dataesr/scientific-paragraphs-categorization huggingface dataset:
Chemicals Chlorpyrifos (purity: 99. 8%) was purchased from LGC Standards (Molsheim, France). It was dissolved in commercially available rapeseed oil as vehicle and administered by gavage (at a dose of 1 mg/kg bodyweight/day) to the animals in the CPF1 group. This dose corresponds to (i) the maximum daily intake and (ii) the oral ''no observed effect level'' for maternal toxicity, as indicated by the inhibition of cholinesterase activity  [11]. Animals in a control (CPF0) group were gavaged with a volume of 1 mL/ kg bodyweight of vehicle. The high-molecular-weight fluorescent marker fluorescein isothiocyanate-dextran 4 kDa (FITC-Dextran) powder was purchased from Sigma Aldrich (Saint Louis, MO, USA) and dissolved in pure water to a concentration of 80 mg/mL.
Experimental conditions The day of parturition was considered to be postnatal day 0 (PND0). From days 0 to 21 of gestational period, dams received daily doses of 1 mg CPF/kg bodyweight (in the CPF1 group) or vehicle (in the CPF0 group). Pups were thus exposed in utero and then postnatally through their maternal milk (from PND0 to D21). At PND1, each pup's legs were tattooed with a special veterinary ink (Centravet, Nancy, France) for identification and monitoring. The pups in each litter were counted, sexed, and weighed at PND0 (CPF0, n = 26; CPF1, n = 30). The animal body weight was recorded at PND1, 2, 5, 7, 10, 13, 16, 19, and 21. Two different developmental time points were studied: D21 (weaning) and D60 (adulthood). At D21, intestinal permeability was studied in some pups (CPF0: n = 12; CPF1: n = 17).
The other pups were weaned, separated from their mothers, and divided into groups (with three pups per cage) until the age of 60 days (CPF0: n = 14; CPF1: n = 13). From D21 to D60, the animals were individually gavaged with either vehicle alone (for CPF0) or 1 mg/kg/day CPF (as previously described for the dams). At D60, intestinal permeability was investigated in the same way as at D21 and the animals were then all euthanized. Again, different intestinal segments (ileum and colon) and sterile organs (spleen) were removed under sterile conditions. Given that similar patterns were obtained for all sterile organs, only results for the spleen are presented below.
Statistical analysis Values are expressed as the mean6SEM. Intergroup comparisons were performed using a Kruskal-Wallis test. If the Kruskal-Wallis test result was statistically significant, a Mann-Whitney test was then performed. The threshold for statistical significance was set to p,0. 05 for all tests. Only the p value for the Mann-Whitney test is reported below. Translocation data were analyzed using a x2 test for comparison of scores (positive vs. negative samples) between groups. All analyses were performed with StatView software (version 5. 0, Abacus Concepts Inc. , Berkeley, CA, USA).
Evaluation of intestinal permeability with FITC-dextran The FITC-dextran-associated fluorescence peak was clearly observed in blood from CPF-exposed animals but not in blood from control animals (Figure  2 ). ThisCPF1 vs. CPF0difference was statistically significant at D21 (p,0. 05). Moreover, FITC-dextran entered the blood quickly in CPF1 rats tested at D21. Comparison of the corresponding time points at D21 and D60 in the CPFexposed groups revealed that FITC-dextran was first detected after323. 0614. 4 min in D21 animals and after 356. 361. 7 min in D60 animals. Furthermore, FITC-dextran was detected in a significantly higher proportion at D21 than at D60 (p,0. 05).
Gene expression of TJ proteins We observed differences in gene expression between exposed and non-exposed pups (Figure  3 ). At weaning, we observed a decrease in ZO-1 mRNA expression in ileum and colon (p,0. 05 for both) in CPF1 animals compared to CPF0. Claudin 4 mRNA expression was lower in the ileum (p, 0. 05) in CPF1 animals than in CPF0 animals. Both claudin 1 and occludin did not display any variations in mRNA expression at D21. At D60, no variation of mRNA expression was observed for ZO-1, claudin 1 and claudin 4. mRNA expression of occludin rose in ileum (CPF1 vs. CPF0, p,0. 05).
