dataesr/scientific-paragraphs-categorization huggingface dataset:
Experimental Procedures 2. 1. Animals. Data were collected from 11 pcd3j (C57BL/6-Agtpbp1 pcd-3j /J, Stock # 003237) knock out mice and 11 wild type (wt) littermates (6 mice of each genotype for immunocytochemistry and 5 mice of each genotype for biochemistry) of both types of sex at the age of 4 months. Animals were genotyped as juveniles by PCR as described on The Jackson Laboratory's website (Genotyping protocol database of the Jackson Laboratory). They had free access to food and water and were maintained on a 12/12 h light-dark cycle under conditions of constant temperature (22 ∘ C). All animals used in this study were treated in agreement with the German law on the use of laboratory animals. The ethical guidelines of the laboratory animal care and use committee at the University of Leipzig were followed.
Quantification. To estimate molecular alterations in pcd mice frontal sections were investigated and PN-bearing neurons of nucleus interpositus and nucleus dentatus of the DCN and the nucleus vestibularis lateralis were analyzed. The sections were labeled with anti-human aggrecan antibody (HAG7D4), the most comprehensive marker for PNs and additionally with GAD65/67 or vGlut1 and 2 for double immunofluorescence counting. The tissue was analyzed with a Zeiss Axiovert 200 M microscope equipped with a motorized stage (Märzhäuser, Germany) with MosaiX software and by means of a CCD camera (Zeiss MRC) connected to an Axiovision 4. 6 image analysis system (Zeiss, Germany). Counts were performed using the optical fractionator method  [19, 37]  on a Zeiss Axioskop 2 plus microscope (Jena, Germany) equipped with a motorized stage (Märzhäuser, Wetzlar, Germany), a Ludl MAC 5000 controller (LEP, The contours of the DCN were outlined in the Stereo Investigator program using a 10x lens, cell diameter determination and synapse counts were performed using an oilimmersion 63x lens (1.
For discontinuous SDS-Page the supernatant containing 35 g proteins was mixed with 1x SDS sample buffer and denaturized at 70 ∘ C for 15 min. The proteins were separated on a 10% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Perkin Elmer, Rodgau, Germany). Blots were blocked with 1% BSA in Tris-buffered saline containing 0. 05% tween for 1 h, washed, and incubated with primary antibodies (Table  1 ) diluted in blocking solution overnight at 4 ∘ C. Blots were washed and incubated with HRP-conjugated secondary antibodies (Table  2 ) for 1 h. HRP activity was detected using ECL Western blotting (Amersham Biosciences) and scanned with DNR Bio-Imaging System and analyzed by using software TINA. The ratios of optical density of the investigated proteins were normalized to -actin.
Acknowledgments The project was supported by the  German Research Foundation  (through  Priority Program 1608 ;  Mo2249/2-1 ), the  EU-COST Action   BM1001  " Brain Extracellular Matrix in Health and Disease ", the  Alzheimer Forschung Initiative e. V. (AFI # 11861 ), and the  European Union  and the  Federal State of Saxony  to  Markus Morawski  as well as the  Institute Curie  and the  ANR-12-BSV2-0007 Grant  to  Carsten Janke.
Subjects were asked to stay in the middle of an experimental room in quiet upright stance, barefoot and keeping their arms along their body. The subjects wore a special spectacle upon which one could easily put on a 1-diopter vertical prism base down if the case. Amplitude of rotation has been determined, while subjects were asked to rotate their head to the right, then to the left alternately three times without forcing the movement, each time with a stop at the center (i. e. , in a straight ahead position), the shoulder girdle stable. Head rotation on the trunk was measured with the CODAMOTION active markers (Version 6. 70. 16-CX1-CodaSharc V3. 02, Charnwood Dynamics Ltd. , Rothley, UK) placed on the head and at the shoulder girdle level for 3-D recording (see Figure  1 ), and the analysis was done via Matlab software at the faculty of medicine of Paris Descartes.
