dataesr/scientific-paragraphs-categorization huggingface dataset:
491 imilarly, the macrocyclase PatG (Section 3. 19, InterPro family IPR034056) has been used in vitro with synthetic peptides containing ncAAs (e. g. diamino propionate, ornithine, and aminohexanoic acid) including the N-terminal residue that forms the lactam. Although the reaction is slow,  519  diverse circular peptides could be produced. Furthermore, full reconstruction of the cyanobactin pathway has been accomplished. 301 rimming the essential RSs allowed a drastic reduction of the structural peptide to approximately one third of the original length. Sequential addition of the enzymes showed a specic order for the system where the cyclodehydratase ThcD, leader peptidase PatA, macrocyclase PatG and prenyltransferase TruF (InterPro family: IPR031037) worked step-wise to completely transform and decorate the core peptide.
coli. Twelve new improved variants of microcin J25, against one or both strains, with up to three simultaneous mutations, were isolated. 562 In another approach, the lasso peptide astexin-1 has been proposed as a suitable candidate for library screening (including surface exposed libraries, vide infra) owing to the demonstration of high exibility in the C-terminal region of its precursor peptide. Fusions of the precursor with GFP or leucine zipper protein A1 were still modied, indicating that a bulky C-terminal protein domain does not abolish production. 563 acrocyclic peptide libraries have been developed using the fungal machinery of the cyclic hexapeptide cycloamanide in E. coli. 233 The POPB catalyzing the cyclization reaction (Section 3. 18; InterPro family: IPR002470) was used with a set of substrate variants with different lengths fused to the maltose binding protein (MBP), and peptides up to 16 amino acids were cyclized in vitro.
Each of these sections was diversied with modules originating from twelve lanthipeptides, both class I and class II, for which the structure and mechanism of action was (partly) known, connected by designed linkers that confer exibility resembling the hinge region of nisin. Although the Nterminus was limited to conserved sequences from nisin and gallidermin to keep a proper cleavage site for the protease NisP (InterPro family: IPR008357)  311  and to facilitate NisBC modication (Section 3. 1. 1),  501 the remaining regions were designed with larger variability. 567 A library encoding all possible combinations of these modules was assembled by controlled pore glass synthesis combined with a mix-and-split approach (ca. 6000 possible genes). The library was cloned and maintained in E. coli and used to transform L.
Leader peptide recognition 4. 1. 1 The RiPP precursor recognition element (RRE). A large fraction of class I lanthipeptide precursor peptides contain a highly conserved F-D/N-L-N/D motif in their LPs. The rst co-crystal structure of a RiPP biosynthetic enzyme with a LP bound (PDB: 4WD9) revealed how NisB, the dehydratase in nisin biosynthesis (Section 3. 1. 1), recognizes this motif within the NisA precursor peptide.
Acknowledgments  12 References 11  Acknowledgments RiPP research in the laboratories of the corresponding authors is supported by grants from the  National Institutes of Health  ( GM123998  to DAM,  AI144967  to WAV and DAM,  GM058822  to WAV, and  5T32-GM070421  to IRR), the  EU FW7 programme SynPeptide  (to MML, OPK and JP), the  EU   Horizon 2020 programme Ras4Biotech  (to AJvH), the  Swiss National Science Foundation  ( 407240_1167051  to JP), the  Dutch Research Council  ( NWO-ALWOP-214  to JV), a  European Molecular Biology Organization Long-Term Fellowship  ( ALTF 344-2018  to TAS), and the  Howard Hughes Medical Institute  (to WAV). 12 References.
4. 1 1 Leader peptide recognition 4. 1. 1 The RiPP precursor recognition element (RRE). A large fraction of class I lanthipeptide precursor peptides contain a highly conserved F-D/N-L-N/D motif in their LPs. The rst co-crystal structure of a RiPP biosynthetic enzyme with a LP bound (PDB: 4WD9) revealed how NisB, the dehydratase in nisin biosynthesis (Section 3. 1. 1), recognizes this motif within the NisA precursor peptide.
