References of "De Pauw, Edwin"
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See detailIdentification of proteins from wild cardoon flowers (Cynara cardunculus L.) by a proteomic approach
Ben Amira, Amal; Bauwens, Julien ULg; De Pauw, Edwin ULg et al

in Journal of Chemical Biology (in press)

Proteomic approach was applied to identify total proteins, particularly the enzymatic content, from wild cardoon flowers. As the selection of an appropriate sample preparation method is the key for ... [more ▼]

Proteomic approach was applied to identify total proteins, particularly the enzymatic content, from wild cardoon flowers. As the selection of an appropriate sample preparation method is the key for getting reliable results, two different extraction/precipitation methods (trichloroacetic acid and phenol/ammonium acetate) were tested on fresh and lyophilized flowers. After two-dimensional electrophoresis (2D–E) separations, a better protein pattern was obtained after phenol extraction from lyophilized flowers. Only 46 % of the total analyzed spots resulted in a protein identification by mass spectrometry MALDI-TOF. Four proteases (cardosins A, E, G, and H), which have become a subject of great interest in dairy technology, were identified. They presented molecular weights and isoelectric points very close and high levels of homology between matched peptides sequences. The absence of the other cardosins (B, C, D, and F) could be an advantage, as it reduces the excessive proteolytic activity that causes bitter flavors and texture defects, during cheese making. [less ▲]

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See detailSeparation, identification and quantification of peptidoglycan fragments by zwitterionic hydrophilic interaction chromatography and capillary electrophoresis coupled to mass spectrometry
Boulanger, Madeleine ULg; Delvaux, Cédric ULg; Raymackers, Alice ULg et al

Poster (2017, June 05)

Bacterial peptidoglycan-derived muropeptides and peptides are soluble fragments acting as messengers in diverse cell-signalling events. As the peptidoglycan wall is a key target of antibiotics, bacteria ... [more ▼]

Bacterial peptidoglycan-derived muropeptides and peptides are soluble fragments acting as messengers in diverse cell-signalling events. As the peptidoglycan wall is a key target of antibiotics, bacteria have developed specific resistance mechanisms based on the detection of these fragments inside their cytoplasm. In our model strain, Bacillus licheniformis, the peptidoglycan dipeptide m-A2pm-D-Glu triggers a beta-lactamase induction. However, the nature and the concentration of cytoplasmic peptidoglycan fragments leading to the dipeptide formation are unknown. Additionally, the muropeptides sensing is involved in the innate immune response toward bacterial invasion and is therefore of considerable importance in eukaryotes self-defence functions. In this context, the development of reliable analytical methods aiming to identify and quantify those fragments in complex samples are of major interest. [less ▲]

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See detailMass spectrometry imaging of small xenobiotics on Danio rerio : influence of molecular profiles modification as potential localization asset
Tiquet, Mathieu ULg; Muller, Marc ULg; De Pauw, Edwin ULg

Poster (2017, June 02)

MALDI Mass spectrometry often fail to locate small xenobiotics present in low concentration in tissues due to ion suppression effect. This new method compare tissues of contaminated zebrafish to controles ... [more ▼]

MALDI Mass spectrometry often fail to locate small xenobiotics present in low concentration in tissues due to ion suppression effect. This new method compare tissues of contaminated zebrafish to controles with the statistical tool called receiver operating characteristic. Results cannot directly locate the xenobiotic but can indicate which tissues are affected by the contamination and thus give a hint on the biolocalization. [less ▲]

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See detailCoulombic driven multi-conformational aspects of oligorotaxane switches studied by ion mobility mass spectrometry and molecular dynamics
Hanozin, Emeline ULg; Mignolet, Benoît ULg; Morsa, Denis ULg et al

Conference (2017, June)

Introduction Artificial Molecular Machines (AMMs), such as Mechanically Interlocked Molecules (MIMs) and foldamers, have recently raised tremendous interest due to their unique properties. Under the ... [more ▼]

