dataesr/scientific-paragraphs-categorization huggingface dataset:
A limited number of signalling pathways are involved in the specification of cell fate during the development of all animals. Several of these pathways were originally identified in Drosophila. To clarify their roles, and possible cross-talk, we have built a logical model for the nine key signalling pathways recurrently used in metazoan development. In each case, we considered the associated ligands, receptors, signal transducers, modulators, and transcription factors reported in the literature. Implemented using the logical modelling software GINsim, the resulting models qualitatively recapitulate the main characteristics of each pathway, in wild type as well as in various mutant situations (e. g. loss-of-function or gain-of-function). These models constitute pluggable modules that can be used to assemble comprehensive models of complex developmental processes.
, in prep. ). Because signalling pathways are known to play important roles in these developmental processes (see e. g. ref. 34-38 and references therein), these models contain several signalling components (e. g. Wg, Hh, Notch, and a limited number of downstream components) belonging to different signalling pathways. Noticeably, although Drosophila has been a prominent model system to decipher cell signalling, in particular in the context of development, bona fide dynamical models for these pathways are still largely lacking. Furthermore, information about pathway organisation and function is spread across many publications and various databases (for links pointing to Drosophila signalling pathways in public knowledge repositories, see Table  1 ). In particular, Flybase and Interactive Fly provide rich textual descriptions of each pathway.
However, computational biologists must perform substantial analyses to convert this vast array of information into a format that can be used to derive formal graphical and dynamical models. There is thus a clear need for reference models for each of the recurrently used signalling pathways in Drosophila, which would undoubtedly constitute a valuable resource for the modelling of developmental networks. To address this issue, we have developed reference logical models for the major Drosophila developmental pathways, with extensive references as hyperlinks to relevant articles and database entries. These pathway models can be used as building blocks to assemble more comprehensive models, as we are currently doing for mesoderm specification during Drosophila embryogenesis (Mbodj et al. , in prep.
Paper Molecular BioSystems essential dynamical properties, including the stable states, are preserved upon model reduction. 47 he software GINsim (for Gene Interaction Network simulation) implements the logical formalism and various analysis tools, including stable state computation and model reduction. 48 Nsim further supports the annotation of regulatory graphs with free text and URLs, thereby enabling extensive documentation of the considered regulatory elements and interactions. Once a model is defined, the user can launch simulations, which are presented in the form of state transition graphs. GINsim also Fig. 2  Logical models of Drosophila Hh, Notch, JAK/STAT and Dpp signalling pathways. Ellipses and rectangles denote Boolean and multilevel nodes, respectively. Red blunt and green normal arrows denote activatory and inhibitory interactions, respectively, while the blue dot arrows denote dual effects of the source nodes on their targets.
e. the transcriptional factors AOP and PNT). Examples of simulation results for EGF and FGF pathway models are given in Table  3. Finally, Fig. 4A  shows our current logical model for the Drosophila Toll signalling pathway,  68, 69  which is involved in early dorso-ventral embryo differentiation. The Toll pathway also plays a key role in innate immunity. Indeed, it can be activated upon infection by various kinds of pathogens, a process involving complex signals converging onto the production of Spaetzle (SPZ), the main ligand of Toll in Drosophila (Fig. 4B ). At this stage, however, our logical model does not yet account for potential differential effects of SPZ depending on immunological challenges. All nine pathway models are provided in ESI † (ZGINML files, which can be opened using the public software GINsim), along with extensive documentation (included in the model files, but also provided in html and pdf formats).
Molecular BioSystems Paper components by Boolean variables. This is certainly an oversimplification, which could be refined as novel data become available, thanks to the recent technological advances (transcriptomics, proteomics, interactomics, or high-throughput functional assays such as RNAi screens). Furthermore, the mutant developmental phenotypes used to identify pathway components may only reflect extreme situations (loss-of-function or gain-of-function). This presumably led to overlook many potential unknown pathway actors and regulators, which can now be identified using high-throughput methods. 76 he pace of acquisition, the quantity and the nature of the resulting data pose novel challenges to biologists. Indeed, efficient exploitation of the resulting datasets and their integration with relevant lower-throughput results is far from trivial.
To do so, several groups set to progressively complete and extend canonical pathways with novel interactions and components identified by large-scale interaction screens. 77, 78 Although still in its infancy, there is no doubt that integration of large-scale datasets along with more detailed molecular studies will open novel avenues for signalling pathway mapping and modelling. In this respect, the collection of signalling pathways proposed here may be considered as seeds to build more comprehensive maps and ultimately delineate more quantitative and predictive models.
