dataesr/scientific-paragraphs-categorization huggingface dataset:
Bionomics During this research, A. dryadina was found in a rather flattened part of the uppermost area of a mountain ridge, with high and predominantly uniform forest cover dominated by oaks (Quercus costarricensis) at 3000-3100 m a. s. l. Individuals are very actively patrolling. Both males and females fly rather rapidly, but not as fast as some other Andean satyrines, such as Corades. On the wing, they resemble somewhat some species of Pronophila or Pseudomaniola, with the difference that they move invariably high above the ground, usually in the canopy, compared to other Andean or Mesoamerican satyrines which can be observed in the subcanopy but frequently descend to ground level. Perching coupled with territorial behavior among the Pronophilina satyrines is typical of very fast flying species in Corades and Junea, but is also present in some Steremnia Thieme, Daedalma, and Thiemeia.
They were not attracted to traps placed at 1-2 m baited with dog excrement, which otherwise proved effective with other local satyrines. They were never observed on hilltops or other prominent topographical features, such as outstanding trees, contrary to territorial Corades and Junea, or non-territorial Steremnia, Pedaliodes or several other genera in the Andes. Male individuals were seen engaging in short interactions consisting of following one another for a short while, probably identifying whether the followed individual was a female, but no contest behavior was observed. No courtship or mating was observed. Individuals of A. dryadina spent most of the time in the canopy and were extremely reluctant to approach ground level, which was observed only above a boggy clearing covered partially with Vaccinium and Hypericum bushes.
Even then, individuals overflew the plants but never approached within 2 m of the ground. DeVries (1987) reports A. dryadina as a rare butterfly. This is certainly not the case in the Cerro de la Muerte, where by the end of June and beginning of July A. dryadina was arguably common, and also the most obvious butterfly considering it was very active, patrolling, and a large butterfly that could unlikely be confused with any other species. In February-March (dry season), only one individual of A. dryadina was observed in the same area where they were common in June-July (rainy season), which indicates that the species is possibly strongly seasonal. Such important seasonal fluctuation in abundance is uncommon among tropical Pronophilina and has been reported so far only for species occurring in páramo grassland, for example, Steromapedaliodes (Pyrcz et al 2017).
Discussion The currently available morphological and genetic data resulted in incongruent inferences about the relationships among the studied species. In addition, the two sequenced genes COI and RpS5 also yielded different results, segregating either the two species of Arhuaco within the Pronophila clade, placing Arhuaco ica as sister to Pronophila + Arhuaco dryadina or indicating A. ica as sister to J. dorinda, and even placing Pseudomaniola gerlinda within the Pronophila clade. Although only two genetic markers were studied here, the phylogenies within this section of Pronophilina seem unresolved, and the monophyly of Arhuaco should be considered as uncertain. Interestingly, however, the molecular results strongly indicate a clade composed of Pronophila, Arhuaco, Junea, and Pseudomaniola, and, if adopting a broader taxonomic approach, all these could be lumped within a larger genus Pronophila.
These results, incidentally, agree with morphological evidence which indicates close relationships among these genera, possibly forming a monophyletic group. Putative synapomorphies, especially in the wing shape and color pattern involving the so-called elements of the nymphalid groundplan sensu Schwanwitsch  (Nijhout 1991) , suggest a closer relationship of A. ica with A. dryadina than with any other genus or species of the subtribe Pronophilina and indicate that Arhuaco is indeed monophyletic. The broken median band with the basal edge connected to the postbasal line is a rare distortion of the nymphalid groundplan termed "Pierellization" by Schwantwitsch, described for the Haeterini genus Pierella  (Nijhout op. cit. ). Such a Pierellization in the two species of Arhuaco is a unique state in any genus or species of Pronophilina and was considered to be a strong generic synapomorphy  (Pyrcz 2004 ).
