dataesr/scientific-paragraphs-categorization huggingface dataset:
Parasites and trails were then stained with anti-P30 antibodies and visualized with an inverted laser scanning microscope (Eclipse Ti, Nikon). Images were analysed using Metamorph and ImageJ software. Numbers of helical and circular trails associated with parasites were scored in 30 fields. T. gondii video microscopy. Time-lapse video microscopy was conducted with the DeltaVision s Core microscope using a Â 40 immersion lens. Freshly lysed RH Dhxgprt, ku80::diCre or TgAMA1 KO were added onto HFF, HeLa or U373 monolayer grown in glass dishes (ibidi; m-Dish 35 mm, high ). Forty hours post inoculation, invasion of freshly egressed parasites was observed. Normal growth conditions were maintained throughout the experiment (37 °C; 5% CO 2 ). Images were recorded at one frame per second. Further image processing was performed using ImageJ 1.
Acknowledgements We thank the  Center for Human Immunology at Institut Pasteur  for support in conducting this study, the  Center of Production and Infection of Anopheles (CEPIA-Institut Pasteur)  for mosquito rearing, the ISO 9001 qualified CYBIO FACS facilities of the  Institut Cochin  for support,  Eloise Galtier  for FACS acquisition, and  Ming Kalanon  for critical reading of the manuscript. This work was supported by funds from the  Pasteur Institute , the  French National Research Agency , the EviMalar, the  Wellcome Trust , and the  Fondation pour la Recherche Medicale  (FRM team support, Barriers and Pathogens). D. Y. B. was a recipient of a  Manlio Cantarini Fellowship , N. A. is supported by an  EviMalaR PhD  fellowship (European FP7/2007-2013, grant number  242095 ), V. L. is supported by an  EC Marie Curie integration grant  ( PIRG05-GA-2009-249158 ), M.
Expression of cell surface receptors and intracellular proteins Multiparametric flow cytometry now allows for the measurement of up to 12 fluorescent signals in a single tube. Hence, even with limited blood samples, multiple phenotypic and functional parameters of circulating NK cells can be analyzed (Table  1 ). Mean fluorescence intensity is generally used to quantify the level of expression of each receptor. If both the cytofluorometer and the staining procedure are well calibrated, data from patients analyzed on different occasions can be compared with accuracy. This information can relate to:.
Acknowledgements We wish to thank the principal investigators and site staff of all the research centres who participated in the study. We thank the staff of CROS NT S. r. l. , Verona (I), in charge of the statistical analysis and the staff of  CROMSOURCE S. r. l. , Verona (I)  for the study management and study sites monitoring activities. We thank  Stefano Rovati  and  Arturo Lanzarotti  (IBSA Institut.
tachinoides and G. fuscipes fuscipes  [4, 5]. Although recent, large-scale field data on tsetse distribution are lacking  [6] , entomological prediction maps  [7]  and recent unpublished updates of global livestock distribution datasets  [8]  indicate that an area of 65,000 km 2 may be infested by tsetse flies and that one million cattle may be at risk of tsetse-transmitted trypanosomosis. In addition to AAT, human African trypanosomosis (HAT), or sleeping sickness, also continues to impact public health in Chad, with 2,980 cases reported from the years 2000 to 2009  [9]. HAT cases in Chad are caused by Trypanosoma brucei gambiense and they are reported from the southern foci of Bodo and Moissala, associated to the rivers Mandoul and Nana Barya  [9]. Overall, 465,000 people distributed over an area of 14,000 km 2 are estimated to be at risk of contracting HAT  [10].
The full model was y i j = d i j + e i j , where y i j was the observed count, assumed to follow a Poisson distribution conditionally on trap j and explanatory variables; e i j was the residual error. The explanatory variables were the time (in months) from the beginning of the treatment, the treatment type (negative control i. e. without any insecticide treatment or footbath), and the region (Sarh or Moundou). Data analysis was performed with the R software  [26]  and the additional package lme4  [27].
Patients All HIV-positive patients followed in Cayenne, Kourou, and Saint Laurent du Maroni Hospitals between 1 January 1992 and 31 October 2008 were enrolled in the French Hospital Database for HIV (FHDH). Time-independent variables such as sex, nationality, mode of acquisition of HIV, and timedependent variables such as age, CD4 cell counts, CD8 counts, HIV-1 viral loads, antiretroviral treatments, and clinical events are routinely entered by trained clinical studies technicians. Diagnoses are coded according to the 10th International Classification of Diseases. Specific details such as the location of herpes zoster could not be obtained from the database.
