dataesr/scientific-paragraphs-categorization huggingface dataset:
1(ϩ) to produce pcDNA-␤4GalT7 as previously described  (31). Mutations were constructed using the QuikChange site-directed mutagenesis kit, employing pcDNA-␤4GalT7 or pET-␤4GalT7 as template. Mutants were systematically checked by double strand sequencing. The human decorin cDNA sequence (GenBank accession number NM_001920. 3) was cloned by PCR amplification from a placenta cDNA library (Clontech). For heterologous expression in eukaryotic cells, the full-length cDNA sequence was modified by PCR to include an AflII site, a Kozak consensus sequence at the 5Ј end, a sequence encoding a His 5 tag, and an XhoI site at the 3Ј end. This sequence was subcloned into pcDNA3. 1(ϩ) to produce pcDNA-decorinHis as previously described  (31). Expression and Purification of the Soluble Form of h␤4GalT7-A single colony of E.
The supernatant was analyzed by high performance liquid chromatography (HPLC) with a reverse phase C18 column (xBridge, 4. 6 ϫ 150 mm, 5 m, Waters) using Waters equipment (Alliance Waters e2695) coupled to a UV detector (Shimadzu SPD-10A). Kinetic parameters were determined by nonlinear least squares regression analysis of the data fitted to the Michaelis-Menten rate equation using the curve-fitter program of Sigmaplot 9. 0 (Erkraft, Germany). In Vitro Competition Assays of h␤4GalT7 Activity by C4-modified Xylosides-The in vitro inhibition assays of the wild-type GST-h␤4GalT7 were carried out using 0. 2 g of purified protein incubated for 30 min at 37 °C in a 100 mM sodium cacodylate buffer, pH 7. 0, 10 mM MnCl 2 , with 0. 5 mM 4-MUX and 1 mM UDP-Gal, in the presence of concentrations from 0 to 5 mM 4H-Xyl-NP, 4H-Xyl-MU, or 4F-Xyl-MU.
Quantification of the reaction product was carried out by HPLC, as described above. The enzyme activities were reported as a function of the logarithmic values of inhibitor concentration. IC 50 values were determined by fitting the experimental dose-response curves using the curve-fitter program of Sigmaplot 9. 0 (Erkraft, Germany). K i values were calculated from IC 50 values according to the Cheng-Prusoff's equation  (42, 43). In Cellulo Analysis of GAG Chains Biosynthesis by Na 2 [ 35 SO 4 ] Incorporation-GAG chains biosynthesis using 4-MUX as primer substrate was determined with CHOpgsB-618 cells (American Type Culture Collection). Cells were cultured in Dulbecco's modified Eagle's medium/F-12 (DMEM/F-12) (1:1), supplemented with 10% fetal bovine serum (Dutscher), penicillin (100 units/ml)/streptomycin (100 mg/ml), and 1 mM glutamine, then transfected with the wildtype or mutant pcDNA-␤4GalT7-Myc plasmid or with the empty pcDNA3.
The ratio of viable cells upon the total number of cells was determined using the cell counter TC20 (Bio-Rad) in the presence of a vital marker (trypan blue). In Cellulo Analysis of Decorin Core Protein Glycosylation-CHOpgs-B618 cells stably transfected with pcDNA-decorinHis encoding the human decorin core protein  (31, 44)  were transiently transfected with pcDNA3. 1 or with recombinant vector encoding either the wild-type or mutated h␤4GalT7-Myc as described above. 48 Hours following transfection, the cell medium was collected, concentrated by centrifugation at 4 °C for 15 min at 3000 ϫ g, using the Amicon Ultracell 30 MWCO concentrating system (Merck Millipore, Germany), and submitted to SDS-PAGE (25 g of protein/well). The glycosylation level of the decorin core protein was monitored by immunoblot using a 1/5,000 dilution of primary polyclonal anti-human decorin antibody (VWR) and a 1/10,000 dilution of secondary anti-goat antibody coupled to horseradish peroxidase (Sigma), then quantified using ImageJ software.
