dataesr/scientific-paragraphs-categorization huggingface dataset:
Mechanisms leading to expression of Tn and STn O-glycans in cancers include somatic mutations and/or epigenetic silencing of the private chaperone Cosmc responsible for O-glycan elongation  10  as well as relocation of GalNAc-Ts from Golgi to endoplasmic reticulum  11. More recently, we have shown that the truncated O-glycophenotype appear to directly induce oncogenic features with enhanced growth and invasion  12. GalNAc-T isoforms contain an N-terminal catalytic domain adopting a GT-A fold and a unique C-terminal lectin domain, classified as a carbohydrate-binding module (CBM) 13 in the CAZy database  3  , with a b-trefoil fold that are connected through a short flexible linker  [13] [14] [15] [16]. A key structural feature of these enzymes in catalysis is the existence of a flexible loop formed by residues Arg362 to Ser373 (residue numbers in GalNcAc-T2) that adopts different conformations during the catalytic cycle and renders the enzyme catalytically inactive or active  16.
1a, b ). Within the asymmetric unit (AU) of I4 1 crystals, one molecule of GalNAc-T2 is arranged as a dimer with another molecule from the neighbouring AU (Fig. 1a ). These compact dimeric forms are consistent with previous structures of GalNAc-T2 in complexes with UDP and Mn þ 2 (protein data bank (PDB) entry 2FFV  14  ), and UDP or UDP-GalNAc/Mn þ 2 and peptides (PDB entries 4D0T, 4D0Z and 4D11 (ref. 16)). However, members of this family of enzymes are proposed to be monomers in solution  16, 24, 25  (discussed below). The crystal structures show that the typical GT-A fold is located in the N-terminal region and the lectin domain is located in the C-terminal region (Fig. 1a ). The density for all the glycopeptides and UDP/Mn þ 2 was well defined in most monomers (Fig. 1b ). These results are further supported by tryptophan fluorescence spectroscopy experiments showing that peptides/glycopeptides can bind fairly well to GalNAc-T2 in the absence or presence of UDP/Mn þ 2 (Supplementary Table  2 ).
The dissociation constants (K d s) of the peptides were in the low mM range and very similar among them except for the naked peptide MUC5AC, in which the K d in the absence of UDP/Mn þ 2 is fourfold better than in the presence of the nucleotide (Supplementary Table  2 ). The structures also feature a flexible loop that can oscillate between closed and open conformations (Fig. 1c, d ) and is associated with the active and inactive states of the enzyme, as described in previous studies  14, 16. We also reported that the UDP-GalNAc moiety was a key factor to maintain the flexible loop in a closed conformation regardless of whether the peptide was present or not (Fig. 1c  and PDB entry 4D0T)  16. On the contrary, previous structures containing UDP (Fig. 1c  and PDB entries 2FFU  14  , 4D11 (ref.
16) and 2FFV  14  ) including our complex of GalNAc-T2-UDP-MUC5AC-13 (Fig. 1c, d ) exhibit a mix of loop conformations (semi-open, open and closed) along the catalytic cycle (Fig. 1c )  16. Furthermore, the flexible loop dynamics are coupled to the key catalytic residue Trp331 mobility that can adopt 'in' (inside of the active site) and 'out' (outside of the active site) conformations, which in turn are associated to the active or inactive states of GalNAc-T2, respectively (Fig. 1d ). Interestingly, MUC5AC-13 was bound to an active form of GalNAc-T2, whereas MUC5AC-Cys13 and MUC5AC-3-13 were unpredictably trapped bound to the inactive form of GalNAc-T2 (Fig. 1b-d ). In these cases, the flexible loop was found in an open conformation, different from the one previously described for the GalNAc-T2-UDP complex (PDB entry 2FFV  14  and root-mean-square deviation (RMSD) of 4.