(D. ) Number of substitutions per site among promoter sequences between strains within SmPoMuc groups (S on Yaxis, colour-codes described above), along the sequence alignment (nucleotide positions on X-axis as above). * are the substitutions between the two strains positioned within regions conserved among groups of promoters. The number of substitutions among SmPoMuc promoters between the two strains varied from 0 in Group 2 to 8 in Group 3. 1(r1-r2). No substitution was observed in TATA signals and TSS sites between the two strains. The sequences have GenBank accession number JQ615951 to JQ615965 (See Table  S1  for details). doi:10. 1371/journal. ppat. 1003571. g003 SmPoMuc group 1 promoter population sequence difference between IC and C strains is not higher than the average genome-wide difference Promoter diversity within strain and divergence between strains of SmPoMuc group 1 genes were similar to those of 14 microsatellite loci that can be used to reflect genome-wide diversity and divergence  [25].
Therefore, the chromatin structure analysis was performed on the promoter regions of SmPoMuc groups 1, 3. 1 and 3. 1(r1-r2). ChIP experiments were performed using an antibody that recognised Histone 3 acetylated on lysine 9 (H3K9Ac) and Histone 3 tri-methylated on lysine 4 (H3K4Met3) which are euchromatic marks and an antibody that recognised H3 trimethylated on lysine 9 (H3K9Met3), which is a heterochromatic mark. Immunoprecipitation with the antibody that targets H3K4Met3 did not show any enrichment in the SmPoMuc region tested for either the IC or C strains whereas controls, aTub (Smp_090120. 2) and 28S (Z46503. 1) were positive (data not shown). The H3K4Met3 mark is usually very sharp and difficult to localise by target approach. Both SmPoMuc group 1 and 3. 1(r1-r2) from the IC strain displayed a higher level of H3K9Ac compared to the C strain (fig.
This was not the case. In summary, all lines of evidence point towards a chromatin-based regulation of SmPoMuc expression. The chromatin configuration was further investigated by ChIP analysis using antibody that recognises heterochromatic and euchromatic marks. ChIP results clearly demonstrate that different epigenetic marks occur on the SmPoMuc promoter of group 1 and group 3. 1(r1-r2) between the IC and C strains likely resulting in a different chromatin configuration. On these loci, chromatin is indeed more enriched in H3 acetylated on lysine 9 in the IC compared to the C strain and less enriched in the opposite mark, H3 trimethylated on lysine 9. Therefore, the local chromatin structures differ between the two strains for groups 1 and 3. 1(r1-r2) and are consistent with expression data as stronger acetylation correlates with enhanced expression.
PCR screening for promoters of SmPoMuc genes, cloning and sequencing We searched for sequences of promoter regions of SmPoMuc genes in the genomic database of the S. mansoni NMRI strain (assembly version 3. 1) using BLAST searches. Contigs matching to SmPoMuc genes were assembled with the Sequencher software (Gene Codes Corporation) to recover the sequences of the promoter regions of the genes. From the BLAST search and manual assemblage of relevant contigs, scaffolds of promoter regions were constructed for the different SmPoMuc genes in groups 1-4. Primers were designed on these contigs to amplify the promoter regions of the different SmPoMuc genes in the C and IC strains of S. mansoni. The DNA templates to generate PCR products were either genomic DNA (C and IC strains), a BAC library (NMRI strain) or a phage library (IC strain).
Genomic DNA was extracted from adult worms as described previously  [8]. The production of the phage library is described below. Promoter regions were amplified using the Advantage 2 PCR Enzyme System (Clontech) (Table  S1  for primer sequences, amplified fragment lengths and sources of DNA). PCR products were either cloned into pCR-XL-TOPO (TOPO TA Cloning kit for sequencing, Invitrogen) and plasmid DNA was purified using the Wizard Plus SV Miniprep DNA purification system (Promega), or sequenced directly. We sent PCR amplificons or plasmids containing the promoter regions to GATC (GATC Biotech, Germany) for cycle sequencing in both directions and performed primer walking up to 2. 0 kb upstream of the transcription start sites (TSS) of SmPoMuc genes (for primer sequences see Table  S2 ). We checked trace data and aligned nucleotide sequences manually using the BioEdit software.
mansoni IC and C strains Sequence annotation and promoter prediction. The 59UTR and ORF were previously characterised using 59RACE-PCR experiments  [56]. The core promoter including a TATA box and the TSS was predicted using Neural Network Promoter Prediction Tool (http://www. fruitfly. org/seq_tools/ promoter. html) [57]. We identified repetitive elements in the promoter region sequences using the CENSOR software  [58]. We searched for duplications, recombinations and gene conversions using dot plots among sequences and the programs RDP3  [20]. SmPoMuc promoter sequences were annotated using CLC Sequence Viewer v6. 5. 1 (CLC Bio 2011). We colour-coded paralogous sequence blocks, portions of repetitive elements, duplications and recombination to visualise the evolution of paralogous and orthologous SmPoMuc promoter sequences.
