By its creation in 1989, IMGT® marked the advent of immunoinformatics, which emerged at the interface between immunogenetics and bioinformatics. IMGT® is specialized in the immunoglobulins (IG) or antibodies, T cell receptors (TR), major histocompatibility (MH), and proteins of the IgSF and MhSF superfamilies. IMGT® has been built on the IMGT-ONTOLOGY axioms and concepts, which bridged the gap between genes, sequences, and three-dimensional (3D) structures. The concepts include the IMGT® standardized keywords (concepts of identification), IMGT® standardized labels (concepts of description), IMGT® standardized nomenclature (concepts of classification), IMGT unique numbering, and IMGT Colliers de Perles (concepts of numerotation). IMGT® comprises seven databases, 15,000 pages of web resources, and 17 tools, and provides a high-quality and integrated system for the analysis of the genomic and expressed IG and TR repertoire of the adaptive immune responses. Tools and databases are used in basic, veterinary, and medical research, in clinical applications  and in antibody engineering and humanization. They include, for example IMGT/V-QUEST and IMGT/JunctionAnalysis for nucleotide sequence analysis and their high-throughput version IMGT/HighV-QUEST for next-generation sequencing , IMGT/DomainGapAlign for amino acid sequence analysis of IG and TR variable and constant domains and of MH groove domains, IMGT/3Dstructure-DB for 3D structures, contact analysis and paratope/epitope interactions of IG/antigen and TR/peptide-MH complexes and IMGT/mAb-DB interface for therapeutic antibodies and fusion proteins for immune applications (FPIA).IMGT For the first time, immunoglobulin (IG) or antibody and T cell receptor (TR) variable (V), diversity (D), joining (J), and constant (C) genes were officially recognized as “genes” as well as the conventional genes  more than 450 million years ago and is found in all extant jawed vertebrate species from fishes to humans. Understanding the basis for adaptive immunity, at the level of cell populations, individual cells, and molecules, has been a major focus of immunology in the past century  and MH superfamily (MhSF) and related proteins of the immune system (RPI) of vertebrates and invertebrates , and three-dimensional (3D) structures. IMGT® is the acknowledged high-quality integrated knowledge resource in immunogenetics for exploring immune functional genomics. IMGT® comprises seven databases   (IMGTtebrates . IMGT® puctures) – 14, 17 uctures) – 30, anductures) , the IMGormatics –47. Its CA, USA) , 49 and CA, USA) , 51.® data are based on IMGT-ONTOLOGY  (® standardized labels (DESCRIPTION)   , IMGT® sRIPTION) , IMGT® sICATION) , IMGT unICATION) –66, and ICATION) –70  and from IMGT/GENE-DB .The fundamental information generated from these IMGT-ONTOLOGY concepts, which led to the IMGT Scientific chart rules is reviewed. The major IMGT/V-QUEST –20 for t/V-QUEST , 22, IMG/V-QUEST , 24 and /V-QUEST , IMGT/Hi/V-QUEST , 26, IMG/V-QUEST , 27, 28 /V-QUEST –13 and i/LIGM-DB , the IMG/GENE-DB , the IMG® approach is of major interest for bridging knowledge from IG and TR repertoire in normal and pathological situations  were precisely defined  in 2010More than 325 IMGT defined . They reStandardized keywords are assigned at each step of the molecular synthesis of an IG. Those assigned to a nucleotide sequence are found in the “DE” (definition) and “KW” (keyword) lines of the IMGT/LIGM-DB files . They ch®, see footnote text 1, IMGT Education > Aide-mémoire > Amino acids). The AA changes are described according to the hydropathy (3 classes), volume (5 classes), and IMGT physicochemical classes (11 classes) (®: very similar (+ + +), similar , dissimilar , and very dissimilar (− − −).The 20 usual AA have been classified into 11 IMGT physicochemical classes (IMGTclasses) . For exaclasses) . Four ty® standardized labels (277 for sequences and 285 for 3D structures) were precisely defined  for any locus , for any gene configuration , and for any species (from fishes to humans). In higher vertebrates, there are seven IG and TR major loci . The IG major loci include the IGH, and for the light chains, the immunoglobulin kappa (IGK), and the immunoglobulin lambda (IGL) in higher vertebrates, and the immunoglobulin iota (IGI) in fishes .The IMGT-ONTOLOGY CLASSIFICATION axiom was the trigger of immunoinformatics’ birth. Indeed the IMGT® in 1989, at New Haven during the Tenth Human Genome Mapping Workshop (HGM10), the standardized classification and nomenclature of the IG and TR of humans and other vertebrate species have been under the responsibility of the IMGT Nomenclature Committee (IMGT-NC). IMGT® gene and allele names are based on the concepts of classification of “Group,” “Subgroup,” “Gene,” and “Allele”  corresponding to the encoded isotypes , instead of using the letter C. IG and TR genes and alleles are not italicized in publications. IMGT-ONTOLOGY concepts of classification have been entered in the NCBO BioPortal.Since the creation of IMGT“Allele” . “Group”® IG and TR gene names  Metathesaurus. AA sequences of human IG and TR constant genes  were provided to UniProt in 2008. Since 2007, IMGT® gene and allele names have been used for the description of the therapeutic mAb and FPIA of the WHO–INN Programme  > Locus and genes > Gene tables]. It can also be applied to the human genes in databases, which deal with humans and other vertebrate species .The basis for the nomenclature of the MH of newly sequenced genomes has been set up on the same concepts. In IMGT® Rosetta stone” that has bridged the biological and computational spheres in bioinformatics –J rearrangements .The V-domain strands and loops and their delimitations and lengths, based on the IMGT unique numbering for V-domain –64, 66, ., Kabat  or Choth., Kabat , are avatandards , 62–70  encompasses positions 27–38, the C′C″ loop (or CDR2-IMGT in a V-DOMAIN) positions 56–65, and the FG loop (or CDR3-IMGT) positions 105–117. In a V-DOMAIN, the CDR3-IMGT encompasses the V–(D)–J junction that results from a V–J or V–D–J rearrangement , 55 and 66 (anchors of the C′–C″ loop or CDR2-IMGT in V-DOMAIN), 104 and 118 (anchors of the FG loop or CDR3-IMGT in V-DOMAIN). The CDR3-IMGT anchors are highly conserved, they are C104  and F118 or W118 (J-PHE or J-TRP in G strand). The JUNCTION of an IG or TR V-DOMAIN includes the anchors 104 and 118, and is therefore two AA longer than the corresponding CDR3-IMGT (positions 105–117).The loop and strands are visualized in the IMGT Colliers de Perles – 70, whiIn biological data, the lengths of the loops and strands are given by the number of occupied positions . Four of them are highly conserved and hydrophobic  and are ® definitive system, the C-domains (C-DOMAIN and C-LIKE-DOMAIN) are delimited taking into account the exon delimitation, whenever appropriate. As for the V-domain, this IMGT® genomic approach integrates the strands A and G, which are absent of structural alignments.In the IMGT® definitive system, the G-domain includes the G-DOMAIN of the MH  , 66 ® deH Figure , 97. TheA G-domain Figure  compriseThe G-domain