Time-course experiments were carried out and the reactions were analysed and evaluated by MALDI-TOF-MS. The specific activities under linear conditions were inferred from the mass spectrometry data (see Methods for details). One unit of enzyme is defined as the amount of enzyme that transfers 1 mmol of GalNAc in 1 min using the standard reaction mixture and conditions. The velocity values were obtained from three independent experiments and errors are o20%. close to UDP (Fig. 3a ). Particularly, Ser5 is quite close to UDP (the distance between the Ser5 oxygen and the b-phosphate is 3. 81 Å) and is potentially in a position to attack the anomeric carbon of UDP-GalNAc (see Fig. 3c  for further information). This structure is compatible with the fact that GalNAc-T2 prefers to glycosylate N-terminal acceptor sites of glycopeptides efficiently, but the structure itself does not explain why Thr3 is the most favourable acceptor site, as shown earlier by kinetic studies, or why glycosylation optimally takes place at acceptor sites located ten residues N-terminal of an existing site of glycosylation  17, 23.
(c) Overlay of one of the monomers of GalNAc-T2 containing UDP-GalNAc (PDB entry 4D0T  16  ) in the active site with GalNAc-T2-UDP-MUC5AC-13 complex. The structure shows that Ser5 is close to the anomeric carbon of UDP-GalNAc. (d) Free energy landscapes of peptide binding for each one of the three-acceptor sites. (e) Comparison between a reactive conformation of the peptide MUC5AC-13 for the Thr9 (yellow) and Thr3 (grey) acceptor sites. active site was monitored by metadynamics using two collective variables. The first variable was the distance between the centre of mass of the UDP-GalNAc and the centre of mass of the side chain of the peptide acceptor sites (Thr3, Ser5 or Thr9). This variable measures directly the penetration of the peptide into the active site. The second collective variable was taken as the RMSD of the peptide structure and measures changes in peptide conformation.
4a , top-right panel and Supplementary Fig. 3a ) and (iii) extended structures exhibiting fully separated domains (Fig. 4a , bottom panel and Supplementary Fig. 3b ). Our AFM experiments (Fig. 4a , bottom panel and Supplementary Fig. 3b ) support, for the first time, that the extended state visualized in the crystal structure of GalNAc-T2 in complex with UDP and the EA2 peptide (PDB entry 2FFU  14  ; Fig. 1c ), in which the domains are separate, is not an artefact of the crystallographic conditions. The highly compact and compact species show height values of E8-10 nm (Fig. 4a , top panel), whereas the average height for the extended species is smaller, reaching E5 nm (Fig. 4a , bottom panel). The size of the separated domains in the images is barely larger than that estimated from the crystal structure (3.
5 and 4. 7 nm for the lectin and the catalytic domains, respectively, in PDB entry 2FFU  14  ) mainly as a result of hydration  29. The intermediate sizes observed for the compact species thus suggest a slight overlap between both domains of the enzyme. The enzyme in the apo form or bound to UDP or UDP-GalNAc in the presence of Mn þ 2 adopts either the very compact or the compact structure (Fig. 4a , top panel). However, the three different conformational states are present when the enzyme is either bound to EA2 or to glycopeptides such as MUC5ACs-Cys9 (see sequence in Supplementary Table  1 ) or MUC5AC-13 alone or in combination with UDP/Mn þ 2 (Fig. 4a ). Although our data does not provide relevant information of the distribution between different conformational states, clearly demonstrates the highly dynamic nature of the GalNAc-T2 enzyme.
GalNAc-T2 monomeric compact conformations. To quantify the GalNAc-T2 conformational states in solution, we measured SAXS data for a large number of conditions. The capacity to describe this data with available crystallographic structures for the monomeric and dimeric forms of GalNAc-T2 was tested with the programme CRYSOL  30. The poor agreement of either structures to the experimental SAXS curves (see Supplementary Table  3 ), prompted us to study the enzyme flexibility using the Ensemble Optimization Method (EOM)  31. Ensemble analysis of solution scattering curves for the apo enzyme at different concentrations revealed a unique distinguishable bimodal distribution of monomeric compact and extended structural ensembles that have not been found before in other types of proteins by using this method (Fig.