HBEC support the proliferation of activated T cells As optimal T-cell activation and differentiation in vivo requires long-lasting T-APC interaction, a classical in vitro conjugate forming assay was adapted to assess the ability of HBEC to form conjugates with T cells  [28]. Red fluorescently labeled (PKH26) CD4 + or CD8 + T cells were incubated in suspension with green fluorescently labeled (PKH67) HBECs with the adherence between HBEC and T cells examined using flow cytometry. Conjugates were determined to be cells positive for both PKH26 and PKH67. Interestingly, both CD4 + and CD8 + T cells form conjugates, i. e. cell doublets in suspension, with control (data not shown) and cytokine activated HBECs, as shown by flow cytometry (Fig. 2E , F respectively). There were higher percentages of T cell/HBEC conjugates seen when the HBEC were cytokine activated (3.
The study was funded by the  National Health and Medical Research Council of Australia  (http://www. nhmrc. gov. au/)project grants  1009914  and  571014  to VC and GEG; 1028241 to JW, VC and GEG; and  1022368  to GEG; fellowship 571397 to JW. Support was also provided through Rebecca L. Cooper Foundation equipment grants (http://www. cooperfoundation. org. au/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Acknowledgements This work was supported by  INRIA ,  ERC-NERVI  number  227747 ,  KEOPS ANR-CONICYT  and  European Union  Project #  FP7-269921  ( BrainScales ),  Renvision  grant agreement N  600847  and  Mathemacs   FP7-ICT_2011. 9. 7. Published: 8 July 2013.
Finally, intensity ratings were obtained every 30 stimuli with short breaks in between electrical stimulations. The EEG was recorded continuously using Neuroscan (now Charlotte, USA) amplifiers during the electrical stimulation from 60 electrodes, referenced to Cz, using a standard EEG cap (Easy Cap, Falk Minow Services, Germany) based on an extended International 10-20 system. Eye movements were also monitored. All electrode impedances were kept ,5 kV. After analog filtering (0. 1-100 Hz), the EEG was sampled at 500 Hz. The recordings were subsequently re-referenced to a linked ears reference. In this study, only data from 9 electrodes were processed: F3, Fz, F4, C3, Cz, C4, P3, Pz, and P4 according to the extended International 10-20 System of Electrode Placement. These electrodes were chosen because they are situated above important regions of pain processing, attention, and depression (frontal, central, and parietal brain regions).
1 simulated values (multiple imputation) yielding m completed datasets. Each dataset has been analyzed with standard statistical complete-data methods (i. e. mixed linear model) and results of each data analysis are combined according to Rubin's rules for multiple imputation inference  [24]. Fully Conditional Specification (FCS) is a multiple imputation method which does not require assumptions about the joint distribution of the variables (i. e. multivariate normality). Instead of the joint model, conditional distributions (regression imputation models) for each variable with missing values, involving all other variables as predictors, have to be specified. Imputation under FCS is done by iterating over all conditionally specified imputation models, each iteration consisting of one cycle through all variables with missing values (Gibbs sampling procedure)  [25].
Genetic analysis We identified the GNRH1 c. 91C. T (p. R31C) mutation in two unrelated French families with nCHH. Demographic, clinical, biological and genetic data are reported in Table  1. Interestingly, the two pedigrees are very different in terms of presentation (Fig. 1 ). In family 1, nCHH is sporadic. The boy (II. 1) presented at 19 years old with a failure to progress through puberty. At physical examination he had a partial pubertal development with 12-mL bilateral testes volume. He had no olfactory impairment. Hormone assays revealed very low testosterone levels and low gonadotropin levels. No secondary causes were found for central hypogonadism (see Table  1 ). GNRH1 heterozygous mutation in this boy was de novo, as ascertained by micro-satellites analysis. In the second family the GNRH1 mutation was present in heterozygosis and segregated with disease.
Predictive analysis The GNRH1 c. 91C. T nucleotide substitution did not create any donor or acceptor aberrant splice site according to prediction tools with AlamutH software. This nucleotidic substitution induces a missense at codon 31, replacing Arginine-31 by a Cysteine in the pre-pro-GnRH (GNRH1 p. R31C). Prop v. 1. 0b predicted that this missense did not abolish proconvertase-dependent cleavage site for pre-pro-GnRH maturation into the decapeptide (amino-acids 24 to 33). Alignment with orthologs revealed conservation of arginine in position 8 of GnRH in mammals (Fig. 1 ). This amino acid exchange was classified as deleterious by various in silico prediction tools (see methods section). More importantly, experiments on the substitution of arginine 8 with a variety of amino acids demonstrate that it is crucial for binding and signaling  [10].
