dataesr/scientific-paragraphs-categorization huggingface dataset:
Data Analysis Experimental data were routinely expressed as means ± SEM from at least three independent experiments. Statistical analysis of quantitative data was performed by Student's t-test, which is applicable to small size samples  [44] , or analysis of variance (ANOVA) followed by Dunnett's or Tukey's post-hoc test, using the GraphPad Prism software (GraphPad software, La Jolla, CA, USA). The criterion of significance was p < 0. 05.
Expression of Drug Transporter mRNAs in HuH-7 Cells Expression levels of hepatic drug transporter mRNAs were determined in HuH-7 cells and compared to those found in freshly isolated human hepatocytes. As shown in Figure  1a , SLC transporters were very poorly expressed in HuH-7 cells comparatively to hepatocytes. Indeed, HuH-7 cells failed to express the sinusoidal uptake transporters NTCP, OATP1B1 (SLCO1B1), OAT2 (SLC22A7) and OCT1, which, by contrast, were highly expressed in human hepatocytes, in agreement with previous data  [45]. OATP1A2 (SLCO1A2) mRNAs were also not detected in HuH-7 cells, knowing, however, that they were similarly not present in human hepatocytes (Figure  1a ). With respect to the sinusoidal transporters OATP1B3 and OATP2B1 (SLCO2B1) and to the canalicular SLC transporter multidrug and toxin extrusion protein 1 (MATE) 1 (SLC47A1), they were expressed in HuH-7 cells, but at significantly lower levels than those found in hepatocytes.
Supplementary Materials: Copies of the 1 H-, 13 C-NMR for compounds 3, 4, and 7, and MS spectra of 7, as well as the molfile for 7, can be found at http://www. mdpi. com/1422-8599/2016/4/M909/s1. Acknowledgments: One of us (C. D. D. ) wishes to thank the "Fondation Benianh International" and the "Ministère de l'Enseignement Supérieur et de la Recherche Scientifique de Côte d'Ivoire" for grants. Financial support of this program carried out under the French National Cancer Institute "Cancéropôle Grand Ouest" by contract "Ion Channel-Network CGO 2012", is gratefully acknowledged. The authors are grateful to the assistance.
Dissociation constant (K D ) was estimated using the Kaleidagraph software package, by plotting the absorbance intensities at 413 and 433 nm against the inhibitor concentration. Since the K D values of the azole drugs were not markedly higher then the OleP concentration used, data were fitted with the quadratic equation: ΔAUobs ¼ ΔAUmax 2 E 0 ½ þ I 0 ½ þ K D -E 0 ½ þ I 0 ½ þ K D ð Þ 2 -4 E 0 ½ I 0 ½ ð Þ 1 2 ð1Þ where ΔAU obs is the absorbance difference, ΔAU max is the maximum absorbance difference extrapolated to infinite inhibitor concentration, and [E 0 ] and [I 0 ] are the enzyme and the inhibitor analytical concentrations, respectively. Data were also globally fitted with the programme Prism (Graphpad, Fig. 2 ).
Inhibitor binding kinetics The time evolution of the OleP interaction with CTZ and KC was followed by monitoring the absorbance increase at 433 nm on a SX18-MV stopped-flow apparatus (Applied Photophysics, Leatherhead, UK) using symmetric mixing mode. OleP, at a final constant concentration of 2. 5 μM, was mixed with CTZ and KC diluted to the desired final concentration, ranging from 2. 5 to 20 μM. Measurements were carried out in 50 mM Hepes, 0. 2 M NaCl and 1% DMSO, pH 7. 5, at 288 K. Control experiments indicated that the presence of 1% DMSO had no significant effects on the kinetic traces. The kinetic curves correspond to the average of at least three experiments, and they were satisfactorily fitted by a single exponential function using both the software provided by Applied Photophysics and the Kaleidagraph software package to obtain the observed rate constant, k obs.
