dataesr/scientific-paragraphs-categorization huggingface dataset:
The crystal structure of the PfGATase domain was determined by the molecular replacement method with the program PHENIX  31  using the refined structure of the GATase domain from E. coli 11 as search model. The structure was refined with PHENIX  31  , and visualized with COOT  32. The refined structure was validated with PROCHECK 33 before depositing. Crystals of PfGMPS_C89A in complex with Glutamine were obtained by the hanging drop vapour diffusion method at 292 K, in 0. 1 M Tris-HCl pH 8. 5, 22. 5% (w/v) PEG 1000 and 10 mM Gln. Ligand-free PfGMPS crystallized in 20% (w/v) PEG 3350, 0. 2 M Diammonium tartrate and 5 mM NaCl. PfGMPS_C89A/Gln and PfGMPS crystals were flash-cooled in a stream of nitrogen gas after soaking for few minutes in their respective reservoir solutions supplemented with 10% (v/v) ethylene glycol before data collection.
A complete data set was collected to 3. 15 Å resolution for the PfGMPS_C89A/Gln complex. Data on the ligand-free enzyme was collected to 3. 6 Å resolution. All data were processed with programs from the XDS package  30. Initial phases for PfGMPS_C89A/Gln were obtained by molecular replacement with residues 6-233 and 235-555 from PDB 3UOW as independent search models, yielding an initial map of suitable quality for iterative, manual model building in COOT  32  with interspersed cycles of automated refinement and phase improvement in PHENIX  31. For PfGMPS, molecular replacement was done using EcGMPS 11 as a template. Root mean square deviations calculated from Ca's with the program SuperPose (1. 0)  34. Small angle X-ray scattering. SAXS data (PfGMPS and PfGMPS_C89A) were collected at the ESRF BM29 beamline on samples concentrated between 1.
1 and 11 mg ml À 1 in 20 mM Tris pH 7. 4, 50 mM NaCl. As no concentration dependence effects were observed, data at 11 mg ml À 1 were processed for further analysis. Initial processing was done using PRIMUS  35  and P(r) analysis carried out using GNOM  36. Ab initio models of PfGMPS or PfGMPS_C89A were generated from the experimental data using DAMMIF  37. Models were superimposed onto the crystal structures using SUPCOMB  38. Characterization of GATase domain rotation. Two dynamic domains, which correspond to the GATase and ATPPase structural domains, were identified by the program DynDom 39 from pdb files (PfGMPS and PfGMPS_C89A/Gln) containing the monomers of each structure. Hereafter the 'SSM superimpose  40  ' option in COOT  32  was used to superimpose PfGMPS and PfGMPS_C89A/Gln structures, holding the ATPPase domain fixed.
This is in agreement with the experimental SAXS and crystal structures which clearly shows that the dimerization takes place via the ATPPase domain. After superposing the ATPPase domains the change in orientation of the GATase domains in the PfGMPS and PfGMPS_C89A/Gln structures was established using the 'SSM' option in COOT  32  , which gave the rotation matrix and translation vector from which the polar rotation angles and translation along the rotation axis (3 Å) were obtained (o ¼ 34°(angle with the z-axis); j ¼ À 158°(angle between the x-and the y-axis); k ¼ 85°(rotation angle)). The rotation axis runs through Lys104 and Ile29. Molecular dynamics and minimization. Minimizations of each model (410,000 steps with the conjugated gradient algorithm) were done using the Sybyl-X 2. 0 software package, elaborated by Tripos (now CERTERA).
The Tripos force field was applied with Gasteiger-Marsilli partial charges and a dielectric constant of 80 to simulate an implicit water phase (the dielectric constant of water is 20. 10 at 20 °C). No restraints were applied to our models. This step principally refines and corrects the positions of amino acid side chains. The resulting model was inserted into a parapipedic TIP3P solvent box by using the add solvation box module of the VMD 1. 9. 1 software. A distance of 15 Å was used for delimiting surfaces of the protein and that of the solvent box. Conditions for neutralizing charges were computed before the adding of sodium and chloride ions at concentrations similar to the salt concentrations used in the enzymatic assays. One thousand minimization steps of the model were done using the NaMD 2.
8b software 41 before molecular dynamics simulations. Two-dimensional root mean square calculations were performed to determine three key conformation frames resulting from each of the eight dynamics, and selected on the basis of its position along the rotation pathway. Alanine scanning was computed and DDG variations for each residue were evaluated for each of the three selected key frames. Then the mean value of the DDG of these latter was calculated for each of the eight conformation frames. The lowest energy conformation, frame number 8 (final conformation), was set to 0 kcal mol À 1. Computing was performed on a 144 xeon core CPU cluster supercomputer (SGI Altix), and simulations were done at constant temperature (300 K) and pressure (1 atm) by implementing the widely used CHARMM 27 force fields.
