dataesr/scientific-paragraphs-categorization huggingface dataset:
Genotype concordance between WES and genotyping data was verified, and no inconsistencies were found. Population Genetics Analyses. 166,496 variants in common between Cilento villages and TSI, were selected for the analyses. A Principal Component Analysis (PCA) was performed on Campora, Gioi, Cardile, and TSI with PLINK 1. 9 software  44  , using the 106,828 common (MAF > 5%) and the 59,668 rare and low-frequency (MAF ≤ 5%) variants separately. LD-based demographic inference for the Cilento and TSI populations was performed in the NeON R-package  45  , reconstructing the long term effective population size (Ne) from 5000 to 30000 years ago. To detect the ROH, 60,837 among the 106,828 common variants were selected to be independent using the LD pruning option of PLINK 1. 9: In windows of 50 SNPs, LD is calculated between each SNP pair, if for a pair of SNPs, the LD r 2 exceeds 0.
Imputation quality. We sought to verify if the use of the Cilento WES data as a local reference panel (LRP) in addition to the 1KG_Ph3 1 could improve the accuracy of genome-wide imputation. To do this, we performed two imputations: the first using only the 1KG_Ph3 as reference panel, the second using a combination of both the 1KG_Ph3 and a Cilento LRP of WES data (1KG_Ph3 + WES). Variants from both Illumina 370 K and OmniExpress arrays and WES on chromosome 10 were filtered for genotyping call rate ≥95% and MAF ≥ 0. 01. Mendelian errors in the genotyping data were located with PLINK 1. 9 and set to missing. Two individuals were excluded from this analysis for having array genotyping rates below 70%. Haplotypes were reconstructed in two groups, one for each genotyping platform. To create the LRP for Cilento, WES data and SNPs present on both genotyping arrays were merged for the 245 exome sequenced individuals with high quality genotypes  47.
Acknowledgements We address special thanks to the populations of  Campora, Gioi, and Cardile  for their participation in the study. We thank dr. Debora Chirico , dr. Michelina De Cristofaro , dr. Raffaele D'Urso  and dr. Giovanni D' Arena  for Cilento data collection. We thank  Mariarosaria Aletta  for bibliographic support. ESGI-The research leading to these results has received funding from the  Seventh Framework Programme  [FP7/2007-2013] under grant agreement no. 262055. This work was supported by grants from the  Italian Ministry of Universities  and  CNR  ( Interomics Flagship Project ,  PON03PE_00060_7 ), the  Assessorato Ricerca Regione Campania  ( POR CAMPANIA 2000/2006 MISURA 3. 16 ). A. F. H. was funded by an international Ph. D. fellowship from  Sorbonne Paris Cité  (convention  HERZI15RDXMTSPC1LIETUE ) and by the  Fondation Recherche Médicale  (convention FRM  FDT201805005384 ).
The wide extracellular part of the pore is lined with the residues Thr21, Val47, Met17, Ser54, Asn55, Tyr10 and Tyr13. The shoulder and the following part of the pore are paved with fragments of lipid molecules (Supplementary Fig. 9 ). The comparison of the pores inside the oligomers formed by microbial rhodopsins with known structure shows the uniqueness of the channel in OLPVRII pentamer (Supplementary Fig. 8 ). Remarkably, however, a similar pore organization is observed in the transmembrane domains of pentameric Ligand-Gated Ion Channels (pLGICs) (Supplementary Fig. 10 )  24. Indeed, the ring of five negatively charged amino acids (Glu222 in each subunit) plays the role in the ion selectivity of a structurally characterized pLGIC from Gloeobacter violaceus (GLIC). Even more notably, the crystal structure of GLIC (PDB ID: 4HFI) contains similar pentagons of water molecules inside the transmembrane channel, presumably involved into hydration of the permeation ion, that are followed by the extremely hydrophobic region plugged with the carbon tails of detergent molecules  24.
Compared to other rhodopsins, the length of the protein is reduced due to extremely short N-and C-termini as well as short loops that lack defined secondary structure. Because the differences in the structures of the OLPVRII and Halobacterium salinarum bacteriorhodopsin (bR) are likely to reflect functional differences, we compare the structures of bR and OLPVRII in detail. The assembly of OLPVRII helices into a bundle differs from that in bR (PDB ID 1C3W), with RMSD of the backbone atoms positions of about 1. 8 Å. Importantly, in OLPVRII, helix C is considerably longer, whereas helix F, conversely, is shorter than the corresponding helices of bR (Supplementary Fig. 12 ). The extracellular part of helix G is shifted inward for about 4 Å and the tilt and positions of other helices are also slightly altered in OLPVRII compared to bR (Supplementary Fig.
