Synthesis and evaluation of boronic acids as inhibitors of Penicillin Binding Proteins of classes A, B and C.
Zervosen, Astrid ; Bouillez, André ; et al
in Bioorganic & Medicinal Chemistry (2012), 20(12), 3915-24
In response to the widespread use of beta-lactam antibiotics bacteria have evolved drug resistance mechanisms that include the production of resistant Penicillin Binding Proteins (PBPs). Boronic acids are ... [more ▼]
In response to the widespread use of beta-lactam antibiotics bacteria have evolved drug resistance mechanisms that include the production of resistant Penicillin Binding Proteins (PBPs). Boronic acids are potent beta-lactamase inhibitors and have been shown to display some specificity for soluble transpeptidases and PBPs, but their potential as inhibitors of the latter enzymes is yet to be widely explored. Recently, a (2,6-dimethoxybenzamido)methylboronic acid was identified as being a potent inhibitor of Actinomadura sp. R39 transpeptidase (IC(50): 1.3muM). In this work, we synthesized and studied the potential of a number of acylaminomethylboronic acids as inhibitors of PBPs from different classes. Several derivatives inhibited PBPs of classes A, B and C from penicillin sensitive strains. The (2-nitrobenzamido)methylboronic acid was identified as a good inhibitor of a class A PBP (PBP1b from Streptococcus pneumoniae, IC(50)=26muM), a class B PBP (PBP2xR6 from Streptococcus pneumoniae, IC(50)=138muM) and a class C PBP (R39 from Actinomadura sp., IC(50)=0.6muM). This work opens new avenues towards the development of molecules that inhibit PBPs, and eventually display bactericidal effects, on distinct bacterial species. [less ▲]Detailed reference viewed: 41 (15 ULg)
A peptidoglycan fragment triggers beta-lactam resistance in Bacillus licheniformis.
Amoroso, Ana Maria ; ; et al
in PLoS Pathogens (2012), 8(3), 1002571
To resist to beta-lactam antibiotics Eubacteria either constitutively synthesize a beta-lactamase or a low affinity penicillin-binding protein target, or induce its synthesis in response to the presence ... [more ▼]
To resist to beta-lactam antibiotics Eubacteria either constitutively synthesize a beta-lactamase or a low affinity penicillin-binding protein target, or induce its synthesis in response to the presence of antibiotic outside the cell. In Bacillus licheniformis and Staphylococcus aureus, a membrane-bound penicillin receptor (BlaR/MecR) detects the presence of beta-lactam and launches a cytoplasmic signal leading to the inactivation of BlaI/MecI repressor, and the synthesis of a beta-lactamase or a low affinity target. We identified a dipeptide, resulting from the peptidoglycan turnover and present in bacterial cytoplasm, which is able to directly bind to the BlaI/MecI repressor and to destabilize the BlaI/MecI-DNA complex. We propose a general model, in which the acylation of BlaR/MecR receptor and the cellular stress induced by the antibiotic, are both necessary to generate a cell wall-derived coactivator responsible for the expression of an inducible beta-lactam-resistance factor. The new model proposed confirms and emphasizes the role of peptidoglycan degradation fragments in bacterial cell regulation. [less ▲]Detailed reference viewed: 29 (4 ULg)
MICRO-H2 – Microbiological production of hydrogen: study of microalgal and bacterial processes
Calusinska, Magdalena ; Joris, Bernard ; Wilmotte, Annick et al
Poster (2011, September 07)
The project MICRO-H2 aims to study and exploit the microbial (bacterial and algal) production of hydrogen (H2). In addition to building a competence centre around the H2 production by microorganisms and ... [more ▼]
The project MICRO-H2 aims to study and exploit the microbial (bacterial and algal) production of hydrogen (H2). In addition to building a competence centre around the H2 production by microorganisms and the molecular monitoring of the processes, this project tries to address two main socio-economic issues. First, transport and many economic activities will be based on hydrogen energy in the near future. Secondly, many researches and technology developments deal with renewable resources. Therefore, a new integrated technology for a sustainable development should be promoted. Photofermentation and dark-fermentation are the most promising ways to produce biohydrogen. The main advantage of the first process is the complete conversion of substrate, if any, to hydrogen. However, present H2-production rates by microalgae remain low. Therefore, a better understanding of the microalgal hydrogen metabolism and rate improvements by genetic engineering are needed. On the other hand, dark-fermentation achieves at present far higher H2-production rates, but improvements are expected through monitoring and optimisation of bacterial diversity and activity. The objectives about bacterial H2 production were to increase knowledge, stability potentialities and investigation skills about the consortia of bacteria involved in bioreactors treating wastewater rich in carbohydrates to produce biohydrogen. The project focused mainly on the study of the potentialities of different consortia, with a focus on Clostridium strains. Concerning the microalgal production of H2, the objectives were to increase knowledge on the metabolic interactions that determine H2 evolution at the cellular level and to produce new strains with increased ability for H2 production in the two-stage process. [less ▲]Detailed reference viewed: 48 (9 ULg)
Study of the interactions between the late stage proteins of Escherichia coli divisome assembly.