1B ). Kinetic Properties of the Human Recombinant h␤4GalT7 Mutants Expressed in E. coli-To assess the functional importance of the residues of the acceptor binding site highlighted by our model, we carried out point mutagenesis and analyzed the consequences of conservative and non-conservative mutations on the kinetic parameters of h␤4GalT7 expressed and purified from recombinant E. coli cells. The wild-type enzyme and engineered mutants were produced as truncated fusion proteins lacking the 60 N-terminal amino acids (including the trans-membrane domain and part of the stem region) linked to GST and purified by affinity chromatography (data not shown). This led to 1. 0 to 2. 5 mg of pure protein per liter of culture for wildtype and mutant h␤4GalT7. Kinetic assays were performed using 4-MUX as acceptor substrate, which allowed quantification of the transfer reaction product by UV detection coupled to HPLC.
The expression level of enzymes was checked by immunoblot analysis using ImageJ software. As shown in Fig. 2 , an additional upper band at ϳ39 kDa was observed (Fig. 2 , inset). This ϳ39-kDa band can be attributed to the N-glycosylated form of the enzyme as it disappears upon peptide N-glycosidase F digestion (Fig. 2 , inset, left panel). Both bands were taken into account to quantify the total enzyme expression level. The results indicate that all mutants considered in these experiments were expressed at a similar level to that of the wild-type protein (Fig. 2 , inset, right panel). As shown in Fig. 2 , the GAG synthesis level in cells expressing wild-type h␤4GalT7 was about 7-and 9. 5-fold higher in the presence of 5 and 10 M 4-MUX, respectively, than in the absence of acceptor substrate, indicating that CHOpgsB-618 cells expressing h␤4GalT7 are able to prime efficiently GAG chains synthesis from 4-MUX, in agreement with previous studies  (17, 31).
2 ). However, the substitution of Tyr 196 to phenylalanine induced a more dramatic effect on the glycosylation of decorin than on the in vitro or in cellulo activity toward 4-MUX. Furthermore, results shown in Fig. 3  indicate that conservative mutation Y199F allowed recovery up to 70% of the decorin glycosylation level compared with cells expressing the wild-type h␤4GalT7. We also assessed the role of the His 195 residue in the glycosylation process of decorin. The results shown in Fig. 3  indicate that none of the mutations, H195A, H195Q, or H195R, significantly affected the level of decorin glycosylation. These data are consistent with the results obtained on the in vitro and in cellulo activity of the enzyme toward 4-MUX. Together, mutagenesis experiments indicate that modification of the amino acid side chain at position 195 did not greatly affect xyloside binding and galactosyltransferase activity of h␤4GalT7.
4H-Xyl-MU was able to inhibit up to 50% of the initial activity at a 2 mM concentration (Fig. 4B ), with an IC 50 value of about 1 mM and a K i value of about 0. 5 mM (Table  2 ). To test whether hydrogen bond formation between 4-MUX and the protein via His 195 is important for the inhibitory potency, we synthesized 4H-Xyl-NP, which the aglycone struc-FIGURE 3. Effect of wild-type and mutated h␤4GalT7 expression on decorin core protein glycosylation in CHOpgsB-618 cells. Cells stably expressing the human recombinant decorin core protein were transfected with the recombinant vector encoding either the wild-type (WT) or mutated h␤4GalT7. The decorin glycosylation level was monitored by immunoblot then quantified using ImageJ software, as described under "Experimental Procedures. " Immunoblot analyses of the decorin core protein glycanation level are shown as inserts for CHOpgsB-618 cells transfected with empty (pcDNA) or with the recombinant vector coding for WT, Y194A, Y194F, Y196A, Y196F, Y199A, or Y199F h␤4GalT7 (left panel), and for WT, R270A, R270K, R226A, R226K, H195A, H195Q, or H195R h␤4GalT7 (right panel).