On the other hand, the two species do not present any apparent putative synapomorphic characters enabling to associate them with the genus Pronophila, presenting rather some features of other genera, in particular of Junea and Pseudomaniola. Ecological and behavioral data also indicate closer affinities between A. ica and A. dryadina than with any species of Pronophila. They both occur in the same kind of habitat, high elevation forests, at higher elevations than Pronophila, usually close to the upper forest limit. They also both have an unusual predilection among the Pronophilina for flying in the high canopy and not necessarily in association with bamboo thickets, in contrast to Pronophila which are invariably found in the vicinity of bamboo and generally in the forest understory. At this stage, there is not enough evidence to ascertain the monophyly of Arhuaco, since it is not supported by molecular data.
It would be, however, premature to synonymize Arhuaco with Pronophila given sharp-cut morphological  differences between the two. More data are needed, in particular the use of more genetic markers, and the inclusion of even more species of Pronophila as well as other species of Junea and Pseudomaniola in the analysis. It is possible that early-stage data would shed some light on the affinities of Arhuaco and Pronophila, as they have on the status of the genus Daedalma, which has very particular larvae compared to other related genera  (Pyrcz et al 2011) , but given the scarcity of Arhuaco ica in the field, and the difficulties in finding pronophiline larvae, and rearing them, requiring in situ work, it will be a very difficult task. The disjunct distribution of Arhuaco in Costa Rica-Panama and northern Colombia, if the genus is confirmed as monophyletic, would be challenging to explain from a biogeographical and evolutionary perspective.
In any case, the timing of such a scenario does not predate the Pleistocene, which is some 1. 85 MYA. It is congruent with the distribution of most Central American species of Pronophilina, an overwhelming majority of which are closely related to north Andean taxa, including Lymanopoda euopis Godman & Salvin, Pedaliodes ereiba Godman & Salvin, Pedaliodes lithochalcis Butler & Druce, Eretris suzannae DeVries, or Pronophila timanthes Salvin  (Casner & Pyrcz 2010 , Pyrcz & Viloria 2012). These are mostly mid-to lowaltitude species whose ancestors presumably dispersed relatively recently from the Andes. However, the presence of the high elevation specialist A. dryadina, and indeed of the endemic Mesoamerican genus Drucina represented by one Costa Rican species D. leonata Butler 1872, and by D.
Acknowledgments Research permits of TP in Costa Rica were issued by the resolution  ACOPAC-IV-001-16  ( Evaluación de  las estructuras genitales de las mariposas hembras en la resolución de problemas filogenéticos y alfa-taxonómicos de las subtribus Euptychiina y Pronophilina en la  Reserva Forestal Los Santos, Pacífico Central de Costa Rica). We would like to thank  Marianne Espeland  ( Forschungs Museum Koenig, Munich ) for attempting to obtain additional DNA sequences of Arhuaco ica.
Cyclin G-bound genes are enriched in PRC1 and Asx Using published datasets, we analysed the correlation between regions bound by Cyclin G in +/UAS-CycG ΔP ; da-Gal/+ imaginal discs and those enriched in PRC1, or PR-DUB components, RNAPolII, or H3K27me3 in wild type wing imaginal discs (S15 Table ). Cyclin G-bound regions were significantly exclusive from H3K27me3, corroborating previous results  [31]. The same comparisons were performed gene-wise and gave the same results (Fig  8 ). Importantly, 78% of Cyclin G-bound genes were also bound by RNAPolII and Pc. We cannot exclude that Cyclin G might co-localize with PRC1 in some cells and with RNAPolII in others. Alternatively, Cyclin G-bound genes might be simultaneously bound by PRC1 and RNAPolII. Considering RNAPolII as a proxy for transcriptional activity, and given that PRC1 has the ability to block transcriptional initiation  [39] , these genes would be most probably paused.
