dataesr/scientific-paragraphs-categorization huggingface dataset:
Multiple-sequence alignment and phylogeny of EgP1 and Pro_7066 The non-redundant protein sequence database (https:// www. ncbi. nlm. nih. gov/protein/), the Microbial Eukaryote Transcriptome Sequencing Project (MMET) (7. 8 ϫ 10 6 sequences) and the E. gracilis strain Z transcriptome (http:// euglenadb. org/) 3 have been interrogated with PSI-BLASTP and tBLASTN (threshold 0. 0001) using the EgP1 amino acid sequence as a query (File S2). Several hundred sequences of bacterial origin with Ͼ30% sequence identity to EgP1 were identified in the non-redundant protein sequence NCBI database. Protein sequences of eukaryotic origin with 50 -62% sequence identity to EgP1 (93-97% query coverage, E-value ϭ 0) were identified in the MMET database and E. gracilis Z strain transcriptome. A reconstructed molecular phylogeny showed that these EgP1 orthologous sequences formed a discrete clade (Fig.
The EgP1 sequence was then used to interrogate a propriety metagenome database, identifying a second bacterial ␤-1,3-D-glucan phosphorylase candidate, designated Pro_7066. Both proteins were successfully expressed as recombinant proteins in E. coli, which allowed us to characterize their functions. Although more closely related to GH94 than to other GH families, EgP1 and Pro_7066 have no significant sequence identity to any characterized GH94 sequences, which suggests that they belong to a new CAZyme family, namely GH149. In vitro characterization confirmed the function of EgP1 and Pro_7066 as ␤-1,3-D-glucan phosphorylases. EgP1 and Pro_7066 used Glc and longer ␤-1,3-linked gluco-oligosaccharides as acceptors in the reverse phosphorolysis reaction. This substrate specificity is slightly different from the previously described ␤-1,3-D-glucan phosphorylases, where substrate chain length was used to distinguish between different groups of the phosphorylases (i.
The origin of Euglenophyceae GH149 ancestral is unclear; however, the position of the Euglenophyceae clade within bacteria might suggest a lateral gene transfer from bacteria to the Euglenophyceae. A similar horizontal gene transfer from a bacterium to E. gracilis was predicted for mitochondrial trans-2-enoyl-CoA reductase  (49). Nevertheless, it is important to note that bacteria are overrepresented in most available sequence databases compared with eukaryotes, and therefore the prevalence of the bacterial orthologs in our study is unsurprising. Increasing the availability of sequences from the diversity of unicellular eukaryotes is essential for investigating evolutionary origins. A predicted targeting peptide in EgP1 caused aggregation of the EgP1 recombinant protein when expressed in E.
5. 2. 8 (www. MaxQuant. org), 3 and the database search was performed with the merged higher-energy collisional dissociation and collision-induced dissociation peak lists using Mascot version 2. 4 (Matrixscience, London, UK). The search was performed on a E. gracilis protein database  (14)  with a precursor tolerance of 6 ppm and a fragment tolerance of 0. 6 Da. The enzyme was set to trypsin/P with a maximum of 2 allowed missed cleavages. Carbamidomethyl (C) was set as a fixed modification, and oxidation (M) and acetylation (protein N terminus) were used as variable modifications. The Mascot search results were imported into Scaffold version 4. 4. 1. 1 (www. proteomesoftware. com) 3 using identification probabilities of 99 and 95% for proteins and peptides.
Bioinformatics analyses Orthologous sequences to EgP1 were obtained from the nonredundant protein sequence database (https://www. ncbi. nlm. nih. gov/protein/), the Marine Microbial Eukaryote Transcriptome (MMET) Sequencing Project), and the E. gracilis Z strain transcriptome (http://euglenadb. org/) using BLASTP or tBLASTn with E-value score of 0. 0001 or more. Multiple-sequence alignments of amino acid sequences were performed using Clustal Omega (56) (www. clustal. org/omega, 3 version 1. 2. 2) with the default settings and edited with trimAl version New ␤-1,3-glucan phosphorylase family 1. 2 using a heuristic automated method  (57). The alignments were visualized by Jalview (version 14. 6. 4) (58). Phylogenetic trees were reconstructed from a matrix of 331 unambiguously aligned amino acids from 325 species using PhyML version 3.
5, 150 mM NaCl, 1 ml/min. Fractions containing the proteins were pooled and concentrated to 10 mg/ml using an Amicon Ultra-15 30,000 molecular weight cutoff concentrator. The proteins were stored in aliquots at Ϫ80 °C until required. The EgP1 nucleotide sequence was deposited to GenBank TM with accession number MG516599.
