dataesr/scientific-paragraphs-categorization huggingface dataset:
Given the extremely short half-life of superoxide that dismutes quickly into H 2 O 2 , the site of its production determines its biological impact. The effect of HO-1 on Nox4 subcellular localization was assessed in the C-20/A4 chondrocytes. Consistent with data shown in HEK293 cells  [27] , transfection of Nox4GFP fusion gene led to endoplasmic reticulum (ER) and plasma membrane localization of the fusion protein in C-20/A4 chondrocytes by confocal microscopy (Figure  4C ). Plasma membrane localization of Nox4GFP was confirmed by using 8E9 antibody that recognizes an extracellular epitope of Nox4 protein  [27]  and that was added before cell permeabilization (Figure  4C , right panel). After 48h incubation with 25 mM CoPP-IX, a clear induced expression of HO-1 was evidenced in C-20/A4 chondrocytes that was colocalized with Nox4GFP fusion protein (Figure  4C ).
In the absence of a known molar extinction coefficient at 426 nm (e 426 ) value for Nox4, we estimated the amount of Nox4/p22 phox dimer (cyt b) of Nox4 using the e 426 value of the cytochrome b 558. 22 pmol of cyt b per mg protein extract was initially found in WT cells and was increased up to ,70 pmol cyt b per mg of proteins in Nox4 transfected cells (Figure  5B  and 5C ). In addition, overexpression of HO-1 slightly decreased the cyt b amount (from 70 to 50 pmol/mg of protein). Conversely, no spectra were observed upon SA treatment (Figure  5B  and 5C) which is correlated to an absence of ROS production under those conditions (unpublished data). Taken together, these data show that the presence of heme molecules is necessary for the Nox4/p22 phox heterodimerization. However, despite a significant inhibitory effect of HO-1 on Nox4 activity, the maturation of the Nox4/p22 phox heterodimer appears not significantly affected by HO-1.
(F) Total Nox4 expression was assessed by Western Blot with 8E9 antibody on 150 mg of protein from a 1% (v/v) Triton X-100 extract. Actin protein served as loading control. Results are representative of three independent experiments. doi:10. 1371/journal. pone. 0066478. g004 C-20/A4 chondrocytes. Similar data were previously reported in which the Heme oxygenase isoform HO-2 was shown to decrease Nox4 activity in microvasculature  [41]. To our knowledge, it is the first time that HO-1 is reported as a regulator of Nox4 activity. We studied the HO-1 capability to inhibit Nox4 activity in the IL-1b pathway leading to events that participate to the progression of OA. We showed that inducing or overexpressing HO-1 proteins strongly reduces the matrix metalloproteinase MMP-1 secretion and chondrocyte cell death, therefore acting potentially to prevent cartilage degradation.
Acknowledgments The authors thank  Dr. Alexeı ¨Grichine  for his support in confocal microscopy experiments,  Pr. Karl-Heinz Krause  for kindly providing Nox4 HEK293 T-REx TM Nox4 cells and  Pr. Mary Goldring  for the generous gift of the C-20/A4 cell line. The authors thank also  Pr Xavier Ronot ,  Pr Bertrand Toussaint ,  Pr. Francois Rannou ,  Dr. Corinne Dupuy ,  Dr Marie He  ´lene Paclet and  Dr. Patricia Sansilvestri-Morel  for helpful discussion.
Patients Patients included in this study were men and women between 39 and 79 years old, treated with bevacizumab for brain-or breast-cancer (n = 17). For the pharmacodynamic study, patients with breast cancer were excluded (n = 3) and clinical data was confidential for one brain tumor patient. Analysis was therefore performed on 13 glioma patients. All patients provided written informed consent for participation in this study (CBR. GSI. ENR. 003 V4, Grenoble University Hospital). The informed consent document and the study as a whole were approved by the hospital ethics committee (COMITE DE PROTECTION DES PERSONNES).
Bevacizumab Assay Procedure Bevacizumab was assayed by suspension array technology. Briefly, 5,000 VEGF-beads were deposited per well in Bio-Plex pro flat-bottom plates (Bio-Rad). Beads were washed twice with 75 ml of wash buffer (PBS-0. 05%Tween-20, pH 7. 4) on a wash station (Bio-Plex Pro II; Bio-Rad). Beads were then incubated with appropriately PBS-diluted serum for 30 min under slow agitation at RT. Beads were then washed three times on a wash station and incubated with phycoerythrin-conjugated (PE) anti-human IgG (Sigma-Aldrich) for 30 min under slow agitation at RT. Beads were once again washed three times on a wash station. PE binding was measured on a Bio-Plex 220 (Bio-Rad) and analyzed using Bio-Plex Manager 5. 0 software (Bio-Rad). Bevacizumab used as standard was purchased from Roche-Genentech, and stored at 4uC.