Dony, Nicolas ; Brasseur, Robert ; Joris, Bernard
Poster (2011, August 16)Detailed reference viewed: 40 (4 ULg)
Genetic diversity and amplification of different clostridial [FeFe] hydrogenases by group-specific degenerate primers
Calusinska, Magdalena ; Joris, Bernard ; Wilmotte, Annick
in Letters in Applied Microbiology (2011), 53
Aims: The aim of this study was to explore and characterize the genetic diversity of [FeFe] hydrogenases in a representative set of strains from Clostridium sp. and to reveal the existence of neither yet ... [more ▼]
Aims: The aim of this study was to explore and characterize the genetic diversity of [FeFe] hydrogenases in a representative set of strains from Clostridium sp. and to reveal the existence of neither yet detected nor characterized [FeFe] hydrogenases in hydrogen-producing strains. Methods and Results: The genomes of 57 Clostridium strains (34 different genotypic species), representing six phylogenetic clusters based on their 16S rRNA sequence analysis (cluster I, III, XIa, XIb, XIV and XVIII), were screened for different [FeFe] hydrogenases. Based on the obtained alignments, ten pairs of [FeFe] hydrogenase cluster-specific degenerate primers were newly designed. Ten Clostridium strains were screened by PCRs to assess the specificity of the primers designed and to examine the genetic diversity of [FeFe] hydrogenases. Using this approach, a diversity of hydrogenase genes was discovered in several species previously shown to produce hydrogen in bioreactors: Clostridium sartagoforme, Clostridium felsineum, Clostridium roseum and Clostridium pasteurianum. Conclusions: The newly designed [FeFe] hydrogenase cluster-specific primers, targeting the cluster-conserved regions, allow for a direct amplification of a specific hydrogenase gene from the species of interest. Significance and Impact of the Study: Using this strategy for a screening of different Clostridium ssp. will provide new insights into the diversity of hydrogenase genes and should be a first step to study a complex hydrogen metabolism of this genus. [less ▲]Detailed reference viewed: 27 (2 ULg)
Unexpected tricovalent binding mode of boronic acids within the active site of a penicillin binding protein.