After 72 h, we washed HeLa cells with PBS and fixed in 220uC methanol for 5 min. Cells were washed twice with PBS and counterstained with DAPI (100 mg/ml) for 10 sec and mounted with fluorescent mounting medium (Dako). Fluorescence was observed with a Zeiss Axioskop2 (Zeiss) using a camera Leica DC350FX coupled to imaging software (Leica FW4000).
Chromatin status of SmPoMuc promoter region by bisulfite treatment Bisulfite genomic sequencing was carried out as described in  [68] ) on gDNA extracted from miracidia from the NMRI strain. Amplification was performed using primers BS. IC-1-Group1/ 1111-1715. 48f GATATGTTTTAAGAAGTAGAAAAGAATA-TT, BS. IC-1-Group1/1111-1715. 508r ATAAAAATTTTACA-ACCACCTACTC and BS. IC-1-Group3. 1/421-952. 29f AT-TGTTTTTTTTAATTTTAGATATGTTTTA and two rounds of PCR. 1 ml of each PCR products were cloned into the TOPO TA vector (Invitrogen) and sequenced. In-vitro methylation with M. SssI (NEB) was done as recommended by the supplier. A total of 20 sequences (7 M. SssI treated positive controls and 13 target miracidial gDNA) were aligned with the genomic sequence from GenBank (Bioedit) to visualise the sites of methylated cytosine.
qPCR analysis qPCR amplifications were performed with 2. 5 ml of immunoprecipitated DNA or cDNA in a final volume of 10 ml on a LightCyclerH 480 II Real Time instrument (1. 5 ml H 2 0, 0. 5 mM of each primer, 5 ml of master mix). The following protocol was used: denaturation, 95uC for 10 minutes; amplification and quantification (40 times): 95uC for 10 seconds, 60uC for 10 seconds, 72uC for 20 seconds; melting curve, 65-97uC with a heating rate of 0. 11uC/s and continuous fluorescence measurement, and a cooling step to 40uC. For each reaction, the cycle threshold (Ct) was determined using the ''2nd derivative'' method of the LightCyclerH 480 Software release 1. 5. PCR reactions were performed in duplicate and the mean value of Ct was calculated. Correct melting curves were checked using the Tm calling method of the LightCyclerH 480 Software release 1.
The number of substitutions among SmPoMuc promoters between the two strains varied from 0 in Group 2 to 8 in Group 3. 1(r1-r2). No substitution was observed in TATA signals and TSS sites between the two strains. The sequences have GenBank accession number JQ615951 to JQ615965 (See TableS1for details). doi:10. 1371/journal. ppat. 1003571. g003.
The funders are L'Agence nationnal de la recherche through the  Program Schistophepigen  ( ANR-07-BLAN-0119-02 ) and the program  EPIGEVOL  ( ANR-2010-BLAN-1720-01 , http://www. agence-nationale-recherche. fr/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Compositional analysis and quantitation using HILIC/FD and ESI-MS of 2-aminobenzamide labeled structures The PNGase F-released N-glycans from rhFVIIa were analyzed after 2-aminobenzamide (2-AB) labeling, using hydrophilic interaction liquid chromatography (HILIC) for quantitation and compositional analysis by MS. Isolated fractions collected from HILIC were also investigated by MS using an offline nanoelectrospray ionization (nano-ESI) ion source and an Orbitrap mass spectrometer giving mass accuracies better than 5 ppm. The initial pool of derived glycans was also analyzed online on a reduced diameter HILIC capillary column coupled to an Orbitrap via the standard ESI ion source. Carbohydrate compositions were deduced by comparison of experimental with theoretical masses generated by the Glycoworkbench tool software.
Four days after birth, females were milked and selected according to several criteria such as milk volume and rhFVII antigen level to ensure high and constant protein productivity. Milk was then collected from Days 5 to 22. New females (F"x" + 1) were constantly reintroduced into the herd to replace old ones and to ensure a long-term uniform herd, consisting of female rabbits having similar history and productivity. For glycosylation consistency evaluation, data were collected from homogeneous minipools of six milking females of the same breeding generation, i. e. same founder (F"x"), sharing the same number of lactations (L"x") and during the same lactation period (D"x"). The study of these homogeneous minipools allowed us to evaluate putative variations excluding heterogeneity inherent to individual rabbits.
