dataesr/scientific-paragraphs-categorization huggingface dataset:
The aim of the present post hoc analysis was to help guide clinicians on the efficacy and safety profile of sequential therapies based on the initial treatment choice. Response to prior sunitinib or cytokine therapy was not associated with longer PFS or OS in patients administered second-line axitinib or sorafenib. Similar to these findings, a retrospective analysis of an international database of 464 patients with mRCC also found no correlation between response to firstline and second-line VEGF-targeted therapy (Al-Marrawi et al, 2013). Together, these data suggest that lack of response to a VEGF-targeted agent in the first-line setting does not preclude positive clinical outcomes with a second-line VEGF-targeted agent, which is consistent with RCC being predominantly a VEGF-driven disease.
This may reflect higher potency of axitinib compared with sorafenib in patients with slower progressing disease. Similar to the results reported here for patients treated with sunitinib followed by axitinib or sorafenib, an association between PFS during first-line and second-line VEGF-targeted therapy was not observed in the retrospective database analysis of patients with mRCC  (Al-Marrawi et al, 2013). In addition, smaller tumour burden following treatment with sunitinib or cytokines was associated with longer OS with either second-line axitinib or sorafenib. Longer PFS was also seen in patients who had smaller vs larger tumour burden following sunitinib therapy, but reached significance only in the sorafenib arm. Other retrospective studies have identified a correlation between tumour burden and survival in previously treated patients with mRCC.
ACKNOWLEDGEMENTS This study was sponsored by  Pfizer Inc. We thank  Glen I Andrews, MS,  of  Pfizer Inc  for review of the manuscript. Medical writing support was provided by  Joanna Bloom, PhD , of  Engage Scientific Solutions , and was funded by  Pfizer Inc. BE has served as an advisor for Bayer,  Pfizer , and  Novartis  and has received honoraria from  Bayer ,  Roche ,  Pfizer ,  Genentech ,  Novartis , and  AVEO. MDM has served as an advisor for  Pfizer ,  Novartis ,  AVEO ,  Genentech , and  Abbott  and has received research funding from  GlaxoSmithKline ,  Genentech ,  Pfizer ,  Novartis ,  Eisai ,  Bayer , and  Abbott. RJM has served as an advisor for  Pfizer ,  Genentech , and AVEO, received research funding from  Pfizer ,  Novartis , and  GlaxoSmithKline , and provided paid expert testimony for  Pfizer.
ACKNOWLEDGMENTS This work is supported by the grant  ANR-09-BLAN-0245-03  from the  Agence Nationale de la Recherche  ( ANR , project  SENZO ) and the grant  ANR-11-BSV7-010-02  from the  Agence Nationale de la Recherche  ( ANR , project  CAROLS ). We apologize to all authors whose work could not be cited due to space constraints.
Quantification of DARPP-32 lesions and formation of inclusions The loss of DARPP-32 expression was analyzed by collecting digitized images of twelve sections per animal (separated by 300 mm) with an optic bench and by quantifying the lesion areas in mm 2 with image analysis software (MCID Core 7. 0, InterFocus Imaging, GE Healthcare Niagara Inc. ). Lesion areas in each section were determined as regions poor in DARPP-32 staining relative to the surrounding tissue. The lesion volume for each animal is expressed in mm 3 , calculated as the sum of the total lesion area in mm 2 of all sections multiplied by the inter-section distance (300 mm). For estimation of the number of ubiquitinpositive HTT inclusions, 12 serial coronal sections of the striatum (separated by 300 mm) were scanned with a 10x objective using a Zeiss Axioplan2 imaging microscope equipped with an automated motorized stage and acquisition system (Mercator Pro V6.
Four weeks post-injection, the mice were sacrificed and the GFP-positive area of the striatum was grossly punched out from 1 mm-thick striatal slices. HTT mRNA was measured by RT-qPCR from these punches to measure the average silencing obtained in the striatum (i. e. infected/treated and non-infected cells) and evaluate treatment efficiency, which can be expected with this gene transfer approach. The data gathered from this in vivo experiment and those presented in Figures  3 4 are therefore complementary and provide two distinct types of information on the allele-specific silencing. In striatal punches from BACHD mice, the amounts of mutant HTT mRNA (Fig. 5A ) were lower (around 50%) in mice treated with each of the four shSNPs than shCtrl. There was no significant difference in the mRNA levels of the neuronal marker Neuronal Nuclei (NeuN) between any of the samples (Fig.