Introduction Artificial Molecular Machines (AMMs), such as Mechanically Interlocked Molecules (MIMs) and foldamers, have recently raised tremendous interest due to their unique properties. Under the influence of an appropriate stimuli (pH, redox potential, light…), such molecules are able to reversibly switch between distinct conformational states. Scientists may capitalize on such exclusive properties to get a better understanding of the biomacromolecular level or to design innovative “smart” materials. At the interface between foldamers and MIMs, oligorotaxanes exhibit a spring-like folded secondary structure with remarkable mechanical and physicochemical properties. In the present study, we use ion mobility coupled with mass spectrometry (IM-MS) to probe the conformational states of differentially charged oligorotaxanes in the gas phase. Method Oligorotaxanes are donor-acceptor polymers composed of a π electron-donating dumbbell over which a discrete number of π electron-accepting tetracationic cyclophanes are threaded. The numerous intra-molecular interactions provide them a highly-stabilized rigid rod-like structure in solution. We use IM-MS as implemented in the Synapt G2 HDMS (Waters, Manchester, UK) to investigate the structure of the ionized oligorotaxanes. Our purposes are to probe (i) the different populations of stable conformers generated according to the charge state z and (ii) the reversibility of an electron-driven or thermal-driven conformational change in the gas phase implemented via an electron transfer or collisional activation process prior to the mobility separation. Our experimental observations are supported by electronic structure optimizations at the PM6 and DFT levels coupled with Born-Oppenheimer Molecular Dynamics simulations. Preliminary data Our results highlight a progressive elongation of the oligorotaxane structure with increasing charge numbers until it reaches a maximum extension state. Matching the experimental data with theoretical simulations, we find that the oligorotaxanes adopt an entropically-favored globular shape at low z. As z increases, coulombic repulsions occurring between the cyclophanes gradually outweigh the stabilizing π-stacking interactions and force the structure to elongate. This process occurs in a multistep fashion, each corresponding to a distinct group of helical-shaped conformers, before it eventually results in a fully stretched structure. On the other hand, our results also highlight that a charge reduction driven by a non-dissociative electron transfer process leads to a refolding of the structure so that it adopts a size similar to its electrospray-generated counterpart when the appropriate number of electrons is added. This observation may be imparted to the gradual decrease of the Coulomb repulsions between the cyclophanes mediated through increasing numbers of transferred electrons. These results suggests that the transition from one conformer to another is reversible so that the electrostatic balance between the cyclophanes may be used to further tune the structural state adopted by this artificial molecular switch. The second stimulus relied on collisional activation whose inelastic component provides a way to build up energy into the accessible vibrational degrees of freedom. The conformational landscapes of such-activated oligorotaxanes ions were found unchanged in term of collision cross section position but the repartition of population was altered with a promotion of the most elongated conformer, provided the absence of selective fragmentation. Altogether, these results highlight the feasibility of handling the elongation state of oligorotaxanes in the gas phase through appropriate inputs and underline its conformational reversibility properties. Novel aspect Stimuli-induced reversible conformational rearrangements of innovative AMMs studied by IM-MS and molecular dynamics in the gas phase. [less ▲]

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See detailDiscovery and characterization of EIIB, a new α-conotoxin from Conus ermineus venom by nAChRs affinity capture monitored by MALDI-TOF/TOF mass spectrometry
Echterbille, Julien; Gilles, Nicolas; Araoz, Romulo et al

in Toxicon (2017), 130

Animal toxins are peptides that often bind with remarkable affinity and selectivity to membrane receptors such as nicotinic acetylcholine receptors (nAChRs). The latter are, for example, targeted by α ... [more ▼]

