dataesr/scientific-paragraphs-categorization huggingface dataset:
ac. uk. Information about curation is provided at http://www. ebi. ac. uk/intact/complex/documentation/. Extensive documentation and training material on how to best use our resource is available at http://www. ebi. ac. uk/training/networks. Cu-D484 Nucleic Acids Research, 2015, Vol. 43, Database issue ration groups interested in capturing macromolecular complex data or experimental interaction data who would like access to the editorial tool are encouraged to contact IntAct to discuss this further (intact-help@ebi. ac. uk).
These data indicate that the expression of ddHA GCN5b(E703G) protein diminishes acetylation on histone H3. We reasoned that if the growth arrest of Shield-treated ddHA GCN5b(E703G) parasites was due to hypoacetylation of histones, then inhibition of lysine deacetylases (KDACs) should help restore replication. We therefore incubated ddHA GCN5b(E703G) parasites with 500 nM Shield in combination with increasing sublethal concentrations of either apicidin or TSA, two independent, broad-spectrum KDAC inhibitors. Parasite plaque assays were performed one week post-infection. Consistent with results seen in Fig. 1 , virtually no plaques were evident in infected monolayers that contained Shield with no KDAC inhibitor (Fig. 4 ). However, Shield-treated ddHA GCN5b(E703G) parasites were able to resume proliferation with the inclusion of either KDAC inhibitor in dose-dependent fashion (Fig.
Genome-wide localization of GCN5b To identify genes regulated by GCN5b, we performed a genome-wide ChIP-chip analysis on HA-tagged GCN5b expressing parasites. Immunoprecipitated DNA associated with GCN5b was identified following hybridization to custom Nimblegen microarrays that tile the entire Toxoplasma genome. Three replicates were performed. GCN5b was detected at 195 tachyzoite genes in all 3 replicates and 1090 genes in two ChIP-chip replicates, when 0. 05 was used as the FDR for significant peaks (Supplemental Dataset S1). Although we observe variability in the location of GCN5b peaks, we detect a statistically significant overlap between the three ChIP-chip replicates that is not present when we randomize the peak positions for statistically significant peaks (FDR,0. 05; see methods for details).
Overall, the ChIP-chip results establish that GCN5b is present within or near the loci of tachyzoite genes involved in a wide variety of cellular functions (see Supplemental dataset S1 for breakdown of KEGG and GO classifications of the 195 genes identified in all 3 replicates). Many of the genes are annotated as hypothetical genes of unknown function in the ToxoDB. The high confidence genes coinciding with GCN5b localization are associated with gene expression and RNA processing, as well as metabolic genes, rather than genes linked to virulence. Although GCN5b did not show a detectable preference for functional regions of the genome, there was a significant association with genes containing introns versus those that do not. By hypergeometric test, each of the three ChIP-chip replicates has a statistically significant (p,0.
GCN5b interacts with a large number of novel proteins, including AP2 factors As GCN5 KATs lack DNA-binding domains, the complex must be recruited to target genes by virtue of a DNA-bound transcription factor, e. g. GCN4 in Saccharomyces cerevisiae or its human counterpart, ATF4  [31]. However, this well-conserved class of transcription factor is not present in Apicomplexa. We therefore performed biochemical purifications of the GCN5b complex from nuclear fractions of intracellular tachyzoites to define the KAT's interactome. Two independent co-immunoprecipitations were performed using RH parasites stably expressing HA-tagged GCN5b. Supplemental dataset S2 is a complete list of proteins identified in each pull down experiment. As expected, GCN5b itself as well as the known interacting coactivator protein, ADA2-A  [20] , were identified in each purification.
Associations between GCN5 and AP2 proteins have yet to be demonstrated for any species, including plants. To validate that GCN5b and these AP2 factors reside in the same complex, we endogenously tagged AP2IX-7 and AP2X-8 with a C-terminal 36HA tag (Fig. 6A ). Reciprocal co-immunoprecipitation of each AP2 factor pulled down GCN5b and many of the other proteins seen in the previous GCN5b IPs (Supplemental dataset S2). We refer to proteins that were pulled down consistently in all three purifications as the GCN5b/AP2 ''core complex'' (Table  2 ). To further confirm the association of GCN5b with these AP2 factors, we performed Western blots for GCN5b in AP2IX-7 HA and AP2X-8 HA immunoprecipitates. We also endogenously tagged AP2X-5, an AP2 factor that was not seen in the GCN5b IPs, to serve as a control.
