positions with opportunistic access to females; green dots = 'peripheral males that typically lacked access to harems (the 'peripheral' males shown on the upper layer of the network were never observed interacting with alphas). Each arrow represents a directed interaction, drawn from a winner to a loser. The thickness of the arrows is proportional to the number of observed competitive events between two males (mean ± s. d. = 1. 95 ± 2. 02 events; range 1-19; 1170 competitive events; see Methods for details on data collection). Each alpha male interacted with 37.
Recording calls To provide a large dataset of male vocalizations for acoustic analysis, we opportunistically recorded males from 5 to 15 m during stereotyped displays using a Neumann KMR 82i Condenser Shotgun Microphone (with Rycote suspension and windscreen) connected to a Fostex FR-2 Field Memory Recorder (24-bit/48 kHz). Additionally, to determine call source levels, we used a calibrated Brüel and Kjaer 4189 condenser microphone (with UA-1650 windscreen) held at 1 m from, and on-axis with, the head of the animal. Signals were received by a Brüel and Kjaer 2250 sound-level metre (24-bit/48 kHz). Recordings were obtained throughout the breeding season. To determine the stability of an individual's call across different behavioural states, the social context of every recorded vocalization was categorized as either directed (emitted towards another male) or non-directed (produced when not interacting with another individual).
8 calls individual -1 , range: 9-25 calls individual -1 ). We selected acoustic parameters that could be applied to all calls. To assess temporal features, we used Avisoft SAS LAB PRO to perform a pulse train analysis on the normalized envelope of the main (rhythmic) portion of the call, excluding introductory and terminal snorts (smooth: 41 pts, frequency range 0-6 kHz), and measured the following parameters: call duration (s), total number of pulses (n) and the average repetition rate (pulse rate, Hz; figure  3a ). The transient and broadband structure of the entire call precluded a traditional analysis of energy distribution among frequencies. Several spectral features were measured over the same portion of the call with the SEEWAVE R package  [29] : the centroid of the frequency spectrum (Hz), the 25%, 50% and 75% frequency quartiles (1st quartile: 'Q25', 2nd quartile: 'Q50' and 3rd quartile: 'Q75', in Hz), the frequency bandwidth within which the energy falls within 12 dB of the maximal frequency peak (Hz), and the frequency of maximal energy (Hz; figure  3b ).
Individual signatures To determine whether there were reliable differences among the calls of individuals, we used a cross-validated and permuted discriminant function analysis (pDFA  [31, 32] ; customized script written in R). A fitting dataset (two-third of the calls from each individual) was used to generate linear discriminant functions on the basis of the acoustic features describing the calls. The remaining onethird of the calls were used as a cross-validation set to measure the percentage of correctly classified vocalizations. The mean effect size was calculated from 100 random iterations. From the cross-validation results, we extracted a confusion matrix to show the conditional probability that a call emitted by the individual i was in fact emitted by j: confusion(i, j) = p(i|j). To determine the significance of the effect size calculated by the cross-validation step, we created datasets where the identity of calls was randomly permuted between individuals (pDFA).
Acknowledgements. We gratefully acknowledge the efforts of  Selene Fregosi , who contributed to behavioural data collection, analysis of social dominance and experimental design;  Brandon Southall  who contributed to acoustic recording and source level measurements;  Roger Mundry  for statistical advice, and  Rob Harcourt ,  Burney Le Boeuf ,  Bruce Lyon  and several anonymous referees for their helpful comments on a previous version of the manuscript. We thank  Trashon Boudreaux ,  Roxanne Beltran ,  Claire Nasr ,  Bradley Funari ,  Ari Friedlaender ,  Patricia Morris ,  Guy Oliver  and  Brendan Wakefield  for technical and logistical support. We also thank  Dan Costa  and  Dan Crocker  for coordinating research activities.
Statistical analysis Data of the animal studies were analyzed with the Kruskal-Wallis test followed in case of significance (p < 0. 05) by the Mann-Whitney U test (two-sided) to compare each treated group with its vehicle control. Statistical treatments were performed with the Statview ® 5 software (SAS Institute Inc. , USA) and values of P ≤ 0. 05 were considered significant. The Prism software (5. 0d; La Jolla, CA) was used for graphical representation.