Acknowledgements This work was supported by the  ERA-NET New Indigo NPP program 'Plasfalsyn'. We thank the  European Synchrotron Radiation Facility (ESRF)  for access to beamlines ID29,  ID14  and  BM29  and the SOLEIL synchrotron for access to PROXIMA1. S. Shivakumaraswamy  acknowledges  UGC, Government of India for Junior and Senior Research Fellowships.
Professional divers should sometimes immerse in dirty waters, such as divers working in harbours. There is in Brest a military port and a repair shipyard, where divers work and may be exposed to chemical products or microorganisms. Thought, a very few studies is available on professional diver infectious diseases. Here is evidence: search on Medline database ("Diving" [Mesh] AND "Infectious Disease Medicine" [Mesh]) shows no results. It is generally accepted that divers may have cutaneous infectious diseases. A study about Pseudomonas aeruginosa infections among divers was conducted more than 15 years ago  [1]. More studies are available about infectious diseases in recreational divers, mainly about external otitis  [2, 3] , or conjunctivitis  [4]. Infections could sometimes be more serious.
Acknowledgements We gratefully acknowledge the  Russian Foundation for Basic Research  (project  14-03-91342 ), the  Deutsche Forschungsgemeinschaft  (project  857/18-1 ) and the  Ministry of Education and Science of the Russian Federation  (project 4. 619. 2014 /K) for financial support. N. S. thanks the  Alexander von Humboldt Foundation  for a postdoctoral fellowship. A. Prokop gratefully acknowledges support from  Dr. Kleist-Stiftung ,  Berlin.
Detection of Apoptosis and Necrosis HK-2 cells were assessed for apoptosis and necrosis using Cell Meter Annexin V Binding Apoptosis Assay Kit (AAT Bioquest), according to the manufacturer's instructions. For this, cells were detached from slides using trypsin and then trypsin was inactivated with epithelial medium supplemented with FBS 10%. After gently centrifugation and washing with PBS, Annexin V-iFluor 488 (1:400) and Iodure propidium (1:400) were added to the cell suspension for incubation in the dark for 15 min at 37°C. Then, 0. 3 ml of ice-cold 1× annexin-binding buffer was added, and induction analysis was conducted by flow cytometry (BD FACSVerse, BD Biosciences) with BD FacsSuite software (BD Biosciences) using FL1 and FL2 ranges for Annexin V-iFluor 488 and Iodure propidium, respectively.
Renal Histology and Immunohistochemistry Kidneys were collected from each mouse included in the renal function study. Tissues were fixed in Carnoy's solution for 24 h, embedded in paraffin and micrometer sections were cut. For histological analysis, renal sections were subjected to periodic acid-Schiff staining (PAS), scanned using a Nanozoomer 2. 0 RS (Hamamatsu Photonics SARL) and treated with the Morpho-expert image-analysis software (version 1. 00, Explora Nova) for renal corpuscle surface quantification. For immunohistochemistry analysis of primary cilia, renal sections were deparaffinized in toluene and rehydrated from xylene through a series of graded ethanol washes. After treatment with sodium borohydride 1% (71321 Sigma, 20 min at 4°C) and washes in PBS-Tween 0. 01%, sections were permeabilized in PBS-Triton X-100 0.
Statistical Analysis Statistical analyses were performed using GraphPad Prism 5. 0 for Windows (GraphPad Software Inc) and a Mann-Whitney's test was performed to compare two groups (FSS 0. 5 Pa versus FSS 0 [4-7 experiments] or UNx versus sham [6 mice per group]). All data are expressed as mean ± SEM. p<0. 05 was considered as statistically significant.
In Vivo Effect of Increased Urinary FSS Finally, we evaluated in vivo the effect of increased urinary FSS. We used an animal model where increased FSS was induced in proximal tubule by increased urinary flow following hyperfiltration. For this, C57BL/6 mice were uninephrectomized (UNx) by removing the right kidney. The left kidney was harvested 8 months later to analyse tubular epithelial markers. As expected  [22] [23] [24] , total GFR was maintained within the normal range in UNx subjected animals through adaptive increased single kidney (sk) GFR (  (Fig  7B ). Taking into account the observations in vitro, these data suggest that increased FSS in vivo is associated, as well, with a reduction of expression of epithelial markers.