Study population We recruited 10 normal-weight (18. 5 kg/m 2 <body mass index (BMI) <25. 0 kg/m 2 ) and 20 obese (grade I obesity, BMI 30. 0-34. 9 kg/m 2 ) post-menopausal women, aged between 50 and 64 years. They had had a stable weight for at least six months and had no personal or familial history of diabetes, no inflammatory pathology (high-sensitivity C-reactive protein (hs-CRP)< 7 mg/mL), no smoking habits and no treatment that could interfere with insulin sensitivity. Their physical activity level was estimated using Voorrips questionnaire  [28]. Dietary intakes were assessed by self-administered questionnaire in which the subjects indicated the amount of each beverage and food they had drunk and eaten and the cooking method  [29]. These data were reported during three consecutive days, including one Saturday or Sunday to take into account potential different food habits during the weekend.
Questionnaires were then analyzed using GENI software (Micro6, Villiers-lès-Nancy, France). Results are expressed as calorie intake (Kcal) and quantity of proteins, fat and carbohydrates in grams per day. Blood pressure was measured after a 15-min rest period using an automated device. Insulin sensitivity was first assessed by the homeostasis model assessment of IR (HOMA IR ) index in order to classify the subjects in CT, OIS (HOMA IR <2. 7) and OIR (HOMA IR >2. 7) groups  [30]. All clinical investigations have been conducted according to the principles expressed in the Declaration of Helsinki. The study protocol was approved by the local Ethic Committee of CHRU Montpellier (Comité de Protection des Personnes Sud-Méditerranée IV) (number 2011. 01. 04) and informed written consent was obtained from all the participants.
Nuclei were stained by 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, Lyon, France), used at a 1:2000 dilution in Mowiol mounting medium (2. 4 g Mowiol 4-88 from Sigma-Aldrich, 6 g glycerol, 6 mL distilled water, 12 mL 0. 2 M Tris-Cl bufferÁ pH 8. 5). Adipocyte size was estimated using ImageJ software (National Institutes of Health, USA, available online at http://rsbweb. nih. gov/ij/index. html). Muscle and SAT sections were observed with a Zeiss AxioImager M1 microscope (Zeiss, Le Pecq, France) and TIFF images were captured with an Axiocam MRm Zeiss using Axiovision 4. 7 (05-2008) software.
Specific bands were visualized with a LI-COR Odyssey CLx Imaging System (LI-COR Biosciences). Protein expression levels were quantified using ImageJ software. Finally, levels of protein expression in each sample were corrected using the quantification of the corresponding loading control (Akt for P-Akt or α/β-tubulin for IκBα) analyzed on the same membrane. Total RNA purification, cDNA synthesis and quantitative real-time polymerase chain reaction (qPCR) Total RNA was isolated from skeletal muscle (50 mg) and SAT (100 mg) samples using RNeasy Fibrous Tissue and RNeasy Lipid Tissue Mini Kits (Qiagen Sciences, Courtaboeuf, France), respectively. cDNA was generated from 1 μg RNA by reverse transcription using the Verso cDNA kit (ThermoFisher Scientific) in incubation volume of 20 μl. cDNA amplification was then performed by real-time qPCR using the LightCycler 1 480 SYBR Green I Master kit (Roche Diagnostics Limited, Rotkreuz, Switzerland) and specific primers for sequences of interest (i.
Statistical analysis The non-normal distribution of our data was assessed with the Shapiro-Wilk test. Mann-Whitney tests were performed for group comparison according to the non-normal distribution of our data. Wilcoxon test was used for paired sample comparison (basal and insulin-stimulated P-Akt/Akt levels). We considered that significance was reached when P 0. 05. Correlations were determined by Spearman analysis. Statistical tests were performed using R software (version 3. 1. 0, 2014-04-10, The R Foundation for Statistical Computing, Vienna, Austria).