Twelve of the 20 subunits compromising the GCN5b core complex were detected as acetylated in a previous study (shaded gray in Table  2 ). Up to four proteins in the GCN5b complex, including GCN5b itself as well as TAF1/TAF250  [34] , contain bromodomains that recognize acetylated lysine residues  [35]  (Supplemental dataset S2). The high degree of acetylated subunits and the presence of multiple bromodomain modules in the complex supports the idea that they participate in intracomplex protein-protein interactions through the binding of acetylated lysines  [36].
The dynamic exclusion option is enabled. Once a certain ion is selected once for MS/MS in 7 sec, this ion is excluded from being selected again for a period of 15 sec. Mgf files were created from the raw LTQ mass spectrometer LC-MS/MS data using Proteome Discoverer 1. 2 (ThermoScientific). The created mgf files were used to search the Toxo_Human Combined database  [45]  using the in-house Mascot Protein Search engine (Matrix Science) with the following parameters: trypsin 2 missed cleavages; fixed modification of carbamidomethylation (Cys); variable modifications of deamidation (Asn and Gln), pyro-glu (Glu and Gln) and oxidation (Met); monoisotopic masses; peptide mass tolerance of 2 Da; product ion mass tolerance of 0. 6 Da. The final list of identified proteins was generated by Scaffold 3.
1 of the Toxoplasma ME49 genome with a total of 732,672 toxo-specific probes with average spacing of probes every 86 bp using an estimated genome size of 63 Mb. Chip design is available under GEO identifier GPL15563 and GPL15564 and the data is supplied in supplemental dataset S1. H3K4me3, H3K9ac, H3K4me1, H3K9me2, and CenH3 data are also accessible at www. toxodb. org. Hybridization to arrays was performed using standard Nimblegen protocols at Nimblegen or in the Albert Einstein College of Medicine Epigenomics Facility as described in  [26]  with an input DNA and experimental DNA hybridization performed simultaneously for each experiment on the same chip. ChIP-chip analysis was performed three times. Microarrays were scanned once with excitation at 635 nm for the immunoprecipitated DNA and 532 nm for total genomic DNA.
Reproducibility of GCN5b peaks was judged using the ''makeVennDiagram'' function from the ChIPpeakAnno package of the Bioconductor project  [46]. We first selected those peaks that have an FDR below 0. 05. We then identified peaks from different replicates that overlap by 50 nucleotides or more. Finally, we used the hypergeometric distribution to calculate the significance of the overlap. The probability of finding the number of overlapping peaks by chance was calculated as: 1 vs 2 7. 33 E-105; 2 vs 3 2. 56 E-19; 1 vs 3 0. 0005. Association between GCN5b and specific genes was established by using the ''findOverlappingPeaks'' function from the ChIPpeakAnno package of the Bioconductor project. We used the gene annotations from ToxoDB, release 6. 1, ME49 strain. Gene associations for each replicate were determined independently.
Both randomization tests were performed 1,000 times. For examination of GCN5b peaks with introns or genes with introns, gene models and annotations from www. toxodb. org V6 were used. Significance of overlaps was determined by hypergeometric test for intron-containing genes (5,989) versus intronless genes (2,083). To further test this, we selected random peaks from each experiment rather than only the significant peaks (FDR,0. 5%). We repeated the same test with the randomized peaks. Using the same methods, we also tested for an association between GCN5b binding with introns or exons. We also tested for an association with active promoters (as defined by dual marking with H3K9ac and H3K4me3  [26] ), active coding regions (defined by the H3K4me1 mark  [26] ), or centromeres  [27]  and measured the distribution of GCN5b peaks from the inferred transcription start site using data from Yamagishi et al.
