dataesr/scientific-paragraphs-categorization huggingface dataset:
Invading cells were counted at 10x magnification in brightfield on an Olympus BX51 microscope in three different fields per membrane, and both the peripheral and the central areas were evaluated. Experiments were duplicated for each condition and repeated three times. Migration was demonstrated using 100-mm non-pyrogenic dishes  [26]. Briefly, when HPNE/shScr and HPNE/shSMAD4 cells approached 100% confluence, the cell monolayer was scratched using a P200 micropipette tip. Cells were washed in PBS twice and incubated in 5% FBS medium for 20 hours with or without TGF-b (10 ng/ml) to allow them to migrate and close the wound. Three random images were taken at the time of the scratch and at 20 hours (Olympus IX71 inverted microscope, 46 magnification). The migration rate was determined as the ratio of distance of the wound's gap at 20 hours versus at 0 hours using Adobe Photoshop software (San Jose, CA).
Immunohistochemical staining Tissues for this study were obtained from patients with primary pancreatic tumors and incorporated into our patient-direct xenograft program  [29]. The tissues were embedded and cut into sections 4 mm thick for immunohistochemical study at the Clinical Core Laboratory of MD Anderson. Immunohistochemical staining for SMAD4 and N-cadherin was done using a Lab-Vision 480-2D immunostainer (ThermoFisher, Fremont, CA). All reactions were visualized with diaminobenzidine as a chromogen. Positive and negative controls were included in each run for each antibody used. Isotype controls for all antibodies were negative. Images were captured with an Olympus DP72 camera and CellSens software (Center Valley, PA) on an Olympus BX51 microscope at 10X magnification.
In contrast, knock down of SMAD4 in HPNE cells had no notable effect on the protein level of the mesenchymal protein vimentin or the epithelial protein b-catenin (data not shown). It also had no effect on the mRNA levels of CDH1, VIM, BCAT, and FN1. Western blot results demonstrated that proteins from the canonical TGF-b pathway, phospho-SMAD2 and phospho-SMAD3, and non-canonical pathways, such as phospho-AKT, phospho-MEK 1/2, and TAK1 showed similar expression patterns after TGF-b treatment in HPNE, HPNE/Scr, and HPNE/shSMAD4 cell lines, suggesting they were unaffected (Figure  S1a  and 1b ). p21, another downstream protein in the TGF-b pathway, showed the same pattern of expression in migration distance at the time of the scratch and after 20 hours. Three random images (46) were taken at these time points, and migration rate was determined as the ratio of distance at 20 hours versus 0 hours in the wound's gap using Adobe Photoshop software.
Acknowledgments The study was supported by  Viragh Family Foundation , and  Various Donors in Pancreatic Cancer Research. It was also supported by the  NIH/ NCI  under award number  P30CA016672  of the Core Facilities and  Services CCSG of Flow Cytometry and Cellular Imaging Facility, Houston. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Statistical analysis Apparent prevalences and their confidence intervals were estimated using the generalized estimating equation models to adjust for correlations within communities. True prevalences were calculated using the Rogen-Gladen estimator. Intra cluster correlations were estimated using a 1-way mixed effect ANOVA model. All statistical analyses were performed using the R statistical software version 3. 0. 2. ArcMap v10. 3 was used to map prevalence estimates in different villages.
Acknowledgements The research leading to these results has received funding from the  European Union 's  Seventh Framework Program  ( FP7/2007-2013)  under grant agreement n°221948, ICONZ (Integrated Control of Neglected Zoonoses) to CW and JDK and  Carnegie-Makerere University Next Generation of African Academics  under grant number  NGAA-2010-2012  to MD. Authors wish to acknowledge the assistance of Tororo District Veterinary officer;  Dr. Tegule Mukonge  and that of the other  Tororo District Department of Production  staff for the assistance they offered during collection of cattle blood samples. We also acknowledge  Ward Bryssinckx of Avia-GIS, Belgium  for helping with use of ArcMap software in general mapping and spatial data analysis.