Sequencing products were analyzed with an automated capillary sequencer (ABI PRISM 3130xl Genetic Analyzer; Applied Biosystems, Foster City, CA). Electropherogram-derived sequences were compared with NCBI references using SeqScape Software 2. 6 (Applied Biosystems, Foster City, CA). Microsatellites genotyping was performed to confirm filiation (Powerplex 16 SystemH, Promega, Madison, WI). Total RNA was extracted from cells (WBC or cells in culture) with the TrizolH reagent (InVitrogen, Cergy Pontoise, France). RT-PCR and direct cDNA sequencing was performed as previously reported  [26, 27]. Complete CDS of GNRH1 transcripts (NM_0001083111. 1 and NM_000825. 3) were analyzed with primers for RT-PCR and sequencing in Table  S1.
In silico prediction of peptide cleavage was performed by means of Prop v. 1. 0b ProPeptide Cleavage Site (http://www. cbs. dtu. dk/ services/ProP). Prediction of protein function after selective amino acid substitution was obtained by means of AlamutH (Interactive Biosoftware, Rouen, France), AlignGVD (http://agvgd. iarc. fr/ agvgd_input. php/), Polyphen2 (http://genetics. bwh. harvard. edu/ pph2/), SIFT (http://sift. jcvi. org/) and PANTHER Coding SNP Analysis tool (http://www. pantherdb. org/tools/csnpScoreForm. jsp).
1 M). Cell-bound radioactivity was counted in a gamma counter and IC-50 values were calculated using Graphpad Prism (GraphPad Software Inc, San Diego). To determine whether R31C GnRH binds covalently to the GnRH-R, transfected COS-7 cells were incubated with WT GnRH (10 28 M), R31C GnRH (10 25 M) or HEPES-DMEM-BSA alone (2 h, 4uC), washed with HEPES-DMEM-BSA (1 ml) and incubated in HEPES-DMEM-BSA (5 ml, 1 h, 4uC) to allow  dissociation of non-covalently bound peptide, before the binding assay was performed as above. Serum Responsive Element (SRE) luciferase assay luc2P/SRE/Hygro plasmid (Promega, Madison, WI) was used to test luciferase production in response to MAP kinase activation as a reporter gene system. HEK293T cells (1. 2610 4 cells/well) were seeded 72 h before testing in high-glucose Dulbecco's minimal essential medium (DMEM, Invitrogen, Cergy Pontoise, France) containing 2 mM glutamine, 100 IU/mL penicillin, 100 mg/mL streptomycin, and 10% heat-inactivated fetal calf serum at 37uC in 96-well plates.
Twenty-four hours before testing, cells were co-transfected in serum free OptiMEM, using Lipofectamine 2000 (Invitrogen, Cergy Pontoise, France) with the plasmids for human GnRH receptor, luc2P/SRE/Hygro and pMIR-REPORT TM beta-galactosidase vector (Applied Biosystems, Foster City, CA). WT GnRH, R31C, WT+R31C or vehicle were added at different dilutions (from 10 210 to 10 26 M). After 5 h-incubation cells were harvested and assayed for luciferase activities as previously described  [32] , using a luminometer (Victor, Perkin Elmer, Waltham, MA). To standardize for transfection efficiency, the relative light units were normalized by the galactosidase activity at optical density. EC-50 and Emax values were calculated using Graphpad Prism (GraphPad Software Inc).
Inositol phosphate (IP) accumulation COS-7 cells were transiently transfected by electroporation with human GnRH-R DNA (10 mg/15 cm dish), seeded into 12-well plates and radiolabelled by overnight incubation with myo-[3H]Inositol (0. 5 mCi/well, American Radiolabeled Chemicals, St Louis, Mo). Radiolabelled cells were washed and incubated (30 min, 37uC) in IP medium (HEPES-DMEM-BSA supplemented with 10 mM LiCl), then stimulated (60 min, 37uC) with various concentrations of WT GnRH or R31C GnRH or equal concentrations of WT GnRH and R31C GnRH. Incubations were stopped by removal of the medium and cells were lysed by addition of formic acid (1 ml, 10 mM). IP were extracted from cell lysates using Dowex 1 X8-200 chromatography and counted using a liquid scintillation counter (Pack ard). EC-50 and Emax values were calculated using Graphpad Prism (GraphPad Software Inc)  [33].