This work was supported by the  BBSRC Institute Strategic Program on Understanding and Exploiting Metabolism (MET)  (Grant  BB/J004561/1 ); the  BBSRC ,  EPSRC , and  InnovateUK: IBCatalyst  (Grant  BB/M02903411 ); the  John Innes Foundation ; and the  Royal Thai Government Scholarship program. The authors declare that they have no conflicts of interest with the contents of this article. Author's Choice-Final version free via Creative Commons CC-BY license. This article contains Tables  S1-S3 , Figs. S1-S9 , and Files  S1-S4. The nucleotide sequence(s) reported in this paper has been submitted to the Gen-Bank TM /EBI Data Bank with accession number(s)  MG516599.
All data was reviewed by the senior nephrologist. Compliance with the prescribed diet was assessed separately for the two main levels of protein restriction (0. 8 g/kg/day and 0. 6 g/kg/day). For the logistic analysis, only patients with at least 3 months of follow-up were included. Good compliance was defined as protein intake equal to or below prescription; a tolerance interval of 20% was accepted for "0. 6 diets" and of 15% for "0. 8 diets". The definition was based on the Maroni-Mitch formula when available, or using the patient's food diary, assessed by an experienced dietitian, in the other cases.
ACKNOWLEDGEMENTS Rosaria Meccariello ,  Rosanna Chianese ,  Roberto Coccurello ,  Andrea Viggiano  and  Riccardo Pierantoni  designed the study;  Rosanna Chianese ,  Roberto Coccurello ,  Andrea Viggiano ,  Marika Scafuro ,  Marco Fiore ,  Giangennaro Coppola ,  Francesca Felicia Operto ,  Sophie Layé , and  Rosaria Meccariello  draft the manuscript;  Rosanna Chianese  and  Rosaria Meccariello  designed and prepared figures;  Silvia Fasano ,  Riccardo Pierantoni ,  Andrea Viggiano  and  Rosaria Meccariello  designed and prepared the graphical abstract;  Silvia Fasano ,  Riccardo Pierantoni  and  Rosaria Meccariello  critically revised the manuscript; all the authors approved the final version of the manuscript.
The blots performed with sheep antibodies were washed four times with TBS-T and incubated for 1. 5 h at room temperature with secondary anti-sheep horseradish peroxidase (HRP)conjugated antibody diluted 1:5,000 (Santa Cruz Biotechnology) in blocking solution. The membranes were washed six times before signal detection by chemiluminescence using the Luminata Forte Western HRP substrate (Merck Millipore). The following commercial HRP-conjugated antibodies were used: anti-Flag 1:5,000 (Sigma), anti-Myc 1:1,000 (Sigma), and anti-␤-actin 1:2,500 (Santa Cruz Biotechnology). Band analysis. Immunoblots were scanned using the C-Digit (Li-Cor) blot scanner. Densitometric analysis was performed using Im-ageStudioLite software. Figures were generated using Illustrator (Adobe). Intracellular chloride concentration.
GRANTS This work was supported by the collaborative grant no. 188712  from  CONACyT (Mexico)  and  ANR-12-ISVS1-0001-01  (France) to  G. Gamba  and  J. Hadchouel , the grant  IN207716  from  DGAPA, UNAM  to  G. Gamba , and the  NIDDK   RO1  grant no. DK051496-15  to  D. Ellison ,  G, Gamba , and  J. Hadchouel.
We reasoned that if these sequences were acquired from a bacterial source other than Chlamydiales, then deleting Chlamydiales from the alignment should strengthen the relationship with these bacteria, given that related genomes from these donors are in our dataset (i. e. , we cannot account for missing data). Surprisingly, this deletion increased both bootstrap and Bayesian support for Archaeplastida monophyly and attracted the long-branched Cyanidiophytina to this supergroup (BV = 47; BP = 0. 92; Supplementary Figs  S9  and S10 ), arguing against a non-chlamydial source for gene products derived from the bacterial Men pathway of menaquinone synthesis. OSB diffuses out to the peroxisome where it is converted to DHNA (1. 4-dihydroxy-2-naphthoate) in 3 steps (catalyzed by MenE, MenB and DHNAT) and diffuses back to the plastid.
