dataesr/scientific-paragraphs-categorization huggingface dataset:
Endothelial cells are activated by T. congolense, T. vivax, T. b. gambiense but not T. b. brucei To determine whether African trypanosomes can activate endothelial cells, primary cultures of BAE were first cocultivated with four species of African trypanosomes: BSF of T. congolense (two isolates IL3000 and STIB910), T. vivax (Y486), T. b. gambiense (1135 and LiTat) and T. b. brucei (AnTat 1. 1 and 427). BAE were previously cultivated for 24 h in culture medium without serum to avoid the interference of serum components with activation of NF-kB. NF-kB translocation to the nucleus was studied by indirect immunofluorescence as an indicator of cell activation. For BAE in culture medium alone (control cells), NF-kB staining was mainly limited to the cytoplasm (data not shown). In the presence of T.
congolense, T. vivax and T. b. gambiense, the nucleus of the majority of BAE became stained (Fig. 1A ). Interestingly, staining was limited to the cytoplasm in the presence of T. b. brucei. The percentage of activated cells (with stained nuclei) was determined by counting approximately 500 cells for each condition and at various time points, in order to establish the activation kinetics. Data was then normalized to the control by retrieving the percentage of cells activated in culture medium alone (control) representing the background activation (Fig. 1B ). 7862. 96% of BAE were activated in the presence of LPS (positive control). Activation by both T. congolense and T. b. gambiense started after 6 h of coculture, with 4563. 2% and 48. 5610. 9% of cells activated respectively, whereas T. vivax activated BAE only after 16 h of coculture, with 35.
NF-kB activation induces a pro inflammatory response in endothelial cells In order to determine the consequences of NF-kB activation on the endothelial cell, three different markers, potentially regulated by the NF-kB factor, were studied: i) production of pro-inflammatory cytokines IL-1b and IL-6; ii) modification of the expression pattern of adhesion molecules ICAM-1 and VCAM-1; iii) and nitric oxide production by NO synthase. These assays were performed on M BM and Lung, the two cell lines most activated by T. congolense and for which commercial antibodies are extensively developed. IL-1b and IL-6 produced in the coculture media were measured at different time points (Fig. 3A ). As controls, endothelial cell lines were stimulated by 5 ng/ml of TNFa for 4 h (data not shown), or unstimulated to determine basal level of IL-1b and IL-6 (data not shown).
Activation is probably mediated by a lectin/sialic acid interaction To further investigate involvement of the lectin domain of TS family members in NFkB activation, we produced a recombinant catalytically inactive mutant of TcoTS-A1 bearing a Tyr 438 : His 438 substitution with an intact lectin domain, based on the construction of a mutant TS of T. cruzi, described previously  [13, 38]. Absence of SA and TS activities of the recombinant mutant TS was verified by fluorometric assay (data not shown). Inactivation of the catalytic site had minor effects on the activation capacity of TcoTS-A1, with 58. 666. 3% of BAE activated, compared with 68. 664% by the active form TcoTS-A1 (Fig. 8A ). BAE cells were also incubated with different lectins. WGA, Mal, and SNA, which recognize sialic acid residues, were able to activate BAE, whereas ConA and succinylated WGA, which have no binding specificity for sialic acid, were not (Fig.
1% Triton X-100 0. 1% BSA). DNA was stained by adding 1 mg/ml 49,69-diamidino-2-phenylindle (DAPI) for 5 min, and slides were mounted in Vectashield (Vector Laboratories). Cells were observed with a Zeiss UV microscope and images were captured using a MicroMax-1300Y/HS camera (Princeton Instruments) and Metamorph software (Universal Imaging Corporation).
Flow cytometry analysis of adhesion molecules expression M Lung and M Bone Marrow endothelial cell lines were cocultured with T. congolense for 6, 16 and 24 h. Culture medium was removed and cells washed then detached by trypsin treatment. Endothelial cells Fc receptors were blocked by adding mouse serum (1:10) for 30 min. Cells were then incubated with rat monoclonal antibody anti-CD31 APC conjugated (eBioscience) and rat mAb anti-ICAM1 or anti-VCAM1 FITC conjugated (Biolegend) for 30 min on ice, then washed in PBS. Data were collected on a FACS Canto (Becton Dickinson) flow cytometer and analysed using the FACSDiva software. Cells were gated for forward and side-angle scatters and 10 000 fluorescent particles of each gated population were analysed. The analytical method was described in the results.