Structure-guided Inhibitors of h␤4GalT7 To complement the in vitro assay, we assessed the ability of the synthesized xyloside derivatives to inhibit GAG chain biosynthesis in cellulo. Addition of 4H-Xyl-NP produced a moderate but significant 20% decrease of GAG chain synthesis in CHOpgsB-618 cells, when used at 50 and 100 M (Fig. 5A ). This correlated with the weak in vitro inhibition level obtained with this compound. The compound 4H-Xyl-MU allowed a larger inhibition of GAG chain synthesis, with up to 30% reduction of the GAG synthesis rate at similar concentrations (Fig. 5B ). The best inhibitory effect was observed when performed in the pres-   ence of 4F-Xyl-MU leading to up to 50% inhibition of the initial GAG chain synthesis rate at 100 M concentration (Fig. 5C ). Interestingly, preliminary results indicated that 400 M 4F-Xyl-MU inhibited the initial decorin glycosylation rate by about 50%, without affecting the viability of CHOpgsB-618 cells (data not shown).
* This work was supported by Agence Nationale pour la Recherche (ANR) Meca-GT  Grant  ANR-13-BSV8-0011-01 ,  ANR GAG-Sorting  Grant  ANR-13-JS07-0004-01 ,  Labex SynOrg  Grant  ANR-11-LABX-0029 ,  International Associated Laboratory CNRS-Université de Lorraine-University of Dundee, SFGEN ),  Région Lorraine-FEDER  ( Glyco-Fluo ) grant, grants from the  Région Lorraine  (to N. R. and S. F. G. ), and the AMSEDgenetique association. Author's Choice-Final version full access.
Therefore, it is essential to determine the structure parameters that are required to obtain the desired PBG. For this purpose, plane wave expansion (PWE) calculations  [37, 38]  were carried out using a photonics design automation software 'RSoft'. PWE method offers a fast and accurate technique to determine the photonic band diagram, and therefore the PBG, of PhCs. Before proceeding, it is essential to note that, hereafter, transverse magnetic (TM) polarization is defined as the electric field being parallel to the rods/holes; while transverse electric (TE) polarization is define as the electric field running around the rods/holes. Figure  8  shows the photonics band diagram obtained at λ = 375 nm for a hexagonal distribution of ZnO nanorods having radius on period ratio (r/a) of 0. 3. The x-axis represents the wave-vector k (kx, ky), where Γ (0,0), M (π/a, / 3a  ), and K (4π/3a, 0) are the symmetry points of the hexagonal lattice in reciprocal space.
Acknowledgments This work was mainly supported by the  ANR  Project  ULTRAFLU  ( BLAN08-1_320541 ). We would like to thank the  ULTRAFLU  project partners,  Vincent Sallet ,  Pierre Galtier  and  Corinne Sartel  from  Groupe d'Etude de la Matière Condensée (GEMaC ),  Licinio Rocha  and  Vesna Simic  from  Commissariat  à l'énergie atomique et aux énergies alternatives (CEA) and  Christophe Galindo ,  Laurent Divay  and  Pierre Le Barny  from  Thales Research and Technology (TRT).
For the higher simulated pressure of 1 × 10 -4 mbar the FWHM behavior is similar to the case of lower space charge values, although sharply rising at about 2×10 5 ions. Since the simulation of ∼ 10 5 ions without Coulomb scaling with current GPU technology is not feasible because of computation time limitations, these effects will require improvements in simulation speed for accurate predictions under the given experimental conditions. It was found to be of significant importance as a system-663 atic effect in the WITCH on-line experimental data. Its 664 eigenfrequency was studied under different space-charge 665 conditions. It was found that it rises proportionally with 666 the number of ions, as is expected from theory. This result 667 provides an essential correction to the on-line retardation 668 spectrum data of WITCH  [44].
