Patricia L Whetzel

Nigam H Shah

Natalya F Noy

Clement Jonquet

Adrien Coulet

Nicholas Griffith

Cherie Youn

Michael Dorf

Mark A Musen

Ontology Web Services for Semantic Applications

Introduction

Researchers have turned to the Semantic Web to integrate, summarize, and interpret disparate knowledge. Ontologies provide the domain knowledge to drive such data integration and information retrieval, on the Semantic Web. The successful creation of semantic applications in the health and life sciences requires services that provide software applications with access to ontologies over the Web. The National Center for Biomedical Ontology (NCBO), one of seven National Centers for Biomedical Computing created under the NIH Roadmap, has developed BioPortal, which provides access to one of the largest repositories of biomedical ontologies both via Web browsers and Web services (via RESTful services). The BioPortal Ontology Web services allow programmatic access, download and traversal of ontologies in software applications, the NCBO Annotator Web service "tags" text automatically with terms from BioPortal ontologies, and the NCBO Resource Index provides an ontology based search of public data resources.

The Ontology Web services provide access to ontologies, their characteristics, information about different versions, ontology downloads, navigation of the class hierarchy and details of each class. Developers can to embed this functionality in software applications, such as the Microsoft Word 2007's Ontology Add-in, used to mark up a research article at the time of writing or ISAcreator, used to annotate experimental metadata.

The NCBO Annotator Web service processes text to recognize relevant biomedical ontology terms. Users can customize the Web service to limit results to a particular ontology (e.g. SNOMED CT) or to a certain UMLS semantic type (e.g. T017 for 'Anatomical Structure'). The concept recognition engine, MGREP, was developed by the National Center for Integrative Bioinformatics and is combined with BioPortal Ontology Web services to create the NCBO Annotator service to make the task of creating ontology-based annotations accessible for any biomedical researcher.

BioPortal indexes several biomedical data repositories available online (e.g., GEO, ClinicalTrials.gov, PharmGKB) on the basis of their textual metadata, and links their records to ontology terms. These linkages take advantage of the semantic relationships in BioPortal, including subsumption relationships among ontology entities and mappings between entities in different ontologies. The NCBO Resource Index Web service allows biomedical investigators to use ontology terms to search programmatically the resulting index of online public repositories. For example, one can search for all experiments and clinical trials corresponding to 'malignant melanoma' from GEO and ClinicalTrials.gov.

BioPortal offers researchers a one-stop shop on the Web for biomedical ontologies. The Web services provided by BioPortal are available at www.bioontology.org/wiki/index.php/NCBO_REST_services and the BioPortal technology is open-source and domain-independent.

BioPortal Ontology Library

Ontology Recommender Web Service

• Description • The Ontology Recommender helps users identify biomedical ontologies or terminologies that are most relevant to their data based on analysis of free text or keywords. The underlying method uses annotations generated with the NCBO Annotator Web service to score the ontologies. Higher scores correspond to ontologies with the highest number of annotations. • For more information, see: http://www.bioontology.org/wiki/index.php/Ontology_Recommender_Web_service

Acknowledgements

BioPortal is developed by the National Center for Biomedical Ontology (NCBO), one of seven National Centers for Biomedical Computing under the NIH Roadmap. BioPortal is developed in conjunction with partners at the University of Victoria and the Mayo Clinic. For more information on NCBO and working with the Center, visit http://www.bioontology.org/ or email support@bioontology.org.

BioPortal Web Services


Patricia L Whetzel

Natasha F Noy

Nigam H Shah

Benjamin Dai

Michael Dorf

Nicholas B Griffith

Clement Jonquet

Michael J Montegut

Daniel L Rubin

Cherie H Youn

Natasha F Noy

Bioportal

Natalya F Noy

Mark A Musen

BioPortal: A Web Repository for Biomedical Ontologies and Data Resources

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Biomedical ontologies provide essential domain knowledge to drive data integration, information retrieval, natural-language processing, and decision support. The National Center for Biomedical Ontology, one of the seven National Centers for Biomedical Computing created under the NIH Roadmap, is developing BioPortal, a Webbased system that serves as a repository for biomedical ontologies. BioPortal defines relationships among those ontologies and between the ontologies and online data resources such as PubMed, ClinicalTrials.gov, and the Gene Expression Omnibus (GEO). BioPortal supports not only the technical requirements for access to biomedical ontologies either via Web browsers or via Web services, but also community-based participation in the evaluation and evolution of ontology content. BioPortal enables ontology users to learn what biomedical ontologies exist, what a particular ontology might be good for, and how individual ontologies relate to one another. BioPortal is available online at the following location: http://bioportal.bioontology.org/.

Acknowledgements

BioPortal is developed by the National Center for Biomedical Ontology (NCBO), one of seven National Centers for Biomedical Computing under the NIH Roadmap. BioPortal is developed in conjunction with partners at the University of Victoria and the Mayo Clinic. For more information on NCBO and working the Center, visit http://bioontology.org/ or email support@bioontology.org.

BioPortal Ontology Library

Current Set of Mappings in BioPortal

BioPortal has more than 30,000 mappings in its repository, involving 20 ontologies:

Community Feedback

Members of biomedical research communities can use the Notes functionality in BioPortal to • Provide feedback to ontology authors • Reach consensus on ontology decisions • Review ontology components

Ontology Peer-Review

BioPortal enables ontology users to provide feedback on ontologies in BioPortal. Ontology reviewers can provide the following information • General review and rating of the ontology • Usage information

• Which applications have successfully used the ontology?

• What problems were encountered? • Coverage

• Does it cover the domain properly?

• Are there major gaps?

• Are some parts better developed than others? • Concept-specific comments

• Are there problems with specific concepts?

• What alternative definitions should be used? • Web of Trust mechanism will enable users to specify reviewers that they trust, or to create filters to see reviews from particular groups (e.g., OBO Foundry members).

BioPortal Web Services

Access ontology content via REST web services

Ontology-Indexed Data Resources

Find biomedical data resources indexed by ontology terms • Current resources: Gene Expression Omnibus (GEO), ArrayExpress, ClinicalTrials.gov, ARRS GoldMiner

Filter by category


Isabelle Accoceberry

Ce ´lia Couzigou

Vale ´rie Fitton-Ouhabi

Nicolas Biteau

Thierry Noe ¨l

Challenging SNP impact on caspofungin resistance by full-length FKS1 allele replacement in Candida lusitaniae

Objectives: A strain of the opportunistic pathogenic yeast Candida lusitaniae was genetically engineered for fulllength replacement of the FKS1 gene encoding the target of echinocandin antifungals in order to assess the impact of FKS mutations on echinocandin resistance and reduced echinocandin susceptibility (RES).

Methods: FKS1 allelic exchange was achieved by transforming C. lusitaniae with two DNA fragments covering the entire FKS1 ORF. Both fragments overlap a 40 bp region where SNPs or small indels of interest were inserted.

To target integration at the FKS1 locus, each DNA fragment was fused with split auxotrophic markers of which complementary truncated parts were previously inserted into the chromosomal regions flanking FKS1, allowing selection on minimal medium.

Results: Three SNPs described in the FKS1 hotspot (HS) regions HS1 or HS2 of clinical isolates of Candida albicans were expressed at an equivalent position in C. lusitaniae and were confirmed to confer either reduced susceptibility (F641V) or full resistance (S645P and R1361G) to caspofungin. The F659 deletion reported in an FKS2 allele of Candida glabrata and the naturally occurring P660A substitution in FKS1 of Candida parapsilosis were shown to confer a 256-fold and 6-fold increase in caspofungin MIC, respectively, when introduced into an FKS1 allele of C. lusitaniae.

Conclusions:

We have successfully developed a C. lusitaniae strain for the expression of full-length FKS1 alleles harbouring known mutations contributing to reduced susceptibility or resistance to caspofungin, thus opening the way for the screening of other FKS1/FKS2 mutations potentially involved in RES.