Reads were aligned with the D. melanogaster genome (dm6, r6. 07) using TopHat 2 (v2. 0. 10)  [59]. Unambiguously mapping reads were then assigned to genes and exons described by the Ensembl BDGP5 v77 assembly, by using the "summarizeOverlaps" function from the "GenomicAlignments" package (v 1. 2. 2) in "Union" mode  [60]. Library size normalization and differential expression analysis were both performed with DESeq 2 (v 1. 6. 3). Genes with adjusted p-value below 0. 05 were retained as differentially expressed  [61]. Gene Ontology analysis was performed using DAVID  [62]. For RT-qPCR validations, RNAs were extracted from wing imaginal discs and treated with Turbo DNAse (Ambion). cDNA were synthesized with SuperScript II Reverse transcriptase (Invitrogen) using random primers. RT-qPCR experiments were carried out in a CFX96 system (Bio-Rad) using SsoFast EvaGreen Supermix (Bio-Rad).
1% BSA, Roche complete EDTA-free protease inhibitor cocktail. For immunoprecipitation, 3 μg of anti-Myc antibody (Abcam 9132) were added per tube. Two biological replicates were performed for +/UAS-CycG ΔP ; da-Gal4/+ and one for da-Gal4/+. Library preparation and Illumina sequencing were performed at the ENS Genomic Platform (Paris, France). Libraries were prepared using NEXTflex ChIP-Seq Kit (Bioo Scientific), using 38 ng of IP or Input DNA. Libraries were multiplexed by 10 on one flowcell run. 75 bp single read sequencing was performed on a NextSeq 500 device (Illumina). Reads were filtered by the "fastq_quality_filter" command from the "fastx-Toolkit" package (http://hannonlab. cshl. edu/fastx_toolkit/), using a threshold of 90% bases with mapping quality ! 20. Reads are shown on S18 Table.
Those that successfully passed the filtering step were aligned to the D. melanogaster genome (dm6, r6. 07) using Bowtie 2 (http://bowtie-bio. sourceforge. net/bowtie2/ ) (v 2. 1. 0) with default parameters  [65]. Peaks were called by MACS2 (v 2. 1. 0) by comparing each ChIP to its input library, with fragment size fixed at 110 bp and otherwise default parameters  [66]. Peak reproducibility between the two biological replicates was then analysed with the IDR method (https://www. encodeproject. org/software/idr/) [67]. Briefly, an IDR score was assigned to each peak by the "batch-consistency-analysis" function, using the recommended parameters for MACS peaks (peak ranking based on p-value). Peaks below the 0. 05 threshold were considered reproducible. The overlapping reproducible peaks from both replicates were fused using the BEDtools suite "merge" function  [68] , resulting in the final list of peaks kept for subsequent analysis.
Cyclin G-bound genes were defined as genes from the genome annotation file (dm6, r6. 07) which overlapped at least one of these Cyclin G peaks, as obtained by the BEDtools suite "intersect" function. For ChIP-qPCR validations, ChIPs were performed similarly with the anti-Myc antibody. Rabbit IgG (Diagenode) were used as negative control (mock). qPCR experiments were carried out in a CFX96 system (Bio-Rad) using SsoFast EvaGreen Supermix (Bio-Rad). Three biological replicates-three technical replicates per biological replicate-were performed for each antibody and for the Input. Sequences of primer couples are listed in S16 Table. Data were normalized against Input chromatin. Heatmaps and aggregation plots of Cyclin G signal over gene bodies and TSS were generated using the ngsplot package. (https://github.
Genomic association Genomic loci enriched for Polycomb (Pc), Posterior Sex Comb (Psc), Polyhomeotic (Ph), RNA Polymerase II (RNAPolII) and H3K27me3 in wild type imaginal discs of third instar larvae were retrieved from GEO (GSE42106)  [70] [71]  (H3K27me3_WholeWingDisc GSM103 2567, PcRJ_AnteriorWingDisc GSM1032571, PcRJ_PosteriorWingDisc GSM1032574, Ph_WholeWingDisc GSM1032576, PolII_WholeWingDisc GSM1032577, Psc_WholeWing-Disc GSM1032578. Binding sites for Pc in the whole wing disc were defined as the overlap between Pc binding sites in the anterior and posterior wing disc compartment, as obtained by the BEDtools "intersect" function. For Asx and Calypso, the bed files were a kind gift from Dr. J. Mu ¨ller  [34]. The mappability file for dm6 genome with 25 nt reads (the smallest size in the compared data) was generated using the Peakseq code (http://archive.