Statistical analyses Only nonparametric tests were used. Friedman's test was used to compare three or more matched groups and Kruskall-Wallis test for unmatched groups. These analyses were followed by Dunn's post comparison test. Differences were significant when p,0. 05 (*p,0. 05, **p,0. 01, ***p,0. 001). Statistical analyses were performed using GraphPad Prism version 5. 0d (GraphPad Software Inc. , San Diego, CA). HEK293T cells were transiently transfected with GNRHR and SRE-coupled luciferase reporter gene, and then exposed to four 90 min-spaced pulses of 10 nM WT, 10 nM R31C and 10+10 nM WT+R31C. After 5 minutes exposure to each ligand, cells were washed, and a subsequent pulse was given 90 minutes after. Five hours after the last pulse, cells were harvested for luciferase assay. Luciferase activity arbitrary units obtained by luminometry (a.
Acknowledgments We are grateful to  Dr. Alain Deroussent  ( Gustave Roussy Institute and CNRS, Mass spectrometry platform IFR54, Villejuif, France ) for technical support on MS platform; we also thank  Isabelle Boucly  and  Lenaig Lamour  for genetic sequencing technical support and  Chantal Denoyelle  for helping in LH dosages. We finally thank  Dr. Adela Voican  for helping to edit the manuscript.
Electrophysiology Whole cell current recordings. All electrophysiological experiments were conducted on cortical embryonic neurons seeded at a density of 1,000,000 cells/35-mm dish after 10-12 days of culture. Recordings were performed in whole cell configuration of the patch clamp technique  [22]. Each current was evaluated by using a RK 400 patch clamp amplifier (Axon Instrument, USA), low-pass filtered at 3 kHz and digitized at 10 kHz using a 12-bit analog-to-digital converter digidata (1322 series, Axon Instrument, USA). All current amplitudes are expressed in current densities. Results are expressed as mean 6 standard error of the mean (SEM). Patch clamp pipettes were pulled using vertical puller (PC-10, Narishige) from borosilicate glass capillaries and had a resistance of 3-5 MV. The bath solution contained (in mM) 140 TEA-Cl, 10 BaCl 2 , 1 MgCl 2 , 4 4-AP, 10 HEPES, 10 Glucose, 10 27 TTX (pH 7.
35 with TEA-OH). The pipette solution contained (in mM) 110 CsCl, 20 TEA-Cl, 3 Na 2 -ATP, 3. 5 MgCl 2 , 10 EGTA, 10 HEPES (pH 7. 25 with Cs-OH). All experiments were performed at room temperature  (21-22uC). Stimulation protocols and data acquisition were carried out using a microcomputer (Dell Pentium) with a commercial software and hardware (pClamp 8. 2). Calcium currents were recorded by voltage clamp steps to membrane potentials of -70 to +50 mV in 10 mV steps applied from a holding potential of 280 mV. Duration of depolarization pulses were 500 ms, and the pulse cycling rate was 2 s. Current amplitudes were evaluated at different potentials (230, 220, 210, 0, 10 and 20 mV). In all electrophysiological experiments, we used the different drugs at the following concentrations: mapacalcine, 1 mM, nifedipine, 1 mM, SNX485, 250 nM, v-conotoxin GVIA, 1 mM, and calcicludine, 75 nM.
After this delay channels were stimulated for 15 new cycles. Data are expressed in current densities percentage 6 SEM. The number of tested cells was 10(n = 10) per group. Whole cell NMDA/AMPA recordings on mouse cortical neurons. NMDA/AMPA recordings were performed on neurons after 12-15 days of culture. Cells were seeded at a density of 1,000,000 cells/35 mm dish and cultured at 37uC in humidified incubator in an atmosphere of 95% air/5% CO 2. NMDA/AMPA current recordings were realized using the gap free mode in the whole cell configuration of the patch clamp technique. In brief, after the whole configuration obtained, neurons were continuously clamped at 280 mV and the different drugs were tested. Extracellular solution contained (in mM): NaCl 150, KCl 5, HEPES 10, CaCl 2 0. 2, glucose 10 and sucrose 10(pH 7.