Sample preparation and X-ray data collection Crystallization of unliganded OleP and in complex with inhibitors was carried out using the hanging-drop vapour-diffusion methods at 293 K, and 19 mg/ml of the purified protein in 20 mM Tris-HCl and 200 mM NaCl, pH 8. 0. Crystals were obtained for the ligand-free OleP and by co-crystallization of the CTZ-bound form, using saturating concentration of inhibitor (2 mM), but diffraction at high resolution was observed only for crystals of the CTZ-OleP complex, grown in 1. 6 M Ammonium Sulphate, 0. 1 M Hepes Sodium Salt pH 7. 5, 2% (w/v) PEG 1000 (AmSO 4 Suite n°83, Qiagen). 20% PEG 200 was used as cryoprotectant. Diffraction data of CTZ-OleP crystal were collected at 100 K at ELETTRA (Trieste, Italy), beamline 5. 2R, XRD1, using PILATUS detector. Data was indexed, scaled and integrated using the XDS package  [15].
Structure determination and refinement The determination of the initial crystallographic phases was performed by molecular replacement using MolRep  [16]  from the CCP4 suite  [17]. The OleP amino acid sequence was submitted to BLAST search algorithm, to identify in the PDB database the best model for Molecular Replacement. Given the 48% sequence identity, the coordinates of the homologous P450 MycG (PDB ID: 2Y46) have been used as search model. The residues of the starting molecular model were renumbered and renamed using Chainsaw  [17]. Iterative manual and automated refinement of the structure was carried out using Refmac5 in CCP4  [18]. Manual adjustment was performed using COOT with the Fo-Fc map contoured at 1σ and the 2Fo-Fc map at 3σ. Waters were added into the Fo-Fc map, contoured at 3σ, using the Find Waters tool of COOT.
5% of reflections was excluded from refinement and used for the R free calculation  [19]. Secondary structure assignment was performed using the Kabsch and Sander algorithm, and the geometrical quality of final models was assessed using Procheck  [20]. Ramachandran statistics indicate no outliers for the CTZ-OleP structure. The first 13 N-terminal residues in CTZ-OleP are missing in the model due to insufficient electron density. Final crystallographic statistics are shown in Table  1 , data extend to 2. 65 Å resolution with I/σ of 2. 1 in the last resolution shell (2. 81-2. 65 Å). All figures were produced with PyMOL (http://pymol. sourceforge. net). The atomic coordinates and structure factors for CTZ-OleP structure have been deposited in the RCSB Protein Data Bank (accession code 4XE3).
5 in the presence of 0. 2 M NaCl at 298 K (Fig. 2 ). In order to assess the binding mechanism of OleP to both CTZ and KC we followed the binding kinetics of OleP to both inhibitors at 288 K (at 298 K the reaction was too fast for full detection, data not shown) and association (k on ) and dissociation (k off ) rate constants were determined by using a stopped-flow apparatus. Quasi-pseudo-first-order binding experiments were performed by mixing a constant concentration of OleP (2. 5 μM) with increasing concentration of CTZ and KC, ranging from 2. 5 to 20 μM. Under all conditions, the kinetics of formation of the complex with the inhibitor was fitted by a mono-exponential function, indicating a one-step mechanism, without evidence for the accumulation of detectable intermediate (Fig. 3 , Inset).
Comparison with other macrolide epoxigenases OleP represents the fifth macrolide epoxigenase from actinomycete organism that has been structurally characterized. The closest structural homologous (48% of identity, Figure  S3 ) is the P450 MycG (Dali Database  [31] ), whose coordinates have been used as molecular replacement model for OleP (see Section n. 2, PDB ID: 2Y46). MycG is a multifunctional monooxygenase from Micromonospora griseorubida involved in mycinamicin biosynthesis  [12]. It performs sequential reactions of hydroxylation and epoxidation on two sites, a tertiary allylic C-H bond and an olefin, of two metabolic intermediates of the mycinamicin pathway, respectively mycinamicin IV (M-IV) and V (M-V) (Fig. 1C ,  [12] ). Both M-IV and M-V are considerably different from the OleP proposed substrates, 8.