Pooling strategy and consistency of successive batches To obtain an homogeneous raw material for the production of batches, milk was always collected and pooled randomly, from the day-to-day production of the herd, hence, avoiding any selective pooling of milk, as for instance from the beginning or the end of a lactation. Approximately 90 rabbit females were milked to provide the raw material necessary for a 150 L-batch size. Figure  7  shows the N-oligosaccharide profiles obtained from consecutive batches obtained at various scales (7. 5-150 L) during preclinical development and produced using milk collected from generation F1-F3 and from lactation L2-L5. All the chromatograms can be perfectly overlaid; these data emphasize the perfect batch-to-batch reproducibility of the N-oligosaccharide content and validate the pooling strategy.
Inter-animal variability has been excluded by the constitution of homogeneous minipools. The effects of consecutive lactations, generations as well as the time course of lactation were specifically evaluated. Changes of glycosylation patterns during the time course of lactation were predominantly evidenced. The level of sialylated structures was shown to decrease only slightly during the lactation course. In consequence, no reduction of the timeframe for milk collection was necessary to avoid the collection of an undersialylated product. The very low SD calculated with all the data attests that sialylation is remarkably reproducible whatever the milk pool. A decrease in sialylation or an increase in the protein isoelectric point during the time course of lactation has been previously observed for different mammals  (Aoki et al.
2. 1. 18). The digestion efficiency was assessed by using 2 µg of a pure bi-α2,3-sialylated biantennary N-glycan (A 2 G 2 S 2 ) standard as a control. The exoglycosidase was next removed by a cold alcoholic precipitation, and the supernatant containing the digested products was vacuum-dried before HILIC analysis. Nanoelectrospray ionization-tandem mass spectrometry analyses of permethyl oligosaccharides. MS analyses of the permethyl N-glycans were performed using a LTQ-Orbitrap hybrid mass spectrometer fitted with a nano-ESI ion source (Thermo Fischer Scientific, Bremen, Germany) under the control of the Tune Plus 2. 4 software. Samples were infused at 20-80 nL/min from a 75% (v/v) aqueous methanol containing 5 mM sodium acetate through a 0. 69 µm ID nano-ESI uncoated borosilicate glass emitter tip (New Objectives, Woburn, MA).
MALDI-TOF analyses of the permethyl oligosaccharides. MALDI MS and MS/MS experiments were performed on an Autoflex II instrument (Bruker Daltonics, Bremen, Germany) using the Flex Control 2. 5 software. The desorption as well as the laser-induced dissociation of the molecular ions were carried out by a nitrogen laser irradiating at a frequency of 50 Hz and at 20-30% and 40-70% of maximum laser power, respectively. In MS, the mass spectrometer was operated in either positive or negative reflectron ion mode by delayed extraction with an accelerating voltage of 25 kV. The pulse delay time was set to 350 ns to obtain the best resolution and mass accuracy for the molecular ions of m/z comprised between 1000 and 3000 Da. In MS/MS, the accelerating voltage was first set to 8 kV and the precursor ions of interest, accompanied by their in-source-generated daughter ions, were selected using a deflector ion gate.
Strikingly, in human HDACs, only the flipped-in conformation is observed and appears to be fully constrained. Structure-based chemical screening further yielded a set of smallmolecule inhibitors that, compared to generic HDAC inhibitors, showed decreased affinity for human HDACs, while retaining affinity towards smHDAC8. Notably, one of these inhibitors, J1075, not only forces the phenylalanine into the flipped-out conformation, but also induces apoptosis and mortality in schistosomes. Our data thus confirm the validity of the structure-based approach searching for new epigenome-targeting drug leads against schistosomiasis, but also provides a proof of concept for the treatment of other neglected diseases caused by eukaryotic pathogens.
The most striking and certainly least expected difference concerns the smHDAC8 F151 side chain that is turned away  from the catalytic pocket and is inserted into a smaller hydrophobic pocket formed by loops that surround the active site (Figure  3A ). In contrast to this flipped-out conformation of F151, the side chain of its human counterpart, hHDAC8 F152, is turned towards the active site adopting a flipped-in conformation in all inhibitor-and substrate-bound hHDAC8 structures deposited in the Protein Data Bank (PDB) (Figure  3B ). Although this change could be due to the presence of inhibitors bound to hHDAC8, favoring a flipped-in conformation, careful inspection of hHDAC8 structures revealed that human F152 cannot adopt a flipped-out conformation. Indeed, this conformation is incompatible with the conformation of the L31 side chain that is itself locked in this conformation by its surrounding neighbors (Figure  3B ).