In the Roche-Genentech study, slight differences in clearance were noted as a function of these factors  [13]. However, other factors should also be studied. For example, recycling of the neonatal Fc receptor (FcRn) is a common rescue pathway for monoclonal antibodies that leads to a long half-life  [18]. In 2008, Lu et al. [6]  described a mathematical model to address the variability of bevacizumab PK based on five parameters (body weight, gender, albumin, alkaline phosphatase, and SGOT). They found weight and gender to be the covariates with the greatest influence on bevacizumab PK  [6]. Nonetheless, these mathematical models have limits as they cannot take all possible covariates into account. For example, serum VEGF concentrations could affect bevacizumab clearance. To check this, we measured serum VEGF levels, but no correlation with PK was found (data not shown).
cRNA yield and Cy3 and efficiency of Cy5 incorporation into the cRNA were determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Palo Alto, CA). For each sample, a total of 825 ng of cRNA was fragmented using the Agilent Gene Expression Hybridization Kit, and then Cy3 cRNA from a A549 pUse sample and Cy5 cRNA from a A549 Bcl-2 sample were hybridized together on one array. The hybridization was performed in a hybridization oven (Robbins Scientific, Sunnybrook, CA) at 65°C at 10 rpm for 17 hours. Slides were scanned using an Agilent DNA microarray scanner at 5-μm resolution and Cy3/Cy5 intensity data were extracted using Agilent's Feature Extraction software including quality control based on internal controls. Transcriptome analysis was performed using Agilent Genespring GX11 Software.
Statistical Analysis Each experiment was performed at least in triplicate. Data were expressed as means ± SEM. Statistical significance was tested using Student's t test for comparisons between means. A significant difference between two conditions was recorded for P <. 05 or P <. 01. Kaplan-Meier survival curves were plotted using GraphPad Prism 4 (GraphPad Software, San Diego, CA) and analyzed using the logrank (Mantel-Cox) test.
MTAs Exert Higher Antiproliferative Effects in Bcl-2-Overexpressing Lung and Breast Carcinoma Cells We selected three tumor cell lines for their different tissue origins (lung, breast, and neuroblastoma). From these different cell lines, we generated various cellular models overexpressing Bcl-2 to investigate the role of Bcl-2 in the anticancer activity of MTAs. Protein expression levels were measured in whole cells to verify transfection success (Figure  1A ) and in isolated mitochondria to ensure Bcl-2 functional localization (Figure  1B  for A549; data not shown for MCF-7 and SHEP). We verified that with no treatment, doubling times of Bcl-2-overexpressing cells were not modified compared with pUsetransfected cells (Figure  W1 ). The impact of Bcl-2 overexpression on MTA activities was first evaluated with cell proliferation assays in lung carcinoma A549 cells.
We confirm here its implication by using a bim promoter plasmid carrying two mutated FoxO binding sites. Inhibition of SGK has been linked to a decrease in FoxO3a phosphorylation, which results in its activation through its relocalization from cytosol to nucleus  [28, 29]. Our data support the triggering of such a signaling pathway in Bcl-2overexpressing A549 cells, since we recorded a decrease in SGK mRNA levels, visualized FoxO3a nuclear accumulation, and measured the up-regulation of its well-known target MnSOD. Furthermore, we previously showed that Bcl-2 inhibits the transcriptional activity of p53  [45, 46] , which could lead to a decrease in SGK expression  [47, 48]. In response to Bcl-2 overexpression, we thus hypothesize that Bim is upregulated as a result of the increase in FoxO3a activity, allowed by p53 and SGK inhibition.
Acknowledgments The authors thank  C. Ferlini  ( Rome, Italy ) for the Bcl-2 pUse and the empty plasmids,  A. Vasquez  ( Paris, France ) for the pcDNA 6 and pcDNA 6 -Bim, and  J. Ham  ( London, United Kingdom ) for the Bim-pGL3 and Bim-luc dm. The authors also thank  Charles Prevôt  for technical assistance in flow cytometry analysis,  Elise Termine  ( Genomic Platform IFR125, Marseille, France ) for microarray analysis, and  Alain Desobry  for technical assistance.