Immunohistochemical assessment of ZO-1 and claudin 4 To investigate the distribution and localization of ZO-1 and claudin-4 in the gastrointestinal tract, we used rabbit polyclonal primary antibodies directed against epitopes in the proteins' Cterminal domains. No labeling was observed on control slides for which the primary antibody had been omitted. We found that ZO-1 and claudin-4 were expressed in the two intestinal segments studied (ileum and colon). In D21 animals, ZO-1 was found to be expressed in the ileum and colon (Figure  4-A ). We observed that ZO-1 was located at the apical cell membrane and the lateral cell membrane in the tissues of CPF0 animals. The labeling appeared as a marked apical belt and well-defined points especially in the ileum and less marked in the colon (Figure  4-A ).
In CPF1 animals, ZO-1 staining appeared as a discontinuous belt and faint points in the ileum. The CPF0 and CPF1animals did not differ in terms of the distribution and intensity of labeling of ZO-1 in the colon (Figure  4-B ). Claudin-4 staining displayed a different distribution pattern, which was located more diffusely within the cells in the ileum and the colon. More intense TJ staining was observed in the ileum of CPF0 rats. In the ileum, claudin-4 was predominantly located in the microvillus surface of epithelial cells. At D21, the CPF0 and CPF1 animals did not differ clearly in terms of the distribution of claudin-4 in epithelial cells (data not shown). In D60 rats, the CPF0 and CPF1 animals did not differ in terms of the distribution and intensity of labeling of ZO-1. In CPF0 rats, ZO-1 staining was barely visible and weak in the ileum.
However, ZO-1 was clearly visible on top of villosities on the apical surface of colonic epithelial cells in CPF0 animals (Figure  4-C ). In CPF1 animals, ZO-1 staining was weakly visible in the ileum and almost undetectable in the colon (Figure  4-D ). As had been seen inCPF0 rats at D21, claudin-4 was essentially located in the cytoplasm of cells in this group at D60. In the ileum, the surface epithelium was strongly stained both in CPF0 and CPF1 rats. In the colon, claudin-4 staining was weak in CPF0 rats and barely visible in CPF1 rats (data not shown).
Bacterial translocation Aerobes and anaerobes were better able to translocate to the spleen (Figure  5 ) in CPF1 rats than in CPF0 rats (with contamination rates of up to 50% and up to 20%, respectively). At weaning (D21), we observed greater bacterial translocation of total aerobes and total anaerobes in CPF1 pups than in CPF0 pups (50%, vs. 0%; p,0. 001; 50% vs. 10%; p,0. 05, respectively). Concerning aerobes, Enterococcus spp. was found in 20% of the CPF1 pups (vs. 0% of the CPF0 pups) and Staphylococcus spp. was found in 30% of the CPF1 pups (vs. 0% of the CPF0 pups; p, 0. 10). For anaerobes, Bacteroides fragilis was found in 30% of the CPF1 pups (vs. 0% oftheCPF0 pups; p,0. 10) and Clostridium spp. was found in 20% of the CPF1 pups(vs. 0% of the CPF0 pups). At D60 (adulthood), the extent of translocation was lower than atD21 (weaning).
Total aerobes were found in 40% of CPF1 rats (vs. 0% of the CPF0 pups, indicative, p,0. 1) but neither Enterococcus spp. nor Staphyloccus spp. had translocated. Hence, the observed translocation must have been due to other species. We observed translocation of total anaerobes in 50% of the CPF1 animals (vs. 20% of CPF0 rats) and translocation of Bacteroides fragilis in 10%of the exposed-animals (vs. 0% of controls). Again, other species of bacteria must have contributed to this translocation.