Calcein (15 mM) was encapsulated within the vesicles to enable bursting under laser irradiation (488 nm, 5-10 % laser intensity). Movies of pore bursting were obtained at the highest achievable speed of the Leica SP5 resonant scanner (100 frames/s). The extracted frames of the explosions were processed with ImageJ freeware to track and measure membrane pore growth. For each glucose concentration, the experiment was repeated on an average of ten vesicles of equivalent sizes, and the results were plotted on a master curve (horizontal error bars    From the fits derived from the experimental data (Eq. (  1 )), the effective diffusion coefficients Deff could be calculated by fixing the value of the critical radius rc to 0. 38 μm for each viscosity. In agreement with the model proposed by Mabrouk et al.
Images were captured on a Zeiss Laser Scanning 700 inverted confocal microscope equipped with Zen Black control software (Zeiss, Oberkochen, DE) and processed using Velocity (Perkin-Elmer). Figures were compiled using Adobe CS6 (Adobe, Kist, SE). Fluorescence-Activated Cell Sorting (FACS) Sham-operated and HI animals were deeply anesthetized and sacrificed via decapitation at 24 h, 72 h, and 7 days after surgery. Brains were rapidly removed, hemispheres separated, and transferred to ice-cold Hibernate-A (Invitrogen). In order to generate sufficient material for downstream analysis, corresponding hemispheres from two animals were pooled. Single cells suspensions were obtained through enzymatic dissociation in Ca 2+ and Mg 2+ free Hank's Balanced Salt Solution (HBSS) containing 0. 01% papain (Bionordika, Stockholm, SE), 0.
5 µg/10 6 cells, cat. no 141708, BioLegend) and PE/Cy7 anti-mouse/human CD11b antibody (0. 25 µg/10 6 cells, cat no 101216, BioLegend). Stained samples were supplemented with 10 ml staining buffer, pelleted (350 g, 5 min), re-suspended in 500 µl staining buffer, and kept on ice until FACS sorting. Cells were sorted on a FACS Aria II Cell Sorter (BectonDickinson) equipped with a 100 µm nozzle. Primary gates were established on forward/side scatter plots (Figure  2A ) using appropriate isotype controls. Cells positive for CD11b (Figure  2B ) were further analyzed for their expression of CD86 and CD206 in the contralateral non-injured (Figure  2C ) and ipsilateral injured hemisphere (Figure  2D ). Gating strategies were based on previous studies  (Bedi et al. , 2013). Total cell number per sorted population was recorded and representative data was captured for the first 20,000 cells.
Galectin-3 Protein Analysis Cell pellets were lysed using a Bio-Plex Cell Lysis Kit (BioRad) in accordance with manufacturer's instructions. Briefly, lysing solution was added and cells resuspended by repeated pipetting before agitation on a microplate shaker (150 rpm, 4 • C, 15 min). Samples were then centrifuged (4000 g, 10 min) and supernatants collected and stored at -20 • C. Protein concentration was measured using a Pierce BCA Protein Assay Kit (Thermo Scientific) as outlined in the manufacturer's instructions. Galectin-3 protein was measured using a Galectin-3 enzymelinked immunosorbent assay (ELISA) Kit (cat. no 12727, BG Medicine). Data is presented as total amount target protein per ml (pg/ml) or as normalized to protein concentration and presented as pg target protein per µg total loaded protein.
Statistical Analyses All data are presented as group mean ± SEM. Data from males and females were combined in each group as breakdown by sex revealed no sex-specific expression of any of the markers analyzed. qRT-PCR and FACS data were assessed by two-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. Galectin-3 protein concentrations over time were assessed by Kruskal-Wallis followed by Dunn's multiple comparison test for each cell population vs. sham. Differences were considered significant at * p ≤ 0. 05; * * p ≤ 0. 01; * * * p ≤ 0. 001. Analyses were performed using Prism (Graphpad, v. 6. 05).