Excitation models and production rate determination For all molecules, we used the latest spectroscopic data available in the JPL  (29)  or CDMS  (30)  database, both for line identification and computation of line strengths. In our model, the population distribution of the ground vibrational state of molecule slowly evolves from LTE at the temperature T gas maintained by collisions with neutrals and electrons close to the nucleus [(  11 ),  (12 ) , and references therein] to fluorescence equilibrium in less dense parts of the coma. Collisional rates and electron density are based on a water production rate of 5 × 10 29 molecules s -1. We derived T gas = 73 K from the relative line intensities of the strong methanol lines (figs. S1 and S2). For the other molecules, the line intensity ratios (or rotational temperature) agree with the model [for example, T rot (CH 3 CHO) = 67 ± 15 K for 68 K predicted].
Computational details In order to asses the quality of the functions mentioned above as bonding descriptors, we have computed s(r), t LOL (r), t bose (r) and ELF for several chemically representative systems in their ground states: N 2 and F 2 as molecules representing apolar covalent and charge-shift bonding type respectively, H 2 O and CH 3 F, as examples of polar covalent bonds, LiH and LiF as examples of ionic bonds, and 1,2-ethanediol as an example of NCI. The wavefunctions were calculated at the restricted Hartree-Fock level with the 631g++dp basis as implemented in GAMESS code  [28]. The 1,2-ethanediol geometry has been optimised with the CCSD(T)-F12a/cc-pVDZ-F12 method as implemented in MOLPRO 2010. 1  [29]. All the functions herein presented, were computed with our CHECKIN code, developed explicitly for this work and available upon request.
Acknowledgements The authors wish to thank  Professor Andreas Savin  for his endless strength and discussions. This work was supported partially by the framework of CALSIMLAB under the public grant  ANR-11-LABX-0037-01  overseen by the  French National Research Agency (ANR)  as part of the  "Investissements d'Avenir" program  (reference:  ANR-11-IDEX-0004-02 ).
Calculating the Poisson's ratio for off-axis combination of longitudinal axis and transverse direction is not especially complex, but often tedious and repetitive. The method has been systematically implemented in the ElAM software package  19. For a typical calculation, the three angles (θ, φ, χ) describing the axis vector  and direction vector  each varies every degree (see Fig. 2 ). This generates an excess of 23 million sets of off-axis elastic constants, from which the extrema and averages necessary to establish the auxetic typology can be determined.
Methods In order to extend our sample of materials, we calculated the elastic properties of all-silica zeolites as well as α-quartz and αcristobalite using classical and ab initio simulations. Crystallographic information files (. cif) of the 206 known zeolite frameworks were obtained from the international zeolite association  23. The classical calculations were performed with GULP v3. 1  24  , using the five well established potential models of Catlow, Gale, Matsui, Sastre and Van Beest  [25] [26] [27] [28] [29]. These models have different characteristics (partial/formal charges, shell models) and have been shown in a recent review to perform well for elastic properties of α-quartz  30. The first principles calculations were performed with the CRYSTAL09 code  31  within the Density Functional Theory Theory (DFT) approach, using the B3LYP hybrid functional  32  with empirical correction for the dispersive interactions  33  , full symmetry and all electron localized basis sets  34.
The stiffness matrixes for each structure † were used to build a database for the software ElAM  19  to examine. ElAM calculates the off axis elastic properties, allowing for the identification of interesting properties such as a negative Poisson's ratio in particular axes as well as establishing the auxetic typology. Not all pure silica frameworks could be relaxed. Depending on the potential models, between 186 and 189 were optimised with classical simulations. DFT simulations led to elastic constants for a subset of 121 frameworks, the remaining being too large for the computer resources available  9.
We have designed a typology for auxetic materials, classifying materials with distinctions in the number of axes where a negative Poisson's ratio can be found, and the degree for which each axis is auxetic. The system has been demonstrated as a tool for comparing groups of materials and their auxetic properties, and the benefits of distinguishing between levels of auxeticity have also been explored, further highlighting the extraordinary properties of α-cristobalite. The elastic properties of a large number of pure silica zeolite frameworks have been calculated and classified with the proposed typology. After comparison of the auxetic classifications with a database of 471 general materials, the pure silica zeolites are shown to be marginally more auxetic, but follow the same trends. We found that the JST frameworks has great potential for complete auxeticity, and have proposed an explanatory mechanism.