Introduction

The echinocandins caspofungin, micafungin and anidulafungin are structurally related acylated cyclic hexapeptides having concentration-dependent antifungal activity against Candida spp. and Aspergillus spp. [START_REF] Denning | Echinocandin antifungal drugs[END_REF] They are now the first-line treatment for invasive candidiasis in most patients. They act as non-competitive inhibitors of the fungal b(1,3)-glucan synthase, thus disturbing and inhibiting fungal cell wall synthesis. [START_REF] Denning | Echinocandin antifungal drugs[END_REF] b(1,3)-Glucan synthase consists of a cytoplasmic regulatory protein and membrane-bound Fksp catalytic subunits, which may be encoded according to the fungal species by several FKS paralogue genes. In yeast species belonging to the CTG clade of Candida, e.g. Candida albicans, the FKS1 gene that encodes the catalytic subunit targeted by echinocandins is essential, [START_REF] Douglas | Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-b-D-glucan synthase inhibitors[END_REF] whereas in Candida glabrata [START_REF] Katiyar | Fks1 and Fks2 are functionally redundant but differentially regulated in Candida glabrata: implications for echinocandin resistance[END_REF] and Saccharomyces cerevisiae, [START_REF] Kurtz | Lipopeptide inhibitors of fungal glucan synthase[END_REF] the catalytic subunits are encoded by the FKS1 and FKS2 genes, which are at least partially functionally redundant. Besides a tolerance phenomenon mediated by calcineurin, high-osmolarity glycerol (HOG) and protein kinase C (PKC) cellular stress response pathways leading to increased chitin synthesis [START_REF] Munro | The PKC, HOG and Ca 2+ signalling pathways co-ordinately regulate chitin synthesis in Candida albicans[END_REF] and cell wall remodelling, [START_REF] Quile`s F | AFM combined to ATR-FTIR reveals Candida cell wall changes under caspofungin treatment[END_REF] the main mechanism of echinocandin resistance involves non-synonymous mutations of the FKS genes, which decrease the binding affinity of the target protein to the echinocandin inhibitor. [START_REF] Perlin | Mechanisms of echinocandin antifungal drug resistance: mechanism of echinocandin drug resistance[END_REF] The two so-called hotspot (HS) regions, HS1 and HS2, were identified as the regions of the FKS genes containing all resistance mutations. [START_REF] Park | Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates[END_REF] However, in clinical strains resistant to echinocandins, the HS1 and HS2 regions are the only regions that have been screened for genetic variability because of the large size of the FKS genes (~6 kb). Accordingly, very little information is known about the genetic variability of FKS regions outside the HSs and about their possible contribution to echinocandin resistance. The use of next-generation sequencing for clinical molecular diagnosis in the near future is expected to provide extensive information about the overall genetic variability of FKS genes. This will raise the question of the contribution of new genetic polymorphisms to the resistance or reduced susceptibility to echinocandins (RES), as reported for a third HS region identified in the FKS1 gene of S. cerevisiae and C. glabrata. [START_REF] Johnson | New Fks hot spot for acquired echinocandin resistance in Saccharomyces cerevisiae and its contribution to intrinsic resistance of Scedosporium species[END_REF] Generally, the impact of a missense mutation is demonstrated at the molecular level by expressing an allele containing the suspected mutation in a biological model, such as S. cerevisiae. However, because of the large size of the FKS genes, the genetic models developed so far have been restricted to the extrachromosomal expression of chimeric S. cerevisiae FKS genes recombined with the HS1 region that is suspected of being involved in resistance. [START_REF] Garcia-Effron | A naturally occurring proline-toalanine amino acid change in Fks1p in Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis accounts for reduced echinocandin susceptibility[END_REF][START_REF] Katiyar | Role for Fks1 in the intrinsic echinocandin resistance of Fusarium solani as evidenced by hybrid expression in Saccharomyces cerevisiae[END_REF][START_REF] Asner | Acquired multidrug antifungal resistance in Candida lusitaniae during therapy[END_REF][START_REF] Dudiuk | Molecular confirmation of the relationship between Candida guilliermondii Fks1p naturally occurring amino acid substitutions and its intrinsic reduced echinocandin susceptibility[END_REF] In this work, we report the development of a new expression system based on the replacement of the full-length FKS1 allele in Candida (teleomorph Clavispora) lusitaniae, an opportunistic pathogenic yeast belonging to the CTG clade. Its haploid genome can be genetically engineered easily. [START_REF] El-Kirat-Chatel | A two-step cloning-free PCRbased method for the deletion of genes in the opportunistic pathogenic yeast Candida lusitaniae[END_REF][START_REF] Franc¸ois | Development of an integrative transformation system for the opportunistic pathogenic yeast Candida lusitaniae using URA3 as a selection marker[END_REF] This recently allowed us to develop an expression system to screen the impact of mutations in the essential gene ERG11 on azole antifungal susceptibility. [START_REF] Accoceberry | A CTG clade Candida yeast genetically engineered for the genotype-phenotype characterization of azole antifungal resistance in human-pathogenic yeasts[END_REF] Since deletion of FKS1 in C. lusitaniae is not viable, the replacement of FKS1 was based on the recombination of two linear DNA fragments encompassing the entire ORF and overlapping a small 40 bp region where SNPs or small indels of interest were inserted. Correct integration at FKS1 was selected by the functional reconstitution of auxotrophic markers implanted in the upstream and downstream regions of FKS1. As a proof of concept, we successfully expressed FKS mutations in C. lusitaniae that have already been described in clinical isolates of C. albicans, C. glabrata and Candida parapsilosis.

Materials and methods

Yeast strains and culture conditions

The different strains used and constructed in this study are listed in Table 1. The C. lusitaniae WT strain CBS 6936 [START_REF] Durrens | Genome sequence of the yeast Clavispora lusitaniae type strain CBS 6936[END_REF] was used for gene cloning and as a susceptible reference strain for antifungal susceptibility testing. The double auxotrophic mutant 6936 trp1D, ura3D, [START_REF] El-Kirat-Chatel | A two-step cloning-free PCRbased method for the deletion of genes in the opportunistic pathogenic yeast Candida lusitaniae[END_REF][START_REF] Gabriel | Deletion of the uracil permease gene confers cross-resistance to 5-fluorouracil and azoles in Candida lusitaniae and highlights antagonistic interaction between fluorinated nucleotides and fluconazole[END_REF] bearing a 798 bp deletion in the phosphoribosyl-anthranilate isomerase gene and a 360 bp deletion in the orotidine 5 0 -phosphate decarboxylase gene, respectively, was used for transformation experiments.

Yeasts were cultivated in liquid YPD medium (1% yeast extract/2% peptone/2% dextrose) at 35 • C under agitation (250 rpm). In transformation experiments, selection was achieved on synthetic YNBS medium [0.67% yeast nitrogen base without amino acids (Difco Laboratories)/2% glucose/ 1 M sorbitol]. When needed, uracil (50 mg/L), tryptophan (50 mg/L), 5-fluoroorotic acid (0.8 g/L) and/or 5-fluoroanthranilate (0.3 g/L) were added at the indicated concentration. RPMI 1640 (Sigma), supplemented with 2% glucose and buffered to pH 7.0 with 0.165 M MOPS, was used for antifungal susceptibility testing. Solid media were obtained with 2% agar (Sigma).

Antifungal susceptibility testing

In vitro caspofungin susceptibility was determined using Etest (bioMe´rieux, France) according to the manufacturer's instructions. For each FKS mutation expressed in C. lusitaniae, caspofungin MIC was determined for eight prototroph clones selected after transformation and confirmed for having acquired the mutation of interest by nucleotide sequencing. For the WT strain and clones transformed with an FKS1 allele conferring RES (F634V and P642A), MIC was confirmed by the CLSI broth microdilution method [START_REF]Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts-Third Edition: Approved Standard M27-A3[END_REF] using RPMI supplemented with different concentrations of caspofungin (Merck & Co., Kenilworth, NJ, USA), from 0.03 to 16 mg/L.