Animal toxins are peptides that often bind with remarkable affinity and selectivity to membrane receptors such as nicotinic acetylcholine receptors (nAChRs). The latter are, for example, targeted by α-conotoxins, a family of peptide toxins produced by venomous cone snails. nAChRs are implicated in numerous physiological processes explaining why the design of new pharmacological tools and the discovery of potential innovative drugs targeting these receptor channels appear so important. This work describes a methodology developed to discover new ligands of nAChRs from complex mixtures of peptides. The methodology was set up by the incubation of Torpedo marmorata electrocyte membranes rich in nAChRs with BSA tryptic digests (>100 peptides) doped by small amounts of known nAChRs ligands (α-conotoxins). Peptides that bind to the receptors were purified and analyzed by MALDI-TOF/TOF mass spectrometry which revealed an enrichment of α-conotoxins in membrane-containing fractions. This result exhibits the binding of α-conotoxins to nAChRs. Negative controls were performed to demonstrate the specificity of the binding. The usefulness and the power of the methodology were also investigated for a discovery issue. The workflow was then applied to the screening of Conus ermineus crude venom, aiming at characterizing new nAChRs ligands from this venom, which has not been extensively investigated to date. The methodology validated our experiments by allowing us to bind two α-conotoxins (α-EI and α-EIIA) which have already been described as nAChRs ligands. Moreover, a new conotoxin, never described to date, was also captured, identified and sequenced from this venom. Classical pharmacology tests by radioligand binding using a synthetic homologue of the toxin confirm the activity of the new peptide, called α-EIIB. The Ki value of this peptide for Torpedo nicotinic receptors was measured at 2.2 ± 0.7 nM. [less ▲]

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See detailDiversity in sequences, post-translational modifications and expected pharmacological activities of toxins from four Conus species revealed by the combination of cutting-edge proteomics, transcriptomics and bioinformatics
Degueldre, Michel; Verdenaud, Marion; Garikoitz, Legarda et al

in Toxicon (2017), 130

Venomous animals have developed a huge arsenal of reticulated peptides for defense and predation. Based on various scaffolds, they represent a colossal pharmacological diversity, making them top ... [more ▼]

Venomous animals have developed a huge arsenal of reticulated peptides for defense and predation. Based on various scaffolds, they represent a colossal pharmacological diversity, making them top candidates for the development of innovative drugs. Instead of relying on the classical, low-throughput bioassay-guided approach to identify innovative bioactive peptides, this work exploits a recent paradigm to access to venom diversity. This strategy bypasses the classical approach by combining high-throughput transcriptomics, proteomics and bioinformatics cutting-edge technologies to generate reliable peptide sequences. The strategy employed to generate hundreds of reliable sequences from Conus venoms is deeply described. The study led to the discovery of (i) conotoxins that belong to known pharmacological families targeting various GPCRs or ion-gated channels, and (ii) new families of conotoxins, never described to date. It also focusses on the diversity of genes, sequences, folds, and PTM's provided by such species. [less ▲]

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See detailOLFM4, KNG1 and Sec24C identified by proteomics and immunohistochemistry as potential markers of early colorectal cancer stages
QUESADA-CALVO, Florence ULg; MASSOT, Charlotte ULg; Bertrand, Virginie ULg et al

in Clinical Proteomics (2017), 24(9),

Abstract Background: Despite recent advances in colorectal cancer (CRC) diagnosis and population screening programs, the identification of patients with preneoplastic lesions or with early CRC stages ... [more ▼]

Abstract Background: Despite recent advances in colorectal cancer (CRC) diagnosis and population screening programs, the identification of patients with preneoplastic lesions or with early CRC stages remains challenging and is important for reducing CRC incidence and increasing patient’s survival. Methods: We analysed 76 colorectal tissue samples originated from early CRC stages, normal or inflamed mucosa by label-free proteomics. The characterisation of three selected biomarker candidates was performed by immunohisto‑ chemistry on an independent set of precancerous and cancerous lesions harbouring increasing CRC stages. Results: Out of 5258 proteins identified, we obtained 561 proteins with a significant differential distribution among groups of patients and controls. KNG1, OLFM4 and Sec24C distributions were validated in tissues and showed differ‑ ent expression levels especially in the two early CRC stages compared to normal and preneoplastic tissues. Conclusion: We highlighted three proteins that require further investigations to better characterise their role in early CRC carcinogenesis and their potential as early CRC markers. [less ▲]