Lignocellulolytic enzymes, including cellulases, hemicellulases, and lignin-degrading enzymes (including Ligninolytic peroxidases and laccases), are classified in the CAZy database  [10]. The scientific literature related to their characterization is broad and diverse for enzymes derived from terrestrial species. By contrast, very little is known about the enzymatic mechanisms employed by mangrove-derived fungi to break down plant biomass and their adaptation to marine conditions, especially to high salt concentrations. In a previous study, we showed that the presence of sea salt modified the composition of secreted lignocellulolytic enzymes, with increased secretion of xylanases and cellulases, and lower production of oxidoreductases belonging to the auxiliary activities (AA) class of the CAZy database  [11].
Specific reverse transcription of poly-A mRNA, followed by double-stranded cDNA synthesis and amplification, was performed on 500 ng of total RNA using the Mint-2 cDNA synthesis kit (Evrogen, Moscow, Russia). The optimal number of PCR cycles to maintain a balance between transcript representation and nonspecific background amplification during the cDNA amplification was estimated to be 27 cycles. The resulting cDNAs, previously purified using a phenolchloroform protocol  [37] , were used as templates to specifically capture by hybridization and sequence expressed fungal genes encoding DyPs. [38] ) and the specialized RedOxiBase (http://peroxibase. toulouse. inra. fr/, accessed on 19 April 2021;  [39] ). This sequence data set was used to design 69 (70 bp long) degenerate capture probes using the KASpOD software (https://g2im.
In the present work, the SWARM aggregation distance equaled to 3. Chimeras were removed using VSEARCH  [45] , and low abundance sequences accounting for less than 0. 005% of the dataset were filtered out. This whole procedure was performed using the pipeline FROGS  [46]. To be able to compare samples, a normalization procedure was applied to randomly resample down to 32,556 sequences per sample. A Wilcoxon test was used to statistically evaluate the differences between the alpha diversity indices calculated (Shannon index (H'), complement of the Simpson index (1-D), and Evenness (J')). The effect of environmental factors (tree, season, and depth) on the composition of expressed fungal genes encoding DyPs were tested using nonparametric permutationbased multivariate analysis of variance (PERMANOVA, adonis function;  [47]  based on abundance dissimilarity (Bray-Curtis) matrices).
These analyses were performed using the VEGAN package (http://cran. r-project. org, accessed on 19 April 2021) in R. The sequence data generated in this study were deposited in the EMBL-ENA public database (PRJEB43346 for the first sampling campaign (March) dataset and PRJEB43343 for the second sampling campaign (November) dataset). Concerning the HiSeq raw data for full-length cDNA reconstruction through gene capture by hybridization enrichment, adapter sequences were eliminated using Cutadapt  [48]. Sequence quality was evaluated with Trimmomatic  [49] ; bases with quality lower than 20 were eliminated and only sequences longer than 60 bases were kept. Trimmed sequences were then assembled using IDBA-UD  [50]  (default parameters) and resulting contigs were further assembled with CAP3  [51]  (default parameters) to obtain longer contigs.
A similarity search between a custom peroxidase database and contigs was performed using DIAMOND  [52] , with the BLASTx command in "sensitive" mode (i. e. , a maximal E-value of 1 × 10 -5 and a minimal identity of 50%). Matching sequences were further analyzed to search the DYP peroxidase domain using ScanProSite  [53]  and the Prosite database  [54]. The ProtParam tool (http://web. expasy. org/protparam/, accessed on 19 April 2021) was used to predict the theoretical pI, molecular mass, and molar extinction coefficient of DyP. For sequence comparison, BlastP was used to search for sequences with similarity to DyP1 in the UniProtKB/Swiss-Prot database (http://www. uniprot. org/blast, accessed on 19 April 2021). The search parameters were scoring matrix BLOSUM 62, gapped alignment allowed, and cut-off E-value 0.