Zervosen, Astrid ; Herman, Raphaël ; Kerff, Frédéric et al
in Journal of the American Chemical Society (2011)
Boronic acids bearing appropriate side chains are good inhibitors of serine amidohydrolases. The boron usually adopts a tetrahedral conformation, bound to the nucleophilic serine of the active site and ... [more ▼]
Boronic acids bearing appropriate side chains are good inhibitors of serine amidohydrolases. The boron usually adopts a tetrahedral conformation, bound to the nucleophilic serine of the active site and mimicking the transition state of the enzymatic reaction. We have solved the structures of complexes of a penicillin-binding protein, the DD-peptidase from Actinomadura sp. R39, with four amidomethylboronic acids (2,6 dimethoxybenzamidomethylboronic acid, phenylacetamidomethylboronic acid, 2-chlorobenzamidomethylboronic acid, and 2-nitrobenzamidomethylboronic acid) and the pinacol ester derived from phenylacetamidomethylboronic acid. We found that, in each case, the boron forms a tricovalent adduct with Ogamma of Ser49, Ser298, and the terminal amine group of Lys410, three key residues involved in the catalytic mechanism of penicillin-binding proteins. This represents the first tricovalent enzyme-inhibitor adducts observed by crystallography. In two of the five R39-boronate structures, the boronic acid is found as a tricovalent adduct in two monomers of the asymmetric unit and as a monocovalent adduct with the active serine in the two remaining monomers of the asymmetric unit. Formation of the tricovalent complex from a classical monocovalent complex may involve rotation around the Ser49 Calpha-Cbeta bond to place the boron in a position to interact with Ser298 and Lys410, and a twisting of the side chain amide such that its carbonyl oxygen is able to hydrogen bond to the oxyanion hole NH of Thr413. Biphasic kinetics were observed in three of the five cases and details of the reaction between R39 and 2,6-dimethoxybenzamidomethylboronic acid were studied. Observation of biphasic kinetics was not, however, thought to be correlated to formation of tricovalent complexes, assuming that the latter do form in solution. Based on the crystallographic and kinetic results, a reaction scheme for this unexpected inhibition by boronic acids is proposed. [less ▲]Detailed reference viewed: 52 (15 ULg)
Novel FISH and quantitative PCR protocols to monitor artificial consortia composed of different hydrogen-producing Clostridium spp.
; Joris, Bernard ; Wilmotte, Annick et al
in International Journal of Hydrogen Energy (2011), 36
The use of an artificial consortium composed of selected hydrogen-producing species, instead of a natural anaerobic sludge, has been proposed for biohydrogen production. In order to monitor such a ... [more ▼]
The use of an artificial consortium composed of selected hydrogen-producing species, instead of a natural anaerobic sludge, has been proposed for biohydrogen production. In order to monitor such a consortium composed of different Clostridium spp., new protocols were tested for two different assays, FISH and qPCR. New species-specific FISH probes and qPCR primer sets were developed and optimised for three strains: Clostridium butyricum, Clostridium felsineum and Clostridium pasteurianum, that were used in a consortium. Application of a fast two-step FISH protocol, with pre-treatment step at 90 C for 5 min and a subsequent hybridisation step at higher temperature (55 C) for 20 min resulted in a much shorter analytical time compared to the standard FISH procedure (46 C for 2e3 h) and gave a high hybridisation performance. Moreover, to accurately quantify each microorganism by qPCR assay, two innovations were tested: the direct use of cell lysates (omitting the DNA extraction step) and the use of two alternative molecular markers, recA and gyrA. These markers are present in single copies in the genome, whereas there are multiple copies of the ribosomal operons. This resulted in the development of accurate, reliable and fast FISH and qPCR assays for routine monitoring of the dynamics of artificial hydrogen-producing microbial consortia. Moreover, both techniques can be easily adapted to new Clostridium strains. [less ▲]Detailed reference viewed: 60 (6 ULg)
Second-generation sulfonamide inhibitors of D-glutamic acid-adding enzyme: activity optimisation with conformationally rigid analogues of D-glutamic acid.
; ; et al
in European journal of medicinal chemistry (2011), 46(7), 2880-94
D-Glutamic acid-adding enzyme (MurD) catalyses the essential addition of d-glutamic acid to the cytoplasmic peptidoglycan precursor UDP-N-acetylmuramoyl-l-alanine, and as such it represents an important ... [more ▼]
D-Glutamic acid-adding enzyme (MurD) catalyses the essential addition of d-glutamic acid to the cytoplasmic peptidoglycan precursor UDP-N-acetylmuramoyl-l-alanine, and as such it represents an important antibacterial drug-discovery target enzyme. Based on a series of naphthalene-N-sulfonyl-d-Glu derivatives synthesised recently, we synthesised two series of new, optimised sulfonamide inhibitors of MurD that incorporate rigidified mimetics of d-Glu. The compounds that contained either constrained d-Glu or related rigid d-Glu mimetics showed significantly better inhibitory activities than the parent compounds, thereby confirming the advantage of molecular rigidisation in the design of MurD inhibitors. The binding modes of the best inhibitors were examined with high-resolution NMR spectroscopy and X-ray crystallography. We have solved a new crystal structure of the complex of MurD with an inhibitor bearing a 4-aminocyclohexane-1,3-dicarboxyl moiety. These data provide an additional step towards the development of sulfonamide inhibitors with potential antibacterial activities. [less ▲]Detailed reference viewed: 16 (1 ULg)
Production et purification de l’amylolysine, un lantibiotique produit par Bacillus amyloliquefaciens GA1
Arguelles Arias, Anthony ; Ongena, Marc ; et al
Poster (2011)Detailed reference viewed: 13 (1 ULg)
Small molecule inhibitors of peptidoglycan synthesis targeting the lipid II precursor.