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See detailInactivation of the β(1,2)-xylosyltransferase and the α(1,3)-fucosyltransferase genes in Nicotiana tabacum BY-2 cells by a multiplex CRISPR/Cas9 strategy results in glycoproteins without plant-specific glycans
Mercx, Sébastien; Smargiasso, Nicolas ULg; Chaumont, François et al

in Frontiers in Plant Science (2017), 8

Plants or plant cells can be used to produce pharmacological glycoproteins such as antibodies or vaccines. However these proteins carry N-glycans with planttypical residues [β(1,2)-xylose and core α(1,3 ... [more ▼]

Plants or plant cells can be used to produce pharmacological glycoproteins such as antibodies or vaccines. However these proteins carry N-glycans with planttypical residues [β(1,2)-xylose and core α(1,3)-fucose], which can greatly impact the immunogenicity, allergenicity, or activity of the protein. Two enzymes are responsible for the addition of plant-specific glycans: β(1,2)-xylosyltransferase (XylT) and α(1,3)- fucosyltransferase (FucT). Our aim consisted of knocking-out two XylT genes and four FucT genes (12 alleles altogether) in Nicotiana tabacum BY-2 suspension cells using CRISPR/Cas9. Three XylT and six FucT sgRNAs were designed to target conserved regions. After transformation of N. tabacum BY-2 cells with genes coding for sgRNAs, Cas9, and a selectable marker (bar), transgenic lines were obtained and their extracellular as well as intracellular protein complements were analyzed by Western blotting using antibodies recognizing β(1,2)-xylose and α(1,3)-fucose. Three lines showed a strong reduction of β(1,2)-xylose and α(1,3)-fucose, while two lines were completely devoid of them, indicating complete gene inactivation. The absence of these carbohydrates was confirmed by mass spectrometry analysis of the extracellular proteins. PCR amplification and sequencing of the targeted region indicated small INDEL and/or deletions between the target sites. The KO lines did not show any particular morphology and grew as the wild-type. One KO line was transformed with genes encoding a human IgG2 antibody. The IgG2 expression level was as high as in a control transformant which had not been glycoengineered. The IgG glycosylation profile determined by mass spectrometry confirmed that no β(1,2)-xylose or α(1,3)-fucose were present on the glycosylation moiety and that the dominant glycoform was the GnGn structure. These data represent an important step toward humanizing the glycosylation of pharmacological proteins expressed in N. tabacum BY-2 cells. © 2017 Mercx, Smargiasso, Chaumont, De Pauw, Boutry and Navarre. [less ▲]

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See detailProteomic differential distribution of 53BP1 in serrated and conventional adenomas validated by histological characterisation
QUESADA-CALVO, Florence ULg; Merli, Angela-Maria ULg; MASSOT, Charlotte ULg et al

Poster (2017, February 10)

INTRODUCTION: Sessile serrated adenoma/polyp (SSA/p) is a precancerous lesion, mostly located in the right side of the colon (cecum, ascending and transverse colon). The difficulty is to visualize this ... [more ▼]

INTRODUCTION: Sessile serrated adenoma/polyp (SSA/p) is a precancerous lesion, mostly located in the right side of the colon (cecum, ascending and transverse colon). The difficulty is to visualize this lesion during colonoscopy because of its subtle appearance. MATERIAL AND METHOD: We compared proteomes of serrated polyps (SSA/p) and conventional adenomas using residual human formalin fixed paraffin embedded (FFPE) samples. FFPE-FASP method was applied on samples before label free proteomic analysis. Immunohistochemistry (IHC) characterisation of one candidate marker was performed for tissue validation on an independent set of samples including: conventional adenomas (low and high-grade dysplasia), serrated polyps (hyperplastic polyps, SSA/p and traditional serrated adenoma) and finally normal colon (taken at the margin of colorectal cancer (CRC) or of diverticular disease). RESULTS: Proteomics provided 765 proteins (out of 5992 proteins identified) significantly discriminating conventional adenomas from serrated lesions. We selected 53BP1 (Tumor suppressor p53-binding protein 1) among these for IHC validation, because of its tumor suppressor gene function and role as a mediator of DNA damage checkpoint. 53BP1 appeared significantly up-regulated in proteomes of low and high grade adenomas compared to these of normal tissue and SSA/p. 53BP1 IHC signal was located in the nucleus and the percentage of positive nucleus decreased in serrated polyps, especially in crypts and in the border epithelium, confirming part of the proteomic results. CONCLUSION: This study highlights potential marker proteins, including 53BP1 from which IHC signal was strongly decreased in some serrated polyps. The loss of 53BP1 has been associated with tumour progression and poor prognosis, while little is currently known about its involvement in precancerous CRC lesions. 53BP1 decrease of expression in the nucleus and therefore possible loss of function in some epithelial cells could reflect important changes occurring during dysplasia to neoplasia progression in serrated lesions. [less ▲]