Computing details Data collection: CrysAlis PRO (Agilent, 2012); cell refinement: CrysAlis PRO (Agilent, 2012); data reduction: CrysAlis PRO (Agilent, 2012); program(s) used to solve structure: SIR97  (Altomare et al. , 1999) ; program(s) used to refine structure: SHELXL2013  (Sheldrick, 2008) ; molecular graphics: ORTEPIII  (Burnett & Johnson, 1996)  ORTEP-3 for Windows  (Farrugia, 2012) ; software used to prepare material for publication: SHELXL2013  (Sheldrick, 2008). where P = (F o 2 + 2F c 2 )/3 (Δ/σ) max < 0. 001 Δρ max = 0. 13 e Å -3 Δρ min = -0. 14 e Å -3 Extinction correction: SHELXL2013  (Sheldrick, 2013) , Fc * =kFc[1+0. 001xFc 2 λ 3 /sin(2θ)] -1/4 Extinction coefficient: 0. 050 (2) Absolute structure: Refined as an inversion twin. Absolute structure parameter: 0. 0 (3).
Recordings from cells whose identity was ambiguous were not included in subsequent analyses. 4 mM QX-314 was added to the intracellular solution in some experiments to block voltage-gated sodium channels. All drugs were obtained from Sigma (St. Louis, MS, USA). Standard single and dual whole-cell recordings from 24 to 29 hpf larvae were obtained using an Axopatch 200B and a Molecular Devices CV 203BU headstage amplifier (Molecular Devices). Data were acquired at 40 kHz and low-pass filtered at 10 kHz. Cells were held near their resting potential at -65 mV under voltage clamp unless otherwise specified. A maximum of three neural recordings were obtained from each embryo. Electrophysiological analyses were performed offline using Clampex 10. 2 and Clampfit 10. 2 software (Molecular Devices).
The recordings were not analyzed if the resting membrane potential was more positive than -40 mV or if the input resistance was below 500 M. Following each recording, a series of fluorescent images of the rhodamine-filled cell and its axonal projections as well as brightfield images were collected with a QImaging camera (model 1394, QImaging Corporation Canada) using Micro-Manager software (http://www. micro-manager. org/). Images were inverted and brightness/contrast was adjusted using Adobe Photoshop CS2 (Adobe Systems, Inc. , San Jose, CA, USA).
STATISTICAL ANALYSES SPSS 21 (IBM) was used to assess data for statistical significance. All data sets were initially assessed for normality with the Shapiro-Wilk Test. For independent data sets with only two groups we used the Student's t-test or Mann-Whitney U test, and for data from multiple groups we used a one-way ANOVA with Tukey's post hoc test to look for differences between conditions (p < 0. 05). In data obtained from the same group under different conditions, the Paired-samples t-test or repeated-measures variation of ANOVA was used. A Pearson statistic or Spearman rank rho was used to assess correlations for parametric or nonparametric data, respectively. Statistical significance is represented in the graphs as * * * for p < 0. 001, * * for p < 0. 01, and * for p < 0. 05, and individual p-values and numbers of embryos per condition are provided in figure legends and the text.
Error bars in bar graphs indicate the SE, and results are described as the mean ± SE. Excel was used to create all graphs except the box plots, which were created online using the BoxPlotR application (http://boxplot. tyerslab. com/). In these plots, box limits indicate the 25th and 75th percentiles as determined by R software, center lines show the medians, whiskers extend 1. 5 times the interquartile range from the 25th and 75th percentiles and outliers are represented by open circles.
51; p = 0. 08; N = 9). AMPAergic I-V curves were obtained by recording AMPAergic mEPSCs at holding potentials ranging from -80 mV to +80 mV in 20 mV steps in order to determine the degree of rectification. As expected, AMPAergic mEPSC amplitudes decreased as holding potentials moved from -80 mV toward 0 mV, the approximate cationic reversal potential (Figure  3G ; N = 3). However, no outward-going AMPAergic mEPSCs were seen beyond the cationic reversal potential, despite the large driving force present at +80 mV. Despite having a larger signal to noise ratio, evoked AMPAergic EPSCs onto CoPAs also failed to reverse at positive holding potentials (N = 3; data not shown), demonstrating a strong inward rectification of glutamatergic currents, consistent with the presence of calcium-permeable AMPARs that should be sensitive to external polyamine block  (Man, 2011).