gersteinlab. org/proj/ PeakSeq/ Mappability_Map/Code). The overall size of the mappable genome was used as the effective genome size for the GAT software (https://github. com/AndreasHeger/gat) to assess the significance of the overlap between peaks of Cyclin G and other factors  [72]. As GAT performs a two-tailed test, it displays low p-values both for significant overlap and exclusion (as between Cyclin G and H3K27me3). Gene overlap significance assessment was made as follows: under the null hypothesis, genes that are enriched for Asx, Calypso, Pc, Psc, Ph, RNAPolII or H3K27me3 in wild type imaginal discs of third instar larvae should not exhibit any bias towards Cyclin G targets. Thus, the overlap between n enriched genes and K Cyclin G targets genes should be explained by random sampling without replacement of n genes within the total amount N of D.
Acknowledgments We thank  Dr. E. Mouchel-Vielh  and  Dr. J-M. Gibert  for stimulating discussions and critical reading of the manuscript, the  Bloomington Stock Center  for fly strains,  Dr. J. Mu  ¨ller for the alleles of Asx and caly and for the Asx and Calypso ChIP bed files,  Dr. M. Dominguez  and  Dr S. Juarez-Carreño  for Gal4 drivers,  Dr. M. Gho  for the flp line.
This work was supported by grants of the  Centre National de la Recherche Scientifique (CNRS ) http://www. cnrs. fr/index. html, Sorbonne Universite(SU)  http://www. sorbonne-universite. fr/ Universites http://www. sorbonneuniversites. fr (grant  SU-14-R-CDV-05-1 ),  Fondation ARC pour la Recherche sur le Cancer  https://www. fondation-arc. org(grant  PJA20131200314 ) to FP, Fondation pour la Recherche medicale https://www. frm. org(grant  FDT20160435164 ) to JD,  Agence Nationale de la Recherche ,  France Ge  ´nomique national infrastructure https://www. france-genomique. org/spip/spip. php?article82&lang=fr (grant  ANR-10-INBS-09 ) to SLC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Le X was therefore selected for the structural study to determine the molecular basis of the thermodynamic behavior. Structural Analysis. The three fluoro-RSL proteins were cocrystallized with the Le X tetrasaccharide using the vapor diffusion method and yielded diamond-shaped crystals diffracting to high resolution (1. 15 to 1. 35 Å). All structures belonged to the F23 space group with two independent monomers in the asymmetric unit (Table  S4 , PDB ID 5O7W, 5O7V, and 5O7U). The presence of the 3-fold axis resulted in the generation of two trimers (A and B), which are similar to the RSL native structure (2BT9) with RMSD lower than 0. 2 Å (Figure  3a ). The electron density of the Trp residues in the binding sites clearly indicated the presence of the fluorine at each expected position (Figures  S6-S8 ).
1 min, 60-5% B; 20. 1-29 min, 5% B. The maXis II ETD mass spectrometer (Bruker, Bremen, Germany) was operated with the captive spray source in positive mode with following settings: mass range, 300-3000 m/z; 1 Hz; source voltage, 1. 6 kV; dry gas flow, 3 L min -1 ; 180 °C. The protein mass spectra were deconvoluted by the data analysis software, using the MaxEnt2 algorithm. The following main parameters were applied: charge carrier, H + ; m/z range, min. 800 to max. 2000; minimum instrument resolving power was set to 50 000. For peak detection SNAP algorithm with the following parameters was used: quality factor threshold 0. 9, S/N threshold 2, and maximum charge state of 12. Amino Acid Sequence Analysis by LC-MS/MS of RSL Species. Twenty micrograms of RSL protein species was reduced with tris(2carboxyethyl)phosphine (TCEP, final 10 mM) and alkylated with chloroacetamide (final 40 mM) for 10 min by shaking at 550 rpm at 95 °C.