1. DyP1 was aligned with 241 DyP sequences identified in Agaricomycotina genomes available at the MycoCosm portal (https://mycocosm. jgi. doe. gov/mycocosm/home, accessed on 19 April 2021) and GenBank using MUSCLE as implemented in MEGA X (https://www. megasoftware. net/, accessed on 19 April 2021)  [55]. A maximum likelihood phylogenetic tree was then constructed by MEGA X using the WAG evolutionary model with gamma-distributed rate variation and the amino acid frequencies of the dataset.
Diversity and Capture of Fungal DyP Encoding cDNAs The diversity of expressed genes encoding fungal DyPs was investigated in surface and deeper mangrove sediments beneath A. marina and R. stylosa trees during the wet (March) and dry (November) seasons. Sediment samples were collected in three independent plots (A, B, and C) in the A. marina (A) and R. stylosa (R) pristine areas. This expressed gene diversity was evaluated using a metabarcoding approach (Illumina Miseq) on environmental cDNAs previously enriched in DyP sequences by gene capture by hybridization. The normalized DyP dataset consisted of 781,344 sequences distributed among 25 different operational functional units (OFUs), corresponding to 25 putative DyP encoding cDNAs (Table  S2 ). The number of OFUs per sediment sample varied from one to 10.
DyP diversity, estimated with the Shannon index, was systematically higher in the surface layers during the wet season (Figure  1 ; Supplementary Table  S2 ). The highest DyP diversity was observed for the surface layers beneath the R. stylosa area during the wet season. By contrast, the DyP diversity was systematically lower in the surface layers during the dry season (Figure  1A ; Table  S3 ). Regarding the distribution of OFUs, the most abundant one (OFU1) was detected in all sediment samples and represented 51100% of all sequences in 70% of the samples (Figure  1B ). The full-length cDNA sequence of OFU1 was obtained from the HiSeq dataset after gene capture by hybridization enrichment. The difference in the composition of expressed DyPgenes assessed through a nonparametric multivariate analysis of variance (PER-MANOVA) highlighted a strong seasonal effect (P= 5.
8 × 10 -5 , R 2 = 0. 178; Table  1 ). Tree species (P= 0. 032, R 2 = 0. 062) and sediment depth (P= 0. 021, R 2 = 0. 072) had a lower impact on the composition of the expressed fungal genes (Table  1 ). Regarding the distribution of OFUs, the most abundant one (OFU1) was detected in all sediment samples and represented 51,100% of all sequences in 70% of the samples (Figure  1B ). The full-length cDNA sequence of OFU1 was obtained from the HiSeq dataset after gene capture by hybridization enrichment. The difference in the composition of expressed DyP-genes assessed through a nonparametric multivariate analysis of variance (PERMANOVA) highlighted a strong seasonal effect (P = 5. 8 × 10 -5 , R 2 = 0. 178; Table  1 ). Tree species (P = 0. 032, R 2 = 0. 062) and sediment depth (P = 0. 021, R 2 = 0.
Phylogenetic Analysis We decided to study the most widely represented peroxidase in mangrove soils, OFU1, which we called DyP1. A BlastP search was conducted against the UniProtKB/Swiss-Prot database using DyP1 as the query. As a result, DyP sequences from fungal species of the orders Auriculariales, Sebacinales, and Geastrales, included in the subphylum Agaricomycotina, emerged as the proteins with the highest amino acid sequence identities to DyP1 (Table  2 ). DyP1 then underwent a phylogenetic analysis with 241 DyP sequences from 88 fungal species belonging to 15 orders of this subphylum available at the JGI-DOE MycoCosm portal  [38]  and GenBank. Sequences within the resulting phylogram (Figure  2 ) can be sorted into the seven evolutionary clusters previously described by  Linde et al.
Decolorization of Industrial Dyes Because of its potential for dye decolorization, DyP1 activity was tested on five more industrially relevant dyes, belonging to five different chemical classes: acidic, basic, reactive, vat, and disperse dyes. As shown in Table  5 , DyP1 was highly active on Reactive Black 5 (RB5) dye (32. 3% of decolorization) and to a lesser extent on Acid Black (AB) and Disperse Blue 79 (DB). By contrast, TvDyP1 from T. versicolor (GenBank accession numbers 19415892) was active only against AB, though with 75% of decolorization.