Acknowledgements This work was supported by the  Cancé ropôle du Grand Ouest, -Valorisation des produits de la mer ,  Glycoconjugués et Cancer‖, France (Head: Philippe Bougnoux, Inserm, Université de Tours, France ). We wish to thank  Haidar El-Hali ,  Oceanium, Dakar, Senegal , for help in harvesting Axinyssa djiferi. Pierre Guenot ,  Philippe Jehan  and  Fabian Lambert ,  Centre Ré gional de Mesures Physiques de l'Ouest, CNRS , and  Université de Rennes-1, France , for ESI-MS experiments. The first biological evaluations were performed under the auspices of the  French National Scientific Research Centre  within a network called Research Group on Natural Products managed by  Thierry Sé  venet ( GDR G1206 ,  CNRS, ICSN, Gif s/Yvette, France ). We also wish to thank  Mr. Benoî t Serive ,  University of Nantes , for his help in lipid fractionations and  Vony Rabesaotra ,  Faculty of Pharmacy, Nantes , for GC-MS experiments.
Introduction Macromolecular crystals typically contain a substantial amount of disordered solvent, ranging from approximately 20 to 90% of the crystal volume, with a mean of 55%, in the Protein Data Bank (PDB;  Bernstein et al. , 1977; Berman et al. , 2000). Anisotropy in the diffracted intensities is another common feature of macromolecular crystals that arises from various sources including crystal lattice vibrations  (Shakked, 1983; Sheriff & Hendrickson, 1987). When modelling diffracted intensities, for example in structure refinement or automated model building, it is therefore critical to account for these two phenomena (see, for example,  Jiang & Bru ¨nger, 1994; Urzhumtsev & Podjarny, 1995; Kostrewa, 1997; Badger, 1997; Urzhumtsev, 2000; Fokine & Urzhumtsev, 2002a; Fenn et al. , 2010).
An alternative approach which divides the resolution range uniformly on a logarithmic scale ln(d)  (Urzhumtsev et al. , 2009)  efficiently solves this problem. The flowchart of the algorithm is shown in Fig. 2. This scheme allows the higher resolution bins to contain more reflections than the lower resolution bins and more detailed binning at low resolution without increasing the total number of bins. An additional reason for using logarithmic binning is that the dependence of the scales on resolution is approximately exponential (see previous sections), which makes the variation of scale factors more uniform between bins when a logarithmic binning algorithm is used. Table  1  compares binning performed uniformly in d À3 and in ln(d) spacing for three data sets (PDB entries 3hay, 1kwn and 3gk8).
Systematic tests We evaluated the performance of the new scaling protocol by applying it to approximately 40 000 data sets selected from the PDB. The structures were selected by evaluating all PDB entries using phenix. model_vs_data (Afonine et al. , 2010)  and excluding all entries for which the recalculated R work was greater than the published value by five percentage points. To score the test results three crystallographic R factors (46) were computed using all reflections, using only low-resolution reflections and using only high-resolution reflections. Lowresolution reflections were selected using the condition d min > 8 A ˚but selecting at least the 500 lowest resolution reflections. High-resolution reflections were taken from the highest resolution bin. Each of the three anisotropic scaling methods (poly, exp anal and exp min ) was tested independently within each run.
Note the difference in the optimal U cryst values and the corresponding R factors. The parameterization of the total model structure factor (1) does not make any assumption about the shape of k mask ; for example, it does not assume it to be exponential (10). This provides an opportunity to explore the behavior of k mask as a function of resolution and compare it with k mask obtained via (10). Fig. 6  illustrates the differences between the two methods of determining k mask for six representative PDB entries selected from approximately 40 000 entries after inspection of the k mask values. We observe that the plots of the values obtained using our new approach are in general significantly different from the exponential function. This observation is in line with Fig. 1  of  Urzhumtsev & Podjarny (1995).
The new method is implemented in the cctbx project (Grosse-  Kunstleve et al. , 2002)  and is used in a number of PHENIX applications since v. 1. 8 of the software, most notably phenix. refine (Afonine et al. , 2005b (Afonine et al. , , 2012)) , phenix. maps and phenix. model_vs_data (Afonine et al. , 2010). The cctbx project is available at http://cctbx. sourceforge. net under an opensource license. The PHENIX software is available at http:// www. phenix-online. org. All results presented are based on PHENIX v. 1. 8. 1.