Acknowledgments The authors thank  Eric Panader  and  Andre Roques  for technical help in development of the shear stress flow device ( Atelier Mécanique ,  Université Paul Sabatier ,  Toulouse France ),  Christiane Pecher  and  Alexia Zakaroff-Girard  for technical assistance and expertise in flow cytometry ( Cytométrie et Tri cellulaire, Plateforme Imagerie-TRI Genotoul, Toulouse France ),  Frédéric Martins  and  Jean-José Maoret  for technical assistance and expertise in qPCR ( GeT-TQ, Plateforme Génome et Transcriptome (GeT) Genotoul ,  Toulouse France ),  Christine Delage  and  Denis Calise  for technical facilities in animal experimentation (Experimental Microsurgery on rodents,  Plateforme Anexplo Genotoul ,  Toulouse France ) and  Stéphanie Gayral  and  Céline Mias  ( INSERM U1048-I2MC ,  Toulouse France ) for help with the immunostainings.
coli MLST Database (http://mlst. warwick. ac. uk/mlst/dbs/Ecoli). According to MLST analysis, the E. coli isolate was attributed to ST1284. This ST has been identified previously in E. coli isolates producing extended-spectrum β-lactamase recovered from animals  [9]. In summary, this report documents the emergence of NDM-5 among E. coli ST1284 isolated for the first time from a companion animal in the North African countries.
After digestion, cells were washed and stained as mentioned, and post-fixed in 2% PFA. Cells from BAL fluid or digested lungs were labeled with CD45, CD11b, CD11c, and Ly6G (BD Biosciences), stained with Cell Viability Stain (eBiosciences), and counted using LSR Fortessa cell analyzer (BD Biosciences) and analyzed using FlowJo VX software (Tree Star). Total leukocytes were gated as CD45 + cells, while neutrophils were gated as CD11b high , CD11c low , and Ly6G + cells. For histopathology studies, lungs from a subset of immunosuppressed BALB/c mice were fixed in formalin. Thin sections were stained with hematoxylin and eosin (H&E) or periodic acid Schiff (PAS) to visualize fungal elements. Caspase-3 immunohistopathology was performed using an anti-capsase-3 antibody (Institute for Research in Immunology and Cancer, Montreal, Canada).
Statistical analysis For all mouse lung data analysis, unless indicated on the legend, the Mantel-Cox log-rank test with pairwise comparison using Bonferroni correction was applied. For non-parametric data with multiple comparisons, the Krustal-Wallis rank analysis, was performed, followed by Dunn's test for pairwise comparison. For all other data analysis, statistical significance was determined by applying one-way ANOVA, partitioned with pairwise comparison using Tukey's test, and Bonferroni correction applied where applicable. All statistical analyses were performed either with Prism (GraphPad, Inc. ) or SAS (SAS Institute, Inc. ) with significance determined at p < 0. 05. For all statistical analysis, n. s. denotes statistically no significant differences found.
Acknowledgments We thank  Dr. Robert A. Cramer  ( Dartmouth College, USA ) for his guidance on the CGD mouse experiments. We also like to thank  Dr. Adilia Warris  ( University of Aberdeen, UK ), Dr. Stephanie Henriett  ( Radboud University, The Netherlands ), and  Adrian Zelazny  ( US National Institute of Health, USA ) for providing us with an A. nidulans clinical isolate from a CGD patient.
The latter two prosthetic groups are attached to threonine residues in subunits NqrB and NqrC  [6]. Flavinylation of these subunits is catalyzed by a Mg 2+ -dependent flavin transferase (ApbE) using FAD as substrate  [7]. This type of covalent bonding of the flavin is rare and can be found only in NQR subunits and related bacterial proteins such as RnfD and RnfG subunits of sodiumdependent NADH:ferredoxin oxidoreductase (so called RNF complex)  [8] , regulator of NO reductase transcription (NosR)  [9] , soluble cytoplasmic fumarate reductase (KPK_2907)  [10] , and urocanate reductase (UrdA)  [11]. Such unusual type of flavin covalent bonding implies an atypical structure of the flavin-binding pocket. However, there were no data on proteins containing covalently bound FMN residue in structural databases.