Acknowledgments We are grateful to  C. Kieda  ( Orleans, France ) for providing endothelial cells. We are grateful to  S. Claverol ,  AM. Lomenech  and  JW. Dupuy  from the  Genomic Platform at Bordeaux 2 University  for their technical support in mass spectrometry analyses. We are grateful to  N. Biteau  and  J. Izotte  ( UMR5234 ,  Bordeaux2 ) for technical help and to  D. Pillay  and  L. Rivie  `re for their critical reading of the manuscript.
This work was supported by the  French National Centre for Scientific Research (CNRS) , the  Ministere de l'Education Nationale de la Recherche et de la Technologie , the  Conseil Regional d'Aquitaine and the Laboratoire d'Excellence (LabEx) ParaFrap  ( French Parasitology Alliance for Health Care)   ANR-11-LABX-0024. This research was also supported by the  Global Alliance for Livestock Veterinary Medicine (GALVmed)  with funding from the  UK Government's Department for International Development (DFID)  as part of GALVmed's  Animal African Trypanosomosis Program (DFID Programme: Controlling African Animal TRYPANOSOMOSIS [AAT] Aries code   202040-101 ), and by  CEVA santeanimale (Libourne, France). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
In this paper, we have therefore refined and simplified the non-invasive enzymatic determination of spent embryo culture medium, based largely on the work of Leese and Barton  [26] , for use in a modern fluorescence plate reader. In vitro produced bovine embryos have been used as a model species. As well as describing the technique, we have measured the depletion of glucose and pyruvate, and the production of lactate on the same cohort of embryos in individual culture throughout preimplantation development and compared these results to established equivalent data as a means of validating the method. In addition, we have measured the depletion and appearance of a mixture of amino acids by HPLC to generate a comprehensive picture of metabolite consumption and release (CORE) by embryos in vitro.
Experimental design and Statistical analysis Glucose and pyruvate consumption, production of lactate and amino acid consumption/production/turnover by embryos are expressed as pmol per embryo per hour 6 SEM. Embryos were assayed at one developmental stage only in order to minimize the stress of single culture. In the prospective studies, pyruvate alone was measured in triplicate to provide additional confidence in the results. Significant differences in metabolic profile (glucose, lactate and pyruvate) were tested by Student's t-test and one-way analysis of variance (ANOVA) followed by Fisher's LSD test post-hoc using StatviewH (version 5. 1). Following normality tests, amino acid data were tested by Kruskal-Wallis tests followed by Mann-Whitney-U tests post hoc since they were not normally distributed.
During blastocyst expansion, more glucose was depleted than could be accounted for by lactate appearance, suggesting that glucose may be diverted away from aerobic glycolysis. The fate of this glucose is unclear, however it is likely that it is either oxidized completely via the TCA cycle or utilized in one of the other glucose-consuming pathways such as the Pentose Phosphate Pathway to provide biosynthetic precursors. This mirrors the pattern seen in porcine embryos  [39] , but differs slightly from the picture for human embryo metabolism  [35]. In addition, the current study has measured the metabolism of a mixture amino acids, again collecting data from single embryos representing a variety of preimplantation developmental stages. Amino acid metabolism, as indicated by both consumption and release, increased throughout development especially during blastocyst expansion and hatching.
The elevated phospho-STAT1 concentrations in the presence of this potent kinase inhibitor, detected both in cytosolic and nuclear extracts of F364A-expressing cells, 8] [29]  However, the complex phenotype of STAT1-F364A is achieved at the expense as described and subsequently fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature (RT). Nuclei were stained with 5 μg/ml Hoechst 33258 (Sigma) for 10 min at RT. The samples were finally mounted in fluorescence mounting medium (Southern Biotech) and GFP epifluorescence visualized using a Leica DM5000B microscope. Images were obtained with a CCD camera and further processed with the Leica QWin software. Immunocytochemistry. Untagged STAT1 was immunocytochemically detected in transfected U3A cells expressing either wild-type or mutant STAT1.
36  Replacement of the native phenylalanine residue at position 364 for alanine was achieved by site-directed point mutagenesis using the QuikChange II kit from Stratagene, as recommended by the manufacturer. The F364A mutation was introduced in all three expression vectors pSTAT1-GFP, pcDNA-STAT1 and pSTAT1-NES-GFP. Mutations were verified by standard didesoxy termination DNA sequencing (Seqlab). Fluorescence microscopy. The intracellular distribution of GFP-tagged fusions of STAT1 was determined by means of fluorescence microscopy. Transiently transfected cells were treated for luciferase (Promega) and β-galactosidase activities. For each STAT1 variant and stimulation mode, six independent samples were tested and the experiment was repeated at least three times. Data were normalized for the expression of β-galactosidase.