Surprisingly, CPF did not seem to impact on the intestinal distribution of claudin 4 -at least in immature animals. In D60 animals, ZO-1 and claudin 4 staining was much weaker in CPF1 rats than in CPF0 rats; this might be due to downregulated expression and/or altered localization. These observations could be related to Gearhart's study that showed that chlorpyrifos inhibit kinesin-dependent microtubule motility and Grigoryan's study that showed a tubulin polymerization disruption by chlorpyrifos oxon  [22, 23]. As a consequence, proteins synthetized and involved in tight junction formation might be restrained to the cytoplasmic space rather than moving along microtubules to tight junction space. The cholinergic signaling is also involved in the regulation of the barrier function. As described by Cameron and Perdue  [24] , HRP uptake as a marker of transepithelial transport is stimulated by the M3 muscarinic agonist bethanechol.
Imaging for calculation of the below-knee arterial calcification score Tibial artery calcium scoring was performed after scanning with a 128-slice multidetector CT scanner (Somatom Definition Flash, Siemens Healthcare, Forchheim, Germany) in the craniocaudal direction, from the bottom of the patella down to the ankle region. Contrast agent was not used. Crosssectional slices (with: 3 mm) were analyzed individually. The analysis was performed with a commercially available software package (Heartbeat CaScore, Philips Healthcare, Eindhoven, The Netherlands) by radiologists who were not aware of the results of the clinical examination or laboratory assays, On cross-sectional images, areas of calcification along below-knee arteries with a density ≥130 Hounsfield units and a surface >1 mm 2 were identified automatically.
Unadjusted (Table  1 ) and adjusted (Table  2 ) logistic regression analyses were performed to evaluate the association between the peripheral calcification score (as categorized by the median) and dp-ucMGP. A p value ≤ 0. 05 was considered to be statistically significant. All statistical analyses were performed using SPSS software (version 13. 0, SPSS Inc. , Chicago IL, USA) for Windows (Microsoft Corp. , Redmond WA, USA).
Acknowledgments The authors thank  Lilly Company , the  University of Lausanne , and the clinical staff of the  Center for Clinical Investigations Paris-Est , as well as the Diabetes and Cardiology Departments from the  AP-HP Pitié-Salpêtrière Hospital in Paris  for their participation in this project. This study was supported by a fund from  Lilly Company. The company was involved neither in the design of the study nor in data collection. The research activities of C. E. Aubert were supported by a doctoral research scholarship from the  University of Lausanne.
Acknowledgements This work was supported by  Agencia Nacional de Promoci on Científica y Tecnol ogica  ( PICT 00479/06 ),  CONICET  ( PIP 0598 ),  Universidad Nacional de La Plata  (Project  11/X548 ), and  Bordeaux Science Agro ,  Minist ere de l'Agriculture Français. P Carasi  is a fellow of CONICET;  G De Antoni  is a researcher of the  Comisi on de Investigaciones Científicas de la Provincia de Buenos Aires (CIC-PBA) ;  M Serradell  is a member of the  Carrera de Investigador Científico y Tecnol ogico of CONICET. MC Urdaci  is researcher of  Bordeaux Science Agro, Universit e de Bordeaux.
MATERIALS AND METHODS Except when mentioned, all chemicals were purchased from Sigma. Cloning and Site-directed Mutagenesis of ZgLamA GH16 -The gene encoding the putative laminarinase ZgLamA (locus identifier: Zobellia_2431, GenBank TM accession number CAZ96583) was cloned as by Groisillier et al. (28). Briefly, primers were designed to amplify the coding region corresponding to the catalytic module of LamA (forward primer ggggggggatccgcctttaataccttagtgttttcaga, reverse primer cccccccaattgttatt-gatagatccttacatagtctatttc) by PCR from Z. galactanivorans genomic DNA. After digestion with the restriction enzymes BamHI and MfeI, the purified PCR product was ligated using the T4 DNA ligase into the expression vector pFO4 predigested by BamHI and EcoRI, resulting in a recombinant protein with a N-terminal hexahistidine tag (plasmid pLamA cat ).