Within Tetractinellida p , we did not recover monophyletic Astrophorina or Spirophorina. Our results also reaffirmed the monophyly of order Poecilosclerida (excluding Desmacellidae and Raspailiidae), and polyphyly of Hadromerida and Halichondrida. Conclusions/Significance: These results, using an independent nuclear gene set, confirmed many hypotheses based on ribosomal and/or mitochondrial genes, and they also identified clades with low statistical support or clades that conflicted with traditional morphological classification. Our results will serve as a basis for future exploration of these outstanding questions using more taxon-and gene-rich datasets.
portol. org) employs a variety of tools to integrate morphological and molecular data and to expand the diversity of sponge taxa used to elucidate all levels of sponge phylogeny. In this study, we report findings based on a significant expansion (38 new samples from 38 species representing 30 families, including 21 families newly sampled) of the nuclear housekeeping gene dataset first developed for metazoan-wide phylogenetic and molecular dating analyses  [45, 46]  and later applied by Sperling et al. [18, 44]  to sponges, with a thorough taxonomic vetting process and a slightly modified phylogenetic analysis focused on relationships within Demospongiae p.
Results Extraction of high quality RNA for subsequent cDNA synthesis and cloning was a significant hurdle, curtailing use of some samples (e. g. , lithistids), even though a large number of archived specimens were available for potential study  [47]. Several hundred cDNAs were cloned and sequenced, but only 159 usable sequences were generated due to the amplification of non-sponge contaminants (Tables  1 2 ). We evaluated single gene phylogenies (ALD, ATPB, etc. ) including all the members of each gene family that could be identified in GenBank (via reciprocal blasting) to identify and remove potential paralogs. Our dataset for phylogenetic analysis contains 2,033 amino acid characters and a total of 68 sponge species representing 48 of 137 accepted and recently proposed families of Porifera  [38, 40, 48] , including 51 species from 37 of 91 families recognized for Demospongiae (Table  1 ).
The most appropriate models of amino acid evolution, as determined by ProtTest  [49]  for the various datasets (i. e. , all genes, each individual gene, etc. ), nearly always involved some variant of the LG matrix  [50]  (Table  3 ). Maximum likelihood mapping, performed for each gene under the best fitting model, among those implemented in Treepuzzle  [51] , showed that each of the seven considered genes convey enough phylogenetic signal to be considered potentially useful phylogenetic markers to resolve the relationships within Demospongiae (Figs. S1, S2, S3, S4, S5, S6, S7). Bayesian cross-validation  [52]  analyses showed that the CAT based models (CAT and CAT-GTR) fit our dataset significantly better than any empirical site-homogeneous time reversible model tested (WAG+G, and LG+G). Cross-validation also showed that the CAT-based models fit the data better than the more complex site-homogeneous time reversible model: the mechanistic amino acid-GTR (Table  4 ) model.
Higher NA  (1)  Formerly identified as Darwinella muelleri (Darwinellidae) in  Sperling et al. (2007) ; specimen from the Gulf of Mexico. ( ) 2 Formerly identified as Dysidea camera in  Sperling et al. (2007) , and as Dysidea sp. in GenBank. ( ) 3 Formerly identified as Chondrilla sp. in  Sperling et al. (2007)  and as Chondrilla nucula in GenBank. ( ) 4 Formerly labeled as Clypeatula cooperensis in  Sperling et al. (2004)  and Ephydatia cooperensis in GenBank, but synonomized with Ephydatia fluviatilis in WPD. (      ( ) 12 Formerly identified as Damiria sp. in  Sperling et al. (2009)  and in GenBank. ( ) 13 Formerly identified as Tethya aurantia in  Sperling et al. (2009) , and as Tethya actinia in GenBank. ( ) 14 Formerly identified as Spirastrella coccinea in  Sperling et al. (2009)  and in GenBank.
( ) 15 Formerly labeled as Microciona prolifera in  Peterson & Butterfield (2005)  and in GenBank, and as Clathria (Microciona) prolifera in Sperling et al. (  2009 ). ( ) 16 Derived from genomic traces, as reported in  Sperling et al. (2007). ( ) 17 Derived from genomic traces, as reported in Sperling et al. (  2010 ). ( ) 18 Derived from genomic traces, as reported in  Sperling et al. (2009). doi:10. 1371/journal. pone. 0050437. t001 That said, just five of the resolved nodes in the Bayesian analysis contradict those in the ML-based topology and none of these have pp values. 0. 90. The partitioned ML analysis of the combined data had the same topology as that found when assuming a single model of amino acid evolution (LG+F+G). Additionally, no major differences were found when comparing a Bayesian analysis performed under LG+G, the ML analysis performed using LG+F+G, and the ML analysis performed using multiple partitions.