1% formic acid; 0-2 min, 4% B; 2-90 min, 4-25% B; 90-95 min, 25-95% B, 96-110 min, 95% B; 110-110. 1 min, 4% B; 110. 1-125 min, 4% B. The sample was ionized in the nanospray source equipped with stainless steel emitters (Thermo Fisher Scientific) and analyzed in a Thermo Orbitrap velos pro mass spectrometer in positive ion mode by alternating full scan MS (m/z 300 to 2000, 60000 resolution) in the ICR cell and MS/MS by CID of the 20 most intense peaks in the ion trap with dynamic exclusion enabled. The LC-MS/MS data were analyzed by searching a homemade database containing all common contaminants with Proteome Discoverer 1. 4 (Thermo Fisher Scientific) and Mascot 2. 4. 1 (MatrixScience, London, UK). Carbamidomethylation on cysteine was entered as fixed and oxidation on methionine and fluorination on tryptophan as variable modification.
Detailed search criteria were used as follows: trypsin or chymotrypsin; maximum missed cleavage sites, 2; search mode, MS/MS ion search with decoy database search included; precursor mass tolerance, ±10 ppm; product mass tolerance, ±0. 7 Da; label free quantitation of precursor ions was performed with Proteome Discoverer 1. 4. Peptide precursor ion intensities were normalized on total RSL protein intensity and compared for each peptide over the different protein species. Differential Scanning Fluorimetry (DSF). DSF was performed with a 7500 Real Time PCR System (Applied Biosystems PerkinElmer Corp. , Foster City, CA). Protein samples of 3. 5-11. 0 μM were measured in PBS (9. 55 mM Na 2 HPO 4 , 136. 89 mM NaCl, 2. 68 mM KCl, 1. 47 mM KH 2 PO 4 ) with 8× Sypro Orange (Sigma-Aldrich) in the presence (8 μM) or absence of D-(+)-mannose (Carl Roth).
ACS Chemical Biology Articles Differential Scanning Calorimetry (DSC). DSC was performed with a VP-DSC (MicroCal, Inc. , Northampton, MA). Protein samples were prepared in 20 mM Tris/Cl, 100 mM NaCl, 1 mM αMeFuc, pH 7. 5, with concentrations of 1. 5 mg mL -1 for RSL and RSL[5FW] and 1. 0 mg mL -1 for RSL[4FW] and RSL[7FW]. Measurements were performed at 30 psi pressure and a scan rate of 1 °C min -1. Data was analyzed with the MicroCal Origin software (VP-DSC version). Glycan Microarray Analysis. The lectins were biotinylated using the EZ-Link Sulfo-NHS-LC-Biotinylation Kit (Thermo Fisher), according to the manufacturers recommendations. The glycan microarrays contained 317 carbohydrate ligands and were printed as described previously. 36 The microarray slides were blocked with blocking buffer (50 mM ethanolamide in 50 mM borate buffer, pH 8.
Fluorescent measurements were performed using the ScanArray 4000 Microarray Analysis System (PerkinElmer, Waltham, MA). Fluorescence intensities were quantified using the ScanArray Express Microarray Analysis System 4. 0 (PerkinElmer) and the data was further analyzed with Microsoft Excel (Microsoft Corporation, Redmond, WA). Isothermal Titration Calorimetry (ITC). ITC experiments were performed on a MicroCal iTC200 microcalorimeter (Malvern Instruments Ltd. , Worcestershire, UK). The experiments were carried out at 25 °C. αMeFuc (Tokyo Chemical Industry Europe), HType2, and Le X tetrasacharides (ELICITYL, Crolles, France), as well as the lyophilized proteins, were dissolved in the same buffer (20 mM Tris/Cl, 100 mM NaCl, pH 7. 5). Protein concentrations in the microcalorimeter cell (204 μL) varied from 0.