This work was supported by the  Agence Nationale de la Recherche  [ ANR-2006-MRAR-043-01 ],  FP6-NeuroNe  [LSHM-CT-2004-512039],  FP6-Clinigene [LSH-2004-1. 2. 4-3 ], the  Atomic Energy Commission ,  FP6 STEM-HD ,  DIM-STEM POLE Region Ile-de-France fellowship  to [F. B-R] and the  Swiss National Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Methods Data analysis. All statistical analyses in this study were performed using R; the specific methods and packages are detailed in the appropriate sections. Chemical screening assay. This assay was performed as previously described  14  : 240 ml of exponentially growing fmc1D yeast, adjusted to an OD 600 of 0. 2, was spread homogeneously using sterile glass beads (B3 mm diameter) on a square Petri dish (12 Â 12 cm) containing rich solid yeast-peptone-glycerol (YPG) medium (1% (w/v) BD Bacto yeast extract, 2% (w/v) BD Bacto peptone, 3% (v/v) glycerol). Sterile filters were placed on the agar surface, and 2. 5 ml of each compound from the Prestwick chemical library dissolved in DMSO was applied to the filters, with DMSO separately applied as a negative control. The plates were incubated at 37 °C for 6 days and scanned using a Snap Scan1212 (Agfa).
Only tag intensities above 8 (a cutoff set from visual inspection of the intensity distributions as separating a low-from a high-intensity subpopulation) were considered above background. The intensity distributions of the up-and down-tag populations in each microarray were shifted by a separate constant so that all intensity distributions above background have equal medians. Differential strain enrichment between DMSO and NaPT treatment was determined using limma's moderated t-test  41  treating tag as an additive covariate to control for difference in hybridization efficiency between up and down tags. P values were corrected for multiple testing with the Benjamini-Hochberg method  42  , all using the R/ Bioconductor package limma. The fold changes calculated can be found in Supplementary Data Set 2.
2c ) was assessed by western blotting using a polyclonal antibody directed against the DHFR moiety of the preprotein. Statistical analysis of import dynamics. For each substrate (Fig. 2a ,b), three independent import time series were obtained (Supplementary Data Set 3). Within each series, the band intensities corresponding to the imported, protease-resistant proteins were normalized by dividing by the intensity for DMSO at the final time point. Next, a linear model with intercept 0 was fitted by least square regression in the presence or absence of NaPT, separately. The ratio of the fitted slopes represents how the rate of import was affected by the presence of NaPT. To assess statistical significance, the asymptotic likelihood ratio test was applied, comparing the goodness of fit of this model with two slopes against a null model assuming a common slope to both conditions (presence or absence of NaPT).
The specific activity of cytochrome c oxidase was measured using a combination of 12. 5 mM ascorbate and 1. 4 mM N,N,N',N'-Tetramethyl-p-phenylenediamine dihydrochloride (TMPD) to reduce cytochrome c and in the presence of 4 mM CCCP as uncoupler. All of these measurements can be found in Supplementary Data Set 4. ATP synthesis and hydrolysis. For ATP synthesis rate measurements, mitochondria were incubated as for oxygen consumption experiments. The reaction was started by the addition of 4 mM NADH and 1 mM ADP and stopped by addition of 3. 5% perchloric acid and 12. 5 mM EDTA. The samples were then neutralized to pH 6. 5 by the addition of KOH and 0. 3 M MOPS. ATP was quantified in a luciferin/ luciferase assay (Perkin Elmer) with an LKB bioluminometer. ATP hydrolysis rates were measured at pH 8.
4 with a previously described procedure  55. Oligomycinsensitive ATP synthesis and hydrolysis rates were measured by adding oligomycin (20 mg mg À 1 of protein) to estimate the relative amounts of fully assembled F 1 F 0 -ATP synthase. These measurements can be found in Supplementary Data Set 4. Statistical analysis of bioenergetics data. For measurements of respiration rates (whole-cell and isolated mitochondria), specific cytochrome c oxidase activity, and ATP synthesis and hydrolysis, statistical significance of the differences between strains carrying the Tim21 overexpression versus empty plasmid was assessed using a one-sided t-test assuming equal variance in both the groups. Two biological replicates were measured per strain. Technical replicates, which displayed lower variance than biological replicates, were summarized by their median value before testing.
NaPT solutions in DMSO were diluted 1:1,000 in the medium. DHLA at 100 mM or the same volume of DMSO were used as positive and negative controls, respectively. After 6 days of incubation with the drugs, cell proliferation was estimated by cell counting using the Scepter (Millipore). Experiments were performed at least three times per condition and the values obtained can be found in Supplementary Data Set 1. Cell counts in each condition were expressed as a proportion of cell counts in DMSO control experiments ('Percentage of live cells (relative to untreated)'). For statistical analysis, an analysis of variance model was fitted with intercept at 100% growth, and significance of the effect for each concentration was assessed by the t-test using the R package lmtest. TIMM21 overexpression experiments were performed using JCP239 eb13.