strands, turns, and helix and their delimitations and lengths, based on the IMGT unique numbering for G-domain , 66 are Two conserved cysteines, CYS-11 (in the A-strand) and CYS-74 (in the helix) Table , where t® definitive system, the G-domains (G-DOMAIN and G-LIKE-DOMAIN) are delimited taking into account the exon delimitations, if appropriate. Alignment sequence comparison with previously identified genes is used when genomic data are not available, as recently done for the rainbow trout (Oncorhynchus mykiss) MH1 and MH2 .In the IMGT® Web site at http://www.imgt.org, is a generic tool, which allows the users to draw IMGT Colliers de Perles , alignments, and identification of the genes and alleles with the closest germline V, D, and J genes, analysis of the junctions, analysis of somatic hypermutations, and AA changes and, if the option “Search for insertions and deletions” was selected, identification of insertions and deletions (indels) and their correction. Customized parameters and results provided by IMGT/V-QUEST and IMGT/JunctionAnalysis have been described elsewhere –20.The IMGT/V-QUEST reference directory sets against which the IMGT/V-QUEST is running include IMGT reference sequences from all functional (F) genes and alleles, all ORF and all in-frame pseudogenes (P) alleles. By definition, the IMGT reference directory sets contain one sequence for each allele. By default, the user sequences are compared with all genes and alleles. However, the option “With allele *01 only” is useful for: (i) “Detailed view,” if the user sequences need to be compared with different genes, and (ii) “Synthesis view,” if the user sequences, which use the same gene need to be aligned together  , 19.® Web site). Links to the IMGT/V-QUEST reference directory sets are available from the IMGT/V-QUEST Welcome page  or available data. Thus, a clonotype may denote either a complete antigen receptor , or only one of the two chains of the receptor , or one domain , or the CDR3 sequence of a domain. Moreover the sequence can be at the AA or nucleotide (nt) level, and this is rarely specified. Therefore, IMGTiguously .®, the clonotype, designated as “IMGT clonotype (AA),” is defined by a unique V-(D)-J rearrangement  and a unique CDR3-IMGT AA (in-frame) junction sequence ” is “unique” for a given dataset. For that reason, each “IMGT clonotype (AA),” in a given dataset, has a unique set identifier (column “Exp. ID”) and, importantly, has a unique representative sequence (link in column “Sequence ID”) selected by IMGT/HighV-QUEST among the “1 copy” “single allele” (for V and J), based on the highest percent of identity of the V-REGION (“V%”) compared to that of the closest germline, and/or on the sequence length (thus the most complete V-REGION) .The number of the “1 copy” “single allele” sequences not selected as representative. These sequences differ from the representative sequence by a different  length, and/or by sequencing errors in the V-REGION (lower “V%” of identity) or in the J-REGION, and/or by nt differences in the CDR3-IMGT. Sequences with nt differences in the CDR3-IMGT are identified as “IMGT clonotypes (nt),” the nt differences resulting from sequencing errors or, if this can be proven experimentally, from molecular convergence. For a given “IMGT clonotype (AA),” the number (nb) of different CDR3-IMGT (nt) or “IMGT clonotypes (nt),” the CDR3-IMGT sequence (nt) and the nb of different nt in the CDR3-IMGT are reported in the results .The number of the “1 copy” “several alleles (or genes)” sequences that have the same V and J allele as the IMGT clonotype (AA), among their IMGT/HighV-QUEST results.The number of “More than 1” (including those of the IMGT representative sequence) for each retained “1 copy” of steps 1 and 2 .Clonal expression is the number of sequences that can be assigned to each IMGT clonotype (AA). In our procedure, the high-quality and specific characterization of the “IMGT clonotype (AA)”  remains ® standardized approach allows a clear distinction and accurate evaluation between the clonal diversity [nb of “IMGT clonotypes (AA)”], and the clonal expression  ”] . These ae (AA)”] . The useClonal diversity is also visualized in the online results with histograms, which represent the number of IMGT clonotypes (AA) per V, D , and J genes (in pink) . Clonal ® online tool for the analysis of AA sequences and 2D structures of V and C-domains  and of G-domains (for MH and other MhSF) , ORF, and in-frame pseudogene (P) genes. As IMGT® alleles are characterized at the nucleotide level, identical sequences at the AA level may therefore correspond to different alleles, in the IMGT domain reference directory. The sequences can be displayed by querying IMGT/DomainDisplay (see footnote text 1).The IMGT domain reference directory is the IMGT reference directory for V, C, and G-domains. Sequences are from the IMGT Repertoire  and from® structure database, provides IMGT® annotation and contact analysis of IG, TR, MH, IgSf, and MhSF 3D structures, and paratope/epitope description of IG/antigen and TR/pMH complexes  are identified with the integrated IMGT/DomainGapAlign ”]. “Domain pair contacts” refer to contacts between a pair of domains or between a domain and a ligand. Clicking on “DomPair” gives access to the contacts between AA for a given “Domain pair contacts.” Contacts between VH and the Ligand  and the V-KAPPA and the Ligand (Ag) of an IG/Ag complex are shown in Figure In IMGT/3Dstructure-DB, all contacts are described as atom pair contacts. Atom pair contacts are obtained by a local program in which atoms are considered to be in contact when no water molecule can take place between them , 12. AtoClicking on “R@P” gives access to the IMGT identity card of a given residue  at a given position or Residue@Position. The IMGT R@P card can also be accessed from the AA sequences of the IMGT/3Dstructure-DB card or from the IMGT Colliers de Perles, by clicking on one AA. In an IMGT R@P card, the Residue@Position is defined by the IMGT position numbering in a domain, or if not characterized, in the chain, the AA name (three-letter and between parentheses one-letter abbreviations), the IMGT domain description and the IMGT chain ID, e.g., “103 – TYR (Y) – VH – 1hzh_H” –13. The In an IG/Ag complex, the AA in contact at the interface between the IG and the Ag constitute the paratope on the IG V-DOMAIN surface, and the epitope on the Ag surface. Similarly, in an TR/pMH complex, the AA in contact at the interface between the TR and the pMH constitute the paratope on the TR V-DOMAIN surface, and the epitope on the pMH surface. For IG/Ag and TR/pMH, the paratope and epitope are displayed in Contact analysis, but for each V-domain, separately. Clicking on the “Paratope and epitope” tag , gives access to “IMGT paratope and epitope details”, which are described in a standardized way. Each AA that belongs to the paratope is defined by its position in a V-DOMAIN. Each AA that belongs to the epitope in an IG/Ag complex is defined by its position in the chain in the 3D structure or, if the antigen belongs to an IgSF or MhSF protein and if the epitope is part of a characterized V, C, or G-domain, by its position in the domain according to the IMGT unique numbering. The epitope in a TR/pMH complex includes AA of the peptide and of the two G-DOMAIN helices.