04% (w/v) of MLG degradation products) that were added to 1 l of reservoir solution containing 100 mM MIB buffer, pH 4. 0, 17% of PEG 1500, and 10% of glycerol in hanging drops at 12 °C. Prior to flash-freezing in a nitrogen stream at 100 K, single crystals were quickly soaked in the same crystallization solution supplemented with 10, 20, or 12% glycerol, respectively. Diffraction data for ZgLamA GH16 were collected on Beamline ID23-1 at the European Synchrotron Radiation Facility (Grenoble, France). The diffraction data for the two complexes, ZgLamA GH16-E269S ⅐laminarin and ZgLamA GH16-E269S ⅐MLG, were collected on Beamline ID29 (European Synchrotron Radiation Facility). X-ray diffraction data were integrated using Mosflm (32) and scaled with Scala  (33). The structure of ZgLamA GH16 was determined by molecular replacement with MolRep (34) using the chain A of RmLamR from Rhodothermus marinus (PDB code 3ILN  (35)  as a starting model.
The structure of ZgLamA GH16 was manually built using COOT  (36). For both complexes of ZgLamA GH16-E269S , the structures were also determined by molecular replacement, but using the coordinates of the chain A of ZgLamA GH16. For all the structures, the initial molecular replacement solutions were further refined with the program REFMAC5 (37), alternating with cycles of manual rebuilding using COOT. A subset of 5% randomly selected reflections was excluded from computational refinement to calculate the R free factors throughout the refinement. The addition of the ligand sugar units for the complexed structures was performed manually using COOT. Water molecules were added automatically with REFMAC-ARP/wARP and visually verified. The final refinement was carried out using REFMAC5 with TLS, anisotropic B factors, and NCS restraints and Babinet scaling.
Data collection and refinement parameters are presented in Table  1. The atomic coordinates and structure factors of ZgLamA GH16 , ZgLamA GH16-E269S ⅐laminarin and ZgLamA GH16-E269S ⅐MLG (codes 4BQ1, 4BOW, and 4BPZ, respectively) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics (Rutgers University, New Brunswick, NJ). Sequence Analysis and Structure Comparison-The family GH16 laminarinases with known three-dimensional crystal structures were selected in the CAZY database  (38) , and their amino acid sequences were recovered in the PDB. These sequences and the ZgLamA GH16 sequence were aligned using MAFFT  (39). The multiple sequence alignment was manually refined using Bioedit, based on the superimposition of the structures of the different laminarinases.
The crystal structure of ZgLamA GH16 was compared with those of the laminarinase PcLam16A from Phanerochaete chrysosporium (PDB code 2W39)  (40)  and of the lichenase from Fibrobacter succinogenes (PDB code 1ZM1) (41) using COOT and PyMOL. For phylogenetic analysis, close homologues of ZgLamA GH16 and RmlamR were selected on the basis of a BlastP search on GenBank TM NR. The selected proteins were subjected to multiple sequence alignment using MAFFT  (42) , with the iterative refinement method and the scoring matrix Blosum62. These alignments were manually edited using Bioedit (© Tom Hall). The phylogenetic tree was derived from the refined alignment using the maximum likelihood method with the program MEGA 5  (43). The reliability of the trees was tested by bootstrap analysis using 100 resamplings of the data set.
7 mM (1. 57 mg⅐ml Ϫ1 ), respectively) (Fig. 2B ). Taking into account these two parameters, ZgLamA GH16 has a catalytic efficiency (k cat /K m ) 22-fold higher on laminarin than on MLG. The nucleophile candidate Glu-269 was successfully replaced by a serine, by site-directed mutagenesis. The mutant variant ZgLamA GH16-E269S was also produced in E. coli BL21(DE3) in a soluble form with high yield (110 mg/liter of culture) and purified by nickel affinity chromatography. ZgLamA GH16-E269S (10 nM) was assayed on laminarin and MLG with reducing sugar assay as for ZgLamA GH16 , but no enzymatic activity was detected even after 24 h of incubation (data not shown), confirming the involvement of Glu-269 in the catalytic machinery of ZgLamA. Hydrolysis Pattern of Laminarin and MLG Oligosaccharides by ZgLamA GH16 -The hydrolysis of laminarin and MLG by ZgLamA GH16 was monitored by FACE for 1 h and 30 min, respectively (Fig.