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See detailIdentification of proteins discriminating inflammation induced dysplasia from simple inflammation in ulcerative colitis by laser capture microdissection and label free proteomics – a pilot study
Merli, Angela-Maria ULg; QUESADA-CALVO, Florence ULg; MASSOT, Charlotte ULg et al

Conference (2017, February 09)

Chronic colonic inflammation in ulcerative colitis (UC) may induce dysplasia, which can itself progress and transform into neoplasia. Diagnosis of dysplasia in UC remains difficult particularly when ... [more ▼]

Chronic colonic inflammation in ulcerative colitis (UC) may induce dysplasia, which can itself progress and transform into neoplasia. Diagnosis of dysplasia in UC remains difficult particularly when tissue inflammation is present. The aim of this retrospective pilot study was to highlight proteins specifically associated with inflammation induced dysplasia in UC. We performed a pilot experiment on 15 Formalin-Fixed, Paraffin-Embedded (FFPE) samples isolated from 5 cases of UC patients with a Polypoïd Pedunculated dysplasia (UC-PP). We compared the proteomes of the UC-PP, the inflammatory (UC-I) and the normal (UC-NL) tissues of each patient. We performed Laser Capture Microdissection (LCM) in order to collect only epithelial cells, avoiding inflammatory infiltrating ones. Label free proteomic analysis using a 2D-nanoUPLC coupled with a hybrid Quadrupole-Orbitrap was applied, as well as differential analysis on the paired samples. Immunohistochemistry (IHC) characterisation of one of the selected proteins of interest was used for validation. Out of 985 quantified proteins, 7 were found significantly more abundant in UC-PP compared to UC-I tissues, with 6 being only detected in UC-PP using proteomics. One of these is Solute Carrier Family 12 member 2 (SLC12A2), also known as Na-K-2Cl co-transporter 1 (NKCC1), a protein involved in ionic balance, in T-cell migration promotion and in some features involved in cancer development like proliferation, migration or invasion. IHC results obtained were in correlation with proteomic results and showed that SLC12A2 was more abundant in UC-PP tissue than in UC-I and UC-NL tissues, with a signal clearly delimiting the dysplastic region from the surrounding inflammatory tissue. This pilot experiment shows a different proteomic profile in inflammation-associated dysplasia and simple inflammation. This should be replicated using other types of dysplasia in IBD. SLC12A2 could be a potential biomarker of inflammation-associated dysplasia. [less ▲]

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See detailPeptidoglycan fragments separation and identification by zwitterionic hydrophilic interaction chromatography and capillary electrophoresis coupled to mass spectrometry
Boulanger, Madeleine ULg; Raymackers, Alice ULg; Delvaux, Cédric ULg et al

Poster (2017, February 08)

Bacterial peptidoglycan-derived peptides and muropeptides are soluble unique fragments acting as messengers in diverse cell-signalling events. As the bacterial peptidoglycan wall is a major target of ... [more ▼]