“Renumbered IMGT file” allows to view (or download) an IMGT coordinate file renumbered according to the IMGT unique numbering, and with added IMGT specific information on chains and domains (added in the “REMARK 410” lines (blue online), and identical to the “Chain details” annotation).“IMGT numbering comparison” provides, per domain, the IMGT DOMAIN numbering by comparison with the PDB numbering, and the residue (three-letter and one-letter names), which allows standardized IMGT representations using generic tools Figures A and 4A.Tools associated to IMGT/3Dstructure-DB include IMGT/ StructuralQuery  and IMGT® criteria of standardized identification  is only available in the corresponding IMGT/3Dstructure-DB cards, if the antibodies have been crystallized.The objective of antibody humanization is to graft at the DNA level the CDR of an antibody V-domain, from mouse (or other species) and of a given specificity, onto a human V-domain framework, thus preserving the specificity of the original (murine or other species) antibody while decreasing its immunogenicity . IMGT/Do®, see footnote text 1, The IMGT Biotechnology page > Antibody humanization > Alemtuzumab).Analyses performed on humanized therapeutic antibodies underline the importance of a correct delimitation of the CDR and FR. As an example, two AA changes were required in the first version of the humanized VH of alemtuzumab, in order to restore the specificity and affinity of the original rat antibody. The positions of these AA changes (S28 > F and S35 > F) are now known to be located in the CDR1-IMGT and should have been directly grafted, but at the time of this mAb humanization they were considered as belonging to the FR according to the Kabat numbering . In contAllotypes are polymorphic markers of an IG subclass that correspond to AA changes and are detected serologically by antibody reagents . In therThe allotypes G1m, G2m, and G3m are carried by the constant region of the gamma1, gamma2, and gamma3 chains, encoded by the IGHG1, IGHG2, and IGHG3 genes, respectively . The gamIn the IGHG1 CH1, the lysine at position 120 (K120) in strand G corresponds to the G1m17 allotype  Figure D. The isIn the IGHG1 CH3, the aspartate D12 and leucine L14 (strand A) correspond to G1m1, whereas glutamate E12 and methionine M14 correspond to the nG1m1 isoallotype  Table 99. A glyc® information system, which are at the origin of immunoinformatics, have provided the concepts, the knowledge environment, and the informatics frame for a standardized and integrated analysis of IG, TR, and MH, extended to other IgSF   , 72–74 iia (CLL) , 22 are ia (CLL) , 74 and rofiling .® standardization allows repertoire analysis and antibody humanization studies to move to novel high-throughput methodologies with the same high-quality criteria. The CDR-IMGT lengths are now required for mAb INN applications and are included in the WHO–INN definitions , phage displays, combinatorial libraries] and antibody humanization  –77, 82. initions , bringin® databases and tools are encouraged to cite this article and to quote the IMGT® Home page, http://www.imgt.org. Online access to IMGT® databases and tools are freely available for academics and under licenses and contracts for companies.Authors who use IMGTThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Classical models to explain thymic CD4 vs. CD8 fate determination have included a stochastic selection model or instructional models. The latter are based either on strength of signal or duration of signal impacting fate. More recently, differential co-receptor gene imprinting has been shown to be involved in expression of transcription factors impacting cytotoxic T cell development. Here, we address commitment from a structural perspective, focusing on the nature of co-receptor binding to MHC molecules. By surveying 58 MHC class II and 224 MHC class I crystal structures in the Protein Data Bank, it becomes clear that CD4 cannot bind to MHC I molecules, nor can CD8αβ or CD8αα bind to MHC II molecules. Given that the co-receptor delivers Lck to phosphorylate exposed CD3 ITAMs within a peptide/MHC (pMHC)-ligated TCR complex to initiate cell signaling, this strict co-receptor recognition fosters MHC class-restricted SP thymocyte lineage commitment at the DP stage even though both co-receptors are expressed on a single cell. In short, the binding preference of an αβTCR for a peptide complexed with an MHC molecule dictates which co-receptor subsequently binds, thereby supporting development of that subset lineage. How function within the lineage is linked further to biopotential fate determination is discussed.Since the discovery of co-receptor dependent αβTCR recognition, considerable effort has been spent on elucidating the basis of CD4 and CD8 lineage commitment in the thymus. The latter is responsible for generating mature CD4 helper and CD8αβ cytotoxic T cell subsets. Although CD4 The involvement of CD4 and CD8 in antigen-specific T cell recognition, despite their invariant structures, suggested that CD4 and CD8 might function as co-receptors in cognate recognition   and thaton assay . Kinetic−CD8− double-negative (DN) cells to differentiate into CD4+CD8+ double-positive (DP) cells, facilitating TCRα gene rearrangements and thus generating a large αβTCR repertoire among DP thymocytes . In contrast, CD8 uses its two dimeric Ig-like domains to clamp onto the CD loop of the MHCI α3 domain in a fashion similar to an antibody binding to an antigen –7.We have systematically surveyed herein 58 MHCII structures and 224 MHCI structures in the protein data bank (PDB). The structure of human CD4 in complex with HLA-DR1 (PDB code 3S5L) was taken as a reference . All othk in the complex structure of D10 TCR/I-Ak (1D9K) based on the two membrane-proximal α2/β2 domains, several of which are pictured in Figure u/TCR complex (1U3H), I-Ek (1IEA), and CD4/I-Ak complex (1JL4). The RMSD value for 178 overlaid Cα atoms ranges from 0.63 to 1.1 Å, indicating that the two membrane-proximal domains as a structural unit have a conserved relative angle. The same calculation was carried out for MHCI molecules. A representative group of MHCI molecules was superimposed onto H2-Kb in the H2-Kb/CD8αα complex structure (1BQH) based on the two membrane-proximal β2M/α3 domains. These include human HLA-A (1HHJ), HLA-B (1AGB), HLA-C (1IM9), HLA-A/TCR complex (1AO7), HLA-A2/CD8αα complex (1AKJ), as well as mouse H2-Kb/TCR complex (2CKB) and mouse H2-Db (1QLF). The RMSD value for about 180 Cα atoms ranges from 0.73 to 1.49 Å, indicating that the two membrane-proximal domains as a structural unit also have a relatively fixed angle. Notably, the RMSD value for MHCI is slightly larger than that of MHCII, consistent with previous observation that MHCI α3 domain has a small angular variation relative to the rest of MHCI molecule (b in its CD8 complex (1BQH) for comparison. If the MHCII β2 domain is