In contrast to AFM, SAXS is also able to identify a significant population of dimers that supports the dimeric arrangement found for our crystal structures, with the exception of the PDB entry 2FFU  14  , in which the enzyme is monomeric (we used the PISA server to determine the quaternary structure of GalNAc-T2 in the crystals). The SAXS fits with EOM were notably better when the three crystallographic dimeric arrangements were combined with the monomeric forms (Fig. 4b , Supplementary Figs  3c, 4  and 5 , and Supplementary Methods). In particular, three different pools were built consisting in 10,000 monomeric conformations that were separately enriched with 100 copies of the theoretical curves of each of the three dimeric arrangements tested (see Supplementary Methods). The best fit was achieved when the most compact dimer that possesses a buried area of 1,689 Å 2 (PDB entry 2FFV  14  ) was used.
Dimers visualized in crystal structures with a tetragonal space group appear to be the most flexible compact structures of all dimers with E750 Å 2 of buried area. This latter arrangement may explain why this particular dimer can bind glycopeptides. In all three cases, the dimers display similar contacts between the lectin domains and the lectin of one monomer with the catalytic domain of a second monomer (Fig. 4b  and Supplementary Fig. 3c ). Notably, the GalNAc-T2-UDP-MUC5AC-13 complex with an R g of 26. 41 Å is one of the most populated compact molecules in solution unlike less representative structures such as the one found for the PDB entry 2FFU 14 with R g of 30. 5 Å (Fig. 4b ). This result exemplifies that GalNAc-T2 preferentially adopts conformations that might be important for catalytic aspects of this enzyme.
5a )  32. The model predicts, at a qualitative level, the bimodal nature of the R g distribution revealed by SAXS data (Fig. 5b ). It is noteworthy that our structure of GalNAc-T2-UDP-MUC5AC-13 complex is located in the left peak. (Right panel) Of the three crystallographic dimers tested, the SAXS data fit better with the dimer belonging to the PDB entry 2FFV 14. (c) R g distributions for the monomeric forms derived from the EOM analysis displaying the decrease of the relative population of the extended conformation upon addition of the glycopeptide. GalNAc-T2 is at a fixed concentration of 5 mg ml À 1 (black is for the enzyme without the peptide) with increasing amounts of the MUC5AC-13 peptide (1 (blue) and 2 mM (red) of the MUC5AC-13 peptide. (d) GalNAc-T2 is in equilibrium with an ensemble of compact and extended structures, and dimeric forms.
6 M ammonium sulfate, 100 mM sodium chloride and 100 mM HEPES (pH 7). The crystals were cryoprotected in saturated lithium sulfate and frozen in a nitrogen gas stream cooled to 100 K. Structure determination and refinement. All data were processed and scaled using the XDS package  34  and CCP4 software  35. Relevant statistics are given in Table  1. The crystal structures were solved by molecular replacement with Phaser  35  and using the PDB entry 4D0T as the template. Initial phases were further improved by cycles of manual model building in Coot  36  and refinement with REFMAC5 (ref. 37). The final models were validated with PROCHECK  38  and model statistics are given in Table  1. The AUs of the tetragonal crystals contain 1 molecule of GalNAc-T2 (Fig. 1 ). Coordinates and structure factors have been deposited in the Worldwide Protein Data Bank (wwPDB, and see Table  1  for the pdb codes).
The data analysis was performed in Prism (GraphPad software) considering a model with a single binding site (see equation (1), where F0 is the intrinsic fluorescence of the enzyme in the absence of quencher (Q), F1 is the observed fluorescence at a given quencher concentration, f a is the fractional degree of fluorescence and K d is the dissociation constant. 1 À F1 F0 ¼ f aÃ½Q Kd þ ½Q ð1Þ Analysis of the enzymatic reaction by MALDI-TOF-MS. We carried out mass spectrometry analyses to determine the transfer activity of GalNAc-T2 apo form and mutants using UDP-GalNAc and the MUC5AC and MUC5AC-13 acceptor peptides. In vitro glycosylation assays were performed as product development assays in 50 ml buffer (25 mM cacodylic acid sodium, pH 7. 4, 10 mM MnCl 2 , 0. 25% Triton X-100), 5 mM UDP-GalNAc (Sigma), 0.