The C-terminal region is composed of a catalytic module of glycoside hydrolase family 16 (GH16). B, ZgLamA GH16 is compared with all the laminarinases structurally characterized so far. The ␣ helices and ␤ strands are represented as helices and arrows, respectively, and ␤ turns are marked with TT. This sequence alignment was created using the following sequences from the Protein Data Bank: laminarinase 16A from P. chrysosporium (2W39, residues 1-298), LamR from R. marinus (3ILN, residues 2-249), BglF from Nocardiopsis sp. F96 (2HYK, residues 4 -244), endo-1,3-␤-glucanase from Cellulosimicrobium cellulans (3ATG, residues 1-255), TM_0024 from Thermotoga maritima (3AZX, residues 7-256), Tpet_0899 from Thermotoga petrophila (4DFS, residues 15-264), LamA from Pyrococcus furiosus (2VY0, residues 10 -261), and endo-1,3-␤-glucanase from Streptomyces sioyaensis (3DGT, residues 6 -276).
The interaction of the hydroxyl group Glc3 O6 with the carbonyl group of Trp-264, observed in the laminarin complex (Fig. 5 , A and B), is replaced by the formation of a hydrogen bond between the hydroxyl group Glc3 O3 and Glu-263 O ⑀2 in the MLG complex (Fig. 7 ). A structural water molecule (HOH139 in chain A and HOH166 in chain B) is also involved in the stability of the MLG complex. Comparison of ZgLamA GH16-E269S Substrate Complexes with Other ␤-Glucanase Complexes-The ZgLamA GH16-E269S com- plexes have been compared with other GH16 ␤-glucanases for which complex structures are available: the laminarinase PcLam16A from the terrestrial fungus P. chrysosporium (PDB code 2W39, EC 3. 2. 1. 6) (40)  and the lichenase TFs␤-glucanase from the bovine rumen bacterium F. succinogenes (PDB code 1ZM1, EC 3.
B and C, structural comparison of the laminarinases ZgLamA GH16-E269S (B) and PcLam16A from the terrestrial fungus P. chrysosporium (C) bound to laminarin oligosaccharides (G3G3G3G and G3G, respectively). PcLam16A (PDB code 2W39) has 27% identity with ZgLamA GH16. The amino acids are colored as follows: in red, the catalytic residues conserved in the family GH16; in purple, the residues involved in laminarin recognition conserved between the two proteins; and in blue, the residues that differ in the recognition pattern. The asterisk refers to the mutation of the Glu-269 into a Ser-269 in ZgLamA GH16-E269S.
succinogenes (C) bound to a MLG trisaccharide (G4G3G). TFs␤-glucanase (PDB code 1ZM1) has 23% identity with ZgLamA GH16. The amino acids are colored as follows: in red, the catalytic residues conserved in the family GH16; in purple, the residues involved in MLG recognition conserved between the two proteins; and in blue, the residues that differ in the recognition pattern. The asterisk refers to the mutation of the Glu-269 into a Ser-269 in ZgLamA GH16-E269S. TFs␤-glucanase and ZgLamA GH16 have been both crystallized with a trisaccharide G4G3G, but they only share a partially conserved subsite Ϫ1 in common (Fig. 6 ). The groove of TFs␤glucanase is with a straight topology, well adapted to bind linear MLG. A similar binding in ZgLamA GH16 would provoke a steric clash between the oligosaccharide and the additional loop of this laminarinase.