Using Primer 3 software (Applied Biosystems), gene-specific primers for three cDNAs from known STAT1 target genes (irf1, mig1 and gbp1) as well as for stat1 and gapdh were designed. In order to amplify fragments of about 200 bp in length, we used the following primer pairs: IRF1F, 5'-AGC TCA GCT GTG CGA GTG TA-3'; IRF1R, 5'-TAG CTG CTG TGG TCA TCA GG-3'; MIG1F, 5'-CCA CCG AGA TCC TTA TCG AA-3'; MIG1R, 5'-CTA ACC GAC TTG GCT GCT TC-3'; GBP1F, 5'-GGT CCA GTT GCT GAA AGA GC-3'; GBP1R, 5'-TGA CAG GAA GGC TCT GGT CT-3'; GAPDHF, 5'-GAA GGT GAA GGT CGG AGT C-3'; GAPDHR, 5'-GAA GAT GGT GAT GGG ATT TC-3'; STAT1F, 5'-CCG TTT TCA TGA CCT CCT GT-3'; and STAT1R, 5'-TGA ATA TTC CCC GAC TGA GC-3'. Polymerase chain reactions were performed by a denaturation step at 95°C for 15 min, which was followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 30 sec.
Based on these observations, we propose to designate these heteroglycans as ''glucan-chitosaccharides''. In response to microbial attack, plants secrete a variety of chitinases and glucanases that degrade the cell wall of the invading pathogen  [36]. However, there are no reports to date that describe the production of b-1,6-glucanases by plant hosts. In order to investigate whether A. euteiches is able to secrete this type of enzymes, we measured various glucanase activities in culture filtrates of the pathogen. A low level of b-1,6-glucanase activity was detected in addition to b-1,3 and b-1,4 glucanase activities (Table  S1 ). Analysis of the hydrolysis products by high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) revealed the liberation of oligomers from pustulan, a linear b-1,6-glucan, thus indicating the presence of an endoactive b-1,6-glucanase (data not shown).
In addition, two partial unigenes deduced from A. euteiches Expressed Sequence Tags  [37] , Ae_12AL6320 and Ae_1AL0240, were found to possess a glycoside hydrolase GH30 family domain (E-value,e 230 ) which occurs in most of the functionally-characterized b-1,6-glucanases according to the carbohydrate-active enzymes database (CAZY,  [38] ). Both unigenes were represented by at least one expressed sequence tag from an A. euteiches -M. truncatula interaction cDNA library, suggesting that the corresponding bglucanase isozymes are produced in planta. Thus, A. euteiches itself might release glucan-chitosaccharides from its cell wall during the interaction with its host. Functional studies of the Ae_12AL6320 and Ae_1AL0240 gene products might reveal their possible involvement in this process.
5% pustulan (Calbiochem) for b-1,6-glucanase measurement, and 1% colloidal chitin prepared according to  [68]  for chitinase activity. The free sugar reducing ends were quantified by colorimetry  [69] , and the endo-or exo-action of the enzymes was assessed by HPAEC-PAD analysis of the hydrolysates as described in  [14]. Specific activities were calculated after measuring protein contents according to  [70] , and expressed either in nkat. mg 21 or in U. mg 21 , where 1 U is the amount of enzyme required to release 1 mmol of glucose reducing-sugar equivalents per min at 40uC. Purity of the enzymes was checked by measuring the various glycoside hydrolase activities using the assays described above. The data showed that the cellulase from Megazyme was a pure b-1,4endoglucanase while Westase contained both b-1,6-endoglucanase and b-1,3-exoglucanase activities, which were purified as follows: 1 gram of powder was suspended in 100 ml of 10 mM Tris-HCl pH 8, incubated 1 h at room temperature under constant stirring, and centrifuged at 3,500 g for 10 minutes.