In spite of 3D structures of four different NqrC-like proteins having been solved (PDB: 3LWX, 3DCZ, 2KZX, and 3O6U), all these structures were determined for flavin-free, apo-forms of the corresponding proteins. Just recently, a 3D structure of holoNqrC from V. cholerae has been reported  [5]. The reason of FMN covalent binding in NqrC is not currently clear. Therefore, to answer this question, we developed a method for production of apo-and holo-forms of the water soluble flavin-binding domain of the NqrC subunit (NqrC') based on heterologous expression of the nqrC' gene in Escherichia coli cells in the absence or in the presence of co-expressed gene of flavin transferase, respectively  [7]. In the present work, flavin binding properties of apo-and holo-forms of V. harveyi NqrC' were investigated by measuring of apoNqrC' affinity to FMN and structural analysis of the holo-form of this protein.
It cannot be excluded that properties of [NqrC'-noncovalently bound FMN] complex differ from those of holoNqrC' with covalently bound FMN, and only covalent bonding of FMN residue is capable to provide a strong quenching of protein and flavin fluorescence. To test this possibility, the noncovalent binding of free FMN to apoNqrC' was also studied by isothermal titration calorimetry. However, mixing of apoNqrC' with FMN did not result in any significant heat changes. The observed effect was <<0. 1 kcal/mol of FMN (data not shown), which is more than 100-fold lower comparing to FMN binding by flavoproteins  [12, 13]. Thus, affinity of apoNqrC' to free FMN is very low, and formation of a phosphoester bond between Thr229 and FMN residues is essential for further binding of the isoalloxazine moiety of the flavin.
Structure similarity search The search in PFAM database (http://pfam. xfam. org/) shows that holoNqrC' belongs to the FMN binding family (PFAM ID accession code PF04205) with a high E-Value of 2. 6e -16 and bit score of 59. 5. Interestingly, this family does not belong to 23 structural clans described for flavin-dependent proteins  [15]. Besides structures of NqrC' from V. harveyi (current work) and V. cholerae  [5]  there are only 4 PDB structures of proteins from PF04205 family (3LWX, 3DCZ, 2KZX, and 3O6U). Two of them (2KZX and 3O6U) are uncharacterized proteins from Clostridium thermocellum and Clostridium perfringens, respectively; 3DCZ is a putative RnfG subunit of electron transport complex from Thermotoga maritima. The last one (3LWX) is the flavin-binding domain of the NqrC subunit from Parabacteroides distasonis.
) with a 1. 4-ml cell at 12°C. Before experiments, apoNqrC' was dialyzed against 50 mM potassium phosphate buffer (pH = 7. 0). All samples were degassed before the experiment. Reference titrations injecting FMN to the buffer were carried out and the results were used as a reference for the FMN-apoNqrC' titrations. A typical injection schedule included the addition of 15 μl samples of 0. 5 mM FMN to 50 μM apoNqrC' with 30 injections at three minute intervals. For the first injection only 5 μl of FMN was added and the corresponding data point was deleted from the analysis. Data analysis was carried out using MicroCal Origin 7. 0 software with the "one set of sites" model.
The work was supported by the program "Chaires d'excellence" edition 2008 of ANR France and CEA(IBS) -HGF(FZJ) STC 5. 1 specific agreement. Part of this work was supported by BMBF (PhoNa -Photonic Nanomaterials) and Russian Federal Target Program "Research and Development" (№ 14. 587. 21. 0004, unique identifier RFMEFI58714X0004). The work used the platforms of the Grenoble Instruct centre (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Acknowledgments The diffraction experiments were performed on the beamlines ID23-1 and ID29 at the  European Synchrotron Radiation Facility (ESRF), Grenoble, France. We are grateful to  ESRF Structural Biology group  for providing assistance in using these beamlines. We acknowledge the use of the  HTX Laboratory at the EMBL Grenoble Outstation (France). We are indebted to  Dr. V. N. Orlov  for help with the ITC measurements.
MATERIALS AND METHODS Data The data from 223 patients enrolled in the IMMC38 trial, meant to assess the relationships between categorized CTC count (<5 or 5 per aliquot) and survival in patients with mCRPC, were used. 13 Patients were treated by chemotherapy and/or hormonotherapy; the administration dates were collected, but no treatment doses or pharmacokinetic outcomes were available. CTC counts per aliquot and PSA concentrations were measured at different timepoints along treatment. Characteristics of patients are described in Table  1. A median of four CTC values and four PSA titers was available per subject until six months after treatment initiation. The median CTC count was 2 CTC/7. 5 mL, with a range of 0-6,437 CTC/7. 5 mL, and the median PSA concentration was 116 ng. mL 21 with a range of <0.