0476 to 0. 0554 mM and between 0. 242 and 0. 266 mM for measurements with αMeFuc/HType2 and Le X , respectively. Twenty injections with 1 μL of sugar solutions (1 or 1. 5 mM of αMeFuc and HType2 or 5 mM of Le X ) were performed at intervals of 120 s with stirring at 750 rpm. The obtained data was integrated and fitted to a theoretical titration curve with Origin software (OriginLab Corporation, Northampton, MA) applying the one-set-of-sites model. The protein concentration was determined using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). An extinction coefficient of 43. 68 × 10 4 M -1 cm -1 was used. For the variants, the concentration was adjusted in the software to obtain the binding site stoichiometry of n = 2, that is, one inter-and intramonomeric binding site per monomer as confirmed by the crystal structures.
Crystallization and Structure Determination. The three fluorinated RSL samples were dissolved in 20 mM Hepes 7. 5 and 150 mM NaCl to 8 mg mL -1 and cocrystallized with 1 mM Le X tetrasaccharide (Ellicityl, Crolles, France). Crystallization screening was performed using the hanging drop vapor diffusion at 19 °C. Diamond shaped crystals were obtained in a few days from solution 1-8 and 1-9 of the Morpheus screen or 1-30 from the Midas screen (Molecular Dimension Ltd. , Newmarket, UK). Single crystals were directly mounted in a cryoloop and flash frozen in liquid nitrogen. RSL[7FW] diffraction data were collected at 100 K at the European Synchrotron Radiation Facility (Grenoble, France) on BM30A-FIP using an ADSC Q315r detector, while those for RSL[4FW] and RSL[5FW] were collected at Synchrotron Soleil (Saint Aubin, France) on Proxima 1 using a Pilatus 6 M detector.
The data were processed using XDS,  40  and their quality statistics are summarized in Table  S4. All further computing was performed using the CCP4 suite unless otherwise stated. 41 The RSL[7FW] and RSL[5FW] structures were solved by molecular replacement (MR) using PHASER and the coordinates of chain A from PDB-ID 2BT9 to search for two copies in the asymmetric unit. 42 After initial rebuilding with ARP/WARP,  43  the model was refined with restrained maximum likelihood refinement using REFMAC 5. 8,  44  iterated with manual rebuilding in Coot. 45 The RSL[4FW] structure was also solved by MR using PHASER, but the coordinates of the protein chain A of RSL[5FW] were used as the search model. No rebuilding with ARP/WARP was required. Anisotropic refinement was introduced at the end of the refinement of all structures.
Five percent of the observations were set aside for cross-validation analysis, and hydrogen atoms were added in their riding positions and used for geometry and structure-factor calculations. Incorporation of the ligand was performed after inspection of 2F o -DF c weighted maps. The 7FW ligand library was constructed with Acedrg. 46 Water molecules, introduced first with ARP/WARP and then automatically using Coot, were inspected manually. The model was validated with the wwPDB Validation server (http://wwpdb-validation. wwpdb. org), and the glycans were checked with Privateer. 47 The coordinates were deposited in the Protein Data Bank under codes 5O7U, 5O7V, and 5O7W, for the structures of RSL[7FW], RSL[5FW], and RSL[4FW], respectively. We clearly observed the electron density of the carbohydrate ligand in both binding sites of all structures with the exception of chain A of RSL[4FW], where a glycerol molecule was present in the intramonomeric site.
The electron density in the intermonomeric site was of higher quality than in the intramonomeric site in all structures, and the tetrasaccharide was distinctly defined. In contrast, some parts were conformationally disordered in the intramonomeric sites. Molecular Modeling. The crystal structure of wild-type RSL complexed with αMeFuc was used as a starting reference to build all models (PDB ID 2BT9). 17 The fluorinated tryptophans were parametrized for compatibility with the AMBER-ff14SB force field, 48 using available standard parameters for fluorine for bonding and van der Waals parameters. Fluorination is expected to induce a large modification of the π electron density on the indole ring. 14 Therefore we performed quantum chemistry calculations in order to obtain representative atomic charges for each fluorinated tryptophan.