4 software. Spectra were recorded in the positive ion mode and the raw spectra were processed by Bruker FlexAnalysis 3. 4 software. The specific activities under linear conditions were inferred from the mass spectrometry data. One unit of enzyme is defined as the amount of enzyme that transfers 1 mmol GalNAc in 1 min using the standard reaction mixture. Computational details. MD simulations of the enzyme were performed with the Amber11 software package. The protein was modelled with the FF99SB force field, whereas all carbohydrate molecules were modelled with the GLYCAM06 force field  41. A snapshot of the equilibrium MD simulation was taken for the metadynamics simulations. Additional details are in Supplementary Methods. AFM imaging. AFM measurements were performed using a MultiMode 8 AFM system (Bruker).
Images were taken using the Tapping Mode with V-shaped silicon nitride cantilevers with integrated pyramidal 2 nm ultrasharp tips exhibiting a spring constant and a frequency of 0. 03 N m À 1 and 15 kHz, respectively (MSNL-D; Bruker Probes). Additional details are in Supplementary Methods. SAXS and data analysis. SAXS measurements were performed at the European Molecular Biology Laboratory on the storage ring PETRA-III (DESY-Hamburg) on the P12 beamline equipped with a robotic sample changer and a PILATUS-2M. The analysis of the SAXS data was performed by using the EOM  31. Additional details are in Supplementary Methods. Coarse-grained model. We used as a template the crystal structure of the Gal-NAc-T2-UDP-MUC5AC-13 complex to generate our coarse-grained model. The protein was treated as two interacting rigid domains (the catalytic and the lectin domains) joined by a 12-residue semi-flexible linker that is described by a semielastic WLC model  32, 42, 43  (Fig.
Protein, flexible loop, nucleotides, GalNAc moiety and peptides are coloured in grey, yellow, brown, orange and green, respectively. The active and inactive states are indicated as closed and open conformations, respectively. (d) Close-up view of GalNAc-T2-MUC5AC-Cys13, GalNAc-T2-MUC5AC-13-UDP and GalNAc-T2-UDP (PDB entry 2FFV 14  ) complexes. The colours are the same as shown above. The loop in which Trp331 is located is in black. In the latter complex, UDP is shown as inverted relative to the most frequent position found for UDP and has been proposed to be a conformation ready to depart from the active site 14, 16. Trp331 is depicted as black carbon atoms and adopts 'in' and 'out' conformations.
Overall structures of GalNAc-T2 with different R g s are shown with lectin and catalytic domains in red and yellow, respectively, and flexible linker is shown in grey. It is noteworthy that our structure of GalNAc-T2-UDP-MUC5AC-13 complex is located in the left peak. (Right panel) Of the three crystallographic dimers tested, the SAXS data fit better with the dimer belonging to the PDB entry 2FFV 14. (c) R g distributions for the monomeric forms derived from the EOM analysis displaying the decrease of the relative population of the extended conformation upon addition of the glycopeptide. GalNAc-T2 is at a fixed concentration of 5 mg ml À 1 (black is for the enzyme without the peptide) with increasing amounts of the MUC5AC-13 peptide (1 (blue) and 2 mM (red) of the MUC5AC-13 peptide. (d) GalNAc-T2 is in equilibrium with an ensemble of compact and extended structures, and dimeric forms.
Acknowledgements We thank synchrotron radiation sources DLS (Oxford),  ALBA (Barcelona)  and  PETRA-III (DESY/Hamburg ), and in particular beamlines I03 (experiment number  MX8035-21  and  MX8035-24 ), I04-1 (experiment number  MX8035-17 ),  XALOC  and  P12  ( SAXS-172 ), respectively. We thank  ARAID , the  MEC  ( BFU2010-19504 ,  CTQ2013-44367-C2-2-P ,  CTQ2011-25871 ,  BIO2010-14983 ,  CTQ2012-36365  and  MAT2012-38318 ), the  DNRF  ( DNRF107 ), the  DGA  ( B89  and  B18 ), the  Generalitat de Catalunya  ( 2014SGR-987 ),  ANR-CHEX-2011  and  ATIP-Avenir (PB)  for financial support. The research leading to these results has also received funding from the  FP7  (2007-2013) under BioStruct-X (grant agreement  N°283570  and  BIOSTRUCTX_5186 ). We also acknowledge the computer support, technical expertise, and assistance provided by the  Barcelona Supercomputing Center-Centro Nacional de Supercomputacion (BSC-CNS ).