Effect of Hydrogen Peroxide on DyP1 Activity Although hydrogen peroxide is the electron acceptor of peroxidases, these enzymes are known to lose activity in the presence of H 2 O 2 , through a mechanism known as suicide inactivation  [71]. The optimum concentration of H 2 O 2 was determined by incubating the reaction mixture with different concentrations of H 2 O 2 , ranging from 0. 1 to 5 mM, and the highest DyP1 activity was recorded for 0. 25 mM H 2 O 2 (Figure  6A ). Above this concentration, DyP1 residual activity decreased gradually, up to 30% at 5. 0 mM H 2 O 2. Disperse Blue 79 (DB). By contrast, TvDyP1 from T. versicolor (GenBank accession numbers 19415892) was active only against AB, though with 75% of decolorization.
The color of both branches and enzymes indicates the order of the fungal species they belong to according to the legend, where the total DyP cDNA number is shown (including eight from GenBank) followed by the number of genomes for each order. The positions of the characterized DyPs from A. auricula-judae, B. adusta, Polyporaceae sp. (Irpex lacteus), M. scorodonius, T. albuminosus, P. ostreatus, T. versicolor, P. sapidus, and C. trogii are indicated with blue arrows.
2. 3. Probe Design, cDNA Capture by Hybridization, and High-Throughput Sequencing of Fungal DyP cDNA First, 1267 publicly available fungal DyP DNA coding sequences were identified by BLAST searches and collected from GenBank (http://www. ncbi. nlm. nih. gov/genbank/, accessed on 19 April 2021), the Joint Genome Institute Mycocosm database (https:// mycocosm. jgi. doe. gov/mycocosm/home, http://www. ncbi. nlm. nih. gov/genbank/, accessed on 19 April 2021;.
DyPs deposited from the UniProtKB/Swiss-Prot database presenting the highest amino acid sequence identities (47-59%) with DyP1 from mangrove soils (the number of residue pairs considered for each comparison is shown in brackets). The DyP entry names of 12 enzymes are indicated together with the fungal species they belong to, from the orders Auriculariales (Exidia glandulosa and A. auricula-judae), Sebacinales (Piriformospora indica and Serendipita vermifera), and Geastrales (Sphaerobolus stellatus).
Acknowledgments: The authors are grateful to  MN Rosso ,  INRAE Aix-Marseille University , and  Igor V Grigoriev ,  Joint Genome Institute , for providing access to genome sequence data for Trametes betulina BRFM 1801, Trametopsis cervina  BRFM1824 , Trametes cingulata  BRFM1805 , Trametes gibbosa.
Funding: This research was funded by the  ECosphère Continentale et Côtière MicroBien program  ( EC2CO ,  2017-2019 ) and by the  Agence Nationale de la Recherche  (project  PeroxiDiv   ANR-RF-2015-01 ). D. L. and F. J. R-D. were supported by  H2020 BBI-JU  (https://www. bbi-europe. eu)EnzOx2 ( H2020-BBI-PPP-2015-2-720297 ; https://www. enzox2. eu)project and  GENOBIOREF  ( BIO2017-86559-R ) project of the  Spanish Ministry of Science & Innovation  (co-financed by  FEDER  funds).
Human Subjects Human studies were carried out in the Sleep Clinic of the Hospital Universitario Miguel Servet, a large teaching university hospital in Zaragoza, Spain, as part of the EPIOSA study (NCT02131610)  [35]  and in a multicenter trial conducted by the Spanish Sleep Network to assess the efficacy of CPAP in women with OSA (NCT02047071;  [36, 37]. All studies were approved by the appropriate human subject bioethics committees and all participants provided signed informed consents. Adult patients (mean age: 48. 5 ± 6. 7 years; male:female: 1:1; range: 20-65 years) with polysomnographically or polygraphically diagnosed OSA at baseline (V1) and after 12 months of adherent CPAP treatment (6. 0 ± 2. 1 h/night throughout the duration) (V2) were selected into this study (n = 26). Data from all sleep studies were scored using AASM guidelines  [38]  by trained personnel that were blinded to the aims or nature of the current study.