Supporting Information Dataset S1 ChIP-chip of GCN5b. Three replicates of GCN5b chromatin immunoprecipitations were hybridized to a genomewide chip. Genes identified in each of the replicates are indicated by an X for each replicate. Version 6. 1 Toxoplasma identifications were used. GO terms as provided at ToxoDB. org are indicated. KEGG pathways and GO terms are summarized for the 195 genes identified by all 3 replicates of ChIP-chip. (XLSX) Dataset S2 Proteomics of GCN5b, AP2IX-7, and AP2X-8 identifies a core macromolecular complex. Two replicates of GCN5b pull-downs were performed. Proteins detected in one (1/ 2) or both (2/2) of the GCN5b IPs are indicated as such. Proteins that were also identified in either of the AP2 pull-downs are indicated by an X. All proteins were identified by at least 2 independent peptides that were assigned with 95% confidence using the Mascot search of the EPIC-DB custom Toxoplasma database  [45].
Cells were stained for F-actin using Phallotoxin (Invitrogen) for 20 min. Finally, the cells were incubated for 5 min at room temperature with Fluoroshield with DAPI (Sigma-Aldrich), mounted and examined with a fl uorescent microscope. CellZscope Measurements : The CellZscope (Nanoanalytics) measures the frequency-dependent impedance of barrier-forming cell cultures grown on permeable membranes and provides the TER as output. Impedance of cell layers, maintained as above, was measured in complete DMEM. Council ERC-2010-StG Proposal No 258966 (IONOSENSE), FP7-People-2011-IIF, a Marie Curie post-doctoral Fellowship (J. R. ), as well as a joint grant from the Conseil Regional de Provence Alpes Côte d'Azur and CDL Pharma (S. T. ), and funding from ANRT and Microvitae (P. L. ). The authors would like to thank Prof.
Both gating parameters such as time and voltage of the pulse, and acquisition parameters like the sampling frequency, can be set separately for each transistor. In the experiments presented here, the current in the channel was measured at V ds = -0. 2 V and each channel was sampled at 60 Hz. The gate voltage was applied using a NI PXI-6289 modular instrument. A 2 s pulse was applied on the gate every 30 s, at V gs = 0. 3 V. All of the measurements were triggered through the built-in PXI architecture. The recorded signals were saved and analyzed using customized LabVIEW software. Data Analysis : Data analysis was performed using a customized Matlab program to isolate and fi t the time constant for each pulse. The time constant is extracted by performing a least-squares fi t of the data from each pulse current response to Equation 1.
A background is subtracted by taking into account the time response with no cells and after scratch. The data are then normalized using the following equation: NR = ( T no cells -T)/ (T no cells -T cells ), where T cells refers to the Tau value in response to the application of the gate voltage of a barrier forming monolayer, and T no cells refers to the Tau value in response to the application of the gate voltage of no barrier, with the dataset subsequently normalized to a to [0,1] scale. Cell Culture : Caco-2 cells from ECACC (catalog no. 86010202) were seeded at a density of 1. 5 × 10 4 cells/insert. Cells were routinely maintained at 37 °C in a humidifi ed atmosphere of 5% CO 2 , in DMEM (Advanced DMEM (Dulbecco's modifi ed Eagle medium) Reduced Serum Medium 1X, Invitrogen) with 2 × 10 -3 M Glutamine (Glutamax-1; Invitrogen), 10% fetal bovine serum (Invitrogen), and Pen-strep (5000 [U mL  -1  ] Penicillin-5000 [µg mL  -1  ] Streptomycin; Invitrogen).
The Hot Ion Analyser (HIA), part of the Cluster Ion Spectrometry experiment, has the objective to measure the three-dimensional velocity distributions of ions. Due to a variety of factors (exposure to radiation, detector fatigue and aging, changes in the operating parameters, etc. ), the particles' detection efficiency changes over time, prompting for continuous in-flight calibration. This is achieved by comparing the HIA data with the data provided by the WHISPER (Waves of HIgh frequency and Sounder for Probing of Electron density by Relaxation) experiment on magnetosheath intervals, for the high-sensitivity section of the instrument, or solar wind intervals, for the low-sensitivity section. The paper presents in detail the in-flight calibration methodology, reports on the work carried out for calibrating HIA and discusses plans to extend this activity in order to ensure the instrument's highest data accuracy.
An in-flight calibration is needed as well whenever the HIA operating point is changed by commands from ground. The multipoint character of Cluster and its complex payload made it possible to asses the HIA's in-flight performance at an unprecedented level of accuracy. The calibration methodology to be presented in this paper was developed by taking advantage of the large crosscalibration effort carried on in the framework of ESA's Cluster Active Archive (CAA) program. Before the CAA initiative, the resources allocated to this activity were relatively small when compared with the complexity of the work; also, the less accurate Cluster Prime Parameter data set has been used as a reference for the total electron density  (Vallat, 2001). The CIS experiment is prepared by an international consortium, under the principal responsibility of Institut de Recherche en Astrophysique et Planétologie (IRAP) in Toulouse (formerly Centre d'Etude Spatiale des Published by Copernicus Publications on behalf of the European Geosciences Union.