Using the Arabidopsis thaliana plastid proteome as query. This plastid proteome includes proteins encoded by both the plastome and the nuclear genome. We find that 17 of these proteins are of unambiguous chlamydial origin, compared to 6 from all proteobacterial clades, that are the third-most frequent sources of LGT to this compartment. Th s result holds, despite the fact that the protein database for Proteobacteria is >34-fold larger than for Chlamydiales (Supplementary Fig. S26  and Supplementary Table  1 ). We demonstrate here an additional significant contribution of Chlamydiales to menaquinone metabolism in Archaeplastida. These genes impact the synthesis of both the core quinone and the isoprenoid (prenyl) side chain. Most importantly, we provide evidence that plastid genes encoding enzymes of the Men pathway in Cyanidiophytina are most likely of chlamydial (and not cyanobacterial [endosymbiont]) origin.
Methods Phylogenetic analysis. We searched for genes encoding menaquinone biosynthesis enzymes by using several conserved protein sequences as BLAST queries against the following groups of interest: Rhodophyceae, Chloroplastida, Glaucophyta, Chlamydiae, and Cyanobacteria. In those organisms that did not yield homologs in these searches and that presumably lacked the standard Men pathway, we further checked their absence by using ghostkoala (http://www. kegg. jp/ghostkoala/) and thereby generating a KEGG annotation to make sure that we retrieved all possible Men pathway enzymes. We then double-checked for the presence of the expected catalytic domains in sequences retrieved by BLAST using the Interpro database (https://www. ebi. ac. uk/interpro/). For proteins containing several catalytic domains (e.
When we considered a set of sequences to be of sufficient high quality we re-aligned with MUSCLE, carried out a block selection using BMGE with a block size of four and the matrix BLOSUM30. The trees were generated using Phylobayes  46  under the CAT + GTR model  47. These models take site-specific evolutionary properties (such as rate and profile) into consideration and generally fit sequence data significantly better than one-matrix models (e. g. , LG). For all of the analyses, we set up two chains that run in parallel and assessed convergence using the 'bpcomp' and 'tracecomp' functions available in Phylobayes 3. 3. Convergence assessments were carried out based on sampled trees and parameters (taking one from every 10 trees) following burnin equal to 20-25% of the entire length of the chain.
The consensus trees were generated with the 'bpcomp' function. We considered a phylogenetic analysis to be acceptable when maxdiff <0. 3 and effective size >50 for all parameters, according to the Phylobayes user manual. The consensus tree was then used to map bootstrap support values from 100 replicates using IQ-TREE  48  under the CX(10 to 30) models  49  , depending on the number of sites in each alignment. For model selection we followed a rule of thumb where the X value is around ten times under the number of the aligned dataset amino acid positions in order to avoid parameterization overfitting. For concatenation analysis we selected taxa according to single gene phylogenies performed for MenD and MenF, we aligned separately sequences from both proteins with MUSCLE, selected the block with BMGE (using the same settings as previously described), and construct phylogeny with IQ-TREE.
lyrata in the genus Arabidopsis), the species (e. g. , A. thaliana) with largest number of sequence were retained, whereas others (e. g. , A. lyrata) were removed. We incorporated all available Chlamydiae protein sequences downloaded from NCBI Genbank (May, 2017) and red algal sequences from Porphyridium purpureum  50  and from MMETSP database  51  (e. g. , Rhodosorus marinus). To reduce sequence redundancy in the database, we clustered highly similar sequences (identity ≥85%) among taxa from each phylum (e. g. , Chlamydiae and Alphaproteobacteria), retained the longest sequence and removed all other related sequences in the same cluster using CD-HIT v4. 5. 4  52. This step enhanced sequence diversity from a given phylum by avoiding sequence sampling from closely related taxa. Phylogenomic analysis was carried out as described previously  53.
A. thaliana plastid proteomes collected in a previous study  31  were used as query to search against the aforementioned database using BLASTp (e-value cut-off = 1e-05). The top 1,200 significant hits (sorted by bit score) for each query sequence were recorded. Representative sequences were selected (allowing up to 6 sequences for each phylum) on a first-come-first-serve basis to create a taxonomically diverse sample. The BLASTp hits were then re-sorted by sequence identity (of the query-hit alignment) followed by representative sequence sampling in the same way. The two sets of representative sequences, together with the query, were then aligned using MUSCLE version 3. 8. 31  54 under default settings and trimmed using TrimAI version 1. 2  55  in an automated mode (-automated1). The resulting alignments containing at least three bacterial sequences were used to build phylogenetic trees using IQtree  48.