196/0. 232 0. 149/0. 178 0. 156/0. 185 No. of atoms Protein 4,023 4,040 3,991 Glycopeptide 114 129 107 Sulfate 60 155 110 Ethylenglycol 56 - - UDP - - 25 Mn þ 2 - - 1 Water 556 800 614 B-factors (Å 2 ) Protein 22. 41 19. 03 26. 71 Glycopeptide 33. 81 19. 46 19. 29 Sulfate 56. 01 61. 48 69. 44 Ethylenglycol 38. 75 - - UDP - - 21. 88 Mn þ 2 - - 19. 29 Water 33. 51 35. 06 39. 02 R. m. s deviations Bond lengths (Å) 0. 019 0. 012 0. 013 Bond angles (°) 2. 027 1. 613 1. 627 PDB ID 5ajn 5ajo 5ajp UDP, uridine diphosphate. *Highest resolution shell is shown in parenthesis. wR merge ¼ ShklSi |I(hkl)i-[I(hkl)]|/Shkl Si I(hkl). zR work ¼ Shkl|F(hkl) o -[F(hkl) c ]|/Shkl F(hkl) o ; R free was calculated as R work , where F(hkl) o values were taken from 3% of data not included in the refinement.
Model fitting and selection Model parameters for the Gal apagos phylogenetic data set were estimated via maximum likelihood in the DAISIE package. We compared equal-rates DAISIE models that assume that all lineages share the same dynamics to differential-rate models that distinguish DF-type species (defined as those mainland finch species that have the potential to produce a DF-type radiation) from the remaining bird taxa in the archipelago (non-DF-type species), thus allowing the DF radiation to differ in one or more parameters that govern the macroevolutionary process (Table  S8 ). While it is possible that not all non-DFtype species share the same parameters, we chose to compare DF and non-DF-type species because we were interested in the specific question of whether DFs are an exceptional radiation.
We therefore refitted the three best models assuming P DF-type values varying between 0. 001 and 0. 28 (the maximum value is the P DF-type in South America if only lineages present on the Gal apagos are taken into account), in order to examine the effect of P DF-type on our main conclusions. There are currently no molecular data available for one of the species in our data set -Progne modesta. To evaluate the effect of the colonisation time of P. modesta on our parameter estimates, we refitted the best model with varying colonisation times of P. modesta between 0. 01 and 4 Mya.
2015). Hence, phylogenetic data sets with across-lineage sampling of members of a higher taxon, ecological guild or trophic level comprising independent colonising lineages in the same insular system are still rare, especially for remote settings. At present, the Gal apagos avian data set is the only one that meets these criteria. While it has only a moderate number of avian species compared to other more species-rich communities, we have made a thorough examination of the ability of our framework to correctly estimate parameters and distinguish between models, which convincingly showed that our data set is large enough to produce robust conclusions. We hope that the availability of DAISIE will encourage island biologists to collect phylogenetic data sets that encompass whole communities and include estimates of stem ages of independent island lineages, thus enabling tests of equilibrium dynamics over evolutionary time scales on a variety of insular systems.