Identification of Microglia/Macrophage Phenotypes by Immunohistochemistry The presence of different cell phenotypes was confirmed by confocal analysis on immuno-stained histological sections. Cells, expressing Iba-1 (microglia/macrophage marker), but neither CD86 nor CD206, were readily observed in injured areas (Figure  3A ) as well as in the contralateral hemisphere (data not shown). Iba-1 positive cells with strong expression of CD86 were also found in the ipsilateral hemisphere (Figure  3A ). In white matter areas, such as the corpus callosum (Figure  3B ), Iba-1/CD206-positive cells were noted adjacent to Iba-1 positive cells clearly expressing both CD206 and CD86. Interestingly, Iba-1 positive cells expressing CD206 only, were mainly found in the meninges (Figure  3C ) or the contralateral hemisphere (data not shown).
FUNDING The studies were supported by funding from the  Swedish Research Council  ( VR2012-2992 , CM), Government grant in Public Health Service at the  Sahlgrenska University Hospital  ( ALFGBG-142881 , CM, KS),  European Union  grant  FP7 , (Neurobid,  HEALTHF2-2009-241778 , CM), the  Leducq foundation  ( DSRR_P34404 , CM, HH),  Åhlén Foundation (CM) ,  Olle Engkvist Byggmästare (CM) , the  Swedish Brain Foundation  ( FO2013-095 , CM),  Torsten Söderberg  ( M98/15 , CM),  Wilhelm  and  Martina Lundgren Scientific Foundation (KS) ,  Linnéa and Josef Carlsson Foundation (KS). ), the  Swedish Childhood Cancer Foundation (MB) , the  Frimurare Barnhus Foundation (MB)  and  Lions Cancer Foundation  (MB).
Material and Methods Participants We report a longitudinal prospective long-term study of 438 HD gene carriers from the Predictive Biomarkers for Huntington's disease protocol (NCT01412125), which was approved by the ethics committee of Henri Mondor Hospital (Créteil, France) in accordance with EU and French bioethics laws. All HD gene carriers gave written informed consent. They were heterozygous for the Htt gene (> 36 CAG repeats in mHtt) and aware of their genetic status. They had no other neurological conditions or long-term experimental treatment (e. g. cell transplantation). Data were collected from 1994 to 2011, at eight centers from the French Speaking Huntington's Disease Group (Angers: 24%, Bordeaux: 7%, Créteil: 34%, Lille: 4%, Lyon: 1%, Marseille: 12%, Paris: 11%, Strasbourg: 7%), and centralized at the National Reference Centre for Huntington's disease in Créteil.
0 (SD = 3. 2; range: 1 to 19 visits). Thirty-two HD gene carriers were seen only once. Data were recorded for 2185 visits. The mean duration of follow-up was 4. 3 years for the whole cohort (SD = 3. 0; range: 0 to 15. 5 years), and 4. 8 years (SD = 2. 9) years when excluding patients assessed once. The date at onset was available for 86. 53% of the HD gene carriers. It corresponds to the appearance of the first symptoms, and it was determined (observed) by the clinician (93. 14%), or, if missing, by the family (5. 28%), or, if missing that as well, by the participant (1. 58%).
Demographics and characteristics of COMT polymorphism groups at the first visit. The χ 2 goodness-of-fit test was computed to compare the distribution of COMT genotypes in the 438 HD gene carriers and in the control group. Stability of the genotype frequency was assessed through the Hardy-Weinberg test in controls. We assessed whether baseline characteristics of HD gene carriers were similar in the different COMT polymorphism samples (Met/Met, Val/Val and Met/Val), by first assessing the differences between groups for each score of the UHDRS at the first visit. Demographic data and clinical characteristics of the sample (N = 438) at the first visit were compared between groups, with a Pearson's χ 2 tests for qualitative variables and a one-way ANOVA for quantitative variables. For variables with significant difference between groups, student's t-tests (or Welch's tests in cases of unequal variances) were performed with Bonferroni correction for multiple pairwise comparisons (see S1 Table for the same comparisons in the subgroup included in the longitudinal analysis).