34 ± 0. 11 (point of half decay ± standard error). The corresponding pKa values for bR are 12. 2 and 2. 7  36, 37  , whereas for PR, the pKa of the proton acceptor is 7. 68  38. These notable differences in pKa appear to stem, first, from the absence in viral rhodopsins of the histidine that is conserved in PRs and forms a hydrogen bond with the proton acceptor thus decreasing its pKa, and, second, the amino acid substitutions A215M and L93S, compared to bR, that alter the properties of the RSB. The photocycle kinetics of monomeric OLPVRII expressed in the C41 strain and reconstituted into soybean lipid vesicles was measured at pH 7. 5. Transient absorption changes after laser illumination are shown in Fig. 5d  at three characteristic wavelengths, 410, 530, and 550 nm. The dataset is best fitted by seven exponentials.
Methods Protein sequence alignment and phylogenetic analysis. The dataset of group II viral rhodopsins was constructed by searching the NCBI nonredundant protein sequence databases along with the TARA 49 metagenomic sequences using BLASTP and TBLASTN. An alignment of 213 identified complete or partial sequences of group II viral rhodopsins including five previously reported was constructed using MUSCLE  50  with default parameters. The conservation of the functionally important amino acids was investigated using the full sequence alignment (Supplementary Data 1). For the sake of clarity, for the Supplementary Fig. 1  we used a reduced number of sequences. To obtain a representative set of 25 TARA metagenomic sequences, we used the CD-HIT suite  51  with default parameters and 60% identity cutoff.
Then we combined 25 representative sequences with five sequences reported before and aligned them using MUSCLE at default parameters. Phylogenetic reconstruction for the Supplementary Fig. 2  was performed using PHYLIP Neighbor Joining method as implemented in the UGENE software  52  with the following parameters: Jones-Taylor-Thornton model, transition/transversion ratio = 2. 0, no gamma distribution applied. The unrooted phylogenetic tree was visualized using iTOL  53. The full list of rhodopsin sequences that were used for sequence alignment and phylogenetic tree construction is given in the Supplementary information. For Supplementary Fig. 3 , reference rhodopsin superfamily proteins were collected from refs. 16, 54. Sequence names contain Genbank accessions after "@" symbol. GenBank Identification (GI) numbers are shown in parentheses for metagenomic sequences used in ref.
16. Protein sequences were aligned using the MUSCLE with default parameters  50  , columns containing a large fraction of gaps (greater than 30%) and nonhomogenous columns defined as described previously  55  were removed from the alignment. The tree was reconstructed using PhyML  56  with LG substitution model, gamma-distributed site rates, empirical amino acid frequencies and a Bayes branch support values. The root of the tree was enforced between heliorhodopsins and the rest of the microbial rhodopsins  54. The tree in Newick format and the multiple protein alignment used for the reconstruction can be found at ftp://ftp. ncbi. nih. gov/pub/yutinn/viral_rhod_2019/. Cloning. We optimized the nucleotide sequence of OLPVRII (UniprotID F2Y2Z0) for E. coli expression using the GeneOptimizer™ software (Life Technologies, USA) and synthesized it commercially (Eurofins).
We analyzed the data sets using the global multiexponential nonlinear least-squares fitting program MEXFIT as was reported earlier  39. We measured the photocycle kinetics of OLPVRII at pH 7. 5 and temperatures between 0 and 30 °C with 10°intervals. For the experiments solubilized monomeric protein expressed in C41 strain was reconstituted into soybean lipid vesicles. Along with this experiment we conducted additional tests. For the SE1-expressed protein we measured the photocycle of both monomers and pentamers for the protein in three different environments: solubilized in DDM, reconstituted into soybean lipids and nanodiscs, and did not observe any significant photocycle differences. The photocycle of the monomers in detergent was shorter (35 ms comparing to 70-100 ms in the other environments), but the order and appearance of the intermediates were the same for all samples.
The fact that photocycles measured for a membrane protein in different environments are very similar is not unique to OLPVRII. Whereas the replacement of the native lipid matrix by detergents significantly changes the kinetics of bR, in opposite, the photocycles of pHR and pSRII were found almost identical upon solubilization in DM  60, 61. Moreover, as we observed the deprotonation of the Schiff base at higher pH values, to ensure the robustness of the data we additionally measured the photocycles of the detergentsolubilized OLPVRII monomers at pH 8. 0, 7. 0, and 6. 0. The protein demonstrated identical behavior at these pH values. BLM measurements. The BLM setup was similar to that described by Bamberg and co-workers  38, 62. Optically BLMs with an area of ∼10 -2 cm 2 were formed across a hole between the two compartments of a cuvette filled with an electrolyte solution (100 mM Tris-HEPES, initial pH 7.