Acknowledgments We thank  Biotica Technology , Ltd. , UK and  Steven G. Kendrew  for the kind gift of the plasmid pSGOleP, a pUC18 cloning vector containing the nucleotide sequence coding for OleP. We are grateful to synchrotron ELETTRA (beamline 5. 2R ,  XRD1 ,  Trieste, Italy)  and to  Maurizio Polentarutti  for providing assistance in using beamline. The research leading to these results has received funding from the  European Commission Seventh Framework Programme -Research Infrastructure Action  ( FP7/2007-2013)  under grant agreement n°2 83570. This work was also supported by the " Progetto Premiale 2012: Avanzati sistemi biosensoristici per la diagnosi e il follow up della malattia celiaca " from the  CNR  and from the following grant and institution:  Sapienza University Grant   2014prot.
Acknowledgements. This study was funded by  EDF and Andra. We would like to thank  Sabine Maestri  for her great help with the isotopic measurements. We thank the following research teams for having kindly put at our disposition their long-term experiments and associated data:  INRA-Agro-Paristech ECOSYS  and  INRA-UVSQ GCVG (Les Closeaux SolFit experiment in Versailles) ;  INRA Unité Expérimentale Forêt Pierroton and INRA ISPA  (Pierroton forest reserve and fertilization experiment);  OSU-Pytheas  ( O3HP   LTER in Saint-Michel l'Observatoire ). We sincerely thank the anonymous referees for their relevant suggestions to improve the manuscript. This is a LSCE contribution no. 5959. Edited by:  S. Bouillon Reviewed  by: two anonymous referees.
All three BNIPs demonstrated a potent inhibitory effect on parasite growth, with a 50% inhibitory concentration (IC 50 ) within the nanomolar range (Fig. 1B ; Table  1 ). BNIPDabut was the most active compound, with a mean IC 50 (Ϯ standard deviation [SD]) of 2. 4 Ϯ 1. 0 nM, similar to that of the reference drug pentamidine (mean IC 50 [Ϯ SD], 2. 9 Ϯ 0. 7 nM; Table  1 ). Since this class of compounds has been described as a group of inhibitors of the L. infantum Silent information regulator 2-related protein 1 (LiSir2rp1; GenBank accession number AAN39039. 1) (15), we evaluated whether inhibition of the T. brucei orthologue TbSir2rp1 (GenBank accession number AAX70528. 1) is a possible mechanism of action. Whereas BNIPDabut was shown to inhibit the NAD ϩ -dependent deacetylase activity of TbSir2rp1 with an IC 50 (Ϯ SD) of 155 Ϯ 42 M, suggesting that this is not the major mechanism of action (data not shown), LiSir2rp1 was inhibited with an IC 50 (Ϯ SD) of 35.
Similarly to pentamidine, BNIPDabut was more stable than BNIPDahep and BNIPDaoct; 95% to 100% of the drug was not metabolized (Table  3 ). This high metabolic stability is an indicator that the molecule is not easily subjected to common inactivation or loss of potency by reactions catalyzed by liver enzymes and is kept intact in circulation for longer periods. To determine the pharmacokinetics of BNIPDabut, a 10-mg/kg dose was administered to BALB/c mice by intravenous injection. Five minutes later, a concentration of 58 nM was achieved and, during the following 24 h, remained higher than 41 nM (data not shown); thus, the concentration was approximately eight times higher than the calculated IC 90. Taking into consideration the previous results, we chose   (22). After transfection, clones were screened for bioluminescent signal, and the ones that expressed the highest levels were selected.