Acknowledgements This work was supported by the  Engineering and Physical Sciences Research Council  [Grant number  EP/G064601/1 ] ], and  GENCI-IDRIS HPC resources  [project i2014087069]. Notes and references a  College of Engineering, Mathematics and Physical Science, University of Exeter ,  EX4 4QF , UK. b  PSL Research University, Chimie ParisTech -CNRS, Institut de Recherche de Chimie Paris ,  75005 ,  Paris, France † Electronic Supplementary Information (ESI)  available: Detailed exposition of the auxetic typology and elastic constants for all calculated zeolites. See DOI: 10. 1039/c000000x/ ‡ Corresponding author: a. s. h. marmier@exeter. ac. uk.
X-ray crystallographic studies Data for 1, 2, 3 and 5 were collected on a Enraf-Nonius CAD4 diffractometer with graphite-monochromated Mo Ka radiation (l = 0. 71073) source, and data for 1ÁTHF were collected using a Agilent Technologies Xcalibur Gemini ultra diffractometer with a Cu Ka radiation (l = 1. 54184) source at 173 K using an Oxford Cryosystems Cobra low temperature device, operating in o scanning mode with C and o scans to fill the Ewald sphere. The programs used for control and integration were Collect, 22 Scalepack and Denzo. 23 Absorption corrections were based on equivalent reflections using SADABS. 24 Data for 4 were collected and processed by the UK National Crystallography Service at the University of Southampton. 25 The crystals were mounted on a glass fibre with silicon grease, from dried vacuum oil kept over 4 Å molecular sieves in an MBraun glovebox under Ar.
All solutions and refinements were performed using the WinGX or Olex2 packages and software therein. All non-hydrogen atoms were refined with anisotropic displacement parameters and all hydrogen atoms were refined using a riding model. Disordered solvent molecules were modelled using the SQUEEZE 26 function in PLATON. 27 Crystal structure and refinement data are given in Table  S1  of the ESI. † CCDC 1051779-1051784.
The PDB database contains VEGFR2 TK complex 1Y6A possessing N-aryl-5-aryloxazol-2-amine ligand AAZ (Fig. 1 ) determined as a powerful VEGFR2 inhibitor (IC 50 : 22 nM). Ligand AAZ was prepared in five steps in a low overall 10% yield mostly due to the problematic oxazole-2-amine fragment formation  [12]. Low yields of oxazole-2-amine formation (1e58 %) have been also described  [13, 14]. Moreover AAZ contains N-aryloxazol-2-amine part that uses to liberate from connection with oxazole by influence of nucleophilic reagent (e. g. amines, alkoxides etc. ). Nucleophilic attack on C(2) of AAZ oxazole ring resulting in toxic aniline 26 (Scheme 1). (unpublished results) Low yielding synthesis of Naryloxazol-2-amines, their problematic stability and potential toxicity resulting from releasing aniline inspired us to develop novel, stable and synthetically more feasible VEGFR2 modulators based on the oxazole/1,2,3-triazole isosteric replacement.
In case of PDB: 3VO3 a pyrazole ring does not bind the VEGFR2 TK directly. It is exposed towards the solvent accessible part of the protein  [19]. Therefore triazole core that replaces a pyrazole ring can have the same position. Compound 7 is a close analogue of sunitinib that is an active base of the drug Sutent (Pfizer Inc. ). Sunitinib inhibitory activity IC 50 ¼ 39 nM (VEGFR2 TK) was determined  [25]. The structure 7 possesses N-1,2,3-triazolylethyl group instead of N,Ndiethylaminoethyl group present in sunitinib (Fig. 3 ). Recently an X-ray structure of VEGFR2 TK/sunitinib complex (PDB: 4AGD) appeared  [26]. From its analysis is clear that the N,N-diethylamino group in sunitinib is exposed out of the VEGFR2 protein and represents only a group improving the ligand pharmacokinetic profile.