1-17,800 ng. mL  21. A large proportion of CTC counts equal to 0 was observed (40%). The limit of quantification for PSA concentration was 0. 1 ng. mL 21. 18 PSA observations below limit of quantification, representing 0. 1% of the observed values, were fixed at limit of quantification/2 in the dataset. 19 eventy percent of the patients discontinued the study at 180 days. Since the time-to-dropout seemed to be independent of PSA and CTC counts (visual inspection), the probability of dropout was not taken into account in the model, but the model-based analysis was limited to a sixmonth treatment duration to reduce potential statistical biases. Individual kinetic profiles were heterogeneous, as illustrated in Supplementary Figure  S1a, b. Different types of profiles were observed: some with parallel PSA and CTC kinetics, others with divergent evolutions.
External validation of the CTC kinetic model is planned using another mCRPC database. Nevertheless, this model is the first to quantify the dynamic links between the kinetics of PSA and CTC count during treatment in patients with mCRPC. It could serve as a general framework that could be applied to other cancer types in order to compare the properties of CTCs. Application of the CTC kinetic model in patients treated with other drugs is also under consideration. Finally, the mixed effect model allows identifying covariates and quantifying their impact on CTC kinetics. In particular, pharmacogenomic data could be tested to identify patients for whom CTC counting is the most sensitive. In the future, the present model will be challenged to establish a link between a CTC kinetic parameter and survival and to compare the sensitivity and specificity of PSA and CTC count for predicting treatment efficacy.
The parameters applied were two rounds of 12 1-s pulses (each pulse at a 10% duty cycle) at 20% power (protocol adapted in our lab from reference 42). Flow cytometric analysis. For flow cytometric analysis, cells were diluted in filter-sterilized PBS to remove particles. Fluorescence was mea-sured on a CyFlow Space cytometer (Partec, France). For details of the parameters used to collect fluorescence and the software used to analyze data, see the supplemental material. The subpopulations were identified by using histograms or cytograms. YFP-or mCherry-expressing cells were identified as cells giving a higher signal intensity than the reporterless cells used as a control. Data were analyzed by using statistical tables that indicate the percentages of fluorescent cells determined by each detector.
Microscopy. Cells were observed with a Zeiss AxioObserver. Z1 inverted fluorescence microscope equipped with a Zeiss AxioCam MRm digital camera and Zeiss fluorescence filters. For the filters used to image fluorescence, see the supplemental material. Images were processed with the Zeiss ZEN software package. Wild-type cells containing no fluorescent fusions were analyzed under each culture condition (liquid culture, biofilm, or insect cadaver) to determine the background fluorescence of YFP or mCherry and the optimal exposure time. Slides for time-lapse microscopy were prepared according to the protocols described in references 16 and 27, with some adjustments. An LBPpolyacrylamide solution (1% LBP, 10% polyacrylamide solution [acrylamide/bisacrylamide ratio, 37. 5:1], 0. 1% ammonium persulfate, 2 l of N,N,N=,N=-tetramethylethylenediamine [TEMED]) was used to prepare the solid medium.
According to the previously mentioned protocol, a 125-l GeneFrame (Thermo Scientific ABgene) was filled with the LBPpolyacrylamide solution. After polymerization, 1. 5-mm-wide strips were cut and washed four times for 30 min in LBP diluted 1:100. Two strips were then symmetrically placed in a gene frame in the first and third quarters of the width of the frame. A 2-l volume of an exponentially growing cell suspension (optical density at 600 nm of 1) diluted 20ϫ were pipetted onto the strips before the frame was sealed with a coverslip. Bt cell development at 30°C in a temperature-controlled chamber was monitored with the microscope described above. Phase-contrast and fluorescence images were taken every 15 min for 20 h. Nucleotide sequence accession number. The DNA sequence of the codon-optimized mCherryLGC gene has been submitted to GenBank and assigned accession number KJ547675.
ACKNOWLEDGMENTS We thank  Christina Nielsen-Leroux  and  Stéphane Perchat  for discussion and  Christelle Lemy  and  Christophe Buisson  for technical assistance. This study was funded by the  French Agence Nationale de la Recherche  (Cell. com;no. ANR-09-Blan-0253 ). We gratefully acknowledge funding from the  DIM Astrea (French regional program :  Ast11 0137 ) for the CyFlow Space flow cytometer used in this work. D. L. , L. S. , M. G. , and E. V. are responsible for the concept and design of the study. E. V. , L. S. , and D. L. designed the experiments. E. V. performed the experiments. E. V. and L. S. analyzed the data. E. V. , L. S. , M. G. , and D. L. wrote the paper.