dataesr/scientific-paragraphs-categorization huggingface dataset:
Discussion The present work describes the development, optimization, and validation of a micro assay for the quantitation  of 21 free amino acids in human and mammalian milk using UPLC-ESI-MS/MS. Whereas FAA have been quantitated in human and cow milk with numerous analytical methods in earlier studies, the current report is first to provide data for rat milk free amino acids. In the current study, to ensure accurate quantitation of amino acids in biological samples, all validation steps were carried out in the milk samples, and the matrix effect was measured by the calculation of recovery for all FAA. The high recoveries (>95%) obtained in milk are similar to those achieved using other methods  (Agostoni et al. 2000; Carratù et al. 2003). In addition, the limits of detection (LOD) in human milk (between 0.
Acknowledgments This work was funded, in part, by a grant from the  Region Pays de la Loire  (for the creation of the "Human milk" biocollection at  Nantes University Hospital ). The salary of Emmanuelle DESNOTS was directly supported by this programme. The authors are grateful to the staff of the  Clinical Investigation Centre "Mother and Child" at the University Hospital in Nantes , especially  Dr Cécile BOSCHER (MD ) and  Arnaud LEGRAND  for their invaluable help in the management of the biocollection, to  Hélène BILLARD  for the preparation of the human milk samples from the biocollection, and also to  Evelyne BERNARDIN  for editing the bibliography.
The fMRI data were collected in 96 volumes/session with a 36 axial-slice matrix 2D SE/EP sequence (Matrix: 64 × 64; TR: 3 s; TE: 35 ms; FA: 90 • ; voxel size: 3 × 3 × 3. 75 mm). Total duration of the functional sessions was 24 min. In the 6 min immediately following, a high-resolution T1-weighted image of the brain (3D IR/GR sequence: TR = 2180ms/TE = 3. 93 ms) was acquired. During the functional sessions, subjects were instructed to breathe through the mouth without concomitant nasal airflow (velopharyngeal closure, as described above), were not cued for any stimulus presentation and were not aware of the identity of the stimuli. They were not asked to perform any detection or cognitive tasks during stimulus presentation. For each session, following the 6 on/off-blocks, participants were asked to evaluate the stimulus in terms of intensity (on a scale from "0" = "not perceived" to "10" = "extremely intense") and of pleasantness (on a scale from "-5" = "extremely unpleasant" to "+5" = "extremely pleasant").
One intensity rating and one pleasantness rating were collected for each session. Statistical analysis of fMRI data used SPM8 software (Statistical Parametric Mapping; Wellcome Department of Cognitive Neurology, London, UK) implemented in Matlab 7. 1 (MathWorks Inc. , Natick, MA, USA). Spatial pre-processing comprised registering, realignment with motion parameters included later in the model, co-registration between functional and structural images, normalization in stereotaxic space, and smoothing by means of a 7 * 7 * 7 mm 3 FWHM Gaussian kernel  (Ashburner and Friston, 2003) ; first-level statistical analysis was then implemented with canonical hemodynamic response functions. For each subject, the following individual contrasts were performed: ["Odors" vs. "Air"], ["CO 2 " vs. "Air"], ["Cong" vs.
, 2004; Seo et al. , 2010; Seo and Hummel, 2011). Analysis of chemosensory  event-related potentials revealed that both binary mixtures (congruent and incongruent) induced shorter N1 and P2 latencies and smaller N1 amplitudes compared to their individual components. Although studies in the non-chemosensory domain showed an effect of congruency on the N400 component of event-related potential  (Kutas and Federmeier, 2011) , the present EEG study did not show any direct temporal difference between congruent and incongruent mixtures. Because CSERPs were recorded from a small number of electrodes, it is not unlikely that spatial differences exist but are not reflected by our EEG measures. Indeed, fMRI data revealed that congruency affected the spatial processing of chemosensory cross-modal integration.
Definitions. Initially, the doctors recorded the weight and the loss of weight (normal, mild, or severe malnutrition). Digestive pathology and treatment (type of surgery) were prospectively collected in the software. Malnutrition treatment (enteral or parenteral nutrition, nutritional assistance) and nutritional cocktails data (volume, calories, calorie-nitrogen ratio, glucose-lipid ratio, nitrogen, and electrolytes) were equally recorded. Once the weight, height, and loss of weight expressed as a % of usual weight related to the duration of weight loss were registered, the Body Mass Index was automatically obtained allowing for the calculation of the Nutritional Risk Index and subsequently the risk of malnutrition. According to these data, the software was able to calculate the patient's level of energetic needs (Total Energy Expenditure, calculated from Resting Energy Expenditure, resulting from Harris and Benedict formula, corrected by a coefficient ranging from 1.