Experiments were performed at least three times per condition and three different wells were counted for each line in each experiment; the values obtained can be found in Supplementary Data Set 1. Cell counts in each condition were expressed as a proportion of the cell counts following the first 24 h of growth in Fig. 3d. To confirm TIMM21 overexpression, 10 mg protein of cell lysates from JCP239 cells transduced with overexpression constructs were separated on SDS-PAGE and subjected to western blot analysis using anti-TIMM21 (anti-C18orf55) affinity-purified rabbit polyclonal antibodies (Proteintech Europe) and revealed by enhanced chemiluminescence.
Acknowledgements We thank  I. Perschil ,  P. Jakob ,  S. Clauder-Mu ¨nster ,  N. Ezkurdia ,  J. Kirkpatrick  and  J. Krijgsveld  for technical assistance; as well as  N. Pfanner ,  B. Guiard ,  G. Manfredi ,  C. Hughes  and  A. Chacinska  for materials and discussion. We would like to dedicate this work to the memory of  Andy Robertson , who not only provided useful comments on the manuscript, but also contributed tirelessly to professional development and diversity in the biological sciences. This study was technically supported by the  European Molecular Biology Laboratory's Electron Microscopy Core Facility  and the  Stanford Genome.
Like CERT PH domain, EDR2 PH domain preferentially binds to PI4P  [19]. Nevertheless, the mechanisms by which lipid-binding domain containing proteins regulate PCD in plants are largely unknown. Here, we analyzed sequence conservation in the VAD1 family and identified an uncharacterized conserved domain we designated as VASt (VAD1 Analog of StAR-related lipid transfer). Using sequence-and structure-based phylogenetic analyses we demonstrate that this domain is present in all major eukaryotic lineages but no molecular function has been assigned to it. VASt is related to Bet v1-like, a superfamily including lipid-and hormone-binding domains. The VASt domain will be referred to with accession number PF16016 in release 28. 0 of the Pfam database  [20]. These findings open new perspectives for the functional analysis of VASt-domain containing proteins such as A.
Proteins in the VAD1 family contain an uncharacterized conserved domain To get insights into VAD1 putative biochemical function, we analyzed protein sequence conservation among VAD1 homologs. First, to identify VAD1 homologs, we used the full length sequence of VAD1 protein in stringent phmmer searches against the Uniprot database. We identified 13 AtVAD1 homologs with e-value below 1e -100 across twelve angiosperm plant species, including monocots (Brachypodium distachyon, Musa acuminata, Oryza brachyantha, Oryza sativa, Setaria italica, Sorghum bicolor) and eudicots (Arabidopsis lyrata, Glycine max, Ricinus communis, Solanum lycopersicum, Vitis vinifera). In this stringent approach, all species showed a single VAD1 copy except G. max that had three copies. The retrieved homologs showed at least 55% identity and all contained a clearly identified GRAM domain.
The VASt domain is conserved among Eukaryotes Since none of AtVAD1 homologs had characterized biochemical functions, we extended our search of related protein domains using Hidden Markov Models (HMM) to get insights into the VASt domain putative function and evolution. For this, we built a HMM using the 13 plant homologs of AtVAD1 VASt domain. To highlight important sequence motifs in this HMM, we examined the corresponding sequence logo (Figure  2A ). Amino acids W61, R71, P116, F121 and I160 were strongly conserved in the model, suggesting an important contribution of these residues to the protein function. To identify protein domains related to the VASt domain, we searched the Uniprot database with the VASt HMM model. After three iterations of jackhammer search, we retrieved 452 hits distributed exclusively in eukaryotic proteins, including yeasts and other Fungi, Oomycetes, Mammals, and Plants (Additional file 3).
Identification of VAD1 homologs and conservation analysis in plants The VAD1 protein sequence [Uniprot: F4HVW5] was used as a query for a profile Hidden Markov Model (HMM) search with phmmer  [44]  against the Uniprot database using Blosum62 matrix. Hits with e-value 1e -100 or less were selected and manually curated resulting in the identification of 13 homologs, exclusively from plants. Sequences were aligned using MAFFT version 7  [21]  with default parameters. Amino acids conservation score, calculated according to  [45] , was plotted as a moving average using a sliding window approach with window size 10 and steps of size 1. Protein sequence features retrieved through the phmmer search were manually mapped along the alignment. AtVAD1 gene model (At1g02120. 1) was retrieved from TAIR (www.