All BLM experiments were carried out at room temperature. Signal recording and shutter/laser-triggering was carried out by Pclamp8 software via a Digidata 1200 Interface (Axon Instruments). Crystallization and structure determination. The crystals were grown using the in meso approach 63 , similar to our previous studies  10, 21. We mixed the solubilized protein with premelted monoolein at 42 °C (Nu-Chek Prep) to form a lipidic mesophase. Final protein concentration in the phase was 20 mg/ml. Then we spotted 150 nl drops of mesophase on a 96-well LCP glass sandwich set (Paul Marienfeld GmbH, Germany; bottom slide is covered with a 0. 2 mm high spacer that has 96 recesses of 5 mm diameter) and overlaid them with 400 nl of precipitant solution using NT8 crystallization robot (Formulatrix). The crystals grew at 22 °C temperature and appeared in 6-12 weeks.
The best crystals were obtained with a protein concentration of 25 mg/ml and 0. 2 M sodium malonate, pH 4. 6, 15% PEG 550 MME. All crystals were harvested using micromounts (MiTeGen, USA) and were flash-cooled and stored in liquid nitrogen for further crystallographic analysis. X-ray diffraction data were collected at P14 beamline of the PETRAIII, Hamburg at 100 K, with an EIGER 16M detector. We processed diffraction images with XDS  64  and scaled the reflection intensities with AIMLESS from the CCP4 suite  65. The crystallographic data statistics are presented in Supplementary Table  1. Reference model (archaerhodopsin-2, PDB 2EI4) for molecular replacement was chosen with the MoRDa pipeline. Initial phases were successfully obtained in P2 1 space group by an Automated Model Building and Rebuilding using Autobuild.
Acknowledgements We are grateful to  Dr. Igor Melnikov  for obtaining the original molecular replacement solution. We acknowledge the  Structural Biology Group of the European Synchrotron Radiation Facility (ESRF)  and The  European Molecular Biology Laboratory (EMBL) unit in Hamburg at Deutsche Elektronen-Synchrotron (DESY)  for granting access to the synchrotron beamlines. This work was supported by the  common program  of  Agence Nationale de la Recherche (ANR), France and Deutsche Forschungsgemeinschaft (DFG) ,  Germany  ( ANR-15-CE11-0029-02/FA 301/11-1 ), by the  DFG Research Unit FOR 2518 (DynIon , project  P4  to JPM,  MA 7525/1-1 ), and by funding from  Frankfurt: Cluster of Excellence Frankfurt Macromolecular Complexes  by the  Max Planck Society  (to E. B. ) and by the  Commissariat à l'Energie Atomique et aux Energies Alternatives (Institut de Biologie Structurale)-Helmholtz-Gemeinschaft Deutscher Forschungszentren (Forschungszentrum Jülich ) Special Terms and Conditions 5.
1 specific agreement. This work used the platforms of the Grenoble Instruct-ERIC center (ISBG;  UMS 3518 CNRS-CEA-UJF-EMBL ) within the  Grenoble Partnership for Structural Biology  (PSB). Platform access was supported by  FRISBI  ( ANR-10-INBS-05-02 ) and GRAL, a project of the  University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS  ( ANR-17-EURE-0003 ). Spectroscopic characterization, time-resolved absorption spectroscopy, and crosslinking experiments were supported by  RSF-DFG  grant ( Helmholtz-RSF Joint Research Groups grant  (RSF No. 19-44-06302 )). D. B. was supported by grant  ANR-14-CE09-0028. F. R. -V. was supported by grant  VIREVO CGL2016-76273-P (AEI/FEDER, EU ) (co-founded with FEDER funds). D. J. M. was funded by the  Deutsche Forschungsgemeinschaft  under  Germany's Excellence Strategy-EXC 2155 "RESIST"-Project ID 39087428.
In particular, chemical shifts of the side chain could be used to determine the absolute configuration of C-24 in several sterols owing to their chemical shifts being insensitive to structural changes remote from the asymmetric carbon  [29]. The lowest energy conformation of the core ring system was more quickly located by seeding the potential energy surface scan with initial coordinates available in X-ray crystallographic CIF files associated with formerly reported cycloartanes  [30] [31] [32]. Subsequent 13 C NMR data comparison of the two possible epimers against the experimental dataset resulted in the prediction of the 24R configuration with a quantifiable confidence of 99. 2%. Accordingly, compound 1, namely nerviside I, was identified as 3β-O-d-xylopyranosyl-1α,24R,31-trihydroxylcycloartan-28-oic acid.