superimposed onto the MHCI α3 domain, the rest of the MHCII molecule has about a 38° rotation relative to the counterpart of MHCI. This manifests as a larger opening between α2 and β2 domains in MHCII compared to between β2M and α3 in MHCI, a geometric requirement for CD4-binding as shown in Figure We have superimposed a representative group of MHCII molecules onto mouse I-Amolecule . We furtFigure Among class II MHC-like molecules, it is interesting to note that the HLA-DM (1HDM) is structurally distinct from other class II MHC molecules  Figure . HLA-DM d . The very tip amide nitrogen forms bifurcated hydrogen bonds to the Ser37 side chain of the CD8α subunit (in yellow) and the main chain oxygen of CD8β subunit’s Pro102 (in gold). Interestingly, we have noticed that the these hydrogen bonds pull the side chain of Gln226 into an extended conformation such that the elongated aliphatic portion of this Gln226 side chain packs onto the aromatic ring of CD8α Tyr55. This hydrophobic interaction conceivably contributes a significant amount of binding energy. Remarkably the Ser37 and Tyr55 are conserved only in CD8α, but not in CD8β subunit, which structurally positions CD8α subunit at the bottom and CD8β subunit on the top shown in the Figure d are shared in all known CD8αα/MHCI complex structures. Upon CD8 binding to MHCI, the MHCI molecules’ buried surface area for structures of HLA-A2/CD8αα, H-2Kb/CD8αα, TL/CD8αα and H-2Dd/CD8αβ is 1074, 1557, 1645, and 781 Å2, among which a single Gln226 residue alone contributes 16, 11, 10, and 23%, respectively. Interestingly for the H-2Dd/CD8αβ complex, since only the MHCI α3 domain is involved in binding, the buried surface area is smallest but with Gln226 makes the highest contribution.Of note, there is a very similar CD loop in the β2 domain of MHCII molecules . HoweverIn summary, whereas MHCII molecules have their α2/β2 domains arranged in a conserved and opened conformation suitable for CD4 N-terminal domain insertion for binding, MHCI molecules are unique in having a key invariant Gln226 at the tip of their α3 domain’s CD loop. The latter is well-positioned to extend into the crevice formed between two CD8 subunits for specific interactions. These observations apply to the 58 MHCII and 224 MHCI structures of both human and murine origin examined.+CD8low). If during positive selection, one MHCII-restricted TCR on the CD4+CD8low cell surface encounters an MHCII molecule that presents a self-peptide on the stromal cell, the “snug” fit of CD4 into the MHCII molecule’s α2/β2 domains on the cell surface as described above brings Lck into close proximity with exposed CD3 cytoplasmic tail ITAMs in the TCR complex. If successful ITAM phosphorylation and downstream activation persists, transcriptional factors like GATA3 and Th-POK are induced and that induction promotes CD4+ T cell differentiation by preventing Cd4 gene silencing  and MHCII (α1β1 domains) molecules extending from the stromal cell surface. The interacting domains of both TCR and MHC molecules have no intrinsic structural differences that distinguish the two classes of MHC interactions; nor are TCR complex components distinct in overall structural features vis-à-vis the individual subunits . It is dilencing . This suAs both human and mouse TCRs manifest ∼10-fold greater 3-D binding affinities for pMHCI compared to pMHCII cognate ligands , 55, theSince the introduction of TCR transgenic mouse studies, it has been clear that the interaction of the TCR in a DP thymocyte with a specific thymic pMHC molecule determines differentiation of that DP thymocyte to either a CD4 or CD8 SP thymocyte . The parThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Restorative proctocolectomy with ileopouch-anal anastomosis (IPAA) is the treatment of choice for intractable or complicated ulcerative colitis(UC). Elderly patients often present with acute colitis requiring emergent subtotal colectomy(SC). Frail patients are at risk of developing septic complications related to the closed rectosigmoidal stump, often requiring formation of a second stoma to be reversed at the time of completion proctectomy. This carries nuisance to such exhausted patients. We propose a simple and inexpensive trick to avoid the need for creating a mucous fistula.IPAA was performed as a 3-stage procedure in emergency settings. The rectosigmoidal stump was closed and placed subcutaneously; skin was closed over it. After SC, if patients showed signs of stump-related pelvic sepsis, a lavage of the rectal stump with povidone iodine solution and with saline was carried out as a rescue treatment aiming to avoid the need of opening the rectal stump to drain sepsis.Thirty-five patients underwent SC for UC between 1987 and 2012. The skin was closed over the closed stump in the 20. Seven patients out of these 20 experienced early stump-related septic complication. In five cases, we were able to avoid opening of the rectal stump, and a second stoma was unnecessary. After opening the closed stump in the remaining ones, a prompt improving of symptoms was observed.Rectal washout was well tolerated and avoided a second stoma in five out of seven patients, with better quality of life and body perception after IPAA surgery. This is relevant when dealing with geriatric patients, needing to completely recover before undergoing completion proctectomy. Restorative proctocolectomy with ileopouch-anal anastomosis (IPAA) is the treatment of choice for intractable or complicated ulcerative colitis(UC). However, when nutritional status and haemoglobin levels are poor or if patient is receiving treatment with steroids pouch fashioning should be delayed and an urgent/emergency subtotal colectomy(SC) with terminal ileostomy and closure of a long rectal (sigmoid) stump is to be preferred -3. This We herein describe a simple method to avoid a second stoma in UC patients undergoing SC with suspicion of early septic complication related to the retained diseased stump.SC was performed via median laparotomy or laparoscopy with a median mini-laparotomy to exteriorize the colon; a terminal ileostomy was fashioned on a site marked preoperatively by scratching the skin with a small needle; in laparoscopy patients, ileostomy was exteriorized through an adequately widened trocar incision. The rectosigmoidal stump was closed with a linear stapler with or without hand-sewn additional stitches, and placed subcutaneously in left iliac fossa or at the end of the median laparotomy incision. In patients undergoing laparoscopy, this was passed through a trocar incision in left iliac fossa. Skin was always closed over it. At the end of the procedure, two silicone drains were placed in the abdominal cavity, positioning one next to the colonic stump.