Optimal titration of CPAP was implemented by using auto-CPAP (Autoset-T; ResMed, Sydney, Australia), according to the guidelines of the Spanish Sleep and Breathing Group  [39]. Fasting blood samples were drawn at V1 and V2 using a 21G butterfly needle into ethylenediaminetetraacetic acid (EDTA) (PreAnalytix GmbH, Bene, Hombrechtikon, Switzerland). Plasma was separated by centrifugation and stored at -80 • C until analyses. A detailed description of Materials and Methods is provided in the Supplementary Materials. These data include exosome isolation, characterization from plasma and cell culture conditions, in vitro proliferation and migration, Western blotting, semi-quantitative reverse transcription-PCR (qRT-PCR), and miRNA isolation from exosomes derived melanoma cells.
Exosomes, either from plasma or cell cultures, were quantified using either enzymatic fluorescent assays (FluoroCet #FCET96A quantitation kit; System Biosciences, Mountain View, CA, USA) according to the manufacturer's protocol, or with the Spectradyne nCS1 nanoparticle analyzer (Spectradyne, Cherry Hills, CA, USA) which employs resistive pulse sensing technology, and further analyzed for selective subpopulation surface markers using FACS analysis (FACSCalibur, BD Biosciences), whereby purified exosomes were incubated with magnetic beads 9. 1 µm with exosomes markers under gentle agitation at 4 • C overnight according to manufacturer's recommendations. Control experiments were also performed with all the reagents and beads, but in the absence of exosomes. Twentyfive thousand events were computed, and data were analyzed using FlowJo Software (Tree Star, Inc, Ashland, OR, USA).
The cells were allowed to migrate for 40 h from the starved medium side to the lower compartment while using 10% FBS as chemoattractant. Cells were stained with 0. 2% crystal violet solution (Sigma-Aldrich, St. Louis, MO, USA) and phase contrast images of the migrated cells were imaged using NIS-Elements AR vs. 3. 10 imaging software. Sections were visualized with a Nikon Elipse Ti microscope using 4× objectives and photographed with a Cool SNAP EZ camera. The migration rate was determined by counting the number of cells contained in the photos of 5 random-selected different fields at original magnification by operators who were blinded to the experimental conditions.
p-AMPK and p-AMPK Activator Western Blots Human melanoma cells (CRL-1424) were grown as described above in 12-well plates in 10% FBS for 24 hr. Culture media were then replaced with 10% depleted FBS, and the cells were used under two different conditions. First, cells were treated with exosomes derived from OSA-V1, OSA-V2 (equal number of exosomes) or without exosomes for 24 h. Second, cells were treated with 15 µM of p-AMPK activator (PF06409577, Sigma-Aldrich (St-Louis, MO, USA) for 4 h, and equal numbers of exosomes were added to each individual well-plate for 24 h. Protein cell lysed concentrations were quantified using the BCA kit (Life Technologies, Rand Island, NY, USA). Equal amounts of total protein were electrophoresed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE, [10% and transferred into nitrocellulose membrane (Millipore, Billerica, MA, USA)].
The membranes were blotted with 5% skimmed milk and subsequently probed overnight at 4 • C with primary antibodies. The membranes were incubated with polyclonal p-AMPK ((Phospho-AMPKα (Thr172) (40H9), Cell Signaling, Danvers, MA, USA)) and AMPK polyclonal antibody (AMPKα, D5A2, Cell Signaling, Danvers, MA, USA) overnight at 4 • C, then washed three times for 10 min each with 25 mM Tris-HCL, pH 7. 4, 3. 0 mM KCl, Cancers 2021, 13, 4781 6 of 25 140 mM NaCl, and 0. 05% Tween-20 (TBST), incubated with anti-rabbit immunoglobulin G:HRP-linked antibody (Cell Signaling Technology; 1:2000 dilution in blocking buffer with gentle agitation for 1 h at room temperature), and finally immunoreactive bands were visualized using an enhanced chemiluminescence detection system (Chemidoc XRS+; Bio-Rad, Hercules, CA, USA), and quantified by the Image Lab software (Bio-Rad, Hercules, CA, USA).