Derouaux, Adeline ; ; et al
in Biochemical Pharmacology (2011), 81(9), 1098-105
Bacterial peptidoglycan glycosyltransferases (GTs) of family 51 catalyze the polymerization of the lipid II precursor into linear peptidoglycan strands. This activity is essential to bacteria and ... [more ▼]
Bacterial peptidoglycan glycosyltransferases (GTs) of family 51 catalyze the polymerization of the lipid II precursor into linear peptidoglycan strands. This activity is essential to bacteria and represents a validated target for the development of new antibacterials. Application of structure-based virtual screening to the National Cancer Institute library using eHits program and the structure of the glycosyltransferase domain of the Staphylococcus aureus penicillin-binding protein 2 resulted in the identification of two small molecules analogues 5, a 2-[1-[(2-chlorophenyl)methyl]-2-methyl-5-methylsulfanylindol-3-yl]ethanamine and 5b, a 2-[1-[(3,4-dichlorophenyl)methyl]-2-methyl-5-methylsulfanylindol-3-yl]ethanamine that exhibit antibacterial activity against several Gram-positive bacteria but were less active on Gram-negative bacteria. The two compounds inhibit the activity of five GTs in the micromolar range. Investigation of the mechanism of action shows that the compounds specifically target peptidoglycan synthesis. Unexpectedly, despite the fact that the compounds were predicted to bind to the GT active site, compound 5b was found to interact with the lipid II substrate via the pyrophosphate motif. In addition, this compound showed a negatively charged phospholipid-dependent membrane depolarization and disruption activity. These small molecules are promising leads for the development of more active and specific compounds to target the essential GT step in cell wall synthesis. [less ▲]Detailed reference viewed: 84 (15 ULg)
Structure-Guided Design of Cell Wall Biosynthesis Inhibitors That Overcome beta-Lactam Resistance in Staphylococcus aureus (MRSA).
; Amoroso, Ana Maria ; et al
in ACS Chemical Biology (2011)
beta-Lactam antibiotics have long been a treatment of choice for bacterial infections since they bind irreversibly to Penicillin-Binding Proteins (PBPs), enzymes that are vital for cell wall biosynthesis ... [more ▼]
beta-Lactam antibiotics have long been a treatment of choice for bacterial infections since they bind irreversibly to Penicillin-Binding Proteins (PBPs), enzymes that are vital for cell wall biosynthesis. Many pathogens express drug-insensitive PBPs rendering beta-lactams ineffective, revealing a need for new types of PBP inhibitors active against resistant strains. We have identified alkyl boronic acids that are active against pathogens including methicillin-resistant S. aureus (MRSA). The crystal structures of PBP1b complexed to 11 different alkyl boronates demonstrate that in vivo efficacy correlates with the mode of inhibitor side chain binding. Staphylococcal membrane analyses reveal that the most potent alkyl boronate targets PBP1, an autolysis system regulator, and PBP2a, a low beta-lactam affinity enzyme. This work demonstrates the potential of boronate-based PBP inhibitors for circumventing beta-lactam resistance and opens avenues for the development of novel antibiotics that target Gram-positive pathogens. [less ▲]Detailed reference viewed: 52 (5 ULg)
New noncovalent inhibitors of penicillin-binding proteins from penicillin-resistant bacteria.