®) (1%): this is completed in approximately 2 hours. Finally, slow washout with abundant saline is carried out. During night-time, the catheter is removed and washout is suspended. The procedure is repeated the next day, but PVI is diluted at 0.1%; saline solution is subsequently infused continuously (approximately 8 hours). The washout can be repeated for additional 12 hours.If signs of local stump-related sepsis occur during postoperative observation  count), without signs of abdominal involvement , we use the following approach. A soft, silicone three-way catheter, size Ch. 22-24 is introduced through the anus with the patient in Sims’ position and the balloon is insufflated with 30 ml of saline. Then, the patient is placed in mild Trendelemburg position (approximately 30° from the floor) and rectal washout is started through the afferent channel of the catheter with 1000 ml of saline plus 10 ml of Povidone-Iodine solution  connected with a drain bag.During the whole procedure it is mandatory to evaluate general health condition and to assess at least daily the serological status . Antibiotic is continued until normalization of WBC and remission of fever. The drains are to be kept in place. Most important, the patient should receive enteral nutrition as soon as possible, with high caloric and protein intake. If there is no response within 12 hours, it is safe to remove cutaneous sutures over the stump, opening it when needed draining sepsis through this mucous fistula. Washout can be continued with the opened mucous fistula: we are used to place a drainable 1-piece drainable transparent pouch , both in fever and in WBC. Intermittent washout was suspended uneventfully after a mean of 36 (range 24-48) hours. There was no need for opening the tip of the rectal stump in the follow-up and they all underwent successfully pouch surgery after a mean of 7.3 (range 6-8) months. One of the two patients who needed opening of the rectal stump was at risk of developing pelvic septic complication due to marked fragility of the rectum and to poor health status; the other one had persistent fever and high WBC after 12 hours. After opening the closed stump, a prompt improving of symptoms was observed in both. In one of these two patients rectal washout wash carried out after placing a drainable bag on the opened stump, and it succeeded in avoiding a permanent mucous fistula.SC is the mainstay procedure for complicated and fragile UC patients. It is wise to leave a long rectal/rectosigmoidal stump and to place it subcutaneously in left iliac fossa or at the end of the median laparotomy incision3]: it w: it w3]:Although controversial, the use of intraoperative PVI rectal washout is a widely performed procedure in rectal cancer surgery, and studies reported the efficacy of careful mechanical lavage of the rectal stump in eradicating intraluminal cancer cells shed ,6. Some In our experience, PVI alone was well tolerated and neither systemic nor local side-effects were observed. It was effective in avoiding a second stoma in five out of seven patients, with better quality of life and body perception after IPAA surgery. In one patient with a mucous fistula washout allowed avoiding the formation of a long-term draining stoma. Geriatric patients need to recover completely their physical as well as their mental status before undergoing completion proctectomy with IPAA. Avoiding a second stoma contributed to achieve the aim.Anyway, we find it prudent to proscribe PVI if there is an history of allergic reaction to iodine-containing compounds, thyroid diseases, renal insufficiency, and established systemic sepsis.IPAA: ileopouch-anal anastomosis; UC: ulcerative colitis; SC: subtotal colectomy; WBC: white blood cells; PVI: Povidone-Iodine solution.Authors have no competing interests to disclose.GP designed the study, and wrote the draft of the manuscript. GS and GC collected data and participated in the design of the study. SC and FS conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Frabin, together with, at least, FGD1, FGD2, FGD3 and FGD1-related Cdc42-GEF (FRG), is a member of a family of Cdc42-specific gua-nine nucleotide exchange factors (GEFs). These proteins have multiple phosphoinositide-binding domains, including two pleckstrin homology (PH) domains and an FYVE or FERM domain. It is likely that they couple the actin cytoskeleton with the plasma membrane. Frabin associates with a specific actin structure(s) and induces the direct activation of Cdc42 in the vicinity of this structure(s), resulting in actin reorganization. Furthermore, frabin associates with a specific membrane structure(s) and induces the indirect activation of Rac in the vicinity of this structure(s), resulting in the reorganization of the actin cytoskeleton. This reorganization of the actin cytoskeleton induces cell shape changes such as the formation of filopodia and lamellipodia. IntroductionMolecular structures of frabin and other related GEFsTissue distribution, subcellular localization and splicing variants of frabin and other related GEFsCellular activities of frabin and other related GEFsRoles of the domains of frabin in cellular activitiesMode of action of frabin in cell shape changesMode of activation of frabin and FRGFGD4/frabin and FGD1 in human diseasesInvolvement of Conclusions and perspectivesRho family small GTP-binding proteins (G proteins), including Cdc42, Rac and Rho, regulate the actin cytoskeleton-dependent cellular activities, including cell shape changes, cell migration, cell adhesion and cytokinesis , 2. ThesLike other G proteins, the Rho family members have two interconvertible forms: GDP-bound inactive and GTP-bound active forms , 2, 5. TGEFs for the Rho family members share two conserved domains: a Dbl homology (DH) domain, for which the Dbl oncogene product is the prototype, and a pleckstrin homology (PH) domain adjacent the DH domain , 2. ReceFGD1, which contains DH and PH domains, was discovered by positional cloning as the gene responsible for faciogenital dysplasia (FGDY) [FGD2 and FGD3 were identified by genetic searches as FGD1 homologues [Many GEFs for the Rho family members were originally identified as oncogenes , 2; in ca (FGDY) . FGD1 sha (FGDY) . Subsequmologues , 10. We mologues . Like FGmologues –13. HereFrabin, FGD1 , FGD2 99, FGD3 , FGD5, FDH domains encode a principal GEF catalytic unit and are required to stimulate GDP release from the Rho family small G proteins , 2. PH dFYVE domains have been found in many proteins to be involved in membrane trafficking and phosphoinositide metabolism and have been shown to specifically interact with phos-phatidylinositol 3-phosphate (PI3P) . A largeThe amino acid sequence of the DH domain of FRG shows significant homology to those of FGD1 and frabin . ConsistFrabin is expressed in all rat tissues, including the heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis [d testis . In cultd testis . The orid testis . This spFGD1, FGD2 and FGD3 are mainly expressed in restricted tissues, particularly in the bone, spleen and skeletal muscle, respectively , 10, 25.