Their in vitro growth was compared with that of wild-type parasites and was found to be similar (data not shown). In vitro detection limits were also analyzed for BSFs in a 96-well plate and determined to be approximately 2,500 cells (data not shown). Female BALB/c mice were inoculated intraperitoneally with 10 4 BSFs. Three days after infection, five groups of mice (n ϭ 4 per group) were treated intravenously with (i) saline, (ii) pentamidine at 2. 5 mg/kg/day, (iii) dimethyl sulfoxide (DMSO) at 16. 7%, or (iv) BNIPDabut at a dose of 10 or 20 mg/kg/day. Pentamidine was administered for 4 days, while BNIPDabut and the respective vehicle (DMSO at 16. 7%) were administered for 6 days (Fig. 3A ). No adverse effects were observable following any administration regimen. Treatment efficacy was followed through whole-animal live imaging using an IVIS Lumina LT (PerkinElmer).
Differential scanning calorimetry (DSC) DSC analysis of IND, BSA, IND:BSA-PMs and the solid dispersions were performed using differential scanning calorimeter (Mettler Toledo, DSC822e, UK). Samples (2-5mg) were weighed and crimp-sealed in aluminium pans with the led pierced to ensure constant pressure. Analysis was carried out under nitrogen gas flow (20 mL/min) over a temperature range of 25 °C to 300 °C and at a heating rate of 10 °C/min. The obtained thermograms were analysed using STAReSW 10. 00 software.
X-ray Diffractometry IND, BSA, IND:BSA-PMs and the solid dispersions were also characterised by X-ray diffraction through Bruker diffractometer (Bruker AXS D8 Advance; Bruker Corporation, Billerica, MA, USA). The diffractometer operated at room temperature and at 40 kV. The scanning diffraction angle (2θ) ranged from 2° to 45° with a step size of 0. 1°. The data were collected and analysed using DIFFRAC plus XRD commander software (Bruker Corporation).
, 2012; Trivedi et al. , 1999). IND solubility was also evaluated in a phosphate buffer (pH 7); although the IND solubility increased significantly to 472 mg/L (data not shown), this was significantly lower than the solubility enhancements obtained in BSA solutions. -IND-BSA solid dispersions IND-BSA solid dispersions were prepared by both spray drying and freeze drying techniques using IND-BSA solution at (20:1 MR) which is 5 folds higher than the association stoichiometry (4:1) described in the previous section. Fig. 5a  shows that IND solubility increased significantly after being formulated in SD-sol and FD-sol compared to that gained by the physical mixture (P<0. 001). Furthermore, FD-sol resulted in significantly higher solubility (P<0. 05) compared to that of SD-sol (Fig. 5a ). IND solubility was improved ~20,000 fold by SD-sol and ~24,000 folds by FD-sol when BSA is added at 3.
In order to test this hypothesis, IND solubility was evaluated in phosphate buffer (pH 7). Although IND solubility increased significantly in phosphate buffer (up to 472 mg/L) (data not shown), this was significantly lower than the solubility enhancements obtained in BSA solutions (Fig. 4 ). This suggests that IND enhanced solubility in BSA solutions is not mainly dependent on the dissociation of IND molecules and that other solubility enhancing mechanisms could be involved, i. e. salt and binding complex formation. It has been reported that several IND molecules may bind to one albumin molecule, suggesting that IND:BSA binding process might be more complicated than the simple accommodation of an IND molecule in the hydrophobic cavity of albumin  (Curry, 2002; Ghuman et al. , 2005). Several interactions may contribute to the IND:BSA binding process, including hydrophobic interactions, hydrophilic interactions and salt bridges formation  (Bogdan et al.
Acknowledgments The work is supported by  R01NS078307  (N. M. , D. J. , and C. G. );  Federation Françoise Recherché Epilepsies  ( FFRE-TF1  to N. M. ). UH4TR000491 (D. J. and C. G. );  R01NS43284 ,  R41MH093302 ,  R21NS077236 ,  R42MH093302 , and  R21HD057256  (D. J. );  Brain and Behavior Foundation (NARSAD) ,  American Heart Association  ( 13SDG13950015 ), and  Alternative Research Development Foundation  Grant (C. G. ).