DNA extraction, PCR amplifications and nucleotide sequencing

Genomic DNA was extracted by a glass bead method for yeast cell disruption, as previously described. [START_REF] El-Kirat-Chatel | A two-step cloning-free PCRbased method for the deletion of genes in the opportunistic pathogenic yeast Candida lusitaniae[END_REF] The high-fidelity DNA polymerase Pfu (Promega) was used to amplify DNA fragments by PCR for cloning steps and for overlapping PCR. Routine PCRs were performed with GoTaq DNA polymerase or GoTaq Long for PCR targets up to 5 kb (Promega), as recommended by the supplier. All primers used in this study (Table S1, available as Supplementary data at JAC Online) were synthesized by Eurofins MWG Operon (Europe). When required, PCR products were sequenced by Eurofins MWG Operon using the BigDye Terminator v3.1 kit (Applied Biosystems).

Molecular constructions and yeast transformations

To construct the DNA cassettes, DNA fragments were generated by PCR using primers having a 15 bp overlap homology at their ends and were assembled and cloned using the In-Fusion VR HD Cloning Kit (Clontech), as previously described. [START_REF] Accoceberry | A CTG clade Candida yeast genetically engineered for the genotype-phenotype characterization of azole antifungal resistance in human-pathogenic yeasts[END_REF] Yeast strains were transformed by electroporation, [START_REF] Franc¸ois | Development of an integrative transformation system for the opportunistic pathogenic yeast Candida lusitaniae using URA3 as a selection marker[END_REF] slightly modified by adding 1 M sorbitol to the lithium acetate buffer, using 1 to 2 l g of transforming DNA per experiment. Prototrophic transformants were selected on YNBS selective medium after 3 days of incubation at 35 • C.

Southern blotting

Approximately 10 l g of C. lusitaniae DNA was digested with the appropriate restriction enzyme, separated by electrophoresis in a 1% agarose gel and transferred onto nylon membranes (Hybond N+; Roche Molecular Biochemicals). Hybridization was carried out with digoxigenin (DIG)-labelled probes synthesized with a PCR DIG probe synthesis kit (Roche Molecular Biochemicals), as specified by the supplier. F1 TRP1, ura3D IG5 0 FKS1::TRP1, IG3 0 FKS1::URA3 this study F1 trp1D3 0 ,URA3 F1 TRP1, URA3 IG5 0 FKS1::trp1D3 0 , IG3 0 FKS1::URA3 this study F1 trp1D3 0 ,ura3D5 0 F1 trp1D3 0 ,URA3 IG5 0 FKS1::trp1D3 0 , IG3 0 FKS1::ura3D5 0 this study IG5 0 FKS1 and IG3 0 FKS1 correspond to the intergenic (IG) region located upstream (5 0 ) and downstream (3 0 ) of FKS1, respectively. D3 0 and D5 0 mean that the relevant gene was deleted in its 3 0 or in its 5 0 part, respectively.

Results

Accurate annotation of the FKS1 gene of C. lusitaniae

Comparison of BLAST sequences using the

Design of a C. lusitaniae strain for the homologous expression of full-length mutated FKS1 alleles

An initial difficulty for replacing the FKS1 allele in C. lusitaniae is that the gene is essential, as in C. albicans. [START_REF] Douglas | Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-b-D-glucan synthase inhibitors[END_REF] Attempts to obtain a deletion mutant in C. lusitaniae have failed until now, in spite of generally having a good rate of homologous recombination during transformation experiments, from 25% to 100% according to the locus targeted. [START_REF] El-Kirat-Chatel | A two-step cloning-free PCRbased method for the deletion of genes in the opportunistic pathogenic yeast Candida lusitaniae[END_REF][START_REF] Franc¸ois | Development of an integrative transformation system for the opportunistic pathogenic yeast Candida lusitaniae using URA3 as a selection marker[END_REF] The haploidy of C. lusitaniae represents an additional obstacle for working with essential genes.

A second difficulty is the large size of FKS1, which requires the generation and manipulation of large DNA fragments in vitro; in the present case, 5661 bp for the ORF, plus ~0.4 kb for each 5 0 -UTR and 3 0 -UTR (where UTR stands for untranslated transcribed region), and 1.5 kb for the functional genetic marker allowing the selection of the yeasts having achieved FKS1 allele replacement. Using the 'split-marker' strategy developed in S. cerevisiae, [START_REF] Fairhead | New vectors for combinatorial deletions in yeast chromosomes and for gap-repair cloning using 'split-marker' recombination[END_REF] we conducted a preliminary experiment in C. lusitaniae where we successfully repaired a large 990 bp deletion in the URA3 gene of a mutant strain after transformation with two independent linear DNA fragments. Each fragment carried half of the URA3 coding region, and had extremities allowing the fragments to first recombine with each other before integrating the locus by recombination with the chromosomal regions flanking the deletion (data not shown). We therefore decided to replace the FKS1 allele with two recombinant DNA fragments, targeting their integration at the FKS1 locus by the concomitant reconstitution of two auxotrophic markers in the upstream and downstream intergenic (IG) regions of FKS1 (Figure

Engineering the C. lusitaniae strain for FKS1 allele replacement

All genetic modifications were performed in the strain 6936 trp1D, ura3D. First, the C. lusitaniae TRP1 gene was integrated into the FKS1 upstream IG region of strain 6936 trp1D, ura3D (Figure 1a), using a DNA cassette made of the TRP1 gene with its own 5 0 and 3 0 regulatory regions flanked on both sides by DNA fragments (~400 bp) homologous to the FKS1 upstream IG region. One transformant, named F1 TRP1, ura3D, was selected. Second, according to the same principle, the URA3 gene was introduced into the FKS1 downstream IG region of the strain F1 TRP1, ura3D (Figure 1b), leading to the selection of a prototrophic transformant F1 TRP1, URA3. Third, the 3 0 half part of the TRP1 gene was removed from the strain F1 TRP1, URA3 by replacing TRP1 with a DNA cassette made of the 5 0 half part of TRP1 flanked by DNA fragments homologous to the FKS1 upstream IG region (Figure 1c). Tryptophan auxotrophic clones were selected by 5-fluoroanthranilic acid resistance and one of them, named F1 trp1D3 0 , URA3, was retained for further manipulation. Fourth, using the same strategy, the 5 0 half part of the URA3 gene was removed from the strain F1 trp1D3 0 , URA3, to yield the strain F1 trp1D3 0 , ura3D5 0 , which was selected among the clones resistant to 5-fluoroorotic acid (Figure 1d).

At each step of the molecular modifications, correct targeted integration of the different DNA cassettes was verified by PCR and nucleotide sequencing. The genotype of the selected transformants (Table 1) was also verified by Southern blotting analysis of EcoRV-digested genomic DNA using the 3 0 TRP1, 5 0 URA3, 3 0 URA3 and FKS1 probes (Figure S1) to exclude clones exhibiting any additional or incorrect integration events. Finally, we verified that the molecular modifications achieved in the strain F1 trp1D3 0 , ura3D5 0 did not alter its susceptibility to echinocandin drugs; both CLSI and Etest methods gave a caspofungin MIC of 0.125 mg/L, which was identical to that of the WT strain CBS 6936. Accordingly, strain F1 trp1D3 0 , ura3D5 0 was used in the rest of the study for all FKS1 allele replacement experiments.

Expression of full-length mutated FKS1 alleles in C. lusitaniae

Replacement of the resident WT FKS1 allele of the strain F1 trp1D3 0 , ura3D5 0 by an FKS1 allele containing any SNP of interest was achieved by co-transformation with two recombinable DNA fragments overlapping the FKS1 gene and its regulatory sequences. To ensure their correct targeted integration at the FKS1 locus, each fragment was fused to the complementary part of one of the auxotrophic markers inserted in the upstream and downstream regions flanking FKS1. To allow crossover to take place and to reconstitute functional TRP1, FKS1 and URA3 genes, the transforming DNA fragments contained an overlapping region of 166 bp in the TRP1 region or 211 bp in the URA3 region, and 40 bp in the FKS1 region where the SNP was introduced (Figure 2). When the two DNA fragments were co-transformed into the F1 trp1D3 0 , ura3D5 0 strain, they recombined in cellulo to integrate the FKS1 locus, thereby allowing selection on YNBS medium of prototroph transformant yeast cells that had integrated the new FKS1 allele containing the SNP of interest. Selected clones were then screened for the presence of the point mutation by DNA sequencing and their caspofungin susceptibility pattern was determined. According to the position of the SNP of interest, 40% to 75% of the transformants tested carried the desired point mutation. The other clones were prototrophic, but the molecular events leading to integration of the DNA fragments took place outside the overlapping region where the mutation was introduced.