The lowest energy conformation of the core ring system was more quickly located by seeding the potential energy surface scan with initial coordinates available in X-ray crystallographic CIF files associated with formerly reported cycloartanes  [30] [31] [32]. Subsequent 13 C NMR data comparison of the two possible epimers against the experimental dataset resulted in the prediction of the 24R configuration with a quantifiable confidence of 99. 2%. Accordingly, compound 1, namely nerviside I, was identified as 3β-O-D-xylopyranosyl-1α,24R,31-trihydroxylcycloartan-28-oic acid. Molecules 2019, 24, x FOR PEER REVIEW 3 of 8 nervisides A-H  [7, 8] , even though derivatization-based NMR spectroscopy affords reliable outcomes as to this specific point  [9, 10, 27]. To assign the absolute configuration at C-24, 13 C NMR chemical shift calculations of simplified bicyclic models only including cycles C and D were performed using electronic structure methods of the lowest-energy conformer of both C-24 epimers.
The spectral position of triterpene carbon side-chain bands does not vary over extensive sets of derivatives involving the central ring system  [28]. In particular, chemical shifts of the side chain could be used to determine the absolute configuration of C-24 in several sterols owing to their chemical shifts being insensitive to structural changes remote from the asymmetric carbon  [29]. The lowest energy conformation of the core ring system was more quickly located by seeding the potential energy surface scan with initial coordinates available in X-ray crystallographic CIF files associated with formerly reported cycloartanes  [30] [31] [32]. Subsequent 13 C NMR data comparison of the two possible epimers against the experimental dataset resulted in the prediction of the 24R configuration with a quantifiable confidence of 99.
The lowest energy conformation of the core ring system was more quickly located by seeding the potential energy surface scan with initial coordinates available in X-ray crystallographic CIF files associated with formerly reported cycloartanes  [30] [31] [32]. Subsequent 13 C NMR data comparison of the two possible epimers against the experimental dataset resulted in the prediction of the 24R configuration with a quantifiable confidence of 99. 2%. Accordingly, compound 1, namely nerviside I, was identified as 3β-O-D-xylopyranosyl-1α,24R,31-trihydroxylcycloartan-28-oic acid. O 11 , 693. 4219 ). This hinted that 2 differed from 1 by a supplementary acetyl group. Accordingly, both the 1 H and 13 C NMR spectroscopic data were very similar between the two compounds, but the 1 H NMR spectrum of 2 revealed one more methyl group resonating at δ H 1.
General NMR spectra were performed on a Bruker AM500 FT-NMR spectrometer (500 MHz for 1 H NMR and 125 MHz for 13 C NMR). The ESI-HRMS data were generated with a Bruker MicroTOF-QII spectrometer (Bremen, Germany). Open-column chromatography was performed on silica gel 40-63 µm phase (Merck, Darmstadt, Germany) and reversed-phase C 18 (Merck, Darmstadt, Germany). TLC analyses were carried out on precoated silica gel 60 F 254 (Merck, Darmstadt, Germany), and spots were visualized by spraying the plates with 10% H 2 SO 4 solution followed by heating.
Taylor dispersion analysis: experimental conditions TDA was performed on an Agilent 7100 CE instrument (Waldbronn, Germany) using µ-SIL-DNA neutral coated fused silica capillary (Agilent technologies, Waldbronn, Germany), having a length of 45 cm (36. 5 cm to detector) ×75 µm I. D. The carrousel was thermostated at 37°C using an external circulating water bath 600F from Julabo (Germany). Between each analysis, capillaries were rinsed with the corresponding mobile phase (10 min). Samples were injected hydrodynamically at the inlet end of the capillary (50 mbar, 2 s, 16-22 nL). The temperature of the capillary cartridge was set at 37°C. The solutes were monitored at 193 nm and 226 nm. The taylorgrams were recorded with the Chemstation software, and exported to Microsoft Excel for subsequent data processing.
Implementation of Hybrid Automaton With HyTech HyTech  (Henzinger et al. , 1997 ) is a model checking tool developed for the formal verification of real-time systems. It is suitable for modeling of Linear Hybrid Automata and has a powerful set of analysis commands as well as parametric synthesis capability. Using HyTech for modeling of BRN, each gene is modeled by an automaton. Thus, the current state of the BRN would be given by the current state of all the automata considered together. Example 4. The code for implementation of toy example of hybrid automaton geneQ shown in Figure  6  in HyTech is given below. Keyword loc is used to specify the state of automaton (e. g. , q 0 ), keyword while is followed by the invariant condition (e. g. , hq <= dpq1), keyword wait is used to give the rate of increase or decrease during the transition (e.