The majority of these proteins are Glycoside Hydrolases belonging to families  5, 8, 9, 10, 11, 18, 26, 27, 44, 48  and 74 of the CAZy database (http://www. cazy. org)  [8]. Sixty two ORFs that potentially encode dockerincontaining proteins were recently found in the genome sequence of Clostridium cellulolyticum strain ATCC 35319 (http://www. ncbi. nlm. nih. gov; GI: 220927459). All these genes are largely distributed on the chromosome, except two large clusters. The cip-cel cluster begins with the gene encoding the scaffolding protein CipC  [9]  followed by the gene encoding the major cellulosomal cellulase Cel48F and nine other dockerin-bearing enzymes. The xyl-doc cluster encodes 14 cellulosomal hemicellulases  [10]. How the organism regulates the expression of such large number of genes for biomass degradation remains a standing question.
RT-PCR Analytic RT-PCRs were performed from 100 ng of total RNA with the Titan One Tube RT-PCR kit (Roche Applied Science) as previously described  [13]. Products were displayed on a 2% agarose gel and visualized by staining with ethidium bromide. For quantitative RT-PCRs, 500 ng to 1 mg of total RNAs were reverse transcribed using Superscript III (Invitrogen) and 100 ng of random primers according to the manufacturer procedure. The cDNAs were mixed along with 150 nmoles of primers (Table  1 ) and were then subjected to qRT-PCR using an Eppendorf Mastercycler ep realplex and SYBR Premix Ex Taq, according to the manufacturer specifications (Takara). The rpoD gene target was used as an internal control. Data analysis and normalization were performed with the software supplied with the Mastercycler.
cel5A transcription cel5A was found to be transcribed as a monocistronic transcription unit of approximately 1. 8 kb (Fig. 1A ). The length of the mRNA is consistent with the length of the cel5A ORF (1425 bases) located upstream a putative Rho independent transcription termination site  [23]. The location of the transcription initiation site was investigated using three different methods with independent mRNA preparations from cells grown on cellulose containing medium. A RT-PCR analysis showed that it is located upstream position 2381/ATG (Fig. 2A , Fig. 3B ). A primer extension analysis, carried out using the radiolabeled primers A-R1 and A-R2 (Fig. 3A ) showed that three products were obtained with A-R2, whereas no short elongation product was obtained with A-R1 (data not shown). This analysis revealed two main 59 ends: one might correspond to a start located at 2383/ATG (Fig.
3B ), which Table  1. Cont. is consistent with the result obtained by RT-PCR analysis, and the second is located at 2327/2328/ATG. Two faint bands corresponding to minor mRNA species were observed. It can be noticed that these 59ends are located upstream and downstream of a putative secondary structure which might interfere with the extension procedure. The longest mRNAs was detected by RACE (GeneRacer kit, Invitrogen), in addition to several shorter mRNAs corresponding to the mRNA species revealed by primer extension (data not shown). cel44O and cel5I transcription cel44O and cel5I are adjacent divergent genes. Their ORFs (2586 and 2793 bases, respectively) are separated by 871 bp. They are both transcribed as monocistronic transcription units of 2. 7 and 2. 9 kb-long mRNAs, respectively (Fig.
The 59-end extremities, detected using primer extension experiments on another set of mRNAs (Fig. 4A  and C ), are close to the promoter region located by RT-PCR in the case of cel44O whereas they are distant in the case of cel5I. Several cel5D messengers were detected on Northern blot (Fig. 1D ). The largest one of around 3 kb-long might carry cel5D (ORF of 1. 75 kb) and the two genes located downstream from cel5D: a transferase (ORF of 0. 33 kb) and an integrase gene (ORF of 0. 66 kb). A Rho independent terminator with a low stability (DG = 211. 2 kcal/mol, calculated with the program RNAfold) was found at 56 bases after the cel5D stop codon and might stop the transcription in a fraction of transcripts producing a shorter transcript (Fig. 1D ). The transcription initiation site was localized using RT-PCR (Fig.
In contrast, an unrelated DNA probe failed to compete (data not shown), which indicated that GlyR2 binds specifically to the cel5D and to its own gene promoter regions. To determine whether in vivo, GlyR2 serves as a transcription regulator of cel5D, we constructed an insertional mutant of the glyR2 gene using the ClosTron method  [19]. cel5D messengers were analyzed from the WT strain and the glyR2 mutant, using quantitative RT-PCR on total RNAs purified from cellulosegrown cells. As shown in figure  7 , in the absence of GlyR2, the level of transcription of cel5D is about 16 times lower than in the WT strain. This suggests that GlyR2 acts as a transcriptional activator of cel5D.