(i) Cdc42-dependent, (ii) Rac-dependent and (iii) Cdc42/Rac-independent.Expression of the full-length frabin induces the formation of filopodia and the activation of JNK through the activation of Cdc42 in fibroblasts –13 Fig.. FurtherLike frabin, FGD1 and FGD3 exhibit Cdc42-dependent cellular activities: they induce the formation of filopodia and the activation of JNK in fibroblasts , 10, 30.A fragment of frabin containing the DH domain and the first PH domain shows Cdc42-specific GEF activity in a cell-free assay system, but this fragment does not induce the formation of filopodia or lamellipodia or the activation of JNK in fibroblasts –13. For As described above, the expression of Cdc42 CA or Rac CA, or both, does not result in the generation of microspikes in MDCK cells . HoweverOn the other hand, the FYVE domain and the second PH domain, in addition to the DH domain and the first PH domain, are necessary for the formation of lamellipodia and the activation of JNK, suggesting that the association of frabin with membranes is required for these activities , 13 Fig. Thus, dvia mDia2. The cooperation of Cdc42-independent and Cdc42-dependent actin reorganization finally induces the formation of filopodia. This newly formed actin structure further recruits frabin in a positive feedback cycle to lengthen the filopodia.Cell shape change has long been thought to be determined by the cytoskeleton reorganization beneath the plasma membrane. However, this idea could be revised by the discovery of several membrane-deforming proteins (MDPs), such as the BAR domain-containing endophilin and EFC domain-containing FBP17, which bind to WASP and WAVE proteins . It is cFrabin is also recruited to a specific membrane structure(s) through the region including the DH domain, the first PH domain, the FYVE domain and the second PH domain . Like BAIt is unclear how external or internal stimuli transduce the signals to activate frabin and FGDs; but our recent preliminary results show that some phosphoinositides have a potency to stimulate the GEF activity of frabin. It has also been shown that frabin is recruited by the activation of phosphatidylinositol 3-kinase (PI3 kinase) during parasite invasion . Frabin In contrast to frabin and FGDs, the mode of activation of FRG is well studied. FRG is involved in the signalling pathway from the endothelin A receptor to JNK . In thisCryptosporodium parvum (C. parvum), an intracellular parasite, is one of the most commonly reported enteric pathogens worldwide. Frabin was shown to mediate the cellular invasion of this parasite [C. parvum recruits PI3 kinase to the host cell–parasite interface, an event that then results in the recruitment of frabin, leading to the activation of Cdc42 and subsequent host–cell actin reorganization during cellular invasion.Many pathogen microbes including viruses, bacteria and parasites utilize host cell actin for multiple actions such as attachment, entry into cells and movement within and between cells . Cryptosparasite . As descRecently, mutations in FGD4/frabin have been identified to be responsible for the Charcot-Marie-Tooth (CMT) disorder type 4H , 45. CMTFGD1 gene induce alternations in the size and shape of a number of small bones and cartilage elements [FGD1 is initially expressed during the onset of ossification and its expression is limited to the ossifying skeletal components, including the cranio-facial bones [FGD1 was originally determined by positional cloning to be the gene responsible for an X-linked skeletal dysplasia, FGDY . Mutations in the elements . The caral bones . This exWe have described here our current knowledge of frabin and other related Cdc42-specific GEFs, including FGD1, FGD2, FGD3, FGD5, FGD6 and FRG. They all have multiple phosphoinositide-binding domains, including two PH domains and an FYVE or FERM domain. It is likely that they couple the actin cytoskeleton with the plasma membrane and regulate the membrane-dependent actin reorganization. Frabin associates with the specific actin and membrane structures and activates Cdc42 and Rac in the vicinity of these structures, resulting in the reorganization of the actin cytoskeleton and the plasma membrane. Thus, it is becoming clear how frabin induces cell shape changes. However, important questions still remain to be solved: (1) how external stimuli and intracellular signals induce the activation of frabin, (2) the physiological significance of the presence of frabin-β and -γ, (3) how frabin induces the activation of Rac and (4) how frabin regulates myelination. To address these issues, the FAB domain and a fragment containing the mutated DH domain, the first PH domain, the FYVE domain and the second PH domain may be useful because they have DN effects. Further studies are necessary for a better understanding of the modes of action and activation of frabin.


Pseudomonas genome to allow the determination of the type of pyoverdin produced, the update of syringafactin structure and the identification of novel putative products.Nonribosomal peptides represent a large variety of natural active compounds produced by microorganisms. Due to their specific biosynthesis pathway through large assembly lines called NonRibosomal Peptide Synthetases (NRPSs), they often display complex structures with cycles and branches. Moreover they often contain non proteogenic or modified monomers, such as the D-monomers produced by epimerization. We investigate here some sequence specificities of the condensation (C) and epimerization (E) domains of NRPS that can be used to predict the possible isomeric state (D or L) of each monomer in a putative peptide. We show that C- and E- domains can be divided into 2 sub-regions called Up-Seq and Down-Seq. The Up-Seq region corresponds to an InterPro domain (IPR001242) and is shared by C- and E-domains. The Down-Seq region is specific to the enzymatic activity of the domain. Amino-acid signatures (represented as sequence logos) previously described for complete C-and E-domains have been restricted to the Down-Seq region and amplified thanks to additional sequences. Moreover a new Down-Seq signature has been found for Ct-domains found in fungi and responsible for terminal cyclization of the peptides. The identification of these signatures has been included in a workflow named Florine, aimed to predict nonribosomal peptides from NRPS sequence analyses. In some cases, the prediction of isomery is guided by genus-specific rules. Florine was used on a  Nonribosomal peptides (NRPs) are microbial secondary metabolites which are important pharmaceutical natural products. The unique Norine database contains all available information on the structure and functions of all known NRPs such as the antibiotic penicillin precursor ACV (Norine ID : NOR00006), the immunosuppressive cyclosporin (NOR00033-63) and the biosurfactant surfactin (NOR00211-219. NOR00847-860) LCL domains catalyse the condensation between two L-monomers, the dual C/E-domains are capable of both epimerization and condensation leading to a bond between a D- and an L-monomer. The DCL domains also catalyse the condensation between a D- and an L-monomer but it is not responsible for epimerization. When present at the beginning of a NRPS, the C-starter domains catalyse the condensation of a lipid moiety or salicylic acid derivative onto the first monomer of the peptide chain. Finally, at the last position in fungal NRPSs, Ct-domains are responsible for both the release and cyclization of the peptide The basic function