Bacterial peptidoglycan-derived peptides and muropeptides are soluble unique fragments acting as messengers in diverse cell-signalling events. As the bacterial peptidoglycan wall is a major target of antibiotics, bacteria have developed specific resistance mechanisms based on the detection of such fragments. In addition, the muropeptides sensing is involved in the innate immune response toward bacterial invasion and is therefore of major importance in the eukaryotes self-defence functions. In Bacillus licheniformis 749/I, the peptidoglycan dipeptide m-A2pm-D-Glu triggers beta-lactam resistance via the induction of a beta-lactamase, BlaP. This induction process relies on a complex regulation system for which the nature and the concentration of peptidoglycan fragments leading to the formation of dipeptide moiety inside the cytoplasm are unknown. In this context, the development and the validation of a reliable method to identify and quantify those cytoplasmic fragments is of major interest. Conventionally, the peptidoglycan is first digested by mutanolysin in order to generate muropeptides which are subsequently analyzed by reversed-phase liquid chromatography (RP-LC, C18). However, this technique is not effective enough to separate the peptides that, as a result, are eluted in the flow through . In this work, we developed two novel analytical separation methods, namely capillary electrophoresis (CE) and zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) both coupled to mass spectrometry (MS), aiming at overcoming the drawbacks encountered in traditional separation techniques. Both methods show great results in the identification of peptidoglycan fragments in complex samples. CE analysis lead to muropeptides and peptides separation whereas ZIC-HILIC only retains peptides. Nevertheless, the latter has been optimized and validated for the cytoplasmic peptidoglycan peptides identification and quantification. Althogether, ZIC-HILIC-MS and CE-MS have proved to be powerful analytical tools for the identification and quantification of peptidoglycan fragments in complex matrix samples. Further optimizations are still ongoing for the analysis of muropeptides, which hopefully will lead to the identification and quantification of cytoplasmic peptidoglycan fragments composition during the Bacillus licheniformis 749/I BlaP beta-lactamase induction process. [less ▲]

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See detailIdentification of proteins discriminating inflammation induced dysplasia from simple inflammation in ulcerative colitis by laser capture microdissection and label free proteomics – a pilot study
Merli, Angela-Maria ULg; QUESADA-CALVO, Florence ULg; MASSOT, Charlotte ULg et al

Poster (2017, February 01)

Chronic colonic inflammation in ulcerative colitis (UC) may induce dysplasia, which can itself progress and transform into neoplasia. Diagnosis of dysplasia in UC remains difficult particularly when ... [more ▼]

Chronic colonic inflammation in ulcerative colitis (UC) may induce dysplasia, which can itself progress and transform into neoplasia. Diagnosis of dysplasia in UC remains difficult particularly when tissue inflammation is present. The aim of this retrospective pilot study was to highlight proteins specifically associated with inflammation induced dysplasia in UC. We performed a pilot experiment on 15 Formalin-Fixed, Paraffin-Embedded (FFPE) samples isolated from 5 cases of UC patients with a Polypoïd Pedunculated dysplasia (UC-PP). We compared the proteomes of the UC-PP, the inflammatory (UC-I) and the normal (UC-NL) tissues of each patient. We performed Laser Capture Microdissection (LCM) in order to collect only epithelial cells, avoiding inflammatory infiltrating ones. Label free proteomic analysis using a 2D-nanoUPLC coupled with a hybrid Quadrupole-Orbitrap was applied, as well as differential analysis on the paired samples. Immunohistochemistry (IHC) characterisation of one of the selected proteins of interest was used for validation. Out of 985 quantified proteins, 7 were found significantly more abundant in UC-PP compared to UC-I tissues, with 6 being only detected in UC-PP using proteomics. One of these is Solute Carrier Family 12 member 2 (SLC12A2), also known as Na-K-2Cl co-transporter 1 (NKCC1), a protein involved in ionic balance, in T-cell migration promotion and in some features involved in cancer development like proliferation, migration or invasion. IHC results obtained were in correlation with proteomic results and showed that SLC12A2 was more abundant in UC-PP tissue than in UC-I and UC-NL tissues, with a signal clearly delimiting the dysplastic region from the surrounding inflammatory tissue. This pilot experiment shows a different proteomic profile in inflammation-associated dysplasia and simple inflammation. This should be replicated using other types of dysplasia in IBD. SLC12A2 could be a potential biomarker of inflammation-associated dysplasia. [less ▲]