Proof of concept: impact of FKS mutations known to be associated with in vitro caspofungin resistance or RES

Our genetic model was first challenged with three mutations previously reported in the FKS1 alleles of clinical isolates of C. albicans, resulting in amino acid changes phenylalanine to valine at position 641 (F641V) in HS1, serine to proline at position 645 (S645P) in HS1 and arginine to glycine at position 1361 (R1361G) in HS2 (Figure 3).

Expression of the mutated FKS1 allele bearing the F634V mutation (equivalent to F641V) in the C. lusitaniae strain F1 trp1D3 0 , ura3D5 0 conferred an intermediate level of resistance to caspofungin (MIC " 1.5 mg/L), while introduction of S638P (equivalent to S645P) or R1352G (equivalent to R1361G) mutations conferred full resistance (MICs ≥32 mg/L; Figure 4).

Our model was then challenged with a mutation reported in C. glabrata. Unlike other Candida species, mutations conferring resistance to caspofungin also affect FKS2, with a 2-fold greater frequency than that of FKS1 mutations. Accordingly, we introduced a mutation specific to the FKS2 gene of C. glabrata into the FKS1 gene of C. lusitaniae at the equivalent position. The mutation consisted of the deletion of a phenylalanine at position 659 (Figure 3). Expression of the FKS1 F634del allele in C. lusitaniae resulted in an increase in caspofungin MIC from 0.125 to 32 mg/L (Figure 4). Resulting genetic map of the FKS1 locus after FKS1 allelic exchange. The size of the overlapping regions that were used to allow crossover to take place is indicated. An SNP is represented by an asterisk. Lastly, our system was tested with the naturally occurring P660A polymorphism reported in the FKS1 alleles of all isolates of the C. parapsilosis species complex. [START_REF] Garcia-Effron | A naturally occurring proline-toalanine amino acid change in Fks1p in Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis accounts for reduced echinocandin susceptibility[END_REF] This mutation was shown to be responsible for higher MICs of all echinocandins for C. parapsilosis, compared with other Candida species (Figure 3). [START_REF] Garcia-Effron | A naturally occurring proline-toalanine amino acid change in Fks1p in Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis accounts for reduced echinocandin susceptibility[END_REF] Introduction of the equivalent mutation P642A into the C. lusitaniae FKS1 allele gave a 6-fold increase in caspofungin MIC (0.125 to 0.75 mg/L; Figure 4). This confirmed the role of the P660A substitution in RES and showed that this reduced susceptibility can be transferred to other species of the CTG clade.

Discussion

The main molecular mechanism leading to resistance to echinocandins or RES is mediated by non-synonymous mutations affecting the Fksp subunits of b(1,3)-glucan synthase. [START_REF] Douglas | Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-b-D-glucan synthase inhibitors[END_REF][START_REF] Park | Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates[END_REF] The resistance mutations known so far are located within two HS regions. [START_REF] Perlin | Mechanisms of echinocandin antifungal drug resistance: mechanism of echinocandin drug resistance[END_REF][START_REF] Park | Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates[END_REF] However, echinocandins are non-competitive inhibitors and do not interact directly with the catalytic site. This suggests that mutations outside HS regions can contribute to resistance and RES, as already shown in S. cerevisiae. [START_REF] Johnson | New Fks hot spot for acquired echinocandin resistance in Saccharomyces cerevisiae and its contribution to intrinsic resistance of Scedosporium species[END_REF] One way to demonstrate the involvement of a mutation in resistance or RES is to express the mutated allele and the WT allele from which it derives in cells having an isogenic background and to compare echinocandin MICs. The S. cerevisiae expression system was based on an fks1 deletion mutant that was complemented by the expression of a WT or mutated FKS1 allele cloned in a 2 l ori episomal plasmid. [START_REF] Park | Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates[END_REF] Nucleotide modifications were introduced into the FKS allele as point mutations, or as a chimeric construction involving a S. cerevisiae FKS allele recombined with the HS region of an FKS allele suspected of conferring resistance to a clinical isolate. [START_REF] Park | Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates[END_REF][START_REF] Johnson | New Fks hot spot for acquired echinocandin resistance in Saccharomyces cerevisiae and its contribution to intrinsic resistance of Scedosporium species[END_REF][START_REF] Katiyar | Role for Fks1 in the intrinsic echinocandin resistance of Fusarium solani as evidenced by hybrid expression in Saccharomyces cerevisiae[END_REF][START_REF] Asner | Acquired multidrug antifungal resistance in Candida lusitaniae during therapy[END_REF][START_REF] Dudiuk | Molecular confirmation of the relationship between Candida guilliermondii Fks1p naturally occurring amino acid substitutions and its intrinsic reduced echinocandin susceptibility[END_REF] FKS1 expression in S. cerevisiae was facilitated by the fact that an fks1D mutant is viable owing to the presence of the functionally redundant paralogue gene GSC2 (alias FKS2). [START_REF] Parent | Calcineurin-dependent growth of an FK506-and CsA-hypersensitive mutant of Saccharomyces cevevisiae[END_REF] Functional redundancy has also been reported in the related opportunistic pathogenic yeast species, C. glabrata, [START_REF] Katiyar | Fks1 and Fks2 are functionally redundant but differentially regulated in Candida glabrata: implications for echinocandin resistance[END_REF][START_REF] Niimi | Reconstitution of high-level micafungin resistance detected in a clinical isolate of Candida glabrata identifies functional homozygosity in glucan synthase gene expression[END_REF] whose prevalence of clinical isolates resistant to echinocandins has increased in recent years. [START_REF] Perlin | Mechanisms of echinocandin antifungal drug resistance: mechanism of echinocandin drug resistance[END_REF][START_REF] Healey | Prevalent mutator genotype identified in fungal pathogen Candida glabrata promotes multi-drug resistance[END_REF] This made allelic replacement possible at the chromosomal level in C. glabrata by integrating one FKS copy fused to an auxotrophic marker to select the transformants. [START_REF] Niimi | Reconstitution of high-level micafungin resistance detected in a clinical isolate of Candida glabrata identifies functional homozygosity in glucan synthase gene expression[END_REF] However, interpreting the effect of an FKS mutation on RES in C. glabrata can be hampered by the presence of the WT FKS paralogue. [START_REF] Katiyar | Fks1 and Fks2 are functionally redundant but differentially regulated in Candida glabrata: implications for echinocandin resistance[END_REF] Most of the invasive yeast infections are caused by Candida species belonging to the CTG clade, of which C. albicans is the most prevalent. This phylogenic group of yeasts is characterized by the fact that FKS1 is an essential gene whose deletion is not compensated by FKS2. [START_REF] Mio | Cloning of the Candida albicans homolog of Saccharomyces cerevisiae GSC1/FKS1 and its involvement in b-1,3-glucan synthesis[END_REF] Engineering C. albicans to express a mutated FKS1 allele was achieved with an integrative plasmid carrying a copy of the mutated FKS1 allele designed to recombine to the chromosomal FKS1 locus. [START_REF] Park | Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates[END_REF] However, the diploidy of C. albicans can constitute an issue for testing echinocandin resistance because of the difficulty of obtaining homozygous mutants.