This morphological modification, which is typical of CSC differentiation,  33  was not observed in DCA-treated NSC cultures (data not shown). To evaluate whether DCA has an effect on differentiation, the expression of the stem cell marker nestin and the lineagespecific differentiation markers GFAP (astrocytes), Tuj (neurons) and Rip (oligodendrocyte) were analyzed in control and DCAtreated glioma spheroids and NSCs. As shown in Figure  2c , nestin expression was drastically reduced in DCA-treated glioma spheroids, whereas that of Tuj and Rip was markedly increased upon this treatment. In contrast, the expression of these markers was not altered in NSCs (Figure  2c ). Immunocytochemical analyses of the neurospheres confirmed these results (Supplementary Figure  S5 ), suggesting that DCA induced cell differentiation of CSCs contained in glioma spheroids into neural progenitor phenotypes.
Materials and Methods Materials. Cell culture material was obtained from Gibco (Life Technologies, Cergy Pontoise, France). Unless stated otherwise, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). The references of antibodies and the dilutions used are indicated in Supplementary Data. Results shown are the mean values of at least three independent experiments (±S. D. ) unless expressively mentioned. The ImageJ software (freely available online rsbweb. nih. gov/ij/) was used to quantify images. Cell culture. Glioma primary cultures (named P7 and M7) were obtained from Sprague-Dawley rats following antenatal ENU induction as described in Morfouace et al. 21 Cells were either maintained in FCS-containing medium as adherent cells or cultured as neurospheres in FCS-free defined medium (DMEM 1 g/l glucose, 2 mM L-glutamine, N2-and B27-supplement, 2 mg/ml heparin, 20 ng/ml EGF and 25 ng/ml bFGF, 100 U/ml penicillin and 100 mg/ml streptomycin).
75 g/l, corresponding to an average ingestion of 80 mg/kg per day (Morfouace et al. 21 ). After 37 days, tumors were removed, included in a paraffin block and sliced. Immunostaining was performed as described below for neurospheres. Flow cytometry. Cells were dissociated, washed and marked as described by Sergent-Tanguy et al. 37  Data acquisition was performed on a FACScalibur (Becton Dickinson, Le Pont-de-Claix, France). Data were analyzed using the CellQuest software (Becton Dickinson). Immunostaining. For immunostaining, neurospheres were dissociated and 50 ml were spotted onto Superfrost slides. The cells were then fixed with 4% PKM2 activity is controlled by its substrate and oscillates between a dimeric (low activity) form and a tetrameric (high activity) form. The tetrameric form of PKM2 that produces pyruvate from phosphoenolpyruvate (PEP) is the main form in normal differentiated tissues and proliferating cells.
MR imaging was performed within 3 months after the onset of the disease in different hospitals using either a 3T Verio (Magnetom, Siemens Medical Solutions, Erlangen, Germany) or a 3T Achieva (Philips Medical Systems, Best, The Netherlands) scanner according to a multi-center protocol. The protocol contained axial 2D T1 SE sequences, pre, 5 minutes post Gd injection and 24 hours post USPIO injection, with TR/TE 500/ 8. 4 ms, T2 TSE imaging with TR/TE 6530/84 ms, axial 2D FLAIR with TR/TI/TE 10000/2600/80 ms and 3D FLAIR with TR/TI/TE 5000/1800/273 ms. A 256|256mm 2 Field Of View was used to cover the whole brain with 44 3-mm 2D slices or a 1|1|3mm 3 voxel size for 3D acquisitions. The imaging data used in this study was acquired at baseline (m 0 ), after 3 months (m 3 ), 12 months (m 12 ) and 24 months (m 24 ).
However, the patients presenting at least either one C 2 or C 3 lesion pattern seem to have more hypointense lesions by m 24 , this is probably related again to the fact that these patterns indicate high activity  [18]. Beyond the clinical validation, the classification method itself is significantly novel and of general relevance independently from the specific data-set. The tensor-like representation for lesions is motivated by the rotationally invariant eigenvalues and the analogy to PCA. The detected clusters proved to be statistically significant in our dataset. However, both the tensor-like and volumetric representation can oversimplify the complexity of the lesion enhancement even if still informative. To complement this representation a clear indication of the hollowness of the lesion has been added in such a way to discriminate focal from ringing-like lesions.
ROS analysis The oxidative fluorescent dye dihydroethidine (DHE, Sigma-Aldrich) was used to evaluate in situ formation of ROS by a method described by Dal-Ros et al. [37]. Unfixed pancreatic islets were cut into 4 mm-thick sections, treated with DHE (2. 5 mM), incubated in a light protected humidified chamber at 37uC for 30 minutes. The level of ROS was determined using microscope and whole fluorescence of islets was quantified with the microscope software (NIS-Elements BR, Nikon Instruments Inc. , Champigny-sur-Marne, France). The fluorescence intensity of the islets was quantified in fifteen arbitrarily selected islets and the mean value for each islet was calculated.