Conservation and phylogenetic analyses of VAD1 VASt domain The precise boundaries of the newly identified VASt domain were set based on a conservation score >4 among plant AtVAD1 homologs and to include well-conserved N-terminal residues F1, D7 and P11, delimiting a 193 amino acids domain. The ungapped alignment of the 193 amino acids domain of VAD1 VASt domain with its 13 homologs was used as entry for HMM searches with jackhmmer  [44]  using Blosum90 matrix against NR database, with cut off e-value of 1e -10. The final list of proteins containing VASt domains was obtained after 3 iterations of jackhammer search using all hits from previous iteration as a seed. Sequence logo of the 452 VASt domains alignment was done with LOGOMAT-M  [46]. To built the VASt domains phylogenetic tree, we selected all hits from 17 fully sequenced species representative of all major Eukaryotic lineages as follows: Homo sapiens and Mus musculus (Mammals), Caenorhabditis elegans and Drosophila melanogaster (Other Metazoans), Arabidopsis thaliana and Oryza sativa (Tracheophyta), Physcomitrella patens (Briophyta), Saccharomyces cerevisiae, Aspergillus nidulans, Neurospora crassa and Ustilago maydis (Fungi), Phytophthora infestans and Thalassiosira pseudonana (Stramenopiles), Entamoeba histolytica and Dictyostelium discoideum (Amoebozoa), Leishmania major (Kinetoplasts), Tetrahymena thermophila (Ciliates).
Acknowledgments We thank  Penny Coggill  for comments on the manuscript and referencing of our work in the Pfam database. This work has been performed in the LIPM, part of the  "Laboratoire d'Excellence" (LABEX)  entitled  TULIP  ( ANR-10-LABX-41 ). SR is supported by a  Marie Curie   CIG grant  (contract  334036 ) and a  Starting Grant  from the  European Research Council  (ERC, contract  336808 ).
1_FLAG-MCS(MB) expression vector. MYC-tagged versions of HCV proteins (MYC-Core, MYC-NS3/4A, MYC-NS2, and MYC-NS5B) were cloned into pcDNA3. 1_ MYC-MCS expression vector using EcoRV/HindIII enzymes. STAT1 and viral ORFs were amplified via PCR from the human fetal brain cDNA library (OriGene, Lake Forest, CA) and from JFH-1 (Core, NS2) or HCV 1b genotype (Con1 isolate, GenBank TM accession number AJ238799) by using KOD polymerase (EMD Millipore, Billerica, MA). STAT1, NS3/4A, and NS3/4A S139A constructs were cloned using a recombinational cloning system (Gateway, Invitrogen) into pDONR207 or pDONR223. After validation by sequencing, these ORFs were transferred by means of Gateway recombinational cloning from pDONR207 or pDONR223 to the expression vector pCI-neo-3xFLAG (3xFLAG fusion, a gift from Y.
Jacob). The mutant NS3/4A S139A was generated via site-directed mutagenesis using the Quickchange kit (Stratagene, La Jolla, CA, USA). The pISRE-Fluc cis-reporter plasmid (Stratagene) containing the interferonstimulated response element (ISRE) upstream of the firefly luciferase (Fluc) gene was used in combination with the pRL-SV40 plasmid (Promega, Madison, WI). pRL-SV40 contains the SV40 enhancer upstream of the Renilla luciferase (Rluc) gene and was used as an internal control reporter. Antibodies to FLAG and MTHFD1 were purchased from Sigma (St-Louis, MO, USA, F3165 and HPA001290); FKBP5, EXOC7, MYC, STAT1, and XPO1 were purchased from Santa Cruz Biotechnology (Dallas, TX sc-13983, sc-365825, sc-789, sc-346, and sc-74454); EGLN1, KPNB1, and NS3 were purchased from Abcam (Toronto, Ontario, Canada, ab4561, ab7811, and ab13830); DDX3X and ILF2 were purchased from Bethyl Laboratories (Montgomery, TX A-300 -474A and A303-147A); Core was purchased from ABR-Affinity Bioreagents (Golden, CO, USA, MA1-080); HSP90 was purchased from Cell Signaling Technology, Inc, (Danvers, MA 4874); HSD17B12 was purchased from NovusBiologicals (Littleton, CO NBPI-81567); and ACTIN was purchased from Chemicon International (Billerica, MA MAB1501R).