-Due to the penetrating particles from the radiation belts, the HIA measures a high background around perigee passes. A similar effect may also occur during some intense solar particle events. -The detection of low-energy ions may be affected by the spacecraft charging to a positive floating potential that repels these ions. -On some occasions, instrument artifacts (wrong time tagging, sudden density drops, high-voltage discharges, wrong discriminator levels, etc. ) may occur. These events are listed on the CIS Data Caveats list, available on the instrument's web page. Since the beginning of the mission, the HIA sensors were operational only on C1 (Cluster 1) and C3. Since November The HIA detection efficiency as a function of position (polar angle θ) and particle energy E is given by the formula (see  Bosqued, 2000 , reporting on HIA's ground calibration) Eff(θ, E) -1 = Norm_θ • Cheff(θ) • A • E + B T 0 + T 1 • E t + T 2 • E 2 t The first part of the RHS describes the position (anode)dependent efficiency, with Norm_θ designating the anode normalization coefficients (one for each sensitivity side) and Cheff(θ ) the relative anode-dependent efficiency coefficients.
-There is a symmetric anode-dependent efficiency evolution with time, and therefore the relative anodedependent efficiency coefficients Cheff(θ ), determined in the preflight tests, have not changed. -The coefficients A, B and T 0,1,2 describing the efficiency energy dependence do not change as well and assume the values determined in the preflight tests. -The WHISPER data are well-calibrated and free of errors (at least in the statistical sense). The traces in the WHISPER spectrograms are correctly assigned to some characteristic frequencies in plasma, from where electron density can be inferred. In these circumstances, the WHISPER data can be taken as reference. The first two assumptions greatly simplify the calibration task, basically implying that only two coefficients (one for each sensitivity side) are needed to correct the HIA efficiency.
g. , the discussion about the HIA measurements in the plasma-sheet environment in Sect. 6) but so far no careful study addressing this problem has been carried out. However, it seems that a change in the efficiency energy dependence is a second-order effect, at least in the plasma environments where the two HIA sides are calibrated (see the statistical studies presented in Sect. 5). One particular aspect that might be of concern is the role of ion composition in the HIA-WHISPER data comparison. The prevalent minor ions in solar wind and magnetosheath plasma (the environments where the two HIA sides are calibrated; see next sections) are the α particles. If one considers a mixture of protons (number density N p , mass m p , and electric charge q p ) and α particles (with corresponding parameters N α , m α , and q α ), then for a detector like HIA, unable to discriminate between the ion species, the number density reported by the instrument will be (see e.
-Intervals are not on the Data Caveat list (see Sect. 3). For the selected intervals, the WHISPER density data are requested from the instrument team, which either decides to regenerate it for the purpose of HIA calibration or to revalidate the already available CAA data set. Figure  3  illustrates one example of an interval selected for the HIA-WHISPER density comparison. The four panels at the top represent the type of plot routinely produced for the identification of calibration intervals. First, the HIA energy spectrograms in three ranges, i. e. , above 2 keV, the entire energy range, and below 100 eV, are shown in order to identify intervals that better comply with the requirement that virtually all particles are detected by the instrument. In the fourth panel the HIA (red) and WHISPER (black) raw density data are shown.
Here a few sudden HIA density drops can be seen; these signatures are not present in the WHISPER data and are interpreted as instrument artifacts. The next panel compares the two densities after the data processing has been performed, which includes the removal of instrument artifacts, discarding of short intervals with rapid variation in density, data filtering, averaging and interpolation, etc. The sixth panel checks the plasma gyrotropy as measured by the quantity (p ⊥2 -p ⊥1 )/[(p ⊥2 + p ⊥1 )/2], with p ⊥1 and p ⊥2 being the plasma thermal pressure along two orthogonal directions in the plane perpendicular to the magnetic field. The relatively small deviations from gyrotropy, e. g. , around or below 5%, provides supporting evidence of the HIA's symmetric anode response for this interval.