Acknowledgments We thank  Odile Valette  for expert technical assistance and  Henri-Pierre Fierobe ,  Sandrine Page  `s,  Stephanie Perret  and  Pascale de Philip  for fruitful discussions. We are grateful to  Yann Denis  from the transcriptome facilities of  IMM  and to  Jean-Raphael Fantino  from the  Biosensor lab  for technical advices.
The non-saturable uptake component can be estimated from the uptake in the presence of an excess of substrate. The initial uptake rates for saturable components were determined by multiplying the cell-to-medium ratios for saturable component by the substrate concentration in the transport buffer  [9, 10]. The data were fitted to the following equation to estimate the kinetic parameters by means of nonlinear least-squares regression analysis with Prism software (Graphpad, San Diego, CA, USA): V ¼ V max Â s K m þ s where V is the initial uptake rate of substrate (nmol/min/ mg protein), s is the substrate concentration in the medium (μM), K m is the Michaelis-Menten constant (μM) and V max is the maximum uptake rate (nmol/min/mg protein). V max / K m (pmol/min/mg protein/μM = μL/min/mg protein) values were calculated as the uptake clearance for the saturable transport component.
Acknowledgements The authors would like to thank  Prof Margareta Hammarlund-Udenaes  ( Department of Pharmaceutical Bioscience, Uppsala University, Uppsala, Sweden ) for helpful discussion. We also would like to thank  Prof. Sumio Ohtsuki  ( Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan ) for mediating the supply of hCMEC/D3 cells. This work was supported in part by a  Grant-in-Aid for Scientific Research  provided by the  Ministry of Education, Culture, Sports, Science and Technology , and also by the  Japan Society for the Promotion of Science (JSPS)  and  Centre National de la Recherche Scientifique (CNRS)  under the  Japan-France Basic Scientific Cooperation Program.
). Two sequenced H. pylori strains 26695 (ATCC 700392) which were positive for homA and negative for homB, and J99 (ATCC 700824) which was positive for both homA and homB were used as reference strains  [70, 107]. Seven H. pylori clinical strains were used. The latter strains were all homA negative. The strain 417/02 contained 2 copies of homB at the homA and homB locus (GenBank accession nos. EF648377 and EF648378, respectively). All of the other 6 strains carried a single copy of homB at the homB locus (strains 771/99, 1776/05, 559/02, 351/99, 36/00 and 565/99 (GenBank accession nos. EF648357, EF656577 to EF656579, EF648369 and EF648370). DM, double mutant; NS, not significant; SM, single mutant; WT, wild-type. (B) Adherence of H. pylori wild-type (WT) and single homB knockout mutant (SM) strains to AGS cells.
Acknowledgments The authors wish to thank  Jonathan Ferrand  ( INSERM U853 University Bordeaux Segalen, Bordeaux, France ) for supplying the images presented in Figure 2B of this review. The work regarding HomB protein was supported in part by the  "Institut national de la santé et de la recherche médicale" (INSERM) , the  University Bordeaux Segalen , the  Conseil Régional d'Aquitaine (Bordeaux, France) ,.
The molecular weight markers were 1 kbp DNA Ladder (0. 5 to 10 kbp, NEB) and 100 bp DNA Ladder (0. 1 to 1. 51 kbp, NEB). Amplicons generated by PCR were purified using the GeneCleanH Turbo kit (MPBio), and cloned into the pGEM-T Easy vector (Promega) following the manufacturer's instructions. Plasmid DNA from positive clones was extracted using the QuickLyseH kit (Qiagen) and sequenced by Eurofins MWG Operon (Germany). Sequences were assembled, and analysed using the BLASTN program  [27]  at the National Center for Biotechnology Information (NCBI, http://blast. ncbi. nlm. nih. gov/).
Purification of Bacteriocin KM432Bz Purification of bacteriocin KM432Bz was guided by monitoring at each step the inhibitory activity against Lact. sakei subsp. sakei CIP 103139. The SepPak C18 fraction corresponding to 40% isopropanol, which was the most active against Lact. sakei subsp. sakei, was used for further purification by RP-HPLC (Fig. 2 ). Peakassociated fractions were hand-collected and tested for activity against Lact. sakei subsp. sakei and Listeria ivanovii subsp. ivanovii CIP 78. 42. Only one peak retained antimicrobial activity on sensitive strains, with an inhibition zone of 14 mm against L. ivanovii subsp. ivanovii. Separation of bacteriocin KM432Bz by SDS-PAGE yielded a single peptide band of 3-4 kDa active against Lact. sakei subsp. sakei (data not shown).