To overcome the problems resulting from FKS redundancy or diploidy, we developed in this work a strain of the CTG clade haploid yeast species C. lusitaniae as an alternative model for fulllength FKS1 replacement. Several studies previously showed that echinocandin MICs were from 4-fold to 8-fold higher for C. lusitaniae than for C. albicans. [START_REF] Pfaller | In vitro susceptibility of invasive isolates of Candida spp. to anidulafungin, caspofungin, and micafungin: six years of global surveillance[END_REF][START_REF] Pfaller | Wild-type MIC distributions and epidemiological cutoff values for the echinocandins and Candida spp[END_REF] However, most clinical isolates exhibit low echinocandin MICs with more than 90% of the isolates being inhibited by concentrations lower or equal to 1 mg/L for anidulafungin and 0.5 mg/L for caspofungin and micafungin. [START_REF] Lockhart | Candida lusitaniae MICs to the echinocandins are elevated but FKS-mediated resistance is rare[END_REF] Here, we used a strain derived from the type strain CBS 6936 of C. lusitaniae, [START_REF] Durrens | Genome sequence of the yeast Clavispora lusitaniae type strain CBS 6936[END_REF] which has a low caspofungin MIC of 0.125 mg/L and whose susceptibility remained unchanged after the genetic modifications performed in both IG regions flanking the FKS1 gene. We used caspofungin because of its availability in powder form that allowed MIC determination to be performed both by the CLSI microdilution method and by Etest, notably in the case of RES.

Our expression system was challenged with known mutations reported and validated from different genes (FKS1 or FKS2), different regions (HS1 or HS2) and different Candida species. When introduced at an equivalent position into the FKS1 gene of C. lusitaniae, the FKS1 S645P and R1361G of C. albicans, [START_REF] Park | Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates[END_REF] the latter mutation being initially discovered in the FKS1 R1361G of Candida krusei, [START_REF] Park | Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates[END_REF] and the FKS2 F659del of C. glabrata 28 conferred high caspofungin MICs, while the FKS2 F641V of C. glabrata [START_REF] Garcia-Effron | Correlating echinocandin MIC and kinetic inhibition of fks1 mutant glucan synthases for Candida albicans: implications for interpretive breakpoints[END_REF] and the FKS1 P660A of C. parapsilosis 10 conferred RES. Most importantly, the caspofungin MICs conferred to C. lusitaniae were fully comparable to those for the clinical isolates from which they were originally reported. In conclusion, we have successfully developed a C. lusitaniae strain for the expression of a single chromosomal copy of an essential FKS1 essential gene that allows any new genetic polymorphism involved in echinocandin resistance or RES in Candida species to be screened.

WT F634V S638P

  of C. lusitaniae (GenBank accession number OVF07437) in the genome of the CBS 6936 strain (GenBank accession number LYUB02000000[START_REF] Durrens | Genome sequence of the yeast Clavispora lusitaniae type strain CBS 6936[END_REF] ) on scaffold 13 (locus tag A9F13_13g01430). The intronless ORF of 5661 nt encodes a predicted protein of 1886 amino acids showing 82.9% and 71.8% identity with the orthologous protein of C. albicans (strain SC5314, GenBank accession number XP_721429) and of C. glabrata (strain CBS 138, GenBank accession number XP_446406), respectively. The genomic map surrounding the FKS1 gene of strain CBS 6936 was comparable to that of the C. lusitaniae strain ATCC 42720 (GenBank accession number NW_003101577, scaffold 2, FKS1 encompasses CLUG_01697 to CLUG_01702 due to sequence errors). Synteny with C. albicans SC5314 was conserved. A gene encoding the putative eukaryotic translation initiation factor 2A is located 2221 bp upstream of the C. lusitaniae FKS1 gene and a gene encoding a putative a-1,6-mannosyltransferase is located 382 bp downstream.

Figure 1 .

 1 Figure 1. Schematic representation of the molecular modifications performed at the FKS1 locus of C. lusitaniae to obtain the strain F1 trp1D3 0 , ura3D5 0 . (a) FKS1 locus of the strain 6936 trp1D, ura3D. (b) Successive integration of the TRP1 and URA3 genes in the upstream and downstream regions flanking FKS1, respectively. (c) Deletion of the 3 0 coding region and the 3 0 -UTR of the TRP1 gene, and of the 5 0 -UTR and the 5 0 coding region of the URA3 gene. (d) Resulting genetic map of the FKS1 locus of strain F1 trp1D3 0 , ura3D5 0 . Genes are not represented at the same scale.

Figure 2 .

 2 Figure 2. Strategy used to replace the resident FKS1 allele of the strain F1 trp1D3 0 , ura3D5 0 by an FKS1 allele bearing a mutation. (a) Replacement was performed by transforming strain F1 trp1D3 0 , ura3D5 0 simultaneously with two linear DNA fragments covering the entire FKS1 ORF with its regulatory regions, each fragment being fused at one extremity to the complementary part of the genetic marker implanted in the IG region of FKS1. (b)Resulting genetic map of the FKS1 locus after FKS1 allelic exchange. The size of the overlapping regions that were used to allow crossover to take place is indicated. An SNP is represented by an asterisk.

Figure 3 .

 3 Figure 3. HS1 and HS2 amino acid sequences of Fks proteins. (a) Fks1p from C. albicans and C. parapsilosis and Fks2p from C. glabrata. The amino acid substitutions expressed in Fks1p of C. lusitaniae are shown below the underlined WT residue. A deletion is represented by D. Numbers flanking the HS sequence correspond to the positions of the first and last amino acids of the HS relative to the whole Fksp sequence. (b) Corresponding positions of the amino acids that were substituted in the C. lusitaniae Fks1p in this study are indicated by filled circles.

Figure 4 .

 4 Figure 4. Etest determination of caspofungin susceptibility of the C. lusitaniae strain F1 trp1D3 0 , ura3D5 0 expressing a WT Fks1 protein and expressing Fks1 proteins containing different amino acid substitutions. For each FKS1 allele expressed, caspofungin MIC was determined for eight independent clones after having controlled at the molecular level that they had integrated the SNP of interest. The MIC is indicated by a white arrow.

Table 1 .

 1 Origin and genotype of C. lusitaniae strains

	Strain	Origin	Genotype a	Reference(s)

a

Acknowledgements

We would like to thank Ray Cooke for proofreading the manuscript prior to submission and Merck Sharp & Dohme Corp. for providing caspofungin.

Funding

This work was supported by grants from the University of Bordeaux and the Centre National de la Recherche Scientifique (CNRS).

Transparency declarations

None to declare.

Author contributions


Y He

G Ducrozet

N Hoffmann

John Michael Dudley

Amin Chabchoub

Galilean-transformed solitons and supercontinuum generation in dispersive media

Galilean-transformed solitons and supercontinuum

generation in dispersive media.

Introduction

The nonlinear Schrödinger equation (NLSE) is a comprehensive framework which describes the dynamics of wave pulses beyond surface gravity water waves [START_REF] Henry | Nonlinear Dynamics of Deep-Water Gravity Waves[END_REF][START_REF] Osborne | Nonlinear Ocean Waves and the Inverse Scattering Transform[END_REF]. For instance, in Kerr media, plasma, and Bose-Einstein condensates [START_REF] Remoissenet | Waves called solitons: concepts and experiments[END_REF][START_REF] John M Dudley | Instabilities, breathers and rogue waves in optics[END_REF][START_REF] Carr | Spontaneous soliton formation and modulational instability in bose-einstein condensates[END_REF]. Since the proof of NLSE-integrability [START_REF] Shabat | Exact theory of two-dimensional self-focusing and one-dimensional self-modulation of waves in nonlinear media[END_REF], several key wave envelope solutions have been derived and discussed within the context of modulation instability, see [START_REF] Nail | Solitons: nonlinear pulses and beams[END_REF][START_REF] Yuri | Optical solitons: from fibers to photonic crystals[END_REF][START_REF] Malomed | Soliton management in periodic systems[END_REF][START_REF] Mark | Nonlinear dispersive waves: asymptotic analysis and solitons[END_REF][START_REF] Js He | Generating mechanism for higher-order rogue waves[END_REF][START_REF] Onorato | Rogue waves and their generating mechanisms in different physical contexts[END_REF][START_REF] Dudley | Rogue waves and analogies in optics and oceanography[END_REF]. Besides the time-reversal symmetry [START_REF] Chabchoub | Time-reversal generation of rogue waves[END_REF], which has been proven to be useful for applications [START_REF] Ducrozet | Experimental reconstruction of extreme sea waves by time reversal principle[END_REF], another invariant operation is the Galilean transformation (GT) [START_REF] Nail | Solitons: nonlinear pulses and beams[END_REF][START_REF] Dysthe | Note on breather type solutions of the NLS as models for freak-waves[END_REF][START_REF] Widjaja | Absence of galilean invariance for pure-quartic solitons[END_REF]. That said, introducing a Galilean velocity (GV) to a NLSE pulse is colligated to a carrier frequency-shift to satisfy symmetry [START_REF] Sulem | The nonlinear Schrödinger equation: self-focusing and wave collapse[END_REF]. As such, the GT has been legitimately considered not to be applicable and relevant for dispersive physical systems [START_REF] Nail | Solitons: nonlinear pulses and beams[END_REF][START_REF] Ci Christov | An energy-consistent dispersive shallow-water model[END_REF][START_REF] Duran | On the galilean invariance of some nonlinear dispersive wave equations[END_REF].