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See detailHigh-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery
Turchetto; Sequeira, Ana Filipa; Ramond, Laurie et al

in Microbial Cell Factories (2017), 16(6), 1-15

Animal venoms are complex molecular cocktails containing a wide range of biologically active disulphide-reticulated peptides that target, with high selectivity and efficacy, a variety of membrane ... [more ▼]

Animal venoms are complex molecular cocktails containing a wide range of biologically active disulphide-reticulated peptides that target, with high selectivity and efficacy, a variety of membrane receptors. Disulphide-reticulated peptides have evolved to display improved specificity, low immunogenicity and to show much higher resistance to degradation than linear peptides. These properties make venom peptides attractive candidates for drug development. However, recombinant expression of reticulated peptides containing disulphide bonds is challenging, especially when associated with the production of large libraries of bioactive molecules for drug screening. To date, as an alternative to artificial synthetic chemical libraries, no comprehensive recombinant libraries of natural venom peptides are accessible for high-throughput screening to identify novel therapeutics. RESULTS: In the accompanying paper an efficient system for the expression and purification of oxidized disulphide-reticulated venom peptides in Escherichia coli is described. Here we report the development of a high-throughput automated platform, that could be adapted to the production of other families, to generate the largest ever library of recombinant venom peptides. The peptides were produced in the periplasm of E. coli using redox-active DsbC as a fusion tag, thus allowing the efficient formation of correctly folded disulphide bridges. TEV protease was used to remove fusion tags and recover the animal venom peptides in the native state. Globally, within nine months, out of a total of 4992 synthetic genes encoding a representative diversity of venom peptides, a library containing 2736 recombinant disulphide-reticulated peptides was generated. The data revealed that the animal venom peptides produced in the bacterial host were natively folded and, thus, are putatively biologically active. CONCLUSIONS: Overall this study reveals that high-throughput expression of animal venom peptides in E. coli can generate large libraries of recombinant disulphide-reticulated peptides of remarkable interest for drug discovery programs. [less ▲]

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See detailProteomic response of Macrobrachium rosenbergii hepatopancreas exposed to chlordecone: Identification of endocrine disruption biomarkers?
Lafontaine, Anne ULg; Baiwir, Dominique ULg; Joaquim-Justo, Célia ULg et al

in Ecotoxicology and Environmental Safety (2017)

The present work is the first study investigating the impacts of chlordecone, an organochlorine insecticide, on the proteome of the decapod crustacean Macrobrachium rosenbergii, by gel-free proteomic ... [more ▼]

The present work is the first study investigating the impacts of chlordecone, an organochlorine insecticide, on the proteome of the decapod crustacean Macrobrachium rosenbergii, by gel-free proteomic analysis of the hepatopancreas protein expression variations in organisms exposed to three environmental relevant concentrations of chlordecone (i.e. 0.2, 2 and 20 μg/L). Results revealed that 62 proteins were significantly up- or down-regulated in exposed prawns compared to controls. Most of these proteins are involved in important physiological processes such as ion transport, defense mechanisms and immune system, cytoskeleton dynamics, or protein synthesis and degradation. Moreover, it appears that 6% of the deregulated protein are involved in the endocrine system and in the hormonal control of reproduction or development processes of M. rosenbergii (e.g. vitellogenin, farnesoic acid omethyltransferase). These results indicate that chlordecone is potentially an endocrine disruptor compound for decapods, as already observed in vertebrates. These protein modifications could lead to disruptions of growth and reproduction of M. rosenbergii, and therefore of the fitness population on the long-term. Besides, these disrupted proteins could be suggested as biomarkers of exposure for endocrine disruptions in invertebrates. However, further investigations are needed to complete understanding of action mechanisms of chlordecone on proteome and endocrine system of crustaceans. [less ▲]