Analysis of the Gene Cluster Encoding Leucocin B-KM432Bz The nucleotide sequence of the bacteriocin KM432Bz gene cluster was aligned with the partial leucocin gene clusters in Genbank (data not shown), using Genbank accession number M64371 [1088 bp-leucocin A (lcnA) and immunity protein gene from Leuc. gelidum UAL187, the amino acid sequence of which will be called leucocin A-UAL187 below], L40491 [4300 bp-leucocin A ATP-dependent translocator (lcaC) and secretion protein (lcaD) genes from Leuc. gelidum UAL187], AB499610 [780 bp-leucocin A gene encoding LccA (lccA) from Leuc. pseudomesenteroides QU15, the amino acid sequence of which will be called leucocin A-QU15 below], and S72922 [804 bp-leucocin B and immunity protein genes from Leuc. carnosum Ta11a, the amino acid sequence of which will be called leucocin B-Ta11a below].
This alignment permitted to design primers specific to different regions of the gene cluster (Table  2 ). PCR amplification and sequencing allowed assembly of a 5. 5 kb-sequence containing the gene cluster involved in the production of bacteriocin KM432Bz (Genbank accession number JQ904469, Fig. 5 ). At first the leader peptide of bacteriocin KM432Bz was identified from translation analysis of the gene cluster sequence and showed to have an identical amino acid sequence to that shared by leucocin B-Ta11a and leucocin A-QU15 (Fig. 4 ), the latter having also been isolated from a Leuc. pseudomesenteroides strain. Because leucocin A from Leuc. gelidum UAL-187 and leucocin B from Leuc. carnosum Ta11a have been described long before the peptide from strain QU15, we assume that the bacteriocin from Leuc.
pseudomesenteroides QU15 has been wrongly assigned to leucocin A and should have been called leucocin B. We therefore named the bacteriocin leucocin B-KM432Bz. Since the gene nomenclature for bacteriocins is heterogeneous, the genes were named here following the nomenclature used for the mesentericin Y105 gene cluster  [31, 32] , which is the most complete gene cluster available in databases (GenBank accession number X81803) for such class IIa bacteriocins, using lcn as an abbreviation for leucocin. The general organization of the gene cluster encoding leucocin B-KM432Bz is shown in Fig. 5  compared to those of mesentericin Y105 and leucocin A-UAL187. The gene clusters follow the same pattern of organization. Two genes encode the precursors (mesY/lcaA/lcnB) and the putative immunity proteins (mesI/lcaB/lcnI) of mesentericin Y105, leucocin A-UAL187 and leucocin B-KM432Bz.
Acknowledgments We are grateful to  Yann He  ´chard for the generous gift of the Leuc. mesenteroides  Y105  and L. monocytogenes rpoN -strains, and to  Josef Deutscher  and  Eliane Milohanic  for providing L. monocytogenes AML73 and the DmanL and DmanM mutants. We thank  Svetoslav Dimitrov Todorov  for providing strain KM432Bz. We thank  Jacques d'Alayer  ( Plateforme d 'analyse et de microsequencage des proteines,  Institut Pasteur)  for the N-terminal sequencing and the mass spectrometry facility ( Plateforme de spectrometrie de masse ) at the  National Museum of Natural History. We thank  Manon Vandervennet  and  Gerard Gastine  for careful technical assistance with bacteriology and  Severine Zirah  for skilful assistance with mass spectrometry.
Data represent means 6 SEM of 6 to 8 mice per group. *p,0. 05, and **p,0. 01 versus the corresponding sham operated group; +++p,0. 001 versus the indicated I-R group. doi:10. 1371/journal. pone. 0075581. g005 peroxide/O-dianisidine dihydrocholoride, was added to each well. Optical density readings were taken for 3 minutes at 30 seconds intervals at 450 nm using a microplate reader NOVOstar TM (BMG Labtech). Activity was normalized to the sample protein concentration determined with a BCA kitH (Pierce) and expressed as mU/mg protein.
Standards Standards: all compounds were mixed together in MeOH to a first calibration solution of 2000 ng/mL. Then, a series of dilution were prepared in MeOH (1000 ng/mL, 500 ng/mL, 250 ng/mL, 125 ng/mL, 62. 5 ng/mL, 31. 25 ng/mL, 15. 6 ng/mL, 7. 8 ng/ mL, 3. 9 ng/mL). IS was added to each level at a final concentration of 200 ng/mL. This lead to 10 working calibration standards at 500 ng/mL, 250 ng/mL, 125 ng/mL, 62. 5 ng/mL, 31. 25 ng/mL, 15. 6 ng/mL, 7. 8 ng/mL, 3. 9 ng/mL, 1. 95 ng/mL and 0 ng/mL containing 200 ng/mL of IS. Data were acquired in MRM mode with optimized conditions (fragmentors and collision energy). Peak detection, integration and quantitative analysis were done using Mass Hunter Quantitative analysis software (Agilent Technologies).