Homology of the HDAC8 structures from these different species based on sequence alignment and our structural data did not suggest any amino acid substitution in these enzymes that could prevent, directly or indirectly, F151 to adopt its flipped-out conformation. These observations are of paramount importance since they imply that smHDAC8-specific inhibitors should also inhibit the corresponding enzymes of S. japonicum, S. haematobium and C. sinensis, and possibly those of other trematode species that affect different populations worldwide. Moreover, S. haematobium and C. sinensis are also linked with the progression of cancer (reviewed in  [50] ). Hence, development of drugs fighting these infections should also have an implication in cancer prevention. Strikingly, smHDAC8 S18 but also smHDAC8 H292 are conserved in the recently published HDAC8 sequences of the cestodes Echinococcus multilocularis, Echinococcus granulosus and Taenia solium  [51]  that also cause serious human diseases (Figure  S7 ).
Modeling of the structures of HDAC8 from E. multilocularis, E. granulosus and T. solium reveals that the active sites of these enzymes are highly conserved compared to that of smHDAC8. Specifically, as for the various schistosome species, the flipped out conformation of the phenylalanine corresponding to smHDAC8 F151 should also be conserved in these enzymes. In addition, the position of the histidine corresponding to smHDAC8 H292 should be the same as in the schistosome enzyme. These results suggest that the specific drugs developed against smHDAC8 could in fact be used in a pan-platyhelminth treatment strategy. Taken together, our results provide the molecular basis for specific targeting a parasitic epigenetic enzyme to cure parasitic diseases by combining high resolution structural data with biochemical and in vivo studies.
For qRT-PCR (minimum of 3 experiments) total RNA was extracted using the RNeasy Mini Kit (Qiagen). Total RNA (100 ng) was used to synthesize cDNA with Superscript III cDNA Synthesis kit (Life Technologies). S. mansoni cytochrome oxidase was used as an endogenous normalization control in all tested samples (GenBank AF216698) (COX Forward 59-TACGGTTGGTGGTGTCACAG-39 and COX Reverse 59-ACGGCCATCACCATACTAGC-39). Quantitative RT-PCR was carried out using Power SybrGreen PCR Master Mix (Life Technologies) in an AB7900 Real Time PCR System (Life Technologies). Negative controls used were GFP and two internal controls assessing both possible genomic DNA contaminations (no reverse transcriptase) and purity of the reagents (no cDNA). Each experiment was repeated 3-5 times and analyzed using the DDCt method  [53].
Crystallization and data collection Crystallization trials were performed using both sitting and hanging drop vapor diffusion techniques. Diffraction-quality crystals were obtained at 17uC after three to four days by mixing equal volumes of smHDAC8 with reservoir solution composed of 21% PEG 3350 (Fluka) and 0. 2 M Na + /K + L-tartrate. The smHDAC8/SAHA complex was formed by incubating the smHDAC8 protein (2 mg/ml) with SAHA (5 mM in DMSO) at 4uC for 1 h. Crystals were grown in the same conditions as described for native smHDAC8. Crystals of the complexes of smHDAC8 with the inhibitors M344, J1038 and J1075 inhibitors were produced by soaking native smHDAC8 crystals in mother liquor supplemented with the corresponding inhibitor (10 mM resuspended in DMF) for 20 hours. Crystals used for X-ray data collection were briefly transferred in reservoir solution supplemented with 20% glycerol and flash-frozen in liquid nitrogen.
Crystal structures solved in this project have been deposited under the codes 4bz5, 4bz6, 4bz7, 4bz8 and 4bz9 in the Protein Data Bank. Alignments were generated with JalView  [61] , structural panels in figures were generated with PyMOL (DeLano Scientific), and schematic interactions of the inhibitors with smHDAC8 were generated with LigPlot  [62] ).
Virtual screening Virtual screening was carried out using the native smHDAC8 structure as template. We screened the Enamine purchasable compound library (1. 587. 660 compounds) for potential smHDAC8 inhibitors by a stepwise virtual screening procedure. First we screened for compounds containing hydroxamic acid groups or derivatives which are known as zinc binding motifs. The molecular weight was restricted to 300 Daltons to identify smaller lead-like compounds. In total 25 molecules were retrieved, their 3D structures were generated within MOE 2008. 10 (Chemical Computing Group, Montreal, Canada) and docked into the smHDAC8 structure using GOLD4. 1 docking program  [63]. ChemScore was used as scoring function due to the success of ChemScore in earlier HDAC dockings  [64]. The docking region was defined within a radius of 15 A ˚around the zinc ion.