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See detailProteomic signatures reveal a dualistic and clinically relevant classification of anal canal carcinoma
Herfs, Michael ULg; Longuespée, Rémi ULg; Quick, Charles et al

in Journal of Pathology (The) (2017), 241

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See detailEcotoxicoproteomic assessment of the functional alterations caused by chronic metallic exposures in gammarids
Gismondi, Eric ULg; Thomé, Jean-Pierre ULg; Urien, Nastassia et al

in Environmental Pollution (2017)

Very few ecotoxicological studies have been performed on long-term exposure under controlled conditions, hence limiting the assessment of the impact of chronic and diffuse chemical pressures on the health ... [more ▼]

Very few ecotoxicological studies have been performed on long-term exposure under controlled conditions, hence limiting the assessment of the impact of chronic and diffuse chemical pressures on the health of aquatic organisms. In this study, an ecotoxicoproteomic approach was used to assess the integrated response and possible acclimation mechanisms in Gammarus fossarum following chronic exposures to Cd, Cu or Pb, at environmentally realistic concentrations (i.e. 0.25, 1.5 and 5 µg/L respectively). After 10-week exposure, changes in protein expression were investigated in caeca of control and exposed males. Gel-free proteomic analyses allowed for the identification of 35 proteins involved in various biological functions, for which 23 were significantly deregulated by metal exposures. The protein deregulation profiles were specific to each metal, providing evidence for metal-specific action sites and responses of gammarids. Among the tested metals, Cu was the most toxic in terms of mortality, probably linked with persistent oxidative stress. Moulting and osmoregulation were the major biological functions affected by Cu in the long-term. In Pb-exposed gammarids, significant deregulations of proteins involved in immune response and cytoskeleton were observed. Reproduction appears to be strongly affected in gammarids chronically exposed to Cd or Pb. Besides, modified expressions of several proteins involved in energy transfer and metabolism highlighted important energetic reshuffling to cope with chronic metal exposures. These results support the fact that metallic pressures induce a functional and energetic cost for individuals of G. fossarum with potential repercussions on population dynamics. Furthermore, this ecotoxicoproteomic study offers promising lines of enquiry in the development of new biomarkers that could make evidence of long-term impacts of metals on the health of organisms. [less ▲]

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See detailIdentification and quantification of peptidoglycan cytoplasmic fragments by LC-MS
Boulanger, Madeleine ULg; Raymackers, Alice ULg; De Pauw, Edwin ULg et al

Poster (2016, November 16)

In Bacillus licheniformis 749/I, the induction of BlaP beta-lactamase relies on a complex regulation system. During this process, the intracellular repressor BlaI is inactivated by a dipeptide coming from ... [more ▼]

In Bacillus licheniformis 749/I, the induction of BlaP beta-lactamase relies on a complex regulation system. During this process, the intracellular repressor BlaI is inactivated by a dipeptide coming from the peptidoglycan (PG) degradation via an “AND Gate” regulation. This regulation involves the cellular stress induced by the beta-lactam, the membrane receptor BlaR1 and the PG turnover. Briefly, the induction occurs when the extracellular domain of BlaR1 is acylated by the antibiotic which leads to a reorganization of the transmembrane segments and the receptor autocleavage. Simultaneously, the beta-lactam partially inhibits the penicillin-binding protein 1 (PBP1), triggering increased PG turnover and accumulation of PG fragments. Some of these fragments could enter in the cytoplasm and undergo enzymatic degradations which lead to the formation of the pro co-activator (tripeptide L-Ala-D-Glu-m-A2pm). This pro co-activator generates the co-activator, the dipeptide D-Glu-m-A2pm. Nowadays the nature and the concentration of PG fragments inside the cytoplasm are unknown. Therefore, the development and the validation of a zwitterionic hydrophilic interaction liquid chromatography coupled to electrospray ionization mass spectrometry method (ZIC-HILIC-MS) are required in order to identify and quantify those cytoplasmic fragments. [less ▲]

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