Consequently, the emergence of PUUV at the patient's place of residence or the presence of another hantavirus was suspected. Therefore, the serum sample collected on day 4, which had been stored at -20 °C, was sent to the National Reference Centre for Hantavirus and tested for the presence of hantavirus RNA. The assay was positive using pan-hantavirus nested reverse transcription polymerase chain reaction (RT-PCR) targeting a part of L segment, Murinae-borne hantavirus nested RT-PCR targeting a part of S segment, and HTNV as positive control  [3, 4]. Amplicons were sequenced and a BLAST search indicated that the sequences were very close to those of Seoul (SEOV) strains (data not shown). Then the complete S coding domain sequence was recovered via three nested RT-PCRs with in-housedesigned primers (protocol available on request from the authors), producing three overlapping amplicons (GenBank accession number KC902522).
The amino acid sequence was identical to those of SEOV strains found in the United Kingdom, Vietnam, South Korea and China (GenBank accession numbers JX879769, AB618112, NC_005236 and GU592947, respectively). It was also identical to the partial S coding domain sequence obtained from the SEOV strain detected in rodents in France  [5]. Using MEGA version 5. 1  [6] , phylogenetic analysis based on S coding domain sequence confirmed that the strain -named REPLONGES/Hu/ FRA/2012/12-0882 -belonged to SEOV species, especially to the main phylogroup, which includes strains from Asia and the rest of the world  [7]  (Figure ).
The mentioned tetraspanin homologs have not been characterized in schistosomes yet. Many heat shock protein genes were identified within the data sets of the individual Herb A-and the combined treatment. Among these was a hsp70 homolog (Smp_106930), which we selected as a representative of transcriptionally enhanced hsps following both inhibitor treatments. Furthermore, IPA of the data sets of both treatments identified hsp70 within network 1, which includes molecules involved in protein folding, in each case. An impact of Herb A on protein folding processes was shown previously to result in the transcriptional activation of hsp70  [42]. Other molecules identified by IPA were the sodium/potassiumpump (Na/K-pump; Smp_015020) and cathepsin S (Smp_139240). As a member of network 3 the Na/K-pump was detected with an enhanced transcription following TRIKItreatment.
886). The validation of the microarray data of females treated with both inhibitors included the transcriptionally enhanced genes calmodulin-4 and hsp70 (see Fig. 6A ). A repressed transcription was detected for the genes coding for a protein similar to fs800, the eggshell precursor protein, tetraspanin 1, SmTYR1 and cathepsin S (Fig. 6B ). An increase of transcript levels was confirmed for calmodulin-4 and for hsp70, at least in one qPCR experiment. The identified reduction of transcripts was confirmed for Transcriptional changes from the TRIKI-treatment of adult schistosome females detected in qPCR and microarray data were calculated as log 2 ratios (treated/control). The investigated genes were SmTbRI, SmActRIIb, a protein similar to fs800, eggshell precursor, and Na/K-pump as representative for enhanced transcription within the microarray data set (A).
The selected genes representing the set of genes with repressed transcription within the microarray data set (B) were calmodulin-4, tetraspanin 18, SmSmad 4, and snurp. For each method three biological replicas were used, each with two technical replicas for the microarray analysis (except microarray 2) and three technical replicas for the qPCR. The mean of the technical replicas was calculated and is presented with standard deviations. doi:10. 1371/journal. ppat. 1003448. g004 tetraspanin 1, SmTYR1, and cathepsin S. For the gene similar to fs800 an increased transcript level was shown for two biological replicas by qPCR, but for the third qPCR replica and the microarray data a reduction of transcripts was determined. Both results correlated significantly according to Spearman's Correlations Coefficient (r s = 0.
7234). Transcriptional changes from the Herb Atreatment of adult schistosome females detected in qPCR and microarray data were calculated as log 2 ratios (treated/control). According to the microarray analysis SmActRIIb, calmodulin-4, and hsp70 showed enhanced transcription (A), whereas tetraspanin-1, SmSmad 4, and Smp48 were detected as transcriptionally repressed (B). For each method three biological replicas were used, each with two technical replicas for the microarray analysis (except microarray 2) and three technical replicas for the qPCR. The mean of the technical replicas was calculated and is presented with standard deviations. doi:10. 1371/journal. ppat. 1003448. g005.