Data collection The patients were selected prospectively on the basis of the INR measured by the hematology laboratory at Amiens University Hospital. Patients with INR ≥ 5 were included in the study if they had also been treated with VKAs prior to or during hospitalization. Each patient could be included only once. For each patient, the characteristics of bleeding events having occurred during hospitalization (type, site, date of onset, severity, treatment, and outcome) were recorded. Furthermore, the following characteristics were recorded prospectively by questioning the physician, the medical staff, and the patient and consulting the patient's hospital records: (a).
Data analysis Two members of the working group applied the new scale to all patients with 1 or more bleeding ADRs. The scale's reliability was then assessed by calculating 2 measures of inter-rater agreement: -Cohen's kappa (k) for the qualitative assessment of inter-rater agreement. The nonparametric k test is used to estimate the magnitude of the real component of agreement between matched, qualitative judgments. By convention, the degree of inter-rater agreement is considered to be "excellent" for k ≥0. 8, "good" for 0. 61< k < 0. 8, "moderate" for 0. 41< k <0. 6, "poor" for 0. 21< k <0. 4 and "bad" or "very bad" for k 0. 2. [13, 14] able  1  Evaluation of the preventability of a VKA-associated hemorrhage (adapted from Olivier et al). -Spearman's intra-class correlation coefficient for the quantitative assessment of inter-rater agreement (on each item and on the overall score).
ERROR in the CIRCUIT Lastly, 241 bleeding cases were evaluated and classified according to the new, validated scale as "preventable," "potentially preventable," "not preventable," and "unevaluable. " Statistical analyses were performed with SPSS software (version 18. 0, SPSS Inc. , Chicago, IL) for Windows (Microsoft Corp, Redmond, WA). In all tests, the threshold for statistical significance was set to P < 0. 05.
Results Over a 2-year inclusion period, 906 hospitalized patients were included in the study. The patients' characteristics are summarized in Table  2. The mean ± SD (range) age of the study population was 76. 6 ± 12. 4 (19-100), with a median of 77. In total, 439 patients (48. 5%) were male. Some of patients had comorbidities, such as diabetes (28. 6%) and hypertension (58. 9%). [10] The main indication for treatment with VKAs was atrial fibrillation (Table  3 ). Overall, 241 patients (26. 6%) experienced at least 1 clinically apparent bleeding event and 665 did not. Eighty percent of these 241 patients were classified as having experienced a serious ADR. Table  4  summarized the data on the types of bleeding; the most frequent type was subcutaneous bleeding.
The study had some limitations. In view of the present study's design, it was not possible to obtain data on each patient's time in therapeutic range; we only obtained the INR measured in the previous month. The present scale is specific for VKAs and should not be used with for other drug classes. However, it is noteworthy that DOAs are associated with a similar bleeding risk; it might be interesting to adapt the present preventability scale for application to DOA-related bleeding. Fluindione is the most frequently prescribed VKA used in France. It will be necessary to confirm the present scale's validity in another country where other VKAs are used. However, the preventability factors are probably the same. In conclusion, 241 out of 906 hospitalized VKA-treated, atrisk patients (INR > 5) patients (26.
An isotype-matched IgG was used as a non-specific control and binding of antibodies to the cells was evaluated by flow cytometry using 10 000 cells/sample and a BD FACSCanto™ II flow cytometer (Becton Dickinson, San Jose, CA, USA). Cells were gated on forward scattered light (FSC) versus side scattered light (SSC) parameters to select live cells. Data were analyzed using FlowJo software (Tree Star, Inc. , Ashland, OR, USA) and depicted in histograms plotting geometric mean of fluorescence intensity (GFI) on a four-decade logarithmic scale (x-axis) versus cell number (y-axis).
This work was supported by a grant from the  Ministère de l'Enseignement Superieur et de la Recherche  (C. R. ), by  "PROVASC" and "HYPROTEC", grants  from the  Pays de la Loire Region  (S. P. ). It was realized in the context of the  IHU-Cesti, LabEx IGO  and  LabEx Transplantex  projects which received French government financial support managed by the  National Research Agency (ANR)  via the " Investment Into The  Future " programs  ANR-10-IBHU-005 ,  ANR-11-LABX-0016-01  and  ANR-11-LABX-0070. The  IHU-Cesti  project is also supported by  Nantes Metropole  and the  Pays de la Loire Region. This study was also supported by the  "Fondation Centaure" (RTRS)  which supports a French transplantation research network.