FUNDING French  ANR Agency  [ ANR-12-BSV5-0023-01 ]; ' Investissements d'Avenir' LabEx PALM  [ ANR-10-LABX-0039-PALM ];  Blaise Pascal fellowship (French Government)  (to B. S. );  Région Ile-de-France (SETCI) ;  Seoul National University  (Korea). Funding for open access charge: ANR dotation.
A decision regarding the identity of the node calibrated by a fossil has to take into account the uncertainty of its phylogenetic position and the uncertainty of the relationships among extant species. A number of fossil taxa of Magnoliidae have already been included in morphological data matrices and analyzed with a phylogenetic approach taking into account both morphological and molecular data (e. g. ,  Doyle and Endress, 2010; von Balthazar et al. , 2011). These previous studies should, in principle, allow firm calibration of a molecular time scale, which is currently lacking for Magnoliidae  (Forest and Chase, 2009). The purpose of the present study is to provide a reliable calibration scheme for the Magnoliidae, usable as a starting point for any molecular dating study of Magnoliidae or subgroups of this clade, as well as higher-level angiosperm divergence time studies.
A position sister to Lauraceae alone was four steps less parsimonious. However, as discussed for Cohongarootonia (Fossil 7), Lauraceae and Hernandiaceae are included together with Monimiaceae in a well-supported clade, but different analyses have found all possible relationships among the three families. When Doyle and Endress (2010) used a molecular backbone in which Monimiaceae were sister to Lauraceae, the single most parsimonious position of Mauldinia was sister to the whole clade of Hernandiaceae + Lauraceae + Monimiaceae. Doyle and Endress (2010)  did not test the third alternative present in the literature (Lauraceae sister to the remaining two families), but using their data set we find that the most parsimonious position for Mauldinia under this arrangement is also sister to the three living taxa.
Relationships within the compartments of Magnoliidae used by  Endress and Doyle (2009)  and  Doyle and Endress (2010)  to deduce plesiomorphic traits of these supra-specific terminals have not been contradicted by subsequent studies focused on particular families  (Marquínez et al. , 2009; Michalak et al. , 2010; Renner et al. , 2010; Chatrou et al. , 2012). For all these reasons, we argue that the positions of the fossils supported in these studies are up-to-date and can be used with some confidence. Regarding other calibration points, the phylogenetic analyses we have used as a reference for fossil relationships in this paper represent solid advances. However, future analyses with denser taxon sampling and updated morphological and molecular data sets will be required to challenge further, and hopefully confirm, the relationships summarized in this paper.
ACKNOWLEDGMENTS We thank  N. Jud ,  S. Little ,  S. Smith , and  G. Upchurch  for data that helped us in preparing this manuscript. A. Antonelli ,  D. Kspeka ,  L. Hermsen , and  M. A. Gandolfo  are thanked for discussions and support of this study. D. Polly  and two anonymous reviewers are also acknowledged for their helpful comments on this paper. This work was funded by a grant from  Agence Nationale de la Recherche  to H. S. ( ANR-12-JVS7-0015-01 ).
Plasmid construction and verification Plasmids used in this study are listed in Table  1  and oligonucleotide primers used are listed in Table  2. PCR was performed using standard conditions  [20]  with Taq Polymerase (Feldan-bio, Québec, Canada) and the primers listed in Table  1  specific for the wzd, wze and wzb sequences from L. rhamnosus strain ATCC 9595 [GenBank: AY659976] and RW-9595 M [GenBank: AY659979]  [3]. For studying in vitro protein interactions, the amplicons were ligated to the vectors pQE30 and pGEX-6-P and E. coli strains were transformed with the resulting recombinant plasmids by a standard electroporation procedure  [21]. L. lactis subsp. cremoris MG1363 was used as host strain for the Wzy-dependent polymerization complex from L. rhamnosus RW-9595 M. A total of ten plasmids were constructed for expressing Wze and Wzb in conjunction with the wild type or nine mutated versions of the wzd gene in L.