Number of CAG repeats and age at onset. We first assessed the impact of the number of CAG repeats on age at onset in the 379 HD gene carriers with these available data. We used a linear regression model, with age at onset as the dependent variable and the number of CAG repeats as an independent variable. The R 2 value provided by the model is an estimate of the proportion of the variability of the age at onset explained by the number CAG repeats. We also calculated an expected age at onset according to the Langbehn et al. model  [33] , derived from the number of CAG repeats using the formula: expected age = (21. 54 + exp (9. 556-0. 146 Ã CAG)). We evaluated the agreement between this expected age at onset and the age at onset provided in our database by calculating the intraclass correlation coefficient (ICC), a measure for concordance.
To assess the robustness of the results, a sensitivity analysis was performed excluding outliers, on the basis of the number of CAG repeats and of the distribution of dates of visits in our cohort (see S1 Fig and  S2  Fig). The sensitivity analysis included HD gene carriers with a number of CAG repeats between 39 and 49 that were followed in the 20 years after disease onset. Analyses were conducted with R 2. 3 software (http://www. r-project. org/). The R package lcmm was used to perform the longitudinal analysis. All tests were two-tailed. Values of P < 0. 05 were considered significant.
Furthermore, the use of a single language for cognitive testing decreased inter-subject variability in cognitive performance. The HD gene carriers were followed up prospectively for as long as possible, from pre-manifest to advanced stages. Although most of the data was collected between 5 and 15 years after disease onset, it provides a unique continuum of disease progression with enough follow up data to conduct a longitudinal analysis. Results of the sensitivity analysis displayed the same trend as the analysis of the whole sample, however without reaching significance after Bonferroni correction for multiple testing. The latent-class mixed model has the advantage of grouping several tasks within domains and provides a global picture by domain without focusing on specific tasks. This approach, recently developed, is already used in studies evaluating cognitive decline  [45, 46].
A-C Bachoud-Levi is the PI of the biomarker program initially funded by Gis A02216JS/A04159JS and then by Assistance Publique-Hopitaux de Paris (Departement de la Recherche Clinique et du Developpement); she had full access to all of the data for this study and takes responsibility for the integrity of the data and the accuracy of data analysis. She is supported by an interface contract from INSERM and is the Head of the French National Center for Huntington's Disease (funded by the French Ministry of Health). R de Diego-Balaguer was supported by grant PSI2011-23624 from the Ministerio de Economı ´a y Competitividad (MINECO). C Schramm was supported by the NeuroStemcell Consortium (European Community Seventh Framework Program grant agreement no. 222943) and by investment for the future NeurATRIS: Infrastructure de recherche translationnelle pour les biotherapies en Neurosciences (ANR-11-INBS-0011).
Additional support was provided by ANR-10-LBX-0087 IEC and ANR-10-IDEX-0001-02 PSL Ã grants. A Du ¨rr, A Brice, V Damotte, I Rebeix and B Fontaine were supported by IHU grant ANR-10-IAIHU-06. L Cleret de Langavant was supported by an AP-HP/CEA interface contract. The funders had no role in study design, data collection and analysis, the decision to publish, or preparation of the manuscript. Competing Interests: R. de Diego-Balaguer, C. Schramm, I. Rebeix, E. Dupoux, A. Durr, A. Brice, P. Charles, L. Cleret de Langavant, K. Youssov, G. Fenelon, C. Verny, V. Damotte, JP Azulay, C. Simonin, C. Tranchant, P. Maison, A. Rialland, D. Schmitz, C. Jacquemot, B. Fontaine have nothing to disclose. A. C. Bachoud-Levi acted as a consultant once for Teva in 2014 and reports grants from the National Reference Center for Huntington's Disease from the Ministry of Health and from the clinical research directorate (AP-HP).