In this work, we report experimental observations of Galilean NLSE solitons for a wide range of GVs without an external flow forcing [START_REF] Gary Rozenman | Observation of accelerating solitary wavepackets[END_REF]. The experimental data show considerable deviations from hydrodynamic NLSE predictions, nevertheless, a very good agreement with the modified NLSE (MNLSE) [START_REF] Dysthe | Note on a modification to the nonlinear Schrödinger equation for application to deep water waves[END_REF] and the numerically-solved Euler equations using the higherorder spectral method (HOSM) [START_REF] Douglas | A high-order spectral method for the study of nonlinear gravity waves[END_REF][START_REF] Ducrozet | A modified high-order spectral method for wavemaker modeling in a numerical wave tank[END_REF]. It is shown that the type of deviations in wave envelope depends on the sign of GV. For the case of the envelope soliton, the pulse undergoes a strong broadening when GV is negative, whereas a strong self-focusing of wave envelope is observed when the GV is positive [START_REF] Cousins | Unsteady evolution of localized unidirectional deep-water wave groups[END_REF]. Furthermore, we show that for predetermined carrier wave steepness parameters, there are exact GV values for which a Galileantransformed envelope soliton corresponds to an exact multi-soliton solution [START_REF] Satsuma | initial value problems of one-dimensional self-modulation of nonlinear waves in dispersive media[END_REF]. In fact, the higher the value of GV, the higher the order of the multisoliton. As a result, supercontinuum generation is an unavoidable process, which follows as a result of substantial wave focusing and subsequent soliton fission events [START_REF] John M Dudley | Supercontinuum generation in photonic crystal fiber[END_REF][START_REF] Michael | Supercontinuum light[END_REF][START_REF] Chabchoub | Hydrodynamic supercontinuum[END_REF]. We experimentally confirm the hydrodynamic supercontiuum generation from Galilean envelope solitons in form of an irreversible and severe spectral broadening genesis. We also briefly discuss the influence of the GT on the Peregrine soliton dynamics, even though the limited fetch of the experimental facility does not allow the study of emergent and long-ranging complex wave patterns as a result of intrinsic and higher-order modulation instability processes [START_REF] Erkintalo | Higher-order modulation instability in nonlinear fiber optics[END_REF][START_REF] Gelash | Bound state soliton gas dynamics underlying the spontaneous modulational instability[END_REF].

Constructing hydrodynamic Galilean solitons

Our starting point is the dimensional time-like NLSE for deep-water waves. In fact, the weakly nonlinear propagation of a narrowband wave field ψ with dominant wave frequency ω and wave number k while propagating along the longitudinal direction x follows [START_REF] Benney | The propagation of nonlinear wave envelopes[END_REF][START_REF] Zakharov | Stability of periodic waves of finite amplitude on the surface of a deep fluid[END_REF][START_REF] Osborne | Nonlinear Ocean Waves and the Inverse Scattering Transform[END_REF]

i(ψ x + 1 c g ψ t ) - k ω 2 ψ tt -k 3 |ψ| 2 ψ = 0, (1)
where ω and k are connected through the linear dispersion relation ω = √ gk, involving the gravitational acceleration g and t being the time coordinate while c g = ω 2k denotes the group velocity in deep-water. The simplicity of this nonlinear wave framework allows for the understanding of the distinct role of dispersion and nonlinearity in the wave dynamics. Moreover, being an integrable framework [START_REF] Shabat | Exact theory of two-dimensional self-focusing and one-dimensional self-modulation of waves in nonlinear media[END_REF] facilitates the study of coherent structures' behavior in a variety of wave guides, regardless of being either steady or unstable [START_REF] Onorato | Rogue waves and their generating mechanisms in different physical contexts[END_REF][START_REF] Chabchoub | Hydrodynamic envelope solitons and breathers[END_REF]. The dimensional form of the envelope soliton of amplitude a, satisfying Eq. ( 1) reads

ψ S (x, t) = a sech(- √ 2αk(x -c g t)) exp(- iα 2 kx 2 ), (2)
α := ak being the wave steepness. Even though having a simple construction form, such localized wave groups play an important role in understanding the formation of wave coherence in the ocean [START_REF] Slunyaev | Persistence of hydrodynamic envelope solitons: Detection and rogue wave occurrence[END_REF][START_REF] Waseda | Directional coherent wave group from an assimilated nonlinear wavefield[END_REF]. Applying the GT to the sech-soliton yields to the following parametrization

ψ GS (x, t) =a sech(- √ 2αk(x -c g t) - c 2 α 2 kx) exp(- iα 2 kx 2 ) exp( i √ 2cαk(x -c g t) 2 + ic 2 α 2 kx 8 ), (3)
which still satisfies the NLS Eq. ( 1). We refer to [START_REF] Nail | Solitons: nonlinear pulses and beams[END_REF][START_REF] Dysthe | Note on breather type solutions of the NLS as models for freak-waves[END_REF] for GT details, including more convenient theoretical representations in dimensionless physical quantities. Note that the GT compromises a frequency-shift of the carrier and is different from the Tajiri-Watanabe-type construction [START_REF] Tajiri | Breather solutions to the focusing nonlinear schrödinger equation[END_REF], which may cause experimental challenges in optical wave guides.

Examples of dimensional envelope soliton evolution for different GVs and wave steepness values are shown in Fig. 1. We emphasize that when the GV is negative, i.e. c < 0, the wave packet propagates slower, whereas when c > 0 the coherent wave groups evolves faster compared to the pure and non-Galilean soliton. The Galilean-transformed hydrodynamic Peregrine soliton [START_REF] Peregrine | Water waves, nonlinear Schrödinger equations and their solutions[END_REF] is expressed as

ψ GP (x, t) =a(-1 + 4(1 -iωα 2 x/c g ) 1 + 4(cα 2 kx/2 + √ 2kα(x -c g t)) 2 + ω 2 α 4 x 2 /c 2 g ) exp(-iα 2 kx) exp( i √ 2cαk(x -c g t) 2 + ic 2 α 2 kx 8 ), (4)
Both, Galilean-transformed envelope and Peregrine solitons will be tested in a water wave tank. The boundary conditions and times-series applied to the wave maker can be determined from the expression of water elevation to first-order in steepness, as defined by

η(x † , t) = (ψ(x † , t) exp(i(kx † -ωt))), (5)
where x † can be adapted to control the location of wave focusing in the wave guide, i.e. the wave flume in our case, for the case of breathers.