After washes, sections were incubated for 1 h at room temperature with Alexi 488 or Alexi 594 conjugated secondary antibody (1:200; Life Science). For co-localization, spinal NMS were labeled with NeuroTrace 500/525 Green Fluorescent Nissl (1:200, Life Science). To quantify microglial immunoreactivity, microphotographs of the ventral horn gray matter were taken at ×400 and, after defining the threshold for background correction, the integrated density of Iba1 labeling was measured using ImageJ software  (Mancuso et al. , 2012). The integrated density is the area above the threshold for the mean density minus the background. weeks of age, mice with sciatic nerve crush and mice with SCI, were anesthetized and decapitated. The lumbar spinal cord was removed and divided into quarters to isolate the ventral quadrants.
The primary antibodies used were: mouse anti-GAPDH (1:20000, Millipore), rabbit anti-phospho-Ser940 KCC2 (1:1000, Phosphosolutions), rabbit anti-KCC2 (1:500, Millipore), mouse anti-gephyrin (1:1000, BD Bioscience) and anti-GAD65/67 (1:1000, Abcam). Horseradish peroxidase-coupled secondary antibody (1:5000, Vector) incubation was performed for 1 h at room temperature. The membranes were visualized using enhanced chemiluminiscence method and the images were collected and analyzed with a Gene Genome apparatus and Gene Snap and Gene Tools software (Signee), respectively.
REDUCED ACTIVE FORM OF KCC2 IN LUMBAR SPINAL MNs AFTER SCI We first examined KCC2 expression by western blotting in the lumbar ventral spinal cord of animals with SCI at 28 dpi. Compared to naïve animals, we observed a slight reduction of total KCC2 after SCI (around 20%), although the difference did not reach statistical significance (p = 0. 06, data not shown). This tendency is in agreement with previously reported results by. Boulenguez et al. (2010)  after SCI in the rat. To check the activation state of the KCC2, we also performed western blotting for the phosphorylated form of the KCC2 (pKCC2) with a specific antibody against phopho-Ser 940. In contrast to total KCC2 expression, the phosphorylated form was decreased after SCI compared to control samples (p < 0. 05; Figures  4A, B ). Since it has been previously described that KCC2 oligomer/monomer ratio is increased at maturity and correlates with KCC2 activation  (Blaesse et al.
We suggest that this ExPECassociated DNA within the genome of MS499 has been horizontally acquired during its recent evolutionary history, and we speculate that the recruitment of this DNA into the MS499 genome has expanded the niche of this isolate, and allows it (alongside related ST453 isolates) to cause extraintestinal disease. The acquired DNA disproportionately encodes several functional classes of genes, including those involved in adhesion and those directed to the outer membrane or extracellular space. These data help illuminate key components in the genetic basis for extraintestinal pathogenesis in E. coli, and demonstrate the adaptability of this species in its evolution to exploit multiple environments.
Results and discussion MS499 is a member of an emerging ExPEC lineage E. coli strain MS499 (ST453) was isolated from a case of bovine metritis, and previously shown to reproduce extra-intestinal disease in a mouse model of intrauterine infection  [26]. In light of the ability for MS499 to cause extraintestinal disease in animals, we investigated whether E. coli similar to MS499 had been isolated from other extraintestinal infections. We searched the literature for reference to this sequence type (ST453) and found seven recent publications describing eight ST453 isolates, five of which are isolates from human urinary tract infections across the globe (Table  1 ). The remaining three isolates, alongside two further unpublished isolates found in the E. coli MLST database (Ecoli-CHU-52 and Ecoli-CHU-24), are isolated from the faeces of humans or animals in the absence of disease.