The bottom panel shows the N HIA / N WHI density ratio (blue) and its average value for this interval (magenta straight line). The result of comparison can also be shown in the form of a HIA vs. WHISPER density plot, presented in Fig. 4 , where the left panel refers to the event described above. The points are clearly scattered along the regression line (in red) forced to cross the origin; its slope is used to estimate the calibration factor inferred from this interval. The average value of several calibration factors, obtained from different intervals, is then employed to update the calibration files used in processing the HIA data. In spite of the careful selection, it can happen that the HIA-WHISPER data comparison brings inconsistent results on some intervals. Typically a lower (than expected) value of the N HIA / N WHI ratio is attributed to the presence of plasma population outside the HIA detection range, to the spacecraft charging or to events with relatively high abundance of α particles.
Calibration of HIA's LS section With some specific differences, the calibration of the HIA low-sensitivity section follows a similar procedure. One uses solar wind (SW) data and compares the HIA and WHISPER density on carefully selected intervals. The selection is based on the type of plot presented in Fig. 5. The top panel shows HIA (red) and WHISPER (black) raw density data, A number of sudden HIA density drops (instrument artifacts) can be seen here as well. In the second panel the energy spectrogram based on data accumulated on the LS section is shown. For the purpose of calibration, it is desirable to select events with low presence of α particles, (seen in the second panel as the faint green line, at around twice the peak proton energy). Since an accurate calibration of the LS section requires no significant counts accumulated on the HS section (blocked when facing the SW direction, see Sect.
2), the third and the fourth panel present the plasma density and the energy spectrogram based on data accumulated on this side. The bottom panel compares the LS (red) and HS (black) count rates. After the selection of SW intervals suitable for calibration, the raw HIA and WHISPER data are processed in a manner similar to that described in the previous section. For the event presented above, the result of the comparison is shown in the right panel of Fig. 4  in the form of a HIA vs. WHISPER density plot. The points are aligned along the regression line (in red) forced to cross the origin; its slope is taken as the value of the calibration factor corresponding to this interval. By averaging several calibration factors, inferred from different intervals, a value is obtained that will be used to update the calibration files.
Statistic comparison between HIA and WHISPER data To validate the results of calibration methodology presented in Sect. 4, two statistical studies have been carried out, each based on data provided by one HIA sensitivity side. All MS and SW intervals observed in Cluster 1 data between 15 December 2006 and 15 February 2007 have been analyzed in order to compare calibrated HIA and WHISPER density data. Stable detector efficiency is expected in that period since the previous MCP high-voltage increase occurred in January 2006, the next one being performed on 16 February 2007, i. e. , just after the chosen interval. Using selection criteria similar to those presented in Sect. 4, a total of 54 MS intervals sampled by the HIA HS section have been identified, comprising around 55 h of data. For the LS section, 64 SW intervals have been identified, covering around 96 h of data.
Summary and future work The first two panels in Fig. 8 , showing the evolution of HS and LS detection efficiency in the course of the mission, summarize the results of HIA's in-flight calibration activity. Our comments below will refer mainly to the period starting from October 2005, subject to the calibration methodology presented in this paper. The top and middle panels in Fig. 8  refer to the HS and LS sections, respectively. The detector efficiencies shown, obtained by comparing the HIA and WHISPER data densities, are relative to the beginning of the mission. Black lines present the evolution of C1 while the red lines the evolution for C3. Each value of the efficiency is shown by a horizontal segment, with the length indicating its validity period. There are no HIA data on C3 after November 2009 as well as on the C1 LS side after June 2011 (see the last paragraph of Sect.
Acknowledgements. The authors would like to thank the WHISPER team (Principal Investigator,  J. L. Rauch ; former Principal Investigators, P. M. E. Décréau  and  Jean Gabriel Trotignon ) for their effort to ensure reliable electron density data for the HIA calibration. The calibration-routine package is based on the cl (credit to  E. Penou ) and CCAT (credit to  J. McFadden ,  P. Schroeder  and  C. Mouikis ) programs for CIS data analysis. The work has been supported through the project  VALS  (contract  45/19. 11. 2012 ) of the  Romanian Space Agency STAR program , and by the  OTKA  grant  K75640  of the  Hungarian Scientific Research Fund. Edited by:  H. Laakso.