The clade comprising ZgLamA GH16 is rooted by several clades of laminarinases that do not possess with this specific loop (Fig. 7 ). These latter GH16 enzymes likely have a straight groove as observed for the structurally characterized laminarinases (PDB codes 2VY0, 3AZX, 3ATG, 2HYK, 3ILN, and 2CL2). Therefore, the ZgLamA GH16 clade emerged more recently than the clades of laminarinases with straight grooves. Such a hypothesis is also already accepted for the evolution of GH16 enzymes more specific for MLG. Indeed, bacterial lichenases (EC 3. 2. 1. 73) have lost the ␤-bulge of the ancestral catalytic center (conserved in most clan GH-B enzymes: all GH7 and most GH16 enzymes) and thus diverge later from ␤-1,3-glucanases  (49). Interestingly, a new GH16 enzyme from black cottonwood, PtEG16, has been recently characterized as a broad specificity ␤-glucanase with a nearly equal capacity to hydrolyze MLG and xyloglucans.
* This work was supported in part by the  French National Research Agency  with regards to the investment expenditure program  IDEALG under Grant  Agreement  ANR-10-BTBR-04. The atomic coordinates and structure factors (codes  4BQ1 ,  4BOW , and  4BPZ ) have been deposited in the Protein Data Bank (http://wwpdb. org/). 1 Supported by a Ph. D. fellowship from the  French Ministry of Higher Education and Research.
4D ); although in some experiments rSSI was closely to rBE2 and rBE3 protein bands (data not shown). However, SSI dependent activity was restored when recombinant enzymes were mixed prior electrophoresis (Fig. 4E ) showing that the proximities of rBE3 and rBE2 to rSSI were sufficient for a functional interaction. Furthermore, it is clearly visible that the were incubated with antibody against BE3 whereas the nitrocellulose pieces with protein marker below 72 kDa were incubated with antiSSI). Prestained molecular mass marker served as standard. Black triangles indicate the dye front of the gel stripes from the first dimension. (D) rSSI (0. 3 mg) was either mixed in different combinations of rBEs (0. 2 mg each) as indicated. Native PAGE and activity staining was performed as described for (A).
However, for both enzyme reactions, SSI and BE, a glucan acceptor is necessary in a strict sense. Therefore, it is reasonable to predict that endogenous glucans were bound to the enzymes. BE activity in native PAGE is based on stimulating effect of branching enzyme on the phoshorylase a activity. Phosphorylase a elongates glucans in excess of glucose 1-phosphate which are concurrently branched by BE and thereby the number of nonreducing ends increases that can be further used by phosphorylase a. In leaf extracts only BE2 activity was detectable using this assay. BE3 activity was never detected, even when the gel concentration (Fig. 5A ) and/or protein amounts (data not shown) were altered. Similar observations were already reported  [31]. In contrast, using recombinant enzymes both rBE2 and rBE3 formed an activity band in the gel (Fig.
kegg. jp). Protein bound glucans were analysed using HPAEC-PAD. For all protein preparations glucans were detected following isoamylase treatment. For better evaluation glucan chains were grouped in short (DP 2-7), intermediate (DP8-13), and long (DP 14-20) chains (for complete CLD profiles see Fig. S4  in File S1). Note, CLD analyses were limited to DP 20 but also longer chains were detected and account for less than 2% (data not shown). In Table  1  the relative proportion of DP-groups were summarized. Obviously, glucans of both proteins rBE2 and rBE3 contained predominantly short chains that were more accented in rBE3. In contrast, rSSI contained slightly but not significantly more intermediate chain than the shorter DPs. In summary, CLD profiles revealed that the endogenous glucans bound to the enzymes were branched and large.
Acknowledgements Royal Canin contributed to the funding of the study. The study was conceived and the experiments designed by L. J. M. M. , T. A. L. , H. J. W. D. and V. B. The animal facilities were supervised by P. N. Animal experiments were performed by L. J. M. M. , P. B. and  C. D. Analysis  of the data was performed by  L. J. M. M. Reagents , materials and assays were provided by L. J. M. M. , T. A. L. and P. B. The paper was written by L. J. M. M. , and improved by T. A. L. , H. J. W. D. and V. B. before submission. The authors declare no conflict of interest.