COMT polymorphism does not impact age at onset. The number of CAG repeats explains 49. 61% of the variability of age at onset (β coefficient = -2. 07(SE = 0. 11), P < 0. 001). The ICC measuring agreement between expected age at onset by formula (1) and age at onset provided in the database is high for the whole cohort (0. 71: [95% CI 0. 65-0. 76], P < 0. 0001) and in each COMT group (Met/Met ICC = 0. 75 [95% CI 0. 64-0. 83], P < 0. 0001, Met/Val ICC = 0. 70 [95% CI 0. 62-0. 77], P < 0. 0001 and Val/Val ICC = 0. 66 [95% CI: 0. 54-0. 76], P < 0. 0001) (Fig 2).
Acknowledgments We thank  C. Lalanne  for comments relating to methodology and  Page Piccinini  for her language corrections. The French Speaking Huntington group is leaded by  AC Bachoud-Lévi  at the national reference centre for Huntington's disease. Contributors: Angers:  G. Aubin ,  A Olivier ,  P Allain ; Bordeaux:  U Spampinato ,  S Debruxelles ,  B Babiloni ,  C Duché ;  Créteil: M-F Boissé ,  M Couette ,  L Lemoine ,  S Baudic ,  G Morgado ; Lille:  P Krystkowiak ,  M Delliaux ,  K Dujardin ; Lyon:  H Mollion ,  E Broussolle ; Paris:  V Hahn-Barma ; Strasbourg:  N Longato ,  G Rudolf.
Funding: A-C Bachoud-Levi is the PI of the biomarker program initially funded by Gis A02216JS/A04159JS and then by Assistance Publique-Hopitaux de Paris (Departement de la Recherche Clinique et du Developpement); she had full access to all of the data for this study and takes responsibility for the integrity of the data and the accuracy of data analysis. She is supported by an interface contract from INSERM and is the Head of the French National Center for Huntington's Disease (funded by the French Ministry of Health). R de Diego-Balaguer was supported by grant PSI2011-23624 from the Ministerio de Economı ´a y Competitividad (MINECO). C Schramm was supported by the NeuroStemcell Consortium (European Community Seventh Framework Program grant agreement no. 222943) and by investment for the future NeurATRIS: Infrastructure de recherche translationnelle pour les biotherapies en Neurosciences (ANR-11-INBS-0011).
Table. COMT polymorphism does not impact age at onset. The number of CAG repeats explains 49. 61% of the variability of age at onset (β coefficient = -2. 07(SE = 0. 11), P < 0. 001). The ICC measuring agreement between expected age at onset by formula (1) and age at onset provided in the database is high for the whole cohort (0. 71: [95% CI 0. 65-0. 76], P < 0. 0001) and in each COMT group (Met/Met ICC = 0. 75 [95% CI 0. 64-0. 83], P < 0. 0001, Met/Val ICC = 0. 70 [95% CI 0. 62-0. 77], P < 0. 0001 and Val/Val ICC = 0. 66 [95% CI: 0. 54-0. 76], P < 0. 0001) (Fig 2). Number of CAG repeats and age at onset.
4B ) at P56. Taken together, these data indicate that MIUH results in impaired morphological brain development, i. e. , decreased cerebral hemispheric area, decreased gray and white matter areas, and enlarged lateral ventricles, all of which are typically observed in the brains of prematurely born infants. However, MIUH did not cause clear tissue or cellular damage in mature myelin, astrocytes, and axons and did not disturb the gross structure of cortical layers. Changes in protein levels in the sera of dams, amniotic fluid, placentas, and fetal brains. To elucidate the mechanisms underlying prenatal brain injury and to identify the biomarkers of MIUH-induced brain injury, changes in protein levels in the sera of dams, amniotic fluid, placentas and fetal brains were investigated using a multiplex protein assay on E20.