Each sample was prepared according to Agilent's miRNA recommended approach using the one-color technique and profiled on the Agilent human miRNA microarray (Agilent Technologies, Santa Clara, CA, USA)  [47]. Each array consisted of 60-mer DNA probes synthesized in situ that represent 2006 human miRNAs. The labeled RNAs were hybridized to custom human miRNAs microarrays 8 × 60 K (Agilent Technologies, Santa Clara, CA, USA). Following hybridization and washing, the arrays were scanned with an Agilent microarray scanner using high dynamic-range settings as specified by the manufacturer. Microarray results were extracted using Agilent Feature Extraction software (v12. 0) to quantify signal intensities  [32, 47]. The quality control for each miRNA was evaluated based on their Agilent Feature Extraction quality control report.
Target Gene Prediction and Function Enrichment Analysis To identify the underlying target genes of DE miRNAs, the miRWalk2. 0 database ( zmf. umm. uni-heidelberg. de/apps/zmf/mirwalk2/ accessed on 16 July 2021) was used. This database contained a total of 12 bioinformatic algorithms, including DIANA microT (version 4. 0), DIANA microT CDS (version 5. 0), miRanda rel (version 1. 0), mirBridge (version 1. 0), miRDB (version 4. 0), miRmap (version 1. 0), miRNAMap (version 1. 0), PicTar (version 2. 0), PITA (version 6. 0), RNA22 (version 2. 0), RNAhybrid (version 2. 1) and Targetscan (version 6. 2). The Database for Annotation, Visualization and Integrated Discovery (DAVID) online tool (version 6. 8; http://david. abcc. ncifcrf. gov, accessed on 16 July 2021) was used for Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of all target genes of DE miRNAs.
Data Analyses Results are presented as means ± SD, unless stated otherwise. Comparisons of all quantitative data groups were performed using unpaired Student t-tests, non-parametric tests, or ANOVA procedures as appropriate depending on the variable distributions. Statistical analyses were performed using SPSS version 21. 0 (SPPS Inc. , Chicago, IL, USA). For all comparisons, a two-tailed p < 0. 05 was considered to define statistical significance. Cancers 2021, 13, 4781 8 of 25.
Data Analyses Results are presented as means ± SD, unless stated otherwise. Comparisons of all quantitative data groups were performed using unpaired Student t-tests, non-parametric tests, or ANOVA procedures as appropriate depending on the variable distributions. Statistical analyses were performed using SPSS version 21. 0 (SPPS Inc. , Chicago, IL, USA). For all comparisons, a two-tailed p < 0. 05 was considered to define statistical significance.
Melanoma Gene Ontology and KEGG Pathways We next examined the predicted gene targets of the differentially expressed miRNAs in exosomes released after exposures to IH from CRL-1424 and CRL-1675 cells, and based on several prediction tools we identified 8172 gene targets for CRL-1424 and 2073 gene targets for CRL-1625, respectively. To assess the potential biological roles of these exosomal miRNA target genes, we performed GO classification enrichment analysis. For CRL-1424, there were 48 differentially enriched terms involved in biological processes, 42 in the cellular component, and 22 in the molecular functions (Table  S1A -C), respectively, while for CRL-1675 there were 19 involved in biological processes, 8 in cellular component, and 5 in molecular functions (Table  S2A -C). In the KEGG database, we identified several pathways that were differentially and putatively targeted by exosomes from IH-exposed CRL-1424 cells including Ras signaling pathway (hsa04713, p =1.