Compared to our previous report  31  , there is a minor change in Chlamydial-derived proteins. The number of A. thaliana proteins derived from proteobacterial phyla decreased dramatically primarily because the donor phylum was represented by a single sequence in many cases. Note that the extent of sequence sampling is much higher in Proteobacteria than in Chlamydiae (Supplementary Fig. S4 ). The contribution of Chlamydiae to A. thaliana is more pronounced when considering its minimal sequence number in our database when compared to Proteobacteria (Supplementary Fig. S4 ). Metabolite supplementation of the light sensitivity phenotype of Chlamydomonas menaquinone synthesis deficient mutants. The wild-type strain as well as the menaquinone mutants used for growth analysis were cultivated into liquid TAP media at 25 °C under continuous light, for 4 days  16.
Acknowledgements U. C. , S. B. , C. C. , D. K. and M. C. were supported by the  CNRS , the  Université de Lille CNRS , the  ANR grants "Mathtest" "expendo " and "ménage à trois" and the  Région Hauts de France  and  European project Alibiotech. U. C, S. B. , C. C. acknowledge BILILLE  to provide computational resources. D. B. was supported by  NSF  grants  MCB 0946528  and  OA 1416785. P. C. and C. R. were supported through grants  F. R. S. -F. N. R. S. (FRFC 2. 4597 ,  CDR J. 0032 ,  CDR J. 0079  and  Incentive Grant for Scientific Research F. 4520 ) and from  European Research Council  ( H2020-EU   BEAL  project  682580 ). G. G. and T. P. were supported through  SNSF  grants.
Variables displaying statistical significance at the p-value < 0. 25 level in bivariate models were included in the multivariable model. For analyses including the full sample, the country was also included as a covariate. Sensitivity analyses were performed following exclusion of participants who did not recall seeing the FoPL during the survey. A false discovery rate approach was used to take into account multiple comparisons. A p-value below 0. 05 was considered statistically significant. Statistical analyses were carried out using the full sample and by country, for all food categories combined and by individual food category, using SAS Software (version 9. 3, SAS Institute Inc. , Cary, NC, USA).
Acknowledgments: The authors would like to thank all scientists in charge of the translations:  Pilar Galan ,  Karen Assmann ,  Valentina Andreeva , and  Sinne Smed , who contributed to the creation of the different versions of the online survey. We also thank  Mark Orange  for creating the mock packages, and all researchers and doctoral students who tested the online survey. We especially thank  Valentina Andreeva  for editing the paper.
NGS sequencing and genetic analysis Our retrospective study included anonymised data on 697 individuals between January 2015 and May 2017. These patients who had no family history of WD were French residents treated in 18 French university hospitals and had various indications other than WD, hepatic or neurological diseases. After automated DNA extraction (Qiagen, Courtaboeuf, France), the DNA samples were screened by NGS using a panel of various genes which included ATP7B. Library preparations for NGS were established using the sure-lectQXT kit (Agilent, Les Ulis, France) based on a hybrid capture system for sequencing on the Miseq sequencer (Illumina, Paris, France). The bioinformatics pipeline included the miseq-reporter followed by Fastq alignments with SeqNext and visual variant control (JSI Medical Systems, Ettenheim, Germany).
The variants were included if the depth of lecture was greater than 30X with a variant frequency ranging from 0. 40 to 0. 60. The design included the promoter, the 5'UTR region, the 3'UTR region and the coding region of the ATP7B gene (10,000 pb) with Refseq for the nomenclature: ATP7B (NM_000053. 3) for the exonic regions and intronic-exonic boundaries; ATP7B (NG_008806. 1) for exon numbering and the intronic region. Studies of missense variant pathogenicity involved the use of predictive software programs such as Polyphen-2 (http:// genetics. bwh. harvard. edu/pph2/), PhyloP (http://compgen. cshl. edu/phast/), and CADD (http://cadd. gs. washington. edu) and MutationTaster (http://www. mutationtaster. org), while splicing variant pathogenicity was determined used predictive software such as MaxEntScan (http://genes.
mit. edu/burgelab/maxent), Human Splicing Finder (http:// www. umd. be/HSF3/) and NNSPLICE (http://www. fruitfly. org/seq_tools/splice. html). The frequencies of all variants were searched for in Exome Aggregation Consortium (ExAc, exac. broadinstitute. org), Exome Sequencing Project (ESP, evs. gs. washington. edu) and dbSNP (https://www. ncbi. nlm. nih. gov/projects/SNP). We retained variants with an allelic frequency lower than 0. 06% and variants already described in the literature and reported in the Professional Human Gene Mutation Database (HGMD, http://www. hgmd. cf. ac. uk/ac/index. php), the Wilson Mutation Database, University of Alberta, Canada (http://www. wilsondisease. med. ualberta. ca) and in our Lariboisière Wilson's disease Database (Paris), which includes 496 index cases.