; Verlaine, Olivier ; Gerards, Thomas et al
in PloS one (2011), 6(5), 19418
BACKGROUND: Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs beta-lactams inhibit transpeptidase ... [more ▼]
BACKGROUND: Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs beta-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for beta-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs. METHODOLOGY/PRINCIPAL FINDINGS: Our in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains. CONCLUSIONS: We found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria. [less ▲]Detailed reference viewed: 45 (18 ULg)
Antilisterial Activity on Poultry Meat of Amylolysin, a Bacteriocin from Bacillus amyloliquefaciens GA1
Halimi, Badre Eddine ; ; Arguelles Arias, Anthony et al
in Probiotics and Antimicrobial Proteins (2010)Detailed reference viewed: 235 (44 ULg)
The surprising diversity of clostridial hydrogenases: a comparative genomic perspective
Calusinska, Magdalena ; ; Joris, Bernard et al
in Microbiology (2010), 156
Among the large variety of micro-organisms capable of fermentative hydrogen production, strict anaerobes such as members of the genus Clostridium are the most widely studied. They can produce hydrogen by ... [more ▼]
Among the large variety of micro-organisms capable of fermentative hydrogen production, strict anaerobes such as members of the genus Clostridium are the most widely studied. They can produce hydrogen by a reversible reduction of protons accumulated during fermentation to dihydrogen, a reaction which is catalysed by hydrogenases. Sequenced genomes provide completely new insights into the diversity of clostridial hydrogenases. Building on previous reports, we found that [FeFe] hydrogenases are not a homogeneous group of enzymes, but exist in multiple forms with different modular structures and are especially abundant in members of the genus Clostridium. This unusual diversity seems to support the central role of hydrogenases in cell metabolism. In particular, the presence of multiple putative operons encoding multisubunit [FeFe] hydrogenases highlights the fact that hydrogen metabolism is very complex in this genus. In contrast with [FeFe] hydrogenases, their [NiFe] hydrogenase counterparts, widely represented in other bacteria and archaea, are found in only a few clostridial species. Surprisingly, a heteromultimeric Ech hydrogenase, known to be an energy-converting [NiFe] hydrogenase and previously described only in methanogenic archaea and some sulfur-reducing bacteria, was found to be encoded by the genomes of four cellulolytic strains: Clostridum cellulolyticum, Clostridum papyrosolvens, Clostridum thermocellum and Clostridum phytofermentans [less ▲]Detailed reference viewed: 83 (26 ULg)
Standardized evaluation of protein stability.
Thomas, Annick ; Joris, Bernard ; Brasseur, Robert
in Biochimica et Biophysica Acta (2010)
We compare Mean Force Potential values of a large series of PDB models of proteins and peptides and find that either as monomers or polymers, proteins longer than 200-250 residues have equivalent MFP ... [more ▼]
We compare Mean Force Potential values of a large series of PDB models of proteins and peptides and find that either as monomers or polymers, proteins longer than 200-250 residues have equivalent MFP values that are averaged to -65+/-3kcal/aa. This value is named the standard or stability value. The standard value is reached irrespective of sequences and 3D folds. Peptides are too short to follow the rule and frequently exist as populations of conformers; one exception are peptides in amyloid fibrils. Fibrils surpass the standard value in accordance with their uppermost stability. In parallel, we calculate median MFP values of amino acids in stably folded PDB models of proteins: median values vary from -25 for Gly to -115kcal/aa for Trp. These median values are used to score primary sequences of proteins: all sequences converge to a mean value of -63.5+/-2.5kcal/aa i.e. only 1.5kcal less than the folded model standard. Sequences from unfolded proteins have lower values. This supports the conclusion that sequences carry in an important message and more specifically that diversity of amino acids in sequences is mandatory for stability. We also use the median amino acid MFP to score residue stability in 3D folds. This demonstrates that 3D folds are compromises between fragments of high and fragments of low scores and that functional residues are often, but not always in the extreme score values. The approach opens to possibilities of evaluating any 3D model, of detecting functional residues and should help in conducting mutation assays. [less ▲]Detailed reference viewed: 16 (3 ULg)
1,6-AnhMurNAc derivatives for assay development of amidase AmiD.