of a condensation domain is to catalyse the peptide bond formation between two amino-acids linked to their thiolation domains. In the past, various types of C-domains have been distinguished, mostly by multiple sequence alignments combined with phylogenetic studies A key structural feature of nonribosomal compounds is the modification of some of their building blocks during biosynthesis. Additional domains, modifying the monomers during their incorporation, are sometimes present. Among them, the most frequent is the Epimerization domain (E), which modifies an L-monomer into its D-isomer on the α-carbon.Bacillus and Pseudomonas genera include a large proportion of D-monomers Bacillus CLP synthetases harbour E-domains in the modules corresponding to each D-monomer but, surprisingly, no E-domain has been detected in Pseudomonas CLP synthetases. In these assembly lines, epimerization occurs through the dual C/E-domain mentioned above. Such C/E-domains epimerize the activated monomer linked to its cofactor on the T-domain immediately preceding them. Therefore, they are found in the elongation modules corresponding to the following monomer, which can thus be displayed as {C/E-A-T}. Since their initial finding in the arthrofactin (NOR00343) synthetase of Pseudomonas sp.MIS38, the presence of dual C/E-domains has been generalized to all CLP synthetases studied so far in Pseudomonads Pseudomonas genomes with the more frequent strategy involving E-domains as observed for pyoverdin synthesis.Analysis of numerous NRPSs in relation with their products has shown that different microorganisms have developed different strategies to incorporate D-monomers within their active peptides. In a wide range of NRPS an E-domain is located directly after the T-domain, leading to a particular module architecture {(C)-A-T-E} instead of the usual {(C)-A-T} described above. Cyclic lipopeptides (CLPs) produced by species belonging to trans. Until now, only examples of D-alanine loaded by A-domains have been described. The D-Ala is provided by Alanine racemase or Alr (EC 5.1.1.1) that is encoded by a separate gene A third strategy for D-monomer integration into NRPs relies on the recent observation that several adenylation domains are able to directly activate D-monomers both in fungi and bacteria An urgent challenge today is to discover new natural drugs to tackle emerging pathogens and to obtain efficient anti-tumoral compounds. Bioinformatics approaches are largely considered for this purpose as a way to save time during the screening of such molecules. Regarding nonribosomal peptide synthesis, specific tools have been developed for predicting the organization of NRPS modules in terms of domains and the nature of the monomers incorporated by A-domains The work presented herein describes a new strategy for identifying C- and E-domain sub-types leading to the prediction of monomer isomery. As epimerization is essential for architectural diversity of the NRPs, its prediction was considered as a key step of the Florine workflow that was developed to improve structural prediction of peptides from NRPS sequence analysis.Sequences were extracted from universal databases for DNA or protein sequences The catalytic domains occurring in NRPS proteins were identified with widely used tools such as InterProScan at the EBI In summary, the global NRPS architecture was predicted using NRPS-PKS (12), PKS/NRPS analysis et al.LCL, DCL, and dual C/E domains. Because Rausch et al. PseudomonasBurkholderia , BacillusLCL, 19 DCL, 31 E and 34 dual C/E) of added NRPSs are known and specified in the table S1.To study the specificity of the domains described in this article, we performed multiple alignments using the MUSCLE program dual C/E). Except DCL. Remaining domains were classified into LCL group. Finally, a manual cleaning was performed and the classification of domains was checked mainly based on our knowledge about the products. Dual C/E and Ct were classified according to their known activity.NRPS domains were identified based on their InterPro ID . Then, the different sub-types of C-domains were classified according to their neighbourhood. Indeed, if the sequence upstream of a C-domain was less than 50 aa in length, it was considered as a C-starter. If a tandem of C-domains was observed, the first one was considered as an epimerization domain and the second one as a The Down-Seq regions are always delimited between the end of an IPR001242 C-domain and the start of the following domain (A or C). For each group, alignments were performed independently on the Down-Seq regions, and weblogos were designed.−1 is very high compared to the 5.10−6 frequency found in proteins and peptides of the SwissProt database Brevibacillus brevis displaying the following circular sequence  and for which no NRPS gene has been identified yet. In this particular case the presumption for NRPS synthesis is also supported by the presence of the non-proteogenic monomer ornithin.The distribution of D-monomers in NRPs has been studied in the Norine database. We have found 1920 D-isomers among the 11,206 monomers composing the 1164 nonribosomal peptides of Norine, distributed across 213 families. This frequency of 1,7.10Next, we compared the structures and activities displayed by the Norine peptides containing D-monomers with those displayed by the complete set of Norine peptides . All posBacillus licheniformis. This active compound contains 4 D-isomers out of its 12 monomers  is detected in the Down-Seq region of E-domains when the InterProScan search is performed using the TIGR domain database. This short 171-aa domain is described as a non-ribosomal peptide synthase domain, located downstream a condensation domain. However to our knowledge it has never been associated with epimerization domains. Moreover, no counterpart of this domain exists in the TIGR domain database for the Down-Seq region of C-domains.In NRPSs, D-isomer incorporation most frequently relies on the presence of an additional domain, the E-domain, that catalyzes the epimerization reaction. This E-domain is generally located downstream of the A and T domains involved in the monomer activation. We have compared the detection of such E-domains, using dedicated or common tools, in the protein sequence of the three NRPSs  responsible for the synthesis of the well-known antibiotic bacitracin  monomers . All foumonomers . In factmonomers . The Up-Vibrio cholera VibH condensation enzyme, PDB1l5a) can be considered as a pseudodimer with N- and C-terminal structural domains facing each other. However the separation between these two structural domains occurs between two α-helices (α5 and α6) located inside the PF00668 domain after the conserved HHxxDG motif. Thus more than one third of the PF00668 domain is actually part of the C-terminal structural domain We have compared this organization in 2 domains with the 3-D structure of a condensation domain From a functional point of view, the PF00668 (Up-Seq region) contains the solvent channel that drives the extension of the pantetheinyl arm to present the substrates for catalysis and the