Experimental build-up and arrangements

The experiments were carried out in the water wave flume of the University of Sydney, which is 30 meters long, 1 meter high and 1 meter wide, allowing the study of specifically controlled or irregular and random wave trains. A piston-type wave paddle located at one side of the wave tank, as shown in Fig. 2, can generate waves in finite water depth as well as deepwater conditions. The wave paddle can generate wave time-series with a peak frequency ranging between 0.5 to 2 Hz in water depth conditions varying from 0.4 m to 0.9 m. In this study, the carrier wave frequency is fixed at f = 1.25 Hz, which corresponds to a wavelength of λ = 1 m and a wavenumber of k = 2π λ = 2π. The amplitude a of the carrier wave is varied to satisfy wave steepness α values of 0.04, 0.06 and 0.08, respectively. We also ensure that the dominant carrier frequency-shift after the GT, as defined by the GV value c, is within the operational range of the wave maker. The water depth is set to be h = 0.7 m. Hence, kh 1, which satisfies deep-water requirements. Eight capacitance wave gauges with a sampling frequency of 32 Hz can be re-deployed along the flume for a generated time-series to allow not only a very high temporal, but also a spatial resolution considering the repeatability of the experiments.

Experimental results

The focus of our experimental campaign will be on the Galilean-transformed envelope solitons. The case of Peregrine breather is more complex and will be briefly discussed as well.

We recall that both, envelope and Peregrine solitons have been observed in a wide range of physical media [START_REF] Henry | Nonlinear deep water waves: Theory and experiment[END_REF][START_REF] Linn F Mollenauer | Experimental observation of picosecond pulse narrowing and solitons in optical fibers[END_REF][START_REF] Strecker | Formation and propagation of matter-wave soliton trains[END_REF][START_REF] Khaykovich | Formation of a matter-wave bright soliton[END_REF][START_REF] Kibler | The peregrine soliton in nonlinear fibre optics[END_REF][START_REF] Chabchoub | Rogue Wave Observation in a Water Wave Tank[END_REF][START_REF] Bailung | Observation of peregrine solitons in a multicomponent plasma with negative ions[END_REF]. In water waves envelope solitons have been confirmed to be steady even for very large steepness values, even close to the wave breaking threshold [START_REF] Slunyaev | Simulations and experiments of short intense envelope solitons of surface water waves[END_REF][START_REF] Toffoli | Maximum steepness of oceanic waves: Field and laboratory experiments[END_REF]. The carrier wave steepness does not only quantify the degree of nonlinearity of Stokes waves, but also determines the width of a wave pulse or wave packet. Three values of wave steepness have been considered α ∈ {0.04, 0.06, 0.08} as well as negative and positive values of GV. The evolution of envelope solitons subject to GT as propagating over 22 m are shown in Fig. 3. We stress that the wave envelope has been reconstructed from the water surface data using the Hilbert transform [START_REF] Osborne | Nonlinear Ocean Waves and the Inverse Scattering Transform[END_REF]. At first view, the cases for α = 0.04 seem to show a steady evolution of the wave packet. However, since this steepness value is indeed very small, it does not allow for the nonlinear interaction to unfold over the limited fetch. Increasing the value of wave steepness reveals that differently than predicted from the NLSE framework, the Galilean-transformed solitons in a physical water wave guide are not stationary while the type of unsteadiness depends on the sign of GV. In fact, when the GV is negative, we can clearly notice that the solitons subject to GT broaden, whereas when GV is positive, the Galilean-transformed envelope solitons follow a self-compression in form of a breathing process. The latter process becomes clearly visible for the highest steepness and GV values adopted in the experiments.

To examine the physics of these deviations, we performed numerical simulations based on the modified nonlinear Schrödinger equation (MNLSE) [START_REF] Dysthe | Note on a modification to the nonlinear Schrödinger equation for application to deep water waves[END_REF][START_REF] Lo | A numerical study of water-wave modulation based on a higher-order nonlinear schrödinger equation[END_REF][START_REF] Trulsen | Spatial evolution of water surface waves: Numerical simulation and experiment of bichromatic waves[END_REF][START_REF] Shemer | An experimental and numerical study of the spatial evolution of unidirectional nonlinear water-wave groups[END_REF][START_REF] Goullet | A numerical and experimental study on the nonlinear evolution of long-crested irregular waves[END_REF]. We recall that whereas the NLSE can be derived from the Euler equations at third-order in steepness O(α 3 ) [52], the MNLSE is an improvement at the next order O(α 4 ), which accounts for higher-order dispersive effects [START_REF] Dysthe | Note on a modification to the nonlinear Schrödinger equation for application to deep water waves[END_REF][START_REF] Yuri | Dynamics of solitons in nearly integrable systems[END_REF][START_REF] Rogers | Localized pulses for the quintic derivative nonlinear schrödinger equation on a continuous-wave background[END_REF]. The MNLSE simulation results, as revealed in Fig. 4, support the evidence that unsteadiness of GT wave envelope evolution can be attributed to the failure of simplified dispersion relationship in the wave packet evolution description, similarly to the water wave propagation under the action of a uniform and steady current [START_REF] Chiang | Theory and Applications of Ocean Surface Waves: Nonlinear aspects[END_REF][START_REF] David | Linear surface gravity waves on current for a general inertial viewer[END_REF].

Positive values of GVs have a significant influence on the amplitude variations of envelope solitons subject to GT, see Fig. 5. No significant change in steadiness can be noticed for marginal αc values. The observed selfmodulation, which has been also underlined by the MNLSE simulations, for positive GVs is indeed similar to the dynamics of multi-solitons [START_REF] Satsuma | initial value problems of one-dimensional self-modulation of nonlinear waves in dispersive media[END_REF]. The connection between Galilean transformed multi-solitons and Satsuma-Yajima solutions will be further explored in the next Section 5.

The investigation of Galilean-transformed solitons on zero-background are simpler to perform due to the absence of modulation instability [START_REF] Bespalov | Filamentary structure of light beams in nonlinear liquids[END_REF][START_REF] Benjamin | The disintegration of wave trains on deep water Part 1. Theory[END_REF]. Consequently, any yet small perturbation of the background wave can provoke a drastic wave focusing [START_REF] Suret | Single-shot observation of optical rogue waves in integrable turbulence using time microscopy[END_REF][START_REF] Närhi | Real-time measurements of spontaneous breathers and rogue wave events in optical fibre modulation instability[END_REF][START_REF] Chabchoub | Experiments on higher-order and degenerate Akhmediev breathertype rogue water waves[END_REF]. Moreover, the observation of such unstable patterns requires a larger fetch. This makes it challenging to be explored in the University of Sydney wave flume, which is limited to an effective propagation distance of 22 m, considering the frequency range of wave maker and the limited water depth possibilities.

The propagation of a Peregrine breathers being converted according to a GT with negative and positive GVs in the wave flume together with the respective MNLSE expectations are shown in Fig. 6. Indeed, we can ob- serve that the Peregrine perturbation focuses later in the case of a negative GV and earlier for the positive GV while the MNLSE simulations agree well with the observations [START_REF] Slunyaev | Simulations and experiments of short intense envelope solitons of surface water waves[END_REF][START_REF] Shemer | Peregrine breather revisited[END_REF]. However, there is an omnipresent MI dynamics, which cannot be captured within the short fetch. Hence, we extended the propagation distance of the numerical simulations to inspect the longterm behavior of the Galilean transformed Peregrine soliton following the parameters adopted in the experiments, see Fig. 7. The MNLSE simulations suggest that long-term evolution of the Peregrine soliton is indeed complex, particularly, when GV is positive. Considering the Peregrine breather being a particular case of Kuznetsov-Ma [START_REF] Evgenii | Solitons in a parametrically unstable plasma[END_REF][START_REF] Ma | Some solutions pertaining to the resonant interaction of long and short waves[END_REF] and Akhmediev [START_REF] Akhmediev | Generation of periodic trains of picosecond pulses in an optical fiber: exact solutions[END_REF] breathers, an extensive and comprehensive study is required to address and quantify the influence of the GT on solitons on finite background.