Overview of the MS499 genome Our investigation of the MS499 genome sequence  [33]  (accession number JDRV01) revealed that, in addition to the chromosome, this sequence contained several contigs which exhibited marked homology to colV-type plasmids found in a number of ExPECs including APEC O1, S88 and several plasmids isolated from extra-intestinal pathogenic Salmonella enterica serovar Kentucky isolates (Additional file 1), which have been proposed to underpin their virulence  [34, 35]. A similar plasmid was also recently described in an ExPEC isolate from phylogroup C  [25] , indicating that the putative MS499 plasmid could also play a role in virulence. Indeed, many of the traits associated with ExPECs can be conferred when similar plasmids have been experimentally introduced into non-pathogenic E.
coli genome sequences available in the NCBI sequence database at the outset of the study, and approximated their isolation source using the data available at NCBI (Figure  1 ). We partitioned 414 genomes into the ExPEC group, and 365 genomes into the B1 group -further details of the genomes assigned to each group are shown in the table in Additional file 2. Due to the large number of genomes in this analysis bootstrapping of this tree was not possible, so a subset of these sequences were taken for phylogenetic analysis with bootstrapping in order to support the branching pattern, which showed strong support for our tree (Additional file 3). Rather than focusing entirely on previously-defined genes for our comparative analysis, we performed an unbiased analysis in order to identify any region of the MS499 genome which displayed a significantly greater association with sequences in the ExPEC group than those within the phylogroup B1 group.
To do this we investigated the frequency of homologous sequences to 1 kb 'windows' of the MS499 genome in the B1 or ExPEC sequence groups. The resulting data allowed us to calculate the difference in frequency between the two groups, which is expressed on a scale from -100 to +100, representing windows more frequent in B1 than ExPECs (-100 to < 0), regions of no difference between the groups (0) and windows more frequent in ExPECs than B1 ( > 0 to +100). The main panels in Figure  2  represent this frequency difference along the length of the MS499 chromosome (a) and plasmid (b), according to the scale presented to the right of the image. The grey circles on the main panels represent results of a statistical analysis (Fisher's exact test, scale shown on the y-axis) to identify windows significantly associated with either group.
Since regions of no difference (white) do not discriminate between regions of shared commonality (i. e. both groups contain the query window at high frequency) or shared rarity (i. e. both groups contain the query window at low frequency), the top panel for the chromosome and plasmid tracks the total frequency in the B1 group (blue line) and ExPEC group (red line) independently. The data in Figure  2  revealed some striking features of the MS499 genome, with numerous regions clearly associated with either the B1 or the ExPEC group. Our analysis of these data showed that approximately 12. 5% of the MS499 chromosome is significantly enriched in the B1 group -this result was anticipated, since MS499 is a phylogroup B1 strain. These regions encode several metabolic loci (including the hpa and paa aromatic hydrocarbon catabolism loci and several other putative carbon utilisation pathways) and a number of genes involved in the production of extracellular structures including fimbrial and autotransporter genes.
Isolates coloured grey in both channels have either been isolated from a non-intestinal or extra-intestinal source, or no data is available for their source of isolation. The position of MS499 in the dendrogram is marked with an asterisk. A bootstrapped tree using a subset of these isolates is included in Additional file 3. ExPEC-associated, with approximately 54% showing a strong, significant association with ExPECs. A full list of B1-enriched and ExPEC-enriched genes is included in Additional file 4).
Regions of DNA which appear white in the main panel do not differentiate between query windows which are uniformly common or uniformly rare, therefore the line graphs presented above the main panels track the frequency of each window in the B1 group (blue line) and ExPEC group (red line) independently. These data indicate that approximately 4% of the MS499 chromosome (within 52 contiguous regions) and 52% of the putative MS499 plasmid (within 10 contiguous regions) are significantly associated with the ExPEC group (p = < 0. 0000021 for the chromosome, < 0. 00007 for the plasmid). HVH_989, H494, MS182-1 and O08), are all ST453 strains very closely related to MS499 (Additional file 5), whilst O08 was also isolated from extra-intestinal infection  [42]. The increased amount of ExPEC-associated DNA in the MS499 genome relative to the average for phylogroup B1 equates to approximately 100 kbp.
These relationships were confirmed by examining SNPs in the core genome of these sequences using PanSeq  [50]  (Additional file 5). These data supported the validity of our phylogenetic estimate, as the topology of the tree shown in Additional file 5 was very similar to the local topology around MS499 in the tree within Figure  1. Using these genome sequences as close relatives of MS499 we identified regions of the MS499 chromosome or plasmid which were absent in each of these close relatives. We then combined this data with the information on the ExPEC-associated DNA in MS499 to visualise four categories, as shown in Figure  5  (a): (1) regions of the backbone MS499 genome which are found in a relative (white), (2) regions of the backbone genome which are not found in a relative (yellow), (3) ExPEC-associated regions which are found in a relative (grey), and (4) ExPEC-associated regions which are not found in a relative (red).