Simulations The aim of the simulation study was to evaluate and compare the performances of each dose-range and model setting proposition, in terms of selected dose. Based on the motivating illustration, we proposed to plan, conduct and analyse a hypothetical phase I/II dose-finding clinical trial for erlotinib in the paediatric population. We used PK parameters as well as dose-finding toxicity and efficacy clinical trial data for erlotinib obtained from the adult population for extrapolation and bridging. (1) Specification of the dose-range: We hypothesised that the observed AUC in adults was similar in the paediatric population for the three dose-range adjustments LA, AA and MA (linear, allometric and MAs). In previous adult dose-finding studies, the doses ranged from 100 mg to 300 mg, and the MTD was 150 mg.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article:  Caroline Petit  was supported during this work by a grant IDEX from the  Universite´Sorbonne Paris Cite´ (2013, project  24 ). Sarah Zohar ,  Emmanuelle Comets  and  Moreno Ursino  were funded by the  InSPiRe (Innovative Methodology for Small Populations Research)  Project of the  European Union Seventh Framework Programme for Research, Technological Development, and Demonstration  under grant agreement  FP HEALTH 2013-602144. During this work,  Adeline Samson  was partially supported by the  LabEx PERSYVAL-Lab  ( ANR-11-LABX-0025-01 ) funded by the  French program Investissement d'avenir.
ACKNOWLEDGMENTS This study was sponsored in full by  Johnson & Johnson Consumer Companies. The authors fully collaborated in the design, collection of data, data analysis, and interpretation of the data. All authors were involved in reviewing and approving the text, and all authors agreed with the decision to submit the article for publication. The authors wish to thank the families of the study subjects for participating in this study. They would also like to thank  Alex L. Loeb, Ph. D. , and  A. Peter Morello III, Ph. D. , from  Evidence Scientific Solutions, Philadelphia, Pennsylvania , for medical writing and editorial support, which was funded by  Johnson & Johnson Consumer Companies, Skillman, New Jersey.
Definition of LFT abnormalities and DILI The METADAP cohort was not intended to assess liver function and the decision to perform an LFT was not influenced by inclusion in the study. In most cases, DILI related to AD use occurs during the first six months of treatment  [8]. Therefore, we retrospectively collected the LFT [ALT, AST (aspartate aminotransferase), AP, gamma-glutamyl transferase and bilirubin] available at baseline (includes the six months before study inclusion) and during the first six months of AD treatment. Abnormal LFTs were defined by the presence of at least one of the following variables above the ULN provided by the local laboratory: ALT>45 IU/l, AST>40 IU/l, AP>130 IU/l and total bilirubin > 17 μmol/ l. For patients with an abnormal LFT, the available etiologic data were collected: alcohol consumption (quantified as self-reported drinking habits; patients' families were also interviewed, when possible; no blood markers for alcohol consumption such as carbohydrate-deficient transferrin were performed), presence of NAFLD, viral hepatitis [immunoglobulin M antibodies (IgM) to hepatitis A, IgM to hepatitis B core antigen, antibodies to hepatitis C and hepatitis C viral load, IgM to hepatitis E and hepatitis E viral load, IgM to cytomegalovirus, IgM to Epstein-Barr virus, IgM to herpes viruses], ongoing medications, hemochromatosis (serum ferritin level and transferrin saturation), Wilson disease (serum ceruloplasmin level and urinary copper excretion), autoimmune hepatitis (diagnosis based on previously validated criteria)  [14] , bile duct obstruction (abdominal ultrasound), liver ischemia (clinical context and transthoracic echocardiography), hepatic arterial or venous obstruction (abdominal ultrasound examination with Doppler).