Galilean-transformed envelope solitons and multi-solitons

To disclose the relationship between multi-solitons, or Satsuma-Yajima solitons [START_REF] Satsuma | initial value problems of one-dimensional self-modulation of nonlinear waves in dispersive media[END_REF], and the Galilean-transformed solitons, we will assume that both parametrizations are equal at x = 0, for simplicity. We recall that the dynamics of a multi-soliton ψ M S can be triggered by an integer-multiple of an envelope sech-type soliton [START_REF] Michael | Supercontinuum light[END_REF]. That is

ψ M S (0, t) = N ψ s (0, t) = N a sech(- √ 2α k (-c g t)), N ∈ N, (6)
where ψ s (0, t) is a pure envelope soliton of amplitude a , wavenumber k and wave frequency ω . Let us assume that the GT of an envelope soliton of amplitude a, wave number k, and wave frequency ω corresponds to a real-multiple of a soliton of amplitude a , and a wave frequency ω which corresponds to the wave frequency of the Galilean-transformed soliton (incl. the phase-shift), while k = w 2 /g

Rψ S (0, t) = ψ GS (0, t), R ∈ R (7)
Since the water surface elevation to first-order in steepness should be identical, the following holds

η (0, t) = (Rψ S (0, t) exp(i(-ω t))) = (ψ GS (0, t) exp(i(-ωt))) = η GS (0, t), (8)
which is equivalent to:

Ra sech(- √ 2α k (-c g t))) exp(i(-ω t)) = a sech(- √ 2αk(-c g t))) exp(i(-ωt)) exp( i √ 2cαk(-c g t) 2
).

One way to solve this is problem is to set the amplitudes, sech-components, and phases equal. Consequently, we can establish the following set of three equations

       Ra = a α k c g t = αkc g t ω t = ωt + cαkc g t √ 2 . (10)
Using the deep-water expression for the group velocity c g = ω 2k

, we can solve Eqs. [START_REF] Mark | Nonlinear dispersive waves: asymptotic analysis and solitons[END_REF] and get

ω = ω(1 + √ 2 4 cα) and R = (1 + √ 2 4 cα) 3 . (11)
Eq. [START_REF] Js He | Generating mechanism for higher-order rogue waves[END_REF] shows that both, steepness and GV affect the values of R and ω . Moreover, these later parameters α and c can be chosen so that R becomes an integer R = N and as such, Galilean-transformed solitons can define boundary conditions to launch exact multi-soliton orbits. Fig. 8 illustrates the range for the values of ak and c to map the Galilean-transformed solitons to exact Satsuma-Yajima wave groups of order N .

Supercontinuum generation

Having established the relationship between Galilean transformed envelope solitons and higher-order solitons, it is self-evident to discuss supercontinuum generation following a soliton-GT. Supercontiua can be formed as a result of soliton fission of higher-order solitons due to the strong unbalance between nonlinearity and dispersion, which arises from the considerable envelope compression or focusing, i.e. substantial high values of nonlinearity [START_REF] John M Dudley | Supercontinuum generation in photonic crystal fiber[END_REF][START_REF] Dudley | Optical fiber supercontinuum generation[END_REF][START_REF] Michael | Supercontinuum light[END_REF][START_REF] Chabchoub | Hydrodynamic supercontinuum[END_REF].

Recurrent multi-soliton focusing and the formation of supercontinua requires a long fetch. Since these cannot be met by our state of the art facility as illustrated in Fig. 2, we will investigate and explore such dynamics using a numerical wave tank, based on the higher-order spectral model scheme, which solves the Euler equations [START_REF] Douglas | A high-order spectral method for the study of nonlinear gravity waves[END_REF][START_REF] Ducrozet | A modified high-order spectral method for wavemaker modeling in a numerical wave tank[END_REF].

We first consider perfectly recurrent Galilean-transformed envelope solitons to be mapped on the orbit of a multi-soliton as described in the previous section. Simulation results and corresponding spectral evolution are summarized in Fig. 9. The HOSM simulations strikingly confirm several wave focusing recurrence cycles of wave envelope on zero-background focusing, which are clear attribute of Satsuma-Yajima breathers. This is also remarkably noticeable for the order N = 3. One possibility to break this symmetry and induce soliton fission is to increase the order of the multi-soliton solution, i.e. the GV c, or the carrier wave steepness [START_REF] Chabchoub | Hydrodynamic supercontinuum[END_REF]. We stress that wave breaking should be excluded in this process, since the flow is assumed to be irrotational to justify the use of the HOSM. One realization involving an envelope soliton, subject to a GT and meeting an initial multi-soliton profile of the order of 4 is illustrated in Fig. 10. The evolution of the wave envelope shows a clear soliton dominant fission signature into three solitons after the first focusing cycle. The latter mechanism brings along a severe and irreversible spectral broadening, which is a characteristic feature of a supercontiuum emergence.

Conclusion

Galilean soliton dynamics have been investigated in controlled laboratory conditions. Even though the NLSE predicts that the GT should not affect steadiness of the envelope solitons, clear deviations from NLSE predictions have been observed and quantitatively captured by the MNLSE, which takes into account high-order dispersive effects. For positive values of GV, Galilean-transformed solitons exhibit a self-focusing dynamics, which can be in particular cases connected to exact multi-soliton solutions. Higher-order spectral method-based numerical wave tank simulations confirm such distinctive recurrent focusing dynamics of multi-solitons as obtained from GTs, including orders of 2, 3, and 4. In addition, a particular case is considered in which soliton fission has been observed involving a supercontinuum generation, as characterized by a severe and irreversible broadening of wave spectrum. Future work will be devoted to understanding and addressing the role and limitations of MNLSE in the modeling of velocity-translated solitons on zero and particularly finite background. Preliminary observations and simulation results have revealed very complex wave coherence and focusing dynamics as a result of modulation instability at play. We also expect complementary theoretical, numerical, and experimental studies in nonlinear dispersive media other than water waves.

Figure 1 :

 1 Figure 1: Evolution of an NLSE envelope soliton (middle panels), slower analogues with negative GVs (left panels), and faster counterparts with positive GVs (right panels) for three wave steepness values 0.04, 0.06, and 0.08.

Figure 2 :

 2 Figure 2: The University of Sydney wave flume, which operates and piston wave maker.

Figure 3 :

 3 Figure 3: Experimental observations of Galilean-transformed and pure envelope sech-type solitons.

Figure 4 :

 4 Figure 4: MNLSE prediction of pure and Galilean transformed envelope solitons as generated in the wave tank and shown in Fig.3.

Figure 5 :

 5 Figure 5: Amplitude amplification factors of Galilean-transformed envelope solitons with respect to GV αc and the dimensionless evolution distance, as quantified from MNLSE simulations.

Figure 6 :

 6 Figure 6: Galilean-transformed Peregrine evolution in the wave flume (blue lines) for the carrier parameters ak = 0.1, a = 0.01 m and x † = -12 m to observe the maximal compression for the pure case in the middle of the tank. The red lines are the corresponding MNLSE simulations of wave envelope.

Figure 7 :

 7 Figure 7: Long-term MNLSE prediction of Galilean-transformed Peregrine breather for the same parameters as in Fig. 6. Top panels: Envelope evolution. Bottom panels: Corresponding spectral evolution.

Figure 8 :

 8 Figure 8: The order factor N of higher-order solitons corresponding to the Galilean solitons versus GS c and steepness values ak.

Figure 9 :

 9 Figure 9: HOSM simulations of Galilean solitons corresponding to higher-order solitons with order N = 2, 3, 4, carrier steepness of ak = 0.075, and frequency f = 1.25 Hz. Top panels: cα = 0.735, N = 2. Middle panels: cα = 1.251, N = 3. Bottom panels: cα = 1.661, N = 4,

Figure 10 :

 10 Figure 10: HOSM simulations of Galilean solitons corresponding to a multi soliton of order 4 with cα = 1.661, carrier steepness ak = 0.0667 and wave frequency f = 2.035 Hz. The soliton fission is clearly observed.

Acknowledgments J.M.D. acknowledges support from the French National Research Agency: ANR-15-IDEX-0003, ANR-17-EURE-0002, ANR-20-CE30-0004, ANR-21-ESRE-0040.