85 dry objective. Fluorescence intensity for both the CFP and YFP moieties of NupYC2. 1 were collected setting pinhole diameter at 4-5 Airy units, exciting the probe at 458 nm (80% Ar laser) and recording the emitted fluorescence at 470-500 nm and 530-570 nm respectively. Transmitted-light images were acquired simultaneously to confirm cell identity. Images were scanned at a resolution of 5126512 pixels and collected every 5 sec over a period of 15 to 30 minutes following treatment. Average YFP and CFP fluorescence intensities were calculated for each nucleus using Image J software. Values were then exported to a Microsoft Excel spread-sheet in order to calculate the YFP/CFP ratio for each time frame. Ratio values were then plotted over time to obtain a graphical representation of FRET intensity, corresponding to relative Ca 2+ level variations in the nucleoplasm.
Mean values from three biological replicates (6 S. E. ) are given as log2 of foldexpression in elicited seedlings with respect to mock-treated seedlings. The gene set used is the same as in Fig. 5. Asterisks above the bars indicate that the mutant line responded differently compared to the WT line (P,0. 05). Such differences were observed only for the LOX and VR markers and did not concern both mutant lines, thereby showing that NFP is not involved in gene regulation in response to the glucan-chitosaccharide fractions. (PPTX) Table  S1  Glucanase activities present in culture filtrates of Aphanomyces euteiches. Data are the mean6S. E. of 2 independent experiments. Filtrates of early stationary phase cultures were harvested 6 days post-inoculation. Glucanase activities were measured by incubation of culture filtrate aliquots with appropriate carbohydrate substrates and quantification of the liberated reducing ends as described in Materials and Methods.
Acknowledgments We thank  Drs C. Mazars  ( LRSV, Toulouse ) and  C. Gough  ( LIPM, Toulouse ) for providing tobacco cell suspension cultures and M. truncatula A17 nfp-1 and nfp-2 mutant seeds, respectively. We are grateful to  Dr I. Badreddine  and  S. Amatya  ( LRSV, Toulouse ) for preparing A. euteiches culture filtrates,  L. Kamel  ( LRSV, Toulouse ) for participating in A. euteiches cell wall processing,  L. Buon  and  I. Jeacomine  ( CERMAV-CNRS, Grenoble ) for HPAEC-PAD analyses of native glucan-chitosaccharide fractions and assistance in NMR experiments, respectively,  Drs V. Puech  and  M. Bonhomme  ( LRSV, Toulouse ) for assistance in glucanchitosaccharide MS analysis and quantitative data analysis respectively,  S. Vergnes  and  N. Ladouce  ( LRSV, Toulouse ) for assistance in qRT-PCR experiments, and  Drs J.
This work is part of the  ''Laboratoire d'Excellence'' (LABEX)  entitled  TULIP  ( ANR -10-LABX-41 ); it was funded by the  Region Midi-Pyrenees , the  CNRS  (PhD grant  INEE 36  to AN), and the  French Agence Nationale de la Recherche  ( ANR-08-BLAN-0208-01  ''Sympasignal''). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Cotton plants are subjected to the attack of several insect pests. In Brazil, the cotton boll weevil, Anthonomus grandis, is the most important cotton pest. The use of insecticidal proteins and gene silencing by interference RNA (RNAi) as techniques for insect control are promising strategies, which has been applied in the last few years. For this insect, there are not much available molecular information on databases. Using 454-pyrosequencing methodology, the transcriptome of all developmental stages of the insect pest, A. grandis, was analyzed. The A. grandis transcriptome analysis resulted in more than 500. 000 reads and a data set of high quality 20,841 contigs. After sequence assembly and annotation, around 10,600 contigs had at least one BLAST hit against NCBI nonredundant protein database and 65.
Introduction Insects comprise more than one million of the described species. Despite the great diversity of species and the importance of insects, mainly as disease vectors and agricultural pests, attainable molecular biology resources for insect study still need to be increased in order to understand their physiology and biochemistry and to find new targets for biotechnological tools. At least one-third of insect species are beetles, making coleopterans the most diverse order of living organisms. Nevertheless, few data are available for coleopteran molecular resources in the current available databases. The NCBI nucleotide database (http:// www. ncbi. nlm. nih. gov/nuccore/), for example, has currently around 340,000 of total sequences for beetles. Only eight coleopteran species have more than 5,000 sequences deposited: Pogonus chalceus (65779), Dendroctonus ponderosae (41429), Rhynchophorus ferrugineus (27014), Dendroctonus frontalis (20987), T.