Mass calibration used a lock mass from ambient air (protonated (Si(CH 3 ) 2 O) 6 ; m/z 445. 120029) and provided mass accuracy within 15 ppm for precursor ion mass measurements. The dynamic exclusion of previously acquired precursor ions was enabled (repeat count 1, repeat duration of 15 s, exclusion duration of 15 s). MS/MS spectra were acquired in collision-induced dissociation mode using an isolation window of 2 Da, and precursor ions were sequentially isolated and accumulated to a target value of 10,000 with a maximum injection time of 100 ms. Protein Identification and Quantitative Analysis-The centroided MS/MS data were merged into a single peak-list file (Distiller, V2. 4. 2. 0) and searched with the Mascot search engine (version 2. 2, Matrix Science, London, UK) against a concatenated forward and reversed human IPI database (IPI human v3.
54) containing 150,858 forward protein sequences. Mascot was searched with a parent ion tolerance of 10 ppm, a fragment ion mass tolerance of 0. 5 Da, and one miscleavage. Carbamidomethylation of cysteine; oxidation of methionine; deamidation; amd phosphorylation of serine, threonine, and tyrosine residues were specified as variable modifications. The false discovery rate was calculated as the percentage of positive hits in the decoy database versus the target database, and a false discovery rate of 1% was considered for both proteins and peptides. Protein identification is reported only for those assigned with a minimum of two peptides per protein. The data were processed using the Mascot 2. 2 (Matrix Science) search engine. Label-free quantitative proteomics was used to profile protein abundance across sample sets, as reported previously  (22, 23).
Briefly, Mascot peptide identifications were matched to ion intensity (MS peak intensity, minimum threshold: 10,000 counts) extracted from the aligned MS raw data files (tolerances set to m/z ϭ 15 ppm and retention time ϭ 1 min). For each LC-MS run, we normalized peptide ratios so that the median of their logarithms was zero, to account for unequal protein amounts across conditions and replicates. Intensities were summed across fractions for peptides of identical sequences, and protein ratios were calculated as the median of all peptide ratios, while minimizing the effect of outliers. Only proteins defined by two or more peptide quantification events and identified in more than two independent experiments were considered. For each identified host protein, the fold enrichment change was determined from the ratio of the proportion for one target viral protein to the average proportion for other viral baits.
02% sodium azide, and 10% goat serum in PBS and incubated with the following primary antibodies diluted in 5% BSA-0. 02% sodium azide: STAT1 (1:200), NS3 (1:100), and Core (1:200). Cells were then probed with Alexa Fluor-488 and -594 secondary antibodies (Invitrogen) diluted at 1:1,000 in 5% BSA-0. 02% sodium azide, washed, and incubated with Hoechst dye (Invitrogen) at 1 g/ml in PBS. Slides were mounted using DABCO (Sigma-Aldrich) as an antifading agent. Cells were examined via laser-scanning confocal microscopy (Axiovert 100 M LSM510, Zeiss, Toronto, ON, Canada), and images were processed using LSM Image Browser (Zeiss). Functional Enrichment Analysis-The DAVID database was used for functional annotation  (25, 26). The DAVID functional annotation chart tool was used to perform Gene Ontology biological process and InterPro protein domain analysis.
Human Interactome-Human-human protein-protein interactions were downloaded from iRefIndex 9. 0  (28)  and filtered in order to reconstruct a good-quality human interactome. Network Representation-Protein interaction networks were visualized with Cytoscape  (29). Topological Analysis-The R statistical environment was used to perform statistical analysis, and the igraph R package was used to compute network topology measures  (27, 30). Network Interconnectivity Analysis-Statistical significance for the interconnectivity of targeted proteins was assessed via a random resampling testing procedure (n ϭ 10,000 permutations) using R  (27). For each permutation, we randomly extracted from the human inter-actome a number of proteins equivalent to the number of targeted proteins, and the number of shared interactions was determined.
Following individual protein expression, cellular lysates were subjected to IP using a resin coupled to anti-FLAG antibodies. As illustrated in a schematic representation in Fig. 1A , viral protein-containing complexes were isolated following several washing steps and elution with a high-pH buffer. The expression of viral tagged proteins was first confirmed in cell lysates using an anti-FLAG antibody at the expected molecular weight (Fig. 1B ). Following elution with ammonium hydroxide, proteins were subjected to silver staining (Fig. 1C ) to allow visualization of the immunopurification of viral and host protein complexes. The silver-staining analyses of the corresponding protein complexes showed protein bands at the expected molecular weights for 3XFLAG-Core, 1XFLAG-NS2, 3XFLAG-NS3, NS4B-3XFLAG, 3XFLAG-NS5A, and 3XFLAG-NS5B, which were confirmed by Western blot analysis (data not shown).