Oxygen Glucose Deprivation (OGD) model OGD experiments were performed on primary mouse cortical neurons seeded at a density of 1,000,000 cells/35-mm dish between 10-12 days of culture  [23]. The culture medium was replaced by thorough exchange with deoxygenated glucose-free Earl's balanced salt solution (BSS). Composition of BSS solution was (in mM): 140 NaCl, 5. 4 KCl, 1. 2 CaCl 2 , 0. 9 MgCl 2 , 0. 44 KH 2 PO 4 , 4. 17 NaHCO 3 and 0. 34 Na 2 HPO 4. Prior to use, BSS was equilibrated with the anaerobic gas mixture (5% CO 2 , 1. 2% O 2 and 93. 8% N 2 ) by bubbling for 15 min, adjusted to pH 7. 4 if necessary, and heated to 37uC as described in  [24]. Oxygen content of BSS was monitored with two oxygen-sensitive Clark electrodes and the Labchart 7. 02 software (Powerlab, Oxford, UK). Cortical cells were subjected to 120 min OGD.
[Ca 2+ ] i was represented by the relative fluorescence intensity, dF/F0 = (F-F0)/F0, where F is the fluorescence intensity measured after drug application, and F0 is the baseline. 6 different cultures (n = 6) were tested. 2. To correlate the mitochondrial activity measured by OD in the wells to the cell viability, a calibration curve was performed giving the effect of cell number on absorbance at 490 nm. The correlation coefficient was 0. 99, indicating a linear response between cell number and absorbance at 490 nm. Data are expressed as the percentage of cell viability, which is calculated by dividing the absorbance value of mapacalcine/nifedipine-treated samples by that of the untreated control within each group. Neuronal injury was quantitatively assessed by the measurement of LDH release, which provides a measure of cytoplasmic membrane integrity.
100 ml of cell culture medium were transferred from culture wells to 96-well plates and mixed with 100 ml reaction solution according to LDH assay kit. Quantification was done by measuring the Optical Density (OD) 30 min later at 492 nm on a microplate reader (Labsystem Multiscan RC, VWR International, Fontenay sous Bois, France). Background absorbance at 620 nm was subtracted. As recommended by the manufacturer, neurons exposed to a lysis solution (PBS containing 0. 1% Triton X-100) were used as positive control and set as 100% LDH release. Data are expressed as the ratio of LDH efflux/cell viability (n = 4 cultures, 24 wells per experimental group). All experiments were monitored by one researcher blinded to the treatment status. Results corresponded to the mean of four independent experiments with triplicate determination.
OGD induced an increase in LDH release. Addition of either mapacalcine or nifedipine reduced the LDH release/cell survival ratio 24 hours following OGD (Figure  6D ). These data demonstrated that mapacalcine displayed a true protective effect on cortical neuron survival that had undergone an OGD protocol. These protective effects were also evidenced by the calcium measurement technique. In the absence of OGD, the internal calcium concentrations were very similar in the presence or the absence of 1 mM mapacalcine (0. 5160. 01 and 0. 4860. 01, respectively) suggesting no toxic effect of mapacalcine (Figure  7A, B, F ). But when the OGD protocol was applied, the calcium concentration in the vehicle treated cells was largely increased two hours after the end of OGD when compared to the measures performed immediately after OGD (fluorescence ratio of 0.
This work is supported by the  Centre National de la Recherche Scientifique (CNRS) , the  Agence Nationale de la Recherche -Emergence  ( ANR-EMMA 2011-050 ), the  LabEx Ionic Channels Science and Therapeutics  and the ''Fondation  NRJ-Institut de France''. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Acknowledgments We thank  Claudia Farb  for her technical assistance. This research was supported by  P50 MH058911  and  R01 MH038774  grants to J. E. L. J. D. was supported by the 2010  NARSAD Young Investigator Award  from the  Brain & Behavior Research Foundation. V. D. was supported by grants  Memotime  and  TDE  from  Agence Nationale de la Recherche (ANR). V. D. and J. E. L. were recipients of collaborative grants from  CNRS  ( CNRS-NYU-UPS ) and from  Partner University Fund (Emotion & Time).
We also decided to test a higher dose of each compound in order to explore the compounds' safety margins. However, MK801 doses higher than 5 mg/kg could not be used for ethical reasons, as preliminary results showed that treated rats were losing more that 20% of their bodyweight and exhibiting exophthalmia. The higher doses used were thus 5 mg/kg for GK11 and MK801 and 50 mg/kg for Memantine. Additionally, initial experiments revealed that rats treated with 1 mg/kg GK11 did not show any behavioural or histological deficits, so this experimental condition was omitted in later experiments. Behavioural studies. Animals were tested for general behaviour 10 min and 24h after drug administration, using an automated open-field test (Bioseb, France). Locomotive behavior was analyzed during 10 min using Acti-Track software (Bioseb, France) and the maximal and mean speeds were determined.