We apply the proposed Hybrid Process Hitting approach to this network to obtain the Hybrid Automaton for determining the time delays involved in its dynamical evolution. First we note the switch conditions and by using them we model the Hybrid Automaton. Once this Hybrid Automaton model was run in HyTech to compute the time delays, it was observed that the software went Out of Memory. This clearly showed the limitation of HyTech as it was unable to handle 20 Hybrid Automata at one time. The problem was overcome by utilizing the powerful reduction feature of PINT software  (Paulevé, 2017)  which is able to provide Goal-oriented model reduction through its Cutsets feature. Typically a goal is the activation / inhibition of some gene in the modeled Automata Networks for BRNs. From a given initial state, the goal is reachable if a sequence of steps exists which leads to a the state in which goal is present.
After spectroscopic analysis of each product, the diastereomer ent-1 displayed identical NMR data and retention time in HPLC as natural stellettamide A, except for their opposite optical rotation. Therefore, ent-1 was determined as ent-stellettamide A. In summary, the authors achieved the total synthesis of ent-stellettamide A in 15 steps with 11% overall yield. During the synthesis, a new asymmetric [3 þ 2] cycloaddition by using TMSCHN 2 was developed for the rapid access to optically active pyrazolines, which could be further applied for the construction of similar skeletons. The structure of stellettamide B (2) differed from that of stellettamide A (1) on the side chain. In 2001, Kibayashi's group  27  reported the total synthesis of (À)-stellettamide B by a totally different way as Carreira's synthesis for ent-1, especially for the construction of the indolizidine core (Scheme 3).
As we all know that the research of natural products is a labor and resource intensive work, novel and more powerful chromatographic methods should be developed in accelerating the discovery process toward new drug leads from Nature, especially from marine environments which have yet seldom been developed. For example, the LC-MS-based metabolomics approach is a promising way to recognize already known metabolites from marine sponges  105. It may further become crucial in Stelletta-derived drug candidate discovery. Since the high chemical diversity in Stelletta-derived MNPs was established, tandem mass spectrometry could be applied to build molecular networks for new compound searching. Data analysis of MS n fragmentation can be searched and assigned based on the open access databases, such as Global Natural Products Social Molecular Networking (GNPS)  106.
Acknowledgments This paper is dedicated to  Prof. Dr. Yuewei Guo  on the occasion of his 60th birthday, for his pioneer work on Chinese marine natural product chemistry. This work was supported by the  National Natural Science Foundation of China (NSFC , Nos. 41676073 ,  81520108028 ), the  National Natural Science Foundation of China /Centre National de la Recherché Scientifique (NSFC/CNRS)  joint project (No. 81811530284 )," Youth Innovation Promotion Association " (No. 2016258 ) from  Chinese Academy of Sciences , the  SA-SIBS   Scholarship Program , and the  National Key Research and Development Program  ( 2017YFE0103100 ). Figure 16 Stelletta-derived secondary metabolites from 1982 to 2017.
DNA amplification, purification and sequencing protocols are deposited in protocols. io (dx. doi. org/10. 17504/ protocols. io. wpdfdi6). Sequences obtained in this study were submitted to Genbank under the accession numbers indicated in Table  1. The newly-generated ITS and LSU sequences, and homologous sequences retrieved from EMBL/GenBank/DDBJ databases, mainly from  [10, 12, 16, 38] , were separately aligned in MEGA7: Molecular Evolutionary Genetics Analysis version 7. 0  [39]. Alignment gaps were marked with "-" and unresolved nucleotide positions were indicated with "N". Geastrum velutinum was included as outgroup since this species is in section Myceliostroma, subsection Velutina J. C. Zamora, sister clade of subsect. Epigaea  [10]. The maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference analyses are also deposited in protocols.
Results The ITS dataset included 48 sequences of Geastrum specimens, of which 34 were generated in this study (Table  1 ) and 14 obtained from EMBL/GenBank/DDBJ databases. In addition to the new species, sequences were obtained from type collections of five previously described species (Table  1 ). The ITS alignment resulted in 657 unambiguously aligned nucleotide positions (317 constant, 103 parsimony-uninformative, and 237 parsimony-informative). MP analysis resulted in one most parsimonious tree (S1 The evolutionary models chosen by jModel-Test for Bayesian inference were HKY+G for ITS dataset (according to all criteria: AIC, BIC, AICc and DT), and TIM3+I+G for LSU dataset (according to AIC and AICc criteria-BIC and DT suggested TIM3+I, so we decided to use the more thorough algorithm).