We used this topology as the reference point for comparing the different analyses (Fig. 1 ). The Bayesian topology (Fig. 2 ) is highly consistent with the ML-based topology (Table  5 ). Each of the single-gene ML topologies (Figs. S8, S9, S10, S11, S12, S13, S14) differs from that derived from the combined dataset. An ordered ranking of how well the single-gene topologies match our overall hypothesis, based on nodal difference is: PFK, TPI, ALD, MAT, ATPB, CAT and EF1A (Table  5 ). This performance is also reflected in a tabulation of whether notable clades were recovered in the single-gene topologies (Table  6 ), where ATPB, CAT and EF1A recovers less than half of a set of reference clades in the topology based on the combined data. ML analyses serially excluding CAT, EF1A, and ATPB resulted in topologies (Figs.
We first built trees using differently fitting models (WAG, LG, GTR, CAT, and CAT-GTR) and compared their results. This analysis indicated an important area of disagreement with reference to the relationships between Keratosa p and Myxospongiae p (see discussion). We performed a posterior predictive analysis to identify compositionally heterogeneous taxa. This analysis indicated that many taxa in the dataset are, indeed, compositionally heterogeneous (Table  S1 ). The 6-categories Dayhoff recoding strategy is commonly used to ease compositional heterogeneity. We recoded our dataset using the 6-categories Dayhoff strategy and performed   a posterior predictive analysis and found that the Dayhoff recoding eliminated almost all heterogeneity from the data (Table  S2 ). CAT-GTR analyses of the Dayhoff recoded dataset found a tree (Fig.
Sponge Classes Analyses of the seven nuclear housekeeping gene dataset provide strong support for each of the four major clades of sponges assigned the rank of class (Calcarea, Demospongiae, Hexactinellida, and Homoscleromorpha). Because we did not include non-metazoan outgroups our results cannot be used to assess sponge monophyly. Concerning the relationships among the four sponge classes, support is generally poor. Our tree does not recover Silicea p (Demospongiae p +Hexactinellida p ), which has been supported in a great deal of other works based on disparate datasets  [4, 18, 19, 28, 54] , but instead places Calcispongiae p with Hexactinellida p (Figs. 1 2 ), most likely erroneously with low support (bs = 74%; pp = 0. 68). Relationships within Calcispongiae p and Hexactinellida p are consistent with previous analyses  [54] [55] [56].
To be especially useful for rank-based taxonomy and nomenclature, type species within genera and type genera within families (e. g. , our sampling of Spongia officinalis, Halisarca dujardini, and Desmacella pumilio) should be targeted whenever possible. Also, to the extent possible, type species should be collected from their respective type localities for maximum taxonomic and nomenclatural utility. For phylogenetic nomenclature, 'specifiers' (i. e. , species, specimens or apomorphies used in PhyloCode definitions) should be targeted. Of course, when species are used as specifiers (which has so far usually been the case for poriferan names), their name-bearing type specimens are de facto specifiers (PhyloCode, Note 13. 2. 2. ). Nuclear housekeeping gene data strongly support an as yet unnamed clade containing the groups of demosponges with silicamineralized skeletons: Democlavia p , Haploscleromorpha p , and Spongillida p (Figs.
1 2 ), in accordance with other analyses of ribosomal genes  [17] , complete mitochondrial genomes  [43] , and a smaller dataset of nuclear housekeeping genes  [18]. Our ML and Bayesian analyses provide equivocal results concerning the phylogenetic relationships of Keratosa p and Myxospongiae p. A sister group relationship between Keratosa p and Myxospongiae p has been suggested, with only modest support, based on analyses of 18S rRNA genes  [17]  and complete mitochondrial genomes  [43]  but has also been contradicted by earlier Bayesian analyses of nuclear housekeeping genes  [4, 18, 44]. Our ML topology (Fig. 1 ) shows Keratosa p and Myxospongiae p [which both lack mineralized skeletons (with the exception of siliceous microscleres in Chondrilla within Myxospongiae p : Chondrosida)] as a clade that is sister to the mineralized sponges.
Keratosa p This clade is composed of members of the demosponge orders Dictyoceratida and Dendroceratida. Our sampling includes members of five of the six families: Dysideidae, Irciniidae, Spongiidae and Thorectidae in the former, Dictyodendrillidae in the latter. Ribosomal data  [17]  indicate that Dendroceratida is monophyletic, but our results rely on a single genus (Igernella) so we cannot support or refute that result. The nuclear housekeeping gene data also fail to provide support for the monophyly of Dictyoceratida, a result that has also been obtained through the analysis of ribosomal data  [35, 57]. We have conflicting results concerning Dictyoceratida, with our ML-topology (Fig. 1 ) suggesting that dendroceratids are derived from within a paraphyletic Dictyoceratida and the Bayesian tree having a poorly supported monophyletic Dictyoceratida.