ACKNOWLEDGMENTS We thank  Alex Pigot ,  Bart Haegeman ,  Charles Marshall ,  Lisa Becking ,  Will Pearse ,  Menno Schilthuizen ,  Dolph Schluter , the editors and two anonymous referees for valuable comments on the manuscript. Ralph Tiedemann ,  Thijs Janzen ,  Jason Ali ,  Chris Cooney ,  James Rosindell ,  Pablo Vargas ,  Diana Robledo ,  Frida Lona  and  Vincent Savolainen  for help and advice;  Lucinda Lawson  and  Kenneth Petren  for providing Darwin's finch DNA matrices;  Ruben Heleno  and  Steve Arlow  for permission to use their photographs of Gal apagos birds;  Dick Visser  for help with figures; ZEIK ( University of Potsdam ) and  Millipede (University of Groningen ) clusters for computational resources. This work was funded by an  Alexander von Humboldt Postdoctoral Fellowship  to LMV, a  NERC  fellowship to ABP and a  NWO-VICI  grant to RSE.
Acknowledgments The authors would like to thank  Yuewen Gong , B. M. and Ph. D. , Professor, and  Associate Dean  (Research) from the  Faculty of Pharmacy ,  University of Manitoba, Winnipeg, Manitoba, Canada , for the revision. Bingnan Cui. Ph. D. ,  Hua Hua, M. D. ,  Xiaohong Zhang, M. D. ,  Xiaohong Wu, Ph. D. ,  Dong Shen, M. D. ,  Ding Xu, M. D. ,  Zhifang Yan, M. D. ,  Yuming Wang, Ph. D. , and  Xue Zeng M. D. from the  Department of Dermatology of the Guang'anmen Hospital of the China Academy of Chinese Medical Science in Beijing  for the acquisition of clinical data.
Genetic disruption of BASIGIN does not significantly affect MMP2 and MMP9 production by tumour and fibroblastic cells The impact of BASIGIN on MMPs production by tumour cell lines was first evaluated by analysing the MMP2 and MMP9 activities in CM from wt versus BSG -/-tumour cell lines alone, or co-cultured with fibroblasts. Zymogel quantification showed that BSG invalidation does not significantly affect MMP2 and MMP9 production by LS174T (Figure  1C   MMP2 and MMP9 activities were also evaluated in MEF wt versus bsg -/-. We used MEF from BSG-null mice invalidated for BASIGIN by an insertion of a MC1neo box into the gene  [30]. In these cells, as in ZFN invalidated cells, the disruption of BSG gene results in the total loss of the protein (data not shown). As observed in tumour cells, the invalidation of BASIGIN in MEF does not notably modify the level of MMP2 and MMP9 in extra cellular media (Figure  2A.
The same results were obtained for intracellular expression of MMPs by using qPCR analysis. Then, we developed co-cultures experiments between fibroblasts and tumour cells, and measured MMPs production in the extracellular media. We showed that not only did BASIGIN disruption not affect MMPs production, but that co-cultures also did not significantly increase MMPs production in the media. This surprising finding indicates that, contrary to what has been previously reported, fibroblasts and tumour cells do not seem to cooperate to produce more MMPs in the extracellular medium. The use of mouse fibroblasts (MEF) lead to the same result, namely a lack of significant effects on MMPs production while comparing MEF-bsg -/- vs MEF-wt, or in co-culture with tumour cells. How can we then explain these contradictory results? First of all as mentioned above, many previously published data are based on either forced expression (transfection or used of a purified recombinant soluble form of BASIGIN), or with incomplete invalidation (siRNA or antisense).
ACKNOWLEDGMENTS AND FUNDING The team was funded from  Ligue Nationale Contre le Cancer (Equipe labellisée LNCC) ,  Fondation ARC ,  INCa ,  ANR , the  EU-FP7  " METOXIA ",  SERVIER-CNRS, and Centre Lacassagne. IM received a fellowship from  LNCC. We thank  Dr. Scott Kenneth Parks  for editorial correction of the manuscript.
Polyclonal antibodies against human IgG and IgM were from DAKO (Trappes). DSA detection in sera was considered positive when the DSA score (PBMC median of fluorescence X % of positive-PBMC) was !2. The median of fluorescence for IgG and IgM could not be used when comparing new animals with historical controls. Samples were acquired on a BD FACSCANTO TM flow cytometer (BD Bioscience) and analyzed with FlowJo software (FlowJo LLC, Ashland, OR).