Mercier, Frédéric ; Zervosen, Astrid ; et al
in Bioorganic & Medicinal Chemistry (2010), 18(21), 7422-31
Various peptidoglycan fragments were synthesized from two anhydro-muramic acid derivatives protected with a Bn or a PMB group at the 4th position, in homogenate phase or on a solid support. In order to ... [more ▼]
Various peptidoglycan fragments were synthesized from two anhydro-muramic acid derivatives protected with a Bn or a PMB group at the 4th position, in homogenate phase or on a solid support. In order to facilitate HPLC detection, a chromophoric group was attached to the peptide chain. The periplasmic amidase sAmiD of Escherichia coli was used to cleave the amide bond between the lactyl group of the MurNAc and the alpha-amino group of L-Ala where the peptide chain was at least a dipeptide (L-Ala-gamma-D-Glu) amidated by benzylamine on the gamma-carboxyl group of D-Glu. In the presence of a tripeptide chain (L-Ala-gamma-D-Glu-L-Lys) or a tetrapeptide chain (L-Ala-gamma-D-Glu-m-A(2)pm-D-Ala) higher hydrolysis rates were observed. We have also demonstrated that the presence of TNB on the epsilon-amino group of L-Lys only has a small influence on the hydrolysis capacity of sAmiD. [less ▲]Detailed reference viewed: 213 (49 ULg)
Specific Structural Features of the N-Acetylmuramoyl-l-Alanine Amidase AmiD from Escherichia coli and Mechanistic Implications for Enzymes of This Family.
Kerff, Frédéric ; ; Mercier, Frédéric et al
in Journal of Molecular Biology (2010), 397
AmiD is the fifth identified N-acetylmuramoyl-l-alanine zinc amidase of Escherichia coli. This periplasmic lipoprotein is anchored in the outer membrane and has a broad specificity. AmiD is capable of ... [more ▼]
AmiD is the fifth identified N-acetylmuramoyl-l-alanine zinc amidase of Escherichia coli. This periplasmic lipoprotein is anchored in the outer membrane and has a broad specificity. AmiD is capable of cleaving the intact peptidoglycan (PG) as well as soluble fragments containing N-acetylmuramic acid regardless of the presence of an anhydro form or not, unlike the four other amidases, AmiA, AmiB, AmiC, and AmpD, which have some specificity. AmiD function is, however, not clearly established but it could be part of the enzymatic machinery involved in the PG turnover in E. coli. We solved three structures of the E. coli zinc amidase AmiD devoid of its lipidic anchorage: the holoenzyme, the apoenzyme in complex with the substrate anhydro-N-acetylmuramic-acid-l-Ala-gamma-d-Glu-l-Lys, and the holoenzyme in complex with the l-Ala-gamma-d-Glu-l-Lys peptide, the product of the hydrolysis of this substrate by AmiD. The AmiD structure shows a relatively flexible N-terminal extension that allows an easy reach of the PG by the enzyme inserted into the outer membrane. The C-terminal domain provides a potential extended geometrical complementarity to the substrate. AmiD shares a common fold with AmpD, the bacteriophage T7 lysozyme, and the PG recognition proteins, which are receptor proteins involved in the innate immune responses of a wide range of organisms. Analysis of the different structures reveals the similarity between the catalytic mechanism of zinc amidases of the AmiD family and the thermolysin-related zinc peptidases. [less ▲]Detailed reference viewed: 88 (19 ULg)
Interaction of ceftobiprole with the low-affinity PBP 5 of Enterococcus faecium
; Amoroso, Ana Maria ; Coyette, Jacques et al
in Antimicrobial Agents and Chemotherapy (2010), 54Detailed reference viewed: 26 (6 ULg)
Dynamics Characterization of Fully Hydrated Bacterial Cell Walls by Solid-State NMR: Evidence for Cooperative Binding of Metal Ions
; ; et al
in Journal of the American Chemical Society (2010), 132Detailed reference viewed: 71 (4 ULg)