Expression and purification of tagged and untagged proteins Overexpression of the 34 kDa His 6 -tagged Wzd protein from the wzd gene of L. rhamnosus ATCC 9595 was achieved in two E. coli strains, namely BL21(DE3) or C41(DE3) carrying pQEWzd (Figure  2A ). Untagged Wzb and Wze proteins of the predicted molecular mass were obtained in E. coli strain NM522 carrying either pQEWzb (Figure  1B ) or pQEWze. GST-tagged Wze protein was obtained using both E. coli strains C41(DE3) or BL21(DE3) carrying pGSTWze (Figure  2B ). Tyrosine phosphorylation of His 6 -Wzd and GST-Wze expressed in E. coli The phosphorylation state of the expressed proteins was verified prior to testing protein interactions. To detect any autophosphorylation, proteins were incubated with or without ATP. As controls, cell lysates of the host strains BL21(DE3) and C41(DE3) carrying the vector plasmids did not show any tyrosine phosphorylated proteins, with or without added ATP (data not shown).
In the absence of added ATP, the His 6 -Wzd purified from E. coli BL21(DE3) was recognized by the antiphosphotyrosine antibody, which shows tyrosine phosphorylation (Figure  3C ). On the other hand, when purified after expression by strain C41(DE3), the similar amount of His 6 -Wzd was not phosphorylated after incubation either with or without ATP (Figure  3D ), even though the protein was detected at by Western blot using the anti-His-tag antibody (data not shown). Under these conditions, the amount of phosphorylated His 6 -Wzd protein may be below the detection level of our assay. The presence of GST-Wze was confirmed by anti-GST antibody and, as a control, GST expressed alone was not detected by the phosphotyrosine antibody (data not shown). The anti-phosphotyrosine antibody did detect phosphorylated GST-Wze in cell lysates of E.
Strain FF8 T (= CSUR P860 = DSM 28259) was isolated in Dakar, Senegal, from the urine of a 65-year-old man with acute cystitis. This strain shows a similarity of sequence of 16S rRNA of 98. 38% with Weeksella virosa, and its GenBank accession numbers are HG931340 and CCMH00000000. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis had a poor score, ranging from 1. 32 to 1. 56, that did not allow identification of the bacterium. Using a polyphasic study made of phenotypic and genomic analyses, strain FF8 T was a Gramnegative, aerobic rod and a member of the family Flavobacteriaceae. The sequenced genome is 2 562 781 bp with one chromosome but no plasmid. It exhibits a G + C content of 35. 9% and contains 2390 protein-coding and 56 RNA genes, including a complete rRNA operon.
We named this strategy taxonogenomics  [5]. Strain FF8 T (= CSUR P860 = DSM 28259) was isolated from the urine of a 65-year-old man treated at the Hôpital Principal de Dakar, Senegal. That is a Gram-negative bacterium, aerobic, indole negative, nonmotile and rod shaped. This bacterium was cultivated as part of the MALDI-TOF implementation at Hôpital Principal de Dakar aiming to improve the routine laboratory identification of microorganisms  [10]. Here we present a summary classification and a set of features for Weeksella massiliensis sp. nov. , together with the description of the complete genome sequencing and annotation. These characteristics support the circumscription of the species Weeksella massiliensis. Strain FF8 exhibited a 98. 38% 16S rRNA sequence similarity with Weeksella virosa strain DSM 16922 T (GenBank accession number NR_074495), the phylogenetically closest bacterial species with standing in nomenclature.
Extended features descriptions MALDI-TOF protein analysis was performed with a Microflex LT (Bruker Daltonics, Leipzig, Germany), as previously reported  [17, 18]. The scores previously established by Bruker allowing validating (or not) the identification of species compared to the database of the instrument were applied. Briefly, a score of 2. 000 with a species with a validly published name provided allows the identification at the species level; a score of 1. 700 and < 2. 000 allows the identification at the genus level; and a score of < 1. 700 does not allow any identification. We performed 12 distinct deposits from 12 isolated colonies of strain FF8 T. Two microliters of matrix solution (saturated solution of α-cyano-4-hydroxycinnamic acid) in 50% acetonitrile and 2. 5% trifluoroacetic acid were distributed on each smear and submitted at air drying for 5 minutes.