In a second step and to be more stringent, we excluded five variants reported in the literature on WD and that we have classified as VUS according to ACMG guidelines  [10] [11] [12]. The c. 4301C > T (p. Thr1434Met) variant was predicted to be "probably damaging" according to Polyphen-2, but was found in nine subjects which produced a variant frequency of 0. 00645 (9/1394 alleles) in our study, and moreover was never found in our large WD database. Five other subjects harboured four VUS (p. Val536Ala, p. Glu541Lys, p. Met665Ile, p. Val1297Ile) that were predicted to be benign by predictive softwares and were not present in our WD database. Moreover,   the p. Val536Ala variant (c. 1607C > T) (with a MAF = T at 0. 45% in the European population) reached a high frequency (1. 29%) in the Finnish population according to Exac.
In addition p. Val1297Ile (c. 3889G > A) displayed a high MAF = A at 1. 5% in the Asian population  [10, 13]  (Table  1 ). Consequently, ATP7B heterozygous carrier frequency was recalculated to include only heterozygous variants classified as pathogenic  [14] [15] [16] [17] [18] [19]  and likely pathogenic. Eighteen heterozygous mutations were thus found in 22 subjects, yielding a variant frequency of 0. 01578 (22/1394 alleles) or a heterozygous carrier frequency of 1/31 subjects (Table  1 ). All these mutations were deleterious according to the predictive softwares. At a more detailed level, ten missense or splicing mutations were identified in 14 subjects, all of whom were present in HGMD and in our WD database. Among the eight other subjects, one novel deletion, p. (Lys1028-Serfs*40) causing a frameshift mutation in exon 14, one splicing variant in intron 7, and six probably pathogenic missense variants were identified.
Also, p. Thr1434Met should be definitively reclassified as benign, despite the first Turkish publication 18 years ago  [10]. Indeed, its variant frequency was elevated in our study (0. 645%) and in dbSNP databases, its frequency reaching 4% in the African population. Moreover, neither variant was ever found among the 604 WD patients in the Lariboisière WD database. The high heterozygous prevalence of ATP7B observed in France raises the question of the approach to be adopted in the event of a genetic WD finding. Firstly, account needs to be taken of the discrepancies between the clinical and genetic frequencies seen both in France  [9]  and worldwide  [7, 8] , for different reasons. The diagnosis is certainly underestimated, or the pathology may be misdiagnosed. Indeed, in fulminant liver or tardive forms, or with a mild neurological presentation, a diagnosis of WD may not be suggested and the cause of death incorrectly determined.
All patients continued receiving velmanase alfa at the end of study, under compassionate use or after-trial care studies (rhLAMAN-07 and rhLAMAN-09), based on national regulations. In addition, data supporting the use of velmanase alfa as an effective therapy for alpha-mannosidosis have been reported from a 12-month, placebo-controlled, phase III study (rhLAMAN-05 [NCT01681953]; n = 25)  [11]. Patients in the placebo arm (n = 10) switched to velmanase alfa treatment at the end of phase III under compassionate use or after-trial care studies; patients in the active treatment arm could continue receiving treatment either under compassionate use or after-trial care studies. The rhLAMAN-10 study [NCT02478840])  [12]  is the integrated analysis of long-term data (up to 4 years of treatment) from all patients who participated in the early phase and phase III studies, the compassionate use programme and the after-trial care.
Funding sources Phase I/II studies rhLAMAN-02, -03, and -04 and phase III study rhLAMAN-05 were conducted under and co-funded by the EU FP7 project ALPHA-MAN [FP7-HEALTH-2010-261331]. Long-term continuation studies rhLAMAN-07 and -09 were initially sponsored by Zymenex A/S and are currently sponsored by Chiesi Farmaceutici S. p. A. Zymenex sponsored rhLAMAN-10 and the corresponding integrated patient analysis was reported by Chiesi. The sponsor provided study drug, and was involved in the study design, protocol development, regulatory and ethics approvals, safety monitoring and reporting, data management, and data analysis and interpretation. Chiesi Farmaceutici S. p. A. funded third-party writing assistance for the current manuscript, provided by PAREXEL.
Genome Data and Curation of Annotations Our data set consisted of 13 genomes of L. curvatus strains, of which five were complete and eight were draft versions, all available from the Genbank/EMBL databases (table  1 ). All genomes were downloaded to the MAGE annotation platform  (Vallenet et al. 2013 ) and strain-specific genes were all manually curated in order to standardize the comparative analysis. Metabolic pathways were reconstructed using the METACYC database  (Caspi et al. 2016)  or from the literature when indicated.