Down-Seq region forms a part of the structure that interacts with the nascent peptide. We can hypothesize that this region may play a differentiating role between various C- and E- domains. LCL, DCL, E, dual C/E, and Ct (in fungi). Without exception, we have found that all of them have the same architecture with a well-conserved common Up-Seq region, tagged by the presence of a PF00668 domain, and a differentiating Down-Seq region  technology as proposed by NRPSpredictor LCL, DCL, C-starter, Ct) have to be identified by the signatures represented by the WebLogos at WL1, WL2 and WL3 positions within the Down-Seq region , and that this information can direct the type of analysis to perform. For Pseudomonas if a cyclic lipopeptide synthesis is suspected , the occurrence of dual C/E-domains has to be searched among all C-domains. For fungal NRPSs, the presence of a Ct as the last domain favours the prediction for a cyclic peptide.It should be noted that step 3 largely depends on the strain  LCL and DCL is possible to support the D-prediction originally based on the presence or absence of E-domains., Even if the the NRPS/PKS and PKS/NRPS Analysis tools sometimes mention the stereoisomery of the predicted monomer, it should not be considered because it cannot been deduced from Stachelhaus code. For example, a D-Tyr is predicted by the NRPS/PKS tool as the monomer incorporated by the second module of the Bacillus thuringiensis kurstakin synthetase C (KrsC), whereas the PKS/NRPS Analysis tool does not return any prediction for that module. In fact, one D-monomer is present in Kurstakin but the Florine workflow identifies it as a D-Gln associated with the third module of the NRPS DCL-domain in the next module, as identified using their respective WL signatures , which was found to contain several NRPS genes involved in the synthesis of both known and unknown peptides. The complete genome of 6.5 Mb consists of one chromosome and two plasmids which together encode 5763 ORFs Bacillus MycB protein as a query (taxid 223283) we have identified 12 putative NRPS genes distributed over five clusters in the chromosome (NC_004578)  . Similaripr1 and ipr2 genes in Yersinia pestisThe first cluster of genes . This was consistent with our previous observation that the E- and DCL domains are used for incorporation of D-Asp in pyoverdins. In a final step, the predicted peptide was compared to the pyoverdins annotated in the Norine database using the structure search tool. More than 60 different pyoverdins are currently described in the Norine database, all of them displaying one chromophore  linked to a peptide moiety ranging from 5 to 12 monomers. Because of the chromophore, the numbering of each monomer is incremented by one in the final peptide .The second cluster contains genes encoding proteins annotated as parts of a pyoverdin synthetase . One pro peptide . The monLCL domains were identified in the protein sequences (LCL which is correct for only two of them.The third cluster is composed of 2 genes (PSPTO_2829 and PSPTO_2830) encoding 2 proteins (NP_792633 and NP_792634) annotated SyfA and SyfB, including 3 and 5 modules, respectively. The synthetase starts with the first C-domain of SyfA and ends with a tandem of Te domains of SyfB. It has previously been described to produce the syringafactin lipopeptide equences , Table 2LCL domains (The fourth cluster of strain DC3000 only contains one NRPS gene (PSPTO_4699). The protein (NP_794446) is annotated as NRPS terminal component and is organized into 4 modules. Because it starts with a C-domain and no E-domain has been identified, the WL signatures were searched within the four C-domains to define their type. Without any ambiguity, C1 is a C-starter type, C3 is a dual C/E domain and C2 and C4 are  domains . This clLCL type (Cluster 5 consists of 2 genes (PSPTO_4518 and PSPTO_4519) annotated as nonribosomal peptide synthetase initiating- and terminal- components. The first protein (NP_794271) includes one PKS domain and one NRPS {C-A-T} module. None of the WL signatures defined in this study was detected in the Down-Seq region of this C-domain. This can be explained by the hybrid PKS/NRPS organization of the protein. The C-domain directly following the PKS part has probably a new specificity activity correlated with specific signatures. The second protein (NP_794272) harbours three modules and ends with a Te domain. All three C-domains of this protein clearly display WL signatures corresponding to LCL type . TogethePs. syringae pv. tomato DC3000 we have confirmed the presence of genes for yersiniabactin production, defined the type of putative pyoverdin secreted, specified the isomery of the syringafactin monomers and identified two new putative NRPs. The same strategy can now be applied to any genomic data (complete or draft genome) for any microbial strain. Florine is complementary to other tools such as antiSMASH and NapDos and is helpful to extend in the structure prediction of NRPs, especially for putative isomery identification.By this example we have demonstrated the interest of our Florine workflow that includes guidelines for monomer isomery prediction and confrontation with already described NRPs stored in the Norine database. Indeed, in the single genome of Ps. syringae pv. tomato DC3000 is also interesting because it confirms the co-existence in Pseudomonads of at least two strategies for integrating D-monomers into NRPs. The classical way through E- and DCL-domains is encountered in pyoverdin NRPS and the alternative way through dual C/E domains which seems to be mainly restricted to cyclic lipopeptides of Pseudomonads. Further exploration of newly sequenced microbial genomes may lead to the discovery of new strategies for NRP monomer epimerization as suggested by the recent example of lysobactin synthetase from Lysobacter sp. ATCC 53042. This example also shows that our analysis of C-domains which clearly separates the shared common PF00668 domain from a downstream differentiating region, is useful for classifying newly discovered C-domains according to the presence or absence of particular amino-acid signatures.The example of Figure S1Identification of WL signatures within C- and E-domains in bacitracin , syringafactin (SyfA and SyfB) and kurstakin (KrsC) synthetases. The type of C-domain predicted by the various WL signatures is mentioned on the right side and for bacitracin synthetase, the known functional sub-type appears in the description of each domain (first line). Color code : grey for C-starter, yellow for LCL, green for DCL, blue for E, purple for C/E.(DOCX)Click here for additional data file.Table S1Listing of NRPSs added to update the dataset used in this study.(PDF)Click here for additional data file.Table S2Translation of the graphic weblogos into aa signatures for C- and E-domains. At each position the majoritary amino acid(s) is in capital letter(s) and the alternative possibilities are mentioned between brackets. Color code : grey for C-starter, yellow for LCL, green for DCL, Blue for E, purple for C/E, no color for Ct.(XLSX)Click here for additional data file.