Particularly, the next generation sequencing strategy of pyrosequencing using 454  [4, 5]  has been used to study insect vectors  [6] [7] [8] [9]  and to assess insect genes that code for hemolymph and midgut proteins  [10] [11] [12] , metabolic pathway enzymes  [13] [14] [15]  and metagenomics  [16]. Moreover, some transcriptome studies were made in order to provide ESTs datasets of model and non-model insects  [9, [17] [18] [19]  and have become an effective way of assessing gene expression levels. The cotton boll weevil, A. grandis, has been the most important cotton insect-pest in North America. Due to the Boll Weevil Eradication Program, sponsored by the USDA, an integrated pest management strategy has been successful in controlling boll weevil populations  [20]. In South America, nevertheless, the insect populations are still causing great damage to the cotton crops, destroying cotton plant floral buds and bolls.
Assembly, Annotation and Gene Ontology (GO) Contigs were assembled using MIRA v3. 3. 0. 1  [41]. Homology searches (BLASTX and BLASTN) of unique sequences and functional annotation by GO terms (www. geneontology. org), InterPro entries (InterProScan; http:// www. ebi. ac. uk/Tools/pfa/iprscan/), enzyme classification codes (EC) and metabolic pathways (KEGG, Kyoto Encyclopedia of Genes and Genomes; http://www. genome. jp/kegg/) were determined using Blast2GO software suite v2. 4. 3 (www. blast2go. org). Sequences were submitted to NCBI protein nr databank via BLASTx, with e-value threshold of 10 -5. False Discovery Rate (FDR) was used at 0. 05% probability level. GO terms were improved with ANNEX tool  [42] , followed by GOSlim tool available at Blast2GO (goslim_generic. obo) [43]. Combined graphs were constructed at levels 2, 3 and 5 for Biological Process, Cellular Component and Molecular Function categories, respectively.
Sequence alignment, SID phylogenetic analysis and in silico analysis of PAZ Domain contigs Textual and sequence similarity searches were performed in the transcriptome database for genes involved in RNAi mechanism, based on available NCBI Protein Database sequences (http://www. ncbi. nlm. nih. gov/protein). The amino acid sequences of PAZ domains and SID proteins were obtained by in silico translation using TrEMBL (http:// www. expasy. ch/tools/dna. html). Two largest PAZ Domain-containing contigs, here called A_grandis_454_c1018 and A_grandis_454_c4142 were selected for alignment with PAZ domains of argonautes and dicer-like proteins of other organisms including insects (Figure  S1A ). All sequences used for alignment contained full PAZ Domains, including A. grandis contigs and were submitted to ClustalW2 Multiple Sequence Alignment (http://www.
Each experimental unit consisted of 16 female adults, which, after microinjection were kept in a sieved box with 8 non-injected males, in order to allow the collection of laid eggs. Males were previously marked with ink. The sieved box with insects contained artificial diet  [36]  and was maintained at 26 ± 2 °C, 60 ± 10% relative humidity and 12/12-hour day/night photoperiod. Control was performed by microinjection of E. coli gus gene dsRNA, produced as previously described. Analyzed phenotypic parameters were oviposition, egg viability, and adult mortality. Eggs laid by microinjected females were mechanically pierced and kept in artificial diet for 7 days. Neonate larvae development and phenotypes were assessed. Data of three bioassays were applied to variance analysis and Tukey's multiple comparisons test at 5% level of significance.
Sequencing, Assembly and Annotation A cDNA library was constructed after pooling RNA extracted from boll weevil eggs, three larval instars, pupae, male and female adults. cDNA-normalized library 454 runs generated a total of 576,297 ESTs (Table  1 ). The minimal quality standards used in pre-processing provided 310,182 reads, with average read length of 379 bp. These data were deposited in NIH Short Read Archive, with accession number SRA059959. After assembly, 20,384 contigs with average length of 676bp were obtained, with an average depth of coverage of 9. 58 sequences per nucleotide position. Of these, most contigs have length ranging from 300 to 750 bp (Figure  1 ).
Similarity searches and gene ontology analysis After read assembly, contigs were submitted to BLASTx similarity search against NCBI non-redundant protein database (nr) to assess their putative function. Around 10,600 contigs showed at least one hit against nr (Table  1 ). Of these, 84. 9% showed significant blast matches at a cutoff e-value ≤ 10 -3 (Figure  S2 ). Contigs with e-value = 0 were represented at the end of the figure, and correspond to 2. 5% of the total number of contigs. Figure  2  shows the top-hit species after BLASTx similarity search. As expected, 65. 7% of the contigs were similar to T. castaneum sequences. T. castaneum (red flour beetle) is the most important coleopteran of Tenebrionidae family because it attacks stored grain products and is responsible for great loss and damage.