Moreover proposed positions of the above triazole inhibitors in VEGFR2 ATP active site is similar to the poses of ligands from PDB complexes 2P2I (IC 50 : 38 nM), and 3EFL (IC 50 : 3 nM). (not shown) Both VEGFR2 kinase conformers (PDB: 2P2I and 3EFL) used in docking experiments are VEGFR2 inactive (DFG-out) kinases originally accommodating Type II inhibitor  [14]. Considering the data mentioned above, we decided to prepare VEGFR2 TK modulators T1eT7 possessing an internal 1,2,3-triazole core and determine their VEGFR2 TK inhibition potential.
Interaction analysis of AAZ conformers Oxazole VEGFR2 TK inhibitor AAZ was developed by Glax-oSmithKline  [12]. The intermolecular interactions for both AAZ conformers present in VEGFR2 TK complex PDB: 1Y6A are depicted on Fig. 7. Based on the above analysis, we decided to keep the pharmacophorically interesting 5-(ethylsulfonyl)-2-methoxyphenylamine fragment from AAZ ligand in all predicted triazole structures T1eT7. (Fig. 10 ) The proposed intermolecular interactions of triazoles T1eT7 and their poses in VEGFR2 kinase were similar to those known for AAZ ligand from PDB complex 1Y6A. (e. g. T3 from Fig. 12 ).
On the other hand, it was not easy to find triazole structures possessing AAZ-like score and pose by docking. The initial in Silico experiments were performed by an older DOCK software version with a kinase taken from PDB complex 1Y6B [33]. Fig. 6. Structures, VEGFR2 TK IC 50 activities, predicted intermolecular interactions and docking scores of 12 and 14 obtained in VEGFR2 TK variants from PDB: 2P2I and 3EFL, resp. In both cases a triazole fragment in 12 and 14 directly contributes to the ligand/receptor binding (þP: induced dipole, HB: hydrogen bond, PP: stacked interaction). However the position of the triazole ring in 12 and 14 is conformationally blocked by an intermolecular HB with neighbouring NH group. Some 1,4-disubstituted triazole skeletons that fulfilled both required score (AAZ: À53.
6 kcal/mol) and pose conditions were selected: e. g. T3 (score À57. 2), T5 (À52. 9), T6 (À51. 3), T2 (À51. 3), T4 (À49. 2). The triazole core of T1eT7 retains predicted hydrogen bond interaction with the kinase backbone amino acid residue Cys917 an important interaction known from AAZ oxazole ring in PDB: 1Y6A (Fig. 7 ). Pharmacokinetic properties of triazoles T1 e T7 predicted by Molinspiration toolkit supported their drug-likeness [34e38] (see supporting material). Proposed properties for 1,4regioisomeric triazoles T1eT7 were promising. Therefore we decided to perform their synthesis. 1,5-Regioisomeric triazole analogues (Fig. 1 ) of T1eT7 were also docked by the same conditions. All these compounds were less advantageous possessing lower docking scores and several of them did not retain expected AAZlike pose in VEGFR2 TK.
Re-docking experiments In initially performed docking experiments (an older DOCK software) triazoles T2eT6 showed better or similar scores as their oxazole precursor AAZ (vide supra, see part 2. 2 In Silico predictions). These results do not correlate well with obtained IC 50 activities determined from the biological assay. Therefore an additional docking experiment was performed with newer version of the docking software (DOCK 3. 6) on more calibration reliable VEGFR2 kinase taken from PDB: 1Y6B  [76]. By these conditions all triazole structures although still retaining AAZ-like pose in VEGFR2 TK, showed worse score values compared to their oxazole analogue AAZ (Figs. 10  and 11 ). A similar result, disfavouring 1,2,3-triazoles compare to oxazoles, was obtained from docking experiment of isosteric pairs Predicted VEGFR2 TK interactions of the most active 1,2,3triazole